Annual Reports in Medicinal Chemistry, Volume 19

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ANNUAL REPORTS IN MEDICINAL

CHEMISTRY

Volume 19

Academic Press Rapid Manuscript Reproduction

ANNUAL REPORTS IN MEDICINAL CHEMISTRY Volume 19 Sponsored by the Division of Medicinal Chemistry of the American Chemical Society Editor-in-Chief: DENIS M. BAILEY STERLING-WINTHROP RESEARCH INSTITUTE RENSSELAER, NEW YORK

SECTION EDITORS BARRIE HESP WILLIAM T. COMER FRANK C. SCIAVOLINO BEVERLY A. PAWSON ROBERT W. EGAN RICHARD C. ALLEN

ACADEMIC PRESS, INC.

1984

(Harcouft Brace Jovanovich, Publishers)

ORLANDO SAN DIEGO NEW YORK LONDON TORONTO MONTREAL SYDNEY TOKYO

COPYRIGHT 0 1984, 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, OR A N Y INFORMATION STORAGE AND RETRIEVAL SYSTEM,WITHOUT PERMISSION IN WRITINQ FROM THE PUBLISHBB.

ACADEMIC PRESS,INC.

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United Kingdom Edition published by ACADEMIC PRESS. INC. ( L O N D O N ) LTD.

24/20 Oval Road, London N W l 7DX

LIBRARY OF CONQRESS

CATALOG CARD NUMBER:

I SBN 0- 12-0405 19-9 PRINTED IN THE U N m STATES OF AMERICA 84 85 86 81

9 8 7 6 5 4 3 2 1

6 6 - 26 84 3

CONTRIBUTORS PREFACE

xi

...

Xlll

I.

CNS AGENTS

Section Editor: Barrie Hesp, Stuart Pharmaceuticals, Division of ICI Americas, Wilmington, Delaware 1.

Analgesics 0. William Lever, Jr., Kwen-Jen Chang, and John D. McDermed, Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, North Carolina

1

2 . Antianxiety Agents, Anticonvulsants, and Sedative-Hypnotics Joseph P. Yevich, James S. New, and Michael S. Eison, Bristol-Myers Research and Development, Evansville, Indiana

11

3. Antipsychotic Agents Tomas de Paulis and Sten Ramsby, Astra Lakemedal AB, Sweden

21

4. Cognitive Disorders Fred M. Hershenson and John G. Marriott, Warner Lambertl Parke-Davis, Ann Arbor, Michigan

31

5.

Adaptive Changes in Central Nervous System Receptor Systems B. Kenneth Koe and Fredric J . Vinick, Pfzer Central Research, Groton, Connecticut

6 . CNS Autoreceptors as Targets for Drug Design Pieter B. M. W. M. Timmermans, Department of Pharmacy, Division of Pharmacotherapy, University of Amsterdam, Plantage Muidergracht 24, 1018 TV Amsterdam, The Netherlands V

41

51

vi

Contents

11. PHARMACODYNAMIC AGENTS Section Editor: William T. Comer, Bristol-Myers Research and Development, 345 Park Avenue, New York, New York 7. Antihypertensive Agents Peter Sprague and James R. Powell, The Squibb Institute for Medical Research, Princeton, New Jersey

61

8. Cardiotonic Agents Bernd Wetzel and Norbert Hauel, Dr. Karl Thomae GmbH, 0-7950 Biberach, Federal Republic of Germany

71

9. Agents for the Treatment of Peptic Ulcer Disease David E. Bays and Roger Stables, Glaxo Group Research Ltd., Ware, Hertfordshire, England

81

10. Pulmonary and Antiallergy Agents John H. Musser, Anthony F. Kref, and Alan J . Lewis, Wyeth Laboratories, Inc., Philadelphia, Pennsylvania

93

111. CHEMOTHERAPEUTIC AGENTS

Section Editor: Frank C. Sciavolino, Pfizer Central Research, Groton, Connecticut 11, Antibacterial Agents E. S. Hamanaka and M. S. Kellogg, Pfizer Central Research, Groton, Connecticut

107

12. Antiviral Agents James L. Kelley, Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, North Carolina

117

13. Antifungal Chemotherapy Michael B. Gravestock and John F. Ryley, ICI Pharmaceuticals Division, Mereside, Alderley Park, Macclesfield, Cheshire, England

127

14. Antineoplastic Agents Terrence W. Doyle, Bristol-Meyers Pharmaceutical R & D Division, P.O. Box 4755, Syracuse, New York

137

15. Antiparasitic Agents Bernard J . Banks, Pfizer Central Research, Sandwich, Kent, England

147

Contents

vii

IV. METABOLIC DISEASES AND ENDOCRINE FUNCTION Section Editor: Beverly A. Pawson, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 16. Progress in the Development of Antiobesity Drugs Ann C. Sullivan, Susan Hogan, and Joseph Triscari, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 17. Aldose Reductase Inhibitors as a New Approach to the Treatment of Diabetic Complications Christopher A. Lipinski and Nancy J . Hutson, Pfizer Central Research, Groton, Connecticut 18.

Vitamin D: Metabolism and Mechanism of Action Hector F. DeLuca and Heinrich K . Schnoes, Department of Biochemistry, University of Wisconsin-Madison, College of Agricultural and Life Sciences, Madison, Wisconsin

19. Interleukin 2 John J . Farrar, William R. Benjamin, Lorraine Cheng, and Beverly A. Pawson, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey

157

169

179

191

V. TOPICS IN BIOLOGY Editor: Robert W. Egan, Merck Institute for Therapeutic Research, Rahway, New Jersey 20. The Inactivation of Cytochrome P-450 Paul R. Ortiz de Montellano, Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California

20 1

2 1. Phospholipases Eduardo G. Lapetina, Department of Molecular Biology, Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, North Carolina

213

22. Vaccine Synthesis by Recombinant DNA Technology Dennis G. Kleid, Department of Vaccine Development, Genentech Inc., 460 Point San Bruno Blvd., South San Francisco, California

223

23. Collagenases in Tumor Cell Extravasation T. Turpeenniemi-Hujanen, U . P. Thorgeirsson, and L. A . Liotta, Laboratory of Pathology, NCI, NIH, Bethesda, Maryland

23 1

viii

Contents

24. Biology of Leukotrienes William Kreutner and Marvin I . Siegel, Schering-Plough Corporation, Bloomfield, New Jersey

24 1

25. Endogenous Natriuretic Agents Mary Anna Napier and Edward H . Blaine, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey and West Point, Pennsylvania

25 3

VI. TOPICS IN CHEMISTRY AND DRUG DESIGN

Section Editor: Richard C. Allen, Hoechst-Roussel Pharmaceuticals Inc., Somerville, New Jersey 26. Enzymic Methods in Organic Synthesis Mark A. Findeis and George M . Whitesides, Department of Chemistry, Harvard University, Cambridge, Massachusetts

263

27. Stable Isotopes in Drug Metabolism and Disposition Thomas A. Baillie and Albert W, Rettenmeier, Department of Medicinal Chemistry, University of Washington, Seattle, Washington Lisa A. Peterson and Neal Castagnoli, Jr., Department of Pharmaceutical Chemistry, University of California, San Francisco, California

273

28. Drug Discovery at the Molecular Level: A Decade of Radioligand Binding in Retrospect Michael Williams and David C. U'Prichard, Nova Pharmaceutical Corporation, P,0. Box 21204, Wade Avenue, Baltimore, Maryland 29. Forskolin and Adenylate Cyclase: New Opportunities in Drug Design Kenneth B. Seamon, National Center for Drugs and Biologics, FDA, Bethesda, Maryland

30. Recent Progress in the Rational Design of Peptide Hormones and Neurotransmitters Victor J . Hruby, John L. Krstenansky, and Wayne L. Cody, Department of Chemistry, University of Arizona, Tucson, Arizona

VII.

