Annual Reports in Medicinal Chemistry, Volume 12

ANNUAL REPORTS IN MEDICINAL CHEMISTRV Volume 1 2

CONTRIBUTORS

. . . . . . . . . . 298 Baum.T . . . . . . . . . . . . 39 Behling. . R . . . . . . . . . . 309 Beisler. J . A . . . . . . . . . . 120 Bergey. J . L . . . . . . . . . . 39 Bennett. . . . . . . . . . . . 10 Bonney. R . J . . . . . . . . . . 152 Cheng. L . . . . . . . . . . . . 191 Corcoran. J . W . . . . . . . . . 130 Creese. . . . . . . . . . . . . 249 Creger. P. . . . . . . . . . . 278 Davenport. L . . . . . . . . . . 110 Davies. P . . . . . . . . . . . . 152 D r i s c o l l . J . . . . . . . . . . 120 Dybas. R . A . . . . . . . . . . . 234 F i s h e r . . . . . . . . . . . . 140 Flarmn. W . . . . . . . . . . . . 234 Furukawa. T . . . . . . . . . . . 260 Gold. P. . . . . . . . . . . . 30 Gordon. M. . . . . . . . . . . . 20 Gund. P . . . . . . . . . . . . . 288 Hamilton. J . . . . . . . . . . 191 Hauth. H . . . . . . . . . . . . 49 Hinman. . W . . . . . . . . . . 223 H i t e . M. . . . . . . . . . . . . 234 Hoeksema. H . . . . . . . . . . . 110 Hohnke. . A. . . . . . . . . . 9 1 Houlihan. W . J . . . . . . . . . . 10

Abushanab. E

J

G

B

I

L

C

S

M

H

G

E

G

J

L

Jones. J

. B. . . . . . . . . .

............ Kohn. L . D . . . . . . . . . . . L a h t i . R. A . . . . . . . . . . L i p i n s k i . C. A. . . . . . . . . MacKenzie. R . D . . . . . . . . McCandlis. R . P. . . . . . . . McIlhenny. H . M . . . . . . . . Miller. L . L . . . . . . . . . . Nelson. . D. . . . . . . . . . Oronsky. A . L . . . . . . . . . Kariv. E

S

298 309 211

1 91 80 223 201 309 319

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. B. . . . . . . . . . 260 Pohl. L . R . . . . . . . . . . . 319 49 Richardson. B. P . . . . . . . . Schaaf. T . K . . . . . . . . . . 182 60 Shaw. A . . . . . . . . . . . . Sheppard. H . . . . . . . . . . 172 Sih. C . J . . . . . . . . . . . 298 Snyder. S . H . . . . . . . . . . 249 Spatz. D . M. . . . . . . . . . 268 S u l l i v a n . A . C . . . . . . . . . 191 Thornber. . W . . . . . . . . . 60 20 Vida. J . A . . . . . . . . . . . 162 Voorhees. J . J . . . . . . . . . Wang. C . . . . . . . . . . . . 140 70 Wasley. J . W . F . . . . . . . . Webber. J . A . . . . . . . . . 101 Wendt. R . L . . . . . . . . . . 39 Peter. J

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ANNUAL REPORTS IN MEDICINAL CHEMISTRY Volume 1 2 Sponsored by the Division of Medicinal Chemistry of the American Chemical Society Editor-in- Chief: FRANK H. CLARKE CIBA-GEIGY CORPORATION ARDSLEY. NEW YORK

SECTION EDITORS J O H N KRAPCHO

0

J O H N FRANCIS

HANS-JURGEN HESS

0

T. Y. SHEN

ACADEMIC PRESS

@

0 0

GEORGE WHITFIELD RAYMOND COUNSELL

N e w York

A Subsidiary of Harcourt Brace Jovanovich, Publishers

San Francisco

London

1977

Academic Press Rapid Manuscript Reproduction

COPYRIGHT 0 1977, n Y ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. N O PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS. ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, W I T H O U T PERMISSION I N WRITING F R O M T H E PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWI

LIBRARY OF CONGRESS CATALOG C A R D NUMBER: 66-26843

ISBN 0-1 2-04051 2-1 PRINTED IN THE UNITED STATES O F AMERICA

CONTENTS

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Contributors

ii

Preface

xi

I.

CNS AGENTS

Section Editor: John Krapcho, Squibb Institute, Princeton, New Jersey

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1

1.

Antidepressants and Antipsychotic Agents Robert A.. Lahti, The Upjohn Company, KBlamazoo, Michigan

2.

Anti-Anxiety Agents, Anticonvulsants and Sedative-Hypnotics William J. Houlihan and Gregory B. Bennett, Pharmaceuticals Div., Sandoz, Inc., East Hanover, New Jersey

3.

Analgesics, Antagonists, the Opiate Receptor and Endogenous Opioids.. M. Gordon and J. A. Vida, Bristol-Myers Company, Syracuse, New York

4.

Memory and Learning .Animal Models 30 Paul E. Gold, University of Virginia, Charlottesville, Virginia

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

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11.

PHARMACODYNAMIC AGENTS

Section Editor: John E. Francis, CIBA-GE1G.Y Corporation, Ardsley, New York

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5.

Antiarrhythmic and Antianginal Agents Thomas Baum, Robert L. Wendt and James L. Bergey, Wyeth Laboratories, Inc., Radnor, Pennsylvania

6.

Cerebral Trasodilators H. Hauth and B. P. Richardson, Sandoz Ltd., 4002 Basle, Switzerland

7. Antihypertensive Agents

39

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49

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60

Craig W. Thornber and Andrew Shaw, Pharmaceuticals Division, ICI Ltd., Macclesfield, Cheshire, England

vi CONTENTS

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8. Pulmonary and Anti-Allergy Drugs Arnold L. Oronsky" and Jan W. F. Wasley, Pharmaceuticals

70

Division, CIEiA-GEIGY Corp., Summit, New Jersey *Present Address: Lederle Laboratories, Pearl River, New York 9.

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Antithrombotic Agents Robert D. MacKenzie, Merrell-National Laboratories, Division of Richardson-Merrell Inc., Cincinnati, Ohio

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10. Agents Affecting Gastrointestinal Functions Christopher A. Lipinski and Lyle A. Hohnke, Pfizer Inc., Groton, Connecticut

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91

111. CHEMOTHERAPEUTIC AGENTS

Section Editor: George B. Whitfield, The Upjohn Company, Kalamazoo, Michigan

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11.

p-Lactam Antibiotics J. Alan Webber, Eli Lilly and Company, Indianapolis, Indiana

12.

110 Aminocyclitol and Other Antibiotics Herman Hoeksema and Lorraine C. Davenport, The Upjohn Company, Ka lamaz oo , Michigan

13.

Antineoplastic Agents John S . Driscoll and John A. Beisler, National Cancer Institute, N .I.H. , Bethesda, Maryland

14.

Biosynthesis of Antibiotics John W. Corcoran, Department of Biochemistry, Northwestern University Medical and Dental Schools, Chicago, Illinois

15.

Antiparasitic Agents C. C. Wang and M. H. Fisher, Merck Sharp Laboratories, Rahway, New Jersey

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120

. . . . . . . . . . . . . . . . . . . 130

. . . . . . . . . . . . . .Dohme . . .Research ..... &

140

vi i CONTENTS

IV. Section Editor:

METABOLIC DISEASES AND ENDOCRINE FUNCTION Hans-J'Jrgen Hess, Pfizer Inc., Groton, Connecticut

16. Cellular Responses Mediating Chronic Inflammatory Diseases Philip Davies and Robert J. Bonney, Merck Institute for Therapeutic Research, Rahway, New Jersey 17.

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Molecular Mechanisms and Pharmacological Modulation in Psoriasis John J. Voorhees, University of Michigan Medical School, Ann Arbor, Michigan

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162

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172

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182

Activators of Dopamine and f3-AdrenergicAdenylate Cyclases Herbert Sheppard, Hoffmann-La Roche, Inc., Nutley, New Jersey

19. Modulation of the hrachidonic Acid Cascade Thomas K. Schaaf, Pfizer, Inc., Groton, Connecticut 20.

152

Recent Advances in the Etiology and Treatment of Disorders of Lipid Metabolism Ann C. Sullivan, Lorraine Cheng and James G. Hamilton, Roche Research Center, Hoffmann-La Roche, Inc., Nutley, New Jersey

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191

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21.

Drug Metabolism Hugh M. McIlhenny, Pfizer Inc., Groton, Connecticut

V. Section Editor:

T.Y. Shen, Merck

Section Editorial 22.

TOPICS IN BIOLOGY &

Company, Rahway, New Jersey

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Relationships in the Structure and Function of Cell Surface Receptors for Glycoprotein Hormones, Bacterial Toxins, and Interferon Leonard D. Kohn, Sect. Biochem. of Cell Regulation, Lab Biochem. Pharmacol, Nat. Inst. Arthritis, Metab. & Digestive Dis., N .I.H., Bethesda, Maryland

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210

211

viii CONTENTS

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23.

Mineral Metabolism and Metabolic Bone Disease J. W. Hinman and R. P. McCandlis, The Upjohn CO., Kalamazoo , Michigan

24.

Detecting Mutagens Correlations Between the Mutagenicity and Carcinogenicity of Chemicals R. A. Dybas, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey M. Hite, Merck Institute for Therapeutic Research, West Point, Pennsylvania W. Gary Flamm, National Cancer Institute, Bethesda, Maryland

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223

234

25.

Brain Neurotransmitter Receptor Binding and Neuroleptic Drugs 249 Ian Creese and Solomon H. Snyder, Department of Pharmacology and Experimental Therapeutics, Johns Hopkins University School of Medicine, Baltimore, Maryland

26.

Biochemical Aspects of Muscular Disorders 260 James B. Peter, Clinical Immunology Laboratories, Inc. , Santa Monica, California; Department of Medicine, UCJA School of Medicine, Los Angeles, California and Tetsuo Furukawa, Department of Neurology, University of Tokyo, Tokyo, Japan

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VI. TOPICS IN CHEMISTRY Section Editor:

R. E. Counsell, University of Michigan, Ann Arbor, Michigan

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268

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278

27.

Reactions of Interest in Medicinal Chemistry David M. Spatz., Pharmaceutical R&D, Dow Chemical U.S.A., Midland , Michigan

28.

Synthetic Applications of Metalated Carboxylic Acids P. L. Creger, Parke, Davis & Co. , Ann Arbor, Michigan

29.

Computer-assisted Organic Synthetic Analysis Peter Gund, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey

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288

ix CONTENTS

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298

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309

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30.

, , , , Biochemical Procedures in Organic Synthesis Charles J. Sih and Elie Abushanab, School of Pharmacy, University of Wisconsin, Madison, Wisconsin and J. Bryan Jones, Dept. of Chemistry, University of Toronto, Ontario, Canada

31.

Organic Electrosynthesis Larry L. Miller and Esther Kariv, Department of Chemistry, University of Minnesota, Minneapolis, Minnesota and James R. Behling, Department of Chemical Development, Searle Laboratories, Chicago, Illinois

32.

. . The Use of Stable Isotopes in Medicina Chemistry . Sidney D. Nelson and Lance R. Pohl, Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, Bethesda, Maryland

COMPOUND NAME AND CODE NUMBER INDEX

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Annual Reports continues to provide timely reviews on traditional aspects of medicinal chemistry and seeks to describe new approaches to medicinal research which are likely to have increasing importance for drug discovery and development. Many of the chapters discuss the mechanisms of action of drugs and present concepts of the nature of disease which may assist in the design of agents for its control. The importance of drug metabolism for medicinal chemists i s emphasized this year with one chapter describing its use in drug discovery and development and a second chapter on the use of stable isotopes in drug metabolism. A chapter on animal models for memory and learning augments the more traditional CNS topics. Cerebral vasodilators are described this year in the section on pharmacodynamic agents. Other new topics include discussion of the biosynthesis of antibiotics, psoriasis, adenylate cyclases, biochemical procedures for syntheses and computer assisted syntheses. A s expressed in the section editorial in Topics & Biology, chapters are included which describe pathophysiology at the molecular and biochemical level. Of special interest is the chapter on tests for the detection of mutagens and the correlation between mutagenicity and carcinogenicity of chemicals. The Editors of Annual Reports in Medicinal Chemistry continue to welcome comments from its readers. We are especially interested in suggestions for new topics which will make future volumes more useful.

Ardsley, New York June, 1977

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Section I - CNS Agents Editor:

John Krapcho, Squibb Institute, Princeton, N.J.

Chapter 1. Antidepressants and Antipsychotic Agents Robert A. Lahti, The Upjohn Company, Kalamazoo, Michigan

Introduction - This review article covers the pharmacological, biochemical and clinical advances during the past year in the area of antidepressant and antipsychotic agents. Past issues of this series l s 2 as well as reviews of greater depth are recommended to the reader. 3-5 Antidepressant Agents - The past year has produced a number of agents which block either serotonin (5-HT) or norepinephrine (NE) uptake selectively. This direction is an extension of the interest in subclasses of depression based on altered excretion rates of indoles or catechols in depressed patients.6 Pirandamine (L), an indenopyran, has been shown to be a very selective blocker of 5-HT uptake7 using the blockade of brain 5-HT depletion by H75/12 (4-methyl-alpha-ethyl-m-tyramine). A closely related compound, tandamine (2), which is a thiopyranoindole, is a strong blocker of NE uptake but has no effect on 5-HT uptakes and only weakly potentiates the effects of 5-HTP.9 YH3 &:2-CH2-N(CH 312

/

Synthesis of the tandamine analog (3) containing an N-methyl group produced a compound with strong NE uptake blocking activity as well as a strong potentiator of 5-HTP.' A pyranoindole analog (5) of pirandamine was found to be a strong blocker of NE uptake in contrast to pirandamine, but retained the 5-HTP potentiating activity.' Interestingly, the thiopyranoindole (2) and the pyranoindole (5) had very similar pharmacological activity. CH CF3 ,CH2-CH2-N(CH3l2 \3 3 x = s

4

x - 0

Other variations of tandamine (2) in the form of cycloalkylindoles proved to be of considerable interest.lo The structurally closest analog (5) to tandamine was very potent in reversing reserpine ptosis, while the cycloheptyl (5) and cyclopentyl (7) derivatives were progressively weaker

Sect. I

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CNS Agents

Krapcho, Ed.

in reversing reserpine-induced ptosis. All three of these compounds were more potent than imipramine or amitriptyline in reversing ptosis. CH I3 -I"

\""

3' 2

A number of variations of a tricyclic indole structure were prepared. Compound (g) was comparable in activity to imipramine in reversing ptosis and anticholinergic activity. Compound (2), which has the terminal Nbenzyl function, was active in reversing reserpine ptosis and showed no anticholinergic and antihistaminic activity. Compound (10)was significant in that it demonstrated the importance of the location of the side chain, this form being inactive. "

The spiroisobenzofuran piperidine derivative (11)has potency greater than imipramine in reversing tetrabenazine ptosis. l2 H

c1 11 -

Org 6 5 8 2 (12)was found to be a strong, long lasting blocker of 5-HT uptake, five times chlorimipramine, with no apparent effect on NE uptake systems. Compound 12 also caused a decrease in 5-HT turnover and lowered brain 5 - H I M (5-hydroxyindole acetic acid). Deviations from the standard tricyclic structures were frequent during this period as demonstrated by DIV-154 (13)which antagonized reserpine, potentiated the effects of NE, and had little anticholinergic activity.'4 Deximafen (14) was reported to be a potent reserpine and tetrabenazine antagonist with little overt activity of its own.

'=

Chap. 1

Lahti

Antidepressants and Antipsychotic Agents

2

H I

A series of benzofuranyl amidoximes was found to inhibit reserpine ptosis, potentiate yohimbine toxicity, and potentiate the effects of amphetamine. la Compound (15)appeared to be the most interesting structure.

Of a number of phenylcycloalkylamines, compounds (16)and (17) had similar activity in blocking NE and 5-HT uptake and in potentiating the behavioral effects of DOPA (3,4-dihydroxyphenylalanine) and 5-HTP (5hydroxytryptophan). However, compound ( 2 1 , a cyclohexylamine, was inactive except for DOPA potentiation and the cyclohexeneylamine (19)retained l7 the activity of the cyclopentane analogs (16)and (2).

(a)

H-102-09 was tested in parallel with chlorimipramine for its 5HT uptake blocking activity as measured by observing decreases in 5-HT in whole blood.18 Compound 2 was more potent than chlorimipramine in reducing blood 5-HT and initial clinical studies suggest good antidepressant activity. The antianxiety agent, trazodone (G), has also been found to be a blocker of 5-HT uptake into rat brain synaptosomes. Is The activity of trazodone was slightly less than that of chlorimipramine, but it had greater selectivity when NE uptake inhibition was determined.

4

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Br

A drug metabolism study in man20showed that lofepramine (22) was rapidly converted to desmethylimipramine, which probably accounts for the observed blockade of NE uptake.

Numerous studies have attempted to define biochemical parameters which would assist in selecting the method of treatment of depression. Subgroups of depression have been defined by urinary MHPG 21 (3-methoxy-4hydroxyphenylglycol) excretion rates and by the levels of biogenic amines or their metabolites in CSF 22(cerebrospinal fluid). Low urinary MHPG excretors respond well to imipramine, desmethylimipramine and nortriptyline, whereas high MHPG excretors respond well to amitriptyline. 6 9 2 3 Patients with low CSF 5-HIAA respond well to chlorimipramine or 5-HTP but not to nortriptyline. It has been shown that some unipolar depressed female patients have high COMT (catechol-0-methyl transferase) activity in their red blood cells, and that this observation correlates with a poor response to imipramine. 24 On the other hand, high COMT. activity may correspond to high MHPG excretion. This finding supports the previous disclosure that low MHPG excretors respond to imipramine. During the past year several studies have appeared in the literature bearing on the relationship of 5-HT and/or its precursor to depression. Shopsin et al., in two studies, were able to show that the positive therapeutic response of depressed patients to imipramine25 or tranylcyromine 26 could be reversed by co-administration of the tryptophan hydroxylase inhibitor PCPA (p-chlorophenylalanine). These same investigators could not show any effect of alpha-methyltyrosine, a tyrosine hydroxylase inhibitor, on the positive clinical response to imipramine.25 These data seem to indicate that serotonin, rather than catecholamines, is involved in depression. It was reported 27that lithium, ECT and tricyclic antidepressants were all associated with a decreased accumulation of 5-HIAA in CSF of probenecid-treated patients, suggesting that antidepressant therapy may alter

Chap. 1

Antidepressants and Antipsychotic Agents

Lahti

5

5-HT turnover. Others 28 have shown that chlorimipramine lowers CSF 5-HIM in depressed patients, and it was reported that chlorimipramine, amitriptyline, and desipramine all reduce blood tryptophan levels. Earlier it had been mentioned that chlorimipramine lowered whole blood 5-HT. la It has also been shown that chlorimipramine and tryptophan were better in treating depression than chlorimipramine alone,28 however elsewhere it was reported that no significant difference between any of the following treatment modalities was found: chlorimipramine, chlorimipramine plus tryptophan, desipramine plus tryptophan, and chlorimipramine plus desipramine. Investigators30 found no distinct relationship between either total plasma tryptophan or tyrosine and depression. However , they 30 did report that free plasma tryptophan in depressed patients was always higher than controls and returned to normal upon good clinical improvement. In contrast, other investigators found lower plasma tryptophan levels in depressed patients than they did in controls. Utilizing platelets from depressed patients, it was shown 32 that a significant reduction in mean 5-HT and DA uptake occurred in endogenously depressed patients but not in neurotic depressives. In this same study32 it was reported that chlorimipramine caused a rapid reduction in 5-HT uptake, whereas protriptyline administration had no effect. Tuomisto and T~kiainen~~studied the platelet 5-HT uptake system in a well-defined study and found that 5-HT uptake in platelets from depressed patients had one-half the initial uptake value and Vmax of normals; Kms were the same for both groups. Following antidepressant treatment, the Vmax moved towards normal, and the Km remained the same. These results were interpreted to mean that there are less 5-HT transport molecules or some inactive ones in membranes of depressed patients, but those 5-HT transport molecules which are there are completely functional, as demonstrated by the constant Km. Clinical improvement was accompanied by a return of Vmax to normal. Of most interest was the finding that imipramine added in vitro to the platelets increased the Km but had no effect on the Vmax. In summary, the numerous clinical studies cited here and reviewed elsewhere34do little to strengthen the catecholamine hypothesis of depression. The profusion of data relating tryptaminergic systems to depression 31-33 is most interesting and hopefully reflects progress in this area. The abundance of new compounds which are selective NE or 5-HT uptake blockers should do much to confirm or deny the role of biogenic amines in depression and the significance of blocking amine uptake as a mode of action of antidepressants. Antipsychotic Agents - In acute schizophrenia patients, the potent, longtic activity and a high lasting tranquilizer, AL 1965 (23) showed neurole incidence of extrapyramidal side effects (EPS). 32

-6

Sect. I

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Krapcho, Ed.

In acute schizophrenic patients, MJ9022 (24) was a weak, shortlasting neuroleptic. 36 Clinical activity correlated with the blocking of amphetamine stereotypy in dogs.

0

(a),

A simple butyrophenone, azabuperone was found to produce catalepsy, give a positive response in conditioned avoidance responding, and block amphetamine stereotyped behavior in rats. The compound is currently being used clinically as an antipsychotic agent. 37

n 0

25 -

A series of novel neuroleptic agents derived from alpha-tetralone and N-arylpiperazine has been prepared.38 Moderate activity in inhibiting amphetamine hypermobility and in producing hypothermia was found, with compound (26) being the most active. Another N-arylpiperazine analog (27) also had neuroleptic activity in antagonizing amphetamine and apomorphine stereotypy and production of catalepsy. 3s

DBN (28) is an inhibitor of indoleamine-N-methyltransferase and effectively blocks the synthesis of N,N'-dimethyltryptamine (DMT) in vitro and vivo.40 DMT has been considered as an endogenous psychotogenic agent.4Therefore, based on a rationa approach to drug therapy, compound 8 may be an antipsychotic agent.

to

Chap. 1

Antidepressants and Antipsychotic Agents

Lahti

7

Sulpiride (3) has been tested as an antipsychotic agent 4 2 9 4 3 and is atypical in its pharmacological profile. Sulpiride does not block DAstimulated adenylate cyclase either in vitro or in 44 it does not produce catalepsy 4 5 nor antagonize amphetarnineP5- Sulpiride does block apomorphine-induced emesis in dogs44 but only weakly antagonizes other effects of a p ~ m o r p h i n e ,yet ~ ~ it causes an increased turnover of DA vivo. 4 5

w,

The 3H-haloperidol binding assay 4 7 * 4 8 has proven to be a most effective in vitro test system for detecting neuroleptic agents (see Chapter 25). Results from this test correlate well with human clinical doses, 4 7 * 4 8 inhibition of apomorphine stereotypy,47 inhibition of apomorphine-induced emesis in dogs4? and inhibition of amphetamine stereotyped behavior in rats. 4 7 3H-Haloperidol binding data did not correlate well with inhibition of DA-stimulated adenylate cyclase 49 nor with the inhibition of 3H-DA binding. 4 7 ~ 4 8The lack of correlation between inhibition of 3H-haloperido1 and 3H-dopamine binding may be indicative of a two-state model of the receptor49or it may indicate that the two receptors are not the same. 48 The good correlations between most neuroleptic test methods and human clinical dose may reflect the strength of the DA hypothesis 500r the degree to which the hypothesis has not been tested. Although much evidence supports the DA hypothesis of schizophrenia, there have been some viable concerns regarding the theory, such as: the clinical efficacy of clozapine as related by Burki et al., 51 the time dependent effect of neuroleptics on dopamine turnover in psychiatric patients 5 2 and the lack of correlation between results obtained with neuroleptic agents in the 3H-haloperidol and 3H-dopamine binding systems. 4 7 * 4 8 Also, the dopamine blockade hypothesis may explain the effectiveness of the neuroleptic drugs, however, there is little evidence to support DA overactivity in schizophrenia. 53 References

1. P.F. VonVoigtlander, In Annual Reports Medicinal Chemistry, Vol. 11, F.H.Clarke, ed., Academic Press, N.Y., 1976, pp 3-12. I Medicinal Chemis2. W.M. Welch and C.A. Harbert, In Annual Reports & try, Vol. 10, R.V. Heinzelman, ed., Academic Press, N.Y., 1975, pp 2-11. 3. S.W. Matthyse and S.S. Kety, eds. Catecholamines and Schizophrenia, Pergamon Press, Oxford, 1975. 4 . D.M. Gallant, ed. Depression, Spectrum Publications, N.Y., 1976.

-8 5.

6. 7.

a. 9. 10. 11. 12. 13.

14. 15. 16. 17.

18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

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B.I. Hoffrand and G.F.B. Birdwood, eds., Postgrad. Med. J . , 2, Suppl. 3, (1976). J. Mendels, S. Stern and A. Frazer, Dis. Nerv. Syst., 37, 3 (1976). W. Lippmann and T.A. Pugsley, Pharmacol. Res. Corn., 8, 387 (1976). T. Pugsley and W. Lippmann, Psychopharmacologia, 47,33 (1976). W. Lippmann and T.A. Pugsley, Biochem. Pharmacol., 2, 1179 (1976). A.A. Asselin, L.G. Humber, J. Komlossy and M.-P. Charest, J. Med. Chem. , 2, 792 (1976). L. Toscano, G. Grisanti, G. Fioriello and E. Seghetti, J. Med. Chem:, 19, 208 (1976). V.J. Baurer, B.J. Duffy, D. Hoffman, S.S. Klioze, R.W. Kosey, Jr., A.R. McFadden, L.L. Martin, H.H. Ong and H.M. Geyer 111, J. Med. Chem. , 9, 1315 (1976). M.F. Sugrue, I. Goodlet and S.E. Mireylees, Europ. J. Pharmacol., 40, 121 (1976). I.J.E. Boksay and R.O. Weber, Arch. Pharmacol., 293, Suppl. R12 (1976) F.C. Colpaert, F.M. Lenaerts, C.J.E. Niemegeers and P.A.J. Janssen, Arch. int. Pharmacodyn., 215, 40 (1975). A. Areschka, J.M. Mahaux, F. Verbruggen, C. Houben, M. Descamps, M. Broll, J.P. Werbenec, R. Charlier, J. Simiand and P. Eymard, Europ. J. Med. Chem.-Chimica Therapeutica, 2, 398 (1975). B. Carnmalm, T. DePaulis, E. Jacupovic, L. Johansson, U.H. Lindberg, B. Ueff, N.E. Stjernstrom, A.L. Renyi, S.B. Ross and S.O. Ogren, Acta Pharm. Suec., 2, 149 (1975). S.B. ROSS, S. Jansa, L. Wetterberg, B. Fyro and B. Hellner, Life Sci. 19, 205 (1976). E. Stefanini, F. Fadda, L. Medda and G.L. Gessa, Life Sci., 18,1459 (1976). G.P. Forshell, B. Sievers and J.R. Tuck, Europ. J. Clin. Pharmacol., 9, 291 (1976). E. Sacchetti, E. Smeraldi, M. Cognasso, P.A. Biondi and L. Bellodi, Intern. Pharmacopsychiat., 11,157 (1976). M. Asberg, P. Thoren, L. Traskman, L. Bertilsson and V. Ringberger, Sci. , 191,478 (1976). A.P. Ridges, Postgrad. Med. J., 52, Suppl. 3 , 9 (1976). J.R.T. Davidson, M.N. McLeod, H.L. White and D. Raft, Am. J. Psychiat., 133, 952 (1976). B. Shopsin, S. Gershon, M. Goldstein, E. Friedman and S . Wilk, Psychopharmacol. Corn., 1,239 (1975). - B. Shopsin, E. Friedman and S. Gershon, Arch. Gen. Psychiat., 33, 811 (1976). F.K. Goodwin, R.M. Post and T. Wehr, Agents and Actions, 5, 498 (1975). J. Walinder, A. Skott, A. Carlsson, A. Nagy and B.-E. Ross, Arch. Gen. Psychiat., 3,1384 (1976). D.M. Shaw, Postgrad. Med. J . , Suppl. 3 , 47 (1976). P. Niskanen, M. Huttunen, T. Tamminen and J. Jaaskelainen, Brit. J. Psychiat., 128,67 (1976). A.J. Coppen, E.G. Eccleston and M. Peet, Lancet, 2, 60 (1973). C.O.S. Hallstrom, W.L. Rees, C.M.B. Pare, A. Trenchard and P. Turner, Postgrad. Med. J., 52, Suppl. 3, 40 (1976).

s,

Chap. 1

Antidepressants and Antipsychotic Agents

Lahti

9

33. J. Tuomisto and E. Tukiainen, Nature, 262, 596 (1976). 34. D. Luchins, Intern. Pharmacopsychiat., 11,135 (1976). 35. G. Sathananthan, M. Pervez and S. Gershon, Curr. Therap. Res., l9, 516 (1976). 36. G.L. Sathananthan, I. Sanghvi, N. Phillips and S. Gershon, Curr. Therap. Res., 2, 701 (1975). 37. K.S. Raevsky, V.V. Markovich, L.K. Murakhina, L.S. Nazarova, A.M. Likhosherstov and A.P. Skoldinov, Khim. Farmatsevt. Zh., 10,55 (1976). 38. A.M. Eirin, E. Ravina, J.M. Montanes and J.M. Calleja, Europ. J. Med. Chem.-Chimica Therapeutica, 11,29 (1976). 39. G.B. Fregnan, M. Vidali, T. Chieli and I. Bussolera, 6th Intern. Cong. Pharmacol., Helsinki, July 20-25, Abstract 236 (1975). 40. L.R. Mandel, Biochem. Pharmacol., 25, 2251 (1976). 41. R.J. Wyatt and J.C. Gillin, Psychiatric Annals, 5 , 35 (1976). 42. P. Castrogiovanni, G.B. Cassano, L. Conti, C. Maggini, L. Bonollo and P. Sarteachi, Intern. Pharmacopsychiat., 11,74 (1976). 43. M. Nishiura, Curr. Therap. Res., 2, 164 (1976). 44. N. Trabucchi, R. Longoni, P. Fresia and P.F. Spano, Life Sci., 17, 1551 (1976). 45. A. Tagliamonti, G. DeMontis, M.O.L. Vargiu, G.U. Corsini and G.L. Gessa, J. Neurochem., 24, 707 (1975). 46. A.J. Puech, P. Simon and J . R . Boissier, Europ. J. Pharmacol., 36, 439 (1976). 47. I. Creese, D.R. Burt and S.H. Snyder, Science, 192, 481 (1976). 48. P. Seeman, T. Lee, M. Chau-Wong and K. Wong, Nature, 261, 717 (1976). 49. S . H . Snyder, I. Creese and D.R. Burt, Psychopharmacol. Comm., L, 663 (1975). 50. S.H. Snyder, Am. J. Psychiat., 133, 197 (1976). 51. H.R. Burki, E. Eichenberger, A.C. Sayers and T.G. White, Pharmakopsychiat., 8, 115 (1975). 52. R.M. Post and F.K. Goodwin, Science, 190, 488 (1975). 53. T.J. Crow, J.F.W. Deakin, E.C. Johnstone and A. Longden, Lancet, 2, 563 (1976).

Chapter 2

Anti-Anxiety Agents, Anticonvulsants and Sedative-Hypnotics

William J. Houlihan and Gregory B. Bennett Pharmaceuticals Div., Sandoz, Inc., East Hanover, New Jersey Benzodiazepines and Related Compounds Studies i n cats, mice and rats failed to support the interaction of benzodiazepines and mammalian central glycine receptors which had been proposed on the basis of in vitro studies of strychnine binding.lj2 The binding of 11 benzodiazepines to bovine serum albumin (BSA) revealed that most have 2-3 binding sites o n BSA in contrast t o a single site in human SA. Metabolic studies of lorazepam4 (la) in man and animals, pinazepams (&) i n man, diazepam nitrazepam (&)in rabbit urine8 have been reported. N-Desmethyldiazepam (2d) is a new human metabolite of chlorodiazepoxide.g Diazepam and its main active metabolite desmethyldiazepam pass into the breast milk of nursing mothers during repeated oral administration.1° Pharmacokinetic studies of lorazepam’l and chlorazepate12 (Ib) - in humans and diazepam in humans and animals13 have appeared.

(g) in human kidney cortex microsomes6and bile,’and

Lorazepam (la), diazepam and chlorodiazepoxide seem to induce some amnesia in rats as evidenced by learning or noxious events and extinction.14

-X 0 0 0 HO,O Li CH CH CH CH N CH CH CH CH CH CH

Y -

-R i

R2

R -3

0 0 0 0 0 0 0 0 0 0 S

H H H H CHZCECH CHa CH 5 H H H H o r CH1 CH2CH NEt2 CH1 CH,POMe2 CHZCFj

OH C02K OH COzLi CI CI NO2 CI Br NO2 NHOH CI NO2 CI CI

CI H H H H H H H H CI H,CI,F F F H F

-2 Anti-Anxiety Agents - The use of benzodiazepines in the treatment of neurotic anxiety has been reviewed.15 Diazepam (2b) i.v. was beneficial in treating anxiety symptoms associated with vaginismus.16 D-Oxazepam hTmisuccinate (RV1208, I c ) was superior to the racemic form in human 2) at 40-80 mg. and ketazolam’g (4, at 46.9 rng were anxiolytic studies.17 Ripazepamle (Cl-683, clinically effective in treatment of anxiety and bromazepam (&)reduced gastric acid secretion - in anxiety patients uncovered signifirelated in induced anxiety.20A clinical study with BU-1014 (5) cant side-effects that lasted two weeks.*’

Chap. 2

A n t i a n x i e t y Agents, A n t i c o n v u l s a n t s

Houlihan, Bennett

11

In animal studies the 4, 5-dihydro analog of diazepam (2b) was of equal activityZb and CRC 2015

(Id)was especially effective against agressive behavior in

(2)

was clinically useful in the treatment of akinetic and Anticonvulsant Agents - Clonazepam x ~ ~has been recorded as the drug of myoclonic seizures,z intention myoclonus,a tic d ~ u l o u r e u and choice for status e p i l e p t i c u ~ . ~ ~ Diazepam (2b) was completely effective against penicillin-induced convulsions in rabbits.26 The hydroxyaminocompounds 3 were generally less active as anticonvulsants than their nitro analogs against metrazole included convulin mice.u Compound 5 was ca. as potent as diazepam sions28in mice and the pyrrolobenzodiazepines such a s 1 were effective against pentylenetetrazole induced convulsions.29

(2)

M Sedative-Hypnotics - The benzodiazepines have superceded the barbiturates in hypnotic usage - the hypnotic of choice for medical ward patients.31 with diazepam (2b)

Triazolam (U-33,030; 8a) was an effective hypnotic in i n ~ o m n i a c s ~ ~ -0.5-0.6 ~ a t mg, but showed

loss of effectiveness w i x intermediate term usage and its withdrawal was followed by worsening

- at 3 mg and D-40TA39 (8c) - at 2 mg. provided sound sleep for preoperof sleep." EstazoIamM (8b) ative patients. Lorazepam ( l a ) in 2 or 4 mg doses showed hypnotic activity in insomniacsmand good sedation in surgical premedi~ation.4~ Flurazepam (2h) i s useful in the long term treatment of insomnia4zand shows no rebound effect after withdrail.43 Flunitrazepam (2;) has hypnotic activity in man at 2.5 mg, but does not induce physiological sleep.4 Fosazepam (2j) at 60-80 mg decreased sleep onset has been entered in the USAN listand awakening in healthy subjects.4~Quazepam(Sch 16134; ing as a sedative, hypnotic.* Clobazam (9a) at 10-20 mg, but not triflubazam (9b) - was useful for limited sleep difficulties in healthy males."

&)

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A series of aminomethyltriazolyl-, oxotriazolyl- and imidazolybenzophenones were found to be active sedatives, in mice. The potency of triazoles such as lo which can be regarded as prodrugs of 9and & approached that of diazepam."

Non-Benzodiazepine Compounds Anti-Anxiety Agents - Cartazolate (SQ 65, 396; 11) at 100 mg daily was less effective than diazepam at 20 mg on various anxiety rating scales49 anTGPA 2640 (12)at 1100-1300 mg daily failed to alleviate anxiety in anxious non-psych0tics.w Trazodone (13)at75-150 mg daily was equivalent to 30-60 mg of chlordiazepoxide in relieving symptoms caused by anxiety." Evaluation in cats suggests Sch 12679 (14)is effective in reducing many forms of agression? DL-254 (15) - appears similar to diazepam.53-

I?

Chap. 2

A n t i a n x i e t y Agents, A n t i c o n v u l s a n t s

Houlihan, Bennett

12

Anticonvulsants - The diagnosis, treatment and most frequent etiologic factors involved in status epilepticus were reported.% The effect of antiepileptic drugs on epileptogenesis55and the clinical pharmacology of anticonvulsant a g e n t ~ ~ ~ h abeen v e reviewed. Anticonvulsant drugs appear to limit the propagation of seizures through the balance of excitatory glutamate pathways and inhibitory CABA and norepinephrin (NE) pathways.57 A reciprocal relationship between NE levels at receptor sites and audiogenic seizure susceptibility in mice has been observed.% A modified maximal metrazole seizure test in mice that can define three main groups of anticonvulsants has been developed.59 Carbamazepine (16) at plasma levels of 4-10 pg/ml exerted a remarkable drop in seizure frequency,m showed tolerance after six days at 50 mg/kg daily,61 was equivalent to diphenylhydantoin62 and primidone63 (17) in preventing psychomoter seizures and improved alcohol withdrawal symptoms in male outGtients.64 Eterobarb (18) - i s a safe and potent anticonvulsant with a low hypnotic effect.65 The pharmacokinetics of vinylbarbital (19) - in man&,, 11-epoxide, were studied in children.67

08

well as 16 - and i t s major metabolite, the 10,

0

n : H A r

R--NKN-R

0

X

Z

U V

R H CHPCH,

H

R’ CeHs C,Hs CH:CHI

hr Rz X CzHs Hz CzHs 0 C,HS 0

z!

The R isomers of glutarimides such as 20 in mice provide a more rapid onset of action and greater anticonvulsant activity and neurotoxicity than t h e 2 ana1ogs.m Compounds 21-24 were effective in mice against tetrazole induced seizures6~71andTMD (22) significantly reduced grand ma1 convuls i o n Pentetrazole ~ ~ ~ convulsions in mice were protected against by 24T3 TMHT74(25J. &,7521,76 2 and 29 (R = alkyl, R1 = aryl) 2d

-

21

CH3 0 H,p-.NO&H,

CHz

2_2 CH, 0 CH3,CH3

0

2_3 C,H, S

NH

=CHAr

,C-CH,NHNH 0’ SCH,NHNH, 2_5

3

14 -

Sect.

I

- CNS Agents

Krapcho, Ed.

uNHco i

R

CNH iC3H7

Br

5

1 I 0

2_9

2_8

Compound had a better therapeutic index than phenobarbital (PB) but appeared less active7 was comparable to PB against electrochock, but less effective against pentetrazole conand vuIsions.78

2

Aminoethers such as 32 protected against maximal electroshock seizures in rats.79

Several piperazinoimines with anticonvulsant activity such as s m in light sensitive baboons, 348' and @," against tetrazoles, and 3 8 3 against electroshock, were reported. Structurally related imines such as 37 exhibited inhibition of pentylenetetrazole induced convulsions.84

(CH2)3N=CHAr

I

I

(CH,),NXHAr Arz4-CIC6H4

34 0

II

YH2 OCH3

f-3 N NNXHC6H,-4-CN U

X-CN,Br

38

CH i

3_6

HCGC-CCH,CH,CO,H H

e 3_7

C

l

H2NOCH2C02H

@

Chap. 2

A n t i a n x i e t y Agents, A n t i c o n v u l s a n t s

Houlihan, Bennett

15

s),

GAG (RMI 71675; an irreversible GABA transaminase inhibitor, protected mice against audiogenic seizures, thiosemicabazide (TSC) and electroshock.85 The anticonvulsant activity of aminoxyacetic acid (39) is manifested through two mechanisms, one involving GABA metabolism,% and against TSC it hasbeen shown to be fast in onset and of short duration.87 Dipropylacetic acid (40) protected rats against picrotoxin and pentetrazole.m The chelating agent D-penicillamine (41) - markedly reduced seizures in P. Papio baboons, a primate with high serum Zn fevels.89

Lactone 42 proved equivalent to A~-THC(43) against electroshock in rats.% The anticonvulsant activity of A42574 (44) in photosensitive baboons was poor in comparison to its activity in rodents?’

-

Sedative-Hypnotics - The clinical aspects of sleep and psychotropic drugs were reviewed and standardized methodology for measurement was recommended.92 A model study for obtaining a detailed clinical profile of a hypnotic was proposed.” Studies have appeared on the effect of anxiolytic drugs on objective and subjective sleep parameters in healthy normal voIunteers,93 the behavioral performance of habitual long sleepers after an alteration in their sleep schedules,%and the induction of symptoms mimicking those of insomnia by coffee and caffeinegsin normal subjects. Clinical and experimental arguments are summarized in favor of a possible relationship between sleep and memory and between the capacities for learning and paradoxical sleep.%The drug interactions of a number of hypnotics and sedatives in clinical use were reviewed.97 Physostigmine injected during non-REM sleep induced the REM stage, suggesting the role of a cholinergic mechanism in the induction of REM sleep.* The neuroleptic sulpiride (47) at 200-400 mg. i.m decreased time to sleep and awakening, and increased all sleep phases withno significant change in distribution.9 Diphenylhydramine (48) was a safe and effective sleep aid for pediatric patients at 1 mg/kg.’m Single doses of 1-2.5 m g . of nabilone (45) induced sedative effects with no appreciable effect on heart rate, lending support to the hypot&sis that one can separate out the desirable effects of the cannabinoid class of comat 1-5 g orally decreased sleep latency in mild insomniacs and pounds. L-Tryptophan latency and wakenings in normal volunteers.102 Preoperative sedation with oxypertine (50) was achieved at 20 mg oral dosage.Io3Athree week sleep study with mesoridazine (51) gave enzurag- has entered clinical trials.105ing resultslWand a new glutarimide, biglumide (52)

(49)

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46 have shown sedative-hypnotic and analgesic activity in cats and rodents. 3 showed sedation and anticonvulsant activity in mice.lm Kessoglycol mono-

Heterocannabinoids 106-107 Compound

acetate (54) - was reported to have a sedative-hypnotic profile in animals.109

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55. Z. Servit, Cesk, Fysiol., 3,193 (1976). 56. E.F. Hvidberg and M. Dam, Clin, Pharmacokinetics, 1,161 (1976). 57. J. Weinberger, W.J. Nicklas and S. Berl, Nicklas and S. Berl, Neurology, 2, 162 (1976). 58. P.C. lobe, A.L. Picchioni, L. Chin and P.F. Geiger, Fed. Proc., 35, 270 (1976). 59. L.K.C. Desmedt, C.J.E. Niemegeers, P.J. Lewi and P.A.J. Janssen, Arzneim-Forsch., 3, 1592 (1976). 60. F. Monaco, A. Riccio, P. Benna, A. Covacich, L. Durelli, M. Fantini, P.M. Furlan, M. Gilli, R. Mutani, W. Troni, M. Gerna and P.L. Morselli, Neurology, 2,936 (1976). 61. S. Consolo, S. Bianchi and H. Ladinsky, Neuropharmacology, 12,653 (1976). 62. N. Simonsen, P.Z. Olsen, V. Kuhl, M. Lund and J. Wendelboe, Epilepsia, lJ, 169 (1976). 63.E.A. Rodin, C.S. Rim, H. Kitano, R. Lewis and P.M. Rennick, J. Nerv, and Mental Disease, 163, 41 (1976). 64. S.E. Bjorkqvist, M. Isohanni, R. Makela and L. Malinen, Acta Psychiat. Scand., 2,333 (1976). 65. R.H. Mattson, P.D. Williamson and E. Hanahan, Neurology, 2,1016 (1976). 66. D.D. Breimer and A.G. deBoer, Arzneim,-Forsch., 26,448 (1976). 67. A. Rane, B. Hojer and J.T. Wilson, Clin. Pharmacol. Ther., E,276 (1976). 1419 (1976). 68. D.T. Witiak, W.L. Cook, T.K. Gupta and M.C. Gerald, J. Med. Chem., E, 69.I.T.Scoular, P.J. Nicholls and H. J. Smith, Eur. J. Med. Chem. 11,91 (1976). 70. M.J.Korohoda, Z. Klinrok and E. Przegalinski, Pol. J. Pharmacol. Pharm., 28, 329 (1976). 71. M. Chaudhry, S.S. Parmar, S.K. Chaudhry, A.K. Chaturved and B.V. Rama Sastry, J. Pharm. Sci., 65, 443 (1976). 72. C.R. Craig, P. Clin. and B.K. Colasanti, Neuropharmacology 12, 485 (1976). 73. S.K. Chaudhary, S.S. Parmar, M. Chaudhary and J.P. Barthwal, J. Pharm. Sci., 3,1010 (1976). 74. R.S. Misra, J.P. Barthwal, S.S. Parmar, S.P. Singh and V.I. Stenberg, ibid., 65, 405 (1976). 75. S.P. Singh, B. Ali, T.K. Auyong, S.S. Parmar and B. DeBoer, ibid., 65 391 (1976). 0, 76. H. Lauressergues, A. Stenger, M. Culetto, G. Mouzin and H. Cousse, Eur. J. Med. Chem., 1 629 (1976). 77. A.I. Mikhalev, V.K. Kudryashova, M.E. Konshin and V.S. Zalesoy, Kim.-Farm. Zh.,?, 15 (1975). 78. M. Jawdosink, Farm. Ed. Sci., 2,671 (1976). 79. K. Ramabadran, M. Bansinath and M.N. Guruswani, J. Pharm. Sci., 65,1245 (1976). 80. E.K. Killam, Fed. Proc., 2,2264 (1976). 81.A.K. Chaturvedi, B.V. Rama Sastry, A. Chandari and S.S. Parmar, J. Pharm. Sci., 65, 409 (1976). 82. M. Protiva, J. Nemec and Z. Sedivy, Coll. Czech. Chem. Cornmun., fi,1035 (1976). 83. C.R. Ellefson and J.W. Cosic, J. Med. Chem., E, 1161 (1976). 84. S.P. Singh, A. Chaudhari, V.I. Stenberg and S.S.Pqrmar, J. Pharm. Sci., 65, 1678 (1976). 85. P.J. Schechter, M.J.lung, Y. Trainer, B. Lippert and A Sjoerdsma, Fed. Proc.,?, 544 (1976). 86. J.D. Wood and S.J. Peesker, Can. J. Physiol. Pharmacol., 24, 534 (1976). 87. Y. Murakami, M. Abe and K. Murakami, J. Neuro. Chem., 26, 655 (1976). 88. H.H. Frey and W. Loscher, Arzneim.-Forsch.,z, 299 (1976)89.M.C. Alley and E.K. Killam, Pharmacologist, 2,136 (1976). 90.K. Matsumoto, P. Stark and R.G. Meister, J. Med. Chem., g,25 (1977);H Lavid, P. Consroe and A. Straussner, Pharmacologist, 18, 136 (1976). 91. L.W. Mulbry and K.K. Killiam, PTharmacologist, ibid., 18, 136 (1976). 92. W. Baust, Arzneim.-Forsch., 2&, 1039 (1976). 93. B. Saletu, ibid., 2, 1042 (1976). 177 (1976). 94.J.M.Taub and R.J. Berger, Physiology and Behavior, 3, 95. I. Karacan, J. I . Thornby, M. Anch, G.H. Booth, R.L. Williams and P.J. Salis, Clin. Pharacol. Ther., 2, 682 (1976).

Chap. 2

A n t i a n x i e t y Agents, A n t i c o n v u l s a n t s

Houlihan, Bennett

19

96.J.F. Larnbert, Agressologie, 1_7, 1 (1976). 97. S.R. Brown and E.A. Hartshorn, Drug Intelligence and Clin. Pharrnacol., 12,570 (1976). 98. R.J. Wyatt, S. Dawson and J.C. Gillin, Science, 191,1281 (1976). 99.S. Scarone, G . Spoto, G. Penati, R. Canger and E.A. Moja, Arzneirn.-Forsch., 26,1626 (1976). 100. R.M. Russo, V.J. Guraraj and J.E. Allen, 1. Clin. Pharrnacol., lfj,284 (1976). 101. L. Lemberger and H. Rowe, Clin. Pharrnacol. Ther., 2,720 (1976). 102. E. Hartrnann, Monogr. Neural Sci.,, 26 (1976). 103. I.T. Davie and K.B. Slawson, Br. J. Anaesth., 48,915 (1976). 104. K. Adam, 5. Allen, I . Carruthers-Jones, I . Oswald and M. Spence, Br. J. Clin. Pharrnacol.,A 157 (1976). 105. C.Argyropoulos, G . tanner and E. Pasquali, Wien Med. Wochenschr., 12,75 (1976). 106. H.G. Pars, F.E. Granchelli, R.K. Razdan, J.E. Keller, D.G. Teiger, F.J. Rosenberg and L.S. Harris, J. Med. Chern., 2, 445 (1976). 107. R.K. Razdan, B.Z. Terris, H.G. Pars, N.P. Plotnikoff, P.W. Dodge, A.T. Dren, 1. Kynel and P. Sornani, ibid., 19,454 (1976). 108. K. Nagarajan, JYDavid and R.K. Shah, ibid., 19, 508 (1976). 109. K. Takarnura, H. Nabath and M. Kawaguchi, 1. Pharrn. SOC. Japan, 95,1205 (1975).

Chapter 3. Analgesics,Antagonists,the Opiate Receptorand Endogenous Opioids M. Gordon and J. A. Vida, Bristol-Myers Company, Syracuse, New York

The term " o p i a t e receptor'' i s used t o d e s i g n a t e a r e a s of t h e b r a i n h a v i n g a s p e c i f i c a f f i n i t y f o r o p i a t e a g o n i s t s and a n t a g 0 n i s t s . l - 1 7 Endogenous p e p t i d e s c a l l e d enkephalins and endorphins can r e a c t w i t h t h e o p i a t e receptor18919 t o produce o p i a t e e f f e c t s ; t h e e f f e c t s of t h e s e p e p t i d e s a r e a n t a gonized by naloxone. I n e l a b o r a t i o n of t h e o p i a t e r e c e p t o r concept, Feinb e r g e t a1.20 have r e p o r t e d a p o t e n t i a l model e x p l a i n i n g t h e s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s of o p i a t e a g o n i s t s and a n t a g o n i s t s . The f i n d i n g t h a t s y n t h e t i c a n a l g e s i c s l i k e t h e morphinans and t h e benzomorphans a r e capable of i n t e r a c t i n g i n a q u i t e s p e c i f i c way w i t h recept o r s i n t h e c e n t r a l nervous system, depending on t h e i r r e l a t i v e a g o n i s t o r a n t a g o n i s t p r o p e r t i e s , s u g g e s t s t h a t f u r t h e r s t u d i e s w i t h such s y n t h e t i c a n a l g e s i c s may p r o v i d e a s u s e f u l a t o o l i n u n r a v e l i n g t h e m y s t e r i e s of t h e o p i a t e r e c e p t o r as t h e c u r r e n t approaches i n v o l v i n g p e p t i d e r e s e a r c h . 23 The p a s t y e a r has seen t h e i s o l a t i o n from mammals of a number of opio i d s u b s t a n c e s i n a d d i t i o n t o t h e p r e v i o u s l y discovered enkephalins and endorphins ?bAmong t h e s u b s t a n c e s r e p o r t e d are MLC (morphine-like compound, Levy e t a1.25,26), anodynin ( P e r t e t a1.27), MLF (morphine-like f a c t o r , P a s t e r n a k e t a1.281, and p i t u i t a r y o p i o i d p e p t i d e (POP, Goldstein29). A l l of t h e s e are p e p t i d e s with t h e exception of MLC, which appears t o be morphine-like based on t h e f a c t t h a t i t reacts w i t h a n t i b o d i e s generated a g a i n s t morphine; however, t h e e f f e c t s of MLC a r e n o t antagonized by naloxone. A number of known p e p t i d e s have been found e i t h e r t o have p a r t i a l a g o n i s t - l i k e p r o p e r t i e s ( i . e . , s u b s t a n c e P,30 s o m a t o s t a t i n and ACTH31) o r t o a c t as prohormones ( i . e . , f3-1ipotropin32~33). The i n t e n s i v e r e s e a r c h now ongoing i n t h e o p i a t e r e c e p t o r a r e a has l e d t o s u g g e s t i o n s t h a t t h e endogenous o p i o i d s a r e n o t only involved i n analg e s i a b u t may a l s o play a r o l e i n mental d i s e a s e s such a s s c h i z o p h r e n i a and d e p r e s s i o n . Some r e c e n t reviews i n t h e a r e a have been published.21,22Guillemin34has suggested t h a t b r a i n p e p t i d e s are l i n k e d t o s c h i z o p h r e n i a s i n c e t h e c a t a t o n i a produced by t h e s e p e p t i d e s i n animals35 i s very r e m i n i s c e n t of schizophrenic c a t a t o n i a . Naloxone r e v e r s e s t h e c a t a t o n i a and hypothermia produced by b r a i n p e p t i d e s i n animals and i n humans. Indeed G ~ n n e 3 ~ h a s r e p o r t e d t h a t naloxone a n t a g o n i z e s a u d i t o r y h a l l u c i n a t i o n s i n schizophrenic p a t i e n t s . B y ~ hk a s~ suggested ~ t h a t t h e a c t i o n s of morphine (analg e s i a , s l e e p , euphoria, and r e s p i r a t o r y d e p r e s s i o n ) are simulated b y v a r i o u s t r a n s m i t t e r o r modulator s u b s t a n c e s i n t h e b r a i n . Thus, enkephalin i s proposed a s a n e u r o t r a n s m i t t e r and i t s b i n d i n g t o o p i a t e r e c e p t o r s determines mood s t a t e and i n f l u e n c e s r e s p i r a t o r y and s l e e p p a t t e r n s . Lithium may a c t through m o d i f i c a t i o n of t h e binding of t h e endogenous morphine-like subs t a n c e s a t t h e o p i a t e r e c e p t o r . This theory would a l s o p r e d i c t t h e blocki n g of mania and most drug-induced e u p h o r i a s by naloxone. Endorphins. The endorphins have been defined as a c l a s s of opioida c t i n g p e p t i d e s i s o l a t e d from b r a i n , a l l of which seem t o be fragments of f34ip0tropin.22~39 The endor h i n s have t h e f o l l o w i n g s t r u c t u r e s : 4 0 a- Endorphin ( 8- 1ipo t r o pin61-96)

H-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-OH 8-Endorphin (B-lipotropin61-91)

Chap. 3

A n a l g e s i c s , A n t a g o n i s t s , t h e Opiate Receptor

Gordon, Vida

21

a-Endorphin-Leu-Phe-Lys-Asn-Ala-Ile-Ile-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-G~-OH y-Endorphin(8-lipotropin 61-77) H-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH The b r a i n - d e r i v e d 8 - l i p o t r o p i n fragment a-endorphin h a s a n a l g e s i c and t r a n q u i l i z i n g a c t i v i t y i n a n i m a l s , whereas y-endorphin, which d i f f e r s from aendorphin o n l y i n having a l e u c i n e r e s i d u e added t o t h e C-terminal end of a-endorphin, i n d u c e s violen: behavior when i n j e c t e d i n t o animals. F i n a l l y , t h e l a r g e r fragment , 8-endorphin, a l s o produces a n a l g e s i a ,41 profound sedat i o n and c a t a l e p s y 4 2 i n animals. 8-Endorphin h a s 48-400 times t h e analgesic o r c a t a l e p s y producing a c t i v i t y of morphine when i n j e c t e d i n t o t h e brain,43, 44 t h r e e t i m e s t h e a c t i v i t y of morphine when i n j e c t e d i n t r a v e n o u s l y , and i t s e f f e c t s are blocked by naloxone. The i n t e r e s t i n B - l i p o t r o p i n is f u r t h e r s t i m u l a t e d bv t h e f a c t t h a t i t s 61 t o 65 sequence i s i d e n t i c a l t o t h a t of Met-enkephalin.45 However, no p r e c u r s o r h a s y e t been found t o Leu-enkephalin. A t o t a l l y s y n t h e t i c fi-endorphin46 w a s shown t o be i d e n t i c a l w i t h t h e n a t u r a l material b o t h chemically and b i o l o g i c a l l y , u s i n g t h e membrane f r a c t i o n from r a t b r a i n homogenate t o a s s a y f o r r e c e p t o r b i n d i n g and t h e r a t t a i l f l i c k t e s t f o r a n a l g e s i c a c t i v i t y . S y n t h e t i c a-endorphin and yendorphin have been s y n t h e s i z e d 4 7 and t h e s e have been shown t o be i d e n t i c a l w i t h t h e n a t u r a l m a t e r i a l s . I t h a s been r e p o r t e d t h a t 8-endorphin n o t o n l y is a p o t e n t a n a l g e s i c a g e n t b u t a l s o i s c a p a b l e of inducing p h y s i c a l dependence of t h e o p i a t e t y p e . 1 3 , 4 8 - 5 0 ~ 5 2 , 5 3 When i n f u s e d f o r 70 h r s i n t o t h e p e r i a c q u e d u c t a l gray-fourth v e n t r i c l e s p a c e s of t h e r a t b r a i n , methionine-enkephalin and 8-endorphin produce a t y p i c a l morphine-like withdrawal syndromwhen c h a l l e n g e d w i t h n a l o ~ o n e ? 55 ~ . 6-Endorphin can a l s o produce a n a l g e s i a when i n j e c t e d i n t o mice i n t r a v e n o u s 1 56 8-Endorphin i s found i n The 8-endorphin found i n t h e c o n s i d e r a b l e q u a n t i t y i n t h e p i t u i t a r y . 22,3i sheep and camel p i t u i t a r y i s i d e n t i c a l w i t h t h a t found i n man, except t h e l a t t e r h a s t y r o s i n e r e p l a c i n g h i s t i d i n e a t t h e 2 7 t h p o s i t i o n , and glutamic a c i d r e p l a c i n g glutamine a t t h e C-terminal end ( p o s i t i o n 31). 8-Endorphin h a s a h i g h a f f i n i t y f o r b r a i n o p i a t e r e c e p t o r s a s measured by c o m p e t i t i o n w i t h t r i t i a t e d naloxone and dihydromorphine f o r b i n d i n g t o a washedmembrane preparati~n.~ ~ removal of r e s i d u e s 30 and 31 (Gly-Glu) had l i t t l e The e f f e c t and 28 and 29 (Lys-Lys) removal had a profound (20-fold r e d u c t i o n ) e f f e c t on t h e b i n d i n g p r o p e r t i e s of 8-endorphin. Other p i t u i t a r y and b r a i n f a c t o r s d i f f e r from 8-endorphin i n t h e i r g r e a t e r potency,60 d e s t r u c t i o n by t r y p s i n and i n s e n s i t i v i t y t o cyanogen bromide. Thus, t h e e n t i r e endorphin p u z z l e i s a l o n g way from b e i n g s o l v e d . It h a s been s p e c u l a t e d 6 0 t h a t c l a s s i c a l hormonal feedback mechanisms might a c t t o s u p p r e s s endogenous o p i o i d s y n t h e s i s when t h e r e c e p t o r s are occupied by a n endogenous o p i a t e - l i k e morphine 6 1 Enkephalin l e v e l s i n t h e b r a i n of morphine-tolerant r a t s are i n c r e a s e d , and t h e r e i s c r o s s - t o l e r a n c e between morphine and Met-enkephalin. 62 Although t h e block of b o t h o p i a t e and endorphin e f f e c t s by naloxone s h o w s a s i m i l a r i t y between them, t h e r e a r e s t i l l some o b s e r v a t i o n s r e l a t i n g t o t h e o p i a t e r e c e p t o r t h a t cannot be put i n any c o n t e x t . Thus, i t is d i f f i c u l t t o e x p l a i n why naloxone a n t a g o n i z e s t h e a n a l g e s i a produced by f o c a l e l e c t r i c a l s t i m u l a t i o n of t h e b r a i n 6 3 o r by acupuncture,64 y e t does n o t a n t a g o n i z e a n a l g e s i a i n man produced by h y p n o t i c s u g g e s t i o n . Nor does naloxone show any s i g n i f i c a n t d i s r u p t i v e e f f e c t s on shock escape t h r e s h h o l d

22

Sect. I

-

CNS Agents

Krapcho, Ed.

or temperature control under cold stress in rats. On the other hand, naloxone does block food-seeking and water-seeking behavior in hungry orthirsty rats. The fact that biogenic amine modifiers do not affect morphine analgesia and naloxone antagonism by a similar pattern also indicates that our concepts of the opiate receptor interactions are incomplete.65,66 All three endorphin peptides and Met-enkephalin produce in drug-free animals the "wet-dog" shaking seen in opiate withdrawal, and these responses are antagonized by naloxone.43 It has been suggested that $-endorphin and Metenkephalin play a role in growth hormone secretion67 and in cyclic AMp.68 Stimulation of the central gray region in humans has produced marked analgesia which is antagonized by naloxone. This phenomenon has led to speculation that there is ongoing release in the brain of morphine-like compounds which is partially responsible for elevating pain thresholds. However, perception of experimental1 induced pain in human subjects is not altered by naloxone administration.6$ Perhaps an interaction with acet 1 choline, with nor-adrenalin release,51 with the adenylcyclase system57 86 or neural firing38,71 is leading to these complex, unexplained phenomena. 72973 Finally, an electrically induced release of endogenous opiate receptor ligand has been reported, including reversal by naloxone.74 Enkephalins. A subset of the endorphins are called enkephalins.75-80, Although the various enkephalins have agonist activity on appropriate administration,62,90 they are less potent than the endorphins and are thought to be enzymatic degradation products of endorphins, or perhaps even artifacts produced in the isolation technique. Endorphins may be produced by enzymatic cleavage of $-lipotropin, and there is speculation91 that a lack of this enzyme "may be an etiological factor in those psychopathological states for which the exogenous neuroleptics exert an ameliorative influence." Met-enke~haling~(Tyr-Gly-Gly-Phe-Met) and Leu-enkephalin (Tyr-GlyGly-Phe-Leu) have lower in vivo potency of any of the endorphins.94 The enkephalins are quite short-acting in vivo because they are degraded by peptidases in blood and brain.95 Synthetic enkephalins have been prepared which are identical to the natural materials.96997 Goldstein98 has prepared a heptapeptide H-Tyr-Gly-Gly-Gly-Lys-Met-Gly-OH based on its spatial resemblance to morphine. However, its potency was very low. Steric analyses of the enkephalins have been published54,89,99-101,159 and it is concluded that there is no single conformation in solution. A series of enkephalin analogs were synthesized and their affinities for opiate receptors compared. Met-enkephalin has 1f3rd the potency of morphine, but three times the potency of Leu-enkephalin.100,104 A synthetic enkephalin81,102 [D-Ala2]-Met-enkephalinamide (DALA) binds to opiate receptors almost as strongly as Met-enkephalin. Since DALA is not susceptible to degradation by brain enzymes doses of 5-10 mcg cause profound, long-lasting, morphinelike analgesia when injected into rat brain. Resistance to enzyme degradationl03 is retained if D-Alanine is replaced by other D-amino acids, by L-proline or by sarcosine. Replacement of methionine by norleucine gave an analog with about 50% of the potency of the parent, but replacement of glycine or -OH led to marked loss of activity. Substitution of tyrosine by phenylalanine, blocking of the hydroxyl, or removal of the amino group practically abolished activity. Replacement of the Phe- by Tyr- also practically abolished acti82-88

Chap. 3

Analgesics, Antagonists, the Opiate Receptor

Gordon, Vida 2 3

vity. 104,105 The peptides larger than Met-enkephalin are not appreciably more active when measured on the isolated guinea pig ileum. Howeverythe much greater activity of the endorphins compared with Met-enkephalin invivo is probably due to the protective effect of the longer chain. Additional receptor binding sites may also be present within residues 80-91 of LPH (61-91) .Io5 Anodynin. Of major importance to the current of research on opioid peptides in brain is the finding of an endogenous opiate analgesic, anodynin, in human plasma106 and in rat brain. Anodynin differs from the enkephalins in its longer duration of action, its lack of susceptibility to enzymatic splitting, its thin layer mobility, its behavioral effects and its apparant ability to cross the blood brain barrier. It has in common with enkephalin its reversal by naloxone. The pituitary origin of anodynin is suggested by the greatly reduced levels found in rat blood after hypophysectomy. It is unlike enkephalin in this respect. Miscellaneous. A long-acting, polymer-bound form of naloxonelo7 has been prepared. Naloxone, attached to a hydrazine-substituted polysaccharide by a hydrazone bond, antagonizes morphine analgesia more than 25 times longer than free naloxone. Using the spinal dog as a research tool, Martinlo' has postulated at least three different agonist receptor sites, each associated with a particular agonist syndrome. According to Martin's classification: 1) (11) agonist receptor - morphine-like effects like euphoria and sedation 2 ) (K) agonist receptor - nalorphine or cyclazocine-like agonist activity 3) ( u ) agonist receptor - associated with psychotomimetic effects. Cyclazocine and nalorphine are mixed ( K ) and (a) agonist, ethyl keto-cyclazocine is a pure ( K ) agonist, and morphine is a pure (11) agonist. Prodines and Related Structures. Among analogs of the prodine-type,the was found to be half as potent as an analgesic,lOg most potent compound (1) but twice as toxic as alphaprodine (2). The ED50 (tail flick) for L and 2 are 3 . 1 and 1.4 mglkg, respectively. Only one of the three isomers of 4OCOC2H5

A-CH-,

1 -

I

OCOC2H5

b s < c 6 H 5

2 -

'gH5

CH -N 3

CH3

acetoxy-l,2,6-trimethyl-4-phenylpiperidine was an effective analgesic in mice (2.3 x meperidine) as judged by the hot-plate test.l1° Alpha(+)allylprodine was 40 times more potent than morphine, 260 times more potent than (-)allylprodine and 460 times more potent than its f? diastereomer.111 The relative brain levels of meperidine and three N-alkyl homologues determined at equal analgesic iv doses in mice were found to be closely proportional to their ED50 doses in spite of the wide differences in partition coefficients and in rates of metabolic N-dealkylation. It was postulated, therefore, that the observed ED50 potencies provide a fair comparison of the relative receptor affinities of the four homologues.l12

Benzomorphans. The 9f3analog (2)of the isomeric 2,9-dimethyl-Z'-hydroxy-6,7-benzomorphans was 4 times as potent (ED50=l.1 mg/kg) as the 9aanalog by the hot plate test.113

(x)

24

Sect. I

'@ -

-

CNS Agents

3a

R1 = Cii3, R2 = H

3b 3c

R1 = H, C3H7, Rg =R2 CH3 = H

3d

R1 = H, R2 = C3H7

Krapcho, Ed.

nn'

no

The 96-propyl lev0 isomer (&) was considerably more potent subcutaneously than morphine, while thelq$-propyl lev0 isomer (2)was equipotent with morphine as an analgesic. None of the optical isomers suppressed withdrawal signs in monkeys; the 9B-propyl levo isomer exacerbated the withdrawal syndrome, indicating that it possesses some narcotic antagonist activity.l14 The most active compound (3)of six homobenzomorphans was as potent subcutaneously as morphine (measured by pressure stimuli on mouse tail) .1159116 Simplified procedures for the synthesis of the 6,7-benzomorphan series have been described,117Y 118 Several 11-hydroxy and ll-alkoxy2,6-methano-3-benzazocines dis lay as potent analgesic and narcotic antagonist activity as cyclazocine. 1P9 Morphinans. Only one of the two enantiomeric quaternary iodides, Nmethyl-levorphanol exhibited specific opiate effects while the other enantiomer, N-methyl-dextrorphan was, as expected, inactive.12' In a series of 14-hydroxymorphinans, the epimeric isomorphinan (5) was a more potent antagonist than oxilorphan (A) (BC-2605) .lZ1 A review on the harmacology of butorphanol (cyclobutyl analog of 5) has been published12' and clinical investigation of both the oral and parenteral forms is c o n t i n ~ i n g . l ~ 3 - ~ ~ ~ 5 -

OH

no

no

Morphines. It has been shown that removal of the N-methyl group leads to a considerable loss in agonist activity as measured by the guinea-pig ileum method with two exceptions; morphine and normor hbne, as well as codeine and norcodeine, are equiactive in this test.129 Morphine was found to be 6 times more potent as an analgesic and approximately twice as lethal as morphine-6-hemisuccinate after ip administration. 130 Examination of the role of the gut in the metabolism of three phenolic analgesics, dihydromorphine, etorphine (G),and buprenorphine which possess widely differing partition coefficients (K)(heptane/phosphate buffer pH 7.4), revealed that a major pathway (conjugation) exists in the gastrointestinal tract for the protection of the organism from the potential toxic effects of phenolic substances and that a determining factor in

(c),

R=CH3, R =nPr, Xu-CH=CH-, 1

K=

0.15)

R = c H ~R ~~ =, ~ B " ,x = - c H ~ - c H ~ K- ,= i . 78) R=CH CH=CH2, 2

R =n-Bu, X=-CH CH 1 2 2

Chap. 3

Analgesics, Antagonists, the Opiate Receptor

Gordon, Vida

25

the efficiency of this pathway is the lipophilicity of the substance.131 The results of this study suggest that the role of the liver in the deactivation of potent agonists after oral administration has sometimes been overemphasized, for it has been shown that this organ is not essential for the "first pass" metabolism of the lipophilic compounds buprenorphine and etorphine in the rat. Dihydromorphine (K=5.0) appears not to be readily conjugated by rat intestine and the liver must be regarded as the prime site of metabolism of this relatively polar compound.131 The analgesic effects of R+S 218-M (alletorphine)(&) administered in a dose of 0.56 mg/70 kg was as effective as 10.5 mg/70 kg of morphine sulfate. No evidence was found to support the claim that alletorphine (7c) causes less respiratory depression when compared with morphine ~ u l f a t z ~ ~ The relative agonistic and antagonistic potencies of naloxone, naltrexone and their 6-methylene derivatives were evaluated in the guinea-pig ileum. All compounds were competitive antagonists of azidomorphine, the morphine-like agonist used in the experiments. The 6-methylene substitution caused approximately 50 and 100% increases of the antagonistic activity of naloxone and naltrexone, respectively. All compounds have little or no agonistic activity.132 In man, azidomorphine constricted pupils, produced morphine-like subjective effects and euphoria, and suppressed the morphine abstinence syndrome. It was concluded that in man azidomorphine is a typical morphinelike drug.133 The a@) and f3-halomorphides (2) possessing a C-6 axial and C-8 equatorial halide, respectively, were found to be more potent analgesics and also more toxic than morphine.134

8

& ;-H -

.--'H

ao Cl The disposition, metabolism and effects after acute and chronic dosing of naltrexone were investigated in 4 subjects, showing that narcotic antagonism was related to plasma levels of naltre~one.13~ Aminotetralins. In continuing studies on new bridged aminotetralin analgesics, the best activity among esters, N-oxides, and compounds containing a butenyl or a benzo-bridge was seen €or @), which was 2.5 times as potent as m0rphine.13~ Resolution of the more potent analgesics of the bridged aminotetralin type produce WY-16225 (the (-) enantiomer of (11)) which was 8 to 15 times as potent as morphine when administered parenterally. Desocine (11)was judged to be a morphine antagonist slightly less potent but longer acting than nalorphine based on physical dependence studies carried out in morphine-dependent Rhesus monkeys.138 This compound

m, 11 (Dezocine)

26 -

Sect. I

-

CNS Agents

Krapcho, Ed.

is currently undergoing clinical trials. In continuing studies on aminotetralin derivatives the best compound, (g), was found to possess slightly more than half of the oral anal esic potency of morphine as measured by the acetic acid writhing test. 138 fH3

N-CH2 CH=C

12 -

13 NH2

hH3 €I0 CH3

CH3

Miscellaneous. In the seventh publication of ongoing studies on tetrahydro-4,4-di-methylisoquinoline type analgesics, the analgesic activity of (2)was found to be slightly more potent than that of codeine.140 In an attempt to enhance the analgesic activity of fentanyl (g),cyclization of the acyl group with C-2 of the aromatic ring was carried out.Comdid not show an analgesic activity. It did show, howeverystrong pound (g) antihistaminic activity.14P

0

0

CH3

The best analgesic activity of a series of 10,11-dihydrodibenzo[b,f] thiepin derivatives was displayed by (g), without listing a comparative value of a reference standard.142 The compound also displayed other pharmacological activities.

and its dextro isomer exhibited In a series of azabicycloalkanes, (g) analgesic activity comparable to meperidine and morphine, respectively.143 The two compounds also exhibited narcotic antagonist activity. (17) Produced slight physical dependence capacity in the Rhesus monkey. The relative binding affinities of the optical isomers of methadone, a-methadol, a-acetylmethadol and their N-demethylated derivatives to the opiate receptors of rat brain confirmed the agonistic nature of this series of drugs.144 series some com ounds showed analgetic In azabicyclo[3,3,l]non-6-ene activity in the acetic acid induced writhing test.P45 Sufentanyl (R-30,730)(18) was the most potent compound in a novel series of 4-substituted fentanyl derivatives. Sufentanil, which is 4500 times more potent than morphine, has a rapid onset but relatively short duration of action and its margin of safety is reported to be unusuallyhigh (LD /ED50>25000). eR'' orts on research with phenylmorphans,l47 meptazinol (a homopiperidine) , 149 isoxazoloquinazolinones,128 biphenyl Mannich bases,151 59

Chap. 3

Analgesics, Antagonists, the Opiate Receptor

Gordon, Vida

27

methyl methadone diastereoisomers,152 3-isothujone ,153 a-promedol enantiomers, benzoisoquinolines,l55 diphenylethylpiperazines,156 and piritramidel57 have appeared. Cannabinoid Compounds. The continuing studies on heterocyclic and carbocyclic analogs of cannabinoids yielded compounds which show significant analgesic activity. Many of these compounds also exhibit sedative, antihypertensive or anticonvulsant activity.82,93,150,158 References 1. C. Lamotte, C. R. Pert 6 S. H. Snyder. Brain Res. 112, 407 (1976). 2. V. Hollt, I. Haarman 6 H. Herz, Arzneimittel-Forsch. 1102 (1976). 3. G. A. Clay 6 L. R. Brougham, Biochem. Pharmacol. 24. 1363 (1975). 4. R. A. Mannino, Life Sciences, 11. 2089 (1975) 5. E. J. Simon, Bull. N.Y. Acad. Sci. 2,1190 (1975); Brit.J.Pharmaco1. 59,523P (1977). 6. C. B. Pert, M. J. Kuhar 6 S. H. Snyder, Life Sciences 16, 1849 (197517 7. R. Schulz 6 A. Goldstein, Life Sciences la, 1843 (1975). 8. C. G. Jaureguibbery, Biophys. Res. Commun. 2.558 (1976). 9. J . Fishman, E. F. Hahn 6 B. I. Norton, Nature 261, 64 (1976). 10. G. L. Craviso 6 J. M. Masacchio, Life Sciences ?&, 821 (1976). 11. I. Creese. A. P. Feinberg 6 S. H. Snyder, Eur. J. Pharmacol. 36, 231 (1976). 12. C. B. Pert, M. J. Kuhar 6 S. H. Snyder, Proc. Natl. Acad. Sci. 2, 3729 (1976). 13. L. F. Tseng, H. H. Loh 6 S. H. Li, Proc. Natl. Acad. Sci. 12,4187 (1976); Biochem. Biophys. Res. Commun. 14,No. 2 (1977). 14. R. Simantov 6 S. H. Snyder, Proc. Natl. Acad. Sci. 2. 2515 (1976). 15. T. M. Cho, J. S. Cho 6 H. H. Loh, Life Sciences g,231 (1976). 16. I. Creese. Abstr. ACS Eltg. New York, p. MEDI 31, April (1976). 17. W. A. Klee, ACS Mtg. New York p. MEDI 32, April (1976). 18. I. Creese, G. W. Pasternak, C. B. Pert & S. H. Snyder, Life Sciences.E.1837 (1976). 19. C. B. Pert & S. H. Snyder. Biochem. Pharmacol. 25, 847 (1976). 20. A. P. Feinberg, I. Creese d S. H. Snyder, Proc. Natl. Acad. Sci. 2, 4215 (1976). 21. J. L. Marx. Science 193, 1227 (1976). 22. B. M. Cox, S. Gentleman, T. P. Su 6 A. Goldstein, Brain Res. 106. 285 (1976). 23. C. B. Pert, S. H. Snyder 6 P. S. Portoghese, J. Med. Chem. 19, 1248 (1976). 24. L. Terenius 6 A. Wahlstrom, Acta Physol. Scand. E.74 (1975). 25. A. Levy, A. R. Gintzler 6 S. Spector, Fed. Prod. Abstr. 25, 318 (1976) 26. A. R. Ginzler, A. Levy 6 S. Spector, Prod. Natl. Acad. Sci. 2. 2132 (1976). 27. C. B. Pert, A. Pert 6 J. F. Tallmann. Proc. Natl. Acad. Sci. 12, 2226 (1976). 28. G. W. Pasternak, R. Goodman 6 S. H. Snyder, Life Sciences, 16, 1765 (1975). 29. A. Goldstein. Science 193. 1081 (1976). 30. J. M. Steward, Nature 784 (1976). 31. L. Terenius. Europ. J. Pharmacol. 38, 211 (1976). 32. B. M. Cox, A. Goldstein 6 C. H. Li. Proc. Natl. Acad. Sci. 12. 1821 (1976). 33. L. H. Lazarus, N. Ling 6 R. Guilleman, Proc. Natl. Acad. Sci. 77, 2156 (1976). 34. R.Guillemin.1Oth International Congress of Biochemistry, Hamburg, July 26, 1976. 35. C.D. King, J.M. Masseran0,N.N. Santo, E . E . Codd & W.L. Byrne, Fed. Proc. E.570(1976) 36. L. M. Gunne, Science, 193,1229 (1976); L.M.Gunne et al., J.Neurotransmission, InPr. 37. R. Byck, Lancet, 2. 72 (1976). 38. R. A. North, Mature. 266, 460 (1976). 39. Simantov S.H- Snyder, Abstr. ACS Htg. New York. p. ~ D 30, I ~ ~ (1976). ~ i l 40. R * Cuillemin, N. Ling 6 R - Burgus. Comte Rend. Acad. Sci. 282, 783 (1976). 41. c. "* Li* D. Chung 6 B. 2. Donnen, Biochem. Biophys. Res. Comuns. 3,1542 (1976).

a,

x,

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Krapcho, Ed.

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42. 43. 44. 45. 46. 47.

Chap. 3

Analgesics, Antagonists, the Opiate Receptor

Gordon, Vida

2

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Chapter 4 .

Memory and Learning

-

Animal Models

P a u l E. Gold, U n i v e r s i t y of V i r g i n i a , C h a r l o t t e s v i l l e , Va. I n t r o d u c t i o n . T h i s review f o c u s e s p r i m a r i l y on t h e e f f e c t s on memory of hormones and c e n t r a l n o r e p i n e p h r i n e . There h a s been c o n s i d e r a b l e i n t e r e s t l a t e l y i n t h e r o l e s of hormonal and c a t e c h o l a m i n e r g i c systems i n t h e s t o r age of i n f o r m a t i o n provided by a new l e a r n i n g e x p e r i e n c e . Many t r e a t m e n t s , i n c l u d i n g hormones, drugs which a l t e r n e u r o t r a n s m i t t e r f u n c t i o n s , e l e c t r o shock, and l o il i C= + e c t e l e c t r i c a l s t i m u l a t i o n of t h e b r a i n , can modiI !$ I n t h e s e s t u d i e s , a t r e a t m e n t i s t y p i c a l l y adminfy retention. i s t e r e d a t some r e l a t i v e l y s h o r t t i m e a f t e r t r a i n i n g ( e . g . , 0 - 6 h r s ) and animals are t e s t e d f o r r e t e n t i o n a t a l a t e r t i m e ( u s u a l l y more t h a n 24 h r a f t e r t r a i n i n g ) . The g e n e r a l f i n d i n g i s t h a t , i f a t r e a t m e n t a f f e c t s ret e n t i o n , t h e e f f e c t i v e n e s s of t h e t r e a t m e n t d e c r e a s e s as t h e t i m e a f t e r t r a i n i n g i s i n c r e a s e d . Depending on t h e t r e a t m e n t and on t h e s p e c i f i c e x p e r i m e n t a l procedures, t h e e f f e c t , on r e t e n t i o n , may b e e i t h e r impairment ( r e t r o g r a d e amnesia) o r enhancement of memory. The t i m e a f t e r t r a i n i n g d u r i n g which r e t e n t i o n l o s e s i t s s u s c e p t i b i l i t y t o m o d i f i c a t i o n by a p a r t i c u l a r t r e a t m e n t i s termed t h e r e t r o g r a d e amnesia ( o r enhancement) g r a d i e n t . I n t h e p a s t , a g r e a t d e a l of t h e o r e t i c a l i n t e r s i n t h i s r e s e a r c h focused It was thought t h a t t h e on t h e time-dependent n a t u r e of t h e s e e f f e c t s . ” ’ time-course of r e t r o g r a d e amnesia enhancement g r a d i e n t s r e f l e c t e d a biol o g i c a l c o n s t r a i n t on t h e t i m e n e c e s s a r y t o complete memory s t o r a g e , i . e . , t h e t i m e r e q u i r e d t o form long-term memory. A time-constant of t h i s s o r t would have c r i t i c a l i m p l i c a t i o n s i n terms of t h e n e u r o b i o l o g i c a l mechanisms which might b e a s s i g n e d t h e f u n c t i o n of memory s t o r a g e . Obviously, d i f f e r e n t mechanisms of s t o r a g e would be involved f o r a p r o c e s s which r e q u i r e d o n l y a few seconds as compared t o one which r e q u i r e d s e v e r a l h o u r s t o r e a c h completion.

!i;J;3;i,

It i s t h e r e f o r e somewhat u n f o r t u n a t e t h a t t h i s i n f o r m a t i o n i s n o t l i k e l y t o b e forthcoming from s t u d i e s of r e t r o g r a d e amnesia o r enhancement. R a t h e r , i t now a p p e a r s t h a t f o r any l e a r n i n g s i t u a t i o n , t h e t i m e a f t e r t r a i n i n g d u r i n g which memory may b e modified i s determined t o a l a r g e e x t e n t by t h e s e v e r i t y ( i . e . , drug d o s e , e l e c t r o s h o c k i n t e n s i t y , number of animals, t h e g r a d i e n t t r e a t m e n t s ) of t h e t r e a t m e n t . I n some s t u d i e I n humans, t h e r e are may v a r y from a few seconds t o hours o r days. i n d i c a ons t h a t t h e r e t r o g r a d e amnesia g r a d i e n t may b e as l o n g as s e v e r a l years. Because t h e g r a d i e n t s are n o t ” f i x e d ” ( b i o l o g i c a l l y ) f o r a p a r t i c u l a r l e a r n i n g s i t u a t i o n , i t i s p r e f e r a b l e t o t h i n k of t h e time-dependent e f f e c t s on memory as r e f l y S t i n g d e c r e a s i n g s u s c e p t i b i l i t y t o m o d i f i c a t i o n An e x t e n s i o n of t h i s view i s t h a t t h e treatw i t h time a f t e r t r a i n i n g . ments are modulators of memory p r o c e s s i n g , a l t e r i n g t h e a c t i v i t y of systTTs which are r e s p o n s i b l e f o r promoting t h e s t o r a g e of s p e c i f i c information.

e,~sfnf

ft

I n a d d i t i o n t o t h e t h e o r e t i c a l i m p l i c a t i o n s of t h i s r e s e a r c h , t h e r e i s a l s o a p r a c t i c a l advantage t o t h e u s e of t h e g e n e r a l e x p e r i m e n t a l proced u r e s d e s c r i b e d above. S t u d i e s which examine t h e e f f e c t s of a t r e a t m e n t on memory p r o c e s s i n g are perhaps b e s t s u i t e d t o do so i f t h e t r e a t m e n t i s a d m i n i s t e r e d s h o r t l y a f t e r t r a i n i n g . T h i s d e s i g n a s s u r e s t h a t a l l animals

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are u n t r e a t e d a t t h e t i m e of t r a i n i n g . Furthermore, w i t h t h e a d d i t i o n of delayed t r e a t m e n t s which do n o t a l t e r r e t e n t i o n performance, one can conc l u d e t h a t any e f f e c t s on r e t e n t i o n performance are t h e r e s u l t of r e t r o a c t i v e m o d i f i c a t i o n of memory s t o r a g e p r o c e s s i n g and are n o t t h e consequence of a d i r e c t p r o a c t i v e i n f l u e n c e of t h e t r e a t m e n t a t t h e t i m e of memory. Thus, t h e g e n e r a l experimental d e s i g n excludes p o s s i b l e a l t e r a t i o n s i n p e r c e p t i o n o r motor c a p a b i l i t i e s a t t h e t i m e of t r i n i n g o r t e s t i n g as a 3 v i a b l e i n t e r p r e t a t i o n of t h e changes i n r e t e n t i o n .

.

Hormonal manipulation. The g e n e r a l i n t e r e s t i n t h e s t u d y of hormonal manipulation e f f e c t s on memory was f o s t e r e d by t h e r e c o g n i t i o n 1 5 ~ f 6 ~ h ! 18 f7~ systems are r e s p o n s i v e t o many i f n o t a l l t r a i n i n g s i t u a t i o n s and t h e r e f o r e have t h e p o t e n t i a l of p a r t i c i p a t i n g i n b o t h p h y s i o l o g i c a l a d a p t a t i o n t o fgyigjpyi~taldemands as w e l l as t h e b e h a v i o r a l a d a p t a t i o n t o t h e s e demands. Furthermore, t h e ormonal r o l e i n a d a g f i v e 29 b e h a v i o r s may i n c l u d e e f f e c t s on J y x p g , on memory s t o r a g e , o r on maintenance of l e a r n e d responses.

and v a s o p r e s s i n . b&;h2gf 98;lty These literature in t h i s area h a s been reviewed p r e v i o u s l y . studies c l e a r l y i n d i c a t e t h a t ACTH i n j e c t i o n s may d e l a y e x t i n c t i o n of l e a r n e d r e s p o n s e s ( i . e . , a d e c r e a s e i n a p r e v i o u s l y l e a r n e d r e s p o n s e i n t h e absence of reward o r punishment), a l t h o u g h i t i s n o t c l e a r whether t h i s r e s u l t is due t o p o t e n t i a t i o n of p r e v i o u s l y l e a r n e d r e s p o n s e s o r t o impaired a c q u i s i have been t i o n of t h e e x t i n c t i o n t r a i n i n g . These behaviora il:f5gcts observed d u r i n g e x t i n c t i o n gy:f.@-ice r e s p o n s e s as w e l l s e x t i n c 3f rats were t i o n of rewarded behavior. I n one s t u d y f o r example, t r a i n e d t o approach food i n an alleyway. Following t r a i n i n g , a n i m a l s s y n t h e t i c ACTH1-24 ( b o t h of r e c e i v e d d a i l y i n j e c t i o n s of p o r c i n e ACTH (which does n o t have adrenowhich have a d r e n o c o r t i c a l a c t i v i t y ) , ACTHZI;:’ c o r t i c a l stimulating activity) o r corticosterone, prior t o extinction t r a i n i n g . ACTH and i t s a n a l o g s r e t a r d e d e x t i n c t i o n , w h i l e c o r t i c o s t e r o n e a c c e l e r a t e d e x t i n c t i o n , as compared t o p l a c e b o - t r e a t e d c o n t r o l s . I n a d d i on ACTH i n j e c t i o n s may a l t e r a c q u i s i t i o n rates of avoidance t r a i n 35 ing ACTH, ACTH a n a l o g s ,

57;

3nf

.54,

A series of experiments examined t h e r o l e of p i t u i t a r y - a d r e n a l hggmorjys ’ ’ a c q u i s i t i o n and e x t i n c t i o n of c o n d i t i o n e d t a s t e a v e r s i o n l e a r n i n g . I n t h i s b e h a v i o r a l s i t u a t i o n , an animal i s allowed t o i n g e s t a s o l u t i o n w i t h a s p e c i f i c ( t y p i c a l l y novel) t a s t e . A t some t i m e w i t h i n t h e n e x t s e v e r a l h o u r s , t h e animal r e c e i v e s an i n j e c t i o n of l i t h i u m c h l o r i d e o r o t h e r a g e n t . When t e s t e d a t a subsequent t i m e , t h e animal w i l l show an a v e r s i o n f o r t h e s o l u t i o n . The f i n d i n g s of t h i s set of experiments r e v e a l e d t h a t plasma c o r t i c o s t e r o n e l e v e l s i n male rats were e l e v a t e d f o r up t o 4 h r s a f t e r an a p p r o p r i a t e i n j e c t i o n of l i t h i u m c h l o r i d e , i n d i c a t i n g a c t i v a t i o n of t h e p i t u i t a r y - a d r e n a l system under t h e s e t r a i n i n g c o n d i t i o n s . P r e t r e a t m e n t w i t h dexamethosone, a s t e r o i d which i n h i b i t s ACTH s e c r e t i o n , blocked t h e c o r t i c o s t e r o n e r e s p o n s e t o t r a i n i n g and, f u r t h e r , diminished t h e e x t e n t of a v e r s i o n t o t h e test s o l u t i o n as measured a t l a t e r t i m e . Under comparable t r a i n i n g c o n d i t i o n s , ACTH a d m i n i s t e r e d p r i o r t o t h e l i t h i u m c h l o r i d e i n j e c t i o n s had no e f f e c t on a c q u i s i t i o n , a l t h o u g h p o t e n t i a l l y enhanced a c q u i s i -

38

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tion may have been masked by the fact that control animals ingested very little of the test solution. However, ACTH injections administered prior to extinction sessions did delay the recovery from taste aversion. Another set of experiments examined the possible modulatory effects of ACTH on memory processing. The possibility, discussed in the introduction, that some treatments may modulate memory storage predicts that there may be endogenous memory modulatory systems which are activated by those posttrial treatments which act to impair or to enhance memory; one such set of systems might include hormonal responses to training. The rationale guiding these studies is as follows: If, for example, an animal is trained in a one-trial inhibitory (passive) avoidance task, retention performance will vary with the footshock intensity. In addition, the extent of the hormonal response to training with different shock levels will vary. The question posed here was: To what extent is better retention performance after training with high footshock a function of the hormonal response to training? The approach used to address this question was to train animals with a weak footshock and to follow the training with subcutaneous injections of ACTH. The hypothesis guiding this study was that posttrial ACTH injections should enhance retention performance by adding to the normal hormonal consequences of training, thereby mimicking the consequences of more intense training nd ncreasing retention performance. The findings of such experiments are generally consistent with this view. Animals which received immediate posttrial injections of ACTH had enhanced retention performance. Delayed injections were ineffective. Somewhat surprisingly, high ACTH doses produced amnesia. The doses used proved to be critical; the dose-response curve was an inverted-U function. Furthermore, the optimal doses for enhancing retention varied in a meaningful manner with the stress (shock level) used in training. A single dose of ACTH enhanced retention of weak footshock training and impaired retention of strong footshock training. Such data support the view that there is an inverted4 relationship between posttrial circulating ACTH levels (endogenous ACTH + injected ACTH) and memory processing. The inverted-U dosee is characteristic of other treatments which Also, the interaction of the optimally enhance memory.4f'S'''S'3fU'Y; enhancing dose of these drugs and other treatments4~ft~5fr$&;i~~-related stress seems to have some considerable generality. Such findings may be related to the inverted-)J8;e&fisbship between stress and performance which has been noted before, and may suggest that the biological dimension underlying the inverted-U may be hormonally mediated. More generally, these findings support the view that hormonal responses to training may modulate memory storage processing.

