Annual Reports in Medicinal Chemistry, Volume 14

ANNUAL REPORTS IN MEDICINAL CHEMISTRY Volume 14

CONTRIBUTORS Actor. P ........................ 103 Aungst. Bruce J . . . . . . . . . . . . . . . . . 309 146 Baschang. G ..................... Bell. Malcolm R . . . . . . . . . . . . . . . . . 168 Bell. Stanley C . . . . . . . . . . . . . . . . . . . 51 22 Berger. Joel G .................... Buermann. Christine Winslow . . . . . .2 19 Capetola. Robert J . . . . . . . . . . . . . . . . 51 Cayen. Mitchell N . . . . . . . . . . . . . . . 198 Cerami. Anthony ................ 26 I Christiansen. Robert G . . . . . . . . . . . 168 Colonno. Richard J . . . . . . . . . . . . . . . 238 Comer. William T ................. 61 146 Dukor. P ........................ 41 Enna. S . J ........................ Evans. Dale B .................... 81 Fox. Rita ........................ 81 Fung. Ho-Leung . . . . . . . . . . . . . . . . . 309 Ganellin. C . Robin . . . . . . . . . . . . . . . . 91 299 Gund. Peter ..................... Handsfield. H . Hunter ........... 114 81 Hauck. Fred P.................... 22 Iorio. Louis C .................... Kelly. T . Ross . . . . . . . . . . . . . . . . . . 288 Kobylecki. R . J . . . . . . . . . . . . . . . . . . . 31 Koenig. Ronald J ................ 261 Korant. Bruce D . . . . . . . . . . . . . . . . . 238 Kraska. Allen R . . . . . . . . . . . . . . . . . 132 Kripalani. Kishin J . . . . . . . . . . . . . . . 188 Lednicer . Daniel . . . . . . . . . . . . . . . . . 268

Lockart. Royce Z., J r . . . . . . . . . . . . 238 MacKenzie. Robert D . . . . . . . . . . . . . 71 Matier. W . Lesley . . . . . . . . . . . . . . . . 61 McDermed. John . . . . . . . . . . . . . . . . . 12 Migdalof. Bruce H . . . . . . . . . . . . . . . 188 Miller. Richard J . . . . . . . . . . . . . . . . . . . 12 31 Morgan. B . A .................... Morrison. Richard A . . . . . . . . . . . . . .309 Nicolaou. K . C . . . . . . . . . . . . . . . . . . 178 Oronsky. Arnold L ............... 219 Papahadjopoulos. Demetrios . . . . . . 248 Pinder. Roger M . . . . . . . . . . . . . . . . . . . 1 Rifkin. Daniel B . . . . . . . . . . . . . . . . . 229 Ritchie. David M . . . . . . . . . . . . . . . . . 51 Rohrlich. Susannah T . . . . . . . . . . . . 229 Saperstein. Richard . . . . . . . . . . . . . . 209 Schane. H . Philip. J r . . . . . . . . . . . . . 168 Singhvi. Sampat M . . . . . . . . . . . . . . . 188 Sitrin. R . D ..................... 103 Smith. J . Bryan . . . . . . . . . . . . . . . . . 178 Sutton. Blaine M . . . . . . . . . . . . . . . . . 321 146 Tarcsay. L ...................... Taylor. Edward C . . . . . . . . . . . . . . . . 278 Turck. Marvin . . . . . . . . . . . . . . . . . . . 114 103 Uri. J . V ........................ Veber. Daniel F. . . . . . . . . . . . . . . . . 209 Weitzel. Sarah M . . . . . . . . . . . . . . . . 122 Werbel. Leslie M . . . . . . . . . . . . . . . . 122 Worth. Donald F ................. 122

REPORTS IN MEDICINAL CHEMISTRY Volume 14 Sponsored by the Division of Medicinal Chemistry of the American Chemical Society Editor-in-Chief: HANS-JURGEN HESS PFIZER INC. G ROTON, CONNECTICUT

SECTION EDITORS LESLIE HUMBER DENIS BAILEY

JOHN KRAPCHO

JERRY WEISBACH

CHRISTOPHER WALSH

ACADEMIC PRESS

New York

BURT RENFROE

San Francisco

A Subsidiary of Harcourt Brace Jovanovich, Publishers

London

1979

A cadernic Press Rapid Man uscrlp t Reproduction

COPYRIGHT @ 1979, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, O R ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE 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 N W I IDX

LIBRARY OF

CONGRESS CATALOG CARD

NUMBER:66-26843

ISBN 0-12-0405 14-8 PRINTED IN THE UNITED STATES OF AMERICA 79 80 81 82

9 8 7 6 5 4 3 2 1

CONTENTS

CONTRIBUTORS PREFACE

11

ix

I. C N S A G E N T S Section Editor: Leslie G. Humber, Ayerst Laboratories, Montreal, Canada 1.

2.

3. 4.

5.

Antidepressants Roger M . Pinder, Organon International, Oss, The Netherlands Antipsychotic Agents and Dopamine Agonists John McDermed, Wellcome Research Laboratories, Research Triangle Park, North Carolina Richard J . Miller, Department of Physiological and Pharmacological Sciences, University of Chicago, Chicago, Illinois Antianxiety Agents, Anticonvulsants, and Sedative-Hypnotics Joel G. Berger and Louis C. Iorio, Schering-Plough Research Division, Bloomfield, New Jersey Analgetics, Endorphins, and the Opioid Receptor R. J . Kobylecki and B. A . Morgan, Reckitt & Colman Limited, Pharmaceutical Division, Hull, United Kingdom Amino Acid Neurotransmitter Candidates S. J . Enna, Departments of Pharmacology and of Neurobiology and Anatomy, University Texas Medical School, Houston, Texas

11.

1 12

22 31 41

PHARMACODYNAMIC AGENTS

Section Editor: John Krapcho, Squibb Institute for Medical Research, Princeton, New Jersey

51

6. Pulmonary and Antiallergy Drugs

Stanley C. Bell, Wyeth Laboratories, Inc., Radnor, Pennsylvania Robert J . Capetola and David M . Ritchie, Ortho Pharmaceutical Corporation, Raritan, New Jersey V

vi

Contents

7. Antihypertensive Agents W . Lesley Matier and William T. L O ~ L JMead ~ , Johnson Pharmaceuticals, Evansville, Indiana 8. Antithrombotic Agents Robert D. Mac Kenzie, Merrell Research Center, Merrell-National Laboratories, Division Richardson-Merrell, Inc., Cincinnati, Ohio 9. P-Adrenergic Receptor Blockers as Therapeutic Agents Dale B. Evans, Rita Fox, and Fred P. Hauck,* The Squibb Institute for Medical Research, Princeton, New Jersey 10. Histamine Receptors C. Robin Ganellin, The Research,Institute, Smith Kline and French Research Ltd., Welwyn Garden City, Herts, United Kingdom

61

71 81

91

111. CHEMOTHERAPEUTIC AGENTS Section Editor: Jerry A. Weisbach, Smith Kline & French Laboratories. Philadelphia, Pennsylvania 1 I.

12.

13.

14. 15.

Antibiotics P. Actor, R. D. Sitrin, and J . V . Uri, Smith Kline & French Laboratories, Philadelphia, Pennsylvania Chemotherapy of Sexually Transmitted Infections H . Hunter Handsfield, Seattle- King County Department of Public Health, Seattle, Washington Marvin Turck, Harborview Medical Center, University of Washington, Seattle, Washington Antiparasitic Agents Leslie M. Werbel, Donald F. Worth, and Sarah M. Weitzel, Warner-LambertlParke-Da vis Pharmaceutical Research Division, Ann Arbor, Michigan Antineoplastic Agents Allen R. Kraska, Pfizer Inc., Groton, Connecticut Immunostimulants P. Dukor, L . Tarcsay, and G. Baschang, Research Department, Pharmaceuticals Division, Ciba-Geigy Limited, Basel, Switzerland

IV. METABOLIC DISEASES AND ENDOCRINE FUNCTION Section Editor: Denis M. Bailey, Sterling-Winthrop Research Institute Rensselaer, New York *Present address: Sterling Drug, Inc., 90 Park Avenue, New York, New York

I03 114

122

132 146

Contents

16. Chemical Control of Fertility Malcolm R. Bell, Robert G. Christiansen, and H. Philip Schane, Jr., Sterling- Winthrop Research Institute, Rensselaer, New York 17. Prostacyclin, Thromboxanes, and the Arachidonic Acid Cascade K . C. Nicolaou, Department of Chemistry, University of Pennsylvania, Philadelphia , Pennsylvania J . Bryan Smith, Department of Pharmacology and the Cardeza Foundation, Thomas Jefferson University, Philadelphia, Pennsylvania 18. Drug Metabolism Bruce H . Migdalof, Kishin J . Kripalani, and Sampat M. Singhvi, The Squibb Institute for Medical Research, New Brunswick, New Jersey 19. Disorders o f Lipid Metabolism Mitchell N . Cayen, Ayerst Research Laboratories, Montreal, Quebec, Canada 20. Somatostatin Daniel F. Veber, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania Richard Saperstein, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 21. Neutral Proteinases in Rheumatoid Arthritis Arnold L. Oronsky and Christine Winslow Buermann, Medical Research Division, American Cyanamid Company, Lederle Laboratories, Pearl River, New York

vii

168

178

188 1 98

209

2 19

V . TOPICS IN BIOLOGY Section Editor: Christopher T. Walsh, Massachusetts Institute of Technology, Cambridge, Massachusetts 22.

Proteases and Cell Invasion Susannah T. Rohrlich and Daniel B. Riflin, N YU Medical Center, Department of Cell Biology, New York, New York 23. Selected New Developments in the Biochemistry o f Viruses Royce Z. Lockart, Jr., Richard J . Colonno, and Bruce D. Korant, Central Research & Development Department, E. I. du Pont de Nemours & Co., Inc., Wilmington, Delaware 24. Liposomes as Drug Carriers Demetrios Papahadjopoulos, Cancer Research Institute and Department of Pharmacology, Medical School, University of California, San Francisco, California 25. Nonenzymatic Glycosylation Ronald J . Koenig and Anthony Cerami, The Rockefeller University, New York, New York

229 238

248

26 1

viii

Contents

VI. TOPICS IN CHEMISTRY AND DRUG DESIGN Section Editor: Burt Renfroe, Ciba-Geigy Corporation, Ardsley, New York 26. Reactions of Interest in Medicinal Chemistry Daniel Lednicer, Mead Johnson Pharmaceuticals, Evansville, Indiana 27. New Methods in Heterocyclic Chemistry Edward C. Taylor, Department of Chemistry, Princeton University, Princeton, New Jersey 28. Synthetic Approaches to Anthracycline Antibiotics T. Ross Kelly, Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 29. Pharmacophoric Pattern Searching and Receptor Mapping Peter Gund, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 30. Pharmacokinetics and Drug Design Ho-Leung Fung, Bruce J . Aungst, and Richard A. Morrison, School of Pharmacy, State University of New York at Buflalo, Amherst, New York 31. Metals in Treatment of Disease Blaine M . Sutton, Smith Kline & French Laboratories, Philadelphia, Pennsylvania COMPOUND NAME AND CODE NUMBER INDEX CUMULATIVE INDEX

268 278 288 299 309

32 I

330 342

PREFACE

Since its inception, the objective of Annual Reports in Medicinal Chemistry has been to provide brief, critical summaries of highlights of the recent literature in the fields of chemistry, pharmacology, biochemistry and medicine for those interested in the discovery and development of new drugs. The format of organization originally established for this series has admirably served the intended purpose. Accordingly, as in the past, this year’s volume is divided into six sections: CNS Agents; Pharmacodynamic Agents; Chemotherapeutic Agents; Metabolic Diseases and Endocrine Function; Topics in Biology; and Topics in Chemistry and Drug Design. In selecting suitable chapter topics, the editors have sought a balance between areas of long-standing interest in drug research and special topics that may offer opportunities for future drug design and exploration. Similar thinking has guided the selection of subject matter for inclusion in many of the chapters. Thus, the discussions increasingly reflect the view that new breakthroughs in drug discovery are likely to emerge from new conceptual approaches based on a better understanding of disease processes and drug mechanisms. This volume, for the first time, contains a cumulative chapter title index as a convenient reference for quickly locating reviews on special topics that have appeared in past volumes. To the many individuals who contributed to this volume, we extend our sincere thanks.

Groton, Connecticut May 1979

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Annual Reports in Medicinal Chemistry-I4 Section I Editor:

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CNS Agents

L e s l i e G . Humber, A y e r s t L a b o r a t o r i e s , M o n t r e a l , Canada

Chapter 1.

Antidepressants

Roger M. P i n d e r , Organon I n t e r n a t i o n a l , Oss, The N e t h e r l a n d s I n t r o d u c t i o n - Concern o v e r t h e s a f e t y of t r i c y c l i c a n t i d e p r e s s a n t s (TCA)1,2 h a s m a i n t a i n e d t h e impetus t o develop new d r u g s . Compounds reviewed i n 1978 i n c l u d e m i a n s e r i n , 3 , 4 nomifensin , 5 and a l l non-TCA. Spp o s i a covered d ~ t h i e p i n ,m~i a n s e r i n , 8 nomifensin,g and t r a z o d o n e . lo The u s e s of L-tryptophan and 5-hydroxytryptophan 65-HTP) i n d e p r e s s i o n 1 1 and t h e p r o p e r t i e s of t h e new a n t i d e p r e s s a n t s 1 2 - l were summarized. The neuropharmacology of d e p r e s s i o n , 1 7 and t h e mechanism of a c t i o n 1 8 and pharmacok i n e t i c s 1 9 of a n t i d e p r e s s a n t s w e r e reviewed , and t h e monoamine t h e o r y r e a s s e s s e d . 20 P r e v i o u s r e p o r t s 2 ’ t h a t some TCA and non-TCA, l i k e i p r i n d o l and m i a n s e r i n , were i n h i b i t o r s of h i s t a m 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 from mammalian b r a i n i n v i t r o were confirmed f o r a wide range of a n t i d e p r e s s a n t s . 2 2 The e f f e c t appeared t o be mediated by H2-receptors b u t n e i t h e r monoamine o x i d a s e i n h i b i t o r s (MAOI) n o r s e l e c t i v e s e r o t o n i n (5-HT) u p t a k e i n h i b i t o r s were v e r y e f f e c t i v e . However, TCA a l s o blocked histamine-stimu l a t e d c y c l i c GMP f o r m a t i o n i n c u l t u r e d n e r v e c e l l s by a mechanism which involved Hi-recep t o r s . 23 The r e l e v a n c e of t h e s e f i n d i n g s , and indeed of t h e r o l e of h i s t a m i n e i n t h e c e n t r a l nervous sytem, h a s s t i l l t o b e defined. Evidence accumulated i n 1978 f o r a c a t e c h o l a m i n e r e c e p t o r supersens i t i v i t y t h e o r y of d e p r e s s i o n . 18 The t h e r a p e u t i c a c t i o n of a n t i d e p r e s s a n t s may be due t o d e l a y e d p o s t - s y n a p t i c changes i n r e c e p t o r s e n s i t i v i t y , r a t h e r t h a n t o a c u t e e v e n t s l i k e u p t a k e . Various d r u g s , i n c l u d i n g TCA, m i a n s e r i n , v i l o x a z i n e and i p r i n d o l , as w e l l as e l e c t r o c o n v u l s i v e t h e r a p y (ECT), b u t n o t s e l e c t i v e 5-HT u p t a k e i n h i b i t o r s , caused c e n t r a l a l p h a - a d r e n o c e p t o r s u b s e n s i t i v i t y i n r a t s as measured by n o r a d r e n a l i n e (NA)-associated adenyl a t e c y c l a s e o r by r e c e p t o r b i n d i n g . 1 8 I n v i v o , t h e e f f e c t s w e r e a s s o c i ated with chronic but not acute treatment, p a r a l l e l i n g the c l i n i c a l e f f e c t s . MAOI may c a u s e s i m i l a r e f f e c t s on c h r o n i c b u t n o t a c u t e t r e a t m e n t . 18,24-27 Brain NA t u r n o v e r i n r a t s w a s d e c r e a s e d by c h r o n i c d e s i p r a m i n e and o t h e r TCA, b u t u n a f f e c t e d by i p r i n d o l and i n c r e a s e d by m i a n s e r i n . 3 328

(2,

Ri=R2=H) are e f f e c t i v e anS a f e r Drugs - M i a n s e r i n (1)and nomifensin t i d e p r e s s a n t s , s u p e r i o r t o p l a c e b o and comparable t o TCA. There have been with b u t n e i t h e r a c t s any f a s t e r t h a n TCA. no t r i a l s s o f a r comparing Both l a c k c a r d i o t o x i c i t y and have minimal a n t i c h o l i n e r g i c e f f e c t s , and w h i l e e x p e r i e n c e w i t h nomifensin i s l i m i t e d , m i a n s e r i n a p p e a r s t o be s a f e i n overdosage. 29 Mianserin a l s o h a s a n x i o l y t i c a c t i v i t y comparable t o diazepam,3 and can be given as a s i n g l e bedtime dose o r i n d i v i d e d d o s e s . Nomifensin cannot be g i v e n a t n i g h t and i t s s h o r t h a l f - l i f e n e c e s s i t a t e s d i v i d e d d a i l y dosage. T o l e r a t i o n of 1 i s good i n t h e e l d e r l y , and i n a double-blind t r i a l i n g e r i a t r i c d e p r e s s i v e s , 1 w a s s u p e r i o r i n e f f i c a c y and

1

g,

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0-12-040514-8

2 -

Sect. I - CNS Agents

Humber, Ed.

v 1

\CH3

side-effects to amitri~tyline.~' Experimental studies in isolated hearts, intact animals, healthy subjects, depressed patients and patients with cardiac disease showed L to be less cardiotoxic than TCA or maprotiline.3,31-35 Mianserin did not affect peripheral NA mechanisms in humans and did not reverse the antihypertensive actions of adrenergic neuron blockers or propranolol.36 There was no interaction between L and phenprocoumon in cardiac patients receiving anticoagulant therapy .37 The precise mechanism of action of 1 is unclear, but llke amitriptyline it is a central 5-HT antagonist38339 and blocks presynaptic alpha-adrenoceptors.40,41 It may also block postsynaptic alpha-adrenoceptors and inhibit NA uptake at high doses,42 but the roles of these activities in the antidepressant action of 1 have yet to be defined. The cardiac safety of nomifensin was established by measurements of cardiac conduction in depressed patients receiving therapeutic doses over 3 weeks, and it was less toxic than doxepin or amitriptyline in isolated hearts.43 Nomifensin inhibited NA uptake in the same way as TCA but its principal actions may be on dopaminergic (DA) systems, both as an uptake inhibitor and as an agonist.5 The metabolites (&, Ri=OH, R2=H; &, R1= Ri=OH, R2=OCH3) inhibit the uptake of DA and 5-HT, but OCH3, R2=OH; another potential metabolite, the catechol (&, Rl=R2=OH) is a potent DA agonist in behavioral and neurochemical studies.449 45 However , the beneficial effects of 2a in parkinsonism may be due to its improvement of depressive symptomF46

a,

Trazodone (3) is another non-TCA claimed to produce minimal sideeffects.10 It showed a complex pharmacological profile in animals, including tranquilizing and antihypertensive properties, with some selectivity towards inhibition of 5-HT uptake.6 Like mianserin and doxepin it acted at low doses as a central 5-HT antagonist, but at higher doses it was agonistic. Trazodone depressed prolactin and growth hormone secretion in humans, suggesting a possible DA-like action which may explain its beneficial use in parkinsonism.10 In clinical trials in depression, 2 had a broad spectrum of activity, combining rapid anxiolytic effects with a slower antidepressant action comparable to various TCA and superior to placebo.10 Antidepressants and 5-HT - Because of the possibility of reduced cardiotoxicity, interest was maintained in 1978 in compounds which are selective inhibitors of 5-HT uptake, many of which were reported in Volume 13 of this series21 and elsewhere.6 Open clinical studies of zimelidine (4) showed good antidepressant effects after doses of 150 mg daily.47 The claimed rapid onset of action of 5 was not confirmed in the first double-blind trial in hospitalized depressives given single bedtime doses of or amitriptyline for 4 weeks.48 No difference in efficacy emerged, but sideeffects were fewer with 4. A cardiovascular study showed 4 and mianserin

Chap. 1

Pinder 3

Antidepressants

to be safer drugs in depressed patients than TCA, the only effects of being to slow heart rate and increase QT interval.33

4

C

Fluoxetine (5) inhibited 5-HT uptake into platelets and also reduced platelet 5-HT content after chronic administration to healthy volunteers.49 This dose had no effect on the usual pressor responses to NA or tyramine

Q

/=i

5 -

8 injection.

F

9 -

10 -

Inhibition of 5-HT uptake was correlated with plasma levels of

5, and plasma from treated subjects also inhibited 5-HT uptake in platelets

from untreated subjects. Fluoxetine was well absorbed and reached peak plasma levels and steady-state in 6 and 12 hours, respectively.49 It suppressed REM sleep in cats in a dose-dependent way, an effect which was potentiated by 5-HTP.50 Fluoxetine did not block arecoline-induced EEG desynchronization in cats.50 Clovoxamine (5) is the chloro analog of fluvoxamine (I), which has been shown to have antidepressant effects in humans.21 Open clinical studies with 5 showed rapid and persistent benefit in endogenous depression without unwanted side-effects.51 The animal pharmacological profile of 5 was similar to 1 - antagonism of reserpine effects, potentiation of 5-HTP, potent and selective inhibition of 5-HT uptake, and no anticholinergic or cardiotoxic properties.52 Paroxetine (8), an analog of femoxetine, was more potent and longer-lasting as a 5-HT uptake inhibitor than chlorimipramine. Oral administration to rats produced long-lasting potentiation of the effects of 5-HTP.53 Citalopram (9) was about 2-8 times more potent in vivo than chlorimipramine against 5-HT uptake in rat brain or blood platelets but had essentially no effect on uptake of DA or NA.54 In vitro, the two drugs were equipotent and were competitive inhibitors of 5-HT uptake but about 20-35 times more potent than imipramine or amitriptyline. The citalopram metabolites were also active. In healthy subjects, 9 had a long plasma halflife of 1 to 2 112 days.55 Another nitrile, but derived from imipramine, R o 11-2465 (lo), was one of the most potent and selective inhibitors of 5-HT uptake in vivo and in vitro, being up to 20 times more potent than chlorimipramine. 5-HT content in platelets was drastically reduced in rats given 10 for 4 days.56 Compound 10 was 30 times more potent than chlorimipramine in potentiating 5-HTP behavioral syndromes in mice. 56 Other selective inhibitors of 5-HT include CGP 6085A

(g), which

has

4 -

Sect. I - CNS Agents

Humber, Ed.

an oral ED50 in rats of-between 1 and 4 mg/kg.57 NA uptake was unaffected at doses up to 1000 mg/kg.58 Like other compounds, such as fluoxetine and chlorimipramine, 11 was a more potent inhibitor of 5-HT uptake in the brain than in platelets. Alaproclate (12)was a weak competitive inhibitor of 5-HT uptake, being less potent than chlorimipramine in vivo and in vitro, but exhibiting a much greater separation of 5-HT and NA uptake effects.59 CH3 CH3 CH3 I I 12 CH3n X H c 1 0 - c ” 2 7 - 0 -CO-CH-NH2 CH

Alaproclate potentiated 5-HTP syndromes in animals and had no anticholinergic effects. Its proposed pharmacophore also accommodated Org 6582 (13). Compound 13 exerted significant inhibition of 5-HT uptake for at least 48 hours after administration to rats .60 Competitive inhibition by 3 occurred in vitro at concentrations that were substantially less than those required for inhibition of DA or NA uptake, and it was more potent in vivo than fluoxetine and chlorimipramine.60

LM 5008 (14)was the most potent and selective inhibitor of 5-HT uptake of a series of indole derivatives.61 The duration of action and potency in vivo were similar to chlorimipramine. LM 5008 also inhibited platelet 5-HT uptake in rats and decreased platelet 5-HT content after single or repeated dosings.62 Open clinical trials in 40 depressed patients showed a strong and rapid antidepressant effect associated with pronounced inhibition of 5-HT uptake.63 7,N,N-Trimethyltryptamine selectively inhibited 5-HT uptake into rat brain synaptosomes without affecting the uptake of tryptophan, DA or NA.64 5-HT may be involved in the mechanism of action of antidepressants which block central 5-HT receptors at doses below those causing inhibition of 5-HT and NA uptake.38~39 Some TCA strongly and competitively inhibit 5-HT uptake,65 and activity may be higher in the brain than in platelets.58 During clinical improvement some TCA, particularly chlorimipramine, lowered the rate of 5-HT transport into platelets (Vmax), which is already reduced in depression, and increased Km values.65 Mianserin normalized Vmax concomitant with clinical improvement.3 Tricyclics and Analogs - TCA, old and new, were the subject of continuing investigation. Dothiepin, a sulphur isostere of doxepin, is a typical TCAlike inhibitor of both NA and 5-HT uptake.7 Double-blind studies showed dothiepin to be similar in efficacy to TCA after divided or single daily doses, with fewer anticholinergic effects and good tolerance in the elderly. A pharmacokinetic study showed a significant correlation between clinical response to doxepin and plasma steady-state levels of both it and its desmethyl metabolite.66 Lofepramine, an N-(p-chlorobenzoyl) derivative of desmethyl imipramine, had reduced anticholinergic activity in healthy subjects67 and lower cardiotoxicity in animals.68 Double-blind trials in depression failed to reveal any other advantages over imipramine or other TCA.69~70 Amoxapine had antidepressant and neuroleptic effects in animals,

Chap. 1

Antidepressants

Pinder

5

-

but clinical trials showed little difference between it and imipramine and anticholinergic effects were common.71 Amitriptyline-N-oxide differed from amitriptyline in animals only by its decreased peripheral anticholinergic activity and better gastric tolerance.72 Clinical trials showed antidepressant activity together with anxiolytic, sedative and anticholinergic properties. Rapid and quantitative absorption of the N-oxide in humans was followed by hydroxylation at the 10-position, without any substantial conversion to the parent TCA, as was observed with imipramineN-oxide. 73 Side-effects of TCA were compared in double-blind trials in healthy subjects. Marked inhibitory effects on salivary flow declined from amitriptyline through doxepin, imipramine, nortriptyline to desipramine, paralleling subjective reports of anticholinergic side-effects and reported affinities for muscarinic receptors.7 4 Similar rankings for sedation and decrements in psychometric performance were obtained in another study; BW 247 and protriptyline were inactive.75 BW 247 (15) is a ring-opened TCA with few anticholinergic effects and a profile of mixed NA and 5-HT uptake inhibition. It is structurally related to several potential antidepressants, including AHR 1118 (16). Both 15 and 16 are effective antidepressants in man;6 16 showed fewer side-effects than imipramine in a

double-blind trial in depression but was somewhat less effective.'6 Compound 1 7 was the most potent inhibitor of NA and 5-HT uptake in a series of diphenylcycloalkanes, and it also showed some inhibition of DA uptake. 7 caused central stimulation like that of methylphenidate In animals, 1 rather than amphetamine, and it may release DA from granular stores.77 DL 262 (3)inhibited NA and 5-HT uptake in vitro and in vivo, and had an amitriptyline-like profile in p h a r m a c o l o g ~ 7 8 eimidazole analog MCPI (19)is the l.ead compound of a series which blocks NA but not 5-HT uptake in rat brain, does not affect MAO, and possesses weak sedative but not anticholinergic properties. MCPI was too toxic to give to man, but analogs with better tolerance are available.7 9 The maprotiline analog C49802-B-Ba (20) inhibited NA uptake in rat brain and heart in vivo without affecting the uptake of 5-HT.57 The new tetracyclic derivative incazane (21) was about half as potent as imipramine in pharmacological tests, with lower toxicity and little anticholinergic activity.80 It has a slow onset of action clinically, like TCA, and combines antidepressant effects with stimulant properties. The selective inhibition of NA uptake by viloxazine, in the absence of effects on DA or 5-HT uptake, resided in the trans-S-isomer, paralleling the reserpine reversal effects.81 The anorectic drug mazindol was also a potent inhibitor of NA uptake,82 like its structural relative ciclazindol (229.83 In a double-blind trial, 22 was as effective as amitriptyline in ameliorating endogenous depression. Ciclazindol, which lacked significant anticholinergic or cardiotoxic effects, produced fewer side-effects than amitriptyline. Diverse structures included befuraline (DIV 154, 23) an

Sect. 1 - CNS Agents

6 ._

Humber, Ed.

imipramine-like drug of low cardiotoxicity ,84 and EGY-475 (24) which was more potent behaviorally than imipramine and lacked cardiotoxicity and anticholinergic effects .85 Bupropion (25) does not affect up take or reports of release of biogenic amines and does not block ~ ~ 0 . 8 Previous 6 a rapid antidepressant action for 25 were not confirmed in a multicenter double-blind trial in depression, but it was superior to placebo in the absence of marked side-effects.87

I

CH2

-$?% d

CH2-CHOH-CH2-NHCH3

18 -

20 -

n

R-CO - L P C H 2 - P h

23,

R = 2-benzofuranyl

24, R 21 -

=

2-pyridyl

22 -

Antidepressants and DA - Antidepressants acting principally on DA systems include nomifensin (3)and the antiparkinson drug piribedil (26). Piribedil produced moderate antidepressant effects when given in doses of 120240 mg daily, accompanied by increased REM latency and decreased REM sleep.88 In drug-resistant depressives, 26 produced a rapid, transient and sometimes pronounced antidepressant effect but also a consistent syndrome a proven antiof anger and hostility.89 In the rat brain, tandamine depressant in previous studies,21 was more potent in inhibiting NA uptake than DA uptake and had virtually no effect on uptake of 5-IIT.90 The bicyclooctane derivative LR 5182 (28) blocked DA uptake in rat brain synaptosomes and also affected NA uptake at 20-fold higher concentrations.go This high potency and selectivity was lost in vivo, but the potent effects of 28 on DA and NA uptake suggest a possible antidepressant action.

(z),

28 -

29 -

30 -

MAOI - There was renewed interest in MAOI since their combination with TCA =be the only alternative to the use of ECT for drug-resistant patients.91 ECT was superior to combined phenelzine and amitriptyline in severe depression,92 but phenelzine alone was as effective as amitriptyline in depressed probably outpatients.93 Caroxazone, an effective antidepressant drug acts through reversible inhibition of the two known forms of PfAO (A and B) .6

(z),

Chap. 1

Antidepressants

Pinder

7

Lilly 51641 (30) was a selective and long-lasting inhibitor of MAO-A in rat brain and other tissues.94 Compound 2 was the most active of a series of phenethylamines which had selective inhibitory effects on rat brain MAO-A and antidepressant activity in animals.95 Anti-tetrabenazine activity was correlated with inhibition of mouse brain MA0 for a series of imidazoles, and potential antidepressant activity was greatest with 3 2 . 9 6 The oxazolidone toloxatone (33) acted as a specific and reversible MAO-A inhibitor in rats, was clinically effective in depression, and may act per se and through its hydroxylated metabolites.97 Q kC.

c@

( CH3)C hCH22i%H2 CIA'-

H

fH20H c H 3 n N yI o

I

32 -

33 31 OC3H7 Amino Acids - Rapid catabolism by liver tryptophan pyrrolase may be the reason for the equivocal effects of tryptophan in depression, and addition of an inhibitor of this enzyme, allopurinol, to a daily regimen of tryptophan did produce marked and safe benefit in 8 patients with endogenous depression.98 DL-Phenylalanine produced a complete or good response in 1 2 / 2 0 depressed patients whereas 4 patients did not respond; depressed mood, retardation and agitation were preferentially affected.99 Beta-Agonists - The efficacy of beta-adrenergic agonists was confirmed.100 Most beta-agonists show a TCA-like profile in some animal models of depression and open studies in depressed patients given intravenous salbutamol for 10 days demonstrated a rapid antidepressant effect. loo No anticholinergic effects or orthostatic hypotension were observed, but tachycardia was dose-limiting and extrapyramidal signs were common. A controlled trial in 20 severely depressed patients showed a more rapid and effective response to salbutamol 6 mg daily than to chlorimipramine 150 mg daily, both given intravenously.101 There are no reports of the effects of other beta-agonists in depression. Peptides - Brain peptides and their analogs may offer the prospect of a rapidly acting antidepressant.102 103 Beta-endorphin, an opiate-like fragment of the much longer pituitary peptide beta-lipotropin, showed antidepressant effects in pilot trials following intravenous injection. 104 The biphasic pattern of response to melanocyte-stimulating-hormone releasing factor I (MIF-I) , H-Pro-Leu-Gly-NH2 ,2 1 was confirmed in a double-blind trial. Lower doses of MIF-I were more effective than either placebo or higher doses, acting within a few days of starting treatment orally.lO5 Other orally effective analogs are available.21 After initial promising results, most studies with thyrotrophin-releasing hormone (TRH), pGlu-HisPro-NH2, have demonstrated minimal antidepressant effects following single or multiple doses given orally or intravenously.102,106 Some patients with unipolar or bipolar depression or manic-depressive illness show a reduced secretion of thyroid-stimulating hormone (TSH) in response to TRH challenge, and respond to ECT but rarely to drugs.106,107 TRH has been suggested as a useful diagnostic and prognostic tool.107 New Screening Methods

-

New animal models for depression included the

8

-

Sect. I - CNS Agents

Humber, Ed.

bulbectomized rat. Bilateral olfactory bulbectomy results in a syndrome of deficient learning, hyperreactivity and elevated plasma ll-hydroxycorticosteroids, all of which were blocked by pretreatment with TCA and non-TCA, including mianserin and nomifensin, but not by other classes of psychotropic agents.108,109 The activity of Org 6582 (g), a selective inhibitor of the uptake of 5-HT, was also demonstrated in this model. The model appears to depend upon 5-HT systems in the brain, since an identical syndrome was produced by intrabulbar injection of the 5-HT neurotoxin, 5,6dihydroxytryptamine, but not by the DA neurotoxin, 6-hydroxydopamine.ll0 The behavioral despair model forces rats or mice to swim in a narrow cylinder of water from which they cannot escape and they ultimately adopt a characteristic posture of immobility.111 Immobility was reduced by MAOI, TCA and by new drugs like mianserin and nomifensin, as well as by ECT, REM sleep deprivation and an enriched environment. Anxiolytics did not affect immobility, neuroleptics enhanced it, and psychostimulants reduced it but at doses also causing motor stimulation. Another new model utilized the dose-related hypothermia produced in mice by subcutaneous clonidine.1 1 2 Chronic , but not acute pretreatment with TCA, non-TCA o r MAOI, antagonized the response, as did alpha-adrenergic blockers, but not beta-blockers, CNS stimulants, anxiolytics, anticholinergics, analgesics, or selective inhibitors of 5-HT uptake. Chronic imipramine treatment blocked clonidine hypothermia and antagonized clonidine-induced slowing of NA turnover in rat brain, but it did not alter other pharmacological effects of clonidine. A common underlying mechanism of antidepressants may be to alter alpha-adrenoreceptor sensitivity possibly by a presynaptic mechanism. 112 Reserpine-induced ptosis in the rabbit was proposed as a 5-HT dependent test for antidepressant activity.113 Stronger antagonism to ptosis was shown by TCA containing a tertiary amino than a secondary amino moiety, and ptosis was also blocked by selective 5-HT uptake inhibitors, 5-HTP and 5-HT-like drugs, including the new antidepressant LN 5008 (14). MAOI were also effective. Anxiolytics and neuroleptics did not reverse ptosis, nor did TCA acting principally on NA mechanisms. Nevertheless, doubts continue to be cast upon the relevance of models based upon reserpine-like drugs to the therapeutic action of antidepressants. Thus, reversal of tetrabenazine effects, a l s o a reserpine-like drug, appeared t o produce a pattern of stereotyped behavior quite distinct from the behavior of normal control animals.114 References 1. 2.

3. 4. 5. 6. 7.

A.Bonaccorsi and S.Garattini, Gen.Pharmaco1. 9, 81 (1978). J.T.Bigger, S.J.Kantor, A.H.Glassman and J.M.Pere1, in "Psychopharmacology : A Generation of Progress", M.A.Lipton, A.DiMascio and K.F.Killam eds., p. 1033-1046, Raven Press, New York, 1978. Proceedings of a symposium on mianserin, Brit.J.Clin.Pharmaco1. 5, Suppl.1 (1978). R.N.Brogden, R.C.Hee1, T.M.Speight and G.S.Avery, Drugs 273-301 (1978). Symposium on nomifensin, Nouvelle Presse Medicale 7, 2287-2338 (1978). J.van Dijk, J.Hartog and F.C.Hillen, Progr.Med.Chem. 261-320 (1978). Round table discussion on prothiaden, Abstr. 11th Congr.Colleg.1nt.Neuro-Psychopharmacol.(CINP), Vienna, Austria, July 9-14, 1978, p. 58-61, m-316.

2,

15,

Chap. 1 8.

910. 11.

12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

28.

29.

30. 31. 32.

Pinder

Antidepressants

Mianserin : Progress in the pharmacotherapy of depression, Abstr. 2nd Wld.Congr.Biol.Psychiat., Barcelona, Spain, Aug.31Sep.6, 1978, p. 125-126. Neurochemical and clinical aspects of nomifensin, see reference 8, p. 130-132. Trazodone - a new broad-spectrum antidepressant, see reference 7, p. 363-367. G.D'Elia, L.Hanson and H.Raotma, Acta Psychiat.Scand. 57, 239-252 (1978). P.K.Bridges and T.R.E.Barnes, J.Pharmacother. 1,12-19 (1978); Practitioner 2,515520 (1978). Editorial, Brit.Med.J. 1,128 (1978). R.N.Herrington, Mod.Med. g, 72-77 (1978). L.E.Hollister, Ann.Int.Med. 2, 78-84 (1978). J.Ananth, Int-Pharmacopsychiat. 2, 69-93 (1978). A.B.Adolph, E.R.Dorsey and M.J.Napoliello, Dis.Nerv.Syst. 8, 841-848 (1977). F.Sulser, J.Vetulani and P.L.Mobley, Biochem.Pharmaco1. 257-261 (1978). B.Muller-Oerlinghausen, Pharmakopsychiat.Neuro-Psychopharmkol. 2, 55-62 (1978). H.M.van Praag, Acta Psychiat.Scand. 2, 389-404 (1978). 1.Jirkovsky and W.Lippmann, in "Annual Reports in Medicinal Chemistry, Vol. 13, F.H.Clarke ed., Academic Press, 1978, p. 1-10. P.D.Kanof and P.Greengard, Nature r/2, 329 (1978). E.Richelson, Nature 176 (1978). F.Sulser, Pharmakopsychiat.Neuro-Psychopharmakol. 2, 43 (1978) ; Pharmacologist 2, 2 3 (1978). S.Clements-Jewry, Neuropharmacology 2, 779 (1978). D.G.GrahameSmith, A.R.Green and D.W.Costain, Lancet 1,254 (1978). V.Genti1, B.Alevizos and M.Lader, Biochm.Pharmaco1. 2, 1197 (1978). B.N.Rosloff and J.M.Davies, Psychopharmacology 56, 335 (1978). P.Crome and B.Newman, Brit.Med.J. 2, 260 (1977); 1,859 (1978); also in G.J.Drykoningen, R.M.Pinder and J.J.de Ridder, in "Mianserin : Progress in the Pharmacotherapy of Depression", G.J.Drykoningen, W.L.L.Linford Rees and C.Ruiz Ogara eds., Excerpta Medica, Amsterdam, 1979, p- 74-78. J.Kretschmar, in "Mianserin : Progress in the Pharmacotherapy of Depression*' (see reference 31, p. 19-25. B. Harper and I.E. Hughes, Brit.J.Pharmaco1. 2, 651 (1978). L.B. Cobbin and B. Engel, 7th Int.Congr.Pharmacol., Paris, Frwce, July 16-21, 1978, Abstr. 2042,

z,

e,

P. 638. 33. C.D.Burgess,

34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.

S.Montgomery and J.Wadsworth, see reference 32, Abstr. 2569, p.830 and i n Mianserin : Progress in the Pharmacotherapy of Depression ( s e e reference 291,. p. 60-64. M.Peet, P.Tienari and M.O.Jaskari, Pharmakopsychiatrie 10, 303 (1977). H.Kopera and H.Schenk, Deutsche Med.Wochenschr. 2,1373 (1988). K. Ghose, Fortschr.Med. 2, 1343 (1978). H.Kopera, H.Schenk and S.Stulemeijer, Europ.J.Clin.Pharmaco1. 2, 1 (1978). K.Fuxe, S.Ogren, L.Agnati, J.A.Gustafssen and G.Jonsson, Neuroscience Letters 339 (1977): see also reference 24, Abstr. 882, p. 347. J.Maj H.Sowinska, L.Baran, L.Ganacarczyk and A.Rawlow, Psychopharmacology 2, 79 (1978). I.E.Hughes, Brit.J.Pharmaco1. 315 (1978). R.D.Rohson, M.J.Antonaccio, J.K.Saelens and J.Liebman, Eump.J.Pharmaco1. 47, 431 (1978)J.C.Doxey, J.Everitt and G.Metcalf, Europ.J.Pharmaco1. 2, 1 (1978). G.D.Burrows, J.Vohra, P.Dumovic, B.A.Scoggins and B.Davies, Med.J.Austra1. 1,341 (1978). B.Costal1 and R.J.Naylor, J.Pharm.Pharmaco1. 2, 514, (1978). J.A.Poat, G.N.Woodruff and K.J.Watling, J.Pharm.Pharmaco1. 2, 495 (1978). J.Schmitz, see reference 8, p. 131. O.Benkert, G.Laakmann, L.Ott, A.Strauss and R.Zimmer, Arzneh.-Forsch. 2421 (1977)S.A.Montgomery, R.McAuley, G.Rani and D.B.Montgomery, see reference 32, Abstr. 621, p- 246. L.Lemberger, H.Rowe, R.Carmichae1, S.Oldham, R.Crabtree, J.S.Horng, F.P.Bymaster and D.T.Wong, ClhPharmacoLTher. 421 (1978). I.H.Slater, G.J.Jones and R.A.Moore, Neuropharmacology 383 (1978). N.T.Daskapoulos, M.J.Cottereau and P.Deniker, see reference 7, p. 174.

,

2,

z,

E,

50. 51.

9

17,

10 -

Sect. I

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Humber, Ed.

CNS Agents

ArznehK.M.Shasmana, F.C.Hillen, W.J.Vaatsta and J.M.A.Zwagemaker, 52- V.Claassen, T.A.C.Boschman, Forsch. 28, 1756 (1978). 151 (1978) ; Europ.J.Phamaco1. 351 (1978). 53. J.Buus-Lassen, Psychopharmacology 13 (1978). 54. J.Hytte1, Psychopharmacology Europ.J.Clin.Pharmaco1. &, 69 (1978). 55. K.F.Overo, and M.Da Rada, see reference 7, p- 95. 56- W.Haefely, RSchaffner, W.P.Burkard P.A.Baumann, M.Wilhelm, R.Bernasconi and L.Maitre, Europ.J.Pharmaco1. 387 (1977). 57. P.C.Waldmeier, and L.Maitre, see reference 32, Abstr. 491, p. 189. 58. P.C.Waldmeier S.O.Thorberg, S.Bengtsson, A.L.Renyi, S.B.Ross and S.O.Ogren, J.Med.Chem. 2, 448 59. U.H.Lhdberg, (1978) 60. S.E.Mireylees, 1.Gooblet and M.F.Sugrue, Biochem.Phamaco1. 2, 1023 (1978). 61. G.Le Fur and A.Uzan, Biochem.Phmaco1. &, 497 (1977). 62. A.Uzan, G.Le Fur, M.Kabouche and N.Mitrani, see Life Sci 2, 1317 (1978). and LSchott, see reference 8, p. 14% 63. G.Chazot, B.Renaud, J.F.Henry 64. R.A.Glennon, B.Martin, K.M.Hohnson and D.End, Res.Commun.Chem.Pathol.Pharmcc1. 2, 161 (1978). 65. O-Lhgjaerde, see reference 7, p. 3666. V.E.Ziegler, J.T.Biggs, L.T.Wylie, S.H. Rosen, D.J.Hauf and W.Coryel1, Clin.Pharmacol.Ther- 2, 573 (1978). 67. B.Fredholm, L.Herrmenn, E.Rasmussen, C.Sjogren and U.Sawe, see PefeI-enCe7, PO 25568. C.Sjogren, see reference 7, p. 32. 69. G.D. *El& S.Borg, L.Hermn, G.Landin, C.Perris, H.Raotma, G.Roman and B-Swers, Acta Psychiat. Scand. 2, 10 (1977). 70. W.Obermeier and W.Poldinger, 1nt.Pharmacopsychiat. 2, 65 (1w)a 71. E.N. Greenblatt, A.S.Lipp.3 and A.C.Osterberg, Arch,Int.Phsrmacodyn. 233, 107 (1978)72. Nine papers on Adtriptylhe-N-oxide, Arzneh-Forsch. g,18751926 (1978). 73. A.Nagy, Acta PharmacoLToxicol. 42, 58 (1978). 74. R.N.Hostetter, G.R.Peterson, B.Blaekwel1, R.Kuzma and A.Adolphe, Pharmacologist 2, 171 (1978). 75. C.Bye, M.Clubley and A.W.Peck, Brit.J.Clin.Pharmaco1. 6, 155 (1978). 76. D.H.Mielke and D.M.Gallant, Curr.Ther.Res. 734 (1978). 77. B.Carnmalm, S.Ramsby, A.L.Renyi, S.B.Ross, S.O.Ogren and N.E.Sjernstrom, J.Med.Chem. 2, 78 (1978). 78. M.Narnima and M.V.Aylott, see reference 32, Abstr. 490, p. 189. 79. M.B.Wallach, A.P.Roszkowski and L.D.Waterbury, see reference 32, Abstr. 623, p. 247. 80. N.I.Andreeva, see reference 32, Abstr. 626, p. 248. 81. T.P.Blackburn, G.A.Foster, D.T.Greenwood and R.Howe, Europ.J.Pharmaco1. 2, 367 (1978). 82. M.F.Sugrue and S.E.Mireylees, Biochem-Pharmacol. 1843 (1978). 83. K.Ghose, V.A.Rama Rao and A.Coppen, Psychopharmacology 109 (1978). 84. J.Komarek and F.J.Neumann, Arzneh-Forsch. g,2066 (ign). 85. I.Kosoczky, K.Grasser, E-fiszelly, E.Tonscev and L.Petocz, see reference 32, Abstr. 486, p. 187. 86. F.E.Soroko, N.B.Mehta, R.A.Maxwel1, R.M.Ferris and D.H.Schroeder, J.Phm.Pharmaco1. 2, 767 (1977). 87. W.C.Stern, N.Harto and W.Cumings, see reference 7, p. 98. 88. RAPost, R.H.Gerner, J.S.Caman, J.C.Gillh, D.C.Jimerson, F.K.Goodwin and W.E.Bunney, Arch. Gen. Psychiat. 2, 609 (1978). 89. 5.Shopsin and S-Gershon, Neuropsychobiology 1 (1978). 90. B.T.Wong and F.P.Bymaster, Life Sci.2, 1041 (1978). 91. R.S.Goldberg,and W.E.Thornton, J.Clh.Pharmaco1. 18, 143 (1978). 92. J.Davidson, M-Mcbod, B.Law-Yone and M.Linnoila, Arch.Gen.Psychiat. 2, 639 (1978). 93. D.S.Robinson, A.Nies, L.Ravaris, J.0.Ives and D.Bartlett, Arch.Gen.Psychiat. 2, 629 (198). 94. R.W.Fuller and S.K.Henrick, Proc.Soc.Exp.Biol.Med. 158, 323 (198). 95. LAorvall, A-Ask, S-Ogren and S.B.Ross,J.Med.Chem. ,-? I 56 (1978). 96. M.Harfenist, F.E.Soroko and G.M.McKenzie, J.Med.Chem. 2, 405 (1978). 97. J=P.Kan, A.Malone and M-Strolin-Benedetti, J.Pharm.Phamco1. 2, 1% (1978). 98. B-Shopsin, Neuropsychobiology, L l 8 8 (1978). 99. H.Beckmenn and E-Ludolph, Arzneh-Forsch. 2, 1283 (1978). 100. P.Simon, Y .Lecubrier, RJourvert, A.Peuch, J.F.Allilaiz-e and D.Widlocher, Psychol.Med. 2, 335 (1978).

g,

z,

2,

46,

2,

z,

i,

z,

Chap. 1

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101. A.Peuch,

11

D.Widlocher, Y.Lecubrier, H.Frances, R.Jouvert, and P.Simon, see reference 8, p. 158. 102. B.T.Donovan, Psychol.Med. 2, 305 (1978). 103. K.Vereby, J-Volavka and D.Clovet, Arch.Gen.Psychiat. 2, 877 (1978). 104. N.S.Kline, C.H.Li, H.E.Lehmann, A.Lajtha, E.Laski and T.Cooper, Arch.Gen.Psychiat. 2, 1111 (1978). 2,5 (1978). 105. R.H.Ehrensing and A.J.Kastin, Amer.J.Psychiat. 106. H.P.Voge1, O.Benkert, R-Illing, B-Muller-Oerlinghausen and A-Poppenberg, Acta Psychtat-Scand. 56, 223 (1978). 107. C.Kirkegaard, N.Bjorum, D.Cohn and B.Lauridsen, Arch.Gen.Psychiat. 2, 1017 (1978). 108. H.van Riezen, H.Schnieden and A.Wren, Brit.J.Pharmaco1. 521 (1977). 497P (1977). 109. K.D.Cairncross, B.Cox, C.Forster and A.F.Wren, Brit.J.Pharmaco1. 110. A.Wren, C.Forster, B.Cox, K.Cairncross and HSchnieden, see reference 32, Abstr. 2305, p. 738. 111. R.D.Porsolt, G.Anton, N.Blavet and M.Jalfre, Europ.J.Pharmaco1. 2, 379 (1978). 375 (1978). 112. P.F.von Voigtlander, H.J.Triezenberg and E.G.Losey, Neuropharmacology 113. J.Mizoule, N.Mitrani, G.Le Fur and A.Uzan, J.Pharmeco1. (Paris) 8, 269 (1977). 114. P.Willner and D.Clark, Psychopharmacology 2, 55 (1978).

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Annual Reports in Medicinal Chemistry-14

12 Chapter 2.

Antipsychotic Agents and Dopamine Agonists

John McDermed Wellcome Research Laboratories Research Triangle Park, N.C. 27709 and Richard J. Miller Department of Physiological and Pharmacological Sciences University of Chicago, Chicago, Illinois 60637 The term schizophrenia, far from implying a homogeneous syndrome, embraces clinical manifestations ranging from catatonic withdrawal to florid paranoia, whose etiologies are unknown and may not be identical.1’2 There are no laboratory criteria for diagnosis of the disorder, and the subjective criteria are sometimes contr~versial.~Thus it may not be surprising that agreement between diagnosticians has been generally less than 60X4 and that wide variations in course and outcome are seen. Such uncertainties have encumbered the search for characteristic neurochemical lesions. Moreover, investigators must anticipate that the complexity of the brain’s interneural connections will make the crucial distinction between primary causative lesions and secondary reactive ones very difficult. Thus reports of biochemical abnormalities in schizophrenia span a confusing spectrum from prostaglaudin to errors in neurotransmitter metabolism.7 ’ 8 Nevertheless, it is encouraging that neuroleptic (antipsychotic) drugs are clearly effective in symptomatic treatment of schizophrenia .’,lo Among investigators in this field, the working hypothesis which currently enjoys the widest popularity is the dopamine (DA) hypothesis of schizophrenia: since virtually all known antipsychotic drugs block DA activity and since no other common denominator has yet been found, excess central dopaminergic activity may be present in the disorder” ’ l 2 Therefore, this review begins with recent discoveries about DA receptors. Dopamine Receptors. Since the identification of DA receptors at the biochemical’ level, using both adenylate cyclase and radioligand binding systems, it has become clear that a heterogeneity of DA receptors exists. One distinction which can be made is between DA ligand binding sites which are, or are not, linked to adenylate c y ~ l a s e . ~ ~ This ’ * ~ point can be well illustrated by characteristics of receptors in the striatum and nigrostriatal pathway. Kainic acid injection into the striatum destroys striatal neurons on which postsynaptic DA receptors are located while sparing axons which pass through this region and terminals which end within it.14 Such treatment produces a decrease of about 40% in 3H-neuroleptic binding while destroying virtually all o f the DA-sensitive adenylate cyclase activity.I4-l6 This shows that about 60% of the binding sites are not cyclase-linked. Most of the remaining antagonist binding sites are lost following ablation of the cerebral cortex, indicating that they are located on axons and terminals of the cortico-striatal projections.1 5 ’ 1 7 In addition, i t h a s now been shown in vitro that agonist binding to sites linked to adenylate cyclase is modulated by guanyl nucleotides,’8’ l9 whereas binding of antagonists is not. However, after removing the portion of striatal binding sites sensitive to kainic acid lesion, the sensitivity of the remaining sites to guanyl nucleotide modulation is completely lost.20 This implies that the receptors removed by kainic acid are linked to adenylate cyclase, whereas those remaining (on axons and terminals) are not. Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

Chap. 2

Antipsychotic Agents

McDermed, Miller

13

Kebabi an and Cal.ne have lately organized the available evidence into a coherent argument for classification of DA receptors as D-1 or D-2, according to whether or not they are linked to adenylate cyclase.l3 Various criteria for this classification are listed in Table 1. Table 1. DA receptor classification criteria (adapted from ref. 13) D-1 (cyclase-linked) Bovine parathyroid Agonist (pM range) partial agoni st or antagonist Ergots Potent antagonist (nM) or weak agonist (pM) Selective antagonist None known as yet Selective 3H-ligand cis-Flupenthixol

Receptor type Prototypical example Effects of: DA APO

D-2 (not cyclase-linked) Pituitary mammotroph Agonist (nM range) Agonist (nM) Agonist (nM) Sulpiride Dihydroergocriptine

The bovine parathyroid gland is a tissue with a relatively homogeneous population of D-1 (cyclase-linked) receptors. In this tissue DA stimulates the accumulation o f c-AMP, and this event is followed by release of parathyroid hormone from the gland.21 Apomorphine (APO) acts as a pure antagonist in this system. It may be recalled that in the striatum APO in the pM range acts as an agonist in stimulation of adenylate cyclase and behaves as an antagonist at higher concentrations.22 It appears that haloperidol and the other classical neuroleptics block both types of DA receptors. Prolactin secretion from the anterior pituitary is modulated via DA receptors located on the mammotrophs.23’24 Much evidence implies that these DA receptors are not linked to adenylate c ~ c l a s e and ~ ~ ’ hence ~ ~ are D-2 receptors, although one recent study using high concentrations of agonists has reported DA-sensitive adenylate cyclase activity in that tissue.26 Pharmacological specificity of the pituitary DA response clearly distinguishes it from systems linked to adenylate cyclase. Two main classes of drugs permit this distinction. The benzamide antipsychotic sulpiride is among the most potent drugs in producing an increase in prolactin secretion, indicating that it is a potent antagonist of the pituitary DA receptor.24 However, it is quite ineffective in blocking ~ ~ disparate results DA-sensitive adenylate cyclase in the ~ t r i a t u m . Such are also seen with certain ergot alkaloids such as bromocriptine. This compound is nearly inactive in stimulating adenylate ~ y c l a s e but ~ ~ ’ is~ ~ a very potent inhibitor of the release of prolactin, indicating that it is a potent DA agonist on the pituitary D-2 receptors.23 Binding data supplied by Seeman and his colleagues have indicated that in the striatum low concentrations of APO bind primarily to presynaptic receptors while low concentrations of neuroleptics (e.g., haloperidol) bind preferentially to postsynaptic receptors.30 ThGinjection o t 6-OH-DA into the substontia nigra, which destroys presynaptic nerve terminals but spares postsynaptic neurons, decreases the binding of 3H-AP0 and increases the binding of 3H-haloperidol.30 However, it must be noted here that Creese and Snyder have reported that a similar lesion produces increases in both 3H-AP0 and 3H-spiroperidol binding. 2o The technical differences underlying this discrepancy are not yet known. The increased

14 -

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Humber, Ed.

DA receptors and is a model for the postsynaptic dopaminergic supersensitivity that occurs in Parkinson's disease.31 In the past year such drug- or diseaseinduced supersensitivity has been studied by many authors.32-35 Two papers refer to the effects of lithium, a well known treatment for manicdepressive illness, in such models. Rats treated with haloperidol and lithium salts together did not develop the supersentivity which resulted from haloperidol treatment alone.36 Lithium treatment also blocked the development of presynaptic supersensitivity, as detected by electrophysiological measurements. 37 hindi-ng of antagmist-s is due t o increased numbers o f

Earlier, Lee and Seeman reported some preliminary data showing increased numbers of DA receptors in the post-mortem brains of schizophrenic patients.38 This important observation supported the possibility that such receptor supersensitivity underlies the etiology of schizo hrenia. Those authors have now reported an expanded study showing that k - A P O binding (presynaptic) was normal in schizophrenic brains whereas 3Hhaloperidol binding (postsynaptic) was significantly elevated in the caudate and p~tamen.~' The trend was the same in the nucleus accumbens, but in that case the sample size was very small. Owen, et al., have also reported significantly increased levels of 3H-spiroperidol binding iu all three of these nuclei in post-mortem schizophrenic brains, including those from seven patients who had not been medicated for at least a year before death.40 However, Mackay, et al., detected no abnormality in the 3H-spiroperidol binding in samples of nucleus accumbens from the brain of 26 schizophrenic^.^^ Because of the probable importance of limbic areas such as the nucleus accumbens in schizophrenia,42 the question of whether or not increased receptor levels are somehow responsible for the pathogenesis o f this disease must still remain an open question. Antipsychotic Drugs. It is a corollary of the DA hypothesis of schizophrenia that pharmacological screening methods for antipsychotics are usually designed to discover DA blocking agents. Neuroleptics typically increase DA turnover, elevate prolactin levels, compete with DA receptor ligands for membrane binding sites, induce catalepsy and block conditioned avoidance responding ( C A R ) in trained animals. It is widely believed that extrapyramidal side effects (EPSE) are low in antipsychotic drugs with central anticholinergic proper tie^^^'^^ and that sedative and hypotensive side effects may result from a-adrenergic blockade. 44 Newer behavioral models purport to discriminate between drugs with high liability to EPSE and those with low liability. Thus Ljungberg and Ungerstedt have shown, in an APO-induced stereotypy model (rats), that classical neuroleptics (high EPSE) predominantly antagonize the compulsive gnawing while non-cataleptic neuroleptics (low EPSE) block primarily the hypermotility.45 Prediction of EPSE has also been suggested in an APO-induced mouse climbing model. 46 A phenylethylamine-induced stereotypy model for s ~ h i z o p h r e n i aacquires ~~ additional significance from reports that increased levels of this amine occur in some psychotic A survey of potential new neuroleptics reported during the year shows that the large majority are variations on well-known drug classes. A Hoechst-Roussel group has reported a group of 9 new butyrophenones, of which 1 is the most interesting." It is orally effective in blocking

Chap. 2

Antipsychotic Agents

McDermed, Miller

2

CAR i n monkeys ( 0 . 5 x rhlorproniazi.ne) b u t has little effect on APO- or amphetamine-induced behaviors, a combination which was expected to predict effectiveness with freedom from EPSE. A group of 29 butyrophenones generally related to benperidol (&) has been reported51, of which 2b is the most promising. It is very effective orally (10 x haloperidol) at blocking drug-induced stereotypies with less propensity to induce ) a ~ benzamide ~ which is otherwise closely catalepsy. Halopemide ( 2 ~ is related to the butyrophenone 2a. It is a potent inhibitor of APO-induced emesiss3 and increases prolactin secretion in cultured pituitary mammotrophs.54 A preliminary clinical report indicates that it produces low EPSE.52 A large series of papers55 on bromperidol (3a), which is closely - document the clinical efficacy,56 behavioral related to haloperidol(3b),

F

@

C

"

*

q

p

F

F

~

X

4

U

2 -

1 -

a. X=CH2, Y=H, Z=O

b. X=CH2, Y=H, Z=S c. X=NH, Y=Cl, Z=O

N

Q

and biochemical pharmacology,5 7 neuroendocrine effects,58 and pharmacokinetic^'^'^' of this drug. Cloroperone (AHR 6134; 4a) is the chloro analog of lenperone (4b). A summary of the pharmacology and preliminary anti-schizophrenic activity of has appeared.61 Clopimozide (*), the appears to be clinically effective chloro derivative of pimozide and long-acting without excessive side effects .62

(z),

061% ~ ~1 % ~ a.

X=Br

b. X=C1

5

-

a. X=C1 b. X=F

a. X=C1 b. X=H

Patients may occasionally manifest symptoms of both depression and schizophrenia.63 Some recent compounds are active against models of both syndromes and might thus obviate problems of multi-drug interactions. An SKU group has reported a thorough comparison of the pharmacology of fluotracen (SKF 28175; 6) to that of standard antipsychotics and antidepressants and has found-it to possess both types of activity.64 Interestingly, removal of the 10-methyl group greatly enhances the neuroleptic effects and abolishes the antidepressant effects, while addition of a second 10-methyl group removes both types of activity. Amoxapine (7), which was previously known as a clinically effective antidepre~sant;~' has now been shown by a Lederle group to have significant neuroleptic activity in a number of animal models.66 Finally, the antidepressant trebenzomine (CI 686; 8)67 is reported to exhibit clinical antipsychotic efficacy similar to-that of chlorpromazine.68 Its mode of action does

H

Sect. I - CNS Agents

16 -

Humber, Ed.

not appear to be dopamjnergir, as it does not increase DA turnover, stimulate prolactin secretion, or compete for neuroleptic binding sites.6 8

8 -

6 -

Several papers have now appeared from Ayerst groups detailing the effects of modifications to the structure of the clinically effective"' neuroleptic butaclamol Because transposition of chlorine groups can eliminate cataleptic side effects from other tricyclic neuroleptics octoclothepin+doclothepin), a series of chloro derivatives of the (g., butaclamol analog 9b was made.7 0 However, this produced on1 compounds which were inactiveor retained butaclamol-like activity. 7 0 9 T 1 They also made a series of 15 analogs (9c) in which the t-butyl group was replaced by aryl groups.72 This produced a variable pattern of in vivo neuroleptic activities, but generally enhanced the adrenolytic liabilities. By making a series of modifications on ring E , they concluded that the t-but 1 but not the hydroxyl group is required for neuroleptic a~tivity.'~ They also found that ring A of 9a can be moved from the 6,7-position to the 5,6-position without reduction of activity. 74 Based on geometrical analysis of these compounds, they devised a 3-dimensional model of the receptor binding site which includes a primary nitrogen binding site, a naphthalene-sized primary aromatic binding site, and an accessory binding site for the t-butyl group.73'74 A Janssen group has also attempted to use the rigid structure of 9b to infer pharmacologically relevant conformations of more flexible neuroleptics. 7 5 Seeman, et al. , have noted that the stereospecificity of binding to striatal sites is higher 9 a. R=t-Bu for the enantiomers of than for b. R=i-Pr any of 7 other resolved neuroleptic c . R=Ar pairs, probably reflecting in part its rigid structure.7 6

(e).

More data have accumulated on the interesting benzamide group of antipsychotics, of which sulpiride is the best known. It has now been shown in studies o f behavior,77 vasodilation,7 8 and bindingx7 that neuroleptic activity resides primarily in the ( - ) - S enantiomer, whose absolute et_al., configuration was determined by chiral synthesis. l 7 Costall, compared a group of benzamides to a group of conventional neuroleptics for their abilities to differentially antagonize some behavioral models which mesolimbic vs. are believed to have different cerebral substrates, striatal DA systems.79 Some benzamides have also been studied for their ability to antagonize DA-induced renal vasodilation, in which model the order of potencies is not the same as that seen in the CNS.80 Jenner, et a1.,81 have concluded that for a group of 5 benzamide drugs there is little correllation between their clinical characteristics and their activ-

e.,

Chap. 2 ities

in

Antipsychotic Agents

McDermed, Miller 17

behavioral and hiocheniical mcdels of DA antagonism.

Several discussions o f the relation of opioid peptides to psychosis have appeared.82-84 Such studies raise the possibility that novel antipsychotic drugs could be based on the structure of endogenous peptides.” DeWied, _ et al., _ have reported that a peptide corresponding to sequence 62-77 of f3-lipotropin (G, des-tyr’-y-endorphin) exhibits neuroleptic activity in behavioral models. 86 Limited antipsychotic activity of this peptide has now been reported in humans (i.m. i n j e ~ t i o n ) . ~ However, ~ it has also been reported that this peptide does not compete with 3H-neuroleptics for binding sites in any brain areas.88 A year ago there was a provocative report by Palmour and Ervin that the CSF of schizophrenics contains a previously unknown peptide and that this molecule can be removed by h e m o d i a l y ~ i s . ~This ~ would provide a simple explanation for the controversial observations that schizophrenic symptoms are alleviated by hemodialysis but no further data concerning the existence of this endogenous psychotogen have yet appeared. Dopamine Agonists. During the past year a number of new modifications of the structure of DA were reported. It is notable that the effects of structure on activity in a series of compounds lo in the CNS do not parallel those in the periphery. Conipou~id 9was the most potent in inhibiting the cardioaccelerator nerve in the cat, while only 10b showed any central emetic activity (0.07 x APO).’* Compound 10b is o f n t e r e s t as a DA agonist in the renal vascular bed because, although it is much weaker than DA (20 x), it exhibits no a-adrenergic a ~ t i v i t y . ’ ~In a larger series o f compounds 10, all were found to be much weaker agonists than APO in animals with unilateral cerebral le~ions.’~ Compound was selected for more detailed behavioral and metabolic study as a potential anti-parkinson drug.95 Wikstrom, et al. , 9 6 have made the very interesting observation that the phenol IOd, although only 0 . 1 ~as potent as APO in decreasing DOPA accumulationin rat brain, is actually lox more potent than the corresponding catechol 10b in the same model. They saw the same rank order in stimulation of motor activity. It has been reported that aromatic fluorination does not substantially alter the activity o f are two reports in which the conformaDA at vascular r e ~ e p t 0 i - s . There ~~ tion of the ethylamine side chain of DA was constrained by incorporation into cyclopropaneg8 or cyclobutaneg9 rings, but the best compounds were much weaker than DA. V

OH

10 -

a . X=OH, R=R’=Me b . X=OH, R=R‘=Pr c. X=OH, R=Pr, R’=Bu d . X=H, R=R‘=Pr

a . X=8-NH2, Y=Z=H b. X=8-NH2, Y=OH, Z=H C. X=8-NH2, Y=Z=OH d. X=7-OCH3, Y=Z=Cl

a. R=H b . R=CH3

18

Sect. I

- CNS

Agents

Humber, Ed.

Nomifensin (B) is an antidepressant which produces significant DA-mimetic motor effects in animal models.100 It has now been shown is a potent agonist (0.25-0.50x DA) on the that the catechol analog adenylate cyclase from both striatum and nucleus accumbens, whereas is potent in and llb are inactive.lo1 Another group has shown that i n d u G stereotyped behavior upon injection into the nucleus accumbens . l o g Because this effect was blocked by a-methyltyrosine pretreatment, a presynaptic mode of action was inferred. The related compound has shown limited clinical efficacy in the treatment of Parkinson's disease.l o 3 SKU groups have reported that 12a (SKF 38393) is a pew dopaminergic agent with a very unusual profile ofytivity. In the dog (i.v.) it specifically dilates the renal vascular bed without increasing cardiac output or arterial blood pressure,l o * apparently by a direct agonist effect on vascular DA receptors. In the CNS 12a is a particularly potent partial agonist in the striatal adenylate cyclase preparation and is an agonist in rats with unilateral lesions in the substantia nigra.lo5 However, it fails to produce stereotypy in normal rats, is not emetic, and does not affect prolactin levels or DA turnover.lo5 The unusual profile of 12a may be partially explained by considering it an agonist selective fF D - 1 receptors within the classification scheme discussed above. If so, it may become an inyortanl. pharmacological tool. Interest12b has clinical utility as an anti-aggressive inglyio;he dimethyl ether drug.

Many new data have appeared about the dopaminergic properties of 2-aminotetralins. Eighteen of these were X&NR2 evaluated for inhibition of 3H-haloper8 idol binding and stimulation of l3 a * X=5y6-(0H)2y R=Pr striatal adenylate cyclase, and these b. X=6,7-(OH)z, R=H data were compared with in vivo c . X=5-OH, R=Pr behavioral data. l o 7 It was noted in particular that 13a is far more potent (150 x DA) than any other known DA agonist in displacing the antagonist 3H-haloperidol. It is also a potent agonist (1 x DA) in stimulating adenylate cyclaselo7 and has been extensively studied in_vivo _ as an agonist in behavioral models.108-110 However, the best known compound in this series is 13b, This compound is particularly useful f o r also known as ADTN.ll1'll2 defining the agonist-specific component of ligand binding in DA receptor studies,'l3 and when tritiated is a useful ligand in its own right.114'115 Horn, et al., reported that the 0,O'-dibenzoyl ester of 13b is an effective pro-drug with CNS activity because it penetrates the bloodbrain barrier, whereas the free catechol does not. A Burroughs Wellcome group has now resolved 13b and has determined the absolute configurations of the enantiomers o f 3 b and 13c by degradation.l17 Studies of binding, inhibition of prolactinsecretion, l8 stimulation of adenylate cyclase, l9 and renal v a s o d i l a t i ~ nall ~ ~ confirm that the more active enantiomer is (+)-2R-l3b. This corresponds to an absolute configuration which is o p p o s i t e 0 those of the more active enantiomers of 13c and APO. This observation was used to form a hypothesis about the mode of binding of such agonists to DA receptors, in which the required configuration at the chiral center is controlled by the particular location of a hydroxyl group "meta" to the aminoethyl fragment.1 1 7 This proposal may account

Chap. 2

Antipsychotic Agents

McDermed, Miller

19

f o r the inactivities o f iso-APO and a related striicture120 which was recently reported.

Ergots. The ergot alkaloids have generally complex pharmacological profiles,28’121’122 but some with DA agonist properties are therapeutically useful. The best studied of these are lergotrile (14a) and bromocriptine (CB 154; 15a) , 2 9 ’ 1 2 2 - 1 2 4 which are effective in treatment of Parkinson‘s disease. 1m126 Bromocriptine is used to treat excessive secretion of prolactin (=., galactorrhea) and growth hormone (acromegaly). 1 2 7 It is also an effective antihypertensive agent,1 2 8 ’ 1 2 9 though its locus of action is unclear.

14

b

.

C.

( a ) -NHSOzN (CH3)

2

15

c

(cx)-CH~CN

b.

(B)-CHZ-S

c. ( B ) - C H & N + O C H ~

The dopaminergic activities of some newer ergolines (14) and ergolenes (15) have been studied. Compound 14b (CH 29-717) is potent (10 x 15a) insuppression of hyperprolactinemzin rats. 130 In behavioral a n d biochemical models (CM 29-712) is generally bromocriptine-like in ~ ~ ergolene 15b (CF 25-397) induces its DA agonist e f f e c t ~ . l ~ l ’ lThe lesion^,'"^ but exhibits contralateral turning in rats with ni ral 6-0-A An extensive study of few other central DA agonist __ 15c (MPME; PTR 17402) shows that in rat brain it is a DA agonist which is selective for subcortical limbic structures. 133 REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18.

H.M. van Praag, ”Depression and Schizophrenia,“ Spectrum Publications, New York. 1977. H.E. Lehmann in ”Comprehensive Textbook of Psychiatry,” 2nd ed., A.M. Freedman, H.I. Kaplan and B.J. Sadock, Eds., Williams and Wilkins, Baltimore, 1975, p 890. H.G. Pope, Jr. and J.F. Lipinski, Jr.. Arch. Gen. Psychiatry,35, 811 (1978). R.L. Spitzer and P.T. Wilson in “Comprehensive Textbook o f Psychiatry, ” 2nd ed., A.M. Freedman, H.I. Kaplan and B.J. Sadock, Eds., Williams and Wilkins, Baltimore, 1975, p 826. J. Rotrosen. A.D. Miller, D. Mandio, L.J. Traficante and S.Gershon, Life Sci.,23, 1989 (1978). D.F. Horrobin, Lancet 1,936 119771. W.H. Berrettini, W. Prozialeck and W.H. Vogel, Arch. Gen Psychiatry, 35,600 (1978). K.L. Pajari, New Eng. J. Med.. 298, 1150 (1978). (Letter; see also other letters p p 1150-1 152). P. Deniker, Am. J. Psychiatry, 135, 923 (1978). J.M. Davis and R. Casper, Drugs, 14, 260 (19771. A . Carlsson, 8iol. Psychiatry, 73, 3 (19781. T.J. Crow, E.C. Johnstone, A. Longden and F. Owen in “Adv. in Biochem. Psychopharmacol.,” Vol. 19, P.J. Roberts. G.N. Woodruff and L.L. Iversen, Eds., Raven Press, New York, 1978, p 301. J.W. Kebabian and D.B. Calne, Nature.277, 93 (1979). K.P. Minneman, M. Quik and P.C. Emson, Brain Res., 751, 507 11978). R. Schwarcz, I . Creese, J.T. Coyle and S.H. Snyder, Nature,271, 766 11978). S. Govoni, V.R. Olgiati, M. Trabucchi, L . Garau, E. Stefanini and P.F. Spano. Neurosci. Lett., 8, 207 (1978). L. Garau, S. Govoni, E. Stefanini. M. Trabucchi and P.F. Spano, Life Sci., 23, 1745 (1978). N.R. Zahniser and P.B. Molinoff, Nature.275, 453 (1978).

20 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. 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.

Sect.

I - CNS Agents

Humber, Ed.

I. Creese, T. Prosser and S.H.Snyder, Life Sci., 23, 495 (1978). I. Creese and S.H. Snyder in "Catecholamines: Basic and Clinical Frontiers," Vol. 1, E. Usdin. Ed., Pergamon, New York. 1979. p 601. E.M. Brown, R.J. Carroll and G.D. Aurbach, Proc. Nat. Acad. Sci. USA. 74, 4210 (1977). J.W. Kebabian, G.L. Petzold and P. Greengard, Proc. Nat. Acad. Sci. USA. 69, 2145 (1972). M.G. Caron. M. Beaulieu, V. Raymond. B. Gagne, J. Drouin, R. Lefkowitz and F. Labrie, J. Biol. Chem.. 253, 2244 (1978). T.F. Mowles. B. Burghardt. C. Burghardt, A. Charnecki and H. Sheppard, Life Sci., 22, 2103 (19781. R.S. Rappaport and N.H. Grant. Nature, 248, 73 (1974). H.S. Ahn. E. Gardner and M.H. Makman, Eur. J. Pharmacol., 53, 313 (1979). M. Trabucchi, R. Longoni, P. Fresia and P.F. Spano. Life Sci., 77, 1551 (1975). K. Fuxe, 8 . Fredholm, S.Ogren, L. Agnati, T . Hokfelt and J. Gustafsson, Fed. Proc., 37, 2181 (1978). R. Markstein, P. Herrling, H. Burki, H. Asper and W. Ruch, J. Neurochem., 37, 1163 (1978). J,t, Nagy, T. Lee, P. Seeman and H.C. Fibiger, Nature.274, 278 (19781. T. Lee, P. Seeman, A. Raiput, I.Farley and 0.Hornykiewicz, Nature, 273, 59 (1978). J.C. Schwartz, J. Costentin, M.P. Martres. P. Protais and M. Baudry, Neuropharmacol.. 77, 665 (1978). A. Hitri, W. Weiner, R. Borison, B. Diamond, P. Nausieda and H. Klawans, Annals Neurol., 3, 134 (1978). B. Bioulac, G. Boulard, J. Vincent and E Puil, Neuropharmacol., 77, 965 (1978). M. Quik and L. Iversen, Naunyn-Schmiedeberg's Arch. Pharmacol., 304, 141 (1978). A. Pert, J. Rosenblatt, C. Sivit, C. Pert and W. Bunney, Jr., Science.201, 171 (1978). D. Gallager, A. Pert and W. Bunney, Jr.. Nature, 273, 309 (19781. T. Lee and P. Seeman, 7th Ann. Mtg. SOC.Neurosci., Anaheim, Cal., Nov. 6, 1977, Abstr., Vol. 3, no. 1414. T. Lee, P. Seeman, W.W. Tourtellotte, I.J. Farley and 0. Hornykeiwicz, Nature, 274, 897 (1978). F. Owen, T. Crow, M. Poulter, A. Cross, A. Longden and G . Riley, Lancet 2, 223 (19781. A. Mackay, A. Doble, E. Bird, E. Spokes, M. Ouik and L. Iversen, Life Sci., 23, 527 (19781. A. Carlsson, Am. J. Psychiatry, 135, 164 (1978). P.M. Laduron and J.E. Leysen, J. Pharm. Pharmacol., 30, 120 (1978). S.H. Snyder, Drug Therapy (Hosp.), 3, 30 (1978). T. Ljungberg and U. Ungerstedt, Psychophartnacol., 56, 239 (1978). B. Costall, R.J. Naylor and V. Nohria. Eur. J. Pharmacol., 50, 39 (1978). R.L. Borison, H.S.Havdala and 8.1. Diamond, Commun. Psychopharmacol., 2, 209 (1978). M. Sandler, C. Ruthven, 8.Goodwin, H. Field and R. Matthews. Lancet 2. 1269 (1978). M. Sandler, C. Ruthven. B. Goodwin, G. King, 8.Pettit and G. Reynolds, Commun. Psychopharmacol.. 2, 199 (1978). R . Allen, V . Bauer, R. Kosley, Jr., A. McFadden, G. Shutske, M. Cornfeldt, S.Fielding, H. Geyer, Ill and J. Wilker, J. Med Chem., 27, 1149 (1978). M. Sato, M . Arimoto, K. Ueno, H. Koiima, T . Yamasaki. T. Sakurai and A . Kasahara, J. Med. Chem. 21, 11 16 (1978). J. Castaner, Drugs of the Future, 3, 44 (1978). A.J.M. Loonen, I. Van Wiingaarden, P. Janssen and W. Soudijn, Eur. J. Pharmacol., 50, 403 (19781. C. Denef and J.J. Follebouckt, Life Sci.. 23, 431 11978). Acta Psychiatr. Belg., 78(1),Jan.-Feb. (1978). (Many refs. therein; for examples see refs. 56-60 below). W. Poldinger, Acta Psychiatr. Belg., 78, 96 (19781. C.J. Niemegeers, P.M. Laduron and P.A. Janssen, Acta Psychiatr. Belg., 78, 37 (1978). A. Bianchi, F. Drago. P.L. Canonico, V. Guarcello, B. Carpiniello and U. Scapagnini, Acta Psychiatr. Belg., 78, 69 (19781. E. van den Eeckhout and F. Belpaire, Acta Psychiatr. Belg., 78, 64 (1978). L.F. Chasseaud, Acta Psychiatr. Belg., 78, 51 (19781. i. Castaner. Drugs of the Future, 3.445 (1978). L. Floru and J. Tegeler, Arrneim. Forsch., 28, 341 (1978). M. Sim, A m . J. Psychother., 32, 74 (1978). P.J. Fowler, C.L. Zirkle. E. Macko, P.E. Setler, H.M. Sarau, A . Misher and D.H. Tedeschi, Arzneim. Forsch., 27, 1589 (1977). G.L. Sathanathan, R. Matz. M. Thompson and S. Gershon, Curr. Ther. Res., 15, 919 (1973). E.N. Greenblatt, A.S. Lippa and A.C. Osterberg. Arch. Int. Pharmacodyn., 233, 107 (19781. J. Castaner, Drugs of the Future, 3, 556 (19781. H.Y. Meltzer, M. Simonovic. V. Fang, S.Piyakalamala and M . Young, Life Sci.. 23, 605 (1978). J.N. Nestoros, H.E. Lehmann and T.A. Ban. Int. Pharmacopsychiat., 13, 138 (1978). L.G. Humber. N. Sideridis. A.A. Asselin, F.T. Bruderlein and K . Voith, J. Med. Chem.. 27, 1225 (1978). T.A. Pugsley and W. Lippmann. J. Pharm. Pharmacol., 31, 47 (19791. K . Voith, F.T. Bruderlein and L. G. Humber, J. Med. Chem., 27, 694 (1978). L.G. Humber, F.T. Bruderlein, A.H. Philipp, M. Gotz and K. Voith, J. Med. Chem., 22, in press (1979). A.H. Philipp, L.G. Humber and K . Voith, J. Med. Chem., 22, in press 11979). H. Moereels and J.P. Tollenaere, Life Sct., 23, 459 (1978). P. Seeman, K. Westman. M. Protiva. J. Jilek, P. Jain. A. Saxena, N . Anand, L. Humber and A. Philipp, Eur. J. Pharmacol., in press (1979). C.D. Andrews and G.N. Woodruff, Brit. J. Pharmaco\., 64, 434P (1978). L.I. Goldberg, J.D. Kohli. J.J. Litinsky and J. McDermed in "Catecholamines: Basic and Clinical Frontiers," Vol. 1. E. Usdin, Ed., Pergamon, New York, 1979, p 447. 8 . Costall, W.H. Funderburk, C.A. Leonard and R.J. Naylor. J. Pharrn. Pharmacol.. 30, 771 (1978). D. Glock, J.D. Kohli and L.I. Goldberg, Fed. Proc.. in press (1979) P. Jenner, P.N.C. Elliott, A. Clow. C. Reavill and C.D. Marsden. J. Pharm. Pharmacol., 30, 46 (1978). F. Bloom, 0. Segal, N. Ling and R. Guillemin, Science, 194, 630 (1976).

Chap. 2 83. 84. 85. 66. 67. 88. 89.

90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133.

Antipsychotic Agents

McDermed. M i l l e r

21

K. Verebey, J. Volavka and D. Clouet, Arch. Gen Psychiatry, 35, 877 (19781. J.S. Hong, H.T. Yang, W. Fratta and E. Costa, J. Pharmacol. Exper. Ther.. 205, 141 (1978). R.A. Lahti, D.D. Gay and C. Barsuhn, Eur. J. Pharmacol.. 53, 117 (1978). D. De Wied, G. Kovacs, B. Bohus, J. Van Ree and H. Greven, Eur. J. Pharmacol., 49, 427 (1978). W. Verhoeven, H. Van Praag, P. Botter, A . Sunier, J. Van Ree and D. De Wied, Lancet 1, 1046 (1978). J. Van Ree, A. Witter and J. Leysen. Eur. J. Pharmacol., 52, 41 1 (19781. R.M. Palmour and F.R. Ervin, 7th Ann. Mtg. SOC.Neurosci., Anaheim, Cal., Nov. 6, 1977, Abstr., Vol. 3, no.1029. H. Wagemaker, Jr., and R. Cade, Am. J. Psychiatry, 134, 684 (1977). F.K. Port, P.D. Kroll and R.D. Swartz, Am. J. Psychiatry, 135, 743 (1978). J.G. Cannon, F. Hsu, J.P. Long, J.R. Flynn, 8.Costall and R.J. Naylor, J. Med. Chem.,21, 248 (1978). J.D. Kohli, L.I. Goldberg, P.H. Volkman and J.G. Cannon, J. Pharmacol. Exptl. Ther., 207, 16 (1978). J.Z. Ginos, G.C. Cotzias and D. Doroski, J. Med. Chem., 21, 160 (19781. J.Z. Ginos and F.C. Brown, J. Med. Chem., 21, 155, (1978). H. Wikstrom, P. Lindberg, P. Martinson, S. Hjorth, A . Carlsson, U. Hacksell, U. Svensson and J.L.G. Nilsson, J. Med. Chem..21, 864 (1978). L.I. Goldberg, J.D. Kohli, K.L. Kirk and C.R. Crevellng, Fed. Proc.. in press (1979). R.J. Borgman, P.W. Erhardt, R.J. Gorczynski and W.G. Anderson, J. Pharm. Pharmacol., 30, 193 (1978). H.L. Komiskey, J.F. Bossart. D.D. Miller and P.N. Patil, Proc. Natl. Acad. Sci. USA, 75, 2641 (1978). C. Braestrup and J. Scheel-Kruger, Eur. J. Pharmacol., 38, 305 (1976). J.A. Poat. G.N. Woodruff and K.J. Watling, J. Pharm. Pharmacol.,30, 495 (1978). B. Costall and R.J. Naylor, J. Pharm. Pharmacol., 30, 514 (19781. J. Presthus, J. Ankerhus, A. Buren, J. Bottcher, R . Holmsen, 8. Mikkelsen, 6 . 0 . Mikkelsen, R. Nyberg-Hansen, E. Riman, 6. Severin and J. Aarli, Acta Neurol. Scand., 58, 77 (1978). R.G. Pendleton. L. Samler, C. Kaiser and P.T. Ridley. Eur. J. Pharmacol., 51, 19 (1978). P.E. Setler, H.M. Sarau, C.L. Zirkle and H.L. Saunders, Eur. J. Pharmacol., 50, 419 (1978). J.-M. Albert, R . Elie, S.F. Cooper, A. Clermont and Y. Langlois, Curr. Ther. Rsch., 21, 786 (1977). J.G. Cannon, 6. Costall. P.M. Laduron, J.E. Leysen and R.J. Naylor, Biochem. Pharmacol., 27, 1417 (1978). B. Costall. R.J. Naylor and R.T. Owen, Eur. J. Pharmacol., 49, 407 (1978). B. Cootall, R.J. Naylor and R.T. Owen, Eur. J. Pharmacol., 50, 113 (1978). J.D. McDermed, G.M. McKenzie and A.P. Phillips, J. Med. Chem., 78, 362 (1975). A. Davis, P.J. Roberts and G.N. Woodruff, Brit. J. Pharmacol., 63, 183 (1978). A.S. Horn, C.J. Grol, D. Dijkstra and A.H. Mulder, J. Med. Chem., 21, 825 (1978). M. Quik, L.L. Iversen, A. Larder and A.V.P. Mackay, Nature, 274, 513 (1978). I. Creese and S.H. Snyder, Eur. J. Pharmacol.,50, 459 (1978). Y. Clement-Cormier and M. Abel, Res. Commun. Chem. Pathol. Pharmacol., 22, 1 5 (1978). A.S. Horn, D. De Kaste, D. Dijkstra. H. Rollema, M. Feenstra, 8.Westerink, C. Grol and A. Westerbrink, Nature, 276, 405 (1978). J.D. McDermed, H.S. Freeman and R.M. Ferris in "Catecholamines: Basic and Clinical Frontiers," Vol. 1, E. Usdin, Ed., Pergamon, New York, 1979, p 568. H.Y. Meltzer, M. Simonovic, D.W. Nichols, R.J. Miller, J.D. McDermed and V.S. Fang in "Catecholamines: Basic and Clinical Frontiers," Vol. 2, E. Usdin, Ed., Pergamon, New York, 1979, p 1251. C.D. Andrews, A. Davis, H.S. Freeman, J.D. McDermed, J.A. Poat and G.N. Woodruff, Br. J. Pharmcol., 64, 433P (1978). D.E. Nichols, J.E. Toth, J.D. Kohli and C.K. Kotake, J. Med. Chem.. 21, 395 (1978). K. Fuxe, B. Fredholm. L. Agnati, S.Ogren, B. Everitt, G. Jonsson and J. Gustafsson, Pharmacol.. 16, (Suppl. 1), 99 ( 1978). M. Goldstein, J.Y. Lew, S. Nakamura. A. Battista, A. Lieberman and K. Fuxe. Fed. Proc., 37, 2202 (1978). G. D i Chiara, M. Porceddu, L. Vargiu and G. Gessa, Pharmacol., 16 (Suppl. 1) 135 11978). K. Fuxe, 8.Fredholm, S.Ogren. L. Agnati, T. Hokfelt and J Gustafsson, Acta Endocrin., 88 (Suppl. 216). 27 (1978). M. Goldstein, A. Lieberman, A. Battista, J. Lew and F. Hata, Pharmacol.. 16 (Suppl. 1). 143 (1978). A. Agnoli, S.Ruggieri and M. Casacchia, Pharmacol.. 76 (Suppl. 1). 174 (1978). F. Silvestrini, A. Liuzzi and P. Chiodini, Pharmacol., 16 (Suppl. 1). 78 (1978). A. Nilsson and B. Hokfelt, Acta Endocrin., 88 (Suppl. 216). 83 (1978). 8.Clark, G. Scholtysik and E. Fluckiger, Acta Endocrin., 88 (Suppl. 216), 75 (1978). E. Fluckiger, U. Briner, W. Doepfner, E. Kovacs, P. Marbach and H. Wagner, Experientia, 34, 1330 (1978). J. Vigouret, H. Burki, A. Jaton, P. Zuger and D. Loew, Pharmacol., 16 (Suppl. 1). 156 (1978). P.L. Stutz. P.A. Stadler, J. Vigouret and A. Jaton, J. Med. Chem., 21, 754 (1978). K. Fuxe, B. Fredholm, L. Agnati and H. Corrodi. Brain Res., 746, 295 (19781.

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Annual Reports in Medicinal Chemistry-14

Chapter 3.

Anti-Anxiety Agents, A n t i c o n v u l s a n t s , and Sedative-Hypnotics

J o e l G. Berger and L o u i s C . I o r i o Schering-Plough Research D i v i s i o n , Bloomfield, N . J. 07003 Benzodiazepines and R e l a t e d Compounds The n a t u r e of t h e benzodiazepine 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 f u r t h e r by t h e demonstration t h a t a s i n g l e class of b i n d i n g s i t e s e x i s t s i n r a t b r a i n f o r a s t r u c t u r a l l y d i v e r s e series of benzodiazepines.’ The p o p u l a t i o n s i z e , a s measured by t h e number of 3H-diazepam (3H-D, la) b i n d i n g s i t e s , w a s d e c r e a s e d by c h r o n i c t r e a t m e n t w i t h flurazepam ( l j ) . 2 Binding of 3H-D and 3H-flunitrazepam (&) t o t h e i r r e c e p t o r s was shown t o occur i n v i v o as w e l l as i n v i t r o . 3 Regions of t h e r a t b r a i n c o n t a i n i n g t h e h i g h e s t number of 3H-D b i n d i n g s i t e s were t h e c e r e b r a l c o r t e x , hippocampus, and ~ e r e b e l l u m . ~On a c e l l u l a r l e v e l , s t u d i e s on bovine c o r t i c a l p r e p a r a t i o n s i n d i c a t e t h a t t h e benzodiazepine r e c e p t o r is l o c a l i z e d l a r g e l y on t h e a s t r o g l i a l plasma membrane.’ However, s t u d i e s w i t h “nervous” mutant mice s u g g e s t t h a t i n t h i s s p e c i e s t h e r e c e p t o r s a l s o r e s i d e on c e r e b e l l a r P u r k i n j e cells. Ontogenetic s t u d i e s i n d i c a t e t h a t benzodiazepine r e c e p t o r s are a l r e a d y p r e s e n t i n rat b r a i n a t b i r t h , and reach maximal c o n c e n t r a t i o n one week later.’ S e v e r a l r e c e n t s t u d i e s i n d i c a t e t h a t some i n t e r a c t i o n may e x i s t beI t h a s been found t h a t tween t h e benzodiazepine and GABA rece GABA9Yl0 o r t h e GABA-agonist muscimollg c a u s e an i n c r e a s e i n t h e a f f i n i t y of %-D f o r i t s b i n d i n g s i t e which is markedly dependent on t h e p r e s e n c e of NaC1,’ $ l o w h i l e t h e GABA-antagonist b i c u c u l l i n e d e c r e a s e d a f f i n i t y . I n o s i n e and hypoxanthine have been i d e n t i f i e d as endogenous l i g a n d s f o r %-D b i n d i n g sites i n bovine b r a i n , ” as h a s a h e a t - s t a b l e p r o t e i n of M.W.>15,000 i s o l a t e d from rat b r a i n . 1 2 The l a t t e r w a s found t o i n h i b i t GABA b i n d i n g , and hence t h e benzodiazepines may enhance t h e e f f e c t s of GABA by d i s p l a c i n g t h i s endogenous l i g a n d from i t s r e c e p t o r . Recent work i n t h i s area h a s been comprehensively reviewed.13 The f i n d i n g t h a t physostigmine i s a h i g h l y p o t e n t a n t i d o t e i n a c u t e diazepam i n t o x i c a t i o n ” r a i s e s t h e p o s s i b i l i t y t h a t benzodiazepine act i v i t y may a l s o i n v o l v e c h o l i n e r g i c systems. S t u d i e s on t h e pharmacokinetics of f l u n i t r a z e p a m (&) i n man,15 clonazepam (&) i n r h e s u s monkeys,16 and t r i a z o l a m (B) i n dogs17 have appeared. The metabolism of bromazepam (&) i n r o d e n t s h a s been s t u d i e d , and t h e major m e t a b o l i c pathway i s c l e a v a g e of t h e benzodiazepine r i n g a t t h e N1-CZ and N4-C bonds w i t h subsequent r e d u c t i o n and hydroxyl a t i o n of t h e r e s u l t i n g prjmary m e t a b o l i t e 2-( 2-amino-5-bromobenzoyl) pyridine.

*

The s u b j e c t of benzodiazepine dependence has been e x h a u s t i v e l y reviewed,” and t h e r i s k f a c t o r a p p e a r s t o be low. A c o r r e l a t i o n between blockade of p e n t y l e n e t e t r a z o l e ( 1 e p t a z o l e ) Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0-12-040514-8

Chap. 3

Anti-Anxiety Agents, Anticonvulsants

la lb lc Id

c1

CH3

H

H

CH

NO2

CH3

H

CH

CH2CF3

H

F H H

C1 C1

CH2CH20CH2-O H

F

CH

CH3 CH2CgCH

H H

H

CH CH

c1

H

OH

c1

CH

lh

NO2

li

Br

H H

H H

c1 H

CH N

F

CH

c1

lk 11 lm

In lo

CH2CH2N(Et)2 H

NO2

H

H

H

CH

c1

CH3

OH

H

CH

OH

H

CH

H

H

CH

H

c1

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H C1 CH2P(O)(CH3)2 Br CH3 c1

3

CH

c1 C1

If R1

Berger, Iorio

icduced convulsions i n mice and rate c o n s t a n t s f o r t h e NaBH4 r e d u c t i o n of 11 d i f f e r e n t 1,4-benzodiazepinones h a s been demonstrated, sugg e s t i n g a p o s s i b l e involvement of t h e carbonyl group a t t h e r e c e p t o r s i t e . 20

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A n x i o l y t i c Agents The animal pharmacology, metabolism, c l i n i c a l pharmac o k i n e t i c s , and c l i n i c a l a p p l i c a t i o n s of lorazepam (Wy-4036 &) i n v a r i ous a n x i e t y s t a t e s h a s been reviewed.21 Halazepam (Sch 12041, &) i n 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 h a s been shown t o be an e f f e c t i v e a n x i o l y t i c agent a t 40-600 mgfday with r e l a t i v e l y minor s i d e e f f e c t s . 2 2 I n a doub l e - b l i n d t r i a l vs. c h l o r d i a z e p o x i d e , clazepam ( I d ) a t 30 mg d a i l y was only moderately e f f e c t i v e i n d e c r e a s i n g a n x i e t y a m ID-540 (&) w a s shown t o i n c r e a s e peak l a t e n c y of t h e p h o t o p a l p e b r a l r e f l e x i n man, a test which may c o n s t i t u t e a u s e f u l method f o r a s s e s s i n g p o t e n t i a l anxiol y t i c a c t i v i t y i n t h e c l i n i c . 2 4 Pinazepam (If) a e a r e d t o have a g r e a t e r a n x i o l y t i c e f f e c t i n r a t s t h a n diazepam. 29p I n two double-blind a t 20-30 mg d a i l y w a s found h a l f a s p o t e n t c l i n i c a l t r i a l s , clobazam a s diazepam i n t h e r e l i e f of a n x i e t y . 26 I t s neuropharmacological p r o f i l e i n animals h a s been e s t a b l i s h e d , 2 7 and i t s e f f e c t s on psychomotor performance i n man have been s t u d i e d . 2 8 The s t r u c t u r a l l y r e l a t e d OW-8063 (&) a t 66.5 mg d a i l y was judged t o be a n e f f e c t i v e a n t i a n x i e t y a g e n t with e n e r g i z i n g p r o p e r t i e s i n an u n c o n t r o l l e d s t u d y . 29 C o n t r o l l e d s t u d i e s i n normal s u b j e c t s showed c l o t i a z e p i n e (BAYg 5633, 3) a t 10 mg t o be an e f f e c t i v e a n x i o l y t i c . 30

(a)

Sect. I

24 -

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CNS Agents

Humber, Ed.

I n double-blind placebo c o n t r o l l e d s t u d i e s , ketazolam (4) a t about 8 mg appeared t o be of s i g n i f i c a n t b e n e f i t i n r e l i e v i n g a n x i e t y and w a s w e l l - t o l e r a t e d . 3 1 The 2,3-benzodiazepine, t o f isopam (5), w a s claimed t o e x e r t a n x i o l y t i c e f f e c t s i n man w i t h o u t muscle-relaxant o r s e d a t i v e e f f e c t s . 32

'

X

0

J3q I

2a,

X=Cl

2b. X=CF3

Although t h e pyrrolobenzodiazepine w a s a c t i v e i n s e v e r a l animal tests i n d i c a t i v e of a n x i o l y t i c a c t i v i t y , i t w a s c o n s i d e r a b l y less a c t i v e t h a n diazepam i n p r e l i m i n a r y tests i n man. Metabolism s t u d i e s suggested i n a c t i v a t i o n v i a h y d r o x y l a t i o n a t t h e 3 and 11a-positions. Attempts t o i n c r e a s e potency and d u r a t i o n of a c t i o n by b l o c k i n g t h e s e p o s i t i o n s w i t h a l k y l groups gave much less active compounds. 3 3 The t h i e n o d i a z e p i n e Y-7131 (AHR-3219, &) was found t o have p o t e n t a n t i - a n x i e t y , a n t i - c o n v u l s a n t , muscle-relaxant and s e d a t i v e p r o p e r t i e s and e f f e c t s of t h i s i n s e v e r a l animal s p e c i e s . 34 Pharmacodynamics compound on b i o g e n i c amine metabolism36 have been d e s c r i b e d . Syntheses and SAR have a l s o been d e s c r i b e d . 3 7

''

Imidazobenzodiazepine h3' and t h e r e l a t e d pyrrolobenzodiazepine t h e b e s t members of t h e i r r e s p e c t i v e series, having taming and sedative/muscle r e l a x a n t p r o p e r t i e s similar t o diazepam i n r o d e n t s .

8b39 were

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Sleep l a b o r a t o r y s t u d i e s w i t h benzodiazepines have deS e d a t i v e Hynotics f i n e d t h e phenomenon of "rebound insomnia" which o c c u r s on a b r u p t withdrawal of drug. This phenomenon seems t o be a s s o c i a t e d only with t h e f l u n i t r a z e p a m (&) and nitrazepam (lk) b u t short-acting triazolam o r diazepam n o t w i t h t h e l o n g e r a c t i n g flurazepam

(s),

(u)

fH3

0

-6

b"

Cl

Chap. 3

Anti-Anxiety Agents, A n t i c o n v u l s a n t s

Berger, I o r i o

25

Triazolam (9a)(0.125-1.0 mg) w a s a n e f f e c t i v e sleep-inducer i n c o n t r o l l e d s t u d i e s i n insomniacs, 41,42 p r e s u r g i c a l p a t i e n t s , 43 and g e r i a t r i c s . 44 A t e q u i e f f e c t i v e h y p n o t i c doses i n man, t r i a z o l a m (0.5 mg) caused less performance d e f i c i t s t h a n f l u r a z e p a m " ( 3 0 mg) when t e s t e d t h e f o l l o w i n g morning. 45 An open, m u l t i - c e n t e r s t u d y i n d i c a t e d t h a t temazepam (11)a t 10-30 mg w a s an e f f e c t i v e , w e l l - t o l e r a t e d hypn o t i c . 46 However, s l e e p l a b o r a t o r y s t u d i e s w i t h insomniacs i n d i c a t e d no e f f e c t on s l e e p i n d u c t i o n o r m a i n t e n a n ~ e . ~ ~ Oxazepam (h) a t 15-30 m was judged a u s e f u l h y p n o t i c i n c o n t r o l l e d s t u d i e s i n normal s u b j e c t s " and i n geriatric^.^' Flunitrazepam (&) a t 2 mg was a n e f f e c t i v e h y p n o t i c w i t h few s i d e - e f f e c t s i n p s y c h i a t r i c inpatients. at 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 i n d i c a t e t h a t fosazepam 60 mg i s an e f f e c t i v e s e d a t i v e - a n x i o l y t i c . 5 1 Midazolam (10)a p p e a r s t o be u s e f u l a s a n i.v. i n d u c t i o n a g e n t i n a n a e s t h e s i a . 52 WE 941 at 0.1-0.5 mg was s e d a t i v e - t r a n q u i l i z i n g i n normal v o l u n t e e r s . 53

''

(In) (B)

The oxazolobenzodiazepine CS-430 (11)showed s e d a t i v e - a n x i o l y t i c e f f e c t s i n animal s t u d i e s . 54 A s t u d y on t i s s u e d i s t r i b u t i o n of nitrazepam (&) i n r a t s h a s appeared. 55

(a)

A n t i c o n v u l s a n t s - Clobazam blocked c o n v u l s i v e s e i z u r e s i n mice and baboons.56 The 7-bromobenzodiazepine was t h e b e s t of a series i n p r o t e c t i o n a g a i n s t PTZ and maximal e l e c t r o s h o c k (ME'S)-induced c o n v u l s i o n s i n mice. 57

11 -

10 -

12 -

The t r i a z o l e 12 d e r i v e d from t h e corresponding t r i a z o l o b e n z o d i a z e p i n e by f i s s i o n of t h e Cq-Ng bond was t y p i c a l of a series of such compounds i n a n t a g o n i z i n g c o n v u l s i o n s due t o t h i o s e m i c a r b a z i d e , PTZ, o r MES i n mice, b u t n o t i n r a t s . 58 The 4-hydroxylated m e t a b o l i t e s of t r i a z o l a m 9b and a l p r a z o l a m & p o s s e s s a low o r d e r of a n t i c o n v u l s a n t a c t i v i t y i n c o n t r a s t t o the hydroxylated m e t a b o l i t e s of diazepam. 59 Non-Benzodiazepines

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The u s e of a n x i o l y t i c s i n c l i n i c a l p r a c t i c e h a s been A n x i o l y t i c Agents reviewed. 6 o S e v e r a l c l i n i c a l s t r a t e g i e s designed t o d i s t i n g u i s h anxiol y t i c s from n e u r o l e t i c s f l and t o d i f f e r e n t i a t e t r u e a n x i o l y s i s from s e d a t i v e p r o p e r t i e s 9 0 , 6 2 have been d e s c r i b e d . The u s e of animal models f o r t h e i d e n t i f i c a t i o n , 63 d e l i n e a t i o n of mechanism, 64 and c l i n i c a l progn ~ s i of s ~a n ~ x i o l y t i c drugs has been d e s c r i b e d .

26 -

Sect. I

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CNS Agents

Humber, Ed.

Fenobam (McN-3377, 13), which had a pharmacological p r o f i l e i n a n i mals i n d i c a t i v e of a n x i o l y s i s , showed EEG e f f e c t s i n man more t y p i c a l of psychostimulants.66 Of t h e r e l a t e d p y r r o l i d i n y l u r e a s , & w a s t h e most i n t e r e s t i n g p o t e n t i a l a n x i o l y t i c i n e v a l u a t i o n s i n rodents." a t 1-8 mg,6* lenperone (B) a t 6-40 mg,69 b r o f o x i n e AHR-6134 (15%) 60-150 mg,70 and n a b i l o n e (17) a t 0.5-5 mg d a i l y 7 1 doses were e f f e c t i v e a n x i o l y t i c s . Sch 12679 (18)a t 75-225 mg d a i l y was e f f e c t i v e a f t e r 2-4 weeks of a d m i n i s t r a t i o n i n t h e t r e a t m e n t of a n x i e t y n e u r o s i s . 7 2 Centazolone (19)73 was found t o have p o t e n t t r a n q u i l l o s e d a t i v e hypnot i c , muscle r e l a x a n t and a n t i c o n v u l s a n t p r o p e r t i e s i n animals.34 I n nora 1 s u b j e c t s , t h e drug lacked u n d e s i r a b l e s i d e e f f e c t s i n s i n g l e d o s e s (40 mg) and was s e d a t i n g a t 20 rng.75 The e n d o c r i n e - i n a c t i v e L - n o r g e s t r e l (20) showed a n EEG p a t t e r n t y p i c a l of s e d a t i v e - a n x i o l y t i c s . 76

(16)a t

0

a, X-Cl a, X=F

Chap. 3

Anti-Anxiety Agents, Anticonvulsants

Berger, Iorio

27

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Sedative-Hypnotics The sedative-antihistamine trimeperazine (2) at 20 mg was inferior to nitrazepam (5 mg) as a sleep inducer in a double-blind study 7 7 Clinical trials indicate that zopiclone (RP-27 ,267 , 22) is useful as a hypnotic and is as active as nitrazepam. The compound was active in a variety of animal tests with a profile similar to the benzodiazepines. 7 8 The pyrazole 23,79 triazole & and imidazole Z 8 O all potentiated hexobarbital-induced sleep time.

.

-

Compounds reported to block PTZ and/or MES-induced Anticonvulsant Agents convulsions in mice include the benzenesulfonamide derivatives =,81 26, 8 2 27, 8 3 and 28; 8 4 pyranothiophenes and the related pyranothienopyrimidines, the best of which were Be5and 30;86 oxadiazoles, the best of which were and semicarbazide Lg9and thiosemicarbazide 3490; phenothiazine 35;91 quinazolone 3692; novel structures 3 7 , 9 3 , 38,94 39,95 40, 96 and C9?and the non-nitrogen containing compounds 42, gT43, 9Tand 44 * 100 Xilobam (McN 3113, E)was described as a centrally acting muscle relaxant without anxiolytic or sedative properties. l o 1

I

CHa&ORj

I \

NHR

SO2NH2 27 -

*,

R-H;

SO2WH2

30 -

Ryrn-NO2C684

2, ~ = c O c A 3 ; Ri=OC2H5

28 -

R

31 -

32;

R-P-CH~, 2-0CY3

Sect. I

0 w I

cn2

AC'

I

33 -

P

- CNS Agents

Humber, Ed.

Chap. 3

Anti-Anxiety Agents, Anticonvulsants

Berger, Iorio

29 -

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21.

22 * 23. 24. 25. 26. 27. 28. 29. 30. 31.

32. 33. 3&. 35. 36.

37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56.

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u,

u,

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I - CNS A g e n t s

Humber, Ed.

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15,

67,

m,

Annual Reports in Medicinal Chemistry-I4

Chapter 4 .

31 -

Analgetics, Endorphins and the Opioid Receptor

R. J. Kobylecki and B. A. Morgan, Reckitt & Colman Limited, Pharmaceutical Division, Hull, U.K. Introduction - The increased intensity of research stimulated by the discovery and characterization of the endorphins has been maintained throughout 1978. Significant advances in knowledge of the biosynthesis and neurophysiology of endorphins have been made and the first reports of clinical studies have appeared. In the non-peptide field, exciting discoveries on approaches to novel narcotic antagonists have been reported. The proceedings of several symposial-4 have been published, as well as reviews5-8 on research related to processes involving opioid receptors. Endorphins Biosynthesis - The biosynthesis of endorphins has been the subject of intensive investigation. Pulse-chase methodology has been used to show that in rats pars intermedia,g and mouse pituitary tumor cell line AtT20/D-l6v,10 radio-labelled amino acids are incorporated into an ACTH/endorphin precursor ( 3 1 K precursor). This molecule then degrades to an endorphin-containing molecule and an ACTH-containing molecule; both B-endorphin and B-lipotropin (6-LPH) were identified in the endorphin series. Similar processes have been characterized in mouse anterior pituitary,ll rat pituitary,12 and human non-pituitary tumors. 13 No trace of Leu5-8-endorphin was detected during these studies.12314 Studies in rhesus monkeys have shown that the breakdown of the 'stem hormone' ( 3 1 K precursor) can change with age, giving rise to different relative amounts of metabolites.15 The finding that there is a striking increase of B-endorphin in neonate (12 hrs postpartum) monkey pituitary, possibly rendering the fetus insensitive to the assault of parturition, may prove to have great physiological importance. The induction of a withdrawal-like phenomenon in fetal guinea-pig ileum upon naloxone challenge also points to a physiological role for endorphins in pregnancy. 16 Experimental evidence to support the hypothesis that the primary biosynthetic route to Met5-enkephalin is 6-LPH to B-endorphin and eventually to Met5-enkephalin is still awaited. Recent evidence supports the existence of two separate opioid receptor systems in brain; using a combination of anatomical, biochemical and immunological methods, several groups have differentiated enkephalin neuronal systems from 6-LPH/B-endorphin/ACTH positive systems.17,18 Metabolism - It has been shown that aminopeptidase-induced enkephalin degradation proceeds at the same rate whether or not the peptide is bound to its re~ept0rs.l~The degradation rate, however, is a linear function of substrate concentration regardless of the degree of receptor occupancy. It is suggested that these results indicate that enkephalin binding to opioid receptors is coupled to subsequent degradation. The presence has been reported of a high-affinity peptidase in a particulate fraction of mouse striatum which cleaves Leu5-enkephalin to yield the N-terminal tripeptide (Tyr-Gly-Gly) .20 The selective increase in the activity of this peptidase in mouse striatum after chronic morphine treatment suggests that it might be associated with enkephalinergic transmission. Cleavage of the Gly3-Phe4 bond of the enkephalins has also been demonstrated on incubation Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

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with kidney peptidyl dipeptidase (angiotensin converting enzyme). 21 The specific inhibitor SQ 14225 blocked this process. The metabolism of D-Ala2, Met5-enkephalinamide has been investigated in the mouse. 22 Analgesic activity (tail flick) correlated with brain concentration following i.v. administration of the pentapeptide. It has been shown that Dphenylalanine (250 mg/kg, i.p.) gives rise to significant naloxonereversible antinociception (mouse hot plate test). It is suggested that this effect is due to the inhibition of endogenous enkephalin or endorphin metabalism.23 Pharmacology of Endorphins - The potencies of enkephalin analogs in inhibiting neurotransmission in the cat nictitating membrane,24,25 and the rabbit ear artery,24 have been investigated. The rank order of potency of most analogs on the cat nictitating membrane corresponded with that obtained on the guinea-pig ileum, while results with the rabbit ear artery gave a rank order similar to that obtained on the mouse vas deferens. It has been reported that the histamine-H2 antagonist cimetidine at high concentrations can antagonize the actions of opiates such as Leu5-enkephalin and morphine on the guinea-pig ileum.26 An approach to studying the functional role of enkephalin in the nervous system using tissue cultures of mouse spinal cord neurone has been reported.27 The results obtained suggest that Leu5-enkephalin is capable of acting as a neurotransmitter, neurohormone or neuromodulator. The effects of endorphins on a wide range of pharmacological and physiological processes have been investigated. The finding that endorphins induce a range of cardiovascular effects after central administration suggests their involvement in central control of cardiovascular function.28 The reversal of endotoxin-induced hypotension by naloxone implies that endorphins may have a role in the physiology of shock.29 The detection of large amounts of enkephalin in the gut has initiated studies of their effects on gastro-intestinal processes.30 Both Met5-enkephalin and morphine have been shown to increase histamine-induced gastric secretion and gastric mucosal blood f low,31 and to inhibit induced pancreatic bicarbonate secretion in dogs.32 The reversal of these effects by naloxone suggests a mechanism involving opioid receptors. It has been found that morphine and 6-endorphin exert similar effects on the endocrine pancreas.33 These results indicate the presence of opioid receptors on the islets of Langerhans. The observation that naloxone selectively abolishes over-eating in genetically obese mice and rats, and of elevated 6-endorphin concentrations in the pituitaries of these strains, suggests a relationship between 6-endorphin and obesity. Whether this effect is centrally mediated or is a consequence of interaction with gastrointestinal opioid receptors is unclear.34 Various aspects of the endocrinology of the endorphins have been investigated. A variety of enkephalin analogs have been shown to stimulate prolactin,35-37 and growth hormine,35 secretion in vivo. The function of enkephalin in the transmission of nociceptive stimuli has received much attention. The striking similarity between the anatomical distributions of opioid receptor sites, enkephalin and substance P in brain stem and spinal cord, as well as the naloxone-reversible inhibition of substance P release from rat spinal trigeminal nucleus by a variety of opioid agonists, has led to the hypothesis that enkephalin acts as a presynaptic neuromodulator on substance P terminal~.3~This hypothesis is supported by the report that the spontaneous

Chap. 4

Analgetics, Endorphins

Kobylecki, Morgan

33

firing frequency of nociceptive neurones in cat nucleus caudalis was excited by substance P and decreased by Met5-enkephalinY and that the effects of enkephalin were in most cases blocked by naloxone.39 Similar effects have been observed in cat spinal dorsal horn.40 A s a corollary to this hypothesis, substance P should possess analgesic properties. Recent work has shown that substance P could have a dual action on nociception;41 at low doses substance P (1.25-5 ng/mouse, intracerebroventricularly (i.c.v.)) causes naloxone-reversible antinociception, whereas at higher doses (>50 ng/mouse i.c.v.) this effect is lost. However, at these higher doses substance P produced hyperalgesia when combined with naloxone (this effect being greater than in the naloxone control). These rather complex actions may be explained by substance P releasing endorphins at l o w doses, and, at higher doses, directly exciting neuronal activity in nociceptive pathways. Injection of substance P into the periaqueductal gray matter of rats produces naloxone-reversible analgesia.42 The hypothesis that acupuncture produces analgesia through the release of endorphins has received much attention. This view has been supported by the demonstration that CXBK mice that are deficient in opioid receptors show poor electroacupuncture analgesia43 and by the suppresion of naloxone-precipitated withdrawal symptoms in morphine-dependent mice by electroacupuncture.44 It has been reported that rats trained to discriminate morphine from saline will respond to D-Ala2-enkephalinamide similarly as to an internal cue of morphine.45 Studies in Man - Many reports have appeared on the actions of endorphins in man. The enkephalin analog FK 33-824 (&) has been used in two studies. In a single blind volunteer study, 1 (0.1-1.2 mg, i.m.> was found to be free of effects on respiratory rate and blood pressure but gave rise to a feeling of muscular 'heaviness', and in 50-60% of the subjects, 'anaphylactoid' and gastrointestinal symptoms were reported. Dose-dependent stimulatory effects on the secretion of prolactin and growth hormone were also observed; the effect on prolactin apparently was not abolished by pretreatment with nalorphine. Classical morphine symptoms such as changes in emotional behavior or nausea were not observed.46 The hormonal and metabolic responses to 1 were measured in a double-blind tria1.47 Again the analog had a pronounced endocrine effect, raising prolactin and growth hormone levels and lowering LH, FSH and ACTH levels. Low doses of naloxone partially blocked these effects. The metabolic effects evoked by L w e r e similar to those reported for morphine. Since the discovery of the endorphins, there has been much speculation concerning their significance in psychiatric disorders.48949 If increased levels of endorphins are related to abnormal behavior, then it is conceivable that this behavior might be effected by narcotic antagonists. Naloxone was without effect on mood in normal human volunteers.50 However, recent results suggest that it may attenuate symptoms in a subpopulation of patients with bipolar depression.51 The results of a double-blind cross-over study suggest that naloxone is effective in reducing auditory hallucinations in 'some' schizophrenic patients. 103 Preliminary studies with (des-Tyr1)&endorphin (B-lipotropin 62-67) in schizophrenic patients have indicated that this material has neuroleptic a ~ t i v i t y . ~ * ,Various ~~ aspects of the role of endorphins in human pain perception have been investigated. Both endorphin-like54 and enke~halin-like~~ material have been shown to increase in the cerebrospinal fluid of patients undergoing electrical stimulation procedures for the relief of intractable pain. Although naloxone was

34

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-

CNS Agents

Humber, Ed.

without effect on perception of experimentally produced pain, enhancement of clinical pain perception by naloxone in patients responding to 'placebo analgesia' suggests that this effect is produced by the action of endorp h i n ~ . ~However, ~ , ~ ~ the interpretation of these data has been c r i t i c i ~ e d . ~ ~

L

H-Tyr-D-Ala-Gly-Me Phe-Met (0)-01 H-Tyr-D-Ala-Gly-Phe N-MeMet-NH2 1. 3 H-Arg-Tyr-Gly-Gly-Phe-Met-OH Endorphin Analogs - D-Ala2, N-MeMet 5-enkephalinamide (2) has been found to be a potent parenteral analgesic.59 It was 238 times as potent as normorphine in the mouse vas deferens assay, and four times as potent as morphine in the mouse hot plate assay (jump response) after subcutaneous administration. It is claimed that 2 has relatively little respiratory depressant properties or tendency to cause tolerance or physical dependence. A series of Met5-enkephalin analogs extended at the amino terminus with amino acid residues corresponding to 8-LPH have been described.6o Using the guineapig ileum-myenteric plexus assay, potency was found to decrease dramatically on further extension of the 6-LPH 60-65 sequence 2. The Opioid Receptor Conformation Studies of Enkephalins - Investigation of the conformation of the enkephalins and comparisons with non-peptide opiates has continued. In a review of earlier work, it was noted that several groups have assigned incorrect absolute configurations to morphine and enkephalin.61 An X-ray crystallographic study of Leu5-enkephalin has indicated that the peptide adopts a 8-bend conformation with hydrogen bonds between the tyrosine nitrogen and the carbonyl oxygen of phenylalanine, and, between the phenylalanine nitrogen and the carbonyl oxygen of tyrosine.62 Good correlation between the structural features of Met5-enkephalin and a wide variety of non-peptide opioid drugs is obtained when the peptide is assigned a 8-11' conformation.63 Empirical and quantum mechanical methods have been used to identify low-energy conformations of Met5-enkephalin and its D-ala2 analog which may be related to rigid opioids.64 Receptor Models - Conformational analyses of several non-peptide opioid agonists and antagonists offers supportive evidence for the assumption that the essential difference between them arise from subtle conformational changes in the piperidine ring, leading to different interactions with the amine binding site. 65 Quantum-mechanical calculations of six oxymorphone derivatives showed that a multiplicity of low energy conformations exists for various N-substituted derivatives, possibly explaining essential differences between agonists and antagonists.66 Experimental observation of changing agonist/antagonist ratios in some N-alkyl morphines goes some way towards supporting these studies,67 X-ray examination of seven-membered C-ring homologs 4a-4c, and an N-positional isomer showed that the lone electron pair on the nitrogen of 3 and 4c projects away from, and that of 4a and 4d projects toward the benzene ring.65 As these four compounds have potencies between those of morphine and codeine, it must be concluded that N-lone pair orientation does not necessarily account for structurally induced variations of pharmacological properties.

s,

Chap. 4

Analgetics, Endorphins

Kobylecki, Morgan

35

Opioid Receptor Multiplicity - The investigation of the concept of multiple opioid receptors has received further attention. Structure-activity studies with enkephalin analogs have provided support for the hypothesis that analgesia is mediated by the u-receptor.69 It has been found that analgesia without EEG change is observed after injection of Met5-enkephalin into caudal midbrain periaqueductal gray matter, and that seizures and other EEG changes, without analgesia, are seen following injections into the dorsomedial thalamus. It is suggested that seizures are mediated by &-receptors in the dorsomedial thalamus and analgesia by v-receptors in the periaqueductal gray matter. 70 The finding that D-Ala2 Leu5-enkephalinamide binding sites are more numerous than those of dihydromorphine binding sites supports the hypothesis of multiple opioid receptors. 71 Analgetics Novel Antagonist Effects - There have been a number of very interesting developments in this area. A further study has been reported on antagonist series of structure activity in the 4-(3-hydroxyphenyl)-3-methylpiperidine 5.72 Classical antagonist pharmacophores did not increase antagonist activity. Thus, 2, R=CH2-c-C3Hg, (AD50 0.72 mg/kg s.c.) is a less potent antagonist than 2, R=CH3, (AD50 0.24 mg/kg s.c.) (rat tail heat against morphine), but slightly more potent in the Straub tail test. Both diastereoisomers of the propiophenone analog 1,R=PhCOCH2CHz were antagonists, the (+)-isomer being 206 times the potency of the (-)-isomer. The racemate was without measurable narcotic agonist effect in the guinea-pig ileum, and completely antagonized the effects of morphine at 10 ng/ml. Similarly, 3-f3-methyl substitution in ketobemidone, gave a partial agonist of weak antagonist character (AD50 21 mg/kg (rat)). Antagonism has been detected in a fentanyl analog similarly substituted at the 3 position.73 R 34995, (-)-&, is a very potent agonist (ED50 0.0006 mg/kg (rat tail flick)), whereas the (+)-isomer, R 34994, is only a weak agonist (ED50 2 . 2 mg/kg) which shows significant but short acting (<5 min.) antagonism of fentanylinduced respiratory depression. R 34995, (-)-5,shows a long duration of activity, apparently due to long lasting opioid receptor binding. 74 Very potent pure antagonists are also seen in a substituted metazocine series.75

?

R

O'

R

R4

2

4 CH ,R 2 = H, R 3-R4=-CH CH N(CH )3 2 2 J b, R = H,R2 = H, R3-R4= -CH N(CH )CH22 3 c, R 1 = CH , R 2 = CH ,R3-R4 = -CH2N(CH3)CH21 3 2 3 d, R = CH ,R 23, R3-R4 = -N(CH )CH23 3 1 a, R 1

=

6

Sect. I - CNS Agents

36 -

R1 3 2

d, R = CO (CH2)-c-C H

5 9

=

Humber, Ed.

CH 3

)

(+>-Win 42156, &, is equipotent to naloxone as a phenazocine antagonist (AD50 0.008 mg/kg), the (-)-isomer being twice as potent. (-)-Win 42964, 7b, has a similar profile to buprenorphine but was slightly more potent (0.088 vs 0.11 mg/kg) in the acetylcholine test (mouse), and slightly less potent (0.026 vs 0.0043 mg/kg) in the bradykinin test (rat). (+)-Win 43632, 7c, and (?)-Win 4441, are potent pure antagonists, supported by their binding behavior, their in vitro antagonism of ethylketocyclazocine, and their inactivity in the bradykinin test. Resolution of the cyclazocine analog, &, of buprenorphine showed that only the (-)-series had antagonist activity, N-methyl analogs also had some antagonist character.76 Tropanes, 8, were found to be narcotic antagonists devoid of demonstrable analgesic activities.77

z,

-

Tolerance-Dependence Antagonists such as naloxone78 and naltrexone79 inhibited the development of acute dependence to morphine. Adminstration of morphine alone, or in combination with naloxone, in doses producing differing degrees of antinociception, indicates that the physiological stress leading to tolerance/dependence is proportional to the degree of analgesia experienced and not to the amount of morphine present.78 Studies on the narcotic cue may lead to understanding of mechanisms of tolerance.80 It is shown that the response to narcotics in rats changes with age.81-82 Nociceptive stimulation (rat tail clip) is reported to prevent development of tolerance to fentanyl (0.04 mg/kg) given twice daily over 4 consecutive days. Other Chemical Entities - Morphines-Morphinans - A study of the effect of morphine on castor oil induced increase in intestinal transit shows that the antidiarrheal action of morphine has both central and peripheral components.84 (+)-Naloxone has been prepared,85 and in rat brain binding, guinea-pig ileum, neuroblastoma-x-glioma, and hybrid cell adenylate cyclase assays, it had no more than 10-3 - 10-4 x the activity of (-)-naloxone, and can therefore be used to test the stereospecificity of the effects of the latter. Pellets composed of cholesterol, glyceryl stearate and naltrexone implanted S.C. in rats were found to confer blockade of the analgesic effects of morphine for up to 2 months, offering an improved long-term delivery system €or antagonists.86 A 6 8-N mustard analog of naltrexone, 9, covalently binds to the opioid receptor, conferring narcotic antagonist

Analgetics, Endorphins

Chap. 4

Kobylecki, Morgan

37

activity, detectable up to 3 days, without agonist effects. 3-Plethoxy-8oxamorphinans, 10,show pharmacological properties essentially similar to 9-OH benzomorphans.83

a,R=CH2-c-C CH R

1

=

$5 H ,R1=CH ,R32=H b, R = CH c-C 23 5 3

3

a,> R =(CH2)n

OH

A'

R2

= CH

b) R = ( C H 2 ) n k 4 " 3

c)

,R1=H,R2=CH

R1

2 =

R =H,R=CH

0 (On

3

Benzomorphans - The activities of N-alkoxyalkylnormetazocines, 11 a-b, show large variations,89 and the SAR suggests interactions of high stereospecificity for the receptor environment around the N-substituents. The activity of NIH 8933, L & , is shown not to be due to an impurity or to metabolic hydroxylation in vitro, indicating that an oxygen atom is not essential for interaction of an agonist with the opioid receptor.90 Interesting previously-reported 3-benzazocines possessing nitro groups have been shown to be long-acting antagonists, partially antagonizing up to about 50% of the antinociceptive effect of,20 mg/kg of morphine, for over 4 8 hrs. 91 Substituted Piperidines - The pharmacokinetics of fentanyl have been determined by using radioimmunoassay.92,93 In man, at effective analgetic doses, the action of fentanylmay be sufficiently prolonged to cause a delayed respiratory depression, after apparent recovery from anesthesia.94 Phenobarbitone has been shown to enhance the demethylation of meperidine, to the toxic normeperidine, by enzyme induction.95 Some 2,3-dimethyl-3arylpiperidines, 12, with allyl, CH2-c-C3H5, and dimethylallyl N-substituents show a range of antagonist potencies. The f3-2-methyl derivatives showed antagonism of the effects (respiration, righting reflex, rigidity and analgesia) of fentanyl (0.63 mg/kg, s.c.), with a potency similar to that of nalorphine. Substitution of a para hydroxyl for the meta hydroxyl and 12b acted in the led to a fall in antagonist activity. Isomers single dose suppression test as potent pure antagonists. Aromatic esters of 1-methyl-4-piperidinol have a wide range of activity in the range 0.2-1 One derivative, 13 (0.33 x codeine), is reported x codeine (hot plate).97 to be free of morphine-like physical dependence liability in monkeys. Miscellaneous - Several 5-aryl-2-azabicyclo ( 3 . 2 . 1 . ) octanes, 14,are partial agonists with potencies comparable to morphine, the phenethyl derivatives being the most potent .98 Antagonist pharmacophores (CHZ-C-C~H~, dimethylallyl) resulted in little increase in agonist or antagonist potency, while the furylmethyl derivative was a good antagonist without much increase at 50 and 100 mg/kg/day, fails in analgesic potency. The derivative, to substitute for morphine in rats infused with morphine at 50, 100, 4 x 200 mg/kg/day.

=,

Sect. I

38 -

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CNS Agents

Humber, Ed.

An aryloxazine derivative, 15,is reported to be more potent than morphine in the hot plate and phenylquinone writhing tests, and about equipotent with morphine in blocking acetic acid writhing. It did not lead to &I and &, from a tolerance or dependence in rats.99 Two examples,, have shown series of 1,2,3,4,5,6-hexahydro-l,6-methano-3-benzazocines potencies greater than codeine in the hot plate test, while a third example¶ &, was somewhat less potent.100 Antagonist pharmacophores (CH2-cC3H5,C3H7, allyl, dimethylallyl) did not give antagonists. It is reported J & i and 16b will neither support morphine dependence in single dose that j suppression studies, nor precipitate withdrawal symptoms in morphine addicted non-withdrawn rhesus monkeys. Spiro (tetralin-2,2'-pyrrolidine) and spiro (indan-2,2'-pyrrolidine) derivatives of structure 1 7 showed some activity.101 The best derivatives, R1=H, R2=H, Me, n=2, were less potent than morphine. Some long-acting piperidinospiro derivatives of methadone have potencies up to 212 x methadone and durations of action of up to 20.5 hours. 102

a, R

1

=H,R

2

x

16 -

= PhCH2CH2

a.R=H b, R = CH3 C , R = CH2CH2Ph

-

n = 1,2 R1 = H, OCH3,0H

R

3

0

RL = H, CH

3

References 1. 2. 3.

4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14.

nCentrally Acting Peptides", J.Hughes, Ed., Macmi LLan Press , London, 1978. Vhe Endorbins", Advances i n Biochemical Psychopharmacology Vo1.18, E.Costa and M-Trabucci, Eds., Raven Press, New York, 1978. "Characteristics and Function o f Opioids", J.M. van Ree and L.Terenius, Eds., ELsevier/North Holland, 1978. "Factors effecting the action o f narcotics", M.V. Adler , L.Manara and R.Samanin, Eds., Raven Press , k w ork 1978. SH.. Snyder, AmJ.. sychiat .,135 , 645 (1978). L. Terenius, Ann.Rev. PharmacocToxicol., 18,189 (1 978) Drug Metab. Rev., 2, 255 (1978). R. Guillernin, Science, , 390 (1978). P. Crine, C. Gianoulakis, NG.. Seidah , F. Gossard, PD.. Pezalla,M.lis,and M. Chretien, Proc. Natl. Acad. Sci. USA,. 75 , 4719 (1978). R.E.Mains and B.A.Eipper, J.851. Chem., 23,651 (1978). R.G Allen, .E.Herbert, M.Hinnian H. Shibuya and C.B. Pert, Proc. Natl. Acad.Sci.USA., 75,4972 (1 978) M. Hubinstein, S S t e i n and S. Udentriend, Yroc. Natl. Acad. Sci. USA, 2 , 669 (19781,X.Y. Bertagne, WE.. Nicholson, G.O. Sorenson, 0.S. P e t t e r g i l l , C.O. Mount and O.N. Orb, Proc. Natl. Acad. Sci. USA, 75 5160 (1978). N.G. Seidah, C.GianwLakis, brine, M. Lis, S. Benjannet, R. Routhier and M.Chretien, Proc. Natl. Acad. Sci. USA., 75, 3153 (1978).

T

.

Chap. 4 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29; 30. 31 a 32. 33 34. 35. 36.

0

37. 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.

A n a l g e t i c s , Endorphins

Kobylecki, Morgan

R.E. Silman, 0. Holland, T. Chard P.J. Lowry, J. Hope, J.S. Robinson, and G.D. Thorburn, Nature, 276, 526 (1978). W, Kramer and H. Teschemacher, Eur. J. Pharmacot., 49, 445 (1978). S J . Watson, H. Akil, CW. . Richard I l l and JO . . Barchas, Nature, 275, 226 (1978). F. Bloom, E. Battenberg, J. Rossier, N.Ling and R. Guillemin, P r o c x a t l . Acad. Sci.USA., 75, 1591 (1978). M.Knight and W.A. Klee, J.Biol.Chem., 253 , 3843 (1978). B. Malfroy, J.P. Swerts, A. Guyon, B.Pnoques and J.C. Schwartz, Nature 276 523 (1978). E.G. Erdos, A.R. Johnson and N.T. Boyden, Biochem. Pharmacol,, 27 , 843 1 1 m ) . P.F. von Voigtlander and R.A. Lewis, Res. Commun. Chem. Pathor; Pharmacol., & ,I265 (1978). S. Ehrenpreis, J.E. Comaty and S.B. Miles i n ref. 3, p. 417. A.Z. Ronai 1. Berzetei, J.I. Szekely and S. Bajusz i n ref. 3, page 493. J. Knoll, 1. I l l e s and K. Medzihradszky, J. Pharm. Pharmac., 30 , 394 (1978 I. Takayanagi, Y. Iwayama, and y. Kasuya, J. Pharm. Phanac.,2 , 519 (1978 J.L. Barker, DL.. G r u o l , L.M. Huang, J.H. Neale and T.G. Smith i n ref. 3, p. 87. P, Bolme, K.Fuxe, LF., Agnati, R. Bradley and thies, Eur. J. Pharmacol. 9, 319 (1978). JW . . Holaday and A.I Faden, Nahre, 275, 450 S.J. Konturek, Scand. J. Gastroent., 257 S.J. Konturek, W. Pawlik K.M. Walus,a.H. Coy and A.V. Schatly, Proc. SOC. Expt. Biol.Med., 158 156 (19781, g : Konturek, J. Tasler, M Geszkowski, J. Jaworek, D.H. Coy and A.V. Schally, Gastroenterology, 74 , 851 11978). E. Ipp, R. Dobbs a 2 R.H. Unger, Nature 276, 190 (1978). D.L. Margules, B. Moisset, MJ.. Lewis, k. Shibuya and C.8. Pert Science, 2 0 2 ,988 (1978). C.J. Shaar, R.CA.. Frederickson, N.B. Dininger and L. Jackson. L i f e Sciences, 3, 853 (1978). H.V. Meltzer, RJ.. Miller, R.G. Fessler, M. Simonovic and VS. . Fang, L i f e Sciences, 22, 1931 (19781, B.Brown, PW . . Dettnar, P.R. Dobson,A.G.Lynn,G.Metcalf and B.A.Morgan, J. Pharm. Pharmac.,30, 644 (1978). T.M. Jessell and L.L. Iversen, Nature, 268, 549 (1977). R.K. Anderson, J.P. Lund and E. Puil, Can. J. Physiol. Pharmacol., 6, 216 (1978). J.L. Henry i n ref. 3 p. 103. RC.A. Frederickson, V, &@is, C.E. Harrett and JD. . Edwards , Science, 199, 1359 (1978). J.B. Malick and JM . . Goldstein L i f e Sciences, 23, 835 (1978). J.M. Peets and B. Pomeranz Nafure, 273 , 675 (v78). Y.M. Choy W.W. Tso, K.P. tong, K.C.Gn YF.. Tsang, C.Y. Lee, 0. Tsang and H.L. Wen, Bicchem. biophys. Res. Codn., 82 , 305 qi978). R.E. Chipkin JM . . Steward, D.H.Torris and T.J. Crowley, Pharmacul. Biochem. and Behaviour, 9, 129 (1978). 8. von Graffenried, E. Del Pozo, J. Roubicek, E. Krebs, W. Poldinger, P. Burmeister and L. Kerp. Nature, 272 729 (1978). UA . . Stubbs, A.Jones,Zk.W. Edwards, G. Detitala, WJ. Jeffcoate, S.J. Ratter, G.M. Besser, R.S. B l m and KG.M.M. . Alberti, The Lancet, 1225 (1978). LH . . Lindstrom, E. Widerlov, LM . . Gunne A. Wahlstrom and L. Terenius, Acta. Psychiat. Scand., 2 , 153 (1978). L. Terenius i n ref. 3 .p 143. P. Grevert and A. Goldstein, Science, 199, 1093 (1978). L.A. Judd, OS.. Janowsky, D.S. Segal, Huey i n ref. 3 p. 173. JM . . Van Ree, W.MA.. Verhoeven, HM . . van Praag and 0. Oe Wied, i n ref. 3 p. 181. W.MA.. Verhoeven, H.M. van Praag, P.A. Botter, A. Sunier JM . , van Ree and DD. e Wied. The Lancet, 1046 (1978). H. Akil, DE.. Richardson JD . . Barchas, and C.H.Li.Proc. Natl. Acad.Sci. USA, 2 ,5170 (1978). H. Akil, D.E. Richadsun, J. Hughes and JD . . Barchas Science, 201, 463 (1978). JD . . Levine, N.C. Gordon,R.T.Jones, and H.L. Fields, Nature, 2 r 8 2 6 (1978). J.D. Levine, NC . . Gordon, and H.L.Fietds, The Lancet, 654 A. Goldstein and P. Grevert, The Lancet, 1385 (1978). R.C.A. Fredericksun, E.L. Smithwick and R. Shuman i n ref. 3 p. 215. N. Ling, S. Minick and R. Guillemin. , Biochem. Bi0phys.Re.s. Commun. 83, 656 (1978). BE.. Maryanoff and MJ.. Zelesko, J. Pharm. Sci. 67 , 590 (1978) G.D. Smith and J.F. Griffin. Science, l 9 9 , 1214 r 9 7 8 ) . FH . . Clarke, H. Jaggi and R.A. Lovell, J.Med. Chem., 21, 600 (1978). G.H. Loew and S.K. Burt Proc. Natl. Acad. Sci. USA, 75, 7 (1978). V.M. Kolb, J. Pharmaceufical Sci., 67 999 (1978). GH.. Loew and D.S. Berkowitz. J.MecChem., 1,101, (1978).

r,

m.

(19m.

.

2

40 -

Sect, I

-

Humber, Ed.

CNS Agents

82.

J.I. DeGraw, J.A. Lawson J.L. Crase H.L. Johnson, M. Ellis, E.T. Uyeno, G.H.Loew and D.S. Berkotwitz, ibid., , 415 (19f8). S. Shiotani, T. Kometani, Y.litaka and A. Itai, ibid. , 153 (1978). ayward and G.J. Stacey i n ref.3. J.S. Shaw, M.J. Turnbull, A.S. h t t a , J.J. Gormley, C.1. H. Frenk, B.C.McCarty and J.C. Liebeskind, Science, 200, 335 (1978). M.GC.. Gillan, S.J. Paterson and H.W. Kosterlitz i n 2. 3 ps. 475. D.M. Zimmerman, R. Nickander, J.S. Horng and D.T. Wong ,Nature, 275, 332 (1978). J.E. Leysen, P.M. Laduron and C.J.E. Neimegeers, i n r e f 3 p. 4 x JE . . Leysen, and P.M. Laduron , Arch. Int. Phanacodyn., 232, 243 (1978). WF.. Michne, T.R. Lewis, S.J. Michalec, A.K. Piersdn, Gillan, S.J. Patterson, L.E. Robson and H.W. Kosterlitz, i n ref. 3. p. 197. WF. Michne, J.Med. Chem., 21 , 1322 (1978). R.L. Clarke, A.J. Gambino, KK. Pierson and S.J. Daum, ibid., 21, 1235 (1978). C.-L.Wong and G.A. Bentley, European J. Pharmacol., 47 7 i 5 (1978). H.N. Bhargarva, ibid., 0 , 193 (1978). FC . . Colpaert, C n . Niemegeers and P.A. J. Janssen, Neuropharmac,logy, 12,705 (1978). 1. Paul, J. Diaz and B. Bailey, ibid., , 655 (1978). A. Auguy-Vallette, J. Cros , Ch.Gouarderes, R. Gout and G. Pontonnier, Br.J. Pharmacol.,

83

F.C.

84.

J.J.

67. 68. 69. 70. 71 72. 73. 74. 75.

.

76.

n.

18. 79 I 80.

a.

12

63. 303 (1978).

1. J.

86. 87,

AL.. P.S.

92. 93 94 95. 96. 91. 98. 99. 100. 101. 102. 103. 0

185.

M m .

85.

88. 890 90.

P.

21

Colpaert, -C.J.E. Niemegeers and P.A.J. Janssen, European J. Pharmacol. 4'3, 335 (1978). Stewart, NW . . Weisbrodt and T.F. Burks, J. Pharmacol, Expt. Ther., 2 O t 547 (1978). lijima, J.I. Minamikawa, A.E. Jacobson, A. Brossi, K.L. Rice, and W.Axlee, Med. Chem., 2 398 (1978). Misra and Pontani, J. Pharm. Pharmacol., 2 , 325 (1978). Portoghese, D.L. Larson, J.B. Jiang, A.E. Takemori and TP., Caruso, J.Med. Chem.,

, 598 (1978).

a,

Lambert, J.P. Davis, I. Monkovic and A.W. Pircio, ibid., 423, (1978). H. Merz and K. Stockhaus, i n ref. 3 p. 227. A. Goldstein and A. Naidu , Biochem. Pharmacol. 27 1033 (1978). M.J. Strauss, R.R. Bard and 0. S. Robinson, J d e X them., JZ , 139 (1978). G.J.J. Plomp, R.A.A. Maas, B. Kwakernaak and JM . . van Ree, i n ref. 3, p. 231. R. Schleimer, E. Benjamini, J. Eisele and G. Henderson, Clin. Pharmacol. Ther.,Z3, 188 (1978). AP . . Adams and D.A. Pybus, BritHed. J., 279, (1978). J.E. Stambaugh, W .I . Wainer and 1. Schwartz, J. Clin. Pathol., 487, (1978). M.A. Lorio and AF.. Casy, J.Med. Chem., 3, 812 , (1978). JA.. Waters, ibid., 2,628 (1978). H.H.Ong, V.B. Anderson and J.C. Wilder, ibid., 21 , 758 (1978). J. Hache, W. Oiamantis, D. Sofia and G. Streichenberger, Arzneim.- Forsch. 28 , 642 ( 1 9 7 8 ) P.H. Mazzochi and A.M. Harrison, J.Med. Chem. 21 238 (1978). P.A. Crooks and H.E. Rosenberg, ibid., J2 , 5 6 11978). J.M. Frincke, GL.. Henderson, P.A.J. Janssen and C.AM. . Van der Eycken, ibid., 21 474 (1978). S.J. Watson, P.A. Berger, H. Akil, M.J. M i l l s and J.O. Barchas, Science, 3 (1978).

a,

.

Annual Reports in Medicinal Chemistry-I4

Chapter 5.

41 -

Amino Acid Neurotransmitter Candidates

S . J . Enna, Departments of Pharmacology and of Neurobiology and Anatomy, Univ. Texas Medical School, Houston, Tx 77025

Biochemical, electrophysiological and pharmacological studies indicate that certain amino acids may serve as neurotransmitters or neuromodulators in the mammalian central nervous system (CNS). In spite of the impressive amount of research conducted on this subject, the application of this knowledge to the treatment of CNS disorders has been limited due to the paucity of chemical agents which will, after systemic administration, act in a specific fashion on these neurotransmitter systems. A major reason for the delay in the development of effective therapeutic agents is that, of the sixteen electrophysiologically active amino acids present in the mammalian CNS, there are only two, y-aminobutyric acid (GABA) and glycine, for which potent antagonists are known. Without potent, relatively specific antagonists it is virtually impossible to fully characterize the structure-activity requirements of a neurotransmitter system since specificity of action cannot be demonstrated. Thus, much work remains with respect to the chemistry of these transmitter systems. The present communication is intended as a brief overview of the literature pertaining to amino acid transmitter candidates. Interested readers are urged to consult any of a number of excellent Neurotransmitter candidates may be classified as inhibitory or excitatory, depending upon the electrophysiological response to the agent. Inhibitory transmitters cause hyperpolarization or partial depolarization of nerve cells, inhibiting cell firing, whereas excitatory transmitters cause depolarization sufficient to generate an action potential. With regard to amino acid transmitter candidates, twelve substances have been identified in the mammalian central nervous system which, based on electrophysiological data, may be inhibitory, and four which appear to be excitatory3 (Table 1). However, before a substance can be seriously considered as a neurotransmitter candidate, several criteria, relating to the transport, storage, release and termination of action must be fulfilled. Of the agents listed in Table l , five--taurine, glycine, GABA, glutamic acid and aspartic acid--have been studied sufficiently to indicate the likelihood of a neurotransmitter action. These five will be considered in more detail in the following paragraphs. Inhibitory Amino Acids Inhibitory amino acids are sometimes subdivided into GABA-like and glycine-like since, at present, the action of only these two amino acids can be pharmacologically differentiated from one another. Thus, strychnine (17) antagonizes glycine but not GABA, and bicuculline (18)antagonized GABA but not glycine. However, the action of a number of other amino acids is blocked by strychnine or bicuculline, while the action of some, on certain cells at least, is blocked by both agents. Accordingly, bicuculline and strychnine are referred to as GABA and glycine antagonists, respectively, because these two amino acids are the most sensitive Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

TABLE 1 ELECTROPHYSIOLOGICALLY ACTIVE AMINO ACIDS PRESENT IN MAMMALIAN CENTRAL NERVOUS SYSTEM Amino Acid

Structure

Reference

Inhibitory L-a-Alanine B- Alanine Y-Aminobutyric Acid (GABA) y-Amino-6-hydroxybutyric Acid L-Cystathionine 2,4-Diaminobutyric Acid (DABA) Glycine Hypotaurine Imidazole-4-acetic Acid

CH3'CH(NH2) .COOH 1 H2N*(CH2)2.COOH 2 H2N. (CH2) 3 - COOH 3 H2N'CH2.CH(OH)*CH2*COOH 4 HOOC-CH(NH2) ' CH2.S (CH2);' CH(NH2) COOH H2N' (CH2)2'CH(NH2) 'COOH 6 H2N.CH2.COOH 1 H2N' (CH2)2-S02H S

-

a

HOOC 'CH2

23

2

8 9 10,ll 12 13 14 10,15,16

5 12,17

rn n,

n

rt

H

I

c3

Z

Cn P

09 ID

9

3

R v)

Proline

3

L-Serine Taurine

3

18

Excitatory L-Aspart ic Acid L-Cysteic Acid L-Cysteine Sulphinic Acid L-Glutamic Acid

HOOC.CH2*CH(NH2).COOH 13 H03SSCH2-CH(NH2)*COOH H02S*CH2*CH(NH2)'COOH 15 HOOC. (CH2)2'CH(NH2) 'COOH 16

14

19 19 20 19,21

n, rl

"

M

a

Chap. 5

Enna

Amino Acid Neurotransmitters

43

to the action of these alkaloids. Taurine - A glycine-like, sulphur containing amino acid, taurine (12)is found in reasonably high concentrations throughout the mammalian central nervous system and in heart.22*23 In brain, taurine is formed as a result of the decarboxylation of cysteine sulphinic acid (15)to hypotaurine (g), which in turn is oxidized to form t a ~ r i n e . Like ~ ~ other transmitter candidates, taurine is accumulated and released by brain tissue and the accumulation can be inhibited by ouabain. Clinically, significant alterations in taurine leve15gmay be associated with retin is pigmentosa,27 epilepsy,28 mongolism, and possibly heart disease. Reports have suggested that systemic administration of taurine may be efficacious in treating epilepsy. 31,32 Like glycine, the action of taurine is inhibited by strychnine,8 though in other brain areas, such as the cerebral cortex, its action is blocked by both strychnine and bicuculline.' Thus, unlike glycine and GABA, there is no antagonist for taurine which will differentiate taurine receptors from other inhibitory amino acid receptor sites.

%

17 -

H2N. (CH2)3*S03H

H03S * (CH2) 2' CH (NH2) .COOH

20 -

19 -

In this regard, it is interesting to note that another sulphur containing amino acid related to taurine, 3-amino-1-propanesulfonic acid (N),is a potent, and relatively specific, (bicuculline-sensitive strychnine-insensitive) GABA *o receptor agonist ,33,34 which further suggests a lack of specificity for this class of compounds. Other derivatives such as N-methyltaurine, N-carbamoyltaurine, 4-aminobutanesulfonic acid and the taurine metabolite isethionic acid are very weak 18 or ineffective when a lied to cortical neurones or spinal cord. 33 ,35 The structure-activity relationship of the taurine receptor is further complicated by the fact that two other structurally related amino acids, L-cysteic acid (3) and DL-homocysteic acid (20), are potent excitatory agents.33 Thus, further pharmacological studies to characterize the putative neurotransmitter receptor site for taurine must await the discovery of compounds capable o f specifically inhibiting the action of this substance.