WORLDWIDE MARKET INTRODUCTIONS

Section Editor: Richard C. Allen, Hoechst-Roussel Pharmaceuticals Inc., Sornerville, New Jersey

283

293

303

Contents

31.

To Market, to Market-1983 Richard C . Allen, Hoechst-Roussel Pharmaceuticals Inc., Somerville, New Jersey

COMPOUND NAME AND CODE NUMBER INDEX CUMULATIVE CHAPTER TITLES KEYWORD INDEX

ix

313

327 335

This Page Intentionally Left Blank

Allen. Richard C . . . . Baillie. Thomas A . . . Banks. Bernard J . . . . Bays. David E . . . . . Benjamin. William R . . . Blaine. Edward H . . . . Castagnoli. Neal. Jr . . . Chang. Kwen-Jen . . . . . . Cheng. Lorraine Cody. Wayne L . . . . DeLuca. Hector F. . . . de Paulis. Tomas . . . Doyle. Terrence W . . . Eison. Michael S . . . . Farrar. John J . . . . . Findeis. Mark A . . . . Gravestock. Michael B . . Hamanaka. E . S . . . . Hauel. Norbert . . . . Hershenson. Fred M . . . Hogan. Susan . . . . Hruby. Victor J . . . . Hutson. Nancy J . . . . Kelley. James L . . . . Kellogg. M . S . . . . . Kleid. Dennis G . . . . Koe. B . Kenneth . . . Kreft. Anthony F. . . . Kreutner. William . . . Krstenansky. John L . . . Lapetina. Eduardo G . . . Lever. 0. William. Jr. . .

. . . 313 . . . 273

Lewis. Alan J . . . . . . Liotta. L . A . . . . . . Lipinski. Christopher A . . . Marriott. John G . . . . . McDermed. John D . . . . Musser. John H . . . . . Napier. Mary Anna . . . New. James S . . . . . . Ortiz de Montellano. Paul R . . Pawson. Beverly A . . . . Peterson. Lisa A . . . . . Powell. James R . . . . . Ramsby. Sten . . . . . Rettenmeier. Albert W. . . Ryley. John F. . . . . . Schnoes. Heinrich K . . . . Seamon. Kenneth B . . . . Siegel. Marvin I . . . . . Sprague. Peter . . . . . Stables. Roger . . . . . Sullivan. Ann C . . . . . Thorgeirsson. U . P. . . . Timmermans. Pieter B.M. W.M. Triscari. Joseph . . . . . Turpeenniemi.Hujanen. T. . U’Prichard. David C . . . . Vinick. Fredric J . . . . . Wetzel. Bernd . . . . . . Whitesides. George M . . . Williams. Michael . . . . Yevich. Joseph P. . . . .

. . . 147 . . . 81 . . . 191

. . . 253 . . . 273 . . . 1 . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . .

. 191 . 303 . 179

. 21 . 137 .

11

. 191 . 263 . . . . . . .

. . . .

. . . .

. . .

127 107 71 31 157 303 169 117 107 223 41 93 241 303 21 3 1

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. . 231 . . 169 . . 31 . .

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93 253 11 201 191 273 61 21 273 127 179 293 241 61 81 157 231 51 157 231 283 41 71 263 283 11

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PREFACE

Volume 19 of Annual Reports in Medicinal Chemistry contains 30 chapters organized in the traditional format of previous volumes-in sections titled CNS Agents, Pharmacodynamic Agents, Chemotherapeutic Agents, Metabolic Diseases and Endocrine Function, Topics in Biology, and Topics in Chemistry and Drug Design. In addition, this year the editors experiment with a new section that summarizes worldwide first market introductions of therapeutic agents during the previous calendar year. In the first six sections are included a mix of literature updates on a broad sweep of drug research areas, as well as reviews of highly specialized and newly emerging technologies. Topics covered for the first time include CNS autoreceptors, interleukin 2, endogenous natriuretic factors, and enzymic methods in organic synthesis. Phospholipases and collagenases, touched on in earlier Annual Reports, receive in-depth coverage in separate chapters in this volume. Reviews on recombinant DNA technology and the biology of leukotrienes bring the reader up to date in these highly important and active areas. In the new section, Worldwide Market Introductions, the chapter “To Market, to Market” presents an aspect lacking in the previous 18 volumes of Annual Reports-an organized follow-up on market introduction, the ultimate endpoint of the many agents reviewed. The accumulation of data for this chapter proved formidable and while a number of sources were used, it is inevitable that we missed some drugs. We hope we have not made a major omission! Finally, I want to extend my gratitude to the section editors and contributors whose time and effort made Volume 19 possible, and in particular to Martha Johnson, whose assistance was invaluable in preparing the final copy. Denis M . Bailey Rensselaer, New York M a y I984

...

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Section I

-

CNS Agents

Editor: Barrie Hesp, Stuart Pharmaceuticals, Division of ICI Americas, Wilmington, DE 19897 Chapter 1.

Analgesics

0. William Lever, Jr., Kwen-Jen Chang and John D. McDermed Wellcome Research Laboratories, Burroughs Wellcome Co. Research Triangle Park, NC 27709 Efforts toward alleviation of pain continue at an intensive pace at the clinical, pre-clinical and basic research levels. In this review, recent research developments in opioid and non-opioid analgesia are presented, along with selected clinical highlights. Opioid Receptors and Ligands Endogenous opioids and their multiple receptors continue as the focus of vigorous research. The proceedings of the 1983 International Narcotic Research Conferenc were published,’ and reviews were recently p o ided on opioid peptides,2,$ opioid receptors and their functional roles, macology and therapeutic implications of enke halins and endorphins,Shycardiovascular effects of endogenous opioids, opioids and the adrenalpituit ry axis, opioids and the hippocampus,l2 substance in nociception,’$ potential therapeutic roles for opioid antagonists’‘, roles of opioid pept‘des in appetite control,l 5 and autoradiography of opioid receptors. 16

6-8

’’

Biosvnthesis and Metabolism of Opioid Peptideg - Opioid peptide biosynzhesis was summariz in our previous review nd additional overviews of the biosynthesis”-22 and distribution2o 9 7 2%25 of endogeneous opioids have appeared. Several inhibitors of enkephalin convertase, a carboxypeptidase which may liberate enkephalins from immediate precursors, Degradation of enkephalins by peptidases continues were described 2 6 9 2 7 to be s t ~ d i e d , ~ ~ , ~ the ~ analgesic properties of inhibitors of as28z54 Thiorphan, N-[(R,S)-3-mercapto-2-benzylproenkephalin metabolism. panoyllglycine, is an enkephalinase inhibitor (Ki = 3.5 nM) which also inhibits (Ki = 140 nM) angiotensin-converting enzyme (ACE).32 By contrast, the retro-inuerso isomer, retro-thiorphan [(R,S)-HS-CH2-CH(CH2Ph)- N H CO-CH2C02H], does not inhibit ACE but retains potent enkephalinase inhibition (Ki = 6 nM) and has an inuiuo analgesic potencs2(i.c.v.; mouse hot plate, mouse writhing) similar to that of thiorphan. Thiorphan was reported to reduce postmyelographic side effects (headache, nausea, sciatica) when administered (i.v.) to patients prior to m y e l ~ g r a p h y . ~ ~ Oral administration of D-phenylalanine, a carboxypeptidase inhibitor, was found to be of benefit in patients with certain chronic ain complaints; the drug was most effective after 3-5 weeks of therapy.38

’’

Opioid Receptors and Pharmacology - The previously defined opioid receptor sub-types ( P , ~ , K , E , U ) continue to be studied, and an additional binding site, designated the A site, was recently described in rat brain.35 The A

ANNUAL REPORTS IN MEDICINAL CHEMISTRY-I9

Copyright 0 1984 by Academic Press, Inc. All nghts of reproduction in any form reserved. ISBN 0-12-040519-9