'" "

The mechanism by which ACTH acts on learning and memory is not clear. The hormonal action does not, however, appear to depend on adrenocortical activity. Many studies have demonstrated that melanonyte stimulating hormone, which shares a behaviorally active peptide sequence with ACTH, as well as several N-terminal ACTH analogs (e.g.,ACTH4-10 [Met-Glu-His-Phe-ArgTry-Gly]) which have no corticotrophic activity, have effects on learning, memory, and maintenance ~5,lsa;n~$performance which are quite comparable to those of ACTH itself. Recent studies have examined the

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behavioral and biological effects of these a ents. The agents do not appear to affect spontaneous motor behavior59y54 but there is some evidence that the agents may enhance attention in rats55956 and in m a n . 5 7 ~ 5 ~ACT -10 alters the frequency and excitability of hippocampal theta 0 and alters visual evoked responses in rats53 when injected subcutaneously. Some ACTH analogs (e.g., ACT%-g containing amino acid substitutions [Met(O)-His-Phe-dLys-Phe 1) are effective behaviorally at far lower doses than those needed for ACTH itself.61 The peptide doses which are effective appear to be related to an increase in in vitro half-life.61,62 ACTH and some analogs appear to increase brain RNA and protein rnetabolismY63~64~65 glucose metabolism66 and noradrenaline turnover.67~68 Thus, it is clear that the ACTH analogs which enhance learning and memory have the capacity to act on the brain, possibl at the level of the posterior thalamic nucleus parafascicularis ,69 ~7~ but the multiple biological effects complicate the issue of which mechanism of action is related to the behavioral effects obtained with these agents.

2

Other examinations of the relation of ACTH and memory studied the effect of ACTH (or its analogs) on animals rendered amnestic by carbon dioxide exposure (to respiratory arrest) or electroconvulsive shock. If amnestic animals receive an injection of ACT&-10 1 hour prior to retention testing, the amnesia is significantly attentuated.71,72 S Vasopressin. Vasop essin (a posterior pituitary peptide [H-Cls-Tyr-Phehas many behavioral effects comGlu(NH2)-Asp(NH2)-Cys-Pro-Lys-Gly-NH2]) Darable to those observed with ACTH. A posttrial injection of vasopressin enhances retention of inhibitor avoidance training73 and impairs extinction of avoidance training.74r73 In general, the major difference between the effects of ACTH and vasopressin on learning and memory is that ACTH must be administered throughout training or extinction trials to enhance acquisition or retard the development of extinction. However, a single injection of vasopressin prior to training or extinction trials has comparable effects. Thus, vasopressin effects on learning and memory seem to be of much longer duration than those of ACTH.75

8

It was recently reported that a posttrial injection of vasopressin may enhance later retention of inhibitory (passive)avoidance training in rats with hereditary hy othalamic diabetes insipidus that are not able to synthesize v a s o p r e ~ s i n 377~78 .~~ This effect is time-dependent; delayed injections do not affect later retention performance. Furthermore, in normal Wistar rats, intraventricular injections of antibodies to arginine8-vasopressin 30 min prior to or immediately after the training trial produce amnesia in dose-dependent manner.78 Catecholamines. Peripheral catecholamines have generally received less attention than pituitary hormones in terms of involvement in learning and memory. However, the effects on memory of posttrial epinephrine and norepine hrine appear comparable to those obtained with ACTH or vasopressin. 38,79 Epinephrine injections administered shortly after inhibitory

34

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avoidance t r a i n i n g enhance l a t e r r e t e n t i o n performance and yglayed Furtheri n j e c t i o n s ( e . g . , by 2 h r s a f t e r t r a i n i n g ) are i n e f f e c t i v e . more, t h e dose-response c u r v e f o r t h e e f f e c t s of e p i n e p h r i n e on memory i s an inverted-U; high doses produce r e t r o g r a d e amnesia. The memory enhancing e f f e c t s of e p i n e p h r i n e are blocked by p r e t r i a l i n j e c t i o n s of t h e b e t a - a d r e n e r g i c blocking a g e n t , p r o p a n o l o l , b u t a r e u n a f f e c t e d by p r e t r i a l i n j e c t i o n s of t h e alpha-adrenergic b l o c k i n g a g e n t , phenoxybenzamine. Conv e r s e l y , t h e amnesia produced by e p i n e p h r i n e i s blocked by phenoxybenzamine b u t n o t propanolol. Thus, t h e r i s i n g and f a l l i n g p o r t i o n s of t h e inverted-U dose-response curve8pfay be mediated by d i f f e r e n t pharmacologic a l mechanisms of e p i n e p h r i n e . The r o l e of c e n t r a l n o r e p i n e p h r i n e and ca&;h@m&es, particularly i n memory, h a s been i n v e s t i g a t e d i n more d e t a i l . Many of t h e r e c e n t s t u d i e s t e s t i n g t h e view t h a t c e n t r a l e p i n e p h r i n e i s important t o memory p r o c e s s i n g have used s y n t h e s i s i n h i b i t o r s such as d i e t h y l d i t h i o carbamate (DDC), a dopamine beta-hydroxylase i n h i b i t o r . T h i s drug i s a p o t e n t amnestic a g e n t which produces r e t r o g r a d e mnesia i n r a t s , f o r example, even i f i n j e c t e d 24 h r a f t e r t r a i n i n g , 8 ' a t an i n t r a p e r i t o n e a l d o s e (900 mglkg) which r e d u c e s whole b r a i n n o r e p i n e p h r i n e c o n c e n t r a t i o n s t o approximately 15% of c o n t r o l l e v e l s . The drug produces amnesia i n r 85: i n h i b i t o r y avoidance and v i s u a l d i s c r i m i n a t i o n t a s k s . etf"8"r8"ttt8 Furthermore, t h e amnestic e f f e c t s of a p r e t r a i n i n g DDC i n j e c t i o n can be a t t e n u a t g g w i t h immediate p o s t t r i a l i n t r a v e n t r i c u l a r It should be noted t h a t a l t h o u g h t h e s e i n j e c t i o n s of n o r e p i n e p h r i n e . c o l l e c t i v e r e s u l t s are c o n s i s t e n t w i t h t h e view t h a t t h e e f f e c t s of DDC on memory are a consequence of t h e d e c r e a s e i n n o r e p i n e p h r i n e concentrat i o n s , t h e r e a r e o t h e r e f f e c t s of t h e drug which may be important as we8.J. For example, DDC produces a s h o r t - t e r m i n c r e a s e i n whole b r a i n dopamine and, because DDC c h e l a t e s z i n c , t h e drug i n j e c t i o n r e s u l t s 8 & n b l e a c h i n g It i s t h e r e of a s u l f i d e s i l v e r s t a i n i n t h e hippocampal mossy f i b e r s . f o r e p o s s i b l e t h a t t h e e f f e c t s on memory of t h i s drug may b e r e l a t e d n o t only t o t h e i n t e r f e r e n c e with norepinephrine s y n t h e s i s but a l s o t o i n t e r f e r e n c e w i t h t h e a c t i v i t y of o t h e r n e u r o b i o l o g i c a l systems.

"'

The evidence t h a t t r e a t m e n t s which a c u t e l y i n t e r f e r e w i t h c e n t r a l n o r a d r e n e r g i c systems a l s o impair memory p r o c e s s i n g seemwell founded. However, c h r o n i c d e p l e t i o n of c e n t r a l n o r e p i n e p h r i n e ( e . g . , w i t h 6-hydroxydopamine o r l o c u s coeru h y i 8 & e s i o n ) does n o t r e l i a b l y r e s u l t i n impaired acquisition o r retention. T h e r e f o r e , a l t h o u g h t e l e c e p h a l i c nore p i n e p h r i n e i s n o t n e c e s s a r y f o r l e a r n i n g and r e t e n t i o n , i t a p p e a r s t h a t , i f a v a i l a b l e , t h e c e n t r a l n o r a d r e n e r g i c systems may modulate memory processing. Conclusions. There i s now c o n s i d e r a b l e evidence t h a t p e p t i d e hormones and catecholamines have t h e p o t e n t i a l t o a c t on a c q u i s i t i o n , on t h e maintenance of l e a r n e d r e s p o n s e s , and on memory p r o c e s s i n g . The n e u r o b i o l o g i c a l mechanisms by which t h e s e systems a c t i s less clear. The problem i s n o t t h a t c e n t r a l e f f e c t s of t h e hormones and drugs are unknown b u t , r a t h e r , t h a t t o o many e f f e c t s are recognized. T h e r e f o r e , i t i s d i f f i c u l t t o assess which of t h i s l a r g e s e t of e v e n t s mediates t h e hormonal and aminergic

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35 -

modulation of learning and memory. However, we can presumably look to the future for studies which pursue these biological mechanisms, perhaps using the strategy of selectively manipulating a particular consequence of a hormonal or catecholaminergic activity independently to assess which neurobiological effects of these systems are related to learning and memory. References

1. J. L. McGaugh and P. E. Gold, "Neural Mechanisms of Learning and Memory'', The MIT Press, Cambridge, Mass., 1976, pp. 549-560. 2. P. E. Gold, S. F. Zornetzer and J. L. McGaugh, "Advances in Psychobiology, Vol. 2", Wiley Interscience, New York, New York, 1975, pp. 64-75. 3. J. L. McGaugh, Ann. Rev. Pharm., 13,229 (1973). 4. R. P. Kesner and M. W. Wilburn, Behav. Biol., lo, 259 (1973). 5. R. A. Barraco and L. J. Stettner, Psych. Bull., 83, 242 (1976). 6. P. E. Gold and J. L. McGaugh, "Neuropeptides and Behavior", Raven Press, 1977. 7. R. W. Gerard, her. J. Psychiat., 126, 161 (1949). 8. D. 0. Hebb, "The Organization of Behavior", Wiley Interscience, New York, New York, 1949. 9. A. Cherkin, Proc. Nat. Acad. of Science, 63, 1094 (1969). 10. P. E. Gold, J. Macri and J. L. McGaugh, Science, 179, 1343 (1973). 11. C. J. Mah and D. J. Albert, Behav. Biol., 2, 517 (1973). 12. L. R. Squire, Behav. Biol., 12, 119 (1974). 13. J. L. McGaugh and R. G. Dawson, "Animal Memory", Academic Press, New York, New York, 1972, pp. 215-242. 14. P. E. Gold and J. L. McGaugh, "Short-Term Memory", Academic Press, New York, New York, 1975, pp. 355-378. 15. H. Selye, "Stress: The Physiology and Pathology of Exposure to Stress", Acta Medicia Publications, Montreal, Canada, 1950. 16. G. D. Coover, L. Goldman and S. Levine, Physiol. and Behav., 6, 261 (1971). 17. G. D. Coover, L. Goldman and S. Levine, Physiol. and Behav., I , 727 (1971). 18. S. Levine, L. Goldman and G. D. Coover, "Physiology, Emotion and Psychosomatic Illness", Assoc. Scientific Publishers, Amsterdam, North Holland, 1972, pp. 281-296. 19. B. Bohus, "The Hippocampus, Vol. l", Plenum Press, New York, New York, 1976, pp. 323-353. 20. B. Bohus, "Progress in Brain Researchft,Elsevier, Amsterdam, Holland, 1974, pp. 181-183. 21. D. de Wied, "The Neurosciences", The MIT Press, Cambridge, Mass., 1974, pp. 653-666. 22. D. de Wied, A.M.L. van Delft, W.H. Gispen, J.A.W.M. Weijnen, and Tj. B. van Wimersma, "Hormones and Behavior", Academic Press, New York, New York, 1972. 23. P. E. Gold and J. L. McGaugh, "Short-Term Memory", Academic Press, New York, New York, 1975, pp. 355-390. 24. D. de Wied, B. Bohus, Tj.B. van Wimersma Greidanus, ''Progress in

36

25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55.

Sect. I

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CNS Agents

Krapcho, Ed.

Brain Research, Vol. 41", E l s e v i e r , Amsterdam, Holland, 1974, pp. 197201. D. d e Wied, L i f e S c i . , 9, 2, pp. 195-204 (1977). D. d e Wied, " F r o n t i e r s i n Neuroendocrinology", Oxford Univ. P r e s s , London, England, 1968, pp. 97-140. S. Levine, Hormones and Conditioning, Nebraska Symposium on Motivat i o n (1968). R. L. S p r o t t , "Hormonal C o r r e l a t e s of Behavior, Vol. 2", Plenum P r e s s , New York, New York, 1975. F. R. Brush and J. C. F r o e h l i c h , "Hormonal C o r r e l a t e s of Behavior, Vol. 2", Plenum Press, New York, New York, 1975. S. Guth, S. Levine and J. P. Seward, P h y s i o l . and Behav., 1, 195 (1971). P. Garrud, J. A. Gray and D. d e Wied, P h y s i o l . and Behav., 109 (1974). S. Guth, J. P. Seward and S. Levine, Hormones and Behavior, 2, 127 (1971). J. A. Gray, A. R. Mayes and M. A. Wilson, Neurophar., 10, 223 (1971). E. L. D i Gusto, K. C a i r n c r o s s and M. G. King, Psychol. B u l l . , 75,432 (1971). A. M. Schneider, J. Weinberg and R. Weissberg, P h y s i o l . and Behav., 13, 633 (1974). K. Kendler, J. W. Hennessey, W. P. Smotherman and S. Levine, Behav. 225 (1976). Biol., W. P. Smotherman, J. W. Hennessy and S. Levine, Behav. B i o l . , 16,401 (1976). J. W. Hennessy, W. P. Smotherman and S. Levine, Behav. B i o l . , 2, 413 (1976). P. E. Gold and R. van Buskirk, Hormones and Behavior, 7 , 509 (1976). P. E. Gold and R. van Buskirk, Behav. B i o l . , 2, 387 ( i 9 7 6 ) . J. Krivanek and J. L. McGaugh, Psychophar., 303 (1968). J. Krivanek and J. L. McGaugh, Agents and A c t i o n s , 1,36 (1969). J. L. McGaugh and J. Krivanek, P h y s i o l . and Behav., 1437 (1970). J. Krivanek, Psychophar., 9, 213 (1971). P. E. Gold, L. Hankins, R. Edwards, J. Chester and J. L. McGaugh, Brain Res., 86, 509 (1975). V. Bloch, Brain Research, 24, 561 (1970). L. 0. S t r a t t o n and A. J. K a s t i n , Hormones and Behavior, 5, 149 (1974). H. Selye, "The Physiology and Pathology of Exposure t o S t r e s s ' ' , Acta, I n c . , Montreal, Canada, 1950. D. 0. Hebb, "A Textbook of Psychology", W. B. Saunders, P h i l a d e l p h i a , Pa . , 1966. D. E. Berlyne, S c i . Amer., 215, 8 2 (1966). D. de Wied, "The Neurosciences'', The MIT Press, Cambridge, Mass., 1974, pp. 653,666. H. M. Greven and D. d e Wied, "Progress i n Brain Research, Vol. 39", E l s e v i e r , Amsterdam, Holland, 1973, pp. 429-442. 0. L. Wolthuis and D. d e Wied, Pharm. Biochem. and Behav., 6,273 (1976). G. L. Kovacs, I. G a j o r i , G. Telegdy and K. L i s s a k , P h y s i o l . and Behav., 1 3 , 349 (1974). C . A. Sandman, A. J. K a s t i n and A. V. S c h a l l y , J. Comp. and P h y s i o l .

12,

17,

12,

Chap. 4

Memory and Learning

Go Id

37 -

Psych., 9, 54 (1972). 56. C. A. Sandman, W. Beckwith, M. M. Gittis and A. J. Kastin, Physiol. and Behav., 13,163 (1974). 57. L. H. Miller, A. J. Kastin, C. A. Sandman, M. Fink and W. van Veen, Pharm. Biochem. and Behav., 2, 663 (1974). 58. C. A. Sandman, J. M. George, J. D. Nolan, H. van Riezen and A. J. Kastin, Physiol. and Behav., 2, 427 (1975). 59. I. Urban, F. H. Lopes de Silva, W. Storm van Leeuwen and D. de Wied, Brain Research, 3,361 (1974). 60. I. Urban and D. de Wied, Experimental Brain Research, 24, 324 (1976). 61. A. Witter, H. M. Greven and D. de Wied, J. Pharm. and Exp. Therap., 193, 853 (1975). 62. J. Verhoeff and A. Witter, Pharm. Biochem. and Behavior, 5, 583 (1976). 63. W. H. Gispen and P. Schotman, "Progress in Brain Research, Vol. 39", Elsevier, Amsterdam, Holland, 1973, pp. 443-459. 64. W. H. Gispen, D. de Wied, P. Schotman and H. S. Jansz, Brain Res., 2, 341 (1971). 65. M. E. A. Reith, P. Schotman and W. H. Gispen, "Progress in Brain Research, Vol. 42", Elsevier, Amsterdam, Holland, 1975, pp. 195-200. 66. E. R. De Kloet and A. Witter, Proc. 14th Meeting of Med. Biol. SOC. in the Netherlands, 1973. 483 (1973). 67. D. H. G. Versteeg, Brain Research, 2, 68. B. E. Leonard, Arch. Intl. Pharmacol. Therapy, 207, 242 (1974). 69. Tj. B. van Wimersma Greidanus, B. Bohus and D. de Wied, Neuroendo., 14, 280 (1974). 70. Tj. B. van Wimersma Greidanus, B. Bohus and D. de Wied, "Progress in Brain Research, Vol. 41", Elsevier, Amsterdam, Holland, 1974, pp. 429-432. 71. H. Rigter, H. van Riezen and D. de Wied, Physiol. and Behav., 13,381 (1974). 72. H. Rigter and H. van Riezen, Physiol. and Behav., 14,563 (1975). 73. R. Ader and D. de Wied, Psychonomic Science, 29, 46 (1972). 74. D. de Wied, Nature (London), 232, 58 (1971). 75. D. de Wied, B. Bohus and Tj. B. van Wimersma Greidanus, "Progress in Brain Research, Vol. 41", Elsevier, Amsterdam, Holland, 1974, pp. 417-428. 76. D. de Wied, B. Bohus, I. Urban and Tj. B. van Wimersma Greidanus, Brain Research, 85, 152 (1975). 77. Tj. 5 . van Wimersma Greidanus, J. Dogterum and D. de Wied, Life Sciences, 16,637 (1975). 78. Tj. B. van Wimersma Greidanus and D. de Wied, Behav. Biol., 18,325 (1976). 79. P. E. Gold and R. B. van Buskirk, Behav. Biol., 13,145 (1975). 80. R. B. van Buskirk, P. E. Gold and 3. L. McGaugh, "Progress in Brain Research, Vol. 4 2 " , Elsevier, Amsterdam, Holland, 1975. 81. S. S. Kety, "The Neurosciences", Rockefeller Univ. Press, New York, New York, 1970, pp. 324-336. 82. S. S. Kety, "The Chemistry of Mood, Motivation and Memory", Plenum Press, New York, New York, 1972, pp. 65-80. 83. J. L. McGaugh, P. E. Gold, R. van Buskirk and J. W. Haycock, "Progress in Brain Research, Vol. 42", Elsevier, Amsterdam, Holland, 1975.

38 84. 85. 86. 87.

88.

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- CNS Agents

Krapcho, Ed.

C. T. Randt, D. Quartermain, M. G o l d s t e i n and B. Anagnosti, S c i e n c e ,

172, -

498 (1971). L. S t e i n , J. D. B e l l u z z i and D. C. Wise, Brain Research, 84, 329, (1975). M. D. Hamburg and A. Kerr, Pharm. Biochem. and Behav., 2, 489 (1976) J. B. F l e x n e r and L. B. F l e x n e r , Pharm. Biochem. and Behav., 5, 117 (1976). M. E. H a l l , Behav. B i o l . , 145 (1976).

16,

Section I1 - Pharmacodynamic Agents Editor: John E. Francis, CIBA-GEIGY Corp., Ardsley, N.Y.

Chapter 5. Antiarrhythmic and Antianginal Agents Thomas Baum, Robert L. Wendt and James L. Bergey Wyeth Laboratories Inc. Radnor, Pennsylvania 19087

,

Antiarrhythmic Agents

It has long been believed that cardiac arrhythmias result from changes in the conducting properties and/or automaticity of the myocardium. Normally, each cardiac impulse arises in the sinoatrial node in the right atrium and then is rapidly transmitted throughout the atria and via the atrioventricular node, His bundle, bundle branches and specialized conduction fibers (Purkinje fibers) to all regions of the ventricles to assure coordinated activation and contraction, The concentration of potassium is considerably higher and that of sodium lower within cardiac cells than in the extracellular fluid. External membranes of cardiac cells are selectively permeable to ions and contain biochemical pumping mechanisms which can transport ions against electrical and concentration gradients. These factors lead to an electrical potential difference across the membrane with the inside negative with respect to the outside. An impulse can arise in the sinoatrial node or other "automatic" or "pacemaker" cells as a result of slow spontaneous depolarization. Usually, when the level of depolarization reaches a critical "threshold" level, self perpetuating changes occur which tend to depolarize the cell completely and even reverse the potential to inside positive. The juxtaposition of depolarized and polarized regions alters the membrane characteristics of the latter and, in turn, causes their depolarization and results in the development of a propagated impulse. Repolarization occurs after a certain period and the cycle recurs.1-3 As just outlined, both impulse initiation (automaticity) and conduction result from changes in the permeability of the external membrane of cardiac cells to various ions. Normally, impulse propagation in most cardiac cells results from a rapid but brief increase in sodium permeability which permits an influx of that ion and produces rapid depolarization. A s cells become partially depolarized, either as a result of the normal "fast" sodium inward current or for other reasons, other changes in membrane permeability occur leading to a more slowly developing and smaller inward ion flow composed primarily of calcium. In diseased andlor partially depolarized cells this "slow" calcium current may be the only mechanism available for impulse propagation. It may also be responsible for impulse initiation in areas not normally automatic.1-5 The electrophysiological characteristics, e.g.,

excitability,

40

Sect. I1

-

Pharmacodynamic Agents

Francis, Ed

r e f r a c t o r y period, conduction v e l o c i t y , etc., of even t h e normal v e n t r i c u l a r conduction system a r e not uniform. Factors which enhance t h i s d i s p a r i t y f o s t e r t h e development of arrhythmias. 1,395-7 Ischemia, s t r e t c h , catecholamines, potassium, etc. can cause nonuniform o r r e g i o n a l suppress i o n of normal conduction mediated by t h e " f a s t " response and r e s u l t i n t h e predominance of t h e "slow" response with i t s attendant s u s c e p t i b i l i t y t o blockade and automaticity. For example, coronary a r t e r y occlusion i n dogs r e s u l t s i n highly complex arrhythmias. l J 3 r 5 - 7 The i n i t i a l phase occurring within t h e f i r s t few hours i s probably due t o depressed conduct i o n and r e e x c i t a t i o n i n t h e ischemic area. The l a t e r phase s t a r t i n g a t 12 t o 16 h r i s probably t h e r e s u l t of r e p e t i t i v e impulse i n i t i a t i o n i n ischemic Purkinje fibers. Antiarrhythmic drugs have been c l a s s i f i e d on t h e b a s i s of t h e i r e f f e c t s on t h e r a t e of depolarization, a c t i o n p o t e n t i a l duration, i n h i b i t i o n of t h e "slow" current, beta-adrenergic blockade, etc.2 However, it has become apparent t h a t t h e conditions under which drugs a r e assessed, i.e., potassium concentration, perfusion with blood i n c o n t r a s t t o Tyrode's solut i o n o r t h e se of diseased r a t h e r than normal t i s s u e , can a f f e c t r e s u l t s greatly. l j 2 j 8 For example, lidocaine and propranolol markedly depress conduction s e l e c t i v e l y i n ischemic zones but not i n normal regions during t h e e a r l y phase following coronary a r t e r y occlusion i n dogs and thereby suppress arrhythmias .7

-

Clinical reports B e n e f i c i a l e f f e c t s of disopyramide (A) i n various human Prophylacarrhythmias were described i n r e c e n t l y published symposia.g,10 tic administration of t h e compound following myocardial i n f a r c t i o n w a s reported t o reduce t h e incidence of v e n t r i c u l a r arrhythmias as w e l l a s t h e occurrence of r e i n f a r c t i o n during t h e h o s p i t a l stay. l1 M e x i l i t i n e (Kbl 1173, 2) and procainamide were compared i n a 12 day study i n a group of a c u t e myocardial i n f a r c t i o n p a t i e n t s . 12 Both drugs suppressed arrhythmias but t h e former exerted fewer s i d e e f f e c t s . M e x i l i t i n e was a l s o evaluated i n an extended study of 1 t o 16 months.I3 S a t i s f a c t o r y c o n t r o l of a r rhythmias w a s achieved i n 79% of p a t i e n t s , Side e f f e c t s included tremors, dizziness, nausea and blurred v i s i o n , although t h e authors concluded t h a t t h e drug was w e l l t o l e r a t e d i n most p a t i e n t s . A recent symposium d e t a i l e d t h e e f f i c a c y of aprindine (2) i n v e n t r i c u l a r and s u p r a v e n t r i c u l a r arrhythmias. 14 The drug suppressed t h e former i n p a r t i c u l a r . Troublesome neurological s i d e e f f e c t s occurred i n some p a t i e n t s .

I n a recent study intravenous verapamil (&), an agent which i n h i b i t s transmembrane i n f l u x of calcium, e f f e c t i v e l y suppressed s u p r a v e n t r i c u l a r tachycardias but proved much less b e n e f i c i a l i n v e n t r i c u l a r tachycardias15

Chap. 5

Antiarrhythmics, Antianginals

Baum, Wendt, Bergey

41

The o r a l l y active l i d o c a i n e analog t o c a i n i d e ( W-36095, 3 ) suppressed premature v e n t r i c u l a r contra t i o n s following s i n g l e doses as w e l l as during s e v e r a l days of therapy. l-7 Although t h e compound was g e n e r a l l y w e l l t o l e r a t e d , s i d e e f f e c t s o f c e n t r a l nervous system o r i g i n appeared i n some p a t i e n t s a t blood l e v e l s not g r e a t l y above t h e t h e r a p e u t i c range.

Plasma f r e e f a t t y a c i d s (FFA) a r e elevated during t h e e a r l y phase of myocardial i n f a r c t i o n . Rapid reduction of FFA following t h e a d m i n i s t r a t i o n of t h e n i c o t i n i c a c i d analog 5-fluoro-3 hydroxymethylpyridine hydrochloride t o p a t i e n t s with i n f f g c t i o n was a s s o c i a t e d with decreased incidence of w a s r e p o r t e d t o be e f f e c t i v e i n v e n t r i c u l a r arrhythmias. Amiodarone t h e treatment of a t r i a l and v e n t r i c u l a r arrhythmias i n a r e c e n t t r i a l . 19

(6)

(I)

I

FQCHZoH

-1

-6

-

Newer antiarrhythmic agents A comprehensive review of new agents has appeared r e c e n t l y . a > 2 1 R e s u l t s of d e t a i l e d experimental e v a l u a t i o n of s e v e r a l newer compounds were a l s o published recently. BL-3677A (8) suppressed v e n t r i c u l a r arrhythmias produced by ouabain i n dogs and proved t o be more potent than lidocaine.22 It a l s o antagonized v e n t r i c u l a r arrhythmias occurring 24 hours a f t e r coronary a r t e r y occlusion i n dogs. The compound possesses l o c a l a n e s t h e t i c a c t i v i t y and b r i e f l y reduced blood pressure. MK-251 (2) antagonized v e n t r i c u l a r arrhythmias following myocardial i n f a r c t i o n produced by t h e intracoronary i n j e c t i o n of a s c l e r o s i n g agent o r by coronary a r t e r y occlusion i n dogs.23 It increased t h e dose of digoxi n required t o induce c a r d i a c dysrhythmias i n cats. The compound i s reported t o l a c k s i g n i f i c a n t hemodynamic e f f e c t s .

8 -

The aminosteroid ORG 6001 (&) i n h i b i t e d arrhythmias produced by a c o n i t i n e i n rats and by ouabain and coronary a r t e r y occlusion i n dogs.2425 The compound a l s o reduced t h e biochemical changes r e s u l t i n g from myocardial

42

Sect. I1

- Pharmacodynamic Agents

Francis, Ed.

ischemia. ORG 6001 has a tendency to depress myocardial contractile force. Its dimethyl also exerted antianalog (ORG NA 13, arrhythmic activity but could not suppress conduction disturbances produced by ouabain.26

m)

10a R:H

Interest in the antiarrhythmic activity of quaternary amonium compounds continues. Unlike bretylium ( I & its o-iodobenzyl trimethylammonium analog ( UM-360,2 )did not inhibit release of norepinephrine from sym athetic nerve endings and did not cause adrenergic stimulation on its own. UM-360 effectively antagonized ventricular arrhythmias produced by ouabain in dogs, an action bretylium did not possess. Both agents elevated the threshold to electrically induced ventricular fibrillation. The same group of workers also studied the profile of a quaternary propan2-01 derivative (UM-424,12).28 In contrast to tertiary compounds of

,

%’

lla CH2N-R2

gJ

R1: Br I R2:C2H5 CH3

,

generally similar structure UM-424 did not block cardiac @-adrenergic receptors. The compound suppressed ventricular arrhythmias produced by ouabain and coronary occlusion and elevated ventricular fibrillatory threshold. It produced only brief depression of blood pressure and cardiac contractility. The potential role of the calcium current in the genesis of arrhythmias was discussed above. Verapamil (4 which inhibits calcium influx, can suppress experimental arrhythmias.7 , 3 ~ 3 0Another calcium blocker, nifedipine inhibits irregular rhythms produced by calcium in isolated atrial preparations. 3l The calcium antagonist TMB-6(&) was reported to be as effective as lidocaine in suppressing digoxin-induced arrhythmias in dogs.32 As expected, inhibition of calcium influx leads to depression of cardiac contractility and atrioventricular conduction.

1,

(m),

13a 13b _ R1: CH300C QCH3 H3C

H

COOR3

H

C H5\NICH216-00C

NO2

R 2 : N02 R3 : CH3 ICH2)zN(CH31CH2C6H5

G

O

OCH3 C H

/ ‘ZH5 14 -

OCH3

3

Chap. 5

Antiarrhythmics, Antianginals

Barn, Wendt, Bergey

43

The antiarrhythmic activity of newer 8-adrenergic blocking agents was reviewed recently.a?a,w Most 8-blockers exhibit at least some degree of antiarrhythmic activity since catecholamines contribute to the development of experimental and, particularly, clinical arrhythmias. 1,395 In addition, some of these agents affect the conduction system directly. 1,2,7,8,a However, considerable quantitative as well as qualitative differences exist in regard to antiarrhythmic efficacy. McN-2840-46 ( 1 ) suppressed experimental atrial arrhythmias preferentially in dogs% Higher doses were required to antagonize conduction disturbances following coronary occlusion. KT-9067 (16)antagonizes ventricular arrhythmias in do s and exerts membrane depressant activity in isolated Purkinje fibers.8935

$c"HcHzQ

R-818 (lJ) prevented chloroform-induced ventricular fibrillation in mice as well as arrhythmias caused by hydrocarbon-epinephrine, aconitine and coronary occlusion in dogs. The activity of numerous analogs in the mouse chloroform test has been described.37

H

ouaba3P

OCHzCF3

17

(a,

*) were compared to disopyramide The compounds suppressed ventricular arrhythmias in dogs but may have been more toxic than the latter. Preliminary reports on a series of 3-amino-3-methyloxindoles indicate that one member (2) is as effective as lidocaine in the mouse chloroform test and possesses a higher LD50.39 A series of dibenzazepines

(1). 38

NHZOC

R

@R:H

cHZCH3

R:

(CHz)2N(CHJ)CH2C6H5

(s),

(e),

MG 8926 a substance closely related to prenylamine antagonized ventricular arrhythmias which followed abrupt occlusion of a coronary artery in dogs as well as conduction disturbances produced by ouabain in guinea pigs. In general, prenylamine exerted similar effects.&

44

Sect. I1

-

Pharmacodynamic Agents

Francis, Ed.

A group of 2, 2-disubstituted-1,3-benzodioxoles were evaluated f o r t h e i r a b i l i t y t o i n h i b i t arrhythmias i n r a t s r e s u l t i n g from t h e i.v. i n j e c t i o n of calcium chloride, Compound 2 was e f f e c t i v e and possessed a r e l a t i v e l y high t h e r a p e u t i c r a t i 0 . 4 ~

f"3 U H C H 2 C H 2 N HCHCH2R

20a 20b

R: C6Hll R: C6H5

21 -

Antianginal Agents The major determinants of c a r d i a c oxygen consumption a r e h e a r t r a t e , myocardial wall tension ( d i r e c t l y r e l a t e d t o v e n t r i c u l a r c a v i t y pressure and volume) and force and v e l o c i t y of contraction. Under normal condit i o n s , t h e h e a r t e x t r a c t s oxygen a t a near-maximal r a t e and added r e q u i r e ments a r e met by a reduction i n coronary vascular r e s i s t a n c e and an inc r e a s e i n blood flow. I n t h e a t h e r o s c l e r o t i c h e a r t , coronary v e s s e l s i n t h e ischemic region and p a r t i c u l a r l y i n t h e subendocarium a r e already d i l a t e d and t h e i r a b i l i t y t o i n c r e a s e flow by f u r t h e r d i l a t a t i o n i s severely compromised. Thus, under t h e s e conditions, increasing oxygen demand may exceed supply, myoc r d i a l ischemia ensuesand t h e p a t i e n t manifestssymptoms of angina pectoris. &2 Development of novel a n t i a n g i n a l therapy has s u f f e r e d from t h e l a c k of p r e d i c t i v e animal models. Some of t h e more i n t e r e s t i n g newer approaches include d i r e c t measurement of myocardial oxygen t e n s ion 43~44e l e c t r o cardiographic a n a l y s i s i n norma145~46and diseased hearts,b'l. r e g i o n a l vascular r e s i s t a n c e studies,48 blood flow distribution49,50 and f u n c t i o n a l evaluation i n normal h e a r t s 5 1 a s w e l l a s i n animals subjected t o chronic coronary a r t e r y occlusion. 52,53 The c l i n i c a l p r e d i c t a b i l i t y of these models remains undetermined.

-

Organic n i t r a t e s and t h e B-adrenergic r e c e p t o r blocking Clinical reports agents c o n s t i t u t e t h e mainstay of therapy.42~54-58 The mechanism of a c t i o n of n i t r o g l y c e r i n remains uncertain. Although r e c e n t evidence tends t o underline i t s p e r i p h e r a l v a s c u l a r activity,54,55 s e v e r a l s t u d es ave a l s o implicated a d i r e c t coronary a r t e r y o r c o l l a t e r a l response. t 3 9 4 k 9 5 2 9 59960 C l i n i c a l d a t a generally support t h e hypothesis t h a t primary coronary v a s o d i l a t o r s are of l i m i t e d v a l u e i n t h i s d i s e a s e s i n c e v e s s e l s i n ischemic a r e a s a r e already dilated.42 However, b e n e f i c i a l e s u l t s have and been reported i n g a t i e n t s with t h e v a s o d i l a t o r s chromonar (g)E1 l i d o f l a z i n e ( 3 )2,63 .

Chap. 5

Baum, Wendt, Bergey

Antiarrhythmics, Antiangimls

45

F

23 -

Cn3

Perhexilene (24) continues to demonstrate antianginal efficacy.64,65 Although the compound dilates coronary vessels, other mechanisms such as a reduction in exercise tachycardia and myocardial flow redistribution may be responsible for its effectiveness.66,67

24

Favorable clinical results have been reported recently with the caland verapamil (4).70 Again, alcium antagonists n€fedipine (=)a969 though these drugs increase coronary blood flow experimentally, their clinical effects probably result from other mechanisms. Nifedipine can reduce myocardial contractility and oxygen consumption following ntracoronary injection in dogs and can promote collateral de~elopment.~,7~>7~ An analog (YC-93, LJb) produces marked vasodilatation in animals.73 Prenylamine ( M b ) has a l s o been reported to reduce the frequency of anginal epis0des.r

-

Newer experimental agents The dimethyl quaternary analog of propranolol, m-2‘72 (SC-27761, a non beta-blocker, has been shown to reduce myocardial oxygen consumption and infarct size in dogs, presumably via a non-specific re uction in heart rate and contractility.75,79

a),

~OCH2U-f@tkC””,2 CH3

The cardiovascular effects of a new “benign“ coronary vasodilator, cinepazide (26) were recently reported.77~78 The compound increased perfusion of ischemic areas in the dog 25 heart. Specific coronary vasodilation was demonstrated in the dog heart with a new chromonar derivative morocromen (27).79 Coronary activity was also noted in humans following oral administration.79

,

46

Sect. I1

- Pharmacodynamic Agents

27 -

26

CH30

Francis, Ed.

The adenosine derivative, A bott 40557 (28)produced sustained A reduction in feline myocardial coronary vasodilatation in dogs.$0 oxygen consumption was reported with the Russian antianginal agent, chloracyzine .81

(a)

The prenylamine analog MG 8926 (G)inhibited electrocardiographic S-T segment elevation, in dogs manifestations of cardiac ischemia, i.e., following coronary artery

cH3cH200c~H

’*

-

H OH OH

References

M.R.

Rosen, A.L. W i t and B.F. Hoffman, Amer. H e a r t , J., 88, 380 (1974) and i n s u b s e q u e n t i s s u e s p. 515, 664, 798; 8_9, 115 (1975) and p. 253, 391, 526, 665, 804; 9 0 , 1 1 7 (1975) and p. 2 6 5 , 397, 521, 665, 795. 115 (1975). 2. E.M. Vaughan W i l l i a m s , Pharmaool. Therap., P a r t B, I, 3 . A.L. W i t and J.T. B i g g e r , J r . , C i r c u l a t i o n , 52 (Suppl. 111), 111-96 (1975). 4. D.P. Zipes, H.R. Besch, Jr. and A.M. Watanabe, C i r c u l a t i o n , 3, 761 (1975). 5. D.P. Z i p e s , C i r c u l a t i o n , 5 2 (Suppl. III), 111-120 (1975). 6 . H.A. F o z z a r d , C i r c u l a t i o n , 52 (Suppl. 111), 111-131 (1975). 7. J. K u p e r s m l t h , Amer. J. C a r d i o l . , 3, 119 (1976). 8 . B.I. Sasyniuk and R.I. O g i l v i e , i n “AMual Reviews of Pharmacology“, Val. 15, H.W. E l l i o t , R. George and R. Okun, Ed,, Annual Reviews, P a l o A l t o 1975, p. 131. 9. Symposium on Norpace, Angiolow,Z6,(Suppl.) 65 (1975i. 10. Symposium on Disopyramide, J. h t . Med. R e s . , 4 (Suppl. l ) , 1 (1976). 11. G . J e n n i n g s , D.G. Model, M.B.S. J o n e s , P.P. T u r n e r , E.M.M. Besterman and P.H. K i d n e r , Lancet, 1 , 51 (1976). 12. R.W.F. Campbell, R.C. T a l b o t , M.A. D o l d s r , A . Murray, L.F. P r e s c o t t and D.C. J u l i a n , Lancet, 1, 1257 (1975). 1 3 . E.G. T a l b o t , D.G. J u l i a n and L.F. P r e s c o t t , Amer. H e a r t J., 9l, 58 (1976). 1 4 . I n t e r n a t i o n a l Symposium. Pathophysiology and Drug Treatment o f C a r d i a c Arrhythmias. P r e l i m i n a r y Experience w i t h a New A n t i a r r h y t h m i c Drug ( A p r i n d i n e ) , Acta C a r d i o l . , Suppl. 1 8 , 1 (1974). 15. M.K. H e w , B.N. Sixgh, A.H.G. Roche, K.M. Norris and C.J. M e r c e r , Amer. H e a r t J., 90, 487 (1975).

1.

,

Chap. 5 16. 17. 18. 19.

20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

32. 33. 34. 35. 36.

37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. M. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61.

b u m , Wendt, Bergey

9

D.G. McDevitt, A.S. N i e s , G.R. Wilkinson, R.F. Smith, R.L. Woosley and J . A . O a t e s , C l i n . Pharmacol. Ther., 396 (1976). R.A. Winkle, P.J. Meffin, J.W. F i t z g e r a l d and D.C. H a r r i s o n , C i r c u l a t i o n , 54, 8 8 4 (1976). M . J . Rowe, J.M.M. N e i l s o n and M.F. O l i v e r , L a n c e t $ , 295 (1975). M.B. Rosenbaum, P.A. C h i a l e , M.S. Halpern, G . J . Nau, J. P r z y b y l s k i , R.J. L e v i , J.O. L a z z a r i and M.V. E l i z a r i , Amer. J. C a r d i o l . , 3 8 , 934 (1976). P.H. Morgan and I.W. Mathison, J . Pharm. S c i . , 65, 467 (1976). P.H. Morgan and I.W. M a t h i s o n , J . Pharm. Sci. 635 (1976). J.P. B u y n i s k i and R.W. C a r d i e r , J. Pharmacol. Exp. T h e r . , 190, 289 (1974). M.L. T o r c h i a n a , C.A. S t o n e , H.C. Wenger, R. Evans, B. L a g e r q u i s t and T. O'Malley, J . Pharmacol. Exp. T h e r . , 194, 415 (1975). B.B. V a r g a f t i g , M.F. S u g r u e , W.R. B u c k e t t and H. van R i e z e n , J . Pharm. Pharmacol.,27, 697 (1975). R.J. M a r s h a l l and J.R. P a r r a t t , B r i t . J. Pharmacol., 5 5 , 359 (1975). Pharmacol., 54, 3 (1975). W.R. B u c k e t t , F.A. Marwick and B.B. V a r g a f t i g , B r i t . F.J. K n i f f e n , T.E. Lomas, R.E. C o u n s e l l and E.R. L u c c h e s i , J. Pharmacol. Exp. Ther., 192, 120 (1975). F.J. K n i f f e n , S. Winokur, R.E. C o u n s e l l and B.R. L u c c h e s i , J . Pharmacol. Exp. T h e r . , 196, 420 (1976). G.W. A d e l s t e i n and R.R. Dean, i n A n n . Rep. Med. Chem., Vol. 9, R.V. Heinzelman, ed., Academic P r e s s , New York ( 1 9 7 4 ) , p. 67, C.W. Thornber, i n Ann. Rep. Med. Chem,, Vol. 11, F.H. C l a r k e , ed., Academic P r e s s , New York 1976, p. 61. H. Refsum, Acta Pharmacol. T o x i c o l . , 3 7 , 377 (1975). C.Y. C h i o u , M.H. Malagodi, B.V.R. S a s t r y and P. P o s n e r , J. Pharmacol. Exp. T h e r . , E , 444 (1976). W.E. Hageman and T.P. P r u s s , Fed. P r o c . , 3 4 , 776, a b s t r . 3149 (1975). J.K. G i b s o n , P. Somani and A.L. B a s s e t t , The P h a r m a c o l o g i s t , l8, 169, a b s t r . 312 (1976). J.E. Byrne, A.W. Gomoll and G.R. McKinney, J . Pharmaool. Exp. Ther., 200, 147 (1977). J . R . Schmid, B.D. S e e b e c k , C.L. H e n r i e , E.H. B a n i t t and D.C. Kvam, Fed. P r o c . , 34, 775, a b s t r . 3148 (1975). E.H. B a n i t t , W.E. C o p e , J.R. Schmid and A. Mendel, J. Med. Chem., l8, 1130 (1975). C.R. E l l e f s o n and J.W. C u s i c , J . Med. Chem.319, 1345 (1976). 64, 639 (1975). M.J. K o r n e t , P.A. Thio, N. Malone and W.C. Lubawy, J. Pharm. Sci. R.J. M a r s h a l l and J.R. P a r r a t t , B r i t . J . Pharmacol.,59, 311 (19775.A. S a l i m b e n i , E. M a r y h i s i , G.B. Fregnan and M. V i d a l i , Eur. J . Med. Chem.,ll, 539 (1976). J . R . P a r r a t t , Gen. Pharmacol., 6, 247 (1975). M.M. Winbury, B.B. Howe and H.R. Weiss, J . Pharmacol. Exp. Ther., 176, 184 (1971). S.F. K h u r i , J.T. F l a h e r t y , J.B. O'Riordan, B. P i t t , R.K. Brawley, J.S. Donahoo and V.L. G o t t , C i r c u l a t i o n Res., 37, 455 (1975). P.R. Maroko, J.K. Kjekshus, B.E. Sobel, T. Watanabe, J.W. C o v e l l , J . R o s s , J r . and E. Braunwald, C i r c u l a t i o n , 43, 67 (1971). L. S z e k e r e s , V . C S i k and E. Udvary, J . Pharmacol. Exp. Ther., 196, 1 5 (1976). R.J. Lee and S.H. Baky, J. Pharmacol. Exp. T h e r . , 184, 205 ( 1 9 m . M.M. Winbury, B.B. Howe and M.A. H e f n e r , J . Pharmacol. Exp. T h e r . , 168,70 (1969). L.C. Becker, N.J. F o r t u i n and B. P i t t , C i r c u l a t i o n R e s . , 28, 263 (1971). D.C. W a r l t i e r , G.J. Gross and H.F. Hardman, J. Pharmacol. Exp. Ther., 198,435 (1976). 896 (1974). P. Theroux, D. F r a n k l i n , J . Ross, J r . and W.S. Kemper, C i r c u l a t i o n Res., 5, W.M. Fam and M. McGregor, C i r c u l a t i o n Res., l5, 355 (1964). M.V. Cohen, E.H. S o n n e n b l i c k and E.S. K i r k , Amer. J. C a r d i o l . , 244 (1976). W. Ganz and H.S. Marcus, C i r c u l a t i o n , 46, 880 (1972). H.-Juergen E n g e l , P.R. L i c h t l e n and H. Hundeshagen, C i r c u l a t i o n , 2 (Suppl. 11), 11-73, a b s t r . 284 (1976). R.R. M i l l e r , H.G. Olson, C.M. P r a t t , E.A. Amsterdam and D.T. Mason, C l i n . Pharmacol. T h e r . , 1 8 , 598 (1975). W.S. Aronow, P o s t g r a d . Med., 60, 100 (1976). A.S. Nies and D.G. Shand, C i r c u l a t i o n , 52, 6 (1975). R.J. Bache R.M. B a l l , F.R. Cobb, J . C . Rembert and J.C. G r e e n f i e l d , Jr., J. C l i n . I n v e s t . , 55 1219 (1975f. R.E. G o l d s t e i n , E.B. S t i n s o n , J.L. S c h e r e r , R.P. Seningen, T.M. G r e h l and S.E. E p s t e i n , C i r c u l a t i o n , 49, 298 (1974). R.J. Bing, S.R. Bender, M.I. Dunn, G.A. F r y , W.M. h l l e r , S.C.K. Liu, H.S. M i l l e r , J.W. Moses, L.W. Ritzmann, J.P. S e g a l , G . I . S h u g o l l , H. Tillmanns and A. Wallace, C l i n . Pharmacol. Ther., 1 6 , 4 (1974). R.4J.Lehmann and H. Hochrein, Med. K l i n . , z , 189 (1976).

,kK

x,

-

-

62.

Antiarrhythmics, Antianginals

&!i 63. 64. 65. 66. 67.

68. 69. 70. 71.

72. 73. 74. 75. 76.

77. 70. 79. 80. 81.

Sect. I1

-

Pharrnacodynamic Agents

F r a n c i s , Ed.

L. Nordstrom and F. Gobel, C l i n . R e s . , Z , 4738 ( a b s t r . ) (1975). M.J. B r o w n , J.D. Horowitz and M.L. Mashford, Med. J . A u s t r a l . , 1, 260 (1976). H.F. Mizgala, B.R. Chaitman, P. The'roux and G . Convert, C i r c u l a t i o n , s (Suppl. 11) 11-72, a b s t r . 283 ( 1 9 7 6 ) . I.L. Grupp, C.A. Bunde and G . Grupp, J. C l i n . Pharmacol., 3 , 312 (1970). G.A. K l a s s e n F. S e s t i e r , A. L'Abbate R.R. Mildenberger and D.T. Zborowska-Sluis, C i r c u l a t i o n , 54, 14 (1976j. Second I n t e r n a t i o n a l Adalat Symposium. New Therapy of Ischemic Heart Disease, ed. by W, Lochner, W. Braasoh and G. Kroneberg, S p r i n g e r - V e r l a g B e r l i n , 1975. H.B. S n s o h e d e and P.Gmndei, Med. Welt. 26, 1847 (19751. V. Balasubramanian, G.R. Narayanan, P.K. Khanna and R.S. Hoon, P o s t g r a d . Med. J . , 2, 143 (1976). T. Kanazawa, N. Suzuki, E. Ino-oka, T. Takahashi, A. Mori, Y. Maruyama, E. Ashikawa and Y. Koiwa, Arzneim.-Forsch., 24, 1267 (1974). K. Van Ackern, W. Braasch, U.B. B d o k n e r , W. Heger and J. Schmier, Arzneim.-Forsch., 24, 1517 (1974). T. Takenaka, S. Usuda, T. Nomura, H. Maeno and T. Sado, Arzneim.-Forsch., 26, 2172 (1976). H. Tucker, P. Carson, N. Bass and J . Massey, Brit. Heart J . , 36, 1001 ( 1 9 7 q . E.G. Olson, A.V.N. Goodyer, R.A. Langou, L.S. Cohen and S. Wolfson, C i r c u l a t i o n , 53, 501 (1976). B.R. Lucchesi, W.E. Burmeister, T.E. Lomas and G.D. Abrams, J . Pharmacol. Exp. Ther., 199, 310 (1976). U.B. BGckner and J . Schmier, Arzneim.-Forsch,, 26, 1565 (1976). U.B. B d c k n e r , W. D i e t z e , U. M i t t m a n n , J . Schmier and R.H. Wirth, Arzneim.-Forsch., 26, 1569 (1976). K.H. Becker, K.-H. B o l t z e , H.-D. D e l l , H. J a c o b i and P.-R. S e i d e l , N a t u r w i s s e n s c h a f t e n , 63, 148 (1976). S. Afonso, R.R. Henderson, J.D. F o l t s and G.G. Rowe, B a s i c Res. C a r d i o l . , 7 0 , 390 (1975). I.E. K i s i n , B r i t , J . Pharmacol., 58, 189 (1976).

,

-

,

Chapter 6 H. Hauth and B.P.

Cerebral Vasodilators

Richardson, Sandoz Ltd.,

4002 Basle, Switzerland.

Introduction Physiology of c e r e b r a l blood flow -Despite f l u c t u a t i o n s i n systemic blood p r e s s u r e , blood flow t o t h e b r a i n i s maintained w i t h i n very narrow limits by a process known as a u t o r e g u l a t i o n l . This involves changes i n t h e C a l i b e r of t h e c e r e b r a l v e s s e l s and thus i n cerebrovascular r e s i s t a n c e which are brought about by a v a r i e t y of neurogenic and humoral f a c t 0 1 - s ~ ’ ~ .I t i s now g e n e r a l l y believed t h a t t h e c a l i b e r of l a r g e r extraparenchymal v e s s e l s i s c o n t r o l l e d by nerve tone alone, w h i l s t t h a t of t h e s m a l l e r intraparenchymal v e s s e l s i s c o n t r o l l e d by both nerve tone and by l o c a l p r e v a i l i n g metab o l i c conditions such as t i s s u e C02, 02 and H+ concentrations3,5,6, Innervation of t h e extraparenchymal v e s s e l s i s by sympathetic f i b e r s o r i g i n a t i n g from t h e s u p e r i o r c e r v i c a l ganglion and by parasympathetic f i b e r s c a r r i e d i n t h e f a c i a l nerve2r4. Adrenergic b r a i n stem neurones are considered solel y responsible f o r innervation of t h e intraparenchymal vessels6 9 7 . I n vivo and i n v i t r o s t u d i e s with catecholamines have shown t h a t a-adrenergic a g o n i s t s cause c e r e b r a l vasoconstriction w h i l s t P-adrenergic a g o n i s t s cause vasodil a t a t i o n 8 9 9 9x0. I n addition it has been suggested t h a t physiological a c t i vation of t h e parasympathetic component of t h e innervation t o t h e extraparenchymal v e s s e l s a l s o r e s u l t s i n vasodilatationlo-12. As well as t h e s e adren e r g i c and c h o l i n e r g i c mechanisms, it i s p o s s i b l e t h a t t h e v a s o d i l a t o r y polypeptide (VIP) r e c e n t l y i d e n t i f i e d i n cerebrovascular nerves a l s o p l a y s an important p a r t i n t h e r e g u l a t i o n of c e r e b r a l blood flowl3. Patho1op;ical changes i n c e r e b r a l blood flow - A reduction i n regional blood flow has been recorded i n s e v e r a l acute and chronic c e r e b r a l d i s e a s e s . The acute conditions include apoplexy, i n t r a c r a n i a l t u m o r s , head trauma, hypoxia, compression of t h e b r a i n , highly increased i n t r a c r a n i a l pressure and cereb r a l i n f a r c t i o n l 4 , 1 5 . Autoregulation i s u s u a l l y s e v e r e l y impaired because of vasomotor p a r a l y s i s which r e s u l t s from t h e extreme t i s s u e a c i d o s i s prev a i l i n g i n each of t h e s e conditionsl4. Cerebral blood flow i s a l s o reduced i n a v a r i e t y of chronic d i s e a s e s such as s e n i l e and p r e s e n i l e demential6-18, schizophrenial9, and c e r e b r a l a r t e r i o s c l e r o s i s 2 0 . The exact cause of t h e reduced regional c e r e b r a l blood flow i n dementia and schizophrenia i s not c l e a r , b u t may be r e l a t e d t o a primary decrease i n l o c a l neuronal metabolism18,21. I n t h e case of cerebrovascular s c l e r o s i s t h e reduction i s r e l a t e d t o t h e o c c l u s i v e nature o f t h e l e s i o n s and t o t h e reduced r e a c t i v i t y of cereb r a l blood v e s s e l s t o physiological stimuli22. C l i n i c a l aspects of c e r e b r a l v a s o d i l a t o r t h e r a p x -Although c e r e b r a l vasod i l a t o r s normally induce i n c r e a s e s i n c e r e b r a l blood flow under physiolog i c a l circumstances, c l i n i c a l r e s u l t s a r e o f t e n disappointing. Sometimes even a paradoxical reduction i n t h e flow r a t e through a diseased b r a i n area i s recorded, o r a l t e r n a t i v e l y , t h e drug-induced i n c r e a s e i n perfusion may be f a r i n excess of t h e t i s s u e s metabolic requirements. These phenomena, known as t h e ‘ I n t r a c r a n i a l s t e a l phenomenon’ and the’luxury perfusion syndrome’ respectively23 a r e important complicating f a c t o r s i n t h e treatment of c e r e b r a l i s c h e m i a . The t h e r a p e u t i c r a t i o n a l e o f p r e s c r i b i n g vasodila-

so

Sect. I1

-

Pharmacodynamic Agents

F r a n c i s , Ed.

t o r drugs i n t h e l i g h t o f such o b s e r v a t i o n s h a s r e c e n t l y been c r i t i c a l l y reviewed24-28. I t was g e n e r a l l y concluded t h a t t h o s e compounds with a p r i mary v a s c u l a r a c t i o n ( i . e . t h o s e c l a s s i f i e d as v a s o t r o p i c d i l a t o r s below) seldom prove t o be o f s u b s t a n t i a l c l i n i c a l b e n e f i t . Methodolorn f o r t e s t i n g p o t e n t i a l c e r e b r a l v a s o d i l a t o r s -Methods f o r t h e pharmacological and c l i n i c a l t e s t i n g o f c e r e b r o v a s c u l a r a c t i v e s u b s t a n c e s have been d i s c u s s e d i n s e v e r a l r e c e n t r e ~ i e w s 2 6 9 2 9 ~ 3and 0 symposia18,31,32. The a b i l i t y t o d e t e c t changes i n l o c a l c e r e b r a l blood flow r e l a t e d t o inc r e a s e d neuronal a c t i v i t y and glucose metabolism i s a p a r t i c u l a r l y import a n t r e c e n t methodological advance33. Various books22,34,35 and symposia proceedingsl8~31~32~36-40 d e a l i n g w i t h many a s p e c t s of c e r e b r o v a s c u l a r c i r c u l a t i o n have r e c e n t l y been published. S p e c i f i c Ce rebrovasodi l a t i n g Agents B a s i c a l l y two groups of c e r e b r o v a s o d i l a t o r s can be i d e n t i f i e d accordi n g t o t h e i r s i t e o f a c t i o n . I n t h e f i r s t group a r e t h o s e which have primary v a s c u l a r a c t i o n , d i l a t a t i o n u s u a l l y b e i n g e f f e c t e d e i t h e r by a d i r e c t relaxant a c t i o n o f t h e drug on t h e smooth muscle c e l l s i n a r t e r i o l a r w a l l s o r by an i n h i b i t o r y e f f e c t on t h e endogenous v a s o c o n s t r i c t i v e nerve f i b e r s , A m a j o r i t y o f t h e s e agents have been d e v e l o p e d i n i t i a l l y as p e r i p h e r a l V ~ S O d i l a t o r s which have then been t e s t e d f o r t h e i r a b i l i t y t o i n c r e a s e c e r e b r a l blood flow on q u i t e e m p i r i c a l grounds. The second group c o n t a i n s t h o s e which s t i m u l a t e neuronal metabolism, t h e r e s u l t a n t i n c r e a s e i n l o c a l p e r i v a s c u l a r COP production consequently causing v a s o d i l a t a t i o n . The former group w i l l be r e f e r r e d t o as 'Vasotropic d i l a t o r s ' a n d t h e l a t t e r as 'Cerebrometabolic stimulants

.

( A ) Vasotropic d i l a t o r s

Chemistry - O f t h e f i v e compounds d i s c u s s e d i n d e t a i l below t h e f i r s t papav e r i n e , i s a l o n g recognized a l k a l o i d o f t h e b e n z y l i s o q u i n o l i n e group which was o r i g i n a l l y i s o l a t e d from opium. The remaining f o u r compounds are a l l s y n t h e t i c . "here are no s t r i k i n g s t r u c t u r a l s i m i l a r i t i e s between any members o f t h i s group which could i n d i c a t e a s t r u c t u r e a c t i v i t y r e l a t i o n s h i p .

:::y

1. Papaverine

- Papaverine

(L) i s

a non-specific smooth muscle r e l a x a n t . This a c t i o n prnbably r e l a t e s t o i t s p o t e n t a h i l i t y t o i n h i b i t pi,?sphodiesterases41,42. By rel a x i n g va.scular smL, t h muscle and t h u s c a u s i n g v a s o d i l a t a t i o n , p a p a v e r h e i n c r e a s e s rrean c e r e bral blood flow i n both anirnals26,43,44 and rnan45,46. Whether papaverine produces u s e f u l \ CH jO c l i n i c a l improvements i n c o n d i t i o n s of decreased OCH c e r e b r a l blood flow i s n e v e r t h e l e s s d e b a t a b l e , Most s t u d i e s have involved long termpapaverine -1 administration t o g e r i a t r i c patients suffering from 'chronic b r a i n syndrome' due t o l o n g s t a n d i n g c e r e b r o v a s c u l a r i n s u f f i ciency. I n f o u r double b l i n d s t u d i e s papaverine w a s shown t o improve v a r i o u s n e u r o l o g i c a l e n d p s y c h o l o g i c a l s y m p t o r n ~ ~ 7 - 5w0h~i l s t i n two f u r t h e r s t u d i e s no such b e n e f i c i a l e f f e c t s could be demonstrated51.52. Papaverine has n o t

-

Chap. 6

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51

been widely employed i n t h e treatment of acute cerebrovascular disorders , but preliminary s t u d i e s seem t o suggest t h a t it may have b e n e f i c i a l action i n t h e treatment of c e r t a i n sequelae53~54.The r e p o r t e d s i d e e f f e c t s of papaverine therapy are t y p i c a l of those encountered with p e r i p h e r a l vasodilat o r y agents generally.

w~N~cH

2. Bencyclane -The pharmacological p r o f i l e of bencyclane (2) has received comprehensive c o v e r a g e 5 7 ~ 5 ~Basically . bencycl a n e possesses d i r e c t musculotropic spasmolyt i c e f f e c t s l i k e papaverine, although it does not i n h i b i t phosphodi esteras e.d8. Bencyclane may b l o c k t h e uptake of calciumions i n t o vascul a r smoothmuscle c e l l s 5 8 , thereby d i l a t i n g both p e r i p h e r a l and c e r e b r a l v e s s e l s and consequently 2 i n c r e a s i n g cergbral blood flow i n animals59~60andman57,61. Whether bencyclane i s r e a l l y e f f e c t i v e i n i n c r e a s i n g c e r e b r a l blood flow i n s i t u a t i o n s where flow i s p a t h o l o g i c a l l y reduced i s s t i l l questionable62 963. Bencyclane a l s o i n h i b i t s p l a t e l e t aggregation both i n vivo and i n v i t r o , although it i s not c e r t a i n whether t h i s e f f e c t occurs a t t h e dosages c u r r e n t l y used c l i n i c d l y 5 7 ~ ~ ~ -In~a7 double . b l i n d study bencyclane w a s shown t o improve symptoms r e l a t e d t o acute c e r e b r a l ischaemia67, w h i l s t i n two f u r t h e r s t u d i e s no drug r e l a t e d improvement could he demonstrated57~68.Studies i n p a t i e n t s with chronic cerebrovascular i n s u f f i c i e n c y have a l s o y i e l d e d c o n f l i c t i n g r e s u l t s . In a s e r i e s of double-blind Studies bencyclane e i t h e r improved 3. inconsit h e s y m p t o m s 5 7 ~ ~ 9o - 7r ~f a i l e d t o have a clear e f f e c t 5 7 ~ 6 8 ~ 7This stency i s rather d i f f i c u l t t o understand s i n c e dosage, duration of medicat i o n andthe t a r g e t symptoms studied were o f t e n very similar. The negative i n o t r o p i c and chronotropic e f f e c t s of bencyclane on t h e h e a r t and t h e prolongation of t h e r e f r a c t o y period of h e a r t muscle c e l l s may be r e l a t e d t o e f f e c t s on calcium fluxes7

,

.

3. Cyclandelate -Like both papaverine and bencyclane, cyclandelate (2)has d i r e c t smooth muscle relaxant p r o p e r t i e s which account f o r i t s potent v a s o d i l a t i n g effectb75. Its exact mechanism of action i s s t i l l not c l e a r . On t h e b a s i s o f i t s known e f f e c t s on t h e perip h e r a l c i r c u l a t i o n , c l i n i c a l s t u d i e s were i n s t i gated t o t e s t f o r a p o s s i b l e cerebrovasodilating -3 action. The r e s u l t s of a double b l i n d study showed cyclandelate b o t h t o increase b r a i n bloodvolume and t o rectifypathol o g i c a l l y prolonged c i r c u l a t i o n times76. These results were l a t e r confirmed by measurements of c e r e b r a l blood flow employing 133Xe clearance techniques77. An e a r l y double b l i n d study demonstrated both a s i g n i f i c a n t decrease i n mean c e r e b r a l c i r c u l a t i o n time and an improvement i n t h e mental function of e l d e r l y p a t i e n t s receiving cyclandelate compared with those administeredplacebo78. Five similar double b l i n d s t u d i e s on g e r i a t r i c p a t i e n t s have subsequently y i e l d e d extremely v a r i a b l e re~ults79’~3.Those conducted over longer periods were t h e more successful i n showing a drug-related improvement and it has t h e r e f o r e been suggested t h a t cyclandelate may prove more e f f e c t i v e i n t h e prophylaxis o f progressive mental decline than i n i t s treatment.

QE:-m04

52 -

Sect. I1

- Pharmacodynamic Agents

Francis, Ed.

4. B e t a h i s t i n e - B e t a h i s t i n e i s an o r a l l y a c t i v e histamine analogue

(4).

I t s a c t i o n s on p e r i p h e r a l and c e r e b r a l v e s s e l s are thus similar t o t h o s e of histamineand can be antagonized by a n t i h i s t a m i n i c compounds84. I t s predominant vasoaction i s enhancement o f 3 t h e m i c r o c i r c u l a t i o n , presumably by a r e l a x a n t e f f e c t on t h e smooth muscle c e l l s of theprecapil4 lary a r t e r i o l e s , Themolecular mechanism by which histamine i t s e l f induces such e f f e c t s is not fully e l u c i d a t e d , b u t may involve a lowering of t h e i n t r a c e l l u l a r ionized calcium concentration85. Unl i k e histamine, t h e v a s c u l a r a c t i o n s of b e t a h i s t i n e are long l a s t i n g and it does not markedly stimulate g a s t r i c secretion84. B e t a h i s t i n e i n c r e a s e s c e r e b r a l microcirculation thereby i n c r e a s i n g mean c e r e b r a l blood flow i n both animals86 and man87,88. Recent c l i n i c a l r e s u l t s have been v a r i a b l e and o f t e n c o n f l i c t i n g . I n one double b l i n d cross-over study involving p a t i e n t s with dementia r e l a t e d t o v e r t e b r o b a s i l a r insufficiency, b e t a h i s t i n e increased both mean and regional blood flow values and improved t h e n e u r o l o g i c a l and psychological s t a t u s of t h e patientsag. I n a second study, again involving p a t i e n t s with c e r e b r o b a s i l a r i n s u f f i c i e n c y , hut where t r a n s i e n t ischaemic a t t a c k s were t h e t a r g e t symptoms investigated, t h e r e was no such c l e a r drugr e l a t e d benefitgo. Yet another study seems t o suggest t h a t p a t i e n t s experienc i n g v e r t i g o due t o c e r e b r o b a s i l a r i n s u f f i c i e n c y derive b e n e f i t from betahistine91.

5.

Cinnarizine

- Cinnarizine (2)i n c r e a s e s

c e r e b r a l blood flow i n both man92 and a n i d s 9 3by causing c e r e b r o v a s o d i l a t a t i o f i 9 The mechanism of a c t i o n of c i n n a r i z i n e relates t o i t s a b i l i t y t o i n h i b i t depolarization-depend e n t calciumuptake i n t o a r t e r i a l smoothmuscle?? Conversely t h e r e l e a s e of calcium i o n s from bound i n t r a c e l l u l a r s t o r e s within t h e muscle f i b r e s by noradenaline i s unaffected, This sugg e s t s t h a t c i n n a r i z i n e s p e c i f i c a l l y blocks cal-5 cium uptake at t h e o u t e r membrane, thereby preventing o r reducingmuscle tension and so producingvasodilatation. C l i n i c a l r e s u l t s with cinnarizine, as with t h e o t h e r vasotropic d i l a t o r s , have been variable.In t h r e e double b l i n d s t u d i e s conductedon p a t i e n t s with c e r e b r a l a r t e r i o s c l e r o s i s , medication produced a clear-cut inprovement i n t h e i r c l i n i cal status92,93,96. I n a f o u r t h study where c i n n a r i z i n e and digoxin were given t o g e t h e r , t h e combination produced considerably b e t t e r r e s u l t s t h a n c i n n a r i z i n e rilone97. I n c o n t r a s t t o t h e foregoing, a m u l t i l o c a t i o n a l study performed i n B r i t a i n f a i l e d t o demonstrate any b e n e f i c i a l a c t i o of c i n n a r i zine i n p a t i e n t s with longstanding c e r e b r a l a r t e r i o s c l e r o s i s 9 8 : ( B ) Cerebrometabolic s t i m u l a n t s

Chemistry - A l l t h e w e l l e s t a b l i s h e d , c l i n i c a l l y a c t i v e compounds i n t h i s category a r e n a t u r a l l y occurring, or derived from n a t u r a l l y occurring ergot and vincamhe a l k a l o i d s . Although both chemical groups a r e t h e s u b j e c t of i n t e n s e chemical i n v e s t i g a t i o n and molecular manipulation no s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s f o r t h e i r cerebrometabolic s t i m u l a t i n g p r o p e r t i e s have y e t been published.

Chap. 6 1. HyderEine

Cerebral Vasodilators

Hauth, Richardson

53

@ -Hydergine @ (6)contains dihydroergotoxine mesylate a s t h e

a c t i v e p r i n c i p l e , which i s an a s s o c i a t i o n of t h e n e s y l a t e s of dihydroergocornine(6aX dihydroergocri s t i n e (6b),dihydro-a-ergocrypt he (6c), and dihydro-P-ergocryptine (6a)i n t h e r a t i o 3:3:2:1 ( r e f . 9 9 ) . Hyderginea possesses cx-edr e n e r g i c blocking a c t i v i t y and thus induces some degree of v a s o d i l a t a t i o n by i n h i b i t i n g t h e act i o n of noradrenaline t o n i c a l l y r e l e a s e d from nerve endings within v e s s e l walls. However, t h e g r e a t e r p a r t of i t s o v e r a l l p e r i p h e r a l vasodi6 a R = C H C H l a t i n g a c t i o n probably r e s u l t s from a c e n t r 1 2 6 5 i n h i b i t o r y a c t i o n on t h e vasomotor ~ e n t e $ ~ ~ * ’ ~ ~ . b R = CH(CH ) Under conditions of experim n t a l l y reduced cere3 2 b r a l blood flow Hydergine induces d i l a t e c R = CH CH(CH ) 2 3 2 t i o n of t h e b r a i n c a p i l l a r i e s thereby lowering d R = CH(CH3)CH2CH3 vascular r e s i s t a n c e and improving both blood flow and glucose u t i l i z a t i o n l o * . The mechanism by which H y d e r g i n e B effects t h i s i s probably q u i t e d i f f e r e n t from t h a t involved i n i t s d i l a t a t i o n of t h e peripheral c i r u l a t i o n . Based on recent experimental r e s u l t s it has been suggested t h a t Hyderginea p r i m a r i l y prot e c t s neuronal metabolism under conditions of inadequate c e r e b r a l perfusion44r102. The observed i n c r e a s e i n blood flow i s t h u s considered e n t i r e l y secondary t o t h e l o c a l i n c r e a s e s i n CO2 production t h a t accompany improved neuronal metabolisrn102-105.

-

That t h e increased blood flow i s a consequence o f e f f e c t s on neuron a l metabolism and not t h e cause of them i s f u r t h e r supported by t h e ob-s e r v a t i o n t h a t Hydergine i n c r e a s e s t h e reduced EEG a c t i v i t y occurring i n both a n i m a l s and man with pathologically reduced c e r e b r a l blood flow. Simply c o r r e c t i n g t h e flow rate b cerebrovasodilatation with papaverine does not produce such an e f f e c t l o ,107. The exact mechanism by which Hydergine @ s t i m u l a t e s neuronal metabolism i s not c l e a r s b u t may relate t o i t s a b i l i t y t o a c t i v a t e dopamine receptors i n t h e pontomedullary r e t i c u l a r formation o r t o i t s c e n t r a l serotonergic p r o p e r t i e s 1 0 8 , W . Preliminary stud i e s w i t h t h e semi-synthetic dihydrogenated e r got a l k a l o i d s dihydroergonine (DN 16-45”, 7 ) and dihydro-P-ergosin (DQ 27-422, 8 ) suggert t h a t t h e s e compounds may possess c e r e b r a l s t i mulating E r o p e r t i e s similar t o those of Hydergine 10 Recent s t u d i e s on i t s molecular mechanism of a c t i o n suggest an important e f f e c t of Hydergine on neuronal cAMf levelsll0,By i n h i b i t i n g a low I(m-phosphodiesterase, H y d e r gine 03 may prevent neuronal CAMP concentrations R from f a l l i n g excessively i n c e r t a i n pathologid R1 c a l s i t u a t i o n s . Conversely both i n s l i c e s of 1 C2H5 CH(CH~)* r a t c e r e b r a l c o r t e x and i n c a t b r a i n homogenates , norepinephrine-induced i n c r e a s e s i n CAMP l e v e l s 8 CH CH(CH )CH CH aye tagonized by low concentrations of Hyder - 3 gme% ,probably through i t s a b i l i t y t o block

a

t

a .

a

t h e a-adrenergic e f f e c t s of norepinephrine at t h e post synaptic membrane.

54

Sect. I1

-

Pharmacodynamic Agents

Francis, Ed.

Hydergine @ a l s o p a r t i a l l y i n h i b i t s norepinephrine-induced a c t i v a t i o n o f neuronal Na+-K+ ATPase by a s i m i l a r anti-adrenergic mechanismlll. Hyderginea e f f e c t i v e l y decreases c e r e b r a l c i r c u l a t i o n times i n pat i e n t s with prolongedvalues and i n c r e a s e s both c e r e b r a l oxygen u t i l i z a t i o n and CO2 product ion104 ,112,113. Several recent double b l i n d s t u d i e s have demonstrated a u s e f u l action of Hyderginea i n t h e treatment of s e n i l e dement i a . In t h r e e such s t u d i e s HydergineB was shown t o be s i g n i f i c a n t l y bett e r t h a n placeboin ameliorating many of t h e neurological and psychological symptomsl14-116. In two f u r t h e r s t u d i e s conducted over a twelve week period, it was p o s s i b l e t o c o r r e l a t e such improvements i n t h e a t i e n t s ' mental stat u s w i t h drug-related changes i n t h e i r EEGpatternsl0E1l7. I n f i v e of t h e most recent s t u d i e s comparisons with papaverine have been made. I n each case Hydergine @ has been found t o be s u p e r i o r f o r t h e treatment of t h e various mental manifestations studied. In t h e s e s t u d i e s HydergineB ameliorated such symptoms as confusion, i r r i t a b i l i t y and depression, w h i l s t mental a l e r t ness and motivation were c o n s i s t e n t l y irnproved51~118-121, The improvements i n i n t e l l e t u a l function and cognition were always among t h e most s t r i k i n g . H y d e r g i n e h a l s o had a b e n e f i c i a l e f f e c t on c e r t a i n physical symptoms such as f a t i g u e , d i z z i n e s s , n o c t u r a l cramps and anorexia. 2. Nicergoline -Nicergoline

(2)i s

an e s t e r of 10cwnethoxy-dihydrolysergol, I t possesses considerable a-adrenergic blocki n g action and consequently induces d i l a t a t i o n of t h e p e r i p h e r a l microvasculature122. Like Hydergine@ , it i n c r e a s e s c e r e b r a l microcirc u l a t i o n by primary p r o t e c t i v e e f f e c t s on neuron a 1 met a b o l i sm1* 3. Under c ondi t i ons of expe r i mental ischaemin, nicergoline i n c r e a s e s both neuronal glucose uptake and reduces pyruvate and l a c t a t e formation124, I n animals t h e s e eff e c t s a r e r e f l e c t e d i n a more r a p i d post ischae-9 mic recovery of EEG activityl25. I t s molecular mechanism of action seems r e l a t e d t o e f f e c t s on brainATP levels,which a r e more rapidly r e s t o r e d a f t e r experimental ischaemia when pretreatment w i t h nicergoline h a s been performed126 Like Hydergine @ and dihydroergotmine, nicergoline s t i m u l a t e s CAMP accumulation i n r a t c e r e b r a l cortex s l i c e s by an i n h i b i t o r y action on phosphodiesterase, P a r t of t h i s r i s e i n CAMP may be mediated v i a a d i r e c t stimulation of adenylate cyclasel27. Nicergoline a l s o i n c r e a s e s cerebral oxygen and glucose consumption i n e l d e r l y p a t i e n t s s u f f e r i n g from c e r e b r a l a r t e r i o s c l e r o s i s l 2 8 . I n one study it was claimed t h a t nicergoline i n c r e a s e s cerebral blood flow and t h e symptoms a s s o c i a t e d w i t h acute o r chronic reduction of b r a i n perfusion ratesl29. However t h i s must s t i l l remain conjectural s i n c e , although another study has confirmed a drug-induced increase i n c e r e b r a l blood flow i n p a t i e n t s with recent apoplexy, no such increase w a s detected i n those with chronic o r d i f f u s e cerebrovascular pathology130. No c a r e f u l l y c o n t r o l l e d c l i n i c a l s t u d i e s with nice r g o l i n e have yet been reported. In one open, uncontrolled study, f i f t e e n e l d e r l y p a t i e n t s s u f f e r i n g from c e r e b r a l a r t e r i o s c l e r o s i s were considered t o have b e n e f i t e d from n i c e r g o l i n e therapyl31.

Chap. 6 3. Vincmine

Hauth, Richardson

Cerebral V a s o d i l a t o r s

- Vincamine

55

(2) is

a p l a n t a l k a l o i d , which has been shown t o i n c r e a s e c e r e b r a l blood flow i n both man61 and animals132. Recent animal s t u d i e s suggest t h a t v i n c m i n e s t i m u l a t e s neuron a1 metabolism, t h e r e b y i n c r e a s i n g glucose u t i l i z a t i o n and C02 production. The r e s u l t i n g r i s e i n pC02 o f t h e p e r i v a s c u l a r neurones i s t h u s b e l i e v e d t o be t h e d e f i n i t i v e v a s o d i l a t i n g stimulusl33. There lo a r e very few r e c e n t , w e l l documented c l i n i c a l s t u d i e s with vincamine. In one double b l i n d s t u d y conducted on g e r i a t r i c p a t i e n t s s u f f e r i n g advanced c e r e b r a l a r t e r i o s c l e r o s i s , vincamine a p p a r e n t l y improved t h e EEG p a t t e r n as w e l l as t h e a s s o c i a t e d n e u r o l o g i c a l symptoms134. I n a second s t u d y it was claimed t h a t a r e d i s t r i b u t i o n o f c e r e b r a l blood flow t o p a t h o l o g i c a l l y a f f e c t e d areas accompanied such improvements i n c l i n i c d statusl35. Recently t h e chemical, pharmacological and c l i n i c a l a s p e c t s of a new vincamine analogue have been r e p o r t e d ( C a v i n t o n a RGH-4405, 11)136. I t i s a simple e s t e r of apovincaminate. This agent appears t o p o s s e s s u s e f u l c l i n i c a l a c t i o n f o r t h e treatment o f v a r i o u s cerebrovas c u l a r disorders.

-

-

11

( c ) MiscellaneousCompounds B u t a l a m h e (12)i s a compound which induces v a s o d i l a t a t i o n by r e l a x i n g v a s c u l a r smooth muscle and which seems promising c l i n i c a l l y l 3 7 . Dextran, a polysaccharide which improves t h e flow q u a l i t i e s of t h e blood by a volume expansion e f f e c t , and should not perhaps be considered a t r u e c e r e b r a l VRSOd i l a t o r , i s n e v e r t h e l e s s s t i l l one o f t h e most e f f i c a c i o u s compounds f o r t h e treatment of a c u t e c e r e b r a l ischaemial38. Fenoxedil (13)seems t o induce c e r e b r a l v a s o d i l a t a t i o n by both v a s c u l o t r o p i c and cerebrometabolic actions139,140, Isoxsuprine i s an a d r e n a l i n e d e r i v a t i v e w i t h pure P-adren e r g i c s t i m u l a t i n g a c t i o n . I t causes c e r e b r a l v a s o d i l a t a t i o n by t h i s mechrr nism and improves various symptoms r e l a t e d t o decreased c e r e b r a l perfusionl41. Moxisylate (1 ) i s a s p e c i f i c a-adrenergic a n t a g o n i s t w i t h benefic i a l c l i n i c a l a c t i o n d e . Nafronyl o x a l a t e p o s s e s s e s c e r e b r a l me a b o l i c s t i m u l a n t a c t i v i t y and thereby b r i n g s about c e r e b r a l v a s o d i l a t a t i o n 1 3. Nicoi s a primary cerebrometabolic s t i m u l a n t wh'ch improves t i n y 1 a l c o h o l (17) c l i n i c a l symptoms a s s o c i a t e d w i t h decreased c e r e b r a l blood flowlt4. Pentoxif y l l i n e ( g ) , l i k e t h e o p h y l l i n e ( 2 ) p o s s e s s e s phosphodiesterase i n h i b i t i n g a c t i o n . Both compounds relax v a s c u l a r smooth muscle by t h i s mechanism an t us cause v a s o d i l a t a t i o n , They may a l s o have e f f e c t s on neuronalmetabolisml 57. Piracetam ( 2 0 ) i s a primary cerebromet o l i c s t i m u l a n t with claimed e f f i c a c y i n t h e treatment of s e n i l e dementia if%. Proxazole ( 2 1 ) i s papaverinel i k e i n action. I t causes i n c r e a s e s i n c e r e b r a l blood f l o w n 9 and improves t h e symptoms o f cerebrovascular i n s u f f i c i e n c y l 5 0 . Raubasine ( 2 2 ) i s an ct-adren e r g i c blocking agent and p o s s i b l y possesses neurometabolicstinulating p r o p e r t i e s . I n p a t i e n t s w i t h reduced c e r e b r a l blood flow due t o a r t e r i o s c l e r o s i s , raubasine i n c r e a s e s blood flow and improves t h e i r c l i n i c a l s t a t u s l 5 1 , 1 5 2 . V i q u i d i l ( 2 3 ) , i n c r e a s e s c e r e b r a l blood flow b inducing v a s o d i l a t i o n i n a similar G y t o p a p a v e r i n e l 5 1 Hexobendine 2 4 y i s a cerebrometabolic s t i mulant t h a t i n c r e a s e s c e r e b r a l blood flow l 5 b

(2)

(16)

'e

k

E:

56 -

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3

H H"

-

'

Francis, Ed.

Pharmacodynamic Agents

.

C

CH 3ooc

'

H

N3'

d

--2 3

22 CH30 N(CH2)300C CH 3O D -C O O ( C H 2 ) 31N(CH 2 ) 21 CH3d

CH 3

24 -

CH3

\

OCH

3

H

Chap. 6

Cerebral Vasodilators

Hauth, Richardson

57

REFERENCES

1. 2.

3.

4. 5.

6. 7. 8. 9. 10.

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14. 15. 16.

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31. 32. 33. 34. 35.

36. 37. 38. 39.

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Pharmacodynamic Agents

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49, F.H. S t e r n , J. Am. C e r i a t r . SOC., 507 (1970). 50. P. Klaw and M. Wiethaup, Med. Welt, 24, 1413 (1973). 51. A.J. Bazo, J. Am. C e r i a t r . SOC., 1. (1973). 52. L. Lu. B.A. S t o t s k y and J.O. Cole, Arch, Gen. P s y c h i a t r y , 2 284 (1971). 53. A. P i c i n e l l i , G. Neri and A. Aenoli, Minerva Med. &, 4199$;974). 54. A. Agnoli , Minerva Med., 61. 1719 (1976). 8, 292 (1974). 55. H.W. K i a e r , S. Olsen and V. Rbnnov-Jessen, Arch, Pathol., 56. E. Komolos and L.E. P r t o z . Arzneim. Forsch., 20, 1338 (197%. 57. K. Kohlmeyer, Therapiewoche, 5 , 2813 (1974).58. W.R. Kukovetr, G. Pbch, S. Holzmann and E. P a i e t t a Arzneim. Forsch., 3, 722 (1975). 59. H-J. Hapke. Arch. I n t . Pharmcodyn. Ther., 202, 231 (1973). 60.

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Chap. 6

Cerebral Vasodilators

Hauth, Richardson

113. J.P. 114. ll5. 116.

ll7. ll8. ll9. 120. 121. 122. 123.

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a,

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Chapter

7.

Antihypertensive Agents

C r a i g W. Thornber and Andrew Shaw, I C I Ltd., Pharmaceuticals D i v i s i o n , M a c c l e s f i e l d , Cheshire, U.K.

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Introduction Reviews have been published on t h e physiology of c a r d i o v a s c u l a r regulation1 and t h e biochemical e t i o l o g y of hypertension.2 It has been proposed t h a t hypertension r e s u l t s from a repeated sequence of small r i s e s i n blood p r e s s u r e , due t o sympathetic h y p e r a c t i v i t y , which produce changes i n t h e kidney.3 The r o l e s of cadmium, l e a d 4 and catecholamines5 i n t h e development of hypertension and t h e c a r d i o v a s c u l a r and r e n a l a c t i o n s o f p r o s t a g l a n d i n s 6 have been reviewed. The drugs used i n t h e c l i n i c a l management of hypertension have been surveyed.?