''

Glycine - The simplest amino acid, glycine (I)has a unique distribution within the mammalian central nervous system, being highest in the medulla and spinal cord.37 This finding, along with autoradiographic and electrophysiological studies, have suggested that glycine may ser e y 3 s ;here neurotransmitter primarily in the spinal cord and brain stem.' are three major pathways for glycine synthesis; glycine from glucose serine (11);glycine by way of a ph sphorylated pathway;39 glycine by way of a dephosphorylated pathway. 48 Within the central nervous system glycine does not appear to be catabolized as such, but rather the majority

44 -

Sect. I

-

C N S Agents

Humber, Ed.

El,k!

Glycine i s accumulat is i n c o r p o r a t e d i n t o p r o t e i n s and n u c l e o t i d e s . a h i g h a f f i n i t y t r a n s p o r t system i n t o s p i n a l c o r d , pons and medulla, and h a s bee shown t o b e r e l e a s e d from nervous t i s s u e under a p p r o p r i a t e The m a j o r i t y of e x p e r i m e n t a l evidence s u g g e s t s t h a t , i n t h e s p i n a l c o r d , g l y c i n e is t h e preLominating i n h i b i t o r y n e u r o t r a n s m i t t e r a t synapses on motor neurones, s p i n a l i n t neurones, i n d o r s a l column Also, g l y c i n e may serve a s a n u c l e i and medullary r i c u l a r formation." transmitter i n retina." However, as y e t , t h e r e i s l i t t l e evidence d i r e c t l y l i n k i n g n e u r o t r a n s m i t t e r g l y c i n e t o any s p e c i f i c n e u r o l o g i c a l o r p s y c h i a t r i c d i s o r d e r , though a g l y c i n e a b n o r m a l i t y may be a s s o c i a t e d w i t h s p a s t i c i t y 46

condition^.'^ .

''

I n t r a c e l l u l a r recording s t u d i e s with glyc i n e i n d i c a t e t h a t i t hyperpola z e s neurones and i n c r e a s e s membrane conductance. A number of compounds b l o c k t h e a c t i o n of g l y c i n e , w i t h s t r y c h n i n e b e i n g t h e most p o t e n t . Other g l y c i n e thebaine antagonists include diaboline gelsemine, dendrobine, b r u c i n e and 4-phenyl-4While t h e s e a g e n t s f ormyl-N-methylpiperidine. a r e u s e f u l f o r d i f f e r e n t i a t i n g g l y c i n e from GABA receptors, they w i l l a l s o antagonize the inhibit i o n induced by o t h e r amino a c i d s such a s s e r i n e cystathionine t a u r i n e a n d a and B-alan i n e (2). 8

(a),

21 -

(u),

(z),

(z),

S t u d i e s have shown t h t , when i n c u b a t e d und e r t h e proper c o n d i t i o n s , 'H-strychnine b i n d s i n a s e l e c t i v e f a s h i o n t o s p i n a l cord membranes and CH3O cH3%NCH3 t h i s b i n d i n g a p t p a r s t o be a s s o c i a t e d w i t h glycine receptors. The potency of v a r i o u s amino a c i d s t o i n h i b i t 3H-strychnine b i n d i n g p a r a l l e l s 22 t h e i r e l e c t r o p h y s i o l o g i c a l p o t e n c i e s on s t r y c h n i n e s e n s i t i v e r e c e p t o r s , w i t h g l y c i n e and 8-alan i n e b e i n g t h e most p o t e n t . Also, b i n d i n g s t u d i e s w i t h s t r y c h n i n e have i n d i c a t e d t h a t t h e r e c e p t o r f o r t h e a l k a l o i d may be more r e l a t e d t o t h e i o n conductance mechanism of t h e g l y c i n e r e c e t o r If a t h a n t o t h e r e c e p t o r r e c o g n i t i o n s i t e t o which g l y c i n e s i n g l e i o n channel can be a c t i v a t e d by a number of d i f f e r e n t r e c e p t o r a g o n i s t s a c t i n g a t d i f f e r e n t r e c o g n i t i o n s i t e s , t h i s f i n d i n g may e x p l a i n why s t r y c h n i n e i s c a p a b l e of r e v e r s i n g t h e a c t i o n of a number of amino a c i d s . On t h e o t h e r hand, i t i s a l s o p o s s i b l e t h a t a g e n t s such a s 8-alan i n e , p r o l i n e (10)and t a u r i n e , w h i l e perhaps n o t n e u r o t r a n s m i t t e r s thems e l v e s , have some a f f i n i t y f o r t h e g l y c i n e r e c o g n i t i o n s i t e . The l a t t e r e x p l a n a t i o n would seem most l i k e l y i f t h e antagonism of g l y c i n e by s t r y c h n i n e i s a c o m p e t i t i v e phenomenon, a s h a s been s u g g e s t e d by some workers. 49

attache^.^'

A t p r e s e n t t h e r e a r e no s p e c i f i c g l y c i n e r e c e p t o r a g o n i s t s more p o t e n t t h a n g l y c i n e i t s e l f , and t h e r e are no a g e n t s which c a n s p e c i f i c a l l y a l t e r t h e n e u r o n a l metabolism, uptake o r release of t h i s amino a c i d making i t d i f f i c u l t t o s e l e c t i v e l y m a n i p u l a t e t h i s system.

Chap. 5

Amino Acid Neurotransmitters

Enna 45

GABA - Of all the amino acid neurotransmitter candidates, GABA (2) has been the most comprehensively studied. Numerous reviews have been published concerning the biochemical, physiological and pharmacological actions of G B of the most recent of which appeared in Volume 13 of this serie~.'-'~~~''~ Thus GABA will be discussed only in terms of the most recent findings. Electrophysiological and biochemical studies have provided considerable evidence to suggest that GABA is an inhibitory neurotransmitter in the mammalian central nervous system. In addition, studies have indicated that alterations in GABAergic transmission may un erlie the symptoms of numerous neurological and psychiatric disorders.5'-57 Because of these findings, and because the metabolic pathways for the synthesis and degradation of GABA are well defined, numerous compounds have been synthesized which can affect, quite selectively in some cases, GAFb-yynthesis, degradation, transport and postsynaptic receptor activity. Most of the research in this area has been directed towards finding agents which will enhance GABAergic transmission since agents which decrease GABA function, such as the receptor blocker bicuculline, convulsants. Other GABA receptor blocking agents include picrotoxin, ar amethylenedisulphot etramine58359 and some bicyclophosphate esters.66"f In ddition, the GABA receptor appears to be blocked by certain caprolactams,6' such as 4,6,6trimethyl-B-caprolactam and by the alkaloid shikimin (24) - .63

(z),

With regard to direct acting GABA receptor agonists, much work has been devoted over the past few years to synthesizing and studying isoxazole derivatives related to muscimol (3)and to cyclic amino carboxylic acids, such as isoguvacine (26). 64 The reason for the interest in these structures is that muscimol appears to be significantly mor potent than GABA as a GABA receptor agonist.%5,66 As a result of these studies several new, relatively potent and specific, GABA agonists have been synthesized. However, while these compounds have been invaluable in understanding the pharmacological specificity of the GABA system, there is some question as to whether they will be useful therapeutic agents, because of their low lipid solubility and rapid metabolism after systemic administration.67

mRo; 23 -

0

24 -

25 -

26 -

Recent experiments on the physicochemical properties of the GABA receptor have revealed that there is present on the nerve cell membrane a triton-sensitive substance which interferes with the att,,Ft,nt of GABA to its receptor recognition site. Careful biochemical studies have suggested that this triton-sensitive substance modifies the affinity of the GABA receptor site in a non-competitive manner, suggesting that this substance, or modulator, interacts with a membrane component to produce an allosteric change

46

Sect. I

-

CNS Agents

Humber, Ed.

in the conformation of the receptor site.70 Further experiments indicate that benzodiazepines, like triton, can remove this inhibitory substance, with resultant enhanced attraction of the receptor for GABA. Thus, these findings suggest the possibility that the mechanism of action of the benzodiazepines may be to facilitate GABAergic transmission by removing an endogenous modulator of GABA binding from the vicinity of the recognition site. These biochemical findings support earlier electrophysiological results indicating that benzodiazepines enhance GABAergic transmission .7’-73 Thus significant progress has been made with respect to the pharmacology of the GABAsystem, However, it should be pointed out that electrophysiological and biochemical studies have shown that other endogenous amino acids, such as y-amino-B-hydroxybutyric acid (9, 2,4-diaminobutyric acid (6) and imidazole-4-acetic a id (?), can also interact with the GABA receptor and transport sites1 2 y 7 ‘ though they are less potent than GABA. Clarification as to whether these substances, which are present in only minute concentrations in the CNS, are neurotransmitters in their own right must await the development of agents which will specifically antagonize their act ions. Excitatory Amino Acids Glutamic and Aspartic Acids - These two amino acids will be considered together since they have a similar action and distribution in the central nervous system and since there is as yet no antagonist which can totally differentiate the action of these agents. As opposed to the two other endogenous excitatory amino acid candidates, cysteic acid and cysteine sulphinic acid (g), glutamate (16) a d aspartate (13) are found in abundant quantities in the mammalian ES. 75,76 Metabolically, aspartate and glutamate are related and their metabolism is quite complex, Thus, there are undoubtedly several metabolic pools of glutamate and aspartate, in addition to any neurotransmitter pool, making it difficult to study the biochemical aspects of their neurotransmitter action. Nevertheless, it has been shown that both bstances are accumulated into brain tissue by a high affinity processYyYand thy& both can be released from brain tissue by electrical field stimulation. This behavior is characteristic of neurotransmitters. While the regional distribution is fairly uniform for these two substances, there may be significant differences in the conc trations of aspartate and glutamate in certain areas of the spinal cord.” Furthermore, biochemical studies have suggested that aspartate may be concentrated in the interneurons of the polysynaptic reflex arc,8o and that there may be a s ecific glutamate pathway from the cortex to the corpus striatum. 8?,82 With respect to their postsynaptic action, both glutamate and as ar tate evoke an excitatory response from central nervous system neurons.fg9 21 However, while there are a number of agents which will inhibit the excitatory action of these substances, none will completely differentiate between the two. Thus, glutamate-aspartate antagonists incluce DL-a-

Chap. 5

Amino Acid Neurotransmitters

HOOC * (CH2) 2 * C (CH3) (NH2) * COOH

Enna

47

CH3S(O) (NH). (CH2)2*CH(NH2) *COOH

27 -

28 -

29

-

methylglutamic acid (ZJ), L-methionine-DL-sulfoximine L-glutamic acid diethyl este (29), and 1-hydroxy-3-amino pyrrolidone-2 (30). of these compounds only one, the diethyl ester as an inhibitor may be significantly more PO of glutamate than aspartate,b534 whereas the others are essentially equipotent, making it difficult to differnetiate the action of the two amino acids.

(z),

(z),

HO' 30 -

CHTC I

4

CH3 31 -

32 -

Based on structural and electrophysiological similarities, a number of compounds have been proposed as glutamate agonists. Some of the most CH2COOH potent of these substances are kainic acid (31), ibotenic acid cyclo entane glutamate (33) and quisqualic acid (34). g5986 It is noteworthy that electrophysiological experiments suggest that kainic acid, as opposed to the other compounds, may not activate glutamate receptors directly, but rather may, in some way, allosterically sensitize the rece tor to the action of endogenous glutamate.87,g8 While it has been suggested that these compounds are specific agonists for glutamate vis-s-vis aspartate, such specificity cannot be absolutely proven until agents are available which clearly differentiate the excitatory action of glutamate from aspartate.

(s),

HooCcYooH NH2

33 -

0

II -NaCH2*CH(NH2) 0

34 -

It has recently been discovered that some of the excitant amino acids and their analogues, such as kainic acid, are nerve cell toxins. That is, when administered to immature animals, either generation of glutamate or aspartate causes retinal and hypothalamic cellsi' and, when injected into brain, kainic acid causes irreversible destruction of virtually all neuronal cell bodies around the injection These discoveries may be of immense importance *COOH with respect to understanding the etiology of some neurodegenerative disorders since they suggest that an overabundance of excitatory amino acids in the brain may cause progressive cell l o s s . Therefore,

48

Sect. I - CNS Agents

Humber, Ed.

potent, and specific antagonists for excitant amino acids may be capable of preventing, or delaying, some types of neuronal degeneration. Conclusions Substantial evidence has been accumulated suggesting that certain amino acids may serve as neurotransmitters in brain. However, little is yet known about the pharmacology of these systems. One reason for the delayed progress in this area is that, as opposed to other neurotransmitters such as the catecholamines, the transmitter function of amino acids is only one of perhaps several actions of these agents making it difficult to identify the neurotransmitter pool. The structural and electrophysiological similarities of the amino acids are other hindrances to the study of these compounds. Nevertheless, the possibility that some or all may be neurotransmitters is great enough to warrant continued study since there is evidence suggesting that the pharmacological manipulation of amino acid transmitter systems may be beneficial in a variety of CNS disorders. What makes them especially tempting in this regard is the fact that amino acids appear to act as transmitters only in the CNS. Therefore, drugs which will activate or depress these transmitter systems may have little or no direct action on peripheral organs making them somewhat more specific, and therefore less toxic, than other neuropharmacological agents

.

References

1. 2. 3. 4. 5. 6. 7. 8.

N. Davidson, "Neurotransmitter Amino Acids", Academic Press, New York, N.Y., 1976. G.A.R. Johnston, Ann. Rev. Pharmacol. Toxicol., 18,269 (1978). D.R. Curtis and G.A.R. Johnston, Ergeb. Physiol., 69, 98 (1974). F. Fonnum, Ed., "Amino Acids as Chemical Transmitters", Plenum Press, New York, N.Y., 1978. D.R. Curtis and J.C. Watkins, Pharmacol. Rev., lJ, 347 (1965). F. Kofod, P. Krogsgaard-Larsen and J. Scheel-Krueger, Eds., "GABANeurotransmitters", Munksgaard Publ., Copenhagen, Den., 1979. F.E. Bloom, Neurosci. Res. Prog. Bull., 10,124 (1972). D.R. Curtis, L. Hosli and G.A.R. Jonhston, Exp. Brain Res., 5, 1 (1968).

9. 10.

11. 12.

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Amino Acid N e u r o t r a n s m i t t e r s

Enna

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166,

76,

141,

71,

50

Sect. I

-

CNS Agents

Humber, Ed.

56. 57. 58.

Raven Press, New York, N.Y., 1976, p. 479. B. Meldrum, Int. Rev. Neurobiol., 17, 1 (1975). E. Roberts, Biochem. Pharmacol., 2 % 2637 (1974). J.F. Collins, R.G. Hill and F. Roberts, Proc. Brit. Pharmacol. SOC,

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N.G. Bowery, D.A. Brown and J.F. Collins, Brit. J. Pharmacol.

2398 (1975).

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422 (1975).

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

N.G. Bowery, J.F. Collins and R.G. Hill, Nature, 261, 601 (1976). S.J. Enna, J.F. Collins and S.H. Snyder, Brain Res., 1 2 4 , 185 (1977). D. Kerr, B. Dennis, E. Brecher, R. Prager, A . Ward and T. Duong, Brain Res., 110,413 (1976). D.R. Curtis, J. Davies, C. Game, G.A.R. Johnston and R.M. McCulloch, Brain Res., 63, 419 (1973). P. Krogagaard-Larsen, T. Honore and K. Thyssen, IN "GABA-Neurotransmitters", F. Kofod, P. Krogsgaard-Larsen and J. Scheel-Krueger, Eds, Munksgaard Publ., Copenhagen, Den., 1979, in press. S.J. Enna and S.H. Snyder, Brain Res. 115, 174 (1975). P. Krogsgaard-Larsen, G.A.R. Johnston, D.R. Curtis, C. Game and R.M. McCulloch, J. Neurochem., 25 803 (1975). A. Maggi and S.J. Enna, Neuropharmacol., in press. S.J. Enna and S.H. Snyder, Mol. Pharmacol., 13 442 (1977). D.T. Wong and J.S. Horng, Life Sci., 20, 445 (1977). A. Guidotti, G. Toffano, M. Baraldi, J.P. Schwartz and E. Costa, IN "GABA-Neurotransmitters", F. Kofod, P. Krogsgaard-Larsen and J. Scheel-Krueger, Eds., Munksgaard Publ., Copenhagen, Den., 1979, in press. P. Polc, H. Mohler and W. Haefely, Arch. Pharmacol., 284, 319 (1974). T. Tsuchiya and H. Fukushima, Europ. J. Pharmacol.;@, 4 4 (1978). D.W. Choi, D.H. Farb and G.D. Fischbach, Nature, 269, 342 (1977). S.J. Enna and S.H. Snyder, Brain Res., 100, 8 1 (1975). S.I. Singh and C.L. Malhotra, J . Neurochem. 9, 37 (1962). C.J. Van Den Berg, IN "Handbook of Neurochemistry", Vol 3, A. Lajtha, Ed., Plenum Press, New York, N.Y., 1970, p. 355. W.J. Logan and S.H. Snyder, Brain Res., 42, 413 (1972). H.F. Bradford, Brain Res., 2, 239 (1970). L.T. Graham, R.P. Shank, R. Werman and M.H. Aprison, J. Neurochem., 14, 465 (1967). M.H. Aprison and R. Werman, IN "Neurosciences Research," Vol.1, S.Ehrenpreis and O.C. Solnitzky, Eds., Acad. Press, N.Y.,N.Y., 1968, p.143. P.L. McGeer, E.G. McGeer, U. Scherer and K. Singh, Brain Res., 1 2 8 , 369 (1977). J . Kim, R. Hassler, R. Haug and K. Paik, Brain Res., 370 (1977). S. Haldeman, R. Huffman, K.C. Marshall and H. McLennan, Brain Res., 39, 419 (1972). S . Haldeman and H. McLennan, Brain Res., 45, 393 (1972). H. McLennan and H.V. Wheal, Neurosci., Lett., 8, 5 1 (1978). H. Shinozaki and M Ishida, Brain Res., 4 3 5 (1976). J.C. Hall, T. Hicks and H. McLennan, Neurosci. Lett., 8, 171 (1978). H. Shinozaki and I. Shibua, Neuropharmacol., 13, 1057 (1974). J.W. Olney, 0. Lee and V. Rhee, Exp. Brain Res., 6 1 (1971). J.T. Coyle and R. Schwarcz, Nature, 263, 244 (1976). P.L. McGeer and E.G. McGeer, Nature, 263, 517 (1976).

132,

109,

14,

Annual Reports in Medicinal Chemistw-14

Section I1 Editor:

-

51 -

PHARMACODYNAMIC AGENTS

John Krapcho, Squibb I n s t i t u t e f o r Medical Research, Princeton, N . J . 06540 Chapter 6.

Pulmonary and Anti-Allergy Drugs

Stanley C. B e l l , Wyeth Laboratories, Inc., Radnor, Pa. 19087 Robert J . Capetola and David El. Ritchie, Ortho Pharmaceutical C o r p s Raritan, N . J . 08869

-

Introduction The innnunopathologic events involved i n an asthmatic a t t a c k can be generally c l a s s i f i e d a s a Type I inmediate h y p e r s e n s i t i v i t y r e a c t i o n involving s p e c i f i c antigen bridging r e c e p t i v e IgE immunoglobulins bound t o host mast c e l l s and basophils t h a t culminates i n t h e r e l e a s e of various pharmacologic mediators. 1 The mechanisms responsible f o r the r e l e a s e of these mediators have been extensively reviewed. l-3 Traditionally, drugs used i n the treatment of asthma can be conveniently categorized i n t o 4 classes: methylxanthines, @-adrenergic stimulants, glucocorticoids and a n t i c h o l i n e r g i c s .4 I n recent years, however, the c l i n i c a l e f f i c a c y of cromolyn sodium a s a prophylactic a n t i a l l e r g y agent has stimulated a s u b s t a n t i a l research e f f o r t t o f i n d more potent, o r a l l y a c t i v e i n h i b i t o r s of mediator release. One of t h e p r i mary assay systems designed t o evaluate p o t e n t i a l a n t i a l l e r g i c compounds of t h i s type i s t h e passive cutaneous anaphylaxis (PCA) t e s t i n rats. The f a c t t h a t compounds i n t h e PCA t e s t , e . g . , bufrolin and doxantrazole have been less than promising i n c l i n i c a l t r i a l s of asthma, has shed doubt on t h e capacity of t h i s t e s t t o p r e d i c t c l i n i c a l efficacy. Longer treatment times may be necessary t o achieve c l i n i c a l b e n e f i t with t h i s c l a s s of compounds o r perhaps t h e r e a r e a s yet ill-defined pathologic mechanisms involved i n asthma t h a t a r e unaccounted f o r i n experimental models. Given t h e innnunopathologic events involved i n a l l e r g i c diseases and the multitude of mediators released from t r i g g e r e d s e n s i t i z e d cells, f u t u r e trends f o r exploration of drug a c t i o n designed t o i n t e r r u p t o r r e g u l a t e t h i s sequence of events should include: 1) compounds with t h e capacity t o i n h i b i t mediator r e l e a s e , a s well a s antagonize s p e c i f i c mediators through receptor blockade, i . e . , compounds exhibiting pharmacology, 5 2 ) receptor antagonists with broncho s p e c i f i c i t y , S'ty-the development of s p e c i f i c antagonists of I g E binding t o mast c e l l s a n v o r basophils7 and 4) t h e development of compounds with innnunoregulatory a c t i v i t y capable of influencing t h e synthesis ana/or s e c r e t i o n of IgE molecules.8 Although a formidable task, development of compounds of t h i s nature should lead t o r a t i o n a l therapy f o r a l l e r g i c diseases.

-

I n h i b i t o r s of Mediator Release Several reviews have examined t h e r o l e of mediators i n the pathophysiology of asthma and other a l l e r g i c disorders .9 Numerous pharmacologic agents have been introduced which i n h i b i t t h e mechanisms responsible f o r t h e r e l e a s e of these Their a c t i o n s and c l i n i c a l e f f i c a c y have been compared mediators. Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0-12-040514-8

52 -

Sect. I1

-

Pharmacodynamic Agents

t o the prototype drug of t h i s c l a s s

Krapcho, Ed.

- disodium cromoglycate (DSCG) 1.

Analogs of 1 FPL-52757 (2)and FPL-57787 (2) have been reported 11 t o be o r a l 1 e f f e c t i v e i n c l i n i c a l t r i a l s while 4 was i n e f f e c t i v e i n asthmatics.13 A t e t r a z o l e analog u - 3 4 4 ( 5 ) was 4 times as active a s DSCG i n t h r a t PCA and o r a l l y e f f e c t i v e i n 7 of 14 asthmatic p a t i e n t s lZj15 0 these 7 patients, 6 were RAST p o s i t i v e with high serum IgE levels.f6 Although t h e tetrazolecarbonyl d e r i v a t i v e 6 was very potent i n the r a t PCA t e s t by t h e i.v. route (ED 00.03 d g ) , i t was much less potent by o r a l administration ( E D 5 0 6 1 m 4 7 ) . l 7 BenzoC l i n i c a l i n v e s t i g a t i o n reveals comparable a c t i v i t y t o DSCG. pyrones 1 containing a carboxy group i n t h e 6-position and a 2-pyridyl CG i n t h e rat group i n t h e 2-position had comparable i.p. potency t o PCA t e s t and were o r a l l y a c t i v e a t doses of 5-10 &g.

.

El

R-tetrazole, RjMEt R 40NH-tetrazole, R4=OMe 1 R 32-pyridyl, 5 = C O 2 H

1

W-8011 (8) a 3-hydroxymethyl benzopyrone was a c t i v e i n t h e r a t PCA (IDs0=2 m&g p.0.) but inactive i t r o suggesting that t h e a c t i v e form was 3-carboxy-8-methoxychromone 9

%

Me

The t e t r a c y c l i c pyrone PRD-92-M (Q) had a n i.v. ED of 0.5 xWkg i n t h e rat PCA and wa8 only s l i g h t l y e f f e c t i v e i n t h e 1% mediated PCA Compound 19 a l s o i n h i b i t s t h e e f f e c t s of mediators, i n t h e guinea pig. i.e., histamine, 5-HT, bradykinin SRS-A on guinea pig ileum20 and on cardiovascular responses i n Compounds 2 and 12, r e l a t e d i n

Chap. 6

B e l l , Capetola, Ritchie

Pulmonary Drugs

53

s t r u c t u r e t o 10, were reported t o be not only i n h i b i t o r s of mediator 22 release but a l s o antagonists of phamacologic mediators of anaphylaxis. hdoxami.de tromethamine (U-42585E,

a)i s 25OO X more potent than and i s o r a l l y a c t i v e i n I n c l i n i c a l studies, a appears

DSCG i n t h e r a t PCA test23 administered i.v.,

a s c a r i s s e n s i t i v e monkeys a t 5 m&g.24 t o p r o t e c t e x t r i n s i c asthmatics from a l l e r g e n induced bronchoconstrictioadministered by the a e r o s o l route, and 5 of 10 asthmatic p a t i n t s were protected by lodoxamide given o r a l l y a t doses of 1 and 2 rng. 23

R

CN Me

Me0

R=~c(c%oH)~

&l

'4,

16

R=Et

& X=NH

or 0

(a)

i'he e t h y l ester s reported t o be s i m i l a r i n potency t o t h e t r o methamine s a l t C l i n i c a l t e s t i n g of 14 has shown i t t o e f f e c t i v e l y block antigen bro choprovocation i n asthmatic s u b j e c t s a t o r a l doses of 1 and 3 mg; 27,28 however, higher o r a l doses (10 mg) produced i n t o l e r a b l e s i d e e f f e c t s such as: weakness, nausea, diaphoresis, a f e e l i n g of warmth, and i n 2 cases, vomiting and diarrhea. Furan and pyrrole 2-cyanooxamates a 2 9 were l e s s potent than

(a).2i

a.

A series of 2-~arbethoxypyrimidoquinolines, e x e m p l i f i e d by compound potent o r a l a c t i v i t y 3 m and were up t o 400 X a s potent a s DSCG by t h e i.v. r o u t e i n t h e PCA test.30b

16. had

Bufrolin, ICI74917 (Z), although e f f e c t i v e i n a l l e r g e n induced bronchoconstriction i n asthmatic patients, f 1 t o c l i n i c a l l y improve 4 t h e symptoms of asthma over a 4 week period. f f , 1

H02C H

H

ia

II

20

I

Et

(u),

similar i n structure t o The t e t r a c y c l i c pyridone-2-carboxylic a c i d was e q ~ i p o t e n t 3t o ~ DSCG while a sulfon 1 analog (19)was up t o 8 X more potent than DSCG i n the r a t PCA model.33 A s e r i e s of 3-aminoquinolones (ex, 3)proved t o be less e f f e c t i v e than DSCG in t h e r a t PCA test?

54

Sect. I1

- Pharmacodynamic Agents

Krapcho, Ed.

Using molecular o r b i t a l theory, a s i g n i f i c a n t c o r r e l a t i o n was found between t h e LUMO and t h e b i o l o g i c a l a c t i v i t y i n a series of oxamatem, quinaldic, and benzopyran-2-carboxylic acid& This was r a t i o n a l i z e d i n terms of charge t r a n s f e r s t a b i l i z a t i o n of t h e drug-receptor complex. Numerous xanthone d e r i v a t i v e s have undergone c l i n i c a l investigation.

AH-7725 (3) was comparable t o DSCG i n c l i n i c a l t r i a l s , l l while xanoxic a c i d (22) was reported t o be e f f e c t i v e i n exercise-induced asthma.

24

-9

R1=MeCO;

Rl=Me8;

h 12 were

S=H %=hexyl

a c t i v e by t h e i.p. r o u t e i n t h e Derivatives of 21. i.e. r a t PcA t e s t but i n a c t i v e o r a ~ l y . 3 6 The 6-acet 1 analog 24 was r e p o r t t o b e 6 X as potent as DSCG i n t h e r a t PCA test37 and a 6-sulfonimide, Ru31156 (3) was 263 X more potent than DSCG by t h e i.v. r o u t e i n t h e PCA t e s t . The r i n g nitrogen analog Y-9000 was similar i n a c t i v i t y t o DSCG and i n addition was o r a l l y active.3

38

Several c l i n i c a l t r i a l s have shown k e t o t i f e n HC-2O-5ll (27)t o be an e f f e c t i v e antiasthmatic compound and i n f a c t t h e compound has been marketed i n several European ~ o u n t r i e s , ~ ~ 'I~n 7a d d i t i o n t o having a n t i anaphylacti properties, J ' 2 i s a l s o a potent long a c t i n g H receptor antagonisteE8 Ketotifen has a l s o been shown t o i n h i b i t c-hlP phosphoOxatomide (28)presumably inhibits mediator r e l e a s e from diesterase mast cellsag and demonstrates non-competitive antihistaminic a ~ t i v i t y . 5 ~ ~ 5 ~ I n a large c l i n i c a l t r i a l , 28 was e f f e c t i v e i n a l l e r g i c r h i n i t i ~ . ~ ~

I

H

28 -

A series of a n t i a l l e r g i c pyranenamines was developed with t h e a i d of QSAR.52 The c l i n i c a l candidate of t h i s class of cornpound appears of 0.7 mghg by both the i.v. t o be SW-78929-A (3) which has an I D and o r a l routes. Compound 3 a l s o pro%ced a dose dependent i n h i b i t i o n of immunologic r e l e a s e of h i s t mine and SRS-A from passively s e n s i t i z e d rhesus monkey and canine lung. 73

Pulmonary Drugs

Chap. 6

B e l l , Capetola, Ritchie

55 -

30 The t e t r a c y c l i c pyrazole 2 ( t r a n s form) has an o r a l ED50 of 35 m&g i n t h e rat PCA.54~55 I n c o n t r a s t t o DSCG, 2 exhibited a c t i v i t y f o r 6 hours when doses 4 X higher than t h e ED50 were administered. P r e dose of t h e compound increased i t s effectiveness.

(as )

A s e r i e s of quinazolinecarboxylic a c i d s ha e been synthesized and evaluated a s p o t e n t i a l a n t i a l l e r g i c a g e n t ~ . 5 ~ Compound 2 w a s 10-20 X more potent than DSCG o r doxantrazole intravenously, but i n a c t i v e o r a l l y i n t h e PCA t e ~ t . 5 Compound ~ was 5-10 X more potent than DSCG and also w a s o r a l l y inactive,, Synthesis and biologic evaluat i o n of analogs of a r e i n progress.5b

A series of 4-hydroxy-3-nitrocoumarins had potent a c t i v i t y i n t h e These PPA ( p a s s i v e p e r i t o n e a l anaphylaxis) t e s t (about 10 X DSCG). compounds a l s o antagonized the e f f e c t s of SRS-A57 and one of t h e most with an ED50 of .2 pM. potent analogs w a s OH

-

3.3

-Adrenergic Stimulants The a c t i v i t y , potency, a s e l e c t i v i t y of a Ember of $-adrenergic agonists has been reviewed.P' Sympathomknetic bronchodilator drugs are of considerable value i n t h e treatment of some o b s t r u c t i v e airway diseases but s i d e e f f e c t s , p a r t i c u l a r l y on t h e heart, have prompted t h e search f o r new d e r i v a t i v e s i n recent years with higher $,-& s e l e c t i v i t y . With t h e advent of newer agents with high 82 a c t i v i t y , muscle tremor, which i s S2 mediated, appea54-gf be t h e most cornanon s i d e e f f e c t of long term maintenance therapy. The pharmacology and c l i n i c a l e f f e c t i v e n e s s of f e n o t e r o l (34) has been recently reviewed62 with comparisons made t o isoproterenol orciprenaline and salbutamol C l i n i c a l experience has shown t h a t fenoterol, a highly s e l e c t i v e g2 agonist, i s an e f f e c t i v e bronchod i l a t o r with negligable e f f e c t s on t h e cardiovascular system following

(s)

(a).

(s),

56

Sect. I1

-

Pharmacodynamic Agents

Krapcho, Ed.

a e r o s o l administration of usual therapeutic doses, 62

34, R=3,5-di-OH, 3.5, R=3 ,4-di-OH9

Rl=CHM&~CGH4-~-CH Rl=isopropyl

R=3,5-di-OH9 R -isspropyl 1R=4-OH-3 -CH20H, R l = t -bu t y 1

R=3 -OH-4-NHS02Me, R1=CMe2C%C6H5 R=4-C1, R1=t-butyl

s),

Zinterol (M.79184, which i s s t r u c t u r a l l y similar t o 34? i s a long a c t i n g bronchodilator, less potent than isoproterenol, and not highly s e l e c t i v e f o r 8, receptors. The agent i s o r a l l y e f f e c t i v e a t doses as low as 0.25 mg i n asthmatic p a t i e n t s but s i g n i f i c a n t i n c ase i n pulse rate a t s k e w h a t higher doses of .5 mg were observed." AEchloro analog (2)g ~ reported s t o have a 20 X g r e a t e r D/p1 selectivity than isoproterenol.

&:&

Several papers have described the synthesis and biologic a c t of c y c l i c phenethanolamines ( i. e. tetrahydronaphthalenes, ex. &), X that of isoproterenol. some of which have p2

0€1

40 The synthesis and pharmacologic a c t i v i t y of erythro and threoisomers ok trimethoquinol (42) have been studied and t h e a-hydroxy e r y t h r o isomer 41 was shown t o be a more poten adrenoceptor stimulant than t h e threo isomer but less potent than 42. 89 CHOHCH2NHCHMe2

-

HO

HO

R 41 Rs-CHOH-2,3,4( 0%) -7 42 R=-CH2-2,3,4( OMe)32jd

OH

-6

-?

A quinolone ethanolamine d e r i v a t i v e

s

a i s one of

t h e most s e l e c t i v e The campound, cardiac adrenergic

p2 adrenergic receptor stimulants described t o date.T0, T1 l i k e s a l b u f p o l , i s a p a r t i a l agonist on receptors.

81

-

There i s increasing evidence suggesting a r o l e f o r Anticholinergics cholinergic mediated r e f l e x b onchoconstriction as an e t i o l o g i c f a c t o r i n obstructive lung disease.7

3,n

Ipratropium bromide (Sch 1000, 44) continues t o be of c l i n i c a l i n t e r e s t . The compound has been shown t o be superior t o f e n o t e r o l a t doses of 40 pg as an aerosol. Effectiveness i s diminished a t higher

Chap. 6

Pulmonary Drugs

B e l l , Capetola, Ritchie

57

(s)

doses, p o s s i b l y due t o impairment of mucus t r a n ~ p o r t . 7 ~C1-864 i s a broncho-selective a n t i m u s c a r i n i c agent which i s o r a l l y a c t i v e i n animals and compared t o a t r o p i n e has redu ed e f f e c t s on t h e CNS, h e a r t , s e c r e t o r y mechanisms, and p u p i l diameter.

E

Me N‘’

+

CHMe2

Me0 44 -

-

5

Phosphodiesterase I n h i b i t o r CK-0383 ( 4 6 ) was 170 X more p o t e n t and more s e l e c t i v e than theophylline t e s t e d i n guinea p i g t r a c h e a and atrium i n v i t r o p o s s i b l y i n d i c a t i n g phosphodiesterase s e l e c t i v i t y . 75

H

5

X

!I

I

.c1 J

7-Acyl d e r i v a t i v e s of theophylline were prepared as prodrugs. 76 The compound of choice was 7,7’-succinylditheophylline. A t r i c y c l i c h e t e r o c y c l e ( M . J . 12504, 3 )described as having polypharmacology, i s r e p o r t e d t o be more p o t e n t than t h e o p h y l l i n e i n t h e r a t and t o have less CNS and c a r d i o v a s c u l a r s i d e e f f e c t s . The compound a l s o i n h i b i t s mediator r e 1 e a s e . n

-

S e v e r a l r e p o r t s i n d i c a t e t h a t t h e products of a r a c h i Prostaglandins donic a c i d metabolism, i. e., prostaglandins, p r o s t a c y c l i n s and thromboxanes a e involved i n t h e pathophysiology of o b s t r u c t i v e lung d i s e a ~ e . 7 ~ Prostaglandins -~~ of t h e E s e r i e s a r e bronchodilators while prostaglandins of t h e F s e r i e s a r e bronchoconstrictors. S y n t h e t i c prostaglandins similar t o PGE continue t o be of p o t e n t i a l reported i n t e r e s t i n asthma therapy. The s u l f u r containing analog 48 t o be an a c t i v e bronchodilator but much l e s s p o t e n t than PGE1.

w8f

Recent s t u d i e s have suggested that p r o s t a c y c l i n ( PG12) and thromi n h i b i t t h e e l e a s e of histamine and SRS-A from lung boxane (T+) t i s s u e during anaphylaxis.82 These f i n d i n g s may i n d i c a t e a modulating r o l e i n asthma f o r t h e s e compounds.

Sect. I1 - Pharmacodynamic Agents

58 -

Krapcho, Ed.

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I. Hurtado, V.E. Ma>quez and M.J. V
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Sunday and D.J.

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Annual Reports in Medicinal Chemistry-I4

Chapter 7 .

61 -

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

W. L e s l e y Matier and W i l l i a m T . Comer, Mead Johnson P h a r m a c e u t i c a l s Evansville, Indiana

I n t r o d u c t i o n - The s e a r c h f o r new a n t i h y p e r t e n s i v e s and f o r a b e t t e r unders t a n d i n g of e x i s t i n g o n e s r e m a i n s q u i t e i n t e n s e w i t h emphasis t h i s p a s t y e a r on 8 - b l o c k e r s ( s e e C h a p t e r 9 ) and on a g e n t s t h a t a f f e c t t h e r e n i n a n g i o t e n s i n s y s t e m . D i u r e t i c s were reviewed l a s t y e a r and are n o t i n c l u d e d i n t h i s c h a p t e r . S t r u c t u r e s of d r u g s mentioned i n t h i s review are shown o r may b e found i n r e c e n t volumes of Annual R e p o r t s i n M e d i c i n a l Chemistry. r e f l e c t a d e f i n i t e a d o p t i o n by G e n e r a l - C l i n i c a l p a p e r s and reviews'-' p r a c t i t i o n e r s of t h e " s t e p p e d - c a r e " a p p r o a c h t o d r u g s e l e c t i o n mentioned l a s t y e a r . T h i s e m p i r i c a l a p p r o a c h t o t h e r a p y d o e s n o t i d e n t i f y t h e underlying pathophysiology i n individual p a t i e n t s but i s useful f o r c o n t r o l l i n g In c o n t r a s t , some c l i n i c i a n s a d v o c a t e t h e blood p r e s s u r e of most p a t i e n t s . p r o f i l i n g of p a t i e n t s by measurement of hemodynamic and hormonal parame t e r s ; t h e r a p y i s t h e n p r e s c r i b e d a c c o r d i n g t o t h e mechanism r e s p o n s i b l e f o r t h e e l e v a t e d blood p r e s s u r e . "Renin-prof i l i n g " i d e n t i f i e s p a t i e n t s w i t h r e n i n - d e p e n d e n t and t h o s e w i t h volume-dependent h y p e r t e n s i o n ; '/ t h e f o r m e r are t h e n t r e a t e d f i r s t w i t h @ - b l o c k e r s o r i n h i b i t o r s o f t h e r e n i n a n g i o t e n s i n s y s t e m , and t h e l a t t e r w i t h d i u r e t i c s . However, t h i s a p p r o a c h i s n o t universally accepted o r successful. Measurement o f s y m p a t h e t i c n e r v o u s a c t i v i t y by r e c e n t l y d e v e l o p e d , h i g h l y s e n s i t i v e a s s a y s f o r n o r e p i n e p h r i n e (NE) l e v e l s may h e l p d e t e r m i n e t h e e t i o l o g y and b a s i s f o r t h e r a y i n t h e 30-40% of e s s e n t i a l h y p e r t e n s i v e s who are " h y p e r a d r e n e r A p a r t i c u l a r l y good c o r r e l a t i o n h a s been found between b l o o d gic. I t g , l o p r e s s u r e and a c o m b i n a t i o n of plasma NE l e v e l s and r e a c t i v i t y t o exogenous NE. l 1 An improved p r o g n o s i s f o r h y p e r t e n s i v e p a t i e n t s may depend on t h e t y p e of d r u g used f o r t r e a t m e n t i n a d d i t i o n t o good blood p r e s s u r e c o n t r o l . Thus, t h e i n c i d e n c e of f a t a l and n o n - f a t a l c o r o n a r y h e a r t d i s e a s e i s rep o r t e d t o b e s i g n i f i c a n t l y lower i n p a t i e n t s t r e a t e d w i t h a 8 - b l o c k e r t h a n i n a c o n t r o l group.12 However, u s e of p r o p r a n o l o l , i n c o m b i n a t i o n w i t h a t h i a z i d e , r e d u c e s serum h i g h d e n s i t y l i p o p r o t e i n c h o l e s t e r o l and i n c r e a s e s serum t r i g l y c e r i d e s and u r a t e more t h a n methyldopa and t h i a z i d e , o r t h i a z i d e a l o n e ; t h e s e biochemical changes are c o n s i d e r e d t o b e r i s k f a c t o r s f o r c o r o n a r y h e a r t d i s e a s e . l 3 I n r a t s , v a s o d i l a t o r s s u c h as m i n o x i d i l c a u s e c a r d i a c h y p e r t r o p h y which may b e p r e v e n t e d o r r e v e r s e d by methyldopa b u t n o t by p r o p r a n o l o l . I n c r e a s e d c o l l a g e n b i o s y n t h e s i s , due t o h i g h blood p r e s s u r e , c a u s e s s t r u c t u r a l c h a n g e s i n blood vessel w a l l s l e a d i n g t o a n i n c r e a s e d w a l l f l u m e n r a t i o and a l t e r a t i o n of v a s c u l a r e l a s t i c i t y which i s d i f f i c u l t t o reverse; l 5 c h l o r o t h i a z i d e and r e s e r p i n e p r e v e n t o r r e d u c e the collagen formation. l 6 A d r e n e r g i c B l o c k i n g Agents - An e x c e l l e n t r e v i e w of t h e u s e of 8-blocking agents i n hypertension has appeared. Propranolol, t h e sole 8-blocker a v a i l a b l e i n t h e U.S. s i n c e 1967, h a s now been j o i n e d by t h e c a r d i o s e l e c t i v e a g e n t m e t o p r o l o l (LopressorB) which w a s approved f o r u s e i n h y p e r t e n s i o n i n August, 1 9 7 8 . 1 8 A second & b l o c k e r , t i m o l o l (Timoptic@), a l s o rec e i v e d FDA a p p r o v a l b u t o n l y f o r t h e t r e a t m e n t of glaucoma.19 A n i m a l Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

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s t u d i e s on n a d o l o l show no e v i d e n c e of t o x i c i t y o r c a r c i n o g e n i c i t y 2 ' and t h i s a g e n t , a s w e l l as p i n d o l o l , i s expected t o r e c e i v e marketing a p p r o v a l soon.21 The u s e of 8-blockers i n h y p e r t e n s i o n and o t h e r d i s e a s e s t a t e s is d i s c u s s e d i n Chapter 2.

A l l of t h e $-blockers now i n common c l i n i c a l u s e worldwide s e e m t o be e f f e c t i v e i n lowering blood p r e s s u r e b u t t h e mechanism of t h e i r a n t i h y p e r t e n s i v e a c t i o n i s s t i l l u n c e r t a i n . l 7 Although @ - b l o c k e r s i n h i b i t r e n i n release from t h e kidney, probably by an a c t i o n on $ 1 - r e c e p t o r s 2 2 , 2 3 t h e r e i s g e n e r a l l y o n l y a poor c o r r e l a t i o n between d e c r e a s e i n plasma r e n i n a c t i v i t y (PRA) and blood p r e s s u r e d e c r e a s e d u r i n g t h e r a p y . Recent s t u d i e s show a much b e t t e r c o r r e l a t i o n w i t h sympathetic r e s p o n s i ~ e n e s s o~r~ w i t h d e c r e a s e i n sympathetic n e r v e a c t i v i t y (SNA).25-28 T h i s d e c r e a s e i n SNA may b e a n i n d i r e c t r e f l e x e f f e c t due t o dampening 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 o r t o a d i r e c t c e n t r a l a c t i o n . There i s now good e v i d e n c e f o r t h e e x i s t e n c e of c e n t r a l B - r e c e p t o r ~ , ~b u~t , ~ only ~ i n d i r e c t evidence f o r t h e i r involvement i n t h e a n t i h y p e r t e n s i v e a c t i o n of 6-blockers. Direct injection of @-blockers i n t o t h e b r a i n r e s u l t s i n blood p r e s s u r e l o w e r i n g b u t t h i s is n o t n e c e s s a r i l y due t o blockade of B-receptors. 3 0 y 3 1 The a n t i h y p e r t e n s i v e potency of s i x 8 - a n t a g o n i s t s i n man c o r r e l a t e s s i g n i f i c a n t l y w i t h t h e i r a b i l i t y t o penetrate i n t o r a t b r a i n s , but t h i s is n o t conclusive evidence f o r a c e n t r a l mechanism of a c t i o n . 3 2 A v a s o d i l a t i n g a c t i o n of t h e @ - b l o c k e r s a l p r e n o l o l , 3 3 p r o p r a n ~ l o l ~ ~ and o x p r e n o l 0 1 ~h~a s been demonstrated b u t i s probably n o t s i g n i f i c a n t a t t h e r a p e u t i c a l l y u s e f u l d o s e s . I n c o n t r a s t , t h e h y p o t e n s i v e a c t i o n of lab e t o l o l i s p r i m a r i l y due t o a r e d u c t i o n i n p e r i p h e r a l v a s c u l a r r e s i s t a n c e due t o i t s s u b s t a n t i a l a-blocking component i n a d d i t i o n t o a 8-blocking a c t i o n . 3 6 The a c t i o n s of l a b e t o l o l a t t h e membrane l e v e l have been demons t r a t e d . T h i s drug i n h i b i t s t h e b i n d i n g of t h e s p e c i f i c l i g a n d s (-)-[3H]d i h y d r o a l p r e n o l o l and [ 3H] -dihydroergocryptamine t o 6- and a - a d r e n o c e p t o r s of r a t h e a r t and l i v e r p r e p a r a t i o n s and h a s a 10-fold h i g h e r a f f i n i t y f o r ~ pharmacology and t h e r a p e u t i c u s e of t h e 8- t h a n f o r t h e a - r e ~ e p t o r s . ~The l a b e t o l o l h a s been reviewed. 3 8 A s t r u c t u r a l analogue of l a b e t o l o l , RMI81968 ( l ) , i s r e p o r t e d t o have a d i r e c t v a s o d i l a t i n g a c t i o n i n a d d i t i o n t o a- and B-blocking a c t i o n s . 3 9 A s i m i l a r s t r u c t u r e , WIN-40,808-7 ( 2 ) , exhib i t s both $-antagonist and d i r e c t v a s o d i l a t i n g a c t i o n s b u t no a-blockade. I t lowers blood p r e s s u r e i n r e n a l h y p e r t e n s i v e dogs a t 25 mg/kg/day w i t h l i t t l e change i n h e a r t r a t e . 4 0

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2 The v a s o d i l a t i n g and 8-blocking a c t i o n of 4-(2-imidazolyl)phenoxy propranol amines w a s r e p o r t e d l a s t y e a r . A d d i t i o n a l s t u d i e s show t h a t s u b s t i t u t i o n or t h o t o t h e i m i d a z o l i n e group r e d u c e s b o t h t h e d i r e c t v a s o d i l a t o r and i n t r i n s i c sympathomimetic potency.

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The u s e of a-blockers i n t h e t r e a t m e n t o f h y p e r t e n s i o n h a s l o n g been i n disrepute However, t h e d i s c o v e r y of t h e unique a-blocking a c t i o n s of p r a z o s i n h a s g e n e r a t e d renewed i n t e r e s t i n t h i s form of t r e a t m e n t . The pharmacological and c l i n i c a l e f f e c t s of t h i s drug have been reviewed.42 I n comparison t o phentolamine i n a n i m a l s , p r a z o s i n is a more p o t e n t hypot e n s i v e a g e n t , i t h a s less p o t e n t i a l f o r o r t h o s t a t i c h y p o t e n s i o n and i s more s e l e c t i v e f o r p o s t - s y n a p t i c a - r e ~ e p t o r s . ~The ~ l a c k of a d i r e c t v a s o d i l a t o r a c t i o n f o r p r a z o s i n h a s been confirmed i n r a t s , r a b b i t s , and d o g s . 4 4 ~ 4 5 C o n s i d e r a b l e e f f o r t s c o n t i n u e t o t r y t o e x p l a i n t h e absence of r e f l e x c a r d i a c and r e n i n e f f e c t s a f t e r t h i s drug. These e f f e c t s are g e n e r a l l y a t t r i b u t e d t o p r a z o s i n ' s s e l e c t i v e blockade of p o s t - s y n a p t i c ar e c e p t o r s as h a s been shown i n r a b b i t pulmonary a r t e r y and i n However, i n dogs and c a t s , t h i s s e l e c t i v i t y f o r p o s t - o v e r p r e - s y n a p t i c rec e p t o r s i s much l e s s and may n o t e x p l a i n t h e l a c k of c a r d i o s t i m u l a t i o n i n t h e s e s p e c i e s and i n man.49,50 A l t e r n a t i v e l y , t h e l a c k of t a c h y c a r d i a may b e due t o i n h i b i t i o n of t h e enzyme dopamine 8-hydroxylase. 5 1 C l i n i c a l s t u d i e s w i t h t h e p r a z o s i n a n a l o g u e , t r i m a z o s i n , and t h e a - b l o c k e r , i n d o r m i n e , have demonstrated a n t i h y p e r t e n s i v e a c t i o n s i n p a t i e n t s , w i t h o u t The l a t t e r drug shows s e l e c t i v i t y s i m i l a r t o p r a z o s i n tachycardia. 5 2 3 5 3 f o r p o s t - s y n a p t i c a - r e c e p t o r s . 54 955 I n h i b i t o r s of t h e Renin-Angiotensin System - T h i s area w a s reviewed i n det a i l i n t h e p r e v i o u s Annual R e p o r t s . Other u s e f u l reviews on t h e r e n i n a n g i o t e n s i n system ( U S ) , 5 6 ~ 5 7f a c t o r s i n f l u e n c i n g r e n i n r e l e a s e Y 5 *t h e r a t i o n a l e and SARs f o r a n g i o t e n s i n c o n v e r t i n g enzyme (CE) i n h i b i t o r s , 59 and a n i m a l pharmacology60 of c a p t o p r i l have appeared. Of major i n t e r e s t d u r i n g t h e p a s t y e a r h a s been t h e f i r s t r e p o r t s of c h r o n i c t r e a t m e n t of h y p e r t e n s i v e p a t i e n t s w i t h t h e o r a l l y a c t i v e CE i n h i b i t o r , c a p t o p r i l A s e x p e c t e d , t h i s drug i s v e r y e f f e c t i v e i n r e n o v a s c u l a r , malig(2).61-64 n a n t , and e s s e n t i a l h y p e r t e n s i v e s c h a r a c t e r i z e d by h i g h r e n i n l e v e l s . S u r p r i s i n g l y , however, i t is a l s o e f f e c t i v e i n p a t i e n t s w i t h normal r e n i n l e v e l s and even i n some low r e n i n p a t i e n t s . The d o s e s of c a p t o p r i l used i n t h e s e s t u d i e s were 50-100 t i m e s t h o s e p r e v i o u s l y shown t o be r e q u i r e d t o block t h e c o n v e r s i o n of a n g i o t e n s i n I t o a n g i o t e n s i n 11, s o i n h i b i t i o n of t h e CE may n o t c o m p l e t e l y account f o r i t s a n t i h y p e r t e n s i v e a c t i o n . 6 5 C a p t o p r i l w a s w e l l t o l e r a t e d by most p a t i e n t s b u t r a s h and f e v e r o c c u r r e d i n some p a t i e n t s . P a t i e n t s w i t h v e r y h i g h i n i t i a l PRA may b e e x t r e m e l y s e n s i t i v e t o f3 HSCH2CHC-N blockade of t h e RAS and a t l e a s t one e p i s o d e of symptoCOOH m a t i c h y p o t e n s i o n h a s o c c u r r e d a f t e r c a p t o p r i l i n a volume-depleted p a t i e n t . 61 These c l i n i c a l s t u d i e s , as 0 w e l l as animal s t u d i e s ,60 g e n e r a l l y s u p p o r t t h e view -3 XC' H2 t h a t t h e major mechanism of t h e a n t i h y p e r t e n s i v e a c t i o n 4 x-s of c a p t o p r i l i s by s u p p r e s s i o n of t h e RAS w i t h consequent d e c r e a s e i n a n g i o t e n s i n 11. However, p o t e n t i a t i o n of t h e p h y s i o l o g i c a l v a s o d i l a t o r b r a d y k i n i n , from r e n a l o r e x t r a r e n a l s o u r c e s , may a l s o c o n t r i b u t e t o i t s a ~ t i o n . ~I n~ f,a c~ t ,~ t h e hemodynamic e f f e c t s of c a p t o p r i l are s i m i l a r t o t h o s e produced by v a s o d i l a t o r s t h a t a c t on b o t h v e i n s and a r t e r i e s . 6 8

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F i r s t r e p o r t s on t h e p h a r m a c o k i n e t i c s of c a p t o p r i l i n man i n d i c a t e t h a t i t i s w e l l absorbed o r a l l y and i s e x c r e t e d l a r g e l y unchanged w i t h a h a l f - l i f e of 0.9 hr." The d i s u l f i d e i s t h e o n l y m e t a b o l i t e i d e n t i f i e d s o f a r i n man and i n m i ~ e . ~ ' , ~ ~

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o r a l l y a c t i v e CE i n h i b i t o r , SA-291 ( 4 ) , i s t h e most p o t e n t of t h i a z o l i d i n e a n a l o g u e s of ~ a p t o p r i l . The ~ ~ SAR a p p e a r s s i m i found f o r c a p t o p r i l and SA-291 shows t h e same s p e c t r u m o f c a p t o p r i l i n a n i m a l s . 72

The n o n a p e p t i d e t e p r o t i d e (SQ 20,881) a l s o i n h i b i t s CE b u t i t h a s l i m i t e d c l i n i c a l p o t e n t i a l s i n c e i t i s n o t o r a l l y a c t i v e . However, t h i s drug i s v e r y u s e f u l f o r d i a g n o s t i c p u r p o s e s i n r e n i n p r o f i l i n g and f o r emergency t r e a t m e n t o f p a t i e n t s w i t h m a l i g n a n t h y p e r t e n s i o n . 5 6 It h a s been used s u c c e s s f u l l y , by i . v . , i . m . , and S . C . a d m i n i s t r a t i o n f o r 4 months t o t r e a t h i g h r e n i n h y p e r t e n s i o n r e s i s t a n t t o o t h e r d r u g s . 7 3 The a n g i o t e n s i n I1 a n t a g o n i s t s a r a l a s i n ( 'Sar-aAla-ang 1 1 ) i s a l s o u s e f u l f o r r e n i n p r o f i l i n g b u t i s l e s s p r e d i c t a b l e t h a n t e p r o t i d e b e c a u s e of i t s p a r t i a l a g o n i s t a c t i o n . 5 6 S a r a l a s i n i s o n l y e f f e c t i v e i n l o w e r i n g blood p r e s s u r e d i r e c t l y dependent on p r e t r e a t m e n t PRA. T h i s d r u g may i n c r e a s e p r e s s u r e i n some p a t i e n t s o r c a u s e s e v e r e h y p o t e n s i o n i n p a t i e n t s s t r o n g l y dependent on a n g i o t e n s i n I1 f o r blood p r e s s u r e m a i n t e n a n ~ e,75 . ~ ~ Comparis o n of t e p r o t i d e and s a r a l a s i n i n s a l t - d e p l e t e d r a t s f a i l e d t o show any d i f f e r e n c e i n t h e i r blood p r e s s u r e l o w e r i n g e f f e c t s and no a d d i t i o n a l l o w e r i n g o c c u r r e d when t h e y were a d m i n i s t e r e d t o g e t h e r , s u g g e s t i n g t h a t b o t h f u n c t i o n s p e c i f i c a l l y t o d i r e c t l y , o r i n d i r e c t l y , p r e v e n t vasocons t r i c t i o n due t o a n g i o t e n s i n I I . 76 An i n t e r e s t i n g h y p o t h e s i s p r o p o s e s t h a t t h e blood p r e s s u r e d e c r e a s e , f o l l o w i n g CE i n h i b i t o r s o r a n g i o t e n s i n I1 a n t a g o n i s t s , i s caused by a v a s o d i l a t o r a c t i o n of r e n i n i t s e l f on vasc u l a r w a l l s . T h i s a c t i o n is o n l y unmasked when o p p o s i n g v a s o c o n s t r i c t o r e f f e c t s of a n g i o t e n s i n I1 are b l o c k e d . 7 7 C e n t r a l l y A c t i n g Drugs - E v i d e n c e , s u p p o r t i n g t h e v i e w t h a t d r u g s s u c h a s c l o n i d i n e and a-methyldopa r e g u l a t e blood p r e s s u r e by a n a c t i o n on c e n t r a l a - r e c e p t o r s , h a s been r e ~ i e w e d . ~ ' A s u r v e y o f c l i n i c a l s t u d i e s on amethyldopa show t h a t i t c a u s e s v e r y f a v o r a b l e hemodynamic e f f e c t s compared t o B-blockers; i t l o w e r s blood p r e s s u r e m a i n l y by d e c r e a s i n g t o t a l p e r i p h e r a l r e s i s t a n c e w i t h l i t t l e e f f e c t on h e a r t r a t e o r c a r d i a c o u t p u t and u s u a l l y a d e c r e a s e i n PRA.79980 I n a l a r g e group of p a t i e n t s t r e a t e d w i t h methyldopa, t h e major s i d e e f f e c t s were e x c e s s i v e h y p o t e n s i o n and drowsiA p r o d r u g 5 w a s 3 . 2 t i m e s a s p o t e n t a s methyldopa i n SH r a t s , ness.'l p r o b a b l y due t o b e t t e r o r a l a b s o r p t i o n , b u t t h i s i n c r e a s e d potency d i d n o t c a r r y o v e r t o man." The c l i n i c a l d o s e of c l o n i d i n e i s a b o u t 0.3-1.5 mg/day and p a t i e n t s may b e res i s t a n t t o i t s a c t i o n s a t h i g h e r d o s e s , prob a b l y due t o i t s a - s t i m u l a n t , v a s o c o n s t r i c t o r a c t i o n opposing t h e c e n t r a l d e p r e s s o r effect^.'^ A f t e r i.v. i n j e c t i o n of t h i s d r u g i n p a t i e n t s t o g i v e plasma l e v e l s of 0.1-2.2 ng/ml, c l o n i d i n e caused d o s e - r e l a t e d d e c r e a s e s i n blood p r e s s u r e ; however, a t plasma l e v e l s g r e a t e r t h a n 1 0 ng/ml no h y p o t e n s i v e a c t i o n w a s o b s e r v e d . 8 4 S i m i l a r l y , i n r a t s , c l o n i d i n e caused d o s e - r e l a t e d d e c r e a s e s i n blood p r e s s u r e a t 1-10 pg/kg i.v., b u t no a d d i t i o n a l e f f e c t w a s found a t h i g h e r d o s e s . 8 5 Plasma l e v e l s o f c l o n i d i n e a f t e r s i n g l e o r m u l t i p l e d o s e s a l s o c o r r e l a t e d w e l l w i t h s e d a t i o n and d r y mouth c a u s e d by t h e drug.86 M e t a b o l i t e s p r o b a b l y p l a y l i t t l e p a r t i n c l o n i d i n e ' s e f f e c t s s i n c e i t i s e x c r e t e d m a i n l y unchanged i n man; however, i n r a t s , and

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e s p e c i a l l y i n d o g s , i t i s e x t e n s i v e l y m e t a b o l i z e d by h y d r o x y l a t i o n i n t h e a r o m a t i c and i m i d a z o l i n e r i n g s . 8 7 Rebound h y p e r t e n s i o n o c c u r s i n a small p e r c e n t a g e of p a t i e n t s a f t e r sudden 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 . 88 Recent s t u d i e s i n man8’, and rats9’ s u p p o r t t h e view t h a t t h i s e f f e c t i s c a u s e d by release of n o r e p i n e p h r i n e from s y m p a t h e t i c nerve^,^^'^^ p r o b a b l y as a r e s u l t of removing t h e A simidampening i n f l u e n c e of c l o n i d i n e on c e n t r a l n o r a d r e n e r g i c n e u r o n e s . l a r c e n t r a l n o r a d r e n e r g i c e x c i t a t i o n may b e i n v o l v e d i n t h e o p i a t e w i t h d r a w a l syndrome which i s p r e v e n t e d by low d o s e s of c l o n i d i n e , p o s s i b l y by r e p l a c i n g o p i a t e - m e d i a t e d i n h i b i t i o n of c e n t r a l n o r a d r e n e r g i c n e u r o n e s w i t h a 2 - a d r e n e r g i c i n h i b i t i o n i n areas s u c h a s t h e l o c u s c o e r u l e u s . 9 2 Most s t u d i e s s u p p o r t t h e view t h a t c l o n i d i n e and s i m i l a r d r u g s e x e r t t h e i r a n t i h y p e r t e n s i v e a c t i o n by s t i m u l a t i n g c e n t r a l m e d u l l a r y a-adrenoc e p t o r s . C l o n i d i n e a p p e a r s t o a c t d i r e c t l y on t h e a - r e c e p t o r s s i n c e i t s a c t i o n i n r a b b i t s i s n o t a f f e c t e d by d e p l e t i n g c e n t r a l n o r a d r e n e r g i c neur o n s w i t h 6-hydroxydopamine. 9 3 These r e c e p t o r s a r e p r o b a b l y of t h e p r e s y n a p t i c o r a2-type and c l o n i d i n e ’ s a c t i o n s are blocked by t h e s e l e c t i v e a2-blocker m i a n s e r i n e . 9 4 C l o n i d i n e ‘ s s e d a t i v e a c t i o n a l s o a p p e a r s t o b e mediated by c e n t r a l a 2 - l i k e r e c e p t o r s . 9 5 y q 6 S e v e r a l a l t e r n a t i v e mechanisms have been proposed t o e x p l a i n c l o n i d i n e ’ s a c t i o n s . It may l o w e r b l o o d p r e s s u r e and s u p p r e s s s y m p a t h e t i c a c t i v i t y by s t i m u l a t i n g i n h i b i t o r y s e r o t o n i n r e c e p t o r s on s y m p a t h e t i c p r e g a n g l i o n i c n e u r o n e s a t t h e s p i n a l l e v e l of s y m p a t h e t i c i n t e g r a t i ~ n . ’ ~C e n t r a l h i s t a m i n e H 2 - r e c e p t o r s may b e i m p l i c a t e d i n c l o n i d i n e ‘ s a n t i h g K e r t e n s i v e a c t i o n s i n c e i t s t i m u l a t e s H2r e c e p t o r s i n g u i n e a p i g a t r i a y q 9 and i n j e c t i o n of t h e a n t a g o n i s t s c i m e t i d i n e o r metiamide i.c. v . b l o c k e d c l o n i d i n e ’ s h y p o t e n s i v e and bradyc a r d i c a c t i o n s i n c a t s . l o O However, t h e a c t i o n of metiamide may be due t o an a 2 - b l o c k i n g a c t i o n . l o l

6 -

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The c l o s e c l o n i d i n e a n a l o g u e , t o l o n i d i n e , w a s i n t r o d u c e d t o t h e I t i s similar t o c l o n i d i n e b u t no reFrench market d u r i n g t h e p a s t y e a r . bound h y p e r t e n s i o n h a s been r e p o r t e d . l o 2 C l i n i c a l s t u d i e s on guanab e n z , l o 3 , 1 0 4 l o f e x i d i n e , l o 5 g u a n f a c i n e , 1 0 6 - 1 0 9 and UK-14,304 , 1 1 0 show h y p o t e n s i v e a c t i v i t y and s i d e e f f e c t s s i m i l a r t o c l o n i d i n e . The hemodynamic e f f e c t s of g u a n f a c i n e i n r a t s are v e r y s i m i l a r t o c l o n i d i n e b u t i t d e c r e a s e s h e a r t r a t e and PRA more and c a u s e s g r e a t e r rebound h y p e r t e n s i o n and t a c h y c a r d i a a t e q u a l h y p o t e n s i v e d o s e s . l 1 C o n t r a r y t o e a r l i e r expect a t i o n s based on animal s t u d i e s , t h i s d r u g does c a u s e s e d a t i o n and d r y mouth as w e l l as rebound h y p e r t e n s i o n a f t e r sudden t e r m i n a t i o n of t h e r apy. l o 7 , l o 8 A new c l o n i d i n e - l i k e d r u g , 7 , ICI-101,187, i s r e p o r t e d t o b e a s p o t e n t a s c l o n i d i n e i n l o w e r i n g blood-pressure i n a n i m a l s b u t i s o n l y S t r u c t u r a l l y t h i s d r u g i s q u i t e similar t o S o n e - t e n t h as s e d a t i n g . ’ 1 2 (Sandoz 4 4 - 5 4 9 ) , which is an even more p o t e n t h y p o t e n s i v e a g e n t t h a n clonidine i n r a t s . l 1

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66

S e c t . I1

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Pharmacodynamic Agents

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Like c l o n i d i n e , t h e i n t e r e s t i n g drug R-28935 l o w e r s blood r e s s u r e by c e n t r a l l y mediated d e p r e s s i o n of s y m p a t h e t i c n e r v e f u n c t i o n . 1 P 4 I n c o n t r a s t , however, i t i s suggested t h a t t h e a c t i o n of t h i s drug i s due t o i n h i b i t i o n of c e n t r a l n e u r o t r a n s m i s s i o n by blockade of p o s t s y n a p t i c areceptors.’l5 The a c t i o n s of t h e drug u r a p i d i l are complex b u t i t a p p e a r s t o both s t i m u l a t e p r e s y n a p t i c and b l o c k p o s t s n a p t i c a - r e c e p t o r s . 116 A n t i h y p e r t e n s i v e V a s o d i l a t o r s - A common c h a r a c t e r i s t i c of h y p e r t e n s i v e p a t i e n t s , r e g a r d l e s s of t h e e t i o l o g y of t h e i r d i s e a s e , i s abnormally h i g h p e r i p h e r a l v a s c u l a r r e s i s t a n c e . Drugs t h a t a c t d i r e c t l y on p e r i p h e r a l v a s c u l a t u r e t o d e c r e a s e r e s i s t a n c e are, t h e r e f o r e , l o g i c a l a g e n t s f o r t h e t r e a t m e n t of h y p e r t e n s i o n . U n f o r t u n a t e l y , s y m p a t h e t i c r e f l e x a c t i o n s l e a d i n g t o c a r d i a c s t i m u l a t i o n , hyperreninemia and f l u i d r e t e n t i o n l i m i t t h e hypotensive a c t i o n of v a s o d i l a t o r s and t h e y are used mainly i n combin a t i o n w i t h 6-blockers and d i u r e t i c s f o r t h e t r e a t m e n t of more severe hyp e r t e n s i o n . 117-120 A v a s o d i l a t o r t h a t may n o t c a u s e u n d e s i r a b l e r e f l e x c a r d i a c e f f e c t s i s t h e calcium a n t a g o n i s t verapamil. T h i s d r u g lowered blood p r e s s u r e w i t h o u t t a c h y c a r d i a i n one study121 b u t was i n e f f e c t i v e and p o o r l y t o l e r a t e d i n a n o t h e r . 122 A u s e f u l c l a s s i f i c a t i o n o f h y p o t e n s i v e v a s o d i l a t o r s , which e x p l a i n s t h e i r d i f f e r e n t hemodynamic p r o f i l e s , h a s been d e s c r i b e d . These a g e n t s cause a r t e r i o l a r d i l a t a t i o n b u t d i f f e r i n t h e i r v e n o d i l a t i n g and sympathetic s t i m u l a n t a c t i o n s . Of s i x v a s o d i l a t o r s s t u d i e d i n hand v e i n s and forearm a r t e r i e s of v o l u n t e e r s , o n l y hydralazine w a s a s e l e c t i v e arterial d i l a t o r . 24 Various p r o s t a g l a n d i n s (PGs), such as PGE2, and t h e r e c e n t l y discove r e d p r o s t a c y c l i n (PGI2), are p o t e n t , s h o r t - a c t i n g , r e n a l and s y s t e m i c v a s o d i l a t o r s t h a t may have a p h y s i o l o g i c a l r o l e i n t h e c o n t r o l of blood I n c r e a s i n g t h e i n t a k e of l i n o l e i c a c i d , t h e d i e t a r y prep r e s s u r e . 125-127 c u r s o r f a t t y a c i d of endogenous PG b i o s y n t h e s i s , lowered blood p r e s s u r e of mild h y p e r t e n s i v e s . 1 2 8 The hypotensive a c t i o n of t h e PG p r e c u r s o r a r a c h i d o n i c a c i d i n r a t s depends on a m e t a b o l i t e s i n c e i t s e f f e c t i s blocked by endoperoxide cylooxygenase enzyme i n h i b i t o r s . 12’ The PG system may a l s o be involved i n t h e a n t i h y p e r t e n s i v e a c t i o n of h y d r a l a z i n e and d i a z o x i d e . These d r u g s are r e p o r t e d t o s e l e c t i v e l y i n h i b i t t h e f o r m a t i o n of t h e p o t e n t , v a s o c o n s t r i c t o r , m e t a b o l i t e of a r a c h i d o n i c a c i d , thromboxane A2 ( T x A ~ ) , from t h e endoperoxide PGH2, and may s t i m u l a t e PGI2 product i o n . 1299 1 3 0

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A number of i n t e r e s t i n g new a n t i h y p e r t e n s i v e v a s o d i l a t o r s have been r e p o r t e d d u r i n g t h e p a s t y e a r . Tolmesoxide (9) is similar i n potency t o d i a z o x i d e i n DOCA h y p e r t e n s i v e r a t s . I t reduced blood p r e s s u r e by 25-45% a t doses of 20-100 mg/kg p.o.131 and w a s e q u a l l y a c t i v e as a v a s o d i l a t o r i n v e i n s and arteries of v o l u n t e e r s . l Z 4 The h y d r a z i n o p y r i d i n e EU 2540 (10)i s a p o t e n t h y p o t e n s i v e a g e n t i n SH r a t s but c a u s e s i n t e n s e tachyc a r d i a . 132 The cyanoguanidine i s 150 t i m e s as p o t e n t i n SH r a t s as t h e c l i n i c a l l y used a g e n t guancydine; t h e r a t i o n a l e f o r t h e s y n t h e s i s of t h i s

11

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compound w a s based on t h e p r e v i o u s l y o b s e r v e d h y p o t e n s i v e a c t i v i t y of t h e (Sch 21700) i s t h e most pob i o i s o s t e r i c p y r i d y l t h i o u r e a . 1 3 3 Compound t e n t of a new s t r u c t u r a l c l a s s of v a s o d i l a t o r s . I t i s ll t i m e s a s p o t e n t a s h y d r a l a z i n e i n d e c r e a s i n g t h e blood p r e s s u r e of SH rats.134

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The mechanism by which v a s o d i l a t o r s c a u s e v a s c u l a r r e l a x a t i o n i s s t i l l p o o r l y understood. Hydralazine i n h i b i t s contractions of i s o l a t e d r a b b i t a o r t i c s t r i p s and may a c t by r e s t r i c t i n g c a l c i u m e n t r y i n t o K+ d e p o l a r 12 i z e d c e l l s . In v i t r o , as w e l l as in vivo, t h e a c e t o n e hydrazone m e t a b o l i t e and t h e b u t a n o n e hydrazone are more p o t e n t v a s o d i l a t o r s t h a n t h e p a r e n t compound. 1 3 5 Another m a j o r h y d r a l a z i n e m e t a b o l i t e , t h e p y r u v i c a c i d hydrazone, w a s n o t h y p o t e n s i v e i n r a b b i t s . 1 3 6 The a c t i o n of h y d r a l a z i n e on r a t arteries i s blocked by a d e n o s i n e t r i p h o s p h a t e (ATP) and i t i s s u g g e s t e d t h a t t h i s d r u g i n t e r a c t s w i t h s p e c i f i c r e c e p t o r s p h y s i o l o g i c a l l y a c t i v a t e d by ATP o r a r e l a t e d s u b s t a n c e r e l e a s e d from s y m p a t h e t i c n e r v e t e r m i n a l s . 1 3 7 S t u d i e s of t h r e e v a s o d i l a t o r s i n r a t vas d e f e r e n s f a i l e d t o c o n f i r m t h e h y p o t h e s i s t h a t c y c l i c GMP is a m e d i a t o r of . v a s c u l a r smooth muscle r e l a x a t i o n . 1 3 8 The v a s o d i l a t i n g a c t i o n of d i h y d r o p y r i d i n e s , such as n i f e d i p i n e , i s g e n e r a l l y a t t r i b u t e d t o t h e i r c a l c i u m a n t a g o n i s t a c t i o n . However , t h e s t r u c t u r a l l y s i m i l a r d r u g YC-93 and v a r i o u s a n a l o g u e s a r e e x t r e m e l y p o t e n t i n h i b i t o r s of c y c l i c AMP p h o s p h o d i e s t e r a s e , l e a d i n g t o s u b s t a n t i a l i n c r e a s e s i n i n t r a c e l l u l a r c y c l i c AMP which may be a t l e a s t p a r t l y r e s p o n s i b l e f o r t h e i r v a s o d i l a t i n g properties.13'

(CF3)ZA 0 - 4

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R o b e r t D . MacKenzie, Merrell R e s e a r c h C e n t e r , 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 R i c h a r d s o n - M e r r e l l I n c . , C i n c i n n a t i , Ohio 45215

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Introduction Though t h e d e a t h r a t e d u e t o v a s c u l a r d i s e a s e s h a s peaked and s l i g h t l y d e c r e a s e d i n t h e p a s t 2 - 3 y e a r s , t h e y a r e s t i l l t h e number one c a u s e of d e a t h i n t h e U n i t e d S t a t e s and i n t h e t o t a l w e s t e r n c i v i l i z a t i o n . I t h a s been s u g g e s t e d t h a t t h e d e a t h r a t e h a s d e c r e a s e d d u e t o b e t t e r e a r l y c a r e a f t e r a h e a r t a t t a c k o r s t r o k e and/or because people a r e t a k i n g c a r e o f t h e m s e l v e s d u e t o t h e i r a w a r e n e s s of t h e r i s k f a c t o r s t h a t r e l a t e t o t h e development o f v a s c u l a r d i s e a s e . Though n o s t a t i s t i c s a r e a v a i l a b l e , t h e number of p e o p l e who have v a s c u l a r d i s e a s e h a s p r o b a b l v n o t d e c r e a s e d ; o n l y t h e f r e q u e n c y of a c u t e a t t a c k and d e a t h r a t e h a s been a f f e c t e d . Thrombosis o r b l o o d c o a g u l a t i o n components p l a y a r o l e i n t h e development of v a s c u l a r d i s e a s e and i n most c a s e s , t h r o m b o s i s i s a 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 . S e v e r a l good r e v i e w s , h a v e been p u b l i s h e d i n which i n t e r r e l a t i o n s h i p s of a t h e r o s c l e r o s i s and t h r o m b o s i s and o t h e r v a s c u l a r interplays a r e discussed Murano h a s reviewed t h e r e l a t i o n s h i p of a c t i v a t i o n o f c o a g u l a t i o n and Hageman F a c t o r w i t h l y s i s and k i n i n a c t i v i t y , ' and Berghaus h a s reviewed t h e r e l a t i o n s h i p of complement t o blood c o a g u l a t i o n .fi Therapy f o r t h e c o n t r o l o r p r e v e n t i o n o f t h e a c u t e e v e n t s i n d e a t h d u e t o v a s c u l a r d i s e a s e c a n b e a p p r o a c h e d i n s e v e r a l ways. One g e n e r a l a p p r o a c h would be t o c o n t r o l t h e i n t e r a c t i o n of b l o o d components w i t h t h e d i s e a s e d blood v e s s e l o r w i t h e a c h o t h e r . In both t h e s e c a s e s antithromb o t i c a g e n t s of d i v e r s e mechanism s h o u l d be h e l p f u l . Whether c o n t r o l o f f i b r i n formation, f i b r i n o l y s i s o r p l a t e l e t f u n c t i o n is d e s i r a b l e , each p a t i e n t ' s problem would d i c t a t e t h e t h e r a p y b e s t s u i t e d . S e v e r a l r e v i e w s a r e a v a i l a b l e i n which t h e d i f f e r e n t b l o o d c o a g u l a t i o n mechanisms t h a t may be h e l p f u l i n s u c h t h e r a p y a r e d i s c ~ s s e d . ~ Reviews ~ ~ - ~ have a l s o been p u b l i s h e d i n which methodology i s d i s c u s s e d f o r t h e t e s t i n g of compounds1' and f o r t h e d e t e r m i n a t i o n of a b n o r m a l i t i e s i n p l a t e l e t f u n c tion T h i s r e v i e w w i l l r e p o r t on t h e p r o g r e s s made i n u n d e r s t a n d i n g t h e b i o c h e m i s t r y and p h y s i o l o g y of blood c o a g u l a t i o n and t h r o m b o s i s and t h e It i s development o f a c t i v e a n t i t h r o m b o t i c a g e n t s i n t h e l a s t 2 y e a r s . s u g g e s t e d t h a t t h e r e a d e r r e f e r s t o t h e most r e c e n t r e v i e w o f t h i s s e r i e s .I2 P l a t e l e t Aggregation I n h i b i t o r s S i n c e a n t i c o a g u l a n t s such a s h e p a r i n and v i t a m i n k a n t a g o n i s t s h a v e n o t been found t o e f f e c t i v e l y c o n t r o l a r t e r i a l t h r o m b o s i s , t h e main a t t e n t i o n f o r t h e p a s t 15 y e a r s h a s been t o s t u d y p l a t e l e t f u n c t i o n and d e v e l o p compounds t o c o n t r o l t h e r o l e o f t h e p l a t e l e t i n a r t e r i a l thromb o s i s , m a i n l y t h r o u g h t h e c o n t r o l of p l a t e l e t a g g r e g a t i o n o r w h i t e thrombus f o r m a t i o n . The g r e a t e s t a d v a n c e i n t h e l a s t s e v e r a l y e a r s h a s been t h e d i s c o v e r y , s t r u c t u r e d e t e r m i n a t i o n and b i o l o g i c a l c h a r a c t e r Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 012-040514-8

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These two i z a t i o n of thromboxane A, (TXA2) and p r o s t a c y c l i n ( P G I , ) . compounds a l o n g w i t h PGE, and PGFZQ, have common p r e c u r s o r s , t h e endoTXA, ( s y n t h e s i z e d i n t h e p l a t e l e t ) i n d u c e s p e r o x i d e s PGGa and PGH,. p l a t e l e t aggregation, w h i l e PGI, (synthesized i n t h e normal blood v e s s e l ) 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 . S e v e r a l e x c e l l e n t r e v i e w s on t h i s subj e c t , l 3 - I 5 a s w e l l a s g e n e r a l r e v i e w s and m e c h a n i s t i c a r t i c l e s on t h e r o l e of t h e p l a t e l e t i n t h r o m b o s i s have b e e n published.16'2? (cf. also c h a p t e r 1 7 i n t h i s v o l u m e ) . The r e l a t i o n s h i p o f d i a b e t e s m e l l i t u s t o abnormal p l a t e l e t f u n c t i o n due t o a n i n c r e a s e d s e n s i t i v i t y i n t h e second p h a s e o f p l a t e l e t a g g r e g a t i o n r e s u l t i n g from TXA, s y n t h e s i s , h a s b e e n reviewed by C o l w e l l and a s s o c i a t e s .73 ,24 T h e r e a r e a t l e a s t 3 d i f f e r e n t mechanisms by which p l a t e l e t s w i l l a g g r e g a t e ; 1) by p r i m a r y a g g r e g a t i o n i n d u c e d by A D P , 2 ) one o f t h e a g g r e g a t i o n pathways i n d u c e d by thrombin and 3 ) one i n d u c e d by t h e p r o I n h i b i t o r s of p l a t e l e t a g g r e g a t i o n may i n h i b i t t h e d u c t i o n of TXA,. i n d u c e r s d i r e c t l y , i n h i b i t t h e i r p r o d u c t i o n o r i n t e r f e r e w i t h t h e biochemi c a l p r o c e s s i n v o l v e d i n p l a t e l e t a g g r e g a t i o n . The i n h i b i t o r may b e a s p e c i f i c ( d i r e c t ) one o r one t h a t a f f e c t s a m o d i f i e r o f one o f t h e b i o c h e m i c a l s t e p s i n v o l v e d i n a g g r e g a t i o n ( i n d i r e c t ) . The f i n a l e f f e c t may be on t h e f l u x o f c a l c i u m i n o r o u t of t h e c e l l o r i t s membranes. Throm~ - ~ ~ ionoboxane A, h a s been shown t o b e a c a l c i u m i o n o p h ~ r e . ~Calcium p h o r e s such a s A23187 and X 5 3 7 A c a u s e p l a t e l e t a g g r e g a t i o n . 2 8 - 3 0 Calcium f l u x e s and d i s t r i b u t i o n a r e a f f e c t e d w h e t h e r t h e s t i m u l u s f o r a g g r e g a t i o n i s collagen o r epinephrine, thrombin, s e r o t o n i n , o r ADP, t h a t e i t h e r prod u c e s TXA,, lowers c y c l i c AMP l e v e l s , o r " s t a b i l i z e s membranes." The compounds t h a t have been r e p o r t e d t o i n f l u e n c e p l a t e l e t a g g r e g a t i o n r e c e n t l y w i l l be discussed under t h e i r r e s p e c t i v e c l a s s i f i c a t i o n a s f o l l o w s : 1) p r o s t a g l a n d i n s and s y n t h e t i c a n a l o g s , 2 ) n o n - s t e r i o d a l a n t i i n f l a m m a t o r i e s (PG s y n t h e s i s i n h i b i t o r s ) and 3 ) m i s c e l l a n e o u s . P r o s t a g l a n d i n s and S y n t h e t i c A n a l o g s ( A l s o see C h a p t e r 1 7 o f t h i s volume.) B e f o r e t h e d i s c o v e r y of P G I , , t h e p r o s t a g l a n d i n s PGE, and PGD, were cons i d e r e d t o be t h e P G ' s of c h o i c e f o r i n h i b i t i o n o f p l a t e l e t a g g r e g a t i o n . S i n c e t h e y were b i o l o g i c a l l y u n s t a b l e , s y n t h e t i c d e r i v a t i v e s t h a t would be more s t a b l e were c o n s i d e r e d . Now t h a t P G I , h a s b e e n shown t o be t h e most p o t e n t i n h i b i t ~ r ~ l -and " ~ i s a c t i v e when g i v e n i n t r a v e n ~ u s l y ~ ~ - ~ ~ , more s t a b l e d e r i v a t i v e s o f P G I , a r e b e i n g s y n t h e s i z e d . Such more s t a b l e d e r i v a t i v e s a s 6 , 9 - t h i a p r o s t a c y c l i n (1),37 1 3 , 1 4 - d e h y d r o P G I , (2)38 and 5 , 6 a d i h y d r o PGI, (2)"" have been s y n t h e s i z e d and t e s t e d .

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I t h a s been s u g g e s t e d t h a t PGI, 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 by t h e s t i m u l a t i o n of a d e n y l c y c l a s e and t h e i n c r e a s e of c y c l i c AMP l e ~ e l s . ~ O ~ ~ ~ T h i s i s t h e o p p o s i t e e f f e c t of most a g g r e g a t i n g a g e n t s which u s u a l l y d e c r e a s e c y c l i c AMP l e v e l s . A compound SQ 2 2 5 3 6 (4),42 h a s been r e p o r t e d t o i n h i b i t a d e n y l c y c l a s e and lower c y c l i c AMP l e v e l s T h i s compound d o e s n o t a g g r e g a t e p l a t e l e t s , b u t d o e s i n c r e a s e p l a t e l e t a g g r e g a t i o n when c h a l l e n g e d w i t h an aggregating agent.42 This supports t h e t h e o r y t h a t c y c l i c AMP r e g u l a t e s p l a t e l e t a g g r e g a tion.

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 A n t i - i n f l a m m a t o r y Compounds - Most 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 (NSAI) compounds i n c l u d i n g a s p i r i n i n h i b i t t h e c y c l o o x y g e n a s e t h a t c a t a l y z e s t h e f o r m a t i o n of t h e e n d o p e r o x i d e s PGG, and PGH, from a r a c h i d o n i c a c i d . Normally t h i s would i n h i b i t b o t h t h e s y n t h e s i s o f TXA, and t h e second p h a s e of p l a t e l e t a g g r e t a t i o n , b u t i t would a l s o i n h i b i t PGI, f o r m a t i o n a s i t i s a l s o d e r i v e d from PGG, and PG&. The i n h i b i t i o n of PGI, f o r m a t i o n i s c o n s i d e r e d t o be u n d e s i r a b l e b e c a u s e i t i s a p o t e n t i n h i b i t o r o f p l a t e l e t a g g r e g a t i o n . TXA, i s s y n t h e s i z e d i n t h e p l a t e l e t , w h i l e PGI, i s formed i n t h e b l o o d v e s s e l w a l l . A s p i r i n u s a g e h a s been q u e s t i o n e d f o r t h i s r e a s o n . A s p i r i n a f f e c t s t h e p l a t e l e t a t l o w e r d o s e s and f o r a l o n g e r t i m e t h a n i t a f f e c t s o t h e r t i s s u e s . T h e r e f o r e low d o s e a s p i r i n i s u ~ e f u l . ~ 3However, e x p e r i m e n t s have shown t h a t low d o s e a s p i r i n (< 1 5 mg/kg) i n c r e a s e s b l e e d i n g t i m e w h i l e h i g h e r d o s e s ( > 5 0 mg/kg) d o n o t a f f e c t t h e b l e e d i n g t i m e . 4 4 , 4 5 S e v e r a l c l i n i c a l s t u d i e s h a v e been c o n d u c t e d t h a t s u p p o r t t h e c o n c l u s i o n t h a t a s p i r i n i s h e l p f u l i n r e d u c i n g t h e f r e q u e n c y of t r a n s i e n t i s c h e m i a a t t a c k s (TIA) i n m e n . 4 6 ~ 4 7 Women were n o t p r o t e c t e d by a s p i r i n t r e a t ment.4B The e x p l a n a t i o n f o r t h i s s e x d i f f e r e n c e i s u n d e t e r m i n e d , A d d i t i o n a l d a t a were r e p o r t e d on t h e f o l l o w i n g p r e v i o u s l y mentioned N S A I compounds: s u l f i n p y r a z o n e , 4 9 y 5 0 n a p r o x e n Y 5 1 i b u p r o f e n , 5 1 i n d o m e t h a c i n , 5 2 h a l o f e n a t e Y 5 3 and f l u r b i p r o f e n . 5 4 S u l f i n p y r a z o n e was n o t shown t o have a b e n e f i c i a l e f f e c t on p a t i e n t s i n t h e Canadian a s p i r i n sulfinpyrazone stroke However, i n a p r o g r e s s r e p o r t of t h e m u l t i - c e n t e r m y o c a r d i a l i n f a r c t i o n (MI) s t u d y w i t h s u l f i n p y r a z o n e , i t was r e p o r t e d t o g r e a t l y d e c r e a s e m o r t a l i t y of t h o s e p a t i e n t s t h a t had H a l o f e n a t e was r e p o r t e d n o t t o a c t a t t h e c y c l o o x y g e n a s e had a n M I . 5 5 , 5 E s t e p s i n c e it does not i n h i b i t arachidonic acid-induced p l a t e l e t aggreg a t i o n . 5 7 However, i t d o e s i n h i b i t e p i n e p h r i n e and c o l l a g e n - i n d u c e d aggregation, leading to the suggestion t h a t i t i n h i b i t s t h e e a r l i e r s t e p i n which p h o s p h o l i p a s e A, h y d r o l y s e s a r a c h i d o n i c a c i d from phospholipid. 57 Compounds t h a t have r e c e n t l y been r e p o r t e d t o i n h i b i t t h e c y c l o t r i f l u s a l (6),60 p r o oxygenase a r e : t i o x a p r o f e n (EMD 2 6 6 4 4 ) (5),58Y~~ d i f l u n i s a l ( ~ ) , "s u~l i n d a c (?) ( t h e s u l f i d e m e t a b o l i t e o f quazone (l),'I s u l i n d a c which i s t h e a c t i v e s p e c i e s of s u l i n d a c i n man, i s 8 t i m e s more __ , 6 4 p r a n o p r o f e n ,65 and p o t e n t t h a n a s p i r i n ) , 6 3 d i f l u m i d o n e (10) .E6 alchfenac

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S i n c e NSAI compounds i n h i b i t t h e f o r m a t i o n of PGG, and PGH,, t h e i r e f f e c t i v e n e s s i s d u e t o t h e d e c r e a s e i n TXA, f o r m a t i o n . It m y be more d e s i r a b l e t o s p e c i f i c a l l y i n h i b i t TXA, s y n t h e s i s o r a c t i v i t y , so t h a t PGI,, PGEs and PGF,, c a n s t i l l b e s y n t h e s i z e d unimpeded. TXA, h a s c a u s e d M I when i n j e c t e d i n t o r a b b i t s . 6 7 I t a l s o h a s been r e p o r t e d t h a t TXA, may r e a c t w i t h t h e same r e c e p t o r s i n t h e b r a i n , a s t h o s e t o which b e n z o d i a z o p i n e s r e a c t . 6 8 Compounds t h a t were r e p o r t e d t o s p e c i f i c a l l y b l o c k TXA, s y n t h e t a s e a r e : burimamide (13),G9 9 , l l - a z o p r o s t a - 5 , 1 3 - d i e n o i c a c i d ( 14),70 L8027 ( n i c t i n d o l e ) (g) 15-deoxy-9,ll-(epoxyimino) p r o s t a g l a n d i n s (l&),72 i m i d a ~ o l e ~ 3and , ~ ~1-methyl i m i d a ~ o l e (17, ~ ~ 18). I n a s e r i e s of 1 - a l k y l i m i d a z o l e d e r i v a t i v e s , 1 - n o n y l (19)was t h e most p o t e n t i n h i b i t o r and an SAR was e v i d e n t . 7 5 N i c o t i n i c a c i d h a s a l s o been r e p o r t e d t o i n h i b i t TXA, ~ y n t h e s i s . ~ "More work h a s a l s o been done on N-0164.77 P h t h a l a z i n e (EG626) (20) h a s been r e p o r t e d t o i n h i b i t t h e a c t i v i t y o f TXA,, b u t n o t i t s s y n t h e s i s . 7 6 - 8 0 Some r e c e n t e v i d e n c e s u g g e s t s t h a t dipyridamole, p r e v i o u s l y r e p o r t e d t o i n h i b i t phosphodies t e r a s e , i n h i b i t s TXA, s y n t h e s i ~ 9 .8 3~ ~ II

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M i s c e l l a n e o u s Compounds S e v e r a l compounds p r e v i o u s l y r e p o r t e d were f u r t h e r i n v e s t i g a t e d a s i n h i b i t o r s of p l a t e l e t a g g r e g a t i o n and new d a t a r e p o r t e d . These were c a r b e n i c i l l i n and t i ~ a r c i l l i n ,BL3459,85 ~ ~ ticlopidine,'" s u l ~ c t i d i l , ~ ~and - ' ~v i n c a a l k a l o i d s

A m e t a b o l i c a l l y more s t a b l e d e r i v a t i v e o f BL3459, BL4162 ( 2 1 ) , was found t o i n h i b i t c o l l a g e n - i n d u c e d p l a t e l e t a g g r e g a t i o n s i m i l a r t a t h a t of I t i s now u n d e r g o i n g c l i n i c a l e v a l u a t i o n . y93 RL3459

."

a s e d a t i v e - h y p n o t i c , was r e p o r t e d t o i n h i b i t Methaqualone (g), b o t h ADP and c o l l a g e n - i n d u c e d 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 i n human p l a t e l e t - r i c h plasma and i n v i v o i n r a b b i t s . g * P e n t o x i f y l l i n e (23), a m e t h y l x a n t h i n e d e r i v a t i v e , was r e p o r t e d t o i n h i b i t b o t h ADP and s e r o t o n i n - i n d u c e d p l a t e l e t a g g r e g a t i o n i n t h e stumpt a i l monkey .95 M i a n s e r i n (ORG GC94) (%), a c l i n i c a l l y e f f e c t i v e a n t i d e p r e s s a n t s u g g e s t e d a s t h e r a p y f o r m i g r a i n e , h a s been found t 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 , e s p e c i a l l y when i n d u c e d b y s e r o t o n i n .96 S e v e r a l f l a v a n o i d s were 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 d h e s i v e n e s s i n r e p e a t e d l y b r e d r a t s . 9 7 The o r d e r o f p o t e n c y f o r 6 d i f f e r e n t f l a v a n o i d s were d e t e r m i n e d . Venoruton P, (2)was t h e m o s t p o t e n t , w h i l e r u t i n (26) was much l e s s p o t e n t . S i n c e t h e o r i g i n a l c l a s s i c work of Born i n 1963 on a d e n o s i n e and 2 - c h l o r o a d e n o s i n e , many a d e n o s i n e d e r i v a t i v e s have been r e p o r t e d t 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 , e s p e c i a l l y i n d u c e d b y ADP. I n 1977 work was r e p o r t e d on some new d e r i v a t i v e s o f S - s u b s t i t u t e d 2 - t h i o a d e n o s i n e s (2) and N - s u b s t i t u t e d 2 - a m i n o a d e n o s i n e s

(2).98

a 'y 7 - . - O

Cl

Jl

21 -

76 -

S e c t . I1

-

Pharmacodynamic Agents

Krapcho, Ed.

A new compound c a l l e d SH 1117 (29) h a s been r e p o r t e d t 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 and be s y n e r g i s t i c w i t h a s p i r i n s i m i l a r t o d i p y r i d a mole b u t w i t h o u t i t s c a r d i o v a s c u l a r e f f e c t s . 9 9

Many p a p e r s have been p u b l i s h e d r e p o r t i n g t h e s e r i n e p r o t e a s e i n h i b i t o r y a c t i v i t y of a m i d i n e o r d i g u a n i d e d e r i v a t i v e s ( s e e c h a p t e r 2 1 o f t h i s v o l u m e ) . A s e r i e s of a r o m a t i c a m i d i n e compounds was r e p o r t e d t 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 induced by ADP, c o l l a g e n and t h r o m b i n . The most a c t i v e compound was (g).loo

Anticoagulants During blood c o a g u l a t i o n e i t h e r a n i n t r i n s i c ( a l l b l o o d ) s y s t e m o r an e x t r i n s i c ( t i s s u e j u i c e - l i p o p r o t e i n ) s y s t e m i s a c t i v a t e d o I n e i t h e r c a s e t h e pathways meet a t t h e a c t i v a t i o n o f F a c t o r X, f o r m i n g a p r o t e o T h i s enzyme i n t h e p r e s e n c e of c o f a c t o r s ( c a l l y t i c enzyme F a c t o r X cium i o n , p h o s p h o l i p i g and F a c t o r V ) w i l l form t h r o m b i n from p r o t h r o m b i n . H e p a r i n i s a c o f a c t o r f o r a p r o t e i n c a l l e d a n t i t h r o m b i n 111 w h i c h c i r c u l a t e s i n t h e plasma and i s a n i n h i b i t o r of b o t h F a c t o r Xa and t h r o m b i n . A n t i t h r o m b i n I11 n e u t r a l i z e s t h e s e 2 enzymes by m o l e c u l a r c o m b i n a t i o n ; In the p a s t 2 years h e p a r i n i n c r e a s e s t h e r a t e of t h i s n e u t r a l i z a t i o n . More work h a s c o n t i n u e d on t h e mechanism o f b l o o d c o a g u l a t i o n . 1 ° 1 - 1 0 3 d a t a have a l s o been r e p o r t e d on t h e e f f e c t i v e n e s s of m i n i - d o s e h e p a r i n . 1 " ,104,105

.

Due t o t h e lowered l e v e l of a n t i t h r o m b i n I T 1 found i n some fami l i e s l o 6 , o r during treatment with heparin, i t has been suggested t h a t t h e i n t r a v e n o u s a d m i n i s t r a t i o n o f p u r i f i e d a n t i - t h r o m b i n 111 would b e b e n e f i c i a l .Io7 Since heparin i s a hetereogeneous mixture t h a t v a r i e s markedly depending on t h e s o u r c e , f u r t h e r i n v e s t i g a t i o n s on f r a c t i o n s and r e l a t i v e a c t i v i t i e s a r e i n p r o g r e s s . 1 0 " ,109

A s e m i - s y n t h e t i c h e p a r i n o i d , A73025, which i s a g l y c o s m i n o g l y c a n p o l y s u l f a t e of b o v i n e o r i g i n w i t h a n a v e r a g e m o l e c u l a r w e i g h t a b o u t 112 t h a t of h e p a r i n h a s a p o t e n c y of a b o u t 9 0 p/mg b a s e d on It p r o d u c e s good a n t i t h r o m b i n 111 mucosal h e p a r i n . a c t i v a t i o n , b u t h a s l i t t l e e f f e c t on o v e r a l l c l o t t I t i s s u g g e s t e d t o have good p o t e n t i a l a s a ing. heparin substitute The a c t i o n and p r o p e r t i e s of t i o c l o m a r o l ( l ) , a n a n t i - v i t a m i n k -coumarin t y p e compound, h a s been reviewed

Chap. 8

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

MacKenzie

77

F i b r i n o 1y t i c Agent s F i b r i n o l y s i s h a s r e c e n t l y been r e v i e w e d . l , l * *I1' In order f o r f i b r i n o l y s i s t o o c c u r , t h e proenzyme plasminogen must b e a c t i v a t e d t o p l a s m i n by an a c t i v a t o r . S e v e r a l a c t i v a t o r s c a n be formed; one from Hageman f a c t o r components a f t e r i t s a c t i v a t i o n , o r by a t i s s u e a c t i v a t o r r e l e a s e d from normal blood v e s s e l s . U r o k i n a s e i s an a c t i v a t o r c o n s i d e r e d t o be l i m i t e d i n i t s f u n c t i o n t o t h e k i d n e y . S t r e p t o k i n a s e , a s i n g l e c h a i n p r o t e i n from A-hemolytic s t r e p t o c o c c i , i s an a c t i v a t o r t h a t h a s been s t u d i e d f o r many y e a r s and h a s found c l i n i c a l u s e e s p e c i a l l y i n Europe. I t s main weakness i s d e c r e a s e d p o t e n c y d u e t o a n t i b o d y f o r m a t i o n . The u s e of u r o k i n a s e , which i s n o t a n t i g e n i c , h a s been l i m i t e d due t o s u p p l y problems and c o s t o f m a n u f a c t u r e from u r i n e . However, t h e problem may have been s o l v e d due t o a new s o u r c e from k i d n e y t i s s u e c u l t u r e (Abbokinasem) .I2

L i t t l e new work h a s been done i n t h i s a r e a . The i s o l a t i o n and t e s t i n g o f a low m o l e c u l a r w e i g h t s p e c i e s (L-UK) and a h i g h m o l e c u l a r w e i g h t s p e c i e s (H-UK) of u r o k i n a s e h a s been r e p o r t e d . l 1 3 The H-UK had t h e g r e a t e r a c t i v i t y . F u r t h e r work h a s been r e p o r t e d on u r o k i n a s e and i t s s u c c e s s f u l u s e .114 The f i b r i n o l y t i c a c t i v i t y of t h e o n i o n and g a r l i c c o n s t i t u e n t c y c l o a l l i i n h a s been ~ o n f i r m e d . 1 ~A~s p i r i n h a s been r e p 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 , i n d e p e n d e n t of i t s p l a t e l e t e f f e c t s .lle A h e p a r i n - u r e a complex was r e p o r t e d t o enhance f i b r i n o l y s i s , l 1 7 w h i l e e a c h component s e p a r a t e l y had no effect. B e z a f i b r a t e (32)was r e p o r t e d t o s h o r t e n e u g l o b u l i n l y s i s t i m e i n human pa t i e n t s I 1

.

Comment - Many d i f f e r e n t c l i n i c a l t r i a l s a r e i n p r o g r e s s on a v a r i e t y o f a n t i t h r o m b o t i c a g e n t s ( a n t i p l a t e l e t , h e p a r i n , and t h r o m b o l y t i c a g e n t s ) .119 I n t h e p a s t 2 y e a r s p r o g r e s s h a s been r a p i d . A s p i r i n h a s proven t o b e e f f e c t i v e i n r e d u c i n g t h e f r e q u e n c y of T I A . S u l f i n p y r a z o n e may r e d u c e t h e d e a t h r a t e a f t e r M I . An 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 (BL 4 1 6 2 ) d e v e l o p e d s p e c i f i c a l l y f o r t h i s a c t i v i t y , and n o t a l r e a d y u n d e r e v a l u a t i o n or marketed f o r a n o t h e r i n d i c a t i o n , i s b e i n g t e s t e d c l i n i c a l l y . The f u t u r e i s indeed b r i g h t t h a t some of t h e s e compounds w i l l be u s e f u l i n t h e c o n t r o l of t h r o m b o s i s , which w i l l s t i m u l a t e f u r t h e r development of more p o t e n t and s a f e r d r u g s and t o o l s t o e n a b l e u s t o u n d e r s t a n d t h e mechanisms i n v o l v e d i n t h r o m b o s i s and v a s c u l a r d i s e a s e .

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24

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Adv. Exptl.Med. Biol. 104,A . B. The Thrombotic P r o c e s s in A t h e r o g e n e s i s C h a n d l e r , K. E u r e n i u s , G . C . McMillan, C . B. N e l s o n , C . J . Schwartz and S . W e s s l e r E d . , Plenum P r e s s , New York, 1978. 3 . H . R e u t e r and H. L i n k e r , Med. Monatsschr. Pharm. L ( 6 ) 167 ( 1 9 7 8 ) . 4. V a s c u l a r G r a f t s , P. N . Sawyer and M. J. K a p i t t , Ed., A p p l e t o n - C e n t u r y - C r o f t s , New York, N . Y . 1978. 5. C,. Murano, Am. J . Hemat. 5 409 ( 1 9 7 8 ) . 6. G. M u e l l e r Berghaus, K l i n . Wochenschr. 15 ( 1 4 ) 663 (1977). 7. R. D. Rosenberg, Ann. Rev. Med. 2, 367 (1978). 8 . P. J. Hamilton, A . L. S t a l k e r and A. S . D o u g l a s , J. C l i n . P a t h . 2, 609 ( 1 9 7 8 ) . 9 . G . J . S t e w a r t , B r i t . J. Haemat. 40 ( 3 ) , 359 ( 1 9 7 8 ) . Adv. E x p t l . Med. B i o l . 102, H. J. 1 0 . Thrombosis, Animal and C l i n i c a l Models Day, B. A . Molony, E . E. Nishizawa and R. H. Rynbrandt, Ed., Plenum P r e s s , N.Y. 1978. 11. P l a t e l e t F u n c t i o n T e s t i n g in Proceeding from Workshop on P l a t e l e t s h e l d O c t . 18-19 ( 1 9 7 8 ) , H. J. Day, H. Holmsen and M. B. Zucker, Ed., DHEW P u b l i c a t i o n No. (NIH) 78-1087, U . S . Dept. HEW, PHS, NIH. 1 2 . R. D . MacKenzie, Ann. R e p o r t s i n Med. Chem., Vol. 1 2 , F. H. C l a r k e , Ed. 1977, P. 80. Academic P r e s s , New York, N.Y., 13. B. Samuelsson, M. Goldne, E . Granstrom, M. Hamberg, S . H a m r s t r o m and C. Malmsten, P r o s t a g l a n d i n s and Thromboxanes & A n n . Rev. Biochem. 47, 997 ( 1 9 7 8 ) . 14. D . H . Nugteren, Agents and A c t i o n s 8 ( 3 ) , 296 ( 1 9 7 8 ) . 15. A . J. Msrcus, J . L i p i d Res. 2, 793 ( 1 9 7 8 ) . 1 6 . M. A . Packham and J. F . M u s t a r d , Blood 50 ( 4 ) , 555 (1977). 1 7 . H. J. Weiss, New Engl. J . Med. 298 ( 2 4 ) , 1344 ( 1 9 7 8 ) . 18. H. J. Weiss, New Engl. J . Med. 3 ( 2 5 ) , 1403 ( 1 9 7 8 ) . 19. 0. V. M i l l e r , R . A . Johnson and R. R. G o m n , P r o s t a g l a n d i n s 13 ( 4 1 , 599 (1977). 20. M. Lagarde, P. A . Byron, B. B. V a r g a f t i g and M. Dechavanne, B r i t . J. Haemat. 38, 251 (1978). 21. H. Akbar and N. G. A r d l i e , B r i t . J. Haemat. 3 9 , 7 1 (1978). 22. L. A. H a r k e r , R. Ross and J . Glomset, Ann. N . Y . Acad. S c i . 275, 321 (1976). 23. J. A . C o l w e l l , P. V. Halushka, K. E. S a r j i and J . S a g e l , Med. C l i n i c s N. A m e r . 62 ( 4 ) , 753 ( 1 9 7 8 ) . 24. P. V . Halushka, D. L u r i e and J . A . C o l w e l l , New E n g l . J. Med. 297 ( 2 4 ) , 1306 (1977). 25. A , I. A l l y , D. F. H o r r o b i n , M. S . Manku, R. 0. Morgan, M. Karmazyn, R. A . Karmali and S. C. Cunnane, Can. J . P h y s i o l . Pharmacol. 5 6 , 520 ( 1 9 7 8 ) . 26. J . M. G e r r a r d , A . M. B u t l e r and J . C . W h i t e , P r o s t a g l a n d i n s 15 ( 4 ) , 703 (1978). 27. D. S c h u b e r t , M. L a C o r b i e r e , C. W h i t l o c k and W . S t a l l c u p , N a t u r e 273, 718 (1978). 28. H. R. Knapp, 0 . Oelz, L. J . R o b e r t s , B. J . Sweetman, J. A . Oates and P. W . Reed, P r o c . N a t l , Acad. S c i . 74 ( l o ) , 4251 ( 1 9 7 7 ) . 29. K. Komatsu and R. K o d a i r a , J . A n t i b i o t i c s 30 ( 3 ) , 226 ( 1 9 7 7 ) . 30. 0. O e l z , H. R. Knapp, L. J. R o b e r t s , B. J . Sweetman, J. A . O a t e s and P. W . Reed, Prostaglandins ( 6 ) , 1013 ( 1 9 7 7 ) . 31. J. E . T a t e s o n , S . Moncada and J . R. Vane, P r o s t a g l a n d i n s 12 ( 3 ) , 389 ( 1 9 7 7 ) . 32. B. H. Crane, T. L. Maish, Y . T . Maddox, E. J. Corey and P. W. Ranwell, C l i n . Res. 25 ( 3 ) , 337A (1977). 33. Drugs o f t h e F u t u r e 2 ( l o ) , 785 ( 1 9 7 8 ) . 34. R. J . Gryglewski, A . S z c z e k l i k and R. Nizsnkowski, Thrombos. Res. 12 ( 2 1 , 153 (1978). 35. Drugs o f t h e F u t u r e 1, 688 ( 1 9 7 7 ) . 36. A . S z c z e k l i k , R. J . G r y l g l e w s k i , R. Nizankowski, J . M u s i a l , R. P i e t o n and J. Mruk, Pharmacol. Res. Commun. lo ( 6 ) , 545 ( 1 9 7 8 ) . 37. K. C . Nicolaou, W . E. B a r n e t t e , G . P. G a s i c and R. L. Magolda, J. Am. Chem. SOC. 99 (231, 7736 ( 1 9 7 7 ) . 38. J. F r i e d and J . Barton, P r o c . N a t l . Acad. S c i . fi ( 6 ) , 2199 ( 1 9 7 7 ) . 39. G . Togna, C. G a n d o l f i , A. Andreorii, A . F u m a g a l l i , C. P a s s a r o t t i , F. F a u s t i n i and C. P a t r o n o , Pharmacol. Res. Commun. 2 ( l o ) , 909 (1977). 40. R. R. Gorman, S . Bunting and 0 . V . M i l l e r , P r o s t a g l a n d i n s 12 ( 3 ) , 377 (1977). 4 1 . L. C . Best, T. J . M a r t i n , R. G . G . R u s s e l l and F . E . P r e s t o n . N a t u r e 267, 850 (1977).

2.

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

til. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71,

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

79. 80. 81. 82. 83. 84.

Antithrombotic Agents

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E . W. Salzman, D . E . MacIntyre, J . L. Gordon and M. S t e e r , Thromboa. Haemost. 38 ( l ) , 6 (1977). N . L. Baenziger, M. J . D i l l e n d e r and P. W. Majerus, Biochem Biophys. Res. C o m n . 78, 294 (1977). M. L i v i o , S . V i l l a and G . deGaetano, Lancet 1, 1307 (1978). J . O'Grady and S . Moncada, Lancet 2, 780 (1978). W . S . F i e l d s , y . A . Lemak, R.F. -8 ( 3 ) , 3 0 1 (1977). H . J . M . B a r n e t t , New Engl. J. J. G . K e l t o n , J. H i r s c h , C . J . 1073 (1978). M . A l i and J.W.D. McDonald, J .

Frankowski and R. J . Hardy, S t r o k e Med. 299 (21, 53 (1978). C a r t e r and M. R. Buchanan. Blood

(5)

Lab. C l i n . Med. 8 9 , 868 (1977). E . W a l t e r and E . Weber, Arch. Pharmacol. 297, Suppl 2 , R62 (1977). B. A . McIntyre and R. B. P h i l p , Thrombos. Res. 2,67 (1977). A . Rane, C l i n . Pharmacol. T h e r . 23 ( 6 ) , 658 (1978). H . Akbar and N . G . A r d l i e , Thrombos. Haemostas. 38 (31, 612 (1977). J . J. T h e b a u l t , G . Lagrue, C . E. B l a t r i x , L. Cheynier and R. Cluzan, C u r r . Med. Res. w i n . 5 (l), 1 3 0 (1977). The Anturane R e i n f a r c t i o n T r i a l Research Group, New Engl. J. Med. 298, 289 (1978). S . S h e r r y , Muench. Med. Wochenschr. 120 (31), 1015 (1978). C. R. F a v i s and R. W . Colman, J . Lab. C l i n . Med. 2 (I), 45 (1978). E . J a c o b i , G . Leopold, R. L i s s n e r and J. Maisenbacher, I n t . J . C l i n . Pharmacol. l6, 136 (1978). Drugs of the F u t u r e 2 ( 8 ) , 615 (1978). M. L. R u t l l a n t , M . B a r r e l l , J . F e l e z , J . M . Diaz, J . M. V i c e n t e and R. J . G a r c i a , C u r r . T h e r . Res. ( 4 ) , 510 (1977). I . B. Holmes, Arch. I n t e r n . Pharmacodyn. (l), 136 (1977). K. F . Tempero. V . J . C i r i l l o and S . L. Steelman, B r i t . J. C l i n . Pharmacol. 4, 315 (1977). 5. H . Minsker, P. J o r d a n , P. Kling and T. Welch, Thrombos. Res. ll, 217 (1977) R. L. Vigdahl and R. H . Tukey, Biochem. Pharmacol. 26 (4), 307 (1977). K . Goto, H . Mikashima, M. Terasawa and Y . Maruyama, J. Pharm. SOC. Japan 2 (ll), 1 2 1 1 (1977). C . C a n e s t r i n i and R. T e s t a , Pharmacol. Res. Commun. 10 (2), 173 (1978). T . Shimamoto, M. Kobayshi, T . T a k a h a s h i , F . Numano :a S . Morooka, C i r c . 56 ( 4 ) , S u p p l . 3 , 123 (1977). A . I. A l l y , M . S . Manku, D . F . Horrobin, R. A . Karmali, R. 0 . Morgan and M . Karmazin, N e u r o s c i . L e t t . 7 (l), 3 1 (1978). G. A l l a n and K. E . E a k i n s , P r o s t a g l a n d i n s 2 (41, 659 (1978). R. R . Gorman, G . L . Bundy, D. C. P e t e r s o n , F . F. Sun, 0. V . Miller and F . A . F i t z p a t r i c k , P r o c . Natl. Acad. S c i . 24 (91, 4007 (1977). R. J. Gryglewski, A . Zmuda, A . Dembinska-Kiec and E . K r e c i o c h , Pharmacol. Res. Commun. 9 (2), 109 (1977). G. L. Bundy and D . C. P e t e r s o n , T e t r a h e d r o n L e t t e r s I , 4 1 (1978). P. Needleman, A . Raz, J. A . F e r r e n d e l l i and M . Minkes, P r o c . N a t l . Acad. S c i . 3 (4), 1716 (1977). S . Moncada, S . Bunting, K . Mullane, P. Thorogood and J . R. Vane, Prostaglandins ( 4 ) , 611 (1977). H . H . T a i and B. Yuan, Biochem. Biophys. Res. Conrmun. 80 (l), 236 (1978). J. E . Vincent and F. J. Z i j l s t r a , P r o s t a g l a n d i n s 15 ( 4 ) , 629 (1978). K . E. Eakins and P. S. K u l k a r n i , B r i t . J. Phannacol. 6 0 , 135 (1977). T . Shimamoto, Y . Takashima and T . T a k a h a s h i , J a p . J . Pharmacol. S u p p l . , 1 3 0 P (1977). T . Shimamoto, Thrombos. Haemostas. 38 ( l ) , 132 (1977). T . Shimamoto, Blood V e s s e l s ( 1 - 3 z 1 7 0 (1978). S. Moncada and R. Korbut, Lancet,.I 1286 (1978). A . I . A l l y , M. S . Msnku, D. F . Horrobin, R. 0. Morgan, M . Karmazin and R. A . Karmali, P r o s t a g l a n d i n s ( 3 ) , 607 (1977). D . F. Horrobin, A . I . A l l y and M . S . Manku, Lancet 2, 270 (1978). G . J . Johnson, G . H . R . Rao and J . G . White, Am. J. P a t h o l . 85(1978).

22

228

13

27

14

2,

2

80 -

Sect. I1

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Pharmacodynamic Agents

Krapcho, Ed.

85.

M. V , C r i f f i t h s , J. M. F r e d r i c k s o n and M. F. X. Glynn, Arch. O t o l a r y n g . 103, 318 ( 1 9 7 7 ) . 86. J . R. O ' B r i e n , M.D. E t h e r i n g t o n and R. D . S h u t t l e w o r t h , Thombos. Res. ( 2 ) , 245 (1978). 525 ( 1 9 7 7 ) . 8 7 . C. deGaetano and A . E. Cavenaghi, Thrombos. Res. 1010 ( 1 9 7 7 ) . 88. D . C. B . Mills and D . E. MacFarlane, Thrombos. Haemostas. ( 3 1 , 107 (1977). 89. Drugs of Today 9 0 . S . R e t s a s , K. A . Newton, G. Westbury, New Engl. J . Med. 299 ( 6 1 , 310 (1978). 9 1 . J . L. W a u t i e r , B. B o i z a r d , F. Rendu, J . P . Caen, New Engl. J . Med. 299 ( 6 ) , 310 (1978). 92. 3. S . Fleming and J. P. B u y n i s k i , Thrombos. Haemostas. 38 (11, 65 (1977). (I), 95 (1977). 9 3 . S.S. Tang and M. M. F r o i m o v i c , Thrombos. Haemostas. 94. D . G. Mills, C l i n . Pharmacol. Ther. 23 ( 6 ) , 685 ( 1 9 7 8 ) . 95. H . G a s t p a r , J . L. Ambrus, C . M. Ambrus, P. Spavento, F. J . Weber and L. E. T h u r b e r , J . Med. 8 ( 3 - 4 ) , 191 ( 1 9 7 7 ) . 96. N . Bhargava, R. M a r r h i j s e n and E . Schonbaum, Eur. J. Pharmacol. 42 ( 3 ) , 241 ( 1 9 7 7 ) . 97. E. Sempinska, B. K o s t i k a , M. Krolikowska and E . K a l i s i a k , P o l . J . Pharmacol. Pharm. 29 (1) 7 ( 1 9 7 7 ) . 98. K. Kikugawa, H . S u e h i r o and A . Aoki, Chem. Pharm. B u l l . 25 ( l o ) , 2637 ( 1 9 7 7 ) . 9 9 . A . J . Honour, R. D. C a r t e r and J. I . Mann, B r i t . J . Exp. P a t h . 58, 474 ( 1 9 7 7 ) . 1 0 0 . J . D. G e r a t z , R . R. T i d w e l l , K . M. Brinkhous, S . F. Mohammad, 0. Dann and H . Loewe, Thrombos. Haemostas. 39 ( 2 ) , 411 ( 1 9 7 8 ) . 101. W . H . S e e g e r s , Am. J. C l i n . P a t r 6 9 , 367 ( 1 9 7 8 ) . 102. G. B. V i l l a n u e v a and I . D a n i s h e f s k y , Biochem. Biophys. Res. Commun. ( 2 ) , 803 (1977). 103. 0 . R. Odegard and M. L i e , Thrombos. Res. 2, 697 (1978). 104. L. J . Bynum and J . E . Wilson, Am. Rev. Resp. D i s . 117 ( 4 ) , 99 ( 1 9 7 8 ) . 1 0 5 , E . J. Kass, P. Sonda, C . Gershon and C . P . F i s c h e r , J . U r o l . L2_11 ( 2 ) , 186 ( 1 9 7 8 ) . 106. M. Mackie, B. B e n n e t t , D . Ogston and A . S . Douglas, B r i t . Med. J . 1, 136 ( 1 9 7 8 ) . 107. H . 6 . S c h i p p e r , L . H. Kahle, C . S . P. J e n k i n s and J . W . TenCate, Lancet 1, 854 ( 1 9 7 8 ) . 108. R. D . Rosenberg, G. Armand and L. Lam, P r o c . N a t l . Acad. S c i . 75 ( 7 ) , 3065 ( 1 9 7 8 ) . 109. T. W . B a r r o w c l i f f e , E. A . Johnson, C . A . E g g l e t o n and D . P . Thomas, Thrombos. Res. 1 2 , 27 (1977). 110. D. A . Lane, R. M i c h a l s k i , M. E . Van Ross and V . V. Kakkar, B r i t . J . Haemat. 2, 239 (1977). 111. M. Neuman, Drugs of Today (8), 383 ( 1 9 7 8 ) . 1 1 2 . J . H i r s c h , Angiology 28 ( l ) , 1 ( 1 9 7 8 ) . 113. T. Suyama, M. N i s h i d a , Y . I g a and R. N a i t o , Thrombos. Haemostas. 38 ( l ) , 48 (1977). 114. W. T h e i s . A . Kriessmann, L. L u t i l s k y , E . S a u e r and A . W i r t z f i e l d , Deut. Med. Wochenschr. 102 ( 6 ) , 2 1 2 (1977). 1 1 5 . R. K . Agarwal, H. A . Dewar, D. J . Newel1 and B. Das, A t h e r o s c l e r o s i s 2 ( 3 ) , 347 ( 1 9 7 7 ) . 116. L. A . Moroz, New E n g l . J . Med. 296 (10) 525 ( 1 9 7 7 ) . 117. B. A . Kudryashov and L. A . Lyapina, Farmakol. T o k s i k o l . (5), 559 (1977). 118. R. Zimmermann, A t h e r o s c l e r o s i s 2 ( 4 ) , 477 ( 1 9 7 8 ) . 1 1 9 . M . V e r s t r a e t e , Thrombos. Haemostas. 2% ( 3 ) , 759 (1978).

12.

lo,

13

38,

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Annual Reports in Medicinal Chemistry-14

Chapter 9.

81 -

6-Adrenergic R e c e p t o r B l o c k e r s as T h e r a p e u t i c Agents

Dale B. Evans, R i t a Fox, and F r e d P. Hauck* The Squibb I n s t i t u t e f o r M e d i c a l R e s e a r c h , P r i n c e t o n , N . J .

08540

I n t r o d u c t i o n - A h l q u i s t ' s c o n c e p t which c l a s s i f i e d a d r e n e r g i c r e c e p t o r s 1 i n t o two d i s t i n c t g r o u p s p r o v i d e d t h e b a s i s f o r t h e emergence o f a c l a s s o f d r u g s which h a s made s u b s t a n t i a l i m p a c t on t h e u n d e r s t a n d i n g and t h e t r e a t m e n t of a w i d e o r d e r o f d i s e a s e s t a t e s . D u r i n g t h e e a r l y e r a o f Bb l o c k e r s , t h e i r i n i t i a l proposed t h e r a p e u t i c u s e w a s i n t h e t r e a t m e n t of It w a s t h o u g h t t h a t t h e B - r e c e p t o r m e d i a t e d i n c r e a s e s i n angina p e c t o r i s . h e a r t r a t e and m y o c a r d i a l c o n t r a c t i l i t y o b t a i n e d w i t h c a t e c h o l a m i n e rel e a s e were d e t r i m e n t a l i n p a t i e n t s s u f f e r i n g from i s c h e m i c h e a r t d i s e a s e b e c a u s e t h e s e a c t i o n s i n c r e a s e d oxygen demand i n t h e p r e s e n c e o f a l i m i t e d oxygen s u p p l y . The f i r s t $ - b l o c k e r t o b e e f f e c t i v e i n t h e t r e a t m e n t of a n g i n a p e c t o r i s w a s p r o n e t h a l o l . ~3 S h o r t l y a f t e r t h e i n t r o d u c t i o n and s u b s e q u e n t w i t h d r a w a l o f p r o n e t h a l o l from c l i n i c a l t r i a l s b e c a u s e o f c a r c i n o g e n i c i t y , 4 p r o p r a n o l o l w a s developed. Propranolol was extensively e v a l u a t e d and w a s shown t o b e b e n e f i c i a l i n t h e t r e a t m e n t o f a n g i n a pect o r i s . 6 T h i s d r u g h a s s i n c e become one o f t h e most w i d e l y i n v e s t i g a t e d and w i d e l y u s e d P-blocking a g e n t s .

A t t h e same t i m e 6 - b l o c k e r s w e r e b e i n g c o n s i d e r e d as a n t i - a n g i n a l a g e n t s , o t h e r t h e r a p e u t i c u s e s i n c a r d i o v a s c u l a r d i s e a s e s were a l s o b e i n g c o n s i d e r e d . I n a n i m a l s t u d i e s , o b s e r v a t i o n s were made t h a t p r o n e t h a l o l induced b r a d y c a r d i a and t h e r e b y s u g g e s t e d t h e u s e of a-blockers i n t h e treatment of arrhythmias. D i c h l o r o i s o p r o t e r e n o l and p r o n e t h a l o l w e r e demonstrated t o possess antiarrhythmic a c t i v i t y a g a i n s t experimentallyi n d u c e d c a r d i a c a r r h y t h m i a s ;7 9 8 s u b s e q u e n t l y , p r o n e t h a l o l w a s r e p o r t e d t o p o s s e s s a n t i a r r h y t h m i - c e f f i c a c y i n humans. 9 S i m i l a r a c t i v i t y w a s o b s e r v e d 8-Blockers were a l s o c o n s i d e r e d f o r u s e i n t r e a t w i t h p r o p r a n o l o l . 'OY1' ment o f m y o c a r d i a l i n f a r c t i o n . The f i r s t c l i n i c a l t r i a l o f p r o p r a n o l o l i n myocardial i n f a r c t i o n demonstrated t h a t 6-blockers reduced m o r t a l i t y i n t h i s p a t i e n t p o p u l a t i o n . 1 2 The o b s e r v a t i o n t h a t p r o p r a n o l o l l o w e r e d b l o o d p r e s s u r e i n p a t i e n t s who were b e i n g t r e a t e d f o r a n g i n a l 3 and t h e s i g n i f i c a n t a n t i h p e r t e n s i v e a c t i v i t y o b t a i n e d i n c l i n i c a l t r i a l s w i t h prop r a n ~ l o l l~e d~ t, o~ t ~h e u s e o f @ - b l o c k e r s as p r i m a r y d r u g s i n t h e t r e a t ment of h y p e r t e n s i o n . Numerous 8 - b l o c k e r s h a v e b e e n d e v e l o p e d a n d a d d i t i o n a l t h e r a p e u t i c i n d i c a t i o n s h a v e b e e n s u g g e s t e d f o r them s i n c e t h e i n i t i a l development o f p r o n e t h a l o l and p r o p r a n o l o l as c l i n i c a l l y u s e f u l a g e n t s i n t r e a t m e n t o f v a r i o u s c a r d i o v a s c u l a r d i s o r d e r s . The p u r p o s e of t h i s r e p o r t i s t o p r e s e n t an u p d a t e on t h e s t a t u s of t h i s c l a s s o f a g e n t s . A l i s t o f 8 - b l o c k e r s i n d i c a t i n g t h e i r s t r u c t u r e and d e v e l o p m e n t a l s t a t u s and t h e a b s e n c e o r p r e s e n c e o f 6 - r e c e p t o r s e l e c t i v i t y , p a r t i a l a g o n i s t a c t i v i t y and n o n - s p e c i f i c c a r d i a c d e p r e s s a n t p r o p e r t y , is p r e I n a d d i t i o n t o p r o p r a n o l o l , two new Bsented i n Table I f o r reference. b l o c k e r s h a v e now b e e n approved f o r u s e i n man i n t h e U.S. Metoprolol has been approved f o r u s e i n h y p e r t e n s i o n , 1 6 t i m o l o l h a s b e e n a p p r o v e d f o r u s e i n g 1 a u c 0 m a . l ~ I n o t h e r p a r t s of t h e w o r l d , t h e r e a r e a t l e a s t 1 7 Bblockers a v a i l a b l e f o r use.18

*Present address:

S t e r l i n g Drug, I n c . , 90 P a r k Ave., New York, N . Y . Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

82

S e c t . I1

- Pharmacodynamic Agents

Krapcho, Ed.

Pharmacologic P r o p e r t i e s - By d e f i n i t i o n , a l l 8-blockers b l o c k B-adrenerg i c r e c e p t o r s . It i s t h i s a c t i o n which is r e s p o n s i b l e f o r t h e i r e f f i c a c y i n v a r i o u s c l i n i c a l c o n d i t i o n s . 19 Some & b l o c k e r s have s e l e c t i v i t y i n b l o c k i n g sub-types of 8 - r e c e p t o r s . C a r d i o s e l e c t i v i t y (81-receptor f u n c t i o n i s i n h i b i t e d t o a g r e a t e r d e g r e e t h a n 82-receptor mediated responses) a p p e a r s t h e o r e t i c a l l y t o be of c l i n i c a l s i g n i f i c a n c e . I n a d d i t i o n t o claimed b e n e f i t i n a s t h m a t i c p a t i e n t s , 2 0 ~ 2 1c a r d i o s e l e c t i v e 8-blockers are proposed t o o f f e r an advantage o v e r n o n - s e l e c t i v e ones i n p a t i e n t s w i t h p e r i p h e r a l c i r c u l a t o r y i n s u f f i c i e n c y . 22 923 T h e o r e t i c a l l y , t i s s u e blood flow would be l e s s impaired w i t h t h e s e a g e n t s because of reduced e f f e c t s on v a s c u l a r B2-receptors. 8 1 - S e l e c t i v i t y may a l s o b e n e f i c i a l l y i n f l u e n c e t h e e f f e c t s t h a t & b l o c k e r s have i n d i a b e t i c p a t i e n t s . The prol o n g a t i o n of t h e i n s u l i n - i n d u c e d hypoglycemia which i s observed w i t h 8b l o c k e r s i s r e p o r t e d t o b e l e s s w i t h c a r d i o s e l e c t i v e 8-blockers. In a d d i t i o n , t h e h y p e r t e n s i v e response which o c c u r s s e c o n d a r i l y t o i n s u l i n induced hypoglycemia i n t h e p r e s e n c e of 8-blockade h a s been r e p o r t e d t o be l e s s s e v e r e w i t h c a r d i o s e l e c t i v e than w i t h n o n - s e l e c t i v e 8-blockers. 24 The a c t u a l s i g n i f i c a n c e of r e c e p t o r d i f f e r e n t i a t i o n i n t e r m s of common c l i n i c a l p r a c t i c e may be q u e s t i o n a b l e , however, because such s e l e c t i v i t y o c c u r s w i t h low doses and i t i s l o s t w i t h h i g h e r d o s e s which a r e commonly used i n p a t i e n t s . 2 5 3 2 6 Furthermore, t h e t h e o r e t i c a l advantage of s e l e c t i v e 8-blockers relies on t h e assum t i o n t h a t t h e r e is an a b s o l u t e organ s e p a r a t i o n of 8 1 and 8 2 Such an assumption i s tempered by evidence which s u g g e s t s t h a t a given organ p o s s e s s e s b o t h 8 1 and 82 rec e p t o r s , t h e r e l a t i v e p r o p o r t i o n v a r y i n g from organ t o organ.28929

receptor^.^^

A d i f f e r e n t t y p e of s e l e c t i v i t y t h a n t h a t a f f o r d e d w i t h a g e n t s b l o c k i n g p r e f e r e n t i a l l y 8 1 as compared t o B2 r e c e p t o r s h a s been s u g g e s t e d f o r mepindolol. T h i s a g e n t h a s been claimed t o b l o c k c h r o n o t r o p i c 8 1 rec e p t o r s t o a g r e a t e r e x t e n t t h a n i n o t r o p i c 8 1 receptors.30,31 The presumed advantage of t h i s t y p e of agent i s t h a t i t would b e accompanied by a lower r i s k of c a r d i a c decompensation t h a n t h a t a s s o c i a t e d w i t h o t h e r 8b l o c k e r s . Again, such s e l e c t i v i t y may be l o s t w i t h commonly used c l i n i c a l doses. Controversy s t i l l e x i s t s w i t h r e f e r e n c e t o t h e n o n - s p e c i f i c c a r d i o d e p r e s s a n t a c t i o n of 8-blockers a s s o c i a t e d w i t h t h e i r membrane s t a b i l i z i n g p r o p e r t i e s . T h i s a c t i o n i s d i r e c t l y r e l a t e d t o dosage l e v e l s l g and i t may be r e l e v a n t a t high d o s e s encountered i n c l i n i c a l p r a c t i c e . Furthermore, p a t h o l o g i c a l as w e l l as o t h e r f a c t o r s can a l t e r t h e pharmacokinetics of t h e s e a g e n t s and i n f l u e n c e c l i n i c a l r e s p o n s e , 25 Y 32-34 Membrane s t a b i l i z i n g a c t i v i t y of 8-blockers a p p e a r s t o be d i r e c t l y c o r r e l a t e d w i t h t h e i r l i p i d s o l u b i l i t y . 25 Moreover, a b s o r p t i o n , d i s t r i b u t i o n and b i o t r a n s f o r mation c o r r e l a t e w i t h l i p i d s o l u b i l i t y , 1 8 , 3 5 @-Blocking a g e n t s which are more l i p i d s o l u b l e c r o s s t h e blood b r a i n b a r r i e r more r e a d i l y and t h e r e f o r e c a u s e such s i d e e f f e c t s as drowsiness, d e p r e s s i o n , h a l l u c i n a t i o n s and v i v i d dreams.25 The l i p o p h i l i c @ - b l o c k e r s a l s o have t h e c a p a c i t y t o c r o s s t h e p l a c e n t a and can c a u s e p e r i n a t a l c o m p l i c a t i o n s . 36, 37

Chap. 9

8-Adrenergic Receptor Blockers Evans, Fox, Hauck

TABLE I. B-Receptor Blocking Agents Generic Name (Trade) Code Structure Acebutolol (Sectral)

M&B 17803A

Alprenolol (Aptin)

H 56/28

Atenolol (Tenormin)

ICI 66082

Bevantolol (Bevantol)

CI 775

Bucumo1o1

cs

Bufuralol

Ro 34787

-

a b Status Cs PAC MSAd Refe

C3H7CONH 3HCOC&-

Q kCH3

M

+

-

+

38

M

-

+

+

39

M

+

-

-

40

C

+

-

+

41

C

-

-

+

42

c

-

+

+

43

H2CONH2

359

3H*

gR4

Bupranolol K 1 255 (Betadrenol) But idrine

83

M

C 1 & H 3

M

CO 405

(Recetan)

44

+

-

45

0 9 - R 5

Bunitrololf (Stresson)

KO 1366

Bunolol

W 6412A

Butocrolol

P 18

Butoxaminef

BW 64-9

Carazolol

BM 51052

46

N&

j$

c

FR6&

-

*H3E

r’

+

47

-

+

48

-

49

C

-

50

E

+

51

E

-

-

H

Carteolol H

84

Sect. I1

-

Pharmacodynamic Agents

Krapcho, Ed.

Table I (cont.) Exaprolol

MG 8823

Indenolol

YB-2

Iprocrolol

P-16

Labetololg (Trandate)

AH 5158

E

/

&

+

52

E

53

-

yR7

-

E

HO

-

-

3H* OH

-

54

M

-

+

55

C

- + +

56

M

-

-

+

-

ONH2 Mepindolol (Corindolan)

LF 17895 CH3-

k Metipranololh W F B (Disorat) 6453

C”+CH3

CH3

57,58

I

OCOCH3 Metoprolol (Lopressor)

H 93/26

M

Mop ro l o 1 (Moro l o 1)

SD 1601

C

Nadolol (Corgard)

SQ 11725

HO

&

M

_

+

59

+

60

_

-

61

R

15

Nif enalol

INPEA

M

-

+

-

62

Oxprenolol (Trasicor)

Ba 39089

M

-

+

+

63

Pamato lo1

H 104108

c

+

Pargolol

KO 1400

E

-

+

65

Penbutolol

Hoe 893d

c

-

+

66

Pindolol (Visken)

LB 46

M

-

+

67

64

+

Chap. 9

85

8-Adrenergic Receptor Blockers Evans, Fox, Hauck

Table I

(cont.)

Practolol (Era1din)

ICI 50172

Procinolol

SD 212401

Pronethalol (Alderlin)

ICI 38174

Propranolol (Inderal)

ICI 45520

Sotalol (Sotacor)

MJ

Tazolol

RS 6245

Ti m o l o 1 (Blocadren)

MK 950

Tiprenolol

Du 21445

Tolamol o 1 Toliprolol

+

+

MR

-

68

-

69

W

-

-

+

70

M

-

-

+

71

M

_

_

_

72

E

_

_

_

73

M

-

E

-

UK 6558

C

+

KO 592

M

-

R I 1 R 18

9

1999

CH3S02 H

-

-

C

74

O w 3 T 7 3

R1

=

OCH2CHOHCH2NHCH (CH3)

R2

=

OCH2 CHOHCH2NHCH 3H?2(CH :&2-

&SCH3

R

6

=

75

-

-

76

77

CHOH HNHC(CH3)3

7

R7 = CHOHCH NH H C H 2 C H 2 a

2

R

3

=

7

OCH CHOHCH2NC(CH3)3 2

R4 = CHOHCH NHC(CH

2

R5

=

>

3' 3

R8 =

7 bH3 CHOHCH2NHCHCH 1

3

CH2CH3 Rg = OCH2CHOHCH2NHCH

CHOHCH~HCH(cH~) CONH2

a

Status(Developmenta1): E, Experimental; C , Clinical; M, Marketed;

MR, Mark ted with restricted use; W, Withdrawn from study or

gse Ei c in man; Cs, Cardioselective; PA, partial agonist activity; MSA, e pmbrane stabilizing activity' Chemistry andfor gharmacology reference; Selective f o r B2-receptors; &-blocking action; Also known as methypranol and trimepranol.

86

S e c t . I1

-

Pharmacodynamic Agents

Krapcho, Ed.

New Chemical Types - LL 21945 (1)r e p r e s e n t s a s i g n i f i c a n t s t r u c t u r a l d e v i a t i o n from s t a n d a r d 6-blockers.78 The c e n t r a l r i n g s t r u c t u r e is a 9fluorenone. I n a d d i t i o n , t h e secondary hydroxyl group of t h e a l i p h a t i c s i d e c h a i n is e s t e r i f i e d . Presumably, t h e e s t e r group is s l o w l y hydrolyzed d u r i n g b i o t r a n s f o r m a t i o n . T h i s a c t i o n r e s u l t s i n slow release of an a c t i v e m e t a b o l i t e which a c c o u n t s f o r t h e l o n g d u r a t i o n of a c t i o n of LL 21945.79 An oxime d e r i v a t i v e o f 9-fluorenone, IF'S 339 (2), p o s s e s s e s s i g n i f i c a n t 6-blocking a c t i o n w i t h p r e f e r e n t i a l a c t i o n on 6 2 - r e c e p t o r s . 80 Oxime d e r i v a t i v e s of thiochromanones, of t h e g e n e r a l s t r u c t u r e 2, have a l s o been r e p o r t e d t o have 6-blocking a c t i o n . 8 1

I

NOCH2 CHOHCH2 NHR

-

2 -

-

CH~CHOHCH~NHCH(CH~) 5 HO HCH2NH H-CH2CH2-@CH3 HOHCH2NHCHCH3CH2CH2

7

-6

-

OCH2CHOHCH2NHC(CH3) F3C

(-8)Y -

=

CH

(9)Y = N

The s t r u c t u r e of KF-577 (3) d i f f e r s from t h e u s u a l a r o m a t i c r i n g system by t h e i n c o r p o r a t i o n of a b e n z t h i a z o l e r i n g . 8 2 T h i s a g e n t h a s a pharmac o l o g i c a l p r o f i l e similar t o t h a t of p r o p r a n o l o l w i t h t h e e x c e p t i o n of a low l e v e l of c a r d i o s e l e c t i v i t y . 83 The r e l a t e d compound, 2, a b e n z t r i a z o l e a n a l o g , h a s a l s o been r e p o r t e d t o p o s s e s s s i g n i f i c a n t @-blocking a c t i v i t y 84

.

RMI-81968 ( g ) , a 1,3-benzydioxol-5-yl s u b s t i t u t e d a l k y l amine p o s s e s s i n g b o t h 6- and a-blocking a c t i v i t i e s , h a s been r e p o r t e d t o a l s o produce d i r e c t v a s o d i l a t i o n . 85 A similar s t r u c t u r e , 7, W I N 40808-7 ( s u l f i n a l o l ) ,86 p o s s e s s e s v a s o d i l a t o r a c t i o n i n a d d i t i o n t o & r e c e p t o r b l o c k i n g p r o p e r t i e s . 87 Both 6-blocking a c t i o n and v a s o d i l a t o r y a c t i v i t y have been r e p o r t e d f o r 2-phenyl- and 2-pyridyl-4-trifluromethyl-imid a z o l e s , 4 and 2,88389 New Concepts - "Combined a c t i o n ' ' blockade of b o t h 6- and a - r e c e p t o r s as demonstrated w i t h l a b e t o l o l and RMI-81968 h a s been s u g g e s t e d t o o f f e r an advantage o v e r c o n v e n t i o n a l .i3-blockers i n t h e t r e a t m e n t of h y p e r t e n s i o n . 90 Compounds p o s s e s s i n g b o t h 6-blocking a c t i o n and v a s o d i l a t o r a c t i v i t y have been developed w i t h t h e primary goa18gf a t t e n u a t i n g t h e t a c h y c a r d i a associated with v a s o d i l a t o r therapy. D i u r e t i c s have been combined w i t h 6-blockers i n f i x e d combinations

as e x e m p l i f i e d by clopamide and p i n d o l o l ( V i s k a l d i f i ) and by m e t o p r o l o l

Chap. 9

8-Adrenergic R e c e p t o r B l o c k e r s

and h y d r o c h l o r o t h i a z i d e (Co-Betaloem), moderate h y p e r t e n s i o n . 91

Evans, Fox, Hauck

87

f o r t h e treatment of mild t o

A s i m p l e regimen of t r e a t m e n t w i t h one-a-day m e d i c a t i o n i s p o s s i b l e f o r example w i t h a l o n g - a c t i n g p r o p r a n o l o l f o r m u l a t i o n . 92 A s l o w - r e l e a s e o x p r e n o l o l f o r m u l a t i o n h a s been r e p o r t e d t o produce b e t t e r t h e r a e u t i c The r e s u l t s t h a n c o n v e n t i o n a l f o r m u l a t i o n of several 8 - b l o c k e r s . 93-9' a p p a r e n t a d v a n t a g e of t h e s e f o r m u l a t i o n s i s t h o u g h t t o b e d u e t o b e t t e r p a t i e n t compliance. T h i s a d v a n t a g e would a l s o b e s h a r e d b y B-blockers which have an i n t r i n s i c a l l y l o n g d u r a t i o n of a c t i o n . N a d o l o l g i v e n once d a i l y h a s been r e p o r t e d t o b e as e f f e c t i v e as p r o p r a n o l o l a d m i n i s t e r e d ~ i~m i l a r l y , f o u r t i m e s d a i l y i n t h e t r e a t m e n t of a n g i n a p e c t ~ r i s . S s o t a l o l e f f e c t i v e l y c o n t r o l l e d b ood p r e s s u r e i n h y p e r t e n s i v e p a t i e n t s on a o n c e - d a i l y dosage regimen. 9+

O t h e r C l i n i c a l I n d i c a t i o n s - The l i s t of c l i n i c a l i n d i c a t i o n s i n which Bb l o c k e r s have e i t h e r been proven e f f i c a c i o u s o r a r e now b e i n g s t u d i e d cont i n u e s t o grow. I n a d d i t i o n t o t h e i r now a c c e p t e d e f f i c a c y i n a n g i n a , g 8 , 9 9 a r r h y t h m i a s l o 2 and h y p e r t e n s i o n , l o 3 9 m y o c a r d i a l i n f a r c t i o n ,loo,lol o t h e r d i s e a s e s t a t e s c h a r a c t e r i z e d by e x c e s s s y m p a t h e t i c a c t i v i t y s u c h as t h y r o t o x i c o s i s lo5have been t r e a t e d w i t h 8 - b l o c k e r s . B-Blockers h a v e a l s o been r e p o r t e d t o b e u s e f u l d r u g s i n t r e a t m e n t o f m i g r a i n e a t t a c k s ,1069107 a n x i e t y , l o 8 and t r e m o r . lo9 8-Blockers a r e a d v o c a t e d f o r u s e i n c o n d i t i o n s s u c h as s c h i z o p h r e n i a and mania These a g e n t s are p o s s i b l y u s e f u l i n o p i a t e and o t h e r d r u g w i t h d r a w a l . The i n t r o d u c t i o n of B-blockers i n o hthalmology r e p r e s e n t s an i m p o r t a n t advance i n t r e a t m e n t o f glaucoma. Timolol h a s been s t u d i e d e x t e n s i v e l y and i t i s r e p o r t e d t o e f f e c t i v e l y r e d u c e i n t r a o c u l a r p r e s s u r e i n p a t i e n t s w i t h open a n g l e glaucoma o r o c u l a r h y p e r t e n s i o n . 1 1 3 References 1. 2. 3.

4. 5. 6.

7. 8. 9.

10. 11.

12. 13. 14. 15. 16.

R.P. A h l q u i s t , Am. J . P h y s i o l . , 153, 586 (1948). A.C. Dornhorst and B.F. Robinson, L a n c e t , 2, 314 ( 1 9 6 2 ) . G.H. Apthorp, D.A. Chamberlain and G.W. Hayword, B r . Heart J . , 26, 218 (1964). G.E. P a g e t , B r . Med. J . , 2, 1266 (1963). J.W. B l a c k , A.F. Crowther, R.G. Shanks, L.H. Smith and A.C. D o r n h o r s t , Lancet, 1080 (1964). S.C. S r i v a s t a v a , H.A. Dewar and D . J . N e w e l l , B r . Med. J . , 2, 724 (1964) , B.R. L u c c h e s i and H.F. Hardman, J . Pharmacol. Exp. T h e r . , 132, 372 (1961). . - , A. S e k i y a and E.M. Vaughan W i l l i a m s , B r . J . Pharmacol., 3 , 462 (1963). J . P . P . S t o c k and N . Dale, B r . Med. J . , 2 , 1230 (1963). D . J . Rowlands, C. Howitt and P. Markman, B r . Med. J . , 1,1891 (1965). D.C. H a r r i s o n , J . R . G r i f f i n and T . J . F i e n e , N . Engl. J . Med., 273, 410 (1965). P . J . D . Snow, L a n c e t , 2, 551 (1965). B.N.C. P r i c h a r d , B r . Med. J . , 1, 1227 (1964). B . N . C . P r i c h a r d and P.M.S. G i l i a m , B r . Med. J . , 2, 725 (1964). B.N.C. P r i c h a r d and P.M.S. G i l l a m , Am. J . C a r d i o l . , 18,387 (1966). FDC R e p o r t s , 3 ( 3 3 ) , TtiG-5 (1978).

r,

88 -

S e c t . I1

- Pharmacodynamic Agents

Krapcho, Ed.