-2

Sect. I

- CNS Agents

Hesp, Ed.

sites have a regional distribution in brain different from that of p receptors and have a high affinity for 4,5-epoxymorphinans (u., morphine, naloxone) and low affinity for non-epoxymorph‘nan p ligands, K ligands, 6 ligands, and “universal” ( p , 6 , ~ )ligands.j5 Evidence that u receptors may differ from phencyclidine receptors was provided by the report of a naloxone-inaccessible u receptor in rat brain and spinal cord which stereoselectively binds (+)-ethylketocyclazocine and (+)-N-allylnormetazocine [(+)-SKF 10,0471 and has drug selectivity and regiona13$istribution different from those of p, 6 and phencyclidine receptors. Additional studies on u agon t and on their phencyclidine-like behavioral effects were reported.jf-81The benzomorphan Mr 2034 [ ( - ) - N (2-tetrahydrofurfuryl)normetazocinel, originally proposed as a K liga d is more accurately described as a universal ( p , 6 , ~ , 0 ) opioid ligand.42 The nature of opioid binding continues to be e ~ p l o r e d . ~ ~In ’ ~rat ~ brain and spinal cord, p a@jl 6 sites predominate, and K sites represent Both 6 and p receptors mediate antinocionly 10% of total binding. pro il s of i.~and K agonists were ~ e p t i o n . ~ The ~ - ~antinociceptive ~ reported to be distinguishable inuiuo, 55,5‘ and the affinities of K agonists for p and 6 receptors invivo in the rat were assessed.55 Respiratory depression is caused by opioid agonists but may be mediat b a receptor population different from that producing antinociception.5‘-521 Evidence suggesting th t the p sites in mouse vas deferens and guinea pig ileum are different,60,g1 and support for the possibility that opio agonists and antagonists interact with different sites on )1 receptors? were also presented. The antitussive effects of opioids appear to arise from interaction with pharmacological receptors which are less st eoselective and less sensitive to naloxone than the analgesic c tors.g5 Attempts Additional data to purify opioid receptors have progressed slowly. were cited for the coexistence of allosterically coupled morphine an enkephalin receptors within an opioid receptor complex in rat brain.26,67

sQ*gFs

-

Newer Endonenous ODioid Peptides The number of endogenous opioid peptides continues to grow rapidly. Several fragments of proenkephalin A have received recent attention. A u-selective octapeptide fragment, Gly-Gly-Phe-Met-Arg-Arg-Val-NH2, has been isolated from bovine brain6”;nd from h y g n pheochromo~ytoma~~ and has been designated both as metorE, a 25-amino acid fragment, phamide and as a d r e n ~ r p h i n . ~Peptide ~ binds preferentially to 1.1 and K receptors but has minimal analgesic tail flick) in potency (i.c.v., The C-terminal heptapeptide fragmen mous: Met -enke h lin-Argg-Phe7, has e n found in human and rabbit pl sma,72, rat brain 75-iJ5and spinal c~rd,~‘,?~ and lung of several species.78 A unique high affinity binding site was described in rat lung membranes.78 The role of this peptide as a Met-enkephalin pr cursor or a transmitter is speculative. The fragment Met5-enkephalin-Arg&-Gly7-Leu8 is a p- and &-selective ligand which binds poorly to K receptors; synthetic extension to incorporate a Lys9 residue improved K affinity 10-fold but did not alter p or 6 binding or the p,6-~electivity.~~

-

Svnthetic ODioid PeDtide Analonues Structure-activity studies on analogues of the exceptionally selective ligand morphiceptin (TyrPro-Phe-Pro-NH2) were reported.g’ One of the most potent analgesics was PL017 ( Tyr-Pro-NMePhe-D-Pro-NH2). 6o A peptide containing the PL017 sequence at the N-terminus and the dyn-A -17) sequence at the C-terminus had high affinity for both p and K s i t e s 3 Analogues of 8-endorphin containing the dynorphin (5-13) or (6-13) sequences showed excitatory behavioral effects in the mouse t had no analgesic properties in the By contrast, a 8-endorphin analogue mouse tail flick assay (i.c.v. 1 with a dermorphin sequence (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser) in residues

.”

Chap. 1

Analgesics

Lever, Chang, McDermed

3

(1-7)wag2several-fold more potent than B-endorphin (mouse tail flick, A number of additional studies on the dermorphins, potent i.c.v.). Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) originally opioid heptapeptides (u., isolated from the skin o S th American frogs, and their synthetic analogues were reported.83-gH A heptapeptide is not required for high activity. The dermorphin (1-4) analogue H2N-C(NH)-Tyr-D-Ala-Phe-Gly-NHadamantyl, which contains a guanidino N-terminus and an N-adamantyl amide moiety, is a powerful p-agonist (mouse tail flick, i.c.v.) with potency 30,000 and 1000 times that of Met-enkephalin and morphine, r e ~ p e c t i v e l y . ~ ~ New &-selective li ands have emerged, including deltakephalin (TyrD-Thr-Gly-Phe-Leu-Thr),8g certain enkephalin dimers,l7vgo and cyclic enkephalin analogues containing disulfide-linked penicillamine and cystine residues.91 Possibly the most highly 6-selective ligands reported to date are the bis-penicillamine 14-membered cyclic disulfide-containing en~ kephalin analogues DPDPE (la) and DPLPE ( & ) , which gave 6 4 selectivity By contrast, ratios in isolated tissues of 3164 and 1088, respectively. the 14-membered cyclic enkephalin analogue 2, as well as a number of partial retro-inuerso modifications of this cyclic structure, are p-selective ligands 93 Other approaches to conformational restriction were reported 941 95 as were enkephalin analogues incorggrating dehydroamino cyclopropyl-containing amino acids, y g 9 p-nitro substitution in Phe4,100 ketomethylene replacement of the T r-Gly and Gly-Gly amide linka e lo' modifications at the C-terminus,182 S-oxidized m t io nine,'Oj'and phosphonate analogues of the C-terminal residues.7ob -

acid^,^^,^^

!Q

R1 = C02H, R 2 = H

lb

R1 = H, R 2 = C 0 2 H (DPLPE)

(DPDPE)

T y r -D -Ala-G Iy -P he- Leu - NH-C H, C H,-X

QN3

3 X=NH X = NHCO(CH2)2NH

4

NO z

New Ligands, SAR, Structural Studies - Irreversible opioid ligands are of interest as receptor probes. The photolabile enkephalin d r'vatives 3 and and the 2, lo5 the chloromethyl ketone Tyr-D-Ala-Gly-Phe-Leu-CH$l, fentanyl isothiocyanate 5Io7 all appear to label 6 receptors selectively, whereas the etonitazene isothiocyanate 61°7 appears to inactivate only p receptors. A variety of epoxymorphinans with alkylatin groups attached Both B-CNA (1) through amino groups at C-6 are opioid affinity labels.'ol and a-CNA (8) produce irreversible antagonism at p, K and 6 receptors, and a-CNA appears to have an additional irreversible agonist property in the guinea pig ileum assay.lo9 Further studies in the ileum with B-FNA ( 9 ) and B-FOA (lo) led to the suggestigs that 1.1 agonists and antagonists interact with distinct receptor sites. Comparisons of isolated tissue data for various epimeric antagonist affinity labels indicated that the chiralit at C-6 is an important determinant of receptor alkylating The configuration at C-6 was shown to determine the ring C ability.'lo solution conformations of the oxymorphamines: rinh C is a chair in the B-epimer 11 and a twist boat in the a-epimer 12.

4 -

Sect. I

- CNS Agents

Hesp, Ed.