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C e n t r a l Mechanisms A u s e f u l review of the physiology of c e n t r a l c a r d i o v a s c u l a r r e g u l a t i o n has appeared1 and two reviews899 d e a l c o n c i s e l y with t h e c e n t r a l n o r a d r e n e r g i c c o n t r o l of blood p r e s s u r e . Less well-understood a s p e c t s of t h e pharmacology of c e n t r a l c o n t r o l , i n c l u d i n g t h e c e n t r a l e f f e c t s of a n g i o t e n s i n , a r e d e a l t w i t h i n a congress report.10 Clonidine (l)(Boehringer-Ingelheim) has continued t o a t t r a c t of 22 congeners of c l o n i d i n e has c o r r e l a t e d t h e a t t e n t i o n . A stud?l p e r i p h e r a l a - s t i m u l a n t p r o p e r t i e s w i t h physicochemical parameters; however, t h e c e n t r a l hypotensive a c t i o n u s i n g i.v. or i n t r a c e r e b r o v e n t r i c u l a r (i.c.v.1 d o s i n g , could n o t be s o c o r r e l a t e d . Indeed, t h e n a t u r e of t h e c e n t r a l r e c e p t o r s and t h e i r r e l a t i o n s h i p t o p e r i p h e r a l a - r e c e p t o r s remains unclear. It h a s been suggestedl-2 t h a t c l o n i d i n e may a c t i v a t e c e n t r a l H2 r e c e p t o r s , s i n c e i.c.v. metiamide, a s p e c i f i c H2-blocker, was a b l e t o r e v e r s e and antagonize t h e hypotensive b u t not t h e bradycardiac e f f e c t of i.v. c l o n i d i n e i n a n a e s t h e t i z e d rats. Clonidine was a l s o shown13 t o s t i m u l a t e a t r e l a t i v e l y h i g h c o n c e n t r a t i o n an H2-receptor-coupled adenyla t e c y c l a s e i n t h e hippocampus of guinea pigs. However, t h e r e is no evidence t o suggest a h i s t a m i n e r g i c a c t i o n of c l o n i d i n e i n conscious h y p e r t e n s i v e c a t s . 1 4 (Histamine i t s e l f given c e n t r a l l y is p r e s s o r i n conscious c a t s v i a an H1 mechanisml5). The s t r i k i n g f i n d i n g t h a t c l o n i d i n e a c t s p u r e l y a s an a n t a g o n i s t t o noradren l i n e s t i m u l a t e d i n c r e a s e s i n CAMP i n rat b r a i n s l i c e s has been a m p l i f i e d . l Z A comprehensive s t u d y i n r a t s 1 7 o f t h e f u n c t i o n a l and biochemical a c t i o n s of c l o n i d i n e and t h e e f f e c t s o f d i f f e r e n t a n t a g o n i s t s has given f u r t h e r support t o t h e developing concept t h a t pre-synaptic a - s t i m u l a t i o n i s involved i n some responses. The c e n t r a l syrnpatho-inhibitory e f f e c t , however, is probably a post-synaptic action.18 A novel technique i n v o l v i n g t h e measurement of e l e c t r o d e r m a l responses t o demonstrate clonidine-induced c e n t r a l sympathetic i n h i b i t i o n h a s been described.19 The water d i u r e s i s produced i n conscious dogs by c l o n i d i n e appears t o be consequent upon an i n c r e a s e i n r e n a l FGE s y n t h e s i s , which h a s a n a n t i - A D H ( a n t i d i u r e t i c hormone) e f f e c t . 2 0 The a b i l i t y of very low doses of c l o n i d i n e t o reduce v a s c u l a r r e a c t i v i t y h a s a g a i n been demons t r a t e d . 2 1 A well-described c l i n i c a l i n v e s t i g a t i o n of t h e e f f e c t s of 300 p g p.0. c l o n i d i n e i n normotensive v o l u n t e e r s has given u s e f u l d a t a on t h e s e d a t i o n and h y p o s a l i v a t i o n s i d e e f f e c t s a g a i n s t which s i m i l a r a g e n t s

Chap. 7

A n t i h y p e r t e n s i v e Agents

Thornber, Shaw

61 -

might be compared.22 The f i r s t c l i n i c a l i n v e s t i g a t i o n of t h e h y p e r t e n s i v e rebound phenomenon on a b r u p t c e s s a t i o n of c l o n i d i n e t h e r a p y f a i l e d t o f i n d evidence of t h e e f f e c t i n a small number of mild-to-moderate h y p e r t e n s i v e s a f t e r s h o r t term treatment.23

(z)

C l i n i c a l d a t a on BS-100-141 (Sandoz) are now r e a d i l y a v a i l a b l e . 2 4 ~ 2 5 The compound is a s e f f e c t i v e an a n t i h y p e r t e n s i v e i n man a s c l o n i d i n e but t e n times l e s s p o t e n t . S e d a t i o n and dry mouth appeared with t h e same frequency a s with c l o n i d i n e . The c e n t r a l a - s t i m u l a n t a c t i o n of guanabenz (3) i n c a t s appears d i s t i n c t from t h a t of c l o n i d i n e i n t h a t b a r o r e c e p t o r mechanisms a r e not involved i n t h e hypotensive response.26 I n t h e c l i n i c , guanabenz (24-48mg) produced modest blood p r e s s u r e r e d u c t i o n with s e d a t i o n as t h e major s i d e - e f f e c t . 2 7

-2

1 -

-3

C e n t r a l hypotensive mechanisms n o t i n v o l v i n g a - s t i m u l a t i o n remain was l a s t y e a r ' s major development i n t h i s i l l - d e f i n e d . Janssen R28935 a r e a and t h e s u g g e s t i o n has been made r e c e n t l y t h a t c e n t r a l a-blockade is r e s p o n s i b l e f o r t h e hypotensive response t o i n t r a c i s t e r n a l i n j e c t i o n i n r a b b i t s of R28935, BE2254 (5)and phentolamine.28

(4)

The mechanism of t h e c l i n i c a l hypotensive a c t i o n of P-blockers remains c o n t e n t i o u s , but s e v e r a l papers29 have demonstrated f u r t h e r t h e a b i l i t y of c e n t r a l l y - a d m i n i s t e r e d b l o c k e r s t o lower blood p r e s s u r e i n experimental animals. It i s n o t c e r t a i n t h a t t h i s e f f e c t r e s u l t s from P-blockade , however. F u r t h e r evidence t h a t c e n t r a l 5-hydroxytryptamine (5-HT) neurones are involved i n c a r d i o v a s c u l a r c o n t r o l systems i s found i n t h e o b s e r v a t i o n t h a t a c e n t r a l a d m i n i s t r a t i o n of t h e s e l e c t i v e n e u r o t o x i n 5,6-dihydroxytryptamine causes a s i g n i f i c a n t d e l a y i n t h e development of h y p e r t e n s i o n i n young spontaneously h y p e r t e n s i v e rats (SHRS) .3O A f u r t h e r demonstration of t h e centrally-mediated hypotensive e f f e c t of methysergide h a s a p p e a r e d ; 3 l t h i s may n o t be a r e s u l t of 5-HT antagonism. Tetrahydrocannabinol is considered a l e a d f o r novel c e n t r a l l y - a c t i n g hypotensives. C a r d i o v a s c u l a r a c t i v i t y was found f r e q u e n t l y i n a wide-

62

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Francis, Ed.

ranging s e r i e s of v a r i a t i o n s of t h e cannabinoid s t r u c t u r e . 3 2 As a n example, l o n g - l a s t i n g hypotension with bradycardia upon o r a l a d m i n i s t r a t i o n t o SHRs and n e u r o g e n i c a l l y h y p e r t e n s i v e dogs.

-6 produced

Levels of phenylethanolamine-N-methyltransferase (PNMT) have been found t o be s i g n i f i c a n t l y r a i s e d i n d i s c r e t e b r a i n stem r e g i o n s i n b o t h SHR and D O C A - s a l t h y p e r t e n s i v e rats.33 Administration of SKF 7698 (2), a PNMT i n h i b i t o r , normalized p r e s s u r e i n DOCA rats. The p o s s i b i l i t y t h a t t h e e f f e c t was due t o t h e a d r e n o l y t i c a c t i o n of (2) was not excluded. The b e s t of a new s e r i e s o f PNMT i n h i b i t o r s w i t h much reduced a-blocking p r o p e r t i e s i s S U 64139 (81.34

-

I n two h y p e r t e n s i v e rat models, whole b r a i n t y r o s i n e hydroxylase l e v e l s c o r r e l a t e d w e l l with s y s t o l i c blood pressure.35 The h y p e r t e n s i v e response t o t h e phosphodiesterase i n h i b i t o r RA-642 (2) administered i n t h e v e r t e b r a l a r t e r y t o c a t s o r i g i n a t e s i n t h e medulla oblongata36 and is a r e s u l t of a c t i v a t i o n of t h e sympathetic outflow. Conversely, a p r e l i m i n a r y r e p o r t 3 7 s u g g e s t s t h a t c e n t r a l a d m i n i s t r a t i o n of a phosphodies t e r a s e a c t i v a t o r such as imidazole lowers blood p r e s s u r e .

-

The r e g u l a t i o n of r e n i n r e l e a s e ,j8939 i t s The Renin-Angiotensin System biochemistry,40 c l i n i c a l s i g n i f i c a n c e 3 9 , h l and t h e r o l e of t h e r e n i n a n g i o t e n s i n system i n e x t r a - r e n a l t i s s u e 4 2 have been reviewed. The e f f e c t s of a n t i h y e r t e n s i v e a g e n t s on t h e r e n i n - a n g i o t e n s i n system have been surveyed.tT Evidence has been obtained f o r a r e n a l a-adrenergic r e c e p t o r i n h i b i t i n g r e n i n r e l e a s e i n rats44 and' man.45 Dopamine and apomorphine induce r e n i n s e c r e t i o n ; t h e e f f e c t of apomorphine is blocked by pimozide$ I n rnan,47 rats and r a b b i t s 4 8 plasma r e n i n a c t i v i t y is suppressed by indomethacin concomitantly with a r e d u c t i o n i n p r o s t a g l a n d i n s y n t h e s i s . I n

chap. 7

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63 -

Thornber, Shaw

rats and rabbits arachidonic acid stimulates plasma renin activity.48 Lysophosphatidylethanolamine derivatives inhibit renin release in rats. 49 Low renin essential hypertension has been found to be associated with a suppression of sympathetic nervous activity.50 Angiotensin converting enzyme has been re~iewed.5~It is a single peptide chain associated with polysaccharide and contains a zinc atom. On the basis of a suggestion that the active site of this enzyme may resemble that of carboxypeptidase A, novel orally active angiotensin converting enzyme inhibitors have been designed such as 3-mercapto-2-D-methylpropanoyl-L-proline (Squibb 14,225).52 This Compound has a marked and sustained antihypertensive effect on renal hypertensive rats at doses of 3 to lOmg/kg. A new enzyme, tonin, has been described which can produce angiotensin I1 (AII) from AI. Its activity appears to be under the influence of p-adrenergic receptors.53 The structure activity relations of peptide antagonists of the renin-angiotensin system such as substrate analogues, converting enzyme inhibitors and angiotensin I1 antiagonists have been reviewed ,54 and the clinical significance of angiotensin blockade has been surveyed.55 Beta-Adrenergic Blocking Agents (@-Blockers) - Studies on the identification and characterisation of P-adrenergic receptors have been reviewed.56 P-Receptors have been identif'ed in the rat brain57 and localised using a fluorescent p-bl0cker.5~ The clinical pharmacology of P-blockers has been summarised.59 The use of propranolol (ICI) (2) in hypertension has been reviewed.60 It is now available in the U.S.A. for this indication.6la Atenolol (ICI 66082, 'Tenormin' (11)has been launched61b in the U.K. for the treatment of hypertension.62 It is a cardioselective p-blocker without intrinsic sym athomirnetic activity. Its duration of activity allows once a day dosing.6y Bunitrolol (KO 1366 'Stresson')(z) a cardioselective P-blocker has been marketed in Austria by B~ehringer-Ingelheim.~~ The medicinal chemistry of the compounds related to acebutolol (z)(May and Baker) has been described65 and the first clinical report of talinolol (14)(Veb Arzneimittelwerke Dresden) a cardioselective agent has appeared.s ArOCH2CHOHCH2NHR

10 11 12 13

Propranolol Atenolol Bunitrolol Acebutolol 14 Talinolol

15

Ar 1-naphthy1

4-H2NCOCH2-phenyl 2-cyanophenyl 4-butyramido-2-acetyl-phenyl 4-[3-~yclohexylureido-]-phenyl 2-[3-cyanopyridyl]

-R iPr iPr tBu iPr tBu iPr

Although a large number of p-blockers have been investigated in the clinic, their mode of action in hypertension is still unknown.60 It is unclear whether all agents act by the same or the same combination of effects and whether all the observed effects are due to p-adrenergic receptor anta onism. The role of renin continues67 to be argued without new conclusions.68 The central depressor effects have been reviewed.69 It has

64 -

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Francis, Ed.

been suggested t h a t damping s e n s o r y i n p u t t o t h e CNS from t h e h e a r t may d i m i n i s h sympathetic nerve e f f e r e n t a c t i v i t y . 7 0 p-Blockers may a c t by c o n t r o l l i n g t h e s u r g e s i n blood p r e s s u r e r e s u l t i n g from s t r e s s . 7 1 The e f f e c t s of p-blockers on a d r e n e r g i c t r a n s m i s s i o n have been discussed.72a I t has been proposed t h a t p-blockers may a c t by r e d u c i n g n o r a d r e n a l i n e r e l e a s e by b l o c k i n g p r e s y n a p t i c p-receptors mediating p o s i t i v e feedback82b PresynapCic e f f e c t s have a l s o been d e s c r i b e d f o r p r o s t a g l a n d i n s , dopamine, h i s t a m i n e , n i c o t i n e and angiotensin.73 I n a d d ' t i o n t o t h e i r common use i n combination with v a s o d i l a t o r s and d i u r e t i c s ,7i p-blockers have been used with a - a d r e n e r g i c b l o c k e r s 7 5 t h a t t h i s combination would g i v e a similar although i t might be e f f e c t t o a d r e n e r g i c neurone blockers. L a b e t a l o l ( A l l e n and Hanbury 5158) is a n o n - s e l e c t i v e p-blocker w i t h weak a - a d r e n e r g i c b l o c k i n g p r o p e r t i e s . 7 7 It is 4-8 t i m e s more p o t e n t a t t h e (3 t h a n a t t h e a - r e c e p t o r . I n man i t c a u s e s a r a p i d f a l l i n blood p r e s s u r e w i t h reduced p e r i p h e r a l r e s i s t a n c e but l i t t l e change i n c a r d i a c output. The r e l a t ' v e e f f e c t s of t h e a- and p-blockade a p p e a r t o vary w i t h t h e dose used.?& P o s t u r a l hypotension i s noted p a r t i c u l a r l y a t h i g h e r doses but is s a i d t o b e r e d u c e d

(g)

y 3 CHOH-CH2-NH-CH-

(CH2)

17

16 -

w i t h c h r o n i c t r e a t m e n t . It could be a u s e f u l a g e n t i n h y p e r t e n s i v e emergencies and i n p a t i e n t s with moderate h y p e r t e n s i o n who do n o t respond t o p-blockers and d i u r e t i c s . Compounds with P-blocking a c t i v i t y and vasob been p a t e n t e d . d i l a t i n g p r o p e r t i e s such as 2 7 8 a and ~ 7 8 have

-

Prazosin67 ( P f i z e r ) has been introduced61c Antihypertensive V a s o d i l a t o r s i n t o t h e U.S. Unlike o t h e r v a s o d i l a t o r s i t causes a d e c r e a s e i n plasma r e n i n l e v e l s . 7 9 The use of h y d r a l a z i n e has been reviewed,80 and t h e s t r u c t u r e - a c t i v i t y r e l a t i o n s of h y d r a z i n o p y r i d a z i n e s and p h t h a l a z i n e s have been s t u d i e d u s i n g molecular o r b i t a l c a l c u l a t i o n s 8 1 and Hansch a n a l y s i s . 8 2 Animal pharmacology h a s been summarised and t h e f i r s t c l i n i c a l d a t a r e p o r t e d 8 3 f o r L e p e t i t 6150 I n 20 p a t i e n t s i t was found t o b e g t i m e s MF?

(2).

18 -

19 -

more p o t e n t t h a n h y d r a l a z i n e i n l o w e r i n g d i a s t o l i c blood p r e s s u r e b u t o n l y 2-4 times more potent i n r a i s i n g h e a r t r a t e . CL90,394 ( L e d e r l e ) ( x ) h a s a l o n g d u r a t i o n of a c t i o n i n rats, l o w e r i n g blood p r e s s u r e with t a c h y c a r d i a a t 10 mg/kg The v a s o d i l a t i o n and t a c h y c a r d i a a r e p a r t i a l l y b l o c k e d b y p r o p r a n o l o l . B c o i o a - a d r e n e r g i c , neurone o r ganglion blockade was observed and t h e compound d i d not i n h i b i t MAO, t y r o s i n e hydroxylase o r dopade-

Chap. 7

Antihypertensive Agents

Thornber, Shaw

65 -

carboxylase. Bupicomide (5-butylpicolinamide)(Schering 10,595) reduced systolic and diastolic blood pressure in 6 out of 10 patients. Peripheral resistance fell and there was an increase in heart 1-ate.~5There is evidence in rats that fusaric acid (5-butylpicolinic acid) the main metabolite of bu icomide, causes tachycardia by indirect release of catecholamines. 8g Nifedipine ( 'Adalat , Bayer 1040)(B), used for the treatment of angina ,87 is a powerful peripheral vasodilator and has hypotensive properties in man.88 A dose of 3Omg sublingually lowered blood pressure substantially in 14 essential hypertensives for more than 4 hrs. with a rise in heart rate and plasma renin activity. Nifedipine interferes with the transmem ra e calcium flux causing a reduction in vascular smooth muscle tone. $7 8 9 The conformational requirement for dopamine induced renal

vasodilation has been identified9' as the fully extended form related to 21. The mode of action of organic nitrates as vasodilators has been reviewed.91 Some amides of adenosine-5'-carboxylic acid lower blood pressure in SHRs. They may act directly on an adenosine receptor.92 Some vasodilators may act as adenosine deaminase inhibitors.93

-

Miscellaneous Agents - A number of bisbenzyltetrahydroisoquinolines from Thalictmm species have been found to lower bloodTT pressure in dogs.94

Me

0

22

NHCO

R

-@(

23

hides of the pyrrolizidine retronamine, such as 22, have been found to be hypotensive95 and the decahydroisoquinolines 23 lower blood pressure in SHRs.96 A new antihypertensive dopamine+hydroxylase inhibitor, BRL-8242 (24) has been described.97 In metacorticoid and normotensive rats, the hypotensive response to oral administration was dose-related in the range 3-100 mg/kg and was shown to correlate with the reduction of endogenous noradrenaline in the tissues examined. Heart rate was not changed significantly. BRLH 8242 was shown to have Cu++-complexing properties, in vitro.

24 -

66 -

g

Sect. I1

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Pharmacodynamic Agents

Francis, Ed.

Another Beecham compound, BRL 13776 (25) has shown antihypertensive properties due t o noradrenaline depletion in DOCA rats and renal hypertensive cats. 98 Only the medulla/pons region of brain showed significant drops in noradrenaline, in contrast to reserpine. There were no behavioural effects and the compound is being taken to clinical trial. A novel hypotensive peptide, hypotensin, has been isolated from the CH3 C5Hll venom of the Western diamondback rattlesnake.99 It contains approximately 20 amino-acid residues CH3 25 and appears unrelated to the kinins. The hypotensive effect is said to be dose-related after oral administration in normal rats and SHRs and is not consequent upon histamine release. Brief details of a clinical trial of a PGE2 analogue (26) are available.100 Eleven of seventeen hypertensives responded with lower blood pressure to oral doses (10-20 p g ) of &. A recent study101 of the marked antihypertensive properties of the diuretic lndapamide (3) (SE 1520, Servier) in rats and cats shows it to reduce vasc,liLar reactivity on chronic dosing. CH2-(2-naph$hyl)

-8

bci C02Me

-'".\

HO

OH

C l p C o N H

SO2NHz

27

26 -

0

References 11

1.

2.

3.

4. 5. 6.

7. 8. 9. 10.

B. Oberg, Ann.Rev.Physiol., 3, 537 (1976). E. Knobie Ed., Ann. Rev. Inc. Palo Alto. D.G. Beevers, J.J. Brown, R. Fraser, D. Kremer, A.F. Lever, J.J. Morton, J.I.S. Robertson, M.A.D.H. Schalekamp, R.F. Semple and A. Wilson, Essays Med.Biochem., L, l(1976)V. Marks and C.N. Hales Eds. J.J. Brown, A.F. Lever, J.I.S. Robertson and M.A.D.H. Schalekamp, Lancet, i (7971) 1217 (1976). Editorial, Lancet, ii (7997) 1230 (1976). M. Mendlowitz and N.D. Vlachakis, Am.Heart J., 2, 378 (1976). K.V. Malik and J.C. McGiff, Prostaglandins: Physiological Pharmacological and Pathological Aspects, S.M.M. brim Ed., M.T.B. Press,(1976) pp lo3 and 201. J.B. Lee, R.V. Patak and B.K. Mookerjee, Am.J.Med., 60, 798 (1976). L.B. Page, J.M. Yager and J.J. Sidd, Arn.Heart J., 92, 252 (1976). A. Scriabine, B.V. Clineschmidt and C.S. Sweet, Ann.Rev.Pharmaco1. Toxic., 1976 p 113. M. Laubie, B. Delbarre, D. Bogaievsky, Y. Bogaievsky, D. TsoucarisKupfer, D. Senon, H. Schmitt and H. Schmitt, Circ.Res., 38, Supplement I1 11.35 (1976). Proc.VIth.Intern.Congress.Pharmacol., Vol IV, H. Vapaatalo Ed., p49 -

106 (1975).

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Antihypertensive Agents

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B. Rouot, G. L e c l e r c , C-G. Werrnuth, F. Miesch and J. Schwartz, J.Med. Chem., 9, 1049 (1976). 12. H. Karppanen, I. P a a k k a r i , P. P a a k k a r i , R. H u o t a r i and A-L. O r m a , N a t u r e , 259, 587 (1976). 13. Y. A u d i g i e r , A. V i r i o n and J - C . Schwartz, N a t u r e , 262, 307 (1976). 14. L. Finch and P.E. H i c k s , Eur.J.Pharrnacol., 40, 3 6 5 7 9 7 6 ) . 15 L. Finch and P.E. H i c k s , Eur.J.Pharmacol., j6,263 (1976). 16. P. S k o l n i c k and J . W . Daly, Eur.J.Pharrnacol., 3, 11 (1976). 171 N-E. And&, M. Grabowska and V. Strgrnbom, Naunyn-Schmiedeberg’s Arch. Pharrnacol., 292, 43 (1976). 18. W. Kobinger and L. P i c h l e r , Eur.J.Pharrnacol., 40, 311 (1976). 381 (1976). 19 M.C. KOSS, Eur.J.Pharrnacol., 2, 95 (1976). 20. U.B. Olsen, Eur. J.Pharrnaco1. , 21. F. Lornbardi, A. M a l l i a n i , P.Portillo-Nunez, E. Z a i m i s and A. Z a n c h e t t i , B r i t . J. Pharmacol., 57, 448P (1976). 22. C.T. D o l l e r y , D.S. Davies, G.H. D r a f f a n , H.J. D a r g i e , C.R. Dean, J.L. R e i d , R.A. C l a r e and S. Murray, Clin.Pharrnac.Ther., Q, 11 (1976). 23. T.L. W h i t s e t t , S.G. C h r y s a n t , B. D i l l a r d and A.W. Czerwinski, J. Am. Med. ASSOC;, 235, 2717 (1976). 10,69, 73 (1976). 24. A. J g l t t e l a , Eur.J.Clin.Pharrnacol., 109 (1976). 25 * I. Esch, Int.J.Clin.Pharrnacol., 31 (1976). 26. T. Baurn and A.T. S h r o p s h i r e , Eur.J.Pharrnacol., 2, 27- R.S. Shah, B.R. Walker, S.K. Vanov and R.H. H e l f a n t , Clin.Pharrnaco1. Ther. , 9, 732 (1976). 28. W. Hoefke and I. S t r e l l e r , Naunyn-Schmiedeberg’s Arch.Pharrnacol., 294, R 2 1 (1976). L.R. Klevens, J . L Kovacs and R. K e l l y , J.Pharrnacol.Exp.Ther., 29 389 (1976) C.T. D o l l e r y and P.J. Lewis. P o s t g r a d u a t e Med.J., 52, Supplement 4 , 116 (1976) C.S. Sweet and H.C. Wenger, Neuropharrnacology, 15, 511 (1976). J.G. Webb and P.J. P r i v i t e r a , The Pharmacologist,

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3,

2,

s,

2, 135 (1976). R.E.

30

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41. 42.

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431 (1976).

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M . J . Antonaccio and D. Cote., Eur. J.Pharrnacol., 451 (1976). R.K. Razdan, B.Z. T e r r i s , H.G. P a r s , N.P. P l o t n i k o f f , P.W. Dodge, A.T. Dren, J. Kyncl and P. Sornani, J.Med.Chern., 19,454 (1976) s e e a l s o i b i d 445, 461, 549, 552. J.M. Saavedra, H. Grobecker and J. Axelrod, S c i e n c e , 191,483 (1976). R.G. P e n d l e t o n , C. K a i s e r and G. Gessner, J.Pharrnacol.Exp.Ther., 197, 623 (1976). P. R y l e t t , H.G. Dean and M.R. Lee, J.Pharrn.Phamacol., 28, 559 (1976). W. Kobinger, A. Walland and R. Kadatz, Naunyn-Schrniedeberg’s Arch. Pharrnacol. , 105 (1976). A. Walland, Naunyn-Schrniedeberg‘s Arch.Pharrnaco1. , 293, R30 (1976). J.O. Davies and R.H. Freeman, Physiol.Rev., 1 (1976). A. Z a n c h e t t i , A. S t e l l a , G. L e o n e t t i , A. Morganti and L. T e r z o l i , Am.J.Cardiol., 675 (1976). L.T. Skeggs, F.E. D o r e r , J.R. Kahn, K.E. Lentz and M. Levine, Am.J. Med., &, 737 (1976). G.P. G u t h r i e , J. Genest and 0. Kuchel, Ann.Rev.Pharrnacol.,&, 287(1976). D. Ganten, J.S. Hutchins, P. S c h e l l i n , U. Ganten and H. F i s c h e r , C l i n .

-

292,

56,

x,

68

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Pharmacodynamic Agents

Francis, Ed.

Exp. Pharmacol. Physiol., 3, 103 (1976). C . I . Johnston, Drugs, 774 (1976). W.A. P e t t i n g e r , T.K. Keeton, W.B. Campbell and D.C. Harper, Circ. Res., 338 (1976). 45. L. Hsu, J.J. L i l l e y and R.A. S t o n e , Clin. Res. 24, A254 (1976). 46. J.L. Imbs, M. Schmidt, J. Velly and J. Schwartz, Eur.J.Pharmacol., 175 (1916). 47 J.C. F r o h l i c h , J. W. H o l l i f i e l d , J.C. Dormois , B.L. F r h c h , J. S e y b e r t h , A.M. Michelakis and J.A. Oates, C i r . R e s . , 447 (1976). 48. C. Larsson, P. Weber, E. O l i w , M. Hamburg, E. Anggard and B. Samuelsson, Eur.J.Clin.Invest., 327 (1976). 49. J.G. T u r t o t t e , C.S. Yu, H.W. Lee, S.K. Pavanaram, S. Sen and R.R. Smeby, J. Med. Chem., 18, 1184 (1975). 3 9 (1976). 50- 0. Kuchel, Circ. Res., 3, 73 (1976). 51 R.L. S o f f e r , Ann. Rev. Biochem., M.A. O n d e t t i , D.W. Cushman, B. Rubin and E.F. Sabo, 173'd A.C.S. 52 Meeting, New O r l e a n s , March 1977, Abstr. MEDI 23 and 24. 53 J. Genest, W. Nowaczynski, R. Boucher, 0. Kuchel and J.M. Rojo-Ortega, Can.Med.Assoc. J., 421 (1975). 54. G.R. Marshall, Fed. Proc., 35, 2494 (1976). 55 D.H.P. S t r e e t e n , G.H. Anderson and T.G. Dalakos, Am.J.Med., @, 817 (1976). 56 R.J. L e f k o v i t z , L.E. Limbird, C. Mukherjee and M.G. Caron, Biochim. 1 (1976). Biophys. Acta, 57 J.R. Sporn and P.B. Molinoff, J.Cyclic Nucleotide Res., 2 , 149 (1976). (5559) 470 (1976). 58 E. Melamed, M. Lahav and D. A t l a s , Nature, 59. M.E. Connolly, F. K e r s l i n g and C.T. D o l l e r y , Progress i n Cardiov a s c u l a r D i s e a s e s , 2,203 (1976). 60. Ten Years of Propranolol. Symposium. B.I. Hoffbrand, Ed., Postgrad. Med. J., 52, Suppl 4 (1976). 61. P. DeHaen, New Product Survey, 3, (1975); ( a ) August 1976 Suppl. p3. (b) J u l y 1976 Suppl. p5. ( c ) October 1976 Suppl. p2. 62. L. Hansson, A. Westerlund, H. Aberg and B.E. Karlberg, Eur. J. Clin. Pharmacol., 2 , 361 (1976). 63. A.M. H a r r i s , K.V. Woollard and J.A. Tweed, J.Int.Med.Res.,h, 347(1976). 141c (1976). 64. U n l i s t e d Drugs 65. B. B a s i l , J.R. C l a r k , E.C.J. Coffee, R. J o r d a n , A.H. Loveless, D.L. Pain and K.R.H. Wooldridge, J.Med.Chem., 9, 399 (1976). 66. P. Wolf, K. F e l l e r and K. Femmer, Pharmazie, 30, 678 (1975). 67. C.W. Thornber, Ann.Rep.Med.Chem., Vol 11, F.H. Clarke, Ed., Academic P r e s s , New York, N.Y., (1976) p63. 68. G.S. S t o k e s , R.M. Graham, M.A. Weber, D r u g s , l l , Suppl. 1, 150 (1976). J.H. Laragh and F.R.Buhler, Postgrad. Med. J., 52, Suppl.4, 109 (1976). 69. C.T. D o l l e r y and P.J. Lewis i n r e f e r e n c e 60. 70 P. Lewis, Am. J. Med., @, 837 (1976). 71 A.R. Lorimer, F.G. Dunn, J.V. Jones and T.D.V. Lawrie, Am. J. Med., 60, 877 (1976). M.J. Rand, M. Law, D.F. S t o r y and M.W. McCulloch, Drugs, 2, 72 Suppl.1, 134 (1976); ( b ) B. Ablad, B. Ljung and R. S a n n e r s t e d t , i b i d 127 and 158 and B. Ablad, E. Carlsson C. Dahlof and L. Ek i b i d 100. 73. U. Trendelenburg, Acta Endocrinol. 82, 18 (1976).

12,

43. 44.

2,

38,

2,

6,

5,

113,

457,

a,

3,

G)

Chap. 7

74.

75 76. 77 78 79

80. 81. 82.

Antihypertensive Agents

Thornber, Shaw

69 -

P.S. Kincaid-Smith, Drugs 2,Suppl. 1, 78 (1976). N.D. Vlachakis and M. Mendlowitz, J.Clin.Pharmacol., &, 352 (1976). C.T. Dollery in ref 77. D.A. Richards and P. Turner, Eds., Labetalol Symposium, Brit. J. Clin. Pharmacol., 3, Suppl. 3 (1976). (a) D.T. 2,556, 110 (Merck). (b) U.S. 3,931,177 (SKI?). R.M. Graham, M.R. Muir and J.M. Hayes, Clin.Exp. Pharmacol.Physiol.,

-3,

173 (1976).

J. Koch-Weser, New Engl.J.Med., 295, 320 (1976). V.M. Kulkarni, Indian J. Biochem. Biophys., 12,367 (1975). G. Leclerc, C.G. Wermuth, F. Miesch and J. Schwartz, Eur.J.Med.Chem.,

11, 107

(1976).

C. Pasotti, F. Nicronisi, M. Marchetti, A. Manzini and F.B. Nicholis, Farmaco ed Prat., 31, 453 (1976). A. Maseri, A. Pesola, A. L'Abbate, C. Contini and G. Magiri, J.Int.Med.Res., 5,402 (1976). C. DePonti, U. Bardi and M. Marchetti, Arzneim.-Forsch., 26, 2089 (1976). 84. J.L. Grace, R.Z. Gussin, L.M.Lipchuck, L. Ellenbogen and P.S. Chan, Pharmacologist, 2 , 187 (1976). 85 S.G. Chrysant, P. Adamopoulos, M. Tsuchiya and E.D. Frohlich, Am. Heart J., 92, 335 (1976). 86. M.J. Antonaccio, D. Cot< and T. Cavaliere, Clin.Exp.Pharmaco1. Physiol., 3, 199 (1976). 87. W. Lochner, W. Braasch and G. Kroneberg Eds., New Therapy of Ischaemic Heart Disease. 2nd IntiAdalat ' Symposium. Springer Verlag1975. 88. K. Aoki, T. Yshida, S. Kato, K. Tazumi, I. Sato, K. Takikawa and K. Hotta, Jap. Heart J., 17,479 (1976). 89. P. Meisel, M. Rohde, M. Roenfanz, H. Teichmann and R. Fermun, Pharmazie, 2, 417 (1976). 90 H. J. Crumly, R.M. Pinder, W.B. Hinshaw and L.I. Goldberg, Nature,

83.

259,

91 92 * 93 94. 95

(5544) 584 (1976).

P. Needleman, Ed., Handbuch Exp.Pharrnakol., 40, Springer Verlag1975. H.H. Stein and P. Somani, Ann.N.Y.Acad.Sci., 255, 380 (1975). P. Meisel, M. Meisel and A. Grisk, Cor & Vasa, 2 , 56 (1976). W.-N. Wu, J.L. Beal, L.A. Mitscher, K.N. Salman and P. Patil, Lloydia, 2, 204 (1976). P.G. Rao, R.S. Sawhney, O.P. Gupta and C.K. Atal, Indian J. Pharm.,

37,

128 (1975).

I.W. Mathison, P.H. Morgan, N.J. Wojciechowski and J.W. Lawson, Eur. J. Med. Chem., l l , 247 (1976). 97 I.M. Claxton, M.G. Palfreyman, R.H. Poyser and R.L. Whiting, Eur. 179 (1976). J. Pharmacol., 98. J.Melrose, M.G. Palfreyman, R.H. Poyser and R.L. Whiting, Brit.J. Pharmacol., 56, 361P (1976). 99 C.A. Bonilla, FEBS Letters, @, 297 (1976). 100. Y. Shimada, T. Inoue, Y. Ohtsuka, H. Morii and M. Wada, Osaka City Med.J. , 2, 71 (1975) 2 Chern.Abs., &, 10450311 (1976). ~ 2 8 2(1976). 101. L. Finch and P.E. Hicks, Brit.J.Pharmaco1, 9,

96.

x,

Chapter 8. Pulmonary and Anti-Allergy Drugs Arnold L. Oronsky" and Jan W.F. Wasley, Pharmaceuticals Div., CIBA-GEIGY Corp., Summit, NJ *Present Address: Lederle Laboratories, Pearl River, NY Genera/ - Work in the area of immediate hypersensitivity has continued to define the mechanism of mediator release stimulation by the interaction of antigen with specific cell bound immunoglobulin. Characterization of specific mediators such as slow reacting substance of anaphylaxis (SRS-A), eosinophil chemotactic factor of anaphylaxis (ECF-A) and histamine have The interaction of cell bound immunoglobulins IgE with antigen and haptens ultimately leads to the release of mediators of immediate hypersensitivity from these stimulated cells. IgE appears to interact with a specific plasma membrane receptor on the target cell. Characterization of the cellular receptor for IgE was determined using rat peritoneal mast cells and rat basophilic leukemia cells and indicates it has a molecular weight of 60,000.' The suggestion was made that this molecule is a single polypeptide chain. Experiments defining the nature of the cell surface IgE receptor will significantly contribute to the understanding of antigen antibody complex induced release of mediators of allergy and biochemical modifications of this process can now be envisioned. Pharmacologic studies have indicated that cyclic adenosine 3',5'-monophosphate (CAMP)modulates mast cell and basophil secretion.2 Agents that increase cAMP levels usually inhibit mediator release, whereas agents that decrease cAMP enhance release. However, that concept may undergo some modification since rat mast cells challenged with anti IgE antibody or Conconavalin A indicated that histamine release from these cells was preceded by increases in cAMP levels.2 Therefore, cAMP modulation of mediator release appears to be more complex than initially discerned. Eosinophil chemotactic factor of anaphylaxis (ECF-A) is a mediator of immediate hypersensitivity reactions and has been characterized. ECF-A activity resides in two acidic tetrapeptides, Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu and synthetic tetrapeptides with these sequences were preferentially chemotactic for eo~inophils.~ Histamine also modulates human eosinophil migration by interacting with HI and H2 receptors. At higher histamine concentration (> lOS5M)H2 receptor interactions predominate producing inhibition of eosinophil movement. At 1OS6Mor less, H1 receptor activation by histamine resulted in enhanced eosinophil m i g r a t i ~ n . ~ Another mediator of the allergic reaction, SRS-A, thought to be present only in basophils, has now been shown from which it was released by stimulation with calcium ionophore to be present in human leukocytes as A23187. Until now, allergic mediators in primate systems have been derived from tissue mast cells or basophils. Since these mediators also reside in other cell types and may be released by non-immunologic mechanisms, this suggests that these agents may play a more general role in inflammatory processes.5

Clinical - In a recent study total secretory immunoglobin A (IgA) and specific anti-antigen E were measured in nasal washings from ragweed allergic and normal individuak6 None of the data support the hypothesis that allergic individuals are deficient in secretory antibody responses but the data do support the concept that hay fever sufferers belong to a high responder population which genetically responds to low doses of inhalant antigens.6 Other studies have shown that IgE antibodies (usually thought to induce only immediate skin reactions) in combination with appropriate antigen on the surfaces of mast cells or infiltrating basophils cause both immediate and late cutaneous response^.^ These skin responses may be analogous to late onset asthmatic responses induced by inhalation of ragweed and house dust, etc. This technique might serve as a valuable tool in analyzing the anti-asthmatic effects of new pharmacologic agents. Techniques for the diagnosis of anaphylactic sensitivity t o hymenoptera (bees, wasps, ants, etc.) stings have been difficult to establish. Studies with commercially available whole body extracts of hymenoptera for skin testing were unable to discriminate between hypersensitive and control subjects. Use of hymenoptera venom skin tests clearly distinguishes between allergic and normal subjects8 and this venom was made available to the National Institute of Allergy and Infectious Diseases for further uses as a diagnostic material in 1976.8

Chap. 8

Pulmonary and Anti-Allergy Drugs

71 -

Oronsky, Wasley

Befa-Adrenoreceptor Sfimulants - The development of @stimulant bronchodilators, their pharmacology, evaluation, metabolism and structure activity relationships has been r e ~ i e w e d .The ~ goal is s t i l l an orally effective compound which is a specific p-2 adrenoreceptor stimulant with a long duration of action. Terbutaline was marketed in the U.S. in 1974 and has been studied extensively. It is effective by inhalation (0.50 mg)l0 and orally (5 mg)ll in the prevention of exercise-induced bronchospasm and i t s bronchodilating effect is evident for 6 hrs. Orally, peak effect is 2-3 hrs; by inhalation, 5-30 min.12 I (0.25 mg/s.c.) is effective in the management of acute bronchospasm in stable asthmatics.13 Several long term studies of 1, both oral (5 mg t.i.d.1 for 32 months14 and by inhalation (375 p g t.i.d. for 6 weeks)15 showed good efficacy with no drug tolerance1 and only minimal side effects such as occasional trem0r.l Metabolism studies on 3H-terbutaline indicate sulfate and glucuronide conjugation whether administered by i.p. or by inha1ation.l Studies on 1and its prodrug, lbuterol (3show that after 2 the serum concentration of 1 rose more rapidly (30 min.) and to higher levels (4.1 pg/ml) than after 1 itself?8 Structure activity studies indicate p-2 selectivity is greatest when as determined by the amine moiety i s t-butylamine (terbutaline) or p-hydroxy N-t-butylaniline (ME-106) cat soleus muscle, bronchi and heart rate experiments.lg

c)

w,

OH ?2

R2=H

terbutaline

R2=H

ibuterol

OH Salbutamol has been extensively studied by oral (4 mg), inhalation (200 pg),20 intravenous,21 and intramuscular22 administration and found to be effective in the inhibition of exercise induced bronchospasm.20 No drug tolerance was observed in chronically pre-treated guinea pigs.23

4. R=t-butyl; salbutamol 5. R=CH(Me)CHZ-@-OMe;

6. fenoterol (Th 1165a)

salmefamol.

In patients with chronic airways obstruction salmefamol (5,gave greater response than 4, particularly 6 to 8 hr. post dose.24#25Orally (2 mg) 3 caused a fall in diastolic blood pressure, whereas 1 mcdose did not, and both were found equally effective on ventilatory capacity.26 By inhalation fenoterol (6J was equipotent with 4 in the treatment of asthmatic children but caused some increases in pulse rate.27 Orally, 6 caused significant bronchodilatation with rapid onset (30 min.) and long duration (6 hrs.) but showed mild tremors and increased (SKF 40383) in a double-blind study produced safe pulse rates a t higher doses (15, 20 mg).28 Carbuterol and effective bronchodilatation both orally (4 mg) and by inhalation29 (300 pg), but was less potent than $.30

c)

NH2 oAH&l-CMe3 HO

7. carbuterol (SKF 40383)

CHO

CH20

I

1/2 fumarate

OH

HND,ce-cMe3

HO

OMe

HO

8

9. BD4OA

72

Sect. I1

-

Francis, Ed.

Pharmacodynamic Agents

A series of compounds of general structure (81 variously substituted in the benzyl group were claimed to be 10 x 5 in potency as bronchodilators with higher bronchoselectivity (cat soleus muscle prep).31 BD 40A. is more potent than 3 by various routes but i s less /32-~eIective.~~

g)

A series of mono and diesters of N-t-butylarterenol

(10) showed that the monoesters have a moderate degree of

9 OH

OR

0 11

10

activity with rapid onset and a duration of action of 2 hrs. The diesters show marked bronchodilatation by i.v., i.d. and inhalation administration with a 5-10 min. onset of action and lasting 4 hrs. The diester of choice i s 4-Me-CgH4-CO-(BitoIter01).~~ Hydrolysis rates of the esters correlate well with the bronchodilatator activity seen in intact, anaesthetized dogs.33 Bitolterol is less cardiostimulating than ?in dogs a t doses which cause some bron~hodilatation.~~

(11)

Structure activity relationships for a series of sympathomimetic arnines having a carbostyril moiety have been reported.35 The most potent ( 1 1, R3=H, Rq=t-butyl) is 22,400 x isoproterenol in relaxation of guinea pig trachea, and is also more 0-2 selective than Order of activity: R3: H>Me>Et, R4: t-butyI>i-Pr>CMegCHp(d> B z > H . ~Another ~ analog (11, R3=Et, R4=CHMe2) (OPC 2009) is claimed to be more active and more selective than4.36

5

0,

(NAB-35) in Detailed pharmacology, toxicology, pharrnacokinetics and metabolite patterns of clenbuterol rat, rabbit, dog and man have been reported.37 A double-blind study has shown z o r a l l y effective (10 pg t.i.d.) in the treatment of moderately severe asthma.38 ,OH H A OH

H2N cI

WNH-,.., CI

he

12. denbuterol (NAB-351

13. reproterol (D1959)

Detailed accounts have been published of the synthesis, structure activity relationships and pharmacology of a series of bronchospasmolytic 0-phenylethylaminoalkylxanthines, of which (reproterol, D 1959) was i s a 0-2 agonist with minimal CNS or CV side effects and is clinically effective selected for clinical trial. against bronchial asthma and chronic bronchitis (orally or by inhalation) with no tachyphylaxis after 4 weeks.39

13

(13)

Antichofinergics - Although the precise role of anticholinergics in the treatment of asthma is not yet known, they have been advocated as an alternative to 0-agonist therapy in patients with cardiac arrhythmias or angina.40 lpratropium bromide (14) (Sch. 1000, AtroventQ) by inhalation showed bronchodilator activity comparable with isoproterenol, buthad a longer duration of action (4 hr.) with no significant side effect^.^'

Chap. 8

Pulmonary and Anti-Allergy Drugs

73 -

Oronsky, Wasley

OH

\-OH 15. Pamine@

14. SchlOOO Atroved

Side effects of 14 appear minimal, i.e,, dry mouth observed a t maximum doses in some patients.42 14 appears to have bronchospasmolytic action and no secretory inhibition properties a t therapeutic doses, and is without effect on sputum viscosity or volume.42 Detailed studies on the synthesis, general pharmacology and toxicology in rats, dogs, mice and monkeys by various routes of administration of 1 4 have been p~blished.~2 Pharmacokinetic and metabolism studies of E s h o w a t 1/2 i.v. of 1.9 hr. (rat), 3.4 hr. (dog) and 4 metabolites have been isolated and characterized42 Inhalation of atropine (0.1 mg) blocked exercise induced bronchospasm and caused significant bronchodilatation for up to 5 hr.43 Studies conducted in 1949 on methscopolamine in which prolonged efficacy against the bronchospastic effects of methacholine in asthmatic (Pamine@) patients was noted were restated. No cardiovascular side effects but drying of the oropharynx were observed.44

(2)

Prostaglandins - The pharmacologic actions of prostaglandins and their role in allergy has been re~iewed.~5 Inhalation of PGF2, in normal subjects produces 2 qualitatively different airway responses, which may reflect the balance between sympathetic and parasympathetic nervous control of the airways. Diminished 0-receptor activity in asthmatic patients may account for heightened bronchoconstrictor response to PGF2,.46 Plasma levels of 15-keto-l3,74dihydro F2, (a relatively stable metabolite of PGF2,) were measured under various conditions in exercise induced asthmatics, but did not show any significant change, leading to the conclusion that PGF2, does not play a significant role in the aetiology of exercise induced asthma.47 The contractile effects of some cycloendoperoxides (CEP's), intermediates in the biosynthesis of PG's, are 3 to 4 orders of magnitude greater than PGE2 and PGF2, and may represent the active form of PG's in the lung.48.49 The synthesis of ?r ,,..i(CH2)&02H 15-methyl-11-deoxy PGEl (doxaprost, AY-24,559) (16) and i t s C15 epimer have been reported.50 16, togetherwith ?r dc,(CH214Me 1 1- d e o x y P G E l , inhibited hzamine-induced bronchoconstriction in guinea pigs. 16 was 73 and 32 times H M 'e more potent than 11-deoxy PGEl by the aerosol and i.v. HO routes, respectively. 16 also demonstrated a longer duration of effect.5' 16. doxaprost, AY-24,559

B

(17)

Corricosreroids - Beclomethasone dipropionate aerosol has been tested extensively clinically. It is used prophylactically (400 p g per day), not therapeutically, in the treatment of chronic asthma, particularly in children.52 An evaluation of the drug has been published.53 One of the most important clinical advantages is that effectively can replace oral corticosteroids in steroiddependent patients and avoid many of the adverse effects of adrenal s ~ p p r e s s i o n . ~ Most ~ # ~patients ~ with impaired adrenal function due to oral corticosteroids show recovery of adrenal function within 6 months.55 The combination of and disodium cromoglycate (DSCG) showed no additive therapeutic effects.56 Flunisolide (181, when administered as a nasal spray for 4 weeks during the hay fever season in 51 patients, showed significant symptomatic improvement with no systemic steroid effects observed.57

74

Sect. I1

-

Francis, Ed.

Pharmacodynamic Agents

CH20H

CH20COEt

I

c=o &Me ’

0’

,.%.OCOEt !

, Cl F

17. bedornethasone dipropionate Phosphodiesterase lnhibitors - LM 209

18. flunisolide

(19)

has been shown to be a noncompetitive inhibitor of lung phosphodiesterase (PDE) (2.5 x 10-4M) in contrast to theophylline, which is a competitive inhibitor. 19 i s antihistaminic, orally effective and inhibits bronchospasm provoked by histamine, serotonin or citric acid. It is distinguished from other antihistamines by i t s lack of sedation and long duration of action.58 Absorption, distribution and elimination studies in rat and dog after i.v. or oral administration have been reported.59

/ Me l/N=C I

19. LM 209

M ‘e

20. SQ20.009

A pyrazolo[3,4-b] pyridine (20)(SO20,009), a potent inhibitor of CAMP PDE, inhibited antigen induced histamine release from passively sensitized guinea pig lung slices. 2_0 also inhibited the phosphatidylserine plus dextran induced release of histamine from rat peritoneal mast cells. DSCG and doxantrazole also act as inhibitors in this system, but demonstrate cross-reactive tachyphylaxis. 20 shows no tachyphylaxis.60 Serum concentration determinations are important in guiding safe usage of theophylline since a relationship between daily dosage and serum levels of the drug is not predictable.61 Toxic symptoms occur commonly over levels of 25 pg/ml, but are not noted below 15 pg/ml.

(30)

lnhibitors of Mediator Release - A review of several trials of disodium cromoglycate (DSCG) (IntalB) concluded that clinical benefit is obtained in 33-50% of patients.63 Long term studies show that the early response to DSCG is maintained but not heightened over long periods of time (up to 5 ~ r s . 1 The . ~ ~most valuable results of DSCG therapy are (a) a reduction in symptoms of asthma (sneezing and coughing), (b) no side effects, (c) reduced need for oral bronchodilators, aerosolized adrenergics and corticosteroids, and (d) greater exercise tolerance.65 In a perennial allergic rhinitis trial it was found that patients which responded best were female and those who had high IgE levels and a markedly positive skin test to food and epidermoids rather than pollen allergy.66 DSCG (orally) has been found effective in gastritis v a r i o l i f o r m i ~and ~ ~ ulcerative colitis.6E After 10 months on DSCG, one patient developed pulmonary infiltrates with eosinophilia. The symptoms disappeared 2 weeks after the drug was w i t h d r a ~ n . ~ ~ Structure activity relationships for a series of chromone-2-carboxylic acids, of which FPL 52791 (21) showed efficacy in passive cutaneous anaphylaxis (PCA) (rat) both by intravenous and oral administration, have been published. Substitution in ring A shows that 6,E-di-t-butyl (12 x DSCG)>6,8-di-Et>6,8-di-Me>> m o n o s ~ b s t i t u t i o n .Clinical ~~ studies of are in progress. Similarly, in a series of chromones with a tetrazolyl moiety a t the 2 or 3 position, (22) was the most potent in the PCA (rat), both orally and i.v. (-1 1.6 x DSCG). The following structure activity relationships were noted: (a) the 3-tetrazolyl

21

Chap. 8

Pulmonary and Anti-Allergy Drugs

Oronsky, Wasley

75 -

compound was twice the potency of the 2-tetrazolyl isomer, (b) introduction of a methyl group in position 2 of the chromone ring reduced activity, (c) substitution in the phenyl ring showed the following order of potency: 6,8-di-MeX-CI-6-Et>6-NO2-6-OH. The naphthyl analogues, e.g., were also active.71

(3,

he

t-Bu

21. FPL52791

22

23

Metabolism studies o f 24a (W8011) i n rats identified 3 metabolites (24b. c, and 2-hydroxy-3-methoxyacetophenone). Although 3 and b are active in vivo (rat PCA), 24bwas the only compound active in an in vitro system for inhibition of anaphylactic histamine release.72

0

HO 0

24a. R=CH2OH (WE011)

25

b. R=CO2H c. R=H

The disodium-6-phosphatederivative of baicalein (25) inhibited PCA (rat) and experimental asthma caused by passive systemic anaphylaxis in guinea pigs.73 Aseries of benzodipyrandicarboxylic acids (26) has been described and their structure activity relationships in PCA reported. Linear analogs26 were more active than the corresponding angular analogs (27), although substitution effects differ from the linear to the angular series.

26

27

28. PRD92-EA

In 26, introduction of alkyl groups led to enhancement of activity, e.9.. R1, R2=H: i.v. PCA 6 x DSCG; R1=Me, R;=Et: -36 x DSCG, whereas introduction of alkoxy caused a reduction in activity. Conversely, in the angular series 27, introduction of alkyl substituents produced no significant change, but an alkoxy group ortho to a pyrone carbonyl group resulted in a marked increase in activity. Electron withdrawing substituents in either series resulted in loss of activity.74 Structure activity relationships for a series of compounds of which PR-D-92-EA (28) was the most potent (6 x DSCG) i.v. (rat PCA) have been published.75 Simple substitution did not yield compounds with activity significantly enhanced over with the exception of the 3-OCH2CH20H.4-Me compound (-30 x DSCG).75

g,

76

Sect.

I1

-

Francis, Ed.

Pharmacodynamic Agents

Clinical studies on t i x a n ~ x(29) ~ ~(OTMX) by inhalation showed good protection which lasted up to 4 hr. in 5/6 patients with allergic asthma; 2 patients had a delayed (Type Ill) reaction 4-6 hrs. after antigen challenge which was unaffected by 29. No evidence of bronchodilator activity was observed.77 Doxantrazole is 10 x DSCG and 10 x theophylline as a PDE inhibitor, and the anti-allergic activity of 2may be due in part to this ability to elevate intracellular levels of CAMP. Replacement of the tetrazole moiety by carboxyl results in a significant loss of activity (0.2 x The pharmacology of compound A, (311, - indicates a qualitative and

(30)

29. tixanox (OTMX)

30. doxantrazole

31. Compound A

quantitative advantage over DSCG in that (a) it shows bronchodilator activity, (b) inhibits mediator release (50 x DSCG), and (c) is orally effective.79 On intrabronchial administration inhibited the immediate type hypersensitivity to Ascaris antigen in rhesus monkeys (1000 x DSCG). is presently in clinical trial.80 Bufroline (32) (ICl-74917) is 5 x DSCG as a PDE inhibitor and 100 x DSCG in the PCA modeLE1 Clinically, b y inhalation showed almost complete C02H protection against nasal stenosis induced by grass pollen extract in 10 patients.82 Studies Na02C o@ on is related 2 andtoDSCG the continued indicate that occupation tachyphylaxis of a

x; o//

'

3 31

2

0 0

receptor by drug molecules on the mast cell surface, preventing the access of further drug \ NH to these sites.83 32, DSCG and doxantrazole 32. ICI 74,917 cO2 H may a ct by interfering with calcium transport (bufroline) 33 across the mast cell The synthesis of 14C labeled 3 has been described.85 Of a series of compounds related to 32, 3_3 had comparable activity in the rat PCA.86 In EM 15,100 (34) chemical and pharmacological combination of an antihistamine of t h e cyclizine type and shows activity in rat PCA, both orally and by anti-allergic agents of the DSCG type has been achieved.87 intravenous administration; it antagonizes bronchospasm in guinea pigs and prevents anaphylaxis in monkeys C02Na

2

I

34. BM15.100

35. N5l

(35)

induced by ascaris antige11.8~An anthranilic acid derivative N5l is orally effective in rat PCA and it i s claimed to show good results clinically in the prophylactic treatment of asthmatic attacks.@ Detailed pharmacological comparison of 3 with standard anti-inflammatory agents has been published.89

Chap. 8

P u l m o n a r y and A n t i - A l l e r g y D r u g s

O r o n s k y , Wasley

77 -

Structure activity relationships on (36) indicate that several cinnolone-3-propionic acid derivatives are comparable with DSCG in rat PCA, and 36 is also orally effective.go The esters are comparably active with the carboxylic acids (i.v.) due to rapid hydrolysis in viva 3s is not metabolized by 0-oxidation to the

corresponding cinnolone carboxylic acid.go Inhalation or oral administration of the nitroindane dione BRL 10,833 (37) protected approximately 50% of patients against allergen-induced immediate bronchospasm and also showed some protection against the delayed bronchial reaction.g1 A number of and 3-cyano-4-hydroxy coumarins (39) related to have been reported active in 2-cyanoindane-l,3diones rat PCA (i.v. and p.0.). The structural requirements for these new series parallel that already reported for 37.92 Replacement of the hydroxy in by a wide variety of groups resulted in loss of activity.

7

(3) 39

Structure activity relationships for a series of quinolyl oxamic acids (40)with several analogs showing good activity in rat PCA (25 x DSCG) have been published.g3 The most active compounds have the oxamic acid residue a t the 6 or 7 position of the nucleus and for X: MeXI>MeO. The naphthyl analogue (41)showed

(42)

cornparable activity.g3 The N-aryl oxamic acid ester WY-16.922 effectively inhibited reaginic mediated immunologic reactions in skin, lung and mast cell. It is more potent than DSCG and is orally active.g4 Structure activity relationships have been reported for a series of bisoxamic acids of which (43)showed high potency (250 x DSCG) in rat PCA i.v. and also oral activity. The order of activity for substituents at the 3 position of 43 is CN

-N02>>0Me>CONH2>>C02H.86 (0.05 x DSCG) in rat PCA.95

Of a series of 3.5-disubstituted pyrantriones, (44)showed modest activity

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78 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

29. 30. 31. 32. 33. 34. 35. 36.

37. 38.

39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

49. 50. 51. 52. 53. 54. 55. 56. 57. 5B. 59. 61).

61.

62. 63.

64. 65.

Sect. I1

-

Pharmacodynamic Agents

Francis, Ed.

R.A.F. Clark, J.A. Sandler, S.I. Gallin. A.P. Kaplan, J.lmmunol. ll3 137 (1977). M.C. Conroy, R.P. Orange, L.M. Lichtenstein, J.lmmunol., ll3 1677 (1976). T.A.E. Platts-Mills. R.K. von Maur, K. hhizata, P.S. Norman, L.M. Lichtenstein, J.Clin.lnvest.,L7. 1041 119761. G.O. Sollay. G.J. Gleich. R.E. Jordon, A.L. Schroater, J.Clin.lnvast., g, 408 (1976). K.J. Hunt, M.D. Valentine, A.K. Sobotka. L.M. Lichtenstain. Ann.lnt.Mad.,E5, 56 (19761. R.T. Brittain, C.M. Dean, D. Jack, Pharmac.Ther., 82,423 11976). J. Allegra. J. Field, J. Treutlain, M. Gillin, R. Zalis. J.Clin.Pharmaco1.. 3 , 444 (19761. J.L.C. Morse, N.L. Jones, G.D. Anderson. Amer.Rav.Rasp.Dis., L3.89 (1976). J. Allegra. J. Field, J. Trautlein. M. Gillin, R. Zelis, J.Clin.Pharmacol., l6, 367 (19761. H.J. Schwartz, J. Trautlain. A.R. Goldstein. J.Allergy Clin.lmmunol.. 53 516 (1976). H. Formgren, Scan.J.Resp.Dis., E,321 119751. J. Trautlein, J. Allegra. M. Gillin. J.Clin.Pharmacol., 361 (19761. N. Svedmyr, S. Larsson, G.K. Thiringer, Chest, E,479 119761. H.T. Nilsson. B.G. Simonsson, B. Strom. Europ.J.Clin.Pharmaco1.. 1 (1976). Y. Hornblad. E. Ripe, P.O. Magnusson, K. Tegner, Eur.J.Clin.Pharmacol., g,9 (1976). E. Malta. C. Rapar, Clin.Exptl.Pharmaco1. and Physiol.,_3.49 (19761. S. D. Anderson. Am. Rev. Respir.Dis., x4.493 (19761. I.W.B. Grant, Br.J.Clin.Pharmac..?, 509 119761. P. d'A. Semple. J.S. Lagge, T. Habeshaw. Br.J.Clin.PharmacoL,~, 936 (1976). A.A. Anderson, G.M. Lees, Br.J.Pharmacol.,26.331 (1976). LA. Campbell, C.H. Dash, G.J.R. McHardy, M. V. Shoner. Br.J.Clin.Pharmacol.,?, 151 (1976). W. Sillett, C.H. Dash. M.W. McNicol, Eur.J.Clin.Pharmacol.,!, 277 (1976). W. Sillett, C.H. Dash, M.W. McNicol, ibid,?, 281 (1976). M.I. Blackhall. M. Dauth, M. Mahoney, S.R. O'Donnell, Med.J.Aust.,?, 439 (1976). A.M. Gaumei, W.F. Miller, J. Miller, L.R. Gast, Chest. LO. 460 (19761. D.W. Cockcroft. RE. Donevan, G.M. Copland, Curr. Tharap. Res., L9, 170 (1976). A. Funahashi, L.H. Hamilton, Am. Rev. Resp.Dis., x3,398 119761. K. Hermansen. K.Hedegaard, J.J. Larsen, J. Weis, Acta.Physiol.Scand., Suppl. 40, 143 Abstract No. 226 (1976). H. Ida, Arzneim.Forsch..g, 1337 (19761. B.F. Tullar. H. Minatoya, R.R. Lorenz, J.Med.Cham., l9.834 (1976). H. Minatoya. Fed.Proc., 25,802 Abstract No. 3289 (1976). S. Yoshizaki, K. Tanimura. S. Tamada, Y. Yabuuchi, K. Nakagawa. J.MedChem.. 2. 1138 (1976). Y. Yabuuchi, S. Yamashita, S. Tei. Jap.J.Pharmacol..2J (Suppl.) 66 P. Abstract No. 66 (1976). G. Engelhardt, Arzneim.Forsch., 3(7al 1404 and subsequent papers 11976). B. Stenius. P. Tukiainen, H. Poppins, Eur.J.Clin.Pharmacol.,J, 189 (19751. K.H. Klingler. Arzneim.Forsch., 27 ( l a ) 4 and subsequent papars 11977). T. Vlagopoulos. R.G. Townley, S. Ghazanshahi, A. 8ewtra. K. Burke, Ann.Allargy, 56, 223 (19761. H. Yeager, R.M. Weinberg, L.V. Kaufman. S.Katz, J.Clin.Pharmaco1.. 3 l 198 (1976). W. Schulz. R. Banholzar, K.H. Pook. Arzneim.Forsch. 2_6 15al960 and subsequent papars (1976). D.G. Tinkalman, M.J. Cavanaugh. D.M. Cooper, Am.Rev.Resp.Dis.. c 4 . 8 7 (1976). M.S. Segal. Am.Rev. Resp.Dis.. E3,893 (1976). J. R. Vane. J.Allergy Clin.lmmuno1.. g.691 (1976). K. R. Patel, Pactgrad.Med.J.,z, 275 (19761. S. D. Anderson. R. Pojer, I.D. Smith, D. Temple, Scan.J. Resp. Dis., z7, 4 1 (1976). P.J. Kadowitz, 8.M. Chapnick. P.D. Joiner, C.C. Matthews. A.L. Hyman, Clin.Res., 24, 53A (1976). D.8. McNamara. C.A. Gruener, A.L. Hyman. P.J. Kadowitz, Pharmacologist, lJ, 602 (1976). J.F. 8agli. R. Greenberg, N.A. Abraham. K. Pelz. Prostaglandins, Ll. 981 (1976). R. Greenberg, K. Smorong, J.F. Bagli, Prostaglandins, l-1, 961 11976). E. Bondarevsky. M.S. Shapiro. G. Schey, J. Shabor. I. Brudermann. J.A.M.A..=6, 1969 (1976). V.P. Gotz, R.D. Lauper, Drug Intell. and Clin.Pharmacy, L O, 635 (1976). J. Lovera. D.M. Cooper, C. Collins-Williams, H. Levison, J.D. Bailey, R.P. Orange. J.Allergy Clin.lmmunol. L7, 112 (1976). S.A. Spitzer, H. Kaufman, A. Koplovitz, M. Topilsky, I. Blum, Chest, 38 (19761. I. Mitchell, I.C. Paterson, S.J. Cameron, I.W.B. Grant, BritA4ed.J.. S D . 457 (1976). P.C. Turkeltaub. P.S. Norman, S. Crepea. J.Allergy Clin.lmmunol. 597 (1976). A. Uzan. G. LeFur, Arch.int.Pharmacodyn.,E9, 160 (19761. A. Uzan, G. Gueremy. G. LeFur. Xen0biotica.g. 633 (1976). C.A. Free, L.E. Hall, J.D. Shada, Pharmacologist, g,218 (19761. M.H. Jacobs, R.M. Senior. G. Kasslar, J.A.M.A.,235, 1983 11976). M.W. Weinberger. R.A. Matthay. E.J. GinchanskrC.A. Chidsev, T.L. Petty, J.A.M.A.,Z5.2110 (1976). D.P. Nicholson. Heart Lung.3. 71 (1976). W.E. Hermanca. E.B. Brown, Ann.Allergy, 3~6, 423 (1976). A. V. Mascia. E. Friedman, M.A. Kornfield, Ann.AlIergy,L?, 1 (1976).

s,

z,

z,

3

Chap. 8

Pulmonary and Anti-Allergy Drugs

Oronsky, Was ley

79 -

66. R. H. Cohan, F.L. Bloom, R.B. Rhoades, H.J. Wittig, L.D. Haugh. J.Allergy Clin.lmmunol., z8, 121 119761. 67. 68. 69. 70. 71. 72.

73. 74. 75.

76. 77. 78. 79. 80. 81. 82. 83. 84. 85.

86. 87. 88. 89. 90. 91.

92. 93. 94. 95.

964 119761. C. Andre, B. Moulinier, R. Lambert, 8. Bugnon. Lancet,=l, G. DellaCella. L.R. Garibaldi. P. Durand, Lancet,Z9=/1, 1129 119761. U. K. Repo. R. Nieminen, Scan.J. Resp.Dis.. 57, 1 119761. J. Augstein. H. Cairns, A. Chambers. J.W. Burns. H. Radziwonik, J.Pharm.Pharmacol., 23 919 119761. Y. Sanno, J.Med.Chem..2_0. 141 119771. M.D. Melgar, S.Geowe, M.C. Crew, Drug Metab.Disp.._4.368 119761. H. Nagai, K. Osugar, A. Koda. Jap.J.Pharmacol.,~5, 763 119761. J. R. Bantick, H. Cairns. A. Chambers, R. Hazard, J. King, T. Lee, R. Minshull, J.Mad.Chem.. L9, 817 119761. J.P. Devlin. K. Freter, P.B. Stewart, J.Med.Chem.,zO, 205 119771. U.S.A.N.. J.A.M.A.,a6.2543 119761. B. Wuthrich. D. Parrott, Respiration,23,231 119761. J.E. Tateson, D.G. Trist, Life Sci., l8, 1 5 3 119761. H.G. Johnson, C.A. Van Houk, 1nt.Arch.Allergy appl. Immun., 446 119761. ibid. 454 119761. K. Barrett-Bee. W. Hendersion, Biochem.Soc. Trans., 9,699 119761. J.S. Vilsvik, A.O. Jenssen, Clin.Allergy,~,487 119761. P.W. Marshall, D.S. Thomson. D.P. Evans. lnt.Arch.Allergy appl.lmmun., Kl, 274 119761. J.C. Foreman, L.G. Garland, Brit.Med.J.,K3/1.820 119761. D.C.H. Sigg, J.Label.Cpd.Radiopharm., L2, 571 119761. C.M. Hall, J.B. Wright, H.G. Johnson, 10th Middla Atlantic Regional Meeting (Philadelphia Feb. 19761, Abstract No. J.2. 438P 119761. A. Roesch, E. Roesch, Br.J.Pharmacol., A. Koda, H. Nagai. S. Watanabe, Y. Yanagihara, K. Sakamoto, J.Allergy Clin, Immunol.. g7, 396 119761. H. Aruma. K. Banno, T. Yoshimura. Br.J.Pharmaco1.. 53 483 119761. D. Holland, G. Jones, P.W. Marshall. G.D. Tringham. J.Med.Chem.. 1_9, 1225 (19761. R. Pauwels. H. Lamont, M. Vander Straeten. Clin.Allergy, g, 463 119761. D.R. Buckle, 8.C.C. Centello. H. Smith, 8.A. Spicer, J.Med.Chem.,LO, 265 119771. J.B. Wright, H.G. Johnson. J.Med.Chern.,LO, 166 119771. M.E. Rosenthale, A.J. Begany. A. Dervinis. J. Sellstedt. C. Guinosso. M.I. Gluckman, J.Pharmacol.Exp. Ther.. x 7 , 725 (1976). C. R. Willis, K.M. Snader, C.K. Miao, W.L. Mendelson, L. W. Chakrin, 10th Middla Atlantic Regional Meeting (Philadelphia Feb. 19761, Abstract No. J.8.

LO.

z,

57.

Chapter 9 .

A n t i t h r o m b o t i c Agents

Robert D. MacKenzie, M e r r e l l - N a t i o n a l L a b o r a t o r i e s D i v i s i o n of Richardson-Merrell I n c . , C i n c i n n a t i , Ohio

-

Vascular d i s e a s e s a r e t h e primary causes of d e a t h i n westIntroduction e r n c i v i l i z a t i o n . These d i s e a s e s c a u s e about 1 m i l l i o n d e a t h s a y e a r i n t h e lJnited S t a t e s , w i t h a b o u t 25 m i l l i o n people a f f l i c t e d . T o t a l c o s t s of time l o s t and c a r e exceeds 7 b i l l i o n d o l l a r s p e r y e a r . I n Over 90% of t h e v a s c u l a r - r e l a t e d d e a t h s , thrombosis was p a r t of t h e d e a t h - r e l a t e d e v e n t . Though t h e c h r o n i c , i n s i d i o u s development of a t h e r o s c l e r o s i s i s t h e main c a u s e of t h e v a s c u l a r d i s e a s e s , thrombosis i s t h e f i n a l death-producing e v e n t . Good reviews have been p u b l i s h e d on a t h e r o s c l e r o s i s , i n which i t s development and i n t e r a c t i o n s w i t h t h e blood components are discussed."' I n o r d e r t o p r e v e n t t h e a c u t e e v e n t s i n v a s c u l a r d i s e a s e d e a t h , maint a i n i n g a p h y s i o l o g i c b a l a n c e between blood components, blood v e s s e l w a l l , and c i r c u l a t o r y f u n c t i o n i s i m p o r t a n t . Many approaches t h a t may h e l p cont r o l v a s c u l a r d i s e a s e a r e a v a i l a b l e . However, t h e i n t e r a c t i o n of t h e blood components w i t h t h e d i s e a s e d blood v e s s e l w a l l , and t h e i n t e r a c t i o n of t h e blood components w i t h each o t h e r must be c o n s i d e r e d a s a n i m p o r t a n t area t o a t t a c k i n t h e t r e a t m e n t of t h e s e d i s e a s e s . I n t h e s e a r c h f o r weapons t o h e l p r e g u l a t e t h e p h y s i o l o g i c b a l a n c e , a n t i t h r o m b o t i c a g e n t s should p l a y a n important r o l e . T h e r e f o r e development of new and p o t e n t a n t i t h r o m b o t i c a g e n t s would be a n important c o n t r i b u t i o n t o t h e c o n t r o l of vascular disease. S i n c e t h e c o a g u l a t i o n of blood i s a complicated biochemical and b i o p h y s i c a l p r o c e s s i n which d i f f e r e n t blood components i n t e r a c t w i t h each o t h e r and w i t h t h e surrounding d i s e a s e d environment, t h e r e c a n be m u l t i p l e approaches t o a n t i t h r o m b o t i c therapy. The c o a g u l a t i o n p r o c e s s has been reviewed.' The main approaches t o i t s c o n t r o l a r e : 1. C o n t r o l o f f i b r i n formation. 2 . C o n t r o l of f i b r i n o l y s i s a c t i v i t y , s o t h a t formed c l o t s may be l y s e d . 3. C o n t r o l o f p l a t e l e t f u n c t i o n . Previous and more r e c e n t reviews on background i n v a s c u l a r d i s e a s e , a t h e r o s c l e r o s i s , and on a n t i t h r o m b o t i c a g e n t s a r e r e c o m e n d e d . 1-a, B , 9 P r e v i o u s reviews i n t h i s s e r i e s l o - l 6 a r e a l s o recommended. P l a t e l e t Aggregation I n h i b i t o r s The r o l e of p l a t e l e t s i n v a s c u l a r d i s e a s e , b o t h on t h e o r e t i c a l and A r e p o r t on onc l i n i c a l b a s e s , has been d i s c u s s e d i n many reviews.10'20 going c l i n i c a l t e s t i n g of a s p i r i n and o t h e r i n h i b i t o r s has a l s o been summarized .21 C l i n i c a l r e s u l t s w i t h s e v e r a l of t h e o l d e r i n h i b i t o r s ( a s p i r i n , s u l f i n p y r a z o n e , dipyridamole, e t c . ) have been reported.Z2 These i n h i b i t o r s a r e e i t h e r n o t v e r y p o t e n t , have s i d e e f f e c t s a t t h e r a p e u t i c l e v e l s , a n d / o r i n h i b i t o n l y one of s e v e r a l a g g r e g a t i o n mechanisms. Adenosine d i p h o s p h a t e (ADP) , thrombin, e p i n e p h r i n e , c o l l a g e n , and s e r o t o n i n induce p l a t e l e t a g g r e g a t i o n by d i f f e r e n t mechanisms. Compounds t h a t s p e c i f i c a l l y i n h i b i t ADP-induced a g g r e g a t i o n w i l l n o t i n h i b i t thrombin-induced a g g r e g a t i o n . Those compounds t h a t i n h i b i t e p i n e p h r i n e and collagen-induced a g g r e g a t i o n (e.g. nons t e r o i d a l a n t i - i n f l a m m a t o r y a g e n t s )

Chap. 9

Antithrombotic Agents

MacKenz i e

81 -

do n o t i n h i b i t t h e primary phase of ADP o r thrombin-induced a g g r e g a t i o n . T h e r e f o r e , combinations of s p e c i f i c a g e n t s f o r each type may be needed t o g i v e b e t t e r c o n t r o l of i n v i v o p l a t e l e t a g g r e g a t i o n , o r compounds t h a t i n h i b i t a l l systems should be c o n s i d e r e d . The following d i s c u s s i o n has been organized w i t h t h i s i n mind. Each t y p e of compound i s d i s c u s s e d i n t h e m e c h a n i s t i c c a t e g o r y t h a t i t f i t s , when t h i s i s known.

-

A s p i r i n and o t h e r N o n s t e r o i d a l Anti-inflammatory Compounds The n o n s t e r o i d a l a n t i - i n f l a m m a t o r y compounds i n h i b i t t h e p r o d u c t i o n of l a b i l e aggreg a t i o n - s t i m u l a t i n g s u b s t a n c e s (LASS) .23 Most of them have been shown t o i n h i b i t t h e enzyme cyclooxygenase ( s y n t h e t a s e ) t h a t produces the endoperox i d e s PGG, and PGH, from a r a c h i d o n i c a c i d (AA). These endoperoxides a r e Rei n t e r m e d i a t e s i n t h e s y n t h e s i s of p r o s t a g l a n d i n s (PG) E, and F,,.24 c e n t l y Hamberg, Svensson, and Samuelssona6 r e p o r t e d a n o t h e r series of rea c t i o n s i n which endoperoxide PGG, i s a s u b s t r a t e . An enzyme found i n p l a t e l e t s and smooth muscle converted t h e endoperoxides i n t o 2 new f a t t y a c i d d e r i v a t i v e s of which t h e f i r s t i n sequence was v e r y s h o r t l i v e d (about 30 s e c . ) and was a v e r y p o t e n t s t i m u l a t o r of p l a t e l e t a g g r e g a t i o n and smooth muscle c o n t r a c t i o n . T h i s compound (1)i s c a l l e d Thromboxane A, (TBXA,). TBXA, breaks down i n t o Thromboxane B, (TBXB,, o r i g i n a l l y c a l l e d PHD)(2), which i s much more s t a b l e ( s e e Chapter 1 9 ) . I t i s now b e l i e v e d Since n o n s t e r o i d a l anti-inflamt h a t L A S S a s o r i g i n a l l y d e f i n e d i s TBXA,. matory compounds i n h i b i t s y n t h e t a s e , they reduce formation of endoperox i d e s and t h e r e f o r e TBXA, and PG s y n t h e s i s ; t h i s r e d u c t i o n a f f e c t s smooth

CH2

I

CH-CH,

I

b O=P- CH, I 0-

muscle c o n t r a c t i o n and t h e p l a t e l e t "release r e a c t i o n ' ' and second phase p l a t e l e t a g g r e g a t i o n . T h i s new import a n t d i s c o v e r y makes i t p o s s i b l e t o look f o r compounds t h a t w i l l i n h i b i t TBXA, formation, b u t w i l l n o t i n h i b i t PG s y n t h e s i s . Such a compound has been r e p o r t e d , N-0164 (?)."" I t i n h i b i t e d t h e formation of TBXA, from PGG, in vitro. --

Of a l l t h e anti-inflammatory a g e n t s , a s p i r i n i s unique. It a c e t y l a t e s s e v e r a l p r o t e i n s i n the p l a t e l e t . One p r o t e i n i n t h e p a r t i c u l a t e f r a c t i o n , mol. w t . 75,000, i s suggested t o be t h a t which i s r e q u i r e d f o r PGG, b i o ~ y n t h e s i s . ~The ~ e f f e c t of a s p i r i n i s i r r e v e r s i b l e . New normal p l a t e l e t s must be formed and e n t e r t h e c i r c u l a t i o n t o s u p p o r t t h e r e l e a s e r e a c t i o n t o t h e e x t e n t t o cause second phase p l a t e l e t a g g r e g a t i o n (4-7 d a y s ) . Other n o n s t e r o i d a l a n t i - i n f l a m m a t o r y a g e n t s have a n e f f e c t o n l y a s long a s t h e y are p r e s e n t i n t h e c i r c u l a t i o n ( u s u a l l y o n l y 2-4 h o u r s ) and t h e r e f o r e must be g i v e n many t i m e s d a i l y .

82

Sect. I1

-

Pharmacodynamic Agents

F r a n c i s , Ed.

A s p i r i n has been w i d e l y i n v e s t i g a t e d both p r e c l i n i c a l l y and c l i n i Though t h e r e a r e many c l i n i c a l s t u d i e s i n p r o g r e s s Y a 1 no cally.gs sag c o n c r e t e w e l l - c o n t r o l l e d r e s u l t s have a s y e t been completed and r e p o r t e d . The A s p i r i n / S t r o k e S tudy30 conducted through a m u l t i - c e n t e r c l i n i c a l i n v e s t i g a t i o n under t h e a u s p i c e s of N I H has been f i n i s h e d and t h e d a t a i s being compiled. The r e s u l t s 3 1 i n d i c a t e t h e r e i s a s t a t i s t i c a l l y s i g n i f i c a n t b e n e f i c i a l e f f e c t on t h e i n c i d e n c e of t r a n s i t ischemic a t t a c k s (TIA, I n t h i s s t u d y a r e l a t i o n s h i p was sought between a s p i " l i t t l e strokes"). r i n t r e a t m e n t , i n h i b i t i o n of p l a t e l e t a g g r e g a t i o n and p r e v e n t i o n of TIA. A p o s i t i v e r e s u l t would p r o v i d e t h e f i r s t hand evidence f o r a c l i n i c a l l y u s e f u l a n t i t h r o m b o t i c e f f e c t of a s p i r i n . Though many p h y s i c i a n s have p r e s c r i b e d a s p i r i n f o r t h i s t y p e of e f f e c t , even a recommendation o f 1 gram twice weekly f o r p a t i e n t s who have spontaneous p l a t e l e t a g g r e g a t i o n Y 3 aw e s t i l l do n o t know which c o n d i t i o n s would be b e s t b e n e f i t e d by a s p i r i n and which by o t h e r i n h i b i t o r y a g e n t s , Other p r e v i o u s l y r e p o r t e d n o n s t e r o i d a l anti-inflammatory a g e n t s d i s cussed i n r e c e n t l i t e r a t u r e were phenylbu tazone , s u l f inpyrazone , and indom e t h a ~ i n . Work ~ ~ on s e v e r a l p r e v i o u s l y r e p o r t e d p r o p i o n i c a c i d d e r i v a t i v e was ~ ~ e~x t e n d e d , and s e v e r a l new compounds have been r e p o r t e d t o produce i n h i b i t i o n of p l a t e l e t a g g r e g a t i o n such a s : indoprofen3e(&) and and ~ ~ a K - 3 @) 8~8 suprofen3' ( 5 ) . Feprazone (6), 3 7 p y r a z o l i d i n e d i o n e 2, have s t r u c t u r e s more r e l a t e d t o t h a t of phenylbutazone.

&o

S0,Na

a,-

NO

H ,N-

8

-(CH,)a-N(Cak)a

0

O-(CHa)a+i(Cpk).

9

T i l o r o n e (?) a l s o has been r e p o r t e d t o have a n t i - i n f l a m a t o r y a c t i v i t y , 3 * and i n h i b i t s e p i n e p h r i n e and collagen-induced b u t n o t ADP-induced p l a t e l e t aggregati~n.~ R e~c e n t l y Ford-Hutcheson e t a l . r e p o r t e d t h a t t i l o r o n e d i d n o t i n h i b i t pros t a g l a n d i n s y n t h e s i s i n v i t r o i n t h e guinea p i g lung system.q1 Thus t i l o -rone may a l s o e f f e c t t h e p r o d u c t i o n o r a c t i o n of TBXAS

-

In P r o s t a g l a n d i n s and S y n t h e t i c R e l a t e d Compounds p a s t review^^^,'^ p r o s t a g l a n d i n s , n a t u r a l and s y n t h e t i c , have been d i s c u s s e d . O r i g i n a l l y t h e r a t i o n a l e was t o s y n t h e s i z e d e r i v a t i v e s o f PGE,, then t h e most p o t e n t i n h i b i t o r of p l a t e l e t a g g r e g a t i o n known.44 PGE, i s a c t i v e i n v i v o f o r o n l y 5-20 minutes a f t e r i n f u s i o n i s s t o p p e d , and s i d e e f f e c t s l i m i t i t s i n v i v o u s e f u l n e s s a s a p l a t e l e t aggre-

Chap. 9

A n t i t h r o m b o t i c Agents

83 -

MacKenzie

g a t i o n i n h i b i t o r . More r e c e n t l y , i t has been r e p o r t e d t h a t PGD, i s a n even b e t t e r i n h i b i t ~ r . Others ~~ have suggested t h a t s u b s t i t u t i o n i n t h e d i e t of 5,8,11,14-eicosatetraynoic a c i d (TYA) f o r AA would produce i n h i b i t i o n of TBXA, s y n t h e s i s and s t i m u l a t e PGE, s y n t h e s i s . 4 6 The i n h i b i t i o n of TBXA, a n d / o r t h e i n c r e a s e i n s y n t h e s i s of PGE, o r a d d i t i o n of a p r o s t a g l a n d i n t h a t i n h i b i t s p l a t e l e t a g g r e g a t i o n should h e l p c o n t r o l thrombosis. Recently i t has been r e p o r t e d t h a t a new p r o s t a g l a n d i n c a l l e d PGX o r p r o s t a c y c l i n (lo) i s s y n t h e s i z e d from PGG, o r PGH, i n t h e microsomal f r a c t i o n of blood T h i s b i c y c l i c p r o s t a g l a n d i n i s t h e most p o t e n t i n h i b i t o r of p l a t e l e t a g g r e g a t i o n found t o d a t e . J. Vaneea specul a t e s t h a t when p l a t e l e t s bump i n t o h e a l t h y e n d o t h e l i a l c e l l s , they rel e a s e endoperoxides. The h e a l t h y e n d o t h e l i a l c e l l releases a n enzyme t h a t c o n v e r t s t h e endoperoxides i n t o PGX and p r e v e n t s p l a t e l e t aggregation, I n a r e a s of damaged endothelium t h i s enzyme i s a b s e n t , TBXA, i s formed, and p l a t e l e t a g g r e g a t i o n o c c u r s .

P bH

h

It

-

Q l 12

II

-

14 -

13 -

Miscellaneous Compounds S ~ l o c t i d i(g), l ~ ~ an a n t i - s p a s t i c ( v a s o a c t i v e ) a g e n t was r e p o r t e d t o i n h i b i t phospholipid and collagen-induced aggregat i o n . T h i s compound i s s i m i l a r t o dipyridamole b u t more p o t e n t . Halofea h y p o l i p i d e m i ~and ~ ~ u r i c o s u r i c 5 1 a g e n t , has been r e p o r t e d t o nate ( g ) , inhibit platelet S e v e r a l a n t i b i o t i c s of t h e p e n i c i l l i n c a r b e n i c i l l i n type have p r e v i o u s l y been r e p o r t e d t o a f f e c t c o a g u l a t i o n a n d o r p l a t e l e t f u n c t i o n . 5 3 T i c a r c i l l i n (13)was r e p o r t e d t o a f f e c t p l a t e l e t a g g r e g a t i o n i n human p a t i e n t s g i v e n t h e r a p e u t i c l e v e l s of t h e a n t i b i o t i c .5*

s),

a n a t u r a l l y occurring material 2,3-Diphosphoglycerate ( 2 , 3 DPG, t h a t i n c r e a s e s i n people w i t h anemia, was r e p o r t e d t o i n h i b i t t h e second phase of p l a t e l e t a g g r e g a t i ~ n . ~The ~ i n h i b i t o r y e f f e c t was p o t e n t i a t e d by dipyridamole and Vinca minor a l k a l o i d s . r e p o r t e d t h a t n a t u r a l l y o c c u r r i n g polyamines s p e r Rennert e t a l ? midine (Is)and spermine (16)i n h i b i t e d ADP-induced, b u t n o t e p i n e p h r i n e induced p l a t e l e t a g g r e g a t i o n . T h i s i n h i b i t i o n was r e l a t e d t o i n c r e a s e s in t h e s e polyamines i n leukemia, p s o r i a s i s , e t c . , and t o i n c r e a s e s i n b l e e d i n g t i m e s . S y n t h e t i c diamines and polyamines have been r e p o r t e d p r e v i o u s l y t o a f f e c t p l a t e l e t a g g r e g a t i o n . 57 H H,N- (CH, )3 -N- (CH, 1, -N% 15 -

H H H,N-(CH, 13 -N- (CHa )b-N- (CH? 16 -

17 -

84 -

Sect. 11

-

P h a m c o d y n a m i c Agents

F r a n c i s , Ed.

(17)

Ticlopidine was r e p o r t e d t o i n h i b i t ADP-induced p l a t e l e t aggreg a t i o n when administered o r a l l y t o 6 human v o l u n t e e r s . Doses of 2501000 mg/day f o r 1 week gave i n h i b i t i o n s of 40-75%.6e The e f f e c t of d e r i v a t i v e s of imidazo[1,2-~]quinazoline on p l a t e l e t a g g r e g a t i o n was reported.59 Many showed a c t i v i t y , which was n o t l i m i t e d t o 5,6-dihydroimidazo[l,2-c]quinazolines. The most a c t i v e was a 2,3dihydro d e r i v a t i v e (g). These compounds a r e r e l a t e d t o BL-3459, which was p r e v i o u s l y r e ~ i e w e d . 1, 6~0 s 6 1 The i n h i b i t o r y e f f e c t of n i t r o f u r a n t o i n (19)and s e v e r a l a n a l o g s on primary phase of ADP-induced p l a t e l e t a g g r e g a t i o n has been r e p o r t e d . 0 2 The a u t h o r s s u g g e s t t h a t t h e n i t r o group on t h e f u r a n r i n g and the s p e c i f i c arrangement of the two k e t o groups on t h e imidazole r i n g are r e q u i r e d f o r activity. S e v e r a l lactamimides have p r e v i o u s l y been r e p o r t e d t o have i n h i b i t o r y a c t i v i t y on p l a t e l e t a g g r e g a t i o n induced by ADP, e p i n e p h r i n e , c o l l a g e n , thrombin, and serotonin.13,63,64966 A new series of lactamimides was r e c e n t l y r e p o r t e d t o a l s o i n h i b i t p l a t e l e t a g g r e g a t i o n i n v i t r o by t h e same inducers."6 One of t h e s e r i e s , RMI 12,366A ( E ) ,i n h i b i t e d ADP-induced p l a t e l e t a g g r e g a t i o n when given o r a l l y t o guinea p i g s .

19 -

20

Anticoagulants I n the formation of t h e f i b r i n c l o t , t h e r e are numerous s t e p s i n v o l v i n g many d i f f e r e n t procoagulant f a c t o r s . Both i n t r i n s i c and e x t r i n s i c systems, a l o n g w i t h p l a t e l e t f u n c t i o n and blood flow p a t t e r n s , p l a y a r o l e i n t h e formation of t h e f i b r i n c l o t . A t t h e p o i n t of convergence of b o t h i n t r i n s i c and e x t r i n s i c c o a g u l a t i o n systems , t h e f i n a l p r o d u c t i s an enzyme r e f e r r e d t o a s F a c t o r Xa, o r Autoprothrombin C. T h i s enzyme a l o n g P l a t e l e t F a c t o r 3 , and c a l w i t h c o f a c t o r s ( F a c t o r V , phospholipoprotein cium i o n ) c a t a l y z e s t h e formation of the enzyme thrombin from p r o t h r m b i n . Thrombin i n t h e presence of calcium i o n produces t h e f i b r i n c l o t from f i b r i n o g e n . Both F a c t o r Xa and thrombin are s e r i n e c o n t a i n i n g enzymes c l a s s i f i e d a s s e r i n e e s t e r a s e s and p r o t e a s e s . They have a l a r g e p a r t of t h e i r p e p t i d e c h a i n s i n common and have been c o n s i d e r e d as p a r t of t h e same p r e c u r s o r p r o t h r ~ m b i n . ~ ~

-

I n the l a s t s e v e r a l y e a r s , i t h a s become known and more a p p r e c i a t e d t h a t t h e i n v i v o r o l e of h e p a r i n a s a n a n t i c o a g u l a n t may be l e s s important a s an i n h i b i t o r of thrombin and more important a s an i n h i b i t o r of F a c t o r Xa." A c t u a l l y h e p a r i n i s n o t an a n t i c o a g u l a n t , b u t a c o f a c t o r f o r a proa n t i t h r o m b i n 111) i n t h e plasma t h a t n e u t r a l i z e s t e i n (ma g l o b u l i n F a c t o r Xa o r thrombin by m o l e c u l a r combination. Without h e p a r i n t h i s neut r a l i z a t i o n , which i s c o n c e n t r a t i o n dependent, i s slow. I n t h e presence of h e p a r i n , it i s g r e a t l y a c c e l e r a t e d . Heparin has no e f f e c t on thrombin

-

Chap. 9

Antithrombotic Agents

a c t i v i t y i n a p u r i f i e d Fibrinogen-Thrombin-Ca*

MacKenzie

85

system.