1 7 . FDC R e p o r t s , 40 ( 3 8 ) , T&G-6 ( 1 9 7 8 ) . 18. R.P. A h l q u i s t , Am. Heart J. , 97,1 3 7 ( 1 9 7 9 ) . 1 9 . D.G. Gibson i n " C a r d i o v a s c u l a r Drugs'', v o l . 2 ADIS P r e s s , Sydney, 1 9 7 7 , p. 1. 20. M.K. Benson, W.T. Berril, J . M . Cruickshank and G.S. S t e r l i n g , B r . J. C l i n . Pharmacol., 5, 415 ( 1 9 7 8 ) . 21. D.G. McDevitt, B r . J. C l i n . Pharmacol., 5, 97 ( 1 9 7 8 ) . 22. P.D. McSorley and D . J . Warren, B r . Med. J . , 2, 1 5 9 8 ( 1 9 7 8 ) . 23. A . J . M a r s h a l l , C . J . C . B a r r i t t and D.W. B a r r i t t , B r . Med. J . , 1,1 4 9 8 (1976). 24. E d i t o r i a l , L a n c e t , 1, 8 4 3 ( 1 9 7 7 ) . 25. A . Marquez-Julio a n d E.M. S e l l e r s , Can. J. Hosp. Pharm., 179, ( 1 9 7 8 ) . 26. E.T. O b r i e n , Angiology, 2, 332 ( 1 9 7 8 ) . 27. A.M. Lands, A. Arnold, J.P. McAuliff, F.P. Luduna and T.G. Brown, N a t u r e , 214, 597 ( 1 9 6 7 ) . 28. E. C a r l s s o n , B. Abldd, A. Brandstrom and B. C a r l s s o n , L i f e S c i . , 3, 953 ( 1 9 7 2 ) . 29. B. Ablad, I. B o r j e s s o n , E. C a r l s s o n and G. Johnson, A c t a Pharmacol. e t T o x i c o l . , 36 (Suppl. V), 8 5 ( 1 9 7 5 ) . 30. J . B o n e l l i , I n t . J. C l i n . Pharmacol., 15,325 ( 1 9 7 7 ) . 31. J . B o n e l l i , J . I n t . Med. R e s . , 5, 317 ( 1 9 7 8 ) . 32. K.M. P i a f s k y and 0. Borga, C l i n . Pharmacol. T h e r . , 2 2 , 545 ( 1 9 7 7 ) . 33. J . Meier, Acta Med. S c a n d . , 606 ( S u p p l . ) , 6 5 (1977). 34. D.G. Shand i n " C a r d i o v a s c u l a r Drugs", v o l . 2 , ADIS P r e s s , Sydney, 1 9 7 8 , p. 41. 35. D.F. Weetman, Drugs o f Today, 13,260 ( 1 9 7 7 ) . 36. A . Habib and J.S. McCarthy, J. P e d i a t . , 91,808 ( 1 9 7 7 ) . 37. O.M. C o t t r i l , R.G. McAllister, J r . , L. Gettes and J . A . Noonan, J. P e d i a t . , 91, 812 ( 1 9 7 7 ) . 38. B. B a s i l , R. J o r d a n , A.H. L o v e l e s s and D.R. Maxwell, B r . J. Pharmacol. 48, 1 9 8 (1973). 39. B. Ablad, M. Brogard and L. Ek, Acta Pharmacol. T o x i c o l . Suppl. , 25, 9 (1967). 40. A.M. Barrett, J. Carter, J . D . F i t z g e r a l d , R. H u l l and D. LeCount, B r . J. Pharmacol., 48, 340P ( 1 9 7 3 ) . 41. S.G. H a s t i n g s , R.D. Smith, R.M. Corey, A.D. Essenburg, C.E. Pettway and D.K. Tessman, Arch. I n t . Pharmacodyn. T h e r . , 226, 81 ( 1 9 7 7 ) . 42. Y. S a t o , Y. Kobayashi, T. Nagasaki, T. Oshima, S. Kumakura, K. Nakayama, H. Koike and H. T a k a g i , Chem. Pharm. B u l l . , 20, 905 ( 1 9 7 2 ) . 43. T.C. Hamilton and N.W. P a r k e s , Arzneim. F o r s c h . , 27, 1 4 1 0 ( 1 9 7 7 ) . 44. Who C h r o n i c l e , 3,1 1 2 5 ( 1 9 7 2 ) . 45. R. F e r r i n i , G. M i r a g o l i and G. Croce, Arzneim. F o r s c h . , 18,829 (1968). 46. T. Baum, G. Rowles, A.T. S h r o p s h i r e and M . I . Gluckman, J . Pharmacol. Exp. T h e r . , 176,339 (1971). 47. R.D. Robson and H.R. Kaplan, J . Pharmacol. Exp. T h e r . , 175,1 5 7 (1970). 48. M. M a r t i n , Eur. J. Med. Chem-Chim. T h e r . , 9 , 5 6 3 ( 1 9 7 4 ) . 49. B. L e v y , J . Pharmacol. Exp. T h e r . , 151,4 1 7 ( 1 9 6 6 ) . 50. W. B a r t s c h , K. Kietman, H. L e i n e r t and G. Sponer, Arzneim. F o r s t h . , 27, 1 0 2 2 ( 1 9 7 7 ) . 51. y.Yabuuchi and D. K i n o s h i t a , J a p . J . Pharmacol. , 24, 8 5 3 ( 1 9 7 4 ) . 52. M. C a r i s s i m i , P. G e n t i l i , E. G r u m e l l i , E. M i l l a , G. P i c c i o l a and F. Ravenna, Arzneim. Forsch. , 26, 506 ( 1 9 7 6 ) . 53. S . Tachikawa and T. Takenaka, Arch. I n t . Pharmacodyn., 202, 79 ( 1 9 7 3 ) . 54. J. Van Den D r i e s s c h e , T h e r a p i e , 32, 111 ( 1 9 7 7 ) . I

Chap. 9

6-Adrenergic R e c e p t o r B l o c k e r s

Evans, Fox, Hauck

89 -

55. J . B . Farmer, I. Kennedy, G.P. Levy and R . J . M a r s h a l l , B r . J . Pharrnacol. 45, 660 (1972). 56. G u g l e r , L. Kreis and H . J . D e n g l e r , Arzneim. F o r s c h . , 25, 1067 (1975). 57. A . J . J o u n e l a , P . J . P e n t i k a i n e n and P . J . Neuvonen, I n t . J . C l i n . Pharmacol., 16,1 8 3 (1978). 58. S. Z a k h a r i , Eur. J. Pharmacol., 3,2 2 (1974). 59. B. Ablad, E. C a r l s s o n and L . Ek, L i f e S c i . , 12, 107 ( 1 9 7 3 ) . 60. R. F e r r i n i , G. M i r a g o l i and G. Groce, Arzneim. F o r s c h . , 20, 1974(1970). 61. R.J. Lee, D.B. Evans, S.H. Baky and R . J . L a f f a n , Eur. J . Pharmacol., 33, 371 ( 1 9 7 5 ) . 62. XD. F i t z g e r a l d , C l i n . Pharmacol. T h e r . , 10,292 (1969). 63. C. R a p e r and J . Wale, Eur. J . Pharmacol., 5 , 1 (1968). 64. S.G. C a r r u t h e r s , J . P . H o s l e r , P. P e n t i k a i n e n , D.L. A z a r n o f f , C l i n . Pharmacol. T h e r . , 24, 1 6 8 ( 1 9 7 8 ) . 65. I . Matsubara, F o l i a Pharmacol. J a p . , 72, 557 (1976). 66. G. H a r t f e l d e r , G. L e s s e n i c h and H. Schmidt, Arzneim. F o r s c h . , 22, 1903 (1972). 67. J . F . G u i d i c e l l i , H. Schmidt and J . R . B o i s s i e r , J . Phannacol. Exp. T h e r . , 168, 116 (1969). 68. D. Dunlop and R.G. Shanks, B r . J . Pharmacol., 32, 201 (1968). 69. J . R . B o i s s i e r , J . F . G u i d i c e l l i , P. Viars, C . A d v e n i e r , P. M o u i l l e and S . Larno. Eur. J . Pharmacol., 3, 1 5 1 (1971). 70. J . W . Black and J . S . S t e p h e n s o n , L a n c e t , 2 , 311 (1962). 7 1 . J . W . Black, A.F. Crowther, R.G. Shanks, C.H. Smith and A.C. D o r n h o r s t , L a n c e t , 1,1080 (1964). 72. P.M. L i s h , J . H . Weikel and K.W. Dungan, J . Pharmacol. Exp. T h e r . , __ 149, 1 6 1 ( 1 9 6 5 ) . 73. G. V a u g u e l i n , M.L. Lacombe, G. G u e l l a e n , D. S t r o s b e r g and J . Hanoune, Biochem. P h a r m a c o l . , 25, 2605 (1976). 74. A. S c r i a b i n e , M.L. T o r c h i a n a , J . M . S t a v o r s k i , C.T. Ludden, D.H. Minsker and C.A. S t o n e , Arch. I n t . Pharmacodyn., 205, 76 (1973). 179 (1974). 75. J . D . A l l e n and R.G. Shanks, B r . J . Pharmacol., 2, 76. J . A u g s t e i n , D.A. Cox, A.L. H a m . , P.R. Leming and M. S n a r e y , J. Med. Chem., 16,1245 (1973). 77. K. S t o c k and E. Westermann, Biochem. Pharmacol., 2, 227 (1965). 78. R. C l a r k s o n , H . Tucker and J . Wale, Ann. R e p o r t s Med. Chem., 10,51 (1975). 79. Drugs o f t h e F u t u r e , 2, 116 (1977). 80. J . L . Imbs, F. Miesch, J . S c h w a r t z , J . V e l l y , G. L e c l e r c , A. Mann and C . G . Wermuth, B r . J . Pharmacol., 60, 357 ( 1 9 7 7 ) . 81. A. F r a v o l i n i , F. S c h i a f e l l a , G . O r z a l e s i , R. S e l l e r i and I . V o l p a t o , Eur. J . Med. Chem. Chimica. T h e r . , 13,347 (1978). 82. Y . Kasuya, Yen F e i n Chiu-Wei, K. Goto and M. Watanabe, Chem. Pharm. B u l l . , 25, 2105 (1977). 83. Drugs o f t h e F u t u r e , 3 , 456 ( 1 9 7 8 ) . 8 4 . H. Obase, H. T a t s u n o , K. Gato, J . Shigenobu, Y . Kasuya, Y . Yamada, K. F u j i i and S . Yada, Chem. Pharm. B u l l . , 2, 1443 ( 1 9 7 8 ) . 85. R . C . Dage, H . C . Cheng, C . P . Hsieh and J . K . Woodward, Fed. P r o c . 37, 633 (1978). 86. J . Am. Med. ASSOC., 241, 402 (1979). 87. R . E . P h i l i o n , D.K. P h i l l i p s , S.C. Laskowski, D . C . S c h l e g e l , R.R. Lorenz, P.H. Hernandez and H.E. Lape, 1 7 6 t h Am. Chem. SOC. N a t . Mtg., S e p t . 11-14, 1978, Med. Chem. Abst. t 2 4 .

90

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- Pharmacodynamic Agents

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88. J.J. Baldwin, R. Hirschmann, P.D. Lumma, W.C. Lumma and G.S. P o n t i c e l l o , J. Med. Chem., 20, 1024 ( 1 9 7 7 ) . 89. J.J. Baldwin, E.L. E n g e l h a r d t , R. Hirschman, G.F. L u n d e l l and G.S. P o n t i c e l l o , 1 7 6 t h Am. Chem. SOC. N a t . Mtg., S e p t . 11-14, 1 9 7 8 , Med. Chem. Abst. #24. 90. R.N. Brogden, R.C. Heel, T.M. S p e i g h t and G.S. Avery, Drugs, 15,2 5 1 (1978). 91. S c r i p , P26 ( S e p t . 1 6 , 1 9 7 7 ) . 92. J . McAinsh, N.S. Baker, R. Smith and J. Young, B r . J . C l i n . Pharmacol. 6 , 115 ( 1 9 7 8 ) . 93. Watt and T . D . Duncan, New Zealand Med. J., 8 7 , 245 ( 1 9 7 8 ) . 94. W.A. F o r r e s t , J. I n t . Med. R e s . , 5, 1 3 6 (1978): 95. W.A. F o r r e s t , C l i n . Therap., 1,235 ( 1 9 7 8 ) . 96. B. F u r b e r g , A. D a h l q v i s t , A. Raak and U. Urege, C u r r . Med. R e s . Opin., 5 , 388 ( 1 9 7 8 ) . 97. E. G a b r i e l , Curr. Med. R e s . Opin., 2,739 ( 1 9 7 7 ) . 98. B.N. P r i c h a r d , Drugs, 7, 55 (1974). 99. B r . Med. J . , 1,462 ( 1 9 7 8 ) . 100. B.N. Singh, Drugs, 15,218 ( 1 9 7 8 ) . 101. R . J . Lee, L i f e S c i . , 3, 2539 ( 1 9 7 8 ) . 1 0 2 . Proc. Royal SOC. Med., 70, Suppl. 11 ( 1 9 7 7 ) . 103. B.N.C. P r i c h a r d , B r . J. C l i n . Pharmacol., 5, 379 ( 1 9 7 8 ) . 1 0 4 . H . J . Waal-Manning, Drugs, 17,1 2 9 ( 1 9 7 9 ) . 105. S. R u b e n f i e l d , V.E. S i l v e r m a n , K.M.A. Welch, L.E. Mallette and P.O. Kohler, N. Eng. J. Med., 300, 353 ( 1 9 7 9 ) . 1 0 6 . M. Anthony, Drugs, 15,249 ( 1 9 7 8 ) . 107. R.N. Nanda, Headache, 2, 20 (1978). 1 0 8 . M.M. Suzman, P o s t g r a d . Med. J . , 52 (Suppl. 4 ) , 1 6 ( 1 9 7 6 ) . 1 0 9 . I . S e v i t t , P o s t g r a d . Med. J., Z T S u p p l . 4 ) , 1 7 4 ( 1 9 7 6 ) . 110. N . J . Yorkston, P o s t g r a d . Med. J . , 52 (Suppl. 4 ) , 1 7 5 ( 1 9 7 6 ) . 111. J . W . J e f f e r s o n , Arch. Gen. P s y c h i a t . , 33, 993 ( 1 9 7 6 ) . 112. 1.M. Katz, Ann. Ophthalmol., 10, 8 4 7 (1978). 113. R.C. Heel, R.N. Brogden, T.M. S p e i g h t and G.S. Avery, Drugs, l7, 38 (1979).

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Chapter 10. Histamine Receptors C. Robin Ganellin, The Research Institute, Smith Kline and French Research Ltd., Welwyn Garden City, Herts., U.K. Introduction - The classification of histamine H1 receptors' in 1966 and of histamine H2 receptors2 in 1972 reawakened interest in histamine; interest has intensified since the introduction of the specific H2receptor histamine antagonist, cimetidine (TAGAMET), in 1976 for the control of gastric acid hypersecretion and treatment of peptic ulcers. The identification of specific antagonists and selective agonists has given great impetus to pharmacological analysis of histamine actions and in the last few years there has been a rapid growth of the literature on histamine pharmacology. Histamine receptors were reviewed in 1973 before the advent of the H2 antagonist^;^ later developments are described in reference 4, and the subject was mentioned briefly in these reports5 in 1975 and 1977. The classification and distribution of histamine receptors has been discussed by Black6 and reviewed by Chand and E ~ r e . The ~ Physiological Society of Philadelphia held a symposium on histamine receptors in 1977, the proceedings of which are in press.' A symposium was held in 1978 at the VIth International Conference on Medicinal Chemistry, Brighton, England (proceedings to be published). In addition, there have been several symposia on H2-rece tor histamine antagonists, mainly concerned with clinical aspects.9 3 2 Drugs Acting at Histamine Receptors

Agonists - The existence of two receptor populations for histamine raises the interesting question of whether the chemical mechanism of histamine interaction differs between the two receptor types. Some indications of the chemical properties which may differentiate receptor action come from studies of histamine chemistry and from structure-activity considerations of ~0ngeners.l~Histarnine in aqueous solution is a mixture of equilibrating species, viz. ionic forms, tautomers and conformers; I3C nmr stu1 dies14 confirm earlier pKa work" indicating a N'-H:N"-H (structures and 2) tautomer ratio of approximately 4:l for histamine monocation, and a comparable ratio for histamine base.16 The latter result contrasts with crystal structure data17 and molecular orbital prediction^,^'-^^ and may indicate an influence of solvent on tautomer stability. Recent studies of properties pertinent to consideration of ligand-receptor interactions are conformation (MO calculations20-21 and infra-red comparison of solid state and chloroform solutions of histamine base22) , electronic charge d i ~ t r i b u t i o n ,metal ~ ~ c ~ m p l e x a t i o n ,and ~ ~ phospholipid interactions.25 The identification of selectively acting agonists, chemically related to histamine, provides a valuable basis for studying the medicinal chemistry of histamine. Thus, 2-pyridylethylamine (3, R=H) and 2-thiazolylethylamine (4) have appreciable activity as agoKists at H1 receptors and therefore indycate that although the N'-H tautomer (1) - of histamine is Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

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.z

-1 N’-H

tautomer

2 -

-3

4 -

N ~ - Htautomer

likely to be an active form, tautomerism is not a pre-requisite for activity at HI re~ept0rs.l~These compounds are not very active at H2 receptors which suggests that the chemical features of a particular tautomeric structure, or tautomerism, may be important for H2 receptor stimu1ati0n.l~ It has been suggested that the histamine NT-H tautomer may bind to the receptor via hydrogen donor and acceptor bonding and it may also function as a proton transfer agent;13,26 such a mechanism is analogous to the function of imidazole in the histidyl residues of certain enzymes. Molecular orbital calculations provide a similar model.’ The H receptor agonist, di~naprit~~ 2[ S- (3-N,N-dimethylaminopropyl) isothiourea] (5) which lacks an imidazole ring, could nevertheH2N NH less function analogously to histamine since the isothiourea group is a tautomeric amidine and may provide hydrogen donor and acceptor -5 bonding through the amidino nitrogen atoms.27 dimaprit monocation An alternative view regards the sulphur atom as an H-bond acceptor. Isothiourea and imidazole are both planar 6II-electron systems and in the dimaprit-histamine comparison they represent a very interesting and novel example of bio-is~sterism.~~ Dimaprit is highly specific for H2 receptors; in vitro it has, respectively, 17 and 10% of the potency of histamine on the rat uterus and guinea-pig right atrium (H systems) but less than 0.0001% on the guinea-pig ileum (an Hi system),2a a ratio of better than 1:100,000. Dimaprit stimulates gastric acid secretion in the rat, dog and cat, although there are differences in potency. In the dog and cat the maximum secretory response to dimaprit was significantly greater than that obtained to hista~nine.~~,~’ Dimaprit is especially useful for studying H receptor mediated cardiovascular responses.28’3 0 2Dimaprit poses an interesting problem for conformational analysis since the higher and lower homologs are not a ~ t i v e . ~ ~ ~ ~ ~

’*

Substituents in the heterocyclic ring may impart selectivity of action. Thus 4-methylhistamine (6b) is a selective H2-receptor agonist2 (some authors refer to this compouz as 5-methylhistamine), 2-methylhistamine (&) is a relatively selective HI-receptor agonistS2 and their N“-methyl derivative^^^,^^ are also correspondingly selective (numbering according to ref. 3 4 ) . Presumably, the methyl group interferes with molecular function; this may occur as a simple steric inhibition of agonistreceptor ‘fit’ but an alternative explanation is provided by a study of the conformation of methyl derivatives which defined an essential conformation for H1 receptor activity.35 Various ri id analogs of histamine have been tested but none is particularly active.3‘ Branching of the histamine side chain markedly reduces ability to act at either receptor.32 Surprisingly, this does not appear to hold for 4-methylhistamine since a,4-

Chap. 10

Histamine Receptors

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93

-R a M e b H c H d H e H 6 -

H Me Me Et nPr

H H Me H H

I

a H b NH2 c Me

-\

?gN R

7 -

dimethylhistamine (6c) is reported to be selective, having about 5 - 10% of the potency of hEtamine at H2 receptors.37 4-Ethylhistamine (6d) is also a selective H -receptor agonist31 y 3 * but larger 4-alkyl groupshinder agonist f~nction.~g3-(1,2,4-Triazolyl)ethylam~ne (7a) is an agonist at both receptors but, interestingly, a 5-amino substitutent (7b) imparts selectivity towards H2 receptors whereas the 5-methyl derivazve (7c) (structurally analogous to 2-methylhistamine) appears to be specifEally active at H~ receptors.39 Partial Agonists - Increasing the size of side-chain N-substituents 2-pyridylethylamine (3) reduces H1-receptor agonist potency. When R "or R2 = E compounds are partial H1 agonists, but higher alkyl homologs (R1= nPr, R"= H) are purely H1 antagoni~ts.~oAntagonist potency is enhanced by an increase in size of the substituent. A detailed study shows that the substituent makes a major contribution to receptor affinity whereas the pyridylethyl contributes little to affinity.40 Similar substituent effects obtain for the histamine molecule, partial agonism at H1 receptors appearing when cyclohexyl or aryl substituents are incorporated in the 2-positi0n.~~Increase in size of the 4-substituent in histamine reduces H2-receptor agonist potency and, in fact, leads to partial agonism. Thus 4-propylhistamine (6e) has only 7% of histamine's potency on the guineapig isolated right atrium and achieves only 50% of histamine's maximal response.38

f

There have been some interesting developments with guanidine derivatives. e-Guanylhistamine (8a) is a very weak partial Hp a g ~ n i s t . ~ * ~ The homolog (SK&F 91486, 8b) is a more potent partial agonist42b,42c but further extension of the side chain (as in the N-methyl thia-linked anal g, SK&F 92408, &) gives an H antagonist which has no agonist activity.4 2 2 A fascinating compound is ogtained by combining the imidazolylalkyl groups (8b and 13a) into a single guanidine structure viz. SK&F 92676, irnpromidze, ( 9 ) ; t h i s compound is a very potent selective H2-receptor agonist, and is near1 50 times more potent than histamine on the guinea-pig atrium preparati~n.'~ Impromidine is less active on the rat uterus preparation (9 times more active than histamine) and it behaves as a partial agonist,

8 -

-9

94

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Pharmacodynamic Agents

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achieving only 80% of histamine's maximal response, which indicates that the increase in activity relative to histamine is due to increased affinity for H2 receptors rather than increased efficacy. Yet, a chemical group which contributes affinity in antagonist structures apparently converts a weak partial agonist (E)into an agent which has increased efficacy as well as increased affinity; this result has important implications in considering drug-receptor interaction. Impromidine is a potent stimulant of gastric acid secretion and may be useful as a diagnostic agent for the estimation of the maximal secretory capacity of the stom a ~ h s44 . ~ ~ Other amidines behaving like partial agonists are clonidine (lo) and tolazoline (11). Various suggestions have been made that clonidine stimulates CNS H ~ r e ~ e p t o r s , ~ 5 but ' ~ 6 on peripheral tissues clonidine appears to be a very weak partial H2 agonist47 and the studies suggest that the action of clonidine at H2 receptors is complex. Tolazoline has been estimated to have 3% he agonist activity of histamine on the guineapig isolated right atrium4' but it behaves like a partial agonist giving a sub-maximal resp0nse4~r49 Tolazoline may lead to H1 and H2 receptor

..

H

10 -

11 -

12 -

responses,50 and may act via histamine release. 51 The closely related compound, tetrahydrozoline (12), also acts as a histamine-like partial ag~ n i s tand ~ ~this activity apsars to be associated with the d - i ~ o m e rbut ~~ not the 1-isomer.

-

Antagonists Recent structure-activity studies of H1-rece tor antihistamines have been concerned with stereochemical aspects,53 994 partition characteristi~s,~''~~' 56 pattern reco nition,57 affinity contributions to activity4' and association phenomena.' 8 , 5 9 The subject has also been reviewed recently.60 Undoubtedly, the main focus of attention has been on the H2-receptor histamine antagonists. Three compounds have been widely studied, viz. burimamide2 (13b), metiamide" (13c) and cimetidine62 (13d), and their pharmacological pGerties summarizeu3 Their discovery and development at SK&F was the culmination of a research programme initiated in 1964. The search for an antagonist and some of the medicinal chemical approaches used in the structure-activity analysis have been outlined in several Concurrently, other researchers had sought R

CH2XCH2CH2NHCNHMe

I-\ HN

I

-R

X

Y -

a H S NH CH2 S b H 13 c Me S S d Me S N.CN antagonists using the early HI-antihistamine 929F as a starting point; they obtained an antisecretory agent but not an H2-receptor antagonist.66

GN

Chap. 10

Histamine Receptors

Ganellin

95

In structure-activity analyses of the H2-receptor histamine antagonists, physicochemical properties such as acidity, hydrophility, dipole moment and geometry have been emphasized. Thiourea, urea, guanidine, cyanoguanidine and nitroguanidine comprise the variable structural units of a small homogeneous group of compounds (13, Y = S,0,NH,N.CN,N.N02) which provide the basis for analysis of physicGhemica1 properties in relationship to biological a c t i ~ i t y . ~ Thiourea, ~,~~ nitroguanidine and cyanoguanidine are thereby regarded as bioisosteres with respect to action at histamine H2 receptors; the functional similarity between thiourea and cyanoguanidine is emphasized by the parallelism between variously substituted imidazoles, and also from a detailed analysis of their physico~hemistry.~~ The extent of the isosterism is strikingly illustrated by the almost identical molecular arrangements found in crystal structures of metiamide6’ and cimet i d i r ~ e . ~Comparison ~ of CH2 with thioether S linkages by crystal structure and solid-state infra-red spectra suggest that the thioether linkage may increase conformational flexibility and favor folding of the side chain; this effect may contribute to H2-receptor antagonist a~tivity.~’ An interesting correlation is found between crystal stability, m.p. and aqueous solubilities of these compounds.69 Alkyl branching of the methylene adjacent to the imidazole ring lowers activity7’ but whether this is a conformational effect has not been studied. Biological Distribution of Histamine Receptors - In the original classification of histamine receptors1y2 HI-receptors were defined as mediating those actions of histamine that were ilocked competitively by mepyramine,eg. the action of histamine in stimulating contraction of smooth muscle from the gut and bronchi. H2-Receptors were identified from those actions of histamine, refractory to mepyramine but blocked competitively by burimamide, viz. stimulation of rate of the spontaneously beating guinea-pig isolated right atrium (chronotropic effect), inhibition of evoked contractions of the isolated uterus from the rat, and the stimulation of gastric acid secretion. Both receptors appeared to be involved in mediating the depressor effect of histamine on blood pressure, as demonstrated in the cat. There has since been a considerable pharmacological investigation of histamine actions in various systems and species.6’7 The discovery of H2-receptor histamine antagonists has rekindled interest in histamine physiology. Three stimulants of gastric acid secretion, generally regarded as being of physiological importance, are histamine, gastrin and acetylcholine. The H2-receptor histamine antagonists and, to a lesser degree, anticholinergics can inhibit acid secretion induced by all three agents in vivo, and various models have been proposed involving sequential hormonal actions or interacting receptors on the parietal cell.72 To simplify the complex situation in vivo., studies also isolated stomachs,73 mucosal continue with in vitro preparations e sheets,74 or isolated parietal cells.

7;

A comprehensive review of histamine receptors in the cardiovascular system has been published.76 Histamine lowers blood pressure by causing widespread vasodilatation in most animal species but the effects are complex to analyse. It is clear that both H1 and H2 receptors are involved, but their distribution and effect varies depending on the species and particular vascular bed under study. Both types of antagonist have t o be administered together to fully block many of the vascular effects of

96

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histamine. In addition to the well known Hi-receptor effects of histamine in stimulating contraction of smooth muscle from parts of the gastro-intestinal tract, lungs and genito-urinary systems, there have been various reports of inhibitory (relaxant) actions of histamine which are apparently medin the lower esophageal sphincter of the oposiated by H2-receptors e sum,77 bovine stomach, chicken ileum 79 guinea-pig gall bladder ,80 tracincluding man,84 mouse vas deferhea and bronchus of various ens.85 The distribution of receptors is species dependent and in many cases both receptor types appear to be present and causing opposing effects. This raises interesting questions about possible function and requires careful study with respect to both drug concentration and time dependency. The possibility of both types of receptor being present in the guinea-pig ileum may have a bearing on the effect of temperature in apparently altering receptor population.82 In some circumstances, eg. cat bronchus and horse trachea, there may be actions of histamine not mediated by either H1 or H2 receptors.83

.

”

specie^^'-'^

One of the fascinating developments is the discovery of histamine receptors in the immuno-re ulatory system. Histamine has been shown to inhibit its own release,”‘ inhibit T-lymphocyte-mediated cytolysis (indeed, T-lymphocytes may develop H -receptors as they mature) ,86 inhibit enzyme release from 1eukocytes,lo3 inhibit production of migration inhibitory factor,87 modulate eosinophil migration,88 and suppress human lymphocyte responses to mi tog en^.^^ H2-Receptors appear to be involved in mast cells, basophils, T-lymphocytes and neutrophils. This array of histamine effects holds a tantalizing prospect for future drug investigation. In recent years evidence has accumulated to suggest that histamine may act as a neurotransmitter in the brain but direct evidence of a function is still lacking. Histamine is present in the brain, its distribution is uneven and histamine sensitive neurones have been demonstrated using microelectrophoretic measurements. However, administration of histamine, selective agonists, or antagonists have not clearly indicated whether there are CNS histamine receptors resembling the peripheral receptors. The last 2 or 3 years has seen a dramatic change, however, because investigations of CAMP accumulation in brain tissue, and direct binding studies, suggest the presence of both HI- and H2-receptors (see below), Furthermore a fascinating interaction with anti-depressant drugs has emerged.11 3-1 14

-

Chemical Control Substances Pharmacological classification of histamine receptors rests on the use of the specific competitive antagonists, supplemented by studies with relatively selective agonists and, ideally, characterization requires that the agonist - antagonist interaction is shown to be competitive.93 Where histamine is not administered but the possible involvement of endogenous histamine is being investigated, classification poses a special problem. Because drugs (whether agonist or antagonist) may cause effects other than the intended action at receptors, it may be helpful to use chemical controls i.e. compounds which match many of the chemical properties of the active drugs but lack the specific structural properties needed f o r effective receptor interactions, viz. N-telehistamine o r 2- or 4methylhistamines, &, me thylhistamine,29 6b) 4-pyridylethylamine, (14‘3 ($;) (cf. selective H1 agonist 2-pyridylethyl-

Chap. 10

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97

17

15

amine, 2) S-(2-dimethylaminoethyl)i~othiourea,~~(16, SK&F 91487 'nordimaprit') (cf. selective H2 agonist, dimaprit, 2)T-methyl+'13-(imidazol-4-y1)propyll ~ h i o u r e a ~(17, ~ , SK&F ~ ~ 91581, 'norburimamide') (cf. H2 antagonists, burimamide 13b, or Gtiamide 13c).

-

Cyclic Nucleotide Responses - Many H2-receptor mediated actions of histamine have been shown to be associated with increased levels of cyclic adenosine 3' ,5'-monophosphate (CAMP).9 5 Histamine stimulated adenylate cyclase systems from gastric mucosa have been found in most specie^^^,^^ including mang8 and results with antagonists and selective agonists indicate that this is an H2-receptor effect. Most work on histamine and cardiac adenylate cyclase has used the guinea-pig heartg9 and demonstrated that the inotropic effect of histamine (increase in force of contraction) on the isolated heart is closely preceded by increases in CAMP levels; this appears to be H2-receptor mediated. Studies on broken cell preparations indicate regional differences; the ventricle appears to contain an H2-receptor histamine-sensitive adenylate cyclase but in the left atrium, where the inotropic response to histamine is an H1-receptor system, CAMP does not seem to be involved.lOO,lO1 In the right atrium, where the chronotropic response to histamine is mediated by H2 receptors, it has not yet proved possible to identify an associated adenylate cyclase.' O1 Histamine increases CAMP levels in the brainAo2 depending on species, brain region, and method of tissue preparation eg. intact cells (brain slices) versus broken cells (washed membrane preparations) ,103,104 additions of adenosine1o291o5or GTP.106992 The uinea-pi has been most studied and, from the use of specific antagonistsK08~109r'H0 supplemented by selective ago,nists104y106y109y110 it appears that both HI and H2 receptors may mediate CAMP formation. Results have been variable and conflicting, probably due to differences in methodology. In the rat''' and chick'12 the receptors appear to be H2; in the rabbit,lo3 H1. D-Lysergic acid diethylamide, its 2-brorno-derivati~e~and many antidepressant drugs are a l s o potent inhibitors of histamine-sensitive aden late c clase in homogenates of guinea-pig hippocampus or neocortex;10z*92*118,114 they also inhibit dimaprit stimulation and thus appear to act at H2-receptors."" l 3 Histamine increases CAMP formation via H2-receptors in various other systems eg., segments of rat uterus,'l5 canine fat cells (leading to lipol sis),li6 mouse thyroid lobes (to facilitate th roid hormone secretion) ,lY7 glomureli isolated from rat renal cortex,lY8 human leukocyte^'^^ and neutrophils.l o 7 Histamine may stimulate formation of guanosine 3 ' , 5'-monophosphate (cGMP) via an H receptor, eg. in mouse neuroblastoma cells,12o human and guinea-pig lung.1121 Receptor Location and Binding Studies - Mepyramine tritiated in the 5position of the pyridine ring has been synthesizedi2' and specific binding to homogenates from guinea-pig small intestine demonstrated. '23 Inhibition of 3H-mepyramine binding by H -antagonists gave affinity constants in good 1 agreement with in vitro activities for antagonizing histamine stimulated contraction of ileum, suggesting that the observed specific binding is to

98

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histamine HI-receptors. Subsequently, binding of 3Hmepyramine to membrane fractions from guinea-pig or rat whole brains has also been demonstrated.1 2 4 9 1 2 5 Various phenothiazine antipsychotic drugs also bind to the mepyramine site126 in brain tissue but there are indications that 3Hmepyramine binding ma not represent a simple equilibrium with a single set of binding sites.y24 Binding of 3H-cimetidine to homogenates of guinea-pig whole brain,127 or with a particulate fraction of gastric mucosa from rats,128 has been studied but this does not necessarily represent H2-receptor binding. Binding of 3H-histamine to homogenized regions of rat brain,129 or of 14C-histamine to plasma membranes from rat gastric mucosal cells, 3 0 has also been studied, Histamine receptors have not so far been identified by chemical or physical means but there have been attempts13' to isolate and purify the histamine H1-receptor from a membrane fraction of smooth muscle of the cat small intestine. Binding of 3H-histamine to this fraction appears to be to H1-receptors, and is consistent in comparison with 2- and 4-methylhistamines.1 3 2 Affinity labelling with the 8-haloalkylamine, Dibenamine, was thwarted by lack of specificity, and a potential1 more specific alkylating agent (diphenhydramine mustard) was developed.l J 3 However, subsequent work on the guinea-pig ileum with the corresponding aziridine suggests134 that this compound does not alkylate the Hi receptor. Clearly there is a great need for specific affinity labels for both HI- and H2receptors. REFERENCES 1. A.S.F. Ash and H.O. Schild, Brit. J. Pharmacol., 27, 427 (1966). 2. J.W. Black, W.A.M. Duncan, G.J. Durant, C.R. Canellin and M.E. Parsons, Nature (London), 236, 385 (1972). 3. D.M. Paton in "Histamine and Antihistaminics," M. Schachter, Ed., Pergamon, New York, 1973, p3. 4. "Histamine I1 and Anti-Histaminics," Handb. Exp. Pharm. , Vol. 18/2 M.Rocha e Silva, Ed., Springer Verlag,Berlin Heidelberg New York,1978. 5. C.A. Lipinski and L.A. Hohnke in "Ann. Rep. Med. Chem.," Vol. 10, R.V. Heinzelman, Ed., Academic Press, New York, N.Y., 1975, p90. C.A. Lipinski and L.A. Hohnke in "Ann. Rep. Med. Chem.," Vol. 12., F.H. Clarke, Ed., Academic Press, New York, N.Y., 1977, p91. 6. J.W. Black in "Proc. 6th Int. Congr. Pharmacol., 1975," E. Klinge, Ed., 1976, p3. 7. N. Chand and P. Eyre, Agents Actions, 5, 277 (1975). 8. "Histamine Receptors, T 0. Ye1lin,,:dE Spectrum Pub1ications, Holliswood, N.Y., 1979. 9. "International Symposium on Histamine H2-Receptor Antagonists," C.J. Wood and M.A. Simkins, Eds., Smith Kline and French Laboratories Limited, Welwyn Garden City, 1973. 10. Feder. Proc., 35, 1923-1952 (1976). lla. "Cimetidine. Proceedings of the Second International Symposium on Histamine H2-Receptor Antagonists," W.L. Burland and M.A. Simkins, Eds., Excerpta Medica, Amsterdam-Oxford, 1977. llb. "Cimetidine. Proceedings of an International Symposium on Histamine H2-Receptor Antagonists," W. Creutzfeldt, Ed., Excerpta Medica, Amsterdam-Oxford, 1978. 12. Gastroenterol., 74, 338-488 (1978).

.

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13. G.J. Durant, C.R. Ganellin and M.E. Parsons, J. Med. Chem., 18,905 (1975). 14. W.F. Reynolds and C.W. Tzeng, Canad. J. Biochem., 55, 576 (1977). 15. C.R. Ganellin, J. Pharm. Pharmacol., 25, 787 (1973). 16. R.E. Wasylishen and G. Tomlinson, Can=. J. Biochem., 55, 579 (1977). 17. J.J. Bonnet and J.A. Ibers, J. Am. Chem. SOC., 95, 4829(1973). 18. S. Kang and D. Chou, Chem. Phys. Letters, 34, 537 (1975). 19. H. Weinstein, D. Chou, C.L. Johnson, S. Kang and J.P. Green, Mol. Pharmacol., 12, 738 (1976). 20* J.A. Defina and G.N. Vaughan, Austral. J. Pharm. Sci., 6, 15 (1977). 21 G. Simons and E.R. Talaty, J. Am. Chem. SOC., 2, 2407 71977). 22. S.R. Byrn, C.W. Graber and S.L. Midland, J. Org. Chem., 41,2283 (1976). 19, 1250 (19x). 23. W.G. Richards and J. Wallis, J. Med. Chem., 24. P.A. Kramer, J.R. Hazlett and D.O. Kildsig, J. Pharm. Sci., 66, 542 (1977). 25. D. Abernethy, T.J. Fitzgerald and E.J. Walaszek, Biochem. Biophys. Res. Comm., 2, 535 (1974). 26. C.R. Ganellin in "Molecular and Quantum Pharmacology," E.D. Bergmann and B. Pullman, Eds., D. Reidel Publishing Co., Dordrecht-Holland, 1974, p43. 27. G.J. Durant, C.R. Ganellin and M.E. Parsons, Agents Actions, 7, 39 (1977). 28. M.E. Parsons, D.A.A. Owen, C.R. Ganellin and G.J. Durant, Agents Actions, 7 , 31 (1977). 29. D.C. Carter, M. Impicciatore and M.I. Grossman, Agents Actions, 8, 453 (1978). 3 0 . S.B. Flynn, B.M. Johnston and D.A.A. Owen, Br. J. Pharmacol., 61, 101 (1977). 31a. G. Bertaccini, G. Coruzzi and T. Vitali, Pharmacol. Res. Commun., 10, 747 (1978). 31b. M. Impicciatore, G. Morini, F. Bordi and G. Bertaccini, Ital. J. Gastroenterol., 10,10 (1978). 32. G.J. Durant, J.C. Emmett, C.R. Ganellin, A.M. Roe and R.A. Slater, J. Med. Chem., 19, 923 (1976). 33. G. Bertaccini, M. Impicciatore and T. Vitali, Farmaco, Ed. Sci., 31, 934 (1976). 34. J.W. Black and C.R. Ganellin, Experientia, 30, 111 (1974). 35. L. Farnell, W.G. Richards and C.R. GanellinyJ. Med. Chem., 18,662 (1975). 36. E. Lebenstedt and W. Schunack, Arch. Pharm., 310, 455 (1977). 37. M. Impicciatore, G. Bertaccini, M. Chiavarini, E. Molina, T. Vitali and F. Bordi, Farmaco, Ed. Sci., 33, 696 (1978). 38. H.G. Lennartz, M. Hepp and W. Schunack, Eur. J. Med. Chem., 13,229 (1978). 39. L.L. Grechishkin, L.K. Gavrovskaya and V.L. Goldfarb, Pharmacol., 15, 512 (1977). 40. F.G. van den Brink and E.J. Lien, Eur. J. Pharmacol., 44, 251 (1977). 41. P. Dziuron and W. Schunack, Eur. J. Med. Chem., 10,129 (1975). 42a. G.J. Durant, M.E. Parsons and J.W. Black, J. Med. Chem., 18,830 (1975). 42b. M.E. Parsons, R.C. Blakemore, G.J. Durant, C.R. Ganellin and A.C. Rasmussen, Agents Actions, 5, 464 (1975). 28, 183 (1978). 42c. C.R. Ganellin, J. Appl. Chem. Biotechnol., -

100

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42d. G.J. Durant, J.C. Emmett, C.R. Ganellin, P.D. Miles, M.E. Parsons, H.D. Prain and G.R. White, J. Med. Chem., 2, 901 (1977). 43. G.J. Durant, W.A.M. Duncan, C.R. Ganellin, M.E. Parsons, R.C. Blake403 (1978). more and A.C. Rasmussen, Nature (London), 44. R.H. Hunt, J.A. Billings, J.G. Mills, W.L. Burland and G.J. MiltonThompson, Gut, 19, A980 (1978). 45. H. Karppanen, ITPaakkari and P. Paakkari, Eur. J. Pharmacol. , 42, 299 (1977). 46. S.R. Sastry and J.W. Phillis, Neuropharmacol., 2, 223 (1977). 47. M.E. Parsons, Agents Actions, 8, 402 (1978). 48. T.O. Yellin, J.W. Sperow and STH. Buck, Nature (London) 253, 561 (1975). 49. J. Sanders, D.D. Miller and P.N. Patil, J. Pharmacol. Exp. Ther., 195, 362 (1975). 50. S.C. Verma and J.H. McNeill, Agents Actions, 7, 191 (1977). 51. M.J. Hughes and L.J. O'Brien, Agents Actions, 7, 225 (1977). 52. T.O. Yellin, M.S. Katchen, S.R. Lavenhar and ETG.Nelson, Acta. Physiol. Scand. Special Suppl., 219 (1978). 53. N.S. Ham, J. Pharm. Sci., 65, 612 (1976). 54. A.E. Ahmed, P.E. Hanna and V.R. Grund, J. Med. Chem., 2, 117 (1976). 55. R.F. Rekker, W.T. Nauta, T. Bultsma and C.R. Waringa, Eur. J. Med. Chem., 10, 557 (1975). 56. B. Testaand L. Murset-Rossetti, Helv. Chim. Acta., 61, 2530 (1978) 57. A. Cammarata and G.K. Menon, J. Med. Chem., 19, 739 n976). 58. D. Attwood and O.K. Udeala, J. Pharm. Sci., 65, 1053 (1976). 59. J. Gettins, R. Greenwood, J. Rassing and E. Wyn-Jones, J.C.S. Chem. Corn., 1030 (1976). 60. A.F. Casy, p 175,and W.Th. Nauta and R.F. Rekker, p 215, in ref. 4. 61. J.W. Black, W.A.M. Duncan, J.C. Emmett, C.R. Ganellin, T. Hesselbo, M.E. Parsons and J.H. Wyllie, Agents Actions, 3, 133 (1973). 62. R.W. Brimblecombe, W.A.M. Duncan, G.J. Durant, J.C. Emmett, C.R. Ganellin and M.E. Parsons, J. Int. Med. Res., 2, 86 (1975). 63. R.W. Brimblecombe and M.E. Parsons in "Pharmacological and Biochemical Properties of Drug Substances,'' Vol. 1, M.E. Goldberg, Ed., American Pharmaceutical Association. 1977.- I*, 329. 64. C.R. Ganellin, G.J. Durant and J.C. Emmett, Feder. Proc., 35, 1924 (1976). 65. C.R. Ganellin, Farm. Tijds. Belg., 55, 1 (1978). 66. P.E. Cross, R.P. Dickinson, J.E.G. E m p , P.R. Leeming and L.G. Pullman, J. Med. Chem., 2, 1317 (1977). 67. G.J. Durant, J.C. Emmett, C.R. Ganellin, P.D. Miles, M.E. Parsons, H.D. Prain and G.R. White, J. Med. Chem., 20, 901 (1977). 68. C.R. Ganellin, p 251 in ref. 4. 69. K. Prout, S.R. Critchley, C.R. Ganellin and R.C. Mitchell, J. Chem. SOC. Perkin 11, 68 (1977). 70. E. Hsdicke, F. Frickel and A. Franke, Chem. Ber., 111, 3222 (1978). 311, 98 (1978). 71. K. Wegner, E. Fritschi and W. Schunack, Arch.:mrahP 72. A.H. Soll and M.I. Grossman, Ann. Rev. Med., 29, 495 (1978). 73. K.T. Bufice and M.E. Parsons, J. Physiol. (London), 258, 453 ( 976). 74. S.E. Sjostrand, B. Ryberg and L. Olbe, Arch. Pharmacol., 296, 139 (1977). 75. A.H. Soll, J. Clin. Invest., 61, 370,381 (1978). 76. D.A.A. Owen, Gen. Pharmacol., 8, 141 (1977). 77. S . Rattan and R.K. Goyal, J. Pharmacol. Exp. Ther., 204, 334 1978).

=,

Chap. 10 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117.

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102

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118. V.E. Torres, T.E. Northrup, R.M. Edwards, S.V. Shah and T.P. Dousa, J. Clin. Invest., 62, 1334 (1978). 119. L.M. Lichtenstein and E. Gillespie, J. Pharmacol. Exp. Ther., 192, 441 (1975). 120. E. Richelson, Science, 201, 69 (1978). 121a. L. Platshorn and M. K a l G r , J. Clin. Invest., 62, 1113 (1978). 121b. G. Martin and R.H. Fertel, Feder. Proc., 37, 393 (1978). 122. D.H. Marrian, S.J. Hill, J.K.M. Sanders and J.M. Young, J. Pharm. Pharmacol., 30, 660 (1978). 123. S.J. Hill, J.M. Young and D.H. Marrian, Nature (London), 2,361 (1977). 124. S.J. Hill, P.C. Emson and J.M. Young, J. Neurochem., 2, 997 (1978). 125. R.S.L. Chang, V.T. Tran and S.H. Snyder, Eur. J. Pharmacol., 48, 463 (1978). 126. S.J. Hill and M. Young, Eur. J. Pharmacol., 52, 397 (1978). 127. W.P. Burkard, Eur. J. Pharmacol., 50, 449 (1978). 128. G.C. Rosenfeld, E.D. Jacobson and W.J. Thomson, Gastroenterol, 70, 832 (1976). 129. J.M. Palacios, J.C. Schwartz and M. Garbarg, Eur. J. Pharmacol., 50, 443 (1978). 130. R.I. Bersimbaev, Vop. Med. Khim., 22, 728 (1977). 131. M.K. Uchida, Gen. Pharmacol., 9, 145 (1978). 132. M.K. Uchida and K. Takagi, Jap: J. Pharmacol., 27, 9 (1977). 133. K. Ohta, I. Takayanagi and K. Takagi, Pharmacometrics, 11,365 (1976). 134. M.Th.M. Tulp and J. Zaagsma, Eur. J. Pharmacol., 45, 95 (1977).

Annual Reports in Medicinal Chemistry-14

103 -

Section 111 - Chemotherapeutic Agents Editor:

Jerry A. Weisbach, Smith Kline & French Laboratories Philadelphia, PA 19101 Chapter 11. Antibiotics

P. Actor, R. D. Sitrin and J. V. Uri, Smith Kline & French Laboratories Philadelphia, PA 19101 Introduction - This chapter will be limited to antibiotics for which antibacterial activity has been demonstrated. Subjects related to biosynthesis are excluded. A variety of antibiotic topics were presented at the 18th Interscience Conference on Antimicrobial Agents and Chemotherapy, for which abstracts were published. The proceedings of the previous year's 10th International Congress of Chemotherapy cover a variety of clinical topics.' Two books of general interest are concerned with the indexing, isolation, separation and purification of antibiotics. 9 Cephalosporins - Most of the new cephalosporins have been designed to improve the shortcomings of the available products. Some under clinical development are wider spectrum agents possessing antipseudomonal activity and good 8-lactamase stability. Several others show favorable pharmacokinetics and offer the potential for reduced dosage or frequency of dosing. It is worthy of note that no significant orally-active cephalosporins were reported. Interest has continued in cephalosporins with antipseudomonal activity. Cefsulodin (SCE-129, CGP 7174, 1) shows moderate in v i t r o and in vivo activity against P. aeruginosaand gram-positive cocci with poor general activity against gram-negative organisms despite its high resistance to hydrolysis by various B-lactamases.'-' Its pharmacokinetics and metabolism have been studied in experimental animals.',' It is well tolerated in man and achieves good serum levels,exceeding those of carbenicillin upon IM or IV administration. lo,ll An effective clinical response in 54% of 320 patients with various pseudomonal infections was reported.l 2 T-1551 ( L ) , the cephalosporin analog of piperacillin, has good i n v i t r o activity against P. aeruginosa and a broad spectrum of gramnegative bacteria. It is stable to cephalosporinases from gram-negative bacteria, but less s o to penicillinases. l 3 The pharmacokinetic profile is similar to that of cefazolin; however, lower urinary excretion was observed. l 4 Good clinical activity was obtained against most susceptible organisms but only 319 patients with pseudomonal infections responded. l 4 HR756 (cefotaxime, 3 ) has remarkable broad spectrum in v i t r o activity.1 5 - 2 1 B. fragitis and Serratia are relatively resistant," as are pseudomonal strains. Resistance may be conferred by a modification of the permeability barrier. HR756 was not hydrolyzed by Richmond type I, 111, IV and V 8-lactamases and was found to be an inhibitor of 8lactamases.2 4 It offered good protection to mice infected with bacterial organisms." Initial clinical studies' 6-30 show it to be effective upon parenteral administration; however, the relatively low serum levels and half-life may limit its use in the treatment of pseudomonas infections. "9

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

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SCE-963 ( 4 ) 3 1 has broad activity f l J C H 2 - x COOH against bacteria3', '3 PharmacoR X R x kinetics in animals show lower blood JNf-J-coNH, 5 SNTZ 1 O C H levels and urinary HzN SO,Na 'OCH, excretion than with C1 Z C I H I - F on -CHNHC:- sJyl (SMTZ) " C N - C # r SEvz cefazolin. p 3 N P Preliminary clinic1 AH, cal studies are positive to date.35 OH -OCOCH, 7 HOOCl:H=CHSCH,-S A related compound H,N s 'OCH. SCE-1365 (5) is re8 CH,CDCH2SMTZ N -N ported to possess 4 -AN> &H smz broader in v i t r o HaN < ,CH, 9 0 1 CH2CH,N, CH, HNCONH, antibacterial activity with improved 8-lactamase resistance.3 6 Cefazedone (6) a narrow spectrum cephalosporin,3 7 gave low serum levels in man following IM or IV administration but good tissue demonstrated broad spectrum parenteral action penetration.3 8 K13176 (1) in limited laboratory studies.3 9 The synthesis and antimicrobial profile 40 of a series of 7-(B-heteroacylamino)cephalosporins have been described. Of these, the most active member, 8, showed moderate antibacterial activity i n v i t r o and i n v i v o . Several new cephalosporins containing 2pyridinethiol 1-oxide groupings were found to have i n v i t r o antifungal activity.41 None of these compounds protected mice infected with C. albicans, despite being active i n vivo against bacterial species. Of interest are a group of 7-ureidoacetyl cephalosporins in which the L-side chain isomers were potent antibiotics and had greater potency against gram-negative bacteria than the corresponding D-side chain isomers. 5469,613 (2) was the most active member of this series. ~

13i-

0

'

~

fiN,?

2 3 i-

1-y-

CH2,NHx

Ceforanide (BL-S786, 10)a parenteral cephalosporin with high and prolonged serum 11 CH,S NP COOH hHICOOH levels in man,4 3 3 4 4 has broad spectrum i n v i t r oH a ~ t ~i v i t~ y . ~ Initial ~ , ~ ~ clinical 11 ~ ~ ~ N-NO, N ' CH,S y Y N n trials using twice-a-day treatment show COO' h,so,H promise. 4 7 - 5 0 SK&F 75073 (11) is a new ii N -N parenteral broad spectrum cepfialosporin with +HF4 --R C H ~ ~ & ~ > N high and prolonged serum levels in experi'or< CDOH OCH > hHsSD,H mental animals. ' Serum levels and halfHoQFHC~>NH life exceeded those of any reported cephalo. 13 COOH LHIS IN sporin and this is reflected in animal proAH$ tection studies. Despite high binding to COOH serum proteins, it penetrates well into normal and inflamed tissues.'' Thfs compound offers the potential for once-a-day treatment in man. Another long-acting parenteral cephalosporin of note is SK&F 80303 (12) .5 The semisynthetic oxacephalosporins are new chemical structures synthesized from penicillin^.^^ The most promising member of this series, 6059-S (13)has broad Cn v i t r o and in vivo activitys5 and good serum levels in experimental animals following parenteral administration. 5 6 in @ z c o N p p > -

1 - N

N

0

r:

*G4:

Chap. 11

Antibiotics

Actor, Sitrin, Uri

105

Cephamycins - At present, the standard cephamycin is cefoxitin (3) A . review lists the isolated natural variants as well as the various methods for the 7a-methoxylation of cephalosporins ' SK &F 73678 (15 ), CS-1170 (16)and SQ 14,359 (17) - have been studied in the laboratory.-r,59 Basically these have the i n v i t r o and in v i v o properties of OCH, cefoxitin. Compound 17 is claimed to be the most r C ( I N H y S potent. ImmunologiT' and binding properties of o~N.,AcH2c)coNH2 16 to penicillin-binding proteins61 in E. coZi and CXOOH Proteus sp. are different from its demethoxy ana.14 .. log.

.

ucH Rumti

Fp '

Y -N

A. ,;

Penicillins - Interest in the penicillins is centered on broader spectrum, parenteral antispeudoI$ R = -ClI>SCH>CN monal ureido structures. Piperacillin (18) shows 1 7 K = -l~iI-~HCONH2 good i n v i & r o activity, superior to thatof car-. Ll-) benicillin against P. aeruginosa and other gramnegative organisms, but was hydrolyzed by many different 8-lactamases.6 3 Its activity against gram-positive cocci is generally comparable to carbenicillin and ticarcillin.62-64 It is rapidly absorbed and excreted in mice and is protective against a variety of infections. Initial clinical trials suggest that it is safe and of potential value in the treatment of serious bacterial infections.6 5 - 6 7 FurazloR cillin (Bay k 4999, 2) has i n v i t r o activity simico CnnH lar to piperacillin against P. aeruginosa. 6 8 It is I X less active than piperacillin against B. f r a g i Z i s X 5 and shows poor resistance to hydrolysis by staphyloA 1% H -N N-C>H, coccal B-lactamases. 69, 7 0 PL-385 (20) has a spectrum of i n v i t r o activity similar topiperacillin but P o has generally better protective activity in mice.'= n o/-'

CH,S

N,

CH ,

LiIOH

15

R = -IH,SCT,

&

0rob;g-2 ~

19

H

&rTo

-NKA-N-C'i

Other Inhibitors of Cell Wall Synthesis - Work continues with unusual 8-lactam structures, of which ,N thienamycin still shows high promise. The details 0 o f the structure determination of this antibiotic have been p~blished.~' Its total synthesis73 and that of a number of analogs have been described,including dl-descysteaminylthienamy~in~','~and the ring expanded analog, homothienamycin.76 This latter compound shows greatly enhanced chemical stability over thienamycin; however, its biological activity is substantially reduced. Alaphosphin (Ro 03-7008) is a synthetic phosphonopeptide (L-alanyl-'L'-1aminoethylphosphonic acid) which, in antagonist-free medium, is active against many gram-negative bacteria but less so against gram-positive c o c c i . " ~ ~ ~It protects mice infected with E. c o z i and K . aeroyenes and is absorbed rapidly upon parenteral or oral administration to laboratory animals or man. H

:O

OH

O

W C H O

*% do

'Inhibitors of 8-lactamases - New entities have been synthesized or isolated. 6-8CoOH 0 Bromopenicillanic acid is an irreversible 21 22 inhibitor of the 8-lactamase I o f B. cereus and B. Z i c h e n i f o m i s , but less s o of the enzymes of S. aureas and E. c o l i . 7 9 PS-5 ( 21 ) , a new natural product of a Streptomyces sp. A 271, CH,CH, f l C H I C H z N H C O ( X >

oc>

106

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Chemotherapeutic Agents

Weisbach, Ed.

possesses good activity gram-positive and gram-negative organisms and It greatly increases also inhibits 13-lactamases of various bacteria. the activity of ampicillin and cephaloridine vs P. vulgaris. CP-45,899 (22)is a new, synthetic, irreversibly acting Flactamase inhibitor with excellent solution stability.'l It has weak antibacterial activity and potentiates the i n v i t r o and i n V ~ V Oactivities of ampicillin 5 13-lactamase-producing strains. Experiments continue with clavulanic acid whose 13-lactamase inhibitory activity was first d$:s3fr,;bed in 1976. Its total synthesiss2 and that of a number of analogs were reported. A simple and rapid color-inhibition screening method for 8-lactamase inhibitors was described. Beta-lactamases have been reported from yeastssa and B. s u b t i l i s ATCC 6 6 3 3 , a widely used assay ~train.8~~0,~ CH'NH,

!ill,

OH f

26 23 24 -

25 -

I

n

OH Rr=COCH(OH)Cii,SR, Ez=R,=CH,. R.=OH X=H

27 Rl-CH,CHtOH)CH,Cll,NH, R ~ = R I = ! I . RI-OH

X=CH,OH

HIC

OH

Chap. 11

Antibiotics

Actor, Sitrin, Uri

107

Two new mechanisms of resistance to P-lactams have been reviewed.go The "tolerant" 5'. aureus strains are inhibited by low penicillin concentrations, but not by high concentrations, probably due to an excess of an inhibitor of autolysin.9 1 Enzyme preparations, derived from infected, human inflammatory exudate, were found to inactivate several 8-lactams but not other types of antibiotics.92,93 Aminoglycosides - Three semisynthetic analogs warranted extensive i n vivo and i n v i t r o studies. Sch 22591 (5-episisomicin, 23) has increased potency over gentamicin against Pseudomonas, Prouidencia and P. r e t t g e r i , as well as activity against many gentamicin resistant strains.94 Sch 9 6 as did another 21420 (26) shows activity comparable to amikacings~ amikacinanalog UK-18892 (27).', 9 8 New semisynthetic analogs were prepared to circumvent enzymatic inactivation in resistant strains. The 2l-N-methyl derivative of gentamicin Cla (29) was almost as potent as gentamicin Cla and was active against a resistant Providencia containing an aminoglycoside 2'-acetylating [AAC( 2 ' ) ] enzyme. The 3"-de-N-methyl derivatives of sisomicin (24) , gentamicin B (28) and gentamicin C2 (30) and the 3"-N-alkyl analogs ofgentamicin C2 (30) displayed activities similar to their parent compounds. l o o Compound 24 was isolated as a minor component, antibiotic 66-406, from Micromonospora inyoensis.'" The unique dimeric sisomicinlike aminoglycoside 66-40C (31) was used for the synthesis of sisomicin, antibiotic G52, and a series of 6'-N-alkylated sisomicins (2)some of which displayed activity against 6'-acetylase containing strains. ' O 2 Un- l o 4 showed like 5-deoxyneamine, 5-deoxykanamycins A, (32)l o 3 and (33) activity similar to,or weaker than their respective parents. The syntheses of 2"-deoxykanamycin B (34)and 2",3',4'-trideoxykanamycin B (35) were reported. l o 5 N-Alkylation of kanamycins A and B led to less active compounds. ' 2'-N-Ethyl-paromomycin (36) was as active as its parent. l o ' The 4-0-glucosamine unit o f parornomy& was replaced with glucose, mannose and galactose, with only the glucosyl derivative retaining activity. lo' The 2',3'-epimino analogs of neamine (37)and kanamycin B (38) were found to be inactive.log The a- and 8-furanosyl isomers of kanamycin B @)had low activity,l 1 as did other furanosyl neamines and paromamines. ' ' Compound 9, 3',3''-dideoxy butirosin,was less active than 3'-deoxybutirosins A or B. ' 1 2 Employing "mutational biosynthesis", a neamine-negative mutant of B. circuZans was used to prepare from various gentamines, the butirosin analogs, 9, 42, and 43, all with enhanced activity resistant strains.l13 '14 Various axial and equatorial 3',4',5 and 6 amino-deoxy derivatives of neamine and paromamine were described. '' 5-' '' Attempts to overcome enzymatic adenylation of spectinomycin at its 9-position by preparing the 9-deoxy- and 9-epi- derivatives, 44, 45, 46, and 47,resulted in inactive products, 1 1 9 ' 1 2 0 as did preparation of the 7Of the two monoguanidino derivatives of dihydrodeoxy analog, 48. streptomycin, 49 and 50, only 49 was active.'" A newly isolated, plasmid-determined acetyl transferase [AAC(3)-III] inactivates most a m i n o g l y c o s i d e s , i n c l u d i n g gentamicin and apramycin,but not butirosin, amikacin or fortimicin.l Z 3 Earlier data had indicated no cross-resistance of apramycin with other aminoglycosides.'" A broad-

108

Sect. 111 - Chemotherapeutic Agents

Weisbach, Ed.

spectrum enzyme, aminoglycoside nucleotidyl transferase [ANT(4',4")], capable of adenylating most aminoglycosides except gentamicin, sisomicin and apramycin, was isolated.l Z 5 An enzyme from a resistant S. aureus was found to inactivate amikacin by adenylating its 4'-position and phosphorylating its 3'-position. l Z 6 A study of the relationship of aminoglycoside accumulation and inactivation in a resistant E. c o l i was published. ' 2 7 Among the new aminoglycoside-like structures reported are the Ncoardia-produced novel glycocinnamoylspermidines , LL-BM123R (51), y, and y 2 (53) which are by definition not aminocyclitols since they contain no inositol moiety. 1 2 8 All three have broad-spectrum activity' 2 9 and a semisynthetic analog, isopropyl LL-BM123y (54) showed i n v i t r o and in vivo activity equal to or greater than gentamicin. 1 3 0 Structures of several minor, less active, nebramycin factors have been reported. '" Syntheses of dihydrostreptomycin,' 3 2 sorbistin Al , ' and some sorbistin analogs,1 3 4 as well as a new procedure for the preparation of 3'deoxy-agly~osides'~~ were published. The novel use of copper complexation to selectively acylate kanamycin A, the precursor to amikacin, was studied. 3 6 The 13c N M R spectrum of amikacin and its isomers was reported.1 3 7

(z),

A 2-deoxystreptamine (2-DOS) requiring idiotrophic mutant was used to determine the presence of 2-DOS in unknown antibiotic hydrolyzates.' 3 8 A B. eireuzans mutant was isolated which produced ribostamycin free of A rapid luciferase-based assay of gentamicin was reportxylostasin. 13' edml4' Labeled sisomicin was found to be taken up into bacterial cells at a higher rate than gentamicin. '" The binding of aminoglycosides to acidic mucopolysaccharides was studied.' Macrolides - A previously-reported basic 16-membered macrolide antibiotic, de-epoxy-rosamicin (M-4365 G2), is at least as active as erythromycin and josamycin. 1 4 3 Unlike erythromycin, it has i n v i t r o activity against a host of gram-negative bacilli, including indole-positive Proteus spp. and mycoplasmas. It protects mice infected with s. aureus.143 Deltamycins are produced by Streptomyces h a l s t e d i i subsp. deltae. 1 4 4 Deltamycin Ah, the most active component, was identified as carbomycin A. Demycarosylturimycin H is less active than turimycin and erythromycin v s 3. s u b t i l i s . 1 4 5 Inhibition of polypeptide and ribosomal peptidyl-transferase synthesis by demycarosylturimycin is associated with acetylation of the 4"-position. Several diastereomeric l0,ll-epoxyerythromycin B and 10-epi-erythromycin B ' s were synthesized with all being less active i n v i t r o then erythromycin A or B. 1 4 6 A structural revision of the megalomycins by 1 3 NMR ~ and X-ray crystallography was reported.147 A conformational study of oleandomycin and its derivatives was published. 14' Using a blocked mutant of the erythromycin-producing Streptomyces erythreus, biotransformation of lankamycin, darcanolide and ll-acetyllankolide was performed, but the products showed poor activity. Peptides - The structure of the cyclopeptide globomycin was described. 1 5 0 This spheroplast-forming antibiotic with mainly gram-negative activity inhibits the final steps of outer membrane lipoprotein synthesis.15' Teichomycins A1 and A2, phosphorous- and chlorine-containing glycopeptides produced by an Actinoplanes sp., are cell wall active antibiotics with mainly gram-posirive activity.'" Structural work on the thiostrepton-type

Antibiotics

Chap. 11

Actor, Sitrin, Uri

109

peptides multhiomycin,l53 siomycin-A,154 micrococcins P 1 and P Z l s 5 and the thiopeptin~,'~~ was described. The structure of bleomycin was revised, replacing the 8-lactam with an open carboxamide. 5 7 Some analogs of gramicidin S were prepared. 5 0 Studies of the solution conformation of echinomycinlS0 and the effects of bicyclomycin'61 and polymyxin Bi"' on bacterial membranes were published. Ionophores - Several new polyether structures were published, with.most bearing a close structural resemblance to earlier reported compounds. 1 6 3 - 1 6 5 The structures for the lasalocid-like aromatic ionophores noboritamycins A (55) and B isolated from Streptomyces noboritoens i s 1 6 6 and CP-44,161 (57) produced by DactyZosporangiwn sahonewn, 1 6 7 were reported as was that of another nitrogen containing nonpolyether ionophore, x145478 (58) isolated from Streptomyces antiAn organic salt of an optibioticus? 2 cally active amine was used in the X-ray determination of 58 to assign absolute configuration. The structures of two minor boron-containing aplasmomycin components were reported.16' The isolation and properties of ionomycin, a calcium specific ionophore produced by Streptomyces congZobatus, were described,170 as were the details the isolation and characterization of narisin17' and corriomycin. 17' The complete assignment of the 1 3 NMR ~ a El spectrum of lasalocid was given. The 300 MHz-NMRspectrum of septamycin was used to study the solution conformations of the antibiotic and its sodium salt.174 The effects of various ionophores on monovalent cation and divalent cation dislocation in mitochondria176were investigated.

(s),

1.1,

1.11

h

I >I

Miscellaneous Antibiotics - Other primarily antibacterial antibiotics reported in 1978 are listed in Table 1. Structural work was also published 77 for the following previously reported compounds: mimosamycin (2) griseorhodin C (60)17' ristomycin A, 17' striatin A (61),'" isochelocardin and rifamycin R. 1 m Marine sources (62) -,'"pseudomonic acid A and 65.'" have been used to obtain the new antimicrobial metabolites Syntheses of racemic negamycin and its analogs were reported. l e 6 'l e 7

ele4

Table 1

New A n t i b i o t i c E n t i t i e s

Antibiotic

P r o d u c i n g Organism

A32390A AA-57 Aurant i n i n Enaminornycin A Enaminomycins B , C E p i c o r a z i n e s A,B G1499-2 G2201-C Rubradirin B S e t omimyc i n

G+ ,AF Pyrenochaeta sp. G+ ,GStreptomyces sp. G+, Anaerob Bacillus aurantinus G+ ,G- ,AT ,AF Streptomyces baarnensis G+ ,GStreptomyces baarnensis G+ +icoccm n i g m G+ Cytophaga j o h n s m i i G+,GStreptomyces c a t t leya G+ S t rep tomyces achromogenes G+,AT Streptomyces pseudovenezuc !Zae

*G+,G-

(gram-positive,

-negative);

Activity*

AF ( a n t i f u n g a l ) ; AT ( a n t i t u m o r )

Structure

Reference

66

195 196 197 198 198 199,200 201

67 Polyene 68 69,70 71,72 73 74 75

-

202 203,204 205

110

S e c t . 111

In v i t r o and in

- Chemotherapeutic Agents

Weisbach, Ed.

V ~ V Od a t a on a tolypomycin Y d e r i v a t i v e “ ’ w i t h improved a c t i v i t y as w e l l as t h e a n a e r o b i c a c t i v i t y of t i a m u l i n , l B 9were p u b l i s h e d . n o c a r d i c i n A,’91 malonoThe t o t a l s y n t h e s e s of (-) v e r m i c u l i n e , micin, 19’ capreomycin, and e r y t h r o n o l i d e B 1 9 4 were r e p o r t e d .

0

Chap. 11

Antibiotics

Actor, S i t r i n , U r i

References -__ 1.

A b s t r a c t s , l R t h I n t e r s c i e n c e C o n f e r e n c e on A n t i m i c r o b i a l A g e n t s a n d Cliemntlrerrrpy ( 1 8 t h ICAAC), A t l a n t a , Georgia, O c t o b e r 1-4, 1978. C u r r e n t Chernothempy - P r o c e e d i n g s of t h e 1 0 t h I n t e r n a t i o n a l C o n g r e s s of Chemotherapy, V o l . I and V o l . 1 1 , W . S i e g e n t h . i l e r a n d R.LUthy E d s . , ASM P u b l i c a t i o n s . W a s h i n g t o n , D . C . ( 1 9 7 8 ) . 3. I n d e x a f A n t i b i r i t i c s from A c t i n o m y c e t e s , V a l . I T , H . Umerawa. Ed.. J a p . S c i e n t i f i c Soc. Press, ? h k y o (1978). 4. A n t i b i o t i c s - I s o l a t i o n , S e p a r a t i o n and P u r i f i c a t i o n , . I . Chromatography L i b r a r y , Vol. 1 5 , M . J . Weinsteln and 6 . H. Wapnan, Eds.. Elsevier S c i e n t i f i c P u b l i s h i n g CO., Amsterdam (1978). 5. K , T s u c h l y a . M . Kondo and H . Nagatorno, A n t i m i c r o b . Ag. Chemother. 13. 137 ( 1 9 7 8 ) . i b i d_ . 1 3 , 536 ( 1 9 7 8 ) . 6 . K . T s u c h i y a and M . Konda, _ 7 . M . Kondo a n d K. Tsuchiya, i b i d . 1 4 , 151 ( 1 9 7 8 ) . 8 . K . T s u c h i y a , H . Nagatorno. M. Kondo, Y. Kita and T. Fugona. 3 . A n t i b i o t . 2, 5 9 3 (1978). 9 . s. Tanayama, K. Y o s h i d a and Y . Kunai, A n t i m i c r o b . Ag. Chernother. 14,137 ( 1 9 7 8 ) . 10. K . Mashimo. T. T a g u c h i , Y. Nakana, T. Ynmamoto a n d N . Yarnaguchi. i n R e f . 2 . , p . 841. 11. J , F i l 1 h a a s . P . R . Irniiol a n d W . A. 12. K. Mashimo. 1 8 t h ICAAC, 159 ( 1 9 7 8 ) . 1 3 . S . M i t s u h a s h i , N . M a t s u b a r a , 5. Minami, T. Muraoka, T. YaSuda and I . Saikawa, E d . 1 5 3 , (1978). 14. Y . Ueda. A . S a l t o , M. Dhmori, K. S h l b a , T. Ysmaji and H. I h a r a . g . 1 5 8 ( 1 9 7 8 ) . 1 5 . .I. P e r r o n n e t , R . B U C O U C ~ , R . HeyniPs, 0 . Bomann a n d W . D u r c k h e i m e r , 75 ( 1 9 7 8 ) . 16. Y. A. C h a b b e r t and A. J . L u t z , A n t i m i c r o b . Ag. C h e m a t h r r . 749 (1978). 1 7 . R . Wise, T . R o l l a s o n , M . Logan. .I. M. Andrews and K . A . B e d f o r d , i b i d . 1 4 , 807 ( 1 9 7 8 ) . 18. J . P. Sosna, P. R. Murray and G. M e d c f f , i b i d . 1 4 , 876 ( 1 9 7 8 ) . 1 9 . P. M . Shah. G . T r o c h e and W . S t i l l e . J . A n t i b i o t . 1170 ( 1 9 7 8 ) . 2 0 . R . Vanhoof, .J. P . B u t z l e r and E. Yourassowsky, L a n c e t 2 , 209 ( 1 9 7 8 ) . 21. J . M. T. H a m i l t o n - M i l l e r . W. B r u m f i t r a n d A. V . R e y n o l d s , J. A n t i m i c r o b . Chemother. 5 , 437 ( 1 9 7 8 ) 22. H . C . N e u , N . Aswapokee a n d K. P . F u , 1 8 t h ICAAC, 77. ( 1 9 7 8 ) . 23. Y. A . C h r b b e r t , A . Dan8 Van and R . L a b i a , sii. 76 ( 1 9 7 8 ) . 24. K. P. Fu a n d H. C . Neu, A n t i m i c r o b . Ag. Chernother. 1 4 . 322 ( 1 9 7 8 ) . 25. 8. S c h r i n n e r , N . Klesel. M. L i m b e r t a n d A. I.utz, 1 8 t h ICAAC, 8 0 ( 1 9 7 8 ) . 26. w . M a r g e t , B. Belohrad.iky and R. ROOS, g . 296 ( 1 9 7 8 ) . 2 7 . K . H. S p i c z y . W . C r a n i n g e r , H. P i c h l o r and J , L a r k n e r , E.297 ( 1 9 7 8 ) . 28. P . M . S h a h , E. R. H e l m m d W . S t i l l r , 298 ( 1 9 7 8 ) . 29. C . K. D a i k o s , J. K o s m i d i s , H. Giamarellou. 8. D r a n l d i s and C. H. S t a t h a k i s . .&i 299 ( 1 9 7 8 ) . 30. P . H i n n i n e n a n d P. T o i v a n e n , E .300 ( 1 9 7 8 ) . 31. M . Numrlta, I . Minamida. M. Yamaoka, M . S h l r a i s h i . T. Miyawaki. H . Akimoto, K . N a i t o and M. K i d a , J. A n t i b l o t . 21, 12h2 (1978). 3 2 . K. T s u c h i y a , M. K i d a , M . Konda, H . Ono. M . T a k e u c h i and T. N i s l i i , A n t i m i c r o b . Ag. Chemother. 14, 5 5 1 ( 1 7 1 6 ) 33. K . T s u c h i y a . M. Kondo. Y. K i t a , Y. Noji, M . T a k e u r h i a n d T. Fugona, J . A n t i b i o t . 2,1272 ( 1 9 7 8 ) . 34. s. Tmayama. T. Kondo and Y . K a n a i , jbid. & 703 ( 1 9 7 8 ) . 35. K . S h i m i z u , I8Lli ICAAC, 160 ( 1 9 7 8 ) . 36. M . O c h i a i . A . Morimoto, T. Okeda, Y. M a t s u s h i t a , 0 . A k i , M. Kida and K. Okonagl, 150 (1978) 37. E . D i n g e l d e i n . H. W a h l i g and R. Bergmnnn, i n R e f . Z . , p . 8 3 2 . 38. c. L e a p o l d , W. Ungetlium, .I. P a b s t and E. D i n g c l d e i n , A N . p. 8 3 1 39. I . d e C a r n e r i . 6. M e i n a r d i , G. M o n t i , G . N a n n i n i and E. P e r r o n e , 1 8 t h ICAAC, 1 4 8 ( 1 9 7 8 ) . 40. . I . Numata, M . Yamaaka, 1. Minamida, M. K u r i t a n i and Y. I m a s h i r a , J. A n t i b i o t . 2. 1245 ( 1 9 7 8 ) . 41. J . V . Uri. P . A c t o r . L. P h i l l i p s and J . A. Weli,bach, i b G . 21, 5 8 0 ( 1 9 7 8 ) . 42. H. Breuer. 11. 0. T r e u n s r , H . J. S c h n e i d e r , M. G . Yome and H . I . Basch. i b i d . 31. 54h f 1 9 7 R I . 41. R . D. Smyth, M. P f r f f e r , 0. R . Van Harken, C . H. H o t t e n d o r f and A . G l i c k T m t h ICAAC, 1 5 6 ( 1 9 7 8 ) . 44. M. P f e f f e r , D . R. Van Harken a n d R. D . Smyth. 157 ( 1 9 7 8 ) . 45. s. Shadomy. G. Wagncr a n d M . C a r v e r , A n t i m i c r o b . A g . Chemother. 412 ( 1 9 7 8 ) . 46. R . J . G o e r i n g , C . C . S a n d e r s and W . E. Sanders, J r . , J. A n t i b i o t . 2 , 363 ( 1 9 7 8 ) . 47. M . J. R a f f . .J. C . Melo. R. V a r g h e ~ e . C . H. Chum and J . S u m m e r s g i l l , 1 8 t h ICAAC. I 6 1 ( 1 9 7 8 ) . 48. K. B u r c h . D . Polilod. I,. D . S o r a v o l a t z . D . K i a n i . T . Madhavan. E . L . [!\,inn. . , E . F i s h e r a n d R . D e l Busto. i b i d . I 6 2 (1978). 49. F. B. L u t z , .Ir. a i ~ dW. J . Magabgah, aij. 1 6 3 ( 1 9 7 8 ) . 50. R . J. W a l l a c e . J r . and R. R . M a r t i n , 1%. 301 ( 1 9 7 8 ) . 51. P . Actor, J. V . Url. I . Z a j a c , .I. R . G u u r i n i , L . P h i l l i p s , 0. H . P i t k i n , D. A. B e r g e s , 6 . L. Dun", .I. R . E . Hoover and J . A . Weisbach. A n t i m i c r o b . Ag. Chemother. 12, 784 ( 1 9 7 8 ) . 52. A. P. I n t o c c i a , S. S. W a l k e n s t e i n . C. J o s e p h , R. W i t t r n d o r f , C . Girman, D . T. Walz, P. A c t o r , and J. W e i s b a c h , 1. A n t i h i o t . 31, 1188 ( 1 9 7 8 ) . 53. J . V . U r i , P. A c t o r . .I. R . Guarini, L . P h i l l i p s . D. P i t k i n , 6 . L. Dunn, T. P a l a n s k y , J . R . E . Hoover and J . A . Weisb.ich, 1 8 t h ICAAC, 149 ( 1 9 7 8 ) . Hamashima, M . , M . N a r i s n d a , M. Y o s h i n k a , S . Uyeo, T . T s u j i , I . Kikkawa and W. N a g a t a , A b s t r . 1 7 6 t h Mtg. ,4. Am. Chem. Sor. ( 1 7 6 t h ACS),Mtami Beach, F l a . September 11-17, 1978. MEDI 1 4 . 5 5 . T. Y o s h i d a , M. Narisada, S. M a t s u u r a , W . N a g a t a and S . Kuwahara, 1 8 t h ICAAC, 1 5 1 ( 1 9 7 8 ) . 56. S . M a t s u u r a . T. Y o s h i d a , K. Suge no, Y. H a r a d a , M. Harada and S . Kuwahara, g . 152 ( 1 9 7 8 ) . 57. T. H i r a o k a , Y. Sugimura, T. S a l t " and T . K o b a y a s h i , H e t e r o c y c l e s S, 719 ( 1 9 7 7 ) . 5 8 . J. V. U r l . P. A c t o r , J . R. C u n r i n i , L. P h i l l i p s , 0. P i t k i n , R . M. DeMariniS a n d J . A . W e i s b a c h , J . Antlhiot. 82 ( 1 9 7 8 ) . 5 9 . H. H . Gadcbusch. R . Schwind. P. Lukaszow, R . Whitney a n d R . J . McRipley, i b i d . 31, 1 0 4 6 ( 1 9 7 8 ) 6 0 . M . Iwata and T . M a t u h a s i .. J a e. . J. E m . Med. 48.. 401 (1978). . 6 1 . 5 . Ohya, M . Yamazki. 5. Sugawara, S. Tumaki and M. M a t s u h a s h i , A n t i m i c r o b . Ag. Chemother. 3, 780 ( 1 9 7 8 ) . 6 2 . L . V e r b i s t , i b i d . 1 3 , 349 ( 1 9 7 8 ) . 6 3 . K. P. Fu a n d H. C. N m , i b i d . 1 3 , 358 ( 1 9 7 8 ) . 6 4 . G. P. Bade" and B. LeBlanc. _ i b i d_ . 1 4 . 71: (1178). 65. W. L. H e w i t t . W. Murohv. D . wan^ and.D. .I. Winston. 1 8 t h ICAAC. 1 6 7 ( 1 9 7 8 ) C . M . D i c k i n k n , D. G . ' i r o l l e r , 'R. L. Greenman a n d ' T . A. Hoffman, 166 ( 1 9 7 8 ) . 66. 61. F. 8 . L u t z , .I=. and W . . I. Mogabzab, Lbid. 303 (1978). 1 4 , 549 ( 1 9 7 8 ) 68. R . Wise, _ I . M. Andrews and K . A . B e d f o r d , A n t i m i c r o h . Ag. Chemother. 69. F. H. K s y s e r , 1 8 t h ICAAC. 137 ( 1 9 7 8 ) . 138 (1978). 10. K . Kung, K . P. Fu a n d H. C. Neu, 71. S. Nakamura, Y . T a k a s e , A . Minami. M. I s h i y a m a . K . N a k a t a , N . Kurobe and M. S h i m i z u , E.30 ( 1 9 7 8 ) . 7 2 . G . A l b e r s - S c h a n h e r g . R. H. Arisan, 0. D . H e n s e n s , J . H i r s h f i e l d , K. H o o g s t e e n , E. A . Kaczka, R . E . Rhodes, .1. S . Kahan, F. M. Kahan, R . W . R a t c l i f f e , E . W a l t o n , I.. J . R u s w i n k l e , R. 8 . Morin and R. C. C h r i s t e n s e n . .J. Am. Chem. Snc. 6491 (1978). 0. B. R . J o h n s t o n , S . M. S c h m i t t , F . A. B o u f f a r d and R . C,. C h r i s t e n s e n , i b i d . 100, 313 ( 1 9 7 8 ) . 73. 2.

14.

m.

1,

u.

a.

m.

E,

x.

u.

w.

x.

111

Sect. 111 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85.

86. 87. 88.

89. 90.

91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106.

-

Weisbach, Ed.

Chemotherapeutic Agents

D. H. Shlh, .I. Hannah and B. G. Christensen, ibid. 100, 8004 (1978). L. D. Cama and 8. 6 . Christensen, ibid. 100, 8006 (1978). T. N. Salzmann, R. W . Ratcliffr and 5 . G . Christensen, 176th ACS, MEDI 1 2 (1978) Editorial. Lancet 1.. 314 (1978). . , .I. G. All&. F. R.-Atherton, M . 1. Hall, C. H. Hassall. S. W. Holmes, R. W. Lambert, 1. J . Nisbet and P. S. Ringrose, Nature 72,56 (1978). R. F. Pratt and M. J . Loosemore, Pror. Nat'l. Acad. S c i . USA 75. 4145 (1978). K. Okamura, S. Hirata. Y. Okumura, Y. Fukagawa, Y. Shimauchi,T. K O U ~ O ,T. Ishikura and J . Lein, J. Antibiot. 2. 480 (1978). A. R. English, J. A. Retsema, A. E. Girard, J , E. Lynch and W. E. Barth, Antimicrob. Ag. Chemother. 14. 414 (1978). P. H. Bentley, M. L. Gilpin, E. Hunt, 6 . Brooks and I . I . Zomaya, Abstr. 174th Mtg. Am. Chem. S a c . , Chicago, Ill. August 28 - September 2, 1977, MEDI 43. P. H. Bentley and E. Hunt, J.C.S. Chem. Comm. 518 (1978). S. W. Elson and R. S . Oliver. J . Antibiot. 2, 586 (1978). P. C. Cherry, G. I. Gregory. C. E. Newall, P. Ward and N. S. Watson, J.C.S. C h e m . Comm. 467 (1978). P. C. Cherry, C. E. Newall and N. S . Watson, ibid. 469 (1978). .I. v. uri, P. ~ctor.and J. A. Weisbach, J . Antibiot. 21: 789 (1978). R . J. Mehta and C. H. Nash. ibid. 31, 239 (1978). 1. V. Uri. P. Actor and J . A. Veishach, ihid. 31, 1304 (1978). M. Finland, New Engl. J . Med. 99.770 (1978). L. D. Sabath, N. h'heelrr, M. Laverdiere. D. Blazevic and 8. J. Wilkinson, Lancet 1,443 (1977). P. Barnes and P. M. Waterworth, Br. Med. J. 1,991 (1977). J . deLouvois and R. Hurley, i h g . 1, 998 (1977). J. A. Waitz, 6 . H. Miller, E. Moss, Jr. and P. J . S . Chi", Antimicrob. Ag. Chemother. g , 41 (1478). T. L. Nagabhuahon. A. 8. Cooper, H. Tsai, P. J. L. Unniels and 6 . H. Miller, J . Antibiot. 21, 681 (1978). 6 . H. Miller, P. J . S. Chi" and J . A. Waitz. ibid. 31, 688 (1978). S . Jevons, H. E. Cheeseman and K. W. Brammer, Antimicrob. Ag. Chcmotlier. 14,277 (1978). R . J . Andrews. K. W. Brammrr. H. E. Cheeseman and S . Jevons, ibid. 14. 846 (1978). H. Matsushima and Y. Mari, J . Antiblot. 2.621 (1978). T. L. Nagabhushan. 1. J . Wright, A. 6. Cooper, W. N. Turner and G. H. Miller, ihid. 31, 43 (1978). M. Kugelman, R. S. laret. S. Mittelman and W. Gau, ibid. 31, 643 (1978). D. H. Davies. A. K. Mallams. M. Counelis, 0. Loebenbcrg. E. L. Moss. J r . . and J. A . Wuitz, J . Med. Chem. 21, 189 (1978). C. Kavadias, P . Dextrazc, R. Masse and B. Bellcnu. Can. J . Chem. 55, 2086 (1978). T. Suami, S. Nishiyama, Y. Ishikawa and E. Umemura, B u l . Chem. S O C . J a p . 2,2354 (1978). R. J . Reid, S. A. Mizsak, L. M. Raineke, c. E. Zurenko and R. J . Magerlein, 176th ACS, MEDI 18 (1978). S. Nakagawa. S. Tada, Y. Abe, H. Yamashita, K. Fujisawa, T. Naito and H. Kawaguchi, J . Antihiot. 31. 675 (1978). G. Cassinelli, C. Franceschi, 6 . Di Colo and F. Arcamone, x d .5.379 (1978). G. Cassinelli, P. Julita, and F. Arcamone, ibid. 3 1 , 3R2 (1978). V. Kumar and W. A. Remers. AhStr.17hth Mtg. Am. Chrm. S o c . . Anaheim, ralifornia, March 13-17, 1978, MEDT 37. R . D. Sitrin. D. J. Cooper and .J. A. Weisbach, J . O r g . Chem. 41, 3048 (1978). S. H an e s s i an . T. Ogawa, and T. Takamoto, Can. .I. Chem. 6. IS= (1978). T. Tsuchiya, I. Watanabc, F. Nakamura, M. Hamada and S. Umezawa, J. Antibiot. 2. 933 (1978). K. Takeda, A. Kinumaki, H. Haydsaka, T. Yamaguchi and Y. It", ibid. 31, 1031 (197R). K. Takedn, A. Kinumaki. S. Okuno, T. Matsushita and Y. Ito, g b f i . 31. 1039 (1978). S. Nisiiiyema, Y. Ishikawa, M. Yamazoki and T. Suami, Rul. Chem. S O C . J a p . 2.5 5 5 (1978). T. Suami. S. Nishiyama, Y. Ishikawa, and S . Katsura, Carbohyd. Res. 65, 57 (1978). F. R. Pfciflcr. S. J . Schmidt, C. M. Kinzig. J . R . E. Hoover and J . A. Weisbach. 176th ACS. MEDl 19 (1978). S . Hanessinn. R. tlssse and 1 . Nakaxawa. Cnn. .I. Chem. 5 6 , 1509 (1978). L. Foley, J. T. S . 1.1" and N. Wcigele. 1 . Antihiat. s 9 7 9 (1978). Ref. 119, p . 985. W. Rosenbrook, Jr.. R. E . Carney, R. S. Egan, R. S. Stanaszek, M. Cirouic, T. Nishinaga, K. Mochida and Y. Mori, ihid. 31, 451 (1978). T. Usui, T . Tsuchiya and S . Umezawa. iLid. 31, 991 (1978). J . Davies and S . O'Connor, Antimicrob. Ag. Chcmother. 14. 69 (1978). 1 . R. Walton, .I. Antimicrob. Chemother. 4. 709 (1978). P. Santanam and F. H. Kayser. J . Antiblot. 2.343 (1978). S . loda, S. Nakagawe, T. Naito and H. Knwaguchi, Tetrahedron Lett. 3917 (1978). P. Dirkie, L. E. nryan, and M. A. Plrksrd, Antimicrob. Ag. Chemother. 14, 569 (1978). C. A. Ellestad. D. B. Cosulich, R. W. Broschard, J . H. Martin. M. P. liunatmann, 6 . 0. Morton, J. E. Lancaster, W. Fulrnor and F. M. Lovrll, J . Am. Chem. SDC. loo, 2515 (1978). .I. H. Martin, M. P. Kunstmann, F. Barhatschi, M. Hertz. G. A. Elleslad, M. Dan", G. S . Redin, A. C. bornbusti and N. A. Kuck. .J. Antthiot. 31. 348 (1978). 31. i;ii5 (1978). N. A. Kuck and G. S. Redin, K. F. K m h , K. E. Merkel. S. C. O'Connor, J . L. Occoiowttz, 3 . W. Paschal. and D. E. Dorman, J. Org. Chem. 42, 1430 (1978). T. Yamvsaki. T. Tsurhtya and S. Umezava, J . Antibiot. 21, 1213 (1978). T. Ogawa. K. Katana snd M. Matsut. Carboliyd. Res. C13 (197R). M. M. Panpipam, R . Bugianesi. E. Walton and T. Y. siten. ibid. 65. 121 ( 1 9 1 8 ) . 1. Cleaphax, J . M. Delaumeny. S. D. Crra, A. Rolland a n d T R d i i a n d . .l.C.S. Chem. Comm. 773 (1918). S. Hanessian and G. Pstil. Tetmhedron Lett. 1035 (1978). S. Toda, S . Nakagawa, 'T. Naito and H . Kawaguchi, 3913 (1978). 8. K. Lee, R. G . Condon, M. Patel, E. M. Oden and .I. A. Waitz, Intimicrab. Ag. Chemother. 14, 157 (1978). T. Fujiwara, T. Tanirnoto, K . Matsumoto and E. Kando, J. Antibiot. 2,966 (1978). L. Nilsson, Antimicrab. Ag. Chemother. 14, 812 (1918). B. K. Lee, R. G. Candon, H. Mundyyer a n d x . J. Weinstein, J . AntibioL. 2,141 (1978). T. Degiichi, A. Ishii and M. Tanka, ibid. 31, 150 (1978). T. Yamaguchi, H. Hayasaka, H. Yoshlda.T.Tatsushitn, A. Yamabe and S . Uhshima, ibid. 2_1, 433 (1978). Y. Shimauchi, K. Knbo, K. O s u m i , K. Oknmara, Y. Fukagawa, T. I s h i k u r n and J. k i n, ibid. ?_?, 270 (1978). I . Haupt, H. Fricke, J. Cerna a d 1. Rychlik, ibid. 31, 610 (197Rj. J . Tadanirr, J . R . Martin, A. W. Goldstein m d E. A. tlirner, J . O r g . Chem. 43, 2371 (1978). . .I. B. norton, T. T. Thang, G. Lukacs, S . h u r a . P. Bartner, D. L. Borler. R. brambilla, A ~ K Mallamn, P. Reichert, F. D. Sanailio. H. Surprenant and C. Tomalesky. .J. Am. Chem. S o c , . loo, 6h3 (1978). ~

1117. 108.

109. 110.

111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125.

126. 127. 128. 129. 130.

131. 132. 133. 114. 135. 1i i i

.

137. 138. 139. 140. 141. 142. 143. 144. 145. 14b. 147.

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m,

m.

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Chap. 11

Actor, Sitrin, Uri

Antibiotics

H . Ogura. K . F u r u h a t a , Y . Harada and Y . I i t a k a , i b i d . 1 0 0 , 6733 ( 1 9 7 8 ) . A. W. G o l d s t e i n . 8. S . E g a n , S . 1.. M u e l l c r . J . R X H a r t K a n d W. K e l l e r - S c h i r r l e i n . J . A n t i b l o t . &. 63 ( 1 9 7 8 ) . 150. M . Nakajime. M . I n u k a i , T . H a n e i s h i , A. T c r a h a r a , M . h r a i , T . K i n o s h i t a and C . Tsmura, i b i d . 426 ( 1 9 7 8 ) . 151. N . I n u k a i . M . T n k e o c h i , K . Shimizu 2 n d M. A r a i , i h i i l . 31, 12113 ( 1 9 7 8 ) . 170 ( 1 9 7 8 ) . 152. M. R . E a r d o n e , M . P a t e r n o s t e r and C. C o r o n e l l i , 1 5 3 . T. Endo and H. Y o n e h a r a , iLi4. 2,623 ( 1 9 7 8 ) . 154. A. O l e s k e r , L . V a l e n t e , L. R a r a t a , C . I.uk:ics, W. r . H a l l , K . T o r i , K. Tokura, K. Okahc, M. Ebata and H. O t s u k a , J . C . S . C h c m . Comm. 577 ( 1 9 7 8 ) . 155. B. Id. B y c r o f t and M. S. Cowland, g. 256 ( 1 9 7 8 ) . 156. t l . U . Hsnsens and C . A l b e r s - S c h o n b e r g , T e t r a h c d r o n L e t t . 3649 ( 1 9 7 8 ) . 157. T. T a k i t a , Y . Mursoka. T . N a k a t a n i , A. F u j i i , Y . Umezawa, H . Naganawa, and H. Umezawa, J . A n t i b i o t . 31, 8 U I (1978). 1 5 8 . K . S a t o , K. Ileda. M. Kondo, H. Aoyagi and N . Icumiyo, B u l l . Chem. S o t . J a p . 5 1 , 1830 ( 1 9 7 8 ) . 159. K . Nonaka, K . S a t o , S . Terada. T. Kato and N . Izumiya, i b i d . 2127 (1978);160. H . T . Cllcung, .I. r e c n e y , G , C . K. R o b e r t s , 0 . 11. W i l l i s s r , C. U g h e t t o , and M . J . Wnring, J . Am. C i i e m . sot. g i ! , 46 ( 1 9 7 ~ ) . 161. A . Sonieya.. M . I s e k i and N . Tanaka, J . A n t i b i o t . 3J. 712 ( 1 9 7 8 ) . 162. D. A. Brown and J . C . T s m g , I - h s . LJ, 603 (1978). 163. M. S h i r o . 11. Nakai, K . Nagashima and N. T w j i , . I . C . S . Ci~ern. C c ~ r n . 682 ( 1 9 7 8 ) . 1 6 4 . J . L. Occolowitz. D . E. Dorman .and R. L . Hamill, i s . 683 ( 1 9 7 8 ) . 165. H. S e t o , K . Mizoue, N . W a k e , M . Yamagishi, T . M i z u t a n i , H. Hara, and S. Omura, J . A n t i b i o t . 31, 929 ( 1 9 7 R ) . 166. C. K r l l r r - J u s l r o . H. D. King, M . Kulm, H . L o o s i i and A . von Wartburg, i b i d . 3 1 , 820 ( 1 9 7 8 ) . 167. J. Tone, R . Shibakawa, H. Maeda, K . I n u u e , S . Nishiyanm. M. I s h i g u r o , W. P . C u l l e n , J . B . Routirn, L. R. C h a p p e l , C . E. M o p p e t t , M. T. Jefferson and W. D. C e l m e r , 1 8 t h I C M C , 1 7 1 ( 1 9 7 8 ) . l h 8 . J. W. W e s t l e y , R . H. Evans, J r . , C.-M. L i u , T. Herman" and J . F. B l o o n t , J. Am. Chem. Soc. loo, 6784 1 1 9 7 8 ) . 169. K . S a t o , 'T. Okaadki, K. Maeda and Y. Okami, J . A n t i b i a t . 3J. 632 ( 1 9 7 8 ) . 170. W. L i u , D. S. S l u s a r a c h y k , G . A s t l e , W. H. Trejo. W . E . Brown and E. Meycrs, 31, 8 1 5 ( 1 9 7 8 ) . 1 7 1 . D. H. Berg and R . L. H a m i l l , Lkii. i (1978). 172. A. Imada, Y . Nozaki, T. Ha,egawa, E. M i e u t a , S . 1 ~ a r a s . i a n d M . Yoneda, i b i d . 31, 7 ( 1 9 7 8 ) . 173. H . Sero. J . W. W e s t l e y and R . G . P i t c h e r , 289 (1978). i b i d . 31. 294 ( 1 9 7 8 ) . 174. N . A . R o d i o s and M . .J. 0 . A n t e n i s . -~ 1 7 5 . M . M i t a n i and N . O t a k e , i b l d . 3 1 , . 7 5 0 ( 1 9 7 8 j . 176. R e f . 1 7 5 , p. 888. 177. H. Fukumi, F. Maruyama, K. Yushida, M. A r e i . A . Kubo and T. A r a i , g . 2.847 ( 1 9 7 8 ) . 970 ( 1 9 7 8 ) . 178. K . E c k a r d t , D . T r e s s e l t and W. I h n , i b i d . 179. A. N e s z m i l y i , F. S z t a r i r s k a i , A. L i p z a n d R . ~ o & r . G. 974 (1978). 180. 11.-J. H e c h t , C . H o f l e and W. S t e g l i c h , J . C . S . Chcm. Comm. 6 6 5 ! l ( J 7 8 ) . i n i . E. B e r n s c e i n . D . T . W . C I u , S . N . Huckin. U . L . Garmaisc. R . S . E ~ a c ,T. J , P e r u n , W. Rosenhrriok, J r . , and R. E. C a r n e y , J . Org. Chem. 42, 2855 ( 1 9 7 8 ) . 182. R . C. A l e x a n d e r , .J. P . C l a y t o n , K. Luk, N. H. Rogers and T. 3. King, J. C. S . P e r k i n I , 561 ( 1 9 7 8 ) . 183. E . M u r t i n e l l i , P. A n t a n i n i , R . C r i c c h i o , C. L a n c i n i and R . J . W h i t e , .I. A n t i b i o t . 2. 9 4 9 ( 1 9 7 8 ) . 184. 6 . T. C a r t e r and K . L. R i n e h a r t . J r . . J . Am. Chem. so?. 100. 7441 ( 1 9 7 8 ) . 185. M. T. Chrng ,and K. L . Rinehart, .IT., i b i d . 100, 7409 (197R). A n t i b l o t . g . 725 ( 1 9 7 8 ) . 186. W. S t r r i c h e r , H . Reinshagen and F. Tu;&;sk<,J. 1 8 7 . W. V . Currvn and J. H . Boothe, i b i d . 31, 914 ( 1 9 7 8 ) . 188. L . S t a n z a n i , A . P. V e n t u r i n i and V . Mantovani, i l i i d . 31, 1195 ( 1 9 7 8 ) . 189. H . Werner. 6 . L a b e r , E . Schiitze. C . Krasemann a n d P. Yay, i b i d . 31, 756 ( 1 9 7 8 ) . 190. K. F. B u r r i , R. A. C d r d o n ~ . W. Y. Chen .and P. Rose", J . Am. Chem. S O C . LO!, 7069 ( 1 9 7 8 ) . 191. G . A . K op p e l , I.. McShane. F. .Jose and R. D. 6 . Cooper, i b i d . 100, 3931 ( 1 9 7 8 ) . 192. 1. L. van d e r Barn, J. W. F. K . Barnick a n d F. B i c k e l h a u p t , T e t r a h e d r o n 2,223 ( 1 9 7 8 ) . 193. S. Nomoto, T. Teshimn, T. Wakamiya and T. S h i b a , 211, 921 ( 1 9 7 8 ) . 1 9 4 . E. . I . C o r e y , S . K i m , S . Yoo. K . C. N i c o l a a u , L. S. M e l v i n , J r . . D . J . B r u n e l l e . J . R . F a l c k , E. J. T r y b u l s k i , R. L e t t and P . W. S h e l d r a k e . J . Am. Chem. Sac. loo, 4620 ( 1 9 7 8 ) . 195. C . G . M a r c o n i , B . B. ? l o l l o y , R. Nagarujan, J . w. M a r t i n . J . R . D e e t e r and J. L. Occolowitz. J. A n t i b i o t . 27 (1Y78). 1 9 6 . S . Aizawa, H . Akutsu, T. S a t o m i , S. Kawabata a n d K. S a s a k i . i b i d . 31, 729 ( 1 9 7 8 ) . 197. T. N i s h i k i o r i , R . M a m m a . R. O i w n , M. K a t a g i r l , J. Aoaya, Y . Iwai and S. Omura, i b i d . 3 1 , 525 ( 1 9 7 6 ) . 198. Y . I t o h , T . H e n c i s h i , M . A r a i . T. Hata. K . Aiha and C . Tamura, L b s . 838 ( 1 9 7 8 ) . 199. G . Deffieun, M. R a u t e , R . Baute a n d M . F i l l e a u , g . 31, 1102 ( 1 9 7 8 ) . 200. 6 . DefFlcux, M. Filleau,ond R . B m t e . ijJi. 2 ,1106 ( 1 9 7 8 ) . 201. J. R . Evans, E. .I. N a p i e r and R. A . F i e t t a n , l b i d . 9 5 2 (1978). 102. M . N o b l e , U . Nohl'e and R. A . F l e t t o n , i b i d . 3 T i 5 ( 1 9 7 8 ) . 203. H. Hoeksem.4. C. Lewis. S . A. Mizsak. .J. A. S h i l e v,. . U . R . Wait. H. A . Whale" and C. E . Zurenko. i b i d . 31. 945 ( 1 9 7 8 ) . ' 204. H. Hoekserne. C. C h i d e s t e r , S. A. Mizsak and L. Huczynskyj, i b i d . 31, 1067 ( 1 9 7 8 ) . 205. S. O m U T a , H . Taoaka. Y . I w a i , K . N i s h i g a k i , J . Awaya, Y . T a k a h a s h i and R . Masuma, z d . 2,1091 (1978). 148. 149.

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113

114 -

Annual Reports in Medicinal Chemistry-14

Chapter 12. Chemotherapy of Sexually Transmitted Infections

H. Hunter Handsfield Seattle-King County Department of Public Health, Seattle, Washington 98104 Marvin Turck Harborview Medical Center, University of Washington, Seattle, WA 98104 This chapter is a review of the antimicrobial susceptibilities of the causative organisms and the treatment of the sexually transmitted infections most prevalent in North America and Western Europe, gonorrhea, nongonococcal urethritis, genital herpes virus infections, and trichomoniasis. Because of their relative rarity or because no important advances in therapy have been made in the past decade, syphilis, lymphogranuloma venereum, chancroid, and granuloma inguinale are not considered. Gonorrhea Penicillins - For a 30-year period ending in the early 1970s, there was a gradual increase in the prevalence of strains of Neisseria gonorrhoeae with low level resistance to the penicillins, with consequent periodic increases in the minimal doses necessary for adequate treatment. Prior to 1955, 99.4% of g. gonorrhoeae isolates in the United States had minimum inhibitory concentrations (MIC) of penicillin G of 0 . 0 3 mcg/ml,’ and aqueous procaine penicillin G (APPG) 1.2 million U intramuscularly (IM) in a single dose cured 195% of patients with uncomplicated an0 enital infection. By 1975, only 31.0% of isolates had MICs 50.03 mcg/ml,’ 17.4% had MICs 20.5 mcg/ml,2 and APPG 4.8 million U IM, with probenecid 1.0 gm orally, was required to cure 96.8% of patients with uncomplicated infection.3 In the past five years, susceptibility to the penicillins has remained stable,2 probably because the recommended doses are now sufficiently high to prevent further , selection of more resistant strains.4 Ampicillin2 and amoxicillin5 are effective alternatives to penicillin G Many other penicillins (talampicillin, pivampicillin, carbenicillinYgbacampicillin,g and others) are active in vitro and have been reasonably successful in limited clinical trials, but offer no important advantages over penicillin G, ampicillin, or amoxicillin. Penicillin V and the enicillinase-resistant penicillins are less active against g. gonorrhoeaeyY0 and their use has been associated with unacceptable clinical results. The U.S. Public Health Service Center €or Disease Control (CDC) recomendsll APPG 4.8 million U IM, ampicillin 3.5 gm orally in a single dose, or amoxicillin 3.0 gm orally in a single dose for uncomplicated anogenital infection; each is given with 1.0 gm of probenecid orally. Pharyngeal gonococcal infection responds well to APPG-probenecid , but less reliabl to ampicillin-probenecid or amoxicillin-probenecid in single doses. ,16 Ampicillin-probenecid is associated with slightly higher failure rates compared to APPG-probenecid in anorectal infection,l3 especially in homosexual men. Single dose therapy with any of the penicillins is inadequate for ascending genital infection in women (pelvic inflammatory disease); this complication is usually treated with 7-10 day courses of parenterally administered penicillin G and/or oral am~icil1in.l~The strains of 2. gonorrhoeae that cause over 90% of cases of hematogenously disseminated gonococcal infection have retained susceptibility to ~0.06mcg/ml penicillin G,15 making this syndrome unique among bacteremic diseases in Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-1’2-040514-8

Chap. 1 2

Sexually Transmitted Infections Handsfield, Turck 115

that short (3-day) courses of intravenous (IV) penicillin GI6 or oral therapy alone (ampicillin 2 gm daily in divided doses for 7-10 days)17 are appropriate and uniformly effective, if endocarditis or meningitis is not present. In 1976, Falkow and his colleagues18 predicted the appearance of plasmid-mediated beta-lactamase production by E. gonorrhoeae, a prediction that was soon realized.", 2o Fear that penicillinase-producing 3. gonorrhoeae (PPNG) would become a major public health problem has stimulated a search for additional alternatives to the penicillins. Although PPNG accounts for up to 25% of gonococcal isolates in some areas (notably the Philippines, Southeast Asia, and parts of Africa), they remain rare inmost other areasY2O including North America and most areas of Europe. Other Beta-lactam Antibiotics - Several newer cephalosporins and cephamycins have been tested clinically in gonorrhea. Cefazolin, cefoxitin, cefuroxime, cefamandole, and others are effective in vitro,l0y21 but have given variable results in clinical trials.22-24 Cefuroxine is the most effective, with cure rates in uncomplicated infection of 98-100% when 1.0-1.5 gm is given IM with probenecid 1.0 gm orally, and is effective against PPNG.25 Tetracyclines - Susceptibility of g. gonorrhoeae to the tetracyclines is linked to low level penicillin resistance. Gradually increasing resistance to the tetracyclines occurred through 1972, but since then there has been little change, with 86.0% of strains currently having tetracycline HClMICs c l . 0 mcglml and 14.0% having MICs 22.0 mcg/ml.2 The tetracyclines are not clinicall effective in single doses.26 The regimen recommended in 1974 by the CDC,2V tetracycline HC1 1.5 gm orally as a loading dose, followed by 0.5 gm orally four times daily for four days (total 9.5 gm), results in a cure rate of 96.2% in uncomplicated anogenital infection.3 The 1979 recommendationsll have eliminated the loading dose, with consequent reduction of nausea and vomiting that tended to limit patient compliance, and extended the duration of treatment to five days; cure rates with this regimen are comparable.28 Other tetracyclines, including doxycycline,29 minocycline,30 and others, also are effective. PPNG are often highly resistant to thetetracyclines, with 39% and 2 1 % of isolates having tetracycline HC1 MICsL2.O mcg/ml and 24.0 mcglml, respectively.20 Aminocyclitols - Spectinomycin HC1 in a single dose of 2 . 0 gm IM cures 94.8% of patients with uncomplicated anogenital i n f e ~ t i o n . ~ .MICs ~ ~ are virtually always (32 mcg/m12,31 and have not changed appreciably since introduction of this drug for clinical use in 1971. Resistance of E. gonorrhoeae to .spectinomycinis genetically independent of penicillin andtetracycline re~istance,3~ and spectinomycin is the recommended treatment for gonorrhea that has not responded to a penicillin or tetracycline," as well as for patients with allergy or idiosyncratic intolerance to these agents. It is also the agent of choice for infections caused by PPNG.20 Resistance to spectinomycin is rare;2,31,33 however, absolute resistance can be induced in a single step by serial passage in media containing subinhibitory concentrations of the drug,34 and it has been recommended that its use be limited to the above indications, in the hope that inductio of a resistant population of gonococci can be delayed or avoided.33s32 Although originally effective, streptom cin is no longer useful for gonorrhea, due to a high level of resistance.I0 Gentamicin, kanamycin, tobramycin, amikacin,

116

Sect. 111

-

Chemotherapeutic Agents

Weisbach, Ed.

and other aminocyclitols are effective in vitro," and some have been used successfully in limited clinical tria1~7~,36ut none has important advantages. Miscellaneous Agents - Originally susceptible, N. gonorrhoeae is nowresistant to the sulfonamides (sulfamethoxazole MIC 2 5 mcglml in 40% of isolates) , l o and is relatively resistant to trimethoprim (MIC 225 mcg/ml in 36% o f isolates),1° but synergism is present, and treatment with sulfamethoxazole and trimethoprim has resulted in cure rates nf ~ 9 4 % when givenin various multiple-dose regimens for at least two days.37938 This combination is also effective against PPNG.20 Erythromycin was recommended by the CDC through 197427 as an alternative to the penicillins during pregnancy, but recently has been found to result in a failure rate of 24X when atotal of 9.0 gm was given orally over four days,39 and erythromycin is no longer recommended.11 Rifampin has been used successfully, 40 but is not recommended, largely for ecological reasons and the likelihood of induction of resistance. Chloramphenicol is effective,lobut is inappropriate because of potential serious toxicity. Nongonococcal Urethritis The nongonococcal urethritis (NGU) syndromes are sexuallytransmitted infections that are as fre uent as gonococcal urethritis.in the United States and Western Europe.%l Approximately 40% of NGU is due to Chlamydia trachomatis.42-44 The etiologies of the remainder are uncertain; &plasma urealyticum ("T-strain mycoplasma") may be an importaz$ cause of most of the r e m a i x ~ d e r , ~although ~ , ~ ~ conflicting data exist. Uncontrolled studies recently suggested a role for Corynebacterium genitalium type 1 .45 The same strains of C. trachomatis that cause NGU cause most acuteepididymitis in men under age 35,"' symptomatic cervicitis,43 neonatal inclusion conjunctivitis,47 and infantile nasopharyngitis and pneumonia 48 and have been implicated in nongonococcal pelvic inflammatory disease.29 Other serotypes cause endemic trachoma and lymphogranuloma venereum. U.urealyticum infection has not been proven as a cause of syndromes other than NGU, but has been statistically linked with prematurity and other puerperal complications.50 Tetracyclines - The tetracyclines are the agents of choice for the treatment of NGU.41,51 -In vitro, minocycline (MIC and MBC 20.03 m ~ g / m l )and ~~ ~ ~ more active aganist C. trachodoxycycline ( M E and MBC (0.03 m ~ g / m l )are matis than is tetrac cline HC1 (MIC 20.06 and MBC (0.125 m~g/rnl)5~and other tetracyclines.g3,54 g. urealyticum is most susceptible to minocycline a92 doxycycline, 85% of strains having MICs to both agents of 51.0 When continued for 27 days, several different regimens of tetramcgfml. cyclines have been equally successful in inducing clinical responses in NGU, including tetracycline HC1 0.5 gm four times daily43951 and minocycline 100-200 mgm experience with doxycycline is limited.58 The rates of persistence or relapse within six weeks of treatment have been <17% for chlamydia1 NGU and 40-50% for nonchlamvdial NGU,57 and NGU associated with neither C. trachomatis nor 1 . urealyticum may have still higher rates of treatment failure.44 The optimal duration of tetracycline therapy is debated; some studies suggest lower rela se or recurrence rates when therapy is prolonged to two or three weeks ,g7959 p 6 0 but more carefully controlled studies have shown no additional benefit when treatment was ex-

Chap. 12

Sexually Transmitted Infections Handsfield, Turck

117

tended beyond 7 days .44,61~62 Moreover, symptoms of NGU usually subside within the first 5-7 days of therapy,44,51 after which patient compliance is often uncertain. Other Agents - Erythromycin in a dose of 0.5 gm four times daily for 7 days is usually given for NGU when tetracycline is contraindicated. Cure rates and relapse rates are comparable to those seen with the tetracyclines$4 but the total experience is much less than with the tetracyclines and there are no studies correlating success rates with C. trachomatis or U. ureal ticum isolation. C. trachomatis is generally susceptible (MIC 3.0 mcg/ml. Nevertheless, er thromycin usually eradicates urethral colonization with urealyticum.6qb As with tetracyclines, there are no controlled studies demonstrating additional therapeutic benefit beyond seven days treatment.

1

v.

Aminocyclitols in general, and spectinomycin in particular, are effective in no more than half the patients with NGU,42 an observation that correlates with the fact that g. urealyticum is s ~ s c e p t i b l ebut ~ ~ C. trachomatis is not .52353 Conversely, sulfonamide therapy is successful in chlamydial NGU, but not in NGU associated with urealyticum. The differential response to sulfisoxazole or spectinomycin has been used to differentiate chlamydia1 from ureaplasmal NGU,42 and a combination of these two agents deserves a clincial trial as an alternative to the tetracyclines.44 C. trachomatis and V. ureal ticum are individually susceptible to several other antimicrobial agents, -but all are clinically ineffective or have not been subjected to controlled studies.

u.

Genital HerDes Virus Infections Sexually transmitted infections with Herpes simplex (Herpesvirush%-

type 2 is epidemic in Europe and North America. The importance of e) this infection is compounded by its recurrent nature, its probable etiologic association with squamous cell carcinoma of the cervix, its association with infrequent but devastating neonatal infections, and the lack of effective prophylaxis or therapy. Despite initial optimistic reports, all regimens that have been subjected to rigorously controlled clinical trials have demonstrated no benefit, either with regard to speed of healing or frequency of recurrences.64 Immunization with smallpox vaccine, an illdefined herpes vaccine , bacillus Calmette-Guerin (BCG) ,65 and other agents is clearly ineffective.64 Levamisole , an antihelminthic agent causing nonspecific enhancement of cell mediated immunity, is without benefit .66 Various topically applied agents that have been ineffective in properly controlled studies include diethyl ether ,67 cytosine arabinoside,68 adenine arabinosideY69and idoxuridine,7o althou h the latter agent in dimethylsulfoxide solution may hold some promise;7f further trials are underway. Photodynamic inactivation of Herpes simplex by exposure in vitro to light in the presence of various heterocyclic dyes led t o several clinical trials of exposure of genital lesions to light after application ofneutral red 0. Early reports were optimistic, but this treatment isclearlynot beneficial,72 73 and theoretically could enhance herpes-induced carcinogenesis.74 9

118

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Weisbach, Ed.

Several newer agents with antiviral activity are in various stagesof testing in animal models and in clincial trials, including interferon,75 various interferon inducers15 azauridine, arabinosyl hypoxanthine, trifluorothymidine, phosphonoacetic acid, and others.64 Most promising, however,may be acycloguanosire (9-(2-hydroxyethoxymethy1)guanine)and its triphosphate,76,77 which act by selectively inhibiting thymidine kinase to a greater degree in herpes-infected than innoninfected CH2-O-CH2-CH20H mammalian cells, potentially allowing for heretofore unrealized therapeutic ratios. Acycloguanosine

‘

Trichomoniasis Vaginitis due to the parasite Trichomonas vaginalis has beensuccessfully treated for more than 15 years with metronidazole in multiple dose regimens totalling 5-20 gm over 5-10 days, with cure rates of 90-95% when male consorts were treated simultaneously.78 Virtually all strains of T. vaginalis are inhibited by metronidazole 3 . 2 mcg/ml.79 Studies to determine the minimum effective dose have been stimulated by data su gesting significant carcinogenicity and mutagenicity of metronidazole,88 and because of the desirability of single-dose therapy in venereal diseaseclinic populations. The most frequently recommended therapy at the present time is 2.0 gm orally in a single dose, with cure rates equivalent to thosewith multiple dose regimens.81, 82 Other nitroimidazoles, such as misonidazole (I), nimorazole, tinidazole, ornidazole, secnidazole, and carnidazole represent minor modifications, affecting pharmacokinetics rather than trichomonicidal activity, which is a function of electron transfer involving the nitro group of the imidazole ring.83 MICs for T. vaginalis to all of these agents vary between 0.12 and 6.0 mcglml, 8 3 and all appear to be equally effective clinically. The mutagenicity and carcinogenicity of the nitroimidazoles areprobably also a function of the imidazole nucleus,80 and it is not likely that any of these agents will circumvent the question of long term safety. Unfortunately, no other drugs of comparable efficacy are available. R1=-CH2CH20H ; R2=CH3 metronidazole 02N r7 R ’ - N ~ N R~=-CHCH N o ; R ~ = H nimorazole 2 2 u R1=-CH2CH2* SO2 CH2CH3;R2=CH3 t inidazole R2 NO2 OH (1) (11)

m

T

I

R1-N19N

-

I

OH I R ~ = - c HCH. ~ CH2 OCH3 misonidazole

-

R1=-CH2.CH*CH2C1 ; R2=CH3 OH I R2=CH3 R1=-CH2 CH-CH3 ;

-

ornidazole secnidazole

R1=-CH2CH2NH-CS-OCH3 R2=CH3 carnidazole References

1. 3 . E . Martin, Jr., A . Lester, E.V. Price, and J.D. Schmale, J. Infect. Dis., 122, 459 (1970).

Chap. 1 2

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2. H.W. J a f f e , J . W . B i d d l e , C . T h o r n s b e r r y , R.E. Johnson, R.E. Kaufman, G.H. Reynolds, P . J . Wiesner, and t h e C o o p e r a t i v e Study Group, N. Engl. J. Med., 294, 5 (1976). 3. R.E. Kaufman, R.E. Johnson, H.W. J a f f e , C . T h o r n s b e r r y , G.H. Reynolds, P.J. Wiesner, and t h e C o o p e r a t i v e Study Group, N . Engl. J. Med., 294, 1 (1976). 4 . G.A. O l s e n , B r . J . Vener. D i s . , 49, 20 (1973). 5. W.W. Karney, M. T u r c k , and K.K. Holmes, Antimicrob. Agents Chemother., 5 , 1 1 4 (1974). 6 . ?. Al-Egaily, E.M. Dunlop, P. Rodin, and A.D. S e t h , B r . J . Vener. D i s . , 54, 243 (1978). 7. Forstrb'm, B r . J . Vener. D i s . , 50, 6 1 (1974). 8. M. Nelson, S o u t h e r n Med. J . , &, 921 (1973). 9 . L.O. G e n t r y , J . L . Smythe, R.T. Ives, and C . Brady, A b s t r a c t 1 9 8 , 1 8 t h I n t e r s c i e n c e Conference on A n t i m i c r o b i a l Agents and Chemotherapy (1978). 10. M. F i n l a n d , C. G a r n e r , C . Wilcox, and L.D. S a b a t h , J. Am. Vener. D i s . ASSOC., 2, 33 (1976). 11. C e n t e r f o r Disease C o n t r o l , Morbid. M o r t a l . Weekly Rep., 28, 1 3 (1979). 1 2 . J . M . DiCaprio, J . Reynolds, G. F r a n k , J . Carbone, and R. Nishimura, J.A.M.A., 2,1 6 3 1 (1978). 13. E . J . K l e i n , L.S. F i s h e r , A.W. Chow, and L.B. Guze, Ann. I n t e r n . Med., 8 6 , 340 (1977). 14. T G . Cunningham, J . C . Hauth, J . D . S t r o n g , W . N . P . Herbert, L.C. G i l s t r a p , R.H. Wilson, and S.S. K a p p u s , N . E n g l . J . Med., 296, 1380 ( 1 9 7 7 ) . 15. P . J . Wiesner, H.H. H a n d s f i e l d , and K.K. Holmes, N. Engl. J. Med., 288, 1 2 2 1 (1973). 1 6 . R.M. B l a n k e n s h i p , R.K. Holmes, and J . P . S a n f o r d , N . Engl. J. Med.,290, 267 (1974). 1 7 . H.H. H a n d s f i e l d , P . J . Wiesner, and K.K. Holmes, Ann. I n t e r n . Med., 84, 6 6 1 (1976). 18. S . Falkow, L.P. E l w e l l , J . d e G r a f f , F. H e f f r o n , and L. Mayer, i n Sexu a l l y T r n a s m i t t e d Diseases, R.D. C a t t e r a l l and C . S . N i c o l , Ed., Academic P r e s s , London, 1976, p. 120. 1 9 . L.P. E l w e l l , M. R o b e r t s , L.W. Mayer, and S . Falkow, Antimicrob. Agents Chemother., 11,528 (1977). 20. M.S. S i e g e l , C . T h o r n s b e r r y , J . W . B i d d l e , P.R. O'Mara, P.L. P e r i n e , a n d P.J. Wiesner, J . I n f e c t . D i s . , 137, 170 (1978). 21. I. P h i l l i p s and K. Shannon, S c a d . J . I n f e c t . D i s . , [ S u p p l . ] 13,2 3 (1978). 22. R.L. P e r k i n s , R.B. P r i o r , T.M. F i l e , and T.G. S l a m a , A b s t r a c t 2 6 1 , 1 7 t h I n t e r s c i e n c e Conference on A n t i m i c r o b i a l Agents and Chemotherapy (1977). 23. R.B. J o n e s , G.W. Counts, and K.K. Holmes, A b s t r a c t 200, 1 8 t h I n t e r s c i e n c e Conference on A n t i m i c r o b i a l Agents and Chemotherapy ( 1 9 7 8 ) . 24. L. S a r a v o l a t z , T. Madhavan, K. Burch, D. K i a n i , and E . L. Quinn, Abt r a c t 423, 1 8 t h I n t e r s c i e n c e Conference on A n t i m i c r o b i a l Agents and Chemotherapy ( 1 9 7 8 ) . 25. J . D . P r i c e and J . L . F l u k e r , B r . J . Vener. D i s . , 54, 1 6 5 (1978). 26. P . J . Wiesner, K.K. Holmes, P.F. S p a r l i n g , M . J . Maness, D.M. Bear, L . T . Gutman, and W.W. Karney, J . I n f e c t . D i s . , 127,461 ( 1 9 7 3 ) . 27. C e n t e r f o r Disease C o n t r o l , Morbid. M o r t a l . Weekly Rep., 23, 3 4 1 ( 1 9 7 4 ) . 28. F.N. J u d s o n and R. R o t h e r n b e r g , J . Am. Vener. D i s . ASSOC., 2, 56 (1976). 291 29. L.Z. O l l e r , J . C . Edwards, and K.M. Lumb, B r . J . Vener. D i s . , 3, (1973). 30. H. Baytch, Med. J . A u s t . , 1,8 3 1 (1974).

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31. W.W. Karney, A.H.B. Pedersen, M. Nelson, H. Adams, R.T. Pfeifer, and K.K. Holmes, N . Engl. J . Med., 296, 889 (1977). 32. M.J. Maness and P.F. Sparling, J . Infect. Dis., 128,321 (1973). 33. C. Thornsberry, H. Jaffe, S.T. Brown, T. Edwards, J.W. Biddle, andS.E. Thompson, J.A.M.A., 237 2405 (1977). 34. W.M. McCormack and M. Finland, Ann. Intern. Med., 84, 712-(1976). 35. J.L. Fluker and A.B. Hewitt, Br. J. Vener. Dis., 46, 454 (1970). 36. W. Bowie, A.R. Ronald, W. Krywulak, C.Y. Cates, and P. Boutros, Br. J . Vener. Dis., 50, 208 (1974). 37. T.W. Austin, G.F. Brooks, M. Bethel, F.L. Roberts, M. Turck, and K.K. Holmes, J . Infect. Dis., [Suppl.] 128,S666 (1973). 38. F.R. Sattler and J . Ruskin, J.A.M.A., 240, 2267 (1978). 39. S.T. Brown, A.H.B. Pedersen, and K.K. Holmes, J.A.M.A., 238, 1371 (1977). 40. O.P. Arya, S.K. Rao, and E. Nnochiri, Br. J . Vener. Dis., 47,184 (1971). 41. H.H. Handsfield, Med. Clin. N. Am., 62, 925 (1978). 42. W.R. Bowie, S.-P. Wang, E.R. Alexander, J . Floyed, P.S. Forsyth, H.M. Pollock, J.-S.L. Lin, T.M. Buchanan, and K.K. Holmes, J . Clin. Invest. 59, 735 (1977). 43. X K . Holmes, H.H. Handsfield, S.-P. Wang, B.B. Wentworth, M. Turck, J.B. Anderson, and E.R. Alexander, N. Engl. J . Med., 292, 1199 (1975). 44. W.R. Bowie, Sex. Transm. Dis., 1, 27 (1978). 45. G. Furness, A.T. Evangelista, and 2. Kaminski, Invest. Urol., 15,23 (1977). 46. R.E. Berger, E.R. Alexander, G.D. Monda, J. Ansell, G. McCormick, and K.K. Holmes, N. Engl. J . Med. , 298, 301 (1978). 47. E.R. Alexander, J. Chandler, T.A. Pheifer, S.-P. Wang, M. English,and K.K. Holmes, in Nongonococcal Urethritis and Related Infections, D. Hobson and K.K. Holmes, Ed., American Society for Microbiology, Washington, D.C., 1977, p. 148. 48. H.R. Harrison, M.G. English, C.K. Lee, and E.R. Alexander, N. Engl. J . Med., 298, 702 (1978). 49. P.-A. Mhdh, T. Ripa, L. Svensson, and L. Westrom, N. Engl. J . Med., 296, 1377 (1977). 50. G. McCormack, P. Braun, Y.-H. Lee, J . O . Klein, and E.H. Kass, N. Engl. J. Med., 288, 78 (1973). 51. H.H. Handsfield, E.R. Alexander, S.-P. Wang, A.H.B. Pedersen, and K.K. Holmes, J . Am. Vener. Dis. ASSOC., 2, 5 (1976). 52. W.R. Bowie, C.K. Lee, and E.R. Alexander, J . Infect. Dis., 138, 655 (1978). 53. G.L. Ridgway, J.M. Owen, and J.D. Oriel, Br. J . Vener. Dis., 54, 103 (1978). 54. H . J . Blackman, C. Yoneda, C.R. Dawson, and J . Schachter, Antimicrob. Agents Chemother., 12, 673 (1977). 55. M.S. Spaepen and R.B. Kundsin, Antimicrob. Agents Chemother., 11,267 (1977). 56. B.A. Evans, Br. J . Vener. Dis., 54, 107 (1978). 57. J.D. Oriel, P. Reeve, and C.S. Nicol, J . Am. Vener. Dis. ASSOC., 2, 17 (1975). 58. A. Lassus, R.-L. Perko, S. Stubb, R. Mattila, and E. Jansson, Br. J . Vener. Dis., 47,126 (1971). 59. J. John, Br. J. Vener. Dis., 47,266 (1971). 60. J. Schachter, N. Engl. J. Med., 298, 428 (1978).

Chap. 1 2 61. 62. 62a 62b 63. 64. 65.

66. 67. 68. 69. 70.

71. 72. 73. 74.

75. 76.

77.

78. 79. 80.

81. 82. 83.

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A.S. Grimble and K.L. Amarasuriya, B r . J . Vener. D i s . , 51, 1 9 8 (1975). N . Helmy and W. Fowler, B r . J . Vener. D i s . , 51, 336 (1975). P . Braun, J . O . K l e i n , and E.H. Kass, Appl. M i c r o b i o l . , 2, 62 (1970). M.C. Shepard, J.A.M.A., 211, 1335 (1970). Y.-H. Lee, S. A l p e r t , P.E. B a i l e y , A. Duancic, and W.M. McCormack, J . Am. Vener. D i s . Assoc., 1,37 (1974). H.G. Adams, Sex. Transm. D i s . , 5, 160 (1977). L. Corey, W.C. Reeves, L.A. Vontver, E.R. Alexander, and K.K. Holmes, A b s t r a c t 403, 1 6 t h I n t e r s c i e n c e Conference on A n t i m i c r o b i a l Agents and Chemotherapy (1976). T.-W. Chiang and N . Fiumara, Antimicrob. Agents Chemother., 13,809 (1978). L. Corey, W.C. Reeves, W.-T. Chiang, L.A. Vontver, M. Remington, C. Winter, and K.K. Holmes, N. Engl. J . Med., 299, 237 (1978). R. Marks and J . K o u t t s , Med. J . A u s t . , 479 (1975). A.L. H i l t o n , T.E.C. B u s h e l l , D. Waller, and J . B r i g h t , B r . J. Vener. D i s . , 54, 5 0 (1978). P.K. T a y l o r and N.R. Doherty, B r . J. Vener. D i s . , 51, 125 (1975). F.O. MacCallum and B.E. J u e l - J e n s e n , B r . Med. J . , 2, 805 (1966). A.P.C.H. Roome, A.E. T i n k l e r , A.L. H i l t o n , D.G. M o n t e f i o r e , and D. Waller, B r . J . Vener. D i s . , 51, 130 (1975). M.G. Myers, M.N. Oman, J . E . C l a r k , and K.A. A r n d t , N . Engl. J . Med., 293, 945 (1975). K. L i , M.A. J e r k o f s k y , and F. Rapp, I n t . J . Cancer, 15,190 (1975). G.A. Olsen, E.R. K e r n , J . C . O v e r a l l , J r . , and L.A. Glasgow, J . I n f e c t . D i s . , 137, 428 (1978). P.A. Furman, M.H. S t . C l a i r , J . L . Rideout, J.A. F y f e , P.M. Keller, and G.B. E l i o n , A b s t r a c t 62, 1 8 t h I n t e r s c i e n c e Conference on A n t i m i c r o b i a l Agents and Chemotherapy (1978). E.R. Kern, J . T . R i c h a r d s , J . C . O v e r a l l , J r . , and L. A. G l a s g o w , A b s t r a c t 264, 1 8 t h I n t e r s c i e n c e Conference on A n t i m i c r o b i a l Agents and Chemot h e r a p y (1978). M.F. Rein and T.A. Chapel, C l i n . O b s t e t . Gynec., 18, 73 (1975). J . G . Meingassner, L. Havelec, and H. Mieth, B r . J . Vener. D i s . , 54, 72 (1978). C.E. Voogd, J.J. Van D e r S t e l , and J . J . J . A . A . J a c o b s , Mutation R e s . , 26, 483 (1974). E R . Dykers, J r . , N . Engl. J . Med., 293, 23 (1975). N . Kawamura, B r . J . Vener. D i s . , 54, 81 (1978). E d i t o r i a l ( u n s i g n e d ) , B r . J . Vener. D i s . , 54, 69 (1978).

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Chapter 13.

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

Leslie M. Werbel, Donald F. Worth and Sarah M . W e i t z e l , Warner-Lambert /Parke-Davis Pharmaceutical Research D i v i s i o n , Ann Arbor, Michigan 48105 PROTOZOAL DISEASES

General - An extremely u s e f u l i n t r o d u c t o r y t e x t on t h e b i o c h e m i s t r y of p a r a s i t i c protozoa c o n t a i n i n g l i t e r a t u r e through 1976 w a s published recently.’ M a l a r i a - T h i s i s not, as some would have had us b e l i e v e a few y e a r s back, a r a r e human a f f l i c t i o n . I t s i n c i d e n c e i s on t h e rise2 and t h e need f o r improved t r e a t m e n t means is c l e a r l y e v i d e n t from t h e h o r r i f y i n g s t a t i s t i c t h a t one m i l l i o n c h i l d r e n a r e s a i d t o d i e each y e a r i n A f r i c a from m a l a r i a . 3 R e s i s t a n c e , t h e d e c r e a s i n g s e n s i t i v i t y of t h e m a l a r i a p a r a s i t e t o e x i s t i n g drugs, i s a major p r ~ b l e m . ~ Mefloquine ( l ) , one of t h e most s i g n i f i c a n t achievements i n m a l a r i a chemotherapy i n r z c e n t y e a r s , is e f f e c t i v e a g a i n s t l a b o r a t o r y s t r a i n s r e s i s t a n t t o primaquine, c y c l o g u a n i l , pyrimethamine and s u l f a p h e n a ~ o l e ~ -however, ~; c o n c e r n has been voiced f o r c a r e i n i t s a p p l i c a t i o n t o avoid development of r e s i s t a n c e through i n d i s c r i m i n a n t use.g

2

1

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(c

Must r e a d i n g a r e t h e p a p e r s of L. H. Schmidt summarizing h i s development of t h e o w l monkey model 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 d r u g s P. -v i v a x i n f e c t i o n s . ’‘-I2 a g a i n s t Plasmodium f a l c i p a r u m and S t u d i e s have appeared on a promising pyridinemethanol s t r u c t u r a l l y r e l a t e d t o 1, which i s a c t i v e , i n p r i m a t e s a g a i n s t d r u g - r e s i s t a n t s t r a i n s . 1 3 - 1 5 Complete d e T a i l s on t h e h i g h l y p o t e n t diaminoquinazoline f o l a t e a n t a g o n i s t s have appeared.16’17 The r e l a t e d p t e r i d i n e s showed lower a c t i v i t y such a s against experimental i n f e c t i o n s . l9 Although t h e p l a n a r [ l l b e n z o t h i e n o [3,2-f]quinazoline-l,3-diamine analog 3 was devoid o f a n t i m a l a r i a l a c t i v i t y , m recent publications indicate s u c s t a n t i a l a c t i v i t y f o r t h e l,?-dif-) q u i n a z o l i n e s 4. 21’22 aminopyrro lo- ( 3 , ~-

2

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ArCH2-N

J

A

4 6

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. IS8 N 0- 12-0405 14-8

Chap, 13

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(2)

A novel s t r u c t u r a l type w a s r e p o r t e d t o have p o t e n t a c t i v i t y a g a i n s t r e s i s t a n t m a l a r i a s t r a i n s . 2 3 Minor m o d i f i c a t i o n s , i n c l u d i n g removal o f t h e N-OH s u b s t i t u e n t e l i m i n a t e d a c t i v i t y . 2 4 L i m i t e d c l i n i c a l pharmacology, b i o c h e m i s t r y and mode o f a c t i o n s t u d i e s are a v a i l a b l e i n t h e e n t i r e p a r a s i t o l o g y a r e a . Two r e c e n t p a p e r s r e p o r t t h e pharmacok i n e t i c s o f amod i a q u i n e c h l o r g u a n i d e c h l a r a q u i n e p y r i m e t h am in e q u i n i n e and s u l f a d o ~ i n e . ~ ~ ' ~ ~

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L e i s h m a n i a s i s - T h i s d i s e a s e , which g i v e s e v e r y i n d i c a t i o n o f d r a m a t i c a l l y i n c r e a s i n g i n c i d e n c e , and is p r o b a b l y second i n i m p o r t a n c e o n l y t o m a l a r i a o f t h e d i s e a s e s c a u s e d by p r o t o z o a l p a r a s i t e s , i s i n d e s p e r a t e need o f t r e a t m e n t a d v a n c e s . What promises t o b e a v e r y e x c i t i n g t e c h n o l o g i c a l b r e a k t h r o u g h h a s r e c e n t l y been d e s c r i b e d . The c o n c e p t t h a t d r u g s i n c o r p o r a t e d i n t o liposomes c o u l d be c a r r i e d more e f f i c i e n t l y d i r e c t l y t o lysosomes i n t h e r e t i c u l o e n d o t h e l i a l s y s t e m i n which t h e Leishmania p a r a s i t e s r e s i d e p r e d o m i n a n t l y h a s been s u c c e s s f u l l y u t i l i z e d by t h r e e g r o u p s . A d o s e o f 1.0 mg Sb/kg o f P e n t o s t a m i n c o r p o r a t e d i n t o l i p o s o m e s was more t h a n 200 t i m e s a s a c t i v e a s t h e f r e e d r u g a g a i n s t L. d o n o v a n i i n mice.'7 Meglumine a n t i m o n i a t e (Glucantime9) i n c o r p o r a t e d i n t o l i p o s o m e s was more t h a n 300 t i m e s a s e f f e c t i v e a s t h e u n f o r m u l a t e d d r u g a g a i n s t l e i s h m a n i a 1 i n f e c t i o n s i n hamsters." S u b s t a n t i a l improvement i n e f f i c a c y was a l s o shown w i t h a liposome p r e p a r a t i o n o f antimony p o t a s s i u m t a r t r a t e L. donovani i n f e c t i o n s i n againat T h e t e s t s y s t e m used by t h e W a l t e r Reed Army I n s t i t u t e o f R e s e a r c h f o r s c r e e n i n g compounds a g a i n s t l e i s h m a n i a i n f e c t i o n s i n g o l d e n h a m s t e r e h a s been d e s c r i b e d , and d a t a were p r e s e n t e d on a v a r i e t y o f 6,7-30 and 3a m i n o q ~ i n o l i n e s . ~The ~ most a c t i v e a n a l o g , 6, was more t h a n 700 t i m e s as a c t i v e a s t h e meglumine a n t i m o n i a t e (Glucant?me@) s t a n d a r d . CH3

T r y p a n o s o m i a s i s - L i t t l e or no s i g n i f i c a n t advances have o c c u r r e d i n a l l e v i a t i n g e i t h e r t h e human (American - T . c r u z i ; A f r i c a n - T . gambiense, -T . r h o d e s i e n s e ) o r t h e v e t e r i n a r y ( T . b r u -c e i-, T. v i v a x , T . c o n g o l e n s e , T. e v a m i n u m , e q u i p e r d u m ) forms o f t h z d i s e a s e . The v e t e r i n a r y forms pose a p a r t i c u l a r l y s e r i o u s economic problem on t h e A f r i c a n c o n t i n e n t . A s a n example, t r y p a n o s o m i a s i s h a s been i n d i c t e d a s o n e of t h e p r i n c i p a l f a c t o r s r e s t r i c t i n g growth o f t h e l i v e s t o c k i n d u s t r y i n N i g e r i a . 3 2 S u r v e y s o f t h e b i o c h e m i s t r y and chemotherapy o f trypanosome i n f e c t i o n s 3 3 and t r y p a n o c i d a l a c t i v i t y among a n t i t u m o r a n t i b i o t i c s and o t h e r m e t a b o l i c i n h i b i t o r s a 4 have a p p e a r e d . -

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Human I n f e c t i o n s : P a p e r s i n c l u d e d i n a n i n t e r n a t i o n a l c o l l o q u i u m on human A f r i c a n t r y p a n o s o m i a s e s h e l d i n 1976 have been p u b l i s h e d , 3 5 a s have t h e p r o c e e d i n g s o f a n i n t e r n a t i o n a l symposium on Chagas ' d i s e a s e . 3 6

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Two s c r e e n s have been d e s c r i b e d u s i n g T. c r u z i i n t h e Activity was s e e n w i t h 8-aminoquinolines, a 7-aminoquinoline, a n i t r o f u r a n , pme thylbenzyltriphenylphosphonium c h l o r i d e , b i s q u i n a l d i n e s , arsenobenzenes and 2 - n i t r o i m i d a z o l e s . N i t r o h e t e r o c y c l e s have o f f e r e d t h e most f e r t i l e T. c r u z i . Both 2- and 5 - n i t r o i m i d a z o l e d e r i v ground f o r a c t i v i t y a g a i n s t "~ t h e r e a p p e a r s t o be a a t i v e s a r e of i n t e r e s t a g a i n s t T . c r ~ z i " ~ - though d i s t i n c t d i f f e r e n c e i n t h e i r moze of action."' However, even such h i g h l y regarded compounds a s n i f u r t i m o x ( 7 ) r e q u i r e prolonged t r e a t m e n t which, moreover, i s n o t completely e f f e ~ t T v e . ~ ' - " l Veterinary I n f e c t i o n s : Despite previous negative r e s u l t s a g a i n s t T . brucei,*= e f f e c t i v e t r e a t m e n t of T. v i v a x i n f e c t i o n s was a p p a r e n t l y -achieved w i t h s a 1i c y lhydroxamic a c Trichomoniasis - Most o f t h e r e c e n t l i t e r a t u r e i s concerned w i t h s t u d i e s which a t t e m p t t o provide s u f f i c i e n t d a t a t o a l l o w a c h o i c e among s e v e r a l competing n i t r o i m i d a z o l e s . Metronidazole the leader i n the f i e l d , c o n t i n u e s t o be e x p l ~ r e d . ~ ~ 'A" c~l i n i c a l i s o l a t e h a s been r e p o r t e d r e s i s t a n t t o m e t r o n i d a ~ o l e , and ~ ~ o t h e r 5 - n i t r o i m i d a z o l e s were s i m i l a r l y i n e f f e c t i v e . An i n t e r e s t i n g series of a r t i c l e s d e a l s w i t h t h e p r o p h y l a c t i c and t h e r a p e u t i c use of m e t r o n i d a z o l e f o r a v a r i e t y of s u r g i c a l indication^.^' Comparisons of m e t r o n i d a z o l e w i t h p a n i d a ~ o l e ,s~e~c n i d a ~ o l e ,and ~ ~ ornidazole,"" ( 9 ) have appeared. The l a t t e r is of i n t e r e s t because i t demons t r a t e d no z f f e c t on mammalian chromosomes a l t h o u g h i t was mutagenic i n v a r i o u s b a c t e r i a l s t r a i n s . Moreover, no t e r a t o g e n i c i t y o r c a r c i n o g e n i c i t y was observed i n l a b o r a t o r y s t u d i e s , and i n man no e v i d e n c e of a l c o h o l i n t o l e r a n c e was noted.54 High a c t i v i t y was demonstrated n o t o n l y i n human has t r i c h o m o n i a s i s , b u t i n amoebiasis and g i a r d i a s i s . 5 4 T i n i d a z o l e been compared f a v o r a b l y w i t h metronidazole.55

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Much less common is a c t i v i t y found i n s t r u c t u r e s o t h e r t h a n of t h e n i t r o h e t e r o c y c l e t y p e . A c t i v i t y was r e p o r t e d r e c e n t l y f o r t h e a n t h r a quinonebisamidinea 115’ and t h i a d i a z o l e 12.59 M

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Amoebiasis - L i t t l e new h a s appeared i n t h i s a r e a d u r i n g t h e p a s t two y e a r s . S e v e r a l reviews, e s s e n L i a l l y c l i n i c a l , have been published.60-62 E f f o r t s continue primarily with o t h e r nitroimidazoles t o achieve superi o r i t y t o metronldazole. Tinidazole (10) i s r e c e i v i n g s u b s t a n t i a l f a v o r a b l e a t t e n t i ~ n . “ ~ - “Novel ~ b i s - a g d i n e s of 2,6-diaminoanthraquinone r e l a t e d t o 11 have shown a c t i v i t y comparable t o t h e a c t i v i t y of metron i d a ~ o l e ~ ’a’g~a i n s t E. h i 6 t o l y t i c a c e c a l i n f e c t i o n s €ti r a t s and h e p a t i c i n f e c t i o n a i n hamsters- ‘The compounds were nonmutagenic i n t h e Ames and dominant l e t h a l t e s t s . G i a r d i a s i s - The i n f e c t i o n of t h e g a s t r o i n t e s t i n a l t r a c t o f man by G i a r d i a l a m b l i a known a s g i a r d i a s i s o r l a m b l i a s i s a p p e a r s e i t h e r t o be , i n c r e a s i n g i n p r e v a l e n c e o r i s being recognized more r e a d i l y . Metron i d a z o l e and q u i n a c r i n e a r e probably used most f o r treatment, however more e f f e c t i v e , b e t t e r t o l e r a t e d t h e r a p y would be d e s i r a b l e . T i n i d a z o l s (10) is r e c e i v i n g i n c r e a s i n g l y f a v o r a b l e r e p o r t s : 72-75 i t is, however, n o t y p p r o v e d i n t h e United S t a t e s . Toxoplasmosis - T h e o b l i g a t e i n t r a c e l l u l a r p a r a s i t e Toxoplasma g o n d i i i s r e s p o n s i b l e f o r s i m i l a r , a l t h o u g h v a r i a b l e l e v e l s o f , d i s e a s e response i n a w i d e v a r i e t y o f h o s t s i n c l u d i n g man. The d i s e a s e s t a t e may n o t be recognized u n t i l some v i t a l o r g a n i s compromised, and u s u a l l y . o n l y t h e a c u t e form responds t o m e d i c a t i o n s i n c e t h e e n c y s t e d p a r a s i t e s a r e a p p a r e n t l y r e s i s t a n t t o d r u g s which combat t h e t r o p h o ~ o i t e s . ~The ~ organism i s thought t o be c l o s e l y r e l a t e d t o t h e Coccidia, and most usef u l a g e n t s and on-going e f f o r t s seem t o c e n t e r on t h o s e s t r u c t u r a l c l a s s e s which i n h i b i t t h e f o l i c a c i d c y c l e . Thus, a sulfamethoxydiazine-pyrimethamine combination h a s been recommended f o r t r e a t m e n t of t h e a c u t e d i s e a s e i n pregnancy. 77 S tud ies w i t h 2- s u l f amoy Id iaminod ipheny 1 s u 1f one con t inue t o appear b u t t h e c l i n i c a l s i g n i f i c a n c e of t h i s a g e n t i s n o t ~ l e a r . ~ ” ~ ’ An e a r l y r e p o r t o f t h e t h e r a p e u t i c p o s s i b i l i t i e s of chemical immunomodulation of t h e c e l l u l a r immune response i n toxoplasmosis u t i l i z i n g l e v a m i s o l e i s of interest.’O C o c c i d i o s i s - C o n t r o l of this p a r a s i t e , p a r t i c u l a r l y i n t h e p o u l t r y i n d u s t r y , i s a problem of enormous commercial s i g n i f i c a n c e . The tremendous a b i l i t y of t h e p a r a s i t e t o d e v e l o p r e s i s t a n c e t o most known s t r u c t u r a l types81-84 p r o v i d e s t h e major impetus f o r f u r t h e r r e s e a r c h . Drug t r e a t m e n t o f p o u l t r y , c a t t l e , sheep, g o a t s , p i g s , dogs and r a b b i t s h a s been reviewed.85 Of i n t e r e s t i s t h e r e p o r t t h a t no r e s i s t a n t i s o l a t e s o f Eimeria t e n e l l a were found i n l i t t e r samples from b r o i l e r p r o d u c t i o n f l o c k s b e i n t r e a t e d w i t h monensin.86 A r p r i n o c i d [9- (2-chloro-6-fluorobenzyl ) a d e n i n e r e p o r t e d a s a new a g e n t l a s t y e a r , r e c e i v e d a f a v o r a b l e r e p o r t f o r t r e a t ment o f broiler^.'^ A v a r i e t y o f s t r u c t u r a l types such a s ethanolamines,88 pyridine-3-sulfonamide- l-oxides,8” N - p h e n y l p y r i d o n e h y d r a z o n e ~and ~~ riboIt is f l a v i n analogs” have been r e p o r t e d t o have a n t i c o c c i d i a l a c t i v i t y . n o t known, however, i f any of t h e s e w i l l have any p r a c t i c a l c l i n i c a l utility

7

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HELMINTH DISEASES

F i l a r i a s i s - Recent books are concerned w i t h c o n t r o l measuress2 and v a r i o u s a s p e c t s o f Bruntan f i l a r i a s i ~ . ' ~ A new s c r e e n i n n method i n v o l v e s t h e t r a n s p l a n t a t i o n of g d u l t Brugia pahangi i n t o t h e p e r i t o n e a l c a v i t y of jirds.'* T h e f r u s t r a t i o n e x t a n t i n d i s c o v e r i n g new d r u g s f o r t r e a t m e n t o f o n c h o c e r c i a s i s , f o r which no convenient l a b o r a t o r y s c r e e n e x i s t s , s 5 is exemplified by t r i a l s i n humans of m e t r o n i d a z o l e , t i n i d a z o l e , mebendazole t r i c h l o r o p h o n e , oxamniquine, and p y r a n t e l pamoate. None of t h e s e a g e n t s showed evidence of s u b s t a n t i a l a ~ t i v i t y . ' ~ Reports of t h e e f f e c t s of d i e t h y l ~ a r b a m a z i n e , ~e~~ 'u~r a~m i n , ' ~and l e v a m i s ~ l e , " g~i v e n t r a n ~ e p i d e r m a l l y ' ~ o r d i r e c t l y on t h e eye,s7'sa e f f e r encouragement t h a t more u s e f u l methods of a d m i n i s t r a t i o n of known f i l a r i c i d e s may be found.