7 -

R1= N(CH2CH2C11 R2=H, R 3 = C H 2 d 2‘

(P-CNA)

8

R’= H, R2= N(CH2CH2C1)2, R3= C H 2 U

(a-CNA)

9

R1= NHCOCH= CHC02Me, R2=H, R3= C H 2 - a

10 R 1 = NHCOCH= CHC02Me, R2: 11 R1= NH2, R2= H, R3= Me 12 R1= HI R2= NH2, R3=Me

H, R3= Me

(P-FNA)

( 0 ~ 0 ~ )

Of four pentazocine analogues in which the N-alkyl group was folded was the most potent ( . 5 X back into 6- or 7-membered riyj$,lFgmpound pentazocine, mouse writhing). . Conjugate addition of alkyl groups to the 8-position of codeinone gave a series of analgesics in which the 8 - a - e p i m ~were ~ ~ generally more potent (mouse tail flick) than the 8-BSyntheses of two isomeric groups of thiazolomorphans were epimers. reportjTd o which 14 was the most potent (10 X codeine, mouse writhing) 5:11g Compound 14 is a 9-B-epimer and is somewhat more potent than the 9-a-epimer, in contrast with precedents from the benzomorphans,

a

.

u

14

15

There wer r rts of further modifications of ring C of morphine and From a large series of active compounds oxet n codeine . 1’i7-ci88 was about 700 X more potent (mouse writhing, s.c.) than morphine. Translocation of the hydroxyl group from the 4- to the 1-position destroyed activity in N-meth lmorphinan-6-ones.120 S ntheses of r’ng-C-contracted 6,14-exo-ethen m rphinans,12’ 2‘-thiobenzomorphans,12’ morphinanones,82j and more exotic heterocyclic analogs12‘ were described without indications of biological activity. A series of fentanylenkephalin hybrids were essentially inactive in isolated tissue assays. 125

?18

The roles of hydrophobic and hydrophilic (amphiphilic) domains of the &endorphin surface in relation to ph s’cochemical,biochemical, analgesic and binding activities were explored. ‘2‘,127 Studies on four large synthetic peptides designed to simulate or modify the surface properties of &endorphin led to the conclusion that exact residue homology may be less important to bioactivity than the gross physicochemical properties of the binding surface. In a complementary approach, a 40-residue peptide designed to mimic the binding site of opioid receptors was synthesized. 128 It displayed modest but significant affinity for several opioid peptides and discriminated them from non-opioids. (Leu-enkephalin, KD = 5.8 x

Chap.

Analgesics

1

Lever, Chang, McDermed

2

Non-Opioid Analgesia Direct modulation of opioid pathways has been thetmajor focus of modern analgesic research, but other modalities for antinociception appear to involve such pathways only indirectly or not at all. Stress, of which traditional Chinese acupuncture and modern electroacupuncture may be considered special cases, is one such mode. Early reports of naloxone blockade of acupuncture analgesia suggested mediation by endogenous opioids.129 However, more recent studies contradict this hypothesis. 130 A superficially related clinical procedure for control of chronic pain is electrical transcutaneous or spinal cord nerve stimulation. A doubieblind placebo-control d study indicated that such analgesia is not The neurophysiological mechanisms mediating naloxone-reversible. stress-induced autoanalgesia complex and depend subtly on the paraElectrical stress-induced analge meters of the applied stress. tg3 animals apparently involves both opioid and non-opioid substrates. Front foot shocks in rats produced naloxone-blocked morphine-tolerant analgesia (tail flick) unaffected by h pophysectomy, and therefore describable as opioid, non-hormonal.13y However, similar shocks to the hind feet gave analgesia unaffected by naloxone, tolerance, or morphtS3 Moreover, the hypophysectomy and therefore non-opioid, non-hormonal. footshock regimen influenced the nature of antinociception: prolonged intermittent shocks produced opioid-dependent analgesia, whereas br'ef continuous shocks produced non-opioid analgesia (rat tail flick). 134 esions of the raphe nucleus attenuated the latter but not the ~ ~ ~ ~ though ~ ~ other ~ ' studies i 3 h have i licated the raphe nucleus in mediation of both types of analgesia. 1%

'3'

's5

79,

GABA-ergic mechanisms have been implicated in antinociception. In normal volunteers the GABA agonist THIP (16)produced analgesia against dental pain without tolerance, atory depression, or other characteristics of opiate therapy. resg3S In an open study of cancer patients with chronic pain refractory to mild analgesics, THIP (20 mg i.m.) gave subjective pain control, but side effects (blurred vision, sedation) were dose-limiting.137 The GABA;ergic benzodiazepine midazolam (IJ) given intrathecally to anaesthetized dogs strongly att n ated nociceptive sympathetic reflexes to electrical nerve stimulation,'38 Baclofen and other GABA agonists have a so en found to be central antinociceptives in exThese effects were generally naloxone-in perimental animals. sitive, though indi ct effects o opioid substrates may be present.785GABA analgesia may"? o r may not144 be cross-tolerant to morphine.

139-1''

*N \

16

17

HO

18 -

Intrathecal administration of the -agonist clonidine attenuated nociceptive responses in rats and cats.9L45 Attempts to characterize further the receptors involved as a l or a2 gave inconclusive results.146 Studies with monoamine depletors indicated that intact spinal adrenergic and serotonergic fibers are 'nvolved in a complex way in expression of morphine analgesia in rats. 147 Adrenergic systems may mediate foot shock analgesia (rats, tail flick).148 A study in terminal cancer patients

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Sect. I

- CNS Agents

Hesp, Ed.

indicated effective control of chronic pain by epidural labetalol, a mixed a- and 8-antagonist though the result might have been due to a local anaesthetic effect. Several studies indicate that opioids acti ate erotonergic neurons and increase 5-HT turnover in the spinal cord. 130-155 Intrathecal 5-HT produced antinociception in mice (tail flick).153 The role of serotonergic neurons in opioid analgesia may be modek-dependent.152. Central cholinergic fib rs may also mediate analgesia,l5 and both m ~ s c a r i n i c l ~ ~ and nicotinicl5' agonists produce analgesia in rodents by central mechanisms. There is evidence that cholinergic connections m d'ate the expression of both endogenous and exogenous opioid analgesia.754,157 Meptazinol is one drug in clinical use (videinfra) which appears to take fortuitous advantage of the opioid-cholinergic connection. In rodent antinociceptive models it displays ph r acology appropriate for an opiate partial agonist and a cholinomimetic.?58 The analgesic properties of cannabinoid derivatives are well known, though no such drugs have yet been demonstrated to be substantially free from the CNS side eff ct to which cannabissativa owes its popularity as a substance of abuse. 15% 7 Igo However, cannabinoid analgesia has appeal due t failure to induce to its opioi independent mechanism and its con dependence.1 g i . A series of preliminary reports'ig'"985 have described the syntheses of analgesics modelled on 9-nor-9-8-hydroxyhexa drocannabinol; compound 18 was the most potent member (>60 X morphine). %1 Clinical Hinhliahts ODioid Analgesics - Clinical evaluation of the newer opioi agonistantagonist ugs continues, and theirl@algesic efficacy,lg6 phar aand dependence profileslgl9 were cokinetics,%7 receptor pharmacology, recently revi w d. A review on the pharmacology and therapeutic efficacy of nalbuphin 778 and a compilation of clinical experiences with injectable meptazinol'71 were ovided. Human pharmacokinetic data for meptazi 01 were reported. 1729 lSi5 Efficacy studies with epidura pentazocine,174 epidural buprenorphine,175 and parenteral d e ~ o c i n e 'for ~ ~ postoperative pain relief were reported. Oral cirayadol was superior to codein relief of advanced cancer ain. 177 The clinical pharmacokinetics77p;nd applications in anesthesiaq7g of fentanyl and its derivatives were a1 additional reports on enkephalin analogues reviewed. 8 ' e % ' For relief of severe episiotomy pain, metkephamid appeared. 1 acetate (140 mg, i.m.) was superior to mepiridine (100 mg, i.m.) but minor side effects wer more frequent; a lower dose (70 mg, i.m.) of metkephamid was ineffegbive.'80 The preclinical pharmacology of metkephamid was Intrathecal administration of D-Ala2-D-Leu5-enkephalin described. (DADL) to a single patient showed that DADL could provide a powerful, long-lasting analgesic effect, and also is ?@able of producing profound, naloxone-reversible respiratory depression.