Mini-dose o r subcutaneous h e p a r i n was r e p o r t e d on b r i e f l y i n p r e v i o u s reviews .13,14 I n 1975-76 more c l i n i c a l e v a l u a t i o n s have been rep ~ r t e d . ~ ” These ~ ~ smaller doses w i t h lower and more s u s t a i n e d e f f e c t are e f f i c a c i o u s i n v i v o f o r u s e b e f o r e s u r g e r y and o t h e r thrombotic c o n d i t i o n s t o p r e v e n t venous thrombosis and pulmonary embolism. T h i s new form of dosing was advanced by t h e development of new u n d e r s t a n d i n g of mechanism and development of new methods f o r measuring a c t i v i t y . Such t h e r a p y e l i minates t h e n e c e s s i t y of l a b o r a t o r y monitoring and the danger of b l e e d i n g . A b e t t e r understanding of t h e mechanism of a c t i o n of a n t i - v i t a m i n K compounds has been gained. They lower blood l e v e l s of a n a c t i v e prothrombin molecule by p r e v e n t i o n of $+ uptake on t h e g l u t a m i c a c i d r e s i dues i n t h e molecule t h a t a l l o w s Ca binding.72 These compounds a r e d i s ~ ~ new compounds of t h i s type a r e recussed i n a review by O ’ R e i l l ~ . No ported

.

S e v e r a l new compounds have been r e p o r t e d t h a t a f f e c t t h e formation of a f i b r i n c l o t . Aromatic d i a m i d i n e s , such as 2, were r e p o r t e d t o i n h i b i t s e v e r a l p r o t e o l y t i c enzymes i n c l u d i n g thrombin.74 Concanavalin A (a g l o b u l i n p r o t e i n from t h e j a c k bean) i n h i b i t s f i b r i n formation by i n h i b i t i n g t h e l i p o p r o t e i n c o f a c t o r i n the p r o d u c t i o n of thrombin and t h u s dec r e a s i n g t h e r a t e of thrombin production.75 S e v e r a l a n t i b i o t i c s ( p e n i c i l l i n s and c e p h a l o s p o r i n s ) have been r e p o r t e d t o a f f e c t f i b r i n c l o t formahas been shown t o t i o n a s w e l l a s p l a t e l e t f u n c t i o n . Cephalothin d e l a y f i b r i n p o l y m e r i z a t i o n and thus prolong t h e a c t i v a t e d p a r t i a l thrombp l a s t i n t i m e (APTT) and thrombin t i m e t e s t s . 7 6

(11)

The phospholipase A, from YiDera Berus venom was p u r i f i e d and i t s p r o p e r t i e s were determined77 (mol. w t . 13,400 and i s o e l e c t r i c p o i n t 9 . 2 ) . T h i s enzyme hydrolyses t h e a c y l group a t t h e 2 p o s i t i o n of p h o s p h o l i p i d , t h e r e b y reducing t h e procoagulant a c t i v i t y of t h e p h o s p h o l i p o p r o t e i n cof a c t o r i n thrombin p r o d u c t i o n .

A v e r y simple compound, c y a n a t e , was found t o i n h i b i t c o a g u l a t i o n when used f o r t r e a t m e n t of blood from sickle c e l l anemia p a t i e n t s . 7 B It decreased t h e l e v e l s of F a c t o r V and X a c t i v i t y (19 and 36% r e s p e c t i v e l y ) , i n h i b i t e d F a c t o r s VII, IX, and X I ( 6 3 - 7 5 % ) , and i n h i b i t e d thrombin 80%. F i b r i n o l y t i c Agents The p h y s i o l o g i c F i b r i n o l y s i s has p r e v i o u s l y been reviewed .7@ s e o p r o c e s s i n which a f i b r i n c l o t i s d i s s o l v e d and t h e r e g u l a t i o n of t h a t p r o c e s s i s n o t completely understood. The main proenzyme i n t h e blood i s plasminogen, a B, g l o b u l i n , which when a c t i v a t e d i s converted t o t h e f i b r i n l y s i s enzyme plasmin, a B, g l o b u l i n . Plasmin i s u s u a l l y bound by many o t h e r p r o t e i n s and i n a c t i v a t e d i n t h e plasma. I f t h i s d i d n o t o c c u r , p r o -

Sect. I1

86 -

-

Pharmacodynamic Agents

F r a n c i s , Ed.

t e o l y s i s of blood p r o t e i n s and exposed c e l l s would be a c o n t i n u a l occurrance. P h y s i o l o g i c a l l y t h e plasminogen i s trapped w i t h i n t h e c l o t and a n a c t i v a t o r i s r e q u i r e d t o permeate the c l o t t o a c t i v a t e the l y s i s enzyme. A c t i v a t o r s a r e always p r e s e n t i n blood a t v e r y low c o n c e n t r a t i o n s . One of t h e important a c t i v a t o r s comes from t h e e n d o t h e l i a l c e l l s of t h e blood v e s s e l w a l l . The a b i l i t y t o c o n t r o l t h e l e v e l of a c t i v a t o r s o r reduce any abnormal i n h i b i t i o n of l y s i s i s t h e aim of r e s e a r c h toward f i b r i n o l y t i c agents

.

S i n c e l i p i d ( l i p o p r o t e i n ) i n h i b i t l y s i s , a g e n t s t h a t lower l i p i d l e v e l s have a normalizing e f f e c t on decreased f i b r i n o l y s i s a c t i v i t y due t o e l e v a t e d l i p i d s . Even t h e removal of normal l i p i d c o n t e n t from plasma w i t h chloroform i n c r e a s e s l y s i s a c t i v i t y . Vasoactive a g e n t s have p r e v i o u s l y been reportede' t o s t i m u l a t e a c t i v a t o r r e l e a s e r e s u l t i n g i n enhanced l y s i s a c t i v i t y of s h o r t d u r a t i o n . The normal t i s s u e l y s i s a c t i v a t o r s a r e p r o t e i n s . One such a c t i v a t o r from the kidney is urokinase. This enzyme has n o t been found systemically, b u t only i n the kidney and u r i n e . I t d i r e c t l y a c t i v a t e s plasminogen t o plasmin by h y d r o l y s i s of a small p e p t i d e from t h e plasminogen molecule. Other known enzymatic a c t i v a t o r s a r e s t r e p t o k i n a s e and b r i n a s e , which have been discussed elsewhere.82,B3 urokinase can be i s o l a t e d from u r i n e i n small amounts. This has made i t a v e r y expensive m a t e r i a l . Abbott Labor a t o r i e s has developed a t i s s u e c u l t u r e technique i n which kidney c e l l s a r e grown under e x a c t i n g c o n d i t i o n s and urokinase is i s o l a t e d . * * Recently Abbott L a b o r a t o r i e s made a r e q u e s t t o t h e FDA f o r a l i c e n s e t o s e l l t h i s form of u r o k i n a s e c a l l e d Abbokinase@). This process should i n c r e a s e the a v a i l a b i l i t y o f u r o k i n a s e and d e c r e a s e i t s c o s t . Another a g e n t r e c e n t l y r e p o r t e d i s hementerin i s o l a t e d from the B r a z i l i a n blood sucking l e e c h Haemerteria l u t z i . e 6 The a c t i v i t y of t h i s m a t e r i a l i s s i m i l a r t o t h a t of s t r e p t o k i n a s e , i . e . i t i s a c t i v e i n t h e human c l o t system, b u t n o t i n t h e bovine system. There a r e many s y n t h e t i c chemicals t h a t have been r e p o r t e d t o i n c r e a s e l y s i s a c t i v i t y , chloroform being one of t h e e a r l i e s t reported.e" More r e c e n t l y a c i d anti-inflammatoryYB7 a n t i d i a b e t o g e n i c , e e v a s o a c t i v e , e l and d i u r e t i c a g e n t s ,se and a n a b o l i c s t e r o i d s 8 * have been r e p o r t e d t o have some e f f e c t on the l y s i s system. Even whiskey ( a l c o h o l ) has been r e p o r t e d t o enhance l y s i s . e l Hedner, e t a l . e a r e p o r t e d t h a t e t h y l o e s t r e n o l (8 mg/ day) i n c r e a s e d spontaneous l y s i s i n 45 p a t i e n t s w i t h deep venous thrombos i s and pulmonary embolism a f t e r 3 months of treatment. E u p h i l l i n (aminop h y l l i n e ) was r e p o r t e d t o i n c r e a s e abnormally low f i b r i n o l y s i s i n human p a t i e n t s and i n r a b b i t s w i t h venous thrombosis.e3 When 40 mg of f u r o s e mide was i n j e c t e d i . v . i n t o 33 h e a l t h y people, t h e e u g l o b u l i n l y s i s t i m e was shortened.e4 Since t h i s a c t i v a t i o n was n o t found i n p a t i e n t s w i t h uremia and nephrectomy, a n i n t a c t r e n a l system appears necessary. Many s y n t h e t i c anti-inflammatory compounds have p r e v i o u s l y been rep o r t e d t o have " f i b r i n o l y t i c a c t i v i t y . " The e x a c t mechanism of a c t i o n of t h e s e compounds (mostly i n v i t r o ) , i s n o t known. Some have been shown t o i n h i b i t i n h i b i t o r s p r e s e n t i n plasma o r serum,e6 Usually t h e s e compounds

Chap. 9

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produce b i p h a s i c r e s p o n s e s . Types of compounds r e c e n t l y c i t e d i n c l u d e s u b s t i t u t e d i n d o l e s , such a s 2 3 , g e 2-phenethynylcyclopropanecarboxylates , such a s 24,g7 branched benzoic a c i d s , such a s and two seriesgg rel a t e d t o t h e bis(tetrahydroisoquinoline)bisobrin,IE t h e b i s (benzylamine) ( E ) ,and a combination of t h e s e two s t r u c t u r e s , such a s 3.

I t has been r e p o r t e d l o o t h a t i n g e s t i o n of onions enhances f i b r i n o l y s i s . I n an a t t e m p t t o i s o l a t e and c h a r a c t e r i z e t h e a c t i v e components , one a c t i v e component A t 125 mg/ has been i d e n t i f i e d as c y c l o - a l l i i n (s),lol CH, CWH s u b j e c t , i t enhanced l y s i s by 42% i n t h e e u g l o b u l i n l y s i s t i m e t e s t . Steam d i s t i l l e d and e t h e r e x t r a c t e d onions enhanced a c t i v i t y 50-87%. Other s u l f u r c o n t a i n i n g o i l s 28 were a l s o d i s c u s s e d . Since they d i d n o t have a n e f f e c t on t h e i n v i t r o hanging c l o t method b u t were a c t i v e & t h e a u t h o r s suggested t h a t e i t h e r t h e s e s u b s t a n c e s weaken blood c l o t s by opening d i s u l f i d e c r o s s l i n k s between f i b r i n molecules through a d i s u l f i d e exchange r e a c t i o n and t h e r e f o r e r e n d e r them more s u s c e p t i b l e t o n a t u r a l f i b r i n o l y s i s , o r they may s t i m u l a t e l i b e r a t i o n of a c t i v a t o r .

(1

w,

Summary - Many d i v e r s e n o v e l compounds t h a t i n h i b i t d i f f e r e n t p l a t e l e t f u n c t i o n s show g r e a t promise, n o t o n l y f o r p o t e n t i a l a n t i - t h r o m b o t i c a g e n t s , b u t a l s o f o r more s p e c i f i c e f f e c t s on p r o s t a g l a n d i n a n d / o r thromboxane A, s y n t h e s i s , and s e r o t o n i n o r calcium uptake and r e l e a s e . Many a c t i v e compounds can be used a s t o o l s i n t h e s e a r c h toward a more complete unders tending of the p h y s i o l o g i c i n t e r a c t i o n s of t h e h e m o s t a t i c mechanisms. T h i s b e t t e r understanding would l e a d t o t h e development and use of more p o t e n t and s e l e c t i v e s y n t h e t i c compounds i n t h e i n h i b i t i o n of p l a t e l e t a g g r e g a t i o n and f i b r i n formation, and i n t h e enhancement of f i b r i n o l y s i s I t is hoped t h a t f o r t h e c o n t r o l of b o t h a r t e r i a l and venous thrombosis. some o f t h e s e new compounds w i l l be e v a l u a t e d c l i n i c a l l y i n t h e n e a r future

.

Acknowledgement: I wish t o thank t h e l i b r a r y s t a f f of M e r r e l l - N a t i o n a l L a b o r a t o r i e s , e s p e c i a l l y Mrs. Emily Rahe, f o r t h e i r h e l p i n t h e l i t e r a t u r e s e a r c h f o r t h i s review.

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Sect. I1

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References I.

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Thomas, S p r i n g f i e l d , T l l . , 1963, p. 261-267. 88. R.N. B a n e r j e e , \ r . Kumar, S.R. Rao, A.L. S a h n i , M. Arya, and J . Bardhan, D i a b e t o l o g i a 11. 105 (1975). 89. H.D. Bruhn, Thrombos. D i a t h e s . Haemorrh. ( S t u t t g . ) 22, 672 (1975). 9 0 . J.F. Davidson, 6.A. McDonald, and J . N . Conkie, S y n t h e t i c F i h r i n o l y t i c Thrombolytic Agents, K . N . v o n k a u l l a and J . F . Davidson, E d . , C . C . Thomas, S p r i n g f i e l d I l l . , 1975, p.328. 45. 46. 47 * 48. 49.

136,

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.

71,

0

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90 91. 97. 93.

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2,

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Pharmacodynamic Agents

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917 ( 1 9 7 5 ) .

U. Hedner, I . M . N i l s s o n , S. I s a c a o n , B r i L . Med. J. 2, 729 ( 1 9 7 6 ) . E.K. Bogomolova, A.A. Sukhaiiov, L.P. YasLrebova, and L.A. Yampolskaya, K l i n i c h . Med.

53,

51 ( 1 9 7 5 ) . 9h. R.D. Bruhn, C. F r i c k e , J. S c h m f t t , and tl. N i c d e r m a y e r , Med. K l h . 7 0 , 1 1 2 5 (1975). 9 5 . L’. Raunigarlen, L . I . P r i e s t r r , D.W. S t i l l e r , and A.E.W. Duncan, Thrombos. D i a t h e s . Haemorrh. ( S t u L t g . ) 3, 495 ( 1 9 7 0 ) . 96. H . I t i i o n , 11. D e v o g e l a e r , E . VanDurnie, M . Descanips, R. R r o t c l l e , R. C h a r l i e r , and M . C o l o t , l u r . J . Med. Cliem. lo, 276 ( 1 9 7 5 ) . 9 7 . M. Yoshimotc, K . N . v o n K a u l l a , and C . Hmscli, J. Med. Cliem. g ,9 5 0 ( 1 9 7 5 ) . 98. L.J. L o e f f l e r and S.B. D e l o i c f i c e , J. Pharm. S c i . 64, 117@ (1975). 9 9 . L . J . F l i e d i i e r , J r . , M . J . Myers, J . M . S c h o r , and I . J . P a c h L e r , J o Med. Cheii.. 19, 202 ( 1 9 7 6 ) . 1 0 0 . I . S . Menon, R.Y. K e n d a l , H . A . Dewar, and D . J . N e w e l l , B r i t . Med. J. 3, 351 ( 1 9 6 8 ) . 101. K.T. A u g u s t i , M.E. Benalm, H.A. Dewar, and R . V i r d e n , A t h e r o s c l e r o s i s 2, 409 ( 1 9 7 5 ) .

Chapter 10. Agents Affecting Gastrointestinal Functions Christopher A. Lipinski and Lyle A. Hohnke, Pfizer Inc., Groton, Conn. 06340

Introduction - Gastrointestinal drug research continues to highlight the development o f histamine (H2) receptor antagonists for use in treating peptic ulcer disease and other secretory related disorders. The introduction o f Tagamet (cimetidine), the first clinically acceptable histamine-H2 blocker, culminates the search for a safe and effective gastric acid antisecretory drug. Prostaglandin antisecretory drug development continues t o be hindered by the presence o f side effects. However, commercial interest in this class o f agents continues and improved target selectivity could lead to rapid clinical advances. Developments in understanding histamine receptors and the pharmacology o f histamine antagonists along with progress in prostaglandin research are the focus of this review. The gastric acid antisecretory structure-activity relationships and clinical testing of gastrointestinal peptide hormones are also updated with a view toward avoiding overlap with other excellent summaries. Detailed reviews of the gastrointestinal actions o f prostaglandin^'^^^^^^)^ and gut hormones617 have been published recently along with the proceedings o f a second international symposium on H2 antagonists.' An excellent overview o f the pharmacological and clinical profile of carbenoxolone was published9 recently and will not be treated in this chapter.

'

Histamine Agonist activity - Theoretical molecular orbital calculations" along with X-ray data' indicate that histamine monocation exists as the NT-H tautomer while histamine free base exists as the N"-H tautomer. Deprotonation o f the ethylamine side chain nitrogen is electronically equivalent t o interaction of the charged side chain nitrogen with an anionic binding site and leads t o a proton shift from the NT-H t o the N'-H tautomeric form o f the imidazole ring.12 In a model proposed for H2-agonist activity,l21l3 histarnine NT-H tautomer binds to a receptor site via hydrogen bond donor and acceptor bonding. Interaction o f the charged ethylamine side chain nitrogen with an anionic site triggers the tautomer change and leads to H a onist activity. Removal o f the tautomeric H by methylation o f either the NT-H or Nn-H histamine t a u t ~ m ~ r or f 4 replacement of the imidazole ring by nontautomeric ring systems such as pyridine or thiazole13 leads to loss of H2 agonist activity. Conversion o f the histamine side chain Nuamino group into a guanidine moiety leads t o a molecule having the correct NT-Himidazole ring tautomer for receptor binding but having a side chain moiety markedly different in basicity and geometry from that o f histamine. This compound and i t s homo side chain analog16 are partial agonists at the histamine Hyreceptor and at high doses inhibit histamine-stimulated acid secretion. Methylation o f the histamine imidazole ring at C4 or bismethylation at C4 and Na leads t o compounds that exist predominantly as NT-H tautomers at physiological pH and have selective histamine H2-agonist activity. 14

..

H7 histamine monocation

L

H7 n = 2 , 3 partial

H2 agonists

Tolazoline and the structurally related imidazoline tetrahydrozoline may be H2 receptor agonists since tolazoline-induced acid stimulation in the dog can be blocked by burimamide, metiamide and cimetidine' and both imidazolines induce characteristic H2-agonist effects on isolated guinea pig atria preparations which can be blocked by metiamide.' Clonidine, like the imidazolines, has a-adrenergic agonist activity

92

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Francis, Ed.

Pharmacodynamic Agents

along with H2 agonist activity" and stimulates acid secretion in anesthetized rats. However in conscious rats clonidine and other structurally related imidazolines and pyrrolidines possess gastric acid antisecretory and antihypertensive activit The antisecretory effect of clonidine in rats may be explained by a centrally mediated H2-agonist effect which results in acid inhibition. In anesthetized animals the central effect is masked and only acid stimulation caused by a peripheral H agonist effect i s observed. Among a series of and 2-(2,6-dichlorophenylimino)pyrrolidine clonidine analogs 2-(2,6-Dimethylphenylimino)imidazoline (II) are2xirticularly effective antisecretory agents in conscious rats with minimal antihypertensive activity.

o l

6)

4 / \

X = CI, Y = N H Clonidine X=CH3,Y=NH(I) X = CI, Y = CH2 (11)

N64 c,i n 67,68 3-edeine 6 h i 0 m y c i n , 172 ~ ~have appeared. 0

I

C-peptide

0

H

C-poptide

Chap. 14

Biosynthesis of Antibiotics

Corcoran

135 -

The phenoxazinone r i n g system o f t h e ACTINOMYCINS (19)i s a p p a r e n t l y formed f r o m k y n u r e n i n e and 3-oxykynurenine w i t h m e t h y l a t i o n t a k i n g p l a c e a t a l a t e s t a g e 73. Reports o n amino a c i d v a r i a t i o n and t h e mechanism o f b i o s y n t h e s i s o f t h e D-amino a c i d s p r e s e n t i n t h e s i d e c h a i n s c o n t i n u e t o appear 74 375 376,77,78. I'DOLMYcIN (3) i s formed f r o m p y r u v a t e , and two enzymes a c t i v e i n i n i t i a l stages o f i t s b i o s y n t h e s i s have been s t u d i e d . They a r e a transaminase and a c m e t h y 1 t r a n s f e r a s e . The h y p o t h e t i c a l r o u t e t o i n d o l m y c i n i s by i n d o l e pyruvate, 3-methyl - i n d o l e p y r u v a t e , i n d o l m y c e n i c a c i d (reduced a l p h a 0x0 group) and f i n a l l y indolmyci'n which p r o b a b l y t a k e s i t s a m i d i n e group f r o m an a r g i n i n e m o l e c u l e 79. The c l o s e l y r e l a t e d [ p y r r o l o (1,4) benzodiazepines] 8 1 8~2 a n t i t u m o r a n t i b i o t i c s , anthram c i n toma m c i n n u r e n i n e p a t way , and s i b i r o m c i n a r e formed f r o m t r y p t o p h a n ( v i a t e t y r o s i n e an m e t h i o n i n e - d e r i v e d methyl groups

+

+y-h

The b i o s y n t h e s i s o f t h e BETA LACTAM ANTIBIOTICS ( p e n i c i l l i n s o r enams and c e p h a l o s p o r i n s o r c e hems) has been s t u d i e d i n t e n s i v e l y . The s y n t e s i s o f t h e b a s i c systems by + Cep i-a l o s p o r i u m species which make p e n i c i l l i n N (21) and c e p h a l o s p o r i n C (22)seems t o be f r o m a l i n e a r t r i p e p t i d e , d e l t a L-aamino-adipyl-L-cysteinyl -D-val i n e (23) 8 3 . S t u d i e s w i t h s p e c i f i c a l l y trit i a t e d l ' + C - l a b e l e d t r i p e p t i d e and a l s o w i t h t h e same c o n t a i n i n g s t e r e o s p e c i f i c a l l y l a b e l e d v a l i n e ( c h i r a l methyl groups) have s e v e r e l y r e s t r i c t e d t h e m e c h a n i s t i c p o s s i b i l i t i e s f o r t h e c y c l i z a t i o n steps. Among o t h e r observat i o n s , t h e Val i n e methyl groups a r e i n c o r p o r a t e d w i t h o u t r a n d o m i z a t i o n 8 3 , 84. Presumably t h e r e i s a common pathway i n p a r t f o r t h e f o r m a t i o n b o t h o f t h e penam and cephem a n t i b i o t i c s . Model chemical r e a c t i o n s a r e a t l e a s t c o m p a t i b l e w i t h t h e p o s s i b i l i t y t h a t t h e beta l a c t a m system i s formed by e i t h e r n u c l e o p h i l i c a t t a c k o f amide n i t r o g e n ( f r o m t h e v a l i n e ) on a t h i o a l d e h y d e ( c y s t e i n y l group) o r a l t e r n a t e l y by o x i d a t i o n o f t h e same amide n i t r o g e n f o l l o w e d b y i t s n u c l e o p h i l i c d i s p l a c e m e n t by an a n i o n generated a t t h e beta carbon o f t h e c y s t e i n e r e s i d u e 85. I t i s c l e a r t h a t t h e 0 - c o n f i g u r a t i o n o f t h e aminoadipyl s i d e c h a i n i s generated a t a l a t e stage i n t h e b i o s y n t h e s i s s i n c e t h e LLD t r i p e p t i d e i s a r e q u i r e d p r e c u r s o r . L a t e stages i n t h e b i o s y n t h e s i s o f t h e cephem a n t i b i o t i c s a l s o have been s t u d i e d and seemingly deacetoxy c e p h a l o s p o r i n C i s formed f i r s t , f o l l o w e d by o x i d a t i o n t o d e s a c e t y l c e p h a l o s p o r i n C and t h e n ac e t y l a t i o n t o c e p h a l o s p o r i n CE6. NH,,

f-

R'

H

136 -

Sect. I11

-

Chemotherapeutic Agents

Whitfield, Ed.

Recent r e p o r t s c o n c e r n i n g b i o s y n t h e s i s o f p u r i n e and p y r i m i d i n e - c o n t a i n i n g a n t i b i o t i c s deal w i t h PUROMYCIN (an O-methyl t r a n s f e r a s e t h a t promotes t h e l a s t s t e p i n t h e b i o g e n e s i s o f puromycin by m e t h y l a t i n g t h e t y r o s y l p h e n o l i c group) 8 7 and t h e POLYOXINS (24). The l a t t e r a r e an i n t e r e s t i n g f a m i l y o f a n t i b i o t i c s c o n t a i n i n g thymine o r d e r i v a t i v e s o f i t w i t h o x i d i z e d methyl groups. A novel pathway f o r t h e b i o s y n t h e s i s o f t h e thymine m o i e t y has been proposed one t h a t i s independent o f t h e well-known thymid l a t e synthetase. The a l d o n i c sugar r e s i d u e i s d e r i v e d f r o m L-glutamate 9i and t h e unique a c i d , 3 - e t h y l idene-L-azetidien-2-carb o x y l i c a c i d p r e s e n t i n p o l y o x i n s A,F,H, and K comes from L - i s o l e u c i n e go. Presumably carbamoyl phosphate i s a l s o r e q u i r e d f o r t h e s y n t h e s i s . 0

I1

I .-C+J,") I II HO-CH

NHZ-CH

23

HOOH iOH ~ I

~ H ~ O - C - N H ~

II

ribose

(3) has appeared These p y r a z o l o p y r i m i d i n e a n t i t u m o r a n t i b i o t i c s a r e made f r o m l y s i n e (two carbons), glutamate, and r i b o s e . A t l e a s t t h r e e n i t r o g e n s i n c l u d i n g those i n the pyrazole r i n g a r e derived from l y s i n e .

A s e r i e s of papers on t h e b i o g e n e s i s o f t h e FORMYGINS

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9 2 93,94, ~

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6. 7. 8.

g.,

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Chap. 14

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30. 31. 32. 33. 34. 35. 36.

Biosynthesis of Antibiotics

137 -

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.

.

-

(z),

(E), 4,

37.

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Sect. 111

38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

61. 62.

63. 64 65. 66. 67. 68. 69. 70. 77.

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J. W. Westley, W. Benz, J. Donahue, I:. H. Evans, Jr. C. S c o t t , A. Stempel, and J. Berger, J. A n t i b i o t . 27, 744, 1974. J. W. Westley, R. H. Evans, Jr., G. Harvey, R. G. P i t c h e r , D. L. Pruess, A. Stempel and J. Berger, J. A n t i b i o t . 27, 288, 1974. H. Pape, Arch. M i k r o b i o l . 82, 254, 1972. H. Pape, Arch. M i k r o b i o l . 85, 233, 1972. P. S t a h l and H. Pape, A r c h r M i k r o b i o l . 85, 239, 1972. J. F. M a r t i n and L. E. McDaniel B.B.A. 186, 1975. Z. Vanek, J. Tax, J. C u d l i n , M. Blumauerova, N. Steinerova, J. Mateju, I . Komersova, and K. S t a j n e r , Proc.-Int. Syrnp. Genet. I n d . Microorg., 2ND, 473, 1976. P. M i l l e r and J . T Hash, Methods Enzymol., 43, 603, 1975. P. M i l 1e r and J. H. Hash, Methods Enzymol ,, 606, 1975. M. S. A l l e n , A. M. Becker and R. W. Rickards, Aust. J. Chem. 29, 673, 1976. K. T. Suzuki, and S. Nozoe, Bioorg. Chem. 3, 72, 1974. R. C. P a u l i c k , M. L. Casey and H. W. W h i t l o c k , J. Am. Chem. SOC. 98, 3370, 1976. A. Jones and L. C. V i n i n g , Can. J. M i c r o b i o l . 22, 237, 1976. M. H. G. Munro, M. Taniguchi, K. L. R i n e h a r t , F., and D. G o t t l i e b , Tetrahedron L e t t . (31), 2659, 1975, L. A. Kominek and O T K . Sebek, Dev. I n d . M i c r o b i o l . 15, 60, 1974. L. A. Kominek and H. F. Meyer, Methods Enzymol. 43, m 2 , 1975. A. M. Nadzan and Kenneth L. R i n e h a r t , Jr., J. Am. Chem. SOC. 98, 5012, 1976. G. Murphy and F. Lynen, Eur. J. Biochem. 58, 467, 1975. U. Hornemann, J. P. Kehrer, C. Nunez, R. R a i n e r i and Y. K. Ho, Dev. Ind. M i c r o b i o l . 15, 82, 1974. U. Hornemann, J. P. K x r e r , and J. H. Eggert, J. Chem. SOC., Chem. 1045, 1974. Commun. U. Hornemann, J. P. Kehrer, C. S. Nunez, and R. L. R a n i e r i , J. Amer. Chem. SOC. 96, 320, 1974. U. HornemanE and J. H. E g g e r t J. A n t i b i o t , 28, 841, 1975. U. Hornemann, and M. J. Aikman, J. Chem. SocTChem. Commun. (?), 88, 1973. P. Pfaender, D. S e c h t , G. H e i n r i c h E. Schwarz, E. Kuhnle, M. and M. M. S i m l o t , FEB! L e t t . 32, 100, 1473. H. Rieder, G, H e i n r i c h , E. Breuker, M. M. S i m l o t , and P. Pfaender Methods Enzymol., 43, 548, 1975. A. L. DeMain, and D. I. C. Wong, I n t . Symp. Genet. I n d . Microorg., 2ND ed., K. D. MacDonald, Academic Press, New York, N. Y., 1976. T. Zimmer and S. G. Laland, Methods Enzymol. 43., 567, 1975. H. Paulus, Methods Enzymol. 43., 579, 1975. S. G. Lee and F. Lipmann, MeBods Enzymol. p3., 585, 1975. 0. J. Hook and L. C. V i n i n g , Can. J. Biochem. 51, 1630, 1973. D. J. Hook and L. C. Vining, J. Chem. Soc., ChG. Comun. 185, 1973. Z. Kurylo-Borowska, Biochim. Biophys. Acta 339, 31, 1975. Z. Kurylo-Borowska, Methods Enzymol. , 43, 5 x 1975. J . H. C a r t e r 11, R. H. Du Bus, J. R. Dyer, J. C. Floyd, K. C. Rice, 13, 1227, 1974. and P. D. Shaw, B i o c h e m i s t r y -

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a

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139

Corcoran

J. H. C a r t e r , 11, R. H. Du Bus, J. R. Dyer, J. C. Floyd, K. C. Rice, and P. D. Shaw, B i o c h e m i s t r y 13, 1221, 1974. R. B. Herbert, Tetrahedron LeE.(5J), 4525, 1974. A. B. Mauger, and Edward Katz, Arch. Biochem. Biophys. 181, 1976. 9, T. Yajima, K. Mason, and E. Katz, A n t i m i c r o b . Agents Chemother. 224, 1976. T. Yajima, K. T. Mason, and E. Katz, A n t i m i c r o b . Agents Chemother. 7, 773. 1975. W. 5. May, Jr. J. V. Formica, A n t i m i c r o b . Agents Chemother. 5, 296, 1974. J. E. Walker, S . O t a n i and D. Perlman, FEBS L e t t . 20, 162, 1972. M. K. Speedie, U. Hornemann, and H. G. Floss, J. B z l . Chem. 250, 7819, 1975. C. J. Chang, H. G . Floss, L. H. H u r l e y , and M. Zmijewski, J. Org. Chem. 41 , 2932, 1976. L. H u r E y , N. Das, C. G a i r o l a , and M. Zmijewski, Tetrahedron L e t t . 1419, 1976. L. H. H u r l e y , M. Zmijewski and C.-J. Chang, J. Amer. Chem. SOC., 97, 4372, 1975. P. A. Fawcett, J.J. Usher and E. P. Abraham, I n t . Symp. Gent. Ind. Microorg. , 2ND, 129, 1976. N. Neuss, Methods Enzymol 43, 404, 1975. A. I . S c o t t , S . E. Yoo, S.-K Chung, and J . A. Lacadie, Tetrahedron 1137, 1976. Lett. M. L i e r s c h , J. Nuesch, and H. J . T r e i c h l e r , Proc.-Int. Symp. Genet. I n d . Microorg., 2ND, 779, 1976. B. M. P o g e l l , Methods Enzymol , 43, 508, 1975. 141, K. Isono, and R. J. S u h a d o l n i k , A r c h . B ochem. Biophys. 1976. S. Funayama, and K. Isono, B i o c h e n i i s t r y 14, 5568, 1975. 14, 2392, K. I s o n i , S. Funayama, and R. J. Suhado z k , B i o c h e m i s t r y 1975. K. Ochi, S . K i k u c h i , S . Yashima, and Y. Eguchi, J. A n t i b i o t , 29, 638, 1976. K. Ochi, S. Yashima, Y. Eguchi, J . , A n t i b i o t . 28, 965, 1975. K. Ochi, S. Iwamoto, E. Hayase, S. Yashima, a n T Y . Okami, J. A n t i b i o t . 27, 909, 1974. T. Wakashiro, M. H o r i , and H. Umezawa, J. ki3~a!?b1.

176,

(g),

.

(E),

173,

&t!buld!?g:

Chapter 15. C. C. Wang and

A n t i p a r a s i t i c Agents

M. H. F i s h e r , Merck Sharp & Dohme Research L a b o r a t o r i e s , Rahway, New Jersey

General - A compendium o f chemotherapeutic agents f o r p a r a s i t i c p r o t o z o a and h e l m i n t h s o f dogs and c a t s was p u b l i s h e d . 1 H o s t - p a r a s i t e r e l a t i o n s h i p s were reviewed. 2 The proceedings o f an i n t e r n a t i o n a l conference on c h e m t h e r a p e u t i c agents f o r p a r a s i t i c diseases appeared.3 Nucleoside analogs as a n t i p a r a s i t i c agents were reviewed.4 Malaria

-

Reviews appeared on chemotherapy and p r o p h y l a x i s 5 and drug r e s i s CDC m a l a r i a g u i d e l i n e s 7 were p u b l i s h e d . A t e s t was d e s c r i b e d u s i n g PZasmodiwn cynomoZgi i n rhesus monkeys.8 A method f o r p r e d i c t i n g antima1a r i a 1 a c t i v i t i e s o f a r y l amidinoureas f r o m t h e i r p h y s i cochemi c a l p r o p e r t i e s was d e ~ c r i b e d . 9 ~ 1 0M e f l o q u i n e (WR142,490) I was shown t o be a c t i v e a g a i n s t c h l o r o q u i n r e s i s t a n t s t r a i n s of PZasmodiwn faZciparwn i n man f o r t r e a t m e n t l l o r p r o p h y l a x i s 1 2 u s i n g s i n g l e o r a l doses o f 1 o r 1.5 g.

HO-'o

The s y n t h e s i s o f new u i n o l i n e methanols,l3,14 phenanthrene m e t h a n o l ~ , 1 5 ~ and 1 ~ naphthalene methanolsl continued, aimed a t f i n d i n g compounds o f h i g h a c t i v i t y and low p h o t o t o x i c i t y . A g a i n s t PZasmodium berghei i n mice, I 1 was a c t i v e a t 2.5 mg/kg and p h o t o t o x i c a t 100 mg/kg; I 1 1 was t h e most a c t i v e o f t h e b e n z o q u i n o l i n e s cF3 b u t i t s p h o t o t o x i c i t y was n o t determined. Among t h e phenanthrene methanols, IV was a c t i v e a t 20 mg/kg and CF3 1 V a t 1.25 mg/kg. The naphthalene methanol V I was c u r a t i v e a g a i n s t P. berghei i n mice a t 10 mg/kg. P h o t o t o x i c i t y appeared t o be absent from t h e s e r i e s . H

CHOHCH~NBU~

CHOHCHzNX2

R7 R8

R6

I 1 RE I11

= 4-ClPh; R3 = F R6 = R8 = C1; R7 = H R2 = 2,4-Me2Ph; R3 = CH3 R6 = C1; R7Rg = benzo

CHOHCH2NBu2 (-1

VI

C1

R4

IV R2

= R4 = C1; R6 = SCH3 R7 = H; X = Bu V R2 = R4 = CF3; R6 = R7 = C1 X = Pr

The diastereomers o f phenanthrene methanols and q u i n o l i n e methanols showed s t r i k i n g d i f f e r ences i n a n t i m a l a r i a l a c t i v i t y , l 8 p o s t u l a t e d t o r e l a t e t o t h e d i s t a n c e between oxygen and t h e nona r o m a t i c n i t r o g e n atoms. No e x c i t i n g new s t r u c t u r e s were d e s c r i b e d b u t a c t i v i t y was found i n (4-oxo-2-oxazolin-2-yl)piperazines~~and 1,2,4triazines.20

Chap. 15

Antiparasit ic Agents

Wang, Fisher

14 1 -

Ribosomes o f PZasmodiwn k n o w l e s i were i s o l a t e d and c h a r a c t e r i z e d r e c e n t l y b y Sherman e t a l . 2 1 These ribosomes sedimented i n t h e 80s range and c o u l d be d i s s o c i a t e d i n t o 60s and 405 s u b p a r t i c l e s . The r i b o s o m a l RNA had a low % G+C o f 37% and had s i z e s o f 24.2s and 16.6S.22 The ribosomes demonstrated h i g h a c t i v i t y i n p o l y ( l J ) - d i r e c t e d s y n t h e s i s o f p o l y p h e n y l a l a n i n e and were s t r o n g l y i n h i b i t e d b y lO-4M o f n u c l e o c i d i n , c h l o r t e t r a c y c l i n e , e t h i d i u m , puromycin, c y c l o h e x i m i d e o r b e r e n i l . 2 3 S i m i l a r s t u d i e s have been a l s o c a r r i e d o u t on PZasmodiwn Zophurae, and s i m i l a r p r o f i l e o f d r u g s e n s i t i v i t i e s were demonstrated. 24 Most o f t h e well-known a n t i m a l a r i a l drugs t e s t e d showed no s i g n i f i c a n t i n h i b i t o r y a c t i v i t y i n t h i s i n v i t r o assay. However, c l i n d a m y c i n , a halogenated l i n c o m y c i n analog, has been shown t o be a c t i v e a g a i n s t d i f f e r e n t species o f m a l a r i a i n c l u d i n g s e v e r a l c h l o r o q u i n e - r e s i s t a n t s t r a i n s of P. falciparm.25,26,27 F u r t h e r s t u d i e s by Powers e t aZ.28 on P. k n o w l e s i i n rhesus monkeys i n d i c a t e d t h a t c l i n d a m y c i n and i t s N-demethyl-4'-pentyl analog caused d i s i n t e g r a t i o n and disappearance o f t h e p a r a s i t e ribosomes. The phenomenon c o u l d be i n t e r p r e t e d as t h e mode o f a c t i o n o f t h e drug. Since l i n c o m y c i n i s n e i t h e r e f f e c t i v e c l i n i c a l l y a g a i n s t m a l a r i a 2 8 n o r a c t i v e a g a i n s t e u k a r y o t i c microsomal ribosomes ,29 t h e a c t i v i t y o f c l i n d a m y c i n suggests t h e p o s s i b i l i t y o f uncovering drugs w i t h a narrow spectrum o f s e l e c t i v i t y such t h a t o n l y p r o t e i n s y n t h e s i s b y p l a s modial ribosomes would be i n h i b i t e d . Such an e x p e c t a t i o n has been buoyed by t h e a c t i v i t y a g a i n s t c h l o r o q u i n e - r e s i s t a n t m a l a r i a d i s c o v e r e d i n minoc y c l ine,30,31 a d e r i v a t i v e o f t e t r a c y c l i n e . Jacobs and Koontz32 r e c e n t l y examined t h e r a t e o f development o f r e s i s t a n c e t o c l i n d a m y c i n o r m i n o c y c l i n e i n P. berghei and found i t much s l o w e r t h a n t o c h l o r o q u i n e , q u i n i n e o r p y r i methamine. Clindamycin, doxymycin, t e t r a c y c l i n e and s p i r a m y c i n a l l showed causal p r o p h y l a c t i c a c t i v i t y a t t h e t i s s u e stage o f P. berghei development. 33 The continuous c u l t i v a t i o n o f P. f a l c i p a r m i n human e r y t h r o c y t e s has been r e c e n t l y accomplished b y Trager and Jensen.34 The p a r a s i t e , o r i g i n a l l y d e r i v e d from an i n f e c t e d Aotus t r i v i r g a t u s monkey, propagated o v e r 100 m i l l i o n t i m e s by t h e a d d i t i o n of human e r y t h r o c y t e s a t 3 t o 4-day i n t e r v a l s . The p a r a s i t e c o n t i n u e d t o reproduce a s e x u a l l y w i t h a g e n e r a t i o n t i m e o f about 48 hours and remained i n f e c t i v e t o Aotus. T h i s success may c o n t r i b u t e t o expanded e f f o r t s i n immunological, chemotherapeutic and b i o c h e m i c a l studies o f malaria. H e l m i n t h i a s i s - Reviews appeared on h e l m i n t h i a s i s 3 5 and l a b o r a t o r y methods o f s c r e e n i n g f o r a n t h e l m i n t i c s . 36 Two p u b l i c a t i o n s p o i n t e d o u t t h e r e s i s t a n c e o f f i e l d s t r a i n s 3 7 and s e l e c t e d 1 i n e s 3 8 o f Haemonchus c o n t o r t u s t o benzimidazoles, morantel and l e v a m i s o l e . A method f o r i n d u c i n g H. c o n t o r t u s i n f e c t i o n s i n t h e r a b b i t was described.39 A new, h i g h l y p o t e n t benzimidazole, albendazole40 V I I , was r e p o r t e d t o be a c t i v e a g a i n s t nematodes, cestodes and trematodes. A s i n g l e o r a l dose of 2 . 5 t o 10 mg/kg i n sheep and c a t t l e e l i m i n a t e d 94-100% o f these organisms. I t was a l s o e f f e c t i v e i n chickens, dogs and horses. Oxfendazole V I I I was e v a l u a t e d i n sheep41 and c a t t l e 4 2 showing 92-100% a c t i v i t y a g a i n s t nematodes. Flubendazole I X was r e p o r t e d t o be a c t i v e a g a i n s t e a r l y and l a t e encysted l a r v a e o f T r i c h i n e Z l a cattle,47 s p i r a l i s i n pigs.43 Fenbendazole X was t e s t e d i n sheep,44,45,46

142

Sect. I11

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Chemotherapeutic Agents

Whitfield, Ed.

and p i g s . 4 8 ~ 4 9 Broad a c t i v i t y a g a i n s t nematodes was r e p o r t e d i n a l l cases. A c t i v i t y i n man a g a i n s t Ascaris , hookworm and Trichuris was r e p o r t e d . 50 Mebendazole X I was r e p o r t e d t o be a c t i v e a g a i n s t Aspicularis tetraphera and Syphacia obvelata i n f e c t i o n s i n m i ce,51 HymenoZepis diminuta i n TriboZiwn confuswn52 and developing l a r v a e o f Dirofilaria i m i t i s i n dogs.53 Oxibendazole X I 1 was t e s t e d i n c a t t l e 5 4 and horses55 showing 84-100% e f f i c a c y a g a i n s t mature p a r a s i t e s .

'0 '9

Newly d e s c r i b e d i m i d a z o t h i a z o l e s i n c l u d e d n i t r a m i s o l e X I 1 1 5 6 and b u t a m i s o l e X I V 5 7 ~ 5 8which was 98-100% e f f e c t i v e a g a i n s t Trichuris vuZpis i n N H dogs, o r a l l y o r subcutaneously. Reduced a c t i v i t y R = S C H ~ C H ~ C H ~ was found f o r o t h e r worms. 0 X I 1 1 R = 3-NO2-CgH4 NHCOOCH3

VII

4

X I V R = 3-(CH3)2CHCONH-C6H4

V I I I R = S-CgHg

p - T o l u o y l c h l o r i d e phenylhydrazone (TCPH) XV was found t o be e f f e c t i v e a g a i n s t nematodes and cestodes o f sheep.59 A t h i a z o l i n e X V I was r e p o r t e d t o be o r a l l y e f f e c t i v e a g a i n s t sheep nematodes.60 FosIt p i r a t e X V I I e l i m i n a t e d Echinococcus granulosus and X I R = C-CgHg Taenia hydatigena i n f e c t i o n s f r o m dogs.61-62 'I1 = 0CH2CH2CH3 N i t r o s c a n a t e X V I I I showed s i m i l a r a c t i v i t y . 6 3 ~ 6 4 Other tapeworm compounds r e p o r t e d were p r a z i q u a n t e l (Embay 8440) X I X e f f e c t i v e i n dogs and cats65 and mpbendazole e f f e c t i v e i n experimental i n f e c t i o n s 6 6 p a r e n t e r a l l y . A n t i b i o t i c S15-1 (SQ21,704), a member o f t h e s t r e p t o t h r i c i n f a m i l y , e l i m i n a t e d tapeworms f r o m c a t s , dogs and sheep.

0I t I X R = C-p-F-C6H4 X R = S-CgHg 0

xv I

xv

XVIII

XVII

:u N'

'd

A novel d i s u l f o n a m i d e XX was r e p o r t e d t o e l i m i n a t e mature and immat u r e FascioZa hepatica from sheep and c a t t l e a t o r a l or subcutaneous doses o f 2.5 t o 30 mg/kg and showed no gross t o x i c symptoms up t o 400 mg/kg i n ~ h e e p . 6 8 ~ 6 9 Diamphenethide X X I was i n a c t i v e a g a i n s t F. hepatica i n t h e r a t , presumably because i t was n o t d e a c e t y l a t e d t o t h e f r e e amine.70 4,4'-Diam i n o d i p h e n y l s u l f i d e X X I I and i t s d i a c e t y l d e r i v a t i v e were 62-100% e f f e c t i v e a g a i n s t 3-week-old F. hepatica i n f e c t i o n s i n sheep. 71

Chap. 15

Antiparasitic Agents

Wang, Fisher

c1 I

c12c=c

PYNH2

H2NS02 w S O 2 N H 2

xx

CH 3CONH 0

0

0

NHCOCH 3

143 -

XX I XXII

The e f f e c t o f i n vivo t r e a t m e n t w i t h mebendazole X I on t h e i n t e s t i n a l u l t r a s t r u c t u r e s o f Ascaris s u m and Syngams trachea was s t u d i e d . 72,73 S i x hours a f t e r t h e onset o f medication,microtubules disappeared f r o m t h e a p i c a l cytoplasm o f i n t e s t i n a l c e l l s i n b o t h p a r a s i t e s . No change i n d i s t r i b u t i o n and number o f m i c r o t u b u l e s i n t h e cytoplasm o f i n t e s t i n a l e p i t h e l i a l c e l l s was observed among h o s t animals under t h e same drug t r e a t m e n t up t o a t l e a s t 24 hours. However, a v e r y c l o s e analog o f mebendazole, oncodazole [R17934, methyl-5- ( 2 - t h i e n y l c a r b o n y l )benzimidazole-2-carbamate] has been p r e s e n t e d r e c e n t l y as an a n t i t u m o r agent.74 The d r u g was capable o f a r r e s t i n g m i t o s i s a t metaphase o f L1210 leukemia a s c i t e s c e l l s i n vivo,75 and i n t e r f e r i n g w i t h m i c r o t u b u l e s i n mammal i a n c e l l s c u l t u r e d i n u i t r o . 76 Furt h e r s t u d i e s i n d i c a t e d t h a t oncodazole b i n d s t o r a t b r a i n t u b u l i n i n a mole t o mole r a t i o and i n h i b i t s t h e p o l y m e r i z a t i o n o f t u b u l i n and t h e b i n d i n g o f c o l c h i c i n e t o t u b u l i n . 1 7 It was a l s o more a c t i v e t h a n c o l c h i c i n e i n promotin! concanaval i n A capping on human polymorphonuclear l e u k o c y t e s . 78 Although i t i s n o t known whether mebendazole has a n t i t u m o r a c t i v i t i e s o r oncodazole possesses a n t h e l m i n t i c a c t i v i t i e s , i t i s n o t u n l i k e l y t h a t each drug may demonstrate b o t h a c t i v i t i e s . I t i s a l s o p o s s i b l e t h a t t h e mechanism o f a n t h e l m i n t i c a c t i v i t y by mebendazole c o u l d be p r i m a r i l y b y i t s a n t i - m i c r o t u b u l e a c t i o n . Since cross- r e s istance between mebendazol e and o t h e r benzimi dazol e a n t h e l m i n t i c s has been r e p e a t e d l y demonstrated i n H. contortus i n l a b o r a t o r y as w e l l as i n n a t u r a l environments,79 i t i s t e m p t i n g t o assume t h a t a l l t h e benzimidazoles a c t by i n h i b i t i n g f o r m a t i o n o f m i c r o t u b u l e s i n t h e p a r a s i t e s . T h i s h y p o t h e s i s i s b e i n g f a v o r e d by t h e a u t h o r s o v e r t h e o t h e r t h e o r y t h a t t h e mode o f a c t i o n o f some benzimidazoles may be by i n h i b i t i n g fumarate reductase80 because o f s e v e r a l reasons: 1 ) ID50 o f t h e i n h i b i t i o n o f t u b u l i n p o l y m e r i z a t i o n b y oncodazole i s 0.63 x 1 0 - 6 ~ whereas ID50 o f t h e i n h i b i t i o n o f fumarate reductase b y t h i a b e n d a z o l e i s about 1 x 10-3M.81 2) Thiabendazole i s much more e f f e c t i v e i n i n h i b i t i n g h a t c h i n g o f t h e p a r a s i t i c nematodes82 than i t s i n h i b i t i o n o f fumarate r e ductase. 3) The c o r r e l a t i o n between in vivo r e s i s t a n c e and r e s i s t a n c e o f fumarate reductase t o t h i a b e n d a z o l e o r cambendazole has been poor. 81 F i n a l p r o o f o r d i s p r o o f w i l l have t o w a i t f o r f u r t h e r s t u d i e s . S c h i s t o s o m i a s i s - I n a s e r i e s o f reviews, c l i n i c a l l y a v a i l a b l e a n t i s c h i s t o soma1 drugs,83 s c r e e n i n g procedures,84 and t h e mutagenic e v a l u a t i o n o f a n t i schistosomal drugs85,86,87,88,89,90 were discussed. The e x p e r i m e n t a l chemotherapy, pharmacology and t o x i c o l o g y o f hycanthone was reviewed.91 S t u d i e s on j u v e n i l e and a d u l t schistosomes i n v i t r o i n d i c a t e d t h a t t h i s t y p e o f e v a l u a t i o n was n o t u s e f u l as a s c r e e n i n g t e c h n i q u e f o r new a n t i s c h i s t o s o m a l drugs. 92 4 - I s o t h i o c y a n a t o - 4 ' - n i t r o d i p h e n y l a m i n e (C9333-G0/CGP4540) X X I I I was found t o d i s p l a y an unusual spectrum o f a n t h e l m i n t i c a c t i v i t y a g a i n s t Nematospiroides dubius i n m i ce and Sehistosoma haemotobiwn, Sehistosoma

144

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mansoni and Schistosoma japonicum i n v a r i o u s h o s t s i n c l u d i n g mice, j i r d s and primates.93 The drug was e f f e c t i v e by o r a l o r p a r e n t e r a l a d m i n i s t r a t i o n i n s i n g l e doses o f 5 t o 120 mg/kg and w e l l t o l e r a t e d i n a c u t e t o x i c i t y t e s t s . I t s e f f e c t i v e n e s s was found t o vary w i t h p a r t i c l e s i z e o f t h e d r u g and t h e age o f t h e p a r a s i t e . 9 4 I t was suggested t h a t a mutagenic m e t a b o l i t e of X X I I I was formed by i n t e s t i n a l b a c t e r i a . 9 4 A group o f t r i s u b s t i t u t e d aminoethane t h i o l s were a c t i v e us. S. mansoni i n mice.% The most a c t i v e was X X I V . 0 2 N o NH-Qcs Trypanosomiasis - Reviews were p u b l i s h e d on t h e XXIII chemotherapy o f A f r i c a n trypanosomiasis96 and Trypanosoma cruzi i n f e c t i o n s . 9 7 A t e s t system Me3CNHCH2CH2SH was developed u s i n g Trypanosoma rhodesiense i n XXIV mice, s u i t a b l e f o r s c r e e n i n g l a r g e numbers o f compounds. 9 8 Some n o v e l n i t r o f u r f u r y l hydrazones were a c t i v e a g a i n s t T. cruzi i n mice.99 D i f f e r e n t i a l s c r e e n i n g r e v e a l e d n i n e compounds a c t i v e a g a i n s t T. c m z i i n c e l l c u l t u r e which were n o n - t o x i c t o A f r i c a n green monkey k i d n e y c e l l s . 1 0 0 The bloodstream forms o f A f r i c a n trypanosomes depend on g l y c o l y s i s ( f r o m glucose t o p y r u v a t e ) f o r energy supply because a m i t o c h o n d r i a 1 r e s p i r a t o r y c h a i n i s l a c k i n g . 101,102 NADH generated by glyceraldehyde phosphate dehydrogenase i s r e o x i d i z e d by an a - g l y c e r o l phosphate oxidase system It has l o n g been p o s t u l a t e d t h a t t h i s o x i unique t o trypanosomes.l03,104 dase s stem s h o u l d p r o v i d e an i d e a l t a r g e t f o r a s e l e c t i v e chemotherapeutic agent.f)03,104 Some a r o m a t i c hydroxamic a c i d s have been shown t o i n h i b i t 02 uptake o f bloodstream trypanosomes a t about 10-4M.105,106 One o f these compounds, s a l i c y l h y d r o x a m i c a c i d (SHAM), was shown t o b l o c k t h e oxidase p a r t o f t h e system w i t h a K i v a l u e o f 2.5 pM.107 However, no t h e r a p e u t i c e f f e c t was observed on Trypanosoma brucei when SHAM was t e s t e d i n uivolO8 up t o a plasma c o n c e n t r a t i o n o f lO-4M f o r 4 hours which suggested t h a t 02 uptake may n o t be e s s e n t i a l f o r t h e s u r v i v a l o f trypanosomes i n t h e bloodstream. S i m i l a r r e s u l t s were o b t a i n e d i n vitro when T. brucei, s u p p l i e d w i t h g l u cose, s u r v i v e d f o r hours a f t e r 02 uptake had been b l o c k e d by SHAM. These f i n d i n g s d e f y t h e g e n e r a l l y accepted g l y c o l y t i c pathway f o r t h e p a r a s i t e which p r e d i c t s no n e t g a i n o f ATP under anaerobic c o n d i t i o n s . 1 0 2 The scheme o f metabolism thus must have been wrong.107 Although t h e c o r r e c t pathway o f anaerobic glucose metabolism i n A f r i can trypanosomes s t i l l remains unknown, t h e p r o d u c t i o n o f e q u i m o l a r amounts o f p y r u v a t e and g l y c e r o l under anaerobic c o n d i t i o n s has been r e p e a t e d l y demonstrated. 107,109,110 Clarkson and B r o h n l l l were a b l e t o u t i l i z e t h e s e f a c t s t o observe immediate l o s s o f m o t i l i t y o f T. brucei in v i t r o i n t h e presence o f 1mM SHAM and 2.5mM g l y c e r o l . I n t r a v e n o u s i n j e c t i o n o f T. brucei i n f e c t e d r a t s w i t h SHAM (96 mg/kg) and g l y c e r o l (276 mg/kg) i m m o b i l i z e d a l l t h e p a r a s i t e s w i t h i n 1 m i n u t e and no i n t a c t m o t i l e p a r a s i t e s were found a f t e r 3 minutes. S i m i l a r e f f e c t s by t h e SHAM-glycerol combination were a l s o observed on T. rhodesiense. However, t h e r e c u r r e n c e o f p a r a s i temia always took p l a c e f o l l o w i n g t h e t r e a t m e n t . Damper and Patton112 s t u d i e d t r a n s p o r t o f pentamidine i n T. brucei

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and i d e n t i f i e d t h e presence o f a h i g h l y s p e c i f i c pentamidine t r a n s p o r t system i n t h e b l o o d t r y p o m a s t i g o t e form. The t r a n s p o r t showed s a t u r a t i o n k i n e t i c s and an average Km v a l u e o f 2.68 pM. S t i l b a m i d i n e , propamidine, h y d r o x y s t i l b a m i d i n e and benzamidine a r e a l l c o m p e t i t i v e l y i n h i b i t o r y w i t h KI values o f 3.5, 3.3, 1.7 and 6.5 pM r e s p e c t i v e l y . SHAM (5mM) and iodoa c e t a t e (1mMf i n h i b i t e d t h e t r a n s p o r t a c t i v i t y b y 78% and 47% s u g g e s t i n g i t s p o s s i b l e dependence on energy s u p p l i e d b y a e r o b i c metabolism. The k i n e t i c s o f uptake was a l t e r e d i n d s k i n e t o p l a s t i c s t r a i n s o f T. brucei which a r e r e s i s t a n t t o p e n t a m i d i n e . f l 3 They showed l o w e r r a t e s and h i g h e r Km f o r pentamidine t r a n s p o r t as d i d T. rhodesiense which i s c h a r a c t e r i s t i c a l l y l e s s s e n s i t i v e t o t h e drug. T h i s a l t e r a t i o n i n uptake may p r o v i d e b a s i s f o r pentamidine r e s i s t a n c e and c r o s s - r e s i s t a n c e among a r o m a t i c amidines.

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Leishmaniasis Over 100 compounds w i t h known a n t i p a r a s i t i c a c t i v i t y were t e s t e d a g a i n s t Leishmania rne&cana mexicana M379, Leishmania tropica major P, and Leishmania donovani HV3 i n c e l l c u l t u r e . l l 4 Several a n t i m a l a r i a l compounds i n c l u d i n g 5- , 6- and 8-aminoquinol i n e s , q u i n a z o l i n e s , amidinoureas and d i h y d r o f o l a t e reductase i n h i b i t o r s showed s u f f i c i e n t a c t i v i t y t o w a r r a n t f u r t h e r study. Trichomoniasis - A r e v i e w o f new a n t i t r i c h o m o n a l agents appeared. 115 An e x p e r i m e n t a l r a t model system was described116 u s i n g a dual i n f e c t i o n o f Candida aZbicans and Trichornonas v a g i n a Z i s as a s c r e e n i n g method. A s o l u b l e complex o f t h e polyene a n t i b i o t i c m e p a r t r i c i n and sodium l a u r y l s u l f a t e was r e p o r t e d t o be o r a l l y e f f e c t i v e i n p a t i e n t s w i t h v a g i n a l t r i c h o m o n i a s i s . 117 A s e r i e s o f p h o s p h o r y l a t e d d e r i v a t i v e s o f c a r b o x y l i c acids118 was found t o have t r i c h o m o n o c i d a l a c t i v i t y .

(2-hydroxyethyl)-2-methyl-5-ni t r o i m i dazol e l , a we1 1M e t r o n i d a z o l e [lknown agent a g a i n s t T. v a g i n a Z i s i n f e c t i o n , l I g i n h i b i t s a n a e r o b i c p r o t o z o a and anaerobic b a c t e r i a i n general b u t has l i m i t e d a c t i v i t y a g a i n s t a e r o b i c organisms.120 As t h e mechanism o f i t s a c t i o n , i t has been suggested t h a t an i n t e r m e d i a t e i n t h e r e d u c t i o n o f m e t r o n i d a z o l e , produced o n l y i n anaerobes, i s bound t o DNA and p r o t e i n and i n h i b i t s subsequent n u c l e i c a c i d synthesis.121 The h y p o t h e s i s gained some s u p p o r t from t h e f i n d i n s t h a t e x t r a c t s o f trichomonads and anaerobic b a c t e r i a reduce t h e drug1 2 b 2 3 and t h a t 02 markedly su presses t h e uptake o f t h e d r u g i n trichomonads and Entamoeba invadens,y24 even though t h e reduced r o d u c t o f m e t r o n i d a z o l e has n o t y e t been c h a r a c t e r i z e d . 121 Recent studies,~25,126,127 however, seem t o c a s t some doubt on t h e p o s s i b l e b i n d i n g o f t h e reduced drug t o DNA. I n g s and Constable found t h a t 14C-metronidazole d i d n o t accumulate a t any p a r t i c u l a r s i t e w i t h i n T. v a g i n a Z i s b u t was e v e n l y d i s t r i b u t e d i n t h e n u c l e u s and c y t o plasm.125 Though t h e r e i s some disagreement on whether t h e r e was any change about t h e n u c l e i o f d r u g - t r e a t e d T. vaginaZis,125,126,127 a t l e a s t two i n v e s t i g a t o r s appeared t o agree t h a t t h e number o f polyribosomes had decreased whereas t h e number o f s i n l e ribosomes i n t h e cytoplasm had i n c r e a s e d as a r e s u l t o f d r u g treatn1ent.725~126 M e t r o n i d a z o l e t h u s may i m p a i r p r o t e i n synt h e s i s i n T. v a g i n a l i s . N i t r o f u r a n s , such as n i t r o f u r a z o n e , n i t r o f u r a n t o i n and

SQ 18506, have

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l o n g been used as u r i n a r y t r a c t a n t i s e p t i c s . 1 2 8 They have been r e c e n t l y i d e n t i f i e d as agents a l s o a c t i v e i n v i t r o a g a i n s t T. v a g i n a Z i s . 1 2 7 ~ 1 2 9 Buchner and Edwards127 i n d i c a t e d t h a t t h e m o r p h o l o g i c a l changes i n T. v a g i n a l i s caused b y n i t r o f u r a n s were v e r y s i m i l a r t o t h o s e by m e t r o n i d a z o l e . McCalla, Reuvers and K a i s e r 1 30,131 demonstrated t h a t t h e a n t i m i c r o b i a l act i v i t y o f n i t r o f u r a n s was l i n k e d w i t h r e d u c t i o n o f t h e n i t r o group and t h e reduced n i t r o f u r a n s i n t u r n i n h i b i t DNA f u n c t i o n and cause chromosome breakage i n b a c t e r i a ; a mechanism v e r y s i m i l a r t o t h a t p o s t u l a t e d f o r metron i d a z o l e though n o t r e s t r i c t e d among anaerobes. However, a n o t h e r b a c t e r i o s t a t i c a c t i v i t y o f n i t r o f u r a n t o i n a t much l o w e r doses (0.5-25 pg/ml) has been n o t e d r e c e n t l y by H e r r 1 i c h and Schweiger. 132 They found i n E s c h e r i c h i a c o l i no e f f e c t by n i t r o f u r a n s on t r a n s c r i p t i o n , b u t t h e drugs i n h i b i t s p e c i f i c a l l y t h e e x p r e s s i o n o f i n d u c i b l e genes, e.g., 8 - g a l a c t o s i d a s e , t r y p t o phanase, g a l a c t o k i n a s e , e t c . S i m i l a r a c t i v i t y was expressed i n t h e E. coZi p r o t e i n s y n t h e s i s assay i n v i t r o , and t h e i n h i b i t i o n o f messenger RNA expression occurred a t the i n i t i a t i o n step, suggesting s e l e c t i v e t r a n s l a t i o n a l control.

A l l t h e evidence reviewed above p o i n t s t o an i n t e r e s t i n g p o s s i b i l i t y t h a t t h e p r i m a r y a c t i o n of m e t r o n i d a z o l e and n i t r o f u r a n s i n T. v a g i n u l i s may be t h e i n h i b i t i o n o f s y n t h e s i s o f p r o t e i n s . Whether r e d u c t i o n o f t h e n i t r o group i s t h e p r e r e q u i s i t e f o r t h i s a c t i v i t y remains t o be seen. C o c c i d i o s i s - Reviews appeared on c o c c i d i o s i s o f sheep133 and p r o p y l a c t i c immunization.134 I n a s t u d y u s i n g r e c e n t f i e l d i s o l a t e s , no a n t i c o c c i d i a l was e f f e c t i v e a g a i n s t a l l i s o l a t e s . 1 3 5 S e q u e n t i a l use o f amprolium, n i c a r b a z i n , u n i s t a t and zoalene a g a i n s t E i m e r i a teneZZa produced a s t r a i n s e n s i t i v e t o n i c a r b a z i n b u t r e s i s t a n t t o t h e o t h e r agents.136 A new a n t i c o c c i d i a l MK-302 XXV was r e p o r t e d t o c o n t r o l E i m e r i a acervuZina, E i m e r i a b r u n e t ti, E i m e r i a m&ma, E i m e r i a neeatpix, and E. t e n e l l a . 137,138 New a n t i c o c c i d i a l ionophores d e s c r i b e d i n c l u d e d n a r a s i n (A-28086) X X V I ,139 w i t h predominantly Nat s e l e c t i v i t y 1 4 0 and CP-39,295141 X X V I I . Monensin was r e p o r t e d t o be a c t i v e i n sheep142 and t o have p r o f o u n d e f f e c t s on rumen m i c r o b i o l o g y . 143,144 F u r t h e r s t u d i e s on p y r i d o x a l analogs showed XXVIII145 and XXIX146 t o have a c t i v i t y a g a i n s t E. a c e r v u l i n a . A s e r i e s o f pyran-3(4H)-onesY exemp l i f i e d by XXX, were a c t i v e a g a i n s t E. a c e r v u l i n a and E. teneZZa.147 The mechanism o f a c t i o n o f q u i n o l o n e c o c c i d i o s t a t s was r e c e n t l y s t u d i e d by Wang i n E. t e n e ~ ~ a . 1 4 8 , 1 4 9 Amquinate, b u q u i n o l a t e , methyl benzoquate and decoquinate were a l l r e v e r s i b l e i n h i b i t o r s o f E. teneZZa r e s p i r a t i o n as w e l l as s p o r u l a t i o n . The ID50 values were 1 t o 2x lO-5M a g a i n s t r e s p i r a t i o n d u r i n g s p o r u l a t i o n and 3 x 10-6M a g a i n s t r e s p i r a t i o n d u r i n g exc y s t a t i o n . R e s p i r a t i o n i n i s o l a t e d E. t e n e l l a m i t o c h o n d r i a was i n h i b i t e d 50% by t h e quinolones a t 3 pmoles p e r mg p r o t e i n a t t h e s i t e n e a r c y t o chrome b. M i t o c h o n d r i a of an E. teneZZa a m q u i n a t e - r e s i s t a n t mutant were a t l e a s t 1 0 0 - f o l d l e s s s u s c e p t i b l e t o t h e quinolones, w h i l e c h i c k e n l i v e r m i t o c h o n d r i a showed no s e n s i t i v i t y t o t h e drugs. I t was t h u s suggested t h a t t h e quinolones may suppress t h e p a r a s i t e s b y i n h i b i t i n g t h e i r m i t o c h o n d r i a 1 r e s p i r a t i o n . It was a l s o p o s t u l a t e d t h a t t h e h i g h frequency o f r e s i s t a n c e

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CH3

CH3 XXVI

XXVI I

dH 3 XXVIII

XXIX

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t o t h e q u i n o l o n e s among coccidial50,151 c o u l d be due t o autonomous g e n e t i c c o n t r o l o f drug-resistance i n mitochondria. The phenomenon o f drug r e s i s t a n c e has been s t u d i e d b y Joyner and N 0 r t o n . 1 5 2 ~ 1 5 3 They found t h a t when two d r u g - r e s i s t a n t s t r a i n s o f E. maxima were passaged t o g e t h e r i n u n t r e a t e d chickens, a t l e a s t some o f t h e r e s u l t a n t o o c y s t s became r e s i s t a n t t o b o t h drugs. A c q u i s i t i o n o f r e s i s t a n c e b y one s t r a i n from t h e o t h e r o c c u r r e d between methyl benzoquate and s u l f a q u i n o x a l i n e o r c l o p i d o l and s u l f a q u i n o x a l i n e b u t n o t between c l o p i d o l and methyl benzoquate. The t r a n s f e r o f drug r e s i s t a n c e d i d n o t t a k e p l a c e between d i f f e r e n t species b u t o c c u r r e d r e a d i l y between d i f f e r e n t v a r i a n t s o f t h e same species 96 hours a f t e r i n o c u l a t i o n o r l a t e r . 1 5 4 Treatment w i t h a c r i f l a v i n had no e f f e c t on t h e t r a n s f e r s u g g e s t i n g g e n e t i c r e c o m b i n a t i o n . D i h y d r o f o l a t e reductase was found and p u r i f i e d from u n s p o r u l a t e d

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oocysts o f E. teneZZa by Wang, e t aZ.155 I t s molecular weight was estimated as 240,000 daltons and i t was s t r o n g l y i n h i b i t e d by pyrimethamine (KI = 3nM). C u l t i v a t i o n of E. teneZZa i n c e l l c u l t u r e s has been w i d e l y u t i l i z e d i n studyin known a n t i c o c c i d i a l s and provided useful i n f o r m a t i o n on these c o m p o ~ n d s . ~ 5 6 ~ 1 5The 7 technique was r e c e n t l y f u r t h e r develo ed f o r t h e primary screening o f compounds i n several laboratories.158,l 9 I n a recent report,l60 however, Ryley summarized t h e r e s u l t s from screening o f 11,550 compounds i n c e l l c u l t u r e s and concluded t h a t t h e screening method i s n o t a s a t i s f a c t o r y o r r e l i a b l e a l t e r n a t i v e t o screening i n chickens. Toxoplasmosis - Using ToxopZasm gondii i n c e l l culture,161 t h e p o l y e t h e r ionophores l a s a l o c i d and monensin were h i g h l y a c t i v e whereas ormetoprim and sulfadimethoxime o r a combination were i n a c t i v e o r weakly a c t i v e . References 1.

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Chemotherapeutic Agents

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1,

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Chap. 15 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161.

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11.

a.

Section IV

- Metabolic Diseases and Endocrine Function

Editor: Hans-Jtirgen Hess, Pfizer, Inc., Croton, Conn.

Chapter 16.

Cellular Responses Mediating Chronic Inflammatory Diseases

Philip Davies and Robert J. Bonney Merck Institute for Therapeutic Research, Rahway, N.J. Introduction - The etiology of chronic inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, and sarcoidosis remains unknown. However, a chronic inflammatory component is recognized to occur in these diseases because of an influx of blood leukocytes and a proliferation of local cells. An examytq of this occurs in diarthrodial joints involved in rheumatoid arthritis where synovial lining cells proliferate, overlying a mass of fibrous tissue, termed pannus, which is infiltrated by numerous lymphocytes, macrophages, and fibroblasts. The synovial fluid of such joints is increased in volume and contains large numbers of polymorphonuclear leukocytes (PMN) , lymphocytes, and macrophages. In addition to the cellular components present at sites of inflammation, the products of various humoral systems associated with inflammatory events accumulate--viz, fibrin, cleavage products of the complement system, kinins, and antigen-antibody complexes. We will restrict our remarks to some cellular aspects of chronic inflammatory processes, giving particular attention to biochemical changes occurring in cells responding to inflammatory stimuli. These changes often lead to the secretion of products which modulate inflammatory processes in various ways. Some inflammatory stimuli interact directly with phagocytic cells, causing the release of their mediators. For example, urate crystals release mediators of gouty inflammation from PMN, while the interaction of toxic materials such as asbestos, silica, and thermophilic yeasts with alveolar phagocytes leads to chronic inflammatory changes in the lung. These are nonimmunologically mediated inflammatory events, and there is no evidence for a primary involvement of lymphocytes in this type of inflammation. More commonly, chronic inflammatory processes are the result of the interaction of a specific immunogenic stimulus with lymphocytes. Further exposure of sensitized lymphocytes to the specific immunogen results in the synthesis and release of inflammatory mediators. These products of stimulated lymphocytes act directly upon target tissues, e.g., lymphotoxin, or alternatively influence the activity of other cells, e.g., migration inhibitory factor (MIF), acting upon macrophages. In order to develop meaningful assay systems to study mechanisms involved in chronic inflammation, cells from cartilage, h n e , normal synovium, and from organized inflammatory lesions as well as leukocytes from spleen, thymus, and macrophages from serous cavities of laboratory animals maintained as stable populations in cell or organ culture have been utilized. Recent improvements in methodology for cell isolation from normal and inflamed tissues have permitted the maintenance of cell culture systems

Chap. 16

Inflammation

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Davies , Bonney

relevant to in vivo situations. This has been achieved by dispersing tissues by the judicious use of connective tissue degrading enzymes rather than mechanical disaggregation or mincing of tissue and incubation with trypsin. Clearly, the complex cellular interactions underlying chronic inflammation are best studied using methods of cell and organ culture where the responses of individual cell types to various stimuli can be defined. While such systems fail to provide a comprehensive model for any chronic inflammatory disease, they do provide definitive information on the response of a given cell type to stimuli introduced into its environment. Much of the information reviewed in this chapter has been derived in this way. Only a judicious combination of these test systems will provide comprehensive assays for the various aspects of inflammatory disease. An extensive background to this brief survey is found i s,fhe proceedings of two recent international meetings on inflammation. Chemotaxis - Chemotaxis is the mechanism by which cells are attracted to sites of inflammation. Leukocytes move in a specific direction established by a concentration gradient of a stimulus generated at a site of inflammation. The Boyden chamber5 still serves as the basic appara us for measuring chemotaxis, although improveme ts have been described and 9 % excellent monograph on a method using radiolabeled cells is in use. the subject of chemotaxis has appeared recently. Activation of the complement systems leads to formation of several molecules with chemotactic activity, including C3a, C5a, and the trimolecular complex C567. Human C3a has been purified, sequenced, and shown to be a cationic geptide containing 77 amino acid residues with a molecular weight of 9,000. Human C5a contains 73 amino acid residues with additional carbohydrate accountirlg for 25% of its molecular weight of approximately 11,000 daltons.

L

The eosinophil chemotactic factor of ana ylaxis (ECF-A) is a preformed 4P product fqund in rat mast cell granules, in human lung, nasa3 polyps, The activity resides in two acidic tetrapeptides, Ala-G1 and PMN. Y4 Ser-Glu and Val-Gly-Ser-Glu, which preferentially attract eosinophils. Synthetic peptides of thislsomposition are equipotent to natural ECF-A bot in vitro and in vivo. ECF-A s h y 2 maximal chemotactic activity at -!I10 M I and concentrations as low as 10 M deactivate eosinophils t ?6:8sequent stimulation by chemotactic concentrations of ECF-A and C5a. The CGQH terminal tripeptide of ECF-A, Gly-Ser-Glu, is only weakly chemotactic but it causes dose-depfgffqt suppression of eosinophil chemotactic responses to Val-Gly-Ser-Glu. Simple formy1 methionyl peptfgegostimulate leukocyte movement and are chemotactic. Becker et a1 have synthesized a series of di-, tri-, and tetrapeptides, most of them being formyl methionyl peptides. These are chemotactic, stimulate phagocytosis of latex particles, and cause the selective release of lysosomal enzymes from PMN in the presence of cytochalasin B. There is a high degree of correlation between the ability of the peptides to enhance PMN movement and cause selective release of acid hydrolases. The presence of the formyl group on the methionine leads to a 3,000- to 30,000-fold increase in chemotactic activity. Further studies

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will determine if these oligopeptides bind to a specific receptor and whether such a receptor is involved in initiating bas$? functions of the PMN, such as chemotaxis and phagocytosis. Hook et a1 have shown that formyl methionyl peptides trigger the release of histamine from basophils. Arachidonic acid released from phospholipids or neutral lipids at sites of inflammation can be converted via the cyclooxygenase system into the biologically active endoperoxide2grostaglandin G 2 as well as several nonprostanoate hydroxy fatty acids. Alternatively, it may be converted by a lipoxygenase into 2-L-hydroxy-5,8,10-14-eicosatetraneoic a23d (HETE). HETE produced by platelet aggregation is chemotactic for PMN (see Chapter 19). Cellular Mediators of Inflammatory Processes Lymphocyte Products - It is sply 10 years since it was realized that lymphocytes are heterogenous in terms of their function and that cellto-cell collaboration b ween lymphocyte subclasses is essential for several proper immune function.% Subsequently it has become clear subsets of lymphocytes recognized by their surface antigens mediate specific functions.

2k~~$

Lymphocytes responding to antigens or mitogens synthesize and secrete macromolecular prog y $ g termed lymphokines that possess a variety of biological activities which are thought to initiate many of the changes seen in delayed hypersensitivity responses. Extreme difficulty h36 been encountered in purifying and characterizing these macromolecules. Some progress has been reported toward the preparation of an antiserum 31 with specificity toward lymphokines that influence macrophage functiogq, and also toward products participating in mixed lymphocyte responses. Antibodies prepared agaiggt guinea pig lymphotoxin did not neutralize mitogenic factor or MIF. Mouse lymphotoxin has been characterize molecule of 41,000 daltons with an isoelectric point of 4.4 to 4 . 8 . g4as a Human tonsillar lymphocytes produce a lymphotoxin of molecular weight of approximately 8 0 , 0 0 0 , while human peripheral blood leukocyte roduce a lymphotoxin with a molecular weight of approximately 45,000. 5p

5

This mater&l has MIF inhibits the amoeboid movement gg macrophages been partially purified from human, guinea pig,37 and murine sources. Its ryjeptor on macrophages is susceptible to inactivation by a-fucosidase. Suppressor lymphocytes4’ control immune responses in part by secretion of an antigegispecific factor which is not active across histocompatibility barriers. This product has a molecular weight between 35,000 and 55,000 daltons and can be completely absorbed with an alloantiseruj2 specific for the I region of the major histocompatibility complex. A soluble immune response suppressor (S@S) is produced by concanavalin Astimulated murine splenic lymphocytes and inhibits plaque-fqping responses of B lymphocytes and cytotoxic lymphocyte responses t alloantigens. SIRS has a molecular weight between 48,000 and 6’7,000,24does

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not contain any immunoglobulin-like material, and is destroyed by proteinases and nucleases. Its inhibitory activity may be mediated by interference with th accessory role played by macrophages in the response of 23 these cells. Macrophage Products - Mononuclear phagocytes maintained in tissue culture synthesize and zyrete a number of products relevant to chronic inflammatory processes. In "0'45 instances these are secreted in direct response to inflammatory stimuli. Neutral proteinase secretion is stimulated by intraperitoneal injection of thioglycollate broth, which acts as a sterile inflammatoqg stimulus. Cells harvested iq8this manner secrete plasminogen Collagenase secretion by activator , elastase , 4 7 and collagenase. mineral oil-induced guinea pig peritoneal macrophages is stimulated by the addition of superna6ants of antigen- or concanavalin A-stimulated 50 guinea pig lymphocytes or directly by bacterial lipopolysaccharid 9 -or Collagenase is secreted by alveolar macrophages frofpgynal rabbits animals sensitized with Freund's complete adjuvant, larger amounts of enzyme being secreted from the latter. Alveolar masZophages secrete collagenase in a latent form under certain conditions, but they also secrete a pg2teinase which activates the latent enzyme under physiological conditions. Macrophages harvested from mice given an intraperitoneal injection of chrysotile asbestos, a potent stimulus of chronic i n f l m a tion, secrete plasminogen activator; but mice injected with latex particles, wQ&ch have little inflammatory activity, do not secrete this enzyme. Neutral proteinase secretioggby macrophages is dependent upon an intact protein synthetic mechanism. Glucocorticoids inhibit secretion of plasminogen activator at doses close to phyFSologica1 levels and corresponding to their anti-inflammatory potencies. Colchic'ne and -? vinblastine inhibit plasminogen activator secretion at 1 x 10 M. Cholera toxin is ay2exftemely potent inhibitor of secretion, reducing release by 90% at 10 M. Nonsteroidal anti-inflammatory drugs have no detectable effects on secretion of plasminogen activator. In contrast to its inhibition of plasminogen activator release by macrophages, colchicine stimulates the secretion of elastase, coll?genase, and a neutral proteinase hydrolyzing azocasein by these cells. Acid hydrolase release from macrophages is not a general response to phagocytic stimuli, as is the case with PMN. Enzyme release by macrophages occurs only with inflammatory stimuli. Stimuli causing lysosomal hydrolase release may be those causing nonimmune-based inflammation, such as Group A streptococcal cell walls, carrageenan, zymosan, asbestos, or products of lymphocxke stimulation such as lymphokines and antigenantibody complexes. In addition, products of the activation of the complement system, C3b in pa$$.is.jlar, induce selective release of acid hydrolases from macrophages. This observation is of particular interest since macrophages synthesize those factors of the alternative pathy$y68f complement required for the conversion of C3 to C3a and C3b. Since several inflammatory stimuli, e.g, bacterial lipopolysaccharides, carrageenan, and zymosan, are known to activate the alternate pathway of complement, the formation of C3b may be an intermediate step for the expression of macrophage functions relevant to its role in inflam-

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matory processes.61 The selective release of acid hydrolases is inhibited by anti-inflammatory drugs under certain conditions. Glucocorticoids inhibit acid hydrokise release from thioglycollate-induced macrophages Preincubation with indomethacin inhibitssfhe release caused by zymosan. of acid hydrolases caused by Group A streptococcal cell walls. Macrophages secrete products which stimulate6Qoth the proliferation of fibroblasts and their synthesis of collagen. Macrophages secrete prostaglandins in response to several types of inflammatory stimuli. Oil-induced guinea pig peritoneal macrophaggg secrete Both PGE2 and PGF when cultured in the presence of lymphokines. thioglycolla%-induced and unstimulated mouse macrophages secrete prostaglandins in response to an inflammatory stimulus such as zymosan but fj9t to a stimulus lacking inflammatory capacity, namely, latex particles. Macrophages play an important ro& in the initiation and modulation of lymphocyte responses to antigen. They secrete fact058 which enhance the respq~s7~of both T lymphocytes6' and B lymphocytes to mitogenic stimuli. ~~ a wide PMN Products - Neutral proteinases of PMN l y s o ~ o m e sdegrade variety of coqtective tissue substrates including elastin, proteoglycan, and collagen. Cathepsin G has been found only in PMN, and elastase from these cells has a substrate73pecificity different from that of pancreatic and macrophage elastase. In addition to their effects on connective tissue components, the neutral proteinases of PMN lysosomes also stimulate the activity of other cells involved in the inflammatory response. Both elastase and cathepsin G from human PMN stimulate the incor76 poration of thymidine by human peripheral blood and splenic lymphocytes. The stimulated lymphocytes are of the B lineage, with no effect seen on T lymphocytes. Basophil Products - Basophilic leukocytes participate in both delayed, cell-mediated, and immediate,homocytotropic antibody-induced hypersensitivity reactions. Basophils contain several pharmac9+ogical mediators that are released in response to inflammatory stimuli. These mediators include histamine, slow-reacting substance of anaphylaxis (SRS-A), ECF-A, and platelet activating factor (PAF). The degranulation procesgBhas been observed morphologically in allergic contact dermatit+3,Q8man. C5a causes the release of histamine from human basophils. The C5ainduced release is dependent upon calcium ions and occurs within 2 minutes of C5a addition, it is additive to that induced by IgE, aRf,&9ere is no cross-desensitization tween the two stimuli of gzlease. Shortterm cultures of human and guinea pig basophils have been establi Basophils from the latter specis5 were shown to synthesize histamine, whereas human basophils do not.

8s

PA!? is secreted from basophils by an IgE-dependent reaction, has a mo lar weight of approximately 300, and is sensitive to phospholipase D. It causes the secretion of vasoactive mines from platelets by a calcium-

8Fu-

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requiring and temperature-dependent process86 which is inhibited by serine protgjnase inhibitors and agents elevating cellular levels of cyclic AMP. Production of Mediators in Response to Inflammatory Stimuli by Organized Tissues Maintained in Culture Organ Culture of Synovium - Organ culture is defined as the cultivation of tissues in a diffegjntiated, fun ional state similar to that €3have used organ culture of their organs of derivation. Fell et a1 techniques to study the physiology and pathology of synovium and cartilage. Cartilage devoid of soft connective tissue responds to complementsufficient antiserum with reggrption of its matrix. The proteoglycan component, but not collagen, is degraded. When synovial soft connective tissue is add$? to the cartilage system, both connective tissue components are degraded. Therefore, cultures of the components of connective tissue can serve as models for the study of proteoglycan and collagen breakdown and the effects of various agents on such processes.

in vitro --

Dispersed Cell Culture of Synovial Tissue - Although organ culture studies yield information regarding mechanisms involved in joint and rheumatoid diseases, they do not allow the determination of which cell type or types are responsible for the observed efffjsts. Organ cultures of human rheumaand certain neutralproteinases 93 toid synoviq4secrete prostaglandins Dayer et al, using crude collagenase followed by trypsin treatment, have dispersed human rheumatoid synovium and initiated cultures containing heterogeneous populations of cells as judged by mor@ological criteria. These cells produce collagenase and prostaglandins, and the synthesis of these products correlated with the numb55 of cells which were adherent, had Fc receptors, and secreted lysozyme. Collagenase pr uction by the 89 cultured cells is stimulated by a human lymphocyte factor.

.

Cultured cells from enzymatically dispersed, carrageenan-induced granulomas display several characteristics of mononuclear phagocytes-namely, Fc regeptors, phagocytic activity, and the constitutive secretion of lysozyme. Acute Phase Protein Production by Liver - The liver synthesizes a number of acute phase proteins in response to ill-defined products of chronic inflammatory lesions. These proteins include haptoglobinlgy~glop1asmin, a-1 trypsin inhibitor, C-reactive protein, and fibrinogen. Identification of the cell type in the liver that is responsible for the synthesis of these proteins remains to be established. The process of acute phase protein synthesis has been examined by two general protocols, (1) liver perfusion and (2) incubation of liver slices.

-

Normal intact fab,I&yer can be successfully perfused 1. Liver Perfusion for 12 hours,100 and John and Miller have demonstrated net biosynthesis of albumin, fibrinogen, a-1 acid glycoprotein, and a-2 (acute phase) globulin. Synthesis of the acute phase proteins can be induced by perfusion medium supplemented with insulin, cortisol, growth hormone, and

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100

2. Liver Slices - Liver slices prepared from rats bearing an inflammatory lesion caused by subcutaneous injection of turpentine show an increased capacity for the incorporaf&n of radioactive amino acids and glucosamine into acute phase proteins. A third approach to study the synthesis of acute phase proteins is to use primary cultures of homogenous preparations of adult liver parenchymal cells which can be maintained in culture without division up to 1 week.1o3t1o4 Employing such a system, the synthesis and secretion of several serum proteins such as albumin, f#f~~gen, transferrin, and a-1 acid qlycoprotein has been demonstrated. In addition, it is npt6possible to isolate parenchymal and Kupffer cells from the same liver. Therefore, isolation and cultivation of these two cell types should allow the determination of the cellular origin of the acute phase proteins as well as the products of inflammatory processes which stimulate their production. References 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17.

M. Schubert and D. Hamerman in "A Primer on Connective Tissue BioChemistry," Lea and Febiger, Philadelphia, 1968. H. Ishikawa and M. Ziff, Arth.Rheum., 19, 1 (1976). C.P. Velo, D.A. Willoughby, and J.P. Giroud, Eds., in "Future Trends in Inflammation," Piccin Medical Books, Paduer, 1974. J.P. Giroud, D.A. Willoughby, and C.P. Velo, Eds., in "Future Trends in Inflammation 11," Birkhauser-Verlag, Basel, 1975. S. Boyden, J.Exp.Med., 115,453 (1962). S.M. Wahl, L.C. Altman, J.J. Oppenheim, S.E. Mergenhagen, 1nt.Arch. Allergy Appl.Imunol., 46, 233 (1973). J.I. Gallin, R.A. Clarkrand H.R. Kimball, J.Imunol., 110,233 (1973). P .C Wilkinson in 'Themotaxis and Inflammation,'! Churchill, Livingston, Edinburgh, 1974. T.E. Hugli, J.Biol.Chem., 250, 8293 (1975). H.N. Fernandez and T.E. Hugli, J.Imunol., 117,1688 (1976). S.I. Wasserman, E.J. Goetzl, and K.F. Austen, J.Immunol., 112, 351 (1974). B.M. Czarnetzki, W. Konig, and L.M. Lichtenstein, J.Immunol., 117, 229 (1976). 72, 4123 E.J. Goetzl and K.F. Austen, Proc.Natl.Acad.Sci.,U.S.A., (1975). S.I. Wasserman, D. Whitmer, E.J. Goetzl, and K.F. Austen, Proc.Soc. Exp.Biol.Med., I&, 301 (1975). S.I. Wasserman, R.N. Boswell, J.M. Drazen, E.J. Goetzl, and K.F. 190 (1976). Austen, J.Allergy Clin.Immunol., R . N . Boswell, K.F. Austen, and E.J. Goetzl, Imunol.Commun., 2, 469 (1976). E.J. Goetzl, Am.J.Path., E l 419 (1976).