-

Other Nematodes - Recent reviews c o v e r chemotherapy i n b o t h humanloo and v e t e r i n a r v L o l D r a c t i c e . Treatment of 400 D a t i e n t s i n f e c t e d w i t h Dracunculus medinensis (Guinea worm) w i t h t h i a b e n d a z o l e was s u c c e s s f u l . lo2 From e x t e n s i v e l a b o r a t o r y comparisonslm i t was concluded t h a t flubenda z o l e and mebendazole possessed comparable a n t i p a r a s i t i c p r o p e r t i e s , b u t t h a t f lubendazole was l e s s t o x i c f o r some h o s t s . P r e l i m i n a r v r e p o r t s i n d i c a t e t h a t t i o x i d a z o l e (13) i s b r o a d l y a c t i v e a g a i n s t g a s t r o i n t e s t i n a l nematodes i n l a b o r a t o r y a n i E l s , sheep and h o r s e s . l o 4 A v a r i e t y of s t r u c t u r e s , including lo5 15,1°6 snd 16,1°7have appeared i n t h e r e c e n t patent l i t e r a t u r e claiming n e s t o c i d a l a z i v i t y .

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S c h i s t o s o m i a s i s - The 7 t h I n t e r n a t i o n a l Congress of Pharmacology i n c l u d e d a symposium o n t h e chemotherapy of s c h i s t o s o m i a s i s l o 8 where work w i t h I A 4,N-oxide ( l'j'), lop t h e diphenylamine 18, 'lo oxamniquine, and p r a z i q u a n t e l l ' ' was r e v i e w e r A l s o addressed were m e s s of r e d u c i n g t h e mutagenic l i a b i l i t i e s of d r u s.lo8 No mutagenic a c t i v i t y was found €or p r a z i q u a n t e l The 4 t h volume of t h e mammoth bibliography113 was i n 2 studies. p u b l i s h e d . A v e r y r a p i d , new s c r e e n i n g method using Schistosoma mansoni i n mice was devigedl'* and v a l i d a t e d w i t h known s c h i s t o s o m l c i d e s . A c t i v i t y a g a i n s t Schistosoma mansond i n mice and monkeys was demonstrated f o r t h e t h i a z e l e 19. L L 3 O t h e r s t r u c t u r e s from t h e r e c e n t l i t e r a t u r e w i t h a n t i and M 21.117 s c h i s t o s o m l a c t i v i t y i n c l u d e t h e fosfomycin d e r i v a t i v e

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Other Trematodes - Recent books concerned p r i m a r i l y w i t h f a s c i o l i a s i s chemotherapy, i n c l u d e d i s c u s s i o n s on i n t e g r a t e d c o n t r o l s t r a t e g f e s , 'I5 and c o l l e c t e d a b s t r a c t s of p u b l i c a t i o n s on n i t r o x y n i l . 1 2 ' Cure r a t e s of 73-90$ were r e p o r t e d € o r 95 p a t i e n t s i n f e c t e d w i t h Paragonimus u t e r o b i l a t e r a l i s a f t e r a s i n g l e 2 mg/kg dose of meniclopholan (%).ICestodes - The problems of human d i p h y l l o b o t h r i a s i s 1 2 2 and echinococc o s F were reviewed. E x c e p t i o n a l a n t i c e s t o d e a c t i v i t y was r e p o r t e d M and t h e f o r praziquantel,124 d i u r e d o s a n (23),125 f u r o d a z o l e (24),126 n i c losamide analog 25. 127 M

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REFERENCES 1.

2.

3. 4. 5.

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W. E. G u t t e r i d g e and G. H . Coombs,"Biochemistry of P a r a s i t i c Protozoa," MacMillan Press Ltd., London, 1977. W. Chin, South. Med. J., 97 (1978). A. P. H a l l , Trans. Roy. SOC. Trop. Med. Hyg., 71, 367 (1977). R. H. G l e w , W. E. C o l l i n s and L. H . M i l l e r , A m T J . Trop. Med. Hyg., 27, 9 (1.978). W . P e t e r s , R. E. Howells, J. P o r t u s , B. L. Robinson, S . Thomas and D. C . Warhurst, Ann. Trop. Med. P a r a s i t o l . , 71, 407 (1977). J. M . G r i n d e l , P. F. T i l t o n and R. D . S h a f f e c J . Pharm. S c i . , 66,

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L. H . Schmidt, R. Crosby, J. Rasco and D. Vaughan, Antimicrob. Agents Chemother., 13, 1011 (1978). R. W . Caldweii and C . B. Nash, Toxicol. Appl. Pharmacol., 40, 437 (1977). W. Peters, J. Portus and B . L. Robinson, Ann. Trop. M e d F P a r a s L t o l . ,

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419 (1977).

L. H. Schmidt, Am. J . Trop. Med. Hyg., 2, 671 (1978). 11. L. H . Schmidt, Am. J . Trop. Med. Hyg., 27, 703 (1978). 12. L. H. Schmidt, Am. J . Trop. Med. Hyg., 718 (1978). 13. R. L. Furner, U.S.N.T. I . S . , A. D. Report 1977, B-AO39668 [Chem. Abstr., 88, 104d(1977)1. Govxep. Ann. Index (U.S.) 77(16), 90 (1977). 14. H. ChungTH. H. G i l l u m and R. S . Rozman, Drug Metab. Dispos., 6, 82 (1978)15. L. H . Schmidt, R. Crosby, J . Rasco and D . Vaughn, Antim€crob. Agents 420 (1978). Chemother., 16. E . F. E l s l a g e r , P . Jacob, J. Johnson, L. M. Werbel and D . F. Worth, J. Med. Chem., 1059 (1978). E . F. E l s l a g e r , J . Davoll, P . Jacob, A. M . Johnson, J . Johnson and L. M. Werbel, J. Med. Chem., 21, 639 (1978). 18. D . F. Worth, J. Johnson, E. F Y E l s l a g e r and L. M . Werbel, J . Med. Chem., 21, 331 (1978). 19. L. M. W=bel, J . Johnson, E . F. E l s l a g e r and D . F. Worth, J . Med. Chem., 337 (1978). 20. J . Johnson, E. F. E l s l a g e r and L. M . Werbel, J. Heterocycl. Chem., 14, 1209 (1977). 21. Belgian P a t e n t 856647 t o American Home Products Corp., January 9 , 1978. 22. U. S . P a t e n t 4118561 t o American Home Products Corp., October 3, 1978. 268 (1977). 23. W. Raether and E . Fink, Tropenmed. P a r a s i t o l . , 24. W. Raether and W. Durckheimer, A b s t r a c t s of t h e Fourth I n t e r n a t i o n a l Congress of P a r a s i t o l o g y , Warsaw, 1978, S e c t i o n D, p 107. W. A. K i t s c h e l , G . V. Hammer and G . A . Thompson, I n t . J. C l i n . 395 (1978). Pharmacol. Biopharni., 26. J . C . C a v a l l i t o , C . A . Nichol, W . D. Bienckman, J r . , R. L. Deangelis, D. R . Stickney, W. S . Simmons and C. W. S i g e l , Drug Metab. Dispos., -6, 329 (1978)27. C . D. V. Black, G . J. Watson and R. J. Ward, Trans. Roy. Soc. Trop. Medm Hyg.3 550 (1977)28. C. R. Alving, E. A. Steck, W. L. Hanson, P. S . Loizeaux, W. L. Chapman, J r . and V. B. Waits, L i f e S c i . , 22, 1021 (1978). R. R. C . New, M . L. Chance, S . C . Thomas a x W. P e t e r s , Nature, 272, 10.

3,

2,

2,

z,

-

28,

g,

71,

55 (1978). W.

-7,

L. Hanson, W. L . Chapman, Jr.

443 (1977).

and K. E. Kinnamon, I n t . J . P a r a s i t o l . ,

Chap. 13

31. 32.

33 * 34.

35 36. 37.

38. 39. 40.

41.

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

Werbel, Worth, W e i t z e l

129

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735 (1975).

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8

E,

6,

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42.

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2,

54.

55.

56. 57. 58. 59. 60.

61.

62. 63 * 64.

9,

-5,

438 (1977).

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-

&,

130 -

65

-

66. 67. 68. 6970.

71. 72.

73

-

74.

Sect. 111

- Chemotherapeutic Agents

Weisbach, Ed.

S . N. Mathur, A. I t i g i , Krishnaveni and V . Rai, J. I n t . Med. Res., 429 (1977). J . N . Scragg and E . M. P r o c t o r , Am. J . Trop. Med. Hyg., 824 (1977). K. M . Ohn and M. Thwe, Mod. Med. Asia, 14, 23 ( 1978). J . S . Welch, B. J . Rowsell and C. F r e e m a c Med. J . Aust., 1, 469 (1978). P . F. Fabio, T . L. F i e l d s , Y. Lin, E. J . Burden, S . C a r a v a j a l , K. C . Murdock and S . A. Lang, Jr., 3. Med. Chem., 21, 273 (1978). U. S . P a t e n t 4118498 t o American Cyanamid Co., October 3, 1978. M. S . Wolfe, N . Engl. J . Med., 298, 319 (1978). N . A. E l Masry, Z. F a r i d and W. F. Miner, Am. J. Trop. Med. Hyg., 27, 201 (1978). A. M. M . J o k i p i i and L. J o k i p i i , Am. J . Trop. Med. Hyg., 758,

-5,

5,

-

7,

( 1978). I . Farahmandian, F. Sheiban and A . S a n a t i , J . Trop. Med. Hyg.,

81,

139 (1978)-

75. 76. 77. 78.

79.

80.

81. 82.

-

83 84.

85 * 86. 87.

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-

18,

76,

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( 1977).

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( 1978).

88. U. 89. 90.

S . P a t e n t 4094976 t o Merck and Co., I n c . , June 13, 1978. Japanese P a t e n t 53065881 t o Sankyo KK, June 12, 1978. A . W. Douglas, M . H. F i s h e r , J . J . F i s h i n g e r , P. Gund, E . E. H a r r i s , G . Olson, A . A. P a t c h e t t and W. V. Ruyle, J. Med. Chem., 20, 939

( 1977).

91. 92

93

-

94. 95. 96. 97. 98.

D. W. Graham, J . E. Brown, W. T. Ashton, R. D. Brown and E . F. Rogers, 1274 ( 1977). Experientia, M. Sasa,"Human F i l a r i a s i s : A Global Survev o f Epidemiology and Control," Univ. Park Press, Baltimore, 1976; F. C . Rodger, EQ, "Onchocerciasis i n Z a i r e , " Pergamon Press, Oxford, 1977.

2,

D . A . Denham and P . B. McGreevy ia "Advances i n P a r a s i t o l s g y , " Val. 15, B. Dawes, Ed., Academic P r e s s , London, 1977, p 243. R. R. S u s w i l l o and D . A . Denham, J. P a r a s i t o l . , 63, 591 (1977). B. 0. L. Duke, Symp. B r i t . SOC. P a r a s i t o l . , 16, F ( 1 9 7 8 ) . 0. Kale, Tropenmed. P a r a s i t o l . , 29, 163 ( 1 9 7 q . J . Anderson and H. Fuglsang, B r i t - J . Ophthalmol., 62, 450 (1978). M. E . Langham, Z. D. Traub and R. Richardson, TropeGed. P a r a s i t o l . ,

Chap. 13

99 * 100. 101.

102. 103.

104.

105. 106. 107. 108. 109.

110. 111. 112.

113. 114. 115.

116. 117. 118. 119.

120. 121. 122. 123. 124.

125. 126. 127.

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

Werbel, Worth, W e i t z e l

131

B . R. Jones, J. Anderson and H . Fuglsang, B r i t . J . Ophthalmol.,

62,

440 (1978).

D. R. Eotero, Ann. Rev. Pharmacol. Toxicol., 18, 1 (1978); E. Stahe1,Dtsch. Med. Wochenschr., 102, 133 (197v. P . S c o t t , N. 2. V e t . J., 25? 3 7 3 7 9 7 8 ) ; E. L. Roberson i n "Veteri n a r y Pharmacology and T h G a p e u t i c s , " L. M. Jones, N . H. Booth and L. E. McDonald, E d s . , Iowa S t a t e Univ. Press, Ames, Iowa, 1977, p 987. S . C. S a s t r y , K. Jaya Kumar and V . Lakahminarayana, J . Trop. Med. Hyg., 32 (1978). D. Thienpont, 0. Vanparijs, C. Niemegeers and R. Marsboom, Arzneim. Forsch., g8, 605 (1978); A . H. M. Raeymaekers, J . L. H. Van G e l d e r L. F. C . Roevens and P. A. J . J a n s s e n , Arzneim. Forsch. 28, 586 (1978). E. P a n i t z , P. J . L. Daniels, D. Loebenberg, M. M. N a f i s x - V and J . A . Waite, E x p e r i e n t i a , 34, 733 (1978); U. S . P a t e n t 4093731 t o Schering Corp., J u n e T , 1978. U. S . P a t e n t 4026938 t o S t e r l i p g Drug, May 31, 1977. German P a t e n t 2541742 t o Hoechst A . G . , March 24, 1977. B r i t i s h P a t e n t 1522076 t o Syntex, August 23, 1978. A b s t r . Symp. Chemother. S c h i s t o s o m i a s i s , 7 t h I n c . Congr. Pharmacol., P a r i s , J u l y 1978. W. A. Reid, P. S . Loizeaux and M . J . Reardon, Am. J . Trop. Med. Hyg., 937 (1977). R. P. Batzinger, E. Bueding, B. S . Reddy and J . H. Weisberger, Cancer Res., 38, 608 (1978). J. Seubert, RTPohlke and F. Loebich, E x p e r i e n t i a , 33, 1036 (1977). J . Obermeier and H. Frohberg, Arch. Toxicol., 38, (1977); L. Machemer and D. Lorke, Arch. Toxicol., 39, 18771978). D. B. Hoffman and K. S . Warren, E d s . , " c h i s t o s o m i a s i s I V : Condens a t i o n s of t h e S e l e c t e d L i t e r a t u r e , 1963-1975," Hemisphere Publ. Corp., Washington, D. C., 1978. W. C. Campbell, E. B a r t e l s and A. C . Cuckler, J . P a r a s i t o l . , 64, 69 ( 1978). R. E. Voightman and P. E. Thompson, J. P a r a s i t o l . , 63, 1053 (1977). P. Chabrier, T. T. Nguyen, C . J . M. Warolin and A . =din, Bull. SOC. P a t h o l . Exot., 70, 166 (1977); German P a t e n t 2632136 t o Agence Nat. d e V a l o r G a t i o n , Feb. 24, 1977. Belgian P a t e n t 857979 t o S t e r l i n g Drug, Feb. 20, 1978. N . 0. Crossland i n "Advances i n Drug Research," Vol. 12, N. J . Harper and A. B. Simmonds, E d s , Academic P r e s s , New York, 1977, p 53. E. L. Roberson i n " V e t e r i n a r y Pharmacology and T h e r a p e u t i c s , " L. M. Jones, N. H. Booth and L. E. McDonald, Eds., Iowa S t a t e Univ. P r e s s , Amos, Zowa, 1977, p 1052. " N i t r o x y n i l - Published Papers 1966-1976," May and Baker, 1977. G. Nwokolo and K. J . Volkner, Am. J. Trop. Med. Hyg., 26, 688 (1977). B. vcnBonsdorff,"Diphyllobothriasis i n Man,"Academic WYs, Lmdon, 1977. R. Bahr and L. Koslowski, D t s c k Med. Wochenschr., 102, 1098 (1977). D . D . Heath and S . B. Lawrence, N. 2. Vet. J., ( 1978). H. Thomas and R. Gonnert, R e s . V e t . S c i . , 20 (1975); H. Thomas and R. Gonnert, 2. P a r a s i t e n k d . , 55, 165 (1978). M. A. Gemmell, P. D. Johnstone a n r G . Oudemans, Res. Vet. S c i . , 25 111 (1978). R. J Alaimo, C. F. Spencer, J. B. S h e f f e r , R. J . S t o r r i n , C . J . Hatton and R. E. Kohls, J. Med. Chem., 21, 298 (1978). S . K. Dubey, A. K. Singh, H . Singh, S . Sharma, R. N . I y e r , J. C . Katiyar, P. Goel and A . B. Sen, J. Med. Chem., 21, 1178 (1978).

81,

26,

149

2,

-'

3,T

132 -

Annual Reports in Medicinal Chemistry-I4

Chapter 14. Antineoplastic Agents Allen R. Kraska, Pfizer Inc., Groton, Connecticut 06340

Introduction - The annual literature covering chemotherapy and imunotherapy of human neoplasms has more than doubled over the past 5 years. The resulting increased medical awareness concerning the therapeutic or palliative effects of agents administered either singly or in combination has served to increase their use. There is hardly a cancer patient who is not referred to an oncologist practicing several protocols of adjuvant therapy. This increased awareness in the applications and limitations of available antineoplastic agents has also served to stimulate clinical trials with new agents and more basic research into their discovery. Several general reviews and texts of the clinical experiences with chemotherapeutants and immunotherapeutants have recently appeared. This is in addition to specific reviews on the anthracycline antibiotic immunotherapy of cancer with levdaunomycin, plat h u m complexes, BCG16 and interferon, 17-" the amisole,1 3 , ' c Corynebacterium parvum, biological activities of muramyl dipeptide,2 2 and inhibitors of chemical carcinogenisis.2 3 $ 2 4 Also appearing this past year were fundamental reviews of the biology of cancer invasion and metastasis, 2 5 concepts of cell cycle kinetics in cancer chemotherapy,26 and new systems for detection of antitumor agents. 2 7 A n especially interesting survey was published on the choice of animal tumors for experimental studies of cancer therapy and a discussion of their validity as models of clinical cancer. 2 8 Particular emphasis was placed on the relevance of immunotherapy experiments and the type of tumor and species studied. The remainder of this chapter deals with the compounds most studied in the clinic and new entities reported to have antitumor activity in 1978.

'-'

'

''

Alkylating Agents and Nitrosoureas - Chloroethyl substituted nitrogen compounds and related types of alkylating agents continued to be one of the most widely used class of compounds for the treatment of cancer. Almost all of the new adjuvant chemotherapy protocols have at least one cycle of cyclophosphamide, &-chloroethyl nitrosourea (BCNU), cyclohexyl chloroethyl nitrosourea (CCNU), methylcyclohexyl chloroethyl nitrosourea (MeCCNU) or the more recently studied 1-(4-amino-2-methyl-5-pyrimidinyl)me thy 1-3- (2-ch loroethy 1 -3-nitrosourea (ACNU ) , a water soluble nitrosourea. ACNU behaves like the lipid soluble nitrosoureas in vivoZ9 and has been found to be of benefit in induction thera for small cell carcinoma of the lung and metastatic pulmonary tumors. Several clinical trials have been conducted with Peptichemio (PTC; 11, a mixture of six synthetic peptides of m-L-phenylalanine mustard (m-SL). This mixture of alkylating agents has been thought to have an antimetabolic effect because of the peptide group. Treatment with 1-1.5 mg/kg/day for 3-5 days (i.v.) for several cycles has been used for the induction phase of therapy in a variety of solid tumors.31 With the same treatment regimen in children with advanced neuroblastoma, 11 of 12 patients showed both objective and subjective improvement, with complete remission seen in two of them. 3 2 Isophosphamide (21,having activity similar to cyclophosphamide, is converted to its active form (2)in vivo by C -hydroxylation. In an effort to study preactivated derivatives of 2, anajogs were C4-hydroperoxylated.

''

Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form rESENEd. ISBN 0-12-040514-8

Chap. 14

Antineoplastic Agents CH~CHCOR~

(CICH2CH2)2N

kHR1

Kraska

R’

R2

H-

-(N02)Arg.Nval -0 H -p-FPhe -A5n -Hi5 -Nval

Ser.p- FPheProp-FPhem-SL.Arg.Lysp-FPhe,Gly-

1 -

133

These c4-hydro-peroxides were as effective for activation and were found more stable than the C -hydroxyl derivatives.j3 Compound 4 (p.0.) was 4 found to be the most effective against L1210 leukemia. Chloroethyl and methylnitrosourea derivatives of thymidine were prepared and tested for their relative alkylating and carbamoylating activities. Based on the experience with streptozotocin and chlorozoticin, active compounds with reduced bone marrow toxicity possess high alkylating and low carbamoylating activities. Compared with BCNU, 2 had twice the alkylating activity with approximately equal carbamoylating activity, and preliminary & work suggested the nucleoside structure enhanced the nitrosourea activity. 3 4 The antitumor effects of CCNU in vivo are thought to be due to hydroxylated metabolites arising via liver enzymes. Selective deuteration of the cyclohexane ring was shown to alter the ratio of 2-, 3- and 4-hydroxylated metabolites ; however, the various deuterated analogs demonstrated antitumor activity similar to CCNU against TLX-5 lymphoma in mice. 3 5 A peptidyl nitrogen mustard Cbz-L-Pro-L-Leu-Gly-LPro-Gly-NHC6H4N(CH 2CH2C1)2 (6) - containing a L-Leu-Gly linkage was prepared to exploit tumor-associated collagenase, an enzyme known to cleave this peptide bond. The resulting Gly-L-Pro-Gly-NHC H N(CH CH2C1l2 was 4 {ban 5, but shown, as expected, to be six times more toxic in6 mice activity against murine sarcoma 1805 tumors gave disappointing results. Mouse liver homogenates, however, were also found to produce the desired cleavage, which explained the lack of tumor-specific activity. 3 6 Quantitative structure activity relationships for a large number of nitrogen mustards were determined. Their rate of hydrolysis was found to correlate with their toxicity and activity against L1210 and P-388 leukemias and Walker 256 tumors. The lipophilic requirements were found to be higher in the case of Walker 256 tumors, suggesting solid tumors may require more lipophilic drugs. 3 7

*

R’

2 -

3 4

R3 R2 -

CI

H

CI

H

OS02CH3

CH3

H OH OOH

AHCON(NO)CH2CH2CI 5 -

134

Sect. I11

-

Chemotherapeutic Agents

Weisbach, Ed.

Natural Products and Semi-synthetics - Several naturally occurring compounds or their derivatives have received considerable attention this past year because of their clinical potential. The epipodophyllotoxins, VP-16,213 ( I ) and VM-26 (g), known to be potent inhibitors of cell division, were found not to be inhibitors of microtubule assembly as are

---

R' LGlycoside

-0

I

8 Glycoside 9 H 10 OH

R2

R3

H H OH H

H H CH3 CH3

R4

CH3 2-thienyl -

0

(33 @ocH3o R3

-Glycoside 0T4R =

OH

HO

0

podophyllotoxins (2) and epipodophyllotoxins (10) that are without the glycoside moiety. It was suggested that VP-16,213 and VM-26 are effective during the s 01: G2 phase of the cell cycle, whereas the others act in the M phase.38 A number of clinical trials have been conducted showing that -epe o f the most active in the treatment of small cell lung VP-16,213 carcinoma. Deacetylvinblastine amide (vindesine, M S ; 11) has undergone several successful Phase 1/11 evaluations, primarily in the treatment of leukemia and lymphoma. 42-44 VDS has been shown to resemble vincristine (VCR; 12) rather than vinblastine (VLB; 2)in animal tumor models, but appears less neurotoxic than VCR. 4 5 VDS given i.p. inhibited Ridgeway osteogenic sarcoma and Gardner lymphosarcoma, whereas VLB exhibMaytansine (141,*' an ansa ited no activity against these tumors. macrolide 100 times more potent than VCR for blocking mitosis, is also undergoing Phase 1/11 evaluation. Four new C-3 esters and six new C-9 ether derivatives were prepared to determine the structural requirements at these positions for antileukemic a~tivity.'~ The C-3 ester was found necessary for anti-P-388 leukemia activity, but changes in the structure

*'

I

r

i

.C0CH3

ti

11 CONHZ

I 2 COOCH3 13 COOCH3

OH OCOCH3 OCOCH3

CH3 CH3 CHO

Chap. 14

Antineoplastic Agents

Kraska

135

of the ester did not significantly alter activity. Ether derivatives at were not as effective as the free hydroxyl. In an effort to determine what portion of the tricyclic ring system of helenalin (15)was responsible for its cytotoxicy, the a-methylene lactone was opened with -dimethylethylene diamine in a Michael-type addition to form a cyclic lactam. This type of structure was active against a Walker 256 ascites tumor when the free hydroxyls were converted to cinnamate esters. s o Hydroxymethylcyclopentenones corresponding to the A ring of helenalin were also active against Walker 256 ascites tumor when converted to either a cinnamate (2)or 3,4,5-trimethoxybenzoate ester. 5 1 Some structural requirements for another class of naturally occurring antineoplastics, were studied, and it was determined that the epthe quassinoids, (17) oxymethano bridge and C-1 and C-12 hydroxyls are necessary for biological C-9

14

16 -

15

a~tivity.'~The therapeutic index of anguidine (2) was increased when the 4-acetate was hydrolysed with mild base to 15-acetoxyscirpenol (19). A T/C (mean survival of treated x 100/mean survival of control) of >200 was seen in a P-388 lymphatic leukemia assay after 9 daily doses of 0 . 2 0.4 mg/kg, which was 2-4 fold lower than the maximum tolerated dose.53 9R4

0

CHzOCOCH3

I

I

k' 1 3 R = -COCH3

17

3 R=H

Anthracyclines - Adriamycin (ADR; 20) has continued to be the dominant force in the expanding use of chemotherapeutic agents. Its wide spectrum of activity coupled with its dose-limiting cardiac toxicity continue to stimulate the search for structurally modified agents with improved biological profiles. Recent reviews on daunomycin (DNR; ADR and related antibiotics " have appeared that discuss their chemistry and pharmacology. N-Trifluoroacetyladriamycin-14-valerate (AD-32; 221, unlike the basic anthracyclines, ADR and DNR, has been shown not to

z),"

136

Sect. 111

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intercalate with DNA and must, therefore, possess a different mechanism for its cytoxicity. 55,56 In comparative studies with ADR, AD-32 at 40 to 60 mg/kg (i.p.1 resulted in cures of up to 90% with no early deaths.” A similar increased lifespan (ILS) was observed for ADR at 4 mg/kg, but only an occasional survivor past 60 days was observed.” Four daily doses of 20-90 mg/kg (i.p.) of AD-32 produced no drug induced deaths in BDFl mice after 60 days, whereas a single 2 0 mg/kg (i.p.1 dose of ADR was 100% The 14-diethoxyacetoxy lethal [mean survival time (MST) of 9 days] . 5 9 derivative of daunorubicin (RP 33921; 2)also showed superior antitumor activity versus ADR in L1210 leukemia with a greater than two-fold increase in therapeutic index. 6 o 4-Demethoxy-adriamycin and 4-demethoxy4’-epiadriamycin were synthesized and found to be 10 times more potent than ADR in L1210, P-388 and Gross leukemias.61 The cardiac toxicity of

ADR has been reported to be associated with its ability to inhibit both cardiac Na-K dependent adenosine triphosphatase enzyme and cellular ion transport. The presence of calcium reverses these phenomena, and for this reason it has been hypothesized that a chelate of ADR might not be cardiotoxic. Quelamycin (241, a triferric chelate of doxorubicine, was prepared and found to retain antitumor activity and at the same time display less haematologic and cardiac toxicity in experimental systems.62 Objective responses were observed in bronchogenic carcinoma and ADRresistant osteogenic carcinoma in a Phase I trial of quelamycin at doses of 125 to 175 mg/m2; however, in contrast to what was observed in experimental systems, acute iron toxicity and acute cardiotoxicity were also observed. 6 3 Carminomycin, a 4-hydroxy derivative of DNR reported less cardiotoxic than ADR, was found to be strongly synergistic with cyclophosphamide against L1210 leukemia in mice. 6 4 An ILS of 63-331% over controls was observed with the combination; neither drug given alone produced an ILS greater than 116. The 13-deoxy analogs of ADR and DNR were prepared and shown to possess similar efficacy and potency to the parent compound^^^ These analogs, which are also reported to be less cardiotoxic resemble aklavin whose structure has recently been determined.d6 A quantitative structure activity relationship was obtained for aromatic substituents in the benzylhydrazone moiety of rubi-

(s),

Chap. 14

Antineoplastic Agents

Kraska 137

dazone, the 13-benzhydrazone derivative of DNR. Cardiotoxicity was found to decrease with increasing lip~philicity.~~

WH COzCH3

\

\

I

OH

0

OH

I

0

25

Purine, Pyrimidine and Folate Antagonists - High dose methotrexate (MTX) followed by folinic acid rescue has found application in a wide variety of tumors. Thymidine (TdR) has also recently been reported effective as a rescue agent for high dose MTX. As well as being useful in MTX therapy, TdR and other pyrimidine nucleosides were found to enhance 5-fluorouracil (FU) activity against advanced CD8F1 murine breast tumors. The most effective combination produced 70% greater inhibition than FU alone.69 TdR was also shown to reverse bone marrow toxicity in L1210 leukemia bearing mice treated with FU.70 The therapeutic index for FU treatment of human cancers has been increased with TdR treatment in several Phase I studies. 71-73 A broader spectrum of antitumor activity was observed versus MTX for a new folate analog, 10-deaza-aminopterin (26). At equal doses, 26 and MTX had increases in lifespan of 171.2% and 149.8% against L1210 leukemia, 64% and 20.9% against Ehrlich ascites carcinoma, and 159.6% and 64% against sarcoma 180. 74 Anhydro-ara-5-fluorocytidine administered in weekly doses of 33-40 mg/kgTi.v.), had significant antitumor activity, 9/17 responses, in patients with adenocarcinoma of the stomach and pancreas. 7 5

(z),

26

27 -

A convenient synthesis was reported for 1,3-%( tetrahydro-2-furanyl)-5-fluorouracil (Thf2-FU), whose toxicity and antitumor activity in mice was were compared with Ftorafur (Thf-FU) and FU.76 The oral LD approximately 3-fold greater than that observed for Thf-FU, ;Rich was much less toxic than FU. Thf -FU slowly hydrolyzed to FU in vivo and showed significant antitumor eff2ects at 0.15-0.45 nrmol/kg (p.0.) against Ehrlich and AH-130 carcinomas, sarcoma 180, Yoshida sarcoma and Walker 256 carcinosarcoma. The most active in a series of 1-alkylcarbamoyl derivatives of FU was the 1-l-butylcarbamoyl compound, which gave an ILS30 of 8.4 mg/kg 2 ' -Az ido-and 2 '-amino-2' -deoxy-B-D-arab inofuranosylcytosine

.

138

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(cytarazid; 28 and cytaramin; 2)used at 40 and 75 mg/kg (b.i.d. for 2 days, 24 hr after tumor inoculation) resulted in long-term survival (>120 days) of L1210 leukemia bearing mice. This efficacy was attributed in part to cytarazid and cytaramin not being susceptible to enzymic deamination under conditions where E-C would be converted to the inactive ara-U. 7 8 Concomitant dosing of glucose (5000 mg/kg; i.p.1 and methyl 1~5-fluoro-1H-2-oxopyr~m~d~n-4-yl~-~-D-glucopyranuronate (FU-0-G, 30, 400 mg/kg; i.v.) in mice challenged with L1210 leukemia increased the ILS to

-

30

3 K=N3

29; K = NHZ -

63% from the 20% observed when FU-0-G was administered alone. The lowering of the pH in tumor tissue by glucose and subsequent increases in the pH-dependent 6-glucuronidase activity in that tissue were used to explain the enhancement of FU-0-G.79 Metal Complexes

-

The clinical use of the inorganic complex a - d i Its broad spectrum of antitumor properties coupled with its limiting toxic effects have continued to prompt efforts at understanding the mechanism of action and improving the therapeutic index of this type of drug. The synthesis of this class of agents has been reviewed" along with the physicpi properties of their interactions with nucleosides and polynucleotides. Additional evidence has been presented for the nucleophilic displacement of chloride or other carrier ligands by guanine bases in vivo as the The G-,trans-d-, and trans-1-1,Zmechanism of DDP activity. diaminocyclohexane (dach) platinum complexes have been prepared and compared in a variety of tumor systems. It was found that the leaving group as well as the geometry of the dach was important €or activity, and Pt (oxalato)(&-dach) was the most efficacious against murine sarcoma 180 (EDgo 0.72 mg/kg) with a therapeutic index range of 14-42.e3 Rhodium and iri ium complexes have also been studied for their antitumor effects; bis-1,5-cyclooctadiene (COD)-dichlorodirhodium, acetylacetonate (acac) norbornadiene rhodium and IracacCOD produced cures at approximately onefourth their toxic doses in mice bearing Ehrlich ascites tumors.e4 Complexes of 6lnercaptopurine and thioguanine with platinum and palladium displayed less activity than the parent purine under the same conditions. 8 5 Gallium nitrate, a heavy metal compound active in animal tumor systems and shown to be nonmyelosuppressive, has provided responses in initial clinical studies in Hodgkin's disease, lymphoma, ovarian cancer and osteogenic sarcoma.' 6 , chlorodiaminneplatinum(I1) (DDP) has continued to expand.

-

-

Miscellaneous Synthetic Agents A series of antimetabolites of coenzyme Q,, inhibited human cancer cell lines 4265 and K562c. A % T/C of 923 was

Chap. 14

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139

observed with the phytyl analog (2) at 50 mg/kg in Walker carcinosarcoma bearing rats.88 The antitumor effect was found to correlate with ability to inhibit succinoxidase and NADH oxidase activity. A similar study was done with 2-alkylmercapto-l,4-naphthoquinones, the most active in this

- R1

32 -

31

S H

34 CI

R2

CI

H

R3 OH 4-Me2NPh-

case being the 2-hydroxy-3-dodecyl analog (32).8 9 The most favorable activity and toxicity for nitrogen containing coenzyme Q analogs in mice challenged with sarcoma 180 was observed for the oxime 33 at 20 mg/kg and Another imine 34 at 90 mg/kg ( % T/C of 304 and 256, respectively). quinone structure, 1,4-dihydroxy-5,8-&[ [2-(hydroxyethy 1)amino] ethyl 1 amino-9,lO-anthracenedione (35) demonstrated potent activity against P-388 leukemia ( % T/C of 299 at 0.5 mg/kg with 4/6 cures) and B-16 melanoma ( % T/C of 503 at 1 mg/kg with 7/10 cures). 9 1 The effects on antitumor activity of a variety of substituents in the 9-anilino ring of 9-anilinoacridines have been studied. The substitution must be in the

H2)2N H (CH2) 2 0 H

~

OH

NH(CH2)2NH(CH2)20H

35 -

5 R = COOH 37; R

= CHZCHZCOOH

para position, the most potent in L1210 leukemia being carboxyl (36) and carboxyethyl (371, and the activities can be correlated with R values obtained from reverse-phase chromatography. 9 2 , g 3 Further work has been done on the structural requirements for &-intercalating, agents by connecting acridine with other intercalating nuclei. 9 4 The most active of these bifunctional intercalaters are the polymethy lene-linked diacridines, whose & I viva activity in P-388 and L1210 leukemias did not correlate with ability to inhibit those cells vitro,” but was inversely correlated with the rate of agglutination of Con A-treated sarcoma 180 cells. Hydroxyurea with a combination of cyclophosphamide and platinum complexes, such as oxalato (cis-dach) platinum, has been found The collective cure rate synergistic in treating L1210 leukemia.9 6 increases from 11% to 53% with inclusion of hydroxyurea in the treatment regimen. A series of amidoximes, structural analogs of hydroxyurea, were evaluated against L1210 and P-388 leukemias, the most active being formamidoxime, acetamidoxime and 2-aminoacetamidoxime. ” The indoloquinoline 38 was not effective against L1210 leukemia, however it was

140

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active at 25-100 mg/kg (i.p.) or 25-200 mg/kg (p.0.) against Ehrlich carcinoma and sarcoma 180 in mice. 9 8 Comparison of S-carbamoyl, Sethylcarbamoyl- and S-chloroethylcarbamoyl-L-cysteine in a variety of mouse and rat tumor models showed the S-ethylcarbamoyl analogs to be the most active (100 mg/kg/day for 6 days, i.p., cured 10/10 mice bearing and t r a n s (40) dioxoEhrlich ascites carcin~rna).~~ The cis (2) piperazines were evaluated with ICRF-159 (41) for their ability to inhiH

X = cis-cyclopropyl

9 X = trans-cyclopropyl

-

-

41 ; X = -CH(CH3)CHz-

38

bit pulmonary metastases of a Syrian hamster lung adenocarcinoma.' O 0 A structural effect was observed in which the Cis derivative was more active than the trans, and 41 actually may have enhanced metastases. There has been a growing literature on prostaglandins ( P G s ) and their effect on tumor growth and inhibition. Aspirin and indomethacin, inhibitors of PG biosynthesis, have been reported to inhibit growth of a 3-methylcholanthrene(Meth A)-induced fibrosarcoma. O 1 Another inhibitor, flurbiprofen, enhanced the activity of chlorambucil on a chemo-resistant line of Walker tumor. In the case of a murine WHT-NC tumor a reduction in growth and ability t o synthesize P G s was observed, as well as increased effects of methotrexate and local radiotherapy) 0 3 Mechanistic evidence suggesting tumors may prevent the effect of a cellular immune response through PG production was provided when PG biosynthesis inhibitors were found to enhance natural and antibody-dependent cell-mediated cytotoxicity against the tumor cell lines T24 and HCV29.104,105 Another possible role played by P G s produced by tumor cells is to affect platelet aggregation, a phenomena associated with enhanced tumor cell metastasis. Two inhibitors of platelet aggregation, ditazol ( 4 2 ) ' 0 6 and RA-233 (43)1°7 have been shown to decrease metastasis with Lewis lung carcinoma and Ehrlich Even C. parvum has been shown to cause carcinoma, respective1y thrombocytopenia, a fact which has been implicated in its antimetastatic

.

Chap. 14

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141

activity.1o8 Quantitative structure activity (QSAR) relationships have been derived for antitumor activity and toxicity for a large number of aryl-3,3-dialkyltriazenes. The close parallel between the two QSARs suggested improvements in potency would result in increased toxicity. 1 0 9 , 1 1 0 Immunotherapeutics - The theory behind immune modulation and the various experimental models of cancer chemoimmunotherapy have been reviewed. 3 'I Reports have also appeared covering effects on tumor immunotherapy and immunity for the biologicals most often associated with immunomodulation, 9 7 Levamisole, the most widely studied synthetic BCG and 5. parvum. in this class, was the subject of two review^.'^^" Additional reviews in and this area were on synthetic immunoregulating molecules in general' the present status of clinical trials in immunotherapy.' BCG immunotherapy of L2C guinea-pig leukemia resulted in increased life expectancy for 66% of treated animals (11/56 survived >15 weeks). ' I 3 A rat mammary adenocarcinoma (13762A) was treated by intratumor administration of 5. -parvum. ' local tumor regressed and regional lymph node metastases were destroyed. 9 In clinical experiments C.?:;rap significantly extended survival in stage I11 lung cancer patients and potentiated the therapeutic effect and reduced the myelosuppression of MeCCNU in stage 111 malignant melanoma. ''I In efforts to obtain BCG-like effects without using whole cells, work has continued with purified cell walls and cell wall extracts of a variety of microorganisms. Mycobacterium bovis cell wall skeleton (250-500 pg) caused 80% tumor regression in a guinea-pig and Coriolus versihepatoma."' Extracts of Lentinus edodes (KS-2) color (PS-K) 1 2 0 and cell wall preparations of Streptococcus (OK-432)120 and Norcardia l2'have all been reported to have antitumor activity. The biological activities of synthetic muramyl dipeptide (MDP; 441, the smallest fragment of a cell wall with adjuvant activity, have been thoroughly reviewed." Both lower and higher 6-0-acyl derivatives of MDP have been synthesized. 1 2 2 They had adjuvant activities similar to the parent compound, but displayed no antitumor activity. The 6-0-mycoloylhowever, did supress tumor growth in mice bearing a Meth AMDP (g), induced fibrosarcoma. l Z 3 Cord factor (46), trehelose 6,6'-dimycolate (P3), and lower homologs such as trehelose 6,6'-dicorynomycolate (471,

'

'

''

when combined with a glycolipid and administered intralesionally to strain-2 guinea pigs with line-10 tumors, eliminated tumors and metasAntitases and resulted in specific imunity to tumor re-challenge. 12' tumor effects and mechanisms of interferon action have received renewed interest and were subjects of several reviews. ''I-" Ether analogs of inhibited growth of the mouse ascites tumors, lysolecithin (48 and 9)

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Chemotherapeutic Agents

Meth A, Ehrlich and sarcoma 1805, when given as a sin le i.p. injection The effect was from 7 days before to 2 days after tumor challenge. 1'5 interpreted as macrophage activation by these agents. 2-Cyanaziridines (50 and 511, a new class of immunomodulating compounds, have been found to increase both the number and mitogenic response of peripheral lymphocytes and increase rejection of a variety of transplanted tumors in rats and mice. 126,127

CH2-0-R

I

;i

k~-OH

N-C-NH2

1 5 :

+

CH3

CH- O-P-OCH2CH2NMe3

I 0-

-

Drug Delivery The most widely studied method of reducing toxicity of antitumor agents has continued to be encapsulation in liposomes. The mechanism of drug toxicity reduction and pharmacokinetics of liposomeencapsulated drugs have been reviewed.' 28, 2 9 Methotrexate in lipid vesicles enhanced absorption in tumor cells in vitro and resulted in 80% tumor reduction in mice bearing a P1798 lym hosarcoma, a tumor resistant The unstable nature of to the same amount of free methotrexate. " O aqueous suspensions of CCNU was overcome and the duration of dryflaction increased, when it was formulated with phosphatidyl choline. Targeting of drugs has also been achieved by linking them to carriers such as DDP and DNA'32 or polylactate,"' daunomycin and dextran, 1 3 4 and mitomycin C and agarose.

'

Anticarcinogens - A great deal of research has continued toward the goal of inhibiting carcinogenesis. The Laboratory of Chemoprevention of the National Cancer Institute was formed to exploit the unusual activity of vitamin A and its analogs, the most exhaustively studied class of compounds in this area. Reviews have appeared covering general inhibitors of chemical carcinogenesis 2 3 t Z 4 and specific classes such as the retinoids. 36 Ro-11-1430, the trimethylmethoxyphenyl analog of N-ethylretinamide; was shown to reduce the radiation-induced transformation of C3H mouse embryo fibroblasts grown in culture.'37 Retinoids have also inhibited transformation caused by sarcoma growth factor produced by murine sarcoma virus-transformed cells. " Mechanistically, retinoids have been shown to stimulate deac lation of cellular lipids 13' and to inhibit ornithine decarboxylase. 14' At low doses (25-300 ug/mouse/day), retinoit lacid has induced cell-mediated cytotoxicity to allogeneic tumor cells. REFERENCES 1 . G . Mathe and L.M. van Putten, Cancer Chemother. Pharmacol., 1,S(1978). 2 . S. K. Carter, Cancer Chemother. Pharmacol., 1,l S ( 1 9 7 8 ) . 3 . R . L. Clark, R.W. Cumley and R . C . Hickey, Eds., "The Year Book of Cancer," Year Book Medical Publishers, I n c . , 1978.

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4. F. M. Schabel, "Antibiotics and Chemotherapy," Vol. 24, S. Karger AG, Basel, 1978. 5. C. J. Williams and S.K. Carter, Brit. J. Cancer, 37, 434(1978). 6. S. M. Mikulski, M.A. Chirigos and F.M. Muggia, Adv. Pharmacol. Chemother., 15, 263(1978). 7. A. Nicolin, Pharmacol. Res. Cormaun., lo, 93(1978). 8. D. L. Morton and J.E. Goodnight Jr., Cancer, 42, 2224(1978). 9. E. S . Newlands, Br. J. Radiol., 51, 756(1978). 10. R. A. Bender, L.A. Zwelling, J.H. Doroshow, G.Y. Locker, K.R. Hande, D.S. Murinson, M. Cohen, C.E. Myers and B.A. Chabner, Drugs, 16,46,(1978). 1 1 . D. D. Von Hoff, M. Rozencweig and M. Slavik, Adv. Pharmacol. Chemother., g , I (1978). 1 2 . S. J. Lippard, Acc. Chem. Res., 11,211(1978). 13. G . Renoux, Pharmacol. Ther., 2 ,397(1978). 14. W. K. Amery and H. Verhaegen, J. Rheumatol., 5, (Suppl 41, 123C1978). 15. L. Milas and M.T. Stout, Adv. Cancer Res., g; 257(1978). 16. R. W. Baldwin and M.V. P i m , Adv. Cancer Res., 28, 91(1978). 17. R. M. Friedman, J. Natl. Cancer Inst., 60, 119ln978). 18. I. Gresser, Texas Rep. Biol. Med., 2, 394(1977). 19. M. A. Chirigos, Texas Rep. Biol. Med., 35, 399(1977). 20. H. Strauder, Texas Rep. Biol. Med., 2 , 429(1977). 21. I. Gresser and M.G. Tovey, Biochim. Biophys. Acta, 516, 231(1978). 22. L. Chedid, F. Audibert and A.G. Johnson, Prog. Allergy, 2 , 63(1978). 23. L. W. Wattenberg, J . Natl. Cancer I n s t . , 60, ll(1978). 24. L. W. Wattenberg, Adv. Cancer Res., 26, 197(1978). 25. I. J . Fidler, D.M. Gersten and I.R. Hart, Adv. Cancer Res., 28, 149(1978). 26. B. T. Hill, Biochim. Biophys. Acta, 516, 389(1978). 27. F. M. Schabel, "Antibiotics and Chemotherapy," Vol. 23, S. Kager AG, Basel, 1978, p.4. 28. H. B. Hewitt, Adv. Cancer Res., 27, 149(1978). 29. F. Shimizu and M. Arakawa, Gann, 2, 545(1978). 30. N. Saijo, Y. Nishiwaki, I. Kawase, T. Kobayashi, A. Suzuki and H. Niitani, Cancer Treat. Rep., 62, 139(1978). 31. J. Otten and R. Maurus, Cancer Treat. Rep., 62, 1015(1978). 32. B. De Bernardi, A. Comelli, C. Cozzutto, G . Lamedica, P.G. Mori and L. Massimo, Cancer Treat. Rep., 62, 811(1978). 33. A. Takamizawa, S. Matsumoto, T. Iwata, I. Makino, K. Yamaguchi, N. Uchida, H. Kasai, 0. Shiratori and S. Takase, J. Med. Chem., 21, 208(1978). 34. T.-S. Lin, P.H. Fischer, G.T. Shiau and W.H. Prusoff, J. Med. Chem., 21, 208(1978). 35. P. B. Farmer, A.B. Foster, H. Jarman and M.R. Oddy, J. Med. Chem., 2,514(1978). 36. M. J. Marquisee and J.C. Kaner, J. Med. Chem., 21, 1188(1978). 37. A. Panthananickal, C. Hansch and A. Leo, J . Med. Chem., 2,16(1978). 38. J . D. Loike, C.F. Brewer, H. Sternlicht, W.J. Gensler and S.B. Horwitz, Cancer Res., 38, 2688(1978). 39. R. D. Tucker, A. Ferguson, C. van Wyk, R. Sealy, R. Hewitson and W. Levin, Cancer, 41, 1710(1978). 40. K. W. Brunner, Abstracts, Chemotherapy Foundation Symposium 111, 38(1978). 41. R. T. Eagan, Abstracts, Chemotherapy Foundation Symposium 111, 41(1978). 42. R. W. Dyke, Abstracts, Chemotherapy Foundation Symposium 111, 33(1978). 43. V. E. Currie, P.P. Wong, I.H. Krakoff and C.W. Young, Cancer Treat. Rep., 62, 1333(1978). 44. I. E. Smith, D.W. Hedley, T.J. Powles and T.J. McElwain, Cancer Treat. Rep., 62, 1427(1978). 45. C. J. Barnett, G.J. Cullinan, K. Gerzon, R.C. Hoying, W.E. Jones, W.M. Newlon, G.A. Poore, R.L. Robison, M.J. Sweeney, G.C. Todd, R.W. Dyke and R.L. Nelson, J. Med. Chem., 21, 88(1978). 46. M. J. Sweeney, G.B. Boder, G . J . Cullinan, H.W. Culp, W.D. Daniels, R.W. Dyke and Gerzon, R.E. McMahon, R.L. Nelson, G.A. Poore and G.C. Todd, Cancer Res., 38, 2886(1978). 47. B. F. Issell and S.T. Crooke, Cancer Treat. Rev., 2, 199(1978). 48. P. Chahinian, C. Nogeire, T. Ohnuma and J.F. Holland, Abstracts, Chemotherapy Foundation Symposium 111, 34(1978). 49. S. M. Kupchan, A.T. Sneden, A.R. Branfman, G.A. Howie, L.I. Rebhun, W.E. McIvor, R.W. Wang and T.C. Schnaitman, J . Med. Chem., 2, 31(1978). 50. K.-H. Lee, T. Ibuka, E.-C. Mar and I.H. Hall, J. Med. Chem., 21, 698(1978). 51. K.-H. Lee, E.-C. Mar, M. Okamoto and I.H. Hall, J. Med. Chem., 2,829(1978). 52. M. E. Wall and M.C. Wani, J. Med. Chem., 21, 1186(1978). 53. C.A. Claridge, W.T. Bradner and H. Schmitz, J. Antibiotics, 21, 485(1978).

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54. J . R. Brown, Prog. Med. Chem., l5, 125(1978). 55. T. F a c c h i n e t t i , A. Mantovani, L. C a n t o n i , R. C a n t o n i a nd M. Salmona, Chem.-Biol. I n t e r a c t i o n s , 20, 97(1978). 56. R . Goldman, T. F a c c h i n e t t i , D. Bach, A. Ray and M. S h i n i t z k y , Biochirn. Biophys . A c t a , 512, .254(1978). 57. A. Vecchi. M. C a i r o , A. Mantovani, M. S i r o n i and F. S p r e a f i c o , Ca nc e r T r e a t . Rep., 62, lll(1978). 58. P r a t e s i , A.M. C asazza and A. DiMarco, Cancer T r e a t . Rep., 62, 105(1978). 59. L. M. P a r k e r , M. Hirst and M. I s r e a l , Cancer T r e a t . Rep., 62, 119(1978). 60. R. M a r a l , J . B . Ducep, D. F a r g e , G. P o n s i n e t and D. R e i s d o r f , C m p t . Rend., 286, 4 4 3 ( 1978 ) 61. A. DiMarco, A.M. C a s a z z a , F. G u i l i a n i , G. P r a t e s i , F. Arcamone, L. B e r n a r d i , G. F r a n c h i , P. G i a r d i n o , B. P a t e l l i and S. P enc o, Ca nc e r T r e a t . Rep., 62, 375(1978). 1185(1978). 62. M. G o s a l v e z , M.F. B l anco, C. Vi vero and F. Valles, Europ. J. Ca nc e r, 63. A. B r u g a r o l a s , N. Pachon, M. Gosal vez, A.P. L l a n d e r a l , A . J . La c a ve , J . M . Buesa and M.G. Marco, Cancer T r e a t . Rep., 3,1527(1978). 64. T. L. Avery and P.G. C ruze, Cancer Res., 38, 2892(1978). 65. T . H. S mit h , A.N. F u j i w a r a and D.W. Henry, J . Med. Chem., 280(1978). 66. V. Kumar, W.A. Remers and R . G r u l i c h , J. A n t i b i o t i c s , 30, 881(1978). Lee, D.W. Henry and G. Zbinde n, J. Med. Chem., 1, 67. G. L. Tong, M. Cory, W.W. 732( 1 9 7 8 ) . 68. S. B. H o we l l , W.D. Ensminger, A. K r i s h a n and E. F r e i 111, Ca nc e r Res., 325(1978). 69. D. S. M a r t i n , R.L. S t o l f i and S. S pi egel m a n, A b s t r a c t s , Proc . Am. Assoc. Ca nc e r Res., 1978; A b s t r a c t 882. 70. F. .I. Cummings and P. C a l a b r e s i , A b s t r a c t s , P r o c . Am. Assoc. Ca nc e r Res., 1978; A b s t r a c t 611. 7 1 . S. Vogel, C. P r e s a n t , C. R a t k i n and C. K l a h r , A b s t r a c t s , Proc . Am. Assoc. Ca nc e r Res., 1978; A b s t r a c t 927. 72. T. M. Woodcock, D.S. M a r t i n , N. Kemeny and C.W. Young, A b s t r a c t s , P r o c . Am. Asaoc. C a n c e r Res., 1978; A b s t r a c t C-179. 73. J . M. Kirkwood and E. F r e i 111, A b s t r a c t s , P r o c . Am. Assoc. Ca nc e r Res., 1978; A b s t r a c t 635. 74. F. M. S i r o t n a k , J.I. DeGraw, D.M. Moccio and D.M. D o r i c k , Ca nc e r T r e a t . Rep., 62, 1047(1978). 75. P. A l b e r t o , M. Rozencweig, D. G a n g j i , A. B r u g a r o l a s , F. C a v a l l i , P. S i e g e n t h a l e r , H.H. Hansen and R. S y l v e s t e r , E ur. J. C a n c e r , 195(1978). 76. M. Yasumoto, I. Yamawaki, T. Marunaka and S. Hashimoto, J. Med. Chem., 738(1978). 77. A. H o sh i , M. I i g o , A. Nakamura, M. Inomata and K. K u r e t a n i , Chem. Pharm. B u l l . , 5 , 161(1978). 78. M. Bobek, Y.C. Cheng and A. B l och, J. Med. Chem., 2, 597(1978). 79. F. Shimizu and M. Arakawa, Gann, 9, 667(1978). 80. J . K. B a r t o n and S . J . L i p p a r d , Ann, N.Y. Acad. S c i . , 686(1978). Chu, S. Mansy, R.E. Duncan and R.S. T o b i a s , J. Amer. Chem. SOC., 100, 81. G.Y.H. 593(1978). 8 2 . S. Mansy, G.Y.H. Chu, R.E. Duncan and R.S. T o b i a s , J. Amer. Chem. SOC., 100, 607( 1 9 7 8 ) . 83. Y. K i d a n i , K. I n a g a k i , M. I i g o , A. Hoshi and K. K u r e t a n i , J. Med. Chem., 3, 1315(1978). 8 4 . T. G i r a l d i , G. S ava, G . M e s t r o n i , G. Z a s s i n o v i c h and D. S t o l f a , Chem.-Biol. I n t e r a c t i o n s , 22, 231(1978). 85. M. Das and S.E. L i v i n s t o n e , Brit. J . C a n c e r , 2, 325(1978). 86. J . Brown, E. S a n t o s , G. Rosen, L. Hel son, C. Young and C. Tan, A b s t r a c t s , P r o c . Am. Asso c . Cancer R es., 1978; A b s t r a c t 798. 87. M. V a l d i v i e s o , G.P. Bodey and E.J. F r e i r e i c h , A b s t r a c t s , Proc . Am. Assoc, Cancer Res., 1978; A b s t r a c t 857. 88. K. F o l k e r s , T.H. P o r t e r , J . R . B e r t i n o , and B. Moroson, Res. Commun. Chem. P a t h o l . P h a r ma c o l., 19,485(1978). 89. T. H. P o r t e r 7 C . S . T s a i , T. K i s h i , H. K i s h i and K. F o l k e r s , A c ta Pharm. Sue c . , 15, 97(1978 1. 90. E . M. H o d n e t t , G. P r a k a s h and J . Amirmoazzami, J . Med. Chem., 21, l l ( 1 9 7 8 ) . Zee-Cheng and C.C. Cheng, J . Med. Chem., 21, 291(1978), 91. R.K.-Y. 92. W. A. Denny, G.J. A t w e l l and B.F. C a i n , J . Med. Chem., 21, 5 ( 1 9 7 8 ) . 93. W. A. Denny and B.F. C a i n , J. Med. Chem., Z l , 430(1978), 94. B. F. C a i n , B.C. Baguley and W.A. Denny, J . Med. Chem., 1, 658(1978). 95. T. K. Chen, R. F i c o and E.S. C a n e l l a k i s , J. Med. Chem., 21, 868(1978).

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a,

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1,

313,

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100. 101. 102. 103. 104. 105. 106. 107. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123.

125. 126. 127. 128. 129. 130. 1 3 1. 132. 134. 135. 136. 137. 138. 139. 140. 141.

Kraska

Antineoplastic Agents

41,

145

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Annual Reports in Medicinal Chemistry-14

Chapter 15.

Immunostimulants

P. Dukor, L. Tarcsay, and G. Baschang, Research Department, Pharmaceuticals Division, CIBA-GEIGY Limited, Basel, Switzerland Introduction - Over the past decade, immunopharmacology has become a viable discipline in its own right. Interest in imunostimulation has been fuelled by advances in bacterial cell wall chemistry, by the rapidly expanding knowledge of endogenous regulators and mediators of lympho-myeloid cell differentiation and cooperation, and by the apparent promise of expermental and clinical tumor immunology. In the context of this brief review, the term "immunostimulant" will be applied in its broadest sense, so as to include immunological adjuvants (substances which are added to antigens in order to increase their immunogenicity), systemic immunopotentiators (pharmacological agents which non-specifically enhance the immunologically specific reactivity o f the host), as well as stimulants of non-specific host resistance (pharmacological agents which augment immunologically nonspecific effector mechanisms via humoral mediators, lymphocyte subsets, and accessory cells). This rather loose terminology is justified by practical considerations. Clearly, many of the presently available immunostimulants are known to affect multiple target sites and to differ in their activity depending on the mode and schedule of their application. Bacterial vaccines and crude preparations of bacterial and fungal origin were the subject of intensive investigation and ever-expanding clinical interest. Co prehensive reviews detailing the current status of immunotherapy with BCGP and C. parvum2 have recently appeared. Also, y4 and a protein-bound streptococcal c u l t u c fluid (OK-432, Pi~ibanil)~ polysaccharide fraction from Coriolus versicolor (PS-K, K r e ~ t i n , )6~ were reported to potentiate immune responses and to inhibit tumor growth by host-mediated mechanisms. Both agents are now available f o r clinical use in Japan. However, such complex biological preparations are beyond the . scope of this chapter which will be restricted to a cursory description of comparatfvely recent findings with chemically well-defined immunostimulants of exogenous origin. Peptidoglycans - Several groups continued to characterize the immunopharmacologicall-10 and antitumor11-12 properties of delipidated cell wall skeleton fragments from various sources. An example is provided by the extensive work on Nocardia water-soluble mitogen which has been reviewed recently.13 NWSM was reported to enhance T cell-mediated immune reactions if admixed to antigen-oil preparations, to stimulate humoral immune responses if administered together with antigen in oil or saline, to trigger the proliferation of macrophage precursor cells, to exhibit mitogenicity for a distinct subset of B lym hocytes in mice, rabbits and man, to induce circulating interferon in man,!4 and--if attached to oil droplets--to exhibit significant antitumor activity in mice and in patients with malignant pleurisy.15 It remains to be determined whether the biological profile of NWSM differs from that associated with other peptidoglycans, and if so, whether this can be attributed to distinct structural features of the NWSM. As is the case with other water-soluble peptidoglycans, NWSM exerts its effects on manifestations of cell-mediated immunity only, if

(m)

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0-12-0405148

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147

administered in certain types of lipid vehicles. However, peptidoglycan fragments acylated with lauric acid were found to promote anti en-induced delayed-type hypersensitivity reactions in the absence of oil .86917 Muramyldipeptides - Further progress in the biologic characterization of the s nthetic glycopeptide N-acetyl-muramyl-L-alanyl-D-isoglutadne (MDP: 1)18-$0 and of many new derivatives has recently been reviewed.21,22 In several systems, muramyldipeptides are known to substitute for the mycobacterial component of complete Freund's adjuvant (CFA). The structural requirements for the induction of manifestations of cell-mediated immunity were investigated primarily in two different models in guinea pigs: Imunization with MDP-supplemented water-in-mineral oil emulsions containing heterologous protein antigens or azobenzenearsonate-N-acetyl-L-tyrosine (induction of delayed type hypersensitivity), 18-21923-35 or encephalitogenic proteins and peptides (induction of experimental aller ic ence halomyelitis) .36,37 A large series of analogs was compared.19,2~~23,38-~1 Derivatives closely related to MDP andfor carrying substituents which may 23,35 3,29 4,27 be ex ected to be readily removed b 5 20,!1,36,37 6 2OY25,26,29,32,36,3Y ;:;Es2!$!7 9,30-10,30 11,30 12,30 i;,30,37 14,30-;5,33 16,33 17,28,33 1 8 , 2 1 , d 19,3rg,3412335), or analogs, where L-A< is G p l a c z by G S e r (GP3,31,37Tr L-Abu ( 2 s 5 showed high activity at least in some of the experimental systems investigated. Intermediate or marginal adjuvanticit was found with 2418,23,24,37 2?9,32,37 2622 27,32 28,21 29,32 3O,37 31 3 3 ~ 2 7 ~ 3 1 ~and 3 7 32.23931932937 y-N-methylamidation oTD-isoGln orD-Glu-kCH3 (33,29 34395-; substitution of D-isoGln or D-Glu by D-isoAsn (35);0,29~37 D-Nle'(36)Tg y-Abu (37),29 D-Ala (38) ,20 or D-Gln (39) ,20921,371: inversion of their configuration (4U,20,23~n~37 4120,27,37), led to inactive roducts. Accordingly, 4220 a n F g 2 9 t o o were devoid of activity. A l s o , 4432 was reported to be inactive. Removal of the essential amino acids oFthe peptide moiety (45i9920,26~36 46 20,37 47?O,37 4820) or opening or elimination of the intact sugar rin&,23 51,25 -952 20,26 53,2G 5429,26,37) completely abolished adjuvant 515y23925577activity

.

Recently, muramyldipeptides proved to be effective in experimental malaria vaccination of primates which has hitherto depended on the use of CFA. Thus, a significant degree of protective immunity was induced in aotus monkeys42 and in macaques43 by Plasmodium falciparum or P . knowlesi merozoites in mineral-oil emulsion supplemented with MDP (1)o r Nor-MDP respectively. Full protection against p. faleiparum in aotus monkeys was achieved by immunization with mature segmenters mixed with 6-0-stearoyl - and liposomes.44 MDP (13)

(z),

Indeed, lecithin-cholesterol liposomes successfully substituted for mineral-oil emulsion in the induction of delayed hypersensitivity: In guinea pigs, administration of liposome-suspensions containing MDP and ovalbumin induced delayed skin reactivity similar to that observed following immunization in MDP-supplemented incomplete Freund's adjuvant (IFA) or in CFA.30,45 However, 6-0-acyl-MDP derivatives of appropriate fatty acid but not c2-c8 (9, 10, showed inchain length (C12-C22, 12,13,g), creased effectiveness in this vehicle.30 Obviously, lipophilic MDP derivatives will to a considerable degree be adsorbed to liposomes.

z),

148

Sect. I11

-

Weisbach, Ed.

Chemotherapeutic Agents B:

A: MurNAc-L-Ala-D-isoG1n MurNAc-L-Ala-D-isoG1n(NH2) MurNAc-L-Ala-D-isoG1n (OCH3) MurNAc-L-Ala-D-isoG1n (L-LysOH) MurNAc-L-Ala-D-isoCln ( L-Lys-D-A1aOH)

24 MurNAc-L-Ala-D-GluOH 25 MurNAc-L-Ala-D-Glu-GlyOH 26 MurNAc-L-Ala-D-Glu-GlyNH2 27 MurNAc-L-Ala-D-Glu(L-LysD-Ala)GlyOH

MurNAc-L-Ala-D-Glu-NHCH3

28 MurNAc-L-Ala-D-Glu-OCH3

MurNAc-L-Ala-D-Glu(OCH3)-0CH3

29 MurNAc-L-Ala-D-Glu-

6-0-Acetyl-MurNAc-L-Ala-D-isoGln

6-0-Butyroyl-MurNAc-L-Ala-D-isoGln 6-0-Octanoyl-MurNAc-L-Ala-D-isoGln 6-0-Lauroyl-MurNAc-L-Ala-D-isoGln 6-0-Stearoyl-MurNAc-L-Ala-D-isoGln 6-0-Docosanoyl-MurNAc-L-Ala-D-isoGln 6-0-Corynomycoloyl-MurNAc-L-AlaD-isoGln 6-0-Nocardomycoloyl-MurNAc-L-Ala-

D-AlaNH2 of MurNAc-L-Ala-D-isoGln MurNAc-D-Ala-D-isoGln 32 MurNAc-Gly-D-isoGln

30 a-Methyl-glycoside

a

I

D-isoGln 6-0-Mycoloyl-MurNAc-L-Ala-D-isoGln 4,6-di-O-Acetyl-MurNAc-L-Ala-

D-isoGln 8-Piethyl-glycoside of MurNAc-L-AlaD-isoGln 8-p-Aminophenyl-glycoside of MurNAcL-Ala-D-isoGln MurNAc-L-Ser-D-isoG1n nMurNAc-L-Ala-D-isoGln nMurNAc-L-Abu-D-isoGln

L-Ala-D-isoGln 0

R=-CH3

:

MurNAc

R=-H

:

nMurNAc

1 2.

nMurNAc-L-Ala-D-Glu-GlyNH2

C:

42 43 44 45

MurNAc-L-Ala-D-isoG1n (NHCH3) MurNAc-L-Ala-D-Glu (NHCH3)NHCHj MurNAc-L-Ala-D-isoAsn MurNAc-L-Ala-D-Nle MurNAc-L-Ala-y-Abu MurNAc-L-Ala-D-Ala MurNAc-L-Ala-D-Gln MurNAc-L-Ala-L-isoG1n MurNAc-L-Ala-L-GluOH MurNAc-L-Ala-L-Gln MurNAc-D-Ala-L-isoG1n MurNAc-Gly-D-Glu-GlyOH MurNAc-L-AlaOH

46 MurNAc-D-AlaOH

47 MurNAc-D-isoGln

48 MurNAc 49 Muraminitol-L-AlaD-isoGln IQ D-Lactyl-L-Ala-D-isoGln 51 D-Lactyl-L-Ala-D-isoGln (L-Lys-D-AlaOH) 52 L-Ala-D-isoGln L-Ala-D-isoGln(L-Lys) 54 L-Ala-D-isoGln (L-Lys-D-AlaOH)

MDP-derivatives with high (A), intermediate/marginal (B)or absent ( C ) adjuvant activity (as assessedby i n d u c t i o n o f m a n i f e s t a t i o n s o f cellmediatedimmunityby MDP-supplemented antigen-water-in-oil emulsions in guinea pigs).

Chap. 15

Immunostimulants

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6-0-acylation with even l o n g e r f a t t y a c i d s f u r t h e r changed t h e b i o l o g i c p r o p e r t i e s of t h e p a r e n t molecule. Unlike MDP (1,and 6-0-stearoyl-MDP , 6-0-nocardomycoloyl- ( 1 6 1 , and 6-0-myco(13),25 6-0-corynomycoloyl- (15) were e f f e c t i v e adjuvants f o r t h e induction of cell-mediated loyl-MDP (17) immunity t o a l l o g e n e i c c e l l s , even i f administered t o g e t h e r with t h e a n t i 7 was much l e s s e f f e c t i v e with r e s p e c t t o ingen i n saline.33 However, 1 duction of humoral antibody formation. On t h e o t h e r hand, x s h o w e d t u m o r suppressive p r o p e r t i e s and e x h i b i t e d a g r e a t l y decreased pyrogenicity. 33 D i r e c t coupling of adjuvant a c t i v e MDP-moietiesto poorly immunogenic antigens may provide an opportunity t o o b v i a t e t h e u s e of adjuvant-antigen mixtures i n complex v e h i c l e s and may f u r t h e r reduce g e n e r a l t o x i c i t y b y f o cussing t h e imrnunoadjuvant t o t h e t a r g e t c e l l clones. Inamodelexperiment, immunogenicity of a MS-2 coliphage c o a t p r o t e i n fragment ( a t t a c h e d t o mult i c h a i n poly-DL-alanyl-poly-L-lysine) was g r e a t l y i n c r e a s e d by covalent bindingof anaturaldisaccharide-tetrapeptide from B a c i l l u s megatherium t o t h e a n t i g e n i c c a r r i e r .46 The k i n e t i c s of c e l l u l a r and humoral immune responses i n guinea p i g s immunized with p r o t e i n antigen i n MDP-supplemented I F A d i f f e r e d from t h o s e observed with CFA: Antibody responses were f a s t e r and h i g h e r , b u t l a s t e d s h o r t e r with MDP than with whole mycobacteria.47 I n t h i s model, MDP o r act i v e analogs favored t h e production of s p e c i f i c IgG2 a n t i b o d i e s . 48 MDP without antigen, i n c o n t r a s t t o CFA ( o r t o lipopolysaccharide) , d i d not a l t e r immunoglobulin l e v e l s i n mice, 48 I f given t o m i c e with a n t i g e n , howe v e r , MDP i n s a l i n e o r i n IFA l e d t o an i n c r e a s e i n IgGl a n t i b ~ d i e s ~ ~ , ~ ~ and w a s claimed t o enhance t h e r i s k of anaphylactic r e a c t i o n s t o t h e immun i z i n g antigen. 49 I n c o n t r a s t t o t h e promotion of cell-mediated immunity ( a t l e a s t by water-soluble MDP d e r i v a t i v e s ) , MDP-mediated p o t e n t i a t i o n of humoral a n t i body responses does not depend on t h e u s e of a l i p i d v e h i c l e . Thus, MDP i n aqueous medium w a s found t o s t i m u l a t e antibody formation of mice, r a t s , and hamsters t o a v a r i e t y of a n t i g e n s , i n c l u d i n g bovine serum albumin?* ,27 *3%50 b a c t e r i a l a-amylase ,25 TIP-ovalbumin ,53 TNP-keyhole ~ v a l b u m i n , ~,51,52 " limpet hemocyaninY53 i n f l u e n z a v i r u s subunit vaccine,54 and, though t o a l e s s e r degree, sheep red blood c e l l ~ ~ ~ , 4 9and ~ 5t 5 o t h e T-independent a n t i gen DNP-Ficoll. 25 The s t r u c t u r a l requirements f o r s t i m u l a t i o n of antibody formation by MDP d e r i v a t i v e s i n s a l i n e a r e s i m i l a r t o , thoughnot i d e n t i c a l with those found f o r induction of delayed h y p e r s e n s i t i v i t y byMDPin water3 2 7 , 521, 629,1 z 7 , a 2 7 , B21,21271 in-mineral-oil emulsions. Thus, 329, 2821, and 2 2 7 displayed both t y p e s of a c t i v i t y , while 252913127 , S29, s 2 9 , 3 6 2 9 , z 2 9 , 4027, 4 1 2 7 , 4.327 and &1 were i n a c t i v e i n e i t h e r system. On t h e o t h e r hand, 2 0 3 4 and 2 4 2 4 promoted antibody formation i n mice, if administered i n s a l i n e , b u t were b a r e l y a c t i v e i n t h e guinea-pig model of delayed-type h y p e r s e n s i t i v i t y . The r e v e r s e w a s t r u e f o r compound 39.2 1 Increased antibody formation i n t h e presence of MDP was a s s o c i a t e d This does not with improved c a r r i e r priming of T-helper lymphocytes. 53 Y~~ imply, however, t h a t such c e l l s r e p r e s e n t t h e primary t a r g e t of t h e MDP effect. The timing of t h e MDP a p p l i c a t i o n r e l a t i v e t o a n t i g e n i c s t i m u l a t i o n appeared t o be c r i t i c a l l w i t h maximum adjuvant e f f e c t s obtained by simultaneous a d m i n i s t r a t i o n of a n t i g e n and MDP. 2 1 35 ~ Moreover, l a r g e s i n g l e doses

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of MDP (10 mg/kg) were r e p o r t e d t o cause tachyphylaxis i n mice , i.e .,tempor a r y unresponsiveness t o t h e immunostimulatory e f f e c t of a second dose of t h e same compound, l a s t i n g f o r a period of more than 4 days.35 Upon systemic a d m i n i s t r a t i o n , muramyldipeptides were shown t o stimul a t e a l s o non-specific h o s t r e s i s t a n c e . 21 MDP and v a r i o u s d e r i v a t i v e s (adwere found t o p r o t e c t ministered i n s a l i n e ) (L, 8, & 2856; a d u l t mice a g a i n s t challenge with 5. neumoniae while 3, 4, 6, 1,21, 25, appeared t o be i n a c t i v e 2, 32, 3 3 , 34, 3 5 , 3 6 , 37, 41, 42,43 4:+ and 20 i n t h i s r e s p e c t . I n t e r e s t i n g l y , adjuvant a c t i v i t y and p r o t e c t i v e p o t e n t i a l of t h e compounds were n o t d i r e c t l y r e l a t e d . Thus, 3, 4, 6 , 1,and 21 d i s played good a d j u v a n t i c i t y , both i n o i l emulsion i n guinea p i g s and i n sal i n e i n m i c e , b u t f a i l e d t o p r o t e c t ; 24 and 2 e x h i b i t e d a n t i i n f e c t i o u s p r o p e r t i e s , b u t v i r t u a l l y no a d j u v a n t i c i t y i n o i l emulsion, although both compounds w e r e found t o p o t e n t i a t e antibody formation, when given t o mice together with antigen i n s a l i n e . P r o t e c t i v e a c t i v i t y (though n o t a d j u v a n t i c i t y i n o i l ) was l o s t by s u b s t i t u t i o n of MDP w i t h a B-p-aminophenylaglycone group, but reappeared following cross-linking o f t h e glycosidated MDP with glutaraldehyde 34 Remarkably , immunologically compromised h o s t s , i . e . a d u l t thymectomized, i r r a d i a t e d and bone-marrow r e c o n s t i t u t e d mice could r e a d i l y be p r o t e c t e d a g a i n s t K l e b s i e l l a i n f e c t i o n b y b o t h 1 and 24. 2 1 Moreover, MDP and some d e r i v a t i v e s (1, 5,4 , but not t h e stereoisomer 31) w e r e found t o p r o t e c t a l s o neonatal and very young mice a g a i n s t b a c t e r i a l ~ h a l l e n g e . ~ 7Compound 5 proved t o most e f f e c t i v e and w a s shown t o be act i v e even when given b y t h e o r a l route. P r o t e c t i v e MDP d e r i v a t i v e s enhanced carbon clearance i n a d u l t mice30,57 and increased t h e e l i m i n a t i o n of bact e r i a from blood i n both a d u l t and newborn hosts.57

s,

=,

.

I n v i t r o , MDP w a s r e p o r t e d t o i n h i b i t t h e migration of guinea p i g -p e r i t o n e a l exudate c e l l s without e x e r t i n g c y t o t o x i c i t y 58 s59 An adjuvanti n a c t i v e stereoisomer had no i n h i b i t o r y p r o p e r t i e s . 58 On t h e o t h e r hand, MDP s t i m u l a t e d human monocytes t o r e l e a s e lymphocyte-activating f a c t o r . 60

.

S i m i l a r l y , when p u r i f i e d mouse macrophages were exposed t o MDP, they were found t o produce a supernatant f a c t o r which r e s t o r e d macrophage-deficient lymph-node c e l l c u l t u r e s . 3 5 Moreover, i n t h e presence of MDP, c u l t u r e d mur i n e spleen c e l l s were r e p o r t e d t o r e l e a s e a s o l u b l e mediator which stimul a t e d antibody responses of o t h e r c u l t u r e d spleen c e l l s .61 Production o f t h i s f a c t o r could be blocked by an antiserum s p e c i f i c f o r mouse macrophages .61 F i n a l l y , MDP w a s a l s o capable of inducing t h e formation of colonys t i m u l a t i n g f a c t o r i n c u l t u r e s of mouse macrophages. 62 I n f a c t , macrophage a c t i v a t i o n may be responsible f o r a host of MDPinduced phenomena which have been observed i n mouse lymphocyte c u l t u r e s : MDP and some of i t s s a l i n e - a c t i v e analogs (I, 4, 5, 8, 24, 2 8 , 3 9 , b u t not 20, 2, 3 3 , 35, 36) enhanced c e l l v i a b i l i t y and i n c r e a s e d t h e number ofbackground hemolytic plaque-f orming c e l l s i n mouse spleen c e l l c u l t u r e s . 6 3 ~ 6 4 A l s o , MDP and nMDP (2) exerted powerful p o t e n t i a t i n g e f f e c t s i n mixed mouse lymphocyte c u l t u r e s s e t up with suboptimal numbers of a l l o g e n e i c s t i m u l a t o r c e l l s . 35 Immunologic r e a c t i v i t y of T-cell d e f i c i e n t mouse spleen c e l l s from C57B1/6 or Balb/c athymic donors could be r e s t o r e d by a d d i t i o n , b u t not by 24 o r 4 0 . 3 5 ~ 6 5The reof MDP o r of i t s s e r y l - d e r i v a t i v e (21) l a t i o n s h i p of t h e T - c e l l s u b s t i t u t i n g and t h e B-cell mitogenic e f f e c t s of MDP w e r e n o t c l a r i f i e d . MDP w a s repeatedly shown, however, t o d i s p l a y d i s t i n c t mitogenicity f o r mouse B-lymphocytes from susceptiblestrains(DBA/2,

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Balb/c , AKR, CBA, but not C57B1/6 or C3H/He) 66 There appeared to be a rather impressive correlation between in vitro activationby differentcompounds and their in vivo adjuvant activity in saline (stimulationb 1 4 -5I 6 , 1, 8, 2, 2 4 , 2 8 , no stimulation by 20, 2, 34, 35, 40, 43).6 Z - I Mediator release from accessory cells may also be responsible for side effects of MDP. Pyrogenicity of MDP and of several MDP derivatives was demonstrated in rabbits.67 Following incubation with MDP , rabbit leukocytes were shown to release pyrogenic material into the supernatant.2 1 Moreover, MDP was found to induce a transitory l e u k ~ p e n i areminiscent ~~ of the leukocyte disappearance reaction associated with the administration of lipopolysaccharides.68 On the other hand, MDP is not immunogenic, and the evidence available to date appears to indicate that MDPdoesnot react with naturally occurring or experimentally induced anti-peptidoglycanantibodies from man and rabbit, respectively.69 In water-in-mineral oil emulsions, though not in saline, MDP was found to induce polymorphonuclear infiltration followed by formation of epitheloid granulomas both at the injection site and in the draining nodes, indistinguishable fromthe tissue reaction to CFA.35,70 In saline, MDP was reported to be absorbed rapidly fromtheinjection site. Following intravenous injection,more than 90% of C1*-labelled material was recovered unchanged in urine.2 1 Cord Factors - The interest in immunostimulant properties and antitumor activities of cord factors (6,6'-diesters of a,a trehalosewithmycolic acids, corynomycolic acids and nocardomycolic acids) has continued to grow. Comprehensive reviews on the chemistry and the biological activities of cord factors and of synthetic analogs were published. Parant et al. reported that a lower homolog of mycobacterial cord factor isolated from Corynebacteriumdiphtheriae (with corynomycolic acids with chain lengths of C28 to C36),as well as some synthetic isomers increasednon-specific resistance of mice against infections with Klebsiella pneumoniae or Listeria monocytogenes with the same efficacy as mycobacterialcord factor (with mycolic acid; chain lengths from Cg5 to ego) .74 Synthetic trehalose diesters produced a weaker and more transient inflammatoryresponse in mice, thanmycobacterial cord factor. Ribi et al. found that mycobacterialcord factor and some natural and synthetic homologs causeregression of established transplants of syngeneic guinea-pig hepatocarcinoma when combined with endotoxin of Salmonella Re mutant.75 Yarkoni et a1 observed regression of established syngeneic murine fibrosarcoma after intralesional administration of 6,6'-di-O-2-tetradec 1 3 hydroxyoctadecanoyl-a,a trehalose, a synthetic cord-factor analog. 7 % - fi-(l+3)-Glucans - A number of naturally occurring~-(1+3)-glucans fromvarious sources (higher plants, fungi, lichen, bacteria and yeasts) are known to stimulate both non-specific host resistance and specificimmunologic reactivity and thereby to exert inhibitory effectsagainsttransplantable tumors 7 7 Investigation of a partially purified particulate glucan preparation from Saccharomyces cerevisiae cell walls has expanded. Glucan is held responsi le for most of the reticuloendothelial-stimulatingpropertiesof zymosan.9 8 The preparation was reportedto activatemacrophages as assessed by

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size, spreading, adherence, chemotactic mobility and morphologic surface characteristics79 and to induce a marked but spontaneously reversible granulomatous response in the liver of rats.80 Glucan-induced enhancement of macrophage and granulocyte production was re ortedly mediated by release of colony-stimulating factor from macrophages.8P In addition, i.v. administration of glucan led to a treatly increased serum 1 sozyme concentration and enhanced clearance of colloidal carbon in rats.83 In glucan pretreated mice survival upon challenge with Staphylococcus aureus was enhanced.82 Similarly, glucan also promoted survival of leukemic mice with experimentally induced staphylococcal infections 83 and afforded non-specific protecC. albicans,85 Cryptococcus neofortion against Sporotrichum schenckiiY64mans and Mycobacterium leprae.86 Dependent on the timing, pretreatment of donor mice with glucan either enhanced or suppressed the response of cultured mouse spleen cells to Concanavalin A and to lipopolysaccharide.87 Glucan-activated peritoneal macrophages from pretreated donors showed increased cytostatic activity for anaplastic mammary carcinoma87 and RI leukemia cells.88 Accordingly , growth of several tumors (s.c. transplanted adenocarcinoma BW10232, anaplastic mammary carcinoma 15091A, melanoma B16 and AKR lymphocytic leukemia,89 as well as i.v. injected methylcholanthrene-induced fibrosarcoma88), was inhibited in glucan-treated mice .89 Interestingly, proliferation of melanoma B16 was suppressed by glucan treatment both of normal and athymic mice,87 indicating that a T-lymphocyte independent, presumably macrophage-mediated mechanism appeared to be operative in these hosts. Concurrent glucan and cyclophosphamide therapy had a significant synergistic therapeutic effect in experimental acute myelogenous Shay leukemia of Long Evans rats and in syngeneic lymphocytic leukemia of AKR mice, and led to a considerable reduction in hepatic metastases.90 Apparently, neither whole body irradiation nor cyclophosphamide treatment markedly interfered with the ability of glucan to render macrophages c y t o t o ~ i c . Glucan ~~ was also shown to exhibit considerable immunoadjuvanticity. Thus, concomitant administration of glucan and poorly immunogenic irradiated L1210 cells in syn eneic mice,92 or of glucan and glutaraldehyde-treated Shay cells in rats ,63 conferred resistance to subsequent tumor cell challenge. However, failure of glucan to act as an immune stimulator in antileukemic vaccination94 or therapy of syngeneic tumor transplantsg5 was also reported. Another group of immunostimulating glucans with anti-tumor activity is derived from mushrooms: Lentinan, isolated from Lentinus edodes (Berk.) Sing, believed to be a B-(l+3)-linked linear glucan, pachyman, from Poria cocos Wolf (Bukuryo), consisting of B-(l+3)-linked glucan chains with 0(1+6)-linked branches, pachymaran, derived from pachyman by Smith degradation, removing the branches and thus converting pachyman to a linear glucan, carboxymethyl- and hydroxyethylpachymaran and -pachyman derived from pachymaran and pachyman by carboxymethylation and hydroxyethylation, respectively.96 Treatment of mice with lentinan, pachymaran, carboxymethylpachymaran and hydroxyethylpachymaran B, but not with pachyman and hydroxyeth lpachyman A , caused host-mediated regression of transplanted sarcoma 180.96957 Also, lentinan was reported to inhibit the growth of transplanted methylcholanthrene-induced fibrosarcoma.88 However , the substances were ineffective in ALS-treated or neonatally thymectomized miceYg7398 ap arently in marked difference to what has been reported of yeast-glucan.!7 Again in con-

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t r a s t t o y e a s t polyglucose, l e n t i n a n f a i l e d t o e l i c i t macrophage c y t o t o x i city," although o t h e r s found i t a c t i v e i n t h i s respect.88 Moreover, l e n t i nan s t i m u l a t e d T-helper c e l l a c t i v i t y l o 0 and r e s t o r e d suppressed T-lymphoc y t e h e l p i n tumor-bearing mice. Lentinan , pachyman , pachymaran , carboxymethylpachymaran and hydroxyethylpachyman A a l l s t r o n g l y augmented t h e formation of a l l o r e a c t i v e murine c y t o t o x i c T-lymphocytes -i n vivo. lo2In v i t r o , l e n t i n a n , pachyman, pachymaran and carboxymethylpachymaran were found t o enhance t h e generation of such e f f e c t o r c e l l s . l o 3 C l e a r l y , t h e r e was no c o r r e l a t i o n with t h e antitumor e f f e c t s i n vivo, where pachyman and hydroxyethylpachyman had been r e p o r t e d t o be i n a ~ t i v e . ' ,97 ~ Also, t h e demo n s t r a t e d c a p a c i t y of a c t i v a t i n g t h e a l t e r n a t i v e pathway of complement appeared not t o be c o r r e l a t e d with t h e tumor-inhibitory p r o p e r t i e s of t h e s e c o m p o ~ n d s . ~ I~t4 remains unclear , whether t h e seemingly d i f f e r e n t modes of a c t i o n of y e a s t and fungal qlucans r e f l e c t d i f f e r e n c e s i n supramolecular s t r u c t u r e o r t h e presence of h i t h e r t o undetected s t r u c t u r a l elements. Recently, a t t e n t i o n w a s drawn t o a r e a d i l y a c c e s s i b l e bacterialpo1y.glucose: A well-defined B- (1-+3) -glucan with (En) 540 from c u l t u r e d A l c a l i genes f a e c a l i s v a r . myxoqenes displayedsignificanthost-mediated i n h i b i t o r y e f f e c t s a g a i n s t v a r i o u s t r a n s p l a n t a b l e tumors. Hydrolytic degradation products (DPnb16) r e t a i n e d some a c t i v i t y . 105 S y n t h e t i c Polynucleotides - S y n t h e t i c double-stranded polyribonucleotide complexes (formed by polymerization ofmononucleotide-diphosphates b y s t r e p tomyces l y s o d e i k t i c u s polynucleotide phosphorylase and subsequentmixing of complementary s i n g l e - s t r a n d e d polynucleotide chains) s t i l l hold important promises f o r f u t u r e c l i n i c a l use. Current advancesin t h e b i o l o g i c a l charact e r i z a t i o n of poly A:U and poly I : C have been t h e s u b j e c t of comprehensive reviews. 9 lo7 Both polynucleotide complexes exert marked s t i m u l a t o r y e f f e c t s on cell-mediated and humoral immunity, and on non-specific h o s t res i s t a n c e t o i n f e c t i o n s and t o tumors. However, poly I : C i s more t o x i c , l o 8 and a l s o more e f f e c t i v e than poly A:U i n i n d u c i n g i n t e r f e r o n release i n v i t r o and -i n vivolo9 except i n man, where l i t t l e o r no c i r c u l a t i n g i n t e r f e r o n i s e l i c i t e d by e i t h e r one of t h e compounds. Recent evidence concurs t o i m p l i c a t e t h e T c e l l a s t h e main t a r g e t of poly A:U. The substance was r e p o r t e d t o a s s o c i a t e with membranes of c o r t i sone s e n s i t i v e ( c o r t i c a l ) mouse thymoc t e s l l o and t o induce t h e s e c r e t i o n D e f i c i e n t immune responses i n of a h e l p e r f a c t o r from T lymphocytes."l newborn and i n aged mice could be g r e a t l y improved bypo1yA:Uorby supern a t a n t from poly A:U t r e a t e d thymocytes.l12 S i m i l a r l y , as demonstrated i n c e l l t r a n s f e r experiments, induction of t o l e r a n c e t o a T-dependent a n t i g e n could be prevented by poly A:U treatment and abrogated by poly A:U induced thymocyte supernatant f l u i d . Depending on t h e r e l a t i v e time of poly A: U a p p l i c a t i o n , generation of h e l p e r o r of suppressor c e l l s was found t o be favored. When poly A : U w a s given t o g e t h e r with t h e immunizing a n t i g e n , enhanced antibody responses were observed,while suppressor c e l l s w e r e a c t i vated i f poly A:U was i n j e c t e d p r i o r t o a n t i g e n i c Moreover, i n t h e absence of a n t i g e n , poly A : U was r e p o r t e d t o promote non-specific while i n t h e presence polyclonal a c t i v a t i o n of c y t o t o x i c T-lymphocytes of a l l o g e n e i c c e l l s , a l l o a n t i g e n - s p e c i f i c T-cellmediated c y t o t o x i c i t y was g r e a t l y augmented by poly A:U. Amplification of mixed-lymphocyte c u l t u r e r e a c t i o n s by poly A:U depended on t h e presenceof adherent cells117 and was

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further enhanced by 2-mercaptoethanol.118 In addition, there is suggestive evidence to indicate that differentiation of both T- and B-lymphocyte prein _ vivo, cursors may be triggered by poly A:U in vitro.l19 Nevertheless, _ __ poly A:U appeared to increase the proportion of T-cell precursors.120 Cell-activation by poly A : U may not be restricted to T lymphocytes. Indeed, earlier observations had demonstrated thatbothpo1yA:Uand poly 1:C were mitogenic for B lymphocytes.121 Very immature B cells were subsequently identified as the susceptible population.122 It is also documented that poly A:U exerts a profound influence on hemopoietic stem-cell differin vitro.123~124 Furthermore, a recent study of entiation both -in vivo and -poly A:U in experimental murine brucellosis suggested that macrophages are directly or indirectly involved in its antibacterial effects.125 Yl26 The cellular targets of poly I:C, on the other hand, may not beidentical with those of poly A:U. Thus, poly I:C, butnotpoly A:U was found to significantly increase natural killer-cell (NK) activity in rat spleen, blood and peritoneal exudate.127,128 This effect was thought to be mediated by since interferon has been shown to enhance NK activity by itself130 while NK activation by poly I:C could be blocked by anti-interferon antibody.127 The potential clinical usefulness of polynucleotides may have been augmented by measures aimed at prolongationof activityand reductionoftoxin-vivo was achieved icity. Increased resistance of poly I:C to hydrolysis _ by adsorption to poly-L-lysine and carboxymethylcellulose (poly ICLC) 131 In contrast to poly I:C, poly ICLC proved to be an effective interferoninducer both in primates131 and in man.lo6 The doses required for this effect were no longer associated with prohibitive side effects.lo6 Poly ICLC also displayed increased adjuvanticity, as demonstrated by potentiation of antibody responses to Venezuelan equine encephalomyelitis virus vaccine132 and to swine influenza virus vaccine133 in rhesus monkeys, and by enhanced immunogenicity of phase I antigen of Coxiella burnetii in guinea pigs. 134 Similarly, inclusion of poly I:C in liposomespotentiated interferon induction and lymphocyte activation,while reducing toxicity.135 Preliminary clinical trials of poly A:U136 and poly I:~137in cancer patients suggested absence of major side effects. A l s o , poly I:C appeared to have restored immune reactivity of previously unresponsive patientsas assessed by in vitro lymphocyte transformationtests. A large controlled clinical trial of p o l y A:U as an adjunct to surgery in patients withmammary carcinoma is still in progress. 138

.

Coenzyme Q - A recent review on biomedical aspects of coenzyme Q (55) has appeared. Earlier work had e s t a b l i s h e d t h a t u b i q u i n o n e s a n d b a c t e r i a l phospholipid extracts of various sources enhance phagocytosis,140 stimulate non-specific host-resistance141I 142 and exert marked adjuvant effects on humoral immune responses to T-dependentand T-independent CH~O (CH2-CH=C-CH2+,H antigens.143 Recently, coenzyme Q8 (n=8) H3 was isolated from E. coli BM 1135.144 At 0 25 mg/kg i.v. 24h before challenge,Q8protected mice against i.v. challenge with 55 S. pyoqenes, E. insidiosa, E. e 0 1 and -

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S. typhimurium. Q4, Q6 and QlO were 5 to 10 times less effective. While Q9 appearstobethe preponderantcoenzyme in mice, in man it is Q l O which was the subject of the earlier research. ,141Q8 was found to enhance the phagocytic activity of macrophages and to maintain their activated state 144 ,145 Q10 was shown to reverse the deficient primary humoral immune response in old mice Consideration of properties of antimetabolites of coenzyme Q in certain animal tumor models are beyond the scope of this review.147,148

.

Amphotericin B - Observations on the immunostimulant properties of the clinically used antifungal polyene antibiotic amphotericin B (AmE!) and its methylester ( M E ) were extended. In mice, thedrugs augmented antibody formation to TNP-HSA with pronounced enhancement of secondary IgG responses, and promoted contact sensitivity to 2,4-dinitrofluorobenzene. 149 y 1 5 0 The immunostimulant effects of AmB and AME were said to be related to membrane sterol binding. Efficacy appeared to depend on both genetic and dietary factors that regulate cholesterol metabolism.I5lThe synergistic action with 1,3-bis(2-chloroethyl)-l-nitrosourea in thetherapy of a transplantable AJ.ZR leukemia was thought to be due to a host-mediated immunologic effect of 152 AmB. Protease Inhibitors - Bestatin (56), an aminopeptidase inhibitor isolated from Streptomyces olivoreticuli has been reported to stimulatecellular and humoral immunity against sheep red blood cells in mice153. Blastogenesis of peri heral lymphocytes from guinea pigs by concanavalin A and PHA was enhanced .H54 Moreover , bestatin appeared to increase the effects of bleomycin and adriamycin in experimental systems.156 Preliminary clinical studies in tumor patients have been carried out Another closely related inhibitor, amastatin increased thenumber ofantibody forming cells against sheep erythrocytes as antigen in mice158.

(s),

(R)(s) OH

(S)

I

CH2-CH-CH-CONH-CH-COH I

NH2

56 -

CH2 I CH I \ CH3 CH3

(R)(S) OH H3C, I CH-CH2-CH-CH-CO-L-Val-L-Val-L-AspOH I H3d NH2

57 -

Lysolecithin Analogs - Theimmunomodulating effectsof avariety of synthetic lysolecithin analogs (58) have been reviewed.159

I 0-

58 -

156

Sect. 111 - Chemotherapeutic Agents

Weisbach, Ed.

Lysolecithin derivatives were found to increase humoral immune responses in mice to bovine serum albumin and to sheep red blood cells.159 Compound 58a was most effective. The substance was also shown to suppress the growth of Ehrlich ascites carcinoma, Lewis lung carcinoma and methylcholanthrene-induced fibrosarcoma Recently , other synthetic lysolecithin analogs were reported to have a marked inhibitory effect on syngeneic transplanted ascites tumorsincluding Meth A, Ehrlich carcinomaand sarcoma 1805. The tumor suppressive effects depended on the substituents R' and R". Compounds 58b and 58c were more effective than derivatives and The generationof cytotoxic macrophages seems to play a major rolein the anti-tumor effects of lysolecithins. Macrophages collected from the peritoneal cavity of mice treated with lysolecithin derivatives killedsyngeneic tumorcells with high efficiency It was suggested that this mechanism might be amplified by a direct cytotoxic effect of alkyl-lysophosphatides, since some of the compounds (58b, 58c) selectively destroyed human leukemic cells in vitro Levamisole and Related Compounds - Recent progress in the immunopharmacological and clinical characterization of the immunoregulatory anthelmintic levamisole (59) has been reviewed.162 Levamisole has been shown to restore compromised responses of polymorphonuclear leucocytes, monocytes, macrophages, and T lymphoc tes in a variety of systems both in vitro and, more Thus , monocyte and neutrophil motility and chemoimportantly,in vivo intrataxis ,164-167 phagocytosis,162 9 1689169 lysosomal enzyme release cellular killingl62,170 and macrophage proliferation162 ,171 could be repaired, enhanced or modulated. T-lymphocyte proliferation 162 9173 formation of E-rosettes Y 174-176 in vitro manifestations of cell-mediated immunity177 I 178 and T-dependent antibody plaque formation179 were reported to be restored. Moreover, in vivo, though notin vitro, levamisolewas found to promote differentiation of pre-T cells to mature lymphocytes, thus mimicking the effect of thymic hormonesl62. Levamisole treatment of congenitally athymic mice induced the expression of T-cell membrane markers on spleen cells179 and inhibited autologous rosette formation.180 Restoration of genetically defective T-helper and suppressor cell function by levamisole administration in vivo was demonstrated by reappearance of responsiveness to sheep red blood cells in athymic animals,l79 and suggested by suppression of auto-immune disease in NZB and NZB/NZW mice,181-184 and of Aleutian mink disease 185 Clinically, cell-mediated immunity was reported to be reconstituted by levamisole therapy in children with primary immune defects. 186 Sera from levamisole-treated mice, rabbits andmanwere foundto yield a dialyzable,complement-unrelated serum factor which did not contain any levamisole metabolites, but which could simulate the biologic effects of levamisole itself 162 This factor enhanced carbon clearance stimulated lymphocyte proliferation in vitro,l87 restored immunological reactivity of athymic mice.179 induced -in vitro maturation of mouse pre-T cells,179 stabilized tumor remission and reduced tumor load in experimental systems.162 The factor could not be detected in mice which failed to respond to treatment with levamisole; but non-responders reacted toserum factorin a similar way as responders treated with levamisole itself.162 A perhaps identical serum factor could also be induced by the sulfhydryl compound diethyldithiocarbamate (65) 179 which appears to share some of the immunoenhancing and immunorestorative properties of levamisole if administered in

.

.

.

,

Chap. 15

Immunostimulants Dukor, Tarcsay, Baschang

59 -

60 -

157

61 -

c1

a::3 CH20H I

HSCH2

62 -

.

63 -

vivo 179 Interestingly, 2-mercaptoethanol which is known to enhance the viability of cultured lymphocytes189 to auqment their reactivity to antigenic stimulation in the absence of macrophages ,190 and to induce polyclonal lymphocyte activation ,191,1g2 exerts its activity through a component in fetal bovine serum which is activated by 2-mercaptoethanol and which can be used in place of macrophages or of serum tosupplement otherwise macrophage- and serum-dependent lymphocyte cultures.193 It is tempting to speculate that a similar mechanism is responsible for the activity of levamisole in vivo. Indeed, levamisole is now thought tobe cleavedin vivo to yield dl-20x0-3- (2-mercaptoethyl)-5-phenylimidazoline (60) , 162whichwas reported to be more potent than the parent compound in enhancing carbon clearance in micelq4. Recent evidence concurs to indicate that both levamisole and thymic factors (Trainin’sthymic humoral factor and G. Goldstein’s thymopoietin) have similar effects on CAMP and cGMP in lymphomyeloid target cells: increase of CAMP in precursor populations, increase of cGMP in mature effector cells .I6*,174,195 In view of the apparent similarities of the invivo and some of the in _ -vitro effects of levamisole and thymic hormones it was proposed that levamisole might (through the intermediate of a metabolite) activate a serum factor which would in turn combine with a cell membrane receptor for thymic hormones. 16 2 The current status of ongoing immunotherapeutictrials withlevamisole in immune deficiency disorders,recurrent or chronicinfections, chronic inflammatory diseases and cancer has been reviewed comprehensively.162 Recent trials indicate that levamisole provides significant therapeutic benefits in rheumatoid arthritis,196-199

158

Sect. 111

-

Chemotherapeutic Agents

Weisbach, Ed.

A levamisole analog containing Se instead of S (Se-levamisole, 61) produced a lesser immunopotentiating effect than the parent compound in a murine skin graft model (C3H donors, B6AF1 recipients). However, in a system involving a y-locus difference (B6AF1 male + B6AF1 female), 9 accelerated rejection, while levamisole was found to suppress it.200 A related compound, 3-(p-chlorophenyl)-2,3-dihydro-3-hydroxythiazole in vitro anti[3,2-u]-benzimidazole-2-acetic acid (Wy-13876) (63) increased -body formation of mouse spleen cells sensitized with sheep red blood cells (SRBC) as measured by a direct plaque assay. The compound did not have a mitogenic effect.201 It was reported that i.p. administration of 63 (50150 mg/kg) enhanced the primary and secondary anti-SRBC response after optimal immunization in mice and raised the graft-versus-host-inducing capaci'ty of splenocytes; no influence on the response to type I11 pneumococcal polysaccharide (a T-cell independent antigen) nor on macrophage cytotoxicity toward SL2 and TLX9 lymphomas could be observed. However, the drug inincreased survival of L1210Ha leukemia-bearing mice, when administered in conjunction with irradiated tumor cells. At 150 mg/kg i.p. one day after tumor transplantation compound 63 exerted an antimetastatic effect in a Lewis lung carcinoma model.202 5-Mercaptopyridoxine (62) was reported to enhance the intensity of the secondary reaction in adjuvant arthritic rats and to augment pertussis vaccine edema. Also, it stimulated lymphocyte responses to Concanavalin A. 203 Another sulfhydryl compound, a-mercaptopropionylglycine (64) , enhanced antibody formation to SRBC in mice when given for five days before immunization. T- and B-cell mitogen reactivity of lymphoid cells from EL4 leukemia-bearing mice was restored and production of cytotoxic antibody to EL4 increased by the administration of 3.204 Other Synthetic Immunostimulants 2-Cyanaziridines - 4-Imino-1,3-diazabicyclo[3.l.O]hexan-2-one (E),(BM 06002; Imexon) was reported to inhibit tumor rowth in various tumor models in the mouse and rat. 50 5 and to increase the resistance of mice to chronic infection with Candida albicans; when combined with a subtherapeutic dose of sulfadiazine. comuound 66 was found t o increase resistance of mice a'gainst' Staphylococcus aureus infections.206 Preliminary 0 clinical trials suggested that 66 stimulates delayed cutaneous hypersensitivity reactions and lymphocyte blastogenic response in tumor patients .207~208


2-[2-cyan-aziridinyl-(l)]-2-[2-carbamoylaziridin-

CH3

67

yl-(1)-propane] (67) , (BM 12531; azimexon) also showed therapeuticeffects in tumor models and experimental infections in mice.209,210 Immunostimulant effects of compound 5 were demonstrated by various parameters, including augmented delayed type hypersensitivity reactions in mice, enhanced human T-lymphocyte transformation in vitro, and increased phagocytosis of latex particles by mouse peritoneal cells.211-213

Chap. 15

Immunostimulants Dukor, Tarcsay, Baschang

159

N-(2-Carboxyphenyl)-4-chloroanthranilicAcidDisodium(CCA) Compound 68) was reported to suppress adjuvant arthritis in rats without anti-inflammatory and immunosuppressive activities.2 14 Surprisingly, oral administration of CCA augmented the number of plaque-forming cells (PFC) in the mouse spleen against both a thymus-dependent (sheep red blood cells) and athymus-independentantigen (bacterial lipopolysaccharide).215 It was COO-Na also reported that CCA prevented development of autoimmune kidney disease in NZB/NZW F1 hybrid mice. CCA treatment inhibited antibody produc68 tion to double-stranded native DNA and led to decreased amounts of IgG deposited in glomeruli; the Con A responsiveness of thymocytes from CCA-treated donors was foundtobe increased. It wassuggested that prevention of autoimmune kidney disease by CCA might be due to activation of suppressor T-cells. 216

d*+

The synthetic alkyldiamine CP20961 ( B ) , w h i c h h a d b e e n i n v e s t i g a t e d e a r l i e r with respect to its interferon-inducing properties,217 ,218 was reported to substitute for mycobacteria in CFA in the induction of adjuvant arthritis and the development of cell-mediated andhumoral immune responses to heterologous lymphoma cells in rats.219

c18H37

\

c18H37’

CH2-CH2-OH

/

N-CH2-CH2-CH2-N

\

CH2-CH2-OH

Conclusions - Due t o the rapidly expanding interest in immunostimulants, there has beena dramatic proliferation of recent papers on immunopharmacology and of molecular entities which have been identified as modulators of the immune response. For obvious reasons, the choice of agents discussed on the foregoing pages had to be arbitrary. Thus, very important classes, such as syntheticpolymers with immunostimulating activity, endogenous hormones and m e d i a t o r s , i n c l u d i n g i n t e r f e r o n andsmall molecular weight synthetic interferoninducers,have notbeen dealt with in this chapter. A particularly important omission is the intriguing topic of the immunotherapeutic potential of established drugs with a presently defined spectrum of non-immunologic indications. Indeed, inhibitors of prostaglandin synthesis, histamine- and catecholamine-receptor antagonists, as well as some regulators of ion transport may yet reveal their immunoregulatory properties.

160 -

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Sect. 111 - Chemotherapeutic Agents

R. G. Webster

W.P. Glezen, C. Hannoun, and W.G.

1 1 9 , 2073 ( 1 9 j 7 ) .

Weisbach, Ed.

Laver, J . Immunol.

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93 (1976). J . Hamuro, M. Rdllinghoff, and H. Wagner, Cancer Res. 38, 3080 (1978). J. Hamuro, H. Wagner, and M. Rdllinghoff , Cell. Immunoc 38, 328 (1978). J. Hamuro, U. Hadding, and D. Bitter-Suemann, Immunology 695 (1978) T. Sasaki, N. Abiko, Y. Sugino, and K. Nitta, Cancer Res. 38, 379 (.1978). A.G. Johnson in "Seminars in Immunopathology", Vol. 3, P. Miescher and L. Chedid eds., 1979, in press. A.G. Johnson in "Innnune RNA", E.P. Cohen ed., CRC Press, Cleveland 1976, p. 17. M.R. Hilleman, J. Infec. Dis. 196 (1970).

34,

.

121,

A.K. Field, A.A. Tytell, G.P. Lampson, and M.R. Hilleman, Proc. Natl. Acad. Sci. 58, 1004 (1967). 110. M.G. Mutchnzk, I.H. Han, and A.G. Johnson, Proc. Soc. Exp. Biol. Med. 154, 264 (1977).

111. 112. 113.

P.H. Bick and A.G. Johnson, Scand. J. Immunol. 6, 1133 (1977). I.H. Han and A.G. Johnson, J. Innnunol. 117,423-(1976). S.L. Fessia and Y.M. Kong, Scand. J. Immunol. 6, 1209 (1977).

Sect. I11

164 114.

-

Chemotherapeutic Agents

Weisbach, Ed.

S.L. Fessia, Y.M. Kong, P.H. Bick, and A.G. Johnson, Eur. J. Immunol.

7, 508 (1977). 115. 116. 117.

C.K. Morris and A.G. Johnson, Cell. Immunol. 2, 345 (1978). P.H. Bick and G. Mbller, J. Exp. Med. 146, 844 (1977). P.R. Narayanan, D.B. Kloehn, and G. Suxaradas, J. Immunol.

121,

2502 (1978).

118. 119.

K.D. Graziano and M.R. Mardiney, Jr., Transplantation 21, 317 (1976). M.P. Scheid, G. Goldstein, and E. A . Boyse, J. Exp. Med, 147, 1727

120.

M. Donner, P. Bischoff, and F. Lacour, Develop. Biol. Stand.

(1978).

38, 159

(1977).

121.

H. Fuhl, W. Vogt, G. Bochert, and T. Diamantstein, Immunology 26,

122.

M.G. Goodman, J.M. Fidler, and W.O. Weigle, J. Immunol.

937 (1974). (1978)

123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134.

.

121,1905

T.A. McNeill, Immunology 21, 7 4 1 (1971). T.A. McNeill and M. Killen, Immunology 21, 7 5 1 (1971). E.D. Madraso and C. Cheers, Immunology 35, 69 (1978). E.D. Madraso and C. Cheers, Immunology 35, 77 (1978). J.R. Oehler and R.B. Herberman, Int. J. Cancer 21, 2 2 1 (1978). J.R. Oehler, L.R. Lindsay, M.E. Nunn, H.T. Holden, and R.B. Herberman, Int. J. Cancer 21, 210 (1978). J.Y. Djeu, J.A.Heinbaugh, H.T. Holden, and R.B. Herberman, J. Immunol. 122, 182 (1979). G. Trinchieri and D. Santoli, J . Exp. Med. 147, 1314 (1978). H.B. Levy, G. Baer, S. Baron, C.E. Buckler, C.J. Gibbs, M.J. Iadorola, W.T. London, and J. Rice, J. Infec. Dis. 132, 434 (1975). W.E. Houston, C.L. Crabbs, E.L. Stephen, and H.B. Levy, Infec. Immunity 14, 318 (1976). E.L. Stepzn, D.E. Hilmas, J.A. Mangiafico, and H.B. Levy, Science

197,

1289 (1977).

R.F. Wachter, G.P. Briggs, and C.E. Pedersen, Jr., Infec. Immunity

22, 627 (1978). 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147.

W.E. Magee, Ann. N.Y. Acad. Sci. 308, 308 (1978). H.J. Wanebo, M. Kemeny, C.M. P i n s 6 Y. Hirshaut, and H.F. Oettgen, Ann. N.Y. Acad. Sci. 277, 288 (1976). M.C. Simmler, M. BruleyZkosset, D. Belpomme, and L. Schwarzenberg, Eur. J. Cancer 13,463 (1977). J. Lacour, F. Lacour, A. Spira, G. Delage, and A.M. Michelson, Bull. Cancer (Paris) 61, 275 (1974). "Biomedical andylinical Aspects of Coenzyme Q", K. Folkers and Y. Yamamura eds., Elsevier,Amsterdam 1977. A.C. Casey and E.G. Bliznakov, Chem. Biol. Interact. 2, 1 (1972). J.H. Heller, Persp. Biol. Med. 16, 1 8 1 (1973). R.M. Fauve and B. Hevin, Proc. Natl. Acad. Sci. 71, 573 (1974). K. Sugimura, I. Azuma, Y. Yamamura, I. Imada, anFH. Morimoto, Int. J. Vitam. Nutr. Res. 46, 464 (1976). L.H. Block, A. Georgopoulos, P. Mayer, and J. Drews, J. Exp. Med.

148,

1228 (1978).

D. Biemesderfer, L.H. Block, P. Male, and M. Kashgarian, Scanning Electron Microscopy 11, 333 (1978). E.G. Bliznakov, Mech. Aging Develop. 7, 189 (1978). T.H. Porter, C.S. Tsai, T. Kishi, H. Kishi, and K. Folkers, Acta Pharm. Suec. 15,97 (1978).

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165

148. K. Folkers, T.H. Porter, J.R. Bertino, and B. Moroson, Res. Comm. Chem. Pathol. Pharmacol. 19,485 (1978). 149. T.J. Blanke, J.R. Little, S.F. Shirley, and R.G. Lynch, Cell. Immunol. 33, 180 (1977). 150. S.H. S t e i G E.J. Plut, T.E. Shine, and J.R. Little, Cell. Immunol. 40, 211 (1978). 151. J.R. Little, E.J. Plut, J. Kotler-Brajtburg, G. Medoff, and G.S. Kobayashi, Innnunochemistry 3, 219 (1978). 152. G. Medoff, F. Valeriote, J . Ryan, and S. Tolen, J. Natl. Cancer Inst. 58, 949 (1977). 153. H. Umezawa, M. Ishizuka, T. Aoyagi, and T. Takeuchi, J. Antibiotics 29, 857 (1976). 154. M. Saito, T. Aoyagi, H. Umezawa, and Y. Nagai, Biochem. Biophys. Res. 526 (1977). Commun. 3, 155. M. Saito, K. Takegoshi, T. Aoyagi, H. Umezawa, and Y. Nagai, Cell. Imunol. 40, 247 (1978). 156. H. Umezawa, Biomedicine 26, 236 (1977). S138 (1977). 157. H. Umezawa, Jap. J. Antibiotics g, 158. T. Aoyagi, H. Tobe, F. Kojima, M. Hamada, T. Takeuchi, and H. Umezawa, J. Antibiotics 31, 636 (1978). 159. P.G. Munder, H . C Weltzien, and M. Modolell in "Immunopathology", VIIth Int. Symposium, Bad Schachen, P.A. Miescher ed., Schwabe, Base1 1976, pp. 411-424. 160. G.S. Tarnowski, I.M. Mountain, C.C. Stock, P.G. Munder, H.U. Weltzien, and 0 . Westphal, Cancer Res. 38, 339 (1978). 161. R. Andreesen, M. Modolell, H . C Weltzien, H. Eibl, H.H. Common, G.W. LtJhr, and P.G. Munder, Cancer Res. 38, 3894 (1978). 162. J. Symoens, M. Rosenthal, M. De Brabander, and G. Goldstein in 3, P. Miescher and L. Chedid eds., "Seminars in Immunopathology", Vol. 1979, in press. 163. J. Symoens and M. Rosenthal, J. Reticuloendothel. SOC. 2, 175 (1977). 164. S.D. Nathanson, P.L. Zamfirescu, J.K. Portaro, J.B. deKernion, and J.L. Fahey, J. Natl. Cancer Inst. 61, 301 (1978). 165. M.T. Kelly, J. Reticuloendothel. S G . 24, 139 (1978). 166. A.G. Mowat, Ann. Rheum, Dis. 37, 1 (19%). 167. A.R. Rabson, R. Anderson, and A. Glover, Clin. Exp. Immunol. 33, 1 4 2 (1978) . 168. L. Molin and 0. Stendahl. Scand. J. Haematol. 19,93 (1977). 169. R.F. Van Ginckel and J. Hoebeke, J. Reticuloendothel. SOC. 17,65 (1975). 170. J.A. Child, S. Martin, J.C. Cawley, and A.T.M. Ghoneim, Biomedicine 29, 159 (1978). 171. M . Pelletier, D.A. Willoughby, and J.P. Giroud, 3 . Reticuloendothel. Soc. 2, 333 (1978). 172. W.C. Gordon and M.D. Prager, J. Natl. Cancer Inst. 61, 415 (1978). 173. R.H. Neubauer and H. Rabin, Clin. Exp. Immunol. 33, 65 (1978). 174. R. Michalevicz, A. Many, B. Ramot, and N. Trainin, Clin. Exp. Immunol. 31, 111 (1978). 175. Lomnitzer and A.R. Rabson, Clin. Exp. Innnunol. 33, 499 (1978). 176. W. De Cock, J. De Cree, and H. Verhaegen, Immunology 35, 223 (1978). 177. M.M. Urist, A.W. Boddie, Jr., C.M. Townsend, Jr., and E.C. Holmes, J. Thorac. Cardiovasc. Surg. 73, 189 (1977). 178. R. Windle, R.F.M. Wood, and P.R.F. Bell, Br. J. Surg. 65, 355 (1978). 179. G. Renoux and M. Renoux, J. Exp. Med. 145,466 (1977).

<

166 180. 181. 182. 183.

Sect. I11

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Chemotherapeutic Agents

Weisbach, Ed.

J. Van Wauwe and G. Van Nijen, Clin. Exp. Immunol. 3 0 , 465 ( 1 9 7 7 ) . A.S. Russel, J. Rheumatol. 3, 380 ( 1 9 7 6 ) . L.W. Klassen, D.R. Budman, D.R. Williams, G.W. Williams, A.D. Stein787 ( 1 9 7 7 ) . berg, and N.L. Gerber, Science 617 K. Hashimoto, Y . Kazuta, T. Miki, and A. Horiuchi, Arerugi 2,

=,

(1978). 184. 185. 186.

187. 188.

J. Zulman, J. Michalski, C. McCombs, J. Greenspan, and N. Talal, Clin. Exp. Imunol. 31, 3 2 1 ( 1 9 7 8 ) . A.J. Kenyon, Fed. Proc. 37, 334 ( 1 9 7 8 ) . C. Griscelli, A.M. Prieur, and F. DaGuillard in "Immune Modulation and Control of Neoplasia by Adjuvant Therapy", M.A. Chirigos ed., Raven Press, New York 1 9 7 8 , p. 165. L. Ivanyi and T. Lehner, Scand. J. Immunol. 6 , 219 ( 1 9 7 7 ) . G. Renoux and M. Renoux in "Control of Neoplgsia by Modulation of the Imune System", M.A. Chirigos ed., Raven Press, New York 1 9 7 7 , p. 1 6 7 .

189. 190. 191. 192.

R.E. Click, L. Benck, and B.J. Alter, Cell. Immunol. 3 , 156 ( 1 9 7 2 ) . C. Chen and J.G. Hirsch, J. Exp. Med. 136,604 (1972): M.G. Goodman and W.O. Weigle, J. Exp. Med. 145,4 7 3 ( 1 9 7 7 ) . T. Igarashi, M. Okada, T. Kishimoto, and Y. Yamamura, J. Immunol.

118, 1 6 9 7 ( 1 9 7 7 ) . 193.

194. 195. 196.

H.G. Opitz, U. Opitz, H. Lemke, G. Hewlett, Id. Schreml, and H.D. Flad, J. Exp. Med. 1 4 5 , 1 0 2 9 ( 1 9 7 7 ) . R. Van GinckeEnd M. De Brabander, J. Reticuloendothel. SOC., in press. G.H. Sunshine, R.S. Basch, R.G. Coffey, K.W. Cohen, G. Goldstein, and J.W. Hadden, J. Imunol. 1 2 0 , 1 5 9 4 (1978). L.A. Runge, R.S. Pinals, S.H.%urie, and R.H. Tomar, Arth. Rheum.

20, 1445 (1977). 197.

J. Scott, P.A. Dieppe, and E.C. Huskisson, Ann. Rheum. Dis.

37, 259

(1978).

198. 199. 200. 201. 202.

Multicentre Study Group, Lancet g , 1 0 0 7 ( 1 9 7 8 ) . Multicentre Study Group, J. Rheumatol. 5, Suppl. 4 , 5 ( 1 9 7 8 ) . R.N. Hanson, R.W. Giese, J.J. GOZZO, and M.A. Davis, Abstracts of the 175th Amer. Chem. SOC. Meeting, Anaheim, March 1 9 7 8 . G.H. Warren, L. Mills, and H. Friedman, Experientia 34, 802 ( 1 9 7 8 ) . A. Tagliabue, G. Allesandri, N. Polentarutti, A. Mantovani, E. Falantano, A. Vecchi, S. Garattini, and F. Spreafico, Eur. J. Cancer 14, 393 ( 1 9 7 8 ) .

203.

209.

E. Arrigoni-Martelli, E. Bramm, and L. Binderup, Drugs Exptl. Clin. Res. 3, 1 9 ( 1 9 7 7 ) . H. Mori, I. Saiki, H. Nagai, and A. Koda, Jap. J. Pharmacol. 27, Suppl. 66P ( 1 9 7 7 ) . U. Bicker and G. Hebold, IRCS Med. Sci. 5 , 4 2 8 ( 1 9 7 7 ) . A.E. Ziegler, U. Bicker, and G. Hebold, Exp. Path. 3, 321 (1977). M. Micksche, E.M. Kokoschka, P. Sagaster, and U. Bicker, IRCS Med. Sci. 5, 1 9 2 ( 1 9 7 7 ) . M. Micksche, E.M. Kokoschka, 0. Kokron, P. Sagaster, and U. Bicker, Oesterr. Z. Onkologie 4 , 29 ( 1 9 7 7 ) . U. Bicker, G. Hebold, A.E. Ziegler, and W. Maus, Exp. Path. _ 15, _ 49

210. 211.

(1978). U. Bicker, A.E. Ziegler, and G . Hebold, IRCS Med. Sci. 5, 377 ( 1 9 7 8 ) . U. Bicker, G. Hebold, and W. Maus, IRCS Med. Sci. 2, 399 ( 1 9 7 7 ) .

204. 205. 206. 207. 208.

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Immunostimulants Dukor, Tarcsay, Baschang

167

212. U. Bicker, G. Hebold, A . E . Ziegler, and V. Haas, IRCS Med. Sci. 6, 455 (1978). 213. B. Bonfert, U. Bicker, G . Hebold, and W. Maus, IRCS Med. Sci. 2, 523 (1977). 214. Y. Ohsugi, S.I. Hata, M. Tanemura, T. Nakano, T. Matsuno, Y. Takagaki, Y. Nishii, and M. Shindo, J . Pharm. Pharmacol. 2, 636 (1977). 215. Y. Ohsugi, T. Nakano, S.I. Hata, T. Matsuno, Y. Nishii, and Y. Takagaki, Chem. Pharm. Bull. Tokyo 25, 2143 (1977). 216. Y. Ohsugi, T. Nakano, S.I. Hata, R. Niki, T. Matsuno, Y. Nishii, and Y. Takagaki, J . Pharm. Pharmacol. 30, 126 (1978). 217. W.W. Hoffman, J . J . Korst, J . F . Niblack, and T. H. Cronin, Antimicrob. Ag. Chemother. 3, 498 (1973). 218. R.G. Douglas, RTF. Betts, R.L. Simons, P.W. Hogan, and F.K. Roth, Antimicrob. Ag. Chemother. S , 684 (1975). 21, 169 (1978). 219. Y.-H. Chang and C.M. Pearson, Arth. Rheum. -

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Metabolic Diseases and Endocrine Function

Editor: Denis M. Bailey, Sterling-Winthrop Research Institute Rensselaer, New York 12144 Chapter 16. Chemical Control of Fertility Malcolm R. Bell, Robert G. Christiansen and H. Philip Schane, Jr. Sterling-Winthrop Research Institute, Rensselaer, New York 12144 Introduction - The results of a survey1 of married women aged 15-44 for the 1973-1976 period indicated the following major contraceptive choices: "pill" ( 2 2 % ) , sterilization (19%, female and male), condom (7%), intrauterine device (IUD) (6%), rhythm (3%), foam ( 3 % ) , diaphragm (3%). Major trends are the increasing use of sterilization and a somewhat decreasing reliance on the "pill". Approximately 600,000 male and 600,000 female sterilizations are performed annually in the U. S. With compliance, the "pill" is an excellent method of family planning with a failure rate of 1 pregnancy or less per 100 women-years. A beneficial effect to undernourished women is a decrease in menstrual blood loss. Compliance can be a problem and missed medications can result in pregnancy. Although the IUD is not as effective as the "pill", compliance is not a problem if the IUD is properly inserted and remains in place. Associated with the use of the unmedicated IUD is a significant increase in menstrual blood The incorporation of Cu or a progestin in these devices has increased their effectiveness.4 , 5 Menstrual blood loss seen with the unmedicated IUD is reduced with Progestasert@, an IUD whose effectiveness approaches that of the Elective abortion as a method of family planning is increasing as a result of the availability of safe and legal abortion services.6 There is one elective abortion in the U.S. (1.3 million in 1977) for every three live births. Since the introduction of the "pill" in 1960, the major advances from a medicinal chemical point of view have been the introduction of prostaglandins (PG's) as abortifacients and the medicated IUD's. Neither has yet had the impact of the "pill" but progress has been reported in the development of PG's and IUD's with improved properties. Most of the recent research with sex hormones deals with previously reported compounds. An important finding in the area of luteinizing-hormone-releasing hormone (LH-RH) analogs is that both agonists and antagonists are potential antifertility agents. There are no reports evaluating this class of drugs as human antifertility agents. The immunological approach to fertilit trol has been re~iewed.~A review of many approaches is available. con-

8

Steroids - Two recent re~iews~,~'summarize the adverse reactions from use of the "pill". The "paper pill", a square of edible cellulose impregnated with the steroids, is advantageous with respect to packaging, transportation, acceptability and storage. The conventional and the "paper pill", each containing 1 5 0 pg of levonorgestrel and 30 pg of ethynylestradiol (EE), were equally effective in suppressing ovu1ation.l' The EE content of the "pill" was lowered to 30 ug without loss of contraceptive Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form resaNed. ISBN 0-12-040514-8

Chap. 16

Control of Fertility Bell, Christiansen, Schane

169

efficacy.12 In a modified sequential oral contraceptive dosage schedule, 50 pg of EE administered for 7 days followed by 50 pg of EE and 1 mg of lynestrenol for 15 days resulted in complete inhibition of 0vu1ation.l~ A useful alternative to the pill especially in women in whom estrogen is contraindicated, is Depo-Provera' (medroxypfogesterone acetatpi which when given i.m. every 3 months is an effective contraceptive. This progestin in large continuous doses induced an increased incidence of mammary cancer in beagle dogs, an effect not reported in other animals or women.15 The drug is not available in the U.S.16 Danazol (1) has been reported to be an oral contraceptive in the rhesus monkeyl7z8 and in women.19 In rats, this substance was a pituC E C H itary gonadotropin inhibitor which neither exhib3 ited estrogenic nor progestational activity but did have weak impeded androgen-like activity.2o Administration of estrogens, either 5 mg of EE or 50 mg of diethylstilbestrol p.0. for 5 days beginning within 3 days of coitus in midcycle, is effective in preventing pregnancy in women. Nausea and vomiting are common side effects. Diethylstilbestrol caused an increased incidence of vaginal adenosis and adenocarcinoma of the vagina and cervix in female offspring of women treated during the first trimester with this drug for other reasons.21 In China, the estrogen anordrin (2) is taken immediately after coitus for prevention o f pregnancy.22 When EE and dl-norgestrel were administered in combination to 608 women shortly after coitus, one pregnancy occurred nu nu OCOC,H,

J

U -

I -

rather than the 12-30 pregnancies expected.23 The antiprogestin R-2323 (norgestrienone, 2) had low contraceptive efficacy in w 0 m e n . ~ 4 , ~It ~ was suggested that the antifertility effects of RMI 12,936 (5) resulted from its antiprogestational activity as well as inhibition of progesterone (P) biosynthesis.26 The abortifacient activity of the cyanohydrin diacetate 5 in the rat may be the result of its transformation to estrogens in the ovary.27 ORF 9326 (6) terminated pregnancy in rats when administered P.O. on days 9-12 of gestation.28 At the effective dose, the compound lacked estrogenic, androgenic, and progestational activities. The spiroether 7

170

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Bailey, Ed.

was an estrogen antagonist in mice and ewes but was not estrogenic in rats. When administered to normally cycling rhesus monkeys, it increased the viscosity of the cervical mucus, a possible antiestrogenic effect, and prevented pregnancy.29

-

Steroidogenesis Inhibitors Aminoglutethimide (8), which blocks the conversion of cholesterol to P, terminated pregnancy in rats3’ but not in baboons.31 The 5,lO-seco steroids 9 irreversibly blockedA5-3-ketoisotestosteroni in vitro and caused a decrease in the weight merase from of male sex accessory organs of the rat, an effect which could be the result of inhibition of androgen biosynthesis.3L Azastene (10)was a competitive inhibitor of P biosynthesis in vitro and disrupted pregnancy in both the rat33 and rhesus monkey.34 This agent blocked human chorionic gonadotropin-induced prolongation of corpus luteum function and lacked hormonal activity.35 Trilostane also a competitive inhibitor of 38R /COCH3

e.

(s),

0

JfJp NH2

H

R=O,

..

‘H R’=-CH2C =C-CH2 or CH=C=CHCH2

-8

hydroxysteroid dehydrogenase, was more effective in blocking adrenal steroidogenesis than in disrupting pregnancy in the rat36 and rhesus mon-

CH3 CH3

‘SR R=-C H -p-NH2 6 4 10 11 12 13 key.37 Pregnancy in the monkey was terminated at a time when the ovaries are required for the continuation of pregnancy as well as when the placenta is capable of maintaining pregnancy in the absence of the ovaries. The estrogen synthetase blocker 12 inhibited mating, ovulation, and implantation in rats.38 Of a series of aromatase inhibitors, j.3- was the most active in vitro.39 ‘0

Prostaglandins - PGF c1 is available on a limited basis in the U.S. for the termination of seconi trimester pregnancy by the intraamniotic route. PGE2 has been approved in the U.S. for the same indication as an intravaginal s u p p o ~ i t o r y . The ~ ~ primary action of the PG’s in humans is believed to be a direct stimulatory effect on myometrial smooth m ~ s c l e . ~ l - 4 ~ The decreases in serum P and estradiol levels are considered a consequence and not a cause of the disruption of pregnancy. It should be remembered that pregnancy termination by luteolysis is only possible during early gestation since the corpus luteum is not required for maintenance of pregnancy after 6 or 7 weeks of estation, the time at which the luteoplacental shift takes p l a ~ e . ~When ~ , administered ~ ~ in the mid-luteal phase

Chap. 16

Control of Fertility Bell, Christiansen, Schane

171

PGFZa is not luteol tic in normal cycling women but is luteolytic in rats and other The results of an international multicenter study were interpreted to indicate that intraamniotic saline and PGF2, (25 mg repeated in 6 hours) are safe and effective for termination of second trimester pregnan~y,~'and that PGF2clis significantly more effective than saline in the termination of pregnancy within 48 hours. Another interpretation of these results pointed out the hazards of PGFZa and intraamniotic saline and advised that, up to the sixteenth week dilatation and evacuation is the most advantageous available method. 51 An advantage of dilatation and evacuation i s the low incidence of incomplete abortion compared with intraamniotic PGF or saline.51 In second trimester pregnancy a single intraamniotic injec&on of 2.5 mg of 15(S)-methyl PGFZ, methyl ester was slight1 more effective than 5 0 mg of PGF2, in inducing abortion within 48 hours.5T About one half of the abortions were incomplete with both drugs. In another multicenter study, a 92% abortion rate, 50% of which were incomplete, was achieved within 30 hours after repeated intravaginal administration of 15(S)-methyl PGFZa methyl ester during the second trimester.53 Vomiting and diarrhea were significantly more common with the PG analog, but the mean incidence of diarrhea and vomiting ranged from 2 to 6 episodes per patient in both s t u d i e ~ . ~ ~ 9 First ~ 3 and second trimester pregnancy was terminated with 15(S)-meth 1 PGFZa methyl ester administered as a single intravaginal suppository.34-56 Intravaginal administration of 16,16-dimethyl PGE2 was as effective in terminating first trimester pregnancy as vacuum aspiration but was associated with a much higher incidence of side effects. The incidence of side effects was lower than with 15(S)-methyl PGF2a methyl ester.57 Summaries of the results of clinical evaluation of PG's for pregnancy termination have been published. 58959

specie^.^'

Sulprostone (CP-34,089, ZK 57,671, SHB 286, 14) was 10-30X active than PGE2 as an antifertility agent in laboratory studies to or less active than PGE in laboratory tests considered to be tive of side effects.60 A3ministration of this agent i.v., i.m. 0

more but equal predicor by the

0

CONHS02CH3

HO

OH

14 -

e

2

HO'

~

6H 15 -

transcervical intrauterine route terminated first and second trimester HO pregnancies in women.61-64 A decrease in the incidence of side effects com.:\MM CHc 0 2 3 pared with other PG's was reported, a particularly significant result since the i.m. route could be advantageous HO' HO CH3 in the developing countries.65 A similar favorable separation of activi16 ties has been reported in the monkey for ONO-802 (15) .66 This PG terminated early regnancy in women when given by the transcervical intrauterine route6Por by pessary.68 In the latter study, complete abortion occurred in 54 of 63 women; nausea and

~

172 -

Sect. IV - Metabolic Diseases, Endocrine Function

Bailey, Ed.

vomiting were reported by one patient. Encouraging results have also been obtained in studies with 16,16-dimethyl PGE2 p-benzaldehyde semicarbazone ester for termination of first and second trimester pregnancies and with the dihomo PG derivative 16 for termination of abnormal intrauterine pregnancies.59 LH-RH Antagonists - Des-Glylo-[Trp2,Leu3,D-A1a6]LH-RH ethyl amide (EA) inhibited ovulation in rats after a single s . ~ .dose of 1.5 mg/rat on the day of r o e s t r ~ s . ~ ID-Phe2 ~ Pro3,D-Trp6 or D-Phe'1LH-W were active at 750 p5.'99 70 Ac- [Pro ,D-Phe2,D-Trp3,D-Trp6]LH-RH and [D-
Chap. 16

Control of Fertility Bell, Christiansen, Schane __ 173

dotro in receptor concentrations in the gonads of both male and female rats.fjo-82 Ovulation inhibition in women was achieved by treatment with [D-Ser(t-Bu)']LH-RH EA (HOE 766) S.C. daily for 22-30 days at a dose of 5 p g commencing within 3 days of the beginning of menstruation.83 Premature luteolysis in women resulted from administration of des-GlylO-LHRH EA S.C. for 5 days during the post-ovulatory phase.84 Administration of LH-RH at a dose of 250 ug several times on one day between the second and seventh day following ovulation caused premature luteolysis in women.85 There was evidence of ovulation on laparotomy in 9 of 10 women after pretreatment with EE every 12 hours on days 10 and 11 or days 9-11 followed by one injection of [D-Leu6]LH-RH EA.86 If these results are confirmed, a method could be available for improving the effectiveness of the rhythm method. As in the above study,86 however, it may be necessary to prime the hypothalamic-pituitary-gonadal system by administration of an estrogen prior to the administration of the LH-RH agonist. The observation that cyclo [ (B-Alal) and (6-Aminohexanoyll),D-Ala6) ]LH-RH were more potent agonists than the uncyclized peptides, although only about 1% as potent as LH-RH, supports an "U" shape conformation for LH-RH.87 Structure-activity relationships of LH-RH and its analogs have been reviewed.88 Male Contraception - The most promising approach to male contraception is suppression of spermatogenesis mediated by inhibition of gonadotropin release by the pituitary gland.89-91 Inhibition of spermatogenesis in men by monthly injections of a combination of Depo-Provera and testosterone enanthate (TE) continues to be studied with the objective of developing the optimum dosage schedule,92-94 Danazol (1)plus testosterone nate or TE caused a variable reduction of spermatogenesis in man. TE administered i.m. biweekly also did not result in uniform azoospermia in a year-long study.97 It is not known if azoospermia must be achieved for complete inhibition of fertility or whether oligospermia would be sufThe use of implants containing androgens is not yet practical since the im lant would be too large emphasizing the need for more active The D-homo steroids 1 7 and 18 are considered possible candidates €or use in implants since they are more effective than testosterone in suppressing pituitary gonadotropin secretion and, as a result, have an appropriate balance of peripheral and central activities.99 Antiandrogens such as cyproterone acetate have not been useful as male antifertility agents since libido is reduced at effective contraceptive doses.lOO The effects of prolonged suppression of spermatogenesis need to be studied and it must be established whether fertility returns upon withdrawal of drug. 97s g 8 a-Chlorohydrin(3-chloropropane-l,2-diol) is an example of a compound that interferes with sperm maturation."' Toxic side COOH effects, particularly in the monkey, precluded trials of the compound in man. The antifertility activity of a-chlorohydrin may be the result of an inhibition of glyceraldehyde-3-phosphate de0 'R& hydrogenase in spermatozoa. The 1 7 R=COC2H5,R'=H S(+)-isomer is more active in vitro and in vivo than the R(-)18 R=H, R' =C = CH c1 isomer. In vitro experiments sug19 gest that conversion to the 1-

androgen^.^^

Sect. IV - Metabolic Diseases, Endocrine Function

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Bailey, Ed.

phosphate is necessary for activity.l02 Some 6-chloro-6-deoxy sugars had a reversible contraceptive action in the male rat and were less toxic than a - c h l ~ r o h y d r i n . ~AF~ ~1312/TS (19)inhibited spermatogenesis in the rat and in the monkey.'04-lo6 Miscellaneous Compounds - Centchroman (20)inhibited pregnancy in rats, mice, do s and monkeys when administered once orally within 24 hours of coitus.1 7 The drug was estrogenic, antiestrogenic and antiprogestational and inhibited ova development in the rat. Coadministration with P and estradiol diproprionate prevented the termination of pregnancy. Centchroman possessed both gonadotropin inhibitory and stimulatory properties in unilaterally ovariectomized rats deR R pending upon the dose. The compound resembles in this respect the structurally related clomiphene ( 2 1 , an antiestrogen and a known ovulation stimulator. Centchroman induced ovu5 H CH30 6 : &' 0 lation in women when administered C ~ H ~ dail log The drug is well-tolera21 C6H5 tedl" and is being studied as a 20 once-a-week contraceptive.'l0 The contraceptive activity was suggested R=CH CH N E ~ ~ R=CH2CH2 2 2 to be a result of an inhibition of implantation and sperm transport. The triazoles 22 and 23, inhibitors of PG metabolism in the rat uterus, placenta, and were completely effective in terminating pregnancy in rats and hamsters when administered S.C. daily during any one of four 5-day periods between days 1 and 12 of pregnancy. These compounds were most effective when administered immediately following implantation; they lacked hormonal or antihormonal activity, and their antifertility effect was not reversed by P.lI2 The closely related 24 given S.C. in

8

Q

7

3 OR

23 n=2,R=CH 3 24 n=2,R=C2H5 25 -

26 -

combination with PGE2 during esfrus blocked ovulation almost completely in the hamster.l15 PGE2 alone was ineffective and 2 alone was weakly active; 24 also hastened ova transport. Indomethacin, a PG synthetase inhibitor suppressed spontaneous and induced ovulation in rats and rabbits,I l b suppressed gonadotropin-induced ovulation in rhesus monkeysll7 and marmosets,118 but failed t o inhibit spontaneous ovulation in rhesus monkeys.119 Studies with indomethacin as an ovulation inhibitor in women were inconclusive120 and in two small studies in women aspirin was ineffective.121,122 The biphenyl derivative 2 was completely effective inhibiting pregnancy in rats on days 4 and 5 at a dose of 5 mg/kg p.0. 6-Chloromelatonin (26) inhibited ovulation in rats when administered i.v. or p.0. on the day of proestrus and was more effective than melatonin, probably as a result of an increased metabolic stability.124

&

Chap. 16

Control of Fertility

Bell, Christiansen, Schane

175

A review of adrenergic innervation of the mammalian oviduct includes a summary of drugs that modif

the sympathetic nervous system and affect ovum transport and fertility.P25 Plants are a relatively untapped potential source of new antifertility agents.126 References

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11,

132,

2.

so.

11.

176 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. 75. 76. 77. 78. 79.

80. 81. 82.

83. 84.

Sect.

Iv -

Metabolic Diseases, Endocrine Function

~

~

Ed. i l

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~

Chap. 16

C o n t r o l of Fertility B e l l , Chrlstiansen, Schane

85. A. Lemay. F. L a b r i e , C. Azadian-Boulanger and J . -P. Raytiand, C . R. Acad. Sc . S e r . D., 28, 527 (1978). 86. J. Z a n a r t u , J . M. R osner, E. Guiloff, A. A. I b a r r a - P o l o , H . D. C r o x a t t o . H . B. E. A g u i l e r a , D . H. Coy and A. V. S c h a l l y , Br. Med. J . . 2. 527 (1975). 87. J. S e p r o d i , D. H. Coy, J. A. Vi l chez-Mart i nez, E . Pedroza. W . - Y . Huang and A. S c h a l l y , J. Med. Chem., 2.993 (1978). 88. J . Sandow, W. Kdriig. R. G e i g e r , R . Uhmann and W. von Rechenberg, Proc . E a s t e r S c i . Univ. Nottingham. 26, 49 (1977). 8 9 . D. M. DeKretser, B u l l . W.H.O.. 56. 353 (1978). 90. J . S e n i o r , Pharm. J . , 341 ( 1 9 7 8 z 91. L. L. Ewing a n d B. R o b a i r e , Annu. Rev. Pharmacol. T o x i c o l . . 18, 167 (1978). 92. 93. 94. 95. 96. 97 98. 99. 100. 101.

Paris. Croxatto, V.

Sch. A g r i c .

11,

B a r t s c h and W. -H. Weiske, C o n t r a c e p t i o n . 6 c (1977). F r i c k , 0. B a r t s c h and W. -H. Weiske, i b i d . , 15, 669 (1977). M v a r e z - S a n c hez. A. Faundes, V. Brache and P . Leon, i b i d . , 635 (1977). D. Skoglund and C. A. P a u l s e n . i b i d . , 1. 357 (1973). U l s t e i n , N. Netto, J . Leonard and C. A. P a u l s e n . i b i d . . 12. 437 ( 1 9 7 5 ) . S t e i n b e r g e r and K. C. Smith. F e r t i l . S t e r i l . , 2.1320 ( E 7 7 ) . J. Hammerstein and J. B r o t h e r t o n , I n t . J . Androl. , Suppl. 2 , 659 (1978). F. Neumann, R. C. Ni ckol son, 8. Schenck and H. S t e i n b e c k , i b i d . . 147 (1978). B. Schenck a n d F. Neumann, i b i d . , 1 5 5 (1978). P. D. C. Brown-Woodman, H. Mohri. T. Mohri. D. S u t e r a nd I . C. White, Biochem. J . , 170 2 3 (1978). 102. R. W. F i t z p a t r i c k , H. J a c k s o n and N . A. Di ckins on. C o n t r a c e p t i o n , g. 477 ( 1 9 7 8 ) . 103. W. C. L. Ford a nd G. M. H. Waites, J . Reprod. F e r t . , 52, 1 5 3 (1978). 1 0 4 . G. Corsi, G. P a l a z z o , C. Germani, P. S. Barcellona a nd E. S i l v e s t r i n i , J . Hed. Chem.. 1 9 , 778 (1976). 105. S. B u r b e r i , B. C a t a n e s e , V. C i o l i , B. S. B a r c e l l o n a and B. S i l v e s t r i n i , Exp. Hol. P a t h o . , 9, 308 (1975). 1 0 6 . F. C o u l s t o n . W. J . Dougherty. R . LeFevre. R. Abraham and B. S i l ~ ~ r s t r i n iI. b i d . . 2 , 3;; (1975). 107. V. P. Kamboj, B. S. S e t t y , H . Chandra, S . K. Roy and A. B. Kar, I n d i a n J . Exp. H i o l . . 15, 1144 ( 1 9 7 7 ). 108. S. Roy, G . L. Kumari, K. Madoiya, V. P r a k a s h and S . Ray. F r r t i l . S t r r i l . , 3. 1108 (1976). 109. H. Chandra, R. C. Srimal, V. P. Kamboj, B. N . Dhawan and N. N . Gupta. I n d i a n 3 . Exp. B i O l . , 2.1 1 7 0 (1977). 1151 (1977). 110. J . S. Munshi, R. K. Nair and P. K. Devi , i b i d . , 111. R. Vaidya. U . J o s h i , P. M e h e r j i , N. Rege, S. B e t r a b e t . L . J o s h i , A. S h e t h a nd P. K . Devi, i b i d . , 2,1 1 7 3 (1977). 1 3 0 (1975). 112. L. J . L e r n e r , G. G a l l i a n i . P. C a r m i n a t i and M. C. Mosca, N a t u r e , 113. L. J . L e r n e r and P. C a r m i n a t i i n "Advances in P r o s t a g l a n d i n and Thromboxane Research". Vol. 2 , 5. Samuelsson and R. P a o l e t t i , Ed., Raven P r e s s , New York, NY, 1976, p 645. 114. L. J . Le r n e r and P. C a r m i n a t i , J . S t e r o i d Biochem., S, 395 (1977). 115. L. J . L e r n e r . C. O l d a n i and A. V i t a l e , P r o s t a g l a n d i n s , 15, 525 (1978). 116. H. R. L i n d n e r , V . Zor, S. Bauminget, A. T s a f r i r i , S . A. Lamprecht, Y. Koch. S. A n t e b i and A. S c h w a r tz i n " P r o s t a g l a n d i n S y n t h e t a s e I n h i b i t o r s " , H. J . Robinson and J . R. Vane, Ed., Raven P r e s s , NY, 1974, p 271. 1 1 7 . E. E . Wallach. A. d e l a Cruz. J. Hunt, K. H. Wright a nd V . C. S t e v e n s , P r o s t a g l a n d i n s . -9 , 645 ( 1 9 7 5 ) . 118. H. Maia, I. Barbosa a n d E. M. Cout i nho, F e r t i l . S t e r i l . , 565 (1978). 119. K. H . Wr ig h t , V. C. S t e v e n s , Y . Hamada and E. E. Wallach. i b i d . , 239 (1978). 120. A. R. Souka, H. Rahman and F. S a l e h , IRCS M e d i c a l S c i e n c e : E n d o c r i n e S e c t i o n , 61 (1978). 1 2 1 . G. Chandhuri and M. G. E l d e r , P r o s t a g l a n d i n s , 11, 727 (1976). 122. F. L . Greenway and R. S. S w e r d l o f f , F e r t i l . S t e r i l . , 30, 364 (1978). 1 2 3 . S. K . Garg, I n d i a n J . Med. Res.. 2,392 (1978). 124. M . E . F la u g h , T. A . C r o w e l l , J. A. Clemans and 8. D. Sawyer, J . Med. Chen., 11. 63 (1979). 1 2 5 . D. M. P a to n , J . H. Widdicombe, D. E. Rheaume and A. J o h n s , Pharmacol. Rev., 2. 67 (1978). 126. D. D. S o e j a r t o , A . S. B i n g e l , M. S l a y t o r and N . R . F a r n s w o r t h , B u l l . W.H.O., 343 (1978).

.,

J . F r i c k . G.

177

J. F. R. M. E.

Is,

-

-

11,

256.

2,

a,

a,

Annual Reports in Medicinal Chemistry-74

178 Chapter 1 7 .

P r o s t a c y c l i n , Thromboxanes and t h e A r a c h i d o n i c Acid Cascade K. C. N i c o l a o u , Department of C h e m i s t r y , U n i v e r s i t y of P e n n s y l v a n i a , P h i l a d e l p h i a , PA 19104

J . Bryan S m i t h , Department of Pharmacology and The Cardeza F o u n d a t i o n , Thomas J e f f e r s o n U n i v e r s i t y , P h i l a d e l p h i a , PA 19107

I n t r o d u c t i o n - The d i s c o v e r i e s of thromboxane A2 (TXA2) by S a m u e l s s o n ' s group i n 1 9 7 5 l and of p r o s t a c y c l i n (PG12) by V a n e ' s g r o u p i n 19762 s t i l l remain t h e most r e c e n t and e x c i t i n g b r e a k t h r o u g h s i n t h e p r o s t a g l a n d i n (PG) a r e a . With t h e s e new members, t h e a r a c h i d o n i c a c i d (AA) c a s c a d e ( F i g . I ) a p p e a r s more c o m p l e t e and i t s p h y s i o l o g i c a l r o l e i s b e t t e r unders t o o d and a p p r e c i a t e d . Thus, TXA2 g e n e r a t e d by p l a t e l e t s promotes a g g r e g a t i o n and c o n s t r i c t s smooth muscle w h i l e PGZ2 produced by v a s c u l a r endot h e l i u m , a s w e l l as by s e v e r a l o t h e r t i s s u e s , i n h i b i t s a g g r e g a t i o n and r e l a x e s smooth muscle. The b i o l o g i c a l l y o p p o s i n g e f f e c t s of t h e s e subs t a n c e s a p p a r e n t l y are of c r u c i a l i m p o r t a n c e t o normal h e a l t h and a n i n b a l a n c e i n t h e i r p r o d u c t i o n may l e a d t o d i s o r d e r s s u c h as abnormal blood p r e s s u r e , a t h e r o s c l e r o s i s , s t r o k e and h e a r t a t t a c k . T h e r e f o r e , t h e s y n t h e s i s of t h e s e compounds and t h e s e a r c h f o r more s t a b l e p r o s t a c y c l i n s and thromboxanes become h i g h l y d e s i r a b l e n o t o n l y t o p r o v i d e u s e f u l b i o l o g i c a l t o o l s , b u t a l s o compounds of t h e r a p e u t i c p o t e n t i a l . The s e a r c h f o c u s e s m a i n l y on s t a b l e and s e l e c t i v e b i o l o g i c a l a g o n i s t s o r a n t a g o n i s t s of t h e n a t u r a l m o l e c u l e s , as w e l l as s e l e c t i v e i n h i b i t o r s o f t h e v a r i o u s enzymes i n v o l v e d i n t h e i r b i o s y n t h e s i s . The main s c o p e of t h i s a r t i c l e i s t o r e v i e w t h e r e c e n t p r o g r e s s i n t h e s y n t h e s i s and b i o l o g y of p r o s t a A r e v i e w on t h e s y n t h e s i s and b i o l o g i c a l c y c l i n s and thromboxanes.