'

- Therapeutic appliNon-Ster%dal Antiinflammatory Drugs (NSA ' O f clinical pharmacokineticsq and mechanism of action189 cations, of the peri herally-acting N AIDS were reviewed, and the pharmacology of and suprofen19s were discussed in detail. Ibuprofen diflunisal (400 mg, p.0.) was more effective than z mepirac (100 mg, p . 0 . ) or aspirin (600 mg, p.0.) against episiotomy pain,182 and rectal indomethacin (100 mglgfight hourly) provided effective analgesia following thoraA dose-r sponse study with i.v. indoprofen for postoperative cotomy pain was reported,19f and in women with moderate to severe cancer pain, indoprofen (400mg, i.v.) appeared equivalent to morphine (10 mg,

-

.

Chap. 1

Analgesics

Lever, Chang, McDermed

1

i.m.). lg5 Additional studies with diflunisal,l g 6 9 l g 7 flurbiprofen,lg8 fendosal, Ig9 and diclofenac2” were reported. Therapeutic applications of 2@ were rsJ&ewed.201 f202 In preclinical NSAIDs in primary dysmenorrh were described as effective studies, propionic acids and 3 NSAIDs with reduced ulcerogenic properties.

Migraine - Headache is the most common pain syndrome for which analgesic medication is applied and is associated with a large number of disorders with different etiologies. Comments here will be restricted to vascular headache, particularly migraine, which is probably a symptom of a reactive compensatory mechanism occurring late in a poorly understoojofathogenetic A classisequence. A provocative review of this topic is available. cal migraine attack begins with a prodromal phase which i vol es regional cerebral vasoconstriction leading to hypoxi ischemia,209-208 and possibly reduced neuronal 5-HT synthesis.208’ Onset of the painful headache phase is marked by vasodilation,206 which may reflect a defective vascular homeostatic mechanism.209 Partly because of a dearth of animal models, treatment has evolved empirically from clinical experience and has traditionally consisted of symptom management.210,21 Most drug treatment in the past focused acutely on the painful vasodilation phase. The efficacy of dihydroergotamine212 apparently results from its vasoconstrictor effects. NSAIDs inhibit prostacyclin biosynthes’s a d reduce the intensity, but not the frequency, of attacks.213,214 Newer therapies have addressed prevention of attacks. The prophylactic effects of B-blockers have been established, though their mechanism of action remains speculative. In a long-term placebocontrolled study of propranolol in 245 migraineurs, more than 70% of the subjects had fewer and less severe attacks, and 46% reported th t their improvement was maintained after discontinuation of medication.$15 Nadolol was shown to similarly prophylactic, whereas pindolol and alprenolol were not.28g Calcium channel blockers are the most recent additions to migraine prophylaxis. Clinical studies with flunarizine, a Ca++ blocker with anti-vasoconstrictive and anti-hypoxic effects in animals, indicate that it is clearly ophylactic, though its effect on severity of attacks is marginal.217,28‘ Small clinical studies with nimodipine, nifedipine, and verapamil indicate efficacy for these also.219 Nimodipine appears to produce the fewest side effects, probably because of its high selectivity for cerebral vasculature. These agents appear to prevent migraine attacks by attenuating Ca++-dependent cerebrai vasoconstriction irrespective of the nature of the triggering constrictive stimulus.220’ References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

F . Porreca and T.F. Burks, Eds., Life Sci., 33 Su 1 I pp 1-782 (1983). Opioid Peptides”, J. Hughes, Ed., Brit. M a . ’ B ~ r f , g1-106(1983). , J.W. Thompson, Brit. Med. J. 288 259 (1984). A. Grossman and V. Clement:JKes, Clin. Endocrinol. Metab.. 12,31 (1983). J.A. Gilbert and E. Richelson. Mol. Cell. Biochem. 55 83 ( 1 9 8 3 r A. Herz, J. Neural. Transm. (Suppl.),3 , 2 2 7 (198i)-’ D.L. Copolov and R.D. Helme, Dru s 26 503 (1983). F.E. Bloorn.Ann. Rev. Pharmaeol.%&Zbl., 23,151 (1983). V. Clement-Jonesand G.M. Besser, Brit. MexBull. 39 95 (1983). J.W. Holaday, Biochem. Pharmacol., 32,573 (1983)’fim Rev. Pharmacol. Toxicol., 23,541 (1983). S. Mousa and D. Couri. Subst. Alcohornctions Misuse 4 1 (1983). W.A. Corrigall, Pharmacol. Biochem. Behav. 18 255 {&3), B. Pernow, Pharmacolog. Rev., 35,85 (1983).’-’

-8 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 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.

Sect. I

-

CNS Agents

Hesp, Ed.

L.F. McNicholasand W.R. Martin, Drugs 27 81 (1984). (a)J.E. Morley and A.S. Levine, Lancet, 1: g 8 (1983);(b) J.E. Morley, A S . Levine, G.K. Yimand M.T. Lowy, Neurosci. Biobehav. Rev., 1,281 (1983). J.K. Wamsley, Pharmacolog. Rev., 35,69 (1983). O.W. Lever,Jr., K.-J. ChangandJ.bMcDermed.Ann. Rep. Med. Chem.,8,51(1983). J.L. Marx, Science, 220,395 (1983). R.V. Lewis and A.S.=ern, Ann. Rev. Pharmacol. Toxicol. 23 352 (1983). S. Udenfriend and D.L. Kilpatrick, Arch. Biochem. Bioph;CiZl, 309 (1983). H. Imura, Y. Nakai, K. Nakao. S. Oki, I. Tanaka, H. J i n g a m i , m o s h i m a s a . T. Tsukada, Y. Ikeda, M. Suda and M. Sakamoto, J. Endocrinol. Invest., ti, 139 (1983). J.Hughes, Brit. Med. Bull., 39,17(1983J. S.F. Atweh and M.J. Kuhar,%t. Med. Bull. 39 47 (1983). D.G. Smyth, Brit. Med. Bull., 2 , 2 5 (1983). ’ -’ A.C. Cuello, Brit. Med. Bull., 39, 11 (1983). L.D. Fricker and S.H. Snyder,T Biol. Chem., 258,10950 (1983). L.D. Fricker,T.H. Plummer. Jr. and S.H. Snyder, Biochem. Biophys. Res. Commun., 111,994(1983). J.-C. Schwartz, S. de la Baume, C.C. Yi. P. Chaillet. H. Marcais-Collado and J. CostenG, J. Neural Transm. (Suppl.), 18,235 (1983). “Degradayon of Endogenous Opioids: Its Relevance in Human Pathology and Therapy,” S. Ehrenpreis and F. Sicuteri, Eds., Raven Press, New York, 1983. P. Chaillet, H. Marcais-Collado. J. Constentin, C.C. Yi, S. de la Baume and J.-C. Schwartz, Eur. J . Pharmacol., 86,329 (1983). KBorin, P. Giustiand L. Cima, Life Sci., 33,1575 (1983). B.P. Roques, E. Lucas-Soroca, P. Chaillet,T Costentin and M:C. Fournie-Zaluski, Prw. Natl. Acad. Sci. USA, 80 3178 (1983). ‘PTbloras, A.M. Bidabe, J.M. Caille, G. Simonnet, J.M. Lecomte and M. Sabathie, Am. J. Neuroradiol., 5,653 I1 ~QRRI ____,.