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18. E. Schiffmann, B.A. Corcoran, and S.M. Wahl, Proc.Natl.Acad.Sci., U . S . A . , 72, 1059 (1975). 19. E.L. Becker, Am.J.Pathol., E, 385 (1976). 20. H.J. Showell, R.J. Freer, S.H. Zigmond, E. Schiffmann, S. Aswanikumar, B. Corcoran, E.L. Becker, J.Exp.Med., 143, 1154 (1976). 21. W.A. Hook, E. Schiffmann, S. Aswanikumar, and R.P. Siraganian, J. Immunol. 117,594 (1976). 71,3400 22. M. Hamberg and B. Samuelsson, Proc.Natl.Acad.Sci.,U.S.A., (1974). 23. S.R. Turner, J.A. Tainer, and W.S. Lynn, Nature, 257, 680 (1975). 24. H.N.Claman, E.A. Chaperon, and K.F. Triplett, Transplant.Rev., 92 (1969). 25. J.F.A.F. Miller and G.F. Mitchell, Transplant.Rev., 1,3 (1969). 26. H. Cantor and E.A. Boyse, J.Exp.Med., 141,1389 (1975). 27. M. Feldmann, P.C.L. Beverley, M. Dunkley, and S. Kontiainen, Nature, 258, 614 (1975). 28. R.L. Lundak, J. Lewis, and G. Granger, J.Invest.Derm., 67,625 (1976). 29. D.C. Dumonde, R.H. Kelley, P.M. Preston, and R.A. Wolstencroft in "Mononuclear Phagocytes in Immunity, Infection and Pathology," R. Van Furth, Ed., Blackwell, Oxford, 1975, p . 675. 30. C. Sorg and B.R. Bloom, J.Exp.Med., 137, 148 (1973). 31. T. Kuratsuji, T. Yoshida, and S. Cohen, J.Immuno1. , 117,1985 (1976). 32. C.L. Geczy, A.F. Geczy, and A.L. DeWeck, J.Immunol., 117,1824 (1976). 33. M.K. Gately, C.L. Gately, C.S. Henney, and M.M. Mayer, J.Immunol., 115, 817 (1975). 34. G. Trivers, D. Braungart, and E.J. Leonard, J.Immunol., 117,130 (1976). 35. S.C. Lee and Z . J . Lucas, J.Immunol., 117 283 (1976). 36. R.E. Rocklin, H.G. Remold, and J.R. David, Cell.Immunol., I,436 (1972). 37. H.G. Remold and J.R. David in "Proc. 6th Leukocyte Culture Conference," M.R. Schwartz, Ed., Academic Press, NY, 1972, p. 283. 38. A.C. Kuhner and J.R. David, J.Immunol., 116, 140 (1976). 39. H.G. Remold J.Exp.Med. 138,1065 (1973). 40. R.W. Dutton Transplant.Rev. , 26, 39 (1975). 41. T. Takemori and T. Tada, J.Exp.Med., 142, 1241 (1975). 42. M. Taniguch , K. Hayakawa, and T. Tada, J.Immunol., 116, 542 (1976). 43. T. Tadakuma and C.W. Pierce, J.Immunol., 117,967 (1976). 44. T. Tadakuma, A.L. Kuhner, R.R. Rich, J.R. David, and C.W. Pierce, J.Immunol., 117,323 (1976). 45. P. Davies and A.C. Allison in "Immunobiology of the Macrophage," D.S. Nelson, Ed., Academic Press, NY, 1976, p. 427. 46. J.C. Unkeless, S. Gordon, and Z.A. Cohn, J.Exp.Med. , 139, 834 (1974). 47. 2. Werb and S. Gordon, J.Exp.Med., 142, 361 (1975). 48. 2. Werb and S. Gordon, J.Exp.Med., 142, 346 (1975). 49. L.M. Wahl, S . M . Wahl, S.E. Mergenhagen, and G.R. Martin, Science, 187, 261 (1975). 50. L.M. Wahl, S.M. Wahl, S.E. Mergenhagen, and G.R. Martin, Proc.Nat1. Acad.Sci.,U.S.A., 21, 3598 (1974). 51. A.L. Horwitz and R.G. Crystal, Biochem.Biophys.Res.Commun., 2, 296 (1976).

160 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.

63. 64. 65. 66. 67. 68.

69. 70.

71 72. 73. 74. 75. 76. 77. 78.

79. 80.

81. 82.

83. 84. 85.

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A.L. Horwitz, J.A. Kelman, and R.G. Crystal, Nature, 264, 772 (1976). H. Birkedal-Hansen, R.E. Taylor, and H.M. Fullmer, Biochem.Biophys. Acta, 420, 428 (1976). J. Hamilton, J.D. Vassalli, and E. Reich, J.Exp.Med., 144,1689 (1976). J.D. Vassalli, J. Hamilton, and E. Reich, Cell., S, 271 (1976). S. Gordon and Z. Werb, Proc.Natl.Acad.Sci.,U.S.A., 73,872 (1976). H.U. Schloremmer, P. Davies, and A.C. Allison, Nature, 261, 48 (1976). H.U. Schloremmer and A.C. Allison, Immunol. 31, 781 (1976). C. Bentley, D. Bitter-Suermann, U. Hadding, and V. Blade, Eur0p.J. Imunol. , 5, 393 (1976). H. Colten, Adv.Immunol., 22, 67 (1976). C. Bianco, E. Eden, and Z.A. Cohn, J.Exp.Med., 144,1531 (1976). P.S. Ringrose, M.A. Parr, and M. McLaren, Biochem.Pharmacol., 24, 607 (1975). C. Finlay, P. Davies, and A.C. Allison, Agents and Actions, 2, 345 (1975). S . J . Leibovich and R. Ross, Am.J.Path., 84, 501 (1976). D.M. Lewis and R. Burrell, Br.J.Indus.Med. , 32, 25 (1976). D. Gordon, M.A. Bray, and J. Morley, Nature, 262, 401 (1976). R.J. Bonney, P. Davies, M.E. Dahlgren, L. Pelus, F.A. Kuehl, Jr., and J.L. Humes, Fed.Proc., 36, 673 (1977). A.S. Rosenthal, Ed. , in "Immune Recognition. Proc. 9th Leukocyte Culture Conference," Academic Press, NY, 1975. I. Gery and R.E. Handschumacher, Cell.Immunol., 2, 162 (1974). D.D. Wood, P.M. Cameron, M.T. Poe, and C.A. Morris, Cell.Immunol., 21, 188 (1976). R.C. Page, P. Davies, and A.C. Allison, Int.Rev.Cytol., in press. B.H. Waksman and Y. Namba, Cell. Immunol., 21 161 (1976). B. Dewald, R. Rindler-Ludwig, U. Bretz, and M. Baggiolini, J.Exp. Med., 141, 709 (1975). J.T. Dingle in "Proteolysis and Physiological Regulation ," K. Brew , Ed., Academic Press, NY, 1976, p. 339. M. Zimmerman and B.M. Ashe, Biochem.Biophys.Acta, 480, 241 (1977). T.L. Vischer, U. Bretz, and M. Baggiolini, J.Exp.Med., 144,863 (1976). R.A. Lewis, E.J. Goetzl, S.I. Wasserman, F.H. Valone, R.H. Rubin, and K.F. Austen, J.Immunol., 114,87 (1975). A.M. Dvorak, M.C. Mihm, Jr., and M.F. Dvorak, J.Immunol., 116,687 (1976). W.A. Hook, R.P. Siraganian, and S.M. Wahl, J.Immuno1. 114,1185 (1975). J.A. Grant, E. Dupree, A.S. Goldman, D.R. Schultz, and A.L. Jackson, J.Immuno1. , 114,1101 (1975). R.P. Siraganian and W.A. Hook, J.Imunol., 116,639 (1976). J.A. Grant, L. Settle, E.B. Whorton, and E. Dupree, J.Imnunol., 117, 450 (1976). J.D. Drobis and R.P. Siraganian, J.Imunol., 117,1049 (1976). S.J. Galli, A.S. Galli, A.M. Dvorak, and H.F. Dvorak, J.Immunol., 117, 1085 (1976). S . I . Wasserman, J.Invest.Derm., 67, 620 (1976).

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143,937 (1976). P.M. Henson and Z.G. Oades, J.Exp.Med. , 143, 953 (1976). H.B. Fell, J.Expl.Biol., 1 (1972). P.J. Lackmann, R.R.A. Coo&s, H.B. Fell, and J.T. Dingle, 1nt.Arch. Allergy Appl.Immun. , 36, 469 (1969). 90. J.T. Dingle, P. Horsfeld, H.B. Fell, and M.E.J. Barratt, Ann.Rheum. Dis. , 34 301 (1975). 91. H.B. Fell and M.E.J. Barratt, 1nt.Arch.Allergy Appl.Imun., 44, 441 (1973). 92. D.R. Robinson, A.H. Tashijian, and L. Levine, J.Clin.Invest., 6, 1181 (1975). 93. E.D. Harris, Jr., and S.M. mane, N.Eng1.J.Med. , 291, 557 (1974). 94. J.M. Dayer, S.M. mane, G.G. Russell, and D.R. Robinson, Proc.Nat1. Acad.Sci. ,U.S.A., 2, 945 (1976). 95. J.M. Dayer, R.G.G. Russell, and S.M. Krane, Science, 195,181 (1977). 96. R.J. Bonney, T.G. Richardson, M.E. Dahlgren, and P. Davies, In Vitro, in press. 97. E. Athineas, J.C. Kukral, and R.J. Winzler, Arch.Biochem.Biophys., 106, 336 (1964). 98. N.H. Lang and H.E. Renchler, Z.Ges.Exp.Med., 130, 203 (1958). 99. R. Asofsky and G.J. Thorbecke, J.Exp.Med., 9, 471 (1961). 100. D.U. John and L.L. Miller, J.Biol.Chem., 241, 4317 (1966). 101. D.U. John and L.L. Miller, J.Biol.Chem., 244, 6134 (1969). 102. J.C. Jamieson, A.D. Friesen, F.E. Aston, and B. Chou, Can.J.Biochem., 50, 856 (1972). 103. D.M. Bissell, L.E. Hammaker, and U.A. Meyer, J.Cell.Biol., E l 722 (1973). 104. R.J. Bonney, J.E. Becker, P.R. Walker, and V.R. Potter, In Vitro, 2, 399 (1974). 105. C.R. Savage and R.J. Bonney, In Vitro, in press. 106. A.C. Munthe-Kaas, T. Berg, and R. Seljelid, Exp.Cell.Res., 99, 146 (1976). 86. 87. 88. 89.

P.M. Henson, J.Exp.Med.,

16 2 -

Chapter 17. Molecular Mechanisms and Pharmacological Modulation in Psoriasis John J. Voorhees, University of Michigan Medical School Ann Arbor, Michigan 48109 Introduction - Psoriasis is a skin disease which occurs in separated patches over the body. Its causes are probably multiple genetic and environmental factors acting in concert.1 Of these factors, the three- to fourfold increased frequency of the cell surface gene products HL-A13 and HL-Al72 and induction of psoriasis by cutaneous trauma3 are well established. However, these established facts do not as yet immediately suggest areas of research with therapeutic application. Therefore, we have been concerned with those aspects of the molecular pathophysiology of psoriasis which are hypothetically amenable to pharmacological modulation. When using this approach, research is not restricted by not knowing which molecular events are primary versus secondary. That is not to say that knowledge of primary versus secondary molecular events is unimportant. Quite the contrary, such knowledge might be essential to successful therapeutics, but such studies at the molecular level are not generally feasible at this time in normal or diseased human skin. An important corollary of this research strategy is that in initiation and/or maintenance of a lesion certain but not all molecular events, whether they are primary or secondary, are critical. We have arbitrarily designated these events as "critical molecular events in pathophysiology". The postulate is that without these events, the lesion would either not develop or would spontaneously disappear, We have attempted to establish what certain of these "critical molecular events in pathophysiology" might be by examining lesional epidermis of psoriasis and comparing it to uninvolved psoriatic epidermis and to epidermis of normal subjects. Much psoriasis research has focused on the epidermis for several reasons. Although the clinical lesion of psoriasis has several compartments (i,e., dermal, vascular, inflammatory, immunologic, nervous, etc.) the epidermal compartment is the only one which can be surgically obtained in sufficient quantity, with reasonable homogeneity, and with minimal scarring of the patient. Epidermal research in psoriasis is reasonable and justifiable because without abnormal epidermal homeostasis, the psoriatic lesion could not exist, or at least would not pose a significant clinical problem. The fact that most, if not all, psoriatic lesions sooner or later spontaneously disappear indicates that certain "critical molecular events" occur spontaneously within the epidermis to either promote or permit the lesion to revert to normal, Such "critical molecular events in pathophysiology" are viewed as ideal candidates for pharmacological modulation. When possible, the pharmacological agents employed should be those which exist naturally in human skin, i . e . , the concept of orthomolecular pharmacology. It seems rational to suggest that in principle, the manipulation of naturally occurring molecules will be a safer approach than the utilization of classical anticancer drugs or agents which are potential mutagens. Those events in the epidermal pathophysiology of psoriasis which we consider critical will be reviewed after which

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their pharmacological manipulation will be discussed. Cell Surface in Psoriasis - The first of these factors which we believe are "critical" is the cell surface. In most, if not all animal cells, the cell surface appears to participate in the regulation of proliferation and tissue cytodifferentiation.4 In psoriasis the lesional epidermal cell surface is markedly reduced by ultrastructural analysis.5s6 This may be a general feature of a stimulated epidermis, thus being nonspecific for psoriasis. Since normal epidermal growth regulation may be impossible without a normal cell surface,5,6 such potential nonspecificity should not diminish the importance of this observation. As mentioned above, HL-A13 and HL-A17 gene products are cell surface constituents which have a three- to fourfold elevated frequency in psoriasis. These HL-A abnormalities may participate in the development of a psoriatic lesion and not be merely genetic markers. Several observations suggest this possibility. The H-2 gene complex in the mouse is analogous to the HL-A gene complex in man. In mice a particular H-2 genotype is one regulator of the level of cellular cyclic AMP.7 The cell surface membrane H-2 phenotype presumably is controlled by a specific H-2 genotype. By analogy, the HL-A genotype in man may do likewise. The abnormal HL-A psoriatic phenotype could alter cell adhesion-mediated growth control in the epidermis. The abnormal HL-A phenotype could alternatively interfere with potential physiological regulation of epidermal cyclic AMP content by endogenous beta catecholamine. We have previously shown that exogenously added beta catecholamine elevates the epidermal cyclic AMP content, an elevation which is blocked by propranolol.8 This is especially interesting in light of the suggested common evolutionary origin of HL-A antigens and beta receptors.9 Furthermore, Svejgaard and RyderlO have postulated that HL-A antigens may be cell surface hormone receptors. If so, certain HL-A phenotypes (i.e., HL-A13 and HL-A17), as well as others as yet unidentified) might lead to reduced binding of endogenous catecholamine to the beta receptor. In fact, such a situation might account for the observed propanolol induced, psoriasis-like rashes11 and the recent observation by Wiley and Weinstein12 of a sixfold increase in the number of epidermal cells synthesizing DNA induced by intradermal injection of propanolol into uninvolved psoriatic skin. These recent observations by Wiley and Weinstein12 are potentially important because, as will be discussed below, we believe the reduction in the function of the epidermal cyclic AMP system is central to the misregulated homeostasis of psoriatic epidermis.8 It will be important to determine whether the elevation in the number of DNA synthesizing epidermal cells in response to propanolol occurs in all psoriatic patients or specifically in those who are HL-A13 or HL-A17 or some as yet unknown HL-A phenotype. Cyclic Nucleotides in Psoriasis - In 1971 Voorhees and Duel1 proposed the first working model of a potentially deranged cyclic AMP system in psoriasis.13 The model was based on the fact that three characteristic features of the lesional epidermis of psoriasis are glycogen accumulation,8 decreased terminal differentiation8 and increased proliferation.14 In other tissues and experimental systems, cyclic AMP was capable of reversing

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these three abnormalities.8 In the model, reduced cyclic AMP function could either initiate a lesion, permit maintenance of a lesion, or both.13 Since the absence of these three abnormalities describes a normal epidermis, a key postulate of the model is that normal function of the cyclic AMP system is necessary for normal epidermal physiology, a major feature of which is regulated growth. In 1973 this model was modified to include the observed elevation of cellular cyclic GMP levels in association with induced cell proliferation. l5 Our present formulation suggests that reduced cyclic AMP function and elevated cyclic GMP function, or altered function associated with a decrease in the cyclic AMP/cyclic GMP ratio, is central to the deranged epidermal homeostasis characteristic of a psoriatic lesion. Since 1971 these possibilities have been explored in several laboratories, including our own, have been recently reviewed in detail elsewhere,l6 and will only be summarized here. At this time no direct, clear data are available which demonstrate a role for either cyclic AMP or cyclic GMP in epidermal physiology. However, a considerable amount of indirect, inferential and circumstantial evidence points to a probable highly significant involvement of cyclic AMP in epidermal physiology. This, along with the fact that cyclic AMP is a pivotal effector molecule in the physiological regulation of most tissues, makes it probable that epidermis will not turn out to be an exception. The role of cyclic GMP in cell physiology in general is currently unclear and thus the role of cyclic GMP as a proliferative effector in epidermis is highly speculative at this time. An elevation of cyclic AMP in epidermis 3 vitro thus far has proven to be an inhibitory signal for cell proliferation.8917918 However, due to the high concentrations of cyclic AMP elevating drugs employed in these studies in vitro, it is by no means certain what a given cellular level of cyclic AMP does in in vivo epidermis. It is possible that the metabolic status of the epidermis can at one time perceive a given cyclic AMP concentration as a proliferative signal and at another time interpret the same cyclic AMP content as an inhibitory proliferative signal. Furthermore, although the cyclic AMP content of a tissue is easy to measure and convenient to think about, it is the net function of the cyclic AMP system in the tissue that is important physiologically. The levels of cyclic AMP and cyclic GMP in psoriasis, although known, are disputed.16~19 The net function of dysfunction of the cyclic AMP and cyclic GMP systems in psoriasis has never been investigated. Said differently, cyclic AMP dependent protein kinase activity and substrate phosphorylation can be measured in psoriasis. However, the problems in interpreting such data in terms of physiology and/or pathophysiology are enormous. Thus extensive research will be required before any meaningful conclusions can be drawn about the cyclic nucleotide system in either psoriasis or epidermal physiology in general. Our measurements indicate that the cyclic AMp/cyclic GMP ratio in psoriatic lesions is reduced.16 The level of cyclic GMP is consistently increased16 whereas depending on the study, the level of cyclic AMP is either slightly increased,lg normal19 or modestly reduced.16 Perhaps the most important observation is that whereas most metabolic parameters in psoriasis are unequivocally elevated20r21 as is the case with any acti-

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vated metabolic state, the lesional content of cyclic AMP is either the same or 25% above or below that of uninvolved tissue. Our view is that the cyclic AMP system in a psoriatic lesion needs to be stimulated by additional cyclic AMP to generate an antiproliferative signal in the lesion. Said differently, the normal lesional cyclic AMP content or even a 25% elevation may be insufficient to stimulate the lesional cyclic AMP system to a level of activity which is capable of restoring normalcy to the lesion. This view in pharmacological terms means that if we use drugs or hormones to generate more cyclic AMP in the psoriatic lesion, a therapeutic result may be achieved. This approach is strengthened by several studies. As previously mentioned, work from a number of laboratories8~17~18~22-24 utilizing adult rodent or human epidermis in culture, indicates that cyclic AMP elevation is capable of inhibiting cell division. One study demonstrated the inhibitory effects of cyclic AMP elevating agents on psoriatic epidermal cells vitro.23 Two reports claim the in vitro stimulation of epidermal differentiation by cyclic AMP elevation. 18,25 These reports18,25 although consistent with our cyclic nucleotide model of psoriasis are inadequate because they do not adequately document the direct and specific role of cyclic AMP as an inducer of epidermal differentiation. Cyclic AMP has also been shown to stimulate the disappearance of glycogen from psoriatic lesions in vitro.8 Cyclic AMP is certainly but one possible regulator of proliferation and differentiation in epidermis. However, in several systems cyclic AMP appears to play a major role in growth and differentiation as exhaustively reviewed by Friedman.26 Also, cyclic AMP regulates glycogenolysis in several tissues.8 We thus feel justified in postulating therapeutic effects by raising psoriatic lesional cyclic AMP, which based on the aforementioned studies should reduce proliferation, induce differentiation and reduce lesional glycogen content and in so doing should normalize the lesion. To this end, several preliminary studies have already appeared. Stawiski et a12733 in two separate double blind clinical experiments have shown the superiority of papaverine and d,l-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (R020-1724) when used topically in comparison to control vehicle treatment. R020-1724 at a concentration of 1% was significantly better than 1% papaverine cream but neither was as good as topical triamcinolone. However, experimental determination of the correct drug concentration, dosage schedule and pharmaceutical formulation could transform either of these tools for research into clinical drug candidates. In less well controlled studies topical theophylline, with or without topical dibutyryl cyclic AMP or a cyclic AMP analog, has shown modest efficacy in psoriasis.29,30 An uncontrolled clinical trial of oral theophylline is said to have demonstrated beneficial results in psoriasis.31 In two uncontrolled clinical studies of intramuscular dibutyryl cyclic AMP, studies ins ired by our earlier biochemical studies on cyclic nucleotides in psoriasis,1 Chinese physicians in Shanghai considered the drug effective in 80% of 69 patients treated.32,33 In the Chinese studies, reported side effects were insignificant and thus it should be possible to duplicate their study but using a double blind design. In view of the excellent re-

P

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ported results with minimal side effects, such a controlled trial in our view should have a high priority. In light of these promising reports, the strong circumstantial evidence that cyclic nucleotides are significantly involved in the pathophysiology of psoriasis, and the certainty that it will be several years before definitive data are available on the role of cyclic nucleotides in psoriatic pathophysiology, it seems reasonable to suggest that further clinical pharmacology utilizing the cyclic AMP approach is warranted at this time. This seems especially important since conventional systemic treatment (anticancer drugs, glucocorticoids and oral psoralen plus ultraviolet light type A) either has known serious side effects or serious potential sequelae. Since cyclic AMP is the second messenger of many peptide hormones and neurotransmitters, one’s initial reaction to cyclic AMP therapy for any disorder might be negative on the grounds that cyclic AMP would activate multiple organs. However, local and systemic therapy of asthma, which in our view is probably largely the result of elevated cyclic AMP levels, has been used for years with great benefit and minimal side effects when administered by a physician versed in the pharmacology of sympathomimetic agents. Clearly several approaches to the cyclic AMP therapy of psoriasis are possible. Epidermis accumulates cyclic AMP in vitro when exposed to isoproterenol, epinephrine, norepinephrine, E-type3grostaglandins, adenosine, dopamine, salbutam01~~ and histamine (H2 type). Recent studies by Duell, Terpenning and Collins36 show that epidermis probably contains a beta receptor of the beta2 type. Thus clinical pharmacology using beta2 catecholamine agcnists, such as salbutamol or terbutaline, with or without a cyclic AMP phosphodiesterase inhibitor either topically or systemically appears experimentally feasible. When contemplating such a study, it must be remembered that agonists which stimulate the accumulation of cyclic AMP in several tissues lead to both agonist specific and nonspecific tachyphylaxis.37 According to one group of investigators, the psoriatic epidermal beta receptor is relatively insensitive to cyclic AMP accumula ion induced by beta catecholamine (epinephrine) under in vitro conditions. If this abnormality can be confirmed by other groups, it may suggest that the beta catecholamine approach is less desirable than another agonist such as a suberythema or minimal erythema concentration of topical prostaglandin of the E series. Other potential candidates for evaluation are the newly discovered prostacyclin,39 which is an extremely potent stimulator of cellular cyclic AMP acc~mulation,4~ or a more stable analog of prostacyclin.

”

Cyclic AMP phosphodiesterase inhibitors, with or without an appropriate agonist are also candidates for clinical experimentation. In preliminary work, which requires confirmation, we have reported greater low Km cyclic AMP phosphodiesterase activity in psoriatic lesions than in normal appearing areas in six patients.41 If this is the case in an in vivo lesion and is not an artifact of tissue preparation for biochemical analysis, a cyclic AMP phosphodiesterase inhibitor may be an excellent choice. We have shown that epidermal cyclic AMP phosphodiesterase is inhibited by caffeine, theophylline, diazepam, papaverine and R020-1724.34 The methyl xanthines are poor inhibitors, whereas papaverine and RO20-1724 are potent

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inhibitors leading to pronounced accumulation of epidermal cyclic AMP in vitro. R020-1724 in certain tissues selectively inhibits the hydrolysis of cyclic AMP 3. cyclic GMP.42 If we assume that cyclic GMP plays an important role in the psoriatic process, and if the cyclic AM€' phosphodiesterase inhibitory selectivity exists in epidermis, R020-1724 could be an unusually useful agent. In fact, as mentioned above, in preliminary studies we found that topical R020-1724 produced im rovement in 75% to 77% of 42 patients in two separate double blind studies.s8 Presumably, the improvement is the result of elevated lesional cyclic AMP, although other unknown actions of this agent could have produced the beneficial effect. Tachyphylaxis to agents which raise cyclic AMP was mentioned above.37 This is a serious concern when considering the potentialities of cyclic AMP therapy for psoriasis. The molecular basis of this tachyphylaxis is poorly understood, and what is understood is beyond the scope of this review.37943 Tachyphylaxis in this review should be considered to be synonymous with the term "tissue desensitization". This phenomenon has been known at the clinical level for years in asthma therapy. By proper choice of drug dose and time of administration, it should be possible to determine the proper therapeutic regimen in psoriasis as it has been determined in asthma. By attention to these details, it may be possible to experimentally determine an efficacious drug regimen for psoriasis. However, lack of attention to the tachyphylaxis issue by employing protocols without flexibility of drug dose and time of administration, may cause an experimental agent to be discarded as not sufficiently potent. It is unlikely that a regimen of cyclic AMP therapy for psoriasis will be discovered by the so-called "quick and dirty" approach. Another critical point is the interrelation between cyclic AMP and glucocorticoids. It is quite clear from a number of studies44-47 that in order for cyclic AMP to regulate cell function in a normal manner, a certain critical amount of glucocorticoid must also be exerting or have exerted its action in the same cell. In general, it appears that cyclic AM€' and glucocorticoid induced metabolic signals move the cell in parallel directions but their modes of action appear to be different.46 In fact, in a series of elegant somatic cell genetic analyses, Coffin0 et a147 have shown that no mutable step is common to cyclic AMP and glucocorticoid in the case of lymphoma cell cytolysis produced by these two agents. The necessity of glucocorticoid for normal cyclic AMP function has been termed the "permissive effect" of glucocorticoids.44 It is not known whether this permissive effect can be extrapolated to therapeutics. However, it is possible that for additional cyclic AMP to function in a cell, there must also be additional glucocorticoid. Therefore, it might be that elevation of cyclic AMP in the psoriatic lesion would have, at best, a modest effect in the absence of added glucocorticoid. This concept can best be examined by applying to the skin a suboptimal concentration of a cyclic AMP elevating agent such as theophylline or R020-1724, plus an amount of glucocorticoid which, by itself, has no appreciable effect in psoriasis. One could then observe whether this combination would have a greater therapeutic effect than either agent alone. If s o , such a combination would have the advantage of efficacy without the side effects of long term gluco-

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corticoid therapy (thinning and tearing of the skin) which are frequently seen when glucocorticoids alone are used at therapeutic doses. Arachidonic Acid Transformations - Only infrequently do the patches of psoriasis coalesce to produce total body involvement. The coalescence occurs by the extension of centrifugally enlarging patches. Therefore, it is unlikely that cyclic nucleotides in the skin are regulated by circulating hormones. If this were the case, it would seem that total body psoriasis would be the rule rather than the exception. We have therefore searched for other factors which would be produced locally and also regulate the metabolism of cyclic nucleotides. Psoriasis is induced in the genetically predisposed person1 by cutaneous trauma.3 Trauma to skin and other tissues causes the release of free arachidonic acid from membrane bound phospholipids. Arachidonic acid is then transformed to a series of biologically potent substances, several of which have been discovered by Samuelsson and which are the subject of an excellent review.48 Prostaglandins E2 and F2a (derivatives of arachidonic acid) are elevated only 40% and 86%, respectively, in lesional tissue.49950 However, another derivative of arachidonic acid, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) and free arachidonic acid itself are elevated 82- and 26-fold, respectively.49 Arachidonic acid can stimulate the formation of cyclic GMP51952 and may be able to inhibit the formation of cyclic AMP. Prostaglandins of the E series cannot only stimulate the synthesis of cyclic AMP,49 but on prolonged bathing of a tissue (such as a psoriatic lesion) in prostaglandin, may do just the opposite (i.e., by tissue desensitization37 render the tissue refractory to further production of cyclic AMP by prostaglandin). How or if the 82-fold excess of HETE participates in lesional pathophysiology is unknown. Nevertheless, it is of great interest to note that treatment of psoriasis with topical glucocorticoid reduced the lesional content of arachidonic acid and HETE to normal.50 This presumably is due to inhibition of phospholipase A2 by glucocorticoid. The net result would be a normalization of all components of the arachidonic acid cascade distal to phospholipase A2 and any misregulated cyclic nucleotide metabolism produced by cascade constituents.50 Clearly, new non-glucocorticoid drugs which reduce tissue levels of arachidonic acid and those which would preferentially inhibit the synthesis of HETE by the lipoxygenase will be tried, Perhaps certain of these agents will possess activity against the inflammatory proliferative skin disease psoriasis and other so-called steroid responsive diseases. Polyamines and Psoriasis - The polyamines (putrescine, spermidine and spermine) have been found to be elevated in all proliferating cells thus far examined.53 Some unknown but critical level of polyamine is thought to be necessary for a cell to synthesize DNA. Therefore, reducing the polyamine level in a proliferating cell below some critical point can block cell proliferation.54 Thus we examined the lesions of psoriasis for increased polyamine levels. The levels of all three polyamines55 and the corresponding three biosynthetic enzymes56 were elevated in involved areas in comparison with uninvolved areas. Interestingly, treatment of these patients with topical glycocorticoids markedly reduce the activities of the three poly-

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amine forming enzymes.56 Also, alpha methylornithine inhibited the first (ornithine decarboxylase) of these three enzymes, and methylglyoxyal b i s guanylhydrazone inhibited the two subsequent enzymes (the putrescinestimulated and the spermidine-stimulated S-adenosyl methionine decarboxylase). It may be that either these nonsteroidal agents or others can be used to reduce polyamines to a sufficiently low level in psoriatic patches to prevent DNA synthesis or reduce its rate to normal. It is clear that several experimental approaches to the therapy of psoriasis are available, approaches based on modulation of known misregulated molecular mechanisms. We are currently evaluating some of the approaches discussed in this review. and hope to evaluate others as new agents become available.

REFERENCES 1. W. Watson, H. M. Cann, E. M. Farber and M. L. Nall, Arch. Derm. 105, 197 (1972). 2. A. Svejgaard, P. Platz, L. P. Ryder, L. S. Nielsen and M. Thomsen, Transplant. Rev. 2, 4 (1975). 3. E. M. Farber, R. J. Roth, E. Aschheim, D. D. Eddy and W. W. Epinette, Arch. Derm. 91, 246 (1965). 4. J. J. Voorhees, E. A. Duell, D. A. Chambers and C. L. Marcelo, J. Invest. Dermatol. 67,15 (1976). 5. E. H. Mercer and H. I. Maibach, J. Invest. Dermatol. 51, 215 (1968). 6. G. Mahrle and C. E. Orfanos, Brit. J. Dermatol. 96, 215 (1977). 7. D. Meruelo and M. Edidin, Proc. Nat. Acad. Sci. U S A G , 2644 (1975). 8. J. J. Voorhees, E. A. Duell, M. Stawiski and E. R. Harrell in "Advances in Cyclic Nucleotide Research", Vol. 4, P. Greengard and G. A. Robison, Ed., Raven Press, New York, N.Y., 1974, p. 117. 9. E. Smeraldi and R. Scorza-Smeraldi, Nature 260, 532 (1976). 10. A. Svejgaard and L. P. Ryder, Lancet 2, 547 (1976). 11. H. AE. Jensen, H. I. Mikkelsen, S. Wadskov and J. Sdndergaard, Acta med. scand. 199, 363 (1976). 12. H. Wiley and G. Weinstein, J . Invest. Dermatol. 68, 239 (1977). 13. J. J. Voorhees and E. A. Duell, Arch. Derm. 104, 352 (1971). 14. G. D. Weinstein and P. Frost, Arch. Derm. 103, 33 (1971). 15. J. W. Hadden, E. M. Hadden, M. K. Haddox and N. D. Goldberg, Proc. Nat. Acad. Sci. USA 69, 3024 (1972). 16. J. J. Voorhees in "Annual Review of Medicine", Vol. 28, W. P. Creger, Ed., Annual Reviews, Inc., Palo Alto, California, 1977, p. 467. 17. C. Delescluse, N. H. Colburn, E. A. Duell and J. J. Voorhees, Differentiation 2, 1 (1974). 18. D. P. Chopra, Brit. J . Dermatol. 96, 255 (1977). 19. K. Yoshikawa, K. Adachi, K. M. Halprin and V. Levine, Brit. J . Dermatol. 2, 253 (1975). 20. K. M. Halprin and A. Ohkawara, J. Invest. Dermatol. ,6, 51 (1966). 21. K. M, Halprin and J. R. Taylor in "Advances in Clinical Chemistry", Vol. 14, 0. Bodansky and A. L. Latner, Ed., Academic Press, N.Y., N.Y., 1971, p. 319.

170 22. 23. 24. 25. 26. 27. 28. 29. 30.

31. 32. 33. 34. 35. 36. 37* 38. 39.

40. 41.

42. 43.

44. 45.

46. 47.

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F. Marks and W. Rebien, Naturwissenschaften, 2, 41 (1972). R. A. Harper, B. A. Flaxman and D. P. Chopra, J. Invest. Dermatol. 62, 384 (1974). J, E. Birnbaum, T. M. Sapp and E. L. Tolman, J. Invest. Dermatol. 67, 235 (1976). C. Delescluse, K. Fukuyama and W. L. Epstein, J. Invest. Dermatol. 66, 8 (1976). D. L. Friedman, Physiol. Rev. 56, 652 (1976). M. A. Stawiski, J. A. Powell, P. G. Lang, M. A. Schork, E. A. Duell and J. J. Voorhees, 3. Invest. Dermatol. 64, 124 (1975). M. Stawiski, L. Rusin, M. A. Schork, T. Burns, E. Duell and J. Voorhees, Clin. Res. 24, 267 (1976). P. Laugier, T. Posternak, M. Orusco, G. Cehovic and F. Posternak, Bulletin de la Societe Francaise de Dermatologie et de Sypiligraphie, 80, 632 (1973). F. Posternak, T. Posternak, M. Orusco, G. Cehovic and P. Laugier, Journees Nationales de Dermatologie, 103, 640 (1976). L. Texier, 0 . Gauthier, Y. Gauthier, J.-M. Tamisier and M. -M. Delaunay, Journees Nationales de Dermatologie 103, 645 (1976). Chinese Medical Journal, No. 4, 201 (1974). Chinese Medical Journal, 2(2), 141 (1976). J. J. Voorhees and E. A. Duell in "Advances in Cyclic Nucleotide Research", Vol. 5, G. I. Drummond, P. Greengard and G. A. Robison, Ed., Raven Press, New York, N.Y., 1975, p . 735. H. Iizuka, K. Adachi, K. M. Halprin and V. Levine, Biochim. Biophys. Acta, 437, 150 (1976). E. A. Duell, M. Terpenning and J. Collins, Fed. Proc. 3,346 (1977). B. H. Leichtling, A. M. Drotar, R. Ortmann and J. P. Perkins, J. Cyclic Nucleotide Research 2 , 89 (1976). M. M. Mui, S. L. Hsia and K. M. IIalprin, Brit. J. Dermatol. 92, 255 (1975). S. Moncada, R. Gryglewski, S. Bunting and J. R. Vane, Nature 263, 663 (1976). J. E. Tateson, S. Moncada and J. R. Vane, Prostaglandins g,389 (1977). J. J. Voorhees, N. H. Colburn, M. Stawiski, E. A. Duell, M. Haddox and N. D. Goldberg in "Control of Proliferation in Animal Cells", R. Baserga and 13. Clarkson, Ed,, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y., 1974, p. 635. H. Sheppard, G. Wiggan and W. H. Tsien in "Advances in Cyclic Nucleotide Research", Vol. 1, P. Greengard, R. Paoletti and G. A . Robison, Ed., Raven Press, New York, N.Y., 1972, p. 103. 6. Mukherjee and R. J. Lefkowitz, Proc. Natl. Acad. Sci. USA, 73, 1494, (1976). T. B. Miller, J. H. Exton and C. R. Park, J. Biol. Chem. 246, 3672 (1971). J. H. Exton, N. Friedmann, E. H. Wong, J. P. Brineaux, J. D. Corbin and C. R. Park, J. Biol. Chem. 247, 3579 (1972). E. B. Thompson and M. E. Lippman, Metabolism 23, 159 (1974). P. Coffino, H. R. Bourne, J. Hochman, P. Insel, K. L. Melmon and G. M. Tomkins, J. Invest. Dermatol. 67,648 (1976). B. Samuelsson, E. Granstrom, K. Green, M. Hamberg and S. Hammarstrom,

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in "Annual Review of Biochemistry", Vol. 4 4 , Esmond E. Snell, Ed., Annual Reviews, Inc., Palo Alto, California, 1975, p . 669. S . Hammarstrom, M. Hamberg, B. Samuelsson, E. A. Duell, M. Stawiski and J. J. Voorhees, Proc. Nat. Acad. Sci. USA 72,5130 (1975). S . Hammarstrom, M. Hamberg, E. A. Duell, M. A. Stawiski, T. F. Anderson and J. J. Voorhees, Science, in press (1977). D. B. Glass, W. Frey 11, D. W. Carr and N. D. Goldberg, J. Biol. Chem. 252, 1279 (1977). D. Wallach and I. Pastan, J. Biol. Chem. 251, 5802 (1976). D. H. Russell, Life Sciences 13,1635 (1973). P. S . Mamont, P. Bohlen, P. P. McCann, P. Bey, F. Schuber and C. Tardif, Proc. Natl. Acad. Sci. USA 73, 1626 (1976). D. H. Russell, M. A. Stawiski, E. A. Duell and J. J. Voorhees, Pharmacologist 18,157 (1976). D. H. Russell, W. L. Combest, E. A. Duell, M. A. Stawiski, T. F. Anderson and J. J. Voorhees, Fed. Proc. 36, 970 (1977).

172 Chapter 18. Activators of Dopamine and B-Adrenergic Adenylate Cyclases Herbert Sheppard, Hoffmann-La Roche Inc., Nutley, N.J.

The three major natural catecholamines are epinephrine, from the adrenal medulla, norepinephrine (NE) from specific nerve endings and dopamine (DA) from other nerve endings lacking the enzyme dopamine beta-hydroxylase. These agents are free to act on specific receptors which have been described in pharmacological terms as a,B1 and most recently DA2 types. It should be noted that while DA has been found to be a weak agonist of the a and B receptors, NE is a modest agonist of the DA receptor. Many tissues contain 6 receptors and these are prominantly associated with the relaxation of the smooth muscle of the vasculature and bronchioles as well as the contraction of cardiac muscle3. Recent work has shown them to be involved in the proliferation and differentiation of cell^^'^ as well as the release of’hormones6 and enzymes7. Doparnine receptors in the periphery are also involved in relaxation of certain vascular beds and the release of hormones but most attention has focused on their role in central nervous system function. Insights into the role of DA in Parkinson‘s disease and psychosis have emerged and grown through the use of dopamimetic agents like L-DOPA and DA antagonists such as the antipsychotics*. Subsequent to the findings of the Sutherland group8, evidence from laboratories too numerous to list support the idea that the B-adrenergic receptor is coupled to the generation of cyclic AMP (CAMP) while the a receptor, with few exceptions, is not. The picture with the DA receptor is less clear. Recent neurophysi~logical~’and pharmacological1 evidence suggests that, as with NE responses, there may be at least two types of DA responses. In analogy with the NE responses, it would not be surprising to find one DA response associated with the generation of CAMP and another not. DA receptors coupled to adenylate cyclase (AC) have been described for membrane preparations from a number of tissues and these are discussed in a recent review12. The availability of a DA sensitive AC made it possible to extend structure-activity relationship (SAR) studies to a level comparable to that carried out with the 6 system. The availability of both types of AC coupled receptors made it possible to ascribe the activity of a particular agonist or antagonist to interactions at a level considered to be the earliest in a series of biochemical events leading to the physiological responses generally measured in intact tissues or cells. More recently, the binding of agonists and antagonists to membrane fragments has been used as a more direct measure of interactions with the receptor but it should be kept in mind that binding of substances to membranes include interactions other than that of ligand to cyclase receptor13. It becomes necess8ry, therefore, to correlate the data with some response of these membrane fragments and in this regard the AC serves as an excellent model13. It should also be kept in mind that binding data do not yet distinguish between agonist and antagonist although some modifications

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are being developed which may make that possible14. During the last two years a number of papers have appeared which use AC for drug screening or for mechanism of action studies. Both approaches have generated data which can be helpful for the development of SARs as to potency and specificity. This report will discuss agonists and their inactive analogues and derivatives which have been tested on the B and DA adenylate cyclases and attempt to define structural requirements for enzyme activation. Antagonists, other than those which are derivatives of agonists, will not be covered.

Phenethylamines: I. fi-Adrenergic cyclase

3d:

4

A. Aromatic group: Substitution of the 3-OH group of isoproterenol (ISO) with a methylsulphonamido (MSA) group, -NH-S02-CH3, yielded soterenol, with equal or greater affinity15’16’ l7 but reduced intrinsic activity16’17. Inversion of the 8-OH lowered potency and its removal lowered it even morel6. Deoxysoterenol showed only antagonist activity17. Other 3-MSA derivatives with a cyclopropane (MJ 8798-1) or a 2,2-(CH3) 2-phenethyl (MJ 9184-1) on the nitrogen possessed good agonist activity17. The 3-MSA derivative of DA, however, was inactive with the rat red blood cell (RBC) enzyme15. The 4-MSA derivatives with or OH NHCH(CH3)2 without the 3-OH possessed only antagonist 1 Another agonist with a activity15’l6*l7. nitrogen at position 3 is quinterenol (1)16.

@&

Substitution of an hydroxymethyl group for the 3-OH of IS0 yielded salbutamol which has agonist activity with frog RBC16 but not rat ventricle cyclasel8. The 4-deoxy-IS0 (S-40045-9) and 4-deoxy-N-ethyl-NE (S-4O032-7)l7 were inactive on the frog RBC enzyme. With the rat RBC enzyme 4-deoxy-a-methyl NE (metaraminol) but not 4-deoxy DA (metatyramine) had agonist activitylg. Metaraminol and phenylephrine, however , were only antagonists with the frog RBC17 and rat ventricle cyclaeel8. Interestingly, moving the 4-OH of IS0 to position 5 led to reduced but detectable agonist activitylg. DA was inactive with the RBC enzyme of the frog16’17 but weakly active with that of the rat15. The lack of response to DA by the frog RBC enzyme may be a matter of sensitivity since the affinity of IS0 and other agonists appears to be about ten times greater in the rat RBC cyclase15y16y17. The methylation of either or both agonist activity but did not result in While a methyl group at position 2 had reduced activity19. Activity was also to position 5 or 6 of NYN-(CH3)2-DA or position 6 of DA19.

ring hydroxyls of DA reduced the formation of an antagonist19. no effect, a phenyl group markedly lost If a methyl group was added an OH or MI2 group was added to

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B. Nitrogen substitutions: Alkyl substitution generally increased activity such that ieopropyl>ethyl>methyl>H in the $-OH In the 8-deoxy series, the order of potency was methybethyb isopropyl>propyl>H19. The S-isomers of the 8-OH series were much less potent with methyl and isopropyl derivatives about equiactivel ” and the isobutyl inactive18. N,N-dimethyl DA had reduced activity and the addition of a third methyl group led to inactivity15. The activities of the 8-OH and 8-deoxy compounds generally increased with phenethyl substitutions17’ 20. C. Side chain substitution: As indicated above, inversion of the 8-OH led to compounds comparable in activity t o the 8-deoxy analogues but appreciably below that of the R-conformers. It is clear that the 8-OH is not essential for 8 agonist activity. The insertion of an -0CH2- between the @-carbon and the aromatic ring of I S 0 (MJ-9910) increased the affinity for the frog RBC16 cyclase. The observed reduced intrinsic activity is consietant with the 8-adrenergic blocking activity usually associated with such side chains17,

Substitution on the a-carbon has not been extensively studied but addition of a methyl to either DA or NE resulted in enhanced activity17’19. In the DA series, the increased activity was confined to the S-conf~rmer~~. Addition of an ethyl group to the a-carbon of NE had the same enhancing effect as a methyl group but a reduced effect when added to ISOl8. 11.

The DA cyclase

In general, substitutions on the aromatic ring of DA have changed the agonist potencies in a fashion similar to that observed for the 8-agonists. None of the alterations increased the activity while a marked decrease was noted with a phenyl group at position 2, a methyl group at positions 5 or 6, an OH, NH2, or NO2 at position 6, removal or methylation of either the 3 or 4-OH13, and a CH20H in place of the 3-OH21. The 4-deoxy compound, m-tyramine, was reported t o have either or no22 agonist activity, while metaraminol had nonel9. Substitution of a MSA for the 3-OH group of DA ( M J 7582) reduced activity only slightly19. N-Methylation had little effect on the activity of DA19’22. The addition of two methyl groups reduced activity to different d e g r e e ~ l ~ ’ ~ ~ . N,N,N-Trimethyl DA was reported to be inactive19 or as active as N,Ndimethyl DA22. Agonist potency decreased markedly with an increase in size of the alkyl group19. Surprisingly, N,N-diethyl DA was slightly more potent than N-ethyl DA and N-isoprop 1-N-methyl DA was weakly active compared to the inactive N-isopropyl DAfs Interestingly, replacement of the methyl or n-propyl group of N-methyl-N-n-propyl DA with an n-butyl increased the potency close to that of N-methyl DA23.

.

The @-OH reduced activity such that the rank order of potency was DA>R-NE>S-NE>>R-IS019’2z, Thus the S-conformer of NE does not have the potency of the 8-deoxy compound (DA) as noted with the 8-system.

Activators of Adenylate Cyclases

Chap. 18

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Reducing or increasing the length of the side chain by one carbon resulted in a loss of agonist activity22. Methylation of the ,-carbon also markedly lowered activity1gy22,with the S-isomer being less potent than the R conformerl9. Tetrahydroisoquinolines:

67

w

N 1

-

It has been postulated that the aldehydic products of the oxidation of ethanol and dopamine can condense with dopamine to form 6,'I-dihydroxytetrahydroisoquinoline (THI) with a methyl24925 or 3,4-dihydroxybenzyl20 substituent in position 1, respectively.

8-Adrenergic cyclase: Tetrahydropapaveroline (THP), which repreeents the condensation of DA with 3,4-dihydroxy-phenacetaldehyde, was about as active as NE20. The S-isomer was preferred and N-methylation destroyed activity20. Agonist activity was also lost if the 6,7-hydroxy groups were methylated or a third hydroxyl was introduced in position 5 of the 3,4-dihydroxybenzyl portion20. Activity was not appreciably altered if the benzyl hydroxyls were methylated, replaced by a p-C1 group or only the 3-OH group was removed20. Activity was reduced markedly by substituting a methyl, dimethoxyphenyl, or p-C1-phenethyl group at position 120. The most potent compound of the series and more active than IS0 was trimetaquinol which has a 3,4,5-trimethoxybenzyl substituent at position 116y20. The S-isomer was about 1000 x more potent than the R-conformer20. In many cases, the potency of a THI derivative was greater than that of its open ring analogue20. All B-agonists had reduced intrinsic activity which could be ascribed to their ability to act as antagonists20, as noted earlier for trimetaquinoll6. THI compounds without the 6,7-hydroxyls but with dihydroxy or dimethoxybenzyl substituents at position 1 exhibited only antagonist activity20. As noted for agonist activity, the S-isomer was preferred and N-methylation reduced activity appreciably20. DA cyclase: Most THI derivatives were not DA agonists but several were antagonists. Very weak agonist activity was noted for the N-CH3 derivative of 1-H and 1-CH3-6, 7-dihydroxy-THIZ7 and the nor-1-H compoundz2. Again the S-isomer was more potent but N-methylation tended to increase antagonist activityz0. A benzyl substituent appears to be important for antagonist activity since the 1-methyl and 1-dimethoxyphenyl compounds were inactive20. Aporphines: These compounds are of interest because of the well known action of apomorphine on DA systems in vivo12. Very 2 little work has been reported with respect to the action of these compounds on B-systems other than the inhibitory action of apomorphine (APO) on the rat RBC cyclase28. The action of APO on DA cyclase is one of 11 stimulation at low concentrations and inhibition at high concentration^^^'^^ with the result that the 10 9 intrinsic activity is less than that of DA although

F-

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potency is generally enhanced. The rank order of agonist potency was N-ally1 APO>2-hydroxy-APO=APO>N-desmethyl APO' 5. The N-n-propyl derivative is another potent agonistZ2. However, removal of the 11-OH or its transfer to position 9 (isoapomorphine) resulted in l o s s of agonist Methylation of the 10-OH of APO (apocodeine)20 or of its Nactivity3'. The S-isomer of D O 2 ' , n-propyl analogue abolished agonist activity3'. the APO analogue with the OH groups at 1,2 rather than 10,1130 and bulbocapnine, the 1,2-methylenedioxy-l0-methoxy-S-conformer of A P O , were antagonists20'30. One derivative, (S)-1,2,9,10-tetrahydroxy-noraporphine is a 8agonist31 whose formation from THP was suggested32. Tetrahydroprotoberberines: The formation of tetrahydroprotoberberines (THPB) from THP was also One such derivative was the 2,3,9,10-tetrahydroxy-THPB which had no B-agonist activity3 Racemic 2,3,1O,ll-tetrahydroxy THPB exhibited 8-agonist activity weaker than THP34. A number of other THPB derivrives elicited only DA antagonist activity. 10 Among the 2,3-dihydroxy analogues with 11 hydroxyl groups also at positions 9,lO or lO,ll, the S-conformer was significantly more potent than the R-isomer2'. Steric preference was not evident if methoxy groups were in those positions20. The presence of a 2,3-methylenedioxy group in addition to methoxy groups at 8,10, as in canedine, did not alter potency significantlyz1.

:@?&

.

Miscellaneous compounds:

ET-495

<wNzN G M ~> G D >~ ~ ~~ 2 1 3is1 similar t o t h e s p e c i f i c i t y of t h e ganglioside-TSH i n t e r a c t i o n and s i n c e t h e c l i n i c a l p r e s e n t a t i o n of t e t a n u s i n c l u d e s a syndrome which c l o s e l y resembles t h y r o i d storm. 32-34

-

When r e c e n t l y s t u d i e d , t e t a n u s t o x i n was shown t o i n t e r a c t w i t h thyr o i d plasma membranes. 35-38 More important, t h e p r o p e r t i e s of t h i s i n t e r a c t i o n were shown t o c l o s e l y resemble t h e p r o p e r t i e s of t h e TSH i n t e r a c t i o n w i t h TSH r e c e p t o r s on t h e s e membranes; t h e i n t e r a c t i o n of t e t a n u s t o x i n w i t h thyroid membranes e x h i b i t e d s e v e r a l c h a r a c t e r i s t i c s of i t s i n t e r a c t i o n w i t h n e u r a l t i s s u e ; t e t a n u s t o x i n would n o t bind t o membranes of a t h y r o i d tumor w i t h a TSH r e c e p t o r d e f e c t ; and t e t a n u s t o x i n could s t i m u l a t e thyr o i d hyperfunction i n mice s u b j e c t e d t o c o n d i t i o n s known t o p r e c i p i t a t e t h y r o i d storm. The a b i l i t y of t e t a n u s t o x i n t o i n t e r a c t w i t h thyroid membranes and w i t h TSH r e c e p t o r s on t h e s e membranes, as suggested above, h a s s e v e r a l s i g n i f i c a n t i m p l i c a t i o n s and a p p l i c a t i o n s . An obvious c l i n i c a l i m p l i c a t i o n is t h a t t h e syndrome of sympathetic o v e r a c t i v i t y which can appear i n t e t a n u s is c o n t r i b u t e d t o by t h e a b i l i t y of t h e t o x i n t o induce t h y r o i d hyperfunct i o n . This syndrome would t h u s be analogous i n i t s e t i o l o g i c a l r e l a t i o n s h i p t o t h e t h y r o i d a s i s t h y r o i d storm o r a s i s t h e h y p e r s e n s i t i v i t y t o

Chap. 22

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catecholamines demonstrable in thyrotoxic patients. A second implication is that tetanus toxin will have a receptor structure and mechanism of effecting cell processes analogous to cholera toxin, the glycoprotein hormones, and interferon. It is thus no surprise that tetanus toxin has two different functional subunits, only one of which is responsible for its interaction with gangliosides.39-41 It can be presumed that tetanus toxin will have the common primary sequences within these two subunit or structural components already demonstrated for cholera toxin and the glycoprotein hormones; that tetanus toxin will undergo a specific conformational change upon interacting with the receptor; and that the tetanus toxin action will also require or induce a change in membrane state or a reordering of membrane components. In short, it seems reasonable to presume that tetanus toxin interactions with the TSH receptor on thyroid plasma membranes are analogous to its interactions with neural tissue membranes and that studies which compare the tetanus toxin effects on thyroid membranes with those of TSH will clarify both the mechanism by which tetanus exerts its neurotoxic effects and TSH its thyroid-stimulating effects.35-38 VI. The Mechanism by Which Glycoprotein Hormones, Interferon, and the Bacterial Toxins Effect Cell Changes - Current views regarding the mechanism of action of TSH and other glycoprotein hormones invoke alterations in the concentration of cyclic AMP as the second event in the transfer of information from the hormones to the appropriate ce1l.l That is, the information carried by the hormone is purportedly translated into a "second message" by means of a change in intracellular cyclic AMP concentration. One implication of the schema outlined above is that the increase in intracellular cyclic AMP may not necessarily represent a second message per se, but rather an event which is sequentially or simultaneously coupled to a primary effect of these agents on the membrane. In other words, the common denominator of all of the effectors may be an alteration in the membrane which leads to generalized effects on cellular metabolism, one of which may be an increase in the concentration of intracellular cyclic AMP. Viewed in this manner, it is clear that one means of accomplishing this end could be through alterations in electrochemical ion gradients across the cell membrane. In this regard, the following observations are notable: (i) The Erimary effect of tetanus toxin is an alteration in neural trans(ii) cholera toxin causes a dramatic loss of electrolytes and mission; TSH increases the rate water through the intestinal e p i t h e l i ~ m ; ~(iii) ~ and extent of iodide uptake by thyroid cells;44 (iv) oubain, the classic inhibitor of sodium, Eotassium-stimulated ATPase, blocks the antiviral action of interferon; and (v) electrochemical ion gradients play a primary role in active transport and metabolism in prokaryotes, eukaryotes, and many subcellular organelles. 46 With these items in mind, a recent development is the demonstration that TSH causes an increase in the uptake of the lipophilic cation triphenylmethylphosphonium (TPMP+) when added to cultured thyroid cells or to membrane vesicles derived from these cells under appropriate conditions and that this effect is dependent upon a specific interaction of TSH with its receptor at the cell surface.47 Since uptake of TPMP' and other lipophilic

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c a t i o n s has been shown t o r e f l e c t t h e presence of an e l e c t r i c a l p o t e n t i a l a c r o s s t h e membrane ( i n t e r i o r n e g a t i v e ) , i t i s tempting t o s p e c u l a t e t h a t one of t h e primary e f f e c t s of TSH i s t o hyperpolarize t h e thyroid c e l l membrane. Experiments with thyroid s l i c e s u t i l i z i n g a d i r e c t e l e c t r o physiological approach48 a r e c o n s i s t e n t with t h i s i n t e r p r e t a t i o n , i.e. , TSH a t low concentrations r e s u l t e d i n increased v o l t a g e measurements. Since the e f f e c t of TSH on TPMF'+ uptake precedes t h e e f f e c t of t h e hormone on adenylate cyclase a c t i v i t y , 4 7 t h e p o s s i b i l i t y e x i s t s t h a t a primary mode of a c t i o n of each of t h e s e e f f e c t o r s i s t o a l t e r electrochemi c a l i o n g r a d i e n t s a c r o s s t h e c e l l membrane. This concept i s a t t r a c t i v e i n t h a t i t would s e r v e t o e x p l a i n c e r t a i n o t h e r f i n d i n g s . Thus, hCG causes changes i n a d r e n a l c e l l ion t r a n s p o r t which not only precede adenylate cyclase s t i m u l a t i o n , but occur a t concentrations of t h e e f f e c t o r which have minimal e f f e c t s on cyclase a c t i v i t y ; 4 9 cholera toxin and i t s B p r o t e i n induce a l t e r a t i o n s i n t h e permeability of liposomes r e c o n s t i t u t e d with 51 "receptor" ganglioside i n t h e absence of adenylate cyclase.

-

V I I . S t r u c t u r a l Features of t h e Receptor It has been reported previously t h a t TSH r e c e p t o r s on bovine thyroid plasma membranes can be s o l u b i l i z e d with l i t h i u m d i i o d o s a l i c y l a t e ( L I S ) and t h a t t r y p t i c d i g e s t i o n of t h e solub i l i z e d receptor preparation y i e l d s a 24,000 molecular weight receptor fragment which r e t a i n s s p e c i f i c TSH binding a c t i v i t y . 52'53 Analysis of a p u r i f i e d preparation of t h i s receptor fragment i n d i c a t e d t h a t i t w a s a glycoprotein containing 30% carbohydrate and 10% s i a l i c a c i d by weight. 52'53 The above discussion i n d i c a t e s t h a t gangliosides may be important compon e n t s of t h e TSH receptor and t h a t t h e r o l e of gangliosides i n t r a s m i t t i n g t h e hormonal message t o t h e c e l l machinery i s analogous t o t h e i r r o l e i n t r a n s m i t t i n g t h e message of cholera t o x i n t o c e l l s exposed t o t h i s bact e r i a l product.

Recent s t u d i e s 5 4 have shown t h a t t h e two components of thyroid plasma membranes known t o i n t e r a c t with TSH, t h e glycoprotein with s p e c i f i c TSH binding a c t i v i t y , and t h e gangliosides of t h e thyroid membranes, segregate d i f f e r e n t l y when membranes a r e s o l u b i l i z e d with l i t h i u m d i i o d o s a l i c l a t e . More important, t h e p o s s i b i l i t y r a i s e d i n t h i s study was t h a t t h e lZ5I-TSH binding a c t i v i t y of t h e i n t a c t plasma membrane i s contributed to by both t h e glycoprotein t h e ganglioside components of t h e membrane. Thus, t h e s a l t s e n s i t i v i t y of t h e i n t a c t plasma membrane receptor as w e l l as i t s high degree of hormonal s p e c i f i c i t y i n regard t o o t h e r glycoprotein hormones (LH and hCG, f o r example) appears t o r e f l e c t t h e ganglioside-TSH i n t e r a c t i o n . The conclusion concerning t h e s a l t s e n s i t i v i t y of t h e TSH-ganglioside i n t e r a c t i o n has been confirmed i n d i r e c t 1251-TSH binding experiments using ganglioside-containing liposomes. 55 The high degree of hormonal s p e c i f ic i t y i s evident i n t h e c l o s e c o r r e l a t i o n of i n h i b i t i o n of binding with t h e conformational changes e f f e c t e d i n t h e d i f f e r e n t hormones by t h e d i f f e r e n t gangliosides ( s e e above) and i s i n c o n t r a s t t o t h e l o s s of s e c i f i c i t y f o r LH and hCG exhibited by t h e glycoprotein r e c e p t o r fragment. 5 8 ' 5 3 y 55 I n sum, these d a t a coupled with previous observations concerning ( i ) t h e protease s e n s i t i v i t y of TSH binding and c y c l a s e s t i m u l a t i o n i n c u l t u r e d

Chap. 22

Cell Surface Receptors

Kohn

219 -

thyroid cells56 and (ii) the l o s s of TSH binding and cyclase stimulation in a thyroid tumor whose membranes are deficient in their ganglioside contentY7 suggest that each component contributes to the function of the intact plasma membrane receptor and that the functional transmission of the TSH message to the thyroid cell machinery requires the presence of both a glycoprotein and glycolipid component in the TSH receptor structure.54

It is not clear how the two components function in the binding of TSH and in the transmission of the TSH message to the cell machinery. The binding of TSH may involve both components simultaneously or, more likely at this moment, there may be a sequential interaction with one (the glycoprotein) and subsequent transfer or association with the second (the ganglioside), i.e., one can be the primary interaction site (the glycoprotein) and the other (the ganglioside) can be the obligatory discriminator and coupler to other cell processes.54 The existence of both glycolipid and glycoprotein membrane components with receptor specificity has recently been noted in studies of cryoglobulin interactions with cell membranes.57 It is also evident in the fact that there are both glycolipid and glycoprotein components of the cell with blood group specificity.5 8 The latter precedent is especially pertinent since it suggests that the glycolipid and glycoprotein components of the TSH receptor may contain analogous oligosaccharide structures. The existence of membrane glycoproteins able to specifically bind the effector is not unique to TSH; thus, they have been shown for hCG59 and (in human KB-3 cells). Summary - The details of the schema outlined in the introduction of this review are not defined for each individual agent; however, the relationships are increasingly clear. Although the implications to the pathophysiology of Graves' disease and the mechanism of action of cholera and tetanus toxin are already obvious, it is clear they will extend to other problems. Thus, the observation that contraceptive programs involving the development of antibodies to hCG have also caused significant levels of antibodies to tetanus in some patients, may be the tip of an iceberg, i.e., the implications will extend even to a "normal" disease such as pregnancy. In regard to medicinal chemistry, these relationships clearly imply caution. Yet it is hoped they will hold new opportunities to the development of peptide and carbohydrate analogs which will either block the action of cell membrane effectors which can cause a disease (cholera or tetanus toxin) or mimic the action of effectors regulating body functions (glycoprotein hormones) and blocking viral infestation of the cell (interferon). References

1. G. A. Robison, R. W. Butcher, and E. W. Sutherland, Eds., "Cyc Academic Press, New York, New York, 1971. 2. L. Svennerholm, J. Neurochem., 10, 613 (1963). 3. V. Bennett and P. Cuatrecasas, "Cholera Toxin: Membrane Gang1 and Activation of Adenylate Cyclase," in "The Specificity and Animal, Bacterial, and Plant Toxins," in P. Cuatrecasas, Ed.,

ic AMP," osides Action of Chapman

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and Hall, New York, New York, in press.

4. B. R. Mullin, P. H. Fishman, G. Lee, S. M. Aloj, F. D. Ledley, R. J. Winand, L. D. Kohn, and R. 0. Brady, Proc. Natl. Acad. Sci. U.S.A., 73, 842 (1976). 5. FTD. Ledley, B. R. Mullin, G. Lee, S. M. Aloj, P. H. Fishman, L. T. Hunt, M. 0. Dayhoff, and L. D. Kohn, Biochem. Biophys. Res. Commun., 69, 852 (1976). 6. BFR. Mullin, S. M. Aloj, P. H. Fishman, G. Lee, L. D. Kohn, and R. 0. Brady, Proc. Natl. Acad. Sci. U.S.A., 73,1679 (1976). 7. M. F. Meldolesi, S. M. Aloj, P. H. Fishman, L. D. Kohn, and R. 0. Brady, Proc. Natl. Acad. Sci. U.S.A., 73,4060 (1976). 8. L. D. Kohn, "Horizons in Biochemistry and Biophysics," E. Quagliariello, Ed., p. 123, Addison-Wesley Publishing Co., Reading, Massachusetts, 1977. 9. L. D. Kohn, S. M. Aloj, R. M. Friedman, E. F. Grollman, F. D. Ledley, G. Lee, M. F. Meldolesi, and B. R. Mullin, "Advances in Carbohydrate Chemistry: Symposium on Cell Surface Carbohydrate Chemistry," R. E. Harmon, Ed., Academic Press, New York, New York, in press. 10. B. R. Mullin, T. Pacuszka, G. Lee, L. D. Kohn, R. 0. Brady, and P. H. Fishman, Science, in press. 11. J. Moss, J. C. Osborne, P. H. Fishman, H. B. Brewer, M. Vaughan, and R. 0. Brady, Proc. Natl. Acad. Sci. U.S.A., 74, 74 (1977). 12. D. M. Gill, Proc. Natl. Acad. Sci. U.S.A., 72, 2064 (1975). 13. J . Moss, V. C. Manganiello, and M. Vaughan, Proc. Natl. Acad. Sci. U.S.A., 73,4424 (1977). 14. J. Moss, P. S. Ross, P. H. Fishman, and M. Vaughan, Fed. Proc., 36, 728 (1977). 15. A. Kurosky, D. E. Markel, J. W. Peterson, and W. M. Fitch, Science, 195, 299 (1977). 16. M. 0. Dayhoff, L. T. Hunt, P. J. McLaughlin, and W. C. Barker, "Atlas of Protein Sequence and Structure," M. 0. Dayhoff, Ed., Vol. 5, National Biomedical Research Foundation, Washington, D.C., 1972. 17. G. Lee, A. Eastlake, A. Schechter, J. Gladner, and L. D. Kohn, Biochem. Biophys. Res. Commun., in press. 18. Y. Matuo, M. A. Wheeler, and M. W. Bitensky, Proc. Natl. Acad. Sci. U.S.A., 73,2654 (1976). 19. G. Lee, S. M. Aloj, R. 0. Brady, and L. D. Kohn, Biochem. Biophys. R e s . Commun., 73,370 (1976). 20. G. Lee, S. M. Aloj, and L. D. Kohn, J. Biol. Chem., in press. 21. F. Besancon and H. Ankel, Nature, 250, 784 (1974). 22. F. Besancon and H. Ankel, Nature, 252, 478 (1974). 23. F. Besancon, H. Ankel, and S. Basu, Nature, 2, 576 (1976). 24. R. M. Friedman and L. D. Kohn, Biochem. Biophys. Res. Commun., 70, 1078 (1976) 25. L. D. Kohn, R. M. Friedman, J. M. Holmes, and G. Lee, Proc. Natl. Acad. Sci. U.S.A., 73, 3695 (1976). 26. F. Besancon and H. Ankel, C. R. Seances Acad. Sci. (Paris), in press. 27. V. E. Vengris, F. H. Reynolds, Jr., M. D. Hollenberg, and P. M. Pitha, Virology, 72,486 (1976). 28. E. F. Grollman, G. Lee, J. M. Holmes, R. M. Friedman, and L. D. Kohn, Proc. Natl. Acad. Sci. U.S.A., in press.

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29. R. M. Friedman, E. F. Grollman, E. H. Chang, L. D. Kohn, G. Lee, and F. T. Jay, Tex. Rep. Biol. Med., in press. 30. F. M. Meldolesi, E. F. Grollman, R. M. Friedman, and L. D. Kohn, Biochem. Biophys. Res. Commun., in press. 31. W. E. van Heyningen and P. A. Miller, J. Gen. Microbiol., 24, 107 (1961). 32. S . H. Ingbar, N. Engl. J. Med., 274, 1252 (1966). 33. J. H. Kerr, 3. L. Corbett, C. Prys-Roberts, A. Crampton Smith, and J. M. K. Spalding, Lancet, 2, 236 (1968). 34. C. Prys-Roberts, J. L. Coybett, 3. H. Kerr, A. Crampton Smith, and J. M. K. Spalding, Lancet, 1, 542 (1969). 35. F. D. Ledley, G. Lee, L. D. Kohn, W. H. Habig, and M. C. Hardegree, J. Biol. Chem., in press. 36. W. H. Habig, F. D. Ledley, G. Lee, M. C. Hardegree, and L. D. Kohn, Fed. Proc., 36, 710 (1971). 37. W. H. Habig, M. C. Hardegree, E. F. Grollman, F. D. Ledley, and L. D. Kohn, Endocrinology, in press. 38. W. H. Habig, M. C. Hardegree, M. F. Meldolesi, S. M. Aloj, and L. D. Kohn, Endocrinology, in press. 39. S . van Heyningen, FEBS Letts., 68, 5 (1976). 40. T. B. Helting and 0. Zwisler, J. Biol. Chem., 252, 187 (1977). 41. T. B. Helting, 0. Zwisler, and H. Wiegandt, J. Biol. Chem., 252, 194 (1977). 42. G. N. Kryzhanovsky, Naunyn-Schmiedebergs Arch. Pharmakol., 276, 247 (1973). 43. W. E. van Heyningen, Nature, 249, 415 (1974). 44. R. J. Winand and L. D. Kohn, J. Biol. Chem., 250, 6534 (1975). 45. P. Lebon, M. C. Moreau, L. Cohen, and C. Chany, Proc. SOC. Exp. Biol. Med., 149, 108 (1975). 46. "Cellular Regulation of Transport and Uptake of Nutrients, 1976 Fogarty International Symposium"; J . Cell. Physiol., 9, 495 (1976). 47. E. F. Grollman, G. Lee, F. S . Ambesi-Impiombota, M. F. Meldolesi, S . M. Aloj, H. Coon, H. R. Kaback, and L. D. Kohn, Proc. Natl. Acad. Sci. U.S.A., in press. 48. R. Batt and J. M. McKenzie, "Thyroid Research," J. Robbins and L. E. Braverman, Eds., p. 65, American Elsevier Publishing Co., New York, 1976. 49. C. Levin, M. Dufau, and K. Catt, J. Biol. Chem., 250, 8818 (1975). 50. J . Moss, P. H. Fishman, R. L. Richards, C. R. Alving, M. Vaughan, and R. 0. Brady, Proc. Natl. Acad. Sci. U.S.A., 73, 3480 (1976). 51. J. Moss, R. L. Richards, C. R. Alving, and P. H. Fishman, J. Biol. Chem., 252, 797 (1977). 52. R. L. Tate, J. M. Holmes, L. D. Kohn, and R. J. Winand, J. Biol. Chem., 250, 6527 (1975). 53. R X . Tate, R. J. Winand, and L. D. Kohn, "Thyroid Research," J. Robbins and L. E. Braverman, Eds., p. 57, American Elsevier Publishing Co., New York, 1976. 54. M. F. Meldolesi, P. H. Fishman, S . M. Aloj, F. D. Ledley, G. Lee, R. M. Bradley, R. 0. Brady, and L. D. Kohn, Biochem. Biophys. Res. Commun., in press, 55. S . M. Aloj, L. D. Kohn, G. Lee, and M. F. Meldolesi, Biochem. Biophys.

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Res. Commun., 1053 (1977). 56. R. J. Winand and L. D. Kohn, J. Biol. Chem., 250, 6534 (1975). 57. C. Tsai, D. A. Zopf, R. K. Yu, R. Wistar, Jr.; and V. Ginsburg, Fed. Proc., 36, 709 (1977). 58. E. F. Grollman, A. Kobata, and V. Ginsburg, Ann. N.Y. Acad. Sci., 169, 153 (1970). 59. M. L. Dufau, D. W. Ryan, A. J. Baukal, and K. J. Catt, J. Biol. Chem., 250, 4822 (1975). 60. Axurosky, D. Markel, B. Touchstone, and J. W. Peterson, Proc. ‘Am. SOC. Microbiol., 15, 23 (1975). 61. E. Mendez, C. Y. Lai, and A. Wodnar-Filipowicz, Biochem. Biophys. Res. Commun., 67,1435 (1975).

Chapter 23. Mineral Metabolism and Metabolic Bone Disease J. W. Hinman and R. P. McCandlis, The Upjohn Co., Kalamazoo, Michigan

Introduction - Rapid progress is being made toward a better understanding of mineral metabolism. Advances have been made possible by breakthroughs in several divergent areas of research. Among these are the discovery of the active metabolites of vitamin D (reviewed in this series, Vol. 10, Chapter 30), elucidation of the biochemistry and endocrinology of the calcitropic hormones, the devising of a surgical technique which permits obtaining serial bone biopsies, and histomorphometric methods which allow reliable quantitative assessment of human bone. Biologically active compounds are available for study and the rationale for the preparation of others is forthcoming. The opportunities for the medicinal chemist to contribute to the solution of medical problems involving derangements in mineral metabolism are, therefore, greatly enhanced. The objectives of this review are to outline briefly some new concepts in the field and to provide a key to the pertinent literature. Since the 1930s it has been known that vitamin D is important for normal growth and skeletal development of animals and man. It was recognized that there is a vitamin D requirement for absorption of calcium and phosphorus from the intestinal tract and it was appreciated that maintenance of serum calcium levels within rather narrow concentration limits was critical for normal function and even life itself. But it was not realized until much later that the skeleton plays a vital role in calcium and phosphorus homeostasis and is a dynamic tissue metabolically. The recent flood of knowledge on the biochemistry & endocrinolo y of vitamin D metabolite~l,-~ the prostaglandins parathyroid hormone (PTH), 4 y 6 calcitonin (CT) has enabled the biochemist and (PG) ,7-9 and the cyclic nucleotideslO, cell physiologist to focus attention on the precise mechanisms involved in mineral homeostasis.l 2 Their findings provide the basis for cautious optimism regarding the potential for the development of definitive diagnoses and effective treatments for many forms of metabolic bone disease. Vitamin D Endocrinology - The significance of the active metabolites of vitamin D was aptly expressed by Kodicek13 in 1974 when he wrote, "It was first descriptive, it reached then a physiological niveau, and eventually deepened into basic insights at molecular level and suddenly opened new practical vistas in medicine." Vitamin D must first be metabolically altered before it functions. The functional metabolites14 are formed in organs other than their sites of action. Their rates of synthesis and secretion are feedback regulated and this regulation is effected at least in part by PTH, possibly by CT, other hormones and metabolic regulators and by calcium and phosphorus levels in certain tissues. The active metabolites play important roles in the control of calcium and phosphorus movement from both intestine and bone. They function as major humoral substances in the regulation of serum calcium ~0ncentration.l~It is clear now that vitamin D is not simply a catalytic vitamin substance, but one

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1,25 (OH) 2D :

24,2 5 (OH)2D :

D2 Series

HO\"

R3 D3 Series

whose metabolism is dynamically interrelated with its function in an endocrine system.15 With current knowledge, albeit incomplete, it is possible to speculate with some confidence on how derangements in vitamin D metabolism may be involved in the etiology of certain types of metabolic bone disease. Most of the known metabolites of vitamin D and many analogs and structural variations have been synthesized.14-16 The clinical evaluation of these compounds for safety and efficacy is now progressing rapidly. Biochemical Measurements - Any meaningful evaluation of an experimental drug in the treatment of metabolic bone disease requires dependable methods for measurement of the pertinent minerals and hormones. Atomic absorption methods17 meet the requirement for accurate determination of total serum calcium and magnesium. Since PTH is more responsive to ionized calcium (Cat+) than to total serum calcium,18,19it is fortunate that methods for this measurement have become available recently in the form of specific electrodes.20 Very satisfactory isotopic methodsz1y Z 2 for measuring intestinal calcium absorption are available. Total inorganic phosphorus is usually determined by the method of Fiske and S u b b a R o ~ 724 . ~ ~ Radioimmunoassays (RIAs) reasonably specific for C-terminal and N-terminal portions of human PTH have been developed by several group^.^^-^^ Practical RIAs for human CT have been No successful RIA has been reported, but satisfactory competitive protein-binding radio assays and high pressure liquid chromatography assays have been devised for measurement of the ng/ml. levels of 25-OHD in serum. 30-32 Chromatographic separation of 25-OHD2 and 25-OHD3 has been reported33 but separation is generally not required for most clinical studies. Measurement of the kidney metabolite, 1,25(OH)zD3, is more difficult because of the pg/ml. concentrations encountered, but sensitive radio receptor assays have been devised3493 5 and used to measure serum levels in normal man and in various disease states.36 A method specific for the determination of 24, 25(oH)2D3 was reported37 recently and will undoubtedly be important for certain clinicalevaluations. Since certain prostaglandins, particularly those of the E series, have osteolytic activity comparable to that of PTH, there are situations where measurement of these lipids is useful. RIAs for PGEs and their metabolites have been reported,3 8 although most investigators have had difficulty preparing antisera with the required specificity. Both competitive binding assays39 and RIA40 have been developed for measurement of CAMP. Histomorphometric Measurements

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Determination of the status of the bone

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tissue is vitally important for diagnosis and evaluation of changes induced by experimental treatment. This is accomplished best by direct examination of bone biopsies. Until recently the only surgical procedure for obtaining a bone specimen was the "open biopsy" for removal of a wedge from the iliac crest or a segment of a rib while the patient was under general anesthesia. These procedures were so traumatic and incapacitating for the patient that it was rarely possible to gain consent for a second biopsy. The essentially non-traumatic transilial biopsy technique of Bordier,4 1 which is performed with a trephine and local anesthesia, produces a cylindrical bone sample or core with dimensions of 8-12mm in length and 5-8mm in diameter. This method has made repeated biopsies an entirely feasible procedure for diagnosis and drug evaluation studies. Coincident with this new technique for procurement of human bone biopsies was the development of quantitive methods of bone analysis.l 2 These methods include histochemical analysis of both decalcified and undecalcified42y43bone sections, mi~roradiography,~~ tetracycline labeling45 and autoradiography.42 The latter two techniques require administration of a tetracycline antibiotic or isotopic tracer prior to procurement of the biopsy. Undecalcified thin sections, prepared with the use of a Jung microtome after the bone core is fixed, dehydrated and embedded in metha ~ r y l a t e ,are ~ ~ analyzed by intersect and point count method^^^,^^ which Tetracycline antibiotics deposit permit three-dimensional assessment.4 8 9 4 9 in V ~ V Oin sites of bone formation constituting markers which can be studied in undecalcified sections by fluorescence microscopy.45y47 This represents the safest and best tissue time marker for microscopic measurement of bone formation dynamics. Largely through the application of these new techniques some basic concepts of bone physiology and metabolism are beginning to be appreciated. Normal human bone undergoes four distinct processes: growth, modeling, repair and remodeling.5 0 From fetal development until some time after puberty, growth and modeling are the predominant processes during which bones grow both in length and in width. The modeling process is responsible for insuring proper shape and proportion of the growing skeleton. After maximum growth is achieved, the modeling process ceases, but the skeleton remains metabolically active and undergoes a continuous process of remodeling through resorption and formation. The' process of repair is operative at all stages of life and comes into play whenever a fracture occurs in any member of the skeleton. This process appears to be quite independent of the other two and is usually operative even when modeling or remodeling is defective. Metabolic bone disease in adults nearly always represents some derangement in the remodeling process. This point must be considered in choosing experimental animal models for human disease. For example, the rat cannot serve as an adequate model for the study of human osteoporosis because it never completes the modeling phase in its life cycle. Osteoporosis in man is a disease involving derangement of the remodeling process. Metabolic bone diseases in children are usually more complicated because defects in both the modeling and remodeling processes may be

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involved. The remodeling process, involving continuous resorption of both the mineralized portion of bone and the organic matrix, is coupled with the formation process and must precede it. In other words, in normal bone, formation cannot occur until resorption has taken place. In recent years much has been learned about this microscopic remodeling system which operates continuously in the entire skeleton.12 Bone cells known as osteoclasts are responsible for the resorption process. Formation and mineralization are initiated by cells known as osteoblasts. Modern histomorphometric techniques applied to the cells and surfaces of serial bone biopsies enable researchers to detect and quantitate malfunctions underlying the pathology in various types of metabolic bone disease. With this capability the utility of these techniques in diagnosis and evaluation of treatment are obvious. Non-Invasive Methods of Skeletal Analysis - While information provided by examination of human bone biopsies is invaluable, it is well recognized that the approach has serious limitations. Fortunately, much progress has been made in the development of supplemental non-invasive techniques for assessment of bone mineral content in health and disease. Research initiated years ago by physicists has yielded results which are being widely utilized now by physicians in the study and treatment of metabolic bone disease. The roentgenogram or x-ray was the first of the non-invasive methods in this category, has been used for decades, will never be completely replaced, but suffers from a number of shortcomings. Lack of sensitivity is a serious limitation especially for use in detecting and quantitating skeletal demineralization. Using standard x-ray procedures, it is difficult to detect demineralization until a 30-40% change has taken place. 5 1 The lack of mono-chromicity and dangers from radiation exposure represent further limitations. However, some modern specialized techniques for quantitating changes in metacarpal and phalan eal bones of the hand,52 and others for detection of ectopic calcificationg3 are extremely useful. The photon absorptiometry procedures pioneered by Cameron and S o r e n ~ o n5~5~are , finding widespread use. 56-59 This approach provides a sensitive method for measuring both cortical and trabecular bone mass in the human radius and ulna. Bone mineral mass or content is determined by measuring the photon energy absorbed by bone from a monochromatic gammaray source, usually lZ5I. Standardized instruments are commercially available and are being used in many hospital and research centers. Modifications of the general method are being developed to permit bone mineral content measurements of other parts of the skeleton.59 Total body neutron activation analysis and whole body counting6 permit the determination of the absolute quantity of certain minerals (including calcium and phosphorus) in the entire body with great accuracy and reproducibility. Since absolute measurements of the total body stores of such elements as calcium and potassium have been normalized for size, sex, age, and body habitus in individual patients, estimates of normal, increased and decreased stores of these elements are possible in a wide

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variety of metabolic conditions. Unfortunately, there are very few centers equipped to carry out such sophisticated measurements. Derangements in mineral metabolism frequently lead to metastatic calcification of soft tissue. Bone-seeking radionuclides, such as 99mTclabeled phosphate preparations, are proving particularly useful in detecting extra-skeletal calcification in situations where roentgenographic demonstration is lacking.6 1 Clinical Studies a) Paget's Disease - This fairly rare disease is characterized by bizarre malfunctioning of the bone remodeling process in which both resorption and formation occur at grossly accelerated and unmatched rates.12 Both CT and disodium ethane-1-hydroxy-1,l-diphosphonate (EHDP) have been used in the treatment of this ainful and disabling malady. Combination therapy with human CT and EHDP" appears to offer the best treatment to date. b) Hypoparathyroidism - This disorder is caused by a deficiency of PTH or an impairment of parathyroid functions leading to a state of hypocalcemia. Most cases result from surgical removal of parathyroid glands as the treatment for thyroid or parathyroid malignancy. Ideal treatment for surgical and idiopathic forms would be administration of human PTH. Since this is not available and because prolonged treatment with bovine PTH causes a refractory state due to antibody formation, some form of vitamin D is the only currently available treatment. Clinical experience indicates that pharmacologic doses of vitamin D, 25-OHD363 or 1 , 2 5 ( 0 H ) ~ D ~can ~ ~ produce many of the effects of PTH in the intestines, kidney and bone to stimulate a rise in serum calcium although by somewhat different mechanisms.12 Dihydrotachysterol and 1,25(OH)2D3 offer the advantage of rapid onset of action in initiating treatment in acute post-surgical hypoparathyroidism while in chronic hypoparathyroidism treatment with 25-OHD3 induces good stability of plasma calcium.6 4 c) Renal Osteodystrophy - This term is used to describe the bone disease which accompanies renal insufficiency. Severity ranges from asymptomatic to complete disablement, although abnormality at the cellular level is readily detected even in insipient renal insufficiency.6 5 The incidence of this disease has increased rapidly during the last 15 years as the result of ever increasing availability of dialysis facilities. The consequences of renal failure resulting from disruption of both the excretory and endocrine functions of the kidney include hyperphosphatemia, secondary hyperparathyroidism, hypermagnesemia and impaired intestinal absorption of calcium. Serum calcium levels are usually in the low normal range, but a significant number of these patients are hypocalcemic. Correction of hypocalcemia when it occurs does not necessarily cure the disease. Skeletal demineralization and muscular weakness are evident as the disease progresses. The classical skeletal lesions are osteomalacia (an excess of unmineralized bone matrix or osteoid) and osteitis fibrosa (fibrotic tissue invasion of the bone). The former is believed to be caused by a metabolic resistance to the actions of vitamin D and the latter to the influence of excess PTH.50'66

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Recent studies on the treatment of renal osteodystrophy have concentrated on evaluation of the newly discovered metabolites of vitamin D. Because kidney enzymes are required for in V ~ V Oproduction of 1,25(0H)2D?,l4 many investigators assumed logically that administration of this metabolite would solve the problem of vitamin D resistance. Results to date indicate that lY25(OH)2D3 and the analog, la-OHD3, are remarkably potent in stimulation of intestinal calcium absorption, they lower elevated serum alkaline phosphatase and are of some benefit in control of secondary hyperparathyroidism. 67-72 Hypercalcemia has been enc~untered~~' 70 71 and remineralization of depleted bones has not occurred to the extent anticipated.68,69,72 Studies evaluating pharmacologic doses of 25-OHD3 have produced encouraging preliminary results.7 3 y 74 Some investigators conclude that the osteomalacia of renal failure is caused by a lack of 25-OHD3 rather than a deficiency of 1,25(OH)zD3 alone.75 Very recently results reported on the use of 24,25(OH)zD3 indicate that this metabolite, which is produced from 25-OHD3 in the kidney and elsewhere in man is specifically active in remineralization.76 d) Vitamin D-Dependent Rickets - This form of metabolic bone disease is an inborn error of vitamin D metabolism due to a genetic defect in the 25-hydroxyvitamin D-1-hydroxylase, the renal enzyme responsible for conversion of 25-OHD3 to 1,25(OH)2D3.77 It has been known for years that this rare disorder was manageable with massive doses of vitamin D and recent have shown that 25-0HD3 in doses of 100 to several hundred micrograms/day will provide complete clinical, biological and radiological recovery. Similar results have been reported77 using ca 1 pg/day of 1,25 (OH)2D3, e) Familial Hypophosphatemic Vitamin D-Resistant Rickets - There appear to be several types of hereditary vitamin D-resistant rickets (VDRR) associated with hypophosphatemia due-to diminished tubular reabsorption.of phosphate.e0 This defect may also involve reduced reabsorption of amino acids or glucose. Large doses of vitamin D may in some cases improve the rickets with variable effect on the tubular reabsorption defect.81 Both 25-OHD3 and 1,25(OH)zD3 have been evaluated to some e~tent.'~-~~ Neither compound is highly effective, but large doses of 25-OHD3 have induced beneficial effects in about half of the cases investigated.

f) Anticonvulsant Osteomalacia - This term has been applied to the skeletal demineralization condition which appears to be induced by administration of anticonvulsant drugs, particularly dephenylhydantoin and phenobarbita1.85 Patients, usually epileptics, taking these drugs tend to have lower than normal serum 25-OHD levels and slight hypocalcemia. The incidence appears to be higher among mentally retarded patientse6 and varies from one location to another.85 The disorder is believed to be caused by some abnormality in vitamin D metabolism induced by the action of the anticonvulsant drug on the liver, but the details are not clearly ~ n d e r s t o o d . ~ ~ In many instances the problem has been reported to be minimized by administration of 1000-4000 I.U. of vitamin D per day. However, cases resistant to this treatment have been reported and these responded well to small doses of 25-0HD3.86 Clearly more research is required to solve this con-

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troversial problem. g) Steroid-Induced Osteopenia - Glucocorticoids and synthetic analogs are employed frequently as pharmacologic agents in the treatment of a wide variety of diseases for prolonged periods of time. One of the most serious complications in the use of these steroids is the development of a metabolic bone disease known as steroid-induced osteopenia or osteoporosis. The decrease in bone mass encountered in these situations is believed to depend upon two influences of the steroids: 1) an inhibitory effect on osteoblasts resulting in decreased bone formation and 2) stimulation of mild secondary hyperparathyroidism. The latter is thought to be related to the well established influence of steroid hormones to inhibit intestinal absorption of calcium.8 8 Recent animal studiese9 showed that prednisolone administration caused partial inhibition of the action of 1,25(OH)2D3 upon intestinal calcium transport by induction of an enzyme system which catalyzed conversion of lY25(OH)2D3 to a more polar, biologically-inactive metabolite. A preliminary reportg0 on a clinical study evaluating 25-OHD3 in the treatment of steroid-induced osteopenia claimed that a dose of 40 Uglday for a mean treatment period of 11.3 months led to significant improvement in intestinal calcium absorption, normalization of PTH levels and a 20% increase in mean trabecular bone mass. These results were achieved with patients continuing on their steroid therapy. If these impressive results can be confirmed and expanded, this would represent a significant advance. An effective method for prevention and resolution of steroidinduced osteopenia is important not solely as a benefit for the large population threatened by or suffering from this disorder, but also because this malady is believed to represent a possible model for postmenopausal and senile osteoporosis. Any therapy shown to be effective in the management of steroid-induced osteopenia should be carefully evaluated as a treatment of other forms of osteoporosis. h) Liver Disease & Malabsorption Syndromes - The introduction of the competitive protein-binding radioassay for the circulating metabolite, 25-OHD3, has permitted studies which correlated subnormal serum levels of 25-OHD3 with a variety of pathological conditions. In addition to patients receiving anticonvulsant drugs, it has been found that low levels of 25-OHD are common in patients with derangement of the excretory function of the liver,31 particularly primary biliary cirrhosisg1 and biliary atresia,92 patients with renal stones,9 3 alcoholics,9 3 ulcerative and granulomatous colitis,94 regional enteritis,94 and granulomatous ileocolitis.94 In all of these conditions the finding of subnormal serum 25-OHD concentration is associated with a significant incidence of skeletal demineralization. In patients with various types of inflammatory bowel disease more profound skeletal defects occur in adolescents than in adults. Neonatal hypocalcemia is frequently associated with subnormal 25-OHD serum levels, especially in premature infants.95 From work reported to date, it is clear that hypo-25-hydroxycholecalciferolemia is far from rare, but escaped detection until a suitable diagnostic test became available. Evaluation of treatment of these disorders with exogenous 25-OHD3 and 1,25(OH)2D3 is just beginning.9 1 ’ 9 6

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i) Hypercalcemia - The most common causes of acute hypercalcemia are hyperparathyroidism and neoplastic disease. In the latter case little is known of the mechanism of the hypercalcemia. Only in a few instances have tumors been shown to secrete PTH. Recently prostaglandins have been identified as mediators of hypercalcemia associated with certain human turn or^?^ Administration of PG-synthetase inhibitors, indomethacin and aspirin, caused reduction in the excretion of PGE metabolites and a concomitant fall in serum calcium. Organ cultures of human myeloma cells secrete anosteoclastactivating factor which is different from either PTH or PG.98 The activity of another bone-resorbing factor, complement, is mediated by activation of the PG synthetase system in bone.99 Current findings are consistent with mediation of hypercalcemia not only by PGE2 but also by its endoperoxide or by thromboxane A2.989100 Research on inhibitors of fatty acid cyclooxygenase as agents for treatment of patients with hypercalcemia of neoplasia offers attractive possibilities. References

1. J.L. Omdahl and H.F. DeLuca, Physiol. Rev., 53, 327 (1973). 2. H.F. DeLuca, J. Lab. Clin. Med., 87, 7 (1976). 3. A.W. Norman, R.L. Johnson, T.W. Osborn, D.A. Procsal, S.C. Carey, M.L. Hammond, M.N. Mitra, M.R. Pirio, A. Rego, R.M. Wing and W.H. Okamura, Clin. Endocrinol., 2, Suppl., 1215 (1976). 4. C.D. Arnaud, Am. J. Med., 55, 577 (1973). 5. H.B. Brewer, Jr., T. Fairwell, W. Rittel, T. Littledike and C.D. Arnaud, Am. J. Med., 56, 759 (1974). 6. J. Meienhofer, Ann. Rep. M a . Chem., 11,158 (1976). 7. R.V. Talmage, M. Owen and J.A. Parsons, eds., Calcium-Regulating Kmones, Excerpta Medica, American Elsevier Publishing Co., Inc., New York, 1975. 8. J.W. Hinman, Ann. Rev. Biochem., 3, 161 (1972). 9. B. Samuelsson, E. GranstrEm, K. Green, M. Hamberg and S. Hammarstrtim, Ann. Rev. Biochem., 44, 669 (1975). 10. E. Carafoli, K. Malmstram, E. Sigel and M. Cromptom, Clin. Endocrinol., 5, Suppl., 495 (1976). 11. R.H. Kahn and W.E.M. Lands, eds., Prostaglandins and Cyclic AMP: logical Actions Clinical Applications, Academic Press, Inc., New York and London, 1973. 12. H. Rasmussen and P. Bordier, The Physiological Basis of Metabolic Bone Disease, Williams & Williams, Waverly Press, Inc., Baltimore, 1974. 13. E. Kodicek, Lancet, 1,325 (1974). 14. J.L. Napoli, Ann. Rep. Med. Chem., 10, 295 (1975). 15. H.F. DeLuca, Ann. Int. Med., E, 367(1976). 16. H.F. DeLuca and H.K. Schnoes, Ann. Rev. Biochem., 45, 631 (1976). 17. W. Slavin, Atomic Absorption Spectroscopy, John Wiley & Sons, Inc. New York, 1968. 18. E.W. Moore, J. Clin. Invest., 49, 318 (1970). 19. M. Sore11 and J.F. Rosen, 3 . Pediatrics, 87, 67 (1975). 20. H.D. Schwartz, Clin. Chem., 2, 461 (1976). 21. J. Szymendera, R.P. Heaney and P.D. Saville, J. Lab. Clin. Med., 2, 570 (1970).