(=;zz+ *; GH CH~OCOICH~l,CH, CH2OYOR

-

PhowhvlipsY

Lmoxyd801

HI01 O , , , , ~ c o o H

A,

Amchidom acid I A A I

Phorpholipid enerified arachidonic mid

12 HydroperoxvardchidonIc acid I12 HPAAI

I12 Hydroxyarachidonic acid1 10,

L ti0

OH

AA ~vclmxv~na%e

112HAAl

Primary prostaglandins IPGI PGE,: R 1 = 0. H2 = a OH. H Ibl PGO?: RI - a - O H . H , H2 = 0

(11

OH Thromboxane B 2 ITXBI)

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

Chap. 17

Prostacyclin, Thromboxanes

Nicolaou, Smith

2

properties of PG endoperoxides, thromboxanes and prostacyclins has recently appeared.3 The last PG review in the Annual Reports in Medicinal Chemistry appeared in 1977.4 Synthesis of Prostacyclins - PGT2 (L),its sodium salt (2) and methyl ester (3 ) have been efficiently synthesized from PGF,_* systems. 5-10 Unlike 1 and 3, the sodium salt 2, which is biologicalfy equivalent to PGI2 ' is stable both as a solid and in solution. The 13,14-dihydroprostacyclin methyl ester 4 was also synthesized and found e uipotent to PGI2 methyl ester as an inhibitor of platelet aggregation. l9 The stable prostacyclins 5a5y6, 5b596, 6a5912 and a 5 3 1 2 were synthesized, but showed much less potency than P E as inhibitors of blood platelet aggregation. The A6 isomer (7) of PG? has been reported to be eleven times more potent than PGE (less potent Zhan PGI ) as an inhibitor of platelet aggregation.13 2 synthesized14 and proven to be different The1A7 isomers 8a and 8b were from the "Pace-Gciak and Wolfe PG"15, thus requiring a revision of the structure of this compound. Sih has suggested the structure 7 for the "Pace-Asciak and Wolfe PGtf,16but lH NMR evidence strongly suggests that this PG possesses the ketal structure 910913917, which is obtained from COOR

f

COOCH,

l,R=H 2, R = Na 3, R = CH,

OH

Hd OH

OH 7

Sect. IV - Metabolic Diseases, Endocrine Function

180 -

Bailey, Ed.

PGI methyl ester under anhydrous acidic conditions. To test the inter2 mediacy of 5-hydroxy-PGI in the biosynthesis of PG12 all four isomers 10a-10d were synthesized 8919 and subjected to prostacyclin synthetase.l8 -None of these compounds, however, was converted to PGI by the enzyme 2 proving their non-involvement in the biosynthetic pathway. 18 PGI (13) 3 7 has been synthesized and found to possess similar biological properties as PGI .6b,20 Sulfur-containing prostacyclins 14,21,22 15-17,23 and 182 2024 have been synthesized and found to be considerably more stable than PGI The most extensively studied analog of this series, 6,9-thiaprostacyclin ( 1 4 ) , showed 0.2-0.5 times the potency of PGI as an inhibitor of blood platelet aggregation, but was a constrictor oz the isolated cat coronary artery rather than a dilator.21 The carbocyclic prostacyclin 2 1 and three of its isomers (C-5 and C-15) were synthesized r e ~ e n t l y . ~ ~ - ~ ? -

1

I _

.

OH COOCH,

Hd OH

r/'

OOCH,

13

14. x = s 15ab. X = SO 16, X = SO,

Hd OH

OH 17a (6s) 17b ( 6 R )

18, X = S 19ab. X = SO 20, x = so,

OH

Chap. 17

Prostacyclin, Thromboxanes

Nicolaou, Smith 181

Isomer 21 exhibited higher potency than P G E ~ ,but lower than PG12 as an inhibitor or platelet aggregation and as a dilator of smooth muschle .255 27 The N-containing prostacyclins (22-25) - - have also been reported and compounds 22 and 23 are surprisingly stable and potent biological mimics of PGI2.28-6 ,9-Pyridazaprostacyclin (26) and a number of its derivatives have been synthesized from 6-keto-PE1 (27).29 Both 26 and 27 showed higher potency than PGEl as inhibitors ofplatelet aggregation.29 This first "aromatic" prostacyclin (26) was also found to be a potent dilator of the isolated cat coronary artery.29 The 12-fluoro30-and 14-fluorop r o sta~yclinshave ~ ~ been synthesized and found to be potent agonists of PGI2.30131

COOH

OH

OH

23

22

OH

24

Hd

HO OH

OH

26

27

182 -

Sect. IV

-

Metabolic Diseases, Endocrine Function

Bailey, Ed.

Synthesis of Thromboxanes - TXA2 (%>I still remains elusive to isolation and chemical synthesis. Thromboxane B (TXB2) (%)l, however, with its more accessible structure has been synghesized by several groups and is readily available.32-38 The first TXA analog, pinane thromboxane A? (PTA ) (30), has re2 cently been synthesized in optically active form.39 Unlikethe 15-deoxy PGH analogs 314O and 3z41, which are inhibitors of both thromboxane and 2 prostacyclin synthetasc PTA2 shows remarkable activity as a selective inhibitor of thromboxane synthetase without interfering with cyclooxygenase or prostacyclin ~ynthetase.~' Furthermore, it is a potent antagonist of TXA and various stable endoperoxide analogs with regard to and smooth muscle contraction.39 platelet aggregation 2 so

~~ The methyl ester of PGH 2 -(33) has been chemically ~ y n t h e s i z e dand has43 12-hydroxyarachidonic acid (3).

OH

OH

OH

20

28

OH 31, X-Y = N=N 32, X-Y = H N - 0

30

COOCH,

Y

HO

"%,

OH

33

34

Chap. 1 7

P r o s t a c y c l i n , Thromboxanes

N i c o l a o u , Smith

183

B i o s y n t h e s i s - PG e n d o p e r o x i d e s y n t h e t a s e h a s b e e n e u r i f i e d t o homogenity from t h e s o l u b i l i z e d microsomes of v e s i c u l a r g l a n d s 4-48 and h a s b e e n p a r t i a l l p u r i f i e d from s o l u b i l i z e d microsomes of p l a t e l e t ~ 4and ~ kidney I t s m o l e c u l a r w e i g h t i s a p p r o x i m a t e l y 125,000 d a l t o n s b a s e d medulla.30 on t h e S t o k e s r a d i u s and s e d i m e n t a t i o n c o e f f i c i e n t and i t b e h a v e s as a s i n g l e p o l y p e p t i d e of 6 9 , 0 0 0 - 85,000 d a l t o n s on sodium d o d e c y l s u l p h a t e p o l y a c a m i d e e l e l e c t r o p h o r e s i s i n d i c a t i n g two s u b u n i t s of a p p r o x i m a t e l y The enzyme c a t a l y s e s b o t h c y c l o o x y g e n a s e (AAjPGG2) and e q u a l s i z e . 4',47 p e r o x i d a s e r e a c t i o n s (PGG2+PGH ) and r e q u i r e s hemin o r a s i m i l a r m e t a l l o 2 The p e r o x i d a s e p r o t e i n which i s p o s s i b l y l o s t d u r i n g p u r i f i c a t i o n . 4 7 , 4 8 r e a c t i o n a l s o r e q u i r e s p h e n o l o r a r e l a t e d a r o m a t i c compound ( e . g . t r y p t o p h a n , s e r o t o n i n , e p i n e p h r i n e , h y d r o q u i n o n e ) which may a c t t o s u p p l y e l e c t r o n s . These compounds a l s o p r o t e c t t h e enzyme from s e l f - d e s t r u c t i o n by h y d r o p e r o x i d e s s u c h as H 0 47 o r PGG2.51 2 2 PG e n d o p e r o x i d e E i s o m e r a s e h a s b e e n p a r t i a l 1 p u r i f i e d by s o l u b i l i z a t i o n of microsomes from b o v i n e v e s i c u l a r g l a n d . T2 A s u l p h y d r y l - c o n t a i n i n g compound ( e . g . g l u t a t h i o n e , d i t h i o t h r e i t o l , 2-mercapto e t h a n o l ) i s r e q u i r e d t o s t a b i l i z e t h e enzyme and g l u t a t h i o n e a c t s s p e c i f i c a l l y as a c o - f a c t o r f o r t h e i s o m e r i z a t i o n o f PGH t o PGE. The pathway PGG'15h y d r o p e r o x y PGE'PGE o r i g i n a l l y ~ u g g e s t e d ~ 3seems , ~ ~ u n l i k e l y s i n c e a) PGH r a t h e r t h a n PGG i s t h e p r e f e r r e d s u b s t r a t e , b ) t h e i s o m e r a s e d o e s n o t c o n t a i n p e r o x i d a s e a c t i v i t y and c o n v e r t s PGG o n l y t o 15-hydroperoxy PGE and c ) 15-hydroperoxy - PGE i s n o t a s u b s t r a t e f o r p u r i f i e d PG e n d o p e r o x i d e s y n t h e t a s e and m i g h t w e l l i n a c t i v a t e t h i s enzyme as d o o t h e r hydrop e r o x i d e s . 47 751 Thromboxane s y n t h e t a s e h a s b e e n s o l u b i l i z e d and p a r t i a l l y p u r i f i e d from and bovine56 b l o o d p l a t e l e t s a n d from s h e e p 5 7 and b o v i d 8 l u n g . The enzyme a c t i v i t y a l s o h a s been s t u d i e d i n p l a t e l e t membranes b y s e v e r a l w 0 r k e r s . 5 ~ - ~ Recent ~ s t u d i e s i n d i c a t e t h a t t h e same enzyme cata l y s e s t h e f o r m a t i o n of TXA and hydroxyheptadecatrienoicacid (HHT) i n a 2 b i m o l e c u l a r r e a c t i o n and t h a t t h e f o r m a t i o n of b o t h p r o d u c t s i s i n h i b i t e d i n ~ a r a l l e 1 . ~ 4 , 6 These ~ , ~ ~s t u d i e s a l s o show t h a t t h e f o r m a t i o n o f HHT d o e s n o t i n v o l v e TXA as a n i n t e r m e d i a t e and t h a t TXA i s c o n v e r t e d ex2 2 c l u s i v e l y i n t o TXB PGH3, t h e e n d o p e r o x i d e from e i c o s a p e n t a e n o i c a c i d (C~o:5,5,8,11,14,1?') i s c o n v e r t e d i n t o thromboxane A (TXA ) by p l a t e l e t 3 membranes. Although TXA c o n t r a c t s r a b b i t a o r t a , as d o e s &A2, i t d o e s 3 n o t i n d u c e p l a t e l e t a g g r e g a t i 0 1 - 1 . ~ ~E i c o s a t r i e n o i c a c i d (C20 :3,5 ,8,11) and i t s endo e r o x i d e PGH do n o t i n d u c e p l a t e l e t a g g r e g a t i o n as do AA o r 1' "-" E i c o s a t r i e n o i c a c i d and P G H l a r e c o n v e r t e d o n l y i n low y i e l d t a d e c a d i e n o i c a c i d and h a r d l y a t a l l i n t o thromboxane A N e i t h e r 2-nor-PGH n o r 2-nor-PGH are s u b s t r a t e s f o r 70 2 1 tkrornboxane s y n t h e t a s e a l t h o u g h 2-nor-PGH2 i n d u c e s p l a t e l e t a g g r e g a t i o n .

.

.

and b o v i n e c o r o n a r y a r t e r i e s J 2 ' 74 e f f i P i g a o r t a microsomes 2' The c o n v e r s i o n of AA c i e n t l y c o n v e r t PGH v i a PGI i n t o 6-keto-PGF1,.~ 2 2 i n t o 6-keto-PGF h a s a l s o b e e n o b s e r v e d w i t h microsomes o b t a i n e d from ram75 and b o v i n e 6 s e m i n a l g l a n d s and from s h e e p l u n g . 7 7 P r o s t a c y c l i n b i o s y n t h e s i s i s h i g h e s t i n t h e d u c t u s a r t e r i o s u s , a o r t a and pulmonary a r t e r y of t h e f e t a l lamb78 and h a s been d e t e c t e d i n t h e c o r t e x of r a b b i t h a s been d e t e c t e d i n p e r f u s a t e s and human k i d n e y . " PGI o r 6-keto-PGFla The i n t i m a of r a b b i t and r a t h e a r t 8 8 , 8 1 and r a b b i t and dog lung.82,83 of v e s s e l w a l l s h a s t h e h i g h e s t P G I s y n t h e t a s e a c t i v i t y 8 4 and i t is g r e a t e r 2

184

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- Metabolic Diseases, Endocrine Function

Bailey, Ed.

in rat arteries than veins. 85 There has been active, although not always consistent progress in studying PG synthesis by cells in culture. While mouse macrophophages were found to synthesize PGE2 and 6-keto-PGFla in response to zymosan,86 activated macrophages from rats and guinea pigs were shown to produce TXB2.87 Cultured fibroblasts and smooth muscle cells were found to produce PGI2 by some researchers85989 but had little PGI2 synthetase activity One possible explanation for the discrepanwhen studied by others. cies is that the cultures of macrophages, fibroblasts and smooth muscle cells which produced PG12 were contaminated by endothelial cells A consistent finding has been that endothelial cells synthesize PGI, .41-97 Collectin tubiles from the rabbit renal papillae synthesize 6kketo-PGF1, from AA. 98 Inhibition of Biosynthesis - Inhibition of thromboxane synthetase has received a great deal of attention and a number of inhibitors of this enzyme have been reported. They include substrate analogs such as 9,ll-epoxymethano-15-hydroxyprosta-5,13-dienoic acid55 9 59, 9 11-azo-15-hydroxyprosta5,13-dienoic acid55 61, 9,ll-e ox methanoprosta-5 13-dienoic acids9, 9,llazoprosta-5,13-dienoic acid40,Z;9 p P o 0 , imidazolegg-103 and some l-substituted imidazole derivatives. lo3 Whereas imidazole and its derivatives are weak inhibitors of platelet aggregationg99l0l,Io3 9,ll-azoprosta5,13-dienoic acid is a strong inhibitor.99 It has been suggested that imidazole is only a weak inhibitor of aggregation because it facilitates the movement of intracellular calcium.99 Elabo tion of the discovery that 15-hydroperoxy-AA inhibits PG12 synthetase"' has revealed that 12-hydroperoxy-AA inhibits thromboxane synthetase4', while w-8 and w-10 hydroperoxides of linoleic , dihomo-ylinolenic and AA inhibit prostacyclin synthetase.71 It seems possible that these hydroperoxides may perform a negative feedback role to inhibit excess thromboxane or PGI formation. The effects of the 5-hydroperoxy2 AA forme by rabbit polymorphonuclear leucocytes have not been investigated.1d5 Metabolism - TXB is a poor substrate for the dehydrogenase which acts on 2 PGE2 and PGF2a,and TXB remains the dominating compound in the circulation after its injection.Io3 The major urinary metabolite of TXB2 in the monkey is di-nor-TXB2. Other metabolites, which result from changes in the 6-membered thromboxane ring and &and w-oxidation , have been identified.1°7 108 PGI is rapidly oxidized in vitro by tho 1 -hydroxy-prostaglandin 2 dehydrogenase of lungslog and blood vessels.lloy'll However, it is not a substrate for the lung transport systemlog and is not metabolized by The major inactivation of PG12 appears to occur in the intact lungs. liver and hind quarters.l12 On the other hand, 6-keto-PGFl, is a poor 109,113 substrate for 15-hydroxy-PG dehydrogenasebothin vitro and in Seven urinary metabolites of PGI2 including the di-nor and 19-hydroxy-dinor derivatives of 6-keto-PGF1, and 13y14-dihydro-6,15-diketo-PGFla and w-carboxyl di-nor derivatives of 13,14-dihydro-6,15-diketo-PGFla, have been identified.114

-.

Chap. 17

Prostacyclin, Thromboxanes

N i c o l a o u , Smith

185

References 1. M. Hamberg, J. Svensson, B. Samuelsson, Proc. Nat. Acad. Sci. USA, x,2994(1975). 2. (a) S . Moncada, R. Gryglewski, S . Bunting, J.R. Vane, Nature, 263, 663 (1976); (b) R.A. Johnson, D.R. Morton, J.H. Kinner, R.R. Gorman, J.C. McGuire, F.F. Sun, N. Whittaker, S . Bunting, J . Salmon, S . Moncada, J.R. Vane, Prostaglandins, 12. 915 (1976). 3 . K.C. Nicolaou, G.P. Gasic, W.E. Barnette, Angew. Chem. Int. Ed. Engl., 17, 293 (1978). 4. T.K. Schaaf in Ann. Rep. Med. Chem., H.-J. Hess Ed., 2, 182 (1977). 5. E.J. Corey, G.E. Keck. I. Szekely, J . Amer. Chem. SOC., 99, 2006 (1977). 6. (a) R.A. Johnson, F.H. Lincoln, J.L. Thompson, E.G. Nidy. S.A. Mizsak, U. Axen. J. Amer. Chem. SOC., 99, 4182 (1977); (b) R.A Johnson, F.H. Lincoln, E.G. Nidy, W.P. Schneider, J.L. Thompson, U. Axen, J. Amer. Chem. SOC., 100, 7690 (1978). 7. I. Tomoskozi, G. Galambos, V. Simonidesz, G. Kovacs, Tetrahedron Lett., 2627 (1977). 8. N. Whittaker, Tetrahedron Lett., 2805 (1977). 9. K.C. Nicolaou. W.E. Barnette, G.P. Gasic, R.L. Magolda, W.J. Sipio, J.C.S. Chem. Commun., 630 (1977). 10. K.C. Nicolaou, W.E. Barnette, R.L. Magolda. J . Chem. Res., in press. 2199 (1977). 11. J . Fried, J. Barton, Proc. Nat. Acad. Sci. USA, 2, 12. K.C. Nicolaou, W.E. Barnette, J.C.S. Chem. Commun., 331 (1977). 13. K. Shimoji, Y. Konishi, Y. Arai, M. Hayashi, H. Yamamoto, J. Amer. Chem. SOC., 100, 2547 (1978). 14. J.C. Sih, D.R. Graber, J. Org. Chem., 42, 3798 (1978). 15. C. Pace-Asciak, L . S . Wolfe, Biochemistry, lo. 3657 (1971). 16. C.J. Sih, F.C. Huang, J. Amer. Chem. SOC., 100, 643 (1978). 17. K.C. Nicolaou, W.E. Barnette, R.L. Magolda, Prostaglandins and Medicine, 1. 96 (1978). 18. F.C. Huang, M. Zmijewski, G . Girdaukas, C . J . Sih, Biorg. Chem.. 5, 311 (1977). 19. J.C. Sih, R.A. Johnson, E.G. Nidy, D.R. Graber, Prostaglandins, 15, 409 (1978). 20. E.G. Nidy, R.A. Johnson, Tetrahedron Lett., 2375 (1978). 21. K.C. Nicolaou, W.E. Barnette. G.P. Gasic, R.L. Magolda, J . Amer. Chem. SOC.. 99, 7736 (1977). 22. M. Shibasaki. S . Ikegami, Tetrahedron Lett., 559 (1978). 23. K.C. Nicolaou, W.E. Barnette, R.L. Magolda, J . Amer. Chem. S O C . , 100,2567 (1977). 24. K.C. Nicolaou. R.L. Magolda, W.E. Barnette, J.C.S. Chem. Commun., 375 (1978). 25. K. Kojima, K. Sakai. Tetrahedron Lett., 2743 (1978). 26. K.C. Nicolaou, W.J. Sipio, R.L. Magolda, S.P. Seitz, W.E. Barnette, J.C.S. Chem. Commun., 1067 (1978). 27. M. Shibasaki, J. Ueda, S . Ikegami, Tetrahedron Lett., 433 (1979). 28. G.L. Bundy, J.M. Baldwin, Tetrahedron Lett., 1371 (1978). 29. K.C. Nicolaou, W.E. Barnette, R.L. Magolda, J. Amer. Chem. SOC., 101, 766 (1979). 30. K.C. Nicolaou, W. E. Barnette. R.L. Magolda, P.A. Grieco. W. Owens, C.L.J. Wong. J.B. Smith, M. Ogletree, A.M. Lefer, Prostaglandins, 2, 789 (1978). 31. P.A. Grieco. Y. Yokoyama, K.C. Nicolaou. W.E. Barnette, J.B. Smith, M. Ogletree, A.M. Lefer, Chemistry Lett., 1001 (1978). 3 2 . W.P. Schneider, R.A. Morge, Tetrahedron Lett., 3 2 8 3 (1976). 33. N.A. Nelson, R.W. Jackson, Tetrahedron Lett., 3275 (1976). 34. R.C. Kelly, I. Schletter, S . J . Stein, Tetrahedron Lett., 3279 (1974). 35. S . Hanessian, P. Lavallee. Can. J . Chem.. 55, 562 (1977). 36. E.J. Corey, M. Shibasaki, J . Knolle, Tetrahedron Lett., 1625 (1977). 37. E.J. Corey, M. Shibasaki, J . Knolle, T. Sugahara, Tetrahedron Lett., 789 (1977). 38. 0. Hernandez, Tetrahedron Lett., 219 (1978). 39. K.C. Nicolaou, R.L. Magolda, J.B. Smith, D. Aharoni, E.F. Smith, A.M. Lefer. Proc. Nat. Acad. Sci. USA, in press. 40. R.R. Gorman, G.L. Bundy, D.C. Peterson, W.F. Sun, O.V. Miller, F.A. Fitzpatrick, Proc. Nat. Acad. Sci. USA, E,4007 (1977). 41. F.A. Fitzpatrick, G.L. Bundy, R.R. Gorman, T. Honohan, Nature, 275, 764' (1978).

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186 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. 75, 76. 77. 78. 79. 80 * 81. 82. 83.

Sect. I V

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B a i l e y , Ed.

R.A. Johnson, E.G. Nidy, L. Baczynskyj, R . R . Gorman, J. Amer. Chem. Soc.. 99. 7738 (1977). E.J. Corey, 8. Niwa, J. Knolle, J. Amer. Chem. SOC.. 100, 1942 (1978). T. Miyamoto, S. Yamamoto, 0. Hayaishi, Proc. Nat. Acad. Sci. USA, 2 , 3 6 4 5 (1974). T. Miyamoto, N. Ogino, S. Yamamoto, 0. Hayaishi, J. Biol. Chem., =,2629(1976). M. Hemler, W.E.M. Lands, W.L. Smith, J. Biol. Chem., 251, 5575 (1978). F.J. Van der Ouderaa, M. Buytenhek, D.H. Nugteren, D.A. Van Dorp, Biochim. Biophys. Acta., 487, 315 (1977). N. Ogino, S. Ohki, S. Yamamoto and 0. Hayaishi. J. Biol. Chem., 253,5061(1978). S. Hammarstrom and P. Falardeau, Proc. Nat. Acad. Sci. USA, 76, 3691 (1977). S.C. Bhat. T. Yoshimoto. S. Yamamoto and 0. Hayaishi, Biochim. Biophys. Acta., 529, 398 (1978). R.W. Egan, J. Paxton and F.A. Kuehl. Jr., J. Biol. Chem., 251, 7329 (1976). N. Ogino, T. Miyamoto, S. Yamamoto and 0. Hayaishi, J. Biol. Chem.. 252, 890 (1977). B. Samuelsson and M. Hamberg in "Prostaglandin Synthetase Inhibitors." H.J. Robinson and J.R. Vane, Ed., Raven Press, 1974, p. 107. A. Raz, M. Schwartzman, R. Kenig-Wakshal. Eur. J. Biochem., 70, 89 (1976). U. Diczfalusy and S. Hammarstrom, FEBS Lett., 82, 107 (19771, T. Yoshimoto, S. Yamamoto. M. Okuma and 0. Hayaishi, J. Biol. Chem., 252, 5871 (1977). H.-H. Tai and B. Yuan, Fed. Proc., 36, 309 (1977). P. Wlodawer and S. Hamarstrom, Biochem. Biophys. Res. Commun. 3, 525 (1978). 1432 (1977). F.F. Sun, Biochem. Biophys. Res. Coormun., M.W. Anderson, D.J. Crutchley, B.E. Tainer and T.E. Eling, Prostaglandins, 16. 563 (1978). U. Diczfalusy, P. Falardeau and S. Hammarstrom, FEBS Letts., 84, 271 (1978). P.P.K. Ho, C.P. Walters and H.R. Sullivan,Prostaglandins, 12, 951 (1976). P. Needleman, S. Moncada, S. Bunting. J.R. Vane, M. Hamberg and B. Samuelsson, Nature, 261, 558 (1976). P. Needleman, M.S. Minkes and A. Raz, Science, 193,163 (1976). M.J. Silver, J.B. Smith, C. Ingermary and J.J. Kocsis, ProstaRlandins, i, 863 (1973). 509 (1974). A.L. Willis, K. Comai, D.C. Kuhn and J. Paulsrud, Prostaglandins, M. Hamberg. J. Svensson, T. Wakabayashi and B. Samuelsson, Proc. Nat. Acad. Sci. USA, 2, 345 (1974). P. Falardeau, M. Hamberg and B. Samuelsson. Biochim. Biophys. Acta., 441, 193 (1976). M. Lagarde. A. Gharib and M. Dechavanne, Biochimie.. 2, 935 (1977). A. Raz. M.S. Minkes and P. Needleman, Biochim. Biophys. Acta., 488, 205 (1977). J.A. Salmon, D.R. Smith, R.J. Flower, S. Moncada and J.R. Vane, Biochim. Biophys. Acta., 523, 250 (1978). P.S. Kulkarni, R. Robert, P. Needleman', Prostaglandins,lZ, 337 (1976). A. Raz, P.C. Isakson, M.S. Minkes and P. Needleman. J. -Eel. Chem., 252, 1123 (1977). C.J. Kusting, S. Moncada and J.R. Vane, Prostaglandins, 13, 3 (1977). F. Cottee, R.J. Flower, S. Moncada, J.A. Salmon and J.R. Vane, Prostaglandins. 14, 413 (1977). W.-C. Chang and S.-I. Murota. Biochim. Biophys. Acta., 486, 136 (1977). H.-H. Tai and B. Yuan, A.T. Wu, Biochem. J., 170, 441 (1978). C.R. Pace-Asciak and G. Rangaraj, Biochim. Biophys. Acta., 528, 512 (1978). A.R.Wharton, M. Smigel. J.A. Oates and J.C. Frolich, Biochim. Biophys. Acta., 529, 176 (1978). E.A. M. De Deckere, D.H. Nugteren and F. Ten Hoor, Nature, 268, 160 (1977). P.C. Isakson, A. Raz, S.E. Denny, E. Pure and P. Needleman, Proc. Nat. Acad. Sci. USA., 101 (1977). S. Moncada, R. Korbut, S. Bunting and J.R. Vane, Nature, 273, 767 (1978). R.J. Gryglewski, R. Korbut and A. Ocetkiewicz, Nature, 273, 765 (1978).

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Chap. 17 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104, 105. 106. 107. 108. 109. 110. 111. 112. 113. 114.

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S. Moncada. A.G. Herman, E.A. Higgs and J.R. Vane, Thrombosis Res., 11, 323 (1977). R.A. Skidgel and M. P. Prlntz, Prostaglandins, 16, 1 (1978). J.L. Humes, R.J. Bonney, L. Pelus, M.E. Dahlgren. S.J. Sadowskl, F.A. Kuehl, Jr. and P. Davis, Nature, 269, 149 (1977). S.-I. Murota, M. Kawamura and I. Morita, Blochim. Biophys. Acta., 528, 507 (1978). N.L. Baenziger, M.J. Dlllender and P.W. Majerus. Blochem. Blophys. Res. Commun., 294 (1977). R.L. Tansik, D.H. Namm and H.L. White, Prostaglandins. g,399 (1978). S.-I. Murota, T. Yokoi. I. Morlta and Y. Mori, Blochem. Biophys. Res. Commun., 83, 679 (1978). L.A. Harker, G.E. Striker, R.T. Wall and L.J. Quadraccl, Clln. Res., 25, 318A (1977). B.B. Weksler, C.W. Ley and E.A. Jaffe, J. Clln. Invest., 62, 923 (1978). B.B. Weksler, A.J. Marcus and E.A. Jaffe. Proc. Nat. Acad. Scl. USA, 14, 3922 (1977). V. Tomasl, C. Meringolo, G. Bartolini and M. Orlandl, Nature, 273, 670 (1978). S.R. Saba and R.G. Mason, Thromb. Res., 2. 747 (1974). D.E. MacIntyre, J.D. Pearson and J.L. Gordon, Nature, 271, 549 (1978). R.L. Czervionke, J.B. Smith, J.C. Hoak, G.L. Fry and D. Haycraft, Thromb. Res., I n press. F.C. Grenier and W.L. Smith, Prostaglandins, 16, 759 (1978). F.A. Fltzpatrlck and R.R. Gorman. Blochim. Blophys. Acta., 539, 162 (1978) P. Needleman. B. Bryan, A. Wyche, S.D. Bronson, K. Eaklns, J.A. Ferrendell and M. Minkes, Proptaglandins, 16, 897 (1977). S. Moncada, S. Bunting, K. Mullane, P. Thorogood, J.R. Vane. A. Raz and P. Needleman. Prostaglandins, 2. 611 (1977). P. Needleman, A. Raz, J.A. Ferrendelli and M. Mlnkes, Proc. Nat. Acad. Sci 1716 (1977). USA, 3, T. Yoshlmoto, S. Yamamoto and 0. Hayalshi, Prostaglandins. la, 529 (1978). S. Moncada, R.J. Gryglewskl, S. Bunting and J.R. Vane, Prostaglandins, 12. 715 (1976). P. Borgeat, M. Hamberg and B. Samuelsson, J. Blol. Chem.. 251, 7816 (1976). M. Klndahl, Prostaglandins, l3, 619 (1977). L.J. Roberts, 11, B.J. Sweetman, J.L. Morgan, N.A. Payne and J.A. Oates, Prostaglandins. l3, 631 (1977). L.J. Roberts, 11, B.J. Sweetman and J.A. Oates, J. Biol. Chem.. 253, 5305 (1978). M.J. Hawkins. T.E. Eling. J.B. Smith and K.C. Nlcolaou, Prostaglandins. i n press. F.F. Sun, J.C. McGlff and P.Y.-K. Wong, Fed. Proc., 31, 916 (1978). F.Y.-K. Wong. F.F. Sun and J.C. McGiff, J. Biol. Chem., 253, 5555 (1978). J.M. Armstrong, N. Lattimer. S. Honcada and J.R. Vane, Br. Pharmacol., 62, 125 (1978). C.R. Pace-Asciak, H.C. Carrara and Z. Domazet, Biochem. Blophys. Res. Commun.. Is, 115 (1977). F.F. Sun and B.M. Taylor, Biochemistry. 17. 4096 (1978).

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Chapter 18. Drug Metabolism Bruce H. Migdalof, Kishin J. Kripalani, and Sampat M. Singhvi The Squibb Institute for Medical Research, New Brunswick, NJ 08903 Introduction - Drug metabolism studies of current drugs may provide valuable clues for the design of new, safer drugs with greater specificity of action. This chapter will present selected examples of recent developments in the field of drug metabolism which may be helpful to the medicinal chemist and may provide insight into the current state of theoretical understanding and technological advances in the field. Drug design must not only take into account the chemical and physicochemical properties which relate to intrinsic drug potency, but also those properties which relate to the ability of molecules to reach, and in vivo. Clearly, properties such as remain at, the site(s) of action -rate and extent of absorption, distribution, biotransformation, and excretion have to be considered in drug design. In the past few years the role of metabolic activation in the biogenesis of highly toxic entities has become increasingly clear. A knowledge of which biotransformation pathways tend to produce highly toxic metabolites, and which functional groups or molecular fragments are the best substrates for such bioactivation processes, may be used in the design of drug molecules for better safety as well as for the desired efficacy. Recent Books and Reviews - Recently ublished books contain general information of drug metabolism in mammals? and in man,2 drug disposition during development, biotransformations with emphasis on those aspects which relate to the formation of toxic metabolite^,^-^ the disposition of toxic drugs in many8 bioa~ailability~~and methods and techniques related to drug disposition.l o Other relevant books discuss drug design as related to adverse reactions,l 2 prodrugs,l 3 and sustained and controlled release.l 4 9

The following reviews are also of interest: "Effects of Renal Disease upon Drug D i s p ~ s i t i o n , ~ ~ ~ Metabolism and Pharmacokinetics in "Drug Ma1nutrition,"l6 "Novel Pathways in Drug Metabo1ism,"l7 "Individual Differences in the Disposition of Drugs Metabolized in the Body,"18 "PKa Values of Medicinal Compounds in Pharmacy Practice "Metabolism of Drugs and Other Foreign Compounds by Intestinal Microorganisms "Biliary Excretion of Drugs and Other Xenobiotics , t r 2 1 "Drug-Induced Liver Disease,"22 "Pulmonary Disease and Drug Kinetics, "Some Aspects of Pharmacokinetic and Biotransformation Differences in Human and Mammal Animals,"24 "Disease and Drug Protein Binding,"25 and "Toxic Agents Resulting from the Oxidative Metabolism of Steroid Hormones and Drugs. Methodology - Recently computers have been used by Wipke and coworkers to predict drug biotransformation pathways and products.27 Pang and Gillette have published a series of equations for evaluating sites and pathways of metabolism from blood level data.28 A method for determining binding constants for drug-protein complexes has been reported which utilizes serum albumin immobilized in microparticles of polyacrylamide.29 Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

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Much of the recent development in gas-liquid chromatogra hy (glc) has been in the use of more specific and sensitive detectors.3g-34 Separation of diasterioisomers of B-adrenoceptor antagonists (such as pindolol and propranolol) has been accomplished using high resolution capillary gas chr~matography.~~ Radiochemical glc has been used in the analysis of 3H-ethinyl estradiol in human urine.36 Use of high performance liquid chromatography (hplc) is increasing. An hplc assay of procainamide was developed which requires as little as 20 u 1 of plasma.37 Drug analysis by combined hplc-mass spectrometry has been reported by H e n i ~ n . ~ *A new thin-layer chromatography (tlc) chamber for short bed continuous development is now on the market.39 A spectrodensitometric tlc assay has been reported f o r propranolol ,40 and a fluorescence tlc-densitometric assay has been developed for chlordiazepoxide.4 1

- N ~ t a r ihas ~ ~reviewed the potential utility of prodrugs which exhibit rate-limiting first-order drug delivery. The hemisuccinate ester 1 has been shown to provide protection against first-pass metabolism after oral administration, and has been proposed as a prodrug of propranolol ( 2 ) .43 The prodrug, bitolterol ( 3 ) , the di-p-toluate ester of N-t-butylartereno1 (4) has been shown to provide lon er duration of bronchodilator activity in-dogs than does the parent drug.&4

Prodrugs

Two prodrugs of the analgesic acetaminophen (S), the tetrahydropyranyl ether derivative ( 6 ) and the ethyl vinyl ether derivative ( 7 ) , have been developed to overcome the bitter taste of the compound in-chewable dosage forms.45 The compound, 7,7‘-succinylditheophylline, was found to be a useful controlled-release prodrug of theophylline.46 The feasibility of delivering highly polar drugs to the CNS was demonstrated using 9 as a prodrug for the pyridinium salt 8, which can cross the blood-brain barrier.47 Prolonged antifertility activity for several contraceptive steroids was achieved in animals by subcutaneous injection of sustained release prodrugs in the form of zinc and aluminum tannate salts of tertiary amine derivatives.4 8 OR

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190

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- Metabolic

Diseases, Endocrine Function

Bailey, Ed.

Pharmacokinetics and Pharmacodynamics - A good correlation was shown between the logarithm of blood concentration and the percentage reduction in exercise heart rate for propranolol (2) indicating, for this dru predictability of f3 -adrenoceptor blockade-from blood concentrations.&9.A dose-response relationship was shown between blood levels of d-amphetamine and magnitude of blood pressure and heart rate responses in rats under ganglionic blockage.5 0 The dose-dependent (nonlinear) pharmacokinetics of drugs may have important clinical implications. A disproportionate increase in steady-state plasma concentrations of quinidine was observed in man with increasing doses. 51 Comparing the values of area under the curves of plasma concentration time following oral and intravenous administration of the antiarrhythmic drug disopyramide led to an erroneous estimate of its bioavailability in man, since clearance of the drug was different for the two routes of administration.5 2 Nonlinear elimination kinetics of sulfadiazine in rabbits were attributed to protein-binding interactions between the parent compound and its N-acetyl metabolite.5 3

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A linear relationship was observed between the renal clearance of nadolol (10) and the glomerular filtration rate (GFR) in dogs in different stages OfTxperimentally-induced renal impairment.54 The body clearance of two other B-adrenergic blocking agents, atenolol (11) 5 5 and pindolol (12),56 was also found to correlate with the GFR. ReGction in bioavailazTlity of pindolol in renally impaired patients was attributed to a decrease in drug absorption.56 The rate of elimination of netilmicin, an aminoglycoside was found to decrease proportionally with decreasing renal function.5 3 Pharmacokinetic studies of Lidaprin@ (a trimethoprimesulfametrole combination) in renally impaired patients showed a strong influence of renal function on the accumulation of the metabolized sulfonamide, a weaker influence on trimethoprime, and negligible influence on the free sulfonamide.58

OCH2CHOHCH2NH- ~ - B u

The renal clearance of sulfamethoxazole, but not of its metabolite, N4-acetylsulfamethoxazole, was markedly influenced by urinary pH and urine flow.59 Pharmacokinetic analysis of percutaneous absorption showed evi-

dence of parallel penetration pathways for methotrexate when it was topically applied to hairless mouse skin.60 A recent review summarized the saliva to plasma ratios of a number of drugs and discussed the usefulness of salivary concentrations in therapeutic drug monitoring and in calculation of pharmacokinetic parameters.6 1 Interrelationships among renal hemodynamics, drug kinetics and drug action was recently reviewed.62 The effect of pulmonary disease on disposition kinetics of drugs has been reviewed.23 A mini-review on pharmacokinetic and biotransformation differences among mammalian species suggested that species differences are usually of a quantitative nature, due to such differences as the ratio of body weight to surface area, organ weight

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to body weight, o r presence of differing fat deposits.24 Plasma and Tissue Protein Binding - The extent of, and changes in, the binding of drugs and their metabolites to plasma and tissue proteins can greatly affect their distribution and elimination and may, therefore, influence the extent and duration of effect. A comprehensive review of plasma protein binding of drugs in disease states and its influence on the pharmacologic effects was recently published.2 5 The R (+) enantiomer ofgliflumide (13), a new antidiabetic drug, was less potent in lowering blood glucose rats thangliflumide and had reduced affinity to plasma protein, indicating the binding to receptor sites and to plasma protein was stereospecific.6 3 Protein binding studies of a new cholangiographic agent, iodoxamic acid, to monkey plasma indicated two classes of binding sites, and binding solely to plasma albumin.64 Serum protein binding studies in man, dog, rat, and mouse of the anticonvulsant drug valproate (14)indicated that increased drug bindin was associated with a decrease in the total body clearance of the drug.6f The effect of protein binding on pharmacokinetics and therapeutic activity of several cephalosporin antibiotics was recently reviewed.6 6 Theoretical relationships were presented for the assessment of changes in the binding of drugs to tissues, based on determinations of total clearance, intrinsic clearance, apparent volume of distribution, and biological half-life.67

Drug Interactions - Absorption half-life of the antibiotic, clindamycin, was prolonged in human subjects when a kaolin-pectin antidiarrheal suspension was coadministered, although the extent of absorption remained the same.68 On the other hand, both the rate and the extent of absorption of digoxin were lower when it was coadministered with the same kaolin-pectin suspension.69 Physical absorption and alterations in gut transit time appeared to partly explain the findings. The antitussive activity of dextromethorphan hydrobromide in the unanesthetized dog was faster in onset, greater in intensity, and longer in duration when it was coadministered with salicylamide and acetominophen, due probably to inhibition of the metabolism (conjugation) of dextrorphan, an active metabolite of the parent drug.70 In rats, acute administration of ethanol increased the concentrations of methadone in brain and liver, apparently due to inhibition of methadone metabolism in the liver, whereas chronic ethanol administration had the op osite effect due to increased liver microsomal metabolism of methadone In rat liver preparations, glucuronidation of acetaminophen was inhibited by a variety of drugs, e.g., morphine, dicumarol, hydroxyzine, phenolphthalein, chloramphenicol, and tetracycline.72 In dogs, indomethacin pretreatment inhibited the hemodynamic response to furosemide and reduced the renal and extrarenal clearance of furosemide by approximately 30%, but did not change the proportion of unchanged drug excreted in urine.73 The effects of alterations in blood flow, enzyme activity, and reversible dru binding to blood components on total body clearance have been discussed.78

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Bailey, Ed.

Drug Metabolizing Enzymes - The possible role of excited states of oxygen (e.g., singlet oxygen and the superoxide ion) in cytochrome P-450 linked oxidations, as well as in the induction of the P-450 system by many diverse compounds, has been discussed.75 A good correlation was found between the binding affinity of a wide variety of organic compounds to cytochrome P-450 and their lipophilicity.76 The enzyme system responsible for 7 a-hydroxylation of cholesterol was found to be distinct from the mixed function oxy enases system that catalyzes oxidation of a large number of xenobi~tics.?~In rabbit liver, two stereoselective enzyme systems, o r different conformations of binding of different substrates to the same enzyme system, were suggested to be res onsible for a-carbon oxidation and N-oxidation of ethylamphetamine (15) .T8 Chromatographic and electrophoretic studies indicated extensiveheterogeneity of cytochrome P-450 from rat liver. 79 Dihydropyrimidinase, an enzyme responsible for the hydrolytic ring 1 7 ) , was paropening reactions of dihydrothymine (16) and dihydrouracil (tially purified and characterized fromcalf and rat liver. The enzyme from both sources catalyzed the hydrolytic ring-opening of dihydrouracil, hydantoin (18), - 5-phenylhydantoin (19), - and a-phenylsuccinimide (20). Because many drugs contain either chiral centers, prechiral centers, or both, interest in stereochemical substrate-enzyme interactions, the stereospecificity of biotransformations, and species (and strain) differences in these parameters is increasing. Since enzymes themselves contain chiral centers, differential interaction of R and S isomers of drugs with dru metabolizing enzymes is the rule rather than the exception. BeckettaF reported stereoselectivity in the N-dealkylation, deamination, and formation of the nitrone and secondary hydroxylamine metabolites (+) and ( - ) - N-benzylamphetamine (21) in rabbits. Stereoselectivity has also been observed in the dealkylation of d-, 1-, and d,l-fenfluramine (22), an anorexiogenic agent.82

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Extrahepatic Drug Biotransformation - Although the liver is the major o r gan for biotransformation of xenobiotics, other organs, e.g., kidney, lung, intestine, etc., also possess this capability to varying degrees. Glucuronidation of diethylstilbestrol was shown to occur in all regions

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Drug Metabolism Migdalof, Kripalani, Singhvi

193

of the intestine.8 3 Both glucuronidation of a major digitoxin metabolite and hydrolysis of the glucuronide conjugate took place in various segments of the rat intestine.84 The effect of gut wall metabolism, hepatic elimination and enterohepatic recycling on estimates of bioavailability have been examined from a theoretical standpoint.85 An antischistosomal isothiocyanate (23) was converted to a muta en in six mammalian species by intestinal bacteria (metabolic activation) .86 Oxidative metabolism of polycyclic aromatic hydrocarbons to arene oxides (epoxides) was observed in the lung.87 In the isolated perfused rabbit lun A9-tetrahydrocannabinol was metabolized to at least six metabolites.

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Interspecies Comparative Metabolism - An unusual species difference was reported for absorption of nadolol* (10).Whereas, absorption of an oral dose of nadolol in mice, rats, hamsters, rabbits, monkeys, and man ranged from ca. 8 to 34%, the absorption in the dog was essentially complete. Species differences in metabolism appeared to explain in part the inconsistent and low physiologic response (diuresis) of MK-473 (24) observed in the dog, a species in which minimal MK-473 metabolism was ~ z e r v e d . ’ ~In other species, a number of pharmacologically active diestereoisomeric metabolites were produced by hydroxylation of the cyclopentyl ring. Reduction of the 6-keto group in hydrocodone to the 6-alcohol was stereoselective in various animal species, but not in man.91 In the rabbit and dog, the principal metabolites of isoxepac (25) were the glycine and taurine conjugates, respectively, whereas in the rhesus monkey and man, isoxepac was excreted unchanged or as the glucuronide.

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24

25 -

Biotransformation and Drug Toxicity - In the past few years significant progress has been made in understanding the relationship between biotransformation and toxicity of numerous drugs. Evidence has been presented that biotransformation is responsible for the unusual triphasic lethal in vitro conversion dose curve obtained for d’amphetamine in mice.93 The -of the N-0-glucuronide and N-0-sulfate conju ates of N-hydroxyphenacetin to reactive intermediates has been r e p ~ r t e d . Isolation ~~ of 2-hydroxyphenacetin as a decomposition product also suggested a possible non-arene oxide pathway for the aromatic hydroxylation of certain aromatic amines. Metabolic activation is used routinely in mutagenicity testing, and a method has recently been presented for using metabolic activation in asAwareness of the greater potential for certain sessing cytotoxicity.’ molecular substructures to result in carcinogenicity of drugs containing them has led the FDA‘s Bureau of Foods to publish a list of 13 substructural moieties which enhance the probability of carcin~genicity.~ Alteration of drug biotransformation by structural modification to reduce toxicity has been discussed,13 as have the problems in relating toxicologiA dose dependenc € o r acetaminophencal effects and drug disposition.’ induced renal necrosis in rats has been demonstrated.r8 Differences in the mutagenicity of the 7 , 8 - and 9,lO-dihydrodiols of benzo[a]pyrene have

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been attributed to differences in metabolic pathways .99 Novel Biotransformation Pathways - The recent literature contains examples of some of the newly discovered pathways in addition to those covered in the excellent review by Jenner and Testa.17 Formation of an aliphatic 0-methyl metabolite (26) of the antiinflammatory agent cicloprofen (27) in the rat, has been and it was proposed that it formed via 0-methylation of a dihydroxy intermediate (28). Thiomethyl metabolitrof phenacetin and acetaminophen have been found in dogs and man. l o l An S-methyl metabolite (29) of bromazepam (30), a minor tranquilizer, in the rat was shown to be formed -in vitro by biotransformation of lo2 a mecapturic acid conjugate (2).

R

REFERENCES 1. D.E. Hathway, senior ed., "Foreign Compound Metabolism in Mammals," Vol. 4, A Specialist Periodical Report, The Chemical Society, London 1977. 2. J.W. Gorrod and A.H. Beckett, "Drug Metabolism in Man,!' Taylor and Francis, London 1978. 3 . P.L. Morselli, ed., "Drug Disposition During Development," Spectrum Publications, New York 1977. 4. V. Ullrich, I. Roots, A.G. Hildebrant, R.W. Estabrook, and A.H. Conney, eds., "Microsomes and Drug Oxidations," Pergamon Press, Oxford 1977. 5 . D.J. Jollow, J.J. Kocsis, R. Snyder, and H. Vainio, eds., "Biological Reactive Intermediates: Formation, Toxicity, and Inactivation," Plenum Press, New York 1977. 6 . A. Aito, ed. , ItConjugationReactions in Drug Biotransformations, I t Elsevier/North-Holland, New York 1978. 7. J.W. Gorrod, ed., "Biological Oxidation of Nitrogen," Elsevier/NorthHolland, New York 1978. 8. R.C. Baselt, "Disposition of Toxic Drugs and Chemicals in Man, Volume 1 - Centrally Acting Drugs,tfBiomedical Publications, Canton, Connecticut 1978. 9. J. Blanchard, R.J. Sawchuck, and B.B. Brodie, eds., "Principles and Perspectives in Drug Bioavailability," Karger, New York 1978. 10. E.R. Garret and J.L. Hirtz, eds. , !'Drug Fate and .Metabolism," Vol. 2, Marcel Dekker, New York 1978. 11. A. Frigerio, ed., '%?cent Developments in Mass Spectrometry in Biochemistry and Medicine," Vol. 1, Plenum Press, New York 1978. 12.H. Bungaard, P. Juul and H. Kofod, eds., "Drug Design and Adverse Reaction," Academic Press, New York 1977.

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13. E.B. Roche, e d . , "Design of Biopharmaceutical P r o p e r t i e s through Prodrugs and Analogs," American Pharmaceutical A s s o c i a t i o n , Washington, D . C . 1977. 1 4 . J . R . Robinson, "Sustained and C o n t r o l l e d Release Drug Delivery Systems," Marcel Dekker, New York 1978. 15. M.M. Reidenberg and D . E . Drayer, Drug Metab. Rev. 8 , 292 (1978). 16. K . Krishnaswamy, C l i n . Pharmacokinet. 3 , 216 (19787. 1 7 . P. J e n n e r and B . T e s t a , Xenobiotica 8, 1 (1978). 18. G . Alvan, C l i n . Pharmacokinet. 3 , 15% (1978). 1 2 , 546 19. D.W. Newton and R . B . Kluza, Drug I n t e l l . and C l i n . Pharm. (1978). 20. R.V. Smith, World Rev. Nutr. D i e t , 29, 60 (1978). 2 1 . W . G . Levine, Ann. Rev. Pharmacol. T G i c o l . 18, 81 (1978). 2 2 . H . J . Zimmerman, Drugs 16, 25 (1978). 23. P. duSouich, A . J . McLez, D. Lalka, S. E r i l 1 , a n d M. G i b a l d i , C l i n . Pharmacokinet. 3, 257 (1978). 24. A. Chodera and KT F e l l e r , I n t . J . C l i n . Pharmacol. Biopharm., 357 (1978). 3, 25. J . P . T i l l e m e n t , F . Lhoste,and J . F . G i u d i c e l l i , C l i n . Pharmacokinet. 144 (1978). 26. E . C . Horning, J . - P . Thenot, and E . D . Helton, J . Tox. Environ. Health 4, 341 (1978). 2 7 . M.L. Spann, K . C . Chu, W.T. Wipke, and G . Ouchi, J . Environ. P a t h o l . Tox. 2 , 123 (1978). 28. K.S. pang and J . R . G i l l e t t e , J . Pharm. S c i 67, 703 (1978). 29. A . Kober, B. Ekman, and I . Sjuholm, J . P h a r F S c i . 67, 107 (1978). 30. R . Venkataramanan and J . Axelson, J . Pharm. S c i . 67,201 (1978). 31. A.P. DeLeenheer and C . F . G e l i j k e n s , J . Pharm,. Sci.67, 417 C1978). 32. S.H. Weinstein, M. A l t e r a s and J. Gaylord, J . PharmTSci. 67, 547 (1978) 33. P .T . Funke, M.F. Malley, E . I v a s h k i v , and A . I . Cohen, J . Pharm. S c i . 67, 653 (1978). 34. KG. J e n k i n s and R.O. F r i e d e l , J . Pharm. S c i . 67,1 7 (1978). 35. S. Caccia, C . Chiabrando, P . DePonte, and R. F a n e l l i , J . Chromatograph. S c i . 16,543 (1978). 36. F. Z u l e s k i , A. Loh, and F . J . DiCarlo, J. Pharm. S c i . 67, 1138 (1978). 37. M.A.F. G a d a l l a , G.F. Peng, and W.L. Chiou, J . Pharm. Si. 67 , 869 (1978). 38. J . D . Henion, Anal. Chem. 50, 1687 (1978). 39. P.F. L o t t , J . R . Dias, a n d T . C . Slahck, J . Chromatog. S c i . 16, 571 (1978) 40. B . W . Hadzija and A.M. Mattocks, J . Pharm. S c i . 67, 1307 (1978). 41. S.-R. Sun, J. Pharm. S c i . 67, 639 (1978). 42. R . E . N o t a r i , I n E . B. RocheFed. , "Design of Biopharmaceutical Propert i e s through Prodrugs and Analogs," A.Ph.A., Washington, D . C . 1977, pp. 68-97. 43. Y. Garceau, I . Davis, and J . Hasegawa, J . Pharm. S c i . 67, 1360 (1978). 44. H. Minatoya, J . Pharmacol. Exp. Ther. 206, 515 (19781. 67, 545 45. A . Hussain, P . Kulkarni, and D . P e r r i e T J . Pharm. S c i . (1978). 46. N . Bodor, K . B . Sloan, Y .- N . Kuo and T. Higuchi, J . Pharm. S c i . 1045 (1978). 67, 685 (1978). 47. N . Bodor, R . G . R o l l e r , and S.J. S e l k , J . Pharm. S c i . -

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48. A.P. Gray and T.N. Yamauchi, J. Med. Chem. 21, 712 (1978). 49. J. McAinsh, N.S. Baber, R. Smith, and J . Young, Br. J. Clin. Pharmacol. 6, 115 (1978).

50. L.L. Simpson, J. Pharmacol. Exp. Ther. 205, 366 (1978). 51. Bohme and U. Otto, Europ. J. Clin. PharGol. 12, 73 (1977). 52. J.L. Cunningham, D.D. Shen, I. Shudo, and D.L.Azarnoff, B r . J. Clin. Pharmacol. 5, 343 (1978). 53. P. duSouich7A.J. McLean, D. Lalka, N. Vicuna, E. Chauhuri, and J . L . McNay, J. Pharmacol. Exp. Ther. 207, 228 (1978). 54. S.M. Singhvi, A.F. Heald, B.F.Musy, L.T. DiFazio, E.C. Schreiber, 43, 99 (1978). and J.W. Poutsiaka, Toxicol. Appl. Pharmacol. 55. J. Sassard, N. Pozet, J. MaAinsh, J. Legheand, and P. Zech, Europ. J. Clin. Pharmacol. 12, 175 (1977). 56. D. Lavene, Y.A. Wxss, M.E. Safar, Y. Loria, N. Agorus, D. Georges, and P.L. Milliez, J. Clin. Pharmacol. 17, 501 (1977). 3, 57. J.-C. Pechere, R. Dugal, and M.-M. PecEre, Clin. Pharmacokinet. 395 (1978). 58. G. Hitzenberger, A. Korn, H. Pichler, J . Bonelli, and D. Magometschnigg, Int. J. Clin. Pharmacol. Biopharm. 15, 310 (1977). 59. T.B. Vree, Y.A. Hekster, A.M. Baars, J.E.&sma, and E. Kleijn, Clin. Pharmacokinet. 3 , 319 (1978). 60. S.M. Wallace a d G. Barnett, J . Pharmacokinet. Biopharm. 6, 315 (1978). 61. M. Danhof and D.D. Breimer, Clin. Pharmacokinet. 3, 39 (1978). 62. K.L. Duchin and R.W. Schrier, Clin. Pharmacokinet: 3, 58 (1978). 27, 63. E. Schillinger, I. Ehrenberg, and K. LGbke, Biochem: Pharmacol. 651 (1978). 67, 64. S.K. Lin, A.A. Moss,’R. Motson, and S. Riegelman, J. Pharm. Sci. 930 (1978). 65. W. Loscher, J. Pharmacol. Exp. Ther. 204, 255 (1978). 66. S.M. Singhvi, A.F. Heald, and E.C. S c E i b e r , Chemotherapy 2, 121 (1978). 67. M. Gibaldi, G. Levy, and P.J. McNamara, Clin. Pharmacol. Ther. 24, 1 (19781. 68. K.S. Albert, K.A. DeSante, R.D. Welch,and A.R. DiSanto, J . Pharm. Sci. 67, 1579 (1978). 69. T S . Albert, J.W. Ayres, A.R. DiSanto, D.J. Weidler, E. Sakmar, M.R. Hallmark, R.G. Stoll, K.A. DeSante, and J.G. Wagner, J . Pharm. Sci. 67, 1582 (1978). 67, 761 70. G. Ramachander, F.D. Williams, and J.F. Emele, J . Pharm. Sci. (1978) 207, 123 71. S.A. Borowsky and C.S. Lieber, J. Pharmacol. Exp. Ther. 119781. 206, 233 72. W. Bolanowska and T. Gessner, J. Pharmacol. Exp. Ther. (1978) . 73. J.L. Data, A. Rane, J. Gerkens, G.R. Wilkinson, A.S. Nies, and R.A. Branch, J. Pharmacol. Exp. Ther. 206, 431 (1978). 22, 623 (1977). 74. J.R. Gillette and K.S. Pang, Clin-harmacol. Ther. 27, 1805 (1978). 75. A . J . Paine, Biochem. Pharmacol. 76. K.A.S. Al-Gailany, J.B. Houston, and J.W. Bridges, Biochem. Pharmacol. 27, 783 (1978). 27, 77. K S . Mellon, D.T. Witiak, and D.R. Feller, Biochem. Pharmacol. 1055 (1978).

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78. A.H. Beckett and K . Haya, Xenobiotica 8. 85 11978). 6 , 353 79. M. Warner, M.V. LaMarca,.and A.H. Neims; Drug Metab. Dispos. (1978) . 80. J.H. Maguire and K.H. Dudley, Drug Metab. Dispos. 6 , 601 (1978). 81. A . H . Beckett and G . G . Gibson, Xenobiotica 8 , 7 3 (1978). 82. A. J o r i , P. DiPont, and S. Caccia, XenobioTica 8, 583 (1978). 83. J . Lasker and D.E. Rickert, Xenobiotica 8, 665 (1978). 84. R . F . Volp and G . L . Lage, Drug Metab. Dispos. 6, 418 (1978). 85. K.S. Pang and J . R . G i l l e t t e , J . Pharmacokinet: Biopharm. 6 , 355 (1978) 86. R . P . B a t z i n g e r , E . Bueding, B.S. Reddy, and J . H . Weisburger, Cancer Res. 38, 608 (1978). 87. B.R. Smith, J . H . Maguire, L.M. Ball, and J . R . Bend, Fed. Proc. 37, 2480 (1978). 88. F . P . Law, Drug Metab. Dispos. 6 , 154 (1978). 89. J . Dreyfuss, J . M . Shaw, and J.J. Ross, J r . , Xenobiotica 8 , 503 (1978). 90. A . G . Zacchei, T . I . Wishousky, and L.S. Watson, Drug Metab. Dispos. 6, 313 (1978). 91. E . J . Cone, W.D. Darwin, C.W. Gorodetsky, and T. Tan, Drug Metab. Dispos. 6 , 488 (1978). 92. H.P.A. I i l i n g and J . M . Fromson, Drug Metab. Dispos. 6 , 510 (1978). - 63 93. R . C . James and M.R. F r a n k l i n , Toxicol. Appl. Pharmaczl. 44, (1978). 94. G . J . Mulder, J . A . Hinson, and J . R . G i l l e t t e , Biochem. Pharmacol. 27, 1641 (1978). 95. P. Wiebkin; J . E . Fry, and J . W . Bridges, Biochem. Pharmacol. 27, 1849 (1978) 96. Food Chemical News, p. 16, Oct. 1978. 97. S.D. Nelson, In E.B. Roche, e d . , "Design of Biopharmaceutical Propert i e s through Prodrugs and Analogs," A.Ph.A., Washington, D . C . 1977, pp. 316-343. 98. R . J . McMurtry, W.R. Snodgrass, and J . R . M i t c h e l l , T o x i c o l . Appl. Pharmacol. 46, 87 (1978). 99. D.R. T h a k k e c H. Yagi, R . E . Lehr, W . Levin, M. Buening, A.Y.H. Lu, R . L . Chang, A.W. Wood, A . H . Conney, and D.M. J e r i n a , Molec. Pharmac o l . 14, 502 (1978). 100. S . J . E n , A.V. Dean, K . J . K r i p a l a n i , and A . I . Cohen, Xenobiotica 8, 1 2 1 (1978). 101. A. Klutch, W. Levin, R . L . Chang, F. Vane, and A.H. Conney, Clin. Pharmacol. Ther. 24 287 (1978). 27, 809 102. M. T a t e i s h i , S. S E u k i , and H. Shimizu, Biochem. Pharmacol. (1978).

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Chapter 1 9 .

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M i t c h e l l N . Cayen, A y e r s t R e s e a r c h L a b o r a t o r i e s , M o n t r e a l , Quebec, Canada I n t r o d u c t i o n - The L i p i d H y p o t h e s i s is t h e premise t h a t r e d u c i n g t h e l e v e l s of serum l i p i d s i n p a t i e n t s w i t h h y p e r l i p i d e m i a w i l l l e a d t o a r e d u c t i o n i n t h e i n c i d e n c e of c o r o n a r y h e a r t d i s e a s e (CHD).' In last y e a r ' s chapter of t h i s series, t h e p h a r m a c o l o g i c a l c o n t r o l of serum l i p o p r o t e i n s was reviewed.2 I n t h i s review, r e c e n t developments i n a t h e r o s c l e r o s i s r e g r e s s i o n , h i g h d e n s i t y l i p o p r o t e i n s , and e s t a b l i s h e d and newer a n t i h y p e r l i p i demic a g e n t s a r e o u t l i n e d . Recent work on t h e m e t a b o l i c d i s p o s i t i o n of some of t h e l i p i d l o w e r i n g d r u g s i s d i s c u s s e d . F i n a l l y , a d v a n c e s i n t h e t h e r a p e u t i c c o n t r o l of c h o l e s t e r o l g a l l s t o n e s a r e summarized. R e g r e s s i o n of A t h e r o s c l e r o s i s - The g o a l i n e s t a b l i s h i n g t h e v a r i o u s r i s k f a c t o r s of CHD i s t o d e t e r m i n e whether t h e c o u r s e of t h e d i s e a s e c a n b e modified by a d j u s t m e n t of t h e i n d i v i d u a l ' s l i f e s t y l e . E v i d e n c e h a s begun t o accumulate t h a t human a t h e r o s c l e r o s i s c a n b e a r r e s t e d o r p a r t i a l l y rev e r s e d . 3 Animal models used t o s t u d y t h e reversal of e x p e r i m e n t a l a t h e r o s c l e r o s i s have been r e c e n t l y reviewed. 3 Non-human p r i m a t e s , r a b b i t s , swine, fowl and dogs a r e commonly used. The aims of a n i m a l s t u d i e s a r e t o e x p l o r e t h e mechanisms u n d e r l y i n g r e g r e s s i o n , and t o e s t a b l i s h t r e a t m e n t of t h e human d i s e a s e by d i e t , d r u g s , s u r g e r y a n d / o r e x e r c i s e . ' R e c e n t l y , d i e t - i n d u c e d r e g r e s s i o n h a s been d e m o n s t r a t e d i n swine' and monkeys. 6 , 7 A l f a l f a meal c o u n t e r a c t e d t h e a t h e r o g e n i c i t y of d i e t a r y c h o l e s t e r o l i n cynomolgus macaques. Niceritrol , 8-pyridylcarbinol (2) a n d , t o a lesser d e g r e e , n i c o t i n i c a c i d (NA) (3) p a r t i a l l y r e v e r s e d d i e t induced a t h e r o s c l e r o s i s i n m i n i p i g s . A greater reduction i n the s i z e of c o r o n a r y l e s i o n s i n a t h e r o s c l e r o t i c m i n i p i g s w a s found w i t h a comb i n a t i o n of moderate d i e t p l u s c l o f i b r a t e t h a n w i t h d i e t a l o n e . " Cholestyramine produced a p a r t i a l reversal of a t h e r o s c l e r o s i s i n r h e s u s monk e y ~ . C~ l o f i b r a t e r e t a r d e d t h e development of s p o n t a n e o u s a t h e r o s c l e r o s i s and reduced t h e m o r b i d i t y and i n r e p e a t e d l y - b r e d Sprague-Dawley r a t s , m o r t a l i t y from a c u t e m y o c a r d i a l i n f a r c t i o n induced by i n t r a v e n o u s l y i n jected isoproterenol." The a n t i m e t a b o l i t e s m e r c a p t o p u r i n e and hydroxyu r e a were a n t i - a t h e r o g e n i c i n swine.' It w a s s u g g e s t e d t h a t t h e a n t i a t h e r o g e n i c a c t i o n of p y r i d i n o l c a r b a m a t e and p h t h a l a z i n o l i n r a b b i t s and man was due t o 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 r e s u l t i n g from a n t a g o n i s t i c e f f e c t s on thromboxane-A2."13 On t h e o t h e r hand, t h e a n t i h y p e r t e n s i v e d r u g s h y d r o c h l o r o t h i a z i d e , methyldopa, g u a n e t h i d i n e , m i n o x i d i l o r h y d r a l a z i n e d i d n o t a l t e r t h e s e v e r i t y o r e x t e n t of a t h e r o s c l e r o t i c l e s i o n s i n s t u m p t a i l macaques w i t h h y p e r t e n s i o n (induced by narrowing of t h e r e n a l a r t e r y ) and a t h e r o s c l e r o s i s ( h i g h f a t - c h o l e s t e r o l d i e t ) . 6

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-3 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0-12-040514-8

Chap. 1 9

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Cayen

199

A d e c r e a s e i n serum c h o l e s t e r o l l e v e l s r e s u l t s i n a d e c r e a s e n o t o n l y i n t h e l i p i d c o n t e n t b u t a l s o i n t h e s i z e of e x p e r i m e n t a l l y ' i n d u c e d a t h e r o s c l e r o t i c l e s i o n s . Evidence of t h i s a s s o c i a t i o n i n man h a s been d e m o n s t r a t e d by c o r o n a r y a n g i o g r a p h y . '' Human f e m o r a l a t h e r o s c l e r o s i s r e g r e s s e d i n r e s p o n s e t o d i e t a n d / o r d r u g t h e r a p y ( c l o f i b r a t e , NA, o r c l o f i b r a t e i n combination w i t h neomycin). 1 4 , I s The d e g r e e of r e t a r d a t i o n of a t h e r o s c l e r o s i s w a s d i r e c t l y c o r r e l a t e d w i t h t h e d e c r e a s e i n serum c h o l e s t e r o l l e v e l s . 1 4 , l 6 R e g r e s s i o n i n p a t i e n t s w i t h Type I V h y p e r l i p o p r o t e i n e m i a w a s a l s o a s s o c i a t e d w i t h t h e d e c r e a s e i n serum t r i g l y c e r i d e l e v e l s ; 1 6 however, no such c o r r e l a t i o n w a s found i n a n o t h e r s t u d y . ' '

Serum l i p i d s and l i p o p r o t e i n s - The s t u d y of t h e c o n t r o l o f serum l i p i d l e v e l s and t h e i r r e l a t i o n s h i p t o CHD h a s e v o l v e d t h r o u g h s e v e r a l p h a s e s . I n i t i a l l y , t h e emphasis w a s on serum c h o l e s t e r o l l e v e l s , s t i m u l a t e d no doubt by t h e a v a i l a b i l i t y of methods t o measure c h o l e s t e r o l . Epidemiol o g i c a l s t u d i e s e s t a b l i s h e d c h o l e s t e r o l a s a r i s k f a c t o r of CHD. This was f o l l o w e d by c o n s i d e r a t i o n of serum t r i g l y c e r i d e s , t h e r o l e of which a s an i n d e p e n d e n t r i s k f a c t o r s t i l l r e m a i n i n g t o b e f u l l y e l u c i d a t e d . " Later, i t became a p p a r e n t t h a t e x a m i n a t i o n o f t h e c o n t r o l o f l i p i d t r a n s p o r t i s n e c e s s a r y f o r t h e u n d e r s t a n d i n g and management of l i p i d d i s o r d e r s . A s a n a l y t i c a l methodology became more s o p h i s t i c a t e d , t h e r o l e of i n d i v i d u a l l i p o p r o t e i n s i n a t h e r o s c l e r o s i s began t o b e e v a l u a t e d . Thus, low d e n s i t y l i p o p r o t e i n s (LDL), v e r y low d e n s i t y l i p o p r o t e i n s (VLDL) and chylomicrons a r e a t h e r o g e n i c , w h i l e h i g h d e n s i t y l i p o p r o t e i n s (HDL) a r e A l l t h e l i p o p r o t e i n s a r e m e t a b o l i c a l l y reconsidered anti-atherogenic. l a t e d and i n t e r a c t d y n a m i c a l l y w i t h i n t h e v a s c u l a r system. A s c l i n i c a l e v i d e n c e c o n t i n u e s t o a c c u m u l a t e on t h e i n v e r s e c o r r e l a t i o n between HDL-cholesterol (HDL-C) l e v e l s and t h e i n c i d e n c e of CHD, "-" c o n s i d e r a b l e a t t e n t i o n i s b e i n g d e v o t e d t o t h e m e t a b o l i c c o n t r o l o f HDL and f a c t o r s a f f e c t i n g c i r c u l a t i n g HDL l e v e l s (reviewed i n r e f s . 24-30). Two mechanisms have been proposed ( n o t n e c e s s a r i l y m u t u a l l y e x c l u s i v e ) F i r s t , HDL may f a c i l i t a t e t h e whereby HDL may slow a t h e r o g e n e s i s . 2 3 9 2 8 removal of c h o l e s t e r o l from t h e a r t e r i a l w a l l . 3 1 Th is a c t i o n i s mediated by t h e a c t i o n of l e c i t h i n - c h o l e s t e r o l a c y l t r a n s f e r a s e (LCAT), t h e enzyme which c a t a l y z e s t h e e s t e r i f i c a t i o n of f r e e c h o l e s t e r o l i n t h e HDL p o l a r s u r f a c e . A s t h e c h o l e s t e r o l e s t e r s m i g r a t e t o t h e n o n p o l a r c o r e o f HDL, t h e m o d i f i e d s u r f a c e of HDL c o u l d a c c e p t more f r e e c h o l e s t e r o l from t h e p e r i p h e r a l c e l l s . The p o s s i b l e r o l e of HDL i n c h o l e s t e r o l e x c r e t i o n w a s s u g g e s t e d by a s t u d y which showed t h a t c h o l e s t e r o l from HDL was more r e a d i l y i n c o r p o r a t e d i n t o b i l i a r y c h o l e s t e r o l t h a n c h o l e s t e r o l from LDL. 3 2 Second, HDL may i n h i b i t t h e u p t a k e of a t h e r o g e n i c LDL by t h e a r t e r i a l w a l l . The p i o n e e r i n g work of G o l d s t e i n and Brown h a s e s t a b l i s h e d t h e f u n c t i o n of LDL r e c e p t o r s i n t h e p a t h o g e n e s i s of a t h e r o s c l e r o s i s , 3 3 and i n v i t r o models w i t h v a r i o u s human and a n i m a l c e l l t y p e s have shown t h a t HDL i n h i b i t s LDL b i n d i n g and u p t a k e by t h e c e l l s . z 8 I t r e m a i n s t o b e e s t a b l i s h e d whether m a n i p u l a t i o n of HDL l e v e l s w i l l i n i t s e l f a l t e r t h e development of CHD. The components of HDL a r e d e r i v e d from d i f f e r e n t s o u r c e s . y z 6, 9 7 3 0 9 3 4 HDL ( o r i t s p r e c u r s o r s ) i s produced by b o t h t h e l i v e r and i n t e s t i n e . The s t r u c t u r a l c o m p o s i t i o n of HDL i s r e g u l a t e d by LCAT and l i p o p r o t e i n l i p a s e . T h e r e i s a l s o non-enzymatic t r a n s f e r of a p o l i p o p r o t e i n (apo) C between HDL and VLDL, exchange of f r e e

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cholesterol between HDL and cell membranes, and the association of apo A - I with HDL. Part of circulating HDL is derived from catabolism of VLDL and chylomicrons, and numerous studies have shown reciprocal relationships between HDL-C and serum triglycerides. 9 Agents which induce liver microsomes elevate HDL-C; thus alcohol consumption, exposure to chlorinated hydrocarbon insecticides,3 7 and treatment with phenytoin” caused increased HDL-C. HDL-C was elevated by exercise,3 9 and in men treated with estrogen for prostatic carcinoma.“ HDL-C was lowered by smoking,41,4zdietary carbohydrate,43 and by antihypertensive therapy with a combination of hydrochlorothiazide and propranolol. 44 Oral contraceptives were reported to both increase4’ and decrease4’ HDL-C. Since estrogens elevate and progestins lower HDL-C , the effect of oral contraceptives on HDL-C appears to depend upon the relative estrogen/progestin potencies of the preparations. 4 6 Antihyperlipidemic agents - Clofibrate - A monograph on clofibrate has recently been published.L‘7 The recently completed WHO primary prevention trial showed that clofibrate treatment reduced the incidence of nbn-fatal myocardial infarcts by 25%, but did not alter the incidence of fatal heart attack^.^' In most of the studies reported during the past year, HDL-C was significantly elevated by clofibrate. Whether or not clofibrate elevates HDL-C may depend upon the pretreatment level. Thus, if the initial levels were below 40 mg/dl, clofibrate elevated HDL-C, e.g. from 30.2 to 44.1 mg/d14g or from 37.5 to 4 4 . 9 mg/dl.” On the other hand, in patients who had a myocardial infarction but relatively normal HDL-C, clofibrate did not elicit a statistically significant rise in HDL-C. 5 3 Clofibrate increased apo A-I by 11% and apo A-I1 by 27%, implying a preferential increase in HDL3 (d = 1.125-1.210).50 Combination therapy with clofibrate and NA (or NA prodrugs)49,s4,55 or with colestipols‘~s6produced additive effects on serum lipids in properly selected patients. Thus, clofibrate enhanced the cholesterol lowering action of colestipol in subjects with Type IIa hyperlipoproteinemia, but the combination of the two drugs produced a net increase in serum cholesterol in Type IIb hyperlipoproteinemia.” A preliminary report of the ongoing Stockholm studys4 in survivors of myocardial infarction who were treated with both clofibrate and NA showed no effect on total mortality or on mortality from CHD, but a significant reduction in non-fatal myocardial infarctions was demonstrated. Combinations of clofibrate with a sulfomucopolysaccharide preparation,’‘I phenformin” or etiroxate(umethyl-DL-thyroxine ethyl ester) ’’ lowered serum lipids to a greater extent than did clofibrate alone. The mode of action of clofibrate has yet to be completely elucidated. Sterol balance data in man supported the widely accepted concept that the drug inhibits cholesterol biosynthesis. 6 o However , in rats fed 0.25% clofibrate for 2 weeks, no inhibition in the absolute rates of cholesterol biosynthesis was observed in liver, intestinal wall or adipose tissue ( [’HI 20 incorporation to cholesterol). 6 1 Intralipid clearance from the plasma of rats“ and hyperlipidemic patients62 was enhanced by clofibrate, demonstrating increased fractional turnover rate of endogenous triglyceride. Clofibrate enhanced the activity of post-heparin plasma lipo-

Chap. 19

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201

p r o t e i n l i p a s e a c t i v i t y i n man, b u t had no e f f e c t on h e p a t i c t r i g l y c e r i d e l i p a s e . 6 3 9 6 4 I n a d d i t i o n , c l o f i b r a t e i n c r e a s e d t h e l e v e l s of a d i p o s e t i s s u e l i p o p r o t e i n l i p a s e i n human p a t i e n t s b 2 b u t n o t i n c h o l e s t e r o l - f e d r a t s . 6 1 The d r u g d e c r e a s e d t h e mean molar LCAT r a t e by 10% i n h y p e r t r i g l y c e r i d e m i c s u b j e c t s whose i n i t i a l LCAT r a t e s were r e l a t i v e l y h i g h , b u t d i d not a l t e r t h e mean f r a c t i o n a l LCAT rate." It h a s l o n g been known t h a t c l o f i b r a t e i n c r e a s e s h e p a t i c peroxisomes b u t whether a r e l a t i o n s h i p e x i s t s between peroxisome p r o l i f e r a t i o n and t h e hypolipidemic a c t i o n of t h e drug h a s n o t been e s t a b l i s h e d . S e v e r a l hypol i p i d e m i c c l o f i b r a t e a n a l o g u e s d i d n o t p r o l i f e r a t e h e p a t i c microsomes i n r a t s , t h u s s u g g e s t i n g t h a t b o t h e f f e c t s occur i n d e p e n d e n t l y . 6 5

The m e t a b o l i c d i s p o s i t i o n b 6 and c l i n i c a l p h a r m a c o k i n e t i c s b 7 of c l o f i b r a t e have been r e c e n t l y reviewed. I n h e a l t h y v o l u n t e e r s , t h e plasma e l i m i n a t i o n h a l f - l i f e ( t f ) of c h l o r o p h e n o x y i s o b u t y r i c a c i d (CPIB) , t h e p h a r m a c o l o g i c a l l y a c t i v e form of c l o f i b r a t e , averaged 1 7 h r , independent of dose (500-2000 mg) o r d u r a t i o n of t r e a t m e n t . " The plasma c l e a r a n c e and a p p a r e n t volume of d i s t r i b u t i o n were i d e n t i c a l f o r a l l d o s e s when based on unbound C P I B c o n c e n t r a t i o n s , b u t c l e a r a n c e c a l c u l a t e d from t o t a l CPIB l e v e l s i n c r e a s e d w i t h i n c r e a s i n g d o s e ; t h i s w a s due t o d e c r e a s e d prot e i n b i n d i n g a t h i g h e r plasma c o n c e n t r a t i o n s . 6 8 I n r a t s , t h e serum t $ of CPIB w a s 5 h r , b u t a second e x p o n e n t i a l w i t h l o n g e r t$ o c c u r r e d a t l a t e r time i n t e r v a l s ; t h e t f i n dogs w a s 40-50 h r . 6 9 A t 100 vg C P I B / m l serum, t h e p e r c e n t a g e f r e e CPIB w a s 25, 1 6 and 3% i n r a t s , dogs and man, r e s p . 6 9 Co-administration of t h e a n i o n exchange r e s i n s c o l e s t i p o l o r c h o l e s t y r a mine w i t h c l o f i b r a t e t o human s u b j e c t s d i d n o t a f f e c t t h e b i o a v a i l a b i l i t y of CPIB. 7 0 I n r a t s , c l o f i b r a t e i n c r e a s e d t h e area under t h e serum concent r a t i o n l t i m e c u r v e of NA, and i t w a s concluded t h a t c l o f i b r a t e d e c r e a s e d t h e r a t e of u r i n a r y e x c r e t i o n of NA.71 C l o f i b r a t e d i d n o t modify t h e t$ of a n t i p y r i n e n o r u r i n a r y g l u t a r i c a c i d e x c r e t i o n i n normal s u b j e c t s , t h u s t h e drug i s n o t an enzyme i n d u c e r i n man. 7 2 N i c o t i n i c a c i d (NA) and d e r i v a t i v e s - P a t i e n t s w i t h s e v e r e h y p e r c h o l e s t e r olemia w e r e t r e a t e d w i t h 0.9-1.2 g/day of P - p y r i d y l c a r b i n o l (2)f o r up t o 9 y e a r s ; t h e r a p y reduced serum c h o l e s t e r o l l e v e l s , and no m y o c a r d i a l i n f a r c t i o n w a s r e c o r d e d in t h e 1 2 p a t i e n t s completing t h e s t u d y . 7 3 The a n t i a t h e r o g e n i c e f f e c t s of NA, 2, and two o t h e r NA d e r i v a t i v e s i n r a b b i t s were accompanied by an e l e v a t i o n of t h e HDL/LDL c h o l e s t e r o l r a t i o i n t h e a o r t i c t i s s u e . 74 The mode of a c t i o n of NA7' c o n t i n u e s t o b e t h e s u b j e c t of e x t e n s i v e i n v e s t i g a t i o n . When h y p e r l i p i d e m i c p a t i e n t s were g i v e n 4.5 g NA/day, t h e r e w a s a n i n i t i a l f a l l i n l i p i d s a s s o c i a t e d w i t h b o t h VLDL and HDL; a f t e r t h e f i r s t few d a y s , HDL-C became e l e v a t e d , independent of any change i n VLDL-triglyceride c o n c e n t r a t i o n . 4 8 The same p a t t e r n w a s observed i n p a t i e n t s given c l o f i b r a t e ( 1 . 5 g/day) o r c l o f i b r a t e p l u s 8 - p y r i d y l c a r b i n o l (75 mg/day) ' NA d e c r e a s e d c h o l e s t e r o l b i o s y n t h e s i s and i n c r e a s e d t h e f r a c t i o n a l t u r n o v e r r a t e of plasma t r i g l y c e r i d e s , b u t had no e f f e c t on plasma l i p o p r o t e i n l i p a s e o r LCAT a c t i v i t y ; i t w a s concluded t h a t t h e v a r i o u s e f f e c t s of NA were secondary t o i t s a n t i l i p o l y t i c a c t i o n o n a d i NA d i d n o t a l t e r b i l i a r y l i p i d s i n man, and t h e r e f o r e pose t i s s u e . " should n o t i n c r e a s e t h e r i s k of g a l l s t o n e f o r m a t i o n . 7 7 I n v i t r o , NA i n h i b i t e d thromboxane A2 f o r m a t i o n by p h o s p h o l i p a s e A z i n r a t p l a t e l e t s . 7 8

.

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Apo C-11, the activator of lipoprotein lipase, was absent in a subject with severe hypertriglyceridemia; a transfusion of plasma produced a temporary fall in the patient's circulating triglyceride, due to apo C-I1 in the normal donor's plasma. 79 It was also reported that NA ( 1 2 g/day) increased apo C-I1 in chylomicrons and VLDL of a subject with Type V hyperlipoproteinemia. These studies support the concept that low amounts of apo C-I1 may play a role in the development of hypertriglyceridemia. Ion exchange resins - The bile acid sequestering anion exchange resins (cholestyramine, colestipol and polidexide) continue to show utility in lowering serum cholesterol levels in Type I1 hyperlipoproteinemia.a A large scale trial with patients treated for up to 3 years with colestipol showed lower cardiovascular mortality in men, but not in women.a3 The resins lower LDL-C; a study with colestipol showed no change in HDL-C." The metabolic disposition of oral [14C]colestipol was studied in rats, dogs and man; absorption was negligible and virtually all of the radioactivity was excreted in feces.B4

-

Probucol The pharmacologic and therapeutic properties of probucol (4) have been recently reviewed.B' Probucol lowered plasma cholesterol levels without affecting triglyceride levels." ' 8 6 The mode of action of the drug is unknown. Pharmacokinetic studies in man" showed limited and variable absorption. Upon oral administration, steady state plasma levels were attained only after several months of treatment. Following long-term dosing, the plasma t$ was several weeks. Tissue distribution studies in rhesus monkeys" showed substantially higher tissue than plasma levels; probucol was retained in adipose tissue. N II H (cH,),N-c-N

$H H-C-N ti2

-Sttosterol - Recent reports demonstrated the efficacy of 8-sitosterol in Towering plasma LDL-C in patients with Type I1 hyperlipoproteinemia.8 7 B-Sitosterol obtained from tall oil was found to be effective at 3 glday, a dose substantially lower than that required with older preparations. 8 7 However, in juvenile Type I1 hyperlipoproteinemia, f3-sitosterol lowered LDL-C by only 6% but, in addition, decreased HDL-C by 15%; thus, B-sitosterol was not recommended for the treatment of hypercholesterolemia in children." A potential problem with 8-sitosterol is the increased risk of gallstone formation, in view of the finding of increased saturation index of bile in patients given 3 glday of plant sterols." 9"'

Metformin - The antiatherogenic action of the hypoglycemic biguanide metformin (?) appears to be associated more with alterations in apolipoprotein structure and composition than with lipid lowering effects.9 1 Sixmonth therapy of 254 hyperlipidemic patients with 2.5 g/day of metformin produced an average reduction in serum triglycerides of 57%, but no change in cholesterol levels; metformin therapy improved various hemodynamic

Chap. 1 9

D i s o r d e r s of L i p i d Metabolism

p a r a m e t e r s ( e . g . blood p r e s s u r e , EKG).

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’’

(a),

Bezafibrate - Bezafibrate a n a n a l o g u e of c l o f i b r a t e , had s i m i l a r e f f e c t s on serum l i p i d s and l i p o p r o t e i n s a s d i d c l o f i b r a t e , b u t a t lower The pharmacologic sp‘ectrum of b o t h d r u g s i n r a t s w a s s i m i doses.’3 l a r ’‘,” I n man, b e z a f i b r a t e lowered serum t r i g l y c e r i d e s ; HDL-C i n c r e a s e d by 31%, i n d e p e n d e n t l y of a d e c r e a s e i n VLDL-triglyceride. 9 6 The The d e g r e e of LDL-C d e c r e a s e w a s dependent upon p r e t r e a t m e n t l e v e l s . optimum d o s e w a s 450-600 mg/day, and serum l i p o p r o t e i n c h a n g e s were maint a i n e d d u r i n g one y e a r o f t h e r a p y . 9 7 B e z a f i b r a t e lowered serum l i p i d l e v e l s i n h y p e r l i p i d e m i c d i a b e t i c s . ” A s measured by GLC, ” b e z a f i b r a t e w a s r e p o r t e d t o b e c o m p l e t e l y a b s o r b e d i n man, and had a ti of 2 h r . l o o D e s p i t e t h e s h o r t t $ , t h e a n t i h y p e r l i p i d e m i c a c t i v i t v l a s t e d f o r 24 h r . l o ’

.

’‘

c i ~ c o ~ H c H 2 c DH : , > O O H CH3

-6

C

I

O

CO ~ O ~ : b O C H ( C H 3 ) 2 H3

c

-7

P r o c e t o f e n e - I n a 3-year t r i a l i n 340 p a t i e n t s w i t h Types I I a , I I b and I V h y p e r l i p o p r o t e i n e m i a , p r o c e t o f e n e (7) produced dose-dependent ( 2 0 0 - 4 0 0 mg/ d a y ) d e c r e a s e s i n serum c h o l e s t e r o l (20-36% r e d u c t i o n ) i n Type I1 p a t i e n t s and 20-50% d e c r e a s e s i n serum t r i g l y c e r i d e s i n Types I I b and I V subj e c t s . l o ’ C o l e s t i p o l p l u s p r o c e t o f e n e produced g r e a t e r r e d u c t i o n s i n c h o l e s t e r o l l e v e l s i n Type I1 p a t i e n t s t h a n d i d p r o c e t o f e n e a l o n e . e 2 I n a c o m p a r a t i v e s t u d y , LDL-C i n Types I I a and I I b p a t i e n t s w a s lowered 37% by p r o c e t o f e n e , 27% by b e z a f i b r a t e and 1 8 % by g e m f i b r o z i l (S) l o 4 The t h r e e d r u g s d e c r e a s e d VLDL-triglyceride l e v e l s by 45-58% i n Type I V hyperl i p o p r o t e i n e m i a . O 4 I n man, t h e t $ of p r o c e t o f e n i c a c i d , t h e a c t i v e m e t a b o l i t e of p r o c e t o f e n e , was 24 h r . l o 3

.

T i a d e n o l - T i a d e n o l (9) ( 2 . 4 g / d a y ) lowered serum c h o l e s t e r o l l e v e l s i n Type I I a h y p e r l i p o p r o t e i n e m i a and d e c r e a s e d serum t r i g l y c e r i d e s i n Type I V In vitro, p a t i e n t s ; serum l i p i d s were u n a l t e r e d i n Type I I b p a t i e n t s . l o ’ -tiadenol inhibited l i p i d mobilization as w e l l as phosphodiesterase a c t i v i t y i n r a t e p i d i d y m a l f a t pads.”‘ H O C H2CH2S-( C HQ);-d C H 2 C H 2 O H

E s t r o g e n - Women w i t h Type I11 h y p e r l i p o p r o t e i n e m i a were g i v e n 1 pg/kg/day of e t h i n y l e s t r a d i o l ; VLDL c h o l e s t e r o l , t r i g l y c e r i d e and a p o l i p o p r o t e i n c o m p o s i t i o n became normal. O 7 T h i s w a s i n c o n t r a s t t o t h e u s u a l h y p e r t r i g l y c e r i d e m i c a c t i v i t y of e s t r o g e n i n normal s u b j e c t s , and i t w a s p o s t u l a t e d t h a t , i n t h e Type I11 syndrome, e s t r o g e n i n c r e a s e d t h e r a t e of c l e a r a n c e of VLDL remnants from t h e p l a s m a . l o 7 Postmenopausal women w i t h Type I1 h y p e r l i p o p r o t e i n e m i a were g i v e n 2 mg/day of a n e s t r a d i o l d e r i v a t i v e f o r 6 m o n t h s . l o e LDL-C d e c l i n e d by a n a v e r a g e o f 18%and HDL-C r o s e by 30%, w h i l e serum t r i g l y c e r i d e s were u n a l t e r e d ; t h e d a t a d e m o n s t r a t e a n o t h e r i n d i c a t i o n f o r e s t r o g e n t h e r a p y i n postmenopausal women. l o ’

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Sucrose p o l y e s t e r - I n normal v o l u n t e e r s , d o s e s of 8-25 g/day of s u c r o s e p o l y e s t e r , a l i q u i d non-hydrolyzable u n a b s o r b a b l e l o n g c h a i n f a t t y a c i d ester of s u c r o s e , lowered t o t a l and LDL-C w i t h o u t a l t e r i n g serum trig l y c e r i d e s o r HDL-C. l o 9 The material i n h i b i t e d c h o l e s t e r o l a b s o r p t i o n and i n c r e a s e d t h e e x c r e t i o n of n e u t r a l s t e r o l s and b i l e a c i d s . ' l o

-

(10)

Diosgenin e l e v a t e d HDL-C and lowered LDL-C Sapogenins and s a p o n i n s i n cholesterol-fed rats; t h e agent i n h i b i t e d c h o l e s t e r o l absorption witho u t a l t e r i n g b i l e a c i d turnover."' Combined a d m i n i s t r a t i o n of c l o f i b r a t e and d i o s g e n i n t o r a t s produced combined e f f e c t s on serum l i p o p r o t e i n s . D i g i t o n i n t r e a t m e n t i n h i b i t e d c h o l e s t e r o l a b s o r p t i o n i n r a t s and p r e v e n t e d t h e rise i n serum c h o l e s t e r o l i n c h o l e s t e r o l - f e d cynomolgus monkeys.

''

THD-341 - The t r i c y c l i c d i t e r p e n o i d THD-341 (111, a t a n o r a l dose of 3 mg/kg o r a s 0.001% of t h e d i e t , lowered serum l i p i d s i n c h o l e s t e r o l A t a d i e t a r y l e v e l of 0.01%, THD-341 p r e v e n t e d a o r c h o l a t e f e d rats. '14 t i c atheroma f o r m a t i o n i n c h o l e s t e r o l - f e d r a b b i t s and i n h i b i t e d t h e prog r e s s i o n of e s t a b l i s h e d l e s i o n s i n r a b b i t s f e d t h e c h o l e s t e r o l d i e t . '" C i p r o f i b r a t e - A t o r a l d o s e s of 0.6-3 mg/kg/day, c i p r o f i b r a t e ( 1 2 ) lowered serum c h o l e s t e r o l and t r i g l y c e r i d e l e v e l s i n h y p e r l i p i d e m i c rats.' l 6 When f e d i n t h e d i e t , c i p r o f i b r a t e i n h i b i t e d c h o l e s t e r o l b i o s y n t h e s i s . ' '' ML-1024 - An o x y e t h y l t h e o p h y l l i n e e s t e r of C P I B , ML-1024 (13) lowered serum c h o l e s t e r o l and t r i g l y c e r i d e levels i n r a t s a t s i m i l a r d o s e s a s d i d c l o f i b r a t e ; serum c h o l e s t e r o l l e v e l s were lowered i n c h r o n i c a l l y t r e a t e d minipigs. l 7

C H3 13 (AAS) (14) prevented t h e

L-Ascorbate-2-sulfate - L-Ascorbate-2-sulfate i n c r e a s e i n serum l i p i d l e v e l s and t h e accumulation of c h o l e s t e r o l i n t h e a o r t a of c h o l e s t e r o l - f e d r a b b i t s t o a g r e a t e r d e g r e e t h a n d i d a s c o r b i c AAS had s i m i l a r e f f e c t s on serum l i p i d l e v e l s i n g u i n e a a c i d (AA)."' p i g s , 1 1 9 a s p e c i e s which, l i k e man, does n o t b i o s y n t h e s i z e AA. It w a s

Chap. 19

D i s o r d e r s of L i p i d Metabolism

concluded t h a t t h e h y p o l i p i d e m i c a c t i v i t y of AAS was i n d e p e n d e n t of t h e c o n v e r s i o n of AAS t o A A . l "

Cayen

205 -

C H,OH

I

HCOH

0 0 S03H

HO

14 G a l l s t o n e d i s s o l u t i o n - S i n c e t h e p r e v i o u s d i s c u s s i o n of t h i s t o p i c i n t h i s s e r i e s , l Z oseveral reviews h a v e appeared d e s c r i b i n g t h e m e d i c a l t r e a t m e n t of g a l l s t o n e s , e s p e c i a l l y t h e u s e of o r a l c h e n o d e o x y c h o l i c (chen i c ) a c i d (CDCA). l 2 '-12' Data on t h e s a f e t y and e f f i c a c y of CDCA i n o v e r 1000 p a t i e n t s have accumulated i n t h e p a s t 2 y e a r s ; 2 5 - 1 3 5 one s t u d y comp r i s e d 400 s u b j e c t s . 1 3 0 S u c c e s s r a t e s v a r i e d between 50 and 70%, w i t h s t o n e s d i s s o l v i n g u s u a l l y w i t h i n 4-24 months of t r e a t m e n t . Dose r e l a t e d i n c r e a s e s i n e f f i ~ a ~ y ~and~ f r~e q u,e n ~ cy ~ of ~ s i d ,e e~ f ~ f ~ e , ~ ~ t ~~ ~~ have been shown. The o p t i m a l d o s e i s 750 mg/day o r 15-17 mg/kg/ day; 1 2 S , 1 2 6 , 1 2 8 ,1 3 1 , 1 3 2 o b e s e p a t i e n t s r e q u i r e up t o 20 ~ n g l k g l d a y . ' ~ ~ S i d e - e f f e c t s w e r e minor, t h e most common b e i n g m i l d d i a r r h e a and e l e v a t e d t r a n s a m i n a s e s , which tended t o n o r m a l i z e a f t e r t h e f i r s t month. 1 3 0 , 1 3 7 The mode of a c t i o n of CDCA h a s been reviewed; 12' 9 l Z 2 t h e r e s u l t o f i t s a c t i v i t y i s t o r e d u c e t h e b i l i a r y c o n c e n t r a t i o n of c h o l e s t e r o l r e l a t i v e t o t h a t of b i l e a c i d s and p h o s p h o l i p i d , t h e r e b y r e d u c i n g t h e s a t u r a t i o n i n d e x ( S I ) of b i l e . 1 2 7 A s t u d y on t h e d i s p o s i t i o n of t h e d r u g 1 3 e showed t h a t CDCA w a s c o m p l e t e l y a b s o r b e d ; t h e f i r s t - p a s s h e p a t i c c l e a r a n c e of CDCA a v e r a g e d 62%. CDCA lowered serum t r i g l y c e r i d e s i n h y p e r t r i g l y c e r i d e m i c g a l l s t o n e p a t i e n t s , 12' and h a s been used t o n o r m a l i z e serum l i p i d s i n h y p e r t r i g l y c e r i d e m i a , i n d e p e n d e n t of i t s a c t i o n on g a l l s t o n e s . 13'-14' CDCA d e c r e a s e d t r i g l y c e r i d e b i o s y n t h e s i s i n h y p e r l i p i d e m i a . 3 9 - 1 4 1 CDCA enhanced t h e a n t i h y p e r l i p i d e m i c e f f e c t o f c l o f i b r a t e and reduced t h e c l o f i b r a t e - i n d u c e d i n c r e a s e i n b i l i a r y c h o l e s t e r o l c o n c e n t r a t i o n and S I of b i l e . 1 3 2

Another a g e n t b e i n g i n v e s t i g a t e d f o r g a l l s t o n e d i s s o l u t i o n i s u r s o d e o x y c h o l i c a c i d (UDCA), t h e n a t u r a l l y o c c u r i n g 78-epimer of CDCA. 1 4 2 - 1 4 4 No d i a r r h e a o r e l e v a t e d serum t r a n s a m i n a s e s were found a t d o s e s up t o 1 5 mg/kg/day. 1 4 2 - 1 4 4 The S I of b i l e w a s lowered by smaller d o s e s of UDCA t h a n o f CDCA. 1 4 3 3 1 4 s U n l i k e CDCA, UDCA d i d n o t elevate l i t h o c h o l i c a c i d l e v e l s i n t h e b i l e of r h e s u s monkeys.146 Although enhanced b i l i a r y l e v e l s of u n c o n j u g a t e d l i t h o c h o l i c a c i d were n o t found i n humans g i v e n CDCA, UDCA may t h e o r e t i c a l l y b e s a f e r t h a n CDCA. 1 4 7

~

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References 1. 2. 3.

4. 5. 6. 7. 8.

9. 10. 11. 12. 13.

14. 15. 16. 17. 18.

19. 20. 21 *

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

32. 33. 34.

35. 36.

37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

as,

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c,

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Chap. 1 9

D i s o r d e r s of L i p i d M e t a b o l i s m

Cayen

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51. J . Probstfield. L. Crow, S . Isaacson and D. Hunninghake, Clin. Res.. 2,294A (1978). 52 L. Wallentin, Atherosclerosis, 21. 41 (1978). 53. S.C. Enger, J. Erikssen, V. Johnsen. A. Samuelsen. K. Herbjornsen and E.A. Laws, Artery, 6, 28 (1977). 54. L.A. Carlson, M. Oanielson, I. Ekberg, B. Klintemar and G. Rosenhamer, Atherosclerosis, 2, 81 (1977). 55. W. Schwartzkopff and M. Zschiedrich, Med. Klin., 73. 231 (1978). 56. R. Fellin, G. Baggio, G. Briani, M.R. Baiocchi, E. Msnzato, G. Baldo and G. Crepaldi, Atherosclerosis, 2,241 (1978). 57. W. Schwartzkopff and R. Roetz, Therapiewoche, 28, 2437 (1978). 58. W. Gross, G. Franke and F. Schmidt. Therapiewoche, 2.3550 (1978). 59. H. Kopp. Muench. Med. Wochenschr., 120, 739 (1978). 60. B.J. Kudchodkar, H.S. Sodhi, L. Horlick and D.T. Mason, Clin. Pharmacol. Ther., 22,154(1977). 61. M.A. D'Costa, F.C. Smigura, K. Kulhay and A. Angel, J. Lab. Clin. Med., 90. 823 (1977). 62. K.C. Taylor, G. Holdworth and D.J. Galton, Lancet, 2, 1106 (1977). 63. H. Greten, V. Laible, G. Zipperle and J . Augustin, Atherosclerosis, 563 (1977). 64. J. Boberg, M. Boberg, R. Gross, S . Grundy, J . Augustin and V. Brown, Atherosclerosis, 27, 499 (1977). 65 D.L. Azarnoff, J . Reddy, C. Hignite and T. Fitzgerald, i n "Drug Therapy, Vol. 4", H. Vapaatalo, Ed., Pergamon Press. New York, NY, 1976, p 137. 66. M.N.. Cayen, Quart. Rev. Drug Metabolism Drug Interactions. i n press. 67. R. Gugler, Clin. Pharmacokinetics. 3, 425 (1978), and refs. cited. 68. R. Gugler and J. Hartlapp, Clin. Pharmacol. Ther., 24. 432 (1978). 69. M.N. Cayen, E.S. Ferdinandi, E. Greselin. W.T. Robinson and D. Dvornik, J . Pharmacol. Exp. Ther., 3, 33 (1977). 70. K.A. DeSante, A.R. DiSanto, K.S. Albert, R.D. Welch and T.J. Vecchio, Clin. Pharmacol. Ther., 23, 112 (1978). 71. M.N. Cayen, W.T. Robinson, J. Dubuc and D. Dvornik. Biochem. Pharmacol., i n press. 72. G . Houin and J . P . Tillement, Tnt. J . Clin. Pharmacol. Biopharm., 2,150 (1978). 73. N. Zollner, C. Keller and G. Wolfram, Atherosclerosis, 2,611 (1977). 74. M.R. Parwaresch, H. Haacke and C. Mader. Atherosclerosis, 2, 395 (1978). 75. L.A. Carlson. in "Drug Therapy, Vol. 4", H. Vapaatalo, Ed., Pergamon Press, New York, NY, 1976, p 129. and refs. cited. 76. B.J. Kudchodkar, H.S. Sodhi, L. Horlick and D.T. Mason, Clin. Pharmacol. Ther., %,354(1978). 77. H.Y.I. Mok and S.M. Grundy, Gastroenterology, 73. 1235 (1977). 78. J.E. Vincent and F.J. Zijlstra, Prostaglandins, 2, 629 (1978). 79. W.C. Breckenridge, J.A. Little, G. Steiner, A. Chow and M. Poapst, New Engl. J . Med., 298, 1265 (1978). 80. L.A. Carlson and G. Wahlberg, Atherosclerosis, 2, 77 (1978). 81. G. Ruoff. J . Am. Geriatrics SOC., 2, 121 (1978). 82. J.P. Sauvanet. P. Drouin, G. Debry, L. Mejean and D. Lambert, Nouv. Presse Med., 1, 1362 (1978). 83. A.E. Dorr, K. Gundersen. J.C. Schneider, Jr., T.W. Spencer and W.B. Martin, J . Chron. Dis., 31. 5 (1978). 84. R.C. Thomas, R.S.P. Hsi. H. Harpootlian, T.D. Johnson and R.W. Judy, Atherosclerosis, 29, 9 (1978). 85. R.C. Heel, R.N. Brogden, T.M. Speight and G.S. Avery, Drugs, l5, 409 (1978). 86. W.B. Parsons, Jr., Am. Heart J., 96, 213 (1978). 87. A.M. Lees, H.Y.I. Mok, R.S. Lees, M.A. McCluskey and S.M. Grundy, Atherosclerosis, g,325 (1977). 88. V.H. Kaffarnik, G. Muhlfellner, 0. Muhlfellner, J . Schneider, L. Hausmann, P. Zofel, R. Schubotz and F. Fuchs, Fortschr. Med., 95,2785 (1977). 89. G. Schlierf, P. Oster, C.C. Heuck, H. Raetzer and B. Schellenberg, Atherosclerosis, 30. 245 (1978). 90. D.P. Maudgal, R. Bird, W.S. Blackwood and T.C. Northfield. Brit. Med. J., 2, 851 (19781. 91. R. Paoletti, A. Catapano, G.C. Ghiselli and C.R. Sirtori, i n "Atherosclerosis IV", G. Schettler, Y. Goto, Y. Hata and G. Klose. Eds.. Springer-Verlag. New York, NY, 1977, p517. 92. G. Descovich, U. Montaguti, C. Ceredi, E. Cocuzza and C.R. Sirtori. Artery, 6, 348 (1978). 93. A.G. Olsson, P.D. Lang, S . Rossner, G. Walldius and L.A. Carlson, i n "Atherosclerosis IV", G. Schettler, Y. Goto, Y. Hata and G. Klose, Eds.. Springer-Verlag, New York, NY, 1977. p554. 94. H. Stork, G. Baggio and D. Seidel, ibid., p 547. 95. J . Berndt, R. Gaumert and J . Still, Atherosclerosis, 30, 147 (1978). 421 (1978). 96. A.G. Olsson and P.D. Lang. Atherosclerosis, 2, 97. A.G. Olsson and P.D. Lang, Atherosclerosis, 2, 429 (1978). 98. P. Wahl, C. Hasslacher, P.D. Lang and J. Vollmar, Deut. Med. Wochenschr., 103,1233 (1978). 99. R. Endele, J. Chromatog., 154. 261 (1978). 100. P.D. Lang, W. Bablok, R. Endele, K. Koch, H. Stork and H.A.E. Schmidt, i n "Drugs, Lipid Metabolism and Atherosclerosis", D. Kritchevsky, R. Paoletti and W.L. Holmes, Eds., Adv. Exp. Med. Biol., Vol. 109, Plenum Press. New York, NY, 1978, p 391. 101. P. Oster, G. Schlierf. R. Mordasini, C.C. Heuck, H. Raetzer and B. Schellenberg, ibid., p400. 102. J . Rouffy, C. Dreux. Y. Goussault and R. Dakkak, i n "Atherosclerosis IV", G. Schettler, Y. Goto. Y. Hata and G. Klose, Eds.. Springer-Verlag. New York. NY, 1977. p 546.

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103. J.P. Desager, J . Chromatog., 145,160 (1978). 104. S. Rossner and L. Oro, in "International Conference on Atherosclerosis", L.A. Carlson, R. Paoletti, C.R. Sirtori and G. Weber, Eds., Raven Press, New York, NY. 1978, p 135. 105. G. Crepaldi, G. Briani, U. Senin, U. Montaguti, A. Capurso and A. Bondioli, ibid., p 343. 106. C. Bacciarelli, P, Boni and A. Castelluci. I1 Farmaco. 2.401 (1978). 107. R.S. Kushwaha, W.R. Hazzard, C. Gagne, A. Chait and J.J. Albers, Ann. Int. Med., 87, 517 (1977). 108, M.J. Tikkanen. E.A. Nikkila and E. Vartiainen, Lancet, 2, 490 (1978). 109. C . J . Glueck, F.H. Mattson and R . J . Jandacek, Circulation, 58, 11-170 (1978). 110. J . R . Crouse and S.M. Grundy, Circulation, 58. 11-170 (1978). 111. M.N. Cayen and D. Dvornik. J . Lipid Res., 20, 162 (1979). 112. M.N. Cayen and D. Dvornik, Atherosclerosis,2,317 (1978). 113. M . R . Malinow, P. McLaughlin and C. Stafford, Am. J. Clin. Nutr., 2.814 (1978). 114. H. Enomoto, Y. Yoshikuni, T. Ozaki, R. Zschocke and K. Ohata, Atherosclerosis, 28, 205(1977). 115. H. Enomoto, Y. Yoqhikuni, K. Saito, T. Ozaki, K. Ohata and R. Zschocke. Atherosclerosis, 30, 109 (1978). 116. A. Arnold, J.P. McAuliff, L . G . Powers. D.K. Phillips and A.L. Beyler, Atherosclerosis. 2, 155 (1979). 117. G . Metz. M. SpeCker, W. Sterner, E. Heisler and G . Grahwit. Arzneim.-Forsch., 27. 1173(1977). 118. E. Hayashi, J. Yamada, M. Kunitomo, M. Terada and M. Sato, Japan. J . Pharmacol. 8. 61(1978). 119. E. Hayashi, J . Yamada, M. Kunitomo, M. Terada and Y. Watanabe, ibid.. p 133. 120. A.C. Sullivan, L. Cheng and J . G . Hamilton, in "Annual Reports in Medicinal Chemistry, Vol. 12", F.H. Clarke, Ed., Academic Press, New York, NY. 1977. p 191. 121, R.G. Batey, Drugs, g,116 (1977). 122. A.F. Hofmctnn, An. J. Clin. Nutr., 0, 993 (1977). 123. T. Schersten, Scand. J . Gastroent., 13,129 (1978). 124. B . J . Pearlman and L.J. Schoenfield, Med. Clin. North Am., 62, 87 (1978). 125. M.C. Bateson, P.E. Ross, J. Murison and I.A.D. Bouchier, Gut, l8, 599 (1977). 126. S. Beker, An. J. Gastroent., 68, 456 (1977). 127. J.W. Marks, G.G. Bonorris, A. Chung. M.J. Coyne. R. Okun, J.M. Lachin and L.J. Schoenfield, Am. J . Dig. Dis., 22, 856 (1977). 128. A. Gerolani. H. Sarles, R. Brette, A. Paraf, J. Rautureau, C. Debray, C. Bermann, J.P. Etienne, J.C. Chaput and J.P. Petite, Digestion, l6, 299 (1977). 129. P.N. Maton, J.H. Iser, G.M. Murphy and R.H. Dowling, Gut, l8, A976 (19771. 130. L. Barbara, E. Roda, A. Roda, S . Casanova, D. Festi. C. Sama, G. Mazella and R. Aldini, Hinerva Med.. 68,3355 (1977). 131. P. Bernades. S . Bonfils, M. Bourel, C. Caulin. S . Erlinger, J . P . Ferrier. J. Gastard, R. Lambert. Y. LeQuintrec, H. Michel, P. Paliard, S . Segal, J . Toulet, J . Vilotte and P. Zeitoun, Therapie, 2,405 (1977). 132. M.C. Bateson. P.E. Ross, J . Murison and I.A.D. Bouchier, Brit. Med. J . , 1,1171 (1978). 133. M.C. Bateson, P.E. Ross, J . Murison and I.A.D. Bouchier, Lancet, 1, 1111 (1978). 134. J.L. Thistle. A.F. Hofmann, B . J . Ott and D.H. Stephens, J . Am. Med. Assoc., 239, 1041 (1978). 135. J.W. Marks, J.H. Sherman, G.G. Bonorris, A. Chung, M.J. Coyne and L.J. Schoenfield, Am. J . Gastroent., 69, 160 (1978). 136. J.H. Iser. P.N. Maton, G.M. Murphy and R.H. Dowling, Brit. Med. J.. 1, 1509 (1978). 137. R. Infante, M.E. Bouma, A. Lageron and V.G. Levy, Therapie, 2.401 71977). 138. G.P. van Berge-Henegouwen and A.F. Hofmann. Gastroenterology,12, 300 (1977). 139. N.E. Miller and P.J. Nestel, Lancet, 2, 929 (1974). 140. M.C. Bateson, D. Maclean, J . R . Evans and I.A.D. Bouchier. Brit. J . Clin. Phannacol., 3, 249 (1978). 141. B. Angelin, K. Einarsson and B. Leijd, C l i n . Sci. Mol. Med., 54, 451 (1978). 142. S. Nakagawa, I. Makino, T. Ishizaki and I. Dohi, Lancet. 2, 367 (1977). 143. P.N. Maton, G.M. Murphy and R.H. Dowling, Lancet, 2, 1297 (1977). 144. K. Kutz and A. Schulte, Gastroenterology,73, 632 (1977). 145. J.L. Thistle, J. Turcotte, B . J . Ott, G.L. Carlson, N.F. LaRusso and A.F. Hofmann, Gastroenterology,14,1103 (1978). 146. T. Fedorowski, G. Salen, F.G. Zaki, S. Shefer and E.H. Mosbach, Gastroenterology, 3, 75 (1978). 147. T. Fedorowski, G . Salen, A. Colallilo, G.S. Tint, E.H. Mosbach and J.C. Hall. Gastroenterology,73, 1131 (1977).

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Chapter 20. Somatostatin Daniel F. Veber* and Richard Saperstein** Merck Sharp & Dohme Research Laboratories *West Point, Pennsylvania 19486 and **Rahway, New Jersey 07065 Introduction. The peptide hormone somatostatin has undergone extensive biological and chemical study since its discovery in hypothalamic extracts in 1973.l The availability of synthetic hormone2 has resulted in the recognition of a broad range of biological functions. The synthesis and biological properties of numerous analogs have also been described. Reviews have appeared3,13,24,72,81 and the proceedings of a symposium on somatostatin has been p~blished.~ Biological Findings. Somatostatin was first isolated from sheep hypothalamus and derives its name from its ability to inhibit the The availability of release of growth hormone from the pituitary.' the synthetic peptide has led to the recognition that it expresses inhibitory control over a wide variety of endocrine and non-endocrine organs. The use of a variety of techniques (radioimmunoassay, bioassay, imunocytochemistry) has shown that somatostatin is present in other areas of the brain, spinal cord, the D cells of the endocrine pancreas, stomach, the gastrointestinal tract, cells of the thyroid gland, and in the general c i r c ~ l a t i o n . ~ ' ~The ~ presence of somatostatin in these different areas has led some investigators to suggest that the eptide may function both as a hormone and as a neurotransmitter.'l The original finding that somatostatin, when infused in baboons, caused lowering of blood glucose and concomittant decreases in insulin and glucagon levels,l* has led to its use in studies attempting to define the role of glucagon in normal carbohydrate homeostasis and in diabetes mellitus. Somatostatin has been found to inhibit the secretion of various other hormones such as thyrotropin, gastrin inhibitory peptide (GIP) , vasoactive intestinal peptide (VIP) , pancreozymin, secretin, motilin, gastrin, renin, ACTH in patients with ACTH-secreting pituitary adenoma and ACTH-secretion in vitro and to effect exocrine pancreatic function and various GI function^.^'^^ The physiological significance of these findings is not clear at the present time. From these observations, hypotheses for the development of therapeutic agents based on the actions of somatostatin have emerged. Therapeutic agents could be obtained by the development of analogs of somatostatin having selective inhibitory properties in 1) the endocrine pancreas, 2 ) the pituitary, and 3) the gastrointestinal tract. The short duration of action of somatostatin, however, is a potential limitation to practical therapeutic applications and will require the development of analogs of longer duration of action o r oral activity.3

Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reSeNed. ISBN 0-12-040514-8

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Effects on the Endocrine Pancreas. The most novel, although speculative, potential application of somatostatin relates to its possible use in the treatment of diabetes and its sequelae.22-24 Somatostatin has been used as a valuable tool in the investigation of the relative role of glucagon in carbohydrate homeostasis and in diabetes mellitus. During short-term infusion of somato'statin, hypoglycemia occurs accompanied by a fall in glucagon and insulin levels; cessation of the infusion results in a rebound of all parameters. The fall in plasma glucose levels has been shown to be due to a decrease in hepatic glucose production associated with a sup~ - ~ ~effect most likely is not pression of glucagon ~ e c r e t i o n . ~ This by a direct effect on liver, muscle, or fat tissue.29~30 A report to the contrary has appeared showing in vitro effects of somatostatin on glucagon-stimulated glycogenolysis and gluconeogenesis on isolated liver cells.31 The observed difference may be due to the high concentration of somatostatin used in the latter studies. According to the bihormonal hypothesis of diabetes mellitus as put forth by Unger,32'34 the severity of the hyperglycemia has been attributed not only to the relative or absolute insulin deficiency b u t also tothe hyperglucagonemia which is seen in all forms of spontaneous diabetes in man and chemically induced diabetes in animals. Due to insulin deficiency, glucose utilization is impaired, increased lipolysis may occur, amino acids are released from muscle tissue, and the increased production of glucose and ketones is mediated by glucagon. Withdrawal of insulin from juvenile diabetics and subsequent infusion of somatostatin has been shown to result in the prevention of hypergluconemia, in revention of severe hyperglycemia, and in a rise in B-OH butyrate.3Pt36Zt has been shown, however, that somatostatin most probably cannot reverse existing diabetic ketoacidosis.37 Thus, a glucagon suppressing agent such as somatostatin might prove useful in the treatment of diabetes. Somatostatin has until recently been the only glucagon suppressant available for clinical studies. In spite of the short half life and diverse actions, it has been used in a number of interesting clinical experiments which have attempted to ascertain its utility as an adjunct to the therapy of diabetes. Somatostatin in combination with insulin (at doses which did prevent postprandial hyperglycemia or hyperglucagonemia) completely prevented plasma glucose levels from rising after a meal. 38 Gerich et a139 assessed the effect of somatostatin given along with insulin in insulin requiring diabetics. The patterns of postprandial hyperglycemia and the blood glucose variations throughout the day were better during treatment with lowered insulin doses given along with somatostatin than when only insulin was given. A marked deterioration in the serum glucose pattern was observed when somatostatin infusion was withdrawn. Meissner et a140 reported reduced insulin requirements (38-79%) in juvenile diabetic patients when somatostatin was infused via an artificial pancreas along with insulin. A lowering of postprandial blood glucose as well as fewer fluctuations occurred

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during treatment. Depressions of GH levels as well as decreased circulating glucagon were observed in all patients. In alloxan et a141 have demonstrated improved control of diabetic dogs, Dobbs both hyperglycemia and triglyceridemia when somatostatin analogs were administered along with insulin therapy. Wahren and Felig suggested that the blood glucose lowering effect of somatostatin may be largely due to reduced glucose absorption rather than glucagon upp press ion.^^ Thus, interpretation of the data solely on the basis of glucagon suppression has been questioned. On the other hand, Unger et a1 have cited instances where somatostatin reduces the hy er lycemia of diabetes most likely by In these experiments, attempts glucagon suppression alone.f 3 , f 4 have been made to eliminate glucose absorption as a variable. Somatostatin lowers fasting plasma glucose levels in insulin-deprived diabetic dogs.45 Somatostatin also prevents the rise in glucose levels due to glucagon when alanine is administered parenterally.45 In clinical studies where carbohydrate has been all but eliminated as a factor in the diet, it has been shown that suppression of glucagon along with insulin injections more effective than insulin alone in the maintenance of euglycemia.

ta

The fact that somatostatin effects nutrient absorption may play an important role in future ideas concerning therapy. The treatment of diabetes with insulin may be thought of as a process whereby one increases the rate of removal of nutrients from the circulation. A new mode of therapy with somatostatin as a prototype may equalize the rates of nutrient entry and removal from the circulation as well as suppress glucagon mediated hyperglycemia. A role for endogenous pancreatic somatostatin as a regulator of nutrient influx from the GI tract in coordination with insulin mediated nutrient disposal has been proposed.46 Changes in endogenous somatostatin availability per se may be responsible for some metabolic abnormalities. Although much emphasis has been placed on the development of a somatostatin analog with selected biological activities, an analog which shows insulin, glucagon and growth hormone release inhibiting properties could h e used in the postadolescent insulin dependent diabetic. Suppression of GH release may offer utility for the treatment of diabetes because of possible effects of growth hormone on blood g l u c ~ s e ~ and ~ , the ~ ~ possible role of GH in the development of retinopathies. The desirable properties of a biologically specific somatostatin analog and possible clinical applications have been discussed in detail.49 Effects on the Pituitary. Somatostatin inhibits GH and thyrotropin releasing hormone (TRH)-induced thyroid stimulating hormone (TSH) release from the pituitary. No effects on other pituitary hormones were observed in normal subjects.5o The inhibition of GH release by somatostatin suggests a use in the treatment of acromegaly. The value of GH release inhibition as long-term therpay cannot be predicted.

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A further possible application of GH release inhibition by somatostatin is in the control of diabetic retinopathy. Although the etiology o f diabetic retinopathy remains undetermined, an attractive workin hypothesis is that growth hormone may play a causal role.47y51-86 Evidence against the GH hypothesis has been cited.57

Effects on the Gastrointestinal Tract. Somatostatin inhibits both the gastric secretion induced by gastrin and the release of gastrin,20 which suggests a potential utility for the treatment of ulcers. Some cases of life threatening GI bleeding have been successfully treated by I.V. administration of s~matoetatin.~~A comparative clinical trial of somatostatin with cimetidine in the control of Another important possible application bleeding is in progress .59 is in the treatment of pancreatitis since somatostatin inhibits Recent discussions of potential exocrine pancreatic secretion.60 gastrointestinal applications of somatostatin have appeared.4 Biological Evaluation of Somatostatin Analogs. In order to evaluate synthetic somatostatin analogs biological model systems for the above effects are required. The most widely studied properties of somatostatin analogs relate to the lowering of insulin, glucagon, growth hormone and gastric secretion. No single system has been used routinely for the study of all parameters. Growth hormone release inhibition is most commonly measured in an in vitro primary pituitary cell culture system.’ This assay is closest to measuring receptor interaction since metabolism is minimized and transport problems do not exist. A direct receptor system for somatostatin on -In vivo a pituitary cell system has become available recently.61 evaluation of growth hormone release inhibition is usually studied after subcutaneous administration of somatostatin or analogs in animals havin GH levels elevated by pentobarbital or arginine adminis trat ion.%2,63 An in vivo system for simultaneously measurin the inhibition of glucagon and insulin release in rats both with6f and without65 arginine stimulation has been described. A further test modification involves simultaneous continuous infusion of analog and arginine for 30 minutes.66 These differences in experimental design may lead to apparently different conclusions about the same compound. Somatostatin Analogs. H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Th=-Phe-Thr-Ser-C~s-OH* 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4

Somatostatin * A l l chiral amino acids are of the L-configuration unless otherwise noted. Standard abbreviations for the amino acids have been used throughout. Aha=w-amino heptanoic acid and Abu=y-amino butyric acid.

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Analogs of somatostatin are being prepared in a number of laboratories with the objective of obtaining compounds of therapeutically useful duration of action and/or improved biological specificity. These two objectives are convergent if a mechanism for improved biological specificity involves attaining selective resistance to metabolism in the area of one type of receptor (such convergence has been observed in the oxytocin-vasopressin series and has been reviewed.) 67 The first approach undertaken toward increasing the duration of action of somatostatin analogs was through acylated (e.g., and propionyl) somatostatin derivatives of reduced solubility. These analogs as well as somatostatin given by subcutaneous administration show durations of activity of three hours or more.69s70 Reduction of the rate of metabolism by amino peptidases through the addition of glycine residues (u to 3 ) to the amino terminus has resulted in increased duration. Replacement of various amino acids by the D-enantiomer has also been tried in an attempt to reduce metabolism. Varying degrees of success have been obtained by replacement of Gly-2 by D-Ala and Lys-4, Trp-8, Cys-3, Ser-13, and Cys-14 by the corresponding D-amino acids. Other approaches to increased resistance to enzymatic degradation have included deletion deletion of the of the two amino acids at the amino terminus{3 ensuing amino roup o f C ~ s - 3 ~ 7elimination 3 of the terminal carboxyl of C y ~ - 1 4 , ~and ~ >replacement ~ ~ of the reduceable disulfide by methylene groups.7 4 All of these latter modifications along with substitution of D-Trp in position 8 have been incorporated in analog 1,70375 However, the reason for observed increased duration is not always ~ l e a r . 7 ~ For example, the increased activity of D-Trp somatostatin is observed in vitro as well as in vivo, pointing to factors other than metabolic stability being responsible for the enhancement of activity .76a

'OH

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Another approach t o i n c r e a s e d m e t a b o l i c s t a b i l i t y can be through t h e i n t r o d u c t i o n of conformational c o n s t r a i n t . I n c r e a s e d unders t a n d i n g of t h e importance of conformation i n p e p t i d e b i o a c t i v i t y Polypeptides i n general a r e has occurred d u r i n g r e c e n t y e a r s . 76b l i k e l y t o e x i s t a s a r a p i d l y e q u i l i b r a t i n g m i x t u r e of a number of conformations. I f t h e conformation o r conformations which i n t e r a c t w i t h b i o l o g i c a l r e c e p t o r s happen t o d i f f e r from t h o s e which i n t e r a c t w i t h r a t e l i m i t i n g m e t a b o l i z i n g enzymes ( g e n e r a l l y p e p t i d a s e s ) , t h e n t h e i n t r o d u c t i o n of c o n f o r m a t i o n a l c o n s t r a i n t could r e s u l t i n i n c r e a s e d d u r a t i o n of a c t i o n . I t i s a l s o p o s s i b l e t h a t t h e r e c e p t o r s f o r d i f f e r e n t b i o l o g i c a l parameters w i l l i n t e r a c t d i f f e r e n t l y w i t h v a r i o u s conformations. Thus, i n a d d i t i o n t o t h e more obvious p o s s i b l e i n t r o d u c t i o n of b i o l o g i c a l s e l e c t i v i t y through changes i n chemical (r'eceptor b i n d i n g ) f e a t u r e s , t h i s g o a l might a l s o be achieved through conformational c o n s t r a i n t . Because t h e p o s s i b i l i t y e x i s t s t h a t even t h e s m a l l e s t changes i n a s i d e c h a i n may r e s u l t i n a change i n t h e r e l a t i v e s t a b i l i t i e s of v a r i o u s conformations, i t i s n e c e s s a r y t o discuss 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 somatostatin while a l s o c o n s i d e r i n g p o s s i b l e change i n conformation. Structure modifications i n a p e p t i d e can be placed i n t h r e e c a t e g o r i e s : 1) t h o s e c e r t a i n t o impose a conformational consequence ( a new c o v a l e n t bond i s i n t r o d u c e d ) , 2 ) those l i k e l y t o i n t r o d u c e a conformational consequence, and 3 ) t h o s e u n l i k e l y t o i n t r o d u c e a conformational change. Most modificat i o n s placed i n c a t e g o r y 3 a r e s o placed on t h e b a s i s of b i a s r a t h e r t h a n p r o o f , s i n c e evidence of t h e conformation a t t h e b i o l o g i c a l r e c e p t o r r e g a r d i n g t h e whole molecule would be r e q u i r e d i n o r d e r t o make a p o s i t i v e assignment. No such evidence e x i s t s f o r any p e p t i d e analogs. The change o f c h i r a l i t y on replacement of an L-amino a c i d by i t s enantiomer might be expected t o change s i d e c h a i n r e l a t i o n s h i p s . I n t e r c h a n g e of c h i r a l i t y w i t h r e t e n t i o n of b i o l o g i c a l a c t i v i t y must, t h e r e f o r e , be considered a s o u r c e of p o s s i b l e i n f o r m a t i o n about t h e conformation of t h e n a t u r a l hormone a t t h e b i o l o g i c a l r e c e p t o r . The h i g h potenc of a n a l o g s of s o m a t o s t a t i n having D-tryptophan i n p o s i t i o n 873 can be understood i n terms of a type I B-turn, i n v o l v i n g r e s i d u e s 7 , 8 , 9 , and 10.78 I n t h i s conformation, t h e s i d e c h a i n s of r e s i d u e s 8 and 9 w i l l b e a r an e q u a t o r i a l r e l a t i o n s h i p t o t h e p e p t i d e backbone. A change a t p o s i t i o n 8 t o t h e D c o n f i g u r a t i o n could r e s u l t i n a change i n t h e backbone conformation t o a type 11' Bt u r n , 7 9 t h e r e b y r e t a i n i n g t h e e q u a t o r i a l r e l a t i o n s h i p of t h e s i d e c h a i n s of r e s i d u e s 8 and 9. NMR evidence f o r t h e e x i s t e n c e of such a n e q u a t o r i a l r e l a t i o n s h i p of t h e l y s i n e and t r y p t o p h a n s i d e c h a i n s i n D-tryptophan c o n t a i n i n g a n a l o g s h a s been p r e s e n t e d , based on an u p f i e l d s h i f t of t h e l y s i n e 7-methylene of about 1 ppm and a c o r r e l a t i o n of t h e s h i f t w i t h t h e conformation a t b i o l o g i c a l r e c e p t o r s . 7 8 Since t h e u p f i e l d s h i f t i s n o t observed i n s o m a t o s t a t i n , i t h a s a l s o been proposed t h a t t h e c o n f o r m a t i o n ( s ) which i n t e r a c t w i t h r e c e p t o r s r e p r e s e n t only a minor c o n t r i b u t i o n t o t h e o v e r a l l p o p u l a t i o n of conformations i n t h e n a t u r a l hormone. The change from Type I t o Type 11' r e q u i r e s a 180' f l i p of t h e amide bond s u g g e s t i n g t h a t i t i s n o t d i r e c t l y involved i n r e c e p t o r i n t e r a c t i o n . The u p f i e l d

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s h i f t of t h e y-methylene of l y s i n e i n D-Trp 8 a n a l o g s can supply evidence r e g a r d i n g t h e e f f e c t of o t h e r m o d i f i c a t i o n s on t h e conformation of t h a t p a r t of t h e m o l e c u l e , a n a i d t o drawing s t r u c t u r e a c t i v i t y conclusions. B i o l o o i c a l s e l e c t i v i t y r e s u l t s when D-Cys r e p l a c e s L-C s i n p o s i t i o n 3 f l ( i n h i b i t i o n of i n s u l i n r e l e a s e ) and p o s i t i o n 14x0 ( i n h i b i t i o n of glucagon r e l e a s e ) . No e v i d e n c e e x i s t s r e l a t i n g t o e f f e c t s on conformation o r d i r e c t e f f e c t on r e c e p t o r i n t e r a c t i o n f o r these modifications. Also, t h e b i o l o g i c a l s e l e c t i v i t y i n t r o d u c e d by replacement of L-Lys-4 by D-Lys71 ( s e l e c t i v e i n h i b i t i o n of growth hormone) and i n c r e a s e d potency on replacement of Gly-2 by D-Ala71 have n o t been d e f i n e d a s i n v o l v i n g c o n f o r m a t i o n a l change or o t h e r f a c t o r s . Although i t has been proposed t h a t g a s t r i c s e c r e t i o n i n h i b i t i o n v a r i e s i n a p a r a l l e l f a s h i o n w i t h growth hormone r e l e a s e inhibition,89 t h i s c l a i m i s n o t s u p p o r t e d by o t h e r ~ t u d i e s . ~ ~ ~ ~ ~ Replacement of an amino a c i d by p r o l i n e i n t r o d u c e s conformat i o n a l c o n s t r a i n t by e l i m i n a t i o n of r o t a t i o n about t h e carbonn i t r o g e n bond i n t h e 5-membered r i n g . P r o l i n e i n p o s i t i o n s 5 and 1 3 i s acceptable he r e c e p t o r bound conformation w h i l e i n p o s i t i o n Using t h i s i n f o r m a t i o n , a conformation c a p a b l e 10 i t i s n o t . 6’:7’ of r e c e p t o r i n t e r a c t i o n h a s been proposed (1)65 which i s d i f f e r e n t from conformations proposed based on p h y s i c a l s t u d i e s i n s o l u t i o n . 8 2 T h i s model l e d t o t h e d e s i g n of a n a l o g s of t h e compound 1 having Asn-5 and Thr-12 and Phe-6 and Phe-11 r e p l a c e d by cystin: (2 & 3 , Each of t h e s e a n a l o g s shows a c t i v i t y a t i e a s t as respectively).65 g r e a t as t h a t observed f o r s o m a t o s t a t i n . The monocyclic p r e c u r s o r t o 2 i s a l s o h i g h l y a c t i v e , confirming t h e unimportance of t h e s i d e c h a i n s a t p o s i t i o n s 5 and 1 2 , a s f i r s t observed by replacement of each of t h e s e by a l a n i n e . Replacement of e i t h e r Phe-6 or Phe-11 by Ala i n s o m a t o s t a t i n r e s u l t s i n a s u b s t a n t i a l loss of a c t i v i t y . 8 1 That h i g h a c t i v i t y is s e e n when a d i s u l f i d e bond r e p l a c e s t h e two a r o m a t i c r i n g s ( 3 ) , w h i l e simple removal of t h e s e r i n g s lowers a c t i v i t y , w a s i n t e r p r e t e d by h y d r o p h g t i c bond s t a b i l i z a t i o n of t h e a c t i v e conformation of somatostatin. The h i g h a c t i v i t y of a n a l o g s of reduced r i n g s i z e such as compound 476 i s understood through compound 2, s i n c e t h e r i n g of 4 i s p r e s e n t i n 2. Both 2 and 5 have been r e p o r t e d t o show i n c r e a s e d d u r a t i o n of a c t i o n ; &76 a f t e r subcutaneous a d m i n i s t r a t i o n and 3 a f t e r intravenous administration. The i n c r e a s e d d u r a t i o n of 2 a f t e r intravenous administration l i k e l y r e f l e c t s increased r e s i s t a n c e t o metabolism b u t could a l s o i n v o l v e o t h e r f a c t o r s such a s reduced e x c r e t i o n r a t e . & and t h e a n a l o g having D-Cys i n t h e carboxy-terminal p o s i t i o n a r e r e p o r t e d t o b e r e l a t i v e l y s e l e c t i v e f o r i n s u l i n and glucagon r e l e a s e i n h i b i t i o n , r e s p e c t i v e l y . 76 The l a t t e r i s b e i n g studied clinically.83 Other p o s s i b l y more s e l e c t i v e a n a l o g s of The reduced r i n g s h e were a l s o r e p o r t e d by t h e S a l k group.76 combined c o n c l u s i o n from a n a l o g s 2, 3, and 4 a p p e a r s t o be t h a t most of t h e potency of s o m a t o s t a t i n r e s u l t s from-the s i d e c h a i n s of r e s i d u e s 7-10, a l t h o u g h some c o n t r i b u t i o n t o o v e r a l l potency may r e s i d e outside t h i s region.

216 -

Sect. IV

-

Metabolic Diseases, Endocrine Function

Bailey, Ed.

Cyclo (Aha-Lys-C;s-Phe-Phe-D-Trp-Lys-Thr-Phe-C$s-Ser) Cyclo (Aha-Lys-Abu-C$s-Phe-D-Trp-Lys-Thr-C$s-Thr-Ser) H-C$s-Phe-D-Trp-Lys-Thr-Phe-C$s-OH

4 -

Cyclo (Ala-Gly-C~s-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-C~s)

5

Cycle (~-Abu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Glu)

-6

A bicyclic analog involving acylation of the amino terminal Ala by the carboxyl of Cys-14 (2) has been re orted to be selective for inhibition of growth hormone release.6 3 Other growth hormone selective analogs have been claimed throu h replacement of the Another change in the disulfide bridge by an amide linkage ( 5 ) . nature of the ring of somatostatin through deletion of Asn-5 has shown selective lowering of insulin and growth hormone.85 Relative glucagon and growth hormone selectivity was also claimed on replacement of Lys-4 and Asn-5 by His residues.86 These claims are not based on analysis of relative potencies or ED501s, nor are they supported by statistical analyses. However, selectivity appears to exist at some doses. An analog, (71, which is almost totally selective for inhibition of insulin release, has been reported.76

ht

H-Ala-Gly-C~s-Lys-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-D-S~r-C~s-OH

7 -

The most critical amino acids appear to be Trp-8 and Lys-9, based on various strwture activity studies.71 A variety of substitutions in the indole ring of osition 8 are acceptable and even give analogs In all cases the D-analog is more potent of increased potency.sf than the corresponding L-analog. The nature of the heterocycle is 2 napthyll-L-alanine important, however, since replacement by 3-( 1 results in a loss of activity.88 Conclusion. Possible therapeutic applications of somatostatin analogs exist for the treatment of diabetes through suppression of glucagon release to attain improved control of blood glucose levels. Inhibition of growth hormone release may be of value in the prevention of diabetic retinopathy and in the treatment of acromegaly. Inhibition of gastric secretion may be of value in the treatment of ulcers and gastrointestinal bleeding. The studies of analogs of somatostatin have shown substantial progress toward increased duration of action and improved biological specificity. Analogs of value for human experimentation have been discovered. The progress of the biological and chemical studies sustains the hope of further advances toward therapeutically useful analogs.

Chap. 20

Somatostatin

Veber, Saperstein

217

References 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 31. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49 * 50. 51.

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If?,

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z,

11,

190,

i,

1,

155,

1,

-

2

218 52.

Sect. I V

-

M e t a b o l i c Diseases, E n d o c r i n e F u n c t i o n

B a i l e y , Ed.

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Wyssbrod, and J . D . G l i c k s o n , J. Amer. Chem. SOC., 2, 7326, 1977. 79. 80. M. Brown, J. R i v i e r , and W. V a l e , S c i e n c e . 1 4 6 7 , 1977. 81. W. V a l e , P. B r a z e a u , C. R i v i e r , M. Brown, B. B o s s , J. R i v i e r , R. B u r g u s , N. L i n g , a n d R. G u i l l e m i n , R e c e n t Prog. Horm. Res.. 365, 1975. 82. L.A. H o l l a d a y , J. R i v i e r , a n d D. P u e t t . , Biochem., 4 8 9 5 , 1977. S.R. Bloom, T.E. A d r i a n , A.J. B a r n e s , R.G. Long, J. H a n l e y , C.N. M a l l i n s o n , J.E. R i v i e r , 83. and M.R. Brown, G a s t r o e n t e r . , 3, 1013, 1978. 84. N. G r a n t , D. C l a r k , V. G a r s k y , I. J a u n a k a i s , W. McGregor, a n d D. S a r a n t a k i s , L i f e S c i . , 1 9 , 6 2 9 , 1976. 8 5 . D. S a r a n t a k i s , W.A. McKinley, I . J a u n a k a i s , D. C l a r k , and N.H. G r a n t , i l i n . E n d o c r i n o l . , 5 , 2 7 5 , s, 1976. L e t t e r s , 92, 1 5 3 , 1978. 86. D. S a r a n t a k i s , J. Teichman, R. F e n i c h e l , and E. L i e n , F.E.B.S. 87. C.A. M e y e r s , D.H. Coy, W.Y. Huang, A.V. S c h a l l y , and T.W. R e d d i n g , Biochem., E, 2326, 1978. 9 9 3 , 1978. 88. Y. Yabe, C. M i u r a , Y. Baba, and S. Sawano, Chem. Pharm. B u l l , 8 9 . M.P. Brown, D.H. Coy, A. Gomez-Pan, B.H. H i r s t , M. H u n t e r , C. M e y e r s , J.D. Reed, A.V. S c h a l l y , and 8. Shaw, J . P h y s i o l . , 277, 1 , 1978. 90. M.L. T o r c h i a n a , C.A. S t o n e , P.G. Cook, and S.J. W e i s e , P r o c . SOC. Exp. B i o l . Med., 1 5 4 , 4 4 9 , 1917. D.A.

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219

N e u t r a l P r o t e i n a s e s i n Rheumatoid A r t h r i t i s

Arnold L . Oronsky and C h r i s t i n e Winslow Buermann, Medical Research Divis i o n , American Cyanamid Company, L e d e r l e L a b o r a t o r i e s , P e a r l River, N . Y. I n t r o d u c t i o n - The most w i d e l y h e l d t h e o r y of t h e p a t h o g e n e s i s of RA i s t h a t i n r e s p o n s e t o a s y e t unknown a n t i g e n i c s t i m u l i , lymphoid c e l l s i n t h e synovium form f o l l i c l e s and b e g i n t o produce immunoglobulins s u c h as IgG and IgM (rheumatoid f a c t o r ) . Rheumatoid f a c t o r combines w i t h IgG t o form i n s o l u b l e immune complexes which are found b o t h i n the s y n o v i a l memb r a n e and s y n o v i a l f l u i d . These immune complexes a r e thought t o i n i t i a t e t h e inflammatory r e s p o n s e s commonly a s s o c i a t e d w i t h rheumatoid a r t h r i t i s . Immune complex a c t i v a t i o n of t h e complement c a s c a d e l e a d s t o t h e product i o n of p e r m e a b i l i t y and c h e m o t a c t i c f a c t o r s . These m o l e c u l e s i n t u r n induce t h e a c t i v e m i g r a t i o n of l a r g e numbers of polymorphonuclear l e u k o c y t e s (F")i n t o t h e a f f e c t e d j o i n t s . A s t h e d i s e a s e c o n t i n u e s , mononuclear c e l l s i n c l u d i n g T and B lymphocytes, plasma c e l l s , and macrophages become t h e predominant c e l l t y p e s i n t h e j o i n t space. I n a d d i t i o n t h e s y n o v i a l l i n i n g becomes markedly h y p e r t r o p h i e d . Thickening i s compounded by a l a y er of g r a n u l a t i o n t i s s u e , composed of f i b r o b l a s t s , blood vessels and inflammatory c e l l s , c a l l e d pannus. Pannus may e v e n t u a l l y c o v e r t h e s u r f a c e of t h e c a r t i l a g e , w i t h s i g n i f i c a n t d e s t r u c t i o n o c c u r r i n g u n d e r n e a t h t h e l e a d i n g edge of advancing pannus. A s t h e d i s e a s e p r o g r e s s e s , c a r t i l a g e and bone and tendon undergo s i g n i f i c a n t d e g e n e r a t i v e changes a s s o c i a t e d with severe chronic RA.l,* The d e s t r u c t i o n of t h e j o i n t c o n n e c t i v e tiss u e e l e m e n t s , p r o t e o g l y c a n s and c o l l a g e n , h a s been shown t o b e mediated through t h e a c t i o n of enzymes a t n e u t r a l pH d e r i v e d from t h e i n v a d i n g i n f lammatory c e l l s p a r t i c u l a r 1 PMN3-16 and macro h a g e ~ l ~ - ,~ pl r) o l i f e r a t i n g pannus, 2 , 22-26 ~ a r t i l a g e ~ ~ 7 -and 2 9 bone.30-3f P r o t e o g l y c a n s i n c a r t i l a g e e x i s t a s a g g r e g a t e s of l a r g e m o l e c u l a r weight (MW) p o l y a n i o n i c s p e c i e s of 100 o r more glycosaminoglycan c h a i n s ( p r i m a r i l y c h o n d r o i t i n 6 - s u l f a t e ) a t t a c h e d 0 - x y l o s i d i c a l l y t o t h e serine r e s i d u e of a l a r g e p r o t e i n c o r e . 3 5 Degradation of p r o t e o g l y c a n s a t neut r a l pH u s u a l l y i n v o l v e s p r o t e o l y t i c a c t i v i t y a l o n g t h e p r o t e i n c o r e of t h e molecule.4 T h i s c l e a v a g e l e a d s t o p r o t e o g l y c a n s o l u b i l i z a t i o n releasi n g glycosaminoglycan c h a i n s , which f a c i l i t a t e s t h e s u s c e p t i b i l i t y of c o l Collagen i s t h e major p r o t e i n component of c a r t l lagen t o degradation. l a g e and bone. I n t h e s e s t r u c t u r e s i t exists p r i n c i p a l l y as an i n s o l u b l e The d e g r a d a t i o n of c o l l a g e n a g g r e g a t e w i t h a r e l a t i v e l y long h a l f - l i f e . f i b e r s c a n be i n i t i a t e d by p r o t e a s e s which d e g r a d e t h e n o n - h e l i c a l p e p t i d e r e g i o n s of c o l l a g e n t h e r e b y e l i m i n a t i n g c e r t a i n i n t e r m o l e c u l a r c r o s s l i n k s . Degradation of t h e remainder of t h e c o l l a g e n m o l e c u l e r e q u i r e s t h e a c t i v i t y of a c o l l a g e n a s e which a c t s a t a s i t e s p e c i f i c f o r t h e enzyme. Once t h i s c l e a v a g e o c c u r s , t h e molecule d e n a t u r e s and becomes s u s c e p t i b l e t o non-specif i c p r o t e o l y t i c d i g e s t i o n . 36

PMN - The PMN

i s a p h a g o c y t i c c e l l which c o n t a i n s enzymes t h a t are s t o r e d i n two t y p e s of c y t o p l a s m i c g r a n u l e s . Azurophil g r a n u l e s c o n t a i n l y s o s o m a 1 h y d r o l a s e s , n e u t r a l p r o t e i n a s e s and b a c t e r i c i d a l e l e m e n t s . Specific g r a n u l e s c o n t a i n c o l l a g e n a s e , lysozyme and l a c t o f e r r i n . PMN release t h e c o n t e n t s of t h e i r g r a n u l e s d u r i n g p h a g o c y t o s i s . Release may a l s o o c c u r when t h e c e l l s are t r a p p e d w i t h i n t h e j o i n t s p a c e and undergo spontaneous a u t o l y s i s . 3 ~ 4 Three PMN n e u t r a l p r o t e i n a s e s have t h u s f a r been i s o l a t e d and c h a r a c t e r i z e d ; elastase, c a t h e p s i n G , and c o l l a g e n a s e . Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reSeNed. ISBN 0-12-040514-8

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E l a s t a s e - PMN e l a s t a s e is a s e r i n e p r o t e a s e t h a t is a c t i v e a g a i n s t azoca s e i n , c h l o r o a c etyl-3-naphthoic a c id-0-t o l u i d i d e (ClAc-0-NapAS-D) , e l a s t i n - o r c e i n , N-carbobenzoxy-alanyl-2-naphthol ester, and N-a-tertbutyloxycarbonyl-L-alanine p a r a n i t r o p h e n y l ester. 599, 10 It h a s been demonstrated t h a t elastase c a u s e s complete release of proteoglycan m a t r i x from i n t a c t a r t i c u l a r c a r t i l a g e and can degrade i s o l a t e d a g g r e g a t e s o r s u b u n i t s of proteoglycan i n s o l u t i o n . 4 PMN e l a s t a s e a l s o i n c r e a s e s cell u l a r m i g r a t i o n t o t h e s i t e of inflammation by l i b e r a t i n g complement der i v e d chemotactic $roducts . I 2 It can a l s o c l e a v e i n t e r m o l e c u l a r c r o s s l i n k s of c o l l a g e n . A f f i n i t y chromatography h a s been used t o p u r i f y PMN e l a s t a s e and it has been demonstrated t h a t t h e enzyme i s p r e s e n t a s t h r e e isoenzymes w i t h MW's of 33,000-36,000.1~ Cathepsin G - Leukocytic c a t h e p s i n G is a chymotryptic-like s e r i n e p r o t e a s e t h a t h a s been demonstrated t o break down proteoglycans and t y p e I1 c o l l a g e n from a r t i c u l a r c a r t i l a g e a t n e u t r a l P H . ~ Cathepsin G, u n l i k e elastase, w a s n o t shown t o have any e f f e c t on s o l u b l e t y p e I c o l l a g e n . 4 The p r o p e r t i e s demonstrated f o r c a t h e p s i n G which p e r p e t u a t e t h e inflammatory response a s s o c i a t e d w i t h RA i n c l u d e : (1) c l e a v a g e of t h e t h i r d and f i f t h components of complement, g e n e r a t i n g chemotactic p r o d u c t s ; (2) d i rect s t i m u l a t i o n of c e l l m i g r a t i o n and phagocytosis a t low c o n c e n t r a t i o n s ; and (3) i n h i b i t i o n of c e l l m i g r a t i o n and phagocytosis a t h i g h concentrat i o n s . 1 2 Cathepsin G i s h i g h l y charged and low MW (av. 25,500). P u r i f i c a t i o n , u s i n g a f f i n i t y chromatography on Sepharose-Trasylol has shown c a t h e p s i n G t o e x i s t a s t h r e e isoenzymes.13 Cathepsin G i s a n o n s p e c i f i c p r o t e a s e w i t h a c t i v i t y a g a i n s t s u b s t r a t e s such as a z o c o l l , denatured c a s e i n and ClAc-0-NapAS-D. It can b e d i s t i n g u i s h e d from e l a s t a s e by i t s a c t i v i t y on N-benzoyl-D, L-phenylalanyl-2-naphthyl ester. 13-15 Collagenase - The PMN c o l l a g e n a s e i s a t h i o l - m e t a l l o p r o t e i n a s e t h a t s p e c i f i c a l l y degrades n a t i v e c o l l a g e n f i b r i l s a t n e u t r a l pH.36 Collagenase is s t o r e d w i t h i n t h e s p e c i f i c g r a n u l e s of t h e PMN in b o t h a c t i v e and l a t e n t forms. P u r i f i c a t i o n of PMN c o l l a g e n a s e i n v o l v e s Sephadex G-75 and BioRex-70 chromatography and p r e p a r a t i v e e l e c t r o p h o r e s i s i n polyacrylamide g e l . Two c o l l a g e n a s e s t h u s p u r i f i e d have MW's of 42,000 and 33,000. It h a s been suggested t h a t each of t h e enzymes c o n t a i n s two subu n i t s . The amino a c i d composition of t h e two c o l l a g e n a s e s i s almost identi~al.L ~ a t e n t PMN c o l l a g e n a s e h a s n o t y e t been p u r i f i e d a l t h o u g h p a r t i a l p u r i f i c a t i o n of c o l l a g e n a s e from s y n o v i a l l i n i n g c e l l s i n d i c a t e s t h i s molecule is probably a n enzyme i n h i b i t o r complex. A c t i v a t i o n of l a t e n t PMN c o l l a g e n a s e can occur w i t h n o n s p e c i f i c p r o t e o l y s i s o r proteol y t i c a c t i v i t y derived from RA s y n o v i a l f l u i d . 1 6 MACROPHAGE - Macrophages are a l s o phagocytic c e l l s which e n t e r t h e j o i n t space i n response t o chemotactic s t i m u l i . Upon a c t i v a t i o n t h e y release a v a r i e t y of m e d i a t o r s i n t o t h e l o c a l environment. Macrophages c a n be a c t i v a t e d by inflammatory s t i m u l i such as endotoxins, lymphokines from a n t i g e n - o r -mitogen-stimulated lymphocytes, phagocytosis of non-degradable p a r t i c l e s such as zymosan o r l a t e x and i n t r a p e r i t o n e a l i n d u c t i o n by such exogenous s t i m u l a n t s as t h i o g l y c o l l a t e and carrageenen. Important produ c t s s e c r e t e d as a r e s u l t of a c t i v a t i o n i n c l u d e pyrogens; p r o s t a g l a n d i n s ; Complement components C 2 , C 3 , C4, and f a c t o r B; and p r o t e o l y t i c enzymes a c t i v e a t n e u t r a l pH such as plasminogen a c t i v a t o r , c o l l a g e n a s e , and e l a s t a s e . 17-20 Plasminogen A c t i v a t o r - Plasminogen a c t i v a t o r is p a r t i c u l a r l y i m p o r t a n t i n p e r p e t u a t i n g RA because plasmin formed from plasminogen can

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a c t i v a t e complement which r e s u l t s i n p r o d u c t s that are c h e m o t a c t i c , o p s o n i c , and macrophage a c t i v a t o r s . 1 7 Plasmin i s a l s o i m p o r t a n t i n c o l l a g e n o l y s i s by s e r v i n g a s an a c t i v a t o r of l a t e n t c o l l a g e n a s e . 17 Mouse p e r i t o n e a l macrophage plasminogen a c t i v a t o r s have been found t o be s e r i n e p r o t e a s e s , s e n s i t i v e t o i n h i b i t i o n by d i i s o p r o p y l f l u o r o p h o s p h a t e (DFP) and t h e s p e c i f i c t r y p s i n a c t i v e s i t e b l o c k e r p-nitrophenyl g u a n i d i n o b e n z o a t e Two s p e c i e s of plasminogen a c t i v a t o r w i t h MWs' of 48,000 and (NPGB) 28 ,000 have been i d e n t i f i e d . 18 C o l l a g e n a s e - C o l l a g e n a s e r e l e a s e d from a c t i v a t e d macrophages i s a t y p i c a l metallo-enzyme m o s t l y p r e s e n t i n a l a t e n t form which c a n be act i v a t e d by o t h e r p r o t e a s e s such as t r y p s i n and p l a ~ m i n . ~ , 1 9A c t i v a t e d c o l l a g e n a s e i s i n h i b i t e d by c h e l a t i n g a g e n t s (EDTA, 0 - p h e n a n t h r o l i n e , penic i l l a m i n e ) , d i s u l f i d e r e d u c i n g a g e n t s ( c y s t e i n e , d i t h i o t h r e i t o l ) , and serum a2-macroglobulin. DFP, soybean t r y p s i n i n h i b i t o r (SBTI) , l e u p e p t i n , 4-~hloromercuribenzoate, and NPGB are i n a c t i v e . Elastase and N o n s p e c i f i c N e u t r a l P r o t e i n a s e s - Mouse macrophage elastase i s a l s o a s e r i n e p r o t e a s e r e s p o n s i v e t o DFP and phenylmethylsulf o n y l f l u o r i d e . Mouse macrophage elastase h a s a narrower r a n g e of a c t i v i t y F o r example, i t does t h a n t h e e l a s t a s e s from p a n c r e a s and n e u t r o p h i l s . n o t a t t a c k s y n t h e t i c s u b s t r a t e s such a s Ac(ala)3-4-nitroanilide9 o r It c a n however d e g r a d e e l a s t i n and a z o c a s e i n . 2o A n e u t r a l CUc-ONapAS-D. p r o t e a s e d e r i v e d from r a b b i t bone-marrow macrophages i n c u l t u r e has been It i s metal dependent (C02+, Zn2+) and i n h i b i t e d by EDTA, 1,lOisolated. p h e n a n t h r o l i n e , c y s t e i n e , and serum b u t n o t DFP o r 4-hydroxymercuribenIts a p p a r e n t MW is 1 7 , 0 0 0 . 2 i zoate.