R.C. Balagot, S. Ehrenpreis, J. Greenberg and M. Hyodo, in Ref. 29, p. 207. J. Grevel and W. Sadee, Science, 221,1198 (1983). S.W. Tam, Proc. Natl. Acad. Sci..m6703(1983). D.B. Vaupel, Eur. J. Pharmacol., 269 (1983). B.L. Slifer and R.L. Balster, J. Pharmacol. Exp. Ther., 225,522 (1983). H.E. Shannon, J. Pharmacol. Ex Ther., 225,144 ( 1 9 8 r J.M. White and S.G. Holtzman, Ikychopharrnacol., 80, 1 (1983), J. Pharmacol. Exp. Ther., =,95 (1983). J.M. Moerschbaecher and D.M. Thompson, J. Pharmacol. Exp. Ther., 2 , 7 3 8 (1983). N. Johnson and G.W. Pasternak. Life Sci., 33,985 (1983). J.E. Leysen, W. Gommeren and C.J. Niemegeers, Eur. J. Pharmacol., 87,209 (1983). J.W. Villiger, L.J. Ray and K.M. Taylor, Neuropharmacol., 22,447 (1983). J.-C. Meunier, Y. Kouakou, A. Puget and C. Moisand, Mol. Karmacol., 3 , 2 3 (1983). H.H. Osborne and A. Herz, Eur. J. Pharmacol., 86,373 (1983). E. Castanas, P. Girand, K. Audigier, R. Drissi, rBondouresque, B. Conte-Devolx and C. Oliver, Mol. Pharmacol., 25,38 (1984). K.J. Mack, ATkillian and J.A. Weyhenmeyer, Life Sci., g, 281 (1983). S.L. Hart, H. Slusarczyk and T. W. Smith, Eur. J. Pharmacol., 95,283 (1983). G.S.Ling and G.W. Pasternak, Brain Res., 271, 152 (1983). T.L. Yaksh, J. Pharmacol. Exp. Ther., 226.303 (1983). L.F. Tseng, Life Sci., 32,2545(1983). N. U ton, R.D. Sewe1KndP.S. S ncer, Arch. Int. Pharmacodyn. Ther., 262,199(1983). S.J. &ard and A.E. Takemori, J.!?harmacol. Exp. Ther., 224,525 (1983).P.L. Wood, D. Sanscha in, J W.Richard and M.ThakurFPharmacol. Exp. Ther., 226,545 (1983). A. Pazos and J. Florez,%ur. J: Pharmacol.,Q, 309 (1983). S.J. WardandA.E. Takemori, Eur. J. Pharmacol.,Q, l(1983). G.S. Ling, K. Spie el, S L Nishimura and G.W. Pasternak, Eur. J. Pharmacol., g, 487 (1983). J.W. Holaday, Pasternak and A.I. Faden, Neurosci. Lett., 37,199(1983). K.-J. Chang, E.T. Wei, A. Killian and J.K. Chan J Pharmacomxp. Ther., 227,403 (1983). L.M. Sayre, P.S. Portoghese and A.E. Takemori,%ur. J. Pharmacol., %,159(1983). P.S. Portoghese and A.E. Takemori, J. Med. Chem., 26,1341 (1983). T.T. Chau, F.E. Carter and L.S. Harris, J. PharmacorExp. T h e r . , g , 108(1983). T. Chow and R.S. Zukin, Mol. Pharmacol., @, 203 (1983). T.M. Cho, B.L. Ge, C. Yamato, A.P. Smithand H.H. Loh, Proc. Natl. Acad. Sci. USA, &,5176(1983). R. Rothman andT.C. Westfall, J. Eu’eurobiol.,14,341 (1983). R. Rothman andT.C. Westfall, Neurochem. Res., 8,913 (1983). E. Weber, F.S. Esch, P. Bohlen, S. Paterson, A.D. Corbett, A.T. McKnight, H.W. Kosterlitz, J.D. Barchas and 7362(1983). C.J. Evans, Proc. Natl. Acad. Sci. USA, H. Matsuo, A. Mi ata and K. Mizuno, Nzure, 3 ,721 (1983). M. Westphal, Li, E.P.Heimer and J. Meie9ofer, Biochem. Biophys. Res. Commun., 114,1084 (1983). E.P. Heimer, T.J. Lambros, A.M. Felix, G. Fleminger, C.H. Li, M. Westphal and J. M e i e n h z r , Arch. Biochem. Bio hys., 225,518(1983). J. Cphou, J x n and E Costa, Life Sci. $?, 2589 (1983). H.Y. Yang, P. #anula,J. Tang and E. Costa, J. Neurochem., 3 , 9 6 9 (1983). R.G. Williams and G.J. Dockray, Neurosci., 2,563 (1983). P. Giraud, E. Castanas, G. Patey, C. Oliver and J. Rossier, J. Neurochem. 41 154 (1983). E.A. Majane, M.J. Iadarola and H.-Y.T. Yang, Brain Res.. 264,336 (1983):-’ J. Tang, J. Chou, H.-Y.T. Yan and E Costa. Neuropharmacbl., 22,1147 (1983). J. Tang, J. Chou, A.Z. Zhang, fi.-T. Yan andE. Costa, Life S c i . , B , 2371 (1983). ues Eur. J. P h a r m a c x , a , 147(1983). J.M. Zajac, N. Ling, J. Rossier and B.P. K.-J. Chan and J.-K. Chang, Life Sci., !$%up&. I, 267. (1983). C.H. Li, D.kamashiro, P. Ferrara, L.-FTseng and E.L. Way, Int. J. Peptide Protein Res., 2l,331 (1983) D. Yamashiro, P. Nicolas and C.H. Li, Int. J. Peptide Protein Res., 21,219 (1983). S. Salvadori, G.P. Sarto and R. Tomatis, Eur. J. Med. Chem., 18 483(1983). S. Safvadori, M. Marastoni, R. Tomatis and G.Sarto, FarmacoTS, 153 (1983). K. Darlak, Z. Grzonka, P. Janicki, A. Czlonkowski and S.W. Gumulka, J. Med. Chem., 26,1445 (1983). R. de Castiglione and G. Perseo. Int. J. Peptide Protein Res., 21,471 (1983). G. Feuerstgin and A I. Faden, J. Pharmaiol. Exp. Ther., 226,r51(1983).

s,

-

G.8,

a,

C.d

Chap. 88.

89 90. 91.

1

Analgesics

Lever, C h a n g , McDermed

2

E.C. Degli-Uberti, G. Trasforini. S. Salvadori, R. Tomatis, A. Margutti, M. Bianconi, C. Rotola and R. Pansini, J Clin. Endocrinol. Metab. 56 1032 (1983). J.-M. Zajac, G. G a d , F. k&t, P. Dodey, P. Rossignol and B P. Roques, Biochem. Biophys. Res. Commun., 111, 390 (1983). D. Rodbard, T. Costa, Y Shimohigashi and S. Krumins, J. Rece t Res 3 21 (1983). H.I. Mosberg, R. Hurst, V.J. Hruby, J.J.Galligan, T.F. Burks, 8 . G e e H’dH.I. Yamamura, Life Sci., 32,2565 1l l1 l 0 ~ 9 ) ” ” , .

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. ii7. 118. 119. 120. 121. 122.

123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156.

H.I. Mosberg, R. Hurst, V.J. Hruby, K. Gee, H.I. Yamamura, J.J.Ga1liganandT.F. Burks, Proc Natl. Acad. Sci. USA. 80,5871 (1983). J.M.Berman, M. Goodman, T.M.-D. Nguyen and P.W. Schiller, Biochem. Biophys. Res. Commun ,115,864 (\-””-,. lam T.S. Sudha and P. Balaram, Int. J. Peptide Protein Res., Zl,381 (1983). T. Deeks. P.A. Crooks and R.D. Waigh, J. Med. Chem., 26,762 (1983) Y. Shimohiagashi and C.H. Stammer, J. Chem. Soc., Pe& I. 803 (1983). Y. Shimohigashi, C.H. Stammer, T. Costa and P.F. Vonvoigtlander. Int. J Peptide Protein Res., 22.489 (1983) H. Kimura, C.H. Stammer, Y. Shimohigashi, C. Ren-Lin a n d J . Stewart, Biochem. Biophys. Res.Fommun., 115, 112 (1983). N. Muthukumaraswamy, A.R. Day, D. Pinon, C.S. Liao and R.J. Freer, Int. J . Peptide Protein Res., 22,305 IIBR.?) ,. - - -,.