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54. J.R. Cameron and J . Sorenson, Science, 3,230 (1963). 55. J.R. Cameron, R.B. Mazess and J.A. Sorenson, Invest. Radiol., 2, 11 (1968), 56. D.M. Smith, C.C. Johnston, Jr. and Yu Pao-Lo, J. Am. Med. ASSOC., 219, 325 (1972). 57. R.G. Evens, C.Y.C. Pak, W. Ashburn and F.C. Bartter, Invest. Radiol., 4, 364 (1969). 58. 7.R. Shapiro, W.T. Moore, E. Ferber, C. Epps, G. Aurbach and G.D. Whedon, Clin. Res., 2, 38 (1972). 59. R.B. Mazess, Ed., Third International Conference on Bone Mineral Measurement, Am. J. Roentgenol. Radium Ther. Nucl. Med., 126, 1266 (1976). 60. S.H. Cohn, K.J. Ellis and S. Wallace, Am. J. Med., 57, 683 (1974). 61. G.M. Grames, D.D. Sauser, C. Jansen, R.E. Soderblom, J.E. Hodgkin and M.S. Stilson, J. Am. Med. ASSOC., 230, 992 (1974). 62. D.J. Hosking, J . VanAken, O.L.M. Bijvoet and E.J. Will, Lancet, 1,615 (1976). 63. S.W. Kooh, D. Fraser, H.F. DeLuca, M.F. Holick, R.E. Belsey, M.B. Clark and T.M. Murray, N. Engl. J. Med., 293, 840 (1975). 64. A.M. Parfitt, B. Frame and D. Thomson, Abs., Third Workshop on Vitamin D, p. 188, Asilomar, Calif., Jan. 1977. 65. H.H. Malluche, E. Ritz, H.P. Lange, J. Kutschera, M. Hodgson, V . Seiffert and W. Schoeppe, Kidney Int., 2, 355 (1976). 66. L.V. Avioli and S. Teitelbaum, In Renal Osteodystrophy in the Kidney, Eds. F. Rector and B. Brenner, W.B. Saunders, pp. 1542-1591, 1976. 67. R.G. Henderson, J.G.G. Ledingham, D.O. Oliver, D.G. Small, R.G.G. Russell, R. Smith, R.J. Walton, C. Preston, G.T. Warner and A.W. Norman, Lancet, 1,379 (1974). 68. A.S. Brickman, D.J. Sherrard, J. Jowsey, F.R. Singer, D.J. Baylink, N. Maloney, S.G. Massry, A.W. Norman and J.W. Coburn, Arch. Intern. Med., 134, 883 (1974). 69. A.M. Pierides, M.K. Ward, F. Alvarez-Ude, H.A. Ellis, K.M. Peart, W. Simpson, D.N.S. Kerr and A.W. Norman, Proc. Eur. Dialysis Transplant ASSOC., 12, 237 (1976). 70. A.M. Pierides, W. Simpson, M.K. Ward, H.A. Ellis, J.H. Dewar, D.N.S. Kerr, Lancet, 1,1092 (1976). 71. S. Madsen and K. glgaard, Acta Med. Scand. , 200, 1 (1976). 72. M.W.J. David, T.M. Chalmers, J . O . Hunter, B. Pelc and E. Kodicek, Ann. Intern. Med., 84, 281 (1976). 73. S.L. Teitelbaum, M.J. Bone, P.M. Stein, J . J . Gilden, M. Bates, V.C. Boisseau and L.V. Avioli, J. Am. Med. SOC., 235, 164 (1976). 74. G. Witmer, A. Margolis, 0 . Fontaine, J. Fritsch, G. Lenois, M. Broyer and S. Balsan, Kidney Int., lo, 395 (1976). 75. J.B. Eastwood, T.C.B. Stamp, E. Harris and H.E. dewardener, Lancet, 2, 1209 (1976). 76. P.J. Bordier, P. Marie, L. Miravet, J. Gueris, C. Ferriere, A. Ryckewaert and A.W. Norman, Abs., Third Workshop on Vitamin D, p. 81, Asilomar, Calif., Jan. 1977. 77. D. Fraser, S.W. Kooh, H.P. Kind, M.F. Holick, Y. Tanaka and H.F. DeLuca, N. Engl. J. Med., 2,817 (1973). 78. J. Rosen and L. Finberg, Pediat. Res., 5, 552 (1972). 79. S. Balsan and M. Garabedian, J. Clin. Invest., 2, 799 (1972).

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80. C.E. Dent, Proc. Royal SOC. Med., 63, 401 (1970). 81. T.F. Williams, R.W. Winters and C.H. Burnett, Jr., In The Metabolic Basis of Inherited Disease, 2nd Edition, Eds., J.B. Stanbury, J.B. Wyngaarden and D.S. Frederickson, McGraw-Hill, New York, pp. 11791204, 1966. 82. C.Y.C. Pak, H.F. DeLuca, F.C. Bartter, O.H. Hannsmen, B. Frame, A. Siropoulos and C.S. Delea, Arch. Intern. Med., 129, 894 (1972). 83. J.B. Puschett, M. Genel, H. Rastegar, C. Anast, H.F. DeLuca and H. Friedman, Clin. Pharmacol. Therapeut., 17,202 (1975). 84. R.G.G. Russell, R. Smith, C. Preston, R.J. Walton, C.G. Woods, R.G. Henderson and A.W. Norman, Clin. Sci. Mol. Med., 48, 177 (1975). 85. C.S. Anast, N. Engl. J. Med., 292, 587 (1975). 86. F. Lifshitz and N.K. MacLaren, J. Pediatr., 83, 612 (1973); Pediat. Res. , 1,914 (1973). 87. R. Burt, J.W. Freston and K.G. Tolman, J. Clin. Pharmacol., 16,393 (1976). 88. B.P. Lukert, S.W. Stanbury and E.B. Mower, Endocrinology, 93, 71’8 (1973). 89. M. Carre, 0. Ayigbede, L. Miravet and H. Rasmussen, Proc. Nat. Acad. 2996 (1974). Sci. U.S.A., 2, 90. T.J. Hahn, L.R. Halstead, C.R. Scharp and B.H. Hahn, Abstract 12, 58th Annual Meeting, Endocrine SOC., San Francisco, Calif., 1976. 91. J.B. Wagonfeld, M. Bolt, J.L. Boyer, B.A. Nemchausky, J . YanderHorst and I.H. Rosenberg, Lancet, 2, 391 (1976). 92. F. D a m , J.F. Rosen, M. Roginsky, M.I. Cohen and L. Finberg, J. Pediatr., 9, 1041 (1976). 93. C. Velentzas, D.G. Oreopoulos, L. Brandes, D.R. Wilson and A. MarquezJulio, Ann. Intern. Med., 86, 198 (1977). 94. J.B. Wagonfeld, H.K. Genant, J.C. Mall, M. Bolt, J . VanderHorst and I.H. Rosenberg, Gastroenterology, 68, A-208/1065 (1975). 95. J.F. Rosen, M. Roginsky, G. Nathenson and L. Finberg, A. J. Dis. Child., 127, 220 (1974). 96. S.W. Kooh, D. Fraser and R. Toon, Lancet, 2, 1105 (1976). 97. H.W. Seyberth, G.V. Segre, J.L. Morgan, B.7. Sweetman, J.T. Potts and J.A. Oates, N. Engl. J. Med., 293, 1276 (1975). 98. G.R. Mundy, L.G. Raisz, R.A. Cooper, G.P. Schecter, and S.E. Salmon, N. Engl. J. Med., 291, 1041 (1974). 99. L.G. Raisz, A.L. Sandberg, J.M. Goodson, H.A. Simmons and S.E. Mergenhagen, Science, 185, 789 (1974). 100. D.R. Robinson, A.H. Tashjian, Jr. and L. Levine, J. Clin. Invest., 56, 1181 (1975).

Chapter 24.

W.

D e t e c t i n g Mutagens - C o r r e l a t i o n Between t h e M u t a g e n i c i t y and C a r c i n o g e n i c i t y o f Chemicals

R. A. Dybas, Merck Sharp & Dohme Research L a b o r a t o r i e s , Rahway, New Jersey 07065 M. H i te, Merck I n s t i t u t e f o r T h e r a p e u t i c Research, West P o i n t , Pennsylvania 19486 Gary Flamm, N a t i o n a l Cancer I n s t i t u t e , Bethesda, Maryland 20014

The purpose o f m u t a g e n i c i t y t e s t i n g i s t w o - f o l d . The f i r s t purpose i s t o determine whether a chemical substance has t h e c a p a c i t y t o cause a l t e r a t i o n o f g e n e t i c i n f o r m a t i o n w i t h t h e p o t e n t i a l t o produce h e r i t a b l e g e n e t i c changes i n man. The fundamental concern, t h e r e f o r e , i s t h e r i s k o f m u t a t i o n t o f u t u r e g e n e r a t i o n s as m i g h t r e s u l t f r o m a g e n e t i c a1 t e r a t i o n o c c u r r i n g w i t h i n t h e germ c e l l s o f e i t h e r a male o r female. The o t h e r purpose of m u t a g e n i c i t y t e s t i n g r e l a t e s n o t t o germ c e l l s b u t t o somatic c e l l s , where t h e concern i s one o f cancer. As w i l l be desc r i b e d i n l a t e r s e c t i o n s , t h e r e a r e c o m p e l l i n g reasons which a r e b o t h empiri c a l and t h e o r e t i c a l t o b e l i e v e t h a t c e r t a i n m u t a g e n i c i t y t e s t systems have a c a p a c i t y t o i d e n t i f y chemical carcinogens w i t h a h i g h degree o f accuracy. Recent a t t e n t i o n p a i d t o m u t a g e n i c i t y t e s t systems b y i n d u s t r y and Federal r e g u l a t o r y agencies has i n l a r g e p a r t r e l a t e d t o t h i s l a t t e r purpose. C l e a r l y t h e r e e x i s t s a g r e a t need f o r a short-term, h i g h l y s e n s i t i v e , and r e 1 i a b l e prescreen f o r t h e i d e n t i f i c a t i o n o f p o t e n t i a l chemical carcinogens which a t a p p r o x i m a t e l y one-hundredth t h e c o s t o f l i f e t i m e s t u d i e s i n a n i mals can e f f e c t i v e l y i d e n t i f y p o t e n t i a l c a r c i n o g e n i c hazards. An unders t a n d i n g o f m u t a t i o n and t h e f u n c t i o n i n g o f m u t a t i o n a l t e s t systems depends upon knowledge o f t h e chemical n a t u r e and m o l e c u l a r o r g a n i z a t i o n o f g e n e t i c m a t e r i a l . The g e n e t i c m a t e r i a l o f a l l l i v i n g organisms, w i t h t h e e x c e p t i o n o f c e r t a i n v i r u s e s , i s composed o f d e o x y r i b o n u c l e i c a c i d (DNA). DNA i s an enormously l o n g polymer c o n t a i n i n g as many as 108 n u c l e o t i d e s l i n k e d b y phosphodiester bonds. The development o f a good c o r r e l a t i o n between t h e mutagenic and c a r c i n o g e n i c p r o p e r t i e s o f chemicals would suggest t h e p o s s i b i l i t y t h a t a molecu l a r event may be shared by t h e two phenomena. A t h e o r e t i c a l b a s i s f o r r e l a t i n g mutagenesis and c a r c i n o g e n e s i s has been proposed b y s e v e r a l i n v e s t i g a t o r s based on t h e concept t h a t neoplasms a r i s e from m u t a t i o n s i n somatic c e l l s . 3 , 8 2 ~ 8 3 T h i s has l o n g been a p o p u l a r t h e o r y o f cancer c a u s a t i o n and t h e r e i s i n c r e a s i n g widespread b e l i e f among cancer workers t h a t DNA damage i s i n v o l v e d i n t h e i n d u c t i o n o f cancer. It i s known t h a t c e l l r e g u l a t i o n can be e a s i l y a l t e r e d b y m u t a t i o n and t h a t a h e r i t a b l e change i n c e l l r e g u l a t i o n i s a c h a r a c t e r i s t i c p r o p e r t y o f a cancer c e l l . Other i n d i r e c t evidence accumulated d u r i n g t h e l a s t s e v e r a l y e a r s has begun t o i m p l i c a t e t h e r o l e o f m u t a t i o n s i n c h e m i c a l l y - i n d u c e d ma1 i g n a n t transformation.84,85 However, m u t a t i o n as an e s s e n t i a l s t e p i n c a r c i n o g e n e s i s remains t o be c l e a r l y demonstrated, and a t p r e s e n t , t h e r e l a t i o n s h i p i s m a i n l y d e r i v e d f r o m r e c e n t experimental evidence. 2,55,3,82,83,86-92 There a r e f o u r main n i t r o g e n o u s bases which appear i n DNA--two o f

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them are purines (adenine and guanine) and t h e o t h e r two a r e p y r i m i d i n e s (thymine and cy to s i n e ). I n a l l l i v i n g organisms, w i t h t h e e x c e p t i o n o f cert a i n viruses, DNA molecules occur i n n a t u r e as p a i r e d s t r a n d s i n t h e form o f a double-stranded alpha h e l i x i n which t h e hydrogen bonds o f guanine and adenine i n t e r a c t s p e c i f i c a l l y w i t h c y to s i n e and thymine r e s p e c t i v e l y . The b i o l o g i c a l s i g n i f i c a n c e o f t h i s arrangement i s t h a t one s t r a n d o f t h e DNA double h e l i x can serve as t h e template f o r t h e s y n t h e s i s o f i t s opposite, o r p art ner, strand. The base sequences encode t h e g e n e t i c i n f o r m a t i o n of t h e gene and t h e r e p l i c a t i o n mechanism p e rm i t s t h e propagation o f t h e g e n e t i c material. Were one of t h e nitrogenous bases w i t h i n DNA a l t e r e d (damaged) by chemical r e a c t i o n w i t h a mutagen, such damage c o u l d l e a d t o mutation. For example, a chemical might i n t e r a c t w i t h guanine m o i e t i e s o f DNA a l t e r i n g i t s hydrogen bonding p r o p e r t i e s so t h a t i n s t e a d of forming s p e c i f i c hydrogen bonds w i t h cyto s i n e p a i r i n g occurs w i t h thymine. Another common example o f chemical a l t e r a t i o n of DNA i s t h e loss o f a nitrogenous base through hydrol y s i s o f t h e b e ta g l y c o s i d i c bond. Such a l t e r a t i o n s are r e f e r r e d t o as p o i n t mutations because they may a f f e c t o n l y a s i n g l e o r a few n u c l e o t i d e s w i t h i n a gene c o n s i s t i n g o f hundreds o r thousands o f n u c l e o t i d e s . P o i n t mutations are a w e l l known cause o f mutations which e x i s t w i t h i n human popu l a t i o n s . S i c k l e c e l l anemia i s an example o f a p o i n t mutation t h a t has been w e l l chara c te ri z e d and has a profound e f f e c t upon mutant i n d i v i d u a l s . Other examples o f p o i n t mutations o r single-gene mutations i n c l u d e c y s t i c f i b r o s i s , hemophilia, and Tay-Sachs disease.97 Another major c l a s s o f human mutations a f f e c t s n o t t h e genes per se b u t chromosomes, t h e l a r g e u n i t s i n t o which genes are organized. A s i n g l e chromosome may c o n ta i n as many as 10,000 genes. The core o f a chromosome i s b e l i e v e d t o c o n s i s t e n t i r e l y of DNA and, therefore, i t s backbone i s t h e phosphodiester. Chromosomal mutations i n c l u d e e i t h e r chromosome breaks which would n e c e s s a r i l y i n v o l v e cleavage o f t h e phosphodiester arrangements o f t h e chromosomal segments ( t r a n s l o c a t i o n s , i n v e r s i o n s ) which a r e p redicat ed on a combination o f phosphodiester breaks and r e j o i n i n g s o f phosphodiester bonds. Many o f th e chromosomal mutations o c c u r r i n g i n man i n v o l v e t h e l o s s o f t h e chromosome, which can r e s u l t from e i t h e r breakage o f t h e phosphodiester bond, o r a process r e f e r r e d t o as n o n d i s j u n c t i o n , which does not, as f a r as i s known, i n v o l v e DNA per s e . Other mutations r e s u l t from d u p l i c a t i o n o f p a r t o r a l l o f t h e chromosome. Mutagen Test Systems. Perhaps t h e most w i d e l y used s i n g l e mutagen t e s t system i s t h a t which uses s p e c i a l l y c o n s tru c t e d t e s t s t r a i n s o f SaZmoneZZa typhimuriwn; t h i s t e s t i s f r e q u e n t l y r e f e r r e d t o as t h e "Ames t e s t . " The d e t a i l s o f t h i s t e s t have been e x t e n s i v e l y presented i n t h e l i t e r a t u r e . 1 ~ 2 ~ 3 The p o p u l a r i t y o f t h i s t e s t among s c i e n t i s t s , who are r e s p o n s i b l e f o r t h e e v a l u a t i o n o f t h e p o t e n t i a l mu ta g e n i c i t y o f chemicals and drugs, l i e s i n th e f a c t t h a t i t i s one o f t h e l e a s t expensive mutagen t e s t s and r e q u i r e s minimal amounts o f space and equipment. O f course, a person w e l l s k i l l e d i n m i c r o b i o l o g i c a l procedures i s r e q u i r e d t o supervise t h e d a i l y operations. The Ames t e s t u t i l i z e s several d i f f e r e n t h i s t i d i n e a u x o t r o p h i c mutants

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o f S a l m o n e l l a typhimuriwn. These b a c t e r i a are unable t o c a r r y o u t h i s t i d i n e b i o s y n t h e s i s and t h u s a r e dependent upon an e x t e r n a l source o f t h i s amino a c i d f o r growth. I n t h e presence o f a mutagenic compound t h e s e t e s t s t r a i n s may r e v e r t back t o t h e w i l d type, which a r e a b l e t o s y n t h e s i z e h i s t i d i n e . These s t r a i n s have a d d i t i o n a l m u t a t i o n s i n t h e c e l l w a l l t o a l l o w l a r g e molecules t o permeate t o t h e nucleus, t h e r e b y i n c r e a s i n g t h e i r s e n s i t i v i t y t o these k i n d s o f chemicals. Seven t e s t s t r a i n s o f S. t y p h i m w l i w n a r e c u r r e n t l y b e i n g used and a r e i d e n t i f i e d as f o l l o w s : s t r a i n s TA1530 and TA1535 w i l l d e t e c t base p a i r s u b s t i t u t i o n m u t a t i o n s and s t r a i n s TA1536, TA1537, and TA1538 w i l l d e t e c t f r a m e s h i f t m u t a t i o n s . I n a d d i t i o n , s t r a i n s TA100, d e r i v e d from TA1535, and s t r a i n TA98, d e r i v e d f r o m s t r a i n TA1538, have been f u r t h e r m o d i f i e d t o enhance t h e s e n s i t i v i t y o f t h e s e s t r a i n s t o base p a i r s u b s t i t u t i o n m u t a t i o n s and f r a m e s h i f t mutations, r e s p e c t i v e l y . 5 I n o r d e r t o overcome a m a j o r shortcoming o f t h e b a c t e r i a l mutagen t e s t , namely t h a t mammalian metabolism i s m i s s i n g , most i n v e s t i g a t o r s i n c o r p o r a t e a l i v e r homogenate (termed "S-9") i n t o t h e assay. G e n e r a l l y , t h i s i s prepared from r a t s induced w i t h a p o l y c h l o r i n a t e d b i p h e n y l m i x t u r e (Aroc l o r 1254) o r p h e n o b a r b i t a l . 1 Other i n v e s t i g a t o r s , such as B r u s i c k 6 have used homogenates prepared f r o m o t h e r t i s s u e s and o t h e r species. I n a d d i t i o n t o t h e t e s t s t r a i n s o f s. typhimuriwn, o t h e microbes systems such as Escherichia coZi,7 t h e fungus Neurospora c r a s s a Y 6 and t h e y e a s t Sacchromyces c e r e u i s i a e g have been used as i n d i c a t o r organisms i n m u t a t i o n t e s t s . Two s t r a i n s o f E. c o Z i , namely W3110 (polA+) and t h e DNA polymerase mutant E. c o l i p3478 ( p o l A - ) a r e used t o d e t e c t g e n e t i c damage i n b a c t e r i a b y DNA r e p a i r . C e l l s which have reduced c a p a b i l i t y o f r e p a i r i n g DNA may be more s u s c e p t i b l e t o t h e a c t i o n o f chemical mutagens as d e t e c t e d by i n c r e a s e d h e r i t a b l e change. Such d i f f e r e n t i a l t o x i c i t y i s taken as an i n d i c a t i o n t h a t t h e chemical i n t e r a c t s w i t h t h e DNA o f t h e exposed c e l l s t o produce i n c r e a s e d l e v e l s o f g e n e t i c damage. Sacchromyces c e r e v i s i a e a l l o w s one t o d e t e c t g e n e t i c damage i n a e u k a r y o t i c microorganism whose chromosomes a r e s t r u c t u r a l l y s i m i l a r t o t h e chromosomes o f h i g h e r organisms b u t a r e t o o small t o be observed d i r e c t l y . Many workers f e e l t h a t mutagenic t e s t s w i t h lower forms have l i t t l e r e l e v a n c e t o p o t e n t i a l e f f e c t s i n man. To h e l p b r i d g e t h i s gap, s e v e r a l workers are u s i n g u r i n e and b l o o d f r o m t r e a t e d animals o r exposed humans i n c o n j u n c t i o n w i t h t h e b a c t e r i a l t e s t systems.10,11 The d e t a i l s o f t h e procedures v a r y f r o m i n v e s t i g a t o r t o i n v e s t i g a t o r . Some use u r i n e samp l e s d i r e c t l y ( a f t e r p a s s i n g through a membrane f i l t e r i n o r d e r t o have an a s e p t i c sample) on t h e p l a t e . Others t r e a t t h e u r i n e w i t h g l u c u r o n i d a s e i n o r d e r t o s p l i t conjugated products, w h i l e o t h e r i n v e s t i g a t o r s m i g h t conc e n t r a t e t h e m e t a b o l i t e s t h r o u g h l y o p h i l i z a t i o n o r w i t h t h e use o f Amberl i t e columns. S i m i l a r t r e a t m e n t s a l s o may be done w i t h serum. Neverthel e s s , even t h e s e m o d i f i c a t i o n s do n o t overcome many o f t h e l e g i t i m a t e o b j e c t i o n s t o t h e s e systems, e . g . , dosimetry, p h y s i o l o g i c a l b a r r i e r s , e t c . Host-Mediated Assays. A1 though host-mediated assays were used r a t h e r ext e n s i v e l y a few y e a r s ago t o assess t h e p o t e n t i a l mutagenic e f f e c t s o f chemicals, t h e assay has f a l l e n i n t o some d i s p u t e because o f l a c k o f s e n s i -

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t i v i t y because t h e p h a r m o k i n e t i c s o f t h e t e s t agent i n t h e h o s t were n o t t h o r o u g h l y understood.98 Nevertheless, t h e assay does p r o v i d e a d e f i n i t e b r i d g e between m i c r o b i a l i n d i c a t o r organisms and a mammal. I n t h e r o u t i n e t e s t t h e m i c r o b i a l i n d i c a t o r organism i s i n j e c t e d i n t o t h e p e r i t o n e a l c a v i t y o f a mammal, u s u a l l y a r o d e n t . The h o s t animal i s t h e n t r e a t e d w i t h t h e chemical by any r o u t e o t h e r t h a n i n t r a p e r i t o n e a l l y . Several hours l a t e r t h e h o s t i s k i l l e d and t h e i n d i c a t o r organism i s recovered and scored f o r mutants. T h e o r e t i c a l l y , a comparison can be made between t h e mutagenic a c t i o n o f t h e compound ( 1 ) on t h e organism d i r e c t l y , and ( 2 ) whether t h e h o s t can d e t o x i f y t h e compound o r whether mutagenic p r o d u c t s ( m e t a b o l i t e s ) a r e formed as a r e s u l t o f h o s t metabolism. The h i s t i d i n e auxotrophs o f S. t y p h i m u r i w n ( t h e same t e s t s t r a i n s used i n t h e Arnes t e s t ) have been t h e most e x t e n s i v e l y used. Gene R e p a i r Systems. These systems a r e based on t h e h y p o t h e s i s t h a t DNA r e p a i r i s an i n d i r e c t measure o f g e n e t i c damage.

A system which d e t e c t s DNA damage i n E. coZi by r e p a i r of t h e g e n e t i c damage has been developed.57 I n a d d i t i o n t o t h e use o f b a c t e r i a , mammalian c e l l s i n c u l t u r e have been employed. S t i c h l 2 has d e s c r i b e d an assay i n which c u l t u r e d human s k i n f i b r o b l a s t s a r e used t o measure DNA r e p a i r b y t h e unscheduled uptake o f t r i t i a t e d t h y m i d i n e . The assay i s based on t h e f a c t t h a t DNA damage may be induced by chemical t r e a t m e n t o f t h e c e l l s and t h a t t h i s damage may be measured as an i n c r e a s e i n DNA s y n t h e s i s (DNA r e p a i r ) . Although S t i c h used human f i b r o b l a s t s i n c u l t u r e , o t h e r i n v e s t i g a t o r s have used c e l l l i n e s from Chinese hamsters, kangaroo r a t s , and Muntjac deer. Sega13 has d e s c r i b e d t h e d e t a i l s o f a DNA r e p a i r system i n e a r l y s p e r m a t i d stages o f t h e mouse. W i t h f o u r known mutagens, he has been a b l e t o d e t e c t DNA r e p a i r i n e a r l y spermatids. Dominant L e t h a l Test. Dominant l e t h a l m u t a t i o n i s a g e n e t i c e v e n t t h a t k i l l s t h e i n d i v i d u a l which c a r r i e s i t . The damage, which n o t a b l y c o n s i s t s o f chromosomal-type m u t a t i o n s , i s d e t e c t e d as p r e i m p l a n t a t i o n l o s s o f nonv i a b l e b l a s t o c y s t s and as e a r l y embryonic death.14 F e t a l wastage above t h e spontaneous background r a t e i s a t t r i b u t e d t o dominant l e t h a l m u t a t i o n s s i n c e o n l y t h e males a r e t r e a t e d w i t h t h e t e s t compound; females can be t r e a t e d a l s o , a l t h o u g h t h i s t e s t i s more d i f f i c u l t t o i n t e r p r e t . T h i s t e s t has been w e l l e v a l u a t e d by s e v e r a l i n v e s t i g a t o r s . 15-21 C y t o g e n e t i c Assays. C y t o g e n e t i c assays r e f e r t o t h e general c a t e g o r y o f a wide v a r i e t y o f chromosome t e s t s . Chromosomes a r e i m p o r t a n t i n mutagenic s t u d i e s s i n c e genes a r e p h y s i c a l l y l o c a t e d on t h e chromosomes. Several o f t h e numerous c y t o g e n e t i c assays a v a i l a b l e t o t h e mutagenic i n v e s t i g a t o r a r e d i r e c t l y a p p l i c a b l e t o man. That i s , t h e same procedures t h a t a r e used f o r t r e a t e d animals can be a p p l i e d t o humans. Thus, t h e s e t e s t s h o l d g r e a t promise f o r t h e e v a l u a t i o n o f p o t e n t i a l mutagenic compounds. Perhaps t h e most common c y t o e n e t i c assay i n v o l v e s s h o r t - t e r m (48 o r 72 h o u r s ) c u l t u r e o f lymphocytes.22 F o l l o w i n g w e l l - e s t a b l i s h e d technique, g e n e t i c i s t s have been a b l e t o f u r t h e r d e f i n e i n h e r i t e d m e t a b o l i c d i s o r d e r s i n humans. Next t o t r a n s m i s s i b l e gene mutations, t h e chromosome m u t a t i o n s r e p r e s e n t t h e

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second m a j o r t y p e o f g e n e t i c change. T h i s k i n d o f g e n e t i c a l t e r a t i o n i s u s u a l l y s t u d i e d i n vivo b y u s i n g t h e s h o r t - t e r m l e u k o c y t e c u l t u r e s , o r b y u s i n g bone marrow c e l l s d i r e c t l y . In v i t r o s t u d i e s can be done w i t h v a r i o u s c e l l l i n e s such as those d e r i v e d from Muntjac deer, Chinese hamsters, and mammalian tumor 1 ines. C y t o g e n e t i c analyses a l l o w t h e i n v e s t i g a t o r t o s t u d y t h e c e l l s i n metaphase o r anaphase f o r s t r u c t u r a l changes and r e a r rangements o f t h e i r chromosomes. The occurrence o f such chromosomal aberr a t i o n s m i g h t be c o r r e l a t e d w e l l w i t h t h e a d m i n i s t r a t i o n o f o r exposure t o known chemical mutagens and carcinogens. The d e t a i l s o f s e v e r a l methods f o r these assays have been published.23-27

A r e c e n t a d d i t i o n t o c y t o g e n e t i c analyses i n v o l v e s t h e use o f a s t a i n i n g t e c h n i q u e which can d e t e c t s i s t e r c h r o m a t i d exchanges.28~29 T h i s t e s t g i v e s a v e r y s e n s i t i v e and r a p i d method f o r t h e d e t e c t i o n of chromosome damage b y chemical agents and p r o v i d e s a p o w e r f u l new method f o r det e c t i n g environmental mutagens. S i s t e r c h r o m a t i d exchange i n v o l v e s a symm e t r i c a l exchange a t one l o c u s between s i s t e r chromatids w i t h no v i s i b l e a l t e r a t i o n o f gross chromosome morphology. Cyto e n e t i c procedures have a l s o been a p p l i e d t o s t u d y m e i o t i c chromosomes. 3Oy3Q Such procedures a l l o w one t o s t u d y chromosome changes w i t h i n t h e spermatocyte by d i r e c t v i s u a l i z a t i o n o f chromosome t r a n s l o c a t i o n s . I n an e f f o r t t o s i m p l i f y c y t o g e n e t i c assays, t h e m i c r o n u c l e u s t e s t was r e p o r t e d b y S ~ h m i d . 3 2 ~ 3 3T h i s t e s t i s a r e l a t i v e l y r a p i d i n vivo method d e v i s e d p r i m a r i l y f o r s c r e e n i n g chemicals f o r chromosome b r e a k i n g e f f e c t s . A l l compounds which a r e clastogens ("chromosome b r e a k e r s " ) , t h a t have been s t u d i e d , a l s o cause i n c r e a s e s i n t h e numbers o f bone marrow c e l l s w i t h m i c r o n u c l e i ( s m a l l pieces o f chromatin m a t e r i a l ) . Drosophila. The use o f d r o s o p h i l a ( f r u i t f l i e s ) f o r mutagen s t u d i e s has Few h i g h e r organisms can be r e a r e d i n l a r g e numbers s e v e r a l advantages. as e a s i l y and e c o n o m i c a l l y as can d r o ~ o p h i l a . 3 4 ~ 3 5Thus, one can s t u d y many types o f g e n e t i c a1 t e r a t i o n s : dominant l e t h a l s , p o i n t m u t a t i o n s , chromosome rearrangements, and l o s s o f X o r Y chromosomes. S p e c i f i c Locus Test. The s p e c i f i c l o c u s t e s t i n t h e mouse i s a method f o r d e t e c t i n g and measuring r a t e s o f m u t a t i o n a t s e v e r a l r e c e s s i v e l o c i . The method has been d e s c r i b e d and reviewed e x t e n s i v e l y i n t h e l i t e r a t u r e . 36-38 The method b a s i c a l l y c o n s i s t s o f m a t i n g t r e a t e d and u n t r e a t e d w i l d t y p e mice, e i t h e r male o r female, t o a s t r a i n o f mice homozygous f o r a number o f known r e c e s s i v e genes. The r e c e s s i v e genes a r e such t h a t t h e y a r e r e a d i l y expressed as v i s i b l e phenotypes i n t h e homozygous s t a t e . I f a m u t a t i o n has o c c u r r e d i n any o f t h e t e s t l o c i i n t h e germ c e l l s o f t h e t r e a t e d animals, i t w i l l be d e t e c t e d i n t h e o f f s p r i n g . I f no m u t a t i o n has o c c u r r e d f o l l o w i n g t r e a t m e n t , t h e progenies from t h e cross w i l l be o f t h e w i l d type.39 The main disadvantage o f t h i s t e s t i s t h a t i t r e q u i r e s t h e p r o d u c t i o n o f l a r g e numbers o f mice and consequently c o n s i d e r a b l e space f o r housing. T r a n s l o c a t i o n Test. H e r i t a b l e t r a n s l o c a t i o n s can be measured i n animals. For convenience, male mice a r e u s u a l l y used. The assay c o n s i s t s o f d e t e r -

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m i n i n g t h e a b i l i t y o f a chemical t o induce r e c i p r o c a l t r a n s l o c a t i o n s i n t h e germ c e l l l i n e s o f t r e a t e d m i ~ e . 1 6 ~ 4 0H e r i t a b l e t r a n s l o c a t i o n s can be obs e r v e d i n d i v i d i n g spermatocytes o f F1 males o r i n t h e o f f s p r i n g o f F1 females. The t i e r approach has been suggested as s t r a t e g y f o r The T i e r Approach. s e t t i n g p r i o r i t y when l a r g e numbers o f chemicals must be e v a l u a t e d , o r i f one i s choosing between analogs f o r f u r t h e r d e ~ e l o p m e n t . 4 1 ~ 4 2 These h i e r a r c h a l schemes e x p e d i t e t h e t e s t i n g and e v a l u a t i o n o f g e n e t i c a l l y hazardous substances i n a stepwise f a s h i o n b e g i n n i n g w i t h t h e most r a p i d and i n e x p e n s i v e assays c o n t i n u i n g t h r o u g h t h e more time-consuming and c o s t l i e r t e s t s u n t i l a d e f i n i t i v e assessment a p p r o p r i a t e t o t h e compound i n q u e s t i o n i s determined. The f i r s t t i e r c o n t a i n s s h o r t - t e r m s c r e e n i n g t e s t s w i t h submammalian systems, t h e second t i e r c o n t a i n s s h o r t and l o n g e r t e r m t e s t s w i t h whole animals, and t h e t h i r d t i e r i n v o l v e s a r i s k - b e n e f i t e v a l u a t i o n which may e n t a i l f u r t h e r , more s p e c i a l i z e d t e s t i n g procedures and experiments on t h e d e t a i l e d metabolism o f t h e agent in v i m . I n t h e pharmaceutical i n d u s t r y where a manageable number o f substances a r e e v a l u a t e d f o r p o t e n t i a l use, s e v e r a l t e s t s from t h e v a r i o u s t i e r s may be a p p l i e d , almost s i m u l t a n e o u s l y . Regardless o f which s t u d i e s a r e used, t h e r e s u l t s o f a l l s t u d i e s must be p u t i n t o p r o p e r p e r s p e c t i v e p r i o r t o t h e widespread use o f those sub. stances which have, i n f a c t , g i v e n p o s i t i v e responses i n c e r t a i n mutagenic tests. It i s apparent t h a t chemicals which i n t e r a c t w i t h DNA o r a r e metab o l i c a l l y c o n v e r t e d t o substances which have t h e c a p a c i t y t o i n t e r a c t w i t h DNA t h r o u g h e l e c t r o p h i l i c a t t a c k o r by o t h e r means t o a l t e r DNA have t h e p o t e n t i a l t o induce m u t a t i o n . I t i s a l s o apparent from knowledge o f c h e m i s t r y and b i o c h e m i s t r y t h a t many substances, man-made and n a t u r a l , w i l l have t h i s c a p a b i l i t y e i t h e r d i r e c t l y o r f o l l o w i n g metabolism o f t h e chemical by t h e h o s t organism.

The use o f mutagenic t e s t systems t o i d e n t i f y p o s s i b l e chemical carcinogens i s based on t h r e e m a j o r developments i n t h e f i e l d s o f chemic a l c a r c i n o g e n e s i s and chemical mutagenesis: ( 1 ) t h e r e c o g n i t i o n t h a t chemi c a l carcinogens may be d i v i d e d i n t o two broad categories--precarcinogens and u l t i m a t e carcinogens; ( 2 ) t h e s u c c e s s f u l c o u p l i n g o f m e t a b o l i c a c t i v a t i o n and gene m u t a t i o n s in u i t r o ; and ( 3 ) a background o f i n f o r m a t i o n i n t h e l i t e r a t u r e i n d i c a t i n g t h a t a h i g h number o f chemical carcinogens w i t h g r e a t l y v a r i e d s t r u c t u r e s were mutagenic. The somatic m u t a t i o n concept r e q u i r e s t h a t carcinogens a r e a l s o mutagens and t h a t c a r c i n o g e n i c and mutagenic p o t e n t i a l s o f carcinogens s h o u l d These problems were reviewed b y B u r d e t t e 4 3 i n 1955, a t be c o r r e l a t a b l e . which t i m e he concluded t h a t t h e r e was no c o r r e l a t i o n between t h e mutagenic and c a r c i n o g e n i c a c t i v i t i e s o f t h o s e chemicals which had been assayed f o r b o t h types o f a c t i v i t y . I n 1955, v e r y l i t t l e was known w i t h r e g a r d t o s t r u c t u r e s o f t h e u l t i m a t e r e a c t i v e forms o f chemical carcinogens in v i m , b u t B u r d e t t e r e c o g n i z e d t h a t t h e l a c k o f c o r r e l a t i o n c o u l d be due, among o t h e r f a c t o r s , t o d i f f e r e n c e s i n t h e m e t a b o l i c f a t e s o f t h e chemicals i n t h e d i f f e r e n t organisms used t o assay c a r c i n o g e n i c i t y and m u t a g e n i c i t y .

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I n 1966, t h e s t a t e o f knowledge and c o n f u s i o n o f t h e r e l a t i o n between mutagenesis and c a r c i n o enesis by chemicals had n o t changed much f r o m t h a t desc r i b e d by B u r d e t t e 4 and l e d t h e M i l l e r s 4 4 t o conclude t h a t t h e r e i s no c l e a r l y e s t a b l i s h e d r e l a t i o n s h i p between t h e mutagenic and c a r c i n o g e n i c a c t i v i t i e s o f chemicals. C e r t a i n a l k y l a t i n g agents were shown t o possess b o t h p r o p e r t i e s , b u t s e v e r a l s t r o n g mutagens and s t r o n g carcinogens had been t e s t e d f o r c a r c i n o g e n i c i t y and m u t a g e n i c i t y , r e s p e c t i v e l y , w i t h negative results.

I!

I n r e c e n t y e a r s i t has become e v i d e n t t h a t t h e m a j o r i t y o f chemical carcinogens a r e precarcinogens which must be c o n v e r t e d in vivo t o u l t i m a t e carcinogens. These m e t a b o l i c conversions a r e u s u a l l y mediated b y o x i d a t i v e enzymes and may i n v o l v e t h e f o r m a t i o n o f i n t e r m e d i a t e m e t a b o l i t e s termed p r o x i m a t e carcinogens. A1 though chemical carcinogens comprise a wide v a r i e t y o f s t r u c t u r e s and f o r a l o n g t i m e i t was d i f f i c u l t t o v i s u a l i z e a common denominator f o r c a r c i n o g e n i c a c t i v i t y , i t now appears t h a t F i g . 1.

SCHEME FOR THE METABOLIC ACTIVATION AND DEACTIVATION OF CHEMICAL CARCINOGENS AND FOR THE ROLE OF ULTIMATE CARCINOGENS45 Precarcinogen J.

7 Proximate Carcinogens

U l t i m a t e Carcinogen(s1 ( E l e c t r o p h i l i c , mutagenic) .L

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Inactive Metabolites

N u c l e o p h i l i c N, 0, and S atoms i n macromolecules CNA's, RNA's, p r o t e i n s

Carcinogen Residues Bound C o v a l e n t l y t o I n f o r m a t i o n a l Macromolecules C o n t r o l l i n g C e l l R e p l i c a t i o n .L

.c .L

Carcinogenesis (Mechanisms: g e n e t i c ? , e p i g e n e t i c ? )

Tumors t h e u l t i m a t e c a r c i n o g e n i c forms have t h e common f e a t u r e t h a t most, i f n o t a l l , a r e s t r o n g e l e c t r o p h i l i c reactants.46947 That i s , t h e u l t i m a t e c a r cinogens appear t o c o n t a i n r e l a t i v e l y e l e c t r o n - d e f i c i e n t atoms which can r e a c t c o v a l e n t l y w i t h e l e c t r o n - r i c h o r n u c l e o p h i l i c atoms i n c e l l u l a r components i n c l u d i n g those i n macromolecules such as t h e n u c l e i c a c i d s (DNA and RNA) and p r o t e i n s . The apparent i d e n t i t y o f t h e u l t i m a t e e l e c t r o p h i l i c r e a c t a n t s and t h e u l t i m a t e c a r c i n o g e n i c forms o f chemical carcinogens was f i r s t e v i d e n t f o r t h e s i m p l e c a r c i n o g e n i c a l k y l a t i n g agents. The s i m i l a r i t i e s i n t h e i r r e a c t i o n s t o those o f t h e a l k y l a t i n g agents s t r o n g l y i m p l i c a t e d a l k y l a t i n g i n t e r m e d i a t e s as t h e u l t i m a t e c a r c i n o g e n i c forms o f t h e a1 k y l n i t r o s a m i n e s , a l k y l n i t r o s a m i d e s , d i a l k y l h y d r a z i n e s , d i a l k y l t r i a z i n e s , and cycasin.45 The e l e c t r o p h i 1i c concept o f chemical carcinogenesis was extended

Chap. 24

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Dybas, H i t e , Flannn

Detecting Mutagens

p r i m a r i l y f r o m i n v e s t i g a t i o n s on t h e metabolism of 2 - a c e t y l a m i n o f l u o r e n e (AAF) and 4-methylaminoazobenzene (MAB). The c a r c i n o g e n i c i t y o f t h e arom a t i c amines and amides t h a t have been adequately s t u d i e d depends on t h e i r conversion t o N-hydroxy d e r i v a t i v e s and t h e n on t h e c o n v e r s i o n o f t h e l a t t e r m e t a b o l i t e s t o e l e c t r o p h i l i c metabolites.46,48 The m e t a b o l i c a c t i v a t i o n o f t h e l i v e r carcinogen AAF and i t s p r o x i m a t e m e t a b o l i t e N-hydroxyAAF has been s t u d i e d i n c o n s i d e r a b l e d e t a i l . E s t e r i f i c a t i o n o f N-hydroxyAAF by a 3'-phosphoadenosine-5'-phosphosul f a t e (PAPS)-dependent system i n t h e s o l u b l e f r a c t i o n of r a t 1 i v e r c o n v e r t s N-hydroxy-AAF t o a v e r y r e a c t i v e and mutagenic s u l f u r i c a c i d e s t e r , which a l s o a pears t o be t h e m a j o r u l t i mate c a r c i n o g e n i c m e t a b o l i t e f o r t h e l i v e r . 4 9 - 5 7 O t h e r e l e c t r o p h i l i c metab o l i t e s o f N-hydroxy-AAF ( g l u c u r o n i d e , a c e t a t e , and phosphate) a r e a l s o formed e n z y m a t i c a l l y by v a r i o u s t i s s u e p r e p a r a t i o n s and s h o u l d be c o n s i d e r e d as p o s s i b l e u l t i m a t e carcinogens i n some s i t u a t i o n s . 4 8 ~ 5 2 - 5 4 Fig. 2.

METABOLIC ACTIVATION OF 2-ACETYLAMINOFLUORENE (2-AAF) AND REACTIONS OF THE ELECTROPHILIC INTERMEDIATE WITH CELLULAR CONSTITUENTS50

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l i v e r sulfotrans ferase 0

guanine

o DNA The work o f t h e M i l l e r s 4 4 - 4 8 and others93-96 on t h e a c t i v a t i o n and r e a c t i v i t y o f chemical carcinogens l e d t o t h e r e c o g n i t i o n and acceptance o f t h e f a c t t h a t , w i t h t h e e x c e p t i o n o f d i r e c t a l k y l a t i n g agents, chemicals a r e n o t c a r c i n o g e n i c per se b u t must undergo m e t a b o l i c a c t i v a t i o n by mamm a l i a n enzymes. T h i s knowledge c o n t r i b u t e d g r e a t l y t o t h e development o f t h e c u r r e n t in v i t r o assays s i n c e i t r e v e a l e d t h a t t h e f a i l u r e t o d e t e c t mutagenic a c t i v i t y i n many o f t h e e a r l y g e n e t i c t e s t s c o u l d be d i r e c t l y a t t r i b u t e d t o t h e f a c t t h a t b a c t e r i a do n o t d u p l i c a t e mammalian metabolism i n a c t i v a t i n g carcinogens s i n c e t h e y l a c k many o f t h e a p p r o p r i a t e enzymes U

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r e q u i r e d t o a c t i v a t e t h e chemical t o i t s b i o l o g i c a l l y a c t i v e forms. Conseq u e n t l y , t h e f a i l u r e t o d e t e c t any c o r r e l a t i o n between c a r c i n o g e n e s i s i n animals and mutagenesis i n b a c t e r i a from t h e e a r l y s t u d i e s i s understandable. The f i r s t p u b l i s h e d work i n which t h e mutagenic a c t i v i t y o f metabo1it e s was d e t e c t e d a f t e r m e t a b o l i c a c t i v a t i o n o f carcinogens was b y M a l l i n g 5 5 who used mammalian-liver homogenates t o a c t i v a t e d i m e t h y l n i t r o s a m i n e t o a compound t h a t r e v e r t e d SaZrnoneZZa t y p h i m u r i w n t e s t s t r a i n s . S h o r t l y t h e r e a f t e r , mammalian-liver homogenates were used by Gainer56 t o a c t i v a t e a f l a t o x i n B, t o a compound l e t h a l t o a SaZrnoneZZa t e s t s t r a i n l a c k i n g exc i s i o n r e p a i r , and by S l a t e r 5 7 t o a c t i v a t e d i m e t h y l n i t r o s a m i n e t o a compound l e t h a l f o r E. coZi b a c t e r i a l a c k i n g polymerase I. Subsequently, Ames3 extended t h i s e a r l i e r work by adding human, o r r a t l i v e r homogenates and a TPNH-generating system d i r e c t l y t o p e t r i p l a t e s w i t h h i s SaZrnoneZZa t y p h i murim t e s t s t r a i n s and t h e carcinogen. These procedures have been f u r t h e r m o d i f i e d t o i n c l u d e l i v e r homogenates o b t a i n e d from animals whose 1 i v e r enzymes have been induced w i t h p h e n o b a r b i t a l , A r o c l o r , o r 3-methyl cholanthrene i n order t o provide f o r e f f i c i e n t d e t e c t i o n o f a v a r i e t y o f carcinogens r e q u i r i n g m e t a b o l i c a c t i v a t i o n .

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As a r e s u l t o f t h e advances made i n t h e development o f t h e m i c r o b i a l systems f o r d e t e c t i n g mutagens, a h i g h c o r r e l a t i o n between m u t a g e n i c i t y i n these assays and c a r c i n o g e n i c i t y i n animal s3,55,57,64,71 became e v i d e n t . The f a c t t h a t chemical carcinogens a r e a l s o mutagens s h o u l d n o t be p a r t i c u l a r l y s u r p r i s i n g s i n c e a p p a r e n t l y b o t h b i o l o g i c a l end p o i n t s a r e reached t h r o u g h an e l e c t r o p h i l i c a t t a c k on DNA by t h e p a r e n t compound, i n t h e case of a d i r e c t a c t i n g carcinogen, o r a m e t a b o l i t e form o f t h e chemical. The concept of t h e r e a c t i v e e l e c t r o p h i l i c agent forms t h e r a t i o n a l e b e h i n d t h e u t i l i z a t i o n o f m u t a g e n i c i t y assays i n s c r e e n i n g f o r p o t e n t i a l carcinogens. If chemical carcinogens o r t h e i r e l e c t r o p h i l i c m e t a b o l i t e s i n d u c e g e n e t i c changes which d i r e c t l y o r i n a s s o c i a t i o n w i t h o t h e r c e l l u l a r d y s f u n c t i o n s r e s u l t i n t h e m a l i g n a n t t r a n s f o r m a t i o n of normal c e l l s t o p o t e n t i a l tumor c e l l s , then b y t h e d e t e c t i o n o f mutagenic a c t i v i t y p o t e n t i a l carcinogens c o u l d be i d e n t i f i e d . I n 1975 Ames p u b l i s h e d t h e r e s u l t s o f h i s study o f 300 compounds i n t h e SaZrnoneZZa/microsome m u t a g e n i c i t y t e s t i n which he demonstrated t h a t 90% (156/174) o f t h e carcinogens were a l s o mutagenic i n t h e assay. D e s p i t e t h e severe l i m i t a t i o n s i n h e r e n t i n d e f i n i n g n o n - c a r c i n o g e n i c i t y o f compounds , few non-carcinogens ( < l o % ) showed any degree o f m u t a g e n i c i t y . 58-60 The Ames t e s t has demonstrated t h e a b i l i t y t o d e t e c t a v a r i e t y o f carcinogens as mutagens, such as d i r e c t a1 k y l a t i n g agents, n i t r o s a m i n e s , p o l y c y c l i c hydrocarbons, fungal t o x i n s , a r o m a t i c amines, n i t r o f u r a n carcinogens, a v a r i e t y o f a n t i n e o p l a s t i c agents, and a n t i b i o t i c carcinogens such as a d r i amycin, daunomycin, and mitornycin C. I n a d d i t i o n , most o f t h e known human chemical carcinogens which have been t e s t e d were p o s i t i v e . These i n c l u d e B-naph t h y 1 ami ne , benz id i ne , c iga r e t t e smoke condensates , b isch 1orome t h y l e t h e r , a f l a t o x i n B1, v i n y l c h l o r i d e and 4-aminobiphenyl.58 The p r e s e n t data i n d i c a t e t h a t a p p r o x i m a t e l y t e n p e r c e n t o f t h e c a r -

Chap. 24

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cinogens ( 1 7/175) were non-mutagenic i n t h e t e s t ( T a b l e 1). TABLE 1. o- t o 1 u i d i n e

aurami ne carbon t e t r a c h l o r i d e DOE dieldrin thiourea

NON-MUTAGENIC CARCINOGENS59

e t h y l carbamate 3-amino-l,2,4- t r i azol e phenobarbi t o 1 thioacetamide acetamide ethionine

s a f r o 1e cycasin 4- ami no a n t i py r in e l Y 2 - d i m e t h y lh y d r a z i n e p r o c a r b a z i ne

Some m o d i f i c a t i o n s i n t h e i n v i t r o m e t a b o l i c a c t i v a t i o n system may be necessary f o r t h e d e t e c t i o n o f some of t h e s e carcinogens as mutagens, f o r example, t h e c h l o r i n a t e d hydrocarbons (carbon t e t r a c h l o r i d e , DDE, and d i e l d r i n ) , e t h y l carbamate, s a f r o l e , p r o c a r b a z i n e and 1 ,2-dimethylhydrazine, whilc i n t h e case o f auramine, 4 - a m i n o - a n t i p y r i n e and acetamide, t h e c a r c i n o g e n i c i t y s t u d i e s i n animals have n o t been d e f i n i t i v e . 5 9 3-Amino-1 , 2 , 4 - t r i a z o l e , t h i o a c e t a m i d e and t h i o u r e a possess g o i t r o g e n i c a c t i v i t y and Weissburger has suggested t h a t t h e s e agents cause t h y r o i d tumors t h r o u g h a non-mutagenic mechanism.61 Cycasin, a 8 - g l u c o s i d e o f methylazoxymethanol , i s i n a c t i v e i n t h e Ames procedure because n e i t h e r SaZrnoneZZa n o r mammal i a n m i crosomes cont a i n a 6 - g l u c o s i d a s e necessary f o r c o n v e r t i n g i t t o methylazoxymethanol which does show mutagenic a c t i v i t y i n t h e assay; w h i l e e t h i o n i n e mutagenic i t y may n o t be d e t e c t a b l e i n SalrnoneZZa s i n c e i t may a c t as a c a r c i n o g e n b y e t h y l a t i n g n u c l e i c a c i d a t n a t u r a l m e t h y l a t i o n s i t e s t h r o u g h S-adenosylethionine.59 The Ames t e s t appears t o be h i g h l y s e l e c t i v e f o r t h e d e t e c t i o n o f c a r cinogens. To date , a p p r o x i m a t e l y 108 noncarcinogens (chemical s n e g a t i v e i n t e s t s f o r c a r c i n o g e n i c i t y i n a n i m a l s ) , f a l l i n g i n t o two general c a t e g o r i e s - 62 chemicals, most o f which a r e c l o s e l y s t r u c t u r a l l y r e l a t e d and even i s o m e r i c t o carcinogens, and 46 common biochemicals--have been tested.58-60 About 13% o f these noncarcinogens showed some degree o f mutagenic a c t i v i t y i n t h e assay; however, s i n c e c a r c i n o g e n i c i t y s t u d i e s have been e x t r e m e l y l i m i t e d f o r s e v e r a l o f t h e s e compounds, t h e r e must be doubt as t o t h e c l a s s i f i c a t i o n of t h e s e chemicals as noncarcinogens. Other m i c r o b i a l mutation, r e p a i r and r e c o m b i n a t i o n assays have a l s o been used i n chemical screening, however, t h e d a t a base f o r c a r c i n o g e n i c i t y m u t a g e n i c i t y c o r r e l a t i o n e s t a b l i s h e d w i t h t h e s e i n v i t r o t e s t systems i s r e l a t i v e l y l i m i t e d a t t h i s time.62-70 A t p r e s e n t , t h e Ames SaZmoneZZa assay appears t o be t h e most g e n e r a l l y a p p l i c a b l e screen f o r t h e d e t e c t i o n o f chemical mutagens. No o t h e r mutagenesis assay has been shown t o respond t o such a wide group o f chemical t y p e s as t h e SaZmoneZZa mutants. The i n u i t r o m u t a g e n i c i t y assays w i t h Ames SaZmoneZla typhirnuriwn s t r a i n s have been used t o i n v e s t i g a t e t h e e x t e n t and u n d e r l y i n g b a s i s f o r organ, species, and sex v a r i a b i l i t y i n carcinogen metabolism and s e n s i t i v i t y t o t h e a c t i o n o f chemical carcinogens. R e c e n t l y Weekes and B r u s i c k 7 1 used t h e i n v i t r o system t o compare t h e r e l a t i o n s h i p between t a r g e t organ suscept i b i l i t y f o r dimethylnitrosamine-induced tumors and i n v i t r o m e t a b o l i c a c t i -

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v a t i o n o f d i m e t h y l n i t r o s a m i n e (DMNA) t o a mutagen. DMNA has been shown t o be b o t h mutagenic and c a r c i n o g e n i c f o l l o w i n g m e t a b o l i c a c t i v a t i o n in vivo and mutagenic f o l l o w i n g a c t i v a t i o n in v i t r o . 7 2 ~ 7 3 I n a d d i t i o n , Czygan74 demonstrated t h a t t h e r a t e o f mutagen f o r m a t i o n was l i n k e d t o t h e r a t e o f enzymatic c o n v e r s i o n o f DMNA t h e r e b y p e r m i t t i n g t h e m u t a t i o n assay t o be used as an i n d i r e c t assay f o r t h e enzymatic a c t i v i t y o f a p a r t i c u l a r t i s s u e t o b i o t r a n s f o r m DMNA i n t o a mutagenic ( c a r c i n o g e n i c ) i n t e r m e d i a t e . I n mice, DMNA induces predominantly l i v e r and l u n g tumors w i t h low f r e q u e n c i e s of k i d n e y tumors and r a r e l y , i f ever, tumors o f t h e spleen o r gonads.72~75-77 The data generated by Weekes and B r u s i c k 7 1 demonstrated t h a t a c t i v a t i o n of DMNA t o a mutagenic i n t e r m e d i a t e b y microsome p r e p a r a t i o n s f r o m t h e s e organs o f BALB/cJ, RF/J and C57Blj6J mice show t h e same r a n k i n g ( l i v e r > l u n g > k i d n e y > spleen > t e s t e s ) s u g g e s t i n g t h a t a t l e a s t f o r t h e carcinogen DMNA, t h e l e v e l o f m e t a b o l i c a c t i v a t i o n s o f a t i s s u e may be d i r e c t l y r e l a t e d t o t h e s u s c e p t i b i l i t y o f t h e organ as a t a r g e t f o r tumor i n d u c t i o n . Species d i f f e r e n c e s i n drug metabolism have a l s o been l i n k e d t o observed v a r i a b i l i t y i n t a r g e t organ s e n s i t i v i t y t o t h e t u m o r i g e n i c a c t i o n o f DMNA. I n mice, C3H and Swiss s t r a i n s a r e h i g h l y s u s c e p t i b l e , showing r e n a l tumor i n c i d e n c e s o f a p p r o x i m a t e l y 16 and 11 p e r c e n t r e s p e c t i v e l y , w h i l e BALB/c and RF s t r a i n s a r e q u i t e r e s i s t a n t showing r e n a l tumor f r e q u e n c i e s l o w e r than 4 percent.75-77 Recently, Weekes78 was a b l e t o show t h a t when m i crocome p r e p a r a t i o n s from t h e kidneys o f these f o u r s t r a i n s were compared f o r t h e i r a b i l i t y t o a c t i v a t e DMNA t o a mutagen, t h e r e was an e x c e l l e n t c o r r e l a t i o n between c a r c i n o g e n i c s u s c e p t i b i l i t y and mutagen p r o d u c t i o n . Kidney microsomes f r o m t h e two mouse s t r a i n s e x h i b i t i n g h i g h suscept i b i l i t y t o DMNA-induced r e n a l tumors were c o n s i d e r a b l y more a c t i v e i n f o r m i n g t h e mutagenic/carcinogenic i n t e r m e d i a t e t h a n p r e p a r a t i o n s from t h e two s t r a i n s w i t h lower s e n s i t i v i t y t o DMNA. I n a s i m i l a r f a s h i o n , Weekes and Brusick71 were a b l e t o show t h a t t h e mutagen d a t a from microsome p r e p a r a t i o n s from l i v e r and l u n g t i s s u e s from a l l f o u r s t r a i n s were s i m i l a r as would be expected s i n c e t h e l i v e r and l u n g tumor s u s c e p t i b i l i t i e s o f these mouse s t r a i n s t o DMNA a r e n e a r l y e q u i v a l e n t . These d a t a t e n d t o conf i r m t h e i n i t i a l assumption t h a t t h e f o u r mouse s t r a i n s e x h i b i t d i f f e r e n t l e v e l s of DMNA a c t i v a t i o n and t h a t t a r g e t organism metabolism o f DMNA i s important f o r the i n i t i a t i o n o f neoplasia.71~78 I n a comparison o f t h e m e t a b o l i c a c t i v a t i o n o f d i m e t h y l n i t r o s a m i n e by l i v e r , lung, and kidney microsomes f r o m male and female C57BL/6J mice, t h e e x i s t e n c e o f a sex d i f f e r e n c e i n DMNA a c t i v a t i o n w i t h kidney microsomes was demonstrated i n t h a t t h e female mice c o u l d n o t m e t a b o l i z e DMNA t o a mutagen.71 S i m i l a r sex d i f f e r e n c e s were n o t observed i n s t u d i e s w i t h SpragueDawley o r F i s c h e r r a t s . Hormonal r e g u l a t i o n o f mouse k i d n e y a c t i v a t i o n s was subsequently demo n s t r a t e d by B r u s i c k . 6 N e i t h e r male n o r female k i d n e y p r e p a r a t i o n s were a b l e t o a c t i v a t e DMNA t o a mutagen u n t i l t h e y were 35 days o l d , a t which t i m e t h e a c t i v a t i o n p o t e n t i a l i n male k i d n e y p r e p a r a t i o n s began t o i n c r e a s e . I t was concluded t h a t male hormones m i g h t be r e s p o n s i b l e f o r t h e regul a t i o n s i n c e t h e a c t i v a t i o n t i m i n g (day 35) c l o s e l y c o i n c i d e s w i t h t h e onset of t e s t o s t e r o n e s y n t h e s i s and t h e e x p r e s s i o n o f a d u l t b e h a v i o r i n

Chap. 24

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24 5 -

male mice. I n s t u d i e s w i t h TFM ( t e s t i c u l a r f e m i n i z a t i o n males), t h e a f f e c t e d male animals who appear p h e n o t y p i c a l l y as females were s i m i l a r t o female animals i n t h e i r m e t a b o l i c a c t i v a t i o n o f DMNA t h e r e b y s u b s t a n t i a t i n g t h e hormonal r e g u l a t i o n o f mouse k i d n e y a c t i v a t i o n s . Species, sex, and t a r g e t - o r g a n d i f f e r e n c e s were a l s o n o t e d i n t h e a c t i v a t i o n o f a r o m a t i c amines. These compounds were found t o be a c t i v a t e d t o mutagens b y mouse k i d n e y microsomes t o a g r e a t e r e x t e n t t h a n b y mouse 1 i v e r . W i t h t h e carcinogen 2 - a c e t y l a m i n o f l uorene (AAF) , B r u s i c k 7 9 r e p o r t e d t h a t male mice of s e v e r a l s t r a i n s showed a p p r o x i m a t e l y 10 t i m e s g r e a t e r a c t i v a t i o n o f AAF t o a mutagen by k i d n e y microsome p r e p a r a t i o n s t h a n b y l i v e r p r e p a r a t i o n s . T h i s d i f f e r e n c e was n o t shown f o r o t h e r s p e c i e s such as t h e r a t , guinea p i g , dog, r a b b i t , o r monkey. M i l l e r 8 0 has r e p o r t e d t h a t AAF induces a h i g h i n c i d e n c e o f b l a d d e r tumors i n mice and t h i s o b s e r v a t i o n may be c o n s i s t e n t w i t h t h e i r h i g h r a t e o f k i d n e y a c t i v a t i o n f o r AAF. The mouse k i d n e y a c t i v a t i o n o f AAF has a l s o been shown t o be under hormonal r e g u l a t i o n i n t h a t t h e a c t i v a t i o n p o t e n t i a l o f t h e TFM males was l o w e r than t h e normal male a c t i v a t i o n p o t e n t i a l and almost e x a c t l y as l o w as female mouse kidneys.79 A d d i t i o n a l s t u d i e s d e m o n s t r a t i n g d i f f e r e n c e s i n t a r g e t organ m e t a b o l i c a c t i v a t i o n o f n i trosamine and c h l o r i n a t e d hydrocarbon carcinogens have r e c e n t l y been r e p o r t e d b y Bartsch.81 The use o f in v i t r o m i c r o b i a l mutagenesis assays t o f o l l o w m e t a b o l i c t r a n s f o r m a t i o n of chemical carcinogens ( p r e c a r c i n o g e n s ) has generated data t h a t augments t h e r e l i a b i l i t y o f t h e e m p i r i c a l c o r r e l a t i o n between c a r c i n o genesis and mutagenesis o r i g i n a l l y demonstrated i n t h e r e p o r t s b y Ames. ,58, I n a d d i t i o n , t h e r e s u l t s o f B r u s i c k 7 9 show a q u a n t i t a t i v e r e l a t i o n s h i p between microsomal enzyme a c t i v a t i o n and tumor s u s c e p t i b i l i t y f o r DMNA and AAF i n mice s t r o n g l y i m p l i c a t i n g t h e a c t i v e i n t e r m e d i a t e as b o t h t h e u l t i mate carcinogen and mutagen. The r e s u l t s o f these s t u d i e s p r o v i d e a d d i t i o n a l s u p p o r t t o t h e t h e o r y t h a t a chemical-DNA i n t e r a c t i o n i s a prer e q u i s i t e f o r a t l e a s t some tumor i n d u c t i o n s and t h a t t h e r e i s a f u n c t i o n a l l i n k between mutagenic and c a r c i n o g e n i c events. References 1. 2. 3. 4. 5. 6. 7. 8.

B. N. Ames, J. McCann and E. Yamasaki, M u t a t i o n Res., 31, 347 (1975). B. N. Ames i n "Chemical Mutagens," Vol. 1, A. H o l l a n d e r F E d . , Plenum Press, New York-London, 1971, p. 267. B. N. Ames, W. E. Durston, E. Yamasaki and F. D. Lee, Proc. Nat. Acad. S c i . (USA), 70, 2281 (1973). B. N. Ames, F. D. Lee and W. E. Durston, Proc. Nat. Acad. S c i . (USA), 70, 782 (1973). McCann, N. E. Spingarn, J . Kobori and 6. N. Ames, Proc. Nat. Acad. Sci. (USA), 72, 979 (1975). D. B r u s i c k , K. Bakshi and D. R. Jagannath i n "Proceedings o f t h e In v i t r o M e t a b o l i c Conference, F. J. deSerres, Ed., N o r t h H o l l a n d Publ i s h i n g Co., Amsterdam, 1976, p. 125. G. Mohn, M u t a t i o n Res., 20, 7 (1973). F. J. deSerres and H. V. M a l l i n g i n "Chemical Mutagens," Vol. 2, A. H o l l a n d e r , Ed., Plenum Press, New York, 1971, p. 311.

246 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39, 40. 41.

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R. K. Mortimer and T. R. Manney i n "Chemical Mutagens," Vol. 1, A. Hollander, Ed., Plenum Press, New York, 1971, p. 311. M. S. Legator, J . Connor and M. Stoecker, Annals N. Y. Acad. Sci. , 269, 16 (1975). V. Minnich, M. E. Smith, D. Thompson and S. Kornfeld, Cancer, 38, 1253 (1976). H. F. Stich, P. Lam, L. W. Lo, D. J. Koropatnick and H. C. San, Canad. J. Genetics and Cytology, lJ, 471 (1975). G. A. Sega, J. G. Owens and R. B. Cuming, Mutation Res., 36, 193 (1976). A. J . Bateman and S. S. Epstein i n "Chemical Mutagens," Vol. 2, A. Hollander, Ed., Plenum Press, New York, 1971, p. 541. S. S. Epstein, Environmental Health Perspectives, Issue No. 6, Dec., 1973, p. 23. W. M. Generoso, i b i d . , p. 13. V. A. Ray and M. L. Hyneck, i b i d . , p. 27. S. Green and J . A. Springer, i b i d . , p. 37. W . A. Maxwell and G. W . Newell , i b i d . , p. 47. D. S. Salsburg, i b i d . , p. 51.. R. L. Dixon and J . P. Lee, z b i d . , p. 59. P. S . Moorhead, P. C. Newell, W. J. Mellman, D. M. B a t t i p s and D. A. Hungerford, Exp. C e l l Res. , 20, 613 (1960). L. Schoeller and V. Wolf i n "Chemical Mutagenesis i n Mammals and Man," F. Vogel and G. Rohrborn, Eds., Springer-Verlag, New York, 1970, p. 232. W. W. Nichols, Agents and Actions, 3, 86 (1973). M. W . Shaw, Environmental Health Perspectives, Zoc. c i t . , p. 151. M. M. Cohen and K. Hirschhorn i n "Chemical Mutagens," Vol. 2, A. Hollander, Ed., Plenum Press, New York, 1971, p. 515. K. Hirschhorn, Ann. N. Y. Acad. Sci., 269, 12 (1975). S. A. L a t t , Proc. Nat. Acad. Sci. ( U S A T E , 3395 (1973). P. Perry and H. J . Evans, Nature, 258, 121 (1975). A. Leonard i n "Chemical Mutagen," Vol. 3, A. Hollander, Ed. , Plenum Press, New York, 1973, p. 21. E. Schleiermacher i n "Chemical Mutagenesis i n Mammals and Man," F. Vogel and G. Rohrborn, Eds. , Springer-Verlag, New York, 1970, p. 167. W. Schmid, Agents and Actions, 3, 77 (1973). W. Schmid, Mutation Res. , 31, 9-(1975). S. Zimmering, Environmental Health Perspectives, Zoc. c i t . , p. 111. S. Abrahamson and E. B. Lewis i n "Chemical Mutagens," Vol. 2, A. Hollander, Ed., Plenum Press, New York, 1971, p. 461. W. L. Russell, Cold Spring Harbor Symp. Quant. B i o l . , 16,327 (1951). W. L. Russell i n "Repair from Genetic Radiation Damage and D i f f e r e n t i a l R a d i o s e n s i t i v i t y t o Germ Cells," F. H. Sobels, Ed. , Pergamon Press, London, 1963, p. 206. B. M. Cattanach i n "Chemical Mutagens," Vol. 2, A. Hollander, Ed., Plenum Press, New York, 1971, p. 535. The T e s t i n g o f Chemicals f o r Carcinogenicity, Mutagenicity, and Teratogenicity, M i n i s t r y o f Health and Welfare, Canada, 1973, p. 92. A. Leonard, Mutation Res., 291 (1975). W. G. Flamn, Mutation Res., 3, 329 (1974).

1,

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42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74.

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B. A. Bridges, Environmental H e a l t h P e r s p e c t i v e s , Zoc. c i t . , p. 221. W. J. B u r d e t t e , Cancer Res., 20 (1955). J. A. M i l l e r and E. C. M i l l e r , Lab. I n v e s t . , 217 (1966). J. A. M i l l e r and E. C. M i l l e r i n "Screening Tests i n Chemical Carcinogenesis," R. Montesano, H. Bartsch, and L. Tomatis, Eds., IARC, Lyon, France, 1976, p. 153. J . A. M i l l e r , Cancer Res., 3, 559 (1970). J. A . M i l l e r and E. C. M i l l e r i n "The Jerusalem Symposia on Quantum Chemistry and Biochemistry," Vol. 1, E. 0. Burgmann and B. Pullman, Eds., I s r a e l Academy o f Sciences, Jerusalem, 1969, p. 236. E. C. M i l l e r and J. A. M i l l e r i n M o l e c u l a r B i o l o g y o f Cancer, H. Busch, Ed., Academic Press, New York, 1974, p. 337. J. R. DeBaun, E. C. M i l l e r and J. A. M i l l e r , Cancer. Res., 3, 477 (1970). J. R. DeBaun, J. Y. R. Smith, E. K. M i l l e r and J. A. M i l l e r , Science, 167, 184 (1970). J. H. Weissburger, R. S. Yamamoto, G. M. W i l l i a m s , P. H. Grantham, T. Matsushima and E. K. Weissburger, Cancer Res. , 32, 491 (1972). C. C. I r v i n g , R. A. Veazey and L. T. R u s s e l l , Chem. B i o l . I n t e r a c t i o n s , 1, 19 (1969/70). C. C. I r v i n g and R. Wissman, Cancer Res. , 31, 645 (1971). P. D. L o t h i k a r and L. Luba, Biochem. J. , 124, 69 (1971). H. V. M a l l i n g , M u t a t i o n Res., 13,425 (1971). R. C. Gainer, E. C. M i l l e r and J. A. M i l l e r , Cancer Res., 32, 2058 (1972). E. E. S l a t e r , M. D. Anderson and H. S. Rosenkranz, Cancer Res., 2, 970 (1971). J. McCann, E. Choi, E. Yamasaki and B. N. Ames, Proc. Nat. Acad. S c i . , 72, 5135 (1975). J. McCann and B. N. Ames, Proc. Nat. Acad. S c i . , 73, 950 (1976). B. N. Ames and J. McCann i n "Screening T e s t s i n Chemical Carcinogenes i s , " R. Montesano, H. B a r t s c h and L. Tomatis, Eds., I A R C , Lyon, France, 1976, p. 493. J . H. Weissburger i n "Cancer Medicine," J. F. H o l l a n d and E. F r e i , Eds. , Lea and Febiger, P h i l a d e l p h i a , Pa. , 1973, p. 45. E. E. S l a t e r , M. D. Anderson and H. S. Rosenkranz, Cancer Res. , 31, 970 (1971). B. A. Bridges, Lab. P r a c t . , 21, 413 (1972). T. Nakajima and S. I r v a h a r a , M u t a t i o n Res., 18,121 (1973). T. Kada, K. Tutikawa and Y. Sadaie, M u t a t i o n Res., 16,165 (1972). S. Konso, M u t a t i o n Res., 26, 235 (1974). G. Mohn and J. E l l e n b e r g e r , M u t a t i o n Res. , 19,257 (1973). F. K. Zimmerman, R. Schwaier and U. von Laer, Z. Vererbungsl. , 98, 230 (1966). D. J. B r u s i c k and V. W. Mayer, Environmental H e a l t h P e r s p e c t i v e s , 6, 83 (1973). D. J . B r u s i c k and H. Andrews, M u t a t i o n Res., 26, 491 (1974). U. Weekes and D. J. B r u s i c k , M u t a t i o n Res., 31, 175 (1975). S. Takayama and K. Oota, Gann. , 54, 465 ( 1 9 6 3 . H. V. M a l l i n g , M u t a t i o n Res., 13, 425 (1971). P. Czygan, H. G r e i n i , A. J. Garro, F. H u t t e r e r , F. Schaffner,

Is,

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P. Czygan, H. G r e i n i , A. J. Garro, F. H u t t e r e r , F. S c h a f f n e r , H. Popper, 0. Rosenthal and D. Y. Cooper, Cancer Res. , 3, 2983 (1973). S. Takayama and K. Oota, Gann., 56, 189 (1965). B. T e r r a c i n i , G. P a l e s t r o , M. R. G i g l i a r d i and R. Montesano, B r i t . J . Cancer, 20, 871 (1966). N. K. Clapp, R. L. T y n d a l l and J . A. Otten, Cancer Res., 31, 196 (1971). U. Weekes, J. Nat. Cancer I n s t . , 55, 1199 (1975). D. J . B r u s i c k , T o x i c o l o g y Annual n 9 7 5 ) . E. C. M i l l e r , J. A. M i l l e r and M. Enomoto, Cancer Res., 24, 2018 (1964). H. Bartsch, C. M a l a v e i l l e and R. Montesano i n "Screening Tests i n Chemical Carcinogenesis," R. Montesano, H. B a r t s c h and L. Tomatis, Eds., IARC, Lyon, France, 1976, p. 467. E. C. M i l l e r and J . A. M i l l e r i n "Chemical Mutagens," Vol. 1, A. H o l l a n d e r , Ed., Plenum Press, New York, 1971, p. 83. F. K. Zimmerman, Biochem. Pharmacol., 0, 985 (1971). E. F r e i s e , Brookhaven Symp. B i o l . , 12, 63 (1959). P. J . F i s h i n g e r , S. Nomura, P. T. Peebles, D. K. Haapala and R. H. Bassin, Science, 176, 1033 (1972). B. N. Ames i n "Mutagenic Events o f Environmental Contaminants," M. E. S u t t o n and M. I. Hains, Eds., Academic Press, New York, 1972, p. 57. K. Isono and J . Yourno, Proc. Nat. Acad. Sci., 71,1612 (1974). L. D. K i e r , E. Yamasaki and B. N. Ames, Proc. Nat. Acad. Sci., 7J, 4159 (1974). F. Kae and T. T. Puck, J . C e l l . P h y s i o l . , 78, 139 (1971). D. R. S t o l z , L. A. P o i r i e r , C. C. I r v i n g , H. F. S t i c h , J . H. Weissb u r g e r and H. C. Grice, T o x i c o l Appl. Pharmacol., 3, 157 (1974). T. Yahagi, M. Nagao, K. Hara, T. Matsushima, T. Sugimura and G. T. Bryan, Cancer Res. , 34, 2266 (1974). K. T e r a n i s h i , K. Hamada and H. Watanabe, M u t a t i o n Res. , 31, 97 (1975). D. N. Magee and P. F. Swann, B r i t . Med. B u l l . , 3, 240 (1969). D. V. Parks and R. T. W i l l i a m s , B r i t . Med. B u l l . , 25, 256 (1969). A. H. Connay and J . J . Burns, Science, 178,576 (1972). H. R. Gutmann, D. M a l e j k a - G i g a n t i , E. J . B a r r y and R. E. R y d e l l , Cancer Res., 32, 1554 (1972). W. G. Flamm, J. Assoc. Anal. Chem. @, 668 (1975). L. A. P o i r i e r i n "Screening Tests i n Chemical Carcinogenesis, R. Montesano, H. Bartsch, and L. Tomatis, Eds., IARC, Lyon, France, 1976, p. 15.

24 9 Chapter 25. B r a i n N e u r o t r a n s m i t t e r Receptor Binding and N e u r o l e p t i c Drugs I a n Creese and Solomon H. Snyder Department o f Pharmacology and E x p e r i m e n t a l T h e r a p e u t i c s J o h n s Hopkins U n i v e r s i t y School o f Medicine, B a l t i m o r e , Maryland 21205

The m o l e c u l a r mechanism o f a c t i o n r e s p o n s i b l e f o r t h e e f f i c a c y o f a t h e r a p e u t i c d r u g may be s u g g e s t e d by t h e b i o c h e m i c a l e v e n t which c o r r e l a t e s b e s t w i t h c l i n i c a l potency. The p a s t y e a r h a s s e e n t h e s u c c e s s f u l c h a r a c t e r i z a t i o n of t h e CNS dopaminergic and n o r a d r e n e r g i c a - r e c e p t o r s by means o f b i n d i n g s t u d i e s u t i l i z i n g r a d i o a c t i v e l y l a b e l e d r e c e p t o r - s p e c i f i c ligands. The i n t e r a c t i o n s of n e u r o l e p t i c d r u g s w i t h t h e s e b i n d i n g s i t e s have provided i n s i g h t i n t o t h e way t h a t n e u r o l e p t i c s produce t h e i r unwanted s i d e e f f e c t s a s w e l l a s t h e i r t h e r a p e u t i c mode o f a c t i o n . Such b i n d i n g s t u d i e s may p r o v i d e e x c e l l e n t i n v i t r o s c r e e n s p r e d i c t i v e o f a n t i s c h i z o p h r e n i c p o t e n c y , t h e d e g r e e of e x t r a p y r a m i d a l and autonomic s y m p a t h o l y t i c s i d e e f f e c t s and t h e l i k e l i h o o d of t a r d i v e d y s k i n e s i a f o l l o w i n g c h r o n i c t r e a t n e n t . N e u r o l e p t i c I n t e r a c t i o n s w i t h t h e Dopamine Receptor - Binding s t u d i e s , by d e f i n i t i o n , l a b e l t h e r e c o g n i t i o n s i t e o f a r e c e p t o r . The s t r i a t a l dopam i n e - s e n s i t i v e a d e n y l a t e c y c l a s e , which encompasses b o t h a r e c o g n i t i o n and an e f f e c t o r u n i t , i n i t i a l l y h e l d promise a s a b i o c h e m i c a l marker of t h e dopamine r e c e p t o r which might p r o v i d e a u n i t a r y e x p l a n a t o r y p r i n c i p l e f o r t h e t h e r a p e u t i c mode o f a c t i o n o f n e u r o l e p t i c drugs's2. However, t h e l a c k of c o r r e l a t i o n between c l i n i c a l p o t e n c y and i n h i b i t i o n of dopamine s t i m u l a t e d CAMP p r o d u c t i o n a c r o s s a l l groups of n e u r o l e p t i c d r u g s b e l i e v e d t o a c t by b l o c k i n g dopamine receptor^^,^, h a s i n d i c a t e d t h a t t h e i n t e r a c t i o n s between t h e r e c o g n i t i o n s i t e and t h e a d e n y l a t e c y c l a s e must b e complex5. Two s t u d i e s a t t e m p t e d t o l a b e l t h e r e c o g n i t i o n s i t e o f t h e dopamine r e c e p t o r w i t h r a d i o a c t i v e l y l a b e l e d n e u r o l e p t i c d r u g s b u t were unsuccessf u 1 6 s 7 . T h i s w a s , i n p a r t , b e c a u s e most r a d i o a c t i v e l i g a n d s c a n b i n d t o b i o l o g i c a l membranes i n r e l a t i v e l y n o n s p e c i f i c manners such as by hydrophob i c i n t e r a c t i o n s , i o n i c a t t r a c t i o n o r van d e r Waal's f o r c e s . S i n c e t h e number o f s u c h n o n s p e c i f i c b i n d i n g s i t e s i s v i r t u a l l y i n f i n i t e i t i s d i f f i c u l t t o d i s t i n g u i s h s p e c i f i c r e c e p t o r b i n d i n g ( i n t h e o r d e r of 10-100pmole/ g w e t w t t i s s u e ) from t h e n o n s p e c i f i c background. To overcome t h i s probl e m i t i s n e c e s s a r y t o u s e low c o n c e n t r a t i o n s o f a h i g h s p e c i f i c a c t i v i t y l a b e l e d l i g a n d which h a s h i g h r e c e p t o r a f f i n i t y s o t h a t t h e r a t i o o f s p e c i f i c t o n o n s p e c i f i c b i n d i n g can b e maximized. I t i s n e c e s s a r y t o r i n s e t i s s u e p r e p a r a t i o n s a f t e r i n c u b a t i o n by r a p i d f i l t r a t i o n on g l a s s f i b e r f i l t e r s under vacuum t o remove t h e lower a f f i n i t y , n o n s p e c i f i c a l l y bound ligand8. R e c e n t l y i n o u r s and Seeman's l a b o r a t o r y t h e b i n d i n g o f 3H-haloperid o 1 t o t h e dopamine r e c e p t o r h a s been c h a r a c t e r i z e d g , l O . I n g e n e r a l a c r u d e membrane p r e p a r a t i o n from c a l f s t r i a t u m h a s been u s e d . Binding o c c u r s r a p i d l y a t 37" C and r e a c h e s e q u i l i b r i u m between 1 and 3 min w i t h h a l f maximal b i n d i n g a t t a i n e d a t a b o u t 20 s e c . T h e r e are t h r e e major c r i t e r i a which must be s a t i s f i e d i n o r d e r t o d e m o n s t r a t e t h a t t h e h a l o p e r i d o l

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b i n d i n g s i t e s are i n f a c t t h e p h y s i o l o g i c a l l y and pharmacologically relev a n t dopamine r e c e p t o r . These are 1 ) S a t u r a b i l i t y : l i g a d b i n d i n g must be s a t u r a b l e i n d i c a t i n g a f i n i t e number of b i n d i n g s i t e s . 'H-Haloperidol bindi n g d i s p l a c e a b l e by high c o n c e n t r a t i o n s of dopamine h a s a KD of 1-3 nM de rmined i n both e q u i l i b r i u m and k i n e t i c s t u d i e s s a t u r a t i n g between 10-15 nM However t h e displacement of %-haloperidol w i t h n o n r a d i o a c t i v e halop e r i d o l i n d i c a t e s m u l t i p l e components of b i n d i n g . Between 30-60% of t o t a l s t r i a t a l b i n d i n g (depending on s p e c i e s ) o c c u r s t o t h e s p e c i f i c dopamine receptor, w h i l e t h e remainder o c c u r s t o n o n s p e c i f i c s i t e s i n c l u d i n g o t h e r n e u r o t r a n s m i t t e r r e c e p t o r s and uptake s i t e s f o r which h a l o p e r i d o l h a s been shown t o have low a f f i n i t y . The s p e c i f i c b i n d i n g t o t h e dopamine r e c e p t o r can b e d i s t i n g u i s h e d from such n o n s p e c i f i c binding by pharmacologi c a l t e c h n i q u e s ( s e e below). 2 ) Regional l o c a l i z a t i o n : b i n d i n g s i t e s are expected t o be found only i n areas where t h e n e u r o t r a n s m i t t e r i s p r e s e n t . However, t h i s does not n e c e s s a r i l y imply a p e r f e c t c o r r e l a t i o n between t h e l e v e l of a n e u r o t r a n s m i t t e r and t h e c o n c e n t r a t i o n of i t s r e c e p t o r s i n v a r i ous b r a i n areas. 3H-Haloperidol b i n d i n g i s h i g h e s t i n areas which are known t o have a l a r g e dopamine i n n e r v a t i o n such as t h e c a u d a t e nucleus. Lower numbers of b i n d i n g s i t e s occur i n t h e globus p a l l i d u s , putamen, o l f a c t o r y t u b e r c l e and n u c l e u s accumbensll 3H-Haloperidol b i n d i n g h a s a l s o been d e t e c t e d i n t h e c e r e b r a l c o r t e x 1 2 b u t n o t i n t h e thalamus, hippocampus o r cerebellum where t h e r e i s no known dopamine i n n e r v a t i o n . Seeman h a s r e c e n t l y d e t e c t e d b i n d i n g i n t h e p i t u i t a r y where t h e dopamine r e c e p t o r s i t e s may b e involved i n t h e c o n t r o l of p r o l a c t i n release13. 3) Pharma c o l o g i c a l s p e c i f i c i t y : dopamine a g o n i s t s and a n t a g o n i s t s which d i f f e r i n potency i n i n v i v o , b e h a v i o r a l and pharmacological t e s t s should e x h i b i t p a r a l l e l d i f f e r e n c e s i n potency i n competing f o r 3H-haloperidol i n d i n g s i t e s . This c r i t e r i o n t a k e s on added importance i n t h e c a s e of 'H-haloperi d o l b i n d i n g which o c c u r s t o m u l t i p l e s i t e s . O p t i c a l isomers of neurolept i c drugs which have markedly d i f f e r e n t c l i n i c a l p o t e n c i e s a l s o e x h i b i t i s o m e r i c s p e c i f i c i t y i n t h e i r c o m p e t i t i o n f o r 3H-haloperidol b i n d i n g and can be used t o d e f i n e s t e r e o s p e c i f i c r e c e p t o r b i n d i n g t o t h e pharmacologica l l y r e l e v a n t dopamine r e c e p t o r . One such drug i s butaclamol, a new a n t i s c h i z o p h r e n i c a g e n t which e x i s t s as o p t i c a l isomers, w t h v i r t u a l l y a l l t h e The maximum s t e r dopamine b l o c k i n g a c t i v i t y r e s i d i n g i n t h e (+ -isomerlt. e o s p e c i f i c d i f f e r e n c e between t h e b i n d i n g of jH-haloperidol i n t h e presence of (+)-butaclamol and t h a t i n t h e p r e s e n c e of an e q u a l c o n c e n t r a t i o n of (-)-butaclamol i s thus a measure of t h e s t e r e o s p e c i f i c b i n d i n g of 3H-halop e r i d o l t o t h e dopamine r e c e p t o r . (+)-Butaclamol d i s p l a y s two c l e a r l y d i s t i n c t components i n i n h i b i t i n g 3H-haloperidol b i n d i n g . The high a f f i n i t y component e l i c i t s h a l f maximal e f f e c t s a t about 1 nM w h i l e t h e lower a f f i By o n t r a s t , n i t y component is only apparent a t c o n c e n t r a t i o n s above 1 pM. (-)-butaclamol l a c k s t h e h i g h a f f i n i t y component of i n h i b i t i o n of %-halop e r i d o l b i n d i n g w h i l e i t s lower a f f i n i t y i n h i b i t i o n of 3H-h 1 e r i d o l bindDopamine i n g resembles t h e low a f f i n i t y i n f l u e n c e of (+)-butaclamol maximally reduces h a l o p e r i d o l binding t o t h e same e x t e n t as t h e high a f f i n i t y component of (+)-butaclamol i n h i b i t i o n . The i n h i b i t i o n s by t h e s e maximally i n h i b i t i n g c o n c e n t r a t i o n s of butaclamol and dopamine are n o t a d d i t i v e i n d i c a t i n g t h a t b o t h drugs a r e competing f o r t h e sa e c l a s s of 3H-haloperidol b i n d i n g s i t e s l l . The a g o n i s t s p e c i f i c i t y of 'H-haloperidol b i n d i n g i s c o n s i s t e n t w i t h dopamine r e c e p t o r pharmacology: apomorphine and

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dopamine b e i n g more p o t e n t t h a n (-)-epinephrine and (-)-norepinephrine w h i l e i s o p r o t e r e n o l i s e s s e n t i a l l y inactive', lo9 ll. Dopamine Receptor Binding P r e d i c t s C l i n i c a l and Pharmacological P o t e n c i e s of N e u r o l e p t i c Drugs - An abundance of r e c e n t r e s e a r c h s u g g e s t s t h a t neurol e p t i c drugs may e x e r t t h e i r t h e r a p e u t i c a c t i o n s and i n d c e x t r a p y r a m i d a l While moles i d e e f f e c t s by blocking dopamine r e c e p t o r s i n t h e b r a i n Y,?5 c u l a r modeling i n d i c a t e s how p h e n o t h i a z i n e s can assume t h e p r e f e r r e d conformation of dopamine16, t h e conformation of butyrophenones a t t h e i r recept o r s i t e s i s u n c l e a r . N e v e r t h e l e s s s i n c e b o t h p h e n o t h i a z i n e s and butyrophenones have many b e h a v i o r a l and biochemical e f f e c t s i n common i t h a s been assumed t h a t t h e y e x e r t t h e i r t h e r a p e u t i c e f f e c t s by a similar mechanism. S p i r o p e r i d o l i s t h e most p o t e n t i n h i b i t o r of 3H-haloperidol b i n d i n g t h a t w e have s t u d i e d w i t h a v a l u e of t h e i n h i b i t i o n c o n s t a n t , Ki i n d i c a t i n g 50% r e c e p t o r o c c u p a t i o n , of 0.25 nM. I t h a s a 5-fold h i g h e r a f f i n i t y f o r 3Hh a l o p e r i d o l b i n d i n g s i t e s t h a n f l u p h e n a z i n e , a p o t e n t p h e n o t h i a z i n e , a 40f o l d g r e a t e r a f f i n i t y t h a n chlorpromazine and a 125-fold t o 950-fold g r e a t er a f f i n i t y t h a n t h e weak n e u r o l e p t i c s pipamperone, promazine and promethazinell.