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SYNOVIAL T I S S U E ENZYMES - The i n t e r f a c e between a r t i c u l a r c a r t i l a g e and p r o l i f e r a t i n g pannus r e p r e s e n t s t h e area i n which maximal p r o g r e s s i v e des t r u c t i o n of t h e j o i n t o c c u r s i n RA p a t i e n t s . 2 2 Rheumatoid s y n o v i a l t i s s u e produces l a r g e q u a n t i t i e s of c o l l a g e n a s e . The c o l l a g e n a s e r e l e a s e d by s y n o v i a l c e l l s i s predominantly i n a n i n a c t i v e o r l a t e n t form.36 The i n a c t i v e c o l l a g e n a s e i s p r o b a b l y a n enzyme-inhibitor complex and n o t a t r u e ~ y m o g e n . ~ , 2 425536 , A c t i v e rheumatoid s y n o v i a l c o l l a g e n a s e i s a m e t a l l o p r o t e a s e w i t h m o l e c u l a r weight of 33,000. It i s i n h i b i t e d by chel a t i n g a g e n t s , t h i o l rea e n t s and t h e s e r u m i n h i b i t o r s a2-macroglobulin A monospecif i c a n t i b o d y t o human c o l l a g e n a s e and B l - a n t i c o l l a g e n a s e . 2 t h a s l o c a l i z e d t h e enzyme e x t r a c e l l u l a r l y around c y t o p l a s m i c e x t e n s i o n s of d e n d r i t i c c e l l s and i n t r a c e l l u l a r l y w i t h i n a few macrophage-like and f i b r o b l a s t - l i k e c e l l s .25 Plasminogen a c t i v a t o r s i m i l a r t o t h e macrophage enzyme a p p e a r s t o be p r e s e n t i n c u l t u r e s of RA synovium.2 Rabbit s y n o v i a l f i b r o b l a s t s i n c u l t u r e have been shown t o s e c r e t e a metal dependent n e u t r a l p r o t e i n a s e t h a t d e g r a d e s t h e p r o t e i n c o r e of cart i l a g e p r o t e o g l y c a n s . The enzyme i s found i n a l a t e n t form i n t h e c u l t u r e medium and i s a c t i v a t e d by l i m i t e d p r o t e 0 l y s i s . ~ 3 CARTILAGE AND BONE ENZYMES - N e u t r a l p r o t e a s e s , i n c l u d i n g a c o l l a g e n a s e , a r e r e l e a s e d by c a r t i l a g e c e l l s (chondrocytes) i n t h e p r e s e n c e of p r o d u c t s of l i p o p o l y s a c c h a r i d e s t i m u l a t e d macrophages. They a r e i n h i b i t e d by d i t h i o t h r e i t o l and EDTA, b u t n o t by soybean t r y p s i n i n h i b i t o r . 2 7 Four forms of a metal-dependent n e u t r a l p r o t e o g l y c a n a s e from human a r t i c u l a r c a r t i l a g e have been i s o l a t e d . They have MWs' of 26-28,000 (dimers MW 13-14,000) and r e q u i r e z i n c o r c o b a l t f o r a c t i v i t y , and are t h e r e f o r e i n h i b i t e d by 0 - p h e n a n t h r o l i n e and EDTA. 28 Morphological changes i n c a r t i l a g e from RA

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p a t i e n t s show t h a t p r o t e o g l y c a n i s diminished around most of t h e chondroc y t e s , and d i g e s t e d c o l l a g e n f i b r i l s a p p e a r i n t r a c e l l u l a r l y . 29 These changes s e e m t o c o r r o b o r a t e t h e biochemical e v i d e n c e of d i s s o l u t i o n of p r o t e o g l y c a n and c o l l a g e n by chondrocyte enzymes. Bone r e s o r p t i o n o c c u r s when o s t e o c l a s t s a r e a c t i v a t e d t o produce p r o t e a s e s and c o l l a g e n a s e by p a r a t h y r o i d hormone o r by o s t e o c l a s t a c t i v a t i n g f a c t o r ( d e r i v e d from phytohemagglutinin s t i m u l a t e d lymphocytes). Although t r a s y l o l and soybean t r y p s i n i n h i b i t o r do n o t a f f e c t bone res o r p t i o n a s p e c i f i c c a r t i l a g e - d e r i v e d a n t i - c o l l a g e n a s e seems t o b e a c t i v e . 369 3 1 Two n e u t r a l l y a c t i v e m e t a l l o p r o t e a s e s , d i s t i n c t from c o l l a g e n a s e , have been found i n bone.32 They a r e i n a l a t e n t form and c a n be a c t i v a t e d by e i t h e r 4-aminophenylmercuric acetate o r t r y p s i n . One i s s p e c i f i c f o r g e l a t i n and a z o c o l l , b u t n o t c o l l a g e n , p r o t e o g l y c a n s , o r a z o c a s e i n . The o t h e r d e g r a d e s p r o t e o g l y c a n s , a z o c a s e i n , and a z o c o l l , b u t n e i t h e r c o l l a g e n nor g e l a t i n . 3 2 Mouse and c h i c k bone e x p l a n t s have been shown t o produce a l a t e n t c o l l a g e n a s e . 33,34 I n a d d i t i o n , t h e mouse bone e x p l a n t s produce a l a t e n t n e u t r a l p r o t e i n a s e . Limited p r o t e o l y s i s of t h e s e molecules g i v e s r i s e t o p r o t e o g l y c a n and c o l l a g e n d e g r a d i n g a c t i o n . Latency may b e d u e t o t h e presence of a n enzyme i n h i b i t o r complex. The c h i c k l a t e n t c o l l a g e n a s e h a s an a p p a r e n t MW of 54,000, w h i l e t h e a c t i v e form has an MW of 43,000.33 T r y p s i n a c t i v a t i o n of b o t h l a t e n t mouse enzymes d e c r e a s e s t h e i r MW's from 60-70,000 t o about 40,000. The mouse n e u t r a l p r o t e a s e is i n h i b i t e d by EDTA, c y s t e i n e , and serum.34 INHIBITORS - The s e a r c h f o r new a g e n t s which modify t h e rheumatic p r o c e s s h a s r e c e n t l y been o r i e n t e d toward f i n d i n g i n h i b i t o r s of t h e enzymes d i r e c t l y These i n c l u d e i n h i b i t o r s of t h e s e r i n e involved i n j o i n t d e s t r u c t i o n . ( e l a st a se , c a t heps i n G, plasm inogen a c t i v a t o r ) and m e t a 110 ( c o l l a g e n a se , non-collagenolytic) proteinases. These compounds have been d e r i v e d from b o t h n a t u r a l and s y n t h e t i c s o u r c e s . Serum Many of t h e n a t u r a l l y o c c u r r i n g serum p r o t e i n s t h a t i n h i b i t t h e c e l l d e r i v e d n e u t r a l p r o t e i n a s e s have been p u r i f i e d and w e l l c h a r a c terized.37-40 Both a l - a n t i - t r y p s i n (290 mg%, MW 55,000) and a m a c r o g l o b u l i n (260 mg%, MW 725,000) can i n h i b i t plasmin, c o l l a g e n a s e , efastase, and c a t h e p s i n G, a l t h o u g h t h e r e are d i f f e r i n g o p i n i o n s on t h e e f f i c a c y of a l - a n t i - t r y p s i n a g a i n s t c o l l a g e n a s e . 36-39941-46 Collagenase is i n h i b i t e d (MW 40,000) .38,47 Another r e c e n t l y deby a s p e c i f i c B1-anti-collagenase Cathepsin G i s s c r i b e d i n h i b i t o r of c o l l a g e n a s e i s p l a t e l e t f a c t o r 4.48 a l s o s p e c i f i c a l l y i n h i b i t e d by a2-anti-chymotrypsin (48.7 mg%, MW 69,000) .42 There a l s o e x i s t s a 65-70,000 MW plasmin i n h i b i t o r i n t h e a 2 - f r a c t i o n of Plasmin i n h i b i t i o n a l s o o c c u r s i n t h e p r e s e n c e of a n t i thrombin-heparin complexes. 5 3 Plant I n h i b i t o r s d e r i v e d from p l a n t s o u r c e s i n c l u d e t h e b l a c k bean o r soybean i n h i b i t o r s (Kunitz and Bowman-Birk t y p e s ) ,54-57 l i m a bean i n h i b i t o r , 58 and c h i c k p e a i n h i b i t o r . 5 9 These are a l l u n u s u a l double-headed p o l y p e p t i d e s , w i t h two independent reactive s i t e s , c a p a b l e of i n t e r a c t i n g w i t h both t r y p s i n and chymotrypsin.60-63 They a l s o a r e i n h i b i t o r s of g r a n u l o c y t e elastase and c a t h e p s i n G. 14,60 M i c r o b i a l - Some of t h e most i n t e r e s t i n g p r o t e a s e i n h i b i t o r s have been d e r i v e d from b a c t e r i a l s o u r c e s s u c h as actinomycetes. 64-66 Of part i c u l a r importance t o i n h i b i t i o n of n e u t r a l p r o t e a s e s and c o l l a g e n a s e s are e l a s t a t i n a l , chymostatin, l e u p e p t i n s , and phosphoramidon.

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S i n c e elastase c l e a v e s t h e c a r b o x y l s i d e of a l a n i n e , t h e a l a n i n y l group a t t h e t e r m i n a l carbon of e l a s t a t i n a l [E-(1-carboxy-isopenty1)carbamoyl-a-(2-iminohexahydro-4-pyrimidyl) lycylglutaminyl-alaninal] makes i t a s p e c i f i c i n h i b i t o r of elastase. 64-i6 Chymostatin, N-[ ((S)-l-carboxy-2-phenylethyl)carbamoyl]-a-[ 2-imino hexahydro-4-( S)-pyrimidyl] -L-glycyl-L-leucyl-phenylalaninal, h a s a phenyla l a n i n y l group a t t h e t e r m i n a l c a r b o n , which makes i t s p e c i f i c f o r chymot r y p s i n and c a t h e p s i n G, which cleave t h e c a r b o x y l s i d e of a r o m a t i c amino a c i d s . 64-66 The l e u p e p t i n s a r e i n h i b i t o r s of plasmin, a t r y p s i n - l i k e enzyme. Leupeptins c o n t a i n a r g i n i n y l r e s i d u e s a t t h e i r t e r m i n a l carbon, and i n h i b i t enzymes which c l e a v e a t t h e c a r b o x y l s i d e of b a s i c amino a c i d s s u c h as a r g i n i n e o r l y s i n e . 1 4 , 5 0 The s t r u c t u r e of t h e most a c t i v e l e u p e p t i n m i x t u r e i s propionyl-L-Leu-L-Leu-Argininal and acetyl-L-Leu-L-LeuA r g i n i n a l i n a 3 : l ratio.64-66 The N-phosphoropeptide moiety of phosphoramidon @-[(a-L-rhamnopyranosyloxyhydroxy-phosphiny1)-L-leucyl-L-trypt ophan] ) s p e c i f i c a l l y i n h i b i t s t h o s e m e t a l l o p r o t e a s e s t h a t c l e a v e t h e amino s i d e of hydrophobic amino a c i d s . C o l l a g e n a s e c l e a v e s a t a s p e c i f i c g l y - l e u o r l y - i l e u bond i n c o l l a g e n , and i s weakly i n h i b i t e d by phosphoramidon. 68-66 A new t r y p s i n i n h i b i t o r h a s been i s o l a t e d from Cephalosporium *67 A s t r a i n of Streptomyces produces e l a s n i n (MW 392) which s p e c i f i c a l l y inh i b i t s human g r a n u l o c y t e elastase (50% i n h i b i t i o n a t 1.3vg/ml) b u t is in ef f ec t i v e a g a i n s t p a n c r e a t i c ela s t a s e, t r y p s i n , c hymo t r y p s i n , t hermo l y s i n and papain. 68 Animal T i s s u e I n h i b i t o r s - I n h i b i t o r s of n e u t r a l p r o t e a s e s a r e widel y d i s t r i b u t e d i n animal t i s s u e s . Low m o l e c u l a r weight i n h i b i t o r s of l e u k o c y t e n e u t r a l p r o t e a s e s have been i s o l a t e d from human t r a c h e a l and s u b m a x i l l a r y s i n u s mucosa, 69 and human mucus s e c r e t i o n s . 6 0 , 70 They are double-headed, w i t h two independent r e a c t i v e s i t e s , s i m i l a r t o t h o s e der i v e d from beans. C a r t i l a g e i t s e l f has been shown t o c o n t a i n d i s t i n c t components t h a t i n h i b i t human l e u k o c y t e elastase, c a t h e p s i n G , and c o l l a genase. The material d e r i v e d from b o v i n e n a s a l c a r t i l a g e i s low MW ( 7 , 0 0 0 - s e r i n e p r o t e a s e i n h i b i t i o n , and 22,000-collagenase i n h i b i t i o n ) .71 Other c r u d e c a r t i l a g e d e r i v e d f a c t o r s i n h i b i t c o l l a g e n a s e , r e u l a t e o s t e o c l a s t a c t i v i t y and s i g n i f i c a n t l y s u p p r e s s bone r e s o r p t i o n . 3 0 , 5 1 , 7 2 Another low molecular weight f r a c t i o n of bovine nasal c a r t i l a g e h a s been demons t r a t e d t o i n h i b i t s y n o v i a l c e l l s u r f a c e n e u t r a l p r o t e a s e a c t i v i t y necess a r y f o r p r o l i f e r a t i o n . 73 V a s c u l a r t i s s u e , i n c l u d i n g a o r t i c e ~ t r a c t s 7 ~ and smooth muscle c e l l s i n c u l t u r e 7 5 are s o u r c e s of i n h i b i t o r s of f i b r o b l a s t c o l l a g e n a s e . The a o r t i c t i s s u e material i s less t h a n 10,000 MW. While t h e c u l t u r e d smooth muscle extract h a s n o t been c o m p l e t e l y c h a r a c t e r i z e d i t h a s been shown t o a f f e c t o n l y mammalian and n o t b a c t e r i a l c o l l a g e n a s e , i s s t a b l e t o h e a t and a c i d t r e a t m e n t , and i s s e n s i t i v e t o red u c t i o n f o l l o w e d by a l k y l a t i 0 n . 7 ~ Other i n h i b i t o r s of f i b r o b l a s t c o l l a genase have been demonstrated i n human s k i n f i b r o b l a s t c u l t u r e s , 76 human tendon i n c u l t u r e 7 7 and g i n g i v a l f i b r o b l a s t s . 78 C o l l a g e n a s e i n h i b i t i o n a l s o o c c u r s w i t h p e p t i d e f r a g m e n t s of p r o t e o l y t i c a l l y d i g e s t e d tendon p r o c o l l a g e n . 79 These r e s u l t s s u g g e s t t h a t c o n v e r s i o n of p r o c o l l a g e n t o c o l l a g e n produces p e p t i d e s which p r o t e c t t h e c o l l a g e n molecule from d i g e s t i o n by c o l l a g e n a s e . Basic p a n c r e a t i c t r y p s i n i n h i b i t o r ( t r a s y l o l ) h a s been used i n a f f i n i t y chromatography t o p u r i f y human l e u k o c y t e elastase, however, c o n f l i c -

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t i n g r e p o r t s have appeared concerning i t s i n h i b i t o r y e f f e c t s on human PMN elastase.80 Trypsin, chymotrypsin and e l a s t a s e appear t o be bound a t t h e same s i t e of t h e i n h i b i t o r . 8 0 A review of b a s i c p r o t e i n t r s i n i n h i b i t o r e f f e c t s on plasmin and plasma k a l l i k r e i n h a s a l s o appeared.

Sf

S y n t h e t i c I n h i b i t o r s - S e r i n e p r o t e a s e s such a s elastase and c a t h e p s i n G have a charge r e l a y system a t t h e i r c a t a l y t i c s i t e i n v o l v i n g asp a r t i c a c i d , h i s t i d i n e , and s e r i n e . S p e c i f i c i t y among t h e enzymes arises when secondary amino a c i d s a t t h e a c t i v e s i t e d i f f e r i n p o s i t i o n , s i z e , o r degree of hydrophobicity. The design of i n h i b i t o r s of t h e s e enzymes i s b a s e d on t h e enzyme's s p e c i f i c requirements f o r s u b s t r a t e . 8 2 I r r e v e r s i b l e i n h i b i t i o n o c c u r s when c o v a l e n t bonds form t o t h e h i s t i d i n e a n d / o r s e r i n e m o i e t i e s of t h e enzyme, f a c i l i t a t e d by t h e removal of good l e a v i n g groups such as C1-, F-, and p-nitrophenol. S p e c i f i c i n h i b i t o r s such as p e p t i d e chloromethyl ketones,82 aza-peptide p-nitrophenyl esters, 62 s u l f onyl f l u o r i d e s ( a r y l and peptide)82-84 and l a c t y l d e r i v a t i v e s of 2-aza-alanine peptides85 have been s y n t h e s i z e d i n t h e hope of f i n d i n g s p e c i f i c s e r i n e p r o t e a s e i n h i b i t o r s . T h i o l p r o t e a s e i n h i b i t o r s are being i n v e s t i g a t e d as i n h i b i t o r s of collagenase,86-90 and s u b s t i t u t e d benzamidines may b e of u s e i n i n h i b i t i n g t r y p s i n - l i k e enzymes, such as plasmin. 91-93 a ) Chloromethyl Ketones - Several chloromethyl k e t o n e s (L) have been demonstrated t o be s p e c i f i c i n h i b i t o r s of l e u k o c y t e elastase a n d / o r

k

c a t h e p s i n G. The f i f t h amino a c i d s u b s i t e i s of primary importance and t h e enzyme i n t e r RCO-NH-CH-C-CH2-Cl I1 a c t s w e l l w i t h s u c c i n y l o r methyl' s u c c i n y l groups. The b e s t g r a n u l o c y t e e l a s t a s e O chloromethylketone i n h i b i t o r i s MeO-SucR = p e p t i d e o r amino a c i d kla-Ala-Pro-ValCH2Cl. 82 Granulocyte c a t h e p s i n G i s n o t i n h i b i t e d by Tos-PheCH2C1, b u t Z-Phe CH2C1 and Z-Gly-Gly-PheCH C 1 are f a i r i n h i b i t o r s , w i t h t h e b e s t i n h i b i t o r being Z-Gly-Leu-PheCH2Cl.i2 The carbobenzyloxy group can b e r e p l a c e d by a n a c e t y l group, but Ac-Ala-Ala-Pro-ValCH C 1 i s much b e t t e r t h a n MeO-SucAla-Ala-Pro-ValCH2C1 a g a i n s t c a t h e p s i n G. 8 2 Chloromethyl ketones a l s o react w i t h t h i o l p r o t e a s e s , and could t h e r e f o r e be u s e f u l c o l l a g e n a s e i n h i b i t o r s . 8 2 b) Aza-peptide p-Nitrophenyl Esters - Aza-peptide p-nitrophenyl esters (2) where t h e a-CH i s r e p l a c e d by a n i t r o R gen atom, are i n h i b i t o r s of s e v e r a l s e r i n e prot e a s e s , p o s s i b l y due t o a c y l a t i o n of t h e a c t i v e s i t e serine.82 The most a c t i v e comRCO-NH-N-C-ONp pound Ac-Ala-Ala-Anle-ONp (Anle = -NHN (CH2 CH2CH2CH3)CO-), i n h i b i t s both granulocyte 0 (2) e l a s t a s e and c a t h e p s i n G. Ac-Ala-Aphe-ONp a c y l a t e s c a t h e p s i n G, b u t n o t e l a s t a s e . 8 2 S e r i n e r e s i d u e s i n e l a s t a s e and c a t h e p s i n G c ) Sulfonyl Fluorides are s u l f onylated with compounds such a s phenylmethanesulfonyl f l u o r i d e . 82 Human g r a n u l o c y t e e l a s t a s e r e a c t s f a s t e r w i t h s u l f o n y l f l u o r i d e s , w h i l e p o r c i n e p a n c r e a t i c elastase r e a c t s f a s t e r w i t h chloromethyl k e t o n e s i n d i c a t i n g t h a t t h e two enzymes d i f f e r i n s p e c i f i c i t i e s . Human c a t h e p s i n G, on t h e o t h e r hand, reacts more slowly t h a n bovine a-chymotrypsin A w i t h s u l f o n y l f l u o r i d e s .82, 83 Attempts have been made t o i n c r e a s e t h e s p e c i f i c i t y of s u l f o n y l compounds w i t h r e s p e c t t o d e ree of i n h i b i t i o n of t r y p s i n , plasmin, thrombAn, k a l l i k r e i n and urokinase. Nitrophenyl esters of benzenesulf o n i c a c i d and

<1>

I

I

1

-

g4

Chap. 2 1

Rheumatoid A r t h r i t i s

phenylmethanesulf o n i c a c i d have been s y n t h e s i z e d :

RCH2

a

(3)c o n t a i n i n g

(CH2)n-S03-d:R1 3

Oronsky, Buermann

225

v a r i o u s p o s i t i v e l y charged groups (R = p o s i t i v e l y charged groups; R1, R2 = H o r NO2; n = 0 o r 1)

.B r -

P o s i t i v e l y charged groups i n c l u d e d i m e t h y l s u l f i d e , t h i o u r e a , and i t s Nmethyl d e r i v a t i v e s , trimethylamine, and p y r i d i n e . The b e s t t r y p s i n i n h i b i t o r is 1 - n i t r o p h e n y l 1'-amidinothiomethylphenylethanesulf o n i c a c i d (4),

which shows l i t t l e r e a c t i v i t y toward plasma k a l l i k r e i n , plasmin, thrombin, w i t h i n t e r m e d i a t e i n h i b i t o r y a c t i v i t y d i r e c t e d toward urokinase. The , s h o r t e r compound pnitrophenyl-g-amidinothiomethyl benz e n e s u l f o n a t e does n o t i n h i b i t t r y p s i n , b u t does i n h i b i t thrombin and t o a lesser degree From t h e s e r e s u l t s i t i s suggested t h a t v a r y i n g plasmin and urokinase. t h e p o s i t i v e l y charged groups on t h e s e n i t r o p h e n y l esters of s u l f o n i c a c i d s can l e a d t o s p e c i f i c i t y i n t h o s e i n h i b i t o r s d i r e c t e d toward v a r i o u s t r y p s i n - l i k e s e r i n e p r o t e a s e s .84 d) L a c t y l Derivatives of 2-Aza-alanine P e p t i d e s - S p e c i f i c granul o c y t e elastase i n h i b i t o r s have been found by v a r y i n g s u b s t i t u e n t s on l a c t y l d e r i v a t i v e s of such compounds as Ac-Ala-Ala-Pro-aza-Ala-Lactyl-X. The l a c t y l group can b e changed t o p h e n y l l a c t y l , but n o t a l a n i n e . The b e s t i n h i b i t o r s are t h o s e where X i s -NHNH2 o r -NH2, but t h e y do n o t appear t o be hydrolyzed by t h e enzyme, s o a c y l a t i o n of t h e enzyme probably does not occur .85 e) T h i o l P r o t e a s e I n h i b i t o r s - C y s t e i n e i s a n e f f e c t i v e i n h i b i t o r of c o l l a g e n a s e and i t has been used i n eye drops t o p r e v e n t c o l l a g e n a s e induced c o l l a g e n d e g r a d a t i o n i n t h e cornea.*6 Such compounds a s mercaptoimidazolypropionic a c i d ( 6)87 and Ij- [Ij- (L-3-trans-carboxyoxiran-2-carbony1)-L-leucyl] -agmatine- [from A s p e r g i l l u s j a p o n i c u s ( 7 ) 188-90 are t h i o l protease inhibitors.

(I)

t> N

H

SH 1

CH2-CH-C02H

F;" (CH2

H2NCNH

I

CH2Cmflo2H ) 4NHCOCHNHCO

H

-6

H

7 -

S u b s t i t u t e d Benzamidines - Plasmin and o t h e r t r y p s i n - l i k e esterases r e q u i r e s p e c i f i c i n h i b i t o r s , a s e x e m p l i f i e d by t h e broad s p e c i f i c i t y of some of t h e s u l f o n i c a c i d compounds. S u b s t i t u t e d benzamidines p r o v i d e p o t e n t and s p e c i f i c plasmin i n h i b i t o r s ,91 Compounds such a s , inhibit p l a smin and complement C 1-es t erase.

(s>

226 -

Sect. I V

-

Metabolic Diseases, Endocrine Function

B a i l e y , Ed.

Nn X = alkoxy, H , B r , C H 2 0 H , C 0 2 H , NH2,

NO2

8

Varying degrees of i n h i b i t i o n are n o t e d depending on e l e c t r o n d o n a t i o n from t h e s u b s t i t u e n t and i t s hydrophobicity. 91 S u b s t i t u t e d ( i . e . C:(NH)NJ32, halogen, N 0 2 , e t c . ) benzylphenyl e t h e r s , a,w-dioxyalkanes and a,a’ , a ” - t r i s (phenoxy) m e s i t y l e n e benzamidines have been s y n t h e s i z e d and analyzed € o r t h e i r i n h i b i t i o n of boar a c r o s i n , a t r y p s i n - l i k e p r o t e a s e i n spermatozoa. 92 A l l of t h e compounds a r e more p o t e n t t h a n benzamidine, w i t h t h e most p o t e n t compound being 9. While t h e s e compounds have n o t been t e s t e d a g a i n s t s p e c i f i c enzymes d e r i v e d from inflammatory c e l l s , t h e spectrum of a c t i v i t i e s of t h e s e a g e n t s should be a s c e r t a i n e d . Diarylamidine d e r i v a t i v e s have been i n v e s t i g a t e d as a n t i p r o t e a s e s ; i n 60 out of 62 compounds, one o r both of t h e amidino-substituted a r y l m o i e t i e s included indene, benzimidazole, benzofuran, benzo[b] thiophene and s e v e r a l o t h e r r e l a t e d nitrogen-containing h e t e r o c y c l e s . 93 One outs t a n d i n g i n h i b i t o r of t r y p s i n (Ki 1 . 7 ~ 1 0 - 8 ~ i) s bis(5-amidino-2-benzimidazolelmethane (10). These compounds demonstrate that v a r i a n t s of srnallH

L

molecular-weight i n h i b i t o r s can r e s u l t i n s p e c i f i c changes in e f f i c a c y with r e s p e c t t o i n h i b i t i o n of p r o t e a s e a c t i v i t y . 93 CONCLUSION - The development of i n h i b i t o r s of p r o t e a s e s must t a k e i n t o account such problems a s enzyme and t i s s u e s p e c i f i c i t y , and t o x i c i t y (part i c u l a r l y t h e i n h i b i t o r ’ s a b i l i t y t o a l k y l a t e o r a c y l a t e o t h e r important body c o n s t i t u e n t s ) ; a l t h o u g h in v i v o , t o x i c i t y may b e diminished by homeos t a t i c r e s y n t h e s i s of enzymes and o t h e r p r o t e i n c o n s t i t u e n t s . 94

I n a model of a c u t e inflammatory d i s e a s e , t h e r e v e r s e p a s s i v e Arthus r e a c t i o n , p r o t e a s e i n h i b i t o r s such a s t r a s y l o l and SBTI have been shown t o diminish t h e inflammatory response, p a r t i c u l a r l y accumulation of l e u kocytes and v a s c u l a r p e r m e a b i l i t y . 9 5 T h i s p r o v i d e s support f o r t h e rational t o s e a r c h f o r s p e c i f i c n e u t r a l p r o t e i n a s e i n h i b i t o r s a s new mod a l i t i e s of t h e r a p y i n R.A. See a l s o c h a p t e r 2 3 .

Chap. 21

Rheumatoid Arthritis

Oronsky, Buermann

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W.

2,

84,

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35,

172,

20,

.

z,

x,

c,

199,

a.

171,

103,

148, 84,

253,

3,

261,

227

Sect. I V 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. 75. 76. 77. 78. 79. 80. 81. 82.

83. 84. 85.

86. 87. 88. 89. 90. 91. 92. 93. 94. 95.

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Collen, B. Wiman, Blood, 2,563 (1978). J. Edy, D. Collen, M. V e r s t r a e t e , Proq. Chem. F i b r i n o l y s i s Thrombolysis, 3, 315 (1978). A. C. Teqer-Nilsson, E. Gyzander, U. Hedner, H. Myrwold, H. Noppa, R. Olsson, L. Wallmo, I b i d . , p. 327. N. Aoki, T. Yamanaka, C l i n . Chim. Acta., 84, 99 (1978). N. Semeraro, M. Colucci, P. T e l e s f o r o , D. Collen, B r . J. Haematol., 39, 91 (1978). S . Terada, K. S a t o , T. Kato, N. Izumiya, FEBS L e t t . , 90, 89 (1978). S. o d a n i , T. Ikenaka, J. Biochem. (Tokyo), 747 (19781. S . Odani, T. Ikenaka, I b i d . , 84,1 (1978). C. Toniolo, G. M. Bonora, C. V i t a , A. Fontana, Biochim. Biophys. Acta, 532, 327 (1978). F. C. Stevens, S. WuerZ, J. Krahn, i n "Bayer Symp. V Proteinase I n h i b i t o r s , " H. F r i t z , H. Tschesche, L. J. Greene, E. T n a s c h e i t , Eds. S p r i n g e r Verlaq, B e r l i n , 1974, p. 344. Y. Birk, I b i d . , p. 355. H. S c h i e s s l e r , K. H o c h s t r a s s e r , K. Ohlsson in "Neutral P r o t e a s e s of Human Polymorphonuclear Leukocytes," K. Havemann, A. J a n o f f , Eds., Urban and Schwarzenberq, Munich-Baltimore, 1978, p. 195. I. E. L i e n e r , Proc. E a s t e r Sch. Agric. S c i . , Univ. Nottinqham, 24, 1 1 7 (1978). S. Odani, T. Ikenaka, J. Biochem. (Tokyo), g , 737 (1978). D. L. Hwanq, W-K. Yang, D. E. F o u d , K-T-D. Lin,Plant Physiol., 61, 30 (1978). T. Aoyaqi, Seikaqaku, E, 358 (1978). H. Umezawa, Acta Biol. Med. Gar., 5, 1899 (1977). T. Aoyayi, H. mezawa i n " P r o t e a s e s and B i o l o g i c a l Control", E. Reich, D. B . R i f k i n , E. Shaw, Eds., Cold S p r i n g Harbor Laboratory, Cold S p r i n g Harbor, NY, 1975, p. 429. K. Tsuchiya, T. Kimura, Appl. Environ. Microbiol. S, 631 (1978). S. Omura, H. Ohno, T. Saheki, M. Yoshida, A. Nakaqawa, Biochem. Biophys. R e s . Commun., 83, 704 (1978). H. Teqner, K. Ohlsson i n "Neutral P r o t e a s e s of Human Polymorphonuclear Leukocytes," K. Havemann, A. J a n o f f , Eds., Urban and Schwarzenberq, Munich-Baltimre, 1978, p. 208. H. F r i t z , H. S c h i e s s l e r , R. Geiger, K. Ohlsson, K. H o c h s t r a s s e r , Agents Actions, 8, 57 (1978). P. J. Rouqhley, G. Murphy, A. J. B a r r e t t , Biochem. J . , 169, 721 (1978). F. Wezeman, J. Horton, K. Kuettner, Anat. Rec., 190,624 (1978). 567 (1978). P. G. Burk, V. B. Hatcher, Clin. Res.,.Z&, R. E i s e n s t e i n , S. Goren, B. Schumacher, B. Matijevich, Fed. Proc., 37, 641 (1978). J. C. Nolan, S. Ridge, A. L. Oronsky, L. L. Slakey, S . S . Kerwar, Biochem. Biophys. Res. Commun., E, 1183 (1978). H. G. WelguS, G. P. S t r i c k l i n , A. 2. E i s e n , F. A. Bauer, J. J. J e f f r e y , C l i n . Res., 26 489 (1978). E. D. H a r r i s , C. A. Vater, C. L. Mainardi, I b i d . , p. 623. M. L. Mailman, J. W. Simpson, J. Dent. Res., 264 (1978). Y. Naqai, H. S h i n k a i , Y. Ninomiya, Proc. Jpn. Acad., S e r i e s B, 54, 140 (1978). P. L e s t i e n n e , J. G. B i e t h , Arch. Biochem. Biophys., 190, 358 (1978). H. F r i t z , Proq. Chem. F i b r i n o l y s i s Thrombolysis, 2, 285 (1978). J. C. Powers, 8. F. Gupton, M. 0. L i v e l y , N. N i s h h o , R. J. Whitley i n " N e u t r a l P r o t e a s e s o f Human Polymorphonuclear Leukocytes," K. Havemann, A. Janoff, Eds., Urban and Schwarzenberq, MunichBaltimore, 1978, p. 221. M. 0. L i v e l y , J. C. POWrS, Biochim. Biophys. A c t a . , 525, 171 (1978). S.-C. Wonq, G. D. J. Green, E. Shaw, J. Med. Chem., 21, 456 (1978). M. Zimmennan, B. M. Ashe, C. P. Dorn, S.8. Yanq, H. Jones i n "Neutral P r o t e a s e s of Human Polymorphonuclear Leukocytes," K. Havemann, A. J a n o f f , Eds., Urban and Schwarzenberq, Munich-Baltimore, 1978, p. 234. K. Kusakabe, T. Kishimoto, Y. Mitsui, R. Takashima, Japan. Kokai, 78 18,720 2 1 Feb., ( 1 9 7 8 ) ~Chem. Abstr., g,30799 (1978). J. A. YankeeloV, H. A. P a r i s h , J K . , A. F. S p a t o l a , J. Med. Chem., 21 701 (1978). K. Hanada, M. Tamai, M. Yamayishi, S. Ohmura, J. Sawada, I . Tanaka, Aqric. B i o l . Chem., 42, 523 (1978). K. Hanada, M. Tamai, S. Ohmura, J. Sawada, T. S e k i , I . Tanaka, I b i d . , p . 529. K. Hanada, M. Tamai, S. Morimoto, T. Adachi, S . Ohmura, J. Sawada, I. Tanaka, I b i d . , p. 537. J. M. Andrews, D. P. Roman, Jr., D. H. Binq, M. Cory, J. Med. Chem., 21, 1202 (1978). R. F. P a r r i s h , J. W. S t r a u s , J. D. Paulson, K. L. Polakoski, R. R. Tidwell, J. D. Geratz, F. M. Stevens, I b i d . , p. 1132. R. R. Tidwell, J. D. Geratz, 0. D I M , G. Volz, D. Zeh, H. Loewe, I b i d . , p. 613. H. J. Smith, J. Theor. Biol., 2, 531 (1978). Y. G. Hyman, T. L. Vischer, Agents Actions, 8,532 (1978). D.

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Editor: Christopher T. Walsh, Massachusetts Institute of Technology, Cambridge, MA 02139 Chapter 22.

Proteases and Cell Invasion

Susannah T. Rohrlich and Daniel B. Rifkin, NYU Medical Center, Department of Cell Biology, New York, NY 10016 Introduction - Neoplastic cells display a number of biochemical changes compared to their normal c unterparts. These may include a tered growth controls and requirements,P altered rates of glycolysis,2y3 changes in the composition of the pltsy membrane, including protein&pid and glycolipid mo f ations, ’ increased protfjyf5secretion, cyt0 skeletal changes, increased cell motility, etc. The primary question in cancer biology is what relationship these changes have to those alterations in cell behavior which define neoplasticity. The two features which characterize malignant cells are their invasive and metastatic potentials. While certain normal cells may resemble neoplastic cells with respect to glycolytic rates, motility, and growth controls, only malignant cells are invasive and metastatic. Even cells from benign tumors, which have altered growth controls as evidenced by their tumorigenicity, can be distinguished from malignant cells by their inability to invade and metastasize.

Pi,is

The biochemical alterations which provide malignant cells with the ability to invade adjacent tissues and to spread to other locations are poorly understood and are undoubtedly complex. This complexity manifests itself clearly in the process of metastasis, in which cells must go through a series of steps including tissue invasion, extravasation into blood vessels, thrombus formation, arrest, penetration through the wall of alilood vessel and growth, all in order to develop a secondary tuEach step of this sequence may require the precise temporal mor. expression of specific biochemical functions, and some of these reactions, necessary for a step such as invasion, may be detrimental to other steps, such as the formation of a thrombus and arrest. The process of invasion may be less complex than that of metastasis, since invasion is only one part of the series of steps which comprise metastasis. In fact, cells can display this aspect of the malignant phenotype and not others. Thus, while all metastatic tumors are invasive, not all invasive tumors are metastatic: basal cell carcinomas, for example, are highly invasive but never metastasize. A number of mechanisms have been suggested to explain the process of tumor cell invasion in terms of observed properties of neoplastic

cells. One of these, that tumor invasion is the result of cellular multiplication and mechanical expansion, visualizes tumor infiltration as resembling the growth of a plant root. According to this theory, the pressure from a constantly increasing cell mass is thought to cause Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

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fractures in the tfyue. These fractures would then permit tumor expansion and invasion. This theory is unsatisfactory, however, since there is poor correlation between tumor growth rate and invasive potential. Moreover, certain normal cells such as leukocytes, invade without cell division. A second hypothesis to explain invasion proposes that the invasive potential of tumor cellg8ff$ives from their increased mobility and/or decreased adhesiveness. These two alterations would change the contact relationships of cells, permitting cells to invade surrounding tissues. However, since the increased motility of tumor cells appears to be random, not directional, their increased motility, per se, would not lead to invasion. In addition, many normal cells which are not invasive are quite motile. The destruction of surrounding tissues and/or extracellular matrix by lytic enzymes produced by tumor cells has also been proposed to account for the invasive potential of tumor cells. Hydrolysis of the microenvironment by these cells would destroy the natural, physical , barriers to cell movement, and would presumably allow migration of cells into neighboring sites. Ult structural evidence from in vivo studies $8 For example, in the case of epithelial strongly supports this view. tumors, which are separated from normal tissue by a basal lamina, tumor cell infiltration is always observed to occur in areas of lysis of and damage to the basal lamina. Presumably, the breakdown of the basal lamina, as well as stromal lysis, are mediated by lytic enzymes. The numerous reports of the producb;g:2pf goteases by tumor cells are consistent with such a scheme. Plasminogen Activator and the Transformed Phenotype: In Vitro Studies One protease which increases in many cell types after neoplastic transformation is plasminogen activator (PA). The synthesis of PA may be elevated 50-300 fold after transformation. PA is an active serine protease which cleaves at arginine bonds. The enzyme can be inhibited by di32 i s o p r o p y l f l u o r o p h o s p h a t e , nitrophenyl-guanidobenzoate, and benzamidine. PA efficiently converts the zymogen, plasminogen, to the active protease, plasmin.

plasminogen (inactive)

3.

>

plasmin (ac tive)

No other natural substrates have been found for PA. Plasmin en is a component of plasma and also occurs in extravascular spaces.95 Moreover, since plasmin is a protease with broad substrate specificity and since the amount of circulating plaminogen is relatively high (50 ug/ml), the production of PA may result in extremely high local levels of effective proteolytic activity, due to the amplification achieved by the local conversion of plasminogen to plasmin.

The evidence that increased protease production, especially PA

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production, is related to the malignant phenotype derives from two sources: from in vitro experiments with transformed cells and from experiments with non-malignant cells or tissues which take part in remodeling and/or invasive processes. A large number of experiments in the first category have indicated that properties associated with the transforme$fs$l phenotype in vitro, such as the ability to ff;ow without anchorage, increased Yigration from the edge of a3Yound , decreased cytoskeleta19y5$anization, increased agglutinability and morphological changes, may be mediated by PA and/or plasmin production. In general, the experimental approach has been to show that under conditions of heightened proteolytic activity, the transformed phenotype is accentuated, while under conditions of decreased proteolytic activity, the transformed phenotype is suppressed. Although the bulk of the in vitro experiments show a strong correlation betw A roduction and the transformed phenotype, there are Certain normal cells produce PA, while some transexceptions.5gfj13'-4g formed cells do not; some cells, which synthesize high levels of PA, grow poorly without anchorage, while some cells which grow well without anchorage, appear to produce low levels of PA. Although these examples of exceptional cells cannot be ignored, certain aspects of the experiments which yielded these results are open to criticism. A lack of accurate quantitation, the use of cell populations with variable karyotypes, and the failure to measure cell-associated proteolytic activity could explain some of the discrepancies. In addition, the failure to assess the possible role of protease inhibitors produced by the same cells, and the use of culture media with inappropriate hormonal compositions are possible explanations of results obtained. Non-Malignant Systems for the Study of Cell Invasion - The significance of protease activity during the invasive events of metastasis is further supported by the results from studies of the role of proteases in certain non-malignant biological processes. In vivo and in vitro analyses of inflammatory reactions and normal invasive or remodeling events and their hormonal and/or temporal controls have underlined the correlation between protease secretion and the local breakdown of tissue organization. In all such instances, the event and also the associated proteolysis and tissue breakdown have been found to be coordinately controlled. We devote the remainder of this review to a discussion of some of these studies, since they have not been summarized elsewhere.

1. Ovulation - The release of the ovum during ovulation in the rat is almost cet;bc&ly the result of proteolytic events in the preovulatory follicle. PA has been shown to be produced by granulosa cells in response to exposure in vivo or in vitro to physiological levels of the gonadotropins that induce ovulation. PA production is also stimulated in these cells by the in vitro administration of agents which mimic the effects of gonadotropins by increasing intracellular CAMP levels. vivo, PA is elevated only in granulosa cells from preovulatory follicles, and not in cells from non-preovulatory follicles. The following sequence of events during ovulation in the rat can be postulated based on the precise temporal correlations between gonadotropin stimulation, PA secretion and ovulation. Plasminogen,4gresent in follicular fluid at a concentration equal to that in serum, is activated to plasmin just

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prior to ovulation by the gonadotropin-induced generation of PA in selected follicles. This plasmin dislodges the ovum from Its attachments to the follicle wall and loosens the granulosa cell layer, clearing it completely away from the area of future follicle wall rupture. The basement membrane, connective tissue, and thecal layers of the follicle wall which are thus exposed are then digested by plasmin, perhaps together with collagenase, ultimately allowing the contents of the follicle, including the ovum, to ooze out.

2. Implantation - During a limited period, from days 6-10 of gestation in the mouse embryo,the trophoblast cell is the invasive cell par excellence; it brings about extensive, though localized, destruction of the connective tissue of the uterine wall and invades this tissue to establish an implantation site for the embryo. While the hormonal and/or other con- 47 trols of this precisely timed event are not understood, Strickland et al. have made an in vitro study of proteolytic events associated with preimplantation mouse blastocysts. They found that trophoblast cells produced both cell-associated and secreted PA, and that this activity was detectable only in the four-day period during which the mouse trophoblast is invasive in vivo: it appeared on equivalent gestation day 6 , peaked at day 8, and fell off by day 10. The time-course of PA appearance was unrelated to the emergence of the embryo from the zona pellucida. Microdissection and immunodissection were used to separate trophoblast cells from cells of the inner cell mass in vitro, and the two cell types were cultured separately. The PA was found to be associated only with the invasive trophoblast cells. The authors suggested that the tissue-destructive activity of the trophoblast is probably plasmin-mediated, though the source of the plasminogen remains unclear; relative to PA alone, the plasmin produced by plasminogen activation would provide a tremendous amplification of both the enzymatic activity and the substrate range, in keeping with the extensive tissue destruction observed during implantation. 3. Inflammation - Macrophages arise from precursors in the bone marrow and circulate in the blood as monocytes, but become activated and migratory in response to infection, inflammatory stimuli, and foreign body carcinogens. These stimuli induce young cells to migrate actively to the site of stimulus. To respond properly, these cells must be able to emerge from blood vessels and carve paths to the appropriate tissue spaces, a process which has several features in common with what must occur during tumor cell invasion and metastasis.

Several recent studies of macrophage responses to inflammatory stimuli and suppressants, in vivo and in vitro, have linked the effects of these agents to their ability to modulate the synthesis and secretion of PA by these cells. While normal, unactivated macrophages secrete barely detectable levels of PA, young, newly recruited macrophages from mice with experimentally indugjd inflammations produce and secrete as much as 10 times that amount. Furthermore, in vitro exposure of macrophages to conditioned medium from activated lymphoid cells, to Concanavalin A , known to stimulate several functions in these cells. or to phorbol

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EYY%

myristate acetate (PMA), a potent irritant, inflammatory agent, and promotor, further increases PA synthesis and secretion several-fold.

Suppression of PA synthesis and secretion by macrophages in vitro, and suppression of the recruitment of these cells, in response to inflammatory stimuli, in vivo, can be brought about by physiological levels of anti-inflammatory steroids, such as dexamethasone, hydrocortisone and fludrocortisone. The effectiveness of these steroids in the suppression of PA prodyftion is directly related to their in vivo anti-inflammatory potential. These observations point to the physiological importance o f PA secretion and subsequent plasmin formation by macrophages in the recruitment phase of the in vivo inflammatory response. While other enzymes of these cells, including other proteases, are affected by some of the agents tested, none shows a spectrum of responsiveness to modulating agents that fits as well with the requirements of macrophage migration. Almost identical findings have been reported for PMN leuk95ytes, with respect to their involvement in the inflammatory response 4 . Mammary Gland Involution - During pregnancy, the mammary gland secretory epithelium is elaborated, along with supporting connective tissue, to form ducts and alveoli capable of milk production. At parturition, a period of lactation ensues, and at weaning there is a rapid involution of the mammary gland to its pre-pregnancy state. In mice, as much as 90% of the glandular weight, mostly protein, is removed during one week of involution. Since the rapid and extensive degradative events associated with involution cannot be correlated with any observable changes in lysosomal enzyme activgty or in glandular populations of phagocytic 53 cells, at least in the initial stages, the findings of Ossowski et al. which correlate epithelial PA production with the remodeling events of involution, are of particular interest.

These authors have found an absolute correlation between the onset of involution and heightened PA production by glandular epithelium, both in vivo and in vitro. They found a large peak of PA production within -2 4 h of the cessation of lactation, accompanying the onset of involution, and a gradual decline of glandular PA levels to baseline within the first week of involution. PA production was observed to be temporally coupled to the initiation of involution, even when the onset of involution was moved forward experimentally by the separation of the mother from her litter, or backward, by the administration of oxytocin or corticosteroids, which maintain the gland in its lactating state even

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after weaning. Furthermore, in organ cultures of mammary gland fragments, the same coupling of PA production and gland maintenance or involution was observed. Factors or groups of factors which maintained the differentiated characteristics of the tissue in its lactating state, and which prevented involution, such as hydrocortisone, aldosterone, or polyamines in the presence of prolactin and insulin, prevented PA increases over baseline levels; factors which promoted glandular degeneration in these cultures, such as epidermal growth factor or insulin, especially after prolactin potentiation, induced high PA levels. The specific induction of PA production in secretory epithelial cells in response to signals for mammary gland involution is envisioned by these authors to initiate involution as follows. Secretion of PA leads to increased levels of extracellular PA, which locally activates plasminogen available in the circulation, tissue fluids and milk. Plasmin generated in this manner then degrades, or at least initiates degradation of the basal lamina, the glandular epithelium and any residual milk, and surrounding matrix, restoring the gland to its pre-pregnancy state. Interestingly, preliminary studies of neoplastic mammary tissue have shown both a several-fold increase in the PA levels of this tissue, as compared with i3yoluting normal glands, and a decreased responsiveness to hydrocortisone. These findings lend further support to the notion that it is the l o s s of regulation of PA production (and perhaps of proteolysis in more general terms) that is crucial in generating the pathological remodeling characteristic of the malignant phenotype.

5. Rheumatoid Arthritis - Joint tissue destruction in rheumatoid arthritis, a non-malignant biological process involving major tissue degradation and remodeling, involves plasmin-activated collagenase as well as PA-generated plasmin. Rheumatoid arthritis is characterized by synovial cell proliferation followed by extensive cartilage, bone, tendon, and ligament breakdown in affected joints. An invading, proliferating granulomatous pannus advances by proteolytic destruction of tissue in its path. Dayer et al. have shown that rheumatoid synovial cells in vitro produce elevated levels of collage3Zse (in a latent form) as compared to the same cells from normal joints. Werb et al. have further shown that r&umatoid synovial cells in vitro produce and secrete high levels o f PA. They have constructed in vitro systems to show that collagenase from synovial cells, in its inactive form, binds to collagen types I and 11, the types which occur in articulating cartilage and bone regions. Once bound, the latent collagenase is activated by plasmin. In vivo, plasmin is generated from synovial fluid plasminogen, enriched in rheumatoid fluid, by the PA from synovial cells and perhaps also from infiltrating phagocytic cells. Latent collagenase does not bind a2-macroglobulin. The bound, activated collagenase may also be protected from inhibition by ambient a -macroglobulin and other inhibitors both sterically and by competitive ginding of the inhibitors by plasmin in solution. The correlation between the destructive events of rheumatoid arthritis and the generation of PA, plasmin, and collagenase has been

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further strengthened by the finding that the production of both collagenase and PA by synovial cells in vitro is reduced ma-&edly by glucocorticoids which act as anti-inflammatory agents in vivo. The effective doses and relative efficacies of these modulating agents are identical -in vivo and in vitro. The same relationship between PA production and anti-inflammatory steroids has also been shown for macrophages and PMN leukocytes, as reviewed in section 3 . The collagenase activity seen in rheumatoid arthritis may not be unique to this system. On the contrary, collagen breakdown is important in any major degradative, invasive or remodeling process (also see section 6). The efficiency of plasmin in activating a latent collagenase precursor, and the fact that, where PA is produced, the ready availability o f plasminogen almost certainly assures its local activation to plasmin, make this an extremely attractive scheme to invoke in the foregoing examples of non-malignant remodeling events and also in malignancy. The possibility that increases in collagenase activity also occur in these processes is certainly worth exploring. 6. Resistance of Cartilage to Invasion - A particularly interesting example of the potential involvement of proteases in tumor invasion is the documented resistance of cartilage to tumor infiltration. Numerous descriptions exist of tumors which ha d troyed a portion of bone but It has been speculated that which left adjacent cartilage intact.' 7 9 " the resistance of cartilage to invasion derives from the presence of high levels of inhibi$q;goof plasmin, collagenase and thiol proteases found in this tissue. Crude preparations of protease inhibitors prepared from bovine or human cartilage are claimed to inhibit a variety of processes, includ&pg6sell growth, tumor-mediated bone degradation and tumor cell invasion. The avascular nature of cartilage may also be related to the presence of the same molecules which protect cartilage from tumor invasion, for the procesg40f neovascularization has many aspects in common with tumor invasion. Intact cartilage fragments as well as cartilage extr%S&,are capable of inhibiting angiogenesis in vitro and & vivo Moreover, procedures which extract the protease inhibitors from cartilage render the tissue sugSeptible to both tumor-induced vascularization and tumor invasion.

~

.

These observations, as well as the facts listed below, have led us to propose that both neovascularization and tumor invasion require the production of two proteases: plasminogen activator and collagenase. These points are: 1) Neovascularization always occurs from venules. 68 These structures are known to be leaky, and the leakage of a plasma component, e.g., plasminogen, may be required for the invasive process. 2) The first morphological event observed during neovascularization is the destruction of the basement6gembrane adjacent to endothelial cells, which subsequently migrate. Neighboring endothelial cells then divide to form a new blood vessel. The basement membrane is composed primarily of Type IV collagen and glycoproteins, and the lysis of this structure indicates the production of specific proteases.

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3) The major structural protein of cartilage and many other tissues is collagen. An invasive tumor or endothelial cell would have to produce collagenase to degrade this protein, since collagen is resistant to other proteases. 4 ) Stimulated en t lial cells are capable of synthesizing both PA and procollagenase.~!l,%l The PA produced may convert plasminogen to plasmin, and plasmin may activate procollagenase to collagenase. Thus, we can postulate a mechanism whereby the concerted activities of plasmin and collagenase may degrade the basement membrane glycoproteins and Type IV collagen. Therefore, high local levels of inhibitors of collagenase and plasmin, such as those found in cartilage, might prevent tumor and/or endothelial cell invasion. Conclusion - The experiments discussed throughout this review give strong support to the hypothesis that proteases and, in particular, plasminogen activator are involved in tissue remodeling and invasion. The examples which we have presented comprise but a small sample of the various phenomena which might involve proteases. Processes such as innervation, involution of glands such as the prostate and the thyroid, growth of the mammary gland before lactation and during embryogenesis, the invasion of the pericostal bud, and bone remodeling may involve PA and plasmin either directly or indirectly. Given the proteolytic mechanisms employed for tissue lysis, it is not surprising that pathological situations may develop from the abnormal secretion of PA. One disease which may involve the inappropriate production o f PA is rheumatoid arthritis. Tumor invasion is a second example. The evidence that PA secretion provides the biochemical mechanism for tissue lysis and subsequent tumor invasion during metastasis, while indirect, is strong. This does not mean that other proteases are not involved, either in concert or singly, in invasion by certain tumors or cells. However, most proteolytic assays other than the PA assay are not sensitive enough to detect the small amounts of enzyme produced. Therefore, the most meaningful work has been concerned with PA production. Finally, it is worth noting that the nature of the invasive process, as we present it, is such that invasion could be interfered with by the administration of protease inhibitors of appropriate design. Such molecules with the appropriate tissue and enzyme specificities could potentially provide therapies for a number of normal and pathological condit ions.

REFERENCES 1. "Control of Proliferation in Animal Cells", B. Clarkson and R. Baserga, Eds., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1974. 2. J. Paul, Int. J. Cancer, 1,207-218 (1966). 3. C . E . Wenner, Advanc. Enzymology, 2, 322-356 (1967). 4. R.O. Hynes, Biochem. Biophys. Acta, 458, 73-107 (1976). 5 . G.L. Nicolson, Biochem. Biophys. Acta, 458, 1-72 (1976). 6. A. Fischer, Arch. Entwicklungsmech., 104,210-214 (1925).

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7. A. Fischer, Nature, 157, 442 (1946). 8. J.C. Unkeless, A. Tobia, L. Ossowski, J.P. Quigley, D.B. Rifkin, and E. Reich, J . Exp. Med. , 137, 85-111 (1973). 9:L. Ossowski, J.C. Unkeless, A. Tobia, J.P. Quigley, D.B. Rifkin, and E. Reich, J . Exp. Med., 137, 112-126 (1973). 10. A. Goldberg, Cell, 2, 95-102 (1974). 11. R. Roblin, I.-N. Chow, and P.H. Black, Advances in Cancer Research, 22, 203-260 (1975). 12. K E . Pollack, M. Osborn, and K. Weber, Proc. Natl. Acad. Sci. (U.S.A.), 72,994-998 (1975). 13. R.W. Tucker, K.K. Sanford, and F.R. Frankel, Cell, 13, 629-642 (1978). 14. R.R. BUrk, Proc. Natl. Acad. Sci. (U.S.A.), 70,369-372 (1973). 15. L. Ossowski, J.P. Quigley, and E. Reich in "Proteases and Biological Control", E. Reich, D.B. Rifkin and E. Shaw, Eds., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1975, p. 901-913. 16. I. Zeidman, Cancer Res., 17,157-162 (1957). 17. R.A. Willis, "The Spread of Tumours in the Human Body", Butterworths, London 1973. 18. G. Eaves, J. Pathol., 109, 233-237 (1973). 19. D.R. Coman, Cancer Res., 5, 625-632 (1944). 20. L.M. Franks in "Chemotherapy of Cancer Dissemination and Metastasis", S.Garattiniand G. Franchi, Eds., Raven Press, N.Y. 1973, p. 71. 21. B. Sylvan in "Chemotherapy of Cancer Dissemination and Metastasis I t , S.Garattiniand G. Franchi, Eds., Raven Press, N.Y., 1973, p. 129. 22. D.V. Kazakava and V.N. Orekhovich, J. Biol. Chem., 247, 4224-4228 (1972). 23. F. Yamanishi, M.K. Dabbous, and K. Hashimoto, Cancer Res., 32, 25512560 (1972). 24. B. Sylvan and I. Bois-Svenssen, Cancer Res., 25, 451-468 (1965). 25. H.B. Bosmann and T.C. Hall, Proc. Natl. Acad. Sci. (U.S.A.), 71, 1833-1837 (1974). 26. M.H. Dresden, S.A. Heilman, and J.D. Schmidt, Cancer Res., 32, 993996 (1972). 27. B. Sylvan and I. Bois, Cancer Res. , 3, 831-840 (1960). 28. P.C. Zamecnik and M.L. Stephenson, Cancer Res., 17,326-337 (1947). 29. K. Hashimoto, Y. Yamanishi, M.K. Dabbous, and T. Kanzaki, Cancer Res., 33, 2790-2801 (1973). 30. D.B. Rifkin, J . N . Loeb, G. Moore, and E. Reich, J. Exp. Med., 139, 1317-1328 (1974). 31. J . K . Christman, S. Silverstein and G. Acs in "Research Monographs in Cell and Tissue Physiology, Vol. 2, Proteinases in Mammalian Cells and Tissues," A.J. Barrett, Ed., North Holland Pub. Co., Amsterdam, 1977, p. 90. 32. K. Dan0 and E. Reich in "Proteases and Biological Control," E. Reich, D.B. Rifkin and E. Shaw, Eds., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1975, p. 357-366. 33. H. Green, Cell, 11,405-416 (1977). 34. L. Ossowski, J.P. Quigley, G.M. Kellerman, and E. Reich, J. Exp. Med., 138, 1056-1064 (1973). 35. TPollack, R. Risser, S. Conlon, and D. Rifkin, Proc. Natl. Acad. Sci. (U.S.A.), 71,4792-4796 (1974). 36. R. Pollack and D.B. Rifkin, Cell, 6, 495-506 (1975).

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37. P. Whur, H. Koppel, C. Urquhart, and D.C. Williams, Nature, 260, 709-710 (1976). 37a.M. Weber, Cell, 2, 253-261 (1975). 38. D.M. Mott, P.H. Fabisch, B.P. Sani, and S. Sorof, Biochem. Biophys. Res. Commun., 61, 621-627 (1974). 39. B.A. Chibber, R.M. Niles, L. Prehn, and S. Sorof, Biochem. Biophys. Res. Commun., 65, 806-812 (1975). 40. B.A. Wolf and A.R. Goldberg, Proc. Natl. Acad. Sci., (U.S.A.), 73, 3613-3617 (1976). 41. D.B. Rifkin and R. Pollack, J. Cell Biol., 73, 47-55 (1977). 42. D.B. Rifkin, J. Cell. Physiol. 97, 421-429 (1978). 43. B.A. Wolf and A.R. Goldberg, Proc. Natl. Acad. Sci. (U.S.A.), 75, 4967-4971 (1978). 44. W.H. Beers, S. Strickland, and E. Reich, Cell, 5, 387-394 (1975). 45. S. Strickland and W.H. Beers, J. Biol. Chem. 251, 5694-5702 (1976). 46. W.H. Beers, Cell 5, 379-386 (1975). 47. S. Strickland, E. Reich, and M. Sherman, Cell, 2, 231-240 (1976). 48. J.C. Unkeless, S. Gordon, and E. Reich, J. Exp. Med. 139, 834-850 (1974). 49. J.-D. Vassalli and E. Reich, J. Exp. Med., 145,429-436 (1977). 50. J.-D. Vassalli, J. Hamilton and E. Reich, Cell, 11,695-705 (1977). 51. J.-D. Vassalli, J. Hamilton and E. Reich, Cell, 8, 271-281 (1976). 52. A. Granelli-Piperno, J.-D. Vassalli and E. Reich, J. Exp. Med., 146, 1693-1706 (1977). 53. L. Ossowski, D. Biegel and E. Reich, Cell, 16,929-940 (1979). 54. J.-M. Dayer, S.M. Krane, R.G.G. Russell, and D.R. Robinson, Proc. Natl. Acad. Sci. (U.S.A.), 73, 945-949 (1976). 55. 2. Werb, C.L. Mainardi, C.A. Vater, E.D. Harris, Jr., N. Eng. J. Med., 296, 1017-1023 (1977). 501-517 (1930). 56. TMakrycostas, Frankf. Z. Pathol., 9, 57. K.E. Kuettner, R.L. Croxen, R. Eisenstein, and N. Sorgente, Experientia, 2, 595-597 (1974). 58. K.E. Kuettner, J. Hiti, R. Eisenstein, and E. Harper, Biochem. Biophys. Res. Commun., 72,40-46 (1976). 59. D.B. Rifkin and R.M. Crowe, Hoppe-Seyler's Z. Physiol. Chem., 358, 1525-1531 (1977). 60. P.J. Roughley, G. Murphy, and A.J. Barrett, Biochem. J., 169, 721724 (1978). 61. R. Eisenstein, K.E. Kuettner, C. Neopolitan, L.W. Soble, and N. Sorgente, Amer. J. Pathol., 8, 337-346 (1975). 62. J.E. Horton, F.H. Wezeman, and K.E. Kuettner, Science, 199, 13421345 (1978). 63. K.E. Kuettner, B.U. Pauli, and L. Soble, Cancer Res., 38, 277-287 (1978). 64. J. Folkman in: "Advances in Cancer Research," Vol. 19, G. Klein and S. Weinhouse, Eds., Academic Press, N.Y., 1974, p. 331. 65. R. Langer, H. Brem, K. Falterman, M. Klein, and J. Folkman, Science, 193, 70-72 (1976). 66. N. Sorgente, K.E. Kuettner, L.W. Soble, and R. Eisenstein, Lab. Invest., 32, 217-222 (1975). 67. H. Brem and J. Folkman, J. Exp. Med., 141, 427-439 (1975). 68. D. Ausprunk and J. Folkman, Microvascular Res., 14,53-65 (1977).

Chap. 22

Proteases, Cell Invasion

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69. D.J. Loskutoff and T . S . Edgington, Proc. Natl. Acad. Sci. ( U . S . A . ) , 74, 3903-3907 ( 1977) . 70. % Moscatelli, E. Jaffe, and D.B. Rifkin, Manuscript in Preparation.

240 Chapter 23.

Annual Reports in Medicinal Chemistry-14

S e l e c t e d New Developments i n t h e Biochemistry of V i r u s e s

Royce Z . Lockart, J r . , Richard J . Colonno, and Bruce D. Korant, C e n t r a l Research & Development Department, E. I. du Pont de Nemours & Co., I n c . , Wilmington, DE 19898

I.

Introduction

The g o a l of molecular v i r o l i g i s t s is t o u n d e r s t a n d t h e s t r u c t u r e , e x p r e s s i o n and c o n t r o l of v i r a l genes. C o n t r o l i s known t o be e x e r t e d on t r a n s c r i p t i o n i n i t i a t i o n , messenger RNA (mRNA) p r o c e s s i n g , and p o s t - t r a n s l a t i o n a l c l e a v a g e of v i r a l coded p r o t e i n s . Recent i n f o r m a t i o n on v i r a l genomes, mRNAs, and t h e p r o c e s s i n g of v i r a l p r o d u c t s is b r i e f l y reviewed h e r e . Where p o s s i b l e , novel i m p l i c a t i o n s f o r d i a g n o s i s o r v i r a l c o n t r o l are i n c l u d e d . 11.

R e s t r i c t i o n Endonucleases

R e s t r i c t i o n enzymes are p e r m i t t i n g r a p i d advances i n u n d e r s t a n d i n g Also, s p e c i f i c g e n e t i c f u n c t i o n s c a n be i s o l a t e d and v i r a l DNA genomes. l o c a t e d i n s p e c i f i c fragments of DNA. And t h e c o n s i d e r a b l e v a r i a t i o n t h a t e x i s t s among d i f f e r e n t s t r a i n s of v i r u s e s i s b e i n g r e c o g n i z e d and u t i l i z e d f o r t h e f i r s t time. A . A Genome Map - Probably t h e most complete genome map has been determined f o r t h e small t r a n s f o r m i n g p a p o v a v i r u s SV40. An e x c e l l e n t review c o v e r s t h i s information.’ The v i r u s h a s a DNA genome w i t h an e s t i mated molecular weight of 3.4x106, and t h e complete n u c l e o t i d e sequence of S e v e r a l p r o t e i n s have been matched t h i s DNA w a s r e c e n t l y determined. 3, p r e c i s e l y w i t h t h e i r coding r e g i o n s on t h e DNA and t h e sequences of s e v e r a l The f o l l o w i n g p o i n t s have been e s t a b l i s h e d . o t h e r s have been p r e d i c t e d . 3 y The s i t e of t h e o r i g i n of r e p l i c a t i o n of t h e v i r a l DNA h a s been determined t o be n e a r a 27 b a s e - p a i r palindrome, t h e c e n t e r of which s e r v e s a s t h e Nearby, t r a n s c r i p t i o n only c l e a v a g e s i t e of t h e r e s t r i c t i o n enzyme Bg/I. b e g i n s . Roughly one-half of t h e genome is t r a n s c r i b e d b e f o r e DNA r e p l i c a t i o n b e g i n s t o form two ( e a r l y ) p r o t e i n s . The o t h e r h a l f i s t r a n s s c r i b e d a f t e r DNA r e p l i c a t i o n b e g i n s t o form t h r e e p r o t e i n s which a r e v i r a l s t r u c t u r a l ( l a t e ) p r o t e i n s . ” R e p l i c a t i o n and t r a n s c r i p t i o n occur i n b o t h d i r e c t i o n s from t h e i r s i t e s of o r i g i n . About 15% t o 23% of t h e genome i n formation i s n o t t r a n s l a t e d i n t o p r o t e i n s ; p o s s i b l y i t c o n t a i n s r e g u l a t o r y f u n c t i o n s . 3 ’ 4 The i n f o r m a t i o n f o r t h e two e a r l y p r o t e i n s (20,503 and > 90,000 d a l t o n s ) s t a r t s a t n u c l e o t i d e 80 from t h e c e n t e r of t h e p a l i d r ~ m e . ~The l a r g e e a r l y p r o t e i n i s t h e T a n t i g e n . Direct r e a d i n g of t h e i n f o r m a t i o n f o r t h e smaller e a r l y p r o t e i n s t o p s a t n u c l e o t i d e 602. The l a r g e r p r o t e i n s h a r e s a b o u t one-half of t h e i n f o r m a t i o n of t h e smaller p r o t e i n , t h e n o b t a i n s t h e rest of i t s i n f o r m a t i o n from a non-contiguous A n o n - t r a n s l a t e d r e g i o n of DNA l i e s i n between r e g i o n of t h e D N A . 3 ’ 4 ’ 5 and t h e mRNA f o r t h e l a r g e r p r o t e i n i s processed by s p l i c i n g . 3 ’ 4 ’ 5 The g e n e t i c i n f o r m a t i o n f o r two of t h e t h r e e l a t e v i r a l p r o t e i n s o v e r l a p s . Messenger RNA molecules f o r two of them a l s o are formed by s p l i c i n g . The d i f f e r e n t messenger RNA molecules a p p a r e n t l y c o n t a i n a common 5 ’ l e a d e r sequence. Cowright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

Chap. 23

Biochemistry of V i r u s e s

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B. Locating Gene F u n c t i o n s - The g o a l of mapping a mutant i s t o d e t e r m i n e what f u n c t i o n is a f f e c t e d by t h e m u t a t i o n , and where t h a t funct i o n i s l o c a t e d on t h e p h y s i c a l map. Consequently, t h e p r o t e i n o r p r o t e i n r e g i o n r e s p o n s i b l e f o r t h e f u n c t i o n c a n be determined. The q u e s t t o i d e n t i f y t h e gene f u n c t i o n ( s ) r e q u i r e d f o r v i r a l t r a n s f o r m a t i o n and how t h a t f u n c t i o n i s c o n t r o l l e d h a s been of s p e c i a l i n t e r e s t .

1. T e m p e r a t u r e - s e n s i t i v e Mutants - A number of temperatures e n s i t i v e m u t a n t s of SV40 have been a n a l y z e d . 2 Those found d e f e c t i v e i n t h e complementation A group and t h e p h y s i c a l map r e g i o n s which code f o r t h e e a r l y p r o t e i n s (T a n t i g e n and t ) were a l s o found d e f e c t i v e i n t h e i r a b i l i t y t o transform c e l l s . The t e m p e r a t u r e - s e n s i t i v e t r a n s f o r m a t i o n def e c t w a s r e v e r s i b l e a t permissive temperatures, i . e . , transformed c o l o n i e s appeared. Mutants d e f e c t i v e i n t h e f o r m a t i o n of l a t e p r o t e i n s (viral s t r u c t u r a l p r o t e i n s ) were a b l e t o t r a n s f o r m c e l l s . T h e r e f o r e , f u n c t i o n a l e a r l y p r o t e i n s a r e r e q u i r e d t o e f f e c t and m a i n t a i n t r a n s f o r m a t i o n . ’ The e a r l y p r o t e i n s n e c e s s a r y f o r t r a n s f o r m a t i o n a l s o p e r m i t v i r a l DNA r e p l i c a t i o n and t h e s t i m u l a t i o n of h o s t c e l l DNA s y n t h e s i s . The e x a c t b i o chemical f u n c t i o n s r e q u i r e d f o r t r a n s f o r m a t i o n have n o t y e t been d e t e r m i n e d . 2 . D e l e t i o n Mutants - Another approach h a s been t h e c o n s t r u c t i m of d e l e t i o n mutants by t h e J u d i c i o u s u s e of r e s t r i c t i o n enzymes and The s i t e s of t h e d e l e t i o n s w e r e mapped; i n a p p r o p r i a t e exonucleases.’ ’ some i n s t a n c e s t h e y were widely s e p a r a t e d b u t s t i l l a f f e c t e d a s i n g l e prot e i n . 5 ’ 6 The f u n c t i o n a l a b i l i t y of t h e m u t a n t s w a s t h e n determined. The r e s u l t s , w h i l e a t a n e a r l y s t a g e , are i n agreement w i t h t h e c o n c l u s i o n s reached w i t h t h e t e m p e r a t u r e - s e n s i t i v e m u t a n t s . The e a r l y p r o t e i n s must b e f u n c t i o n a l t o e f f e c t DNA r e p l i c a t i o n , s t i m u l a t i o n of h o s t c e l l DNA s y n t h e s i s and c e l l t r a n s f o r m a t i o n . ’ I n t e r e s t i n g l y , n o t a l l t h r e e of t h e l a t e p r o t e i n s a p p e a r t o be r e q u i r e d f o r v i r u s production.’ Much more w i l l s u r e l y come from t h e s e s t u d i e s .

3 . DNA Fragments - Fragments of DNA c a n b e t a k e n up by c e l l s under p r o p e r c o n d i t i o n s and i n many cases s t i l l e x p r e s s t h e i r g e n e t i c f u n c t i o n s . A fragment of DNA c o n s i s t i n g of 74% of t h e SV40 genome and c o n t a i n i n g t h o s e p a r t s of t h e genome coding f o r t h e earl p r o t e i n s and t h e The minimal o r i g i n of r e p l i c a t i o n w a s c a p a b l e of t r a n s f o r m i n g cells.’ amount of i n f o r m a t i o n r e q u i r e d f o r t r a n s f o r m a t i o n and t h e n a t u r e of t h e product of t h a t i n f o r m a t i o n i s expected from f u t u r e s t u d i e s of t h i s t y p e . T h i s approach h a s been used w i t h t h e a d e n o v i r u s e s , a l s o . I n summary, DNA f r a g m e n t s r e p r e s e n t i n g t h e l e f t t e r m i n a l 4.5% of t h e adenov i r u s genome c a n t r a n s f o r m c e l l s . The t r a n s f o r m a t i o n w a s somewhat abnormal but normal t r a n s f o r m a t i o n w a s a c h i e v e d w i t h t h e l e f t 7.5% f r a g V i r a l messenger RNA molecules w e r e i s o l a t e d from c e l l s t r a n s ment.” formed by fragments. They were t r a n s l a t e d i n v i t r o . Four p r o t e i n s between 33 and 40 thousand d a l t o n s r e s u l t e d from t h e t r a n s l a t e d RNA from t h e c e l l s transformed w i t h a 4.5% fragment. These f o u r and a n a d d i t i o n a l p r o t e i n of 1 9 thousand d a l t o n s r e s u l t e d from t h e mRNAs from t h e normally transformed c e l l s . I t is p u z z l i n g t h a t such l a r g e p r o t e i n s r e s u l t e d a s a l a r g e number of s t o p codons were found when t h e 4.5% DNA fragment w a s The l a r g e number of p r o t e i n s found i n t h i s system i n d i c a t e s sequenced.’ i t s complexity; c o n s i d e r a b l y more work is r e q u i r e d t o u n d e r s t a n d t h e r o l e s of t h e p r o t e i n s i n c e l l t r a n s f o r m a t i o n .

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C. F i n d i n g and Analyzing I n t e g r a t e d Viral Genes - R e s t r i c t i o n enzyme d i g e s t s of t h e DNA from s e v e r a l independent SV40 t r a n s f o r m a n t s were analyzed t o see whether t h e SV40 genome w a s i n t e g r a t e d s i m i l a r l y and a t one o r more s i t e s on t h e c e l l u l a r DNA. Not o n l y d i d i n t e g r a t i o n occur a t more than one c e l l u l a r s i t e b u t d i f f e r e n t s i t e s of t h e v i r a l DNA were j o i n e d t o t h e c e l l u l a r DNA i n each of t h e t r a n s f o r m a n t s . ’

S i m i l a r l y , r e s t r i c t i o n enzyme d i g e s t s of c e l l u l a r DNA have g r e a t s e n s i t i v i t y and t h e r e f o r e d e t e c t e d f o r t h e f i r s t t i m e t h e p r e s e n c e of mouse mammary tumor v i r u s (MMTV) DNA i n i n f e c t e d mammary g l a n d s . ” This p e r m i t t e d t h e d i a g n o s i s of i n f e c t i o n of t h e s e g l a n d s by h o r i z o n t a l l y trarsm i t t e d s t r a i n s of MMTV, i . e . , t h e y could be d i s t i n g u i s h e d from t h o s e endogeneous t o t h e i n f e c t e d mice. T h i s w a s p o s s i b l e because t h e d i f f e r e n t s t r a i n s of MMTV d i f f e r i n t h e i r DNA sequences and t h u s i n t h e fragments produced by s e l e c t e d enzymes. l 1 T h i s i l l u s t r a t e s t h e s e n s i t i v i t y and s e l e c t i v i t y provided by t h i s t e c h n i q u e t o i d e n t i f y s p e c i f i c DNA sequences. D. Comparing Viral Genomes - To e x p l o i t t h e d i a g n o s t i c p o t e n t i a l of r e s t r i c t i o n enzyme mapping, t h e d e g r e e of v a r i a t i o n among t h e s t r a i n s of n a t u r a l l y o c c u r r i n g v i r u s e s must be determined. Comparisons have begun. Comparison of t h e r e s t r i c t i o n fragments o f . 1 0 s t r a i n s of SV40 v i r u s rev e a l e d each s t r a i n t o be unique.’ The DNAs from r a b b i t p o x , v a c c i n i a , cowpox, e c t r o m e l i a , and fowlpox a l l gave d i s t i n g u i s h a b l e p a t t e r n s of fragments when a p p r o p r i a t e l y d i g e s t e d and compared. When Hind I11 f r a g m e n t s of DNA from v a r i o u s p o x v i r u s e s were compared, cowpox, v a c c i n i a , monkeypox, and v a r i o l a were i d e n t i f i e d . Additional d i g e s t i o n w i t h S a l I permitted 4 of 5 s t r a i n s of v a r i o l a v i r u s t o be f u r t h e r d i f f e r e n t i a t e d . 1 3

’

I n o t h e r s t u d i e s , t h r e e s t r a i n s of Epstein-Barr v i r u s ’ 4 and h e r p e s simplex t y p e s 1 and 2 1 5 ’ 1 7each gave unique r e s t r i c t i o n enzyme p a t t e r n s . The h e r p e s simplex case is expected a s t h e two t y p e s of h e r p e s v i r u s e s d i f f e r a n t i g e n i c a l l y . However, a l a r g e number of d i f f e r e n t i s o l a t e s of h e r p e s simplex t y p e 1 y i e l d e d d i f f e r e n t p a t t e r n s of DNA fragments when t h e i r DNAs were fragmented w i t h s e v e r a l a p p r o p r i a t e r e s t r i c t i o n enzymes.’5’ l 6 I s o l a t e s o b t a i n e d y e a r s a p a r t from t h e same i n d i v i d u a l and a s i n g l e i s o l a t e a f t e r numerous p a s s a g e s i n t i s s u e c u l t u r e r e t a i n e d t h e i r unique, c h a r a c t e r i s t i c fragment p a t t e r n s . Thus, t h i s t e c h n i q u e might a i d i n e p i d e m i o l o g i c a l s t u d i e s . Indeed, t h e a n a l y s i s of 1 4 i s o l a t e s from i n f e c t e d i n d i v i d u a l s i n a h o s p i t a l u n i t showed t h a t t h e r e were two separ a t e i n t r o d u c t i o n s of h e r p e s t y p e l v i r u s e s i n t o t h e u n i t . l’

’’

The v a r i a t i o n s found i n t h e genomes of t h e d i f f e r e n t s t r a i n s of a l l t h e v i r u s e s examined, s u g g e s t s t h a t we might e x p e c t t h e s t r u c t u r e s of t h e i r gene p r o d u c t s t o v a r y c o n s i d e r a b l y , a l s o , T h i s could account f o r v a r i a t i o n s i n d i s e a s e s e v e r i t y and s u g g e s t s t h a t a n t i v i r a l chemicals may vary considerably i n effectiveness. 111.

RNA V i r u s Genomes:

S t r u c t u r e s , T r a n s c r i p t i o n , and T r a n s l a t i o n

V i r u s e s which c o n t a i n a n RNA genome c a n b e c l a s s e d as p o s i t i v e and n e g a t i v e - s t r a n d e d v i r u s e s depending on whether t h e i r genome RNA serves d i r e c t l y a s mRNA ( p o s i t i v e ) o r r e q u i r e s RNA t r a n s c r i p t i o n t o s y n t h e s i z e mRNA molecules ( n e g a t i v e ) . Dramatic d i s c o v e r i e s have been made i n e a c h of t h e s e v i r u s group.

Chap. 23

Biochemistry o f V i r u s e s

L o c k a r t , Colonno, Korant

243

A. P o s i t i v e - S t r a n d e d RNA V i r u s e s - The p o s i t i v e - s t r a n d e d RNA v i r u s e s are b e s t r e p r e s e n t e d by t h e p i c o r n a v i r u s e s of which p o l i o v i r u s is a model v i r u s . P o l i o v i r u s c o n t a i n s a s i n g l e - s t r a n d e d genome RNA, which h a s a MW of 2 . 5 ~ 1 0(7700 ~ n u c l e o t i d e s ) , and f o u r v i r a l s t r u c t u r a l p r o t e i n s . Upon e n t r a n c e i n t o a c e l l , t h e genome RNA i s t r a n s l a t e d i n t o one l a r g e polyp e p t i d e which i s c l e a v e d p r o t e o l y t i c a l l y t o produce a l l t h e v i r a l p r o t e i n s ( s e e below). The 5' ends of p i c o r n a v i r u s genome RNAs a r e u n i q u e as t h e y c o n t a i n a c o v a l e n t l y l i n k e d p r o t e i n . l 9 The p r o t e i n (VP ) h a s a MW of 6-12,000, and has b o t h a b a s i c and hydrophobic n a t u r e . 2i,21 It i s l i n k e d t o t h e 5' t e r m i n a l sequence pU-U-A-A-A-A-C-A-G through a tyrosine-0-p-U linkage.

20' 21

The f u n c t i o n a l s i g n i f i c a n c e of VPg h a s n o t y e t been determined b u t i t is b e l i e v e d t o b e involved i n RNA r e p l i c a t i o n . T h i s i s b e c a u s e VPg i s found o n l y i n RNA involved i n RNA s y n t h e s i s o r packaged i n t h e v i r i o n . 2 1 S i n c e no VPg i s found i n v i r a l RNA i s o l a t e d from i n f e c t e d c e l l p o l y r i b o -

somes, 2 1 i t is presumedly removed p r i o r t o o r d u r i n g t r a n s l a t i o n and i s probably n o t r e q u i r e d f o r t r a n s l a t i o n . An enzyme which i s a b l e t o s p e c i f i c a l l y c l e a v e t h e t y r o s i n e - u r i d i n e l i n k a g e l e a v i n g both VPg and t h e genome RNA i n t a c t h a s been i s o l a t e d from t h r e e u n i n f e c t e d c e l l l i n e s and p a r t i a l l y purified

.

The 3' ends of p i c o r n a v i r u s genome RNAs c o n t a i n a s t r e t c h of a d e n o s i n e r e s i d u e s known a s poly(A) 2 4 The genome RNA of p i c o r n a v i r u s e s d i f f e r s from most mRNA molecules i n that i t s 3' poly(A) t a i l i s t r a n s s c r i b e d from a poly(U) t e m p l a t e d u r i n g RNA r e p l i c a t i o n r a t h e r t h a n synthesized post transcriptionally. Recent d e t e r m i n a t i o n s of t h e nucleot i d e sequences of t h e 3' RNA r e g i o n a d j a c e n t t o t h e poly(A) of t e n p i c o r n a v i r u s e s i n d i c a t e no sequence homology between subgroups and o n l y minor sequence homology among t h e v i r u s e s of a p a r t i c u l a r s u b g r o u p . 2 4 T h i s becomes i m p o r t a n t when one r e a l i z e s t h a t t h e 3' t e r m i n a l r e g i o n serves b o t h as t h e i n i t i a t i o n s i t e f o r RNA r e p l i c a t i o n and t h e t e r m i n a t i o n s i t e f o r translation.

.

B. Negative-Stranded RNA V i r u s e s - The n e g a t i v e - s t r a n d e d RNA v i r u s e s are l a r g e r and more complex t h a n t h e p o s i t i v e - s t r a n d e d v i r u s e s . They c o n t a i n a n RNA polymerase which t r a n s c r i b e s t h e i r genome RNAs i n t o f u n c t i o n a l mRNAs. V e s i c u l a r s t o m a t i t i s v i r u s (VSV) is one such n e g a t i v e s t r a n d e d v i r u s . V i r i o n s of VSV c o n t a i n a s i n g l e s t r a n d of RNA w i t h a MW of 4x106 and f i v e s t r u c t u r a l p r o t e i n s . The p r o t e i n s account f o r v i r t u a l l y a l l t h e coding c a p a c i t y of t h e RNA (Review 2 5 ) . S i n c e VSV must undergo RNA t r a n s c r i p t i o n t o s y n t h e s i z e mRNAs b e f o r e t r a n s l a t i o n c a n o c c u r , transc r i p t i o n has received t h e g r e a t e s t a t t e n t i o n .

VSV d i f f e r s from p o l i o v i r u s in s e v e r a l r e s p e c t s . One major d i f f e r e n c e i s t h a t t h e VSV genome s e r v e s a s a t e m p l a t e f o r b o t h mRNA synt h e s i s and RNA r e p l i c a t i o n , i . e . , i n one i n s t a n c e f i v e d i s t i n c t s p e c i e s of mRNA r e s u l t and i n t h e o t h e r a s i n g l e f u l l - l e n g t h copy of t h e genome is formed. The c o n t r o l l i n g mechanism a l l o w i n g e i t h e r t r a n s c r i p t i o n o r r e p l i c a t i o n i s n o t y e t known, a l t h o u g h b r e a k t h r o u g h s have r e s u l t e d from r e c e n t s t u d i e s . The f i r s t w a s t h e u s e of UV i r r a d i a t e d v i r u s t o prime e i t h e r a n RNA polymerase a s s a y 2 o r a coupled transcription/translational system. 2 7 A p o l a r e f f e c t on s y n t h e s i s of mRNA w a s observed. I f t h e f i v e

S e c t . V - Topics i n Biology

244 -

Walsh, Ed.

mRNA s p e c i e s were s y n t h e s i z e d independently a s was b e l i e v e d f o r many year6 t h e n t h e UV i r r a d i a t i o n would show a non-polar e f f e c t . I n s t e a d t h e res u l t s i n d i c a t e t h a t t h e r e w a s a s i n g l e i n i t i a t i o n p o i n t and s e q u e n t i a l s y n t h e s i s of t h e f i v e mRNA s p e c i e s . The s y n t h e s i s of e a c h mRNA species i s dependent on t h e p r i o r s y n t h e s i s of i t s 3' proximal mRNA.

I n a d d i t i o n , RNA s y n t h e s i s i s i n i t i a t e d a t t h e 3' end of t h e genome RNA w i t h t h e s y n t h e s i s of a s m a l l 48 n u c l e o t i d e " l e a d e r RNA."28'29 Det e r m i n a t i o n of t h e complete n u c l e o t i d e sequence of t h e l e a d e r RNA demons t r a t e d t h a t u n l i k e any VSV mRNAs i t c o n t a i n s 50% AMP and a ppA a t i t s 5' e n d . 3 0 Two s e r o t y p e s of VSV and t h e paramyxovirus, Newcastle d i s e a s e v i r u s , s n t h e s i z e l e a d e r RNAs of i d e n t i c a l s i z e and similar b a s e compos f t i o n . 3 Y The VSV l e a d e r RNAs s h a r e 80% b a s e sequence homology which i n d i c a t e s n e a r l y i d e n t i c a l binding and i n i t i a t i o n s i t e s f o r t h e v i r i o n polymerases. 3 2 The q u e s t i o n remains whether t h e f i v e mRNA s p e c i e s arise by a p r o c e s s i n g mechanism a f t e r s y n t h e s i s of t h e l e a d e r RNA o r a s t o p - s t a r t mechanism i n which t h e RNA polymerase r e i n i t i a t e s a t e a c h j u n c t i o n between mRNAs. S i m i l a r s e q u e n t i a l s y n t h e s i s of mRNA h a s a l s o been observed w i t h t h e paramyxovirus, Sendai v i r u s . V i r a l a s w e l l a s e u c a r y o t i c mRNA s t r u c t u r e s have been i n t e n s e l y s c r u t i n i z e d ( s e e Review 3 4 ) . V i r t u a l l y a l l mRNA molecules c o n t a i n a "cap" s t r u c t u r e a t t h e i r 5' t e r m i n i ( F i g u r e 1). 0

Q* W '

a 3

:-

!-

~ O - y F ~ - O 5$'- p - ~

CAP A

3'

OICy,

I

BASE Y

P O H o=p+---

BASE 2

bF i g u r e 1.

S t r u c t u r e of two 5'-caps

o=b-o--I 0-

The cap c o n s i s t s of an i n v e r t e d guanosine r e s i d u e l i n k e d t h r o u g h i t s 5 ' r i b o s e t o t h e 5' r i b o s e of t h e i n i t i a t e d o r f i r s t t r a n s c r i b e d b a s e v i a a The 5'-5' l i n k a g e is i n v e r t e d , relat r i p h o s p h a t e b r i d g e as G p p g v g . ; ; t i v e t o t h e normal 3'-5' p osp o ester bonds i n t h e remainder of t h e p o l y n u c l e o t i d e c h a i n . Most v i r u s e s c a n m e t h y l a t e cap s t r u c t u r e s i n v i t r o i n two p o s i t i o n s - a t t h e 7-methyl p o s i t i o n of t h e capping b a s e and t h e 2I-O-ribose p o s i t i o n of t h e p e n u l t i m a t e b a s e X t o form a Cap A s t r u c t u r e ( F i g u r e 1 ) . I n c o n t r a s t , t h e v a s t m a j o r i t y of v i r a l c a p s t r u c t u r e s synt h e s i z e d i n v i r u s i n f e c t e d c e l l s c o n t a i n a n a d d i t i o n a l 2I-O-ribose m e t h y l a t i o n t o form a Cap B s t r u c t u r e ( F i g u r e 1). Caps have been d e t e c t e d i n almost a l l e u c a r y o t i c v i r a l mRNAs e x c e p t € o r t h e p i c o r n a v i r u s group;

Chap. 2 3

Biochemistry of V i r u s e s

L o c k a r t , Colonno, Korant

245

t h e y d i f f e r only i n t h e p u r i n e o f t h e p e n u l t i m a t e b a s e X and p r e s e n c e of 2-0' m e t h y l a t i o n . The caps on v i r a l RNAs are s y n t h e s i z e d i n one of two ways - e i t h e r w i t h a Gp condensing o n t o a pG as w i t h re0 and v a c c i n i a v i r u s , 3 6 o r a gp condensing o n t o a p p G a s w i t h VSV m R N A s . 3 7 Cons i s t e n t w i t h t h e i r u b i q u i t y i n e u c a r y o t i c mRNAs, c a p s have an i m p o r t a n t r o l e i n t r a n s l a t i o n . T h i s w a s f i r s t demonstrated w i t h r e o v i r u s and VSV mRNAs which r e q u i r e 5 ' t e r m i n a l 7 MeG c a p s f o r e f f i c i e n t t r a n s l a t i o n i n a wheat germ c e l l - f r e e , p r o t e i n - s y n t h e s i z i n g system. * However, t h e dependence of e u c a r y o t i c mRNA t r a n s l a t i o n on c a p s a p p e a r s t o b e n e i t h e r a b s o l u t e n o r u n i v e r s a l s i n c e p o l i o v i r u s RNA i s o l a t e d from polyribosomes c o n t a i n s t h e 5 ' t e r m i n u s pU.' A g r e a t amount of r e s e a r c h h a s been done r e c e n t l y on t h e 5 ' t e r m i n a l b i n d i n g s i t e s of v i r a l mRNA m o l e c u l e s ( s e e Review 3 9 ) . The cap s t r u c t u r e , though involved i n t h e b i n d i n g of mRNA t o ribosomes, i s n o t always prot e c t e d from R N A a s e d i g e s t i o n d u r i n g ribosome b i n d i n g . I n f a c t , d e s p i t e a l a r g e amount of sequencing d a t a , t h e r e q u i r e m e n t s f o r ribosome b i n d i n g and t h e i n i t i a t i o n of p r o t e i n s y n t h e s i s a r e n o t y e t known.

I n i t i a t i o n of t r a n s l a t i o n i s r e s t r i c t e d t o t h e 5' r e g i o n , a l t h o u g h a maximum a l l o w a b l e d i s t a n c e of t h e i n i t i a t i n g t r i p l e t from t h e 5 ' end of t h e RNA h a s n o t been d e f i n e d . I t i s p o s s i b l e t o i n i t i a t e a t an AUG codon l o c a t e d as f a r as 89 r e s i d u e s i n from t h e 5 ' end. Although i n i t i a t i o n of t r a n s l a t i o n i n e u c a r y o t i c systems seems t o be l i m i t e d t o t h e 5' p o r t i o n of t h e mRNA, i t i s clear from examination of t h e a v a i l a b l e sequence d a t a t h a t t h e AUG codon need n o t occur i n a f i x e d p o s i t i o n r e l a t i v e t o t h e 5' end and can e x i s t as c l o s e as 11 n u c l e o t i d e s inbrome mosaic v i r u s m W A 4 ' t o over 200 n u c l e o t i d e s i n SV40 VPI mRNA.41 Likewise, t h e sequences which f l a n k e i t h e r s i d e of t h e AUG codon do n o t seem t o matter. IV.

Enzymatic P r o t e i n Cleavage i n V i r u s R e p l i c a t i o n

A . E x t e n t of P r o t e i n Cleavage i n V i r u s - i n f e c t e d Cells - Probably w i t h o u t e x c e p t i o n among t h e animal v i r u s e s , enzymatic c l e a v a g e ( s i t e s p e c i f i c l i m i t e d p r o t e o l y t i c a t t a c k ) of v i r a l p r o t e i n s o c c u r s i n o r on t h e s u r f a c e of i n f e c t e d c e l l s . The e v i d e n c e c o l l e c t e d f o r some of t h e r e p r e s e n t a t i v e v i r u s e s i n d i c a t e s t h a t t h e c l e a v a g e s are e s s e n t i a l f o r t h e s u c c e s s f u l p r o d u c t i o n of v i r a l progeny (reviewed i n 4 2 ) . A t t h e biochemic a l l e v e l , u n d e r s t a n d i n g t h e n a t u r e of t h e s e c l e a v a g e s is l e a d i n g t o a f u l l e r a p p r e c i a t i o n of t h e r o l e of l i m i t e d p r o t e o l y s i s i n t h e a c t i v a t i o n of enzymes and i n t h e f o r m a t i o n of complex s u p r a m o l e c u l a r b i o l o g i c a l stmt u r e s . These s t u d i e s may a l s o l e a d t o t h e d i s c o v e r y of v i r a l i n h i b i t o r s which act by i n h i b i t i n g t h e r e q u i r e d v i r u s p r o t e i n c l e a v a g e r e a c t i o n s .

The o c c u r r e n c e of v i r u s p r o t e i n c l e a v a g e s and t h e p o s s i b l e r o l e of t h e s e r e a c t i o n s are summarized i n T a b l e I . The most common o b s e r v a t i o n is t h a t l i m i t e d p r o t e o l y s i s o c c u r s a t t h e l e v e l of m a t u r a t i o n ( f o r m a t i o n of i n f e c t i o u s v i r u s p a r t i c l e s ) . M e c h a n i s t i c a l l y , t h e v i r u s s t r u c t u r a l prot e i n p r e c u r s o r is c l e a v e d , and a c t i v a t e d t o a form which makes i t recogn i z a b l e t o v i r a l n u c l e i c a c i d a n d / o r c e l l u l a r membrane r e c e p t o r s . The c l e a v a g e s occur most commonly w i t h i n t h e i n f e c t e d c e l l s , b u t i n s e v e r a l examples f i n a l p r o t e o l y t i c " t a i l o r i n g " o c c u r s a f t e r v i r u s h a s e x i t e d t h e c e l l , and i s i n t h e serum o r some o t h e r e x t r a c e l l u l a r environment.

246

Sect. V

-

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With some animal v i r u s e s , e s p e c i a l l y t h e s m a l l , RNA-containing picorna- and t o g a v i r u s groups, t h e i n t r a c e l l u l a r c l e a v a g e s b e g i n on v e r y l a r g e l'poly p r o t e i n " p r e c u r s o r s , and v i r t u a l l y a l l v i r u s gene p r o d u c t s , i n c l u d i n g n u c l e i c a c i d polymerizing enzymes, are r e g u l a t e d p r o t e o l y t i c a l l y (reviewed i n 4 3 ) . Thus, t h e s e v i r u s e s are examples of p a r a s i t i c a g e n t s whose e n t i r e r e p l i c a t i o n c y c l e i s dependent on s u c c e s s f u l a c t i v a t i o n of s e v e r a l d i s c r e t e p r o t e i n gene p r o d u c t s by l i m i t e d p r o t e o l y s i s .

TABLE 1 C h a r a c t e r i s t i c s of V i r u s Group i n Which P r o t e i n Cleavage Occurs virus

S t r u c t u r a l F e a t u r e s of V i r i o n

Role of Cleavage

Picorna (Polio)

S S ( + ) ~ R N Ap,r o t e i n c o a t .

A l l v i r a l p r o t e i n s are c l e a v a g e p r o d u c t s , most f u n c t i o n s r e g u l a t e d by p r o t o l y s i s , Viral protease i d e n t i f i e d , which processes coat precursor.

Toga ( S i n d b i s )

SS(+) RNA, p r o t e i n c o r e , glycoprotein-lipid envelope.

Similar t o picorna.

Oncorna ( R e t r o ) Rous sarcoma

SS(+) RNA p r o t e i n c o r e , reverse transcriptase. glycoprotein-lipid envelope.

Probably s i m i l a r t o p i c o r n a . Evidence f o r a v i r u s p r o t e a s e , s e n s i t i v e t o -SH p r o t e a s e . inhibitors.

Reo

DS RNA, p r o t e i n c o r e , o u t e r p r o t e i n c o a t , RNA t r a n s c r i p t a s e .

A c t i v a t i o n of t r a n s c r i p t a s e , maturation.

Myxo ( I n f l u e n z a )

S S ( - ) RNA. p r o t e i n c o r e , glycoproteins (hemagglutinin and neuraminidase) and l i p i d e n v e l o p e , RNA t r a n s c r i p t a s e .

A c t i v a t i o n of v i r o n s . (hemagglutinin)

Paramyxo ( S e n d a i )

S i m i l a r t o myxo.

Absolute r e q u i r e m e n t t h a t e n v e l o p e g l y c o p r o t e i n be cleaved f o r i n f e c t i v i t y .

Rhabdo ( V e s i c u l a r stomatitis)

SS (-1 RNA, " b u l l e t - s h a p e ' ' nucleoprotein core, matrix p r o t e i n embedded in l i p i d membrane, o u t e r g l y c o p r o t e i n .

Adeno

DS DNA, numerous p r o t e i n s t r u c t u r e s in c a p s i d .

Maturation. Virus protease?

Pox ( V a c c i n i a )

DS DNA, many p r o t e i n s and l i p i d in c a p s i d .

Maturation.

Herpes simplex

DS DNA, p r o t e i n c o r e , p r o t e i n s and l i p i d in e n v e l o p e .

Papova (SV40)

DS DNA, t h r e e p r o t e i n s i n capsid, histones i n core.

Parvo (Adenoassociated virus)

SS(+) DNA, t h r e e p r o t e i n s .

Removal of " s i g n a l sequence" during t r a n s p o r t t h r o u g h membranes.

on g l y c o p r o t e i n ,

?

Capsid p r o t e i n s y n t h e s i s , T-antigen production, (transformation). Capsid p r o t e i n s y n t h e s i s and assembly.

'Abbreviations: S S - s i n g l e s t r a n d e d , DS-double s t r a n d e d , ( + ) - i n f e c t i o n s (messenger RNA-like), (-)-complementary. t r a n s c r i b e d i n t o messenger RNA.

Chap. 2 3 B.

Biochemistry of V i r u s e s

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O r i g i n s of t h e P r o t e o l y t i c Enzymes

1. Host C e l l P r o t e a s e s used by t h e I n f e c t i n g V i r u s . It is obvious t h a t f o r a complete u n d e r s t a n d i n g of t h e p r o c e s s i n g r e a c t i o n s t h e o r i g i n of t h e p a r t i c i p a t i n g p r o t e a s e ( s ) must b e known. Although unambiguous proof i s s t i l l l a c k i n g , t h e r e are i n d i c a t i o n s t h a t h o s t c e l l proteases p a r t i c i p a t e d i r e c t l y . Most f u l l y d e s c r i b e d i s t h e a c t i v a t i o n by a c e l l membrane-associated p r o t e a s e of a n envelope g l y c o p r o t e i n p r e c u r s o r of t h e paramyxoviruses, e . g . , Sendai v i r u s (reviewed i n 4 4 ) . C e l l s , which appare n t l y l a c k e d c l e a v i n g . a c t i v i t y , produced v i r u s p a r t i c l e s w i t h v e r y low i n f e c t i v i t y . One of t h e envelope g l y c o p r o t e i n s i n t h e s e v i r i o n s w a s an unc l e a v e d form. Growth of t h e v i r u s i n a high y i e l d i n g c e l l gave i n f e c t i o u s v i r i o n s w i t h v i r t u a l l y a l l t h e g l y c o p r o t e i n i n a c l e a v e d form. Cleavage a l l o w s t h e v i r u s t o proceed s u c c e s s f u l l y through t h e e a r l y s t a g e s of i n f e c t i o n t o t h e p o i n t where t h e v i r a l n u c l e i c a c i d i s r e l e a s e d i n t o t h e h o s t c e l l . A compelling f i n d i n g w a s t h a t t r y p s i n , o r o t h e r p r o t e a s e s , c a n r e p l a c e t h e m i s s i n g f u n c t i o n of t h e non-producing c e l l . S i m i l a r r e s u l t s were r e p o r t e d f o r a paramyxovirus, t y p e 1 i s o l a t e d from b r a i n t i s s u e of human p a t i e n t s s u f f e r i n g from m u l t i p l e s c l e r o s i s . The v i r u s , produced i n brain t i s s u e , w a s very l o w i n i n f e c t i v i t y , unless t r e a t e d with t r y p s i n o r mixed w i t h macrophages. I t w a s s u g g e s t e d t h a t t h e i n t r u s i o n of p r o t e o l y t i c macrophages i n t o b r a i n t i s s u e h a r b o r i n g t h e v i r u s c o u l d t r i g g e r o f f a b u r s t of v i r u s a c t i v i t y l e a d i n g t o t h e symptoms of MS.45 2 . Virus-coded P r o t e a s e s . Recent d a t a s u p p o r t t h e view t h a t a p r o t e a s e i s a v i r u s gene p r o d u c t . Examples i n c l u d e t h e RNA tumor v i r u s e s , 4 6 t o g a v i r u s e s , 4 7 ’ 4 8 a d e n o v i r u s e s , 4 9 and p i c o r n a v i r u s e s . 5 0 Although i t i s s t i l l p r o b l e m a t i c whether t h e p r o t e a s e i d e n t i f i e d i s t r u l y of v i r a l o r i g i n ( o r an a c t i v a t e d c e l l enzyme), t h e d a t a c l e a r l y i n d i c a t e a l a r g e q u a n t i t a t i v e i n d u c t i o n of p r o t e o l y t i c a c t i v i t y upon i n f e c t i o n . 5 0 ’ 5 1 Genetic and c e l l - f r e e s t u d i e s 4 ” 5 2 f u r t h e r s u p p o r t t h e virus-coded h y p o t h e s i s . Perhaps t h e most e l e g a n t examples a r e t h o s e i n which messenger RNA from picorna- and t o g a v i r u s e s have been t r a n s l a t e d i n c e l l - f r e e e x t r a c t s t o y i e l d p r o t e a s e s s y n t h e s i z e d de n 0 v 0 . ~ ” 5 2 These s t u d i e s should b e u s e f u l i n f i n a l l y a s s i g n i n g t h e p r o t e o l y t i c a c t i v i t y t o a v i r u s gene p r o d u c t . C. I n h i b i t i o n of t h e P r o t e o l y t i c R e a c t i o n s - The r e a c t i o n s l e a d i n g t o c l e a v e d v i r a l p r o t e i n s may be blocked i n i n f e c t e d c e l l s by a t l e a s t two distinct strategies.

1. The s u b s t r a t e , i . e . , t h e v i r u s p r e c u r s o r p o l y p e p t i d e , may be a l t e r e d so t h a t i t cannot be c l e a v e d . T h i s h a s been accomplished by i n c o r p o r a t i o n of amino a c i d a n a l o g s , s i n c u b a t i o n a t abnormally h i g h temperat u r e s , 5 4 b l o c k i n g g l y c o s y l a t i o n of t h e p r e c u r s o r g l y ~ o p e p t i d e s (~e ~ . g . , by deoxyglucose o r tunicamycin t r e a t m e n t ) , and by z i n c i o n s . 5 6 The l a s t is a p a r t i c u l a r l y s i m p l e , u s e f u l t e c h n i q u e , and i s r e v e r s i b l e i n some examples ?7 2 . Chemical i n h i b i t o r s of p r o t e a s e s may b e used t o i n a c t i v a t e ~ ~ 5, 9 p r e v e n t t h e p a r t i c i p a t i n g enzymes. Iodoacetamide and i ~ d o a c e t a t e 58, p r o c e s s i n g of v i r u s p r o t e i n s . D i i s o p r o p y l f l u o r o p h o s p h a t e , probably acting as an i n h i b i t o r of a s e r i n e a c t i v e - s i t e p r o t e a s e , a l s o p r e v e n t s t h e proc e s s i n g of p o l i o v i r u s p o l y p r o t e i n . 6 o F i n a l l y , chloromethyl k e t o n e s of s e l e c t e d amino a c i d s , p a r t i c u l a r l y t h a t of p h e n y l a l a n i n e , have been w i d e l y used i n s u c c e s s f u l tests t o b l o c k v i r a l p r o t e i n p r o c e s s i n g . 6 0 ’ 6 1 The

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v i r a l p r o t e a s e which c l e a v e s p i c o r n a v i r u s c a p s i d p r o t e i n p r e c u r s o r s i s s p e c i f i c ; l e u c i n e r e s i d u e s d o n a t e t h e new -COOH group (reviewed i n 4 2 ) . Based on t h i s , a r e c e n t s t u d y used a l e u c i n e chloromethyl k e t o n e t o s p e c i f i c a l l y i n h i b i t c a p s i d p r o t e i n p r o d u c t i o n i n p o l i o v i r u s - i n f e c t e d HeLa c e l l s . 5 1 It is p o s s i b l e t h a t , as c a r e f u l c h a r a c t e r i z a t i o n s of t h e v i r u s s p e c i f i c p r o t e a s e s become a v a i l a b l e , s y n t h e t i c e f f o r t s w i l l l e a d t o a n t i v i r a l chemicals which i n h i b i t t h e i r f u n c t i o n . References

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Chapter 24.

Liposomes as Drug Carriers

Demetrios Papahadjopoulos, Cancer Research I n s t i t u t e and Department of Pharmacology, U. of C a l i f o r n i a , Medical School, San F r a n c i s c o , CA 94143 I n t r o d u c t i o n - Liposomes are v e s i c l e s composed of one o r more l i p i d b i l a y e r s completely surrounding a n i n t e r n a l aqueous s p a c e . They are u s u a l l y composed of p h o s p h o l i p i d s e i t h e r i n p u r e form o r i n combination w i t h o t h e r amphipathic molecules such as s t e r o l s , l o n g c h a i n b a s e s o r a c i d s , o r membrane proteins. The s t r u c t u r e of l i p o s p e s varies from l a r g e (0.5+5~) m u l t i l a m e l l a r v e s i c l e s 1 t o s m a l l ( ' ~ 3 0 0A) u n i l a m e l l a r v e s i c l e s . 293 More r e c e n t l y , new methods have been r e p o r t e d d e s c r i b i n g t h e f o r m a t i o n of unilamellar v e s i c l e s of i n t e r m e d i a t e s i z e . 4 , 5 , 6 The g e n e r a l p r o p e r t i e s of liposomes and t h e i r i n t e r a c t i o n w i t h v a r i o u s macromolecules have been d e s c r i b e d i n s e v e r a l reviews. 7 ,8 99 The main f e a t u r e s of liposomes t h a t have made them a v a l u a b l e i n v e s (1) t h e i r c h a r a c t e r i s t i c morphology, t i g a t i v e t o o l are t h e f o l l o w i n g : where a r e l a t i v e l y impermeable membrane completely e n c l o s e s a n aqueous space and (2) t h e i r a b i l i t y t o e n c a p s u l a t e v a r i o u s s o l u t e s p r e s e n t i n t h e aqueous phase d u r i n g t h e i r formation. These c h a r a c t e r i s t i c s prompted t h e i r i n i t i a l d e s c r i p t i o n as a model membrane system1 which w a s followed by an e v e r i n c r e a s i n g number of s t u d i e s i m p o r t a n t i n u n d e r s t a n d i n g b i o l o g i c a l membrane s t r u c t u r e and f u n c t i o n . I t w a s t h e same c h a r a c t e r i s t i c s t h a t a l s o prompted t h e u s e of liposomes as carriers of d r u g s and o t h e r macr om0 l e c u l e s . l o 9 11 3 1 2 The concept behind t h e u s e of liposomes as c a r r i e r s of d r u g s and macromolecules i s r e l a t e d t o an expected p r o t e c t i o n of t h e e n c a p s u l a t e d molecules i n t h e blood stream, a n a l t e r e d t i s s u e d i s t r i b u t i o n and pharmacok i n e t i c s , a s w e l l as an i n c r e a s e d uptake i n t o c e l l s by mechanisms t h a t are n o t normally a v a i l a b l e f o r t h e s e molecules. Some of t h e s e e x p e c t a t i o n s have been v e r i f i e d through s t u d i e s i n v a r i o u s l a b o r a t o r i e s d u r i n g t h e l a s t few y e a r s , Such s t u d i e s have shown t h a t liposome e n c a p s u l a t i o n can a l t e r d r a s t i c a l l y t h e pharmacokinetics and t i s s u e d i s p o s i t i o n of t h e encapsul a t e d s u b s t a n c e s , i t can enhance t h e i r u p t a k e i n t o c e l l s , and i t can i n c r e a s e t h e i r pharmacological e f f i c a c y . S e v e r a l r e c e n t reviews have d i s c u s s e d t h e s e e a r l y r e s u l t s i n c o n s i d e r a b l e d e t a i l . 13-20 A c r i t i c a l e v a l u a t i o n of t h e e a r l y l i t e r a t u r e i n d i c a t e s r e a s o n s f o r both optimism and c a u t i o n i n r e l a t i o n t o t h e f u t u r e u s e of liposomes as drug c a r r i e r s . The s i m p l i c i t y of t h e i n i t i a l concept can l e a d t o an u n d e r e s t i m a t e of t h e t e c h n i c a l d i f f i c u l t i e s of liposomes p r e p a r a t i o n and t h e b i o l o g i c a l c o m p l e x i t i e s i n r e l a t i o n to t h e i n t e r a c t i o n of liposomes w i t h c e l l s i n v a r i o u s t i s s u e s . I t seems c l e a r t h a t t h e u l t i m a t e u t i l i t y of liposomes as drug c a r r i e r s w i l l depend l a r g e l y on f u r t h e r methodologi c a l developments and a b e t t e r u n d e r s t a n d i n g of t h e i r i n t e r a c t i o n s w i t h c e l l s . P r o g r e s s i n t h i s area w i l l e n a b l e us t o produce liposomes w i t h t h e appropriate p r o p e r t i e s s u i t e d t o a p a r t i c u l a r , a p p l i c a t i o n and, thus, optimize t h e i r e f f e c t i v e n e s s . A c o n f e r e n c e on liposomes and t h e i r u s e s i n b i o l o g y and medicine w a s o r g a n i z e d r e c e n t l y under a u s p i c e s of t h e New York Academy of S c i e n c e s . 2 1 The c o n f e r e n c e i n c l u d e d sub j e c t s r a n g i n g from p h y s i c a l c h e m i s t r y of liposomes t o t h e i r i n t e r a c t i o n s w i t h c e l l s i n v i t r o Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12440514-8

Chap. 24

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and their use as drug carriers in tumor-bearing animals. This article will summarize briefly the most significant recent studies. Methodology for Liposome Preparation - An informal agreement was reached on the use of a three-letter acronym to designate the type of liposome such as multilamellar vesicles (MLV) or small unilamellar vesicles (SW) or large unilamellar vesicles (LW) with the chemical composition in parenthesis after the acronym (Ref. 21, p. 3 6 7 ) . The term liposomes is therefore to be used as a generic name to include all types of artificial vesicles composed of phospholipids and other amphipathic lipids. The original liposome preparation of Bangham et all consisting of multilamellar vesicles (MLV) has been admirably suited in defining many membrane properties and was the basis for the development of the sonicated unilamellar vesicles (SW). However, both preparations show a relatively low volume of entrapped aqueous space per mole of lipid and restricted ability to encapsulate large macromolecules. This is because in MLV most of the lipid is participating in the internal lamellae, and the close apposition of the adjacent concentric bilayers restricts the internal water space. In SUV, which are single-compartment vesicles, the ratio of surface area to encapsulated volume is so large that only a small aqueous volume per mole of lipid can be attained. Attempts to circumvent these shortcomings have been only partially successful. The ethanol injection method produces vesicles of about the same size as SW with the same shortcomings.22 The ether infusion technique produces large unilamellar vesicles with high captured volumes per mole of lipid,5 but the efficiency of encapsulation is relatively low. Two recent reports describe technical improvements of the ethanol injection23 and ether injection methods. 24 Other useful techniques for preparing large volume vesicles either use specialized conditions25 or are restricted to a single phospholipid.4 Methods based upon solvent evaporation have been attempted in the past but have resulted in the formation of multilamellar vesicles.26-28 A method designed to form asymmetric vesicles by centrifugation of a suspension of dense aqueous inverted micelles through an organic solvent water interface has been reported.29 This report, however, indicated that the internal volume was small and the vesicles themselves relatively unstable. Techniques based upon the removal of detergents yield vesicles slightly larger than SUV.30 Recent modifications of the cholate detergent dialysis technique of forming small unilamellar vesicles31 include removal of the detergent by gel filtration32 and fast controlled dialysis system.33 These are suitable for membrane reconstitution experiments but, like the S W , fail to encapsulate the aqueous phase efficiently. Recently, a method that combines detergent dialysis and solvent evaporation has been described.34 This technique leaves an equal weight of detergent per phospholipid in the resulting vesicles, making them unsuitable for many biological applications. We have been concerned by the fact that none of the above methods cqn produce liposomes with all the following desirable characteristics: (i) the ability to entrap a large percentage of the aqueous material; (ii) a high aqueous space-to-lipid ratio; and (iii) widely variable chemistry of lipid components without detergents. A new method6 was recently developed which meets to a large extent all the above criteria and is based on evaporation of a water-in-oil emulsion (REV). It is relatively easy

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to establish in most laboratories and produces vesicles of intermediate size (0.2-0.4~), which can encapsulate with high efficiency most watersoluble drugs and large macromolecules. The most recent methodological improvement involves the production of liposomes of well-defined size distribution by extrusion of MLV or LUV through a nucleopore polycarbonate membrane. 35 The pressure extrusion method allows the sequential passage of preformed liposomes through membranes of decreasing pore diameter. During the extrusion, all the lipid material passes through the membrane, but the size of liposomes is reduced to approximately the diameter of the membrane pores.35 This procedure can be applied to any type of liposomes, and it is rapid and simple. If the extrusion is performed in the presence of the mother liquid used for the initial encapsulation, it results in relatively good encapsulation efficiency. Interactions of Liposomes with Cells In Vitro - The first observations on the ability of liposomes to affect cell behavior in vitro include the work of Magee and Miller36 who found that liposomes carrying anti-viral antibody could protect cells against viral infection, the work of Papahadjopoulos et a1,37 who found that liposomes could induce cell fusion without cytotoxic effects, and the work of Gregoriadis and Buckland38 who found that liposomes containing invertase could cause the disappearance of vacuoles of stored sucrose in mouse peritoneal macrophages. The initial observations on the ability of liposomes to induce cell fusion generated interest on the question as to whether liposomes could fuse with the cellular plasma membrane. 12,39940 Such a mechanism of uptake would result in a much more efficient delivery of drugs into the cellular environment, compared to the more conventional endocytotic pathway of pinocytosis or phagocytosis.14 The mechanism of interaction of lipid vesicles with cells in culture has been studied in detail during the last few years in several laboratories, and the results were reviewed recently.l8,19,41 The evidence has been obtained with various types of vesicles and cells under varying conditions, and it therefore seems contradictory in several cases. There is general agreement, however, that liposomes are taken up quite efficiently by cells in culture, that the process of uptake is temperature dependent, but in most cases it does not require metabolic energy, and that the vesicle contents are incorporated along with the lipids. In cases where the uptake has been quantitated in terms of S W liposomes per cell, it appears that several million vesicles can be incorporated within a few hours without overt cytotoxic effects.12 Concerning the mechanism of uptake, the evidence indicates that it could involve an of the following: fusion with the plasma membrane12,42-48 endoc t o ~ i s ~ ~ adsorption to the cell surface50,51 or molecular exchange.43,52 The predominance of any of these mechanisms is controlled by the chemistry and physical properties of the liposomes and perhaps by the cell type and the state of the cell. Since the evidence for most of the mechanism of uptake is indirect and the contribution of each type of uptake is very difficult to quantitate, most conclusions that have been drawn up-to-now should be considered at best only qualitative statements. There is clearly a need for further work in this area, especially in developing quantitative assays for estimating fusion events and distinguishing them from classical endocytosis

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or adsorption followed by molecular exchange at the cell surface. Irrespective of the difficulties in quantitating the contribution from each of the various types of uptake, it is clear that liposomes can be used to enhance drastically the cellular uptake of molecules that are not normally taken up. This aspect of liposome-cell interactions is of obvious importance to their use as drug carriers. The first indication of the dramatic increase of the pharmacological efficacy of an encapsulated drug was the enhancement by 1000 fold of the ability of cyclic AMP to inhibit the growth of 3T3 cells in vitro.12~53 A similar large increase has been observed more recently54 with actinomycin D against a hamster cell line which exhibited resistance to this drug. Increased cytotoxicity following encapsulation has also been observed with cytosine arabinoside triphosphate.55 The last two observations raise the important possibility of using liposomes to circumvent resistance to drugs due to decreased cellular transport as well as to metabolic block. Other important demonstrations of increased cellular uptake in vitro followin liposomal encapsulation include uptake of horseradish peroxidase,56 9 !!!7 hexosaminidase,49 cytoplasmic localization of carboxyfluores~ e i n a, calcium-chelating ~ ~ agent,59 secretion in mast cells induced by calcium-containing liposomes,60 synthetic polynucleotides inducing interferon,61 messenger RNA inducing globin synthesis,62,63 polio virus infecting CHO cells64 and, more recently, isolated viral RNA.65 The last four studies are important as first demonstrations on the use of liposomes for the incorporation of informational macromolecules into the genetic apparatus of cells. Although the quantitative aspects of the uptake of liposomes by various cells and the respective mechanism of uptake have not been studied in detail, it does not appear so far that there is a marked cellular specificity. It would, of course, be of obvious advantage for chemotherapy if liposomes could be constructed that have increased affinity for specific cell types such as tumor cells. Several attempts have been made for & vitro targeting of liposomes with varying degrees of success. The first such experiments were reported by Gregoriadis and Neerunjun66 using antibodies raised against cell surface antigens. The presence of the antibody immunoglobulins resulted in an increased uptake which was cell-specific. Similar results were obtained more recently by Magee et a167 who also reported increased cytotoxicity with liposomes containing both antibody and actinomycin D. Increased cellular uptake has also been obtained with heat aggregated IgM by dogfish macrophages which carry the Fc receptors,57 also with a plant lectin and glycoprotein-containing liposomes by erythrocytes,68 and finally with DNP-containing liposomes and anti-DNP antibody by TNBS-treated c e l l d 9 This last study reported that the increased cellular uptake did not result in enhanced cytoplasmic release of carboxyfluorescein from the vesicles. However, their interactions were only short-timed, and the results do not preclude an enhanced cytotoxic effect over a longer period of time as a result of the antibody-specific enhanced cellular uptake. Observations have also been obtained indicating that the presence of specific glycolipids (such as lactocerebroside) enhances the uptake of liposomes by Hela cells in culture.70

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Tissue Disposition and Pharmacological Effects of Liposomes In Vivo Since the initial publication of studies involving injection of liposomeentrapped substances in vivo ,11,71Y 72 there has been an increasing number of studies on both the altered tissue distribution and also on the increased pharmacological efficacy of encapsulated agents. The subject has been reviewed recently.13 15 ,20. The most important points for future consideration include the permeability properties of liposomes in a physiological environment, their interaction with plasma components, the role of liposome size and chemistry in determining the rate of removal from the circulation and their tissue localization at the cellular level. The permeability of liposomes is an important parameter which depends not only on the innate properties of the lipid bilayer and the encapsulated but also on the interaction of liposomes with plasma components73~74and various cells.75 Our own early studies with liposomes indicated that the presence of a high ratio of cholesterol to phospholipid is essential for preventing various proteins from "penetrating" into the bilayer and increasing their permeability.76 Injection of liposomes composed of phospholipids without cholesterol into mice was found to result in complete release of encapsulated Na+ and other small molecules.77 This effect seems to be related to the interaction of liposomes with plasma proteins and lipoproteins.73,74 The incorporation of a high ratio of cholesterol into liposomes seems to prevent this high degree of leakage, and can also affect drastically the anti-tumor effects of encapsulated ara-C in L-1210 tumor-bearing mice.78 There was a recent proposal that the permeability properties of liposomes in vitro could also be controlled (enhanced) in specific areas of the body by application of hyperthermia.79y80 This possiblity is based on earlier observations showing that liposomes composed of saturated phospholipids exhibit a drastic increase in permeability when the temperature is raised to the critical point (Tm) for the melting of the acyl chains.81 The new technique of gamma-ray perturbed angular correlation seems to be useful for observing the release of liposome contents in vivo.82 The effect of liposome size and chemistry on the rate of clearance from the circulation was demonstrated clearly by Juliano and Stamp83 by the use of well-defined preparations of S U V , which remain in the circulation for much longer eriods of time compared to the large MLV. From this and other studies,84-89 it is clear that blood clearance and tissue distribution of liposomes can be markedly affected by their physicochemical properties. From the work of Kimelberg et a1,88 it appears that encapsulation of methotrexate into SW not only prolongs its plasma clearance rate but allows for a much broader tissue distribution (compared with MLV; see also Ref. 89) and also reduces drastically its metabolic degradation. MLV are usually cleared rapidly by kidney and spleen, although the incorporation of positive charge has been shown to result in lung accumulation.86 The interaction of the drug molecules with the lipid bilayer has also been shown to affect the clearance rates and tissue disposition.90,91 The incorporation of various glycolipids has been shown to result in changes in the tissue disposition of the encapsulated material.92 Finally, recent studies on the effect of size have shown that very large MLV (>lmicron diameter) are retained preferentially in the lung where they retain their contents for relatively long periods of time.93 Lung retention was also achieved by infusion of aerosolized liposome preparations.94 Prolongation

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of the clearance rate in mice of encapsulated maltose in S W has been achieved by the use of dialkyl analogs of phosphatidyl choline.95 A small increase in the uptake of 111I-labelled bleomycin by tumors in mice has been reported after incorporation of immunoglobulins into liposomes, although the uptake of liver and spleen was also augmented.96 The uptake of bleomycin-containing liposomes into neoplasms in human patients has been followed in one reported case.97 Other in vivo studies with antibody bearing liposomes have also been reported.9The role of liposome structure'on their (relatively limited) ability to enhance oral absorption of drugs has been discussed recently.100-103 Localization of the contents of positively charged liposomes in arterial lesions has been reported recently.104

The interaction of liposomes with various plasma components is an important parameter not only in relation to their permeability but also in defining the chemistry of the surface groups available for interaction with cells in various tissues. There is a report indicating that liposomes recovered from plasma have altered electrophoretic mobilities, indicating adsorption of plasma proteins. 105 The same study reported that a2-macroglobulin seems to be specifically recovered with such liposomes. Other studies indicate that lipid material from the liposomes is recovered in the lipoprotein fraction74,106 and that lipid material can exchange between liposomes and cells52Y 107 9 108 especially in the presence of exchange proteins. It is therefore important to consider the question of lipid-exchange between liposomes and various lipoproteins and cells in studies where liposome localization is determined by the appearance of radio-labelled lipid in various tissues. This point is important since localization of such lipid102 may reflect exchange with lipoproteins and may not be strictly indicative of the localization of vesicle contents. In considering tissue localization of liposomes and their contents, the most important question is the bioavailability of such markers. Localization of intact liposomes especially in interstitial spaces in various tissues may not necessarily be accompanied by release of the vesicle contents into intracellular spaces where the drug could act. Techniques should therefore be developed for quantitating not only the tissue localization of vesicle contents but their uptake at the cellular level and their availability for pharmacological action. The pharmacological effects of encapsulated drugs are, of course, the ultimate test of their bio-availability, although the quantitation of the uptake and the mechanism of drug uptake by the recipient cells is difficult to ascertain by such complex biological endpoints. The effects of various pharmacological agents encapsulated in liposomes have been reviewed re~ent1y.l~ ,20 The most recent and exciting application is the dramatic increase of the therapeutic index of antimonial compounds against a model for leishmaniasis disease following liposomal encapsulation. 109-111 In this case, the effectiveness of the liposomes against the parasitic organisms is presumably achieved because of their mutual residence within the reticuloendothelial cells, which represent a natural targeting system. Other recent applications include effects against inflammation with liposomes injected in arthritic joints,ll2 anti-tumor effects of encapsulated methotrexate against lymphosarcomas resistant to this drug , 1 1 3 anti-tumor effects of encapsulated alkylating agentsllh and nucleotide analogs such

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as ara-Cl15 ,116 and alleviation of respiratory distress syndrome, follow-

ing aerosol infusion of dipalmitoyl phospholipids.117 The ability of liposomes to act as adjuvants118 along with other interesting facets relating to immunological properties of liposomes have been reviewed by Tyrell et al.15 Use of liposomes for alleviating enzyme deficiencies by delivering the missing enzymes was the pharmacological application that initiated the activities of several groups in the area of liposomes as delivery vehicles.10 Y 119 Ironically, this has proven a most difficult task, judging from the progress so far.102,103,120 It may be that with the development of methods for the encapsulation and cellular delivery of RNA and DNA62-65,121,122 liposomes could ultimately prove valuable for correcting not only enzyme deficiencies but other genetic defects. Conclusion - From the studies reported so far, it is clear that liposomal encapsulation of drugs and various other macromolecules can have the following important pharmacological effects: (1) increase of cellular uptake of impermeant molecules (shown definitely in vitro) ; (2) decrease of the rate of plasma clearance of molecules with short half-lives; (3) alteration of tissue disposition with variable tissue specificity depending on the properties of the liposomes; ( 4 ) inhibition of the metabolic breakdown of molecules; (5) enhancement of the pharmacological efficacy without overt cytotoxic effects, although the problem of toxicity has been studied in detail only in a few cases.123~124 The studies that have been published so far permit an attitude of cautious optimism, although it may be said that in the past there has been an underemphasis of the possible problems and difficulties.125 There is no doubt that much more work is needed in the areas of increased cellular and tissue specificity and the possible toxicity of liposome-encapsulated drugs. It is also clear that further methodological developments are needed before the liposome system can be utilized at optimal conditions. Liposomes were developed originally as a model membrane system and not as drug carriers. Most of the pharrnacological applications attempted so far have been obtained by using the original preparative methodology, which has been far from optimal. However, the encouraging success that has been achieved so far can be expected to generate increasing interest in further defining the properties of the liposome system and its optimization suitable to each particular application.

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82. 8 3. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110.

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K. J. Huang and M.R. Mauk, Proc. Natl. Acad. Sci. 74, 4991 (1977). R.L. Juliano and D. Stamp, Biochern. Biophys. Res.Tornrnun. 63, 651 (1975). G. Gregoriadis, E.D. Neerunjun, Eur. J. Biochern. 47, 179 ( 1 9 7 a Y.E. Rahrnan and B.J. Wright, J. Cell Biol. 65, 11271975). M.M. Jonah, E.A. Cerny, Y.E. Rahman, x o c h i r n . Biophys. A c t a 401, 336 (1975). I.R. MacDougall, J.K. Dunnick, M.G. McNamee and J.P. Kriss, Proc. Natl. Acad. Sci. 71, 3487 (1974). H.K. Kirnelberg, T.F. Tracy, S.M. Biddlecorne and R.S. Bourke, C a n c e r Research 36, 2949 (1976). R.L. J u l i z o , in: New Approaches t o Membranes in Neoplasia, ed. B. Marchesi; Alan G s s , N.Y. pp. 21 (1976). R.L. Juliano and D. Stamp, Biochern. Pharrnacol 27, 21 (1978). Y.E. Rahrnan, W.E. Kisieleski, E.M. Buess and ETA. Cerny, Europ. J. C a n c e r 11, 883 (1975). M.M. Jonah, E.A. Cerny, Y.E. Rahrnan, Biochirn. Biophys. A c t a 541, 321 (1978). C.A. Hunt, Y. Rustum, E. Mayhew and D. Papahadjopoulos, Drug Dispos. Metab. , In Press (1979). R.L. Juliano, D. Stamp and N. McCullough, Ann. N.Y. Acad. Sci. 308, 411 (1978). D.S. Deshrnukh, W.D. Bear, H.M. Wisniewski and H. Brockerhoft, Bioch. Bioph. Res. Cornrnun. 82, 328 (1978). G. Cregoriadis, D.E. N g r u n j u n and R. Hunt, Life Sciences 21, 357 (1977). A.W. Segal, G. Gregoriadis, J.P. Lavender, D. Tarin, T.J.-Peters, Clin. Sci. and Mol. Med. 51, 421 (1976). J.K. Dunnick, TR. McDougall, S. Aragon, M.L. Goris, J.P. Kriss, J. Nuclear Med. 16, 483 (1975). T. DeBarsy, P. Devos, F. Van Hoof, Lab. Invest. 34, 273 (1976). C. Dapergolas and C. Gregoriadis, The Lancet: 8% (1976). G. Dapergolas, E.D. Neerunjun and G. Gregoriadis, FEBS L e t t e r s Vol. 2,No. 2 (1976). B.E. Ryrnan, R.F. Jewkes, K. Jeyasingh, M.P. Osborne, H.M. Patel, V.J. Richardson, M.H. Tattersall, and D.A. Tyrell, Ann. N.Y. Acad. Sci. 308, 281 (I 978). G.-Gregoriadis, Ann. N.Y. Acad. Sci. 308, 343 (1978). V.J. C a r i d e and B.L. Zaret, Science 198,735 (1977). C.D.V. Black and G. Gregoriadis, Bioch. Soc. Trans. 4, 253 (1976). L. Krupp, A.V. Chobanian, and P.I. Brecher, Bioch.%ioph. Res. Cornrnun. 72, 1251 (1976). D.B. Zilversrnitt. Ann. N.Y. Acad. Sci. 308. 149 (1978). R.A Cooper, E.L. Arner, J.S. W i l e y , x J . Shattil, .J. Clin. Invest. 55, 115 (1975). C.R. Alving, E.A. Steck, W.R. Chapman, V.B. Waits, L.D. Hendricks, G.M. Swartz, W.L. Howson, Proc. Natl. Acad. Sci. (USA) 75, 2959 (1978). C.D. Black, G.J. Watson and R. J. Ward, Trans. R.%c. Trop. Med. Hyg. 71, 550 (1977). R.R.'New., M.L. Chance, S.C. Thomas, a n d W. Peters, N a t u r e 272, 55 (1978). J.T. Dingle, J.L. Gordon, B.L. Hazleman, C.G. Knight, D.PTPage Thomas, N.C. Phillips, I.H. Shaw, J.E. Oliver, G. Jones, E.H. Turner, J.S. Lowe, N a t u r e 271, 372 (1978). M. J. K o a s k i , F. Rosen, R. J. Milholland, and D. Papahadjopoulos, Cancer 38, 2848 (1978). Research -

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R.J. Rutman, C.A. Riter, N.G. Avadhani and J. Hansel, Cancer Treatment Reports 60, 617 (1976). E. Mayhew, D. Papahadjopoulos, Y.M. Rustum and C. Dave, Ann. N.Y. Acad. Sci. 308, 371 (1978). T. K a F o k a and T. Kobayashi, Ann. N.Y. Acad. Sci. 308, 387 (1978). C.J. Morley, A.D. Bangham, P. Johnson, C.D. Thorburn, and G. Jenkin, Nature 271, 162 (1978). A.C. Al&n and C. Cregoriadis, Nature 252, 252 (1974). C. Sessa and G. Weissmann, J. Lipid R e s . x 3 1 0 (1968). C. Weissmann, H. Korchak, M. Finklesteiri, J. Smolen, S. Hoffstein, Ann. N.Y. Acad. Sci. 308, 235 (1978). A.B. M u n k h z e e , S. Orloft, J.D. Butler, T. Triche, P. Lalley, and J.D. Schulman, Proc. Natl. Acad. Sci. (USA) 75, 1361 (1978). R.M. Hoffman, L.B. Margolis, and L.D. Bergelson, FEBS Letters 93, 365 (1978). Bruni, C. Toffano, A. Leon and E. Boarato, Nature 260, 332 (1976). Y.E. Rahman, W.R. Hanson, J. Bharucha, E.J. A i n x o r t h , B. Jaroslow, Ann. N.Y. Acad. Sci. 308, 325 (1978). G. Gregoriadis, Nature 265, 407 (1977).

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Ronald J. Koenig and Anthony Cerami J o s l i n D i a b e t e s Foundation, Boston, Mk and The R o c k e f e l l e r U n i v e r s i t y , NY,NY I n t r o d u c t i o n - The m o d i f i c a t i o n of p r o t e i n s v i a enzymatic g l y c o s y l a t i o n i s a w e l l - d e s c r i b e d a s p e c t of b i o c h e m i s t r y . Indeed, t h e u b i q u i t y of glycop r o t e i n s a l o n e a t t e s t s t o t h e g e n e r a l importance of enzymatic g l y c o s y l a t i o n r e a c t i o n s i n b i o l o g y . R e c e n t l y , however, i t h a s become a p p a r e n t t h a t p r o t e i n s a l s o may b e g l y c o s y l a t e d by nonenzymatic p r o c e s s e s , and t h a t t h e s e r e a c t i o n s may p l a y major r o l e s i n b o t h h e a l t h and d i s e a s e s . The p r o t o t y p e molecule f o r t h i s p r o c e s s must b e c o n s i d e r e d t o b e hemoglobin (Hb AIc), a s t h i s w a s t h e f i r s t p r o t e i n r e c o g n i z e d t o b e nonenzymatiAI c a h y g l y c o s y l a t e d . The c h e m i s t r y and b i o l o g y of t h i s hemoglobin have been t h e s u b j e c t of i n t e n s i v e i n v e s t i g a t i o n , prompted by t h e f a c t t h a t p a t i e n t s w i t h d i a b e t e s m e l l i t u s have e l e v a t e d c o n c e n t r a t i o n s of Hb A Ic' S t u d i e s d i r e c t e d a t u n d e r s t a n d i n g t h i s o b s e r v a t i o n have p r o v i d e d new i n s i g h t i n t o t h e p a t h o b i o l o g y and c l i n i c a l management of d i a b e t e s . Thus, t h i s review w i l l summarize t h e c u r r e n t d a t a concerning t h e nonenzymatic g l y c o s y l a t i o n of p r o t e i n s i n humans and r e l a t e d s p e c i e s and f o c u s t o a l a r g e e x t e n t on Hb AIc. S t r u c t u r e of Hemoglobin A I ~ - The hemoglobins of t h e a d u l t human e r y t h r o c y t e may b e r e s o l v e d by i o n exchange chromatography on t h e p o l y m e t h a c r y l i c a c i d r e s i n BioRex 70 i n t o s e v e r a l d i s t i n c t components.1 Hemoglobin A (Hb A) comprises 90-95% of t h e t o t a l , w h i l e a t l e a s t h a l f a dozen q u a n t i t a t i v e l y Hb AIc i s an a c i d i c minor comminor hemoglobins make up t h e remainder. ponent t h a t comprises 3-5% of t h e t o t a l Hb i n normal i n d i v i d u a l s , and up t o 15% i n p a t i e n t s w i t h d i a b e t e s m e l l i t u s . 2 Hb A and Hb AIc have i d e n t i c a l amino a c i d ~ e q u e n c e s . ~I n i t i a l s t u d i e s 3 determined t h e u n i q u e s t r u c t u r a l f e a t u r e of Hb AIc t o b e a low m o l e c u l a r w e i g h t m o i e t y a t t a c h e d a t t h e NH2 - t e r m i n u s of t h e B c h a i n s v i a sodium b o r o h y d r i d e r e d u c i b l e l i n k a g e , presumed t o b e a S c h i f f b a s e . Mass s p e c t r o s c o p i c d a t a w a s c o n s i s t e n t w i t h a hexose a s t h e b l o c k i n g group and r e c e n t l y t h e s t r u c t u r e 1-amino-1-deoxyfructose h a s been proposed. liberates T h i s w a s based on t h e o b s e r v a t i o n t h a t a c i d h y d r o l y s i s of Hb A 0.25 moles of hexose c o n s i s t i n g of g l u c o s e and mannose i n a 3 : i C r a t i o p e r mole of a , 8 , d i m e r , and t h a t p e r i o d a t e c l e a v a g e of t h e p r o d u c t o b t a i n e d by r e d u c t i o n of Hb Arc w i t h sodium b o r o h y d r i d e (NaBH4) l i b e r a t e s p r i m a r i y 3H-formic a c i d , r a t h e r t h a n 3H-formaldehyde. The u n e q u i v o c a l assignment of t h e 1-amino-1-deoxyfructose s t r u c t u r e was p o s s i b l e by d i r e c t i s o l a t i o n of t h e NH -terminus g l y c o d i p e p t i d e f o l l o w i n g r e d u c t i o n of t h e B c h a i n of Hb AIc wigh NaBH4 and comparison w i t h a u t h e n t i c , s y n t h e t i c compounds usi n g p r o t o n magnetic resonance s p e c t r o s c o p y , g a s - l i q u i d chromatography and t h i n l a y e r chromatography.

t

qc.

Two mechanisms have been proposed t o a c c o u n t f o r B i o s y n t h e s i s of Hb A t h e s y n t h e s i s of Hb Both presume t h e nonenzymatic f o r m a t i o n of a S c h i f f b a s e between a c a r b o h y d r a t e and t h e NH2 - t e r m i n u s of t h e 8 c h a i n s , followed by an Amadori rearrangement t o y i e l d t h e 1-deoxy-1-amino-2-ketose p r o d u c t ( F i g 1).7 I n t h e f i r s t p r o p o s a l g l u c o s e would r e a c t d i r e c t l y w i t h hemoglobin. S i n c e r e d c e l l s are n o t i n s u l i n dependent f o r g l u c o s e u p t a k e , d i a b e t i c s have e l e v a t e d g l u c o s e c o n c e n t r a t i o n s i n e r y t h r o c y t e s and t h i s Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

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H I H-C-N H-fi Amadori rearrangement

e=o

HO-6-H

~ H ~ O H Figure 1 would e x p l a i n t h e i r i n c r e a s e d Hb A l e v e l s . T h i s h y p o t h e s i s would however, Ic make i t d i f f i c u l t t o e x p l a i n t h e a p p a r e n t s p e c i f i c i t y of t h e r e a c t i o n f o r c h a i n s . The NH2- t e r m i n a l v a l i n e of t h e CI t h e NH - terminus of t h e c h a i n &as a lower pk and hence should b e more r e a c t i v e . Furthermore, l y s i n e e-amino groups might b e p r e d i c t e d t o r e a c t , a l b e i t a t a s l o w e r r a t e . Although t h e s y n t h e s i s of Hb A h a s been achieved by t h e i n v i t r o incuba~ l a b o r a t o r i e s found t h i s r e a c t i o n t o b e t i o n of Hb A w i t h g l u c ~ s e ,ot?&r slow and n o t s p e c i f i c f o r t h e B c h a i n NH2-terminus, lo,ll whereas t h e react i o n w i t h glucose-6-phosphate w a s b o t h r a p i d and s p e c i f i c . Thus i t w a s proposed t h a t t h e i n i t i a l r e a c t a n t might b e glucose-6-phosphate, which would combine s p e c i f i c a l l y due t o i t s a f f i n i t y f o r t h e diphospho-glycerate binding p o c k e t , (DPG) and t h a t a phosphatase would t h e n be r e q u i r e d t o complete t h e f o r m a t i o n of Hb I n f a c t , s p e c i f i c a d d u c t s between t h e NH -terminus of t h e B c h a i n s and c a r b o h y d r a t e s were e a s i l y s y n t h e s i z e d 2 -i n v i t r o w i t h any c a r b o h y d r a t e t h a t c o n t a i n e d b o t h a phosphate group and a n unblocked carbonyl ( e . g . glucose-6-phosphate, fructose-6-phosphateY f r u c t o s e 1,6-diphosphate, glyceraldehyde-3-phosphate; b u t n o t glucose-lphosphate, glucose-l,6-diphosphate, UDP-glucose) .I1 The problem w i t h t h i s h y p o t h e s i s would r e q u i r e d i a b e t i c e r y t h r o c y t e s t o have e l e v a t e d glucose-6phosphate l e v e l s , and t h e evidence on t h i s p o i n t i s c o n t r a d i c t o r y . 1 1 , 1 3

A,..lO~ll.

A s o l u t i o n t o t h i s problem may r e c e n t l y have been found. Whereas t h e g l y c o s y l a t i o n of Hb had p r e v i o u s l y been thought t o b e s p e c i f i c f o r t h e B c h a i n t e r m i n u s , r e c e n t d a t a s u g g e s t t h a t Hb A is i t s e l f heterogenous and c o n t a i n s molecules w i t h c a r b o h y d r a t e a t t a c h e d a t v a r i o u s o t h e r amino groups.14 Thus, g l u c o s e may b e t h e r e a c t i n g molecule and t h e a p p a r e n t s p e c i f i c i t y f o r t h e NH -terminus of t h e B c h a i n r e s u l t s from t h e f a c t t h a t t h e adduct a t t h i s p o s g t i o n happens t o have a unique chromatographic mobili t y , whereas g l y c o s y l a t i o n of o t h e r NH2-groups does n o t s i g n i f i c a n t l y a l t e r t h e chromatographic b e h a v i o r of Hb A. s y n t h e s i s i n v i v o have provided a t h e o r e t i I n v e s t i g a t i o n s of Hb A measurements c a l f o u n d a t i o n f o r t h e r e c e n t € $ demonstrated v a l u e of Hb A Ic I n i t i a l s t u d i e s ,I5 i n which wild-type and g e n e t i c a l l y i n c l i n i c a l medicine. d i a b e t i c mice were i n j e c t e d w i t h 59Fe-labeled r e t i c u l o c y t e s , demonstrated t o b e s y n t h e s i z e d i n a slow, n e a r l y i r r e v e r s i b l e r e a c t i o n t h r o u g h o u t Hb A t h e & f e of t h e mature e r y t h r o c y t e . S i n c e e r y t h r o c y t e s are n o t c a p a b l e of p r o t e i n s y n t h e s i s , t h i s h a s

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263 -

t o o c c u r by t h e p o s t - s y n t h e t i c g l y c o s y l a t i o n of already-formed Hb A. Furthermore, when r e t i c u l o c y t e s from w i l d - t y p e mice were i n j e c t e d i n t o synthesis increased 2.7 f o l d d i a b e t i c r e c i p i e n t s , t h e rate of Hb A Ic ( F i g . 2 ) . 1 5 Human Hb AIc h a s a l s o been shown t o b e s y n t h e s i z e d a t a s l o w , c o n s t a n t rate f o r a t l e a s t t h e f i r s t 10 80 days of r e d c e l l s u r v i v a l . 1 6 A s t e a d y s t a t e c o n c e n t r a t i o n of Hb A Ic O - a-o Ob/db i s a p p a r e n t l y a c h i e v e d v i a t h e reCd +/+ placement of o l d e r y t h r o c y t e s l a d e n aw i t h Hb A by new c e l l s r e l a t i v e l y p o o r i n HSCAIc. The a c t u a l Hb AIc 7 c o n c e n t r a t i o n i s a r e f l e c t i o n of r e d N c e l l h a l f - l i v e and t h e r a t e of synt h e s i s , which i n t u r n i s a f u n c t i o n of t h e b l o o d g l u c o s e c o n c e n t r a t i o n . The n e a r - i r r e v e r s i b i l i t y of t h e r e a c t i o n can b e a t t r i b u t e d t o t h e s t a b i l i t y of t h e Amadori r e a r r a n g e m e n t .