J.-L. Fauchere, S. Pfenninger, K.Q. Do, C. Lemieux and P W. Schiller, Helv. Chim. Acta, 66, 1053 (1983). R.G. A1mouist.C.M. Olsen. E.T. Uvenoand L.Toll. J. Med Chem.. 27.115(1984). L Nadasdi, D Yamashiro, C H Lland P Huidobro-Toro. Int J P e z d e Protein Res .21.344 (1983) . _. J.A. Kiritsy-Roy, S.K. Chan and E.T. Iwamoto. Life Sci., 32,889 (1983). L. Kupczyk-Subotkowskaand P. Mastalerz, Int. J. PeptiGProtein Res. 21 485 (1983);P. Mastalerz, L. Kupczyk-Subotkowska. Z.S. Herman and G. Laskawiec. Saturwiss.. 6 r k 6 (1982). C. Zioudiou, D. Varoucha, S. Loukas, N. Nicolaou, R.A. Streaty and W : r K l e e , J. Biol. Chem., 258,10934 (19831. M.Szucs, S.Benyhe, A. Borsodi, M.Wolleman, G.Jancso, J. Szecsi and K. Medzihradszky, Life Sci ,32,2777 (1983). K.C. Rice, A.E. Jacobson, T.R. Burke, Jr., B.S. Bajwa, R.A. Streaty and W.A. Klee, Science.=,314 (1983). P.S. Portoghese and A.E. Takemori, in ”The Chemical Regulation of Biological Mechanisms,” A.M. Creighton and S. Turner, Eds., Royal SocietyofChemistry S ecial Publication No. 42, p. 181 (1983). L.M. Sayre, A.E. Takemori and P.S. Portoghese, J! Med. Chem. 26 503 (1983). L.M. Sayre, D.L. Larson, D.S. Fries.A.E. Takemori andP.S. PorGghese,J. Med.Chem ,26,1229(1983). R.C. Crouch, A.V. Bhatia and O.W. Lever, Jr., Tetrahedron Lett. 24 4801 (1983). M. Hori, T. Kataoka, H. Shimizu, E. Imai, Y. Suzuki and N. Kaw;&ra, Heterocycles, 20,1979 (1983). M. Hori. T. Kataoka, H. Shimizu, E. Imai, Y. Suzuki, N.Kawamura, H. Fujimura, M.h&aki and M.Niwa, Chem. Pharm. Bull. Jap., 31,2520 (1983). F. Ghozland, P. Maroni, I.Tiloria and J. Cros, Eur. J . Med. Chem., Is,22 (1983). K. Katsuura, M. Ohta and K. Mitsuhashi, Chem. Pharm. Bull. J a , 3 0 , 4 3 7 8 (1982). K. Katsuura, K. Yama chi S. Sakaiand K Mitsuhashi, Chem. {harm. Bull. Jap., 3 l , 1518 (1983). D.L. Leland and M.P. J. Org. Chem. ‘48 1813 (1983). M.P. Kotick, J. Org. Chem., 48,1819 (1983).’-’ M.P. Kotick, D.L. Leland, J . r P o l a z z i , J.F. Howesand A. Bousquet, J. Med. Chem., 26.1050(1983). H. Schmidhammer and A. Brossi, J . Org. Chem. 48 1469 (1983). A. Ghosh, D. Portlock, H. Dalzell, C. Malmberg.’P-Herlihy and R. Razdan, J. Org. Chem, 48,4137 (1983). M. Hori, M. Ban, E. Imai, N. Iwata, Y. Baba, H. Fujimura, M. Nozaki and M. Niwa, Heterocycles, 20,2359 (1983). M. BOs, W. Fleischhacker and P. Tschany, Heterocycles, 20,1077 (1983). J. Bosch, A. Doming0 and R. Granados, J. Heterocyclic Chem., 20,887 (1983). M.Y.H. Essawi and P.S. Portoghese, J. Med. Chem., 26,348 (1983). J.W. Taylor, R.J. Miller and E.T. Kaiser, J. Biol. Chem., 258,4464(1983). J . P . Blanc, J.W. Taylor, R.J. Miller and E.T. Kaiser, J. B X C h e m . , *,8277(1983). W. Kullmann, J. Med. Chem. 27 106 (1984). D.J. Mayer, D.D. Price and A: ~ i iBrain , Res., 121,368 (1977). C.R. Chapman, C. Benedetti, Y. Colpitts and R. G l a c h , Pain, 16,13 (1983). T.B. Freeman, J.N. Campbelland D.M. Long,Pain,l7,189(198n. G.W. Terman. J.W. Lewis and J.C. Liebeskind, Brain Res. ’ -’ 260 147 (1983). L.R. Watkins and D.J. Mayer, Science, 216,1185 (1982). J.T. Cannon, J.W. Lewis, V.E. Weinberg and J.C. Liebeskind, Brain Res., 269,231 (1983). L.R. Watkins, E. Young, I. Kinsheck and D.J. Mayer, SOC. Neurosci. Abstr , 340 (1981). T. Lindeburg, S. Folsgard. H. S i l k e n , E. Jacobsen and H. Kehlet, Acta Anaesthesiol. Scand., 27,10(1983). M.Kjaer and H. Nielsen, Br. J . Clin. Pharmacol. 16 477 (1983). D. Niv, J.G. Whitwam and L. Loh, Br. J. Anaest.

1-CH-(CHA0

~ e -

OOH

(CH2)-cn-,. m

36H

2_2

(2)

MDL 899 i s a d i r e c t a c t i n g a r t e r i o l a r v a s o d i l a t o r i n man, but c o a d m i n i s t r a t i o n of a beta-blocker i s n e c e s s a r y t o prevent r e f l e x - r e l a t e d s i d e e f f e c t s . l t g A c l i n i c a l s t u d y w i t h c a d r a l a z i n e (ISF 2469, 22) shows t h i s compound s i m i l a r t o h y d r a l a z i n e w i t h fewer s i d e e f f e c t s . 50 Two o t h e r may owe some of t h e i r acv a s o d i l a t o r s , b u d r a l a z i n e (28) and p i n a c i d i l (2) t i v i t y t o calcium channel blockade. 5 1 52 Anthranilamide 30 (WIN 48,049) lowers blood p r e s s u r e i n monkeys w i t h o u t accompanying t a c h y c a r d i a . This compound a c t s p r i m a r i l y as a d i r e c t v a s o d i l a t o r b u t a l s o has sympatholytic and dopaminergic a c t i v i t i e s . 54 Compounds 2 and 2 a l s o lower blood p r e s s u r e by a p p a r e n t l y d i r e c t v a s o d i l a t o r mechanisms. 55 3 56

n

33 The 0 - s u l f a t e 22 of m i n o x i d i l h a s been proposed as an i m p o r t a n t act i v e m e t a b o l i t e . 5 7 Unlike m i n o x i d i l , 33 i s a c t i v e i n v i t r o and i s more pot e n t w i t h a f a s t e r o n s e t of a c t i o n i n v i v o . Evidence s u g g e s t s t h e dihydroexert t h e i r e f f e c t pyrano and dihydrofuranocoumarin v a s o d i l a t o r s (e.g. by CAMP p h o s p h o d i e s t e r a s e i n h i b i t i o n . 58 F i n a l l y , t h e p i v a l i c a c i d e s t e r of i s o x s u p r i n e 35 h a s a slower o n s e t and l o n g e r d u r a t i o n of a c t i o n t h a n t h e p a r e n t phenoT5’

34)

Calcium Entry Blockers - I n t h e U.S. n i f e d i p i n e , verapamil, and d i l t i a z e m are marketed f o r a n t i a n g i n a l o r a n t i a r r h y t h m i c u s e , though none has FDA approval y e t f o r use i n h y p e r t e n s i o n . The p o t e n t i a l t h e r a p e u t i c use of calcium e n t r y b l o c k e r s (CEB) i n t h e

64

Sect. I1

- Pharmacodynamic Agents

Comer, Ed.

treatment of a t h e r o s c l e r o s i s has r e c e n t l y been reviewed. 6 o Verapamil and n i f e d i p i n e have been shown t o s u p p r e s s a t h e r o g e n e s i s i n c h o l e s t e r o l - f e d r a b b i t s without a l t e r i n g serum c h o l e s t e r o l l e v e l s . 6 1 9 6 2

New evidence s u g g e s t s c e r t a i n CEB a f f e c t n e u r a l r e g u l a t i o n of blood v e s s e l s a t several sites. S t u d i e s i n a n e s t h e t i z e d dogs i n d i c a t e t h a t n i f e d i p i n e i n c r e a s e s and verapamil d e c r e a s e s t h e s e n s i t i v i t y of c a r o t i d s i n u s b a r o r e c e p t o r s a p p a r e n t l y by i n t e r f e r e n c e w i t h a calcium-dependent and a sodium-dependent mechanism, r e s p e c t i v e l y . A similar i n c r e a s e i n baroref l e x s e n s i t i v i t y w a s found i n man t r e a t e d c h r o n i c a l l y w i t h n i f e d i p i n e , which may e x p l a i n why t h e r e i s evidence of sympathetic a c t i v a t i o n d u r i n g i n i t i a l therapy. 6 4 Tachycardia and i n c r e a s e d l e v e l s of plasma norepinephrine a r e found d u r i n g a c u t e b u t not c h r o n i c phases of n i f e d i p i n e t r e a t m e n t . 6 5 During a c t i v a t i o n of p o s t g a n g l i o n i c sympathetic nerves t h e r e i s an e x o c y t o t i c release of norepinephrine which i s dependent upon t h e movement o f calcium a c r o s s t h e n e u r a l c e l l membrane. The e f f e c t s of CEB on stimuluss e c r e t i o n coupling i n sympathetic n e r v e s has been s t u d i e d i n s e v e r a l d i f f e r e n t p r e p a r a t i o n s . Verapamil does n o t a f f e c t t h e n e u r a l release of norepinephrine i n e i t h e r i s o l a t e d c a t h e a r t o r i n the pithed rat.66,67 I n t h e l a t t e r model n i f e d i p i n e b l u n t e d i n c r e a s e s i n blood p r e s s u r e due t o act i v a t i o n of sympathetic n e r v e s b u t d i d n o t a f f e c t c a r d i o v a s c u l a r responses t o exogenous norepinephrine s u g g e s t i n g a p r e j u n c t i o n a l s i t e of i n h i b i t i o n . A similar p r e j u n c t i o n a l CEB e f f e c t w a s found f o r d i l t i a z e m i n guinea p i g m e s e n t e r i c and dog b a s i l a r a r t e r i a l p r e p a r a t i o n s . 6 8 C i n n a r i z i n e and f l u n a r i z i n e were shown more p o t e n t than verapamil, d i l t i a z e m o r n i f e d i p i n e i n reducing t h e potassium-induced uptake of calcium i n a synaptosomal preparation.69

New s t u d i e s have examined t h e i n t e r a c t i o n of v a r i o u s CEB w i t h a-adren o c e p t o r s and t h e i r e f f e c t on p r o c e s s e s mediated by a-adrenoceptors. 70 The (+) and (-) a n t i p o d e s of verapamil, n i c a r d i p i n e and (+) D-600 showed s i m i l a r i n h i b i t i o n of r a d i o l i g a n d binding t o al-adrenoceptors, whereas d i l t i a zem and n i f e d i p i n e showed a v e r y low level of a c t i v i t y . 7 1 A d i f f e r e n t p a t t e r n was s e e n i n r e g a r d t o a2-adrenoceptors. (-) Verapamil w a s t h e most pot e n t i n h i b i t o r of binding w i t h (+) verapamil, ( 2 ) D-600, d i l t i a z e m , and n i c a r d i p i n e showing i n t e r m e d i a t e a c t i v i t y . N i f e d i p i n e was i n a c t i v e . Verapamil, b u t n o t d i l t i a z e m , impairs t h e a2-adrenoceptors p r e s e n t i n r a b b i t hypothalmus which modulate t h e release of norepinephrine. 72 The dihydropyridines nimodipine o r PY-108-068 ( 3 8 ) , a s w e l l a s verapamil nifedipine and d i l t iazem s e l e c t i v e l y i n h i b i t vasoconst r i c t i o n m e d i a t e d by a2 -adreno c e p t o r s and n o t t h a t mediated by a]-adrenoceptors. 73-79 Thus, v a s o d i l a t a t i o n (and presumably a n t i h y p e r t e n s i v e e f f e c t s ) caused by CEB may be due t o impairment of a2-adrenoceptor mediated v a s c u l a r tone. There may be some impairment of v a s c u l a r al-adrenoceptor p r o c e s s e s as w e l l . 78 N i f e d i p i n e dec r e a s e s v a s o p r e s s o r responses t o norepinephrine i n man. *O

(z),

uM8GC

(x),

-

M b H

The dihydropyridine CEB f l o r d i p i n e

(2)h a s

pharmacological proper-

Chap.

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

Sprague, Powell

t i e s u n l i k e o t h e r d i h y d r o p y r i d i n e s . 8 1 F l o r d i p i n e and n i f e d i p i n e showed sim i l a r p o t e n c y i n v i t r o i n r e l a x i n g potassium-depolarized c a n i n e v e i n s b u t f l o r d i p i n e was >lOO-fold less p o t e n t i n c a u s i n g r e l a x a t i o n of a r t e r i a l s t r i p s . F l o r d i p i n e w a s 1000-fold less p o t e n t t h a n n i f e d i p i n e i n c a u s i n g d e p r e s s i o n o f f e l i n e c a r d i a c t i s s u e . Thus, f l o r d i p i n e is a " v a s c u l a r sel e c t i v e ' ' CEB. I n SHR, a 30 mg/kg dose o f f l o r d i p i n e produced blood p r e s s u r e l o w e r i n g which p e r s i s t e d f o r 24 h r s and, i n dogs, o r a l d o s e s from 0.03 t o 3.0 mg/kg lowered blood p r e s s u r e due t o reduced p e r i p h e r a l v a s c u l a r res i s t a n c e . 8 2 The lowered blood p r e s s u r e was a s s o c i a t e d w i t h t a c h y c a r d i a , presumably of r e f l e x o r i g i n . O r a l d o s e s of 200 o r 300 mg lowered blood p r e s s u r e i n normotensive human s u b j e c t s and caused r e f l e x t a c h y c a r d i a . 8 3 I n h y p e r t e n s i v e s u b j e c t s , 60 mg of f l o r d i p i n e lowered blood p r e s s u r e w i t h o u t any accompanying changes i n c a r d i a c r a t e . I n a d d i t i o n t o calcium e n t r y b l o c k a d e , f l o r d i p i n e h a s been found t o i n h i b i t c y c l i c n u c l e o t i d e phosphod i e s t e r a s e . 84 Y 8 5

Another v a s c u l a r s e l e c t i v e d i h y d r o p y r i d i n e , FR34235, (40) i s a b o u t f o u r t i m e s more p o t e n t i n v i t r o t h a n n i f e d i p i n e i n c a u s i n g r e l a x a t i o n i n p o t a s s i u m - d e p o l a r i z e d r a b b i t a o r t i c s t r i p s b u t 50 times less p o t e n t i n causi n