.

I n examining a l a r g e number of p h e n o t h a i z i n e s , butyrophenones and o t h e r n e u r o l e p t i c a g e n t s both w e and Seeman have demonstrated t h a t t h e r e i s an e x c e l l e n t c o r r e l a t i o n between t h e molar pharmacological p o t e n c i e s of t h e s e a g e n t s i n animals and man and t h e i r a f f i n i t i e s f o r 3H-haloperidol binding sites17,18,19. The a f f i n i t i e s of t h e drugs t h a t w e 100 have examined f o r 3H-haloperido1 b i n d i n g s i t e s c o r r e l a t e h i g h l y w i t h t h e i r molar potency 0 ' i n antagonism of b o t h apomorTHIORILIAZINE 0 phine-induced s t e r e o t y p y ( r = 0.94, p s t e m , c o r r e l a t e s c l o s e l y with 3H-haloperidol b i n d i n g s i t e a f f i n i t y (r=0.93, p p>m. groups o f dimethylbenzoic a c i d s r e a c t i n t h e o r d e r : --

acozNa aco2 @IcozH d Na

230'

Na QJco2Na

co,_

H02 C

___c BuLi

Br

- 1000

COCeHs

Li

COZLi

cH3Qc02HC H 3

3 25'

C,HSBr

'

cH3Q?c02H C5H11

Nearly a l l common mono- and d i s u b s t i t u t e d a c e t i c a c i d s , i n c l u d i n g s e v e r a l f u n c t i o n a l l y s u b s t i t u t e d examples, have been r e p o r t e d t o m e t a l a t e . Unsaturated a s w e l l a s s a t u r a t e d a l i c y c l i c c a r b o x y l i c a c i d s form d i a n i o n s , b u t cyclopropane c a r b o x y l i c a c i d i s e x c e p t i o n a l . Low y i e l d s of a l k y l a t e d p r o d u c t s a r e produced,'4'20 s u g g e s t i n g incomplete d i a n i o n formation.

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Grignard r e a g e n t s and u n s u b s t i t u t e d a l k a l i m e t a l amides a r e suff i c e n t l y s t r o n g bases t o remove p r o t o n s from a, B- and B,y-unsaturated c a r b o x y l i c a c i d s . Formation o f d i a n i o n s of u n s a t u r a t e d c a r b o x y l i c a c i d s was f i r s t demonstrated21 by t r a p p i n g t h e i n t e r m e d i a t e s w i t h methyl iodide. Subsequent i s o l a t i o n of t h e p r o d u c t s r e v e a l e d t h a t & s u b s t i t u t i o n had occurred. More r e c e n t l y , a r e l a t i v e l y l a r g e number o f examples h a s been i s s t a b l e i n s t r u c t u r e s i n which t h e i n ~ e s t i g a t e d . ~ Unsaturation ~-~~ double bond i s l o c a t e d a t p o s i t i o n s remote t o t h e carboxyl g r o ~ p . ~ ~ ’ ~ ~ Malonic a c i d s 5 and malonic a c i d esters,2e likewise, form carbanions which can be a l k y l a t e d . T r i a n i o n s of malonic a c i d s can be formed by treatment w i t h n - b u t y l l i t h i u m i n t h e absence of an m i n e .

-

CH3CH( C02H)2

3 BuLi,

c

CH3C( C O 2 ) 2

1’-

Li3+

( C%)2C( COEHI2

CH31

76%

IV. Reactions of Metalated Carboxylic Acids - Metalated c a r b o x y l i c a c i d s r e a c t w i t h most e l e c t r o p h i l e s if t h e d i a n i o n is a s s o c i a t e d w i t h a l k a l i m e t a l c a t i o n s . However, Cu( I) d i a n i o n 8 2 r e a c t r e a d i l y o n l y w i t h a c t i vated a l l y l i c o r benzylic h a l i d e s .

A. Alkylation: 1. Alkyl Halides - A l k y l a t i o n is, perhaps, t h e most usef u l s y n t h e t i c r e a c t i o n of metaIated c a r b o x y l i c a c i d s . The r e a c t i o n proceeds smoothly and i t produces p r e p a r a t i v e y i e l d s o f p r o d u c t s f o r a v a r i e t y of f u n c t i o n a l l y d i f f e r e n t a l k y l h a l i d e s , including examples suscept i b l e t o e l i m i n a t i o n . 1 ° The r e a c t i o n i s n o t s e n s i t i v e t o t h e l e a v i n g grou n o r i s i t s e n s i t i v e t o s t e r i c e f f e c t s a s t h e following examp l e ~ ” ’ ~ le ~ ’i l l u s t r a t e . The Haller-Bauer procedure f a i l s where two of

(5)

PhCHZC1

(7) C4H9-OTs

+

-1

f

-1

90%

c 4H9

/%

87%

C02H

92%

Chap. 28

Metalated Carboxylic Acids

Creger

281 -

t h e Cr-substituents a r e l a r g e r than e t h y l , and it i s advantageous t o i n s t a l l t h e l a r g e s t s u b s t i t u e n t s f i r s t . 2 7 As i l l u s t r a t e d i n eq 8, l a r g e , h i g h l y branched c a r b o x y l i c a c i d s can be prepared e f f i c i e n t l y from metala t e d c a r b o x y l i c a c i d s , and s e q u e n t i a l a l k y l a t i o n does n o t depend upon t h e o r d e r i n which s u b s t i t u e n t s a r e i n s t a l l e d , l6 D i a l k y l a t i o n o f monosubs t i t u t e d a c e t i c a c i d s i s n e g l i g i b l e i n most c a s e s ( e q 9 ) . l3 Lower memb e r s o f t h e homologous s e r i e s o f a l i p h a t i c c a r b o x y l i c a c i d s a f f o r d lower y i e l d s , and small amounts o f d i a l k y l a t e d p r o d u c t s a r e produced a s a re-

s u l t of incomplete m e t a l a t i o n caused by poor s o l u b i l i t y o f t h e c a r b o x y l a t e s a l t s . This complication can be minimized by u s e of HMP a s cosolvent16 o r by s u i t a b l e c a t i o n changes.28 2 - S u b s t i t u t e d t e t r a l o n e s and 2-subs t i t u t e d indanones can be prepared r e a d i l y i f t h e p r o d u c t s of eq 4 and 5 a r e c y c l i z e d . l 4 A l k y l a t i o n r e a c t i o n s of c a r b o x y l i c a c i d s performed w i t h a r y l h a l o a l k y l e t h e r s and 0, m-dihaloalkanes have p r a c t i c a l s i g n i f i c a n c e . The p r o d u c t s produced from t h e s e r e a c t i o n s have a n t i h y p e r l i p i d e m i c prop- have been e r t i e ~ , ~ and~ two - ~ drugs, ~ gemfibrozil, 2 -, and gemcadiol, 3, studied c l i n i c a l l y .

Dianions derived from a, p- o r p, y-unsaturated c a r b o x y l i c a c i d s a l k y l a t e a t t h e mcarbon i f t h e dianions a r e associated w i t h a l k a l i metal cation^.^^-^^ However, h i g h p r o p o r t i o n s of y - s u b s t i t u t i o n a r e observed when Cu( I) d i a n i o n s a r e employed.22 The p r e p a r a t i o n o f d l l a n c e o l i l l u s t r a t e s s y n t h e t i c use o f y-alkylation,22 and t h e p r e p a r a t i o n

L

‘ W C O 2

7

1

1’ cu2

~

B

r

CUI 1

o f polyene, 4, i l l u s t r a t e s ~ a l k y l a t i o no f~ d ~i a n i o n s o f u n s a t u r a t e d c a r b o x y l i c acids.

282 -

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C o u n s e l l , Ed.

Br

+ dl-lanceol Ph

Ph

-4

2. Epoxides - The f i r s t i n d i c a t i o n o f t h e s y n t h e t i c u t i l i t y o f m e t a l a t e d c a r b o x y l i c a c i d s r e s u l t e d from e f f o r t s t o p r e p a r e s t e r o i d a l a l d o s t e r o n e i n h i b i t o r s from s p i r o e p o x i d e s . 39 Model s t u d i e s i n d i c a t e d t h a t t h e r e a c t i o n f a i l s a s a r e s u l t of s e v e r e s t e r i c h i n d r a n c e i n e i t h e r t h e epoxide o r c a r b o x y l i c a c i d and t h a t m o n o s u b s t i t u t i o n o c c u r s f o r t h e same reason.39 The r e a c t i o n h a s been used i n a key s t e p o f an e l e g a n t s y n t h e s i s of v e r n o l e p i n , 5. *’ Forcing c o n d i t i o n s a r e r e q u i r e d , and, i n c o n t r a s t t o a c i d d i a n i o n s , anions of u n a c t i v a t e d e s t e r s f a i l t o r e a c t w i t h epoxi d e s . 39’ 41

-5 -

B. Addition t o Carbonyl Compounds Addition o f m e t a l a t e d c a r b o x y l i c a c i d s t o aldehydes and k e t o n e s a t k o r d s a u s e f u l and v e r s a t i l e a l t e r n a t i v e t o t h e Reformatsky r e a c t i o n . = The r e a c t i o n i s s e n s i t i v e t o s t e r i c e f f e c t s

A B

)= 0 +

FCOz]

‘-Li+M+

- 9-FH B

-6

C

i n both r e a c t i o n p a r t n e r s , b u t t h e method h a s a r e a l s y n t h e t i c advantage because p-hydroxy a c i d s a r e n o t o t h e r w i s e r e a d i l y a c c e s s i b l e . Although a - a d d u c t s predominate, a d d i t i o n o c c u r s t o both f a c e s of 17-keto s t e r o i d s . 4 3 The r e a c t i o n h a s r e s u l t e d i n a v e r s a t i l e and s t e r e o s p e c i f i c o l e f i n s y n t h e s i s . 4 4 Isomers of 6 can b e s e p a r a t e d and degraded v i a pl a c t o n e s , 7, y i e l d i n g h i n d e r e d o T e f i n s , 8, o f known s t e r e o c h e m i s t r y . The r e a c t i o n hTs been used t o i n t r o d u c e a c e t y l s u b ~ t i t u e n t s( e~q~ 10) and i s o p r o p y l i d e n e groups,46 and i t h a s been used f o r t h e p r e p a r a t i o n of

Chap. 28

Metalated Carboxylic Acids

-6

ArS02C1

283 -

Creger

D '

B

8 -

1 c y c l o a l k y l i d e n e cycloalkanes. 47 a c t i o n f o r many a p p l i c a t i o n s .

This sequence should r i v a l t h e W i t t i g re-

'Me S u b s t i t u t e d a c r y l i c a c i d s have been prepared by r e a c t i o n of a c i d d i a n i o n s w i t h monomeric formaldehyde. 48 A v a r i a t i o n of t h e r e a c t i o n D e r m i t s i n s i t u formation o f formaldehyde from anions o f methoxymethyl e s t erZ4-

+ [ RCHC0212-Li2

R CH20

R

H 0 A C 0 2 H

H+

'C02H

d

E s t e r s r e a c t w i t h m e t a l a t e d c a r b o x y l i c a c i d s y i e l d i n g p-ketoacids from which aldehydess0 and ketones51 may be derived. Like the Adamolefin 0

0

Me,CC02Et

+

-1 +

Me3 C 5 C O 2 H

70%

d

s y n t h e s i s , t h e d i a n i o n a c t s a s a r e a c t i v e c a r r i e r of t h e carbon fragment a t t a c h e d t o t h e carboxyl group. I n c a s e s where t h e carboxyl f u n c t i o n i s e v e n t u a l l y eliminated, m e t a l a t e d c a r b o x y l i c a c i d s can a f f o r d u s e f u l synt h e t i c a l t e r n a t i v e s t o Grignard and organolithium r e a g e n t s . Unsaturated e s t e r s and n i t r i l e s r e a c t by 1,4-additionYs1 b u t u n s a t u r a t e d aldehydes undergo 1,g-addition. 42 Carbon d i o x i d e was t h e f i r s t e l e c t r o p h i l e demonstrated t o r e a c t w i t h m e t a l a t e d c a r b o x y l i c acids.' The r e a c t i o n h a s been used t o measure t h e e x t e n t o f m e t a l a t i ~ nand ~ ~ t o p r e p a r e malonic a c i d s which would be d i f f i c u l t t o o b t a i n by o t h e r methods.= Malonic a c i d e s t e r s a r e a v a i l a b l e Ad aman t y 1- C02 H

Adamantyl( C02H)2

30%

by t r e a t m e n t o f a c i d d i a n i o n s w i t h carbon d i o x i d e derivative^^^ o r , equiva l e n t l y , by c a r b o x y l a t i o n of e s t e r anions. 54

ClC02Et (EtO),CO

'

X""'"

85%

C 0 2 Et

T r i m e t h y l s i l y l a c e t i c a c i d forms adducts w i t h carbonyl compounds which undergo spontaneous e l i m i n a t i o n . 5 5 The u n s a t u r a t i o n produced app e a r s s o l e l y i n t h e a, & p o s i t i o n s , b u t E and 2 isomers a r e formed i n

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approximately e q u a l amounts. The r e a c t i o n o c c u r s e f f i c i e n t l y w i t h cyclopentanone a s w e l l . Unfortunately, t h e u t i l i t y o f t h i s d i a n i o n i s res t r i c t e d by i t s l i m i t e d a c c e s s i b i l i l i t y .

+

C7H15CH0

[Me3SiCHC02]2-Li2+

-

s e o + s

88%

C7H1,CH=CHCO,H

~

E : Z = 3:2

84%

H

%C02

C. Oxygenation - Metalated c a r b o x y l i c a c i d s r e a c t i n s t a n t a n e o u s l y w i t h oxygen, and, depending upon r e a c t i o n and workup c o n d i t i o n s , ~ & h y d r o x y - ~ ~ o r a h y d r o p e r o x y c a r b o x y l i c acids57 a r e obtained. I f low temperatures

OH [ C7H15CHC02 1 -Liz+ Et

/

5 1%

C 7 H 1 5 A COzH

d

0

OH

PhACO2H

PhAEt

72%

0 a r e ernployed, t h e hydroperoxide i n t e r m e d i a t e can be converted t o a ketone by treatment w i t h d ime thy1 f onnamide d imethylacet a l . S i l y l a t i o n - Ketene b i s ( t r i m e t h y l s i l y l ) a c e t a l s r e s u l t from t r e a t m e n t D. of a c i d d i a n i o n s w i t h c h l o r o t r i m e t h y l s i l a n e . 55'59 Esters behave analogously.60 S i l y l a t e d k e t e n e a c e t a l s can r e a c t l i k e a c i d d i a n i o n equiva l e n t s . For example, r e a c t i o n w i t h s i n g l e t oxygen was used t o produce t h e f i r s t a-peroxylactone.

Me3cq 1. lo2 OSIMe3 2. MeOH OSFMe3

He3Ct_ C02H 0-OH Me3C

4

YH 0

+ c02

-I 0-0

25'

Non-enolizable aldehydes r e a c t w i t h s i l y l a t e d k e t e n e a c e t a l s by thermal r e o r g a n i ~ a t i o n , ' ~b u t m i l d e r c o n d i t i o n s may be used i n t h e presence of titanium t e t r a c h l o r i d e . 64 ArCHO

+

MoSMe3 10 -

/&

150°

OSiMe3

+ lo

Tic14

Ar

5

C02H

C 0 2H

-78O E. Other E l e c t r o p h i l e s - Amino a c i d s can be prepared e i t h e r by d i r e c t i n t r o d u c t i o n o f t h e amino function,65 o r by e l a b o r a t i n g h i p p u r i c a c i d by a l k y l a t i o n . 6 6 A n i t r o group can be introduced d i r e c t l y , and, a f t e r decarboxylation, n i t r o a l k a n e s a r e obtained. 49 I n a d d i t i o n t o decomposition

Chap. 28 [ PhCHC0212-Li2

-

Metalated Carboxylic Acids

+

+

MeONH2

Creger

PhCHC02H NH2

285 -

55%

68% of hydroperoxides and a-hydroperoxylactones, s u l f e n y l a t e d c a r b o x y l i c a c i d s can be degraded t o ketones. 67'68 The o v e r a l l r e a c t i o n p r o v i d e s another MeSCH2C02H

\ R- X

*

) CHC02H

SMe

c1+

H20'

MeSSMe k 0 2 H example i n which m e t a l a t e d c a r b o x y l i c a c i d s can be used a s r e a c t i v e c a r ri er s of t h e carbon r e s i d u e a t t a c h e d t o t h e carboxyl group; here, a s an a c y l anion e q u i v a l e n t . ( P h e n y l t h i o ) a c e t i c a c i d h a s been recommended a s t h e d i a n i o n i c i n t e r m e d i a t e because of i t s a v a i l a b i l i t y . 6s The l a t t e r can be a l k y l a t e d and used a s a source o f o l e f i n s and epoxides a s w e l l a s ketones. The u t i l i t y o f t h e method has been demonstrated i n t h e s y n t h e s i s

c02cH3

-

V. Conclusions The preceding d i s c u s s i o n should convince t h e r e a d e r t h a t m e t a l a t e d c a r b o x y l i c a c i d s have s u b s t a n t i a l v e r s a t i l i t y i n o r g a n i c s y n t h e s i s on both small and l a r g e s c a l e s . The l i t e r a t u r e c i t e d s u g g e s t s t h a t much more a t t e n t i o n h a s been devoted t o t h e examination o f r e a c t i o n s w i t h v a r i o u s e l e c t r o p h i l e s than t o t h e p r o p e r t i e s and methods o f format i o n of t h e d i a n i o n s upon which p a r t i c u l a r a p p l i c a t i o n s depend f o r sucIt i s l i k e l y t h a t t h e f u t u r e w i l l provide t h e methodology necescess. s a r y for t h e m e t a l a t i o n of d i f f i c u l t c a r b o x y l i c a c i d s , such as, a c e t i c and cyclopropane c a r b o x y l i c a c i d s , and i n s i g h t i n t o t h e s t r u c t u r e s o f t h e

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dianions a s well a s a b e t t e r understanding of the mechanisms of r e a c t i o n s with d i f f e r e n t c l a s s e s of e l e c t r o p h i l e s . References

1. 2.

3.

4.

5. 6. 7. 8. 9.

B. Blagoev and D. Ivanov, Synthesis, 615 (1970). D. Ivanov, G. Vassilev and I. Panayotov, ibid., 83 ( 1975). M. W. Rathke, Org. React., 22, 423 (1975). D. 0. DePree and G. W. Mattson, Ind. Eng. Chem. Prod. Res. Dev., 2, 238 ( 1963). D. 0. DePree and R. D. Closson, J. Amer. Chem. SOC., 80, 2311 (1958). D. 0. DePree, i b i d . , 82, 721 (1960). B. Angelo, B u l l . Soc.-&im. F r . , 1848 (1970). B. Angelo, C. R. Acad. Sci., Ser. C, 276, 293 (1973). A. A. Morton, W. J . LeFevre and I. H e z n b l e i c k n e r , J. Amer. Chem.

-

-

SOC.

10. 11. 12.

13. 14. 15.

16.

, 58,

754 ( 1936).

P. L. E e g e r , i b i d . , 89, 2500 (1967). R. A. Olofson and C. M. Dougherty, i b i d . , 95, 582 (1973). P. L. Creger, Org. Syntheses, 50, 58 ( 1 9 7 0 r P. L. Creger, J. h e r . Chem. Sz., 92, 1397 (1970). P. L. Creger, Abstracts, 11th ACS Mnwest Meeting, Carbondale, Ill., Oct. 30-31, 1975. H. 0. House, W. C. Liang and P. D. Weeks, J. Org. Chem., 39, 3102

-

( 1974) * P. E. P f e f f e r , L. S. S i l b e r t and J. M. Chirenko, Jr., i b i d . , 37,

-

451 (1972).

17. D. 0. DePree, U . S . Patent, 3,108,135 (1963). 18. W. E. Parham and Y. A. Sayed, J. Org. Chem., 39, 2051 (1974). 19* P. L. Creger, J. h e r . Chem. SOC., 92, 1396 (1970). 20. C. Ainsworth and Y-N. Kuo, J. O r g a n z e t a l . Chem., 46, 73 (1972). 21. A. J. Birch, J. Chem. SOC., 1551 (1950). 22. 23. 24.

J . A. 4925 P. E. P. E.

Katzenellenbogen and A. L. Crumrine, J. h e r . Chem. SOC.,

( 1976).

P f e f f e r and L. S. S i l b e r t , J. Org. Chern., 36, 3290 (1971). P f e f f e r , L. S. S i l b e r t and E. Kinsel, T e t r x e d r o n L e t t . , 1163

( 1973)

25. 26. 27 * 28. 29-

30. 3 1. 32.

33.

34 * 35. 36 * 37. 38.

98, -

-

A. P. Krapcho and D. S. Kashdan, i b i d . , 707 (1975). J. E. McMurray and J. H. Musser, J. Org. Chem., 40, 2556 (1975). C. L. Carter and S. N. S l a t e r , J. Chem. SOC., l 3 0 ( 1946). P. L. Creger, U. S. Patent, 3,674,836 ( 1972). P. L. Creger and W. A. Neuklis, U. S. Patent, 3,707,566 (1972). G. W. Moersch and P. L. Creger, U. S. Patent, 3,742,068 (1973). P. L. Creger and W. A. Neuklis, U. S. Patent, 3,759,986 (1973). P. L. Creger and W. A. Neuklis, U. S. Patent, 3,847,994 (1974). G. W. Moersch and P. L. Creger, U. S. Patent, 3,857,884 (1974). P. L. Creger, U. S. Patent, 3,773,946 (1973). G. W. Moersch and P. L. Creger, U. S. Patent, 3,929,897 ( 1975). P. L. Creger, U. S. Patent, 3,930,024 (1975). P. L. Creger, G. W. Moersch and W. A. Neuklis, Proc. Roy. SOC. Med., 69, SUPPI.2, 3 (1976). S. Johnson and L. A. Bunes, J. Amer. Chem. SOC., 5597 (1976).

98,

Chap. 28

39. 40.

41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

51. 52 *

Metalated Carboxylic Acids

287

Creger

-

P. L. Creger, J. Org. Chem., 37, 1907 (1972). S. Danishefsky, T. Kitahara, P. F. Schuda and S. J. Etheredge, J. h e r . Chem. SOC., 98, 3032, 6715 (1976). S. Danishefsky T . y i t i h a r a , M. Tsai and J. Dynak, J. Org. Chem.,

41,

1669 (19761.

W. Moersch and A. R. Burkett, i b i d . , 36, 2403 (1971). Tanabe and R. A. Peters, i b i d . , 36, 2403 (1971). Adam and J - C . L i u , J. Amer. ChemrSoc., 94, 2894 (1972). Caron and J. Lessard, Can. J. Chem., 5l,-l ( 1973). A. P. Krapcho and E. G. E. Jahngen, Jr., J. Org. Chem., 39, 1322

G. M. W. G.

-

( 1974)*

-

A. P. Krapcho and E. G. E. Jahngen, Jr., ibid., 39, 1650 (1974). P. E. P f e f f e r , E. Kinsel and L. S. S i l b e r t , ibid, 37, 1256 (1972). A. G. Schultz and M. H. Bergen, i b i d . , 41, 585 (197g. P. E. P f e f f e r and L. S. S i l b e r t , Tetrahedron L e t t . , 699 (1970). Y-N. Kuo, J. A. Yahner and C. Ainsworth, J. Amer. Chem. SOC., 93, 6321 ( 1971). S. Reiffers, J. S t r a t i n g and H. Wynberg, Tetrahedron L e t t . , 2339

-

( 1971)*

53 * 54. 55.

-

56 57. 58 * 59. 60.

61. 62.

63. 64. 65. 66. 67. 68. 69.

A. P. Krapcho, E. G. E. Jahngen, Jr. and D. S. Kashdan, i b i d . , 2721 ( 1974). S. Reiffers, H. Wynberg and J. Strating, i b i d . , 300 (1971). P. A. Grieco, C-L. J. Wang and S. D. Burke, J. Chem. SOC. Chem. Comun. 537 ( 1975). G. W. Moersch and M. L. Zwiesler, Synthesis, 647 (1971). H. H. Wasserman and B. H. Lipshutz, Tetrahedron L e t t . , 4611 (1975). Y-N. Kuo, F. Chen, C. Ainsworth and J. J. Bloomfield, J. Chem. SOC. Chem. Commun., 136 (1971). C. Ainsworth and Y-N. Kuo, J. Organornetal. Chem., 46, 73 (1972). C. Ainsworth, F. Chen and Y-N. Kuo, 3. OrganometalFChem., 46, 59 ( 1972). W. Adam and J - C . L i u , J. Amer. Chem. SOC., 94, 2894 (1972). W. Adam and H-C. Steinmetzer, Angew. Chem. K t e r n a t . E d i t . Engl., 11, 540 ( 1972). L. Creger, Tetrahedron L e t t . , 79 (1972). K. Saigo, M. Osaki and T. Mukaiyama, Chem. Lett., 163 (1976). S-I. Yamada, T. Oguri and T. S h i o i r i , J. Chem. SOC. Chem. Comun., 623 ( 1972). A. P. Krapcho and E. A. Dundulis, Tetrahedron Lett., 2205 (1976). B. M. Trost and Y. Tamaru, J. Amer. Chem. SOC., 97, 3528 (1975). B. M. Trost and Y. Tamaru, Tetrahedron L e t t . , 3797 (1975). P. A. Grieco and C-L. J. Wang, J. Chem. SOC. Chem. Commun., 714

,

-

( 1975)*

Chapter 29.

Computer-assisted Organic S y n t h e t i c Analysis

Peter Gund, Merck Sharp & Dohme Research L a b o r a t o r i e s , Rahway, N . J . 07065

-

Organic s y n t h e s i s i s s t i l l an experimental s c i e n c e . I n Introduction p r i n c i p l e , however, t h e a p p l i c a t i o n of computers t o t h e c r i t i c a l planning s t a g e of s y n t h e s i s could s u b s t a n t i a l l y i n c r e a s e t h e s y n t h e t i c chemist ' s e f f i c i e n c y .I The thought p r o c e s s e s by which t h e %asters" create a s u c c e s s f u l s y n t h e s i s of a complex s t r u c t u r e remain mysterious: R. B. Woodward won t h e 1965 Nobel P r i z e i n chemistry f o r h i s c o n t r i b u t i o n s t o t h e "art" of o r g a n i c s y n t h e s i s . Corey2 w a s t h e f i r s t t o a t t e m p t a s e r i o u s a n a l y s i s of t h e s e p r o c e s s e s . H e i d e n t i f i e d f o u r s e p a r a t e y e t inter-dependent s t a g e s : choice of t h e molecule t o b e s y n t h e s i z e d , development of an o v e r a l l s t r a t e g y o r p l a n , s e l e c t i o n and o r d e r i n g of t h e i n d i v i d u a l s t e p s , and experimental execution. The s e l e c t i o n and o r d e r i n g s t a g e i s most amenable to computer processing, and t h i s a s p e c t i s r e l a t i v e l y w e l l developed. The s e l e c t i o n and o r d e r i n g of s t e p s should b e done i n a f a i r l y e x h a u s t i v e and unbiased, y e t d i s c r i m i n a t i n g and s y s t e m a t i c manner. Consider a molecule (A) t h a t could b e made from 40 d i f f e r e n t p r e c u r s o r compounds. I f each of t h e s e p r e c u r s o r s i n t u r n could b e made from 40 d i f f e r e n t p r e c u r s o r s , and s o on, then A could be made i n f i v e s t e p s o r l e s s from over 100 m i l l i o n ( 4 0 5 ) p o t e n t i a l s t a r t i n g m a t e r i a l s . 3 A c t u a l l y , thousands of r e a c t i o n s are known - although many are of unknown g e n e r a l i t y - and syntheses of more than f i v e s t e p s are common. The comp u t e r can u n t i r i n g l y a t t e m p t every r e a c t i o n i t "knows" a t v e r y high speeds. 3 Y 4 The s e l e c t i o n and o r d e r i n g of s t e p s should b e unbiased s o t h a t no obvious ( i n r e t r o s p e c t ) approach i s overlooked. An a n a l y s i s i s normally b i a s e d by t h e c h e m i s t ' s previous e x p e r i e n c e , and even by t h e way h e draws t h e t a r g e t molecule. For example, it i s conceivable t h a t t h e f o l l o w i n g two r e p r e s e n t a t i o n s of p a t c h o u l i a l c o h o l could l e a d a chemist t o propose e n t i r e l y d i f f e r e n t s y n t h e t i c approaches (from Ref. 5 w i t h permission):

on

The computer, on t h e o t h e r hand, may b e programmed t o r e c o g n i z e synthet i c a l l y important r e l a t i o n s h i p s , however t h e molecule i s drawn.

Chap. 29

Computer-assisted Syntheses

Gund

289 -

Because of t h e l a r g e number of p o s s i b l e r o u t e s , it is n e c e s s a r y t o d i s c r i m i n a t e between good and bad approaches. The chemist normally d i s c a r d s many p o s s i b i l i t i e s on t h e b a s i s of m e c h a n i s t i c o r g a n i c chemical arguments, and h i s own e x p e r i e n c e . S i m i l a r l y t h e computer may b e programmed t o e v a l u a t e s t e r i c and e l e c t r o n i c e f f e c t s , r e a c t i o n e n e r g e t i c s , i n t e r f e r i n g f u n c t i o n a l i t y , s i d e r e a c t i o n s , and s o f o r t h , i n o r d e r t o d i s c a r d u n l i k e l y approaches. U l t i m a t e l y , powerful s y n t h e t i c s t r a t e g i e s w i l l restrict t h e generat i o n of u n i n t e r e s t i n g r o u t e s and f o c u s on rewarding approaches. Corey2 has c a t e g o r i z e d some s u c c e s s f u l s t r a t e g i e s : avoid o r by-pass s u s p e c t e d poor y i e l d s t e p s , minimize c o r r e c t i o n a l s t e p s , u s e known r e a c t i o n s where p o s s i b l e and s i m p l i f y t h e problem. Problem s i m p l i f i c a t i o n may i n v o l v e r e c o g n i z i n g analogous s y n t h e s e s , p e r c e i v i n g r e a l o r p o t e n t i a l symmetry, r e c o g n i z i n g important s u b s t r u c t u r e s ( s y n t h o n s ) , u s i n g e q u i v a l e n t ( i . e . , e a s i l y i n t e r c h a n g e d ) synthons, p e r c e i v i n g s t e r e o c h e m i c a l r e l a t i o n s h i p s , c o n s i d e r i n g r e a c t i o n e n e r g e t i c s and k i n e t i c s , u s i n g r i n g s o r complexation t o minimize f u n c t i o n a l i t y o r p r o v i d e d i r e c t i o n a l i t y , and p e r c e i v i n g biogenetic p o s s i b i l i t i e s . The f i r s t s t e p s towards c o m p u t e r i z a t i o n of t h e s e s t r a t e ies have been taken. For example, t h e concept of s t r a t e g i c r i n g bondsg e n a b l e s a n a n a l y s i s t o f o c u s on b r e a k i n g bonds which are l i k e l y t o l e a d t o a less complex s t a r t i n g material. The f i n a l r e q u i s i t e f o r t h e s e l e c t i o n and o r d e r i n g of s y n t h e t i c s t e p s is t h a t i t b e performed i n a s y s t e m a t i c manner. While s y s t e m a t i z a t i o n of s y n t h e s i s i s d i f f i c u l t , s e v e r a l a t t e m p t s have been m a d e . 2 ~ 7 ~ 8 Corey's approach2 a p p e a r s t o have been most f r u i t f u l . Logic-Centered Approach t o Organic S y n t h e s i s - I n t h i s approach, formalized by Corey, 2 t h e chemist "works backwards", s y s t e m a t i c a l l y g e n e r a t i n g a l l p r e c u r s o r s which may b e converted i n one s t e p t o t h e prod u c t by known r e a c t i o n s . Each of t h e b e s t p r e c u r s o r s then becomes t h e t a r g e t f o r f u r t h e r p r e c u r s o r g e n e r a t i o n , and s o f o r t h , u n t i l a v a i l a b l e s t a r t i n g materials are o b t a i n e d . Corey l i s t e d twelve s t e p s t o b e followed f o r t h i s type of a n a l y s i s . 2 I n e s s e n c e , t h e s e are t o s i m p l i f y t h e problem; r e c o g n i z e synthons; g e n e r a t e e q u i v a l e n t synthons; add cont r o l synthons; d i s c o n n e c t t h e synthons t o create p r e c u r s o r s ; f o r m u l a t e t h e r e q u i s i t e r e a c t i o n s f o r t h e s e d i s c o n n e c t i o n s ; r e p e a t t h e above s t e p s f o r each i n t e r m e d i a t e and each sequence; c o n t i n u e u n t i l a s u i t a b l e s t a r t i n g p o i n t is reached; remove i n c o n s i s t e n c i e s ; i d e n t i f y unresolved problems; r e p e a t t h e above s t e p s t o g e n e r a t e a l t e r n a t i v e schemes; and r a t e t h e d i f f e r e n t r o u t e s . C y c l i c s t r u c t u r e s r e q u i r e a few a d d i t i o n a l considerations. Not a l l of t h e s e s t e p s a r e n e c e s s a r y f o r developing every s y n t h e t i c p l a n , and n o t a l l t y p e s of s y n t h e t i c problems are amenable t o t h i s approach.

290

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- Topics

i n Chemistry

Counsell, Ed.

N e v e r t h e l e s s , where t h i s procedure is a p p l i c a b l e i t can l e a d t o e l e g a n t s o l u t i o n s t o d i f f i c u l t s y n t h e t i c problems. An example of t h e power of t h e method is found i n Corey's "network a n a l y s i s " of longif 0 1 i n e . ~ More r e c e n t l y , l O a s y n t h e s i s of p o r a n t h e r i n e grew o u t of t h e l o g i c - c e n t e r e d a n a l y s i s summarized below:

0

-T+F 0

0

Corey noted t h a t t h e twelve s t e p s of t h e l o g i c - o r i e n t e d approach resembled a computer program;2 and indeed t h i s formalism served as t h e framework f o r t h e o r i g i n a l Corey and Wipke computer s y n t h e s i s p r o g r a m , l l 2 well. and f o r t h e l a t e r p r o g r a m s 3 ~ 4 ~ 1as Organic Chemical S y n t h e s i s Simulation (OCSS) - T h i s landmark program11 e f f e c t i v e l y removed t h e b a r r i e r between chemist and computer b y t h e u s e of i n t e r a c t i v e computer g r a p h i c s ; t h e chemist could communicate w i t h t h e machine f o r t h e f i r s t t i m e i n t h e language of s t r u c t u r a l diagrams. I n o p e r a t i o n , t h e chemist drew t h e t a r g e t molecule on a n e l e c t r o n i c t a b l e t , and t h e program g e n e r a t e d (under chemist c o n t r o l ) a " s y n t h e s i s tree", where every node r e p r e s e n t e d an i n t e r m e d i a t e s t r u c t u r e , and t h e branches r e p r e s e n t e d s y n t h e t i c r o u t e s f o r o b t a i n i n g t h e t a r g e t s t r u c t u r e . The program emulated many of t h e s y n t h e t i c c h e m i s t ' s planning a c t i v i t i e s , such as molecule p e r c e p t i o n , developing a s t r a t e g y , s t r u c t u r e manipul a t i o n , and p r e c u r s o r e v a l u a t i o n . The p e r c e p t i o n module i d e n t i f i e d f u n c t i o n a l groups, r i n g s , appendages, symmetry elements, and r e l a t e d p r o p e r t i e s of s y n t h e t i c s i g n i f i c a n c e . The s t r a t e g y and c o n t r o l s e c t i o n used h e u r i s t i c p r i n c i p l e s ( ' ' r u l e s of thumb" r e f l e c t i n g e m p i r i c a l knowledge a b o u t r e a c t i o n s ) t o guide p r e c u r s o r g e n e r a t i o n . The manipul a t i o n and c o n t r o l s e c t i o n a p p l i e d symbolic " r e a c t i o n s - i n - r e v e r s e " o r "transforms" t o s y s t e m a t i c a l l y g e n e r a t e p r e c u r s o r s , and t h e e v a l u a t i o n module d e l e t e d poor s t r u c t u r e s and r a t e d t h e remaining ones. The q u a l i t y of s y n t h e s i s a t t a i n a b l e w i t h t h i s e a r l y program is e v i d e n t i n t h e p a t c h o u l i a l c o h o l s y n t h e s i s t r e e l l ( c o p y r i g h t 1 9 6 4 by t h e American Association f o r t h e Advancement of S c i e n c e , reproduced w i t h

Gund

Computer-assisted Syntheses

Chap. 29

..I

OH

29 1 -

Structure index 1.

//I

4

11. 26. 27.

\

n

35-

34

*%

is

51

L o R i s t i c s and H e u r i s t i c s Applied t o S y n t h e t i c A n a l y s i s (LHASA) - T h i s is a second g e n e r a t i o n program developed b y t h e Corey group.13 While OCSS was w r i t t e n i n an obscure computer language (DECAL) f o r a now o b s o l e t e comp u t e r (PDP-l), LHASA i s l a r g e l y w r i t t e n i n PDP-10 FORTRAN and i s more r e a d i l y a d a p t a b l e t o o t h e r computers. LHASA i n c o r p o r a t e s many new and expanded c a p a b i l i t i e s which can o n l y b e mentioned b r i e f l y : t r a n s f o r m s keyed t o p a i r s of f u n c t i o n a l groups o r t o a s i n g l e f u n c t i o n a l group i n t h e t a r g e t m o l e c u l e ; l 4 f u n c t i o n a l group i n t r o d u c t i o n ( i n t h e r e t r o - s y n t h e t i c s e n s e ) ; I 4 f u n c t i o n a l group i n t e r c h a n g e ; l 4 m u l t i s t e p look-ahead ( i . e . , accomplishing subgoal-generated t r a n s f o r m s up t o 1 5 levels deep i n o r d e r t o a l l o w a powerful r e a c t i o n to be used, b e f o r e o t h e r p r e c u r s o r s of t h e t a r g e t are c o n s i d e r e d ) ; I 5 percept i o n of s t e r e o c h e m i c a l i n f o r m a t i o n ; l 5 v e r y e x t e n s i v e and c a r e f u l pro-

292

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gramming of key ring-forming r e a c t i o n s , such a s t h e Diels-Alder r e a c t i o n ; l 5 t h e concept of s t r a t e g i c r i n g bonds t o g u i d e ring-forming r e a c t i o n s ; 6 s t r a t e g i e s keying on r i n g o r branch appendages and using r e c o n n e c t i v e transforms; 1 6 s e q u e n t i a l subgoal-generated f u n c t i o n a l group i n t e r c h a n g e s ; l 7 and f u n c t i o n a l group p r o t e c t i o n and d e p r o t e c t i o n . 1 8 A r e c e n t example17 of LHASA-derived s y n t h e t i c r o u t e s t o s a t i v e n e 8 and a n analog I is shown (from Ref. 1 7 w i t h permission):

b

Q

b

c

I n a d d i t i o n t o i t s u s e i n s y n t h e t i c r e s e a r c h a p p l i c a t i o n s , LHASA h a s proved u s e f u l f o r t e a c h i n g s y n t h e t i c o r g a n i c chemistry.19 I n a n i n d u s t r i a l environment, LHASA i s being used a t DuPont de Nemours & C O . ~ ~ f o r e x p l o r i n g t h e g e n e r a l i t y of r e a c t i o n s , f o r planning t h e s y n t h e s i s of pharmaceuticals and a g r i c u l t u r a l chemicals, and f o r d i s c o v e r i n g novel a p p l i c a t i o n s of s y n t h e s i s to b a s i c i n d u s t r i a l p r o c e s s e s . For t h e l a t t e r purpose, i n d u s t r i a l l y i m p o r t a n t r e a c t i o n s are b e i n g added t o t h e program's chemistry l i b r a r y .

Chap. 29

Computer-assisted Syntheses

S i m u l a t i o n and E v a l u a t i o n of Chemical S y n t h e s i s (SECS)

Gund

-

293 -

T h i s program5, 1 2

was developed by Wipke, working independently of t h e Harvard group, a t P r i n c e t o n and more r e c e n t l y a t t h e U n i v e r s i t y of C a l i f o r n i a a t S a n t a Cruz. SECS i s a l s o w r i t t e n p r i m a r i l y i n FORTRAN f o r a PDP-10 computer, w i t h outp u t v i a t e l e t y p e o r GT40 g r a p h i c s t e r m i n a l . With t h e l a t t e r d e v i c e , t h e t a r g e t molecule i s drawn on t h e s c r e e n u s i n g a l i g h t pen (from Ref. 5 w i t h p u b l i s h e r ' s permission) :

+

SECS i s c u r r e n t l y a c c e s s i b l e over t h e S t a n f o r d U n i v e r s i t y SUMEX-AIM network, t h e Advanced Research Planning Agency network (ARPANET), and t h e F i r s t Data Corporation (Waltham, Mass.) commercial time-shared network. Again, t h e advanced f e a t u r e s of t h i s program can b e l i s t e d o n l y b r i e f l y : p e r c e p t i o n of s t e r e o c h e m i c a l r e l a t i o n s h i p s ; 5921 a s t e r e o c h e m i c a l l y unique molecule naming a l g o r i t h m ; 5922 g e n e r a t i o n and r e c o g n i t i o n of enantiomers; 5921 a u t o m a t i c c o n s t r u c t i o n of a three-dimensional model5 and p e r c e p t i o n of s t e r i c c o n g e s t i o n 5 ~ 2 3and p r o x i m i t y e f f e c t s therefrom; p e r c e p t i o n of a r o m a t i c and h e t e r o a r o m a t i c d i r e c t i o n a l e f f e c t s from Huckel l o c a l i z a t i o n e n e r g i e s ; 1 2 ALCHEM language f o r r e p r e s e n t i n g v i r t u a l l y a l l p o s s i b l e s y n t h e t i c r e a c t i o n s i n E n g l i s h - l i k e s e n t e n c e s ( r e a c t i o n s are keyed by p a i r s of f u n c t i o n a l groups; by s i n g l e groups; o r by v e r y g e n e r a l p a t t e r n s ) ; 1 2 and f u n c t i o n a l group p r o t e c t i o n . 1 2 An example of a SECS d e r i v e d r o u t e t o p r o s t a g l a n d i n Flcl i s shown as d i s p l a y e d i n t h e forward ( " s y n t h e t i c sequence") d i r e c t i o n (from Ref. 5 w i t h permission) :

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-L+

0

f

164

12s

1m

101

46

27

7

11

1

SECS proved to be useful in mechanistic as well as synthetic research. The program was used to systematically generate all WagnerMeerwein products, with elimination of duplicate structures and calculation of strain energies, in order to find the most likely mechanism for acid-catalyzed rearrangement of tetrahydro-Binor-S to diamantane:24

Persistent attempts to analyze the problem by manual methods had previously failed; there are an estimated 40,000 pentacyclotetradecane isomers possible.24 Industry experience with SECS has been fairly widespread, with many companies evaluating the program through the First Data Corporation network. BASF, Merck/Darmstadt, Sandoz and (more recently) Bayer AG have been running SECS in Europe since 1975. SECS is also running at Merck & Co., Inc. 25

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Batch Programs S e v e r a l r e s e a r c h groups have set themselves t h e t a s k of c r e a t i n g b a t c h computer programs f o r s y n t h e t i c a n a l y s i s . S i n c e t h e chemist cannot i n t e r v e n e i n t h e s e programs d u r i n g e x e c u t i o n , t h e program must b e s e l f - d i r e c t i n g i n f u r t h e r p r o c e s s i n g of i n t e r m e d i a t e s t r u c t u r e s . This r e q u i r e s powerful program h e u r i s t i c s t o p r e v e n t t h e program from w a s t i n g t i m e g e n e r a t i n g p r e c u r s o r s of a n u n i n t e r e s t i n g i n t e r m e d i a t e . S i n c e i t is n o t always obvious why c e r t a i n pathways are i n t e r e s t i n g and o t h e r s n o t , t h i s i s a n a r t i f i c i a l i n t e l l i g e n c e problem of more complexity t h a n t h e well-known chess-playing program problem26 - where t h e l e g a l r u l e s are more c l e a r l y d e l i n e a t e d and "winning" s t r a t e g i e s a r e b e t t e r known. The most advanced of t h e b a t c h programs i s probably G e l e r n t e r ' s SYNCHEM,I2s27 w r i t t e n i n P L / I f o r an IBM computer. T h i s program h a s t h e n o v e l c a p a b i l i t y of ending when a known s t a r t i n g material i s r e a c h e d . It r e c o g n i z e s t h i s c o n d i t i o n by g e n e r a t i n g t h e Wiswesser l i n e n o t a t i o n (WLN) n a m e of each p r e c u r s o r and checking i t a g a i n s t a computerized f i l e of a v a i l a b l e compounds from t h e A l d r i c h c a t a l o g . SYNCHEM i s being e v a l u a t e d f o r u t i l i t y i n a n i n d u s t r i a l environment a t L e d e r l e L a b o r a t o r i e s . 1 2 Published s y n t h e t i c a n a l y s e s by SYNCHEM i n c l u d e r o u t e s t o v i t a m i n A27 and t w i s t a n e . 27 An assembly language program w r i t t e n by Bersohn3,12 i s s a i d t o b e c a p a b l e of g e n e r a t i n g 40,000 p r e c u r s o r s t r u c t u r e s p e r cpu minute on a n IBM 3701168. S i n c e no chemist can b e expected t o e v a l u a t e t h a t many s t r u c t u r e s , Bersohn i s implementing s t r a t e g i e s t o upgrade t h e q u a l i t y of p r e c u r s o r s generated. An e a r l i e r program3928 was much slower and more e x p e n s i v e t o run. A group headed by Ugi h a s a s p i r e d t o implement a program which can "create" new r e a c t i o n s . They developed a mathematically based d e s c r i p t i o n of r e a c t i o n s i n which a m a t r i x c o n t a i n i n g a l l atoms and bonds of a l l r e a c t a n t s (an ensemble) was transformed i n t o a m a t r i x of a l l p r o d u c t s and A PL/I program f o r a n IBM computer, i n i t i a l l y named by-products.29 Computers i n Chemistry, Logic O r i e n t e d Planning of Syntheses (CICLOPS) ,30 was developed based on t h i s approach.12 The program's r e a c t i o n l i b r a r y i s f a i r l y g e n e r a l i z e d , s o t h a t " c r e a t i v e " (unexpected) t r a n s f o r m a t i o n s f r e q u e n t l y arise.

Other programs and formalisms have been developed, such as t h o s e by Whitlock, 31 Hendrickson, Sinanoglu, 32 and Barone,33 b u t t h e i r g e n e r a l u t i l i t y f o r s y n t h e s i s remains t o b e demonstrated. Reaction R e t r i e v a l Systems - A c l a s s i c a l a p p l i c a t i o n of computers i n chemistry i s i n f o r m a t i o n r e t r i e v a l , and chemical r e a c t i o n s are amenable t o t h i s t y p e of treatment.34 When a s t r a t e g i c p l a n f o r s y n t h e s i s h a s been e s t a b l i s h e d , t h e r e i s s t i l l a need f o r d e t a i l e d c o n s i d e r a t i o n of r e a g e n t s and r e a c t i o n c o n d i t i o n s - and a Theilheimer? t y p e system may b e b e s t f o r t h i s purpose. Such a f i l e of r e a c t i o n s is t y p i c a l l y searched by t y p e of s t a r t i n g material, t y p e of p r o d u c t , t y p e of r e a c t i o n , o r c o n d i t i o n s . Such a system u s u a l l y c o n t a i n s v e r y s p e c i f i c r e a c t i o n s of

2 96

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unknown g e n e r a l i t y ; t h e s e a r e g e n e r a l l y u n s u i t a b l e f o r t h e more e l a b o r a t e computerized s y n t h e s i s programs d e s c r i b e d above. 25 Extant r e a c t i o n r e t r i e v a l systems i n c l u d e Reactiones Organicae, 35 Derwent Chemical Reactions Documentation S e r v i c e , 36 and a p r o p r i e t a r y system developed by I m p e r i a l Chemical I n d u s t r i e s . 12937 Conclusions - The s y n t h e t i c chemist normally a n a l y z e s a s y n t h e t i c problem u n t i l a r o u t e i s found which l o o k s l i k e i t might work; a f t e r a quick t r i p t o t h e l i b r a r y t o confirm i t s v i a b i l i t y and t o d e t e r m i n e s u i t a b l e r e a c t i o n c o n d i t i o n s , h e goes t o t h e l a b o r a t o r y . Once committed t o a If it f a i l s , r o u t e , he may expend a g r e a t d e a l of e f f o r t t o make i t "go". o r i f h e needs t o scale-up o r t h i n k about p a t e n t s , t h e n t h e chemist w i l l c o n s i d e r a l t e r n a t i v e r o u t e s . C l e a r l y more s y s t e m a t i c ( e s p e c i a l l y comp u t e r - a s s i s t e d ) a n a l y s i s of complex s y n t h e t i c t a r g e t s a t t h e beginning may save wasted l a b o r a t o r y e f f o r t . Computer a n a l y s i s a t a l a t e r s t a g e of t h e s y n t h e s i s might a l s o b e u s e f u l i n overcoming roadblocks. The b a s i c methodology a p p e a r s t o b e w e l l worked o u t . Computer p e r c e p t i o n of s i g n i f i c a n t molecular f e a t u r e s e q u a l s o r exceeds t h e chemist's c a p a b i l i t i e s i n many ways. While t h e computer's knowledge of chemistry i s s t i l l i n f e r i o r t o t h a t of a good s y n t h e t i c o r g a n i c chemist f o r most o r a l l present-day programs, i t should b e k e p t i n mind t h a t t h e computer's knowledge i s cumulative - and expanding r a p i d l y . A t t h i s s t a g e of development, none of t h e computer programs a p p e a r t o b e p a r t i c u l a r l y adept a t s u g g e s t i n g which s p e c i f i c r e a g e n t s and condit i o n s a r e b e s t f o r producing a p a r t i c u l a r product from a s p e c i f i c s t a r t i n g material. On t h e o t h e r hand, they o f t e n can l e a d t h e chemist t o c o n s i d e r n o v e l approaches t o d i f f i c u l t s y n t h e t i c problems. I n t h e long r u n , t h e most complete s y n t h e t i c a n a l y s e s w i l l a r i s e from a p a r t n e r s h i p of chemist and computer, u t i l i z i n g t h e d i f f e r e n t c a p a b i l i t i e s of each t o t h e utmost. References

1. L. H. S a r e t t , speech b e f o r e t h e S y n t h e t i c Manufacturers A s s o c i a t i o n , June 1964; quoted i n Ref. 5, p. 147. 2. E. J. Corey, Pure and Appl. Chem., 19 (1967). 3. Review: M. Bersohn and A. Esack, Chem. Revs., 76, 269 (1976). 4. Review: A. J . Thakkar, F o r t s c h r . Chem. Forsch., 2,3 (1973). 5. W. T. Wipke i n "Computer R e p r e s e n t a t i o n and Manipulation of Chemical Information", W . T . Wipke, S . R. Heller, R. J . Feldmann and E . Hyde, E d i t s . , J . Wiley, N . Y., p. 147 (1974). 6. E. J. Corey, W. J . Howe, H. W. Orf, D. A. Pensak and G. P e t e r s s o n , J . Amer. Chem. SOC., 97, 6116 (1975). 7 . W. Theilheimer, " S y n t h e t i c Methods of Organic Chemistry", 29 Vols , Karger, Basel, New York (1946-75). 8. J . B. Hendrickson, J. h e r . Chem. SOC., 97, 5763, 5784 (1975) and p r i o r papers. 9 . E. J. Corey, M. Ohno, R. B . Mitra and P. A. Vatakencherry, J . A m e r . Chem. SOC., 86, 478 (1964).

14,

.

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E. J. Corey and R. D. B d a n s o n , J. h e r . Chem. SOC., 96, 6516 (1974). See a l s o Chem. and Ind., 1003 (1974). 11. E. J . Corey and W. T. Wipke, Science, 166, 178 (1969). 1 2 . "Computer-assisted Organic Synthesis", W. T . Wipke and W. J. Howe, E d i t s . , ACS Adv. i n Chemistry S e r i e s , i n p r e s s . Symposium p r e s e n t e d a t C e n t e n n i a l ACS Meeting, N. Y., A p r i l 1976. 455 (1971). 13. E . J. Corey, Quart. Rev. Chem. SOC., 14. E. J . Corey, R. D. Cramer 111, and W . J. Howe, J . Amer. Chem. SOC., 94, 440 (1972). 15. E . J. Corey, W. J . Howe and D. Pensak, J. h e r . Chem. SOC., 96, 7724 (1974). 16. E. J. Corey and W. L. Jorgensen, J . Amer. Chem. SOC., 98, 189 (1976). 17. E. J . Corey and W. L. Jorgensen, J. Amer. Chem. SOC., 98, 203 (1976). 18. E. J. Corey, H. W. Orf, and D. A. Pensak, J. h e r . Chem. SOC., 98, 210 (1976). 19. H. W. Orf, J. Chem. Educ., 52, 464 (1975). 20. D. A. Pensak, i n Ref. 12. 21. W. T. Wipke and T. M. Dyott, J . Amer. Chem. SOC., 96, 4825 (1974). 22. W. T . Wipke and T. M. Dyott, J . h e r . Chem. S O C . , 96, 4834 (1974). 23. W. T. Wipke and P. Gund, J . Amer. Chem. SOC., 98, 8107 (1976). 24. T. M. Gund, P. v. R. S c h l e y e r , P. H. Gund and W. T. Wipke, J . Amer. Chem. SOC., 97, 743 (1975). 25. P . Gund, J. D. Andose and J. B . Rhodes, i n Ref. 12. 26. B. Mittman, Datamation, June 1973, p. 84. 27. H. Gelernter, N. S . S r i d h a r a n , A . J. Hart, S . C . Yen, F. Fowler and H. Shue, F o r t s c h r . Chem. Forsch., 41, 113 (1973). 28. M. Bersohn, B u l l . Jap. Chem. SOC., 45, 1897 (1972). 29. J . Blair, J. G a s t e i g e r , C. G i l l e s p i e , P . D. G i l l e s p i e , and 1. Ugi, i n Ref. 5 , p. 129. 30. J. Blair, J. G a s t e i g e r , C. G i l l e s p i e , P . D. G i l l e s p i e , and I Ugi, Tetrahedron, 30, 1845 (1974). 31. P. E. Blower, Jr. and H. W. Whitlock, Jr., J. Amer. Chem. SOC., 98, 1499 (1976). 32. 0. Sinanoglu, J . h e r . Chem. SOC., 97, 2309 (1975). 33. R. Barone, M. Chanon and J . Metzger, Tetrahed. Letters, 2761 (1974). 34. J. V a l l s , i n Ref. 5, p. 83. 35. H. J. Z i e g l e r ( B a s e l ) , Reactiones Organicae, G. Thieme P u b l i s h e r s . 36. Dement P u b l i c a t i o n s , London, England. 37. D. R. Eaken and E. Hyde, Ref. 5 , p. 1.

10.

5,

Chapter 30. Biochemical Procedures in Organic Synthesis Charles J. Sih and Elie Abushanab,* School of Pharmacy, University of Wisconsin, Madison, W I and J. Bryan Jones, Dept. of Chemistry, University of Toronto, Canada. Despite considerable documentation of their value in overcoming difficult problems in organic syntheses, biochemical procedures are still largely ignored by most organic chemists. However, the field and its enormous potential is now generating increasing interest, particularly among chemists engaged in asymmetric syntheses. Biochemically mediated reactions have not been previously reviewed in this series. Accordingly, we have elected to present a broad, and current, perspective of the subject, while including many representative examples of medicinal chemical interest. Scope - There is an enzyme-catal zed equivalent for almost every type of chemically catalyzed reaction.lyS Thousands of microbiological and enzymic transformations of chemical significance have now been documented in excellent monograph^.^ These reviews cover the field in general as well as specialized areas such as steroids, alkaloids, and terpenes. These provide access to m ch of the published data. Current awareness is also easily maintained.' So far, the use of intact microorganisms in organic syntheses has been dominant. However, wholly or partly purified5 and immobilized enzymes6, are gaining rapidly in importance. Advantages - Compared with other catalysts used in organic syntheses, enzymes are exceptional in several respects.8 The spectrum of reactions is very broad.ls2 Furthermore, the generally mild reaction conditions, e.g. , room temperature and neutral pH, minimize problems in sensitive molecules such as epimerization, racemization, and isomerization. However, their main advantage to organic chemists is their specificity.8 Enzymes are usually very selective with respect to reaction type and substrate structure. Of great importance to asymmetric syntheses is their potential for discriminating enantiomers, enantiotopicg,10 groups attached to prochiral centers, and stereoheterotopic (Re/Si)9,10 faces of groups, e.g., CEO and C=C.8-10 For many enzymes,5911 and some microorganisms,1 2 the stereochemistries and specificities of the catalyses are well established, and structures and absolute configurations of products can be projected with confidence. Such knowledge enables preselection of the best enzyme to ensure formation of only the active stereoisomer of a drug, or to effect stereospecific introduction of an isotope prior to metabolism studies. Factors to be considered - Biochemical methods of effecting chemical transformations are often expensive. However, their high "pay-off" can often more than offset this factor, particularly when the target molecules are costly to produce as is the case with most drugs. For example, using chemical reagents, tedious multistep procedures are required to effect controlled oxygenation at unactivated carbon, or to achieve selective oxidation of only one hydroxyl group in a poly-OH molecule, especially if *On sabbatical leave from the University of Rhode Island, 1976-1977.

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transformation of only one enantiomer of a racemate is desired. Such reactions are readily effected enzymically in a single step. For chemists, the most useful enzymes are those which tolerate broad structural variations but retain high stereospecificity. Currently, mammalian enzymes fit these requirements best;8 microorganisms generally have rather narrow structural specificities,l2 and it is often necessary to screen a number of organisms to find the one best suited to the substrate involved. The levels of the desired enzyme in many instances, can then be increased by induction.12 Several factors can have adverse effects on enzymes and microorganisms. These include elevated temperatures, pH changes, added salts, organic solvents, and inhibitors. Information on these aspects is available from several sources.3 3 59 13-15

+

Coenz es - Many enzymes require nonprotein coenzymes for catalytic activiThese are cosubstrates, and must be constantly reconverted into their active form for catalysis to continue. This is not a problem for growing microorganisms since the normal metabolic processes ensure an adequate supply of coenzymes. However, with purified, or immobilized enzymes, maintaining a sufficient concentration of coenzyme can pose a major problem. Coenzymes are expensive16 and it is seldom economically feasible to add them in stoichiometrlc amounts. This is often undesirable for chemical reasons, e.g., the coenzyme may be unstable, or the eventual build-up of high concentrations of its inactive form may induce displacement of an equilibrium reaction in the opposite direction to that desired.5 It is therefore necessary to use catalytic amounts of coenzymes and to ensure that the active forms are continuously regenerated. Some coenzymes present little or no problem in this regard since they are automatically reformed under the normal aqueous reaction conditions or in the presence of oxygen. These include biotin, pyridoxal phosphate (PLP), thiamine pyrophosphate, flavin mononucleotide (F")and flavin adenine dinucleotide (FAD) .I6 Others, such as adenosine triphosphate (ATP), coenzyme A, folic acid, nicotinamide adenine diphosphate [NAD(P)/H], FMNH2 and FADH2, must be continuously reformed from their inactive precursors by incorporating an auxiliary, usually in situ, regenerating system into the reaction mixture.5,16 Recycling of the nicotinamide coenzymes and of ATP have received the most attention so far. The development of economical ATP-recycling systems now seems assured.l6,17 In contrast, despite the considerable effort that has been expended,5916-22 no generally applicable and industrially viable NAD(P) /H recycling systems have been reported. While recycling efficiencies of 2 x lo4 are attainable for brief periods under idealized conditions,l7 10-50 fold turnovers are more normal for preparative-scale oxidoreductions. Nevertheless, the available recycling methods, which have been recently reviewed comprehensively and critically, are more than adequate for research scale applications of NAD(P) -dependent alcohol dehydrogenases.5,23-25 Experimental Procedures - A good knowledge of the principles and techniques of both organic chemistry and biochemistry is highly desirable, but an extensive background in microbiology is not necessary. With purified enzymes, the methods used are essentially those of organic chemistry.5 Carrying out fermentations and working with immobilized enzymes and microorganisms requires that a chemist develop some new skills, which are easily acquired.

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Straightforward illustrative procedures, often written with the chemist in mind, are available.3~7 Some Recent Examples - Since organic chemists have become accustomed to predicting the consequences of using chemical reagents, only biochemical transformations for which the various aspects of specificity can be accurately forecast were considered when the examples cited below were selected. Oxidoreductases - Controlled and stereospecific hydroxylation at unactivated carbon is easily achieved using fungi.12 The chance of finding an appropriate microorganism for a given hydroxylation is improved if a suitable literature analogy can be found. For example, the conversion of L+z26 proved to be a good model for the 3+-527 (prostaglandin synthon).

e-

& c H F

$ o C 6 ) ' . c &

1 HO' 2 3 H08' 4 A rational basis for interpreting the regiospecificity of steroid hydroxylation is emerging.12,28~29 With Calonectria decora and Rhizopus ricans, the orientation of a steroid substrate appears to be controlled by interaction of the oxygen functions with hydrophilic sites (such as A or B) at the active sites of the hydroxylases. Thus 5 and &bind in a similar manner and hydroxylation occurs from the same active site logation to give the llu- or 7u-OH product respectively,28 approximately 5.5 A away from the initial oxygen function. This relationship was also observed in hydroxylations of cyclic alcohols and acylated amines by Sporotrichum sulfurescens.30

a-

1

-A..

..

Immobilization does not destroy hydroxylase activity. 118-Hydroxylation of 17u,21-dihydroxy-pregn-4-ene-3,2O-dione by Culvularia falcata supported in polyacrylamide has been reported.31 This flow procedure was coupled with a polyacrylamide-immobilized A19 2-dehydrogenase of Arthrobacter simplex so that the cortisol produced was converted directly into the more active corticoid, prednisolone. The Al,2-dehydrogenase of A. simplex has also been employed for resolving synthetic steroids and for determining their absolute configurations.32 Hydroxylations of aromatic substrates follow the rules of electrophilic substitution.12 L-Dihydroxyphenylalanine (L-DOPA) ,33 and hydroxylated metabolites acronyane,34 5-anilin0-1,2,3,4-thiatriazoles3~ and umethylfluoren-2-acetic a ~ i d 3have ~ been prepared in this way. Alcohol Dehydrogenases - These enzymes catalyze C=wH(OH) oxidoreductions.

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With few exceptions, Mucor javanicusY5t37 their stereospecificities obey the Prelo rule.g;fi’ Thishas been exploited in several prostaglandin preparatione.I! Considerable control can be exerted over the stereochemistry during oxidoreduction as is demonstrated by the formation of g, 9, and 10 from (5) 7.5 For these and other enzymes sophisticated “diamond lattlce“ activesite models are available for making stereochemical predict ions.5 Large

H 9

H

g

Alcohol dehydro enases are also ideally suited to stereospecific introduction of 2H and H. Elegant enzyme catalyzed exchange processes provide facile routes to the R and S enantiomers of alcohols RCH[2H]OH and of I2-3H] -lac tate, -mala te and -glutamate. 24 Biosynthetically-useful 3H-alcohol enantiomers such as 2 and 12 are also easily preparedS23 An analo ous enzymic reduction is the key step in the recent preparation of (55)-[5- HImevalonic acid.38 Oxidation of (+> 13 to 3 occurs with high regio and enantiomeric specificities.25 Many alcohol dehydrogenases also exhibit enantiotopic specificity of practical value.5 In the reduction of 15 to 16,39 and 17 to the steroid intermediate B , 4 0 and oxidation of 19 to 2 3 2 5 the oxidor&ction is stereospecific for pro-S C=O and OH groups, respect ively

9

9

.

p y $ 2 R1 OH

x

x 15

16

(5) g*: R1

= H, R2 = OH *Horse liver alcohol dehydrogenase (HLAD) +c. - falcata

**M. javanicus arrhizus

-1.

X = 28, 3H

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Hydrolases - Chymotrypsin is the best documented hydrolase. It has a very broad and predictable stereospecificity and i s widely used in resolution of racemic esters.1 L-DOPA has been prepared in this way.41 Several esterases also exhibit enantiotopic specificity.1 This is exploited in the synthesis of R-mevalonolactone 22 from 21.42 Large scale resolution of amino acids is an area where immobilized enzyme technology has triumphed. L-Phe and L-Met are now continuously proimmobilized aminocyclase hydrolysis of duced on a 20 tonlmonth scale their racemic N-acetyl precursors.43 Similarly L-Ly has been obtained by yeast hydrolysis of (2)-a-amino-E-caprolactam. 44 The value of mild and specific enzyme procedures when dealin with sensitive molecules is demonstrated by the conversion of 23 to 24. $5

*

Operation of hydrolases in the reverse direction to effect acylation is also viable, as shown by the preparation of 26 from 25.46 Hog kidney acylase has been used to resolve (2RS,3RS)-valine-[ 2H]3 for cephalosporin biosynthesis .47

1

I-IJ

M ~ O ~ CC O ~ M ~ ’0 0 22 21 -

H 23: R = PhCH2CO 24: R = H -

I

CO2H = H 25: R 26: R = PhCHCO -

ESH

Lyases - These are enzymes that catalyze addition of HX to C=C, C-0, an% C-N bonds. The stereochemistry of addition is established as for all 17 C=C additions studied to date.11 This is again an area where immobilized enzyme methods are superior. Conversion of fumaric acid to L-aspartic48 and L-malic49 acids are two such processes with commercial potential. The most impressive example of lyases in asymmetric syntheses is the synthesis of L-amino acids, 27, using immobilized 8-tyrosinase or tryptophanase.50 Oxynitrilase-catalyzed additions of HCN to aldehydes to give cyanohydrins can be carried out on the kilogram scale. The D-cyanohydrin products are readily converted to other useful optically active synthetic starting materials, e.g., hydroxy acids, hydroxy amines, and acyloins.51 The labelled valines 28 used in cephalos orin and penicillin biosynthetic studies are best prepared enzymatically.52 R = 4-hydroxyphenyl, RH + CH3COC02H + NH4+ + RCH2CHC02Q L - a 3,4-dihydroxyphenylY I 3-indolyl, @NH3 R CO2H 3-(5-hydroxy) indolyl H MI2 I R = 13CH3, C3H3 + R v 0 2 H n HO2C H 28 CH3H Ligases - These mediate C-0, C-S, C-N, and C-C bond formation. Phosphate bond-forming enzymes have been used most widely. Coenzyme A is conveniently made, using a polyacrylamide-supported synthetase to couple ATP and pantethine.53 The use of phosphorylases has also solved some severe

Chap. 30

Biochemical Syntheses

Sih, Abushanab, Jones

303

(po1y)nucleotide coupling problems of nucleic acid syntheses. Examples include syntheses of the termination codons UAA, UAG and UGA.54 Moreover, large (20-30 g) amounts of the poly I - C interferon inducer55 have been prepared using immobilized enzyme techniques. The elegant uses of a T-4 ligase to join polynucleotide phosphate fragments of the alanine t-RNA of yeast and tyrosine suppressor t-RNA of 8.& genes represented key steps in the respective syntheses of these two giant molecules.56 Having surveyed the applications of biochemical systems to organic syntheses, it may be appropriate to follow this up with a discussion of the strategies used in some combined classical-biochemical syntheses. The application of microbes or enzymes for the generation of chiral centers in asymmetric syntheses constitutes one of the most valuable tools available to the synthetic chemist. Although achievement of the desired transformation is heavily dependent upon the judicious selection of microbes or enzymes that possess broad substrate specificities while catalyzing reactions with high degree of stereospecificities, the chemist plays a significant role in designing substrate molecules suitable for biochemical asymmetric inductions. We herein illustrate some approaches to the development of substrate molecules with prochiral centers in some combined asymmetric syntheses. Hopefully, this rationale may also be applicable to other problems. The recent introduction of arylpropionic acids as anti-inflammatory drugs57 has aroused considerable interest in improved methods for their preparation. It is noteworthy that with some arylpropionic acids such as p-isobutylhydratropic acid57 (29) (Motrin) , the individual S (+) and R(-) isomers were essentially biologically equivalent. This equivalence has been shown to be due to the in vivo conversion of the R(-) to the S ( + ) isomer with labelling studies. R(-) -p-isobutylhydratropic acid58 was converted to the s(+) isomer with the loss of two deuteriums suggesting the following pathway of epimerization. E-i-C,$HgC6H4CH(CH3) CO2H 2 P 3 P 2 FD2 02H R(-) + ArCDCOSCoA + ArCCOSCoA ArCCOSX + Ar HCOSCoA + S ( + ) For other anti-inflammation drugs such as Naproxensg and Fenoprofen,60 only the S-isomer is biologically active. Since tedious resolutions are necessary to prepare these compounds, it would be desirable to develop a general asymmetric synthesis of the S ( + ) arylpropionic acids by microbial oxidation of pro-chiral substrates. This concept has been successfully applied in the preparation of a chiral metabolite of 29. Oxidation of one of the enantiotopic methyl groups in 3 with 2. sulfurescens furnished the chiral diol to S-(+) -3-hydroxy-231.61 A similar oxidation of isobutyric acid (%)62 methylpropionic acid (33) has been recently employed in the synthesis of the a-tocopherol synthon (34).63 H RCHzC(-rH I' > -

*

*;,

HO

30: 31:

'CO2H

R R

= =

CH3 CH2OH

32: R 33: R -

=

H

= OH

34 -

304

Sect. VI

- Topics in Chemistry

Counsell, Ed.

One of the key intermediates in Corey's total synthesis of prostag l a n d i n ~is ~ ~the lactone (35) whose optically active form was obtained by resolution of (2) 36. Upon analysis of the sequence of reactions from cyclopentadiene to 35 one would readily visualize the possibility of carrying out microbial resolution of various intermediates along the synthetic pathway. One such resolution has been accomplished by microbial reduction of the (k) ketone 37 with Saccharomyces drosophylarum to yield the (+) exo and (-) endo alcohols (38 and 2). These were separated by chromatography and were oxidized to the optically active ketones (=).65 One of these was converted to 36.64 (t)37: R1 R2 = 0 PhCH2OCH2 38: R1 = H, R2 = OH 39: R1 = OH, Hb 36 R2 R2 = H CHzOCH3

4

a:1::a3

b Hb

-

35

Although these microbial reactions may substitute for chemical resolution methods, they likewise have the inherent disadvantage that only one half of the material is theoretically utilizable, for the substrate is already chiral. It is therefore preferable to design substrates with prochiral centers suitable for microbial asymmetric inductions. Examples that have been applied in the prostaglandin area follow. Reduction of triketone 40 with Dipodascus uninucleatus and Mucor rommanianus furnished the (R) and (S) alcohols (41and 42) respectivel'y7-6b The (R) alcohol (5)was then chemically converted into 43, a key synthon for prostaglandin synthesis via conjugate addition.

( d o

- -

R (CH2)6C02Me H Hb 40 43 41: (R); 42: (S) Since R-% is also an important versatile synthon for prostaglandin synthesis, there has been interest in devising asymmetric methods for its preparation. Japanese workers67 subjected a 1:l mixture of and trans 44 to esterases from baker's yeast and were able to obtain the optically (R,R)-* and (S)-predominant Thus a simultaneous active ( R , R ) - S , kinetic resolution of the diacetate (44) and asymmetric synthesis of the monoacetate (46) were effected by this hydrolysis. These were converted to prostaglandin synthons.68 0

c.

+ 44 -

45 -

46 -

47 -

48 -

Chap. 30

Biochemical Syntheses

Sih, Abushanab, Jones

305

In a related work hydrolysis of the pro-chiral cis-44 using Bascillus &Niger furnished the chiral alcohol (4%6r This was in turn converted to the lactone another prostanoid synthon.70

-tilis var

a,

Although stereoselective chemical reduction of the C-15 carbonyl function in prostaglandins has been achieved,71 it would be desirable to find a microbe capable of catalyzing the reduction of the C-15 carbonyl group stereospecifically yielding the desired (S)-alcohol. Partial success in this area has been achieved when (9 51 was reduced biochemically where Flavobacterium NRRL B-3874 and Pseudomonas x.NRRI, B-3875 produced stereoselectively the (-) and (+) forms of 52,respectively.72 While these transformations proceeded stereospecifically, these enzyme systems require a,Br6,y-unsaturated diketones for reduction. Although it is generally accepted that a 8-unsaturated ketones are not reduced to allylic alcohols biochemically,73 Trechiopora brinkmanii has been found to reduce 15-ketoprostaglandins in the E and F series to the corresponding 15(S) alcohols illustrated by the conversion of 53 to 54.74

=.

Hb

-

53: R1 = R2 0 52: R1 H, Rp = OH 54: R1 = OH, R2 = H The chemical method of choice for the preparatiorof chiral sulfoxides is complicated by the formation of undesirable side products.75 It entails the addition of a Grignard reagent to R-menthyl-L-arenesulfinates76 producing one enantiomer whose antipode has to be obtained by chemical inversion.77 Microbial oxidation of thioethers is, therefore, an attractive method to prepare these chiral reagents. A variety of sulfides have been oxidized by a number of fungi, e.g., As er illus niger, occasionally producing sulfoxides of high optical purity Methyl tolyl 55 as well as benzyl phenyl sulfide (56) have been oxidized to the corresponding R-sulfoxides (57 and 58) in high optical purity. It is clear that a concerted effort in this area is needed to find organisms that produce both R and S sulfoxides of synthetic utility.

++-

Conclusion - The application of biochemical systems to the solution of organochemical problems will continue to expand rapidly, and the few representative examples described herein constitute only the beginning of a very dynamic field. The successful utilization of biochemical chiral reagents is heavily dependent upon the design of suitable substrates, an area where the synthetic chemists can play a major role. It is hoped that before too long chemists will consider biochemical methods a routine powerful addition to their arsenal of synthetic reagents.

3 06

Sect. VI

-

Topics in Chemistry

Counaell, Ed.

References 1. "Application of Biochemical Systems in Organic Chemistry," J.B. Jones, C.J. Sih and D. Perlman, Eds., J. Wiley and Sons, New York, N.Y., 1976, Part I, p. 495. 2. "Enzyme Nomenclature," Elsevier, Amsterdam, 1973. 3. D. Perlman in ref. 1, p . 49 and references therein. 4. E.g., Chem.Abstr.Fermentation sections, etc. Advan.Appl.Microbiol., Prog.Ind.Microbiol., Ann.Repts.of Fermentation Research, etc. 5. J.B. Jones and J.F. Beck in ref. 1, p. 107. 6. O.R. Zaborsky, "Immobilized Enzymes," Chemical Rubber Co., Cleveland, 1973. 7. "Methods in Enzymology," Vol. 44, K. Mosbach, Ed., Academic Press, New York, N.Y., 1977. 8. J.B. Jones in ref. 1, p. 1 and references therein. 9. K.R. Hanson, Ann.Rev.Plant.Physiol., 23, 335 (1972); Ann.Rev.Biochem., -*45 307 (1976). 10. J.B. Jones in ref. 1, p. 479. 11. I.A. Rose and K.R. Hanson in ref. 1, Part 11, p. 507; Accts.Chem.Res., 8, 1 (1975). 12. C.J. Sih and J.P. Rosazza in ref. 1, p. 69. 13. T.E. Barham, "Enzyme Handbook," Vol. 1, 2, Springer-Verlag, New York, N.Y., 1969. 14. "Methods in Enzymology," Vol. 1 et seq., S.P. Colowick and N.O. Kaplan, Eds., Academic Press, New York, N.Y., 1955 to present. 15. R.M.C. Dawson, D.C. Elliot, W.H. Elliot and K.M. Jones, Eds., "Data for Biochemical Research," 2nd Edition, Oxford Univ. Press, Oxford, 1969. 16. W.H. Baricos, R.P. Chambers and W. Cohen, Anal.Lett., 2, 257 (1976). 17. A. Pollak, R.L. Baughn and G.M. Whitesides, J.Arn.Chem.Soc., 99, 000 (1977). 18. K.E. Taylor and J.B. Jones, M.,3, 5689 (1976); Can.J.Chem., 54, 2969, 2974 (1976). 19. R.W. Coughlin and B.F. Alexander, Biotechnol.Bioeng. , 17,1379 (1975). 20. J. Campbell and T.M. Chang, Biochem.Biophys.Res.Commun., 69, 562 (1976). 68, 786 (1976). 21. S.W. May and L.M. Landgraff, 22. M.O. Mansson, B. Mattiasson, S. Gestrelius and K. Mosbach, Biotechnol. Bioeng., 18,1145 (1976). 23. A.R. Battersby and J. Staunton, Tetrahedron, 30, 1707 (1974); A.R. Battersby, P.W. Sheldrake, J. Staunton and D.C. Williams, J.Chem.Soc. (Perkin I), 1056 (1976). 24. H. Gunther, M.A. Alizade, M. Kellner, F. Billes and H. Simon, 2. Naturforech., 241 (1973); M.A. Alizade, H. Simon, R. Bressler and K. Brendel, M., 141 (1975). 25. A.J. Irwin and J.B. Jones, J.Am.Chem.Soc., 98, 8476 (1976); 99, 556 (1977); 2, 1625 (1977). 26. B. Tabenkin, R.A. Lemahieu, J. Berger and R.W. Kierstead, Appl. Microbiol., 17, 714 (1969). 27. S. Kurozumi, T. Toru and S. Ishimoto, Tetrahedron Lett., 4959 (1973).

w.,

x,

m,

Chap. 30

Biochemical Syntheses

Sih, Abushanab, Jones

307

28. E.R.H. Jones, Pure Appl-Chem., 33, 39 (1973); J.W. Browne, W.A. Denny, E.R.H. Jones, G.D. Meakins, Y. Morisawa, A. Pendlebury and J. Pragnell, J.Chem.Soc.(Perkin I), 1493 (1973) and previous papers. 29. D.R. Brannon, F.W. Parrish, B.J. Wiley and L. Long, J.Org.Chem., 32, 1521 (1967). 30. R.A. Johnson, H.C. Murray and L.M. Reineke, J.Am.Chem.Soc., 93, 4872 (1971) and previous papers. 31. K. Mosbach and P. Larsson, Biotechnol.Bioeng., 12, 19 (1970). 32. J. Fried, M.J. Green and G.V. Nair, J.Am.Chem.Soc., 92, 4136 (1970). 91, 6204 33. C.J. Sih, P. Foss, J.P. Rosazza and M. Lemberger, (1969). 34. R.E. Betts, D.E. Walters and J.P. Rosazza, J.Med.Chem., 17,599 (1974). 35. R.J. Theriault and T.H. Longfield, Appl.Microbiol., 25, 606 (1973). 36. E.T. Stiller, P.A. Diassi, D. Gerschutz, D. Meikle, J. Moetz, P.A. Principe and S.D. Levine, J.Med.Chem., 15, 1029 (1972). 37. H. Dutler, J.L.v.d. Baan, E. Hochuli, 2 . Kis, K.E. Taylor and V. Prelog, Eur.J.Biochem., 38, 000 (1977). 38. J.W. Cornforth, F.P. Ross and C. Wakselman, J.Chem.Soc.(Perkin I), 429 (1974). 39. E. Caspi and C.R. Eck, J.Org.Chem., 42, 767 (1977). 40. P. Bellet, G. Nomini and J. Mathieu, C.R.Acad.Sci.Paris Ser.C, 263, 88 (1966). 41. J.H. Tong, C. Petitclerc, A. D'Iorio and N.L. Benoiton, Can.J.Biochem., 49, 877 (1971); M.S. Matta, J.A. Kelley, A.J. Tietz and M.F. Rohde, J. Org.Chem., 2, 2291 (1974). 42. F.-C. Huang, L.F.H. Lee, R.S.D. Mittal, P.R. Ravikumar, J.A. Chan and C.J. Sih, J.Am.Chem.Soc., 97, 4144 (1975). 43. T. Tosa, T. Mori, N. Fuse and I. Chibata, Agr.Biol.Chem., 33, 1047 29 (1971). (1969); M.D. Lilly and P. Dunnill, Proc.Biochem., 44. T. Fukmra, Agric.Biol.Chem., 40, 1687 (1976); T. Fukumura, 40, 1695 (1976). 45. D.A. Self, G. Kay, M.D. Lilly and P. Dunnill, Biotechnol.Bioeng., 11, 337 (1969); G. Kleiner, L.M. Elizarovskaya, M. Mandel and A. Kestner, Tr.Tallin.Politekh.Inst., 383, 31 (1975). 46. W. Marconi, F. Cecere and F. Morisi, German Pat. 246067 (1975); cf., D.R. Brannon, German Pat. 2430952 (1975). 47. R.K. Hill, S. Yan and S.M. Arvin, J.Am.Chem.Soc., 95, 7857 (1973). 48. T. Tosa, T. Sato, T. Mori, Y. Matuo and I. Chibata, Biotechnol. Bioeng., 15,69 (1973). 49. I. Chibata, T. Tosa, T. Sat0 and K. Yamamoto, U.S. Patent 3922195 (1975). 50. H. Yamada and H. Kumagai, Adv. Appl.Microbiol., 19, 249 (1975). 51. W. Becker and E. Pfeil, J.Am.Chem.Soc., 88, 4299 (1966). 5.2. H. Kluender, F.-C. Huang, A. Fritzberg, H. Schnoes, C.J. Sih, P. Fawcett and E.P. Abraham, 96, 4054 (1974). 53. I. Chibata, T. Kakimoto and N. Niehimura, Japan Pat. 75126884 (1975). 54. H.G. Gassen and R. Nolte, Biochem.Biophys.Res.Comun., 44, 1410 (1971). 55. C.H. Hofmann, E. Harris, S. Chodroff, S . Michelson, J.W. Rothrock, 41, 710 (1970). E. Peterson and W. Reuter, E., 56. H.G. Khorana et al., J.Mol.Biol., 72, 209 (1972); J.Biol.Chem., 251, 565 (1976).

u.,

a,

w.,

w.,

308 57 * 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78.

Sect. VI

-

Topics in Chemistry

Counsell, Ed.

S. Wong, Ann.Rep.Med.Chem., 10,172 (1975). W.J. Wechter, D.G. Loughhead, R.J. Reischer, G.J. Van Giessen and D.G. Kaiser, Biochem.Biophys.Ree.Commun., 61, 833 (1974). A.P. Roszkowski, W.H. Rooks 11, A.J. Tomolonis and L.M. Miller, J. Pharmacol.Exp.Ther., 179, 114 (1971). E. Huskisson, J. Wojtulewski, H. Berry, J. Scott, F. Hart and H. Balme, Brit.Med.J., 1,176 (1974). R.A. Johnson, C.M. Hall, W.C. Krueger and H.C. Murray, Biorg.Chem., 2, 99 (1973). C.T. Goodhue and J.R. Schaeffer, Biotechnol.Bioeng., 3,203 (1971). N. Cohen, W.F. Eichel, R.J. Lopresti, C. Neukom and G. Saucy, J.Org. Chem., 41, 3505 (1976). E.J. Corey, N.M. Neinshenker, T.K. Schaaf and W. Huber, J.Am.Chem. SOC., 9 l , 5675 (1969). G. Ambrus, A. Szentirmai, C. Mehesfalvi, G. Kovacs, C. Szanty and L. Novak, Hung.Teljes, 8066 (1974); Chem.Abstr., 81, 49337b (1974). C.J. Sih, J.B. Heather, R. Sood, P. Price, G. Peruzzotti, L.F.H. Lee and S.S. Lee, J.Am.Chem.Soc., 97, 865 (1975). S. Miura, S. Kurozumi, T. Toru, T. Tanaka, M. Kobayashi, S. Matsubara and S. Ishimoto, Tetrahedron, 32, 1893 (1976). T. Tanaka, S. K u r o z d , T. Toru, S. Miura, M. Kobayashi and S. Ishimoto, 32, 1713 (1976). S. Takano, K. Tanigawa and K. Ogasawara, J.Chem.Soc.Chem.Comun., 189 (1976). J.J. Partridge, N.K. Chadha and M.R. Uskokovic, J.Am.Chem.Soc., 95, 7171 (1973). E.J. Corey and H.E. Ensley, J.Org.Chem., 3, 3187 (1973). M. Miyano, C.R. Dorn and F.B. Colton, Chem.Comun., 425 (1971). C.J. Sih and J.P. Rosazza, in ref. 1, p . 75. R.H. Moore, U.K. Patent, Appl. No. 2031 (1973). D.N. Harp, S.M. Vines, J.P. Montillier and T.H. Chan. J. Org. Chem., 41, 3987 (1976). K.K. Anderson, Tetrahedron Lett., 93 (1962). C.R. Johnson and D, McCants, Jr., J.Am.Chem.Soc., 87, 5404 (1965). B.J. Auret et al., Phytochemistry, 9, 65 (1974).

w.,

Chapter 31.