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Hemoglobin A I ~i n t h e Management of Diabetes Mellitus - A d i a b e t i c with s e v e r e ch r o n ic hyperglycemia w i l l s y n t h e s i z e Hb A a t a n abnormally r a p i d r a t e , and'fheref o r e Should h a v e I "0 K) 20 30 40 Y) a v e r y h i g h b l o o d Hb AI c o n c e n t r a t i o n . Doyr An improvement i n d i a b e f i c c o n t r o l would immediately d e c r e a s e t h e r a t e of Figure 2 ( R e p r i n t e d w i t h p e r m i s s i o n from s y n t h e s i s , but o l d red c e l l s with high ref. 1 5 ) l e v e l s of Hb A would remain i n t h e c i r c u l a t i o n u n z f l t h e i r 120 day l i f e s p a n e x p i r e d . P o p u l a t i o n s of r e d c e l l s would be p r e s e n t i n t h e c i r c u l a t i o n measurer e f l e c t i n g t h e mean b l o o d g l u c o s e l e v e l from t h e t i m e of t h e Hb A ment backwards f o r s e v e r a l months. I n o t h e r words, a s i n g l e Hb A: measurement s h o u l d r e f l e c t a p e r s o n ' s mean b l o o d g l u c o s e l e v e l o v e r t h e p r e v i o u s weeks t o months. T h i s h y p o t h e s i s h a s now been s u b s t a n t i a t e d by m u l t i p l e clinical investigations. I n human d i a b e t i c s , Hb AI l e v e l s c o r r e l a t e w i t h f a s t i n g blood and a h i g h d e g r e e . o f c o r r e l a t i o n i s s e e n i n r e s p o n s e t o an o r a l g l u c o s e t o l e r a n c e test.17 When a series of p o o r l y c o n t r o l l e d d i a b e t i c s w a s h o s p i t a l i z e d f o r 3-6 months t o a c h i e v e s t r i c t c a r b o h y d r a t e c o n t r o l (mean f a s t i n g b l o o d s u g a r [FBS] d e c r e a s e d from 343 t o 84 m g j d l ) , t h e dec r e a s e i n Hb A c o n c e n t r a t i o n l a g g e d 3-4 weeks b e h i n d t h e improvement i n I d i - a h e t i c c o n t r o f (Fig,. 3 ) . l 9 To b e t t e r assess mean b l o o d s u g a r l e v e l s , b l o o d s u g a r v a l u e s were measured j u s t p r i o r t o and 1 hour f o l l o w i n g breakf a s t , l u n c h and d i n n e r . These v a l u e s w e r e summed and c a l l e d " g l u c o s e A h i g h l y s i g n i f i c a n t c o r r e l a t i o n w a s found between mean b l o o d brackets''. values s u g a r c o n c e n t r a t i o n s , as a s s e s s e d by g l u c o s e b r a c k e t s , and Hb A Several oulsatient b o t h p r i o r t o and d u r i n g s t r i c t c o n t r o l ( F i g . ,).I9 and m u l t i p l e s t u d i e s have d e m o n s t r a t e d p o s i t i v e c o r r e l a t i o n s between Hb A measurements o v e r t i m e of u r i n a r y s u g a r and low p o s t - p r a n d i a i c b l o o d sugar. 2 0- 2 4

sugar^,^^^^^

S i n c e H b AIc i s a marker f o r c h r o n i c h y p e r g l y c e m i a , i t s c o n c e n t r a t i o n

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should b e e l e v a t e d r e g a r d l e s s of t h e c a u s e of t h e d i a b e t e s . Thus, mice w i t h d i a b e t e s secondary t o s p e c i f i c g e n e t i c l e s i o n s o r a d m i n i s t r a t i o n of t h e p a n c r e a t i c @ - c e l l t o x i n s a l l o x a n and s t r e p t o z o t o c i n a l l have e l e v a t e d l e v e l s are independent of t h e mode of Hb AIc c o n c e n t r a t i o n s . 2 5 Hb A t h e r a p y i n human d i a b e t e s , andl!n i d e n t i c a l twins discordant f o r d i a b e t e s , are e l e v a t e d i n t h e d i a b e t i c s i b l i n g s . 2 6 Thus, b o t h b a s i c and c l i n i c a l d a t a show Hb A levels 120- 0 0 0 r e f l e c t t h e patieng's mean blood - 0 0 e 0 0 0 . . glucose l e v e l f o r t h e preceding 1000 levels weeks t o months. Hb A are n o t i n f l u e n c e d by $art term 8 0 n 0 0 o - 7 f l u c t u a t i o n s i n blood s u g a r . Inf 60o O 0 f r e q u e n t q u a n t i f i c a t i o n of Hb A Ic 0 f 40c o n c e n t r a t i o n i n t h e blood of 0 *. 0 - 3 d i a b e t i c o u t p a t i e n t s can o b j e c t y 200 0 i v e l y and a c c u r a t e l y assess t h e 0 q u a l i t y of c a r b o h y d r a t e c o n t r o l , a f e a t u r e unique t o t h e measurement. T h i s should a l l o w more o p t i m a l a d j u s t m e n t of t h e r a p y f o r Figure 3 (Reprinted w i t h p e r m i s s i o n from r e f . 1 9 ) d i a b e t i c s . Furthermore, Hb AI measured s e r i a l l y i n a p r o s p e c f i v e s t u d y can b e used t o b e t t e r d e f i n e t h e r e l a t i o n s h i p between carbohyd r e t e c o n t r o l and t h e development of t h e s e q u e l a e of c h r o n i c d i a b e t e s .

g Y

D i a b e t i c Sequelae - While t h e development of i n s u l i n r e p l a c e ment t h e r a p y h a s g r e a t l y prolonged the d i a b e t i c ' s l i f e s p a n , i t h a s n o t had a major impact i n t h e p r e v e n t i o n of t h e myriad of s e q u e l a e a s s o c i a t e d w i t h longs t a n d i n g d i a b e t e s . Kidney, r e t i n a , p e r i p h e r a l nerve, l e n s and t h e c a r d i o v a s c u l a r system a l l s u f f e r d y s f u n c t i o n s secondary t o d i a b e t e s . The m u l t i s y s t e m n a t u r e of t h i s d i s e a s e h a s made d i f f i c u l t t h e development of a u n i f y i n g hypothesis t o e x p l a i n t h e s e s e q u e l a e . mIC ! Figure 4 (Reprinted w i t h p e r m i s s i o n from r e f . 1 9 ) "F"1U.+UYlll

1-1

The g l y c o s y l a t i o n of Hb t o form Hb AIc may r e p r e s e n t a model r e a c t i o n t o e x p l a i n t h e e t i o l o g y of many of t h e s e c o m p l i c a t i o n s of d i a b e t e s . It h a s been p o i n t e d o u t t h a t t h e s e s e q u e l a e t e n d t o o c c u r i n t i s s u e which do n o t r e q u i r e i n s u l i n f o r g l u c o s e u p t a k e , and t h e r e f o r e t h a t t h e s e c e l l s w i l l have i n t r a c e l l u l a r g l u c o s e c o n c e n t r a t i o n s i n p r o p o r t i o n t o t h e s e v e r i t y of hyperglycemia. 27 Perhaps p r o t e i n s , o t h e r t h a n hemoglobin are s u b j e c t t o i n c r e a s e d non-enzymatic g l y c o s y l a t i o n i n d i a b e t e s . Such g l y c o s y l a t i o n c o u l d a l t e r t h e s o l u b i l i t y , enzymatic a c t i v i t y , h a l f - l i f e o r o t h e r p r o p e r t y of t h e p a r t i c u l a r p r o t e i n and t h u s account f o r t h e observed c l i n i c a l d y s f u n c t i o n .

Chap. 25

Non-enzymatic G l y c o s y l a t i o n

Koenig, Cerami

265 -

I n s u p p o r t of t h i s i s t h e o b s e r v a t i o n t h a t carbamylation of amino groups by t h e a n t i - s i c k l i n g drug c y a n a t e r e s u l t s i n b o t h p e r i p h r a l neuropathy28 and c a t a r a c t s ,29 t h a t a r e h i s t o l o g i c a l l y s i m i l a r t o t h o s e seen i n d i a b e t e s . It i s noteworthy t h a t t h e cyanate-induced l e s i o n s a r e r e v e r s i b l e upon c e s s a t i o n of drug a d m i n i s t r a t i o n , 2 8 , 2 9 perhaps r e f l e c t i n g t u r n o v e r and implying t h a t t h e l e s i o n s of d i a b e t e s would be r e v e r s i b l e o r a t least cont r o l l a b l e i f euglycemia could be achieved. The rate of r e v e r s i b i l i t y of t h e s e q u e l a e of d i a b e t e s would be a f u n c t i o n of t h e g l y c o s y l a t e d p r o t e i n ' s t u r n o v e r rate i n v i v o . I n a d d i t i o n , nonenzymatic g l y c o s y l a t i o n i s n o t completely i r r e v e r s i b l e and t h e g l y c o groups could e l u t e s l o w l y from prot e i n s w i t h v e r y long b i o l o g i c a l h a l f - l i v e s . I n f a c t , c e r t a i n s e q u e l a e of d i a b e t e s , e . g . , p l a t e l e t h y p e r a g g r e g a b i l i t y , have been shown t o b e revers i b l e upon i n s t i t u t i o n of r i g i d c a r b o h y d r a t e c o n t r o l . 3 0 It s h o u l d be noted t h a t a l t h o u g h Hb AIc h a s a s l i g h t l y e l e v a t e d oxygen a f f i n i t y 3 1 y 3 2 t h i s i s n o t l i k e l y t o c o n t r i b u t e t o c l i n i c a l d i s e a s e s i n c e mutant hemoglobins w i t h much 3;eater oxygen a f f i n i t i e s a r e n o t a s s o c i a t e d w i t h any of t h e s e symptoms. Nonenzymatic G l y c o s y l a t i o n of O t h e r P r o t e i n s - S i n c e any f r e e amino group w i t h a s u f f i c i e n t l y low pK should have t h e p o t e n t i a l f o r forming a S c h i f f b a s e and Amadori rearrangement p r o d u c t , one would p r e d i c t many p r o t e i n s i n a d d i t i o n t o hemoglobin a r e nonenzymatically g l y c o s y l a t e d . T i s s u e s i n which d i a b e t i c s e q u e l a e o c c u r a r e c u r r e n t l y being i n v e s t i g a t e d f o r t h e p r e s e n c e and s i g n i f i c a n c e of such p r o c e s s e s . The p r o t e i n s of t h e l e n s have, t o d a t e , been t h e most thoroughly i n v e s t i g a t e d . S t u d i e s of bovine and r a t l e n s c r y s t a l l i n s have r e v e a l e d nonenzymatic g l y c o s y l a t i o n of l y s i n e €-amino groups i n v i t r o and v i v o i n t h e p r e s e n c e of a h i g h g l u c o s e o r glucose-6phosphate environment. 3 n h i s g l y c o s y l a t i o n i m p a r t s an i n c r e a s e d s u s c e p t i b i l i t y of t h e c r y s t a l l i n s t o undergo s u l f h y d r y l o x i d a t i o n t o form h i g h m o l e c u l a r weight a g g r e g a t e s , which a t t a i n a l a r g e enough s i z e t o d e f r a c t l i g h t and c a u s e an o p a c i t y w i t h i n t h e l e n s . The a d d i t i o n of reducing a g e n t s p r e v e n t s , a s w e l l a s p a r t l y r e v e r s e s , t h e f o r m a t i o n of t h e s e aggregates. The accumulation of s o r b i t o l w i t h i n t h e l e n s has a l s o been o f f e r e d a s a mechanism f o r t h e f o r m a t i o n of d i a b e t i c cataracts.35 A t t h e p r e s e n t t i m e i t i s u n c l e a r what t h e r e l a t i v e r o l e s of t h e s e two mechanism a r e i n t h e g e n e s i s of d i a b e t i c c a t a r a c t s . R e c e n t l y , t h e nonenzymatic l y c o s y l a t i o n of human albumin b o t h i n v i t r o and i n v i v o h a s been d e s ~ r i b e d . ~ ZApproximately 10% of albumin i s glycos y l a t e d a t l y s i n e €-amino groups i n t h e serum of normal i n d i v i d u a l s . The l e v e l of g l y c o s y l a t i o n i n d i a b e t i c s h a s y e t t o be r e p o r t e d , n o r i s t h e s i g n i f i c a n c e of t h i s p r o c e s s i n terms of t h e t r a n s p o r t and o t h e r f u n c t i o n s of albumin known. I t should be p o i n t e d o u t t h a t nonenzymatic g l y c o s y l a t i o n i s n o t unique t o t h e hyperglycemic s t a t e ; r a t h e r , i t simply o c c u r s a t a f a s t e r r a t e t h e more g l u c o s e i s p r e s e n t . Thus, i f p r o t e i n t u r n o v e r w e r e slow enough, cert a i n s e q u e l a e of d i a b e t e s might develop i n non-diabetic i n d i v i d u a l s o v e r a l o n g e r t i m e course. F o r example, s e n i l e c a t a r a c t s might a l s o be caused by g l y c o s y l a t i o n of l e n s c r y s t a l l i n s . I n f a c t nonenzymatic g l y c o s y l a t i o n may p l a y a major r o l e i n t h e normal aging p r o c e s s , s i n c e t h e c o m p l i c a t i o n s of d i a b e t e s are f r e q u e n t l y c o n s i d e r e d t o resemble a c c e l e r a t e d aging.

266 __

Sect. V

- Topics i n Biology

Walsh, Ed.

Furthermore, p r o t e i n s may b e modified by c a r b o h y d r a t e s o t h e r t h a n g l u c o s e . A g a l a c t o s y l a t i o n of l y s i n e r e s i d u e s h a s a l s o r e c e n t l y been i m p l i c a t e d i n t h e g e n e s i s o f c a t a t a c t s of r a t s 'fed a h i g h g a l a c t o s e d i e t . 37 Summary - The tendency of s u g a r s and amines t o condense t o form s t a b l e Amadori rearrangement p r o d u c t s w a s r e c o g n i z e d decades ago a s an i m p o r t a n t r e a c t i o n i n t h e food and a g r i c u l t u r a l i n d u s t r y . Indeed, t h e Amadori rearrangement i s t h e f i r s t s t e p i n t h e M a i l l a r d r e a c t i o n , o r nonenzymatic browni n g of food.38 Only r e c e n t l y , however, h a s t h e r o l e of nonenzymatic glyc o s y l a t i o n i n human b i o l o g y come under i n v e s t i g a t i o n . Hemoglobin w a s t h e f i r s t p r o t e i n shown t o b e modified i n t h i s manner. Glucose reacts t o form a S c h i f f b a s e w i t h NH2Tterminus of t h e B c h a i n of Hb A, and s u b s e q u e n t l y undergoes an Amadori rearrangement t o y i e l d l-aminoThis modified hemoglobin, named Hb AIc, i s a normal r e d 1-deoxyfructose. c e l l constituant present i n increased concentration i n p a t i e n t s with diabete s m e l l i t u s . Hb A i s s y n t h e s i z e d throughout t h e l i f e of t h e e r y t h r o c y t e i n a slow, n e a r l y & r e v e r s i b l e r e a c t i o n . The r a t e of s y n t h e s i s is a funct i o n of blood g l u c o s e c o n c e n t r a t i o n . These p r o p e r t i e s of Hb AI combine t o make i t an i n d i c a t o r molecule whose c o n c e n t r a t i o n a t one p o i n t f n t i m e r e f l e c t s t h e p a t i e n t ' s mean blood g l u c o s e l e v e l f o r t h e p r e c e d i n g month. T h i s g i v e s Hb AIc a unique and i n v a l u a b l e r o l e i n c l i n i c a l medicine, f o r i t is t h e only known parameter t h a t a c c u r a t e l y assesses l o n g term c a r b o h y d r a t e con t r o l

.

While Hb AIc i t s e l f i s n o t l i k e l y t o have d e l e t e r i o u s e f f e c t s , t h e nonenzymatic g l y c o s y l a t i o n of o t h e r p r o t e i n s may r e s u l t i n a l t e r e d enzymatic a c t i v i t y , s o l u b i l i t y , a n t i g e n i c i t y e t c . and t h e r e b y r e s u l t s i n many of t h e c l i n i c a l s e q u e l a e of long-standing d i a b e t e s . I n t h i s r e g a r d , l e n s p r o t e i n s undergo i n c r e a s e d g l y c o s y l a t i o n i n h i g h c a r b o h y d r a t e environments, and s u c h g l y c o s y l a t i o n i n c r e a s e s t h e ease w i t h which t h e p r o t e i n s o x i d i z e t o form h i g h m o l e c u l a r weight a g g r e g a t e s and o p a c i t i e s . S i m i l a r g l y c o s y l a t i o n r e a c t i o n s should occur a t a s l o w e r r a t e i n normal i n d i v i d u a l s and may account f o r many of t h e changes s e e n i n normal a g i n g , e . g . , s e n i l e c a t a r a c t s .

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8. 9. 10. 11.

D.W. A l l e n , W.A. Schroeder and J. Balog, J . Am. Chem., 80, 1628 (1958). L.A. T r i v e l l i , H.M. Ranney and H-T. L a i , N. Engl. J. Med., 284, 353 (1971). W.R. Holmquist and W.A. Schroeder, Biochemistry, 5, 2489 (1966). R.M. Bookchin and P.M. G a l l o p , Biochem. Biophys. R e s . Com., 32, 86 (1968). H.F. Bunn, D.N. Haney, K.H. Gabbay and P.M. G a l l o p , Biochem. Biophys. R e s . Com., 103 (1975). R . J . Koenig, S.H. B l o b s t e i n and A. C e r a m i , J . B i o l . Chem., 252, 2992 (1977). J . E . Hodge, Adv. Carbohyd. Chem., 1 0 , 169 (1955). M.H. Garner, W.H. Garner and F.R.NTGurd, J . B i o l . Chem., 5451 (1973). R . F l u c k i g e r a n d K.H. W i n t e r h a l t e r , FEBS L e t t e r s , 71,356 (1976). D.H. Haney and H.F. Bunn, Proc. N a t l . Acad. S c i . , 73, 3534 (1976). V . J . S t e v e n s , H. Vlassara, A. A b a t i and A . Cerami, J. B i o l . Chem., 252, 2998 (1977).

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267

S. Minakami, C. S u z i k i , T . S a i t o , and H. Yoshikawa, J . Biochem., 58, 543 (1965). M . J . McDonald, R. S h a p i r o , M. Bleichman, J. Solway and H.F. Bunn, J . Biol. Chem., 253, 2327 (1978). H.F. Bunn, R. S h a p i r o , L.M. G a r r i c k , M . J . McDonald, K.H. Gabbay and P. M. G a l l o p , Fed. P r o c . , 37, 1390 (1978). R . J . Koenig and A. Cerami, P r o c . N a t l . Acad. S c i . , 3687 (1975). H.F. Bunn, D.N. Haney, S. Kamin, K.H. Gabbay and P.M. G a l l o p , J . C l i n . I n v e s t . , 57, 1652 (1976). R . J . Koenig, C.M. P e t e r s o n , C. K i l o , A. C e r a m i , and J . R . W i l l i a m s o n , Diabetes, 230 (1976). R . J . Graf, J . B . Halter and D.P. P o r t e , D i a b e t e s , 27, 834 (1978). R . J . Koenig, C.M. P e t e r s o n , R.L. J o n e s , C. Saudek, M. Lehrman and A. Cerami, N. Engl. J . Med. 295, 417 (1976). R. Lanoe, J . S o r i a , N . T h K l t , C. S o r i a , E. Eschwege and G. Tchobrouts k y , L a n c e t , 2, 1156 (1977). K.H. Gabbay, K. H a s t y , J . L . Breslow, R.C. E l l i s o n , H.F. Bunn and P.M. G a l l o p , J . C l i n . Endo. Metab., 44, 859 (1977). B. Gonen, A.H. R u b e n s t e i n , H. Rochman, S.P. Tanega and D.L. Horowitz, L a n c e t , 2, 734 (1977). R . E . Davis, D . J . N i c o l , V . J . McCann and J . S . C a l d e r , Med. J . A u s t r a l i a , 1, 530 (1978). W . J . Tze, K.H. Thompson and J . L e i c h t e r , J . P e d i a t r . , 93, 13 (1978). R . J . Koenig, D. Araujo and A. Cerami, D i a b e t e s , 25, 1 (1976). R.B. T a t t e r s a l l , D.A. Pyke, H.M. Ranney and S.M. Bruckheimer, N. Engl. J . Med., 293, 1 1 7 1 (1975). R.G. S p i r o , D i a b e t o l o g i a , 1 (1976). C.M. P e t e r s o n , P. T s a r i s , A. O h n i s h i , Y.S. Lu, R.W. Grady, A. Cerami and P . J . Dyck, Ann. I n t . Med., 81, 152 (1974). D.H. N i c h o l s o n , D.R. H a r k n e s s , W.E. Benson,and C.M. P e t e r s o n , Arch. O p h t h l . , 94, 927 (1976). C.M. P e t e r s o n , R.L. J o n e s , R . J . Koenig, E.T. Melvin and M.L. Lehman, Ann. I n t . Med. 86, 425 (1977). 1088 (1970). H.F. Bunn and R.W. B r i e h l , J . C l i n . I n v e s t . , M . J . McDonald, M. Bleichman, H.F. Bunn and R.W. Noble, J . B i o l . Chem. 254, 702 (1979). S. Charache, C l i n i c s i n Haematol., 2, 357 (1974). V . J . S t e v e n s , C.A. Rouzer, V.M. Monnier and A. C e r a m i , P r o c . Natl. Acad. S c i . , 2918 (1978). J . H . K i n o s h i t a , I n v e s t . Ophthalmol., 713 (1974). J . F . Day, S.R. Thorpe and J . W . Bayness, J. B i o l . Chem., 254, 595 (1979). V . J . S t e v e n s , V.M. Monnier and A. Cerami, s u b m i t t e d f o r p u b l i c a t i o n (1979). J . Hodge, J. Agr. Food Chem., 928 ( 1 9 5 3 ) .

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268 -

Section VI - Topics in Chemistry and Drug Design Editor:

Burt Renfroe, CIBA-GEIGY Corporation, Ardsley, N.Y. 10502

Chapter 26. Reactions of Interest in Medicinal Chemistry Daniel Lednicer, Mead Johnson Pharmaceuticals, Evansville, IN The selection of novel reactions deemed potentially useful t o medicinal chemists from the great mass of this past years' literature in organic chemistry obviously involves a highly personal view of the field. Some of the seventy odd transforms which follow were picked because they seem to offer more convenient access to important functional arrays, because they lead to moieties which may be associated with biological activity or simply because they involve clever chemistry. REVIEWS - Useful reviews have appeared entitled: "Asymmetric Syntheses:" ' "New applications of Malononitrile in Organic Synthesis,'I3 9 "Intramolecular Ene Reaction in Organic Synthesis,' I 5 and "a-Sulfenylated Carbonyl Compounds in Organic Synthesis.Il6 Additional articles summarize stereospecific olefin syntheses, heterocyclic syntheses by benzyne cyclizations,a and ring transformations of pyrimidines.

'

REAGENTS - E s t e r i f i c a t i o n - Carboxylic acids can be esterified in high yield under very mild conditions by a new procedure using DCC and 4-(Npyrro1idino)pyridine. The procedure gives good yields even with mesitoic acids or with tertiary-butanol. Oxidations - 3,5-Dinitroperbenzoic acid is a stable storable peracid equivalent in activity to trifluoroperacetic acid. A full paper has appeared which gives the experimental details for the a-hydroxylation of carbonyl compounds by treatment of the anions of enol silanes with Mo05'HMPA Anions from carboxylic esters (LiN(iPr12,LDA; -78OC) can be (MOOPH). efficiently, regiospecifically chlorinated or brominated by treatment with respectively CCl4 or CBr4.13 Treatment of enol silanes from conjugated ketones with m-chloroperbenzoic acid (MCPBA) followed by removal of silicon affords the a-hydroxyketones. A new method for transposition of carbonyl groups starts by interception of the intermediate from tosyl hydrazone decomposition with MegSiC1. Oxidation of the silane intermediate with MCPBA gives the transposed product. l 5

'

''

'*

'

+

T S iMe 3

Cyclohexanones can be dehydrogenated to enones by treatment of the corresponding enol silanes with DDQ in the presence of a catalytic amount of the bis-silyl derivative of acetamide. However, the yields fall off drastically when the reaction is applied to cyclopentanones and cycloheptanones. Another new method for dehydrogenation of carbonyl compounds consists of conversion to their pyridine-2-sulfide derivatives, followed by oxidation (MCPBA) , and by mild heat.

Chap. 26

Reactions of Interest

Lednicer

269

Oxidation of primary alcohols carrying unsaturation at the 5 or 6 position by means of the pyridinium chlorochromate provides the cyclized products. The same products are obtained by oxidation of analogously substituted aldehydes, with this reagent.

Reductions - Azides are efficiently reduced to the corresponding amines by 1,3-propylenedithiol. Calcium borohydride (from CaC12 and NaBH4) has been used to reduce unsaturated glutaric monoesters directly to the lac-

Condensations - Methyl ketones are converted to the corresponding vinyl compounds in high yield by treatment with paraformaldehyde in the presence of N-methylaniline trifluoroacetate in THF or dioxane.2 1 The formamide of 2-(N-methyl)aminopyridine has been found to be an efficient reagent for the transfer of formyl groups to Grignard reagents.22 The salt obtained from treatment of 2-trimethylsilyl-N,N-dimethyl acetamide with LDA or BuLi is a stable solid. This gives good yields of acrylamides on reaction with ketones or non-enolizable aldehydes. Li MejSiCHCONMe

>- ,

R2 C=O R2C

=

CHCONMe2

Reaction of the tertiary-butylimine from acetaldehyde with (EtO) 2 POC1, in the presence of two equivalents of base gives the corresponding ylide. This gives good yields of substituted acroleins on condensation with ketones and aldehydes. 2 4 CHjfi=NtBu 7 [ (EtO) 2%H=CNtBu]Li

+ R2C=CHCHO

In a somewhat similar sequence, condensation of the functional ylide below with a ketone leads directly to an enamine. In the specific example at hand the crude product was alkylated without isolation. Hydrolysis of the ketal gives a cyclohexenone v i a the intermediate ket~aldehyde?~

Organoboranes can be efficiently bis-homologated to the corresponding acetic acid derivatives by reaction with the dianion derived from phenoxyacetic acid. The stability of the phenoxide leaving group provides the driving force for the transformation. 2 6

270

Sect. VI

-

Topics in Chemistry and Drug Design

Renfroe, Ed.

- Enol chlorides can be converted to the corresponding ketones in fair yields at room temperature by inclusion of Hg(OAc)2 in the reaction mixture.27 A full paper has appeared which gives experimental details for the conversion of olefins to aminoalcohols by means of osmium reagents.2 8 The side chain characteristic of nonsteroidal anti-inflammatory agents can be efficiently constructed by a novel rearrangement. Treatment of the enamine of aryl ketones with (CbH50)2PON3 leads to the amidine derivative presumably via the triazole. Base hydrolysis gives the desired acid."

Miscellaneous

PROTECTING GROUPS - Cyclic imide disulfides from primary amines can be prepared by the scheme below. Treatment with mercaptoethanol under very mild conditions gives back the starting amine. 3 0 0

.%+

i --ClCSCl 5 5RNHCOEt ) EtONa II

RNH2

R

RNH2

0

-

Acylation of dimethylamine with a hindered phenylcarbonate allows anion chemistry on the methyl groups. 0

II /CH3

0

ArOCN " /CH3

,

-

(

ArOCN3HC'

0

II ,CH3

A r o C N \ ~ ~ 2 ~ ~ ~ 1Ar=

$t

I OH Phosphorus derivatives of imines show reactivity akin to ternary iminium salts, and thus readily add nucleophiles, (CH3MgX;HCN). Isothiocyanates can be obtained under relatively mild conditions, by reduction followed by base and CSZ.~'

\CH~R

Nu R>Npo(C6H512 R

I__,

I

Addition of dimethylamine to acrylates protects the double bond and makes the position c1 to the carboxyl accessible to alkylation. Quaternization of the amine followed by treatment with base (DBU) regenerates a substituted acrylate. 3 3

Chap. 26

Reactions of Interest

Lednicer

271

AROMATIC SUBSTITUTION - Reaction of oxazolines from o-fluorobenzoic acids with organometallic reagents results in net displacement of halogen. Hydrolysis of the heterocycle affords the substituted benzoic acid. 3 4 Condensation of enolates derived from 6-dicarbonyl compounds with o-bromobenzoic acid in the presence of cuprous bromide affords the displacement product directly.

Br The driving force presumably lies in the stability of the halide leaving group. Formal replacement of hydride is possible with an appropriate nucleophile. Reaction of nitrobenzene with the anion from chloromethylphenyl sulfone gives the substitution product in 72% yield as a 1:l ortho to para mixture. Use of bulkier anions gives exclusively para product. When the para position is substituted, the nucleophile enters

ortho,

[.p

02N

9; ] HS02C6H5

-H%

02N~cH2s02C6H5

R

c1

The Claisen rearrangement has only recently been used to provide functionalized derivatives. Thus, mandelate esters react with orthoacetates and orthopropionates to give the corresponding homophthalates.3 7

Condensation of the boron trichloride complex of N-methylanilines with aromatic aldehydes affords o-aminobenzhydrols in moderate yields. Somewhat lower yields of the corresponding ketones are obtained by condensing the adduct obtained from aniline itself with nitriles in the presence of AlCl 3 8 3‘ AHCH3

, a””’ a””” 0 .’ 4

CHOHAr COAr A very mild method for fluorination of the para position of anilines involves formation of the azide &a the diazonium salt followed by treatment with hydrogen fluoride. 3 9 X

X

x

Aromatic iodophenols can be vinylated by ethyl acrylate in the presence of a palladium catalyst.40 In an extension of this work the catalyst

272

Sect. VI

-

Topics in Chemistry and Drug Design

Renfroe, Ed.

was used to vinylate 2-bromothiophene and 3-bromo~yridine.~~

Pd (OAc) 5

Ar 2P 2

Reaction of 3-bromopyridine with ally1 alcohols under similar conditions affords carbonyl compounds which result from migration of the double bond.

0"'. -OH R

Mono-and dialkoxy-N,N-dialkylbenzamides can be lithiated oytho to the amide group by means of sec.BuLi in the presence of MezNCHzCH2NMez. In those cases where the alkoxy and amide groups are disposed 1,3, metallation occurs at the 2-positions. These organolithium reagents react with a variety of electrophiles. FORMATION OF CARBON-CARBON BONDS - The anion derived from the tosylate of CF3CH20H readily adds to ketones or nonenolizable aldehydes. Sequential treatment of the adduct with acid and base gives the substituted pyruvic acid.4 4

>.

'

0

[CF2=COTsLi]

%OH ~y

C=CF2

>-

>b,O,H

1

OTs An interesting method for formation of a-vinylene lactones relies on sulfur for activation of the a-position. Condensation of the anion of the thiocarbonate with a variety of aldehydes gives olefins in moderate to excellent yields with high stereoselectivity.

SC02Et The sulfoxide obtaiiiable in several steps adds the anion from diethyl malonate or ethyl cyanoacetate. The intermediate s u l foxide anion undergoes s ontaneous elimination to afford unusually substituted cyclohexenones.t 6

Alkylation of thiopyrrolidone with ethyl bromoacetate gives the thioether, which undergoes sulfur extrusion with carbon-carbon bond formation on treatment with (CgH5)3P and base.47

In an analogous reaction, open chain thioamides react with ethyl bromocyanoacetate (but not bromomalonate) to give the corresponding vinylidene derivatives

.

Lednicer

Reactions of Interest

Chap. 26

y2

S

RC=

273

,CN

c

R-C \NH* Br <:I2’Et* ‘C02Et 4-Arylisoxazolines, available from 1,3-dipolar addition of nitrile oxides to styrene^,^' can be alkylated by means of LDA. These readily afford 1,3-aminoalcohols on reduction. 5 0 +

0-N

Silyl enol ethers of carboxylic acid esters can be alkylated by means of (CH3)3CCl in the presence of ZnC12 to give a-butylated products.5’ The reaction seems strangely immune to steric hinderance. Silyl enol ethers of ketones undergo an analogous transformation. 5 2 R b ° C H 3 R+CO~CH~ ~ - 0 SiMe3 tBu

,

Thioesters add ethylmagnesium iodide to form a new organometallic reagent. This readily adds to carbonyl compounds to afford the corresponding hydroxyketones on hydrolysis. sEt@ l-R@O, C 6 H 5 7 s E t ---t c 6 H 5 y g ‘gl 2-H20 6 5 0 The anion from theschiffbase of cyclohexanone with optically active

t e r t i a r y butyl a-aminoesters can be alkylated to afford products which are 73-97% optically pure. 5 4

HETEROCYCLES - Isoxazoles can be formed regiospecifically in good yields from oximes by first acylating the dilithio salt with dialkylamides, followed by quenching of the adduct with H2S04. Stereochemistry of the oxime determines regiospecificity. R

Isothiazole-4-carboxylates are obtained starting from amides by first forming the oxathiazolone by means of ClCOSCl. Condensation with methyl acetylene dicarboxylate followed by hydrolysis and decarboxylation completes the sequence. 0

ArCNH2 II

+Arx)=O

1.MeO2CC=CCO Me 2.-co

>

2

Aluminum chloride catalyzed condensation of nitriles with diazoketones affords good yields of oxazoles. 5 7

A general synthesis of 2-amino-3-acylfurans involves acylation of the enolate from cyanoacetone with chloroketones.

Sect. VI - Topics in Chemistry and Drug Design

274 -

Renfroe, Ed.

0

ONa Condensation on the anion derived from the amino-enone with aldehydes or ketones gives the corresponding hydroxyketones. These close to dihydropyrones on treatment with MeOH*HCl.5 9

+1R&;

H

"Me

"Me2 - R

1 R &R

Condensation of N-methylaniline with terminally substituted a-bromo acetoacetates gives the corresponding acrylates. These can be cyclized photolytically to 3-hydroxyindolines. Treatment of the products with Ph (OAc)4 leads to the rearranged indolones.

-

Addition of dimsyl anion to coumarones leads to substituted chromones. These undergo Pummerer rearrangement to the latent aldehydes.

do

0

mxdz

&H2SO<

Ethyl d i e t h y l p h o s p h o n o a c r y l a t e s can be produced by either sulfur6 or selenium6 chemistry. These react with sali.cy1aldehydes to give dihydrobenzopyrones. Condensation with 2-hydroxycyclohexanone gives the hexahydrobenzofuran.

mC02Et f-

RCH4'OzEt PO (OEt)

2

A new synthesis for 2-substituted isoquinolines starts with o-toluonitrile. Base catalyzed acylation followed by ketalization gives the key intermediate. Reduction o f the amine followed by acid cyclization and dehydrogenation (12) gives the heterocycle. 6 4

CN

CH2NH2

Aminoacrylates readily couplewith diazonium salts to form transient imines. These cyclize spontaneously to cinnolines.

C

N CH=CHCE.+

c N + = C -C /COR \\

-0. R & .

+-,

N X=O ,CH2 I Ar -NH SYNTHONS - Sulfur chemistry has provided easy access to acetylenes. Re-

Chap. 26

Reactions of Interest

Lednicer

275

action of phenacylsulfides or their alkylation products with tosylhydrazine gives the corresponding hydrazones. Treatment with CH3Li gives the acetylenes. R ArkCHSCH3 ArCXR ArCbHSCH3 b-

?P

= -

,

fi-NHTS

Enol phosphates of a-ketosulfones provide the key intermediate to alternate schemes leading to acetylenes. Acylation of phenyl sulfones with esters affords the ketone derivatives directly. 6 7 Alternately, the ketones can be obtained by Moffat oxidation of the condensation products from aldehydes with sulfone anions. 6 8 Conversion to the enol derivatives followed by treatment with Na/NH3 or Na(Hg) gives the acetylenes. OR OPO (OEt) II I I RCCHSO~C~H~ R C = C - S O ~ C> -~, H ~ RCECR’ I R’ The recent development of dienes incorporating latent functionality has greatly expanded the utility of Diels Alder approaches to synthesis of complex molecules. Butadiene substituted by silicon at the 2-position is available from 1,4-dichloro-2-butyne by H2PtC12catalyzed reaction with Et3SiH followed by dehydrohalogenation (Zn). The product reacts readily with dienophiles.

>-

C1CH2C=CCH2C1

C1CH2CH=C(SiEt3)CH2C1

+CH2=CH-C=CH2 1

SiEt

3 Pyrolytic scission of cyclobutenes leads to a diene which promises to be useful in a Diels Alder approach to catechols. Heating of the silyl derivative of the acyloin product from dimethyl glutarate in the presence of dienophiles affords adducts of the transient diene. The adduct obtained with dimethyl acetylene dicarboxylate was converted in several steps to phthalic acid derivative. 7 0 O s i M e 3 2 0 ~ m O H

’

OSiMe Me02 OSiMe Me02 OH 3 3 A general procedure has been developed for the preparation of 1,4-dih y d r o t h i o p h e n e - 3 - c a r b o x y l a t e s starting from acetaldehyde-2-thiol. 7 1 The corresponding sulfones extrude SO2 on refluxing in toluene to give the substituted dienes. These readily condense with maleic anhydride.7 2

Nitromercuration of olefins followed by base catalyzed elimination of mercury gives the product of olefinic nitration. 1-Nitrocyclohexene obtained by this scheme readily undergoes conjugate addition of nucleophiles and serves as a dienophile.

a p 2

R=NH ,CH=CH 2 2

ax

p2 -+

NO

Y

276

Sect. VI

-

Topics in Chemistry and Drug Design

Renfroe, Ed.

Monoaeetals of quinones are accessible by Tl(N03)3 oxidation of hydroquinone monomethyl ethers. The unprotected carbonyl group can be condensed with anions to afford products which give substituted phenylacetic acid derivatives on aromatization.7 4 A versatile non-Friedel-Crafts approach to naphthols is provided by condensation of the toluic acid sulfoxide with substituted acrylates or methyl vinyl ketone. Sequential conjugate addition and acylation of the first formed anion leads to the tetralone. Pyrolysis leads to aromatization by l o s s of sulfinic acid. 7 5 SOC6H5 I

A related scheme which leads to hydroxytetralones involves reaction of the anion from a phthalide (LDA) with an unsaturated ester. This reaction too involves a conjugate addition-acylation sequence. 7 6

REFERENCES

1. A.I. Meyers, Accts.Chem.Res. g,375(1978). 2. D. Valentine Jr. and J.W. Scott, Synthesis 329(1978). 3. A.J. Fatiadi, Synthesis 241(1978). 4. A . J . Fatiadi, Synthesis 165(1978). 5. W. Opolzer and V. Snieckus, Angew.Chem.,Int.Ed.in Eng.2, 476(1978). 6. B.M. Trost, Chem.Revs. 363(1978). 7. E.J. Corey and A.K. Long, J.Org.Chem. 43, 2208(1978). 8. S.V. Kessar, Accts.Chem.Res. 11,283(1978). 9. H.C. Van Der Plas, Heterocycles 9 , 33(1978). 10. A. Hassner and V. Alexanian, Tetrahedron Lett. 4475(1978). 11. W.H. Rastetter, T.J. Richards and M.D. Lewis, J.Org.Chem. 43, 3163 (1978). 12. E. Vedejs, D.A. Engler, and J.E. Tetschow, J.Org.Chem. 43, 188(1978). 13. R.T. Arnold and S.T. Kulenovic, J.Org.Chem. 43, 3687(1978). 14. G.M. Rubottom and J.M. Gruber, J.Org.Chem. L x 1599(1978). 15. W.E. Fristad, T.R. Bailey and L . A . Paquette, J.Org.Chem. 43,1621(1978) 16. I. Ryu, S. Murai, Y. Hatayama and N. Sonoda, Tetrahedron Lett. 3455 (1978). 17. P. Dubs and R. Stussi, Helv.Chim. 61, 998(1978). 18. E.J. Corey and D.L. Boyer, Tetrahedron Lett. 2461(1978). 19. H. Bailey, D.N. Standing and J.R. Knowles, Tetrahedron Lett.3633(1978) 20. E. Brown,, J.-P. Robin and R. Dahl, J.Chem.Soc.Chem.Commun. 556(1978). 21. J. Gras, Tetrahedron Lett. 2954(1978). 22. D. Comins and A . I . Meyers, Synthesis 403(1978). 23. R.P. Woodbury and M.W. Rathke, J.Org.Chem. 43, 1947(1978). 24. A.I. Meyers, K. Tomioka and M.P. Flemming, J.Org.Chem. 43, 3788(1978). 25. S.F. Martin and T.A. Puckette, Tetrahedron Lett. 4229(1F8). 26. S. Hara, K. Kishimura and A . Sutuki, Tetrahedron Lett. 2891(1978). 27. S.F. Martin and T. Chou, Tetrahedron Lett. 1943(1978). 28. D.W. Patrick, L.K. Truesdale, S.A. Biller and K.B. Sharpless, J.Org. Chem. 43, 26280978). 29. T. Shioiri and N. Kawai, J.Org.Chem. 43, 2936(1978). 30. G. Barany and R.B. Merrifield, J.Am.Chem.Soc. 2, 7363(1977). 31. D. Seebach and T. Hassel, Angew.Chem.,Int.Ed. 17,274(1978).

s,

Chap. 26

Reactions of Interest

32. 33. 34. 35. 36. 37. 38.

Lednicer

277

B. Krzyzanowska and W.J. Stec, Synthesis 521(1978). L.C. Liu and P. Helquist, Tetrahedron Lett. 3423(1978). A.I. Meyers and B.E. Williams, Tetrahedron Lett. 223(1978). A. McKillop and D.P. Rao, Synthesis 759(1977). J. Grolinski and M. Makosza, Tetrahedron Lett. 3495(1978). R. Ranchers and A.S.-T. Lui, J.Am.Chem.Soc. 100, 4902(1978). T. Sugasawa, T. Toyoda, M. Adachi and K. Sazakura, J.Am.Chem.Soc. 100, 48420978). 39. G. Mulvey, A.M. Mulvey, A.M. DeMarco and L.M. Weinstock, Tetrahedron Lett. 1419(1978). 40. C.B. Ziegler, Jr. and R.F. Heck, J.Org.Chem. 43, 2941(1978). 41. W.C. Frank, Y.C. Kim and R.F. Heck, J.Org.CheE 43, 2941(1978). 42. Y. Tamaru, Y. Yamada and Z. Yoshida, JhOrg.Chem. 43, 3396(1978). 43. S.O. deSilva, J.N. Reed and V. Snieckus, Tetrahedron Lett. 5099(1978). 44. K. Tanaka, T. Nakai and N. Ishikawa, Tetrahedron Lett. 4809(1978). 45. K. Tanaka, N. Yamagishi, H. Uneme, R. Tanikaga and A. Kaji, Chemistry Lett. 197(1978). 46. T.A. Bryson, R.E. Davis and R.B. Gammill, Tetrahedron Lett. 743(1978). 47. H.W. Pinnick and Y.-H. Chang, J.Org.Chem. 43, 4662(1978). 48. H. Singh and C.S. Ghandi, Synthetic Commun. 8, 464(1978). 49. V. Jager, V. Bus and W. Schwab, Tetrahedron Lett. 3133(1978). 50. V. Jager and W. Schwab, Tetrahedron Lett. 3128(1978). 51. M.T. Reetz and K. Schwellnus, Tetrahedron Lett. 1455(1978). 52. M.T. Reetz and W.F. Maier, Angew.Chem.Int.Ed. 48(1978). 53. A.I. Meyers, T.A. Tait and D.L. Commins, TetraGdron Lett. 4657(1978). 54. S. Hashimoto and K. Koga, Tetrahedron Lett. 573(1978). 55. G.N. Barber and R.A. Olofsen, J.Org.Chem. 43, 3015(1978). 56. R.K. Howe, T.A. Gruner, L.C. Carter, L.L. Black and J.E. Franz, J.Org. Chem. 43, 37360978). 57. M.P. D G l e , M. Oppenhuizen, R.C. Elliott and M.R. Buelkins, Tetrahedron Lett. 2247(1978). 58. J.F. Blount, D.L. Coffen and D.A. Katonak, J.Org.Chem. 43, 3821(1975). 59. F.J. Vinick and H.W. Gschwend, Tetrahedron Lett. 315(19z). 60. A.G. Schultz and W.K. Hagmann, J.Org.Chem. 43, 4231(1978). 61. D. Connor, P.A. Young and D. V o n s t r a n d t m a n n F S y n t h e s i s 208(1978). 62. J. Ide, R. Endo, S. Muramatsu, Chemistry Lett. 401(1978). 63. W.A. Kleschick and C.H. Heathcock, J.Org.Chem. 43, 1256(1978). 6 4 . C.K. Bradsher and T.G. Wallis, J.Org.Chem. 43, 3816(1978). 65. C.B. Kanner and V.K. Pandit, Heterocycles 2, 1381(1978). 66. S . Kano, T. Yokomatsu, S. Hibino and S. Shibuya, Synthesis, 305(1978). 67. P.A. Bartlett, F.R. Green I11 and E.H. Rose, J.Am.Chem.Soc. __ 100, 4852(1978) 68. B. Lythgoe and I. Waterhouse, Tetrahedron Lett. 2624(1978). 69. D.G. Batt and B. Ganem, Tetrahedron Lett. 3323(1978). 70. D.R. Anderson and T.K. Koch, J.Org.Chem. 43, 2726(1978). 71. J.M. McIntosh and R.A. Sieler, Can.J.Chem. 56, 226(1978). 72. J.M. McIntosh and R.A. Sieler, J.Org.Chem. 43, 4431(1978). 73. E.J. Corey and H. Estreicher, J.Am.Chem.Soc. 100, 6294(1978). 74. D.J. Hart, P.A. Cain and D.A. Evans, J.Am.Chem.Soc. 100, 1548(1978). 75. F.M. Hauser and R.P. Rhee, J.Org.Chem. 43, 178(1978). 76. N.J.P. Broom and P.G. Sammes, J.Chem.Soc., Chem.Commun. 162(1978).

.

278 __

Annual Reports in Medicinal Chemistry-I4

C h a p t e r 27. New Methods i n H e t e r o c y c l i c Chemistry Edward C. T a y l o r Department of Chemistry, P r i n c e t o n U n i v e r s i t y , P r i n c e t o n , N . J .

08540

I n t r o d u c t i o n - The immensity of t h e l i t e r a t u r e i n h e t e r o c y c l i c c h e m i s t r y p r e c l u d e s a comprehensive t r e a t m e n t i n t h e few pages a l l o c a t e d t o t h i s chapt e r . A s a consequence, w e s u r v e y t h i s y e a r o n l y t h r e e areas of s y n t h e t i c i n t e r e s t - t h e i n t r o d u c t i o n and m o d i f i c a t i o n of s u b s t i t u e n t g r o u p s , new methods f o r r i n g a n n e l a t i o n , and s y n t h e t i c a l l y u s e f u l r i n g t r a n s f o r m a t i o n r e a c t i o n s - and w e w i l l r e s e r v e f o r f u t u r e y e a r s a d i s c u s s i o n o f r e c e n t advances i n s u c h a r e a s a s 1 , 3 - d i p o l a r c y c l o a d d i t i o n r e a c t i o n s , a p p l i c a t i o n s of D i e l s - A l d e r r e a c t i o n s , M i c h a e l a d d i t i o n s , and W i t t i g r e a g e n t s t o t h e c o n s t r u c t i o n o f h e t e r o c y c l e s , and t o p i c s s u c h as N-oxide c h e m i s t r y and t h e u s e of h e t e r o c y c l e s a s r e a g e n t s i n o r g a n i c s y n t h e s i s . S u b s t i t u e n t Group Chemistry - Numerous new methods f o r t h e i n t r o d u c t i o n and m o d i f i c a t i o n of s u b s t i t u e n t s c o n t i n u e t o a p p e a r , r e f l e c t i n g t h e f a c t t h a t t h i s a r e a of h e t e r o c y c l i c c h e m i s t r y c o n t a i n s many of i t s g r e a t e s t s y n t h e t i c c h a l l e n g e s . P y r i m i d i n e s may be brominated under n e u t r a l c o n d i t i o n s by t h e u s e of n i t r o n i u m t e t r a f l u o r o b o r a t e i n s u l f o l a n e . ’ N-Substituted imidazole2-chrboxamides a r e r e a d i l y formed by h e a t i n g i m i d a z o l e an$ l - a l k y l i m i d a zoles with isocyanates i n nitrobenzene o r diphenyl e t h e r . 2- and 4-Metho x y q u i n o l i n e s are c o n v e r t e d i n t o 2- and 4-bromo d e r i v a t i v e s ( r e p l a c e m e n t 0 o f -0CH by -Br) under n e u t r a l c o n d i t i o n s by h e a t i g a t 60-80 w i t h t h e 3 9 An e x t e n s i o n o f t h e complex formed from phosphorus t r i b r o m i d e and DMF. classical maionic ester s y n t h e s i s w i t h h a l o - s u b s t i t u t e d h e t e r o c y c l e s inv o l v e s t h e u s e of i s o p r o p y l i d e n e a l k y l m a l o n a t e s i n a c e t i c a n h y d r i d e ; d o u b l e d e c a r b x y l a t i o n of t h e p r o d u c t s l e a d s t o a l k y l s u b s t i t u t i o n o f t h e h e t e r o 2 A d i f f e r e n t c o n c e p t h a s been a p p l i e d t o t h e s y n t h e s i s o f d i m e t h y l cycle. 2 - t h i e n y l m a l o n a t e ; r e a c t i o n of t h i o p h e n e w i t h d i m e t h y l d i a z o m a l o n a t e i n t h e p r e s e n c e of R h ( I 1 ) a c e t a t e gave a n i n t e r m e d i a t e y l i d which w a s t r a n s f o r m e d i n t o t h e d e s i r e d product (an obvious f n t e r m e d i a t e t o thiophene-2-acetic a c i d ) on h e a t i n g i n e x c e s s t h i o p h e n e .

3-Substituted thiophenes (not r e a d i l y a v a i l a b l e , s i n c e both electrop h i l i c s u b s t i t u t i o n and m e t a l l a t i o n t a k e p l a c e a t p o s i t i o n 2) can b e prep a r e d from the r e a d i l y a v a i l a b l g 3-bromothiophene by Pd(OAc)2-catalyzed This p r o c e s s h a s b e n extended t o t h e reaction with a l l y l i c alcohols. as w e l l as a v a r i e t g p r e p a r a t i o n of 3 - a l k y l p y r i d i n e s from 3-bromopyridine, of v i n y l - s u b s t i t u t e d h e t e r o c y c l e s from the c o r r e s p o n d i n g bromo p r e c u r s o r s .

7

Copyright @ 1979 by Academic Press, Inc. All rightr of reproduction in any form resewed. ISBN 0-12-040614-8

Chap. 27

New Methods in Heterocyclic Chemistry

Taylor

279

3-Substituted furans, which are equally difficult to prepare by direct substitution into the 6uran nucleus, can be obtained by a variety of novel cyclization reactions, or by acid-catalyzed isomerization of the cyc adducts (oxetanes) obtained by photoaddition of furan with aldehydes.$8;11

[g

QH

+ RCHO

Pyrimidyl aldehydes and ketones are conveniently prepared by the reaction of methyl-substituted derivatives with alkyl nitrites; the resulting aldoximes can be converted by hydrolysis to aldehydes, or by dehydration to nitriles, which can be further converted to ketones with Grignard gents, or hydrolyzed and decarboxylated (replacement of -CH3 by -H). lZ:f? The introduction of substituent groups into protonated heterocyclic bases by freT4rqgicals, pioneered by Minisci and well reviewed over the past few years, continues tpgbe actively explored; fy6ther studies on alkylations,”’ l8 acylations, and carboethoxylations have been reported. Various substituents (e.g., phenylsulfonyl, acetyl, phenylmercapto, halogen) are readily displaced by an ips0 substitution process from the 2-pqfition of benzothiazole by the nucleophilic 1-adamantyl and acetyl radicals. Extension of these substituent group interchange processes to other heterocyclic systems could be of considerable synthetic utility. Although the classical method for the preparation of thiolactams involves reaction of lactams with P S in high boiling solvents, a room tem5 perature procedure involving addigion of Et 3N to a suspension of P S and the lactam in acetonitrile i52effective even with sterically hinde?ea secondary and tertiary amides. Phase traqgfer catalysis has been recommended for specific S-alkylation of thiolactams, while specific N-vinylation (of pyridine-2(1H)-thione) has been achieved by reaction with a-halo-cx,@-unsaturated carbonyl compounds, followed by thermal ring opening (with l o s s of 24 C 0 2 ) of the resulting 3-carboxydihydrothiazolo(3,2-a)pyridinium derivatives.

A general procedure for the introduction of substituents in heterocycles is displacement of a good leaving group by an appropriate nucleophile. The most reactive substituent towards displacement by nucleophiles appears to be methylsulfonyl (readily prepared by oxidation of a methylthio subsubstituent); the trimethylammonium group is less reactive, but it is still some 7C)9-1000 times a better leaving group than a chloro or a methylthio group. Although alkylsulfonyl groups are more readily displaced than chloro groups by primary amines or ammonia, secondary and aromatic amines preferentially displace the latter (presumably because of steric hindrance due to a comb3gation of the size both of the alkylsulfonyl group and the nucleophile). Reaction products should be examined with care in such

280

Sect. VI

- Topics in Chemistry and

Drug Design

Renfroe, Ed.

systems, however, *sincemigration of alkylsulfonyl ggmygs under conditions of nucleophilic displacement occurs with some ease. One must also be constantly alert to the intervention of cine or substitution processes (emergence of the attacking nucleophile at a position different from that occupied by the "displaced" leaving group), and to the ubiquitous ANRORC reactions in which ring-opening, ring-closure reactions can introduce many complications (exchange o f endocyclic for exocyclic heteroatoms, Dimroth-type rearrangements, ring feterconversions) into apparently straightforward "displacement" reactions. A substituent which appears to be even more readily displaced by nucleophiles than alkylsulfonyl groups is 0-mesyl (OSO CH ) . Conversion of 3 a cyclic lactam to its 0-mesyl derivative is accompl$shed by treatment with methanesulfonyl chloride/Et N in methylene chloride solution; disp3gcement 3 This takes place readily with dimethylamine (15 min at 20' in dioxane). may prove to be a generally useful procedure for replacement of lactam oxygen by other nucleophiles without resorting to intermediate chloro compounds. Note that 2-trifluoromethanesulfonyloxypyridine (from 2(1H)-pyridone, NaH, and trifluoromethanesulfonyl chloride) is an effective reagent for th52facile acylation of activated aromatic compounds with carboxylic acids.

OMe s

R

R

QB The methylamino grouping in Librium is by initial nitrosation to give the N-nitroso with methylhydrazine ( l o s s of diazomethane); hydrolysis/chlorination seqy5nce which would tionality in the substrate.

CR

ArH

ArCOR

easily displaced by nucleophiles derivative, followed by reaction this procedure avoids the usual have affected the N-oxide func-

NO

Some nucleophilic displacement reactions (particularly those involving ketone enolates and other carbon nucleophiles) proceed routinely in low yield, with poor material balance. There is increasing evidence that these reactions are actually radical chain processes catalyzed, in principle, by a single electron transfer from the anion to the heterocycle. In such cases

Chap. 27

New Methods i n H e t e r o c y c l i c Chemistry

Taylor

281

d e l i b e r a t e a d d i t i o n of a good o n e - e l e c t r o n t r g t s f e r a g e n t , o r i r r a d i a t i o n , might have a s i g n i f i c a n t e f f e c t on t h e y i e l d . An i n g e n i o u s p r o c e d u r e f o r r e p l a c i n g s u l f u r i n a t h i o l a c t a m by a -CH COR group i n v o l v e s a l k y l a t i o n of s u l f u r w i t h an a-haloketone, followed by s u l z u r e x t r u s i o n e i t h e r w i t h t r i p h e n y l phosphine o r by h e a t i n g i n DMF (with o r w i t h o u t added sodium e t h o x i d e ) . Thej5reaction h a s been s u c c e s s f y k l y a p p l i e d t h u s f a r t o i s o q u ~ q o l i n e - l ( 2 H )- t h i o n e , quinazoline-4 (3H)-thione, and A c l o s e l y r e l a t e d intramolecular s u l f u r displacement 6-mercaptopurine. r e a c t i o n w i t h i n t r i g u i n g s y n t h e t i c p o t e n t i a l i s i l l u s t r a t e d by t h e c o n v e r s i o n of t h e S - a l k y l a t i o n p r o d u c t from 6 - c h l o r o p u r i n e r i b o n u c l e o s i d 5 8 a n d c y s t e i n e t o t h e i s o m e r i c N-alkylated p r o d u c t by t r e a t m e n t w i t h a l k a l i .

NH2

OH-

R

R

An e f f i c i e n t two-step p r o c e d u r e f o r t h e i n t r o d u c t i o n of an a c e t i c a c i d s u b s t i t u e n t i n t o t h e 4 - p o s i t i o n of 2-methyl-1(2H)-isoquinolinone i n v o l v e s r e a c t i o n w i t h e t h y l d i a z o a c e t a t e i n t h e p r e s e n c e of a copper c a t a l y s t t o g i v e a m i x t u r e of exo and endo a d d u c t s , which are t h e n isomerized t 3g4carboethoxymethyl-N-methyl-2(1H)-isoquinolino~~ w i t h e t h a n o l i c H C 1 . Uracil The cyclopropane a d d u c t from 5 - a c e t i c a c i d s could b e p r e p a r e d a n a l o g o u s l y . 2-methyl-1(2H)-isoquinolinone and d i c h l o r o c a r b e n e c a n b e r i n g - c o n t r a c t e d t o a n i s o i n d o l e , o r ring-expanded t o a benzazepinone The r e a c t i o n of c a r b e n e s w i t h 40 o t h e r h e t e r o c y c l i c enamides h a s been reviewed.

t- BuO

n ' "Ln3

t-BuOH/EtjN

A s e l e c t i v e p r o c e d u r e f o r t h e r e d u c t i o n of s u b s t i t u e n t a z i d o groups ( a v a i l a b l e by displacement of l e a v i n g groups by a z i d e a n i o n ) t o amino groups, w i t h o u t concomitant r e d u c t i o n of o t h e r r e d u c i b l e groups, i n v o l v e s an aza-

282

Sect. VI

-

Topics in Chemistry and Drug Design

Renfroe, Ed.

41 transfer reaction with acetylacetone.

, l~ k~e n ~ l , and ~~ A ve atile procedure for the introduction of a l k ~ l a 69 epoxyalkyl groups into heterocycles, which involves displacement of leaving groups by Wittig reagents, followed by appropriate manipulation of the resulting ylides, has been applied to the synthesis of various 6-substiE~ted purines designed as inhibitors of adenylsuccinate synthetase and lyase. Ring Annelation Reactions - Many recent developments in synthetic methodology in heterocyclic chemistry have dealt with new methods for annelation of a heterocyclic ring unto a pre-existing ring system. A procedure for exclusive ortho substitution of aromatic amines by (2,3)sigmatropic rearrangement of ylides derived from N-arylazasulfonium salts has been applied to the preparation of indoles, oxindoles, N-methyl-2(1H)-quinolone, 3,4-dihydro-Nz5 methyl-2(1H)-quinolone, isatins, and various condensed indole derivatives.

o-;,,, Et3N

NCS

1then H' R

2-Diethylaminoquinolines are prepared in a single step from acetanilides and phenyl N,N,N',N'-tetraethylphosphorodiarnidahg (from POCl3 by reaction Treatment of m-methoxy or mfirst with phenol, and then with diethylamine). methylacetanilides with the Vilsmeier reagent can be directed t o give either 2-chloroquinolines or 2-chloro-3-formylquinolines; both are ver$9 tile intermediates for further substitution or ring annelation reactions.

2-Arylbenzoxazoles and 2-arylbenzothiazoles are prepared by treatment of a,N-diarylnitrones with 0-methyl diphggylphosphinothioate and phenylphosphonothioic dichloride respectively.

New Methods in Heterocyclic Chemistry

Chap. 27

Taylor

283 -

Addition of thiolacetic acid to acylated p-benzoquinone imines (from oxidation acylated p-phenylenediamines with Pb(0Ac) ) , gives 6-aminobenzo4 thiazoles.

$8

An ingenious procedure for the synthesis of 5H-pyrrolo(3,2-d)pyrimidines involves treatment of a 6-methyl-5-phenylazopyrimidine with t-butoxybis(dimethy1amino)methane or an orthoformate ester, followed by hy96ogenolytic ring contraction of the resulting pyrimido(4,3-~)pyridazine.

0

0

0

By contrast, treatment of 1,3-dimethyl-6-amino-5-arylazouracils with orthoformate esters51followed by reduction with dithionite, yields 8-arylaminotheophy3iines. Many a$j;$onal syntheses of coy$ensed pyrimidine36 5-deazaalloxazines, 5-deazaflavins, azapter idin (p teridines, 57,) involve treatment of other 6-amino and 6-hydrapyrimid0(4,5-~)pyridazines zinopyrimidine derivatives with formylating agents such as DMF acetal or orthoformate esters. An alternate route for the annelation of a pyrrole ring employs the base-catalyzed condensation of furan-, thiophene-, and selenophene-carboxald ydes with ethyl azidoacetate; the bicyclic products are indole isoster5b The isomeric (3,2-b)pyrroles can be similarly prepared from the correses. ponding 2-carboxaldehydes.

H

284

Sect. VI

-

Topics in Chemistry and Drug Design

Renfroe, Ed.

Synthetically Useful Ring Transformation Reactions - Many useful syntheses involve the deliberate transformation of one (presumably more readily available) heterocyclic system into another by a ring contraction, ring expansion, or ring transformation reaction. For example, 4-aminoantipyrines are readily expanded to 5-a no-4(3H)-pyrimidones with NaH, NaNH2, NaOH or NaOEt in refluxing xylene. By contrast, 2,6-disubstituted 4-arylamino-5-cyanopyrimi-

Y4

CH3

base

v

Ph dines undergo ring contraction in high yield w h zinc and acetic acid to give 2,5-disubstituted 3-arylamino-4-cyanopyrroles.

Zn/HOAc

NC

NHAr

____f

H 4-Methoxycoumarin reacts with a number of carbon nucleophites via a ring opening, ring-closing sequence to give 2-substituted chromones. The same

0conceph2has been widely exploited for the conversion of pyrimidines to pyridines

.

A novel pyrimidine-to-pyrimidine conversion exchanges N -C -N of uracil derivatives for a N-C-N fragment of an attacking nucleAphZle! 1,3g3 dimethyluracil, for example, is converted to isocytosine with guanidine. The reaction has unfortunately not yet been successfully applied to fused uracil derivatives.

0

3,4-Diaminofurazan has been elgeloyed as a a synthesis of 2,3-diaminopyrazine; reductive ring @&gds a concept previously employed pteridines,67 and azapteridines,6f'g8 ines,

0

latent tetraaminoethylene in cleavage of the fused furazan the preparation of adenof utilizing the furazan

Chap. 27 ring as

a

New Methods in Heterocyclic Chemistry

Taylor

285

protected o-diamine functionality.

A related synthesis of fused pyrazines employs 3,4-diaminothiophenes (ingeniously prepared from pyridine as depicted below) as latent enediamines; condensation with 1,2-dicarbonyl compounds gives thieno(3,4-b)pyrazines which would appear to $5 potential intermediates to novel pyrazines by reductive desulfurization.

- '3Me2NH HC 1

Q

+ CH2 ' CN

N\ 1

EtsN

@$ Q S C H s

then CHsI

H2N

C2H50

I

I

NC- C= C,

J

S-Na'

NC-

The above example in which a pyridinium ring serves as a latent primary amino substituent is an extension of the classical "Zincke salt" procedure for transfer of the five carbon atoms of the pyridinium ring to an attacking nucleophile. A further example is the conversion of pyridine into 1-substituted 3-formyl-2(1H)-pyridinethiones by reaction of glutacondialdehyde (the hydrolytic ybeavage product of the Zincke salt formed from pyridine) with isothiocyanates.

286

Sect. VI

- Topics in Chemistry and Drug Design

Renfroe, Ed.

REFERENCES 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11.

12. 13.

14. 15. 16. 17.

18.

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

G.-F. Huang and P. F. Torrence, J.Org.Chem., 42, 3821 (1977). E. P. Papadopoulos, J.Org.Chem. , 42, 3925 (1977). T. Yajima and K. Munakata, Chem.Lett., 891 (1977). A. Scoville and F. X. Smith, J.Heterocycl.Chem., 3, 1081 (1977). (a) R. J. Gillespie, J. Murray-Rust, P. Murray-Rust, and A. E. A. Porter, J.Chem.Soc.,Chem.Commun., 83 (1978); ( b ) R. J. Gillespie, A. E. A. Porter, and W. E. Willmott, J.Chem.Soc.,Chem.Commun., 85 (1978). Y. Tamaru, Y. Yamada, and Z. Yoshida, Tet.Lett., 3365 (1977). Y. Tamaru, Y. Yamada, and 2. Yoshida, J.Org.Chem., 43, 3396 (1978). W. C. Frank, Y. C. Kim, and R. F. Heck, J.Org.Chem., 43, 2947 (1978). K. Inomata, S. Aoyama, and H. Kotake, Bull.Chem.Soc.Japan, 51, 930 (1978). A. Zamojski and T. Kozluk, J.Org.Chem., 42, 1089 (1977). T. Kitamura, Y. Kawakami, T. Imagawa, and M. Kawanisi, Syn. Commun., 7, 521 (1977). T. Sakamoto, S. Konno, and H. Yamanaka, Heterocycles, 6, 1616 (1977). H . Yamanaka, H. Abe, T. Sakamoto, H. Hiranuma, and A. Kamata, Chem. Pharm.Bull., 25, 1821 (1977). F. Minisci, Synthesis, 1 (1973). F. Minisci and 0. Porta, Adv. Heterocyclic Chem., 2, 123 (1974). F. Minisci, Topics Current Chem., 62, 1 (1976). T. Caronna, A. Citterio, T. Crolla, and F. Minisci, J.Chem.Soc., Perkin I, 865 (1977); Tet.Lett., 1731 (1976). H. Itokawa, S. Kameyama, T. Inaba, T. Tazaki, R. Haruta, Y . Kawazoe, and M. Maeda, Chem.Pharm.Bull.Japan, 26, 1015 (1978). T. Sakamoto, T. Sakasai, and H. Yamanaka, Heterocycles, 9, 481 (1978). R. S. Min and V. S. Aksenov, Khim. Geterotsikl.Soedin, 2T4 (1976). M. Fiorentino, L. Testaferri, M. Tiecco, and L. Troisi, J.Chem.Soc. Chem.Commun., 316,317 (1977). C. S. Rao, M. P. Dave, P. N. Mody, and A. D. Pandya, Indian J.Chem. B, 14B, 999 (1976). H. J.-M. DOU, P. Hassanaly, J. Kister, and J. Metzger, Phosphorus and Sulfur, 3, 355 (1977). G. A. Ulsaker and K. Undheim, Acta Chem.Scand. B, 31, 917 (1977). G. B. Barlin and A. C. Young, J.Chem.Soc., Perkin I, 1269 (1972) and references cited therein. T. Sawayama, R. Yamamoto, H. Kinugasa, and H. Nishimura, Heterocycles, 8, 299 (1977). J. M. Sprague and T. B. Johnson, J.Am.Chem.Soc., 58, 423 (1936). Y. F. Chi and Y. C. Ling, Sci.Sinica, 1, 205 (1956); 5, 633 (1957). 2. Budesinsky and J. Vavrina, Coll.Czech.Chem.Comun., 37, 1721 (1972). H. C. van der Plas, Accts.Chem.Res., 11,462 (1978). P. K. Bridson, W. Markiewicz, and C. B. Reese, J.Chem.Soc.Chem. Commun., 447 (1977). T. Keumi, H . Saga, R. Taniguchi, and H. Kitajima, Chem.Lett., 1099 (1977). A. Walser and R. A. Fryer, J.Org.Chem., 40, 153 (1975). J. A. Zoltewicz, Top.Curr.Chem., 59, 33 (1975). 571 (1976). H. Singh and K. S. Kumar, Indian J.Chem., ,&I H. Singh, S. S. Narula, and C. S. Gandhi, Tet.Lett., 3747 (1977). H. Vorbruggen and K. Krolikiewicz, Angew.Chem.Intern.Ed.Engl., 15,689 (1976).

Chap. 27 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.

New Methods in Heterocyclic Chemistry

Taylor

287

S. Kirkman and R. Wolfenden, J.Am.Chem.Soc., 100,5943 (1978). H.-P. Soetens and U. K. Pandit, Heterocycles, S , 181 (1977). U. K. Pandit, Heterocycles, 8, 609 (1977). B. Stanovnik, M. Tisler, S. Polanc, and J. Zitnik, Synthesis, 491 (1977). E. C. Taylor and S. F. Martin, J.Am.Chem.Soc., 96, 8095 (1974). E. C. Taylor, M. L. Chittenden, and S. F. Martin, Heterocycles, 1,59 (1973). W. J. Wanner, J. J. M. Hageman, G. J. Koomen, and U. K. Pandit, Rec.Trav.Chim. Pays-Bas, 97, 211 (1978). 42, 3240 (1977) and P. G. Gassman and W.N. Schenk, J.Org.Chem., previous papers in this series. E. B. Pedersen and D. Carlsen, Synthesis, 890 (1977). 0. Meth-Cohn and B. Narine, Tet.Lett., 2045 (1978). R. Nagase, T. Kawashima, M. Yoshifuji, and N. Inamoto, Heterocycles, 8, 243 (1977). W. B. Manning and V. Horak, Synthesis, 363 (1978). R. S. Klein, M.-I. Lim, S. Y.-K. Tam, and J. J. Fox, J.Org.Chem., 43, 2536 (1978). K. Senga, M. Ichiba, H. Kanazawa, S. Nishigaki, M. Higuchi, and F. 5 . , 641 (1978). Yoneda, J.Heterocycl.Chem., l F. Yoneda and M. Higuchi, J.Chem.Soc.Perkin I, 1336 (1977). M. Ichiba, S. Nishigaki, and K. Senga, J.Org.Chem., 43, 469 (1978). M. Ichiba, K. Senga, S. Nishigaki, and F. Yoneda, J.Heterocycl.Chem., 14, 175 (1977). K. Senga, K. Shimizu, S. Nishigaki, and F. Yoneda, Heterocycles, 6, 1361 (1977). K. Mori, K. Shinozuka, Y. Sakuma, and F. Yoneda, J.Chem.Soc.,Chem. Commun., 764 (1978). S. Nishigaki, M. Ichiba, J. Sato, K. Senga, M. Noguchi, and F. Yoneda, Heterocycles, 9, 11 (1978). S. Soth, M. Farnier and C. Paulmier, Can.J.Chem., 56, 1429 (1978) and papers cited therein. T. Ueda, N. Oda, and I. Ito, Heterocycles, 8, 263 (1977). S. Robev, Tet.Lett., 1163 (1978); Dokl.Bolg.Akad.Nauk, 2, 197 (1978). D. T. Connor, P. A. Young, and M. von Strandtmann, Synthesis, 208 (1978). T. Higashino, Y. Iwai, and E. Hayashi, Chem.Pharm.Bull.Japan, 25, 535 (1977) and references cited therein. K. Hirota, K. A. Watanabe, and J. J. Fox, J.Org.Chem., 43, 1193 (1978). N. Sat0 and J. Adachi, J.Org.Chem., 43, 341 (1978). E. C. Taylor, G. P. Beardsley, and Y. Maki, J.Org.Chem., 36, 3211 (1971). G. D. Hartman, S. E . Biffar, L. M. Weinstock, and R. Tull, J.Org.Chem., 43, 960 (1978). E. C. Taylor, S. F. Martin, Y. Maki, and G. P. Beardsley, J.Org.Chem., 38, 2238 (1973). E. C. Taylor and A. J. Cocuzza, J.Org.Chem., in press. Y. Tominaga, H. Fujito, Y. Matsuda, and G. Kobayashi, Heterocycles, 5, 1871 (1977). J. Becher, E. G. Frandsen, C. Dreier, and L. Henriksen, Acta Chem. Scand. B , 31, 843 (1977).

288 -

Annual Reports in Medicinal Chemistry-I4

Chapter 28.

Synthetic Approaches to Anthracycline Antibiotics

T. Ross Kelly, Department of Chemistry, Boston College Chestnut Hill, MA 02167

-

Introduction During the past decade, the anthracyclines, most notably Adriamycin (la, doxorubicin), daunomycin (&, daunorubicin) and carminohave emerged as important chemothera eutic agents for the mycin treatment of a broad spectrum of human cancers.P While la, &, and 3a are presently produced by fermentation, the challenge of developing practical, totally synthetic routes to these molecules, coupled with the quest for superior analogs, has stimulated intense activity on the part of the synthetic community. More than a score of anthracyclines occur naturally,l but apart from early syntheses of the fully aromatic aglycones,l nearly all work to date has been conducted in the context of la3a. Accordingly, this survey will focus primarily on efforts in this area,with emphasis being placed on general strategies rather than operational details. Of necessity, discussion of the preparation of analogs which are actually (or formally) derivable from la-3a is deemed beyond the scope of this review, as is the synthesis of analogs whose structures are only vaguely similar to la-3a. 1 ,X=OCH3, Y=OH y 2,X=OCH3, Y-H

(s),

-

a series (mycins), Z= L-daunosamine (a-anomer)

b series

3,X=OH, Y=H 4,X=Y=H 5,X=H, Y=OH

6, L-daunosamine

(mycinones), Z=OH

Like Gaul, the synthesis of the anthracyclines is divided into three parts: construction of the aglycones (e.g.,adriamycinone, &, daunomycinone, 2, or carminomycinone, 3&), synthesis of the sugar 6 and coupling of the two. residue -

-

Aglycone synthesis The principal synthetic challenges posed by the aglycones include generation of the tetracyclic skeleton, introduction of the A-ring functionality and achievement of the "correct" (i.e., natural) regiochemical juxtaposition of the substituents in the A-and Drings. The demonstration 2-4 that the natural, cis orientation of the 7-and 9-hydroxyl groups is thermodynamically preferred (%6:1 ratio) and that epimerization of the C-7 position is effected by CF3COOH has attenuated the need to address aglycone stereochemistry directly. The recent the 4-demethoxy analogs of & and la,are ca. finding 5-8 that 4a and ten times as potent as &and 2 (they are also more toxic) may well have diminished the practical need for finding an efficient solution to the problem of regiochemistry. Nonetheless, efforts to achieve regiochemically controlled routes have led, inter alia, to a number of new methods of anthraquinone synthesis. Although many of these solutions remain to be applied to the preparation of lb-5b themselves, the underlying con-

*,

Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form resewed. ISBN 0-12-040514-8

Chap. 28

Anthracycline Antibiotics

Kelly

289

cepts have become inextricably intertwined with anthracyclinone synthesis and will be included where appropriate. Interconversions among 1-2 have been developed. Introduction of the C-14 hydroxyl [2a+la,' 2b-tlP (87%), -3(Y=H+Y=OH) ; l o i+5-l11)2 ] has been effected via the C-14 halo derivatives. Demethylation of 2b to 3b (A1C13, %100%)3,13 and meth~lationl~ of (+I-* to (+)-2b (CH2N2,45%) are recorded. Degradation of to 2,l5,l6 also 1 and 2. permitspreparation of l4C-labe1led In large measure syntheses of aglycones have relied on the employment of one (or more) of three general reaction classes: Friedel Crafts alkylations or acylations, nucleophilic condensations, and Diels Alder reactions;l7 the various routes are conveniently discussed under these three headings. (Note: unless otherwise indicated, all compounds described in the section on aglycone synthesis are racemic.) a. Friedel Crafts-type Routes - The first total synthesis of (*)-dauno- was reported in 1973 by Wong and coworkers (Eq. 1) , 2 and was mycinone (2b) (Eq. 1)

n

OH

0

a c series-4-OCHg

0

Z

9c ,Z=H 1G,Z=Br

patterned after their earlier synthesis of k(Z=OCH3) ,I8 as well as model studies by Goodman and colleagues. l9 Although the original sequence(Eq. 1) suffers from a low overall yield (since improved upon14) and lacks regiochemical control, the selective bromination (NBS) of C-7 (and not C-10attributed to steric hindrance) and subsequent R' conversion to 7-hydroxylhave been used in numerous other routes. A refined version of this approach, y 5 employing phthalic anhydride and 11 ,R=H,R'=CH 12, R=CH3, R ' = a (RI2*-(-)-7, provides 7(S) , 9 ( S ) - a in 20% overall yield rbased on (R)-l]. Analogs -11-1S6 (and 1 3 , R 4 1 , R ' =H have been similarthe corresponding glycosides) 14 ,R=H,R'=Cl 15,R=H,K'=-(CH=CH)2ly prepared. In an attempt to overcome the lack of regiochemical control in the Wong route, Kende et a1.21 prepared 16. Photo-Fries rearrangement of 16 proceeded regiospecifically (48% yield, based on recov-

A

OH

16

17, 2-OH, 21, Z-H

18,Z=O; 19, Z=H, OH; %,Z=H2 further elaboration, 17. Introduction of

ery of 53% 16) to give, after the C-9 oxygen was not reported. A similar approach was examined by Sih

290

Sect. VI

-

Topics in Chemistry and Drug Design

Renfroe, Ed.

and coworkers22 who prepared 18 regiospecifically by selective acylation of the non-hydrogen-bonded hydroxyl of the corresponding tetralone. Attempts to effect a Fries rearrangement and subsequent cyclization failed with 18 and 2. Tetracyclic material was achieved with 20, but regiospeciEcity was lost: a 3:2 mixture of 21 and its regiomer was produced in 37% yield. Conversion of 21 to 8c (50%) was accomplished by epoxidation of the enol acetate dericd f r G z . F. Johnson et al.23 have recently developed a regiospecific synthesis of 2 in 20% overall yield from 22 21 (via 8c) is ca. 25%.3,4*2223 via 24. The yield of (*)-2b- from and -

Ro \

(J-f:::: k:::* /

OOCH3

Br

OEt CH3 24 CH30 22 OEt 23 b. Nucsophilic Routes Thesearch for effective solutions to aglycone regiochemistry has led to the development of a number of new methods for the construction of anthraquinones which involve anionic species in the regiochemically determining step. One of the more thoroughly implemented approaches is due to Swenton and associate^.^^ Thus, 26, available in high yield from 25 in two steps (electrochemical methoxylation and transmetalation) , reaxs with dimethyl 3-methoxyphthalate to give 27 regiospecifically. Elaboration of ?7 gives 2 in an overall yield of 14% based on 3-bromo-2,5-dimethoxybenzaldehyde, the precursor to 25. A re-

-

. Li

OCH3

25

OCH3

-

28

lated approach, which does not address regiochemistry, involves the reaction of 28 with dimethyl ~ h t h a l a t e . ~The ~ use of ortho-lithiated benzamides forthe regiospecific construction of anthraquinones (e.g. 29+30u 31) was reported virtually simultaneously from the laboratories of B a l d ~ i n , ~ ~ R a p h a eand l , ~Snieckus,28 ~ but the methods remain to be extended to anthracycline synthesis. OL i I

OCH

1

3

w

OOCH3

\

/

CH,OOC QC00CH3 CH30 0 OCH3 31 OCH3 30 Kallmerten?' and Kende and coworkers3 have examined J I the conjugate addition of and 33 to a number of cyclohex-1-ene esters. Subsequent acylation of the p r o C-ring with the ester moiety and hydroxylation Q to the nitrile (the resulting cyanohydrin emerges upon decomposition as the C-11 oxygen) provide, in due course,% (from 32) and 35 (from 33) regiospecifically; 35 has been converted to (2)-daunomycinone -I

-

32

Chap. 28

Anthracycline Antibiotics

Kelly

291

n CH30 R CH30 34 35 32, R=H ;3 3 ,R=OCH3 (2)in ca. 20% yield (see below). A related reaction, that of 36 with pent-2-enal and NaH, provides the rhodomycinone analogs 37 in 50% yield.32 The preparation of several rhodomycinones from 35 has also been described& Hauser and Rhee33 and Kraus and Su-jim~to~~ haveutilized the conjugate addition of phthalide anions (=,X=CN or S02Ph) to unsaturated ketones for the preparation of naphthalene diols and anthraquinone~~~ via 2.3 5 COOCH3 m t @ @ R '

\

/

\

/

\

\

I

O '

R"

0

OH OH OH R 02 37 36 38 39 In a different approach,36 treatment of 40 (R=H or CH3) with n-C4HgLi gives 41 (55%) via 42; analogous treatment ofFhe anthraquinone corresponding to-g does not result in cyclization. While 41 is obtained quantita~~ acid nor base induces cyclitively from 43 $R=H) with ( C F ~ C O ) Z O ,neither zation of 44 (R =H) or 45.37 The less direct route involving base-catalyzed cyclzation of the leuco derivative of 44 (R2=H) gives 46a after aerial oxidation; the seemingly comparable conversion 44 (R2=OCHg)+leuco+ 0

n

n

\

l'x

\

/

COR' R2 0 OH ~ ~ , R ~ = o cx=-OCH H ~ , 2CH2o41 40,X=Br; G,X=Li; 43,X=H 45,R1=OH, R2=OCH3, X=O 47a virtual1 fails (
OCH3 0

Y

n

48 ,R1=OH,R2=R3=H ~2 ~ , R ~ = o H ,

R2=R3=-(CH 2) 6 ~3 50,R1=OH,R3=H R ~ = (-C H )~4~~

-

R1 0 O H Z R OH 51 ,R1=H,R2=CH2COOMe OH 52 46,R=H; 47,R=OCH3 R3=CH2CH2COCH3 a,Z=O Z=H2 Gtherland and colleagues38 have shown that Marshalk alkylation of 48 (which has been prepared on a 150kg scale)39 with a variety of alde-

;b,

292

Sect. VI

-

Topics in Chemistry and Drug Design

Renfroe, Ed.

hydes can be directed to either the 2 or 3 position with high regioselectivity. The annulated derivative 49 can be prepared either directly from 48 (50%) by reaction with succindialdehydew)or from 5038 (by oxidation and intramolecular Marshalk alkylation) The promise t h z this route holds for regioselective anthracyclinone synthesis is partly exemplified4 by the Triton B-induced conversion of 51 (prepared by successive Marshalk alkylations of quinizarin) to 2 (26%), an analog of c-rhodomycinone. The unusual hydroxylation at C-7 may involve a quinone methide."

.

-

c. Diels Alder Routes The power of the Diels Alder reaction for the expeditious construction of complex carbocycles has not escaped the notice of practitioners of

A:

1-9

I[ !f

;A;

;n;

;I:;]

D C B A , ; v #v; ; :w:;v; I l l OCH3 fe b a 53 56a,R=H;H,R=OCH3 ble constmctions implied by'the vertical lines in 53, six (a-f) have appeared in at least one guise. Unfortunately, however, although Diels Alder routes have led to several efficient aglycone syntheses, they have not, except in one case (below), solved the regiochemical component of the involves the quinizarin quiproblem. The most investigated route A serious complication to this approach is that many dienes nones (e.g. 57 and 58) add preferentially to the "wrong" double bond of 56?44-48 Although several indirect solutions to this diffiGlty OH have been devised [protecting the C-4a,lOa double bond of ' 4 9 4 9 retaining the pro D-ring in latent form use of quinizarin diboroacetate as a dienophileS0 (quinizarin itself does not exhibit dienophilicity)'I], the most direct solution has been to manipulate the substituents in OCH3 OH the diene to promote addition at C-2,3. At this writing three groups have developed dienes which meet this re59 quirement and lead to complete syntheses of (f)-2b or (f)-4b. The first solution, which is also the first reported D i e G Alderbasersynthesis of (*)-daunomycinone (%), is due to Kende et al.3 It emto give, after hydrolysis and tautomerizploys 2-acetoxybutadiene and ation, 35 in 25% overall yield; the regioisomer is produced in similar amount. Reaction of 35 with HCECMgBr followed by hydration gives _sr (40%) which is hydroxylatedat C-7 by an extension of the methods of Goodman and Wong (Br2, hv; AgOOCCF3; CF3COOH) to give (*)-2b (50%). Conversion of 35 to 8c via 60, and thence to was simultaneouay reported by Henry et al.T Whenapplied to %I2 the Kende sequence affords fi in 36% overall Reaction of 56a with 62 yield; C-7 a-hydroxylation (45%) ives (?)-4b.I2 gives 63e8 (comparrthe reaction of 58) which is R2 converzd to 64 in four steps (40% from -1. Surprisingly, attempts to hydrate the double bond of 64 to give (+)-4b directly have failed, but reduction (H2,Pd/C)to 61 provides a less R3 0 OH direct alternative. Chemists at Searle46 have developed a synthesis of (*)-% in 31% overall 60,RLcTHP R2=CN R3=0Me yield from 56a utilizing 65. Introduction of 61, R ~ = H , R ~ ,R& =A~ the C-9 substituents is effected via the ethy0

0

0

4;

I

.

I

#

I

(m),

z. -

(z),

=

-

-

Chap. 28

Anthracycline Antibiotics

Kelly

293

nylcarbinol. The trimethylsilyl group functions as a latent C-7 hydroxyl group, its metamorphosis being precipitated by Pb(OAc)4 oxidation.

n

0

65 TMS 64 The alternate Diels Alder approach to A-ring construction (see *) has been reported by Kerdesky and C a ~ a .Thus ~ ~ 67, prepared via 66 in 73% yield from phthaEc anhydride and 2,3-dimethylhydroquinone, upon treatment with zinc (-66) in the presence of methyl vinyl ketone gives the dimethyl ether of 9-deoxy61 (52%). Hydroxylation at C-9 (t-BuOK, 02, 55%) gives the dimethyl ether of 61 0 OCH3 OCH3 which has been converted to (+)-%. The 66,R=CH3;3,R=CH2Br 68 approach suggested by 53c has been examined by Boeckman et al. :53 in situ generation (170O) of 69 from 70 and trapping with naphthoquinone gives 71 (75%) after oxidation; modifications to incorporate oxygen at C-6 have been unsuccessful. Jung and Lowe") have reported the regiospecific formation of 72 (~70%after oxidation) from the 73 with jugl0ne.4~ cycloaddition of -

c1

62

-

63'

0

c1

0 0 0 0 6H 0 6CH3 OCH3 70 69 71 72 73 Several groupshave investigatedthe route suggested by The most successful effort, that of Wiseman et a155 involves the trapping of 74(R=H) (from tetrabromo-2-xylene and zinc) with 75 (prepared in exceptiona1 yield from the corresponding dimethoxytetralone) to give 76 (33%) 74 (R=OCH3) in which was converted to 61 in three steps (65%). Use of the same sequence results in a mixture of regioisomers. Related approach~ ~ (X=O)57 have been reported, but have not been es using 77 ( X I H ~ )and extended to a total synthesis.

z.

q C H B r

R

'CHBr

74

0 0

75,R=H;75a,R=Ac -

q

c//X

0

76 -

%O

CH30

77 -

The salient finding of FariGa that substituted naphthazarins

(e.g.

78a)58 and their diacetates (e.g., 7sb)59 exist in tautomeric equilibrium (78279) - - and that tautomers 79 can be selectively trapped by Diels Alder in the only regiospecific reactions was exploited by Kelly et al.!O Diels Alder approach to anthracyclinones yet reported? Thus, the a s p -

294 -

Sect. VI

-

Topics in Chemistry and Drug Design

Renfroe, Ed.

metry induced by the two "C"-ring substituents in 80 promotes regiospecific cycloaddition of 80 to l-methoxy3-ethylbutadiene (81) , affording 82. After epoxidation and oxidation , tautomerization (with intramolecular transfer of the directing groups see 83) permits a second, regiospecific Diels 0 OR OR 0 Alder reaction (with l-methoxycyclohexa-lY3-diene), providing in 45% 78 a,R=H;b,R=Ac 2 yield based on 3. Oxidation and

-

82 84 the aromatic D-ring. It is of note that reaction of 81 w i t h 2 gives predominantly the "wrong" regioisomer.51 A variation on this approach by (Eq* 2)

Krohn and Tolkiehn62 (Eq. 2) , which refrains from addressing regiochemical questions,employs 1,3-bis(trimethylsiloxy)butadiene (85) as an A-ring synthon and affords a notably efficient (29% overall yield) synthesis of (*)4b with a virtually stereospecific introduction of A-ring functionality.

-

The unparalleled employ of five Diels Alder reactions (3 normal, 2 retro) in the Kende "isobenzofuran" route12 (Eq.3) provides a fitting conclusion to the discussion of Diels Alder sequences. Although a 1:l mixture of regioisomers is produced, the yield of & is 17% based on

=.

(Eq .3)

140'

0

0

-

-

d. Synthesis of Chiral Aglycones If anthracyclines are to be available by total synthesis, the problem of aglycone chirality must be resolved. Resolution of racemic aglycones in the course of glycosidation is possible, and classical resolutions of (f)-daunomycinone (2b) 3and L6 an intermediate common to several routes,have been attaina. The possibility of developing asymmetrically induced syntheses of chiral aglycones remains, however, largely unexplored. Nonetheless, the only reported efforts in

Chap. 28

Kelly 295

Anthracycline Antibiotics

this area, those of Terashima and a s ~ o c i a t e s ,will ~ ~ be difficult to improve upon. Thus, conversion of 86 to the (-)-proline derivative 87, followed by bromolactonization and reductive dehalogenation provides a 98.5: 1.5 mixture of 9-(R)and 9-(S)-88. Further transformations (H3O+; CH3Li) give (R)-7 in 44% overall yield from 86. The analogous "resolution" of 89 proceezs nearly as well.

n

0

HOOC"

86

0

OCH3

OCH3 88 0 CH3 -89 Syntheses of Daunosarnine and its Isomers Three syntheses of the natural L-antipode of daunosamine (6) have been recorded. The first, by Goodman and colleagues,65 commences with L-rhamnal (90) and proceeds as shown in Eq.4. A second, due to Yamaguchi and Kojima- begins with 92 (from gluCH 1.NZR3 (Eq* 4) 1. ~g*, C H ~ O H 3 2 .MsC1 2.KBH4 3.PhCOOj CH3 6 4.aa. OH-' HO 3.leq. TsCl 4 . CH306H 5.H2 + 90 6.H30 OCH3

87

-

*

cose via the anhydroalloside 91) which upon methanolysis and benzoylation affords the furanoside 93 (41%, accompanied by a similar amount of the corresponding pyranosid3. After conversion of 93 to the 5,6-epoxide (TsC1; CH3O-, 97%) reduction over Raney nickel gives 94 (36%) which yields -6 upon hydrolysis. OOCPh

PhT--phx -

+OCH3 $ H

OCH3

OCH3 - N3 CH3 N3 93 H2 94 The most efficient synthesis of L-daunosamine is that of Horton and W e ~ k e r l e ;it~ ~provides 6 (as its hydrochloride) in 40% yield from readily available methyl a-D-rnannopyranoside (95) Thus butyllithium-induced cleavage6' of the dibenzylidene acetal 96 affords 97 (91%), the oxime

.

) * H

+yhp*R R'O 0

NHAc CH3@ NHAc CH30 98,R=R'=PhCHOz 97 100 96,R=R'=PhCH 99,R=Br,R'=PhCOO (>95%) of which is reduced (LiAlH4) and aczylated to give 98 (87%). After conversion to 100 (70%), stereospecific reduction and hydrolysis give 6. OCH3

95 ,R=R'=H

OCH3

296

Sect. VI

- Topics in Chemistry and Drug Design

Renfroe, Ed.

as well as the r a ~ e m a t e ? ~ - ~ ~ Syntheses of the D-antipode of 5s9-71 have been described. Five of the remaining six configurational isomers (or their derivatives) of 6 have also succumbed to the labors of synthesis: L79-81 a r a b i n o ( K , a c ~ s a m i n e ~ ~ ~ ~ ~ - ~ ~ D - aL-ribo( rabin 102, o , ~ristosamine) ~ D r i b g v and L X y l O ( m ) ;71 101 , ~ 2 , m ,R~,oH,R~,R~,H ~ the D-xylo isomer continues to OH 102,R1=NH2,R3=OH,R2=R4=H be elusive. Glycosidation of ~ ~ ~ , R ~ = N H ~ , R ~ = o H , RE, ~=2 R ~ =and H %with 101 and 102 has been accomplish=

-

Glycosidation Methods The coupling of lb-5b with L-daunosamine (5) constitutes the final stage in total synthesis and has been effected using a variety of methods. It is necessary to protect the hydroxyl and amino substituents in 6 and, in the case of C-14 hydroxylated aglycones (e.g. &) to protect this functionality also: both the C-14 monomethoxytrityl ether (104)4and the dioxolane derivative 1055'84have been employed for this purpose (alter;nately, introduction of the C-14 hydroxyl can be deferred unX

t

-

x , X = B r ,R=pNBz

pNBzo

107,X=Cl,R=pNBz

pNBz

-

Ro NHCOCF3 108,X=a-Cl,R=COCF3 NHCOCF3 - 105 6H 109 ti1 glycosidation has been carried out). The first preparation of &was reported in 1974:85 coupling of & with 3 equiv. 106 under Koenigs-Knorr conditions gives the a-glycoside stereospecifically; deprotection gives & in 50% overall yield. Use of the more stable glycosyl chloride 107 raises the overall yield to 72% (still stereospecific), but again requires 3 equiv. of glycosyl halide.4 Koenigs-Knorr coupling of 106 with 104 is also stereospecific and gives & in 40% overall from E, but the n e G s i t y of using 9 equiv. of 106 is a serious drawback: The Koenigs-Knorr method has also been used for the preparation of a diverse selection of glycosides of Erhodomycinone, but yields and stereochemistry are variable. 87 Subtle changes in the exact structure of the glycosyl halide can have substantial stereochemical consequence^:^^^^^^ use of 108 in place of 107 leadsa4 to & (from 105) and @,but stereospecificity islost in both cases, a 7:3 mixture B-anomers being produced. Although lack of stereospecificity is of acompensated by the generation of additional analogs (some of the B-anomers have significant activity) , the unpredictable stereochemistry of the Koenigs-Knorr method and the need to develop procedures which do not require a large excess of glycosidating reagent have stimulated a search for better methods for stereospecific a-glycosidation. One approach, utilized in the synthesis of the 6'-hydroxy-4'-epi analog of &, employs acid-catalyzed condensation of 2 and glycal 109.5988The outcome (56% overall yield, stereospecific) is superior in terms of both yield and stereochemistry to that realized using the corresponding glycosyl halide under Koenigs-Knorr conditions. More recent work indicates, C H 3 F c 1 however that the silver triflate method of HanessianE9y 4 0 may well have eclipsed earlier procedures. Thus the coupling of &with the 4-deoxydaunosamine R NHCOCF3 derivative N ( 1 . 1 equiv) under the agency of CF3S03Ag provides the corresponding a-glycoside in llo,R=H111,R=0CH3 70% yield.g1 Similarly, reaction of (a-C1 anomer)

and

-

111

Chap. 28

Anthracycline Antibiotics

Kelly 297

with 1.5 equiv. of 2b gives the a-glycoside (70%); 9 2 coupling of 5 and 108 ( 1 . 4 5 equiv.) with CF S03Ag gives the a-glycoside (46%) which affords 4a (37%overall yield).? Additional examples are available. *9 The pre994-96 parations of other analogs are summarized elsewhere. Conclusions - Efforts toward the synthesis of the anthracyclines have provided a chemical harvest which is already bounteous and continues to be reaped. Overall yields for the total synthesis of the intact, chiral natural products la-3a, as well as 5,5a and their analogs, are now on the order of 5% in the best cases. Whether total (or partial) synthesis will emerge as the practical solution for the future is, however, a question whose ultimate answer depends on many as yet incompletely resolved factors which include the relative economics of fermentative and total synthesis, the potential therapeutic superiority of non-natural anthracyclines, and future advances in total synthesis. It is the opinion of the author that at the present time fermentation remains the preparative method of choice, but this verdict is much less certain than it would have been five years ago, Five years hence such a judgment may be indefensible. Synthetic chemists would not be distressed. Acknowledgments - The preparation of this survey has been greatly facilitated by the cooperation of many workers in this field. I thank all of them, but especially Drs. F. Arcamone and J.W. Gillard. Thanks are also due M. Grant, J. Cantwell and K. Barron for their essential contributions. References I.

2.

3.

4. 5,

6. 7. 8.

9. 10. 11. 12. 13.

14. 15. 16. 17.

18. 19. 20. 21. 22. 23. 24.

For recent general reviews see J.R. Brown in "Progress in Medicinal Chemistry," Vol. 15, G.P. Ellis and G.B. West, Eds.. ElsevierfNorth-Holland Biomedical Press, Amsterdam 1978, p. 165: W.A. Remers, "The Chemistry of Antitumor Antibiotics," Vol. 1. Wiley Interscience. Somerset, N.J.. 1979, Chapter 2. C.M. Wong. R. Schuenk, D. Popien and T.-L. Ho, Can.J.Chem.. 51, 466 (1973). A.S. Kende, Y . - p . Tsay and J.E. Mills. J.Am.Chem.Soc.. 98. 1967 (1976). For experimental details see A.S. Kende, J.E. Mills and Y.-8. Tsay, U . S . Patents 4,021.457(May 3, 1977) and 4,070,382(Jan. 24,1978). 98,1969 (1976); T.H. Smith, A.N. Fujiuara, T.H. Smith, A.N. Fujiwara. W.U. Lee and D.W. Henry, W.W. Lee, H.Y. Wu and D.W. Henry, J.Org.Chem. 42, 3653 (1977). F. Arcamone. Lloydia, 40, 45 (1977). F. Arcamone, L. Bernardi. 8. Patelli, P. Giardino, A. DiMarco, A.M. Casazza, C. Soranro and G. Pratesi, Expericncia, 34, 1255 (1978). L.Bernardi. P.Ciardino. B. Petelli and F. Arcamone, Ger.Dffen.. 2,727,341 (Dec. 22,1977) [Chem.Abstr., 88. 120871~ (1978)l. F. Arcamone, L. Bernardi, P. Giardino, B. Patelli, A. DFMarco, G. Pratesi and P. Regiani, Cancer Treatment Reports, 60. 829 (1976). F. Arcamone, G. Franceachi, S. Penco, U.S. Patent 3,803,124 (Apr. 9, 1974); Ger.Offen. 1,917,874 (Nov. 6, 1969). [Chem.Abstr., 73,45799r (1970)l. L.S. Povarov. V.S. Bazhanov and V.F. Vasekina, Antibiotiki, 653 (1977); E.N. Olsufieva and L.S. Povarov, 1085 (1977). A. DiMarco, A.M. Casazza, F. Giuliani, G . Pratesi, F. Arcamone, L. Bernardi, 6. Franchi, P. Giardino, 9 . Patelli and S. Penco, Cancer Treatment Reports, 62, 375 (1978). A.S. Kende, D.P. Curran, Y.-g. Tsay and J.E. Mills, Tetrahedron Lett., 3537 (1977). G. Cassinelli, A. Grein, P. M a h i . A. Suarto, L. Bernardi, F. Arcamone, A. DiMarco, A.M. Casazza, G. Pratesi and C. Soranzo, J.Antibiotics, 21, 178 (1978). R.J. Blade and P. Hodge, J.C.S.Chem.Com.. 85 (1979). S . Penco, G.P. Vicario. F. Angelucci and F. Arcamone. J.Antibiotics, 30, 773 (1977). C.R. Chen, M.T. Fong. A.N. Fujiwara. D.W. Henry, M.A. Leaffer. W.W. Lee and T.H. Smith, J.Labelled Compd.Radiopharm., 14,111 (1978). Claisen rearrangements have also been employed, but usually for the preparation of unsymmetrical starting materials; J.L. Robercs and P.S. Rutledge [Aust.J.Chem.. 30. 1743 (1977)] have examined more direct applications. C.M. Wong, D. Popien, R.Schwenk and J. TeRaa, Can.J.Chem.. 5 , 2712 (1971). J.P. Marsh, Jr., R.H. Iwamoto and L. Goodman, Chem.Commun., 589 (1968). The tetralin (-)-L has been incorrectly represented by the Farmitalia as (S)-L;it should be @)-(-)-Iand ia so indexed by Chemical Abstracts (c.f. the indexing of ref. 7). A.S. Kende, J. Belletire, T.J. Bentley, E.Hume and J. Airy, J.Am.Chern.Soc., 91. 4425 (1975). R.D. Gleim, S. Trenbeath, R.S.D. Hittal and C.J. Sih. Tetrahedron Lett., 3385 (1976). April (1979) (in press). K.S. Kim, M.W. Spat2 and F. Johnson, J.Amer.Chem.Soc.. J.S. Suenton and P.W. Reynolds. loo, (1978) and earlier papers cited therein. See also 17. 945 (1978). M. Braun, Angev.Chem.Int.Ed.Eng1..

c.,

c.,

c..

x,

Sect. VI - Topics in Chemistry and Drug Design

298 -

Renfroe, Ed.

25. J. Alexander and L.A. Mitacher. Tetrahedron Lett., 3403 (1978). 2559 (1978). 26. J.E. Baldwin and K.W. Bair, 3965 (1978). 27. I. Forbes, R.A. Pratt and R.A. Raphael, 28. S.O. desilva and V. Sneickus. H;5103 (1978) and adjoinin& papers. 4557 (1977); xxxx (1979) (in press). 29. K.A. Parker and J.L. Kallmerten, xxxx (1979) (in press). 30. A.S. Kende, J. Rirri and J. Riemer, E., 13, 651 (1965). 31. For a related etudv see 2 . - i . Horii. T. Momoae and Y. Tamura, Chem.Pharm.Bul1. , 4762 (1976). 32. K. Krohn. J.Chem.R;s.(M), 33. F.M. Hauaer and R.P. Rhee, J.Org.Chem., 9. 178 (1978); b) J.Am.Chem.Soc.. w . A p r i l (1979) (in press). 34. G.A. Kraus and H. Suginoto, Tetrahedron Lett., 2263 (1978). 969 (1972). 35. See also W. Treub and C.H. Eugster, Helv.Chim.Acta.. 36. R.J. Boatman, B.J. Whitlock and H.W. Whitlock, Jr., J.Am.Chem.Soc., 99. 4822 (1977); loo, 2935 (1978). 37. F. Sukuki, S. Trenbeath, R.D. Gleim and C.J. Sih, J.Org.Chem., W, 4159 (1978). 712 (1978). 38. L.M. Harwood, L.C. Hodgkinson and J.K. Sutherland, J.C.S.Chem.Com.. For citation and explanation see 39 * BIOS Report 1484, p.22 [available from Library of Congreaa (U.S.A.)]. M.A. Perkins and C.W. Maynard, Jr,, in "The Chemistry of Synthetic Dyes and Pigments," H.A. Lubs,Ed., American Chemical Society Monograph Series, Hafner Publishing Co.. New York, N.Y., 1964, pp. 368, 692. 40. M . J . Morris and J.R. Brown, Tetrahedron Lett., 2937 (1978). 41. K. Krohn and M. Radeloff. Ber.. IlJ, 3823 (1978). 42. H.W. Moore, Science, 197,527 (1977): T.-t. Li and R.H. Ellison, J.Am.Chem.Soc., loo, 6263 (1978). 43. See also U. O'Connor and W. Rosen, Tetrahedron Lett., 601 (1979). 44. H.H. Inhoffen, H. Muxfeldt, V. Koppe and 3. Heimann-Trosien. Ber., 90, 1448 (1957). 45. W.W. Lee, A.P. Martinez, T.H. Smith and D.W. Henry, J.Org.Chem., 4 l 7 2 1 9 6 (1976). 46. R.B. Garland, J.R. Palmer, J.A. Schulz, P.B. Sollman and R. Pappo, Tetrahedron Lett., 3669 (1978). ibid.. 3869 (1976). 47. T.R. Kellv. R.A. Goerner.. Jr... J.W. Gillard and B.K. Prazak. 4457 (1978). 46. T.R. Kelly and W.-G. Tsang, 49. M. Chandler and R . J . Stoodley, J.C.S.Chem.Com., xxxx (1979) (in press). 50. A.M. Birch, A . J . H . Mercer, A.M. Chippendale and C.W. Greenhalgh, J.C.S.Chem.Com., 745 (1977). 51. T.R. Kelly, J.W. Gillard and R.N. Goerner,Jr.. Tetrahedron Lett., 3873 (1976). 52. E.A.J. Kerdesky and M.P. Cave, J.Am.Chem.Soc., loo, 3635 (1978). 2946 (1977). 53. R.K. Boechan, Jr., M . H . Delton, T. Nagasaka and T. Watanabe. J.Org.Chem., 54. M.E. Jung and J.A. Lowe, J.C.S.Chem.Com., 95 (1978). 55. J . R . Wiseman. N.I. French, R.K. Hallmark and K.G. Chiong. Tetrahedron Lett., 3765 (1978). 556 (1978). 56. T. Kametani, M. Takeshita, H. Nemoto and K. Fukumoto, Chem.Pharm.Bull., 57.. M.E. Jung and J.A. Lowe, J.Org.Chem.. 42, 2371 (1977). 58. F. FariWa and J.C. Vega, Tetrahedron L;;t-t., 1655 (1972). 3377 (1970). 59. S. Alvarado, F. Farise and J.L. Martin, 60. T.R. Kelly, J.W. Gillard, R.N. Goerner, Jr., and J.M. Lyding, J.h.Chem.Soc., 99. 5513 (1977). 61. For different possible approaches see D. Rutulo, S. Lee, R. Sheldon and H.W. Moore, J.Org.Chem., 2304 (1978) and B.M. Troat, J. Ippen and W.E. Vladuchick, J.Am.Chem.Soc., 99, 8116 (1977). 62. a ) K. Krohn and K. Tolkiehn, Tetrahedron Lett., 4023 (1978); b) For relaGd studies see F. Fariga and P. Prados, E., 477 (1979). 63. F. Arcamone, L. Bernardi, 8. Patelli and A. DiMarco, Ger.Offen., 2,601,785 (Julv 29, 1976) [Chem. Abstr. E, 142918j (1976)l. 64. S. Terashima, S.-a. Jew and K. Koga, Tetrahedron Lett., 4937 (1978). 65. J.P. Marah, Jr.. C.W. Moaher, E.M. Acton and L. Goodman, Chem.Com., 973 (1967). 66. T. Yamaguchi and M. Kojina, Carbohydr.Res.. 2, 343 (1977). 67. D. Horton and W. Weckerle, 44, 227 (1975); U.S. Patent, 4,024,333 (June 22, 1976). 2612 (1974). 68. A. Klemer and G. Rodemeyer, Ber, 69. A.C. Richardson, Carbohydr.Res., 4. 422 (1967). 41. 84 (1969). 70. H.H. Baer, K. Capek and M.C. Cook, C8n.J.Chem.. 71. J. Boivin, M. Pals and C. Monneret, Carbohydr.Res., 66. 271 (1978). 72. C . M . Wong. T.-L. Ho and W.P. Niemcrura, Can.J.Chem., 53, 3140 (1975). 682 (1978). 73. I. Dyong and R . Wiemann, Angew.Chem.Int.Ed.Eng., 1, 74. S.K. Gupta, Carbohydr.Rea.. 37, 381 (1974). 768 (1975). 75. W.W. Lee, H.Y. Wu, J.E. Chrizenaen, L. Goodman and D.W. Henry, J.Med.Chem., 76. K. Heyns, J.-j. Lim and J.I. Park, Tetrahedron Lett. 1477 (1976). 77. D. Horton, R.J. Sorenaon and W. Weckerle. Carbohydr.Res.. 58. 125 (1977). 78. I. Dyong and H. Bendlin, &, 111, 1677 (1978). 79. W.W. Lee, H.Y. Wu., J.J. Marsh, C.W. Mosher, E.M. Acton, L. Goodman and D.W. Henry, J.Med.Chem., 767 (1975). 80. F. Sztaricskai, I. Pelvis, R. Bognir and G. Bujtia, Tetrahedron Lett., 1111 (1975). 81. F. Arcamone, A. Bargiotti, G. Cassinelli, S. Penco and S. Hanessian. Carbohydr.Res., 5, C3 (1976). 82. D. Horton and W. Weckerle, g.. 5. 227 (1976). 83. H. Baer and F.F.Z. Georges, Can.J.Chem., 55. 253 (1977). 84. F. Arcamone, S. Penco and A. Vigevani, Cancer Chemother.Rep. Part 3. 123 (1975). 85. E.M. Acton, A.N. Fujiwara and D.W. Henry, J.Med.Chem., 17. 659 (1974). 86. A.S. Kende and Y.-g. Tsay, J.C.S.Chem.Com.. 140 (1977)Taee also K. Krohn and A. Roaner, Tetrahedron Lett., 353 (1978). 87. H . S . El Khadem. D.L. Swarte and R.C. Cermak, J.Med.Chm., 2 0 , 957 (1977). 88. F. Arcamone, A . Bargiotti, G. Cassinelli. S. Redaelli, S. Haneasian, A. DlMarco. A.M. Casazza, T. Dasdia, A. Necco, P. Reggianl and R. Supino, 19. 733 (1976). 89. S. Hanessian and 3. Banoub, Carbohyd.Res.. 44, C14 (1975). 90. S. Haneasian and J. Banoub in "Synthetic Methods for Carbohydrates" (ACS Sympoaium Series #39), H.S. El Khadem, Ed., American Chemical Society, Washington, D.C.. 1976, p 36. 91. F. Arcamone, S. Penco, S. Redaelli and S. Hanessian, J.Med.Chem.. 19. 1424 (1976). 92. G. Caaeinelli, D. Ruggieri and F. Arcamone, g., 22 121 (1979). 93. F. Arcamone, L. Bernardi, B. Patelli and S. Penco, iiilgian Patent 842,930 (Oct. 1, 1976) IChemAbatr., 87, 85201k(1977)]. 94. D.W. Henry in "Cancer Chemotherapy" (ACS Symposium Series 4130). A.C. Sartorelli, Ed., American Chemical Society, Washington, D.C. 1976, p 15. 95. H.S. El Khadem and D.L. Swartz, Carbohyd.Res., 65, Cl (1978). 96. E.-F. Fuchs. D. Horton and W. Weckerle. 57, C36 (1977).

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

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I t h a s long been b e l i e v e d t h a t a drug i s a c t i v e because Introduction of t h e p r e s e n c e of c e r t a i n key atoms o r f u n c t i o n a l groupings. E h r l i c h l c a l l e d such e s s e n t i a l f u n c t i o n a l i t y a "pharmacophore", j u s t a s f u n c t i o n a l i t y i m p a r t i n g c o l o r t o a compound i s termed a chromophore. E h r l i c h and o t h e r s s p e c u l a t e d t h a t d r u g s a c t e d by f i t t i n g b i o l o g i c a l "receptors".2

According t o drug-receptor t h e o r y , a b i o a c t i v e molecule i n t e r a c t s w i t h a b i o m o l e c u l a r r e c e p t o r t o form a complex, t h e r e b y e l i c i t i n g a b i o l o g i c a l r e s p o n s e .3 It appears t h a t normal chemical i n t e r m o l e c u l a r f o r c e s a r e s u f f i c i e n t t o e x p l a i n drug-receptor i n t e r a c t i o n s and enzyme c a t a l y s i s w i t h o u t p o s t u l a t i n g a d d i t i o n a l , " v i t a l " f o r c e s . 4 However, t h e o l d "lock and key" p i c t u r e of r i g i d drug f i t t i n g r i g i d r e c e p t o r is g i v i n g way t o one of f l e x i b l e b u t complementary i n t e r a c t i n g u n i t s . 5 For t h e purposes of t h i s c h a p t e r , we d e f i n e a drug a s any subs t a n c e , n a t u r a l o r s y n t h e t i c , which e l i c i t s a b i o l o g i c a l r e s p o n s e ; and a druR r e c e p t o r as any b i o m o l e c u l a r s i t e a t which a drug b i n d s o r is o t h e r w i s e "recognized" i n e l i c i t i n g t h e b i o l o g i c a l response. With t h i s broadened d e f i n i t i o n , a r e c e p t o r may be a p o l y p e p t i d e b i n d i n g s i t e on a membrane; an a c t i v e s i t e of an enzyme; an i n t e r c a l a t i o n o r a l k y l a t i o n s i t e i n DNA; e t c . Whenever a r e c e p t o r "recognizes" a drug, t h o s e s t r u c t u r a l f e a t u r e s of t h e drug which a r e "recognized" make up what may be c a l l e d a pharmacophore. The g e o m e t r i c a l arrangement of t h e s e s t r u c t u r a l f e a t u r e s i s termed a pharmacophoric p a t t e r n . The r e c e p t o r must c o n t a i n c e r t a i n f e a t u r e s complementary t o t h o s e of t h e pharmacophore f o r t h e " r e c o g n i t i o n " t o o c c u r ; t h e arrangement of t h e s e complementary f e a t u r e s i s c a l l e d a r e c e p t o r map. A r e c e n t mathematical model s t r e s s e s t h e symmetry of enzyme-substrate r e c o g n i t i o n ; t h e enzyme "recognizes" a s u b s t r a t e pharmacophoric p a t t e r n , j u s t as t h e s u b s t r a t e "recognizes" a complementary r e c e p t o r p a t t e r n . 6 A s an i n c r e a s i n g numb e r of drug b i n d l n g biomolecules are b e i n g i s o l a t e d and c h a r a c t e r i z e d , i t i s perhaps t i m e l y t o review t h e u t i l i t y f o r drug d e s i g n of t h e c o n c e p t s of pharmacophoric p a t t e r n s e a r c h i n g and r e c e p t o r mapping.

7 A pharmacophoric p a t t e r n h a s been d e f i n e d Pharmacophoric P a t t e r n s a s ''a c o l l e c t i o n of atoms s p a t i a l l y d i s p o s e d i n a manner t h a t e l i c i t s a b i o l o g i c a l response". Such p a t t e r n s have been c l a s s i f i e d as t o p o l o g i c ( g r a p h - t h e o r e t i c o r c o n n e c t i v i t y - b a s e d s u b s t r u c t u r a l f r a g m e n t ) and t o p o g r a p h i c ( g e o m e t r i c , u s u a l l y 3-dimensional) p a t t e r n s . 7 Topographic p a t t e r n s depend on m o l e c u l a r conformation and should be more s u i t a b l e f o r d e s c r i b i n g drug a c t i v i t y even f o r chemically d i s s i m i l a r compounds having t h e same t y p e of b i o a c t i v i t y .

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Topographic pharmacophoric p a t t e r n s may c o n v e n i e n t l y b e d e f i n e d i n terms of atom t y p e s and i n t e r a t o m i c d i s t a n c e . 7 D i s t a n c e geometry has been shown t o u n i q u e l y d e f i n e 3-dimensional molecular s t r u c t u r e s , except f o r t h e i r c h i r a l i t y . 8 P r o p e r t i e s of pharmacophoric p a t t e r n s which have been d e f i n e d i n c l u d e c h i r a l i t y ( s t e r e o c h e m i s t r y ) , Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

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orientation (which face of a 3-D pattern is presented to the receptor), accessibility (which atoms in a pattern are accessible to the receptor), and indefiniteness (for example, when a lipophilic group in the drug can engage more than one hydrophilic region on the receptor).7 Patterns can be made up of atoms of a specific type; of atoms of a general type (e.g., hydrogen-accepting); of atoms with a certain partial charge; of functional groups; and of negative mass (i.e, atoms at a certain position which may interfere with receptor interaction) .7 Pharmacophoric patterns have been proposed as the result of structure-activity studies, modeling studies, mechanistic studies, and enzyme structural studies. While two atom ("distance") and three atom ("triangle") pharmacophoric patterns have been proposed, we would not expect these to be as discriminating as more detailed patterns. Proposed pharmacophoric patterns have been reviewed by Kier,g Korolkovas,lO and Gund.7 Some additional proposed patterns of drug pharmacophores and of complementary receptor maps are listed in Table I. Table I.

Some Recently Proposed Pharmacophoric and Receptor Patterns Bioactivity

Thyroxine Binding Anti-inflammatory (arylacetic) Hypnotic (barbiturate) Insulin Release (sulfonylureas) Acetylcholinesterase Analgesic (opiate) Antimalarial Sweet Taste Dihydrofolate Reductase Inhibition a-Adrenergic Prostaglandin-like

Pattern

References

11,12 Receptor Map 13 Receptor Map Receptor Map 14 Receptor Map 15 Receptor Map 16 Pharmacophore 17,18,19,20,21,22,23 24 Pharmacophore 25 Receptor Map 26 Receptor Map 27 Receptor Map 28 Pharmacophore

One would like an automatic procedure for discovering a common pharmacophoric pattern among a group of similarly acting drugs. The statistical techniques of pattern recognition (nearest neighbor, learning machine, etc.) have been used with limited success to correlate biological activity with molecular geometry-derived information such as a generalized X-ray scattering function (molecular transform).29 However, these techniques appear not yet to have been applied to discovery of pharmacophoric patterns as defined above. Furthermore it may prove difficult to automate the discovery procedure, because of the complicating factors detailed below. Many bioactive molecules are flexible, and their minimum energy conformation need not correspond to the receptor-bound conformation.7 Furthermore, conformational dynamics may be important, with receptor molecule and substrate both changing conformation in order to elicit the biological response.5,30 In some cases, then, a system could show different pharmacophoric patterns for compounds which preferentially bind to the receptor's ground state, its activated state, and an intermediate state (transition state analogs) .4

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It i s r e a s o n a b l e t o suppose t h a t a drug may b e "recognized" by i t s r e c e p t o r , a t l e a s t weakly, i n one of i t s major s o l u t i o n c o n f o r m a t i o n s ; t h i s has been c a l l e d "remote r e c o g n i t i o n of p r e f e r r e d conf ormation",31 A c o l l i s i o n t h e o r y argument and " t h e weak b i n d i n g approximation".32,33 s u g g e s t s t h a t f a s t e s t b i n d i n g would occur f o r a molecule r e a c t i n g from i t s ground s t a t e conformation.34 However, prudence would d i c t a t e t h a t , when l o o k i n g f o r pharmacophoric p a t t e r n s , a l l low energy conformations But s a y , t h o s e w i t h i n 6 k c a l of t h e minimum b e considered.35936 t h i s e x h a u s t i v e approach is i n f e a s i b l e f o r molecules w i t h many r o t a t a b l e bonds, where an a s t r o n o m i c a l number of conformations are possible. I n such cases, s t u d y i n g more r i g i d a n a l o g s can narrow t h e p o s s i b i l i t i e s .37-40

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Once a pharmacophoric p a t t e r n h a s Pharmacophoric P a t t e r n S e a r c h i n g been proposed, i t would be u s e f u l t o l o o k f o r t h a t p a t t e r n i n o t h e r i n o r d e r t o t e s t and r e f i n e t h e p a t t e r n ; t o i d e n t i f y drug molecules a v a i l a b l e compounds which should be t e s t e d f o r t h e b i o a c t i v i t y of i n t e r e s t ; and t o p r o v i d e feedback t o c h e m i s t s s y n t h e s i z i n g new p o t e n t i a l d r u g s . Such a p a t t e r n s e a r c h i n g p r o c e d u r e r e q u i r e s a t h r e e dimensional s t r u c t u r e f o r t h e molecules t o be examined, and a method of d i s c o v e r i n g t h e p r e s e n c e of t h e p a t t e r n i n t h o s e molecules.

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C r y s t a l l o g r a p h i c a l l y determined molecular s t r u c t u r e s have been c o l l e c t e d 4 1 and a r e a v a i l a b l e o v e r a d i a l - u p computer network.42 Other molecules and o t h e r conformations may b e c o n v e n i e n t l y g e n e r a t e d by t h e t e c h n i q u e of computer-assisted m o l e c u l a r modeling. 7937,43944 A p r o t o t y p e computer program t o d i s c o v e r o c c u r r e n c e s of a g i v e n pharmacophoric p a t t e r n i n molecular s t r u c t u r e s , MOLPAT, has been d e s ~ r i b e d . 7 ~ 4 5A s a t e s t of t h e program a h y p o t h e t i c a l a n a l g e s i c pharmacophoric p a t t e r n , c o n s i s t i n g of an a r o m a t i c r i n g and a remote n i t r o g e n atom, w a s e x t r a c t e d from t h e c r y s t a l c o o r d i n a t e s of t h e p r o t o t y p e a n a l g e s i c , morphine. A s e a r c h f o r t h a t p a t t e r n i n o t h e r molecules d i s c l o s e d i t s presence i n t h e c r y s t a l s t r u c t u r e of LSD, a s shown i n F i g . 1.7 LSD does appear t o be a mild a n a l g e s i c . 7 Another r e p o r t e d computer program e x p l o r e s conformations of one s t r u c t u r e t o f i n d s u p e r p o s i t i o n s w i t h another structure.46

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In classical structure-activity Beyond Pharmacophoric P a t t e r n s s t u d i e s , m e d i c i n a l chemists s y s t e m a t i c a l l y v a r y a chemical s t r u c t u r e and, based on r e s u l t i n g b i o a c t i v i t y , draw c o n c l u s i o n s about "essential" s t r u c t u r a l r e q u i r e m e n t s . More r e c e n t l y , q u a n t i t a t i v 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 (QSAR's) have allowed a " f a c t o r i n g o u t " of some of t h e f a c t o r s ( d i s t r i b u t i o n , l i p o p h i l i c i t y , e l e c t r o n i c and s t e r i c e f f e c t s ) a f f e c t i n g bioactivity.47,48,49 While such QSAR's t e n d t o g i v e a r a t h e r hazy view of r e c e p t o r geometry, Hansch and o t h e r s have used i n d i c a t o r v a r i a b l e s ("dummy" v a r i a b l e s ) t o "map" r e c e p t o r s i t e s -49 C o n s i d e r a t i o n s of m o l e c u l a r shape27,50 and pharmacophoric p a t t e r n s 7 9 3 7 I n f u r t h e r development of have l e d t o more s p e c i f i c " r e c e p t o r maps". t h i s i d e a W e i n s t e i n , 5 1 among o t h e r s , h a s c a l c u l a t e d e l e c t r o s t a t i c p o t e n t i a l maps which may e x p r e s s t h e i n i t i a l a t t r a c t i v e p o t e n t i a l between drug and r e c e p t o r . I n a n o t h e r approach, Kier52 and Weinstein51 have c o r r e l a t e d b i o a c t i v i t y w i t h t h e c a l c u l a t e d i n t e r a c t i o n energy between a drug and a n amino a c i d r e s i d u e p o s t u l a t e d t o occur a t t h e r e c e p t o r s i t e . Recently, M a r s h a l l h a s developed a computer-assisted

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method t o superimpose t h e pharmacophoric p a t t e r n s of s i m i l a r l y a c t i n g d r u g s and sum t h e i r molecular volumes i n o r d e r t o d e r i v e an "excluded volume map" of t h e receptor.15,37

/

Me

Fig. 1. Presence of an analgesic pharmacophoric pattern discovered in LSD b y program MOLPAT (from Ref. 7 with permission).

Even more d e t a i l e d t h a n a r e c e p t o r map would be a three-dimensional p i c t u r e of t h e r e c e p t o r . X-ray c r y s t a l l o g r a p h y of i s o l a t e d r e c e p t o r s i s i n c r e a s i n g l y o f f e r i n g j u s t such a p i c t u r e f i r s t a t low r e s o l u t i o n , where t h e contoured s u r f a c e s resemble t h e "blob" of t h e c o n v e n t i o n a l r e c e p t o r s i t e r e p r e s e n t a t i o n ; and f i n a l l y a t atomic r e s o l u t i o n , w i t h and w i t h o u t s u b s t r a t e o r i n h i b i t o r p r e s e n t , so t h a t i n d i v i d u a l a c t i v e s i t e r e s i d u e s may be i d e n t i f i e d and mechanisms of a c t i o n proposed.53 I t is n e v e r t h e l e s s worth s t r e s s i n g t h a t even a c r y s t a l s t r u c t u r e of t h e subs t r a t e - r e c e p t o r complex does not e x p l a i n t h e dynamics of chemical a c t i o n , nor does i t account f o r t h e r e s u l t a n t b i o l o g i c a l a c t i o n . Mechanistic s p e c u l a t i o n based on a c r y s t a l s t r u c t u r e is s t i l l specul a t i o n , and w i l l be judged by how w e l l i t e x p l a i n s o t h e r e x p e r i m e n t a l data.

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Recently, h i g h - q u a l i t y t h e o r e t i c a l c a l c u l a t i o n s have been used t o e x p l o r e t h e e n e r g e t i c s of proposed enzyme mechanisms, u s i n g enzyme c r y s t a l coordinates 954-58 Indeed, simply viewing t h e enzyme c r y s t a l s t r u c t u r e s and "docking" s u b s t r a t e s o n t o them, using i n t e r a c t i v e computer g r a p h i c t e c h n i q u e s , 4 4 h a s been shown t o a f f o r d u s e f u l i n s i g h t s . 5 9 , 6 0

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A p p l i c a t i o n s - From d e t a i l e d i n Table I examples where such Where p o s s i b l e , t h e related studies.

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a number of proposed pharmacophoric p a t t e r n s , as and e a r l i e r r e v i e w s , 7 , 9 , 1 0 w e have chosen t h r e e p a t t e r n s o r r e c e p t o r maps a p p e a r t o o f f e r i n s i g h t s . work h a s been p l a c e d i n t h e c o n t e x t of o t h e r ,

1. B a c t e r i a l P e p t i d e S y n t h e s i s I n h i b i t o r s - The p r o k a r y o t i c ( b a c t e r i a l ) ribosome, which g o v e r n s p r o t e i n s y n t h e s i s , is s t r u c t u r a l l y d i f f e r e n t ( s i m p l e r ? ) t h a n t h e e u k a r y o t i c ribosomes of i t s i n f e c t e d h o s t ; i t is e s t a b l i s h e d t h a t many r e l a t i v e l y n o n - t o x i c a n t i b i o t i c s b i n d s e l e c t i v e l y t o t h e former.61 I n t h e c l a s s i c a l model of t h e mechanism of p r o t e i n s y n t h e s i s , peptidyl-tRNA is h e l d on t h e ribosome a t a p o s i t i o n ("Ps i t e " ) a d j a c e n t t o t h e p o s i t i o n ("A-site") where t h e a t t a c h i n g aminoacyl-tRNA i s bound.62 Based on t h e r e c o g n i t i o n of common topol o g i c a l p a t t e r n s , Harris and Symons had proposed t h a t s e v e r a l a n t i b i o t i c s a c t a t t h e " t r a n s p e p t i d a s e " s i t e (where t h e p e p t i d e bond i s formed).63 B u i l d i n g on t h i s work and a c o n f o r m a t i o n a l s t u d y of l i n c o m y c i n , Cheney proposed a d e t a i l e d t h r e e - d i m e n s i o n a l model f o r t h e n a t u r a l s u b s t r a t e s when bound t o t h e t r a n s p e p t i d a s e s i t e and r e l a t e d t h e a n t i b a c t e r i a l a c t i v i t i e s of l i n c o m y c i n , c h l o r a m p h e n i c o l and e r y t h r o mycin t o t h i s mode1.64 Hahn and Gund found a d i f f e r e n t c o n f o r m a t i o n of c h l o r a m p h e n i c o l which n o t o n l y matched Cheney's model of t h e bound n a t u r a l s u b s t r a t e s , b u t which a l s o c o u l d c o r r e l a t e a g r e a t d e a l of s t r u c t u r e - a c t i v i t y d a t a f o r c h l o r a m p h e n i c o l a n a l o g s .65 However i t is s t i l l c o n t r o v e r s i a l w h e t h e r c h l o r a m p h e n i c o l b i n d s t o t h e A-site, t h e P - s i t e o r both.62 T h i s model of t h e a c t i v e s i t e of t r a n s p e p t i d a s e h a s b e e n used f o r t h e " r a t i o n a l " d e s i g n of a n o v e l a n t i b a c t e r i a l a g e n t . A t r a n s i t i o n s t a t e a n a l o g of t h e n a t u r a l s u b s t r a t e s i n h i b i t s b a c t e r i a l p e p t i d e synthesis i n vitro.66

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I n a n o t h e r a p p l i c a t i o n , a r e c e p t o r map 2. Anti-inflammatory Receptor was d e r i v e d f o r t h e p r o s t a g l a n d i n c y c l o o x y g e n a s e a c t i v e s i t e , by f o l d i n g a r a c h i d o n i c a c i d i n t o a c o n f o r m a t i o n which r a t i o n a l i z e d i t s s t e r e o s p e c i f i c o x i d a t i v e c y c l i z a t i o n t o t h e --peroxide, PGG2 ( F i g . 2a).13 The map accommodated t h e f a t t y a c i d p r e c u r s o r s of t h e PG1, PG2 and PG3 s e r i e s , and w a s consonant w i t h b i o c h e m i c a l s t u d i e s of t h e enzyme. The same r e c e p t o r map c a n be f i t by a n t i - i n f l a m m a t o r y a r y l a c e t i c a c i d s , such a s indomethacin ( F i g . 2b) and p i r p r o f e n , which i n h i b i t t h e enzyme, and a g r e e s w i t h s t r u c t u r e - a c t i v i t y d a t a f o r t h e s e compounds.13 T h i s map, o r a somewhat d i f f e r e n t map due t o S c h e r r e r , 6 7 may p r o v e u s e f u l f o r d e r i v i n g new t y p e s of a n t i - i n f l a m m a t o r y d r u g s . 3. D i h y d r o f o l a t e Reductase I n h i b i t o r s - T h i s enzyme (DHFR) r e d u c e s an e s s e n t i a l cofactor d i h y d r o f o l i c a c i d (1)t o t e t r a h y d r o f o l i c a c i d w i t h i m p o r t a n t r o l e s i n DNA s y n t h e s i s and c e l l growth. DHFR can be w i d e l y d i f f e r e n t i n s t r u c t u r e i n d i f f e r e n t c e l l s , and i n h i b i t o r s which exploit these differences include c l i n i c a l l y useful a n t i b a c t e r i a l , a n t i p r o t o z o a l , immunosuppressant and a n t i n e o p l a s t i c d r u g s . A v a s t amount of s y n t h e t i c a n a l o g work i n t h i s f i e l d h a s been done, much of i t by Baker.68 Hansch and co-workers used d a t a from several g r o u p s and QSAR t e c h n i q u e s t o "map" r e c e p t o r s p a c e f o r DHFR from r a t l i v e r , from

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F i g . 2 . ( a ) H y p o t h e t i c a l r e c e p t o r map of t h e a c t i v e s i t e of p r o s t a g l a n d i n cyclooxygenase, showing p o i n t s of b i n d i n g or r e a c t i v i t y w i t h ( b ) The anti-inflammatory a r y l t h e enzyme s u b s t r a t e , a r a c h i d o n i c a c i d . h e r e , indomethacin which i n h i b i t t h i s enzyme, can f i t acetic acids t h e same r e c e p t o r map i n a way which e x p l a i n s t h e i r 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 (from Ref. 1 3 w i t h p e r m i s s i o n ) .

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For a series b a c t e r i a , and ( i n d i r e c t l y ) from t h e m a l a r i a l p a r a s i t e . 6 9 of 67 q u i n a z o l i n e d e r i v a t i v e s (2)i n h i b i t i n g S t r e p t o c o c c u s faecium, r e g r e s s i o n a n a l y s i s suggested t h a t 4-OH o r 4-SH b i n d s much less

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e f f e c t i v e l y t h a n 4-NH2; 5 - s u b s t i t u e n t s bind t o t h e enzyme hydrophob i c a l l y b u t l a r g e bulk is n o t t o l e r a t e d ; and 6 - s u b s t i t u e n t s are open t o s o l v e n t (whereas they are n o t when b i n d i n g mammalian enzyme). Simon d e r i v e d a comparable r e c e p t o r map of DHFR by a "minimal t o p o l o g i c a l d i f f e r e n c e s " technique. 26 The s t r o n g l y i n h i b i t i n g drug m e t h o t r e x a t e d i f f e r s from t h e enzyme's s u b s t r a t e , d i h y d r o f o l i c a c i d , by exchanging t h e t a u t o m e r i c 4-keto group Spectral studies70 by 4-amino, and by having a methyl group on N-10. and CNDO/2 c a l c u l a t i o n s of model s t r u c t u r e s 7 1 suggested t h a t 1.i s proA calorimetric study supports protonation t o n a t e d more s t r o n g l y a t N-1. of m e t h o t r e x a t e on b i n d i n g t o DHFR, b u t 1 a p p a r e n t l y is n o t p r o t o n a t e d u n t i l t h e r e d u c t i o n s t e p . 72

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Recently t h e r e h a s been a r e s u r g e n c e of i n t e r e s t i n t h i s w e l l s t u d i e d system, due t o t h e c r y s t a l s t r u c t u r e d e t e r m i n a t i o n of methot r e x a t e bound t o E s c h e r i c h i a DHFR,73 and of m e t h o t r e x a t e and n i c o t inamide a d e n i n e d i n u c l e o t i d e phosphate reduced f o m (NADPH) c o f a c t o r bound t o L a c t o b a c i l l u s casei DHFR.74,75 F i g u r e 3 shows a s t e r e o drawing of t h e a-carbon atoms of L. c a s e i DHFR, w i t h t h e methot r e x a t e i n h i b i t o r and NADPH c o f a c t o r i n p l a c e . The m e t h o t r e x a t e i s draped o v e r a pocket i n t h e middle of t h e enzyme, w i t h t h e g l u t a m a t e c h a i n on t h e r i g h t , t h e p-aminobenzoyl group h o r i z o n t a l t o t h e viewer i n t h e c e n t e r , and t h e p t e r i d i n e group a n g l i n g d e e p l y i n t o t h e enzyme's i n t e r i o r . The c o f a c t o r h a s t h e d i h y d r o n i c o t i n a m i d e group j u s t t o t h e l e f t of t h e p t e r i d i n e moiety, d r a m a t i c a l l y p o i s e d t o d e l i v e r a h y d r i d e e q u i v a l e n t d u r i n g r e d u c t i o n of s u b s t r a t e ; t h e phosphonucleotide t a i l of t h e c o f a c t o r c u r l s around t o t h e l e f t and upwards. The s t a g e i s set f o r c o r r e l a t i n g t h e s t r u c t u r e - a c t i v i t y d a t a from hundreds of s y n t h e t i c i n h i b i t o r s w i t h s p e c i f i c i n t e r a c t i o n s on t h e enzyme. The t a s k w i l l n o t be e a s y . Complications i n c l u d e amino a c i d sequence v a r i a t i o n i n DHFR from d i f f e r e n t s p e c i e s ; b i n d i n g of c o f a c t o r ; and a l t e r e d p o s i t i o n i n g of d i f f e r e n t i n h i b i t o r s . There is a l r e a d y e v i d e n c e t h a t t h e p t e r i d i n e r i n g i n m e t h o t r e x a t e may be "turned over" w i t h r e s p e c t t o i t s conformation i n d u r i n g r e d u c t i o n . 74-76 N e v e r t h e l e s s t h e enzyme dihydrof o l i c a c i d (1) s t r u c t u r e "provides an o p p o r t u n i t y f o r r a t i o n a l d e s i g n of a new c l a s s of DHFR i n h i b i t o r s t h a t would i n c o r p o r a t e elements of b o t h s u b s t r a t e and c o f a c t o r i n a s i n g l e molecule".75

F i g . 3. S t e r e o drawing of a-carbon atoms of L. 4 DHFR enzyme w i t h bound i n h i b i t o r (methotrexate: r i g h t c e n t e r of v i e w ) and c o f a c t o r (NADPH: l e f t c e n t e r ) . Stereo viewers a r e recommended, but many people can see t h e 3-D image by p l a c i n g a n index card between t h e images and s t a r i n g , one eye on each image. u n t i l t h e images c o a l e s c e . The bound s u b s t r a t e and c o f a c t o r have oxygen atoms shaded and n i t r o g e n atoms blackened, r e s p e c t i v e l y . Every f i f t h r e s i d u e i s numbered (from Ref. 7 5 with p e r m i s s i o n ) .

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4. Other Examples The d e s i g n of an a n t i h y p e r t e n s i v e a g e n t based on a h y p o t h e t i c a l model of t h e a c t i v e s i t e of a n g i o t e n s i n - c o n v e r t i n g enzyme i s now a well-known s t o r y . 7 7 , 7 8 A s t u d y of b i n d i n g of t h y r o x i n e t o prealbumin u t i l i z e d t h e c r y s t a l c o o r d i n a t e s of t h e p r o t e i n . 1 1 I n h i b i t o r s of t h e oxygen b i n d i n g s i t e of hemoglobin were d e r i v e d as an e x e r c i s e i n r a t i o n a l drug design.79 There are a l a r g e number of p r o p o s a l s of how t h e c o n f o r m a t i o n a l l y f l e x i b l e e n k e p h a l i n s could cause a n a l g e s i a by mimicking t h e o p i a t e s .17-23

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Conclusion Drug r e s e a r c h i s g r a d u a l l y moving away from broad e m p i r i c a l s c r e e n i n g , toward " r a t i o n a l " d e s i g n of i n h i b i t o r s / a c t i v a t o r s of t a r g e t enzymes/receptors. Drug d e s i g n methods which e x p l i c i t e l y c o n s i d e r t h e 3-dimensional s t r u c t u r e of drug a n d / o r r e c e p t o r would seem t o d e s e r v e a p l a c e i n t h e m e d i c i n a l chemist's armamentarium. Pharmacophoric p a t t e r n s e a r c h i n g a t t e m p t s t o i d e n t i f y t h e key geom e t r i c f e a t u r e s of a drug s t r u c t u r e t h a t c a u s e i t t o be "recognized" by i t s "receptor". P o s t u l a t i n g s p e c i f i c i n t e r a c t i o n p o i n t s on t h e r e c e p t o r f o r attachment of t h e drug l e a d s t o a " r e c e p t o r map". Both p a t t e r n and map l e a d t o t e s t a b l e hypotheses, and may be used t o d e s i g n n o v e l s t r u c t u r e s which should mimic a known drug o r i n h i b i t a t a r g e t enzyme. Like any o t h e r " r a t i o n a l " approach t o t h e complex problem of drug d e s i g n , t h e s e methods are f r a u g h t w i t h d i f f i c u l t i e s . A compound " p r e d i c t e d " t o be a c t i v e may prove t o be i n a c t i v e f o r any of t h e u s u a l d i s t r i b u t i o n , e l i m i n a t i o n , metabolism, c o m p e t i t i v e b i n d i n g , reasons e t c . N e v e r t h e l e s s , t h i s approach can b r i n g a new dimension t o t h e chemist's p e r c e p t i o n of 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 and, e s p e c i a l l y i n combination w i t h o t h e r t e c h n i q u e s , may p o i n t t h e way t o n o v e l and e f f e c t i v e drugs.

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Refer en&e s 1. 2. 3.

4. 5. 6. 7.

8. 9.

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14 15. 16. 17. 18. 19.

P. E h r l i c h . Chem. Ber. 9, 1 7 (1909); E. J. Ari&s. P r o g r . Drug Res. lo, 429 (1966). C . J. C a v a l l i t o , i n 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 , Vol. I , C. J. C a v a l l i t o . ed., Pergamon, Dxford, 1973, p. 1. 8. M. Bloom, i n Medicinal Chemistry. 3rd E d i t . , P a r t I , A. Burger, e d . , Wiley, New York. 1970, p. 108. R. Wolfenden, Ann. Rev. Biophys. Bioeng. 2, 2 7 1 (1976). R. J . P. Williams, Angew. Chem. I n t . Ed. Engl. l6, 766 (1977). L. E d e l s t e i n and R . Rosen, J. Theor. B i o l . 73. 181 (1978). P. Gund. i n "Progress i n M o l e c u l a r and S u b c e l l u l a r Biology", Vol. 5 , F. E. Hahn, ed., Springer-Verlag. New York, 1977, p. 1 1 7 . G . M. Crippen, J. Comput. Phys. 24, 96 (1977). L. B. K i e r . i n "Fundamental Concepts i n Drug-Receptor I n t e r a c t i o n s " , J. F. D a n i e l l i , J. F. Moran and D. J. T r i g g l e , Eds., Academic, New York, 1970, p. 15. See a l s o L. 8. Kier. "Molecular O r b i t a l Theory i n Drug Research", Academic, N e w York, 1970. A. Korolkovas, " E s s e n t i a l s of Molecular Pharmacology", I n t e r s c i e n c e , New York, 1970. T. A. Andrea, E. C. J o r g e n s e n , P. A. Kollman, I n t . J. Quantum Chem., Quantum B i o l . S p p . -5, 191 (1978). S. W. D i e t r i c h , M. B. Boger, P. A. Kollman, and E. C. Jorgensen. J. Med. Chem. 20, 863 (1977) P. Gund and T. Y. Shen, J . Med. Chem. 20. 1146 (1977). H . D. H g l t j e , Arch. Pharm. 310. 650 (1977). D. C. Weaver, C . D. B a r r y , M. L. McDaniel, G. R . M a r s h a l l and P. E. Lacy, N a t u r e , submitted A . H. B e c k e t t and A. A. Al-Badr. J. Pharm. Pharmac. 27. 855 (1975). F. H. C l a r k e , H. J a g g i , R. A. L o v e l l . J. Med. Chem. 2, 600 (1978). G . H. Loew and S . K. B u r t , Proc. Nat. Acad. S c i . U.S.A. 11, 7 (1978). G . D. Smith and J. F. G r i f f i n , S c i e n c e 199, 1 2 1 4 (1978).

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f

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

62.

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Conf. Computers i n Chem. Res. and Educ.. L j u b l j a n a , 1973, Vol. 3. E l s e v i e r . Amsterdam, 1 9 7 4 , p . 5/33. K. Sundaram, S . Mahajan and R. K . M i s h r a , P h y s i o l . Chem. Phys.. 5. 469 ( 1 9 7 4 ) . Y . C . M a r t i n , " Q u a n t i t a t i v e Drug Design", M. Dekker. N e w York, 1978. J . G . T o p l i s s and J. Y. Fukunaga, Ann. R e p t s . Med. Chem. 13,292 (1978). C . Hansch, in " C o r r e l a t i o n A n a l y s i s in C h e m i s t r y : Recent Advances". N . B. Chapman and J. S h o r t e r , e d s . , Plenum, New York, 1 9 7 8 , p . 397. P. P a u l i n g , in " P s y c h o t h e r a p e u t i c Drugs", P a r t I , E. Usdin and I . S. F o r r e s t . e d s . , Dekker, New York, 1977, p. 59. H . W e i n s t e i n , R. Osman, W. D . Edwards, and J. P. G r e e n , I n t . J. Quantum Chem., Quantum B i o l . Symp. 5 , 449 ( 1 9 7 8 ) . T . D i p a o l o , L. H . H a l l , and L. B . K i e r , J. T h e o r . B i o l . 2, 295 (1978). B . W . Matthews in "The P r o t e i n s " , 3rd E d i t . , Vol. 3, H . N e u r a t h and R. L. H i l l , e d s . , Academic P r e s s . New York, 1977, p. 403. G . B o l t s , E. C l e m e n t i , M. R a g a z z i , D. S a l v a d e r i , and D. R. F e r r o . I n t . J . Quantum Chem. 14, 815 (1967). M. R . P i n c u s , S. S . Zimmerman, and H . S c h e r a g a , Proc. N a t . Acad. S c i . U.S.A. 2. 4261 (1976). A . Warshel a n d M. L e v i t t , J. Mol. B i o l . 103. 227 ( 1 9 7 6 ) . S . S c h e i n e r and W . N . Lipscomb, P r o c . N a t . Acad. S c i . U.S.A. 3. 432 ( 1 9 7 6 ) . H . M . S o b e l l , C . C. T s a i , S. C. J a i n , and S. G . G i l b e r t , J. Mol. B i o l . 333 (1977). J . M. Andrews, D. P. Roman, Jr., D. H . Bing, and M. Cory, J . Med. Chem. 21, 1202 (1978). R . J . Feldmann, D. H . Bing, B. C. F u r i e and B. F u r i e , P r o c . Nat. Acad. Sci. U.S.A. 5409 ( 1 9 7 8 ) . G . S t G f f l e r and 6 . W. T i s c h e n d o r f , i n "Drug R e c e p t o r I n t e r a c t i o n s in A n t i m i c r o b i a l Chemotherapy", J . D r e w s and F. E. Hahn, e d s . , S p r i n g e r - V e r l a g , Vienna, 1975. p. 1 1 7 . 0. Pongs, in "Drug A c t i o n a t t h e M o l e c u l a r L e v e l " , G . C . K. R o b e r t s , e d . , Univ. P a r k P r e s s , London, 1977, p . 190. R. J . H a r r i s and R . H . Symons, B i o o r g . Chem. 2 , 266, 286 (1973). B . V. Cheney, J. Med. Chem. 17,590 ( 1 9 7 4 ) . F. E . Hahn and P . Cund, in "Drug R e c e p t o r I n t e r a c t i o n s in A n t i m i c r o b i a l Chemotherapy". J. Drews and F. E . Hahn, e d s . . S p r i n g e r - V e r l a g , Vienna, 1975, p . 245. F. S e e l a and V. A . Erdmann, Biochem. Biophys. A c t a 435, 1 0 5 ( 1 9 7 6 ) .

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R. A. S c h e r r e r i n "Antiinflammatory Agents: Chemistry and Pharmacology", Vol. 1, R. A. S c h e r r e r and M. W. Whitehouse, Eds., Academic, New York, 1974, p. 29. B. R. Baker, "Design of A c t i v e - S i t e D i r e c t e d I r r e v e r s i b l e Enzyme I n h i b i t o r s " , Wiley, N e w York, 1967. C. Hansch, J. Y. Fukunaga, P. Y. C. Jow and J. B. Hynes, J . Med. Chem. 3, 96 (1977). M. Poe, J. B i o l . Chem. 252, 3724 (1977). P. Gund, M. Poe, and K. Hoogsteen, Mol. Pharmacol. l3, 111 (1977). S . Subramanian and B. T. Kaufman, Proc. Nat. Acad. S c i . U.S.A. 3201 (1978). D. A. Matthews, R. A. Alden, J . T. B o l i n , S . T. F r e e r , R. Hamlin. N. Xuong, J. Kraut, M. Poe, M. Williams, and K. Hoogsteen, S c i e n c e 197, 452 (1977). D. A. Mathews, R. A. Alden, J . T. B o l i n , D. J. Filman, S . T. F r e e r , N. Xuong, and J. K r a u t , i n "Chemistry and Biology of P t e r i d i n e s " . R. L . K i s l i n k and G. M. Brown, e d s E l s e v i e r , Amsterdam. 1979, p. 465. D . A. Mathews, R. A. Alden, J . T. B o l i n , D. J. Filman. S . T. F r e e r , R. Hamlin, W. G. J Hol, R. L. K i s l i n k , E. J. P a s t o r e , L. T. P l a n t e , N. Xuong and J. Kraut, J. B i o l . Chem., 253, 6946 (1978). J . C. Fontecilla-Camps. C. E. Bugg, C. Temple, Jr., J. D . Rose, J. A. Montgomery, and R. L. K i s l i n k , i n "Chemistry and Biology of P t e r i d i n e s " , R. L. K i s l i n k and G. M. Brown, eds., E l s e v i e r , Amsterdam, 1979, p. 465. M. A. O n d e t t i , B. Rubin, and D. W. Cushman, S c i e n c e 196,441 (1977). M. J . Jung, Ann. Repts. Med. Chem., _12, 249 (1978). P. J . Goodford, i n "Drug Action a t t h e Molecular Level", G. C. K. R o b e r t s , e d . , Univ. Park Press, London, 1977, p. 109.

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Ho-Leung Fung, Bruce J. Aungst and Richard A. Morrison School of Pharmacy, S t a t e U n i v e r s i t y of New York a t B u f f a l o Amherst, N.Y. 14260 I n t r o d u c t i o n - Pharmacokinetics as a s c i e n t i f i c d i s c i p l i n e h a s undergone r a p i d development o v e r t h e p a s t decade. The mathematics and a p p l i c a t i o n s U t i l i z a t i o n of of pharmacokinetics are w e l l d e s c r i b e d i n text-books; 1-3 pharmacokinetic c o n c e p t s and models t o a i d i n t h e i n d i v i d u a l i z a t i o n of dosage regimen i n p a t i e n t s w i t h d i f f e r e n t d i s e a s e s t a t e s is now commonp l a ~ e . ~ , 5The impact of p h a r m a c o k i n e t i c s on d r u g d e s i g n , however, is j u s t b e g i n n i n g t o b e r e a l i z e d . A r e c e n t symposium focused on t h e a p p r o a c h e s used i n t h e d e s i g n o f b i o p h a r m a c e u t i c a l p r o p e r t i e s through p r o d r u g s and analogs. 6 Pharmacokinetic i n p u t i n d r u g d e s i g n r e c o g n i z e s t h e f a c t t h a t , f o r some d r u g s , l a c k of optimum o r d e s i r a b l e t h e r a p e u t i c a c t i v i t y i s n o t due t o an inadequacy i n d r u g - r e c e p t o r s i t e i n t e r a c t i o n , b u t r a t h e r due t o a n i n a p p r o p r i a t e c o n c e n t r a t i o n a n d / o r time-course of drug p r e s e n t a t i o n t o t h e r e c e p t o r . For example, an o r a l anti-inflammatory a g e n t may r e q u i r e a r a t h e r h i g h o r a l dose f o r t h e r a p e u t i c a c t i v i t y i n s p i t e of a low ED50 when i n t r o d u c e d i n t r a v e n o u s l y . A s y n t h e t i c program aimed a t t h e p r e p a r a t i o n of improved d e r i v a t i v e s may b e n e f i t g r e a t l y from comparative p h a r m a c o k i n e t i c d a t a o b t a i n e d a f t e r o r a l and p a r e n t e r a l d o s i n g , a l t h o u g h p a r e n t e r a l u s e of t h e d r u g i n humans may n o t be a n t i c i p a t e d . Data o b t a i n e d from p r o p e r l y designed pharmacokinetic experiments may s u g g e s t t h e r e a s o n ( s ) why a h i g h o r a l dose i s r e q u i r e d , e.g. slow a b s o r p t i o n , poor o r a l a b s o r p t i o n , o r e x t e n s i v e f i r s t - p a s s metabolism. The a p p r e c i a t i o n t h a t s t r u c t u r a l m o d i f i c a t i o n can g r e a t l y a l t e r drug pharmacokinetics i s c e r t a i n l y n o t new. The b a r b i t u r a t e s p r e s e n t c l a s s i c examples of how minor s t r u c t u r a l changes can s i g n i f i c a n t l y a l t e r t h e i r d i s t r i b u t i o n and e l i m i n a t i o n c h a r a c t e r i s t i c s , g i v i n g rise t o a spectrum o f compounds w i t h v a r y i n g d e g r e e s of o n s e t and d u r a t i o n of a c t i v i t y . What i s perhaps n o v e l and e x c i t i n g i s a growing w i l l i n g n e s s on t h e p a r t of i n v e s t i g a t o r s t o probe t h e r e l a t i o n s h i p between p h a r m a c o k i n e t i c p a r a m e t e r s and s t r u c t u r e . The s t a t e of t h e a r t is of c o u r s e f a r from what h a s been achieved i n q u a n t i t a t i v 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 (QSAR). A t p r e s e n t , no u n i f i e d t h e o r y h a s been developed which would p e r m i t p r e d i c t i o n o f pharmacokinetic b e h a v i o r , even a q u a l i t a t i v e one, based s o l e l y on s t r u c t u r a l c o n s i d e r a t i o n s . However, t h e r e have been numerous examples i n t h e p a s t f e w y e a r s i n which c o n s i d e r a b l e improvement of p h a r m a c o k i n e t i c Colc h a r a c t e r i s t i c s have been o b t a i n e d through s t r u c t u r a l m o d i f i c a t i o n . l a t i o n o f t h e s e d a t a may e v e n t u a l l y a l l o w f o r comparison, and ( d a r e w e dream) p r e d i c t i o n , of pharmacokinetic p a r a m e t e r s based on s t r u c t u r e . I n t h e p r e s e n t c h a p t e r , w e review r e c e n t developments which focused on comThe pharmap a r a t i v e pharmacokinetics of s t r u c t u r a l l y s i m i l a r compounds. c o k i n e t i c phenomena d i s c u s s e d h e r e w i l l be c a t e g o r i z e d i n t o ( i ) a b s o r p t i o n , ( i i ) d i s t r i b u t i o n , and ( i i i ) e l i m i n a t i o n . Absorption - Much of t h e e f f o r t i n s y n t h e t i c m e d i c i n a l c h e m i s t r y i n v o l v e s a t t e m p t s t o c o n f e r o r a l a c t i v i t y t o p a r e n t e r a l l y a c t i v e d r u g s . Lack of o r a l a c t i v i t y due t o i n a d e q u a t e c o n c e n t r a t i o n s i n t h e s y s t e m i c c i r c u l a t i o n Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0-12-040514-8

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may be t h e r e s u l t of ( i ) g a s t r o i n t e s t i n a l drug d e g r a d a t i o n , ( i i ) poor a b s o r p t i o n , o r ( i i i ) e x t e n s i v e metabolism d u r i n g t h e f i r s t p a s s a g e of drug through t h e g u t and t h e l i v e r . An i n t e r e s t i n g example of t h e i n t e r a c t i o n s of t h e s e f a c t o r s was provided by t h e a b s o r p t i o n of sodium y-hydroxybutyr a t e (GHB), and i t s l a c t o n e a n a l o g , b u t y r o l a c t o n e (GBL).8,9,10 GHB i s an a c t i v e i n t r a v e n o u s a n e s t h e t i c i n man and h a s been s u g g e s t e d as a p o t e n t i a l l y u s e f u l a n t i - p a r k i n s o n i a n a g e n t because of i t s a b i l i t y t o i n c r e a s e b r a i n dopamine c o n c e n t r a t i o n s when i n j e c t e d i n t o animals. Its o r a l a c t i v i t y a t low doses i s poor because of s u b s t a n t i a l h e p a t i c metabolism d u r i n g a b s o r p t i o n . 8 However, even when o r a l d o s e s are i n c r e a s e d t o t h e e x t e n t where f i r s t - a s s metabolism i s s a t u r a t e d , o r a l h y p n o t i c a c t i v i t y i s s t i l l n o t r e a l i z e d . $ Pharmacokinetic experiments r e v e a l e d t h a t a t t h e s e h i g h doses, t h e b i o a v a i l a b i l i t y of GHB w a s c l o s e t o u n i t y b u t t h e peak plasma c o n c e n t r a t i o n s d i d n o t i n c r e a s e p r o p o r t i o n a t e l y w i t h dose. Appare n t l y , GHB t r a n s p o r t a c r o s s t h e s m a l l i n t e s t i n e became c a p a c i t y - l i m i t e d w i t h i n c r e a s i n g dose and t h e rate of drug a b s o r p t i o n d e c r e a s e d t o s u c h a n e x t e n t t h a t t h r e s h o l d h y p n o t i c plasma c o n c e n t r a t i o n s were n o t a c h i e v e d , even w i t h v e r y l a r g e o r a l d o s e s . GBL w a s found t o h y d r o l y s e r a p i d l y i n plasma, w i t h a h a l f - l i f e of approximately 1 minute. In s p i t e of t h i s r a p i d b i o t r a n s f o r m a t i o n , o r a l a c t i v i t y of GBL w a s almost i d e n t i c a l t o t h a t achieved a f t e r i n t r a v e n o u s Apparently, GBL i s n o t s u b j e c t e d t o any of t h e a d m i n i s t r a t i o n of GHB." p r o c e s s e s which d e c r e a s e peak plasma c o n c e n t r a t i o n s , v i z : g a s t r o i n t e s t i n a l d e g r a d a t i o n , c a p a c i t y - l i m i t e d t r a n s p o r t and f i r s t - p a s s metabolism. The u s e of l a c t o n e a n a l o g s as a g e n e r a l approach t o improve t h e b i o a v a i l a b i l i t y a n d / o r r a t e of a b s o r p t i o n of o t h e r hydroxy-acids, e . g . p r o s t a g l a n d i n s , h a s , however, n o t been t e s t e d .

Drugs c o n t a i n i n g t h e c a t e c h o l and phenol f u n c t i o n a l groups p r e s e n t i n t e r e s t i n g examples of how a b s o r p t i o n and metabolism can b e a l t e r e d through minor s t r u c t u r a l m o d i f i c a t i o n . These groups are v u l n e r a b l e t o conj u g a t i o n , p o s s i b l y b o t h i n t h e g u t and i n l i v e r , and e s t e r i f i c a t i o n o f them could s u b s t a n t i a l l y d e c r e a s e t h e e x t e n t of such m e t a b o l i c i n a c t i v a t i o n . M i n a t o y a l l showed t h a t b i t o l t e r o l , t h e d i - p - t o l u a t e ester of N - t e r t b u t y l a r t e r e n o l , gave good b r o n c h o d i l a t o r a c t i v i t y w i t h a more prolonged d u r a t i o n o f a c t i o n t h a n i t s p a r e n t compound o r i s o p r o t e r e n o l when adminis t e r e d i n t r a d u o d e n a l l y . More i n t e r e s t i n g l y , b i t o l t e r o l appeared t o d i s t r i b u t e p r e f e r e n t i a l l y t o l u n g t i s s u e s as compared t o plasma o r h e a r t , thereby e l i c i t i n g t h e b r o n c h o d i l a t o r a c t i o n w h i l e s i m u l t a n e o u s l y r e d u c i n g u n d e s i r a b l e c a r d i o v a s c u l a r e f f e c t s . The slow i n v i v o h y d r o l y s i s of b i t o l t e r o l a p p a r e n t l y allowed d i s t r i b u t i o n t o t i s s u e s b e f o r e i t s b i o t r a n s f o r m a t i o n , l e a d i n g t o a b e n e f i c i a l s e l e c t i v i t y i n i t s pharmacological a c t i v i t y . Bodor e t a l l 2 p r e p a r e d pro-drugs of L-dopa w i t h a t t a c h m e n t s on t h e t h r e e m e t a b o l i c a l l y s e n s i t i v e c e n t e r s of t h e molecules, v i z : t h e c a r b o x y l i c a c i d , t h e amino and c a t e c h o l groups. Plasma c o n c e n t r a t i o n s of L-dopa and dopamine were o b t a i n e d i n dogs a f t e r o r a l a d m i n i s t r a t i o n of t h e s e d e r i v a t i v e s , which i n c l u d e d c a r b o x y l i c a c i d esters, phenol esters, amides, p e p t i d e s and v a r i o u s combinations of t h e s e f u n c t i o n s . Although some of t h e s e d e r i v a t i v e s could i n c r e a s e t h e b i o a v a i l a b i l i t y o f L-dopa by 2.5-fold, t h e e f f e c t w a s le ss t h a n t h e 5-fold r e d u c t i o n i n L-dopa d o s e requirement which could be b r o u g h t about v i a c o - a d m i n i s t r a t i o n of t h e dec a r b o x y l a s e i n h i b i t o r , L-a-methyl dopa hp f y f i n e . More d r a m a t i c r e s u l t s and adrenalone15 s y n t h e s i z e d w e r e o b t a i n e d w i t h esters of e p i n e p h r i n e

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et a l l 4 showed t h a t t h e d i p i v a l y l ester f o r improved glaucoma t h e r a p y . Wai of e p i n e p h r i n e w a s a b o u t 1 0 t i m e s b e t t e r absorbed t h a n e p i n e p h r i n e , most probably due t o i t s i n c r e a s e d l i p o p h i l i c i t y . Some of t h e a-adrener i c r e c e p t o r b l o c k i n g d r u g s a l s o show c o n s i d e r For example, t h e b i o a v a i l a b i l i t y of o r a l a b l e f i r s t - p a s s metabolism. i) s low and i s s u b j e c t e d t o wide i n t e r - s u b j e c t v a r i a t i o n s . p r o p r a n o l o l (l A major m e t a b o l i t e formed, 4-hydroxy-propranolol, is a l s o an a c t i v e Bet _ a l l 7 found t h a t t h e h e m i s s u c c i n a t e ester of gave b l o c k e r . Garceau _ c o n s i d e r a b l y h i g h e r and l e s s v a r i a b l e o r a l a b s o r p t i o n i n dogs t h a n a corresponding d o s e of 1. I t w a s s u g g e s t e d t h a t b l o c k i n g of t h e hydroxy groups i n 1reduced 0 - g l u c u r o n i d a t i o n d u r i n g f i r s t - p a s s metabolism. Exami n a t i o n of t h e b i o a v a i l a b i l i t y of o t h e r B-blockers, t o g e t h e r w i t h t h a t of 1, (Table I ) , showed t h a t b l o c k i n g of t h e p o s i t i o n p a r a t o t h e amino a l c o h o l s i d e c h a i n , such as i n 4, 5, 6 and 8, l e a d s t o s u b s t a n t i a l S u b s t i t u t i o n of the n a p h t h y l n u c l e u s i n c r e a s e s in o r a l b i o a v a i l a b i l i t y . by an i n d o l e r i n g , a s i n 9, a l s o f a v o r a b l y a f f e c t s b i o a v a i l a b i l i t y . do n o t i n c r e a s e b i o a v a i l a b i l Ortho s u b s t i t u t i o n s , such as i n 2, 2 and i t y significantly.

"

L

1,

@

R

= H, R

R:

= H , R 2 = -OCH2CH 2

4:

R1

=

5:

R1 = -CH 2CH 2OCH3,

R2

6: -

R1

= -H

3: R,I

2, 3, 4, 5, 5

1 -

=

2:

$R2

-OCH2CH(OH)CH2NHCH(CH3)2

R

-

= -CH

CH = CH

2

= CH2

2 -CH2CONH2, R2 = -H

= -NHCOCH3,

R2

=

-H

OH

I

cH3& OCH2CHCH2NHCH2CH20 @

CONH2

I

NHS02CH3

H

8 -

7 Table I:

2

$2mcH(cH3)

9 -

Comparative b i o a v a i l a b i l i t y of B-blockers. Compound

aiprenolol propranolol toiamolol oxprenolol atenolol m e top r o 1o 1 sot a l o l pindolol prac t o l o l

2 1 7

Bioavailability (% of d o s e )

= = =

10 30 30 24-60 3 40

6 -

Do se-dep end en t B i o a v a i l a b i l it y Yes Yes

50

No No No

>, 60 = 100 = 100

No No

= 9 -

16

312

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The a n t i a r r h y t h m i c d r u g l i d o c a i n e (10) i s a l s o s u b j e c t e d t o extens i v e f i r s t - p a s s metabolism. Its m e t a b o l i t e s , monoethylglycinexylidide and g l y c i n e x y l i d i d e , are pharmacologically a c t i v e . The former shows a n t i a r r h y t h m i c and convulsive p r o p e r t i e s and i s a p o t e n t emetic a g e n t i n animals,whereas t h e l a t t e r p o t e n t i a t e s t h e c o n v u l s i v e e f f e c t s of b o t h Tocainide (11) however, i s l i d o c a i n e and monoethylglycinexylidide.18 a p p a r e n t l y n o t s u b j e c t e d t o f i r s t - p a s s metabolism. M e z l e t i n e also has a h i g h o r a l b i o a v a i l a b i l i t y . Both 11 and p o s s e s s a methyl group a l p h a t o t h e amino n i t r o g e n . 10: R -NHCO-,R =H,R =C H 1 2 2 5

(12)

12

11: -

R = -NHCO-,R1=CH

12: -

R = -OCH2-,R1=CH

3 3

,R =H 2

,R =H 2

C o n s i d e r a b l e s u c c e s s h a s been o b t a i n e d i n p r e p a r i n g o r a l l y active d e r i v a t i v e s of p e n i c i l l i n s . Many of t h e s e compounds are p l a s m a - l a b i l e esters, e.g. p i v a m p i c i l l i n and b a c a m p i c i l l i n , b u t a n o n - l a b i l e a n a l o g such as a m o x y c i l l i n can a l s o s u b s t a n t i a l l y i n c r e a s e a b s o r p t i o n Recently, l a c t o n y l esters of a m p i c i l l i n and o t h e r p e n i c i l l i n s , and acyloxymethyl esters of a cephalosporin2' w e r e prepared and found t o have s u p e r i o r o r a l a b s o r p t i o n c h a r a c t e r i s t i c s compared t o t h e i r r e s p e c t i v e p a r e n t compounds. M o d i f i c a t i o n of drug a b s o r p t i o n r a t e can a l s o l e a d t o improvement of t h e r a p e u t i c e f f i c a c y . T h i s i s u s u a l l y accomplished by a l t e r i n g t h e d i s s o l u t i o n c h a r a c t e r i s t i c s of t h e drug v i a f o r m u l a t i o n t e c h n i q u e s r a t h e r than through s t r u c t u r a l m o d i f i c a t i o n . A r e c e n t a t t e m p t of t h e l a t t e r approach i n v o l v e s t h e p r e p a r a t i o n of 4-6-methyl d i g o x i n which showed improved a b s o r p t i o n o v e r t h a t of d i g o x i n i n humans,21s22 b u t n o t g r e a t e r c a r d i a c a c t i v i t y . 21 A s t o i c h i o m e t r i c complex w a s a l s o used t o i n c r e a s e d i g o x i n a b s o r p t i o n r a t e . Higuchi and Ikeda23 prepared a hydroquinone complex of d i g o x i n which i s more r e a d i l y s o l u b l e t h a n d i g o x i n i t s e l f . B i o a v a i l a b i l i t y t e s t i n g i n humans showed t h a t t h i s complex y i e l d e d f a s t e r d i g o x i n a b s o r p t i o n t h a n t h a t provided by a commercial t a b l e t . Decrease i n a b s o r p t i o n r a t e , however, i s d e s i r a b l e i n t h e c a s e of t h e o p h y l l i n e because of t h e n e c e s s i t y f o r f r e q u e n t d o s i n g due t o i t s r e l a t i v e l y s h o r t e l i m i n a t i o n h a l f - l i f e and narrow t h e r a p e u t i c index. et a125 prepared a series of 7-acyl and 7 , 7 ' - a c y l d i t h e o p h y l l i n e Bodor d e r i v a t i v e s . These a u t h o r s suggested t h a t 7 , 7'-succinylditheophylline may have t h e d e s i r a b l e d i s s o l u t i o n c h a r a c t e r i s t i c s f o r i t t o be a u s e f u l c o n t r o l l e d r e l e a s e form of t h e o p h y l l i n e i n vivo. Improvement of d r u g s o l u b i l i t y may a l s o be n e c e s s a r y because an i n et a126 showed t h a t t h e j e c t a b l e form of t h e drug i s d e s i r e d . Bourne _ a n t i t u m o r compound a c r o n i n e can be transformed i n t o t h e s o l u b l e and l a b i l e a c e t y l a c r o n i n i u m s a l t s which may be s u i t a b l e f o r i n t r a v e n o u s use. Absorption k i n e t i c s are an i m p o r t a n t c o n s i d e r a t i o n f o r drugs which produce t o x i c i t y a t t h e a b s o r p t i o n s i t e . Decrease i n g a s t r o i n t e s t i n a l i r r i t a t i o n s o f a c e t y l s a l i c y l i c a c i d w a s accomplished v i a an a n a l o g d i f l u s i n a l , which i s 2',4'-difluoro-4-hydroxy-3-biphenylcarboxylic a c i d 2 7 . This compound a p p a r e n t l y emerged a f t e r pharmacologic s c r e e n i n g

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of s e v e r a l hundred a n a l o g u e s of s a l i c y c l i c a c i d , and i s s a i d t o p o s s e s s i n c r e a s e d a n a l g e s i c and a n t i - i n f l a m m a t o r y a c t i v i t y . T r i g l y c e r i d e s cont a i n i n g a s p i r i n i n p l a c e of one o r more f a t t y a c i d r e s i d u e s of t h e molecule have a l s o been s y n t h e s i z e d . The 1, 3 - d i - f a t t y a c y l - 2 - a s p i r i n g l y c e r i d e appeared t o be p a r t i a l l y absorbed through t h e r a t i n t e s t i n e ; i t d i d n o t produce u l c e r a t i o n i n t h e r a t stomach. D i s t r i b u t i o n - The e x t e n t of d i s t r i b u t i o n of a drug t o e x t r a v a s c u l a r areas can b e r e p r e s e n t e d p h a r m a c o k i n e t i c a l l y e i t h e r by measurement of t i s s u e d r u g c o n c e n t r a t i o n s i n l a b o r a t o r y a n i m a l s , o r by c a l c u l a t i o n of t h e a p p a r e n t volume o f d i s t r i b u t i o n (vd). For t h o s e d r u g s t h a t d i s p l a y monoe x p o n e n t i a l decay, vd i s a p r o p o r t i o n a l i t y f a c t o r r e l a t i n g t h e d r u g c o n c e n t r a t i o n i n serum o r plasma t o t h e amount of d r u g i n t h e body. A l a r g e vd i m p l i e s t h a t t h e amount of drug i n t h e body i s e x t e n s i v e l y d i s t r i b u t e d o u t s i d e t h e plasma s p a c e . Most d r u g s , however, e x h i b i t m u l t i - e x p o n e n t i a l d e l a y and are f i t t e d t o a two-compartment model. I n such cases, vd may b e c a l c u l a t e d by s e v e r a l methods1 producing s u c h volume terms a s Vd(B), V d ( a r e a ) , Vd ( e x t r a p o l a t e d ) and Vd ( s t e a d y s t a t e ) . These volumes o f t e n do n o t have t h e same v a l u e s . However, each can have meaningful a p p l i c a t i o n as described i n d e t a i l elsewhere. 1 The e x t e n t of d i s t r i b u t i o n of a d r u g t o t h e e x t r a v a s c u l a r s p a c e i s i n f l u e n c e d by: ( i ) t h e d e g r e e of i o n i z a t i o n of t h e d r u g , ( i i ) l i p o p h i l i c i t y and t i s s u e l i p i d c o n t e n t , ( i i i ) plasma p r o t e i n and t i s s u e b i n d i n g , ( i v ) t i s s u e p e r f u s i o n , and (v) s p e c i a l i z e d t r a n s p o r t p r o c e s s e s . The r e l a t i o n s h i p s between p a r t i t i o n c o e f f i c i e n t , i o n i z a t i o n , and vd have been examined and mathematical e x p r e s s i o n s d e r i v e d f o r p r e d i c t i n g Vd based on t h e s e physicochemical p r ~ p e r t i e s . ~ gThe e f f e c t s of b i n d i n g on Vd have also been d i s c u s s e d i n a r e c e n t review30 and d e s c r i b e d q u a n t i t a t i v e l y by G i b a l d i and McNamara31. vd i s r e l a t e d t o t h e f r e e f r a c t i o n of drug i n plasma ( f p ) , f r e e f r a c t i o n of drug i n t h e t i s s u e s ( f T ) , volumes of plasma (V,) and t h e t i s s u e compartment (VT), i n t h e f o l l o w i n g manner : f Vd = v + 4 V T fT I n g e n e r a l , o n l y non-ionized, f r e e drug i s a v a i l a b l e f o r t i s s u e d i s t r i b u t i o n and t h e e x t e n t of t i s s u e d i s t r i b u t i o n i s r e l a t e d t o l i p i d s o l u b i l i t y . I n t h e d e s i g n of new d r u g s , t h e u t i l i t y of e s t i m a t i n g vd i s t h a t i t might a f f o r d a q u a l i t a t i v e e s t i m a t i o n of drug c o n c e n t r a t i o n a t t h e s i t e of a c t i o n , whether t h e e f f e c t i s r e l a t e d t o c o n c e n t r a t i o n i n plasma, as f o r w a r f a r i n , 3 2 o r t o t i s s u e c o n c e n t r a t i o n s as w i t h $-methyl-digoxin and i t s m e t a b o l i t e d i g o x i n . 33 For example, a d m i n i s t r a t i o n of t h e methoxymethyl ester of h e t a c i l l i n r e s u l t e d i n more e x t e n s i v e t i s s u e d i s t r i b u t i o n of a m p i c i l l i n t h a n when a m p i c i l l i n i t s e l f w a s given.34 T h i s c o u l d b e pred i c t e d on t h e b a s i s of t h e r e p o r t e d vd v a l u e s of a m p i c i l l i n and t h e h e t a c i l l i n est er which are approximately 30% and 85% of body w e i g h t , r e s p e c t i v e l y . Decreased t i s s u e d i s t r i b u t i o n of adriamycin w a s observed when i t w a s a d m i n i s t e r e d as DNA-complex. 35 Thus, s t r u c t u r a l m o d i f i c a t i o n f o r s e l e c t i v e drug d i s t r i b u t i o n i s a p o t e n t i a l avenue f o r enhancing e f f i c a c y .

3 14

Sect. V I

-

Topics i n Chemistry and Drug Design

Renfroe, Ed.

The two c l a s s e s of p h a r m a c e u t i c a l s which b e s t exemplify t h i s r a t i o n a l e f o r drug d e s i g n a r e t h e a n t i m i c r o b i a l and a n t i t u m o r a n t i b i o t i c s . I n c o n s i d e r i n g t h e d i s t r i b u t i o n of t h e 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 i n t h e body, t h e v e r y i m p o r t a n t r o l e of plasma p r o t e i n b i n d i n g must b e recognized. Both of t h e s e groups of a n t i m i c r o b i a l s d i s p l a y a wide s p a n i n t h e e x t e n t of drug bound t o plasma p r o t e i n s , r a n g i n g from 20 t o 96% f o r t h e p e n i c i l l i n s and 6 t o 96% f o r t h e c e p h a l o s p o r i n s . Accordingly, t h e a p p a r e n t volumes of d i s t r i b u t i o n of t h e s e d r u g s have been c o r r e l a t e d w i t h b o t h p e r c e n t b i n d i n g and l i p o p h i l i c i t y . 36 Binding c h a r a c t e r i s t i c s must a l s o b e c o n s i d e r e d i n i n t e r p r e t i n g i n v i t r o d e t e r m i n a t i o n s of minimum i n h i b i t o r y c o n c e n t r a t i o n s , as o n l y t h e unbound drug i s b i o l o g i c a l l y active.37 Because of t h e v a r i a b l e b i n d i n g and l i p i d s o l u b i l i t y c h a r a c t e r i s t i c s , t h e 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 a l s o e x h i b i t v a r y i n g d e g r e e s of d i s t r i b u t i o n i n t o t i s s u e s i t e s . Drug p e n e t r a t i o n t o t h e i n f e c t i v e s i t e i s a major f a c t o r i n s e l e c t i o n of t h e a n t i b i o t i c of c h o i c e and should a l s o be a major f a c t o r i n d e s i g n of new d r u g s . The v a l u e of pharmacok i n e t i c s i n s u c h s i t u a t i o n s c a n be i l l u s t r a t e d w i t h t h e r e s u l t s of s e v e r a l r e c e n t i n v e s t i g a t i o n s of t h e d i s t r i b u t i o n of c e p h a l o s p o r i n s i n t o i n t e r s t i t i a l f l u i d . Tan and S a l ~ t r o m ,u ~s i~n g a s k i n window t e c h n i q u e , concluded t h a t p e n e t r a t i o n of human i n t e r s t i t i a l f l u i d by However, c e f a z o l i n w a s poor compared t o c e p h a l o t h i n and cefamandole. Carbon e t a139 found t h a t c e f a z o l i n was s u p e r i o r t o cefamandole and cepha l o r i d i n e i n p e n e t r a t i n g r a b b i t i n t e r s t i t i a l f l u i d using t i s s u e cages. I n a d d i t i o n t o t h e s p e c i e s and model d i f f e r e n c e s i n t h e s e two s t u d i e s , only t h e l a t t e r w a s done f o l l o w i n g m u l t i p l e dosing. These d i f f e r e n c e s could be r e s o l v e d by c a l c u l a t i o n of t h e a p p r o p r i a t e p h a r m a c o k i n e t i c parameters. Bergan h a s made such c a l c u l a t i o n s f o r c e f a z o l i n and s e v e r a l o t h e r c e p h a l o s p o r i n s i n humans and found c e f a z o l i n t o b e t h e drug of c h o i c e i n d i s t r i b u t i n g t o t i s s u e f l u i d s . 4 0 T h i s c o n c l u s i o n w a s based on t h e a p p a r e n t volume of t i s s u e compartment, c a l c u l a t e d from vd ( s t e a d y s t a t e ) and a comparison of t h e magnitudes of t h e t r a n s f e r r a t e c o n s t a n t s k12 ( e n t r y i n t o t i s s u e ) and k21 ( e x i t from t i s s u e ) . Of t h e cephalospor i n s s t u d i e d , only c e f a z o l i n had a k12/k21 r a t i o g r e a t e r t h a n u n i t y , a p p a r e n t l y i n d i c a t i n g t h a t c e p h a z o l i n e n t r y i n t o t i s s u e s i s more r a p i d than i t s e x i t . I t should b e mentioned, however, t h a t i n view of t h e v a r i a t i o n s i n l i p i d c o n t e n t and b i n d i n g p r o p e r t i e s of d i f f e r e n t t i s s u e s , such c a l c u l a t i o n s may have somewhat l i m i t e d u s e f u l n e s s i f i n f o r m a t i o n regarding the d i s t r i b u t i o n i n t o a p a r t i c u l a r t i s s u e is desired. Recent advances i n t h e f i e l d of p h y s i o l o g i c a l p h a r m a c o k i n e t i c s , i n which o r g a n blood flows, volumes, and d r u g c l e a r a n c e s are c o n s i d e r e d , have been u s e f u l i n e s t i m a t i n g d r u g c o n c e n t r a t i o n s i n a s p e c i f i c o r g a n . These models have been a p p l i e d f o r t h e most p a r t t o a n t i t u m o r a g e n t s . For example, s u c h a model h a s been used t o show which a n t i c a n c e r a g e n t s might be most e f f e c t i v e i n chemotherapy of o v a r i a n c a n c e r v i a p e r i t o n e a l drug a d m i n i s t r a t i o n . 41 One method t h a t h a s been used t o improve s e l e c t i v e l o c a l i z a t i o n of chemotherapeutic a g e n t s i s v i a enzymatic drug a c t i v a t i o n . 4 2 T h i s i n v o l v e s c o n v e r s i o n of t h e i n a c t i v e p a r e n t compound t o t h e a c t i v e d r u g s e l e c t i v e l y a t t h e s i t e of a c t i o n . I n t h e c o u r s e of d e v e l o p i n g a n t i c a n c e r a g e n t s t o l o c a l i z e i n t h e p r o s t a t e gland, d e r i v a t i v e s of c o l c h i c i n e w e r e made as

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s u b s t r a t e s s p e c i f i c f o r p r o s t a t i c a c i d p h o s p h a t a s e , t h e a c t i v a t i n g enzyme which c o n v e r t s t h e prodrug t o c ~ l c h i c i n e . A ~ ~s i m i l a r mechanism f o r s e l e c t i v e d i s t r i b u t i o n h a s been proposed f o r d e l i v e r y of r a d i o l a b e l e d g l u c o s e t o t h e b r a i n and h e a r t f o r u s e as imaging a g e n t s , w i t h t r a p p i n g 44 of t h e i s o t o p e i n t h e s e t i s s u e s b e i n g r e f l e c t i v e of g l u c o s e u t i l i z a t i o n . I n a d d i t i o n t o t h e p o s s i b i l i t y of improving t h e r a p e u t i c e f f e c t s , s t r u c t u r a l m o d i f i c a t i o n s u t i l i z i n g pharmacokinetic a n a l y s i s of d r u g d i s t r i b u t i o n can e x p l a i n and p o t e n t i a l l y a v o i d t o x i c i t i e s by s e l e c t i v e d i s t r i b u t i o n away from s p e c i f i c t i s s u e s . T h i s h a s been demonstrated i n r e l a t i o n t o aminoglycoside n e p h r o t o x i c i t y , 4 5 and accumulation of B-adrenoc e p t o r a n t a g o n i s t s i n t h e l u n g s . 4 6 However, t h e f u l l p o t e n t i a l f o r t h e u s e of pharmacokinetic d i s t r i b u t i o n volumes i n drug development h a s y e t t o be r e a l i z e d . E l i m i n a t i o n - E l i m i n a t i o n i s d e f i n e d h e r e as e x c r e t i o n a n d / o r metabolism o f i n t a c t drug from plasma. I n g e n e r a l , t h e main e x c r e t i o n r o u t e s are r e n a l and b i l i a r y , and t h e predominant m e t a b o l i c r o u t e i s h e p a t i c . The pharmacokinetic p a r a m e t e r s most widely used t o c h a r a c t e r i z e plasma conc e n t r a t i o n decay are t h e a p p a r e n t f i r s t - o r d e r r a t e c o n s t a n t (K) , eliminat i o n h a l f - l i f e ( t 1 / 2 ) , and c l e a r a n c e ((21). C l e a r a n c e i s d e f i n e d as t h e volume of plasma removed of d r u g i n u n i t t i m e and can b e compared w i t h p h y s i o l o g i c organ p e r f u s i o n rates. For d r u g s w i t h mono-exponential decay, C 1 = Vd K. When more t h a n one r o u t e of e l i m i n a t i o n e x i s t s , t h e sum of i n d i v i d u a l c l e a r a n c e s e q u a l s t h e t o t a l plasma c l e a r a n c e : ‘’total

-

‘‘metabolic

+

‘‘renal

+

‘lbiliary

i‘lothers

S t r u c t u r a l m o d i f i c a t i o n s may a f f e c t one o r more r o u t e s o f c l e a r a n c e , t h e r e b y c r e a t i n g a net: e f f e c t on t o t a l plasma c l e a r a n c e . I n drug d e s i g n , a l t e r a t i o n s i n e l i m i n a t i o n r a t e a r e o f t e n d e s i r e d f o r drugs which are c l e a r e d too r a p i d l y i n t h e body. When m e t a b o l i c c l e a r a n c e i s predominant, examination of t h e r e l a t i v e amounts of metabol i t e s formed may i d e n t i f y t h e l a b i l e o r r e a c t i v e f u n c t i o n a l groups which need t o be modified o r p r o t e c t e d t o d e c r e a s e e l i m i n a t i o n . The p r o s t a g l a n d i n s , f o r example, g e n e r a l l y have a s h o r t d u r a t i o n of a c t i o n due t o r a p i d metabolism and e x c r e t i o n . The main m e t a b o l i c enzymes i n v o l v e d a r e t h e 15-hydroxyl dehydrogenase and B-oxidative systems. A compound t h a t i s n o t a s u b s t r a t e f o r t h e s e systems and y e t r e t a i n s p r o s t a g l a n d i n a c t i v i t y i s o b v i o u s l y d e s i r a b l e . R e c e n t l y , f o r example, cis-A4-15(S)methyl p r o s t a g l a n d i n Flcl w a s found t o meet t h e s e c r i t e r i a . 4 7 A f t e r o r a l a d m i n i s t r a t i o n of t h e methyl ester of t h i s p r o s t a g l a n d i n i n t h e r a t , 4 8 u r i n e w a s v o i d of t h e 15-keto m e t a b o l i t e and showed low l e v e l s of d i n o r and i n t a c t C-20 compounds, s u g g e s t i n g t h a t t h e dehydrogenase s y s t e m w a s n o t o p e r a t i v e f o r t h e a n a l o g , and t h a t B-oxidation w a s s u b s t a n t i a l l y reduced. I n c o n t r a s t t o o t h e r p r o s t a z l a n d i n s , i n t a c t p a r e n t compound could be r e c o v e r e d i n u r i n e . P r o l o n g a t i o n of a n a l g e s i c a c t i v i t y of methadone has a l s o been a t t e m p t e d by r e p l a c i n g t h e m e t a b o l i c a l l y l a b i l e C-6 dimethylamino group w i t h a p i p e r i d i n o s p i r o f ~ n c t i o n . ~ gF r i n c k e e t a l s o gave o r a l d o s e s a t about 0. 2 mg/kg of t h e k e t o n e , a l c o h o l and a c e t a t e forms of t h e p i p e r i d i n o s p i r o d e r i v a t i v e t o mice and found t h e d u r a t i o n of I n c o n t r a s t , o r a l methaa n a l g e s i a t o b e 3.0, 6 . 5 , 20.5 h r , r e s p e c t i v e l y . done a t a dose of 22.5 mg/kg only gave a d u r a t i o n of a n a l g e s i a of 8.0 h r .

3 16 -

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A recent r e p o r t d e s c r i b e d an i n t e r e s t i n g approach w i t h which a marked i n c r e a s e i n t h e d u r a t i o n of a c t i v i t y of t h e enzymes carboxypepti51 d a s e G and a r g i n a s e w a s e f f e c t e d through c o n j u g a t i o n w i t h s o l u b l e d e x t r a n . A s measured by enzyme a c t i v i t y i n t h e blood, t h e n a t i v e enzymes had r e s p e c t i v e h a l f - l i v e s of 3.5 and 1 . 4 h o u r s . When coupled w i t h d e x t r a n , t h e h a l f - l i v e s of t h e s e two enzymes i n blood r o s e d r a m a t i c a l l y t o 1 7 and 1 2 h o u r s , r e s p e c t i v e l y . Attempts t o p r o l o n g drug a c t i v i t y through i n c o r p o r a t i o n of an a l k y l a t i n g group on t h e drug h a s been i n v e s t i g a t e d . The r e a c t i v e group presumably b i n d s w i t h a n u c l e o p h i l e a t o r n e a r t h e r e c e p t o r s i t e , t h e r e b y i n c r e a s i n g d r u g r e s i d e n c e t i m e . Kornet e t alS2 s t u d i e d d e r i v a t i v e s of s u c c i n i m i d e s w i t h a l k y l a t i n g groups a t t h e 2p o s i t i o n and found t h e most s t r i k i n g i n c r e a s e s i n t h e d u r a t i o n of a n t i c o n v u l s a n t e f f e c t w i t h t h e a-bromoamide and maleimidoethyl a n a l o g s . Both t h e s e a n a l o g s have a l o n g e r d u r a t i o n of e f f e c t (> 4 h o u r s ) t h a n t h e c u r r e n t l y a v a i l a b l e phensuximide and methsuximide. The t o x i c i t y of t h e s e a l k y l a t i n g d e r i v a t i v e s , however, is a n obvious concern.

The r e l a t i o n s h i p s between s t r u c t u r e , r e n a l c l e a r a n c e and t o t a l body c l e a r a n c e of series of s u b s t i t u t e d x y l i d i n e s were examined by B e r l i n a f t e r i n t r a v e n o u s i n f u s i o n of t h e s e compounds i n dogs Wahler e t a,' (Table 11). Table 11:

Compound #

11 -

13 14 15 16 17 18 19 -

20 -

Mean r e n a l and t o t a l body c l e a r a n c e of s u b s t i t u t e d x y l i d i n e s i n dogs.

R1 R2

'H 3

C2H5 H CH3 H CH3 CH3 H

R3

H H H H H H CH3 H CH3 H C2H5 H C2H5 H H C3H70 C2H5 C3H70

Renal C l e a r a n c e (ml/min)

14 19

T o t a l Body C l e a r a n c e (ml/min)

48

45 16

55 161 188 172 218

8 3

128

2

301

6

28

260

The d a t a s u g g e s t e d t h a t r e n a l c l e a r a n c e of t h e s e compounds w a s r e l a t i v e l y unimportant. A l k y l s u b s t i t u t i o n a t t h e amide n i t r o g e n a p p a r e n t l y i n c r e a s e d t o t a l body c l e a r a n c e w h i l e s i m i l a r s u b s t i t u t i o n a l p h a t o t h e amino n i t r o g e n d i d n o t s i g n i f i c a n t l y a l t e r t h i s parameter. I n t r o d u c t i o n of a p-OC3H7 moiety, 19 and 20, caused s u b s t a n t i a l i n c r e a s e s i n t o t a l body c l e a r a n c e when compared t o t o c a i n i d e (11). Prodrug f o r m a t i o n h a s been a w e l l - t e s t e d approach t o i n c r e a s e d u r a t i o n of a c t i v i t y of v a r i o u s drugs. A prodrug can b e designed such t h a t t h e r a t e of c o n v e r s i o n t o i t s p a r e n t compound becomes r a t e - l i m i t i n g . This would r e s u l t i n an i n c r e a s e i n t h e a p p a r e n t e l i m i n a t i o n h a l f - l i f e

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of t h e drug. Drugs t h a t p o s s e s s p h e n o l i c o r a l c o h o l i c hydroxyl groups u s u a l l y e x h i b i t s h o r t d u r a t i o n of a c t i o n because of t h e i r h y d r o p h i l i c i t y and t h e i r s u s c e p t i b i l i t y t o a t t a c k by c o n j u g a t i n g enzymes. For esters of t e r b u t a l i n e and apomorphine,55 i n c r e a s i n g s t e r i c h i n d r a n c e i n t h e a c y l moiety was found t o lower t h e i n v i t r o h y d r o l y s i s r a t e i n t h e presence of serum e s t e r a s e s . When i n j e c t e d i n t o mice, t h e d i - i s o b u t y r l apomorphine d e r i ~ a t i v e 5gave ~ d e t e c t a b l e b r a i n apomorphine c o n c e n t r a t i o n s f o r s i x hours compared t o o n l y 90 minutes when apomorphine i s admini s t e r e d . I n c r e a s e i n t h e d u r a t i o n of a c t i o n w a s a l s o observed w i t h t h e phosphate est er of A8-THC57 and t h e d i s t e r o i d y l e n o l esters of 17-6e s t r a d i 0 1 . 5 ~ I n t h e s e compounds, c o v a l e n t bonds were formed a t t h e m e t a b o l i c s i t e , s o t h a t c l e a v a g e of t h e prodrug must occur b e f o r e metabolism can t a k e p l a c e . However, i n h i b i t i o n of metabolism can a l s o be e f f e c t e d through s t r u c t u r e m o d i f i c a t i o n a t n e i g h b o r i n g non-metabolic l o c a t i o n s . For example, f o r m a t i o n of t h e 5 ' - v a l e r a t e e s t e r of 9-B-D a r a b i n o f u r a n o s y l a d e n i n e (ara-A) 59 9 6o a p p a r e n t l y made t h e d e r i v a t i v e r e s i s t a n t t o deamination. A d d i t i o n a l l y , t h i s ester s e r v e d as a competit i v e i n h i b i t o r of ara-A.59 Drug enantiomers may d i f f e r p h a r m a c o k i n e t i c a l l y as w e l l as pharmac o l o g i c a l l y . I n man, R(+) w a r f a r i n showed a s i g n i f i c a n t l y l o n g e r e l i m i n a t i o n h a l f - l i f e than t h e S(-) enantiomer (35 vs. 24 hours)61 and i n rats t h e same t r e n d w a s s e e n f o r t h e congener phenprocoumon.6a S i g n i f i c a n t , though less d r a s t i c , pharmacokinetic d i f f e r e n c e s were s e e n a f t e r a d m i n i s t r a t i o n of racemic amphetamine63 and f e n f l u r a m i n e . 64 I n b o t h c a s e s , t h e r e w a s s i g n i f i c a n t l y more d-enantiomer i n t h e b r a i n and plasma of r a t s a f t e r i n t r a p e r i t o n e a l i n j e c t i o n . Thus, c h o i c e of a p a r t i c u l a r enantiomer f o r c l i n i c a l u s e may be based on pharmacokinetic, a s w e l l as pharmacologic, grounds. T o x i c i t y d i f f e r e n c e s i n a series of a n a l o g s may b e r e l a t e d t o t h e i r r e l a t i v e e l i m i n a t i o n rates. N e t i l m i c i n , a new aminoglycoside a n t i b i o t i c , h a s been shown t o be less n e p h r o t o x i c t h a n gentamicin i n r a t s . 6 5 Pharmac o k i n e t i c a n a l y s i s i n man r e v e a l s t h a t t h e s e compounds have s i m i l a r h a l f l i v e s , w h i l e n e t i l m i c i n shows more e x t e n s i v e e x t r a v a s c u l a r d i s t r i b u t i o n and t h e r e f o r e , a f a s t e r plasma c l e a r a n c e . 6 6 S i m i l a r l y , metformin, a biguanide a n t i - d i a b e t i c , is le ss t o x i c t h a n phenformin r e p o r t e d l y because 1 2 h o u r s ) . 6 7 When a compound causes of i t s s h o r t e r h a l f - l i f e ( 1 . 5 v s . d i r e c t organ t i s s u e damage t h e t o x i c i t y i s more e a s i l y a s s e s s e d . The a n a l g e s i c s acetaminophen and p h e n a c e t i n b o t h appear t o c a u s e t h e i r et a16' h e p a t o t o x i c i t y v i a o x i d a t i o n of t h e amide n i t r o g e n s . Nelson reasoned t h a t t h i s r e a c t i o n could be blocked by N-methylation i n t h e s e compounds. Indeed, N-methylacetaminophen and N-methylphenacetin w e r e found t o have much reduced h e p a t o t o x i c i t y .

-

Summary - I t h a s been shown t h a t pharmacokinetic parameters can b e f a v o r a b l y a f f e c t e d by s t r u c t u r a l m o d i f i c a t i o n w i t h o u t , i n c e r t a i n cases, a l t e r a t i o n i n t h e pharmacological p r o p e r t i e s of t h e p a r e n t drug. D e t a i l e d pharmacokinetic i n f o r m a t i o n o b t a i n e d f o r l e a d compounds may p r o v i d e a v a l u a b l e guide as t o how subsequent s y n t h e s e s should be d i r e c t e d .

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A. Berlin-Wahlgn, J . C . Barkus, J . B . Keenaghan, M. Lebeaux and P.A. Tenthorey, Acta Pharm. S u e c i c a , 417 ( 1 9 7 7 ) . J. K r i s t o f f e r s s o n , L.A. Svensson and K. Tegngr, Acta Pharm. S u e c i c a , 11, 427 ( 1 9 7 4 ) . R . J . Borgman, R . J . B a l d e s s a r i n i and K.G. Walton, J . Med. Chem., 1 9 , 717 ( 1 9 7 6 ) . R. J . B a l d e s s a r i n i , N.S. Kula and G. Walton, Biochem. Pharmacol.,

14,

26, 1 7 4 9 (1977). 57.

H. Yoshimura, K. Watanabe, K. O g u r i , M. F u j i w a r a and S . Veki, J. Med. Chem., 21, 1 0 7 9 ( 1 9 7 8 ) .

320 58. 59. 60. 61. 62. 63. 64. 65. 66. 67.

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R. V i t a l i , S . G l a d i a l i , G. F a l c o n i , G. Celasco, M.A. S a c c a n i and R. G a r d i , J. Med. Chem., 20, 359 (1977). R.A. L i p p e r , S.M. Machkovech, J . C . Drach and W . I . Higuchi, Molec. Pharmacol., 14,366 (1978). D.C. Baker, T.H. H a s k e l l and S . R . P u t t , J . Med. Chem., 3, 1218 (1978). A. Breckenridge, M. O r m e , H. Wesseling, R . J . L e w i s and R. Gibbons, C l i n . Pharmacol. T h e r . , 15,424 (1974). W. Schmidt and E. J'ahnchen, J. Pharm. Pharmac., 2, 266 (1977). A. J o r i , S. Caccia and P . D e P o n t e , Xenobiotica, 8, 599 (1978). A. J o r i , P. De Ponte and S. C a c c i a , X e n o b i o t i c a , 5, 583 (1978). L u f t , F.C., M.H. Yue and S.A. K l e i t , Antimicrob. Agents Chemother. 10, 845, (1976). L . J . R i f f and G. Moreschi, Antimicrob. Agents Chemother., 11,609 (1977). C.R. S i r t o r i , G. F r a n c e s c h i n i , M. G a l l i - K i e n l e , G. C i g h e t t i , G . G a l l i , A. B o n d i o l i and F. C o n t i , C l i n . Pharmacol. T h e r . , 24, 683 (1978). S.D. Nelson, A . J . F o r t e and R . J . McMurtry, R e s . Corn. Chem. P a t h . and Pharmacol., 22, 61 (1978).

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Blaine M. Sutton, Smith Kline & French Laboratories, Philadelphia, PA 19101 Introduction - Inorganic materials, metals and metallic salts have been used in medicine since the dawn of time. Metallic ions have been shown to play vital roles in many biological processes. However, research in developing new metal containing molecules as therapeutic agents has been overshadowed by the vast amount of attention directed to the design of organic molecules with novel biological activity. Nevertheless, in recent years certain metals have been associated with specific therapeutic benefit. Research in the field of bioinorganic chemistry is making increasingly rapid progress with the help of new sophisticated instrumentation and analytical methodology. Earlier work in the use of metals and metal complexes in biology and medicine has been bibliographically described by Ellis.’ The therapeutic use of different metallic compounds as well as the benefit or detriment of their interaction with organic molecules has been reviewed.2 Cleare3 reported on the therapeutic use of metal complexes with special emphasis on the cancer chemotherapeutic utility of platinum complexes. This review will attempt to identify newer metallo-compounds which appear to owe their biological properties to the metal contained therein as well as the status of older ones which have attracted special attention. Since there is little if any structure-activity relationship between the different metals, they will be discussed in the order of their appearance in the periodic table. Group la Lithium - Lithium salts were used indiscriminately over the early part of this century for treatment of gout, epilepsy, insomnia, hypertension and as salt substitutes in cardiac disease. Unfortunately, toxicity associated with lithium salt therapy was not known at that time. Many cases of serious side effects and deaths resulted which led to their disuse. In recent years judicious use of lithium salts, primarily lithium carbonate, for the control of manic symptoms has reestablished their utility in clinical practice. Mania (affective disturbances) recently has been divided into two functional groups, bipolar and unipolar affective disorders. Bipolar disorders include both manic and depressive episodes whereas unipolar disorders show only recurrent depressive episodes. Patients with bipolar disorders usually have an early onset of the illness (20-35 years age) with high incidence of mania and suicide. Some evidence suggests that this condition is hereditary, transmitted through the X-chromosone. On the other hand, onset of unipolar disorders usually occurs at a later age (304 5 ) and hereditary association is less well defined. Recent extensive describe current indications for lithium therapy primarily in bipolar disorders and experimentally in unipolar depression as well as schizo-affective schizophrenia, alcoholism, premenstrual cramps and character disorders. These reports also call attention to the narrow therapeutic index associated with its use and the need for careful monitering of serum levels. Serum levels of 0.6 to 1.5 mEq per liter are usually sufficient for management of symptoms. A dose of 300mg of lithium carbonate t.i.d. or q.i.d. is recommended to maintain these Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

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levels and slow release formulations have been reported to be advantageous dosage forms.7 Levels in excess o f 1.5 mEq often produce toxic side effects leading to hypothyroidism and at times permanent neurologic damage. Lithium also can induce nephrogenic diabetes insipidus by inhibition of antidiuretic hormone (ADH), which gradually disappears when lithium is discontinued. Although the mechanism of beneficial action of lithium carbonate in manic conditions is not well understood, symptom changes have been correlated with cerebral-evoked potentials and EEG changes during treatStabilization of dopaminergic receptor sensitivity in the nigrasment. triatal dopamine pathway has been invoked as contributing to its effect on manic-depressive episodes.

'

Transition Elements - Group VIII-Iron - Current therapy for controllin normally high body iron content was reviewed in vol 13 of this series.f2abIron deficiency anemia remains frequent in many populations including the .affluent countries where dietary deficiencies are.not prevalent. Iron homeostatis is unique in that, unlike other trace metals, it is regulated primarily by absorption and not by excretion. Recent reports detail the mechanism of iron absorption across the intestinal mucosa in the lumen of the small intestine to receptors in the brush border of the mucosal cells and into the cell, followed by transportion of cellular iron into the plasma in combination with various chelating carriers. l 4 Ferrous sulfate remains the cheapest form of supplemental iron, although numerous pharmaceutical forms are available. 9 Parenteral administration of iron salts offers little or no advantage over oral administration and is associated with anaphylactic side effect potential as well as the risk of sarcoma development at the site of injection. However , intravenous administration of dextran iron complex ("Imferon") has been reported to produce a beneficial rise in hemoglobin in patients undergoing regular hemodialysis. l 8 Although ferric salts are not used therapeutically, ferric ion participates in many biochemical events. Physicochemical aspects of iron metabolism and contribution of the Fe(I1) - Fe(II1) redox couple, involving a one electron transfer, have been well described. Aisen19 reported evidence that free radical intermediates, resulting from this redox, may account in part for the toxicity of iron in iron overload. Inonophore A23187 has been reported2 to facilitate transport of Fe (11) across lipid membranes. Although toxicity of A23187 precludes its utilization in any therapeutic form, the potential exists for a non-toxic ionophore with these properties.

'

3

Alksne and Smith2' described the novel use of an iron acrylic compound for the stereotaxic thrombosis of intracranial aneurysms in patients who were poor risk candidates for surgery. The compound polymerized rapidly, did not fragment and was non-toxic. Good results were reported in experimental animals and humans. Osmium - Surgical removal of the synovial membrane (synovectomy) in the affected joints of rheumatoid arthritis (RA) patients is one means of giving relief to, as well as slowing the crippling process in patients with early active RA. Osmium tetroxide injected into the affected joints has been used since 1952 in Europe to perform a synovectomy by chemical means (synoviothesis). Neither procedure gives lasting results. However, the latter can be carried out easily with only a few days hospitalization

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and can be repeated without risk. Recent controlled studies support the benefit of chemical synovectomy.22 , 2 3 The clinical significance of the accompanying superficial damage to articular cartilage has not been determined although the results have not shown harmful late complication^.^^ In contrast knee joint of rabbits with antigen induced arthritis.25 Platinum - Intensive studies of different transition metal complexes in cancer chemotherapy have focused attention on platinum complexes as being of greatest potential. 26-32 Cis-platinumdiaminodichloride (PDD, NSC119875, "Platinol", cisplatin) has been approved recently for treatment of testicular and ovarian cancer as well as showing promise in other neoplastic conditions. 3 3 Its application in therapy is limited by renal, hematologic and "'"\ ototoxicity.3 4 3 3 5 The major limiting toxic effect is renal toxicity which appears to be dose related, rang/Pt, c1 ing from transient elevation of blood urea nitrogen H2N and serum creatinine to irreversible renal insufficiency. Similar toxicity is displayed by mercury and other heavy metals. Mannitol-induced diuresis, previously used to lessen acute renal toxicity produced by mercury, has likewise been shown to reduce the nephrotoxicity of cisplatin both e~perimentally~~ and clinically.3 6 Prehydration and concomitant osmotic diuresis improved the therapeutic index of cisplatin allowing for the administration of larger doses (3mg/kg/iv) which produced encouraging clinical responses in testicular carcinoma, carcinoma of the head and neck and ovarian carcinoma with reduced limiting side effects. Comparative renal pathological studies confirmed that mannitol-induced diuresis considerably decreased the renal toxicity associated with cisplatin therapy. 3 7 Flameless atomic absorption spectrometry has been reported38 to be a convenient and sensitive method for the analysis of platinium in biological material.

/"'

Although the mechanism of antineoplastic action of cisplatin has not been defined, physicochemical studies have shown an interaction with nucleosides, nucleotides, homopolynucleotides and DNA. Cisplatin has been reported to exhibit little binding affinity for thymidine and greater affinity for guanosine, adenosine and cytidine. 3 9 Interaction of cytidine, thymidine, adenosine and guanosine with cisplatin, measured by uv difference spectroscopy, led the authors to suggest that cisplatin bound selectively to GC base-pairs or GC rich regions of DNA as compared to AT regions. Other workers40 using transmission electron microscopy in conjunction with x-ray probe microanalysis have shown that HeLa cells treated in vitro with cisplatin concentrate platinum on the inner side of the nuclear double membrane and in the nucleolus, indicating that cell death may be due to Pt interference at the initiation sites of DNA synthesis. Cisplatin also was reported to decrease the level of testosterone-dependent esterases in kidneys of experimental animals. The association between this induced decrease in testosterone levels and testicular antitumor activity has not been answered. A related compound cis-platinum-(3,4-diaminotoluene)dichloride produced profound changes in the permeability of rat liver mitochondria. Phosphate transport into the mitochondria was inhibited irreversibly. Magnesium and calcium ions were rapidly released. Stimulation of mitochondria1 respiration, similar to the apparent uncoupling of

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oxidative phosphorylation, was observed. Association of these membrane effects with antitumor activity is being studied further.42 Group l b Copper - Copper in the metallic state, as well as different salt forms, has been used since antiquity for the treatment of different inflammatory conditions.43 A copper complex of salicylic acid ("Permalon") has been reported to be of clinical benefit in RA when administered intravenously.44 Copper chelates of a number of non-steroidal antiinflammatory agents (AI) were reported to be more potent and less ulcerogenic than the A1 agents themselves.45y46 Copper ions per se have been reported to afford protection against inflammation in some cases without the assistance of other agents.47 It has been suggested that the A1 drugs exert their beneficial effects by facilitating the release of cupric ions from serum albumin either by a direct competitive complexing mechanism or through a remote mechanism whereby the drug becomes bound to a site remote from the copper ion and facilitates its release by allosteric The beneficial effects of copper salts have been questioned because of conflicting results obtained in rat and guinea pig models of inflammation.52 A computer study has identified the critical chemical feature required in an A1 agent to exploit serum albumin bound copper.53 Although the mechanism of action of copper complexes is unknown, stimulation of superoxide dismutase has been proposed as a contributing factor.54 Gold - Monovalent gold, stabilized as a coordinated salt, was first used forthe treatment of RA by Lande55 as a result of an assumed relationship ,57 early recognition of this between RA and tuberculosis. F~restier's~~ benefit established chrysotherapy as a form of treatment for RA. Since that time the benefit of its use was controversial, until the Empire Rheumatism Council5a in 1960 confirmed the efficacy of gold therapy for RA in a controlled study. In JapanY5' as well as elsewhereY6O gold therapy has been reported to be of special benefit in the treatment of allergic bronchial asthma. Although this therapeutic regimen has not been developed to its fullest clinical utility, recent publications61y 6 2 state that gold preparations inhibit the release of the mediators of immediate hypersensitivity which are concerned with allergic responses. Gold sodium thiomalate ("Myochrysine") and gold thioglucose ("Solganol") are two of the most commonly used salt forms. Numerous recent reports describe conflicting results when treating arthritic patients with gold preparations.63, 64 Although the drugs must be administered intramuscularly for therapeutic effect, a direct correlation between clinical response or toxicity with blood gold levels has not been observed using standard 50mg weekly drug injections.6 5 Adjustment of individual dosage schedules to maintain a serum gold level >300 pg per cent was reported to optimize the therapeutic response.66 Gold therapy has been reported to have a very high toxicity liability. However, careful reevaluation of the statistics leading to that conclusion as well as major changes in use of the drugs and careful laboratory monitoring of treated patients, has allowed its increased use with a reduced likelihood of toxic reactions.67 The exact mechanism whereby gold(1) compounds exert their beneficial effect is not known but numerous activities have been observed (antibacterial activity, inhibition of lysosomal enzymes, stabilization of collagen, inactivation of collagenase, immunological mediation, prostaglandin synthesis inhibition) as being involved in bringing about their therapeutic benefit. 68-71 The

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biochemistry and pharmacology of gold compounds have been reviewed in detai1.72*73 Reports on gold tissue distribution usually focus on its binding with plasma o r serum albumin. However patients undergoing chrysotherapy, accumulate gold in organs of the reticuloendothelial system, such as the liver. Danpure reported that rats concentrated gold in the lysosomes of liver cells.74 In contrast to plasma albumin binding, gold in lysosomal supernatants was bound to molecules of a wide molecular weight range. Shaw has studied distribution of gold in rat kidney cortex cells.75 Unlike cadmium or mercury, gold does not induce a binding protein such as metallothionein. Involvement of glutathione as a nonprotein thiol binding component is undergoing further study. Experimental gold(1) compounds in which the gold has a coordination number of two in contrast to those in clinical use with a coordination number of one produced antiarthritic activity in adjuvant induced arthritis in rats when administered by the oral route.76 Gold sodium thiomalate was effective only when administered parenterally.77 Auranofin [ SK&F D-39162, (2,3,4,6-tetraacetyl-l-thio-~-D-glycopyranosato-5)(triethylphosphine)gold],

a member of this group, was reported to exhibit a unique pharmacological profile when compared to gold sodium thiomalate in a variety of systems.77 Intramuscular administration of gold sodium thiomalate appeared to be less effective in suppressing the lesions of adjuvant induced arthritis in rats, produced relatively higher levels of gold in the sera and kidneys and marked toxicity when compared with an equivalent amount of auranofin administered orally. Auranofin inhibited vitro lysosomal enzyme release from both and human79 leukocytes at concentrations equivalent to those obtained in the blood of patients on clinical trial. Its suppression of lymphocyte functione0 as well as antitumor potential has been reported.8 1 Clinical trials and animal studies indicate that auranofin may prove to be a more effective and less toxic form of chrysotherapy for RA than currently used agents.e2-84 Group 2b Zinc - Much use of zinc sulfate in therapy is a result of the recent recognition that zinc deficiency in man is associated with a variety of toxic phenomena. Differential zinc content in plasma, serum, erythrocytes hair, urine and saliva has been used as an index of deficiency. However, a correlation of zinc deficiencies with specific clinical symptoms has not been unequivocally established. Thus responsiveness to zinc therapy is the most reliable index of zinc deficiency. Recent advances in analytical methods have greatly facilitated measurements of zinc at the metabolic level. The average concentration of zinc in plasma when assayed by modern techniques is approximately 100 ~g/dl.'~ Zinc deficiency in infants and preadolescent children leads to growth retardation, impaired learning and failure of sexual maturation. Other associated abnormalities are impaired taste and/or smell and poor wound healing.86 Low zinc levels in the sera of patients with liver disorders is associated with high urine levels.87 It has been proposed that during acute stress an inflammatory response activates phagocytic cells to release a leukocytic endogenous mediator (LEM) which acts on the liver to increase the movement of zinc from plasma to hepatocytes, thus producing an apparent systemic hypo-

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benefit of zinc therapy in this situation has not ~ i n c e m i a . However, ~~ been determined. Supplemental zinc therapy in malnourished children has been shown t o stimulate growth of the thymus glands.88 It is recommended therapy in post surgical patients and patients with major burns to facilitate wound healing. Zinc sulfate supplementation has been used to treat recurrent idiopathic oral ulcerations (canker sores) where it was most effective in those persons with serum zinc levels 5 100 ~g/dl.~’The benefit of oral zinc sulfate in the treatment of acne was not increased when combined with Vitamin A.90 Prophylactic use of oral zinc as a therapeutic adjunct, in contrast to a trace metal supplement, reduced the frequency of crisis experienced by patients with sickle cell anernia.’l* 92 This was not a double blind study. A reduction in the percentage of irreversibly sickled red blood cells was observed during treatment. The authors suggested that zinc produced its effect by increasing the filterability of partially deoxygenated sickled cells, allowing egress of excess calcium ion. Accumulation of calcium in red blood cells has been associated with the sickling phenomena and damage to the red cell membrane. Uremic impotence in a group of chronic hemodialyzed males was reversed by zinc sulfate treatment. Zinc salts have been recommended as chemotherapeutic agents for animals infected with foot and mouth disease virus.94 These salts were shown to inhibit production of foot and mouth disease virus in tissue culture by interfering with the cleavage of high-molecularweight precursors of FMDV-specific proteins. Local application of silverzinc-allantoinate proved to be a convenient and effective regimen for the treatment of chronic cutaneous leg ulcers in patients who failed to respond to other therapeutic agents, including antibiotics.9 5 Group 5b Vanadium - The observed potent inhibition of (Na,K)-activated renal ATPase by sodium orthovanadate (Na3V04) prompted investigation of its natriuretic and diuretic properties.96y9 7 Intravenous injection of Na3V04 (1.5 pmol) in rats resulted in a prompt and dramatic increase in urine flow and sodium excretion. The action was reversible and the animal model responsive to a second dose. The authors concluded that vanadate was a potent natriuretic diuretic with as yet unknown therapeutic utility. Radioactive Isotopes - Radioactive isotopes of metals are widely used in medical therapy and diagnosis. A tabulated review references this area in detail.98 References 1. “Medicinal Chemistry Reviews”, G. P. Ellis, Ed,, Archon Books, The Shoe String Press Inc., Hamden, Conn., 1972, p 62. 2. D.D. Perrin, Top. Curr. Chem., 64,181 (1963); 3 . M.J. Cleare, Inorg. Perspect. Biol. Med., 1,19 (1977) 4 . J.P. Tupin and J.T. Hopkin, Ration. Drug Thera., 12 (9 , l(1978). 5 . C.K. Cohn, Tex. Med., 73,73 (1977). 6. J. Merry, C.M. Reynolds, J. Bailey and A. Cooper, Lancet, I_, 7894 (1976). 7. T.B. Cooper, G.M. Simpson, J.H. Lee and P.E. Bergner, Am. J. Psychiat. , 135, 917 (1978). 8. C.M. Tonks, Brit. Med. J., 2, 1396 (1977). 9. I. Singer and J . N . Forrest, Jr., Kidney Int., 10,82 (1976).

Chap. 3 1

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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.

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G.R. H e n i n g e r , Arch. Gen. P s y c h i a t . , 35, 228 ( 1 9 7 8 ) . A. P e r t , J . E . R o s e n b l a t t , C. S i v i t , C . B . P e r t a n d W.E. Bunney J r . , S c i e n c e , 201, 1 9 7 1 ( 1 9 7 8 ) . R.W. G r a d G n d A. Cerami i n "Annual R e p o r t s i n M e d i c i n a l Chemistry", Vol. 1 3 , F.H. C l a r k e , Ed., Academic P r e s s , New York, N . Y . , 1978, p 219 H.N. Munro, Fed. PKOC., Fed. Am. SOC. Exp. B i o l . , 36, 2015 (1977). M.C. L i n d e r and H.N. Munro, Fed. P r o c . , Fed. Am. SOC. Exp. B i o l . , 36, 2017 ( 1 9 7 7 ) . Anonymous, Med. L e t t . Drugs T h e r . , 20, 45 ( 1 9 7 8 ) . W.L. C h i o u , S.R. K a y s e r , M. O r r and V . J . S t e l l a , J. Am. Pharm. ASSOC., 1 7 , 377 ( 1 9 7 7 ) . Anonymous, Med. L e t t . Drugs T h e r . , 19, 35 ( 1 9 7 7 ) . B. von H a r t i t z s c h and D.N.S. Kerr, Nephron, 17,430 ( 1 9 7 6 ) . P. A i s e n , C i b a Found. Symp., 51, 1 (1976). S.P. Young and B.D. Comperts, Biochem. Biophys. Acta., 469, 2 8 1 ( 1 9 7 7 ) . J . F . Alksne and R.W. S m i t h , J . N e u r o s u r g . , 47,137 (1977). M. N i s s i l B , H. IsomBki, K. Koota, A. L a r s e n and K . R a u n i o , Scand. J . Rheumatol., 6, 1 5 8 (1977). H. O t t , I . B o u s s i n a and G.H. F a l l e t , Schweiz. Med. Wochenschr., 107, 1165 (1977). M. N i s s i l B , J . A h l q v i s t , Y. C o l l a n , P. Raunio a n d H . I s o m a k i , Scand. J . Rheumatol., 6 , 231 ( 1 9 7 7 ) . K.M.A. A h l b e r g , A . S . H e n r i c s o n , H . T . T e l h a g and F.A. Wollheim, Scand. J . Rheumatol., 1,2 1 (1978). M . J . Cleare, Coord. Chem. Rev., 12, 349 ( 1 9 7 4 ) . M.J. Cleare in "Chemotherapy", K. Hellmann a n d T.A. C o n n e r s , Ed., Plenum P r e s s , New York, N . Y . , 1 9 7 6 , p 1 4 9 . F.K.V. Leh and W. Wolf, J . Pharm. S c i . , 65, 315 (1976). J.L. Marx, S c i e n c e , 192, 774 (1976). B. Rosenberg i n "Advances i n E x p e r i m e n t a l M e d i c i n e and Biology", V o l . 9 1 , G . N . S c h r a u z e r , Ed., Plenum Press, New York, N . Y . , 1 9 7 7 , p 129. A.R. Kraska and J.S. Wolff i n "Annual R e p o r t s i n M e d i c i n a l C h e m i s t r y " , Vol. 13, F.H. C l a r k , E d . , Academic P r e s s , New York, N . Y . , 1 9 7 8 , p 124. S. K i r s c h n e r and S.H. K r a v i t z , Adv. Exp. Med. B i o l . , 91, 151 ( 1 9 7 7 ) . Anonymous, Chem. Week, 1 2 4 , ( 5 ) 42 ( 1 9 7 9 ) . E. C v i t k o v i c , J. S p a u l d G , V. Bethune, J . M a r t i n and W.F. Whitmore, Cancer, 39, 1357 (1977). M. D e n t i n o , F.C. L u f t , M.N. Yum, S.D. W i l l i a m s and L.H. E i n h o r n , Cancer, 4 1 , 1274 ( 1 9 7 8 ) . D.M. Hayes, E. C v i t k o v i c , R.B. Golbey, E. S c h e i n e r , L. H e l s o n and I . H . K r a k o f f , C a n c e r , 2, 1372 ( 1 9 7 7 ) . J.C. G o n z a l e z - V i t a l e , D.M. Hayes, E. C v i t k o v i c and S . S . S t e r n b e r g , Cancer, 39, 1362 (1977). M.F. Pera, Jr. and H.C. H a r d e r , C l i n . Chem., 23, 1245 ( 1 9 7 7 ) . W.M. S c o v e l l and T. O'Conner, J. C l i n . Hematol. and O n c o l . , 2, 487 (1977). M.U.A. Khan and P . J . S a d l e r , Chem. B i o l . I n t e r a c t . , 9, 227 (1978). R.C. Allen, G.R. Gale, P.M. O u l l a a n d A.O. Gale, B i o i n o r g . Chem., 8, 8 3 (1978). A. B i n e t and P. V o l f i n , Biochim. Biophys. Acta., 461, 182 ( 1 9 7 7 ) . M.W. Whitehouse and W.R. Walker, Agents A c t i o n , 8, 8 5 ( 1 9 7 8 ) . J.R.J. S o r e n s o n and W. H a n g a r t e r , I n f l a m m a t i o n , 2, 217 (1977).

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T o p i c s i n Chemistry and Drug D e s i g n

R e n f r o e , Ed.

R.J. S o r e n s o n , J. Med. Chem., 19, 1 3 5 ( 1 9 7 6 ) . J . R . J . S o r e n s o n , J . Pharm. P h a r m a c o l . , 29, 450 ( 1 9 7 7 ) . G.E. J a c k s o n , P.M. May and D.R. W i l l i a m s , J . I n o r g . Nucl. Chem. , 40, 1189 ( 1 9 7 8 ) . A.M. F i a b a n e , and D . R . W i l l i a m s , J . I n o r g . Nucl. Chem., 40, 1195 (1978). A.M. F i a b a n e , M.L. Touche and D . R . W i l l i a m s , J . I n o r g . Nucl. Chem., 4 0 , 1 2 0 1 (1978). G . Arean, G. Kavu and D.R. W i l l i a m s , J . I n o r g . Nucl. Chem., 40, 1221 (1978). M. M i c h e l o n i , P.M. May and D.R. W i l l i a m s , J . I n o r g . Nucl. Chem., 40, 1209 ( 1 9 7 8 ) . A . J . L e w i s , Agents A c t i o n , 8, 244 (1978). G.E. J a c k s o n , P.M. May and D.R. W i l l i a m s , J . I n o r g . Nucl. Chem., 40, 1277 ( 1 9 7 8 ) . U. Weser, C . R i c h t e r , A. Wendel and M. Younes, B i o i n o r g . Chem., 8, 201 ( 1 9 7 8 ) . K. Lande, Muenchen Med. Wochenschr., 74, 1132 ( 1 9 2 7 ) . J . F o r e s t i e r , B u l l . SOC. Med. Hop. P a r i s , 53, 323 (1929). J . F o r e s t i e r , Arq. I n t e r a m e r . Rheumatol., 6, 15 ( 1 9 6 3 ) . R e s e a r c h Subcommittee of t h e Empire Rheumatism C o u n c i l , Ann. Rheum. D i s . , 19, 95 ( 1 9 6 0 ) . T . Miyamoto, S . M i y a j i and Y . H o r i u c h i , Nippon Naika Gakkai Z a s s h i , 63, 1190 (1974). G. F i l i p p , Med. Welt., 42, 2213 ( 1 9 6 0 ) . L.W. C h a k r i n , D.T. Walz, A. M i s h e r , J . Mengel, D. Young, V. Osborne and J.R. W a r d e l l , J r . , P h a r m a c o l o g i s t 15, 220 (1973). T . Nakagawa, M. Hasegawa, K. Kudo, H. O k u d a i r a , T. Miyamoto and Y . H o r i u c h i , Ann. A l l e r g y , 40, 272 ( 1 9 7 8 ) . N . O . R o t h e r m i c h , V.K. P h i l i p s , W. Bergen and M.H. Thomas, A r t h r i t i s Rheum., 19,1 3 2 1 (1976). N.L. G o t t l i e b and A. B j e l l e , Scand. J . Rheumatol., 6, 225 ( 1 9 7 7 ) . A. L o r b e r , C l i n . P h a r m a c o k i n e t . , 2 , 127 ( 1 9 7 7 ) . A. L o r b e r , T.M. Simon, J. Leeb a n d P.E. C a r r o l l , Jr. , J . Rheumatol. , 2 , 401 ( 1 9 7 5 ) . J . M . Gumpel, B r i t . Med. J . , 1,215 ( 1 9 7 8 ) . D. B u r k h a r d t , R.W. S t e p h e n s , P. Ghosh and T.K.F. T a y l o r , A g e n t s A c t i o n s , 8 , 251 ( 1 9 7 8 ) . J . W . Measel, J r . , I n f e c t . Immun., 11,350 ( 1 9 7 5 ) . J . J . Burge, D.T. Fearon and K.F. A u s t e n , J . Immunol., 2,1 6 2 5 (1978). N.S. P e n n e y s , V. Z i b o h , N. G o t t l i e b and S . K a t z , J . I n v e s t . D e r m a t o l . , 6 3 , 356 ( 1 9 7 4 ) . P . J . S a d l e r i n " B i o c h e m i s t r y , S t r u c t u r e and Bonding", Vol. 2, J . D . D u n i t z , Ed., S p r i n g e r - V e l a g , New York, N . Y . , 1976, p 1 7 1 . D.T. Walz, M . J . D i M a r t i n o and B.M. S u t t o n i n " M e d i c i n a l C h e m i s t r y , A n t i i n f l a m a t o r y Agents", Vol 13-1, R.A. S c h e r r e r and M.W. W h i t e h o u s e , E d . , Academic P r e s s , New York, N . Y . , 1974, p 209. K.J. Lawson, C . J . Danpure and D.A. F r y e , Biochem. P h a r m a c o l . , 26, 2417 (1977). H.O. Thompson, J . B l a s z a k , C . J . Knudtson and C.F. Shaw, B i o i n o r g . Chem. , 2, 375 ( 1 9 7 8 ) .

Chap. 31 76. 77. 78. 79. 80.

81. 82. 83.

84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94.

95. 96. 97. 98.

Metals in Treatment of Disease

Sutton

329

B.M. Sutton, E. McGusty, D.T. Walz and M.J. DiMartino, J. Med. Chem., 15,1095 (1972). D.T. Walz, M.J. DiMartino, L.W. Chakrin, B.M. Sutton and A. Misher, J. Pharmacol. Exp. Ther., 197, 142 (1976). M.J. DiMartino and D.T. Walz, Inflammation, 2, 131 (1977). A.E. Finklestein, F.R. Roisman and D.T. Walz, Inflammation, 2, 143 (1977). A. Lorber, W.H. Jackson and T.M. Simon, Abs. 6th Western Regional Meeting, American Rheumation Assoc. , Scottsdale, Arizona 1977. T. Simon and A. Lorber, In Vitro, 14,372 (1978). A.E. Finkelstein, D.T. Walz, V. Batista, M. Mizraji, F. Roisman, and A. Misher, Ann. Rheum. Dis., 35, 251 (1976). D.T. Walz, A. Misher, A.E. Finkelstein, M.J. DiMartino and B.M. Sutton in "Drugs under Experimental and Clinical Research, New anti-inflammatory and Antirheumatic Drugs", Vol. 11, No. 1, A. Bertelli, Ed., J.R. Prous, Barcelona, Spain, 1977, p 47. H. Kamel, D.H. Brown, J.M. Ottaway, W.E. Smith, J. Cottney and A.J. Lewis, Agents Actions, S, 546 (1978). W.R. Beisel, R.S. Pekarek, R.W. Wannemacher, Jr., in "Trace Elements in Human Health and Disease", Vol. I, A.S. Prasad, Ed., Academic Press, New York, N.Y. 1976, p 87, H.H. Sandstead, K.P. Vo-Khactu and N. Solomons, ibid., p 35. J.F. Sullivan and Robert E. Burch, ibid., p 75. M.H.N. Golden, A.A. Jackson and B.E. Golden, Lancet, 1057 (1977). H.W. Merchant, J.P. Gangarosa, A.B. Glassman and R.E. Sobel, South. Med. J . , 70,559 (1977). G. MichaSlsson, L. Juhlin and A. Vahlquist, Arch. Dermatol., 113, 31 (1977). G.J. Brewer, L.F. Brewer and A.S. Prasad, J. Lab. Clin. Med., 90, 519 (1977). G.J. Brewer, E.R. Shoomaker, D.A. Leichtman, N.C. Kurckenberg, L.F. Brewer and N. Meyers, Prog. Clin. Bio. Res., 3, 241 (1977). L.D. Antoniou, R.J. Shalhoub, T. Sudhaker and J.C. Smith, Jr., Lancet, II 895 (1977) J. Polatnick and H. Bachrach, Antimicrob. Ag. Chemotherap., 13, 731 (1978). H.W. Margraf and T.H. Covey, Jr., Arch. Surg., 112, 699 (1977). L.C. Cantley, Jr., L.G. Cantley and L. Josephson, J. Biol. Chem., 2 3 , 7361 (1978). W.E. Balfour, J.J. Grantham and I.M. Glenn, Nature (London), 275, 768 (1978). "The Merck Index", 9th Edition, M. Windholz, Ed., Merck and Co., Inc., 1976, p Misc. 15.

z,

330

COMPOUND NAME AND CODE NUMBER INDEX

A23187, 72, 322 aldosterone, 234 N-alkoxyalkylnormetazocines, 36 A32390A, 109 62, 83, 311 A73025, 76 alprenolol (H 56/28), A74547A, 109 amastatin, 155 bis(5-amidino-2-benzimidazole)methAA-57, 109 AA-344, 52 ane, 226 83 amikacin, 107, 108, 115 acebutolol (M&B 17803A), 1-amino-1-deoxyfructose, 261 acetamidoxime, 139 acetaminophen, 189, 191, 193, 194, 2-aminoacetamidoxime, 139 2-(2-amino-5-bromobenzoyl)pyridine, 317 22 acetaminophen-N-methyl, 317 15-acetoxyscirpenol, 135 4-aminobutanesulfonic acid, 43 11-acetyllankolide, 108 y-aminobutyric acid (GABA), 42, 45 N-acetyl-muramyl-L-alanyl-D-isoglu2‘-amino-2’-deoxy-~-D-arabinofuranotamine (MDP; muramyldipeptide), sylcytosine (cytaramin), 137, 138 147, 148, 149, 150, 151 2-amino-6,7-dihydroxy-l,2,3,4-tetraacetylsalicylic acid, 312 hydronaphthalene (ADTN) , 18 acosamine, 296 aminoglutethimide, 170 acronine, 312 aminoglycoside, 315 actinomycin D, 253 acycloguanosine [9-(2-hydroxyethoxy- y-amino-f+hydroxybutyric acid, 42, methyl)guanine] , 118 46 acycloguanosine triphosphate, 118 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-37,7’-acylditheophylline, 312 AD-32 (N-trifluoroacetyladriamycinnitrosourea, 132 14-valerate), 135, 136 3-amino-1-propanesulfonic acid, 43 adenine arabinoside, 117 2-aminotetralin, 18 adenosine, 75 amitriptyline, 2, 3, 5 72, amitriptyline-N-oxide, 5 adenosine diphosphate (ADP), 75, 76 N-substituted 2-aminoadinosines, 75 ADP (adenosine diphosphate), 72, amodiaquine, 123 amoxapine, 4, 15 75, 76 amoxicillin, 114 adrenalone, 310 adriamycin (doxorubicin), 135, 136, amoxycillin, 312 amphetamine, 317 155, 288, 289, 296, 313 adriamycin-4-demethoxy, 288, 289 d-amphetamine, 190, 192, 193 amphotericin B, 155 ADTN, 18 AF 1312/TS, 174 ampicillin, 106, 114, 115, 312, 313 AH 5158 (labetolol), 84, 86 anguidine, 135 anhydro-~-5-fluorocytidine, 137 AH 7725, 54 9-anilinoacridines, 139 AHR 1118, 5 anordrin, 169 AHR 3219 (Y-7131), 24 AHR 6134 (cloroperone), 5, 15, 26 anthracyclines, 288 aklavin, 136 antibiotic G52, 107 antibiotic 66-406, 107 8-alanine, 42 L-a-alanine, 42 a2-antichymotrypsin, 222 B1-anticollagenase, 222 L-alanyl-’L’-l’aminoethylphosphonic acid (alaphosphin; R o 03-7008), antipyrine, 201 antithrombin 111, 76 105 alaphosphin ( R o 03-7008; L-alanylaplasmomycin, 109 apomorphine, 13, 14, 15, 17, 317 ’L ’-1-aminoethylphosphonic apramycin, 107 105 acid), alaprocate, 4 9-8-D arabinofuranosyladenine, 317 alclofenac, 72, 73 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0-12-M0514-8

COMPOUND NAME AND CODE NUMBER INDEX

arabinoside cytosine, triphosphate, 253 a r a b i n o s y l hypoxanthine , 118 ara-C, 254, 256 arachidonic a c i d , 66, 73, 178, 183 H-Arg-Tyr-Gly-Gly-Phe-Met-OH, 33 arginase , 316 a r p r i n o c i d [9-(2-chloro-6-fluorobenzyl) adenine] , 125 5-aryl-2-azabicyclo (3.2.1.) o c t a n e s , 36 L-ascorbate-2-sulfate7 204 ascorbic acid, 204 L-aspartic a c i d , 42, 46 aspirin, 73, 74, 77, 174 a t e n o l o l ( I C I 66082), 83, 190, 311 a u r a n o f i n [(SK&F D-39162, 2,3,4,6te t r a a c e t y l - l - thio-f3-D-glucopyranosato-5) ( t r i e t h y l p h o s 325 phine)gold] , a u r a n t i n i n , 109 azimexon (BM 12531), 158 a z a - a l a n i n e pep t i d e s , 225 aza-pep t i d e p-ni t r o p h e n y l esters , 2 2 4 , 225 6,9-aza-pros t a c y c l i n , 181 azas tene, 170 aza-uridine, 118 2'-azidw2'-deoxy-f3-D-arabinofuranos y l c y t o s i n e ( c y t a r a z i d ) , 137, 138 9,11-az0-15-hydroxyprosta-5,13-dienoic acid, 184 9 ,l l - a z o p r o s ta-5 ,13-dienoic a c i d , 74, 184 B a 39089 ( o x p r e n o l o l ) , 84 bacampicillin, 1 1 4 , 312 BAYg 5633 ( c l o t i a z e p i n e ) , 23 Bay k 4999 ( f u r a z l o c i l l i n ) , 105 BCG, 132, 1 4 1 , 146 b e f u r a l i n e (DIV 1 5 4 ) , 5 benperidol, 15 benzamidines, 224, 225, 226, 230 benzodiazopines , 74 bes t a t i n , 155 b e v a n t o l o l (CI 775), 83 bezaf i b r a t e , 77 , 203 bicuculline, 45 bicyclomycin, 109 1,3 - e - (2-chloroe t h y l ) -1-ni trosourea, 155 bitolterol, 189, 310 BL 3459, 75 BL S786 (cef o r a n i d e ) , 104

331 -

BL 4162, 75, 77 bleomycin, 109, 155, 255 BM 06002 (4-imino-l,3-diazabicyclo [3.1.0]hexan-2-one), 158 BM 12531 (2-[2-cyan-aziridinyl-(l)]2- [ 2-carbamoylaziridinyl- (1)propane], 158 BM 51052 ( c a r a z o l o l ) , 83 26 brof oxine , bromazepam, 22, 194 b r o m o c r i p t i n e (2-bromo-a-ergocriptine; CB 154) , 1 3 , 19 2-bromo-a-ergocriptine (CB 154, bromocriptine) , 1 3 , 19 6-f3-bromopenicillanic a c i d , 103, 105 15 bromperidol , brucine, 44 bucumolol (CS 359), 83 b u f r o l i n (ICI 74917) , 51, 53 b u f u r o l o l (Ro 34787), 83 b u n i t r o l o l (KO 1366) , 83 b u n o l o l (W 6412A), 83 83 b u p r a n o l o l (K1 255), bupropion, 6 7 4 , 94 burimamide, butaclamol, 16 b u t i d r i n e (CO 4 0 5 ) , 83 butirosin, 107 b u t o c r o l o l (P 18), 83 butoxamine (BW 64-9), 83 N-butyl, N-propyldopamine, 17 BW 64-9 (butoxamine), 83 BW 247, 5 C20:3,5,8,11 ( e i c o s a t r i e n o i c a c i d ) , 183 C20,5,5,8,11,14,17 ( e i c o s a p e n t a e n o i c acid), 183 C49802-B-Ba7 5 110 capreomycin, c a p t o p r i l (SQ 14,225) , 63, 64 c a r a z o l o l (BM 51052), 83 N-carbamoyltaurine, 43 carbenicillin, 75, 103, 105, 114 carbomycin A, 108 carboxymethylpachymaran, 152 316 carboxypep t i d a s e G , N-(2-carboxyphenyl)-4-chloroanthran i l i c a c i d disodium (CCA), 159 carminomycin, 136, 288, 289 118 carnidazole , caroxazone, 6 c a r t e o l o l (OPC l 0 8 5 ) , 83

332

COMPOUND NAME AND CODE NUMBER INDEX

CB 154 (bromocriptine), 19 ciprofibrate, 204 cisplatin (Platinol; NSC-119875, CCA (N-(2-carboxyphenyl)-4-chlorocis-platinumdiaminodichloride), anthranilic acid disodium, 159 323 CCNU (cyclohexyl chloroethyl nitroso- cis-platinumdiaminodichloride (cisplaurea), 132, 133 tin; Platinol; NSC-119875), cefamandole, 115, 314 323 cis-platinum(3,4-diaminotoluene)dicefazedone , 104 cefazolin, 103, 104, 115 chloride, 323 ceforanide (BL-S786), 104 citalopram, 3 103 CK-0383, 57 cefotaxime (HR 756), C1-864, 57 cefoxitin, 105, 115 cefsulodin (SCE-129; CGP 7174), 103 clavulanic acid, 106 cefuroxime, 115 clazepam, 23 centazolone, 26 clindamycin, 191 centchroman, 174 clobazam, 23, 25 cephaloridine, 106, 314 clofibrate, 198, 199, 200, 201, cephalothin, 314 203, 204 CF 25-397, 19 clonazepam, 22 CGP 6085A, 3 clonidine, 8, 64, 65, 66, 94 CGP 7174 (SCE-129; cefsulodin), 103 clopimozide, 15 CH 29-717, 19 cloroperone (AHR 6134), 15 chenic acid (chenodeoxycholic acid), clotiazepine (BAYg 5633), 23 205 clovoxamine, 3 chenodeoxycholic acid (chenic acid), CM 29-712, 13 205 CO 405 (butidrine), 83 chlomiphene, 174 coenzyme Q (ubiquinone), 154, 155 138, 139 chloramphenicol, 116, 303 coenzyme Q l o , chlordiazepoxide, 189 colchicine, 314 chlorguanide, 123 colestipol, 200, 201, 202, 203 chlorimipramine, 3, 4 Concanavalin A , 232 2-chloroadenosine, 75 copper salicyclic acid complex u-chlorohydrin(3-chloropropane-l,2(Permalon), 324 . . Coriolus versicolor (PS-K) , 141 diol), 173 corriomycin, 109 9-(2-chloro-6-f luorobenzyl)adenine Corynebacterium parvum, 132, 140, (arprinocid), 125 141 6-chloromelatonin, 174 chloromethyl pep tides, 224 CP-20,961, 149 3-(p-chlorophenyl)-2,3-dihydro-3CP-34,089 (sulprostone) , 171 hydroxythiazole[3,2-ul-benzCP-44,161, 109 imidazole-2-acetic acid CP-45,899, 106 (Wy-13876), 158 C. parvum, 146 3-chloropropane-1,2-diol(a-chlorohyCS 359 (bucumolol), 83 CS 430, 25 drin), 173 CS 1170, 105 chloroquine, 123 chlorothiazide, 61 Cu, 168 cholestyramine, 198, 201, 202 2-cyanaziridine, 142 chymostatin, 222 2-[2-cyan-aziridinyl-(l)]-2-[2-carbamoylaziridinyl-(1)-propane] CI 686 (trebenzomine), 15 CI 775 (bevantolol), 83 (BM 12531), 158 cyclic AMP, 72, 73 ciclazindol, 5 cycloalliin, 77 cicloprofen, 194 cycloguanil, 122 cimetidine (SK&F 92334), 65, 91, 9 4 , 95, 98, 212

-

COMPOUND NAME AND CODE NUMBER INDEX

333

cyclohexyl chloroethyl nitrosourea cis-dichorodiaminne platinum (11) , (CCNU), 132, 133 138, 142 3 ,3"dideoxybutirosin, 107 cyclopentane glutamate, 47 cyclophosphamide, 152 14-diethoxyacetoxy daunorubicin cyproterone acetate, 173 (W-33921), 136 diethylcarbamazine, 126 L-cys tathionine, 42, 44 diethyldithiocarbamate, 156 L-cysteic acid, 42, 43 L-cysteine sulphinic acid, 42, 43 diethylstilbestrol, 169 1,3-di-fatty acyl-2-aspirin glyccytaramin (2'-amino-2'-deoxy-B-Deride, 313 arabinofuranosylcytosine), diflumidone, 73, 74 137, 138 diflusinal, 73, 311 cytarazid (2'-azido-2'-deoxy-B-Darabinofuranosylcytosine, digitonin, 204 digoxin, 191, 311, 312 137, 138 (-)-[ 3H]-dihydroalprenolo1, 62 cytosine arabinoside, 117 dihydroergocriptine, 13 cytosine arabinoside triphosphate, 253 [ 3H]-dihydroergocryptamine, 62 DABA (2,4-diaminobutyric acid), 46 5,6a-dihydroprostacyclin, 72 13,14-dihydroprostacyclin methyl dacranolide, 108 ester, 179 danazol, 169, 173 daunomycin (daunorubicin), 132, dihydrostreptomycin, 107, 108 5,6-dihydroxy-2-dipropylamino-l,2,3, 135, 142, 288, 289, 296 4-tetrahydronaphthalene, 18 daunomycin-4-demethoxy, 288, 289, 296 5,6-dihydroxytryptamine, 8 daunorubicin (daunomycin), 288, dimaprit, 92 2.89, 296 2,3-dimethyl-3-arylpiperidines, 36 N,N-dimethyldopamine, 17 daunosamine, 288, 295 DDP, 138, 142 diosgenin, 204 6,6'-di-0-2-tetradecyl-3-hydroxyoctadeacetylvinblastine amide (vindecanoyl-a,a trehelose, 151 desine), 134 10-deaza-aminopterin, 137 N,N-dipropyldopamine, 17 de-epoxy-rosamicin, 108 diisopropylfluorophosphate, 230, 247 13,14-dehydroprostacyclin, 72 N,N-dipropyl-m-tyramine, 17 deltamycins, 108 dipyridamole, 74 4-demethoxyadriamycin, 288, 289 disodium cromoglycate (DSCG), 52, 4-demethoxydaunomycin, 288, 289, 53, 54 296 disopyramide, 190 demycarosylturimycin, 108 ditazol, 140 dendrobine, 44 diuredosan, 127 3'-deoxybutirosins A , B y 107 DIV 154 (befuraline), 5 5-deoxykanamycin A , 107 DL 262, 5 2"-deoxykanamycin B y 107 L-dopa, 310 5-deoxyneamine, 107 dopamine, 12, 13, 14, 16, 17, 18, 2-deoxys trep tamine, 108 19 dl-descryteaminylthienamycin, 105 dothiepin (prothiaden), 1, 4 desipramine, 5 dextran iron complex (Imferon), 322 doxantrazole, 51, 55 dextromethorphan hydrobromide, 191 doxepin, 5 doxorubicin (adriamycin), 288, 289, diaboline, 44 296 2,4-diaminobutyric acid (DABA) , 42, doxycycline, 115, 116 46 DSCG (disodium cromoglycate) , 52, diazepam, 22, 24 53, 54 diazoxide, 66 Du 21445 (tiprenolol), 85 dibenamine, 98

334

COMPOUND NAME AND CODE NUMBER INDEX

echinomycin, 109 EG626 (phthalazine), 74 EGY-475, 6 eicosapentaenoic acid (C20:5,5,8,11, 14,17), 183 eicosatrienoic acid (C20:3,5,8,11), 183 elasnin, 223 elastatinal, 222, 223 EMJJ 26644 (tioxaprofen), 73 enaminomycins A, B, C, 109 B-endorphin, 7 (des-Tyrl)-y-endorphin, 17, 32 Leu5-enkephalin, 33 Met5-enkephalin, 33 D-Ala2,Leu5-enkephalinamide , 34 D-Ala2, N-Methyl Met5-enkephalinamide, 33 epicorazines A, B, 109 epinephrine, 72, 73, 310 epipodophyllotoxin, 134 5-episisomicin (Sch 22591), 107 10,ll-epoxyerythromycin B, 108 erythromycin, 108, 116, 117 108 erythromycin A, B, 10-epi-erythromycin B, 108 erythronolide B, 110 17-B-estradiol, 317 S-ethylcarbamoyl-L-cysteine, 140 2'-N-ethyl-paromomycin, 107 ethynylestradiol, 168, 169, 189, 203 etiroxate, 200 EU-2540, 66 exaprolol (MG 8823), 84 fenfluramine, 192, 317 fenobam (McN-3377) , 26 fenoterol, 55, 56 fentanyl, 35, 36 ferrous sulfate, 322 FK 33-824, 32 f lavanoids, 75 flubendazole, 126 flunitrazepam, 22, 24, 25 5-fluorouracid, 137 fluotracen (SK&F 28175), 15 fluoxetine, 2, 3, 4 cis-flupenthixol, 13 flurazepam, 24, 25 flurbiprofen, 73 fluvoxarnine, 3 formamidoxime, 139 fortimicin, 107 fosazepam, 25

FPL-52757, 52 FPL-57787, 52 fructose 1,6-diphosphate, 262 fructose-6-phosphate, 262 furazlocillin (Bay k 4999), 105 furodazole, 127 furosemide, 191 G1499-2, 109 G2201-C, 109 GABA (y-aminobutyric acid), 42, 45 gallium nitrate, 138 gelsemine, 44 gemfibrozil, 203 gentamicins, 107, 108, 115, 317 gliflumide, 191 globomycin, 108 glucan, 153 glucantime (meglumine antimoniate), 123 glucose-6-phosphate, 262 L-glutamic acid, 42, 46 L-glutamic acid diethyl ester, 47 glyceraldehyde-3-phosphate, 262 glycine, 42, 43 glycinexylidide, 312, 316 gold thioglucose (Solganol), 324 gold sodium thiomalate (Myochrysine) , 324, 325 109 gramicidin S, griseorhodin C , 109 guanabenz, 65 guancydine, 66 guanethidine, 198 guanfacine , 65 Na-guanylhistamine, 93 H 56/28 (alprenolol), 83 H 93/26 (metoprolol), 81, 84, 86 H 104/08 (pamatolol), 84 Hageman factor, 77 halazepam (Sch 12041), 23 halofenate, 73 halopemide, 15 haloperidol, 13, 14, 15, 18 HC-20-511 (ketotifen), 54 helenalin, 135 heparin, 76 hetacillin methoxymethyl ester, 313 1,2,3,4,5,6-hexahydro-l,6-methano-3-

benzazocines, 37 hydrocortisone, 234 HHT (hydroxyheptadecatrienoic acid), 183 histamine, 91, 92, 95, 96, 97, 98

COMPOUND NAME AND CODE NUMBER INDEX

335

HOE 766 [D-Ser(t-Bu6) ]LH-RH ethyliodoacetamide, 247 amide, 173 iodoacetate, 247 84 iodoxamic acid, 191 HOE 893d (penbutolol), DL-homocysteic acid, 43 ionomycin, 109 56 ipratropium bromide (Sch lOOO), homothienamycin, 105 HR 756 (cefotaxime) , 103 iprocrolol (P-16), 84 hydralazine, 66, 67, 198 IPS 339, 86 hydrochlorothiazide, 198, 200 iron acrylic compound, 322 isethionic acid, 43 hydrocodone, 193 12-hydroperoxy-arachidonic acid, isochelocardin, 109 184 isoguvacine, 45 15-hydroperoxy-arachidonic acid, isophosphamide, 132 184 isopropyl LL-BM123y, 108 1-hydroxy-3-amino pyrrolidone-2, 47 isoproterenol, 55, 310 12-hydroxyarachidonic acid, 182 isoxepac, 193 y-hydroxybutyrate, 310 K13176, 104 y-hydroxybutyrolactone, 309 kainic acid, 47 kanamycins A, B, 107, 108, 115 6-hydroxydopamine, 8, 65 9-(2-hydroxyethoxymethyl)guanine ketazolam, 24 (acycloguanosine), 118 ketotifen (HC-20-511), 54 KF 577, 86 hydroxyethylpachyman A, 153 hydroxyethylpachymaran B, 152 K1 255 (bupranolol), 83 hydroxyheptadecatrienoic acid (HHT) , KO 592 (toliprolol) , 85 83 KO 1366 (bunitrolol), 183 4-(3-hydroxyphenyl)-3-methylpiperiKO 1400 (pargolol), 84 dines, 34 krestin (PS-K), 146 4-hydroxypropranolol, 310 KS-2 (Lentinus edodes) , 141 L8027 (nictindole) , 74 hydroxyurea, 139, 198 62, 84, 86 labetolol (AH 5158), hypotaurine, 42, 43 lankamycin, 108 IA-4,N-oxide, 126 lasalocid, 109 ibotenic acid, 47 8 4 , 86 LB 46 (pindolol), ibuprofen, 73 ICI 38174 (pronethalol), 81, 85 lenpenone, 26 ICI 45520 (propranolol), 81, 85, 87 lentinan, 152, 153 84 Lentinus edodes (KS-2), 141 ICI 50172 (practolol), ICI 66082 (atenolol), 83 lergotrile, 19 ICI 74917 (bufrolin), 51, 53 leupeptins, 222, 223 levamisole, 117, 125, 126, 132, ICI 101,187, 65 141, 156, 157, 158 ICRF-159, 140 levonorgestrel, 168 idoxuridine, 117 LF 17895 (mepindolol), 82, 84 imexon, 158 LH-RH, 172, 173 imidazole, 74, 184 imidazole-4-acetic acid, 42, 46 Ag[Prol,D-Phe2,D-Trp3,D-trp6]LH-RH, 4-imino-1,3-diazabicyclo[3.l.O]hexan172 2--one(BM 06002), 158 cyclo [ ( B-Alal) ,D-Ala6] LH-RH, 173 cyclo [ (6-aminohexanoyl),D-Alab]LH-RH, imipramine, 3, 5, 6, 8 impromidine (SK&F 92676), 93 173 6 [ D-
i72

336

COMPOUND NAME AND CODE NUMBER INDEX

[D-PheZ,D-Trp3-NE(
COMPOUND NAME AND CODE NLTMBER INDEX

337

monoethylglycinexylidide, 312, 316 OK-432 (Streptococcus, picibanil) , 141, 146 moprolol (SD 1601), 84 multhiomycin, 109 oleandomycin, 108 muranyldipeptide (MDP; N-acetyl-mur- ONO-802, 171 amyl-L-alanyl-D-isoglutamine), OPC 1085 (carteolol), 83 132, 141, 147, 148, 149, 150, orciprenaline, 55 151 OW-8063, 23 muscimol, 45 169 ORE'-9326, Org 6582, 3 , 8 N-0164, 74 ornidazole, 118, 124 nabilone, 26 62, 84, 87, osmium tetroxide, 322 nadolol (SQ 11725), 190, 193 oxamniquine, 126 naloxone, 32 oxatomide, 54 (+)-naloxone, 35 oxazepam, 25 d,l-2-0~0-3-(2-mercaptoethyl)-5(-)-naloxone, 35 naltrexone, 35 phenylimidazoline, 157 oxprenolol (Ba 39089) , 62, 84, 311 naproxen, 73 P-16 (iprocrolol) , 84 narisin, 109 P-18 (butocrolol) , 83 neamine, 107 pachyman, 152 nebramycin, 108 pachymaran, 152 negamycin, 109 84 pamatolol (H 104/08), neomycin, 199 panidazole, 124 netilmicin, 190, 317 84 pargolol (KO 1400), neutral red 0, 117 paromamine , 107 niceritrol, 198 nicotinic acid, 74, 198, 199, 200, paromomycin, 107 paroxetine, 3 201, 202 74 nictindole (L 8027), 84 penbutolol (HOE 893d), nifedipine, 67 penicillin, 312, 314 114, 115 penicillin G, nifenalol (INPEA) , 84 NIH 8933, 36 114 penicillin V, pentostam (stibogluconate sodium), nimorazole, 118 123 nitrazepam, 24, 27 pentoxifylline, 75 4-(3-nitrobenzyloxy)-3,5-diiodobenzamidine, 226 peptichemio, 132 nitrophenylguanidobenzoate, 230 peptidoglycan(s) , 147 PGE, 183 nitroxynil,' 127 noboritamycins A , B y 109 PGE1, 58 181 PGE1-6-ket0, Nocardia water-soluble nitrogen, PGE2, 66, 72, 74, 170, 171 146 PGE2-16,16-dimethyl, 171 110 nocardicin A , PGE2-16,16-dimethyl-benzaldehyde nomifensin, 1, 2, 8, 18 semicarbazone, 172 1-nonylimidazole, 74 norburimamide (SK&F 91581), 97 PGE2-16,16-dirnethyl-p-benzaldehyde semicarbazone ester, 172 Nordardia, 141 PGF1,-+-A4-15 (?,)-methyl, 315 nordimaprit (SK&F 91487), 97 PGFla-+-A4-15 (S)-methyl ester , dl-norgestrel, 169 315 L-norgestrel, 26 PGFla-*-A4-15 (Slmethyl, methyl norgestrienone (R-2323) , 169 ester, 315 normeperidine, 36 PGFla-13,14-dihydro-6,15-diketo, nortriptvline, 5 184 NSC-1168j5 (&-platin, Platinol, cis-platinumdiaminodichloride), PGFla-6-keto, 183, 184 323 PGF2a, 72, 74, 170, 171

338 -

COMPOUND NAME AND CODE NUMBER INDEX

PGF2,-15(S)-methyl, methyl ester, praziquantel, 126, 127 171 prazosin, 63 PGG2, 72, 73, 74, 183 PRD-92-EA, 52 PGH1, 183 primaquine, 122 probenecid, 114, 115 PGH2, 66, 72, 73, 74, 183 PGH3, 183 probucol, 202 PGI1-5-hydroxy, 180 procainamide, 189 PGI2 (prostacyclin), 57, 66, 72, procaine penicillin G, 114 73, 74, 178, 183, 184 procetofene (lipanthyl), 203 PGI2 methyl ester, 179 procinolol (SD 2124-Ol) , 85 prolactin, 13, 14, 15, 18 PGI3, 180 phenacetin, 194, 317 proline, 42, 44 phenelzine, 6 pronethalol (ICI 38174), 81, 85 61, 62, 81, phenformin, 200, 317 propranolol (ICI 45520), phenobarbitone, 36 85, 87, 189, 190, 200, 311, 312 phenprocoumon, 317 propranolol-4-hydroxy, 311 phentolamine, 63 proquazone, 73 phenylalanine, 7 prostacyclin (PG12), 57, 66, 72, 73, 74, 178 phenylethylamine, 14 4-phenyl-4-formyl-N-methylpiperidine, 7-prostacyclin, 179 44 A7 r8-prostacyclin, 179 phenytoin, 200 prostacyclin-6,9-aza, 181 prostacyclin-13,14-dehydro, 72 phorbol myristate acetate (PMA), 233 6,9-desoxa-6,9-methylene-prostacyclin, 180 phosphonoacetic acid, 118 phosphoramidon, 222, 223 prostacyclin-6,9-thia, 180 phthalazine (EG626), 74 prostaglandins, 57 9,11-azo-15-hydroxyprosta-5,13-dienoic phthalazinol, 198 picibanil, 146 acid, 184 picrotoxin, 45 9,11-azoprosta-5,13-dienoic acid, pinane thromboxane A2 (PTAz), 182 74, 184 pinazepam, 23 cis-A4-15 (S)-methyl prostaglandin Fla, 84, 86, 189, 190, - 315 pindolol (LB 46), 311 cis-A4-15 (S)-methyl prostaglandin Fla methyl ester, 315 piperacillin, 105 9,11-epoxy-methano-l5-hydroxy prostapiribedil, 6 pirprofen, 303 5,13-dienoic acid, 184 pivampicillin, 114, 312 9,11-epoxymethanaprosta 5,U-dienoic PL-385, 105 acid, 184 plant source proteinase inhibitors, 15-deoxy-9,11-(epoxyimino)prosta222 glandins, 74 plasmin, 77 13,14-dihydro-6,15-diketo-PGFia, plasminogen, 77 184 platelet factor, 222 16,16-dimethyl PGE2, 171 PMA (phorbol myristate acetate), 16,16-dimethyl PGE2-benzaldehyde 233 semicarbazone, 172 podophyllotoxin, 134 16,16-dimethyl PGE2 p-benzaldehyde polidexide, 202 semicarbazone ester, 172 poly A:U, 153, 154 6-keto-PGE1, 181, 183, 184 153, 154 6-keto-PGF 183, 184 poly I:C, poly ICLC, 154 15-(S)-metkG; PGF2, methyl ester, 171 polymyxin B, 109 85, 311 5-hydroxy-PGI1, 180 practolol (ICI 50172), pranoprofen, 73, 74 prothiaden (dothiepin), 1, 4

COMPOUND NAME AND CODE NUMBER INDEX protriptyline, 5 PS-5, 105 PS-K (Coriolus versicolor; krestin), 141, 146 pseudomonic acid A, 109 PTA2 (pinane thromboxane A2), 182 PTR 17402, 19 pyrantel pamoate, 126 6,9-pyridazaprostacyclin, 181 pyridinolcarbamate, 198 B-pyridylcarbinol, 198, 201 2-pyridylethylamine, 91, 93 pyrimethamine, 122, 123 pyrimethamine-sulfamethoxydiazine, 125 quassinoic, 135 quelamycin, 136 quinacrine, 125 quinidine, 190 quinine, 123 quisqualic acid, 47 169 R-2323 (norgestrienone), R-28935, 65 R-34994, 34 R-34995, 34 RA-233, 140 radioactive isotopes, 326 reserpine, 61 rhodomycinone, 291, 292, 296 ribostamycin, 108 rifampin, 116 rifamycin R, 109 109 ristomycin A, ristosamine, 296 RMI 12,936, 169 RMI 81968, 62, 86 Ro 03-7008 (alaphosphin; L-alanyl'L'-1'-aminoethylphosphonic

339

salicylamide, 191 salicylhydroxamic acid, 124 Sandoz 44-549, 65 saralasin, 64 SCE 129 (CGP 7174; cefsulodin), 103 SCE 963, 104 SCE 1365, 104 Sch 1000 (ipratropium bromide), 56 Sch 12041 (halazepam), 23 Sch 12679, 26 Sch 21700, 67 Sch 22591 (5-episisomicin), 107 SD 1601 (moprolol), 84 SD 2124-01 (procinolol), 85 secnidazole, 118, 124 Se-levamisole, 158 septamycin, 109 L-serine, 42, 43 serotonin, 65, 72, 75 setomimycin, 109 SH 1117, 76 SHB 286 (sulprostone), 171 shikimin, 45 silver-zinc-allantionate, 326 siomycin-A, 109 sisomicin, 107, 108 B-sitosterol, 202 SK&F 28175 (fluotracen), 15 SK&F 38393, 18 SK&F D-39162 [(auranofin, 2,3,4,6tetraacetyl-1-thio-B-D-glucopyranosato-5-)triethylphos-

phine)gold]

,

325

SK&F 73678, 105 SK&F 75073, 104 SK&F 78929-A, 54 SK&F 80303, 104 SK&F 91486, 93 SK&F 91487 (nordimaprit), 97 SK&F 91581 (norburimamide), 97 SK&F 92334 (cimetidine), 212 SK&F 92408, 93 108 SK&F 92676 (impromidine), 93

acid), 105 Ro 34787 (bufurolol), 83 RO 11-1430, 142 RO 11-2465, 3 de-epoxy-rosamicin (M-4365 G2), RP-27,267 (zopiclone), 27 F@-33,921 (14-diethoxyacetoxy daunorubicin) , 136 RS 6245 (tazalol), 85 RU 3116, 54 rubidazone, 136 rubradirin B, 113 rutin, 75 60.59-S, 104 SA-291, 64 salbutamol, 7, 55

sodium orthovanadate (vanadium), 326 somatostatin, 209 sorbistin, 108 sorbistin A1, 108 sotalol (MJ 1999), 85, 87, 311 spectinomycin, 107, 115, 117 spiro (indan-2,2'-pyrrolidines), 37 spiro (tetralin-2,2'-pyrrolidines), 37 SQ 11,725 (nadalol), 84, 87 SQ 14,225 (captopril), 63, 64

340

COMPOUND NAME AND CODE NUMBER INDEX

SQ 14,359, 105 SQ 20,881 (teprotide) , 64 SQ 22,536, 73 SQ 69,613, 104 stibogluconate sodium (pentostam), 123 141 Streptococcus (0K-432), streptomycin, 115 striatin A, 109 strychnine, 44 7,7'-succinylditheophylline, 312 sucrose polyester, 204 sulfadiazine, 190 sulfadoxine, 123 sulfamethoxazole, 116, 190 sulfamethoxydiazine-pyrimethamine, 125 sulfametrole, 190 2-sulfamoyldiaminodiphenyl sulfone, 125 sulfaphenazole, 122 86 sulfinalol (Win 40808-7), sulfinpyrazone, 73, 77 sulfisoxazole, 117 sulfonic acid nitrophenyl esters, 225 sulfonyl fluorides, 224 sulindac, 73 suloctidil, 75 sulpiride, 13, 16 sulprostone (CP-34,089; ZK 57,671; SHB 286), 171 suramin, 126 T-1551, 103 talampicillin, 114 tandamine, 6 taurine, 42, 43 85 tazolol (RS 6245), teichomycin A1, A2, 108 N-telemethylhistamine, 96 temazepam, 25 teprotide (SQ 20,881), 64 terbutaline, 317 testosterone, 173 tetracycline, 115, 116, 117 1,3-e(tetrahydro-2-furanyl)-5-

fluorouracil,

137

A 8-tetrahydrocannabinol phosphate

ester, 317 tetrahydrozoline, 94 tetramethylenedisulphotctramine, THD-341, 204 thebaine, 44 theophylline, 189, 312

thiabendazole, 126 6,9-thiaprostacyclin, 72, 180 2-thiazolylethylamine, 91 thienamycin, 105 S-substituted 2-thioadenosines, 75 thiol protease inhibitors, 224, 225 thiopeptins, 109 thromboxane A1, 183 thromboxane A2 (TxAz), 57, 66, 72, 73, 74, 178, 183 thromboxane A3 (TxA3), 183 thromboxane B2 (TxB~), 182, 183, 184 thymidine, 137 thymopoietin, 157 thyrotrophin-releasing hormone (TRH) , 7 tiadenol, 203 tiamulin, 110 ticarcillin, 75, 105 ticlopidine, 75 timolol (MK 950), 61, 81, 85 tinidazole, 118, 124, 125, 126 tioclomarol, 76 tioxaprofen (EMD26644), 73 tioxidazole, 126 85 tiprenolol (Du 21445), tobramycin, 115 tocainide, 312, 316 tofisopam, 24 tolamolol (UK-6558), 85, 311 tolazoline, 94 toliprolol (KO 592), 85 tolmesoxide, 66 tolonidine, 65 toloxatone, 6 tolypomycin Y, 110 trasylol, 223 trazodone, 1, 2 15 trebenzomine (CI 686), trehelose 6,6'-dicorynomycolate, 141 trehelose 6,6'-dimycolate, 141 TRH (thyrotrophin-releasing hormone, 7 trichlorophone, 126 2",3' ,4'-trideoxykanamycin B, 107 N-trifluoroacetyladriamycin-14-

valerate (AD-32), 135, 136 trifluorothymidine, 118 triflusal, 73 45 trilostane, 170 trimazosin, 63 trimeperazine, 27 trimethoprim, 116, 190

COMPOUND NAME AND CODE NUMBER INDEX

45 YB-2 (indenolol), 84 YC-93, 67 trizolam, 22, 24, 25 yeast glucan, 152 tryptophan, 1, 6, 7 zimelidine, 2 H-Tyr-D-Ala-Gly-Me Phe-Met (O)-ol, zinc sulfate, 325 33 zinterol (MJ 9184), 56 H-Tyr-D-Ala-Gly-Phe N-MeMet-NH2, 33 ZK 57,671 (sulprostone), 171 turimycin, 108 zopiclone (RP-27,267) , 27 TxA2 (thromboxane Az), 57, 66, 72, zposan, 151 73, 74, 178, 183 TxA3 (thromboxane A2), 183 182, 183, TxB2 (thromboxane B2), 184 U-42585E (lodoxamide thromethamine), 53 ubiquinone (coenzyme Q) , 154 UK-6558 (tolamolol) , 85 UK-14,304, 65 urapidil, 66 ursodeoxycholic acid, 205 valproate, 191 vanadium (sodium orthovanadate), 326 venoruton P4, 75 verapamil, 66 (-) verrniculine, 110 viloxazine, 5 vinblastine, 134 vinca alkaloids, 75 vincristine, 134 vindesine (deacetylvinblastine amide), 4,6,6-trimethyl-A3-~aprolactam, 7 ,N ,N- trimethy1tryptamine, 4

134

VM-26, 134 VP-16,213, 134 85 VUFB 6453 (metipranolol), W 6412A (bunolol), 83 W 8011, 52 warfarin, 313, 317 WE 941, 25 Win 40808-7 (sulfinalol), 62, 86 (+)-Win 42156, 35 (-)-Win 42964, 35 (+)-Win 43632, 35 (?)-Win 44441, 35 Wy-4036 (lorazepam) , 23 Wy-13876 (3-(p-chlorophenyl)-2,3-dihydro-3-hydroxythiazole [3,2-a]-benzimidazole-2-acetic acid), 158 X537A, 72 xilobam (McN 3113), 27 xylostasin, 108 Y-7131 (AHR-3219), 24 Y-9000, 54

341

CUMULATIVE CHAPTER TITLES INDEX*

342

Section I

-

CNS Agents

Abuse of CNS Agents

Maxwell Gordon

9,

38

Agents Affecting Appetite

George C. Heil, Stephen T. Ross

8,

42

Agents Affecting GABA in the CNS Jeffrey K. Saelens, Fredric J. Vinick

1 3 , 31 -

Amino Acid Neurotransmitter Candidates

S.

14, 42 -

Analgesic Agents

J. F. Cavalla

4,

37; 1,31

Analgesics

Franklin M. Robinson

6,

34

Analgesics and Narcotic Antagonists

Franklin M. Robinson

7,

31

J. Enna

Analgesics, Antagonists, the Opiate Receptor and Endogenous M. Gordon, J. A. Vida Opioids R. J. Kobylecki, Analgetics, Endorphins & the B. A. Morgan Opioid Receptor Analgetics

-

Strong and Weak

Anorexigenic Agents

1, 40

Louis S . Harris, William L. Dewey

2,

33; 2, 36

George I. Poos

1,

51; 2, 44

Frank P. Palopoli

3,

47; 5, 40

Joel G. Berger, Louis C. Iorio Antidepressant and Antipsychotic P. F. Von Voigtlander Agents Robert A. Lahti

Antidepressants and Stimulants

Antidepressants, Stimulants, Hallucinogens

14, 31 -

Louis S . Harris

Anti-Anxiety Agents, AnticonvulMarvin Cohen sants and Sedative Hypnotics William J. Houlihan, Gregory B. Bennett

Antidepressants

12, 20 -

11, 13 12, 10; 13,21 -

14, 22 11, 3 12, 1 -

Ivo Jirkovsky, Wilbur Lippmann

13, 1 -

Roger M. Pinder

14, -

John Krapcho

5, 1 3 ;

Carl Kaiser, Charles L. Zirkle

7, 18; 8, 11 -

John H. Biel M. A. Davis

1

a, 1 5

1, 12; 1,11 3, 14; 4, 13

*Volumes 1-6 are years 1965-1970. Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040514-8

343 -

CUMULATIVE CHAPTER TITLES INDEX

9, 19

Antiparkinsonism Drugs

Vernon G. Vernier

6 , 42;

Antipsychotic Agents and Dopamine Agonists

John McDermed, Richard J. Miller

13,

Antipsychotic and Anti-Anxiety Agents

Scott J . Childress

1, 1; 2, 1

Irwin J. Pachter, Alan A. Rubin

3 , 1;

1

R. Ian Fryer

5,

5, 1; 6,

1

Charles L. Zirkle, Carl Kaiser

7 , 6;

8,

1

Charles A. Harbert, Willard M. Welch

9, 1;

10,2

Biological Factors in Psychiatric Dennis L. Murphy Disorders

11; 14,1 2

11, 42

Biological Factors in the Major Psychoses

Frederick K. Goodwin, Dennis L. Murphy

10, 39

Hallucinogens

Raj K. Razdan

5 , 2 3 ; 6, 24 -

Paul E. Gold

12, 30 -

Narcotic Analgetics, Endorphins and the Opiate Receptor

David S. Fries

13, 41

Narcotic Antagonists and Analgesics

Robert A. Hardy

8 , 20; -

M. Ross Johnson, George M. Milne, Jr.

10, 1 2 ;

Maxwell Gordon, J u l i u s A. Vida

11, 3 3 -

J . A. Gylys, H. A. Tilson

10, 2 1 -

Richard A. Partyka, Jonas A. Gylys

9, 27

Recent Developments Relating Serotonin and Behavior

Albert Weissman Charles A. Harbert

7 , 47

Sedatives, Hypnotics, Anticonvulsants and General Anesthetics

A. D. Rudzik, W. Fries

7 , 39; -

M. Cohen

10, 30 -

Cornelius K . Cain Carl D. Lunsford

Memory and Learning Models

-

Animal

Opiate Receptor Pharmacological Approaches to Maintaining and Improving Waking Functions Psychomimetic Agents

Sedatives, Hypnotics, Anticonvulsants, Muscle Relaxants, General Anesthetics

9,

11

11,23

8,

29

1, 3 0 ; -

2,

24

3 , 28; -

4,

28

344

CUMULATIVE CHAPTER TITLES INDEX

Skeletal Muscle Relaxants

Robert C. Landes, Roger J. Stopkie, Vincent T. Spaziano

8, 37

Section I1 - Pharmacodynamic Agents Agents Affecting Gastrointestinal William A. Bolhofer, Functions David A . Brodie

1, 99

William A. Bolhofer, Henry I. Jacoby

2 , 91

Hans-Jurgen Hess

4,

Patricia W. ,Evers, Peter T. Ridley

6 , 68;

56

Christopher A. Lipinski. 10, 90; Lyle A. Hohnke Agents for the Treatment of Heart Simon F. Campbell Failure John C. Danilewicz 1 3 , 92 Angina Pectoris and Antianginal Agents

Antianginal Agents Antiarrhythmic and Antianginal Agents

Paul Kennedy, Jr.

1, 7 8

Arch C. Sonntag, Robert I. Meltzer

2 , 69

Arch C. Sonntag

3, 71

W. M. McLamore

5 , 63

Charles F. Schwender

7,

Gilbert W. Adelstein, William B. Lacefield

8 , 63

Gilbert W. Adelstein, Richard R. Dean

9 , 67

Thomas Baum, Robert L Wend t , James L. Bergey

12,

Ralph D. Tanz

1, 85

Charles F. Schwender

6,

Edmond C. Kornfeld

1, 59

John G. Topliss

2 , 48;

Franklin M. Robinson

4 , 47

Fred M. Hershenson

.

Antiarrhythmics Antihypertensive Agents

8,

93

g,9 1

69

39

80

3,

53

5 , 49;

6,

52

Anthony M. Roe

7, 59;

8,

52

John E. Francis

9 , 57

Craig W. Thornber

11,

61

CUMULATIVE CHAPTER TITLES INDEX

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

345 -

C r a i g W. Thornber, Andrew Shaw

12, -

W. L e s l e y Matier, W i l l i a m T. Comer

-

Leonard J . Czuba

7 , 78

Roy G. Herrmann, W i l l i a m B. L a c e f i e l d

8, 73

1 3 , 71; 14, 61

J. S t u a r t Fleming,

f3-Adrenergic Blocking Agents

60

g,99 8 0 ; 14,7 1

John E. MacNintch

9 , 75;

Robert D. MacKenzie

12,

R. C l a r k s o n , H. Tucker, J . Wale

1 0 , 51 -

f3-Adrenergic R e c e p t o r B l o c k e r s a s Dale B. Evans, R i t a Fox T h e r a p e u t i c Agents F r e d P. Hauck

1 4 , 81 -

C a r d i o v a s c u l a r Agents

John E. F r a n c i s

10, 61 -

Cerebral Vasodilators

H. Hauth, B. P . Richardson

1 2 , 49

Edward J. Cragoe, J r . James M. Sprague

1, 67

Edward J . Cragoe, J r . , John B. B i c k i n g

2 , 59

Hans-Jurgen Hess

3, 62

G e r a l d R. Z i n s

6 , 88;

E v e r e t t M. S c h u l t z , R o b e r t L. Smith, O t t o W. W o l t e r s d o r f , J r .

10,7 1

D i u r e t i c Agents

Robert L. Smith, O t t o W. W o l t e r s d o r f , J r . , Edward J. Cragoe, J r . 11, 71; Drugs f o r t h e Therapy of Pulmonary D i s o r d e r s

Thaddeus P . P r u s s , Doming0 M. Aviado

5 , 55

E t i o l o g y of H y p e r t e n s i o n

Donald W. DuCharme

9 , 50

Histamine Receptors

C . Robin G a n e l l i n

14, 91 -

I n h i b i t o r s of t h e Renin-Angiot e n s i n Sys t e m

Miguel A. O n d e t t i , David W . Cushman

13, 82

P l a t e l e t Aggregation I n h i b i t o r s

Leonard J. Czuba

6 , 60

Prostaglandin Structure Activity R el at i o n s h i p s

Thomas K. Schaaf

11, 8 0 -

Pulmonary and Anti-Allergy

Agents Walter T. Moreland

1, 9 2 ;

-

8,

83

13,6 1

2,

83

CUPIULATIVE CHAPTER TITLES INDEX

346

Aubrey A. Larsen, Kendrick W. Dungan

Pulmonary Drugs V a s o d i l a t o r and V a s o c o n s t r i c t o r Agents

S e c t i o n I11

-

S. Tozzi

3 , 84 7 , 89

Ralph E. Giles, David J . Herzig

9 , 85;

Arnold L. Oronsky, J a n W. F. Wasley

11, 51;

S t a n l e y C. B e l l , Robert J . Capetola

13, 51 -

Stanley C. B e l l , Robert J. C a p e t o l a , David M. R i t c h i e

1 4 , 51 -

Aubrey A. L a r s e n , Kendrick W. Dungan

4 , 67

F. P . Hauck, C. N . G i l l i s

4 , 77

lo, 80 12, 7 0

Chemotherapeutic Agents

Aminocyclitol and Other Antibiotics

Herman Hoeksema, L o r r a i n e C. Davenport

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

Dale R. Hoff

1 2 , 110 1, 150; 2, 147 -

Jackson P . E n g l i s h

3 , 140 -

Kenneth B u t l e r , Frank C. S c i a v o l i n o

6 , 99

Frank C. S c i a v o l i n o

7 , 99

K. E. P r i c e , F. L e i t n e r

8 , 104

F. L e i t n e r , C. A. C l a r i d g e

9 , 95

Gerald H. Wagman, Marvin J. Weinstein

10,

Herman Hoeksema, L o r r a i n e C. Davenport

11, 89; -

P. A c t o r , R. D. S i t r i n , J . V. U r i

1 4 , 103

Edwin H. Flynn

1, 109

Lee C. Cheney

2 , 102;

Koert Gerzon, Robert B. Morin

4 , 88

Antibiotics

A n t i b i o t i c s and Related Compounds

109

13,1 0 3

2,

93

347

CUMULATIVE CHAPTER TITLES INDEX

Antifungal Agents

Antineoplastic Agents

Koert Gerzon

5, 75

Robert B. Angier

2, 157

Robert B. Angier, Howard Newman

-

Robert S. Gordee, Marvin Gorman

4, 138

F. E. Pansy, William L. Parker, N. S. Semenuk

5, 129; -

J. Allan Waitz, C. G. Drube

7, 109; 8, 116 -

Smith Shadomy

9,

Smith Shadomy , G. E. Wagner

10, 120

R. Y. Cartwright

11,

Charles W. Young, David A. Karnofsky

2,

Charles W. Young

3, 150

John A. Montgomery

4,

C. C. Cheng

7, 129

C. C. Cheng, Kwang Yuen Zie-Cheng

8, 128

6,

129

107

101; g,113

166

154; 2, 144

John S. Driscoll

9, 139 10, 131; 11,110

John S. Driscoll, John A. Beisler

12, 120 -

A. Bloch

Allen R. Kraska, J. S . Wolff Antiparasitic Agents

3, 145

Allen R. Kraska

13, 120 14, 132

Frans C. Gable

5, 116

M. Hoffer, A. I. Rachlin

7 , 145

M. Hoffer, C. W. Perry

8,

W. C. Campbell, H. Mrozik

9, 115

Edgar J. Martin

10, 154; 11,121

C. C. Wang, M. H. Fisher

12, 140; g,130

141

348

CUMULATIVE CHAPTER TITLES INDEX Leslie M. Werbel, Donald F. Worth, Sarah M. Weitzel

Antiviral Agents

14, 122

Louis S. Kucera, 1, 129 Ernest C. Herrmann, Jr. 2, 122 Ernest C. Herrmann, Jr. Conrad E. Hoffman?

3, 116; 4, 117; 11, 128;

Donald C. DeLong

5, 101 -

Timothy H. Cronin

6, 118;

Andrew R. Schwartz

9, 128

Samuel Baron, George Galasso

10, -

Antiviral and Antitumor Chemotherapy with the Interferon System

Hilton B. Levy

8, 150

@-Lactam Antibiotics

J. Alan Webber

Biosynthesis of Antibiotics

John W. Corcoran

12, 101 12, 130

Chemotherapy of Sexually Transmitted Infections

H. Hunter Handsfield, Marvin Turck

14, 114 -

Host Modulation of Resistance to William Regelson Interferon and Neoplasia Human Antiparasitic Agents

Immunostimulants

Immunotherapy of Cancer Mechanism of Action of Antibiotics

2, 139

7,

119

161

8, 160

Edward F. Elslager

1, 136;

2, 131

Alexander R. Surrey, Allen Yarinsky

3, 126;

4,

P. Dukor, L. Tarcsay, G. Baschang

14, 146 -

Anita Hodson, E. Frederick Wheelock

9, 151

David Vazquez

5, 156

New Concepts in the Chemotherapy Williams Regelson of Neoplasia

10, 142

Structure Activity Relationships L. D. Cama, of "Non-Classical" 8-Lactam B. G. Christensen Antibiotics

13, 149

Synthetic Antibacterial Agents

1, 118 -

Robert G. Shepherd Robert G. Shepherd, Arthur Lewis

2, 112

Leonard Doub

3,

126

105; 5 , 108

349

CUMULATIVE CHAPTER TITLES INDEX Daniel Kaminsky, Maximilian von Strandtmann Section IV

-

5 , 87;

-

6,

108

Metabolic Diseases and Endocrine Function

Activators of Dopamine & 6Adrenergic Adenylate Cyclases

Herbert Sheppard

1 2 , 172 -

Agents Affecting Blood Enzymes

Murray Weiner

1, 233

Agents Affecting Cyclic AMP Levels

Don N. Harris, Nick S. Semenuk, Sidney M. Hess

8, 224

Agents Affecting Thrombosis

Joseph M. Schor

5 , 237

Agents for Treatment of Obesity

Ann C. Sullivan, Lorraine Cheng, James G. Hamilton

L

11, 200

Rex Pinson

1, 1 6 4 ; -

2,

176

George N. Holcomb

3, 156;

4,

164

Michael J. Peterson

6 , 192

Joseph J. Ursprung

1, 178; 2 , 187 -

Charles H. Eades, Jr.

3 , 172;

J. F. Douglas

5 , 180; 6 , 150

Thomas R. Blohm

7, 159; 8 , 183

Cellular Responses Mediating Chronic Inflammatory Diseases

Philip Davies, Robert J. Bonney

12, 152

Chemical Control of Fertility

Malcolm R. Bell, Robert G. Christiansen, 1 4 , 168 H. Philip Schane, Jr. -

Antidiabetic Agents

Atherosclerosis

Chronic Complications of Diabetes Dushan Dvornik

4,

178

13, 1 5 9

H. Samir h e r , Gordon R. McKinney

9 , 203

Cyclic Nucleotides as Mediators of Drug Action

M. Samir h e r , Gordon R. McKinney

10, 192 -

Diabetes Mellitus

Albert Y. Cheng

9 , 182; -

11,170

Disorders of Lipid Metabolism

Gerald F. Holland, Joseph N. Pereira

9 , 172; -

10,182

Mitchell N. Cayen

1 4 , 198 -

James G. Hamilton, Lorraine Cheng, Ann C. Sullivan

11, 180 -

Donald C. Hobbs, Hugh M. McIlhenny

11, 190

Cyclic Nucleotides Discovery

&

Drug

Disorders of Lipid Metabolism: Etiology & Therapy Drug Metabolism

350 -

CUMULATIVE CHAPTER TITLES INDEX

Bruce H. Migdalof

12, 201 13, 1 9 6 -

Bruce H. Migdalof, Kishin J. Kripalani, Sampat M. Singhvi

14,

Immunosuppressive & Immunostimulatory Agents in Rheumatoid Arthritis

Yi-Han Chang

11, 138

Mechanisms of Action of Glucocorticosteroids

Anthony

Fauci

13, 1 7 9

Modulation of the Arachidonic Acid Cascade

Thomas K. Schaaf

12, 182

Molecular Mechanisms & Pharmacological Modulation in Psoriasis

John J. Voorhees

1 2 , 162

Hugh M. McIlhenny

S.

188

Neutral Proteinases in Rheumatoid Arnold L. Oronsky, Arthritis Christine Winslow Buermann

1 4 , 219

Newer Agents for the Treatment of Arthritis

Joseph G. Lombardino

13,

Non-steroidal Antiinflammatory Agents

Robert A . Scherrer

1, 224

T. Y. Shen

3 , 215 2 , 217; -

167

Karl J. Doebel, Mary Lee Graeme, Norbert Gruenfeld, Louis J. Ignarro, Sam J. Piliero, 4 , 207; 5, 225 Jan W. F. Wasley Peter F. Juby, Thomas W. Hudyma 6, 182; 7 , 208 Stewart Wong

8 , 214; 9, 1 9 3 10, 1 7 2

Eugene C. Jorgensen

1, 191

J. W. Hinman, R. M. Morrell

3, 184

Marvin E. Rosenthale Non-steroidal Hormones Antagonists

&

Their

Peptide Hormones

Peptide Hormones of the Hypothalmus & Pituitary

John Morrow Stewart, J . W. Hinman, R. M. Morrell Johannes Meinhofer

5, 210 11, 158

Roger Burgus

-7, 1 9 4

Wilfrid F. White

8 , 204

CUMULATIVE CHAPTER TITLES INDEX Johannes Meienhofer

351 10, 202

Pharmacologic Regulation of Serum Lipoproteins Charles E. Day

13, 184

Prostacyclin, Thromboxanes and the Arachidonic Acid Cascade

K. C. Nicolaou, J. Bryan Smith

14, 178

Prostaglandins Compounds

Jehan F. Bagli

5, 170

Gordon L. Bundy

6, 137;

Richard A. Mueller

8, 172

Richard A. Mueller, Lloyd E. Flanders

9, 162

&

Related

Recent Advances in the Etiology & Treatment of Disorders of Lipid Metabolism

Ann C. Sullivan, Lorraine Cheng, James G. Hamilton

Reproduction

John C. Babcock

Somatostatin Steroid Hormones Antagonists

&

Their

Irving Scheer Irving Scheer, George Karmas

4, 189 -

Daniel F. Veber, Richard Saperstein

14, 209 -

Steroids Steroids & Biologically Related Compounds

Patrick A. Diassi, Leonard J. Lerner Romano Deghenghi

1, 213; 2, 208 3 , 207; 4 , 199

Michael J. Green, Barry N. Lutsky

11, 149

T. L. Popper, A. S . Watnick

5, 192; 6, 162

Duane F. Morrow, Duane G. Gallo

7, 182; 8, 194

Arthur G. Johnson

9, 244

Section V - Topics in Bioloiy Adjuvants to the Immune System Affinity Labeling of Hormone Binding Sites Agents Which Affect Enzyme Activity

John A . Katzenellenbogen 9, 222 A. Horita A. Horita, L. J. Weber

Antiaging Drugs

157

12, 191 1, 205 2, 199 3 , 200

Daniel Lednicer

I,

Jasjit S. Bindra

1, 277 3 , 252 9, 214

352

CUMULATIVE CHAPTER TITLES INDEX

Antimetabolite Concept in Drug Design

Edward F. Rogers

11, 233

Bacterial Resistance to BLactams: The f3-Lactamases

Jed F. Fisher, Jeremy R. Knowles

13, 239

Biochemical Aspects of Muscular Disorders

James B. Peter, Tetsuo Furukawa

12,

Biological Actions of Cyclic AMP Analogs

George I. Drummond, David L. Severson

6 , 215

Brain Neurotransmitter Receptor Binding and Neuroleptic Drugs

Ian Creese, Solomon H. Snyder

12, 249

Cannabinoids: Therapeutic Potentials

Robert A. Archer

9 , 253

Chronopharmacology - Its Implica- Lawrence E. Scheving, tion for Clinical Medicine John E. Pauly

260

Comparative Toxicology

James R. Gillette

11, 2 5 1 11, 242

Current Concepts in Periodontal Disease

Norton S . Taichman, William P. McArthur

1 0 , 228

Current Status of Iron Chelation Robert W. Grady, Therapy Anthony Cerami

1 3 , 219

Current Status of Neurotransmitters

Nicholas J. Giarman, Floyd E. Bloom

3 , 264

Delayed Hypersensitivity: Its Mediation Through Products of Activated Lymphocytes

Ross E. Rocklin

8 , 284

Detecting Mutagens - Correlations R. A. Dybas, M. Hite, Between the Mutagenicity and W. Gary Flamm Carcinogenicity of Chemicals Samson Symchowicz, Drug Metabolism Edwin A. Peers

1 2 , 234 3 , 227;

Jacques Dreyfuss, Eric C. Schreiber

5, 246

Jacques Dreyfuss, Helen Y. Zimmerberg, Eric C. Schreiber

6 , 205

Patrick J. Murphy, Robert E. McMahon

8 , 234

Drug Receptors

Jasjit S . Bindra

8 , 262

Drugs & Deterrence of Alcohol Consumption

Albert Weissman, B. Kenneth Koe

4 , 246

Albert Weissman

3 , 279

Herbert Sheppard

2 , 263

Drugs

&

Memory

&

Learning

Factors Affecting Adrenal S teroidogenesis

5, 259

353

CUMULATIVE CHAPTER TITLES INDEX Fate

&

Distribution of Drugs

Free Radical Pathology: Superoxide Radical & Superoxide Dismutases

D. A. Buyske, D. Dvornik

1, 2 4 7 ; 2 , 237

Irwin Fridovich

10, 257

Glucagon-sensitive Adenyl Cyclase: A Model for Receptors in Plasma Membranes Stephen L. Pohl

6 , 233

5-Hydroxytryptamine & the Central Roberto Levi, Nervous System Jack Peter Green

2 , 273

Immediate Hypersensitivity: I1 Drugs in Clinical Use

Elliott Middleton, Jr., Ronald G . Coffey 8 , 273

Immediate Hypersensitivity: Laboratory Models & Experimental Findings

Michael K. Bach

Immunochemical Mechanism of Drug Allergy Bernard B. Levine Inhibition of Proteolytic Enzymes William B. Lawson Liposomes as Drug Carriers

7 , 238

3 , 240 13, 2 6 1

Demetrios Papahadjopoulos

1 4 , 248

Mechanism-Based Irreversible Enzyme Inhibitors

Robert R. Rando

9,

Mechanisms of Resistance to Antibiotics

Julian Davies

7 , 217

Membrane Regulators as Potential New Drugs T. Y. Shen

11, 210

J . W. Hinman, R. P. McCandlis

1 2 , 223

Barry M. Bloom

1 , 2 3 6 ; 2, 227 -

Gerald T. Miwa, Anthony Y. H. Lu

1 3 , 206

Molecular Bases of Drug Action

H. G. Mautner

4 , 230

Neurotransmitters Revisited

Floyd E. Bloom

4 , 270

Non-enzymatic Glycosylation

Ronald J. Koenig, Anthony Cerami

1 4 , 261

Mineral Metabolism Bone Disease

&

Metabolic

234

Molecular Aspects of Drug Receptor Interactions

Peptide Conformation Activity

&

Biological Garland R. Marshall, Fredric A. Gorin, Michael L. Moore

1 3 , 227

Plasma Membrane Pathophysiology

Donald F. Hoelzl Wallach

10,213

Polyether Antibiotics: Monocarboxylic Acid Ionophores

John W. Westley

10, 246

Prospects for Gene Therapy

Alfred G. Knudson, Jr.

8,

245

354

CUMULATIVE CHAPTER TITLES INDEX

Proteases and Cell Invasion Rational Design of Chemotherapeutic Agents

Susannah T. Rohreich, Daniel B. Rifkin

1 4 , 229 -

Arthur P. Grollman

4 , 218

Recent Advances in Gamete Biology & Their Possible Applications R. B. L. Gwatkin to Fertility Control

10, -

240

Regulation of Cell Metabolism

Charles G. Smith

1, 267

Regulation of Cell Metabolism: Role of Cyclic AMP

Charles G. Smith

2 , 286

Relationship Between Nucleoside Conformation & Biological Act ivity

David C. Ward

5, 272

Relationships in the Structure & Function of Cell Surface Receptors for Glycoprotein Hormones, Bacterial Toxins, & Interferon

Leonard D. Kohn

12, -

Reverse Transcription Inhibitors

M. A. Apple

8 , 251

&

211

Its

Selected New Developments in the Royce 2. Lockart, Jr., Richard J. Colonno, Biochemistry of Viruses Bruce D. Korant

1 4 , 238 -

Selective Enzyme Inhibitors in Medicinal Chemistry

Michel J. Jung

1 3 , 249

Serum Complement System

Harvey R. Cotten

7, 228

Silicon in Biology & Medicine

M. G. Voronkov

1 0 , 265

Some Features of Solute Active Transport Across Biological Membranes

Christopher Walsh

11, 222 -

Structure-Activity Relationship of Adrenergic Compounds That Act on Adenyl Cyclase of the Frog Erythrocyte

Ora M. Rosen

6 , 227

Structure & Biological Activity Miklos Bodanszky, Interrelationships in Peptides Agnes Bodanszky

5, 266

Structured Water in Biological Systems

Donald T. Warner

5 , 256

Transition State Analogs as Enzyme Inhibitors

G. E. Lienhard

7 , 249

Unknown Variable in Sensitization Max Samter, to Drugs: Drug or Host? G . H. Berryman, R. G. Wiegand

2 , 256

CUMULATIVE CHAPTER TITLES INDEX Section VI

-

355

Topics in Chemistry and Drug Design

Advances in Aporphine Chemistry

M. P. Cava, A. Venkateswarlu

4, 331

William I. Taylor

1, 311

Gordon H. Svoboda

3, 358

Raymond W. Doskotch

4, 322

Maurice Shama

5, 323

Alkaloids & Other Natural Products

Stanley L. Keely, Jr., Raymond W. Doskotch

6, 274

Antiradiation Agents

William 0. Foye

1, 324; 2, 330

Edward R. Atkinson

3, 327;

Donald Valentine, Jr., John W. Scott

13, 282

Alkaloids

Asymmetric Synthesis

Biochemical Procedures in Organic Charles J. Sih, Synthesis Elie Abushanab, J. Bryan Jones Chemical Modification of Cyclic Jon P. Miller, AMP & Cyclic GMP Roland K. Robins

12, 298 -

Computer-assisted Organic Synthetic Analysis

12, 288

Peter Gund

11, 291

Cytochrome P-450 Monoxygenases in J. E. Tomeszewski, Drug Metabolism D. M. Jerina, J. W. Daly

9, 290

Drug Binding and Drug Action

Colin F. Chignell

9, 280

Enantioselectivity in Drug Metabolism

Lawrence K. Low, Neal Castagnoli, Jr.

13, 304

Intramolecular Catalysis in Medicinal Chemistry

Richard D. Gandour, Richard L. Schowen

7, 279

Intramolecular Diels-Alder Reaction in Organic Synthesis

Robert G. Carlson

9, 270

Magnetic Resonance Probes of Drug Binding Robert R. Sharp

11, 311

Medicinal Inorganic Chemistry

Robert P. Hanzlik

8, 294

Metal Carbonyls as Reagents & Intermediates for Organic Synthesis

Howard Alper

Metals in Treatment of Disease

Blaine M. Sutton

8, 322 14, 321

John

10, 317 -

Molecular Aspects of Membrane Function New Methods In Heterocyclic Chemistry

S.

Baran

Edward C. Taylor

14, 278

2, 346

356

CUMULATIVE CHAPTER TITLES INDEX

Nucleosides & Nucleotides

Howard J. Schaeffer Thomas J. Bardos

Organic Electrosynthesis

Larry L. Miller, Esther Kariv, James R. Behling

Organocopper Reagents

J. P. Marino

Peptide Synthesis

John Morrow Stewart

Pharmaceutics

J. Keith Guillory

Pharmaceutics

&

Biopharmaceutics Takeru Higuchi, Kenneth F. Finger, William I. Higuchi

Pharmaceutics, Pharmacokinetics & Biopharmaceutics

Physicochemical Parameters in Drug Design

1, 2 9 9 ; 2, 304 3 , 2 9 7 ; 2, 333 12, 10,

309 327

7, 289 6 , 254 1, 3 3 1 ; 2, 340 -

Leslie Z. Benet

-6 ,

Ho-Leung Fung

8 , 332

Edward R. Garrett Oscar E. Araujo

3,

George Zografti, K. C. Kwan

5 , 313

Corwin Hansch

3 , 348

William P. Purcell, John M. Clayton

4 , 314

264;

337;

John M. Clayton, 0. Elmo Millner, Jr.,

William P. Purcell Pharmacokinetics and Drug Design Ho-Leung Fung, Bruce J . Aungst, Richard A. Morrison

5 , 285

14

-9

Pharmacophoric Patter Searching in Receptor Mapping Polymeric Reagents in Organic Synthesis

Peter Gund

-9

Ned M. Weinshenker, Guy A. Crosby

11,

Preparation of Radioisotope Labeled Drugs

Richard C. Thomas

Prodrug Approach in Drug Design

A. A. Sinkula

-7, 296 10, 306 -

Quantitated Structure-Activity Relationships

Arthur Cammarata

6 , 245

-8 ,

Quantitative Drug Design

W. J . Dunn, I11 Richard D. Cramer, I11

Quantitative Structure-Activity Relationships in Drug Design

John G. Topliss, James Y. Fukunaga

14 281

313

11, 301 13, 292

1,259 5,

302

357 -

CUMULATIVE CHAPTER TITLES INDEX Radioimmunoassays

Reactions of Interest in Medicinal Chemistry

F. Kohen, Y. Koch, H. R. Lindner

10,

284

Joseph G. Cannon

1, 3, -

317;

2, k,

Robert A. Wiley

5 , 356;

5,

Herbert T. Nagasawa, John A. Thompson

7 , 269

Herbert T. Nagasawa, Dwight S. Fullerton

8 , 303

Dwight S. Fullerton, George L. Kenyon, Dolan H. Eargle

9,

Mathias C. Lu, D. L. Venton

10, -

274;

11,261

David M. Spatz

1 2 , 268; -

13,272

Daniel Lednicer

14,

Recent Methods in Peptide Synthesis

Brian J. Johnson

5,

Stereochemistry of Drug-Nucleic Acid Interactions & Its Biological Implications

Chun-che Tsai

Steroids

Raphael Pappo

1 3 , 316 2 , 3 1 2 ; 2, -

John S. Baran

4 , 281

Paul D. Klimstra

5 , 296

Synthetic Applications of Metalated Carboxylic Acids

P. L. Creger

12,

Synthetic Approaches to Anthracycline Antibiotics

T. Ross Kelly

1 4 , 288

Synthetic Approaches to Prostaglandins

Udo Axen

3 , 290

Synthetic Peptides

George W. Anderson

1, 2 8 9 ; -

Total Synthesis of 8-Lactam Antibiotics

B. G. Christensen

Edward E. Smissman

R. W. Ratcliffe

Use of Chemical Relationships in Design of DDT-Type Insecticides Robert L. Metcalf Use of Stable Isotopes in Sidney D. Nelson, Medicinal Chemistry Lance R. Pohl Use of Substituent Constants in Drug Design

Corwin Hansch

Vitamin D & Its Metabolites

Joseph L. Napoli

314;

321

284

291

260

268 307

278

11, 9, 12,

307

271

300 319

2 , 347 10, 295

2,

296

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