Organic Electrosynthesis

Larry L. Miller and Esther Kariv, Department of Chemistry University of Minnesota, Minneapolis, MN 55455 and James R. Behling, Department of Chemical Development, Searle Laboratories Chicago, IL 60680 The fundamental question addressed here is, "Can useful or unique reactions be performed in high yield, on a synthetic scale, using electrochemistry?" The answer is "yes". Organic electrosynthesis is not a new technique and there is considerable knowledge of the types of reactions that take place at cathodes and anodes. It is also true, however, that there have been few attempts to develop electrochemical reactions as general synthetic tools and very few attempts to use electrochemistry to synthesize complex molecules. Therefore, electrosynthesis is at present a promising, but seldom tested technique for the synthesis of medicinals. Our view of organic electrochemistry is that it is a unique, nonthermal method for activating molecules. Since the rate of reaction can normally be increased by raising the electrode potential, it is possible to carry out reactions with a high activation energy at low temperatures. Indeed, very highly energetic intermediates can be produced. For example acetonitrile containing a fluoborate electrolyte oxidizes only at potentials above 3.2 V vs a saturated calomel electrode (SCE) reference. This means that oxidations with an activation energy of 74 kcal/mole more endothermic than SCE can be performed without oxidizing the solvent-electrolyte. In this range one can directly oxidize most organic compounds including aliphatic hydrocarbons. Another view is that electrochemistry is an alternative to chemical redox methods. Indeed, in certain cases the products are similar. This is to be expected if the chemical reagent reacts like an electrode via discrete electron transfer steps - not atom transfers. Even here, however, it is not unusual to observe significant differences between chemical and electrochemical processes. A peculiar advantage of electrochemistry is control of the electrode potential. In particular one can adjust the potential to selectively attack the most easily reduced or oxidized moiety in a complex molecule. This technique can also avoid the over-reductions and oxidations produced by chemicals. These views then characterize an electrode as a powerful reagent which, through potential control, has considerable selectivity. An important further consideration is that large amounts of material can be produced. This has been most dramatically demonstrated by the commercial production of adiponitrile. It should be understood that it is not necessary to have an intimate knowledge of the Ilkovich equation or the vagaries of double potential step chronoamperometry to do preparative electrochemistry. Furthermore, it is technically simple. The equipment can be as simple as a DC

310

Sect. V I

- Topics

i n Chemistry

C o u n s e l l , Ed.

power s u p p l y , beaker and e l e c t r o d e s . A d i v i d e d c e l l i s , however, o f t e n i m p o r t a n t and a c c e s s t o a p o t e n t i o s t a t is d e s i r a b l e . The advantage of a c e l l s e p a r a t e d i n t o anode and cathode compartments by a g l a s s f r i t o r membrane i s i l l u s t r a t e d by t h e r e d u c t i o n of a k e t o n e t o an a l c o h o l . Ketone is added only t o t h e cathode compartment and t h e product a l c o h o l i s r e t a i n ed t h e r e . T h i s keeps t h e a l c o h o l from b e i n g o x i d i z e d a t t h e anode and keeps unwanted anodic p r o d u c t s o u t from t h e c a t h o l y t e . A s n o t e d above one of t h e advantages of e l e c t r o c h e m i s t r y i s t h e p o s s i b i l i t y of c o n t r o l l i n g t h e e l e c t r o d e p o t e n t i a l . T h i s can b e accomplished most simply through t h e use of a p o t e n t i o s t a t . Models most u s e f u l f o r s y n t h e s i s are a v a i l a b l e f o r about $3,000. The use of such a machine can b e mastered by anyone who can o p e r a t e a r a d i o and t h e r e i s l i t t l e problem w i t h maintenance. Once t h e p r o p e r p o t e n t i a l h a s been e s t a b l i s h e d i n a small scale ( a few grams) experiment, i t i s n o t n e c e s s a r y o r d e s i r a b l e t o u s e a p o t e n t i o s t a t f o r producing l a r g e amounts of m a t e r i a l . More i n f o r m a t i o n on what can be done and how t o do i t can b e found i n a number of books which have appeared i n t h e p a s t few y e a r s . Thorough coverage of t h e l i t e r a t u r e up t o about 1972 can b e found i n t h e volumes e d i t e d by B a i z e r l o r by Weinberg.2 Useful i n t r o d u c t i o n s f o r medicinal chemists are i n Eberson and S c h a e f e r ' s review3 and i n F r y ' s book.4 I n t h i s b r i e f review w e e n u c l e a t e a few r e a c t i o n s which have been proven t o work f o r complex molecules o r which have demonstrated u t i l i t y f o r t h e prep a r a t i o n of s i m p l e r o r g a n i c compounds. No a t t e m p t h a s been made t o b e comprehensive and only a few l e a d i n g r e f e r e n c e s are i n c l u d e d . Our primary g o a l i s t o i n c r e a s e t h e r e a d e r ' s awareness of e l e c t r o s y n t h e s i s . Carbon-Carbon Bond Formation - A number of anodic and c a t h o d i c coupling r e a c t i o n s are known. The Kolbe o x i d a t i o n o f c a r b o x y l a t e s a l t s and p i n a c o l formation from ketone r e d u c t i o n are f a m i l i a r examples. Somewhat less w e l l known i s t h e r e d u c t i v e c o u p l i n g of a c t i v a t e d a l k e n e s . R-CH=C€I-X

+

2e-

+

2H+

-+

XCR2CHCHCII2X 1

X = CN, COzR, COR

7

R R

This h y d r o d i m e r i z a t i o n p r o c e s s h a s been very thoroughly s t u d i e d by B a i z e r and coworkers and other^.^ A number of examples have been performed and t h e y i e l d s are u s u a l l y >go%. Because v a r i a t i o n s i n y i e l d w i t h many c o n d i t i o n s , e . g . , s o l v e n t , cathode material, have been s t u d i e d and t h e mechanism h a s been e l u c i d a t e d , t h e r e are many p o s s i b i l i t i e s f o r performing t h i s r e a c t i o n i n high y i e l d f o r o t h e r s u b s t r a t e s . One of t h e most i n t e r e s t i n g a p p l i c a t i o n s is i n t r a m o l e c u l a r coupling.6

/ CH-CHC02CH3 (2)

CH-CH2

C02CI-I3

\ CH-CHC0zCH3 Z =

fmgk, f C R p f ,

foCHZCH2Of

Mixed c o u p l i n g h a s a l s o been achieved i n c e r t a i n cases.7 High y i e l d s of mixed dimers can b e o b t a i n e d by s e l e c t i v e l y reducing ( c o n t r o l l e d p o t e n t i a l ) one a l k e n e i n t h e presence of a second which i s more r e a c t i v e as a Michael a c c e p t o r . I n t h e f o l l o w i n g example t h e r e d u c t i o n i s performed a t -1.75 V

Chap. 31

Electrosynthesis

Miller, Kariv, Behling

and the mixed dimer is formed in high yield, CH2xCHNHCOCH3 + CH2sCHCN

-+

NCCH2CH2CH2yHNHCOCH3

1

C02CB3

co2a3

E+

=

-1.7

E

t2

=

-1.9

If the cathode potential used is more negative, more of the symmetrical dimers are obtained in competition. Typical conditions for hydrodimerization involve the use of aqueous solutions containing tetraalkylammonium tosylates (with a cosolvent, if necessary) and alkene. Mercury and lead cathodes give highest yields. The mechanism of hydrodimerization has been elucidated8 and illustrates some general considerations. e2CH2"CIICN;

+ CH2=CHCN -+

-+

-

CH2=CHCN;

NCyHCH2CH2FHCN

2H+

NH(CH2) 4CN

The initial intermediate, as in most reductions, is the anion radical. Its dimerization produces only the most stable 1,4-dianion which is finally protonated. The kinetic acidity near the surface is quite important because at high pH the anion radical will be diverted by protonation before coupling and in aprotic media polymerization results. Anodic coupling of vinyl ethers, phenols and phenol ethers shows considerable promise for the synthesis of natural products and medicinals. Both inter- and intramolecular coupling is possible. Interest has centered on the cyclization of bibenzyls. A simple example is found in the work of Parker and Ronlan.'

Applications to alkaloid synthesis are exemplified by the construction of the morphinan ring systemlOll)and synthesis of the oxocrinine analog (11). OCH 3

-

-2e-

CH3X+ CH3

CH30$

CH3

CH3O OCH 3

I

95%

312 -

Sect. V I

-

C o u n s e l l , Ed.

Topics i n Chemistry

62%

I1 I n each of t h e s e biomimetic examples c y c l i z a t i o n o c c u r s p r e f e r e n t i a l l y p a r a t o an alkoxy l e a d i n g t o cyclohexadienone p r o d u c t s . Oxidation of t h e corresponding phenols u s i n g chemical o r e l e c t r o c h e m i c a l methods g i v e s much lower y i e l d s . B o b b i t t and coworkers have r e p o r t e d a number of i n t e r m o l e c u l a r phenol coupling r e a c t i o n s , t h e most i n t r i g u i n g being a s t e r e o s p e c i f i c phenol c o u p l i n g r e a c t i o n . 1 2 Using a carbon anode they were a b l e t o produce o n l y

one of t h e t h r e e p o s s i b l e d i a s t e r e o m e r i c dimers. This i s c l e a r l y a t t r i b u t a b l e t o r e a c t i o n a t t h e anode s u r f a c e . I t i l l u s t r a t e s t h a t because of t h e heterogeneous and c a t a l y t i c n a t u r e of an e l e c t r o d e r e a c t i o n , one h a s some unique p o s s i b i l i t i e s f o r s e l e c t i v i t y . Very r e c e n t l y i t h a s been shown t h a t e l e c t r o d e s u r f a c e s can b e chemically modified. l 3 Although no u s e f u l r e a c t i o n s have come from t h i s work, i t has been shown t h a t o r g a n i c molecules can b e c o v a l e n t l y a t t a c h e d t o e l e c t r o d e s u r f a c e s and t h a t t h e s e modified s u r f a c e s impart s e l e c t i v i t y t o e l e c t r o c h e m i c a l r e a c t i o n s which i s n o t o t h e r w i s e a v a i l a b l e . Attempts have a l s o been made t o i n c r e a s e t h e s e l e c t i v i t y of e l e c t r o c h e m i c a l react i o n s by adsorbing m a t e r i a l on t h e e l e c t r o d e s u r f a c e . I n p a r t i c u l a r i f c h i r a l a l k a l o i d s are adsorbed on mercury, i t is t h e n p o s s i b l e t o perform t h e asymmetric r e d u c t i o n of p r o c h i r a l ketones t c c h i r a l a l c o h o l s . An o p t i c a l y i e l d of 54% h a s , € o r example, been r e p o r t e d f o r t h e r e d u c t i o n of 4 - a c e t y l p y r i d i n e i n aqueous-ethanol u s i n g s t r y c h n i n e as t h e c a t a l y t i c , c h i r a l reagent. l 4 Cleavage Reactions - Two of t h e most common c a t h o d i c p r o c e s s e s are hydroC-0, C-N, N=N) and c l e a v a g e of C-X g e n a t i o n of double bonds (C-C, bonds.15 V i r t u a l l y any o r g a n i c bromide o r i o d i d e i s e l e c t r o a c t i v e and can b e cleaved under t h e proper c o n d i t i o n s . The p r o d u c t s from RX are g e n e r a l l y RH a n d / o r RR depending on R and t h e c o n d i t i o n s . Cleavage of C 1 , OH, NR2, OR, and CN is a l s o p o s s i b l e , b u t i n t h e s e c a s e s i t i s g e n e r a l l y necess a r y t o have an a c t i v a t i n g group i n t h e molecule. The e l e c t r o c h e m i s t r y of

Chap. 31

M i l l e r , K a r i v , Behling

Electrosynthesis

313

f u n c t i o n a l i t i e s l i k e Ar--CH2X, A r X , -C=C-CH2-X, -COCH2X, -CHX2 have, f o r bond example, r e c e i v e d s t u d y . We show h e r e a few cases of c a t h o d i c C-0 c l e a v a g e .16- 1

*

Ph2C=CII-CH2OH HOCH2-CO-CH-CH2 1

1

OH OH

-+

95%

Ph2C=CH-CH3 -+

QH

CH3-CO-CH-CH2 1

1

OH OH nu

R = CH2CH2CH=C-CH2CH2CH=CCH3

C H C R'

The l a t t e r r e a c t i o n s demonstrate t h e g e n e r a l phenomenon t h a t a p r o t i c s o l v e n t s f a v o r c l e a v a g e and p r o t i c media f a v o r hydrogenation. Both pathways i n v o l v e d i r e c t e l e c t r o n t r a n s f e r from t h e c a t h o d e t o t h e s u b s t r a t e . bond o r b e protonThe r e s u l t i n g a n i o n r a d i c a l can e i t h e r c l e a v e t h e C-0 a t e d on carbon by phenol. The former r e a c t i o n l e a d s t o c l e a v e d hydrocarbon by a second e l e c t r o n t r a n s f e r and p r o t o n a t i o n . The l a t t e r p r o c e s s i s a l s o completed by e l e c t r o n and p r o t o n t r a n s f e r g i v i n g t h e s a t u r a t e d a l c o h o l . A major use of r e d u c t i v e c l e a v a g e i s d e p r o t e c t i o n . T h i s h a s been i n v e s t i g a t e d f o r t h e case of a l c o h o l s and amines which have been p r o t e c t e d as t o s y l a t e s . Using DMF and a Hg p o o l cathode, 70-80% y i e l d s of t h e c o r r e s p o n d i n g a l c o h o l s o r amines are o b t a i n e d from s i m p l e t o s y l a t e s o r ~ ~ altosylamides. A p p l i c a t i o n t o amino a c i d s y n t h e s i s i s s u c ~ e s s f u land though t h i s method would presumably have l i m i t a t i o n s i n p o l y p e p t i d e synt h e s i s , i t i s u s e f u l f o r m i l d , economical, l a r g e s c a l e d e p r o t e c t i o n .

RCH-CO2-

RCH-CO2-

1

NH-TOS

-+

'iH3

Y i e l d s of 65-90% are o b t a i n e d on a 50 g scale f o r f o u r t e e n d i f f e r e n t amino a c i d s . Cleavage of benzyl and carbobenzoxy groups18 i s a l s o p o s s i b l e . These d e p r o t e c t i o n r e a c t i o n s e x i s t as a l t e r n a t i v e s t o c a t a l y t i c hydrogenation and a c i d c a t a l y z e d h y d r o l y s i s . Another a l t e r n a t i v e f o r a l c o h o l d e p r o t e c t i o n i s anodic o x i d a t i o n . 2 o -2eAr-CH2-0-R -ArCHO + ROH The s i m p l e a l c o h o l s are r e l e a s e d i n good y i e l d and p r o v i d e a complimentary t e c h n i q u e which a v o i d s r e d u c t i o n o r h y d r o l y s i s of o t h e r s e n s i t i v e p o r t i o n s of a complex molecule. Another a n o d i c c l e a v a g e r e a c t i o n which h a s been used i s bis-decar-

3 14

Sect. V I

-

Topics i n Chemistry

Counsel1 , Ed.

b o x y l a t i o n . 2 1 I t s u t i l i t y d e r i v e s from use of maleic anhydride a s a d i e n o p h i l e i n t h e c o n s t r u c t i o n of p o l y c y c l i c a l i p h a t i c s . An example is:

"'"&CCI2H

co pH

-"'"a

Anodic S u b s t i t u t i o n - I n t h e p a s t few y e a r s a number of anodic s u b s t i t u t i o n processes have been discovered.22 The scope of t h e s e r e a c t i o n s i s d e f i n e d and o f t e n they provide t h e method of c h o i c e f o r performing t h e p a r t i c u l a r t r a n s f o r m a t i o n . These p r o c e s s e s g e n e r a l l y use non-aqueous s o l v e n t s which have a combination of p o l a r i t y and r e l a t i v e i n e r t n e s s t o o x i d a t i o n . A g e n e r a l scheme i s : RH + Nuc -+ REJuc + H+. Both aromatic and a l i p h a t i c compounds have been s u b s t i t u t e d i n t h i s way and C-0, C-N and C-C bonds can be formed. Considering aromatic s u b s t i t u t i o n , t h e mechanism g e n e r a l l y involves d i r e c t e l e c t r o n t r a n s f e r from t h e aromatic t o t h e e l e c t r o d e . Although t h e timing of n u c l e o p h i l e a t t a c k and l o s s of a second e l e c t r o n h a s n o t been e l u c i d a t e d and t h e importance of t h e s u r f a c e i s g e n e r a l l y unknown, a conceptually u s e f u l mechanism i s :

Ar-NHCOCH3, Ar-02CR and ArCN have been s y n t h e s i z e d i n t h i s way22 and t h e p o s i t i o n of s u b s t i t u t i o n can o f t e n be p r e d i c t e d from c a l c u l a t i o n of charge densities i n the cation radical.

For Ar-0 bond f o r m a t i o n , t h e s o l v e n t i s o f t e n used as t h e n u c l e o p h i l e . Thus, f o r example, a mixture of t r i f l u o r o a c e t i c a c i d and nitromethane i s s u f f i c i e n t l y conductive, does n o t o x i d i z e a t p o t e n t i a l s up t o 2 . 4 V vs Ag/Ag' and t r a p s c a t i o n s formed i n o x i d a t i v e p r o c e s s e s t o form t r i f l u o r o a c e t a t e e s t e r s . Using t h i s medium f o r e l e c t r o l y s i s and an aqueous work-up, i t is p o s s i b l e t o perform aromatic hydroxylation r e a c t i o n s , o f t e n i n y i e l d s of 75x.23 -2eArH Ar02CCF3 ArOH

-

a

Aromatic hydroxylations of any kind t e n d t o be u n s u c c e s s f u l because t h e phenol produced i s more r e a c t i v e than t h e r e a c t a n t , A r H . I n t h i s case, however, only t h e t r i f l u o r o a c e t a t e ester, Ar02CCF3, i s p r e s e n t i n t h e e l e c t r o l y s i s mixture, and i t i s l e s s r e a c t i v e than t h e o r i g i n a l ArH. One of t h e more i n t e r e s t i n g a s p e c t s of t h i s r e a c t i o n i s t h a t aromatic carbonyl compounds can be hydroxylated p r e f e r e n t i a l l y o r t h o , p a r a i n c o n t r a s t t o t h e i r normal meta d i r e c t i n g c h a r a c t e r . Alkoxylation r e a c t i o n s provide a n o t h e r r o u t e t o C-0 bond formation?2 This r e a c t i o n i s l i m i t e d i n scope t o a c t i v a t e d aromatics because of a l c o h o l o x i d a t i o n , b u t a number of u s e f u l s y n t h e s e s have come about. Recent papers have, f o r example, demonstrated t h a t f u n c t i o n a l i z e d quinones can be synt h e s i z e d by blocking t h e carbonyls i n t h i s f a s h i o n 2 4 and t h a t 4,4dialkoxy-2-butenoates a r e a v a i l a b l e from f u r f u r a l . 3s

Chap. 31

Electrosynthesis

Miller, Kariv, Behling

315

-0

R

OCH 9 Another- useful substitution reaction giving, in this case, Ar-C bonds is aromatic cyanation. Due to the ease of oxidation of cyanide, this reaction is limited to activated aromatics, but direct replacement of hydrogen or RO by cyanide is so uncommon this process should have application.26 -2e-&Hc

cH&CH3

___t

CN -

CN Conversion of aliphatic C-H to C-N or C-0 bonds can also be accomplished anodically. Aryl groups, nitrogen and oxygen atoms are relatively easy to oxidize and direct the substitution position

CHO CHO Indeed, the former example illustrates a severe limitation of anodic aromatic substitution. Unactivated aliphatic positions can also be substituted if solvents which are inert to oxidation are employed.27

NHCOCH 3 Construction of Heterocycles and Organometallics - Although there are many odd examples of heterocycle formation, especially from anodic processes, there are only a few which were preconceived and which would seem to have generality. Notable among these is a series of cyclization reactions of o-substituted nitroaromatics studied by Lund and coworkers,28 e.g., n-

K2 H-CH-NO2

1

+

a

50%

R

1

OH R

The tetrazole-formazan conversion can be accomplished oxidatively in very high yield for a number of cases.29

3 16 -

Sect. V I

- Topics

Counsell, Ed.

i n Chemistry

I n o t h e r o x i d a t i v e p r o c e s s e s one simply t a k e s advantage of i n t e r n a l t r a p p i n g of c a t i o n i c s p e c i e s by n u c l e o p h i l e s . 30

Although s t u d i e s d i r e c t e d toward t h e s y n t h e s i s of organometallics have a l i m i t e d scope, two i n t e r e s t i n g and u s e f u l t y p e s of p r o c e s s e s have been developed. A number of r o u t e s t o a l k y l metals have been developed31 and, indeed, t e t r a a l k y l l e a d compounds are produced commercially by an e l e c t r o c h e m i c a l r o u t e . Two r e a c t i o n s a r e shown h e r e i n which organometall i c s are oxidized. The o x i d a t i o n releases a n a l k y l group a t a " d i s s o l v i n d ' anode producing t h e d e s i r e d product. Pb(anode) Hg(anode)

+

+

CH3MgC1

Al(C2Hg)4-

-t

+

(CH3)kPb

IIg(CZHg)2

+

A1(C2H5) 3

The second g e n e r a l scheme i n v o l v e s i n t e r c e p t i o n of reduced metal s p e c i e s formed c a t h o d i c a l l y . For example: Ni(acac) 2

+ 2e-

+

Ni(0)

+ 2(acac)-,

aeac-

w

0-

= CH -C-CH=C-CH

3

3

I n t h i s p a r t i c u l a r example r e d u c t i o n of t h e N i ( I 1 ) complex produces Ni(0) which can be trapped by c e r t a i n l i g a n d s . The competing r e a c t i o n i s format i o n of m e t a l l i c n i c k e l which i s u n r e a c t i v e toward cycloootadiene. Using t h i s method a number of c y c l o o c t a t r e t a e n e (COT) have a l s o been formed. Examples a r e Ni(COT), Fe(COT)*, Ti(COT)2 and C2H5Ti(COT). Summary - The r e a c t i o n s i l l u s t r a t e d h e r e have u s u a l l y been discovered and developed by chemists p r i m a r i l y i n t e r e s t e d i n e l e c t r o c h e m i s t r y , n o t s y n t h e s i s . Thus, few a t t e m p t s have been made t o e l e c t r o s y n t h e s i z e complex molecules o r t o develop t h e r e a c t i o n s i n t h e s e n s e t h a t o t h e r o r g a n i c reacr e a c t i o n s a r e u s u a l l y r e f i n e d f o r s y n t h e t i c purposes. The n e x t few y e a r s should see t h i s s i t u a t i o n change and e l e c t r o d e s w i l l b e more widely used a s powerful, s p e c i f i c and c o n t r o l l a b l e heterogeneous c a t a l y s t s .

Chap. 31

Elec t r o s y n t h e s i s

317

M i l l e r , K a r i v , Behling

1. M.M. B a i z e r , e d . , "Organic E l e c t r o c h e m i s t r y an I n t r o d u c t i o n and a Guide," Marcel Dekker, New York, 1973. 2. N . L. Weinberg, e d . , "Techniques of E l e c t r o o r g a n i c S y n t h e s i s , " Wiley, New York, 1975. 3. L. Eberson and H. S c h a f e r , "Organic E l e c t r o c h e m i s t r y , " Topics i n Current Chemistry, no. 21, Springer-Verlag, New York, NY, 1971. 4. A. J. Fry, " S y n t h e t i c Organic E l e c t r o c h e m i s t r y , " Harper & Row, New York, 1972. 5. Ref. 1, Chapter X I X ; J . P . P e t r o v i e h , M.M. Baizer and M.R. J. Electrochem. SOC., 116,749 (1969). 6. J . P . P e t r o v i c h , J . D . (1966).

Anderson and M.M.

Ort,

B a i z e r , J . Org. Chem.,

2, 3897

B a i z e r , J . P . P e t r o v i c h and D.A. Tyssee, J. Electrochem. SOC., 117 173 (1970); F. L i s k a , V. Dedek and M. N e m e c , C o l l . Czech. Chem. Commun., 39, 689 (1974).

7. M.M.

8. E. Lamy, N. Nadjo and J . M . (1973). 9. A. Rolan, V.D.

Saveant, J. E l e c t r o a n a l . Chem., 42, 189

P a r k e r , J . Am. Chem. SOC., 95, 7133 (1973).

10. L.L. Miller, F.R. (1973).

Stermitz, J.R.

11. S . Tobinaga, Bioorganic Chem.,

F a l c k , J . Am. Chem. SOC.,

i,110

12. J . M . B o b b i t t , I. Nogochi, H. Yagi, K.H. 845 (1976). 13. B.F. Watkins, J . R . 97, 3549 (1975).

95,

2651

(1975). Weisgraber, J. Org. Chem.,

Behling, E. K a r i v , L.L.

41,

Miller, J . Am. Chem. SOC.,

20,

14. J . Kopilov, S. S c h a t z m i l l e r and E. K a r i v , Electrochim. Acta, (1976)

.

535

1 5 . See Ref. 4, Chapters 5, 6 . 16. H. Lund, If. Doupeux, M.A. Michel, G. Mousset and J. Simonet, Electrochim. Acta, 19, 629 (1974). 17. M. Fedoronko, E. F u l l e o v a and E . Linek, C o l l . Czech. Chem. Comun., 114 (1971).

18. V.G.

Mairanovsky, Ang. Chem. I n t . Ed.

(Eng.),

15,281

36,

(1976).

19. T. Iwasaki, K. Matsumoto, M. Matuoka, T. T a k a h i s h i and K. Okumura, B u l l . Chem. SOC. Japan, 46, 852 (1973). 20. L.L.

Miller, J . F . Wolf, E.A.

21. A.F.

V a l l t u r o , G.W.

Mayeda, J . Am. Chem. SOC.,

So and L.L.

3306 (1971).

G r i f f i n , J. Am. Chem. SOC., 87, 3021 (1965).

22. L. Eberson and K. Nyberg, T e t r a h e d r o n , 23. Y.H.

93,

Miller, S y n t h e s i s ,

32,

1,425

2185 (1976). (1976).

Sect. VI

318 -

-

Topics i n Chemistry

24. M.J. Manning, P.W. Raynolds and J.S. 5008 (1976).

Swenton, J. Am. Chem. SOC.,

25. $1. Tanada, Y. Kobatasi and S. T o r i i , J. Org. Chem., 26. S. Andreades and E. Zahnow, J. Am. Ctiem. SOC., 27. J . Y . Becker, L.R. Byrd, L.L. Miller, Y.H. 853 (1975). 28. H. Lund and N.H. and L.G.

Counsell, Ed.

91,

5, 3482

(1976).

4065 (1969).

S o , J. Am. Chem. SOC.,

N i l s s o n , Acta Chem. Scand. B , F e o k t i s t o v , i b i d . , 23, 3482 (1969).

30,

98,

97,

5 (1976); H. Lund

29. M. Lacan, I. Tabakovich and 2. Cekovic, Tetrahedron,

30, 2911

(1974).

30. H. Iwasaki, L.A. Cohen and B. Witkop, J . Am. Chem. S O C . , 85, 3701 (1963). 31. H. Lehmkuhl, S y n t h e s i s ,

1,377

(1973).

Chapter 32. The Use of Stable Isotopes in Medicinal Chemistry Sidney D. Nelson and Lance R. Pohl, Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20014

I. Introduction - Stable isotopes have been used increasingly in recent years in a variety of chemical and biomedical applications. Intensified interest in the use of stable isotopes is reflected by the convening of two international symposia on the subject wi hin the ast two ears.lS2 Expanded use of stable isotopes, especially H, I3C, "N and "0, derives from the development of sophisticated instrumentation (nmr, ir, esr and ms) the greater availability of enriched isotopesY3 and an espousal on ethical grounds for non-radioactive techniques in human studies, particularly for neonates and pregnant women. One report has appeared on the lack of toxic effects observed in vivo with carbon-13 replacement of carbon-12.4 The major disadvantages of the use of stable isotopes is the lack of simple, inexpensive instrumentation for detecting the isotopes and the cost of synthesizing the labeled compounds. These and other advantages and disadvantages of using stable isotopes are discussed more thoroughly in ref. 1 and in an earlier ~ommentary.~

8

Two articles appear in 1975 which cover most of the literature on the use of stable isotopes up to that year. The first was a review of studies with deuterated drugs6 and it also covered the toxicological and therapeutic aspects of deuterium oxide (heavy water). The second article reviewed mass spe~trometry.~ Since the mass spectrometer has been the instrumental tool for most of the research that employs stable isotopes, the article covered many references on stable isotopes as well.

This review is intended to survey those uses of stable isotopes which are of particular importance in medicinal chemistry. These include their use in 1) structure elucidation, 2) studies of drug metabolism, 3) pharmacokinetic analysis and 4) drugs used in therapy. The use of stable isotopes for investigating intermediary metabolism, and their rapidly expanding use in clinical chemistry will not be reviewed since these topics have been covered elsewhere.l.8 Synthesis and biosynthesis with stable isotopes have also been previously surveyed9~10and many articles appear in each issue of the Journal of Labelled Compounds and a few have appeared in & medical Mass Spectrometry in the past year. In addition, one recent article has appeared on the use of plants to continousl produce stable isotope labeled compounds of pharmacological interest.11 11. Use of Stable Isotopes €or Structure Elucidation - Mass spectrometry is commonly used in medicinal chemistry to determine the structure of therapeutic agents and their metabolites. The unequivocal interpretation of the mass spectrum for most classes of compounds requires the use of derivatives which are labeled specifically with stable isotopes. For example, the loss of 16 mass units from hydroxamic acids was shown to be due to the loss of the hydroxylamino oxygen by specific labeling with

320

Sect. VI

-

Topics in Chemistry

Counsell, Ed.

oxygen-18.12 The principal modes of fragmentation of N-acyl derivatives of daunosamine, the glycoside moiety of the antitumor antibiotics daunomycin 13 and adriamycin, were determined using specifically deuterated derivatives. A study of the fragmentation pattern of deuterium and oxygen-18 labeled derivatives of cytosine nucleosides established that a major fragment process occurred that was absent in the spectra of other nucleosides. various fragmeniation pathways for prostaglandins A , B, E, and F were determined by the use of specific derivitization with dg-tetramethylsilane.15 Peptides are becoming increasingly important in medicinal chemistry, and the elucidation of peptide and protein structure has rapidly expanded in the past year. Lithium aluminum deuteride reduction has been used to determine the primary structure of a carboxypeptidase inhibitor from potatoes,16 and combinations of deuterated and ermethylated derivatives have revealed structures of several olig~peptides.~~ The amino acid sequence of other peptides was determined by Edman degradation using p-bromophenylisothiocyanate.18-20 This technique utilized the ion-doublet arising from 79Br and 81Br to unequivocally identify the p-bromophenylthiohydantoins of the amino acids. The use of nmr and ir in conjunction with labeling using stable isotopes is important in solving many structural problems which cannot be readily solved by other methods. This is particularly true in the study of dynamic systems where the structure or conformation of a medicinal agent is subject to change. A relevant example in the literature is the work that has been done on the anticoagulant, warfarin (A).21-22 This compound can potentially exist in solution in several different tautomeric forms. After specifically labeling warfarin in 4-hydro coumarin (L), 2-hydroxychromone ( 2 ) , and the cyclic hemiketal (2) with C in the lactone carbonyl group, the tautomeric equilibrium of warfarin was investigated by13C-nmr analysis. The cyclic hemiketal diastereomers (2) were found to be the predominant tautomers in solution.

s

-3 In otherl3C-nmr studies, the effect of pH on the structure of phenobarbital and diphenylhydantoin23and on the conformation of amino acids and p e p t i d e ~ ~ ~ hbeen a s examined using thel3C-enriched drugs.

Chap. 32

Stable Isotopes

Nelson, Pohl

321 -

Dynamic interactions of medicinal agents with biological macromolecules, such as plasma proteins, receptors, or metabolizing enzymes is another area which can be studied by nmr and ir using drugs specifically labeled with stable isotopes. Several examples exist in the biochemical literature and the procedures can be adapted to the study of organic medicinals. For example, amino acid side-chains have been labeled with C-nmr was then used to elucidate carbon-13 enriched electrophiles and An extension of this technique the reacting side-chain f~nctionality.~~ would be to use active-site directed enzyme inhibitors, some of which are drugs, to probe the active sites of enzymes. Recently, 15N- and 2H-nmr techniques have been applied to studies of macromolecular structure including work on heme-peptide interactions in hemoglobin,26 phospholipid-membrane intera~tions~27~pd the interaction of oxytocin with its protein carrier, neurophysin.

-

111. Use of Stable Isotopes in Studies of Drug Metabolism and Toxicology Since the advent of mass spectrometry (ms) coupled with gas chromatography (gc-ms), stable isotopes have been used extensively in the study of drug metabolism. The use of ms in the field of drug metabolism has been revie~ed,~ and ~ ,both ~ ~ reviews cover the use of stable isotopes. There are several ways in which stable isotopes have been used to study drug metabolism and these will be covered individually.

A. Quantification of Drugs and Metabolites - Stable isotope dilution, a technique in which the unlabeled drug is dosed and the labeled substance serves as carrier and internal standard for measurement of the drug and its metabolites in biological fluids, is the most widely used applicat stable isotopes. This technique has been comprehensively surveyed need not be detailed here. An international symposium on this subject was held in Ghent, Belgium in June 1976 and the proceedings soon will be published (A. DeLeenheer, ed.). Additional new references appeared throughout 1976 in every issue of Biomedical Mass Spectrometry and most other journals relating to drug metabolism. Three articles of special interest to users of stable isotopes and ms for quantitative purposes are 1) "Limits of Detectio of Carbon-13 Labeled Drugs and Their Metabolites in Human Urine",3' 2) "A Comparison of Unlabeled and Labeled In rnal Standards for Quantification by Single and Multiple Ion Monitoring"," and 3) "A Review of the Statistical Considerations Involved in the Treatment of Isotope Calibration Data". 36 Two general methods utilizing the stable isotope dilution technique have been successfully used for the quantification of drugs and their metabolites by ms. The simplest, but least sensitive and specific method, is a non-chromatographic technique that utilizes chemical ionization mass spectrometry (cims) and internal standards labeled with stable isotopes. This method has been used to quantify the antiarrhythmic drugs, l i d o w n e and quinidine and some of their metabolites, in human plasma samples, The second, most widely applied method, utilizes the high resolving power of the gas chromatograph and a technique for selected monitoring(mass

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fragmentography) of the gas chromatographic effluent. This method allows for the quantification of picogram quantities of both endogenous and exogenous substrates, such as prostaglandins, which otherwise cannot be specifically measured in biological samples. Exhaustive review; ,38*39 on the uses of this technique have recently been published, and most examples employ stable isotope dilution assays.

B. Twin-Ion Technique - This technique was used originally to follow the metabolism of chloropromazine, because the ions from the drug and its metabolites which contained chlorine could be easily recognized in a mass spectrum by the natural isotopic pattern of 3 5 ~ 1and 37~1.40 Other investigators soon applied this technique to biotransformation studies of nonhalogen-conta ing compounds by incorporating stable isotopes such as 2H, and into the compound under investigation. Examples otldrugs 13C, that have been investigated bi3this technique include testosterone, nortriptyline 42 d-propoxyphen 4-morpholine-2-piperazinothieno [ 3,2-d] pyrimidineh4 and estrogens.'5 More recently the technique has been used in conjunction with cims to rapidly elucidate the structure of previously unidentified metabolite of warfarin using a 1:l mixture of "C-benzylic warfarin and unlabeled drug. 46 The ion-doublets arising in the chemical ionization mass spectrum of the metabolite helped identify the compound as the benzylic alcohol (5). Similarly, the administration of an equimolar mixture of unlabeled and 1%-labeled phenoxybenzamine to rats, dogs, and man facilitated the identification of urinary metabolites of this antihistamine using gc-cims by virye of the conspicuous equal-intensity ions in the recorded mass spectra.

'%

Application of cims and the twin-ion technique, using *H-labeled compounds, has been made in the past year to better define mechanisms of drug toxicity for acetylhydrazine, a hepatotoxic metabolite of the antituberculosis drug isoniazid, and isopropylh drazine, a hepatotoxic metabolite of the antidepressant drug, i p r o n i a ~ i d . ~ Highly ~ * ~ ~ reactive acylating and alkylating species were generated by the oxidative metabolism of acetylhydrazine and isopropylhydrazine, respectively. These reactive metabolites bound to tissue protein and caused tissue necrosis. The reactive intermediates were trapped using cysteine as an alternate nucleophile and a comparison of the ratio of hydrogen and deuterium in the cysteine adducts to the ratio in the initial substrates showed that the entire acetyl group of acetylhydrazine and the entire isopropyl group of isopropylhydrazine were transferred in the acylation and alkylation process. This suggested the formation of possible cationic or free radical intermediates. Approximately equimolar mixtures of trideuterated diethylstilbestrol and unlabeled diethylstilbestrol also have been used to study the metabolic activation of dieth lstilbestrol to potentially toxic metabolites by rat liver h o m ~ g e n a t e s .Reactive ~~ polar metabolites were converted to non-polar metabolites by enzymatic methylation. The ion-doublets arising in the mass spectra of these derivatives guaranteed that these compounds were metabolites of the substrate and also helped to establish their structures. C. Comparative Metabolism of Enantiomers - Many drugs possess one or more chiral centers and the pharmacological activity of the various isomers may

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differ considerably. This difference is often due to stereoselective metabolism of a particular isomer. An elegant method for investigating the comparative metabolism of enantiomers is to administer a 'pseudoracemate' in which one enantiomer is labeled with a stable isotope. The enantiomeric ratios for the drug and its metabolites can then be determined while the activity of the drug is being monitored. This technique has been applied to investigations of the differential metabolism of the enantiomers and racemate of the psychotomimetic amine, 1-(2 5-dimethoxy-4-methylphenyl)-2aminopropane (5),51-53and amphetamine (5).52 Results in both cases showed that the racemate and individual enantiomers are metabolized differently. For example, comparison of separate incubations of R-amphetamine (R-6) with those of S-amphetamine-d3 ( S - 1 ) and of S-amphetamine (S-6) with those of R-amphetamine-d (R-Z) showed that larger amounts of the N-hydroxyamphet3 amines, 8 and 2, and the alcohols, 10 and 11,were formed from the R-enantiomers. However, when pseudoracemic mixtures of R-A/S-l or R-llS-5 were incubated the metabolites were preferentially formed from the S-isomer. It was concluded that S-amphetamine or one of its metabolites inhibited the metabolism of the R-enantiomer.

Simultaneous measurements of plasma levels of deuterated and unlabeled d- and 1-propoxyphene revealed that the plasma levels of the more analgesically active d-isomer were higher and the half-life was longer.55 On the other hand, simultaneous measurements of (+)- and (-)-propranolol indicated no differences in the rate of elimination of the isomers in dogs.56 Finally, the metabolism of carbon-13 labeled R- and S-a-methyldopa has revealed a high degree of stereoselectivity favoring the S-enantiomer for both transport across the blood-brain barrier and brain decarboxylase activity.57

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D. Use of Isotopes in Studying Enzymatic Mechanisms of Drug Metabolism The most widely used stable isotope in determining enzymatic mechanisms deuterium because of the large mass difference between it and hydrogen. 58" This mass difference leads to differences in the zero-point energy of a bond to deuterium and hydrogen which in turn leads to isotope effects on the rates of metabolic processes where cleavage of a C-H vs. C-D bond is rate-determining. Because the oxidative metabolism of drugs usually involves such a process there are many examples of isotope effects on the metabolism of drugs. 6' Two recent articles have appeared on theoretical treatments of kinetic isotope effects which shoul b of help in designing more interpretable deuterium isotope experiments.8 9 ,%o

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Aromatic hydroxylations do not in general exhibit kinetic deuterium isotope effects since the rate-determing step is the heterolytic cleavage of a carbon-oxygen bond of an intermediate arene oxide.61 Small isotope effects have been found and may have been due to secondary deuterium isotope effects. Such effects have been observed for peracid-type oxidations.62 However, significant isotope effects (%/b = 1.3-1.75) are associated with the aromatic meta- droxylation of benzene substituted with electron-withdrawing substituents!$ possibly indicating a different oxygenation mechanism or change in rate-determing step. Other common pathways of drug metabolism include oxidative 0- and Ndealkylation. Deuterium isotope effects f - 2 have been observed for the 0-demethylation of a variety of anisoles. 84-66 Small isotope effec (SIp-1.3) have been observed for the N-demethylation of amines. ,68 The e fects are exclusive of secondary effects on amine basicity as determined in one of these studies.68 Studies of the oxidative dealkylation by liver microsomes of deuterated N-alkylamphetamines has indicated that a-carbon oxidation was responsible for dealkylation of the S-enantiomers, whereas N-oxidation wgg probably the rate-determining step for dealkylation of the R-enantiomers. Several other studies have b n carried out on Cvs. N-oxidation of amphetamines using atmospheres of fgO and deuterium labeling, and mechanisms have recently been reviewed.70 2

k'i

Two examples of unusually large deuterium isotope effects in biological systems have been observed. The oxidative 0-demethylation of trideuteromethoxy anisole showed an isotope effect of -10 in vitro,65 and the oxidation in vivo of cotinine, a nicotine metabolite, to 3-hydroxycotinine showed an isotope effect of -7 when 3,3-dideuterio-cotinine was used.ll A recent study has employed deuterium labeling to show that the mechanism for the oxidative N-demethylation of nicotine may involve two modes of breakdown for a proposed carbinolamine intermediate, dealkylation with formaldehyde fo mation and dehydration to an iminium ion.72 The formation of such an sp'-hybrid intermediate may help to explain why both a primary and substantial ,R-secondary deuterium isotope effect were observed for the N-deethylation of the antiarrhythmic agent , lidocaine. 73 In contrast, only a primary isotope effect was observed on the rate of oxidative 0-deethylation of deuterated analogs of the analgesic, phenacetin.74 These results indicate differences in the mechanism of oxidative 0- and N-dealkylation. A final example of the use of secondary deuterium isotope effects in studying enzymes involved in drug metabolism revealed an SN-2like transition state for the transfer of a methyl group catalyzed by catechol-0-methyl transferase. 75

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E. Switching of Metabolic Pathways with Deuterium The magnitude of deuterium isotope effects observed in drug metabolism varies considerably and apparently is influenced by the availability of alternate pathways of metabolism. Recent evidence suggests that if a drug is metabolized by multiple alternate pathways, the metabolism may be shifted by deuterium labeling at a site of metabolism.76 Studies both in vivo and in vitro on the effect of deuterium substitution on N-demethylation and on the conversion of a methyl group to a hydroxymethyl group have been investigated

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for antipyrine, and caffeine.76 In both cases examined, deuterium substitution in one position switches metabolism to an alternate position. For example, the metabolism of antipyrine (G), N-C2%-antipyrine and 3-C2H3-antipyrine (14)were compared. The major urinary metabolites of 12 and 13 were 4-hydroxy- and 3-hydroxymethylantipyrine. In contrast, were 4-hydroxyantipyrine and N-demethylantithe major metabolites of pyrine; the metabolism of antipyrine was switched by deuterium substitution from hydroxylation of the methyl group on C-3 to N-demethylation.

(s),

-

12 R = H

5

R='H

'4

These results indicate that the metabolic disposition and thereby the pharmacological activity, whether efficacious or toxicological, could be modified by selective substitution of deuterium for hydrogen. A new combination antibacterial contains 3-fluoro-D-alanine-2-d which is an excellent example of selective deuteration to enhance the pharmacological activity of a therapeutic agent.77 The metabolism in vivo of 3-fluoro-Dalanine is reduced several-fold by substitution of deuterium for hydrogen on the 2-position without loss of antibacterial activity. This enhances the therapeutic index of the compound because metabolism of 3-fluoro-Dalanine leads to the formation of an inactive antibacterial and fluoride which is nephrotoxic.

F. Use of Isotopes to Study Reactive Metabolites - Most drugs are metabolized to inactive and sometimes more active metabolites. However, a few drugs are also metabolized to reactive, often toxic, metabolites which bind to tissue macromolecules (RNA, DNA, and proteins) and can lead to carcinogenesis, mutagenesis, or tissue necrosis.78 Stable isotope labeling has been used to examine mechanisms in the formation of these reactive metabolites. Two examples have already been presented in Section IIIB using the twin-ion technique (ref. 48-50). Mechanisms for the covalent binding of arylating metabolites of the widely used analgesics p-hydroxyacetanilide (acetaminophen) and p-ethoxyacetanilide (phenacetin) to hamster microsomal protein have also been investigated using 1802.79The mechanism of reactive metabolite formation from acetaminophen was found to be different than that for phenacetin in vitro.

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Anti-cancer drugs such as cyclophosphamide (g), aniline mustard, and nitrosoureas are transformed to reactive metabolites which are the toxic species required for their anti-cancer activity.80 Experiments with selectively deuterated analogs of these drugs has distinguished which pathway, among several alternative pathways of metabolism, is responsible for antitumor activity. For example, a deuterium isotope effect was observed for the formation of 4-ketocyclophosphamide formed by the oxidation of the carbon alpha to the phosphoramide nitrogen, but there was no isotope effect on the anti-tumor activity. However, there was a marked effect on the subsequent p-elimination reaction and consequent decrease in anti-tumor activity by deuterium substitution at C-5. Thus, the formation of acrolein and phosphoramide mustard is rate determining for the anti-tumor activity of cyclophosphamide.

(c),

4

3

i

t

The carcinogenicity of dimethylnitrosamine and 4-nitrosomorpholinewas reduced by deuterium substitution for hydrogen on carbon atoms alpha to the amino nitrogen. 81*82 Consistent with the hypothesis that alpha-carbon Oxidation is required for reactive metabolite formation from nitrosamines, there is a substantial primary deuterium isotope effect (%/kD = 3.8) on the rate of dimethylnitrosamine N-demethylation.83 Specific deuteration of 3-methylcholanthrene, a potent polycyclic hydrocarbon carcinogen, showed that oxidation of the 1-carbon atom is critical in the tumor-initiating process in mouse skin.84 Stable isotopes other than deuterium have also been used in some novel approaches for studying reactive metabolite formation. Benzo[a]pyrene, another potent polycyclic hydrocarbon carcinogen, was incubated with cofactors and rat liver microsomes in an atmosphere Of 170 to investigate whether or not 6-oxybenzo[a]pyrene radical was formed.85 'Electron spin

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resonance analysis indicated that very small or undetectable amounts were formed enzymatically. On the contrary, a radical was formed non-enzymatically by air oxidation of benzo[a]pyrene after extraction of the metabolites from the incubation mixture. Infrared analysis was used to determine that the increase in carboxyhemo lobin formation subsequent to the administration of methylene ~hloride-~~C to rats, is caused by its metabolism to carbon monoxide.86

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IV. Use of Stable Isotopes in Pharmacokinetics The quantification of drugs and their metabolites by isotope dilution using gc-ms has been applied by several investigators to study the pharmacokinetics of drug metabolism, distribution, and excretion. The basic approach used in these investigations is illustrated by studies with amphetamine and phentermine!’ caffeine, mephobarbital, secobarbital and antipryine.88 More recently, two elegant adaptations of the general procedure have been used to determine absolute bioavailability89~90and steady state kinetics.9 1 ~ 9 2 The absolute bioavailability of N-acetylprocainamide was determined by administering intravenously the%-labeled drug at the same time the unlabeled drug was given orally.89990 Plasma levels and urinary excretion of both compounds were quantified with the use of a d5-labeled internal standard. Oral absorption was found to be within a range of 75% to 90% in three human subjects and peak plasma levels were attained in 45-90 minutes. The slow absorption rate of N-acetyl-procainamide, coupled with a long half life for elimination plus therapeutically effective plasma levels, make the compound attractive as an oral anti-arrhythmic agent. The stable isotope procedure that was used to determine these parameters offered the advantage of determining the bioavailability and first-pass effects of the drug from a single study by the analysis of only one set of blood samples, and permitted the determination of the kinetics of drug distribution and elimination at the same time that the absorption was under investigation. The steady state kinetics of methadone91 and propoxyphene92 were investigated by substituting the daily dose of the unlabeled compound by a deuterium labeled derivative, and following the plasma half-lives of both drugs. The half-lives of the deuterated derivatives were found to be three to seven times shorter than the unlabeled drugs, which had been administered chronically. These results suggested that deep pools of tissue-bound, unlabeled drug existed which were not readily available to the deuterated derivatives. This experiment illustrates the importance of this technique for investigating the complexities of steady state pharmacokinetics. In summary, stable isotopes are being effectively used in many areas which either directly or indirectly affect research in medicinal chemistry. Hopefully, this review has highlighted those areas of interest to most medicinal chemists.

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References 1. "Proceedings of the Second International Conference on Stable Isotopes" E.R. Klein and P.D. Klein, Eds. National Technical Information Service U.S. Department of Commerce, Springfield, Virginia, 1975. 2. International Symposium on Stable Isotopes: Applications in Pharmacology, Toxicology and Clinical Research, London, Jan. 3-4, 1977. 3. V.L. Arona and C.F. Eck, ref. 1, p. 695. 4. H. Spielmann, H.G. Eibs, D. Nagal and C.R. Gregg, Life Sci., 2, 633 (1976). 5. D.R. Knapp and T.E. Gaffney, Clin. Pharmacol. Therap., 13, 307 (1972). 6. M.I. Blake, H.L. Crespi and J.J. Katz, J. Pharm. Sci. , 64, 367 (1975). 7. A.L. Burlingame, B.J. Kimble and P.J. Derrick, Anal. Chem., 48, 368R (1976). 8. A.M. Lawson, Clin. Chem., 21, 803 (1975). 9. A. Murray, 111, and D.L. Williams, "Organic Synthesis with Isotopes," Parts I and 11, Interscience, New York, 1958. 10. T.J. Simpson, Chem. SOC. Rev., 4, 497 (1975). 11. R.A. Uphaus, J.J. Katz and M.I. Blake, J. Pharm. Sci., 2, 1096(1976). 12. H.A. Akers, C.L. Atkin and J.B. Neilands, Org. Mass Spectrom., @, 259 (1975). 13. A. Vigerani, B. Gioia and G. Cassinelli, Carbohydrate Res., 2,321 (1974). 14. J.G. Liehr, D.L. Von Minden, S.E. Hattox and J.A. McCloskey, Biomed. Mass Spectrom., _1, 281 (1974). 15. B.S. Middleditch and D.M. Desiderio, Adv. Mass Spectrom., 5, 173(1974), 16. H. Nau and K. Biemann, Anal. Biochem., 73, 139, 154, 175 (1976). 17. K.D. Haegele, G. Holzer, W. Parr, S.H. Nakagawa and D.M. Desiderio, Biomed. Mass Spectrom. , 1,175 (1974). 18. H. Tschesche and E. Wachter, Eur. J. Biochem., 16,187 (1970). 19. A. Murai and Y. Takeuchi, Bull. Chem. SOC. Jap., 48, 2911 (1975). 20. M. Schneider and H. Tschesche, H.-S. Zeit. Phys. Chem., 357, 1339 (1976). 21. D.D. Giannini, K.K. Chan and J.D. Roberts, Proc. Nat. Acad. Sci., USA, 71, 4221 (1974). 22. K.K. Chan, D.D. Giannini, A.H. Cain, J.D. Roberts, W.R. Porter and W.F. Trager, Tetrahedron, in press. 23. R.C. Long, Jr., and J.H. Goldstein, J. Magn. Res., 16,228 (1974). 24. S. Fernandjian, S.T. Dinh, J. Savrda, E. Sala, R. Mermet-Bouvier, E. Bricas and P. Fromageot, Biochim. Biophys. Acta, 399, 313 (1975). 25. I.J.G. Climie, D.A. Evans and M. Akhtar, Chem. Commun., 160 (1975). 26. A. Lapidot and C.S. Irving, ref. 1, p . 427. 27. E. Oldfield, M. Meadows and M. Glaser, J. Biol. Chem., ' 2 2 , 6147(1976) 28. J.A. Glasel, V.J. Hruby, J.F. McKelvy and A.F. Spatola, J. Mol. Biol., 79, 555 (1973). 29. B.J. Millard in "Mass Spectrometry," Vol. 3, Specialist Periodical Report, R.A.W. Johnstone, Sr. Reporter, The Chemical Society, London, 1975, p. 339. 30. C. Fenselau, Appl. Spectrosc., 28, 305 (1974). 31. D.J. Jenden and A.K. Cho, Ann. Rev. Pharmacol., 2,371 (1973). 32. M.G. Horning, J. Nowlin, K. Lertratanangkoon, R.M. Stillwell, W.G. Stillwell and R.M. Hill, Clin. Chem., 19, 845 (1973).

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33. A.M. Lawson and G.H. Draffan, Prog. Med. Chem., 12, 1 (1975). 34. G.E. von Unruh, D.J. Hauber, D.A. Schoeller and J.M. Hayes, Biomed. Mass Spectrom., L, 345 (1974). 35. M.G. Lee and B.J. Millard, ibid., 2, 78 (1975). 36. D.A. Schoeller, ibid., 3, 265 (1976). 37. W.A. Garland, W.F. Trager and S.D. Nelson, Biomed. Mass Spectrometry 1 124 (1974). 38. L. Palmer and B. Holmstedt, Sci. Tools, 22, 1215 (1975). 39. F.C. Faulkner, B.J. Sweetman and J.T. Watson, Appl. Spectrosc. Rev., 10, 51 (1975). 40. C.G. Hammar and B. Holmstedt, Anal. Biochem, 2 532 (1968). 41. R.F. Morfin, I. Leav, P. Ofner and J.C. Orr, Fed. Proc. 2, 247(1970). 42. D.R. Knapp, T.E. Gaffney and R.E. McMahon, Biochem. Pharmacol., 2, 425 (1972). 43. R.E. McMahon, H.R. Sullivan, S.L. Due and F.J. Marshall, Life Sci., 12, 463 (1973). 44. A. Prox, A. Zimmer and H. Machleidt, Xenobiotica, 3, 103 (1973). 45. W.E. Braselton, Jr. , J.C. Orr and L.L. Engel, Anal. -Biochem., 64 (1973). 46. L.R. Pohl, S.D. Nelson, W.A. Garland and W.F. Trager, Biomed. Mass Spectrom., 2, 23 (1975). 47. D.R. Knapp, N.H. Holcombe, S.A. Krueger and P.J. Privitera, Drug Metab. Dispos., 4, 164 (1976). 48. S.D. Nelson, J.A. Hinson and J.R. Mitchell, Biochem. Biophys. Res. Commun., 69, 900 (1976). 49. S.D. Nelson, J.R. Mitchell and L.R. Pohl in "Mass Spectrometry in Drug Metabolism," A. Frigerio, Ed., Raven Press, Inc., New York, N.Y., 1977, pp. 237-250. 50. L.L. Engel, J. Weidenfeld and G.R. Merriam, J. Tox. Environ. Health, Suppl. 1, 37 (1976). 51. J. Gal, L.D. Gruenke and N. Castagnoli, Jr., J. Med. Chem., 18, 683 (1975). 52. R.J. Weinkam, J. Gal, P. Callery and N. Castagnoli, Jr., Anal. Chem., 48, 203 (1976). 53. P. McGraw, P.S. Callery and N. Castagnoli, Jr., J. Med. Chem., 0, 185 (1977). 54. J. Gal, J. Wright and A.K. Cho, Res. Commun. Chem. Pathol. Pharmacol., 15, 525 (1976). 55. R.E. McMahon and H.R. Sullivan, ibid., 14,631 (1976). 56. H. Ehrsson, J. Pharm. Pharmacol., 28, 662 (1976). 57. M.M. Ames, K.L. Melmon and N. Castagnoli, Jr., Biochem. Pharmacol., in press. 58. W.P. Jencks, "Catalysis in Chemistry and Enzymology", McGraw-Hill, New York, N.Y., 1969, Chapter 4. 59. D.B. Northrup, Biochemistry, 15, 2644 (1975). 60. W.G. Bardsley and R.D. Waight, J. Theor. Biol., 63, 325 (1976). 61. G.J. Kasperek, T.C. Bruice, H. Yagi, N. Kaubisch and D.M. Jerina, J. Chem. SOC. D, 784 (1972). 62. R.P. Hanzlik and G.O. Shearer, J. her. Chem. SOC., 97, 5231 (1975). 63. J.E. Tomaszewski, D.M. Jerina, and J.W. Daly, Biochemistry, 14, 2024 (1975).

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C. Mitoma, D.M. Yasuda, J. Tagg and M. Tanabe, Biochim. Biophys. Acta, (1967). A.B. Foster, M. Jarman, J.D. Stevens, P. Thomas and J.H. Westwood, Chem.-Biol. Interactions, 2, 327 (1974). K.A.S. Al-Gailany, J.W. Bridges and K.J. Netter, Biochem. Pharmacol., 24, 867 (1975). J.A. Thompson and J.L. Holtzman, Drug. Metab. Dispos., 2, 577 (1974). M.M. Abdel-Monum, J. Med. Chem., 18,427 (1975). P. Th. Henderson, T.B. Vree, C.A.M. van Ginneken and J.M. van Rossum, Xenobiotica, A, 121 (1974). R.T. Coutts, R. Dawe, G.W. Dawson and S.H. Kovach, Drug Metab. Dispos. 4, 35 (1976). E. Dagne, L. Gruenke and N. Castagnoli, Jr., J. Med. Chem., 11,1330 (1974). T-L. Nguyen, L.D. Gruenke and N. Castagnoli, Jr., ibid., 19,1168(1976). S.D. Nelson, L.R. Pohl and W.F. Trager, ibid., 1062 (1975), W.A. Garland, S.D. Nelson and H.A. Sasame, Biochem. Biophys. Res. Comun., 72, 539 (1976). M.F. Hegazi, R.T. Borchardt and R.L. Schowen, J. Amer. Chem. SOC., 98, 3048 (1976). M.G. Horning, K.D. Haegele, K.R. Sommer, J. Nowlin, M. Stafford and J-P. G. Thenot, in ref. 1, pp. 41-54. J. Kollonitsch and L. Barash, J. Amer. Chem. SOC., 2, 5591 (1976). S.D. Nelson, M.R. Boyd and J.R. Mitchell, in "Recent Advances in the Study of Drug Metabolism," ACS Symposium Series, ed. D.M. Jerina, 1977 in press. J.A. Hinson, S.D. Nelson and J.R. Mitchell, The Pharmacologist (Abstract), 18,24 (1976) and Molec. Pharmacol., in press. P.J. Cox, P.B. Farmer, A.B. Foster, E.D. Gilby and M. Jarman, Cancer Treatment Reports, 60, 483 (1976). L.K. Keefer, W. Lijinsky and H. Garcia, J. Natl. Canc. Inst., 2, 299 (1973). W. Lijinsky, H.W. Taylor and L.K. Keefer, ibid., 57, 1311 (1976). D. Dagani and M.C. Archer, ibid., 57, 955 (1976). E. Cavalieri, H. Garcia, P. Mailander and K. Patil, Chem.-Biol. Interactions, 11,179 (1975). A.S. Rispin, H. Kon and D.W. Nebert, Molec. Pharmacol., 12, 476 (1976) V.L. Kubic, M.W. Anders, R.R. Engel, C.H. Barlow and W.S. Caughey, Drug Metab. Dispos., 2, 53 (1974). A.K. Cho, B.J. Hodshon, B. Lindeke and G.T. Miwa, J. Pharm. Sci., 3, 1491 (1973). M.G. Horning, J. Nowlin, M. Stafford, K. Lertratanangkoon, K.R. Sommer, R.M. Hill, R.N. Stillwell, J. Chromat., 112, 605 (1975). J.S. Dutcher, J.M. Strong, W.K. Lee and A.J. Atkinson, Jr., in ref. 1, p. 186. J.M. Strong, J.S. Dutcher, W.K. Lee and A.J. Atkinson, Jr., Clin. Pharmacol. Therap., 2, 613 (1976). E. Anggard, J. Holmstrand, L.M. Gunne, H.R. Sullivan and R.E. McMahon, in ref. 1, p. 117. H.R. Sullivan, P.G. Wood and R.E. McMahon, Biomed. Mass Spectrom., 3, 212 (1976).

136, 566

COMPOUND NAME AND CODE NUMBER INDEX A-28086 (narasin), 146 A-42574, 14 AB-74, 112 Abbott 40557, 46 acebutolol, 201 acetaminophen, 204, 207, 325 acetylhydrazine, 204 ac luraci1, 205 acridines, 122 actamycin, 114 ACTH, 31, 32, 33, 94 ACTH1-24, 31 ACTH1-39, 31 ACTq-9 33 ACTq-16, 31, 32, 33 actinomycin D, 124, 125 actinomycins, 135 actinoxanthin, 115 AD-32, 124 adenosine, 166 adenine arabinoside, 121 adenosine diphosphate (ADP) , 80, 82, 83 adriamycin, 120, 124, 125 ADTN(6,7-dihydroxy-2-aminotetraline), 177 AH-5158 (labetalol), 64 AL-1965, 5, 6 alamethicin, 115 albendazole, 141 alborixin, 115 alletorphine, 25 N-allyl-norapomorphine, 176 amikacin, 110, 111 aminocyclitols, 110, 111, 112 6-amino-dopamine, 173 aminoglycosides, 1l0, 111, 112, 114 aminophylline, 86, 94 aminopolyol, 113 201 aminopyrine, aminodarone, 41 4 amitriptyline, amobarbital, 201 amoxycillin, 102 amphetazmine, 257 amphetamine, 5, 9, 203 amphotericin B, 117 angiotensin, 62, 63 anhydro ara-C, 121 anodynin, 23

331 -

ansamycins, 114, 131 anthramycin, 135 antipain, 261 antipyrine, 7, 9, 204 aplasmomycin, 116 apocodeine, 176 apomorphine, 250, 253 R-apomorphine, 175 S-apomorphine, 176 apomorphine diesters, 204 apramycin, 112, 113 aprindine, 40 ara-C, 121, 125 arachidonic acid, 81, 168 arginine, 94 arginine-8-vasopressin, 33 1-ascorbic acid, 185 asparaginase, 122 aspirin, 80, 81 asterriquinone, 124 asukamycin, 116 atenolol (ICI 66,082), 63 atropine, 94 autoprothrombin C (Factor X,), 84 (doxaprost)3 73 8-aza-6-thioguanine, 122 azabuperone, 6 5-azacytidine, 121 azidomorphine, 25 101 azlocillin, AZQ, 120 B-43, 115 baccharin, 124 bacillomycin B, 115 bacitracin A, 134 baicalein, 75 Bayer 1040 (nifedipine), 65 BC-2605, 24 BCG, 125 BCNU, 120, 125 BD-40A, 71, 72 beclomethasone dipropionate, 73, 74 bencyc lane, 51 benperidol, 251 benzetimide, 202 benzo [alpyrene, 326 benzofuran, 194 benzomorphans, 24 benzothiepins, 26 benzydamine, 185, 186

332

COMPOUND NAME AND CODE NUMBER INDEX

calcium, 94 bestatin, 122 calusterone , 123 betahistine, 52 CAMP, 224 206 bethanidine , candicidin, 132 bicyc lomycin, 117 canedine , 176 biglumide , 15 cannabinoids, 27, 124 biphenylacetic acid, 207 203 canrenone , bisobrin, 87 capreomycin, 115 bitolterol, 72, 204 carbamazepine, 12, 204 BL-3459, 84 carbamoyl mycophenolic acid, 123 BL-3677AY 41 carbazilquinone, 120 BLS-578 (cefadroxil) , 103 carbenicillin, 101, 111 BLS-640 (SKF-60771) (cefatrizine), carbenoxolone, 91, 186 103 carbuterol (SKF 40383), 71 BLS-786, 104 carminomycin, 115, 116, 123 BM-15,100, 76 carnitine, 264 bombesin, 94 cartazolate (SQ 65,396) , 12 boron betaine, 122 Cavintofl (RGH-4405), 55 bottromycin A2, 115 CB 154 (2-brorno-a-ergo~ryptine)~ bretylium, 42 177 brevistin, 115 CCNU, 121 brinase, 86 196 CDC (chenodeoxycholic acid) , BRL-8242, 65 cefaclor (Lilly 99638) , 103 BRL-10833, 77 cefadroxil (BLS-578) , 103 BRL-13776, 66 103 BRL-14151 (clavulanic acid) , 105 cefamandole , cefatrizine (BLS-640) (SKI?-60771) , bromazepam, 10 103, 104 bromazepine , 205 102, 104, cefazaflur (SKF 59962), 2-bromo-a-ergocryptine (CB-154), 105 177 cefazolin, 102, 104 bromoperidol, 251 cefoxitin, 103 BS 100-141, 61 ceftezole (dimethyl cefazolin) , BU-1014, 10 102 bufroline (ICI 74917), 76 cefuroxime , 103 bufuralol , 203 cephalosporin, 302 bulbocapnine, 176 cephalosporin C, 135 bunitrolol (KO 1366), 63 cephalothin, 85 bunolol, 203 cephamycin (7-methoxy cephalosporin), bupicomide (Sch 10,595) , 65 102 buprenorphine , 24 cerexins , 115 (+)-butaclamol, 250, 251, 256 cerulenin, 117 (-)-butaclamol, 250, 256 CGP 4540 (C 9333-G0), 143 butalamine , 55 CGP 9000, 104 butamisole , 142 chenodeoxycholic acid (CDC), 196 butirosin, 110 clofluperol, 251 butorphanol, 24 46 N-n-butyl-N-n-propyl-dopamine, 174 chloracyzine, 115, 133 chloramphenicol, C 2801X, 102 C 9333-GO (CGP 4540), 143 chlorazepate, 10 chlorimipramine, 3, 5 C. parvum, 125 caffeine, 7, 9, 166, 203 chlorocarcins A, By C, 116 chlorodiazepoxide , 10 calcitonin, 94, 223

COMPOUND NAME AND CODE NUMBER INDEX chloroform, 86 chlorpromazine, 251 chloroquine, 188 cholecystokinin octapeptide, 94 choleratoxin, 155 5~-cholest-8(14)en-3~-ol-l5-one, 193 cholestyramine, 193 chromonar, 44 CI-683 (ripazepam) 10 cimetidine (tagamet, SKF 92,334), 91, 92, 93 45 cinepazide , cinnarizine, 52 CL-719, 195 CL-90,394, 64 clavulanic acid (BRL 14151), 105 clenbuterol (NAB-35), 72 clindamycin, 113, 114, 141 clobazam, 11 clofibrate, 192 clonazepam, 11 clonidine, 60, 91, 92, 177, 206 clozapine, 7, 251, 252, 257 cocaine, 202, 203 coenzyme Qlo, 123 colchicine, 124, 155 colestipol, 193 collagen, 80, 82 compactin, 116 Compound A, 76 concanavalin A, 85, 154 conocandin, 116 cordycepin, 121 cortisol, 300 coumermycin A1, 114, 115 coumerose, 114 CP-35,587, 102 CP-38,118, 102 CP-38,295, 115 CP-39,295, 146 CRC 2015, 10 crotepoxide, 125 CS-1170, 105 CT, 223, 227 cyclandelate, 51 cyclo-alliin, 87 cyclobenzaprine, 204 cyclophosphamide, 120, 125, 326 cyclosporin A , 115 153 cytochalasin B y

333 -

cyproheptadine, 204 D-40TAY 11 D 1959 (reproterol), 72 DAM, 22 damavaricin, 114 daunomycin, 133 daunorubicin, 123 , 124 DNB, 6, 7 3-deazauridineY 121 11-deoxy-apomorphine, 176 2' -deoxycoformycin, 121 6-deoxyneomycins, 111 6-deoxyparomomycins, 111 beta-deoxysoterenol, 173 3" -deoxystreptomycin, 112 desipramine, 4, 186 N-desmethyl apomorphine (norapomorphine) , 176 N-desmethyldiazepam, 10 4"-C-desmethylsisomicin, 112 desosamine, 111, 114 dexamethasone, 31, 184 deximafen, 2 D-glucaro-6-lactarny 110 cis-diamminodichloroplatinum 11, 123 20,25-diazacholesterol, 262 diazepam, 10, 11, 12, 166 dibenzazepines, 43 2,8-dibenzyl-cyclooctanone, 194 2,6-dichlorophenylguanidine, 206 2,6-dideoxystreptamine, 111 diethyldithiocarbamate, 34 N,N-diethyl-dopamine, 174 diethylstilbesterol, 261, 322 H-dihyd roa1preno lo1, 255 dihydro-B-ergosine (DQ 27-422), 53 dihydro-5-azacytidineY 121 dihydroergonine (DN 16-457), 53 3H-dihydromorphine, 255 l0,ll-dihydropicromycin, 113 dihydrostreptomycin, 130, 131 6,7-dihydroxy-2-aminotet ra 1ine(ADTN), 177 1,2-dihydroxyaporphine, 176 6,7-dihydroxytetrahydroisoquinoline,

175 5 ,6-dihydroxytryptamineY 61 dimethyl cefazolin (ceftezole), 102

334

COMPOUND NAME AND CODE MIMBER INDEX

3-0,4-0-dimethyl-dopamine, 173 16,16-dimethyl PGE2, 94, 96, 97 4,@-d imethylhis tamine, 91 2,4-dinitrophenol, 263 diphenylhydantoin, 320 diphenylhydramine, 15 2,3-diphosphoglycerate (2,3 DPG) , 83 dipropylacetate, 206 dipropylacetic acid, 14 dipyridamole, 80, 83 disodium cromoglycate, 73, 74, 75, 76, 77 disopyramide, 40, 43 DIV-154, 2 DL-254, 12 DN 16-457 (dihydroergonine) , 53 DNA, 124, 125, 163, 168, 169 L-dopa, 3, 300, 302 dopamine, 166, 173, 250, 253 doxantrazole, 74, 76 doxaprost (AY 24559), 73 doxycycline, 114 2,3 DPG (2,3-diphosphoglycerate), 83 DQ 27-422 (dihydro-@-ergosine), 53 DTIC, 120, 122, 125 ECF-A, 153, 156 echinocandin By 115 efrotomycin, 116 EHDP, 227 5,8,11,14-eicosatetraynoic acid(TYA), 83 ellipticine, 124 Embay 8440 (praziquantel), 142 endorphins, 20, 21 @-endorphin, 20 enkephalins, 20, 21, 22 enomycin, 115 enterocin, 115, 116 epinephrine, 33, 8 0 , 82, 166, 174, 255, 256 (->-epinephrine, 251 ergometrine, 177 erythromycin(s), 113, 117, 131 erythronolide A oxime, 114 estazolam, 11 etamycin, 134 eterobarb, 12 etorphine, 24

ethacrynic acid, 186, 187 ethane-1-hydroxy-1,l-diphosphonate, 227 ethyl apovincaminate, 205 N-ethyl-dopamine, 174 a-ethyl-isoproterenol, 174 a-ethyl-norepinephrine, 174 N-ethyl-norepinephrine, 174 ethyloestrenol, 86 everninomicins, 113 everninomicin B y 117 factor V, 84 factor VII, 85 factor IX, 85 factor Xa (autoprothrombin C), 84 factor XI, 85 feldamycin, 115 f enbendazole , 141 fenoterol (Th 1165a), 71 fenoprofen, 303 fenoxedil, 55 feprazone, 82 fibrinogen, 84 ficellomycin, 116 FL 1060 (mecillinam), 101 fluanisone, 251 flubendazole, 141 flunisolide, 73, 74 flunitrazepam, 11 3-fluoro-D-alanine-2-d, 325 flupenthixol, 255 a-flupenthixol, 251, 255 f3- flupenthixol, 255 3H-naloxone, 255 fluphenazine, 251, 253 flurazepam, 11 fluspirilene, 251 folinic acid, 121 formycins, 136 2-fomylpyridine N-oxide, 122 fortimicins A, B y 112, 113 fosazepam, 11 fospirate, 142 FPL 52791, 74, 75 FR 1923 (nocardicin A ) , 105 FR 10612, 103 5-FU, 125 5-FUDR-5'-phosphate, 121 furosemide, 186, 187, 204 fusaric acid, 65 G-52, 112

COMPOUND NAME AND CODE NUMBER INDEX GAG (RMI 71675),

14

D-galactosamine,

121

garamine, 111 gardimycin, 115 gastric inhibitory polypeptide,

94 gastrin, 92, 93, 94 geldanamycin, 131 gemcadiol , 193 gentamicin, 110, 111, 113 gentamicin B y 111 gentamicin C-complex, 111 gentamicin C2b, 112 gentamicin X2, 111 glucagon, 94 glucocorticoids , 167 glutaminase , 122 13, 132 glutarimide(s) , glutethimide, 206 GPA 2640, 12 gramicidin S, 134 griseoviridin, 115 griseusins A , B y 116 61 guanabenz, I@-guanylhistamine , 91 H-102-09, 3 halofenate, 83, 192 halomorphides, 25 haloperidol, 7, 249, 251 3H-haloperidol, 250, 256 halothane , 204 hamycins , 116 hematoporphyrin, 126 hementerin, 86 heparin, 84, 85 HETE, 154, 168, 182 heterocannabinoids, 15 2-hexadecanoneY 194 hexobendine , 55 n-hexylamine, 202 HHT, 182 histamine, 166 HL-A13, 162, 163 HL-A17, 162, 163 HMG, 195 horhammericine , 124 HR-546, 187 3H-5-HT, 256 5-HTP (5-hydro~ytryptophan)~ 4 HydergineR, 53, 54 hydralazine , 64

335 -

2-hydroxy-apomorphineY 176 20-hydroxycanrenoneY 203 6-hydroxydopamineY 34, 173, 254 5-hydroxytryptamineY 61 4 5-hydroxytryptophan (5-HTP), hyperthermia, 125 66 hypotensin, 1-2743-C, 115 ibuterol, 71 ICI 46,474 (tamoxifen) , 123 ICI 66,082 (atenolol) , 63 ICI 74,917 (bufroline), 76 ICRF 159, 122 ifosfamide, 120 IgE, 156 ilicicolin H, 115, 116 84 imidazo [1,2-c] quinazolines, imipramine, 4 , 186, 260 indapamide (SE 1520) , 66 indoleacryloisonitrile, 116 indolmycin, 135 indomethacin, 62, 82, 94, 183- 186 indoprofen, 82 inosine dialdehyde , 121 insulin, 94 iodipamide, 187 ipratropium bromide (Sch lOOO), 72, 73 iproniazid, 204 , 322 isoapomorphine, 176 S-N-isobutyl-norepinephrine, 174 isoniazid , 204 N-isopropyl-3,4-dihydroxyphenoxypropanolamine (MJ 9910) , 174 N-isopropyl-3,5-dihydroxyphen-

ethanolamine , 173 N-isopropyl-dopamine, 174 isopropylhydrazine, 204 N-isopropyl-N-methyl-dopamine, 174 isoproterenol, 94, 166, 204, 251, 255 R-isoproterenol, 173 S-isoproterenol, 173 isoxsuprine, 55 iturin, 115 Janssen R17934, 123 Janssen R28935, 61 JI-20AY 111 josamycin, 113 juvenimicins, 113

336 -

COMPOUND NAME AND CODE NUMBER INDEX

K-41, 115 K-52, 116 kalafungin, 116 kanamycin, 111 kessoglycol monoacetate, 15 ketazolam, 10 7-ketocholesterol, 193 KM-214, 116 40 : K 1173 (mexi.litine), KO 1366 (bunitrolol), 63 L-8027, 185 L-10492, 186 L-10503, 186 labetalol (AH 5158), 64 laidlomycin, 115 lasalocids, 132 laterosporamine, 116 leuc inostatin, 117 leucomycins, 114, 131 leupeptin, 261 lepetit 6150, 64 LF 178 (lipanthyl), 193 lidocaine, 321, 324 44 lidoflazine, Lilly 99638 (cefaclor), 103 d-limonene, 196, 205, 207 lincomycin, 114 lipanthyl (LF 178), 193 p-lipotropin, 20 lithium, 4, 20 LM 209, 74 lochnericine, 124 lofepramine, 4 lonomycin, 115 lorazepam, 10, 11 LSD (lysergic acid diethylamide), 177, 255 3H-LSD, 256 lucensomycin, 117 D-lysergic acid, 177 lysergic acid diethylamide (LSD), 177, 255 3H-lysergic acid diethylamide (LSD) , 255 lysolipin I, X, 116 macrolide(s), 113, 131 macromomycin B, 124 macrotetrolides, 132 maduramycin, 116 magnamycin, 131 malformin C, 116

marasmic acid, 117 McN-2840-46, 43 ME-106, 71 mebendazole, 142 mecillinam (FL 1060), 101 mefenamic acid, 185 mefloquine (WR142,490), 140 melrosporus, 117 mepacrine, 183 meperidine, 201 mephenesin, 206 mephobarbital, 327 meprobamate, 183 meptazinol, 26, 207 mesoridazine, 15 metaraminol, 173 metatyramine, 173 metformin, 193 methadone, 26, 207, 327 (Me-14C-)-L-methionine, 112 methoprene, 205 methotrexate, 121, 123 7-methoxycephalosporin (cephamycin), 102 5-methoxysterigmatocystin, 124 methsuximide, 202 5-methyl-5-aza-2'-deoxyuridine, 121 3-methylcholanthrene, 326 16s methyl-13-dehydro PGE2, 96 methyldopa, 202, 323 methyl dopamine(s), 173, 174 5,10-methylenetetrahydrofolate, 121 methylenomycin B, 116 methylhistamine(s) 91 2-methyl-L-arginine, 116 methyl p-novioside, 114 N-methyl-N-n-propyldopamine, 174 a-methyl-norepinephrine, 174 a-methylornithine, 169 15R methyl PGE2, 96, 97 15s methyl PGE2, 96, 97 1-methylpseudouridine, 121 9-methylstreptimidone, 132 methyltetracycline(s), 114 metiamide (SKF 92058), 60, 92, 93 metronidazole,) 125, 145 mexilitine (KO 1173), 40 mexlocillin, 101 MG 8926, 43, 46

COMPOUND NAME AND CODE NUMBER INDEX MIF, 152, 154 mimosamycin, 116 minocycline, 111, 141 mitomycin C, 124 MJ-8798-1, 173 MJ-9022, 6 MJ-9067, 43 MJ-9184-1, 173 MK-196, 206 MK-251, 41, 205 MK-302, 146 MK-447, 184 MLC (morphine-like compound), 20 MLF (morphine-like factor), 20 monesin, 115, 131, 146 moperone, 251 morocromen, 45 morphinans, 24 morphi e, 24, 184, 188 Motrib, 303 moxisylate , 55 mycosubtilin, 115 MYaK-38, 82 N-0164, 81, 185, 187 NAB-35 (clenbuterol), 72 nabilone, 15 nafronyl oxalate, 55 naloxone, 20, 21 naltrexone, 24 , 116 184, 185, 303 narasin (A-28086), 115, 131, 146 nebramycin, 112 nectriapyrone, 117 neocarzinostatin, 122 neomycin, 193 neothramycin, 116 N-ethyl sisomicin (netilmicin) , 110, 112 netilmicin, 110, 112 nicergoline, 54 nicotine, 201 nicotinyl alcohol, 55 nifedipine (Bayer 1040) , 42, 45, 65 nikkomycin, 116 nitramisole, 142 nitrazepam, 10 nitrofurantoin, 84 nitrofurazone, 125 nitroscanate, 142

& :::;;;

337 -

nitrosoureas, 120, 121 nocardicin A (FR 1923), 105 nonactic acid, 117 nonactin, 132 norapomorphine (N-desmethyl apomorphine), 176 norocaine, 202, 203 norepinephrine, 33, 166, 174, 255, 256 (-)-norepinephrine, 251 nortriptyline, 4, 322 noviose, 114 novobiocin, 114, 115, 134 nybomycin, 134 nystatin, 117 2-octanone, 194 octapeptin, 115 25-0HD, 224, 229 25-OHD2, 224, 227, 228, 229 ly25(OH)2D3, 224, 228 24 9 25 (OH)2D3, 224, 228 oncodazole, 143 OPC 2009, 72 Org 6001, 41 Org 6582, 2 Org NA 13, 42 OTMX (tixanox), 76 7-oxa-13-prostynoicacid, 187 d-oxazepam hemisuccinate (RV 1208), 10 oxfendazole, 141 oxilorphan, 24 oxisuran, 206 oxypertine, 15 oxytocin, 321 p-3355, 116 PAF, 156 PALA, 122 PamineR, 73 papaverine, 50, 53, 54, 165, 166 parathyroid hormone , 94, 223 patulin, 134 PC-904, 101 penfluridol, 251 D-penicillamine, 14 penicillin(s), 112, 135, 302 pentoxifylline, 55 pepstatin, 261 perhexilene, 45 PGD2, 83 81, 82, 94, 95, 166, 167, PGE(s), 224, 230

338

COMPOUND NAME AND CODE NUMBER INDEX

PGF2a, 81, 97, 168 PGG2, 81, 83 PGH2, 81, 83 PGX (prostacyclin), 83 PHD (thromboxane Bp), 81 phenacetin, 204, 325 phenelzine, 185, 186 phenobarbital, 196 , 206 , 320 phenoxybenzamine, 322 phenoxybenzamine, 34 phenprocoumon, 202 phensuximide, 202 phentermine, 327 phenylbutazone, 82 2-phenyl-dopamineY 173 phenylephrine, 173 phospholipase A2, 85 phytohaemagglutinin, 125 pimaricin, 117 pimozide, 251 pinazepam, 10 pipamperone, 251 piracetam, 55 pirbenicillin, 101 piribedil (ET-495) , 176 piromidic acid, 203 piroxicam, 206 plasmin, 85 plasminogen, 85 platelet factor 3, 84 platinum complex, 120, 123 podophyllotoxin, 124 117 polyenes , polyene macrolide, 132 polyether, 131 polymyxin(s), 114, 115, 134 polyoxins, 136 polypeptin, 115 POP (pituitary opioid peptide), 20 potassium canrenoate, 203 PR-D-92-EAY 75 praziquantel (Embay 8440), 142 prazosin, 64 prednisone, 26 1 prednisolone, 94, 300 prenylamine, 43, 45 primidone , 12 probenecid, 187 probucol, 193 prodines, 23

prolactin, 250 promazine, 251, 257 205 propachlor, propanolol, 34, 63, 94, 163, 201, 203, 323 propoxyphene, 201, 322, 323, 327 N-n-propyl-dopamine, 174 N-n-propyl-norapocodeine, 176 N-n-propyl-norapomorphine, 176 prostacyclin (PGX) , 83, 166, 182, 186 prostaglandin(s), 99, 95, 223 304, 305 prothrombin, 84, 85 protostreptovaricins I-IVY 114 protriptyline, 5 proxazole, 55 202 pseudococaine, pseudoisocytidine, 121 pseudouridine, 121 PTH, 223, 224, 227, 230 PTLA, 195 puromycin, 136 putrescine, 168, 169 pyrenophorin, 117 quassimarin, 124 11 quazepam (Sch 16134) , quinidine, 321 quinomycin, 115 quinterenol, 173 R-818, 43 R-30,730, 26 RA-642, 62 116 rancinamycins, raubasine, 55 reproterol (D 1959) , 72 reserpine, 253 0-retinoic acid, 123 65 retronamide, RGH-4405 (Cavintoa ) , 55 rhodium I1 acetate, 123 ribostamycin, 113 rifampicin, 204 rifamycin(s) , 114, 131 ripazepam (CI-683) , 10 RIT 2214, 101 R M I 12,366A2, 84 R M I 13,640, 195 RMI 14,425, 195 RMI 14,514, 195

COMPOUND NAME AND CODE NUMBER INDEX

RMI 71675 (GAG),

14 RO-07-0582, 125 R0-20-1724, 165, 166, 167 RO-20-5702, 185 rosamicin, 113, 131 rutin, 202 RV 1208 (d-oxazepam hemisuccinate), 10 S15-1 (SQ21,704), 142 S-584, 176 S-40032-7, 173 S-40045-9, 173 S-8527, 193 salbutamol, 71, 166, 173 salicylhydroxamic acid (SHAM) , 144 salmefamol, 71 salsolinol , 175 SC-19,220, 187 SC-28,904, 96 SC-29,333, 96 SCE-100 (tetrahydro cephalexin), 103 Sch 1000 (ipratropium bromide), 72, 73 Sch 10,595 (bupicomide), 65 Sch 12,679, 12 Sch 16,134 (quazepam), 11 Sch 18,640, 115 Sch 22,591, 113 66 SE 1520 (indapamide), secholex, 193 secobarbital, 327 secretin, 94, 95 serotonin, 80, 87 3H-serotonin, 255 , 256 SHAM (salicylhydroxamic acid) , 144 showdomycin, 117 sibiromycin, 135 siomycin A, 115 SIRS, 154 sisomicin, 110, 111, 112 p-sitosterol, 193 SKF 7698, 62 SKF 40,383 (carbuterol), 71 SKF 59;962 (cefazaflur); 102, 104, 105 SKF 60,771 (BLS-640) (cefatrizine), 103 SKF 64,139, 62 SKF 15,073, 104 SKF 91,486; 91

339 -

SKF 92,058 (metiamide), 92, 93 SKF 92,334 (cimetidine, tagamet),

91, 92, 93 somatostatin, 94, 95 sorbistins A 1 , A2, B, 112, 113 R-soterenol, 173 S-soterenol, 173 SPE (sucrose polyester), 195 spectinomycin, 112 spermidine, 83, 168, 169 spermine, 83, 168 spironolactone, 206 spiroperidol, 251 sporamycin, 115 S Q 14,225, 62 SQ 20,009, 74 SQ 21,704 (Sl5-l), 142 SQ 65,396 (cartazolate), 12 SRS-A, 156, 182 ST 155 (clonidine), 91, 92 streptamine, 112 streptokinase, 86 streptomycin, 112, 130 L-streptose, 130 streptovaricins, 114, 131 streptozocin, 94 3H-strychnine, 255 sucrose polyester (SPE), 195 sufentanyl, 26, 27 sulfinpyrazone, 80, 82, 185 0-sulfobenzimide, 206 suloctidil, 83 sulpiride, 7, 15, 94 suprofen, 82 suzukacillin, 115 T-1220, 101 talinolol, 63 tamoxifen (ICI 46,474) , 123 tandamine, 1 TBXA2 (thromboxane A2), 81, 83, 87, 182, 185, 186, 230 TCPH, 142 102 te lampici11in , TEPA, 121 terbutaline , 71, 166 testosterone, 322 tetracaine, 183 tetracyclines, 114, 132 tetragastrin, 93 tetrahydro cephalexin (SGE-loo), 103

340

COMPOUND NAME AND CODE NUMBER INDEX

tetrahydrocannabinol, 61 R-tetrahydropapaveroline, 175 S-tetrahydropapaveroline, 175 tetrahydrozoline, 91 tetrin A, B, 117 tetronic 701, 195 Th 1165a (fenoterol), 71 A~-THC, 205 A9-THC, 14, 188, 205 thiothixene, 255 Cis-thiothixene, 251, 255 theophylline, 55, 165, 166, 167 thienamycin, 105 5-thio-D-glucose, 125 thioridazine, 251, 252 thiostrepton, 115 thrombin, 80 thromboxane A2 (TBXAz), 182, 185, 186, 230 81, 82 thromboxane B (PHD), tiadenol, 163 tibric acid, 193 ticarcillin, 83, 101 ticlopidine, 84 tilorone, 82 tirandamycin, 117 tirandamycin B, 116 tixanox (OTMX), 76 TMB-6, 42 TMD, 13 TMHT, 13 tobramycin, 110, 111, 112 tocainide (W-36095), 41, 205 a-tocopherol, 185, 123 tolazoline, 91 tolypomycin, 131 tomaymycin, 135 tonin, 62 tranylcypramine, 4, 186 trazodone, 3, 12 triamcinolone, 165 triazolam (U-33,030), 11 trif lubazam, 11 trifluoperazine, 251 5-trifluoromethyluridine, 121 trifluperidol, 251 triflupromazine, 251 R-trimetaquino1, 175 S-trimetaquinol, 175 N,N,N-trimethyl-dopamine, 174 tripelennamine, 205

trofosfamide, 120 tryptamines , 202 tryptophan, 5 L-tryptophan, 15 TYA (5,8,11,14-eicosatetraynoic acid), 83 tyrocidine, 134 U-33,030 (triazolam), 11 U-41,792, 194 U-43,120, 116 UM-272, 45 UM-360, 42 UM-424, 4 2 95 ,B ,Y urogastrone , urokinase, 86 vasoactive intestinal peptide, 95 vasopressin, 31, 33 verapamil, 40, 42, 45 verdamicin, 110 vinblastine, 124, 155 vincamine, 55 vinylbarbital, 12 viomycin, 134 Vipera Berus venom, 85 viquidil, 55 virginiamycin M, 117 vitamin D, 223, 227 W-8011, 75 W-36,095 (tocainide) , 41, 205 Grethyl PGE1, 96 warfarin, 320 WB-4101, 255 3H-WB-4101, 254, 255, 256 WR142,490 (mefloquine) , 140 WY- 13,876 , 125 WY-16,225, 25 WY-16,922, 77 xanthinol nicotinate, 193 XK-90, 116 xylocaine, 186 YC-93, 45 zanchol, 196 zinc acetate, 123 339-29, 115 780SE, 193 1294B-2, 116

-

A

8 C D E F 6

7 8 9 O 1 2

n 3 1 4 1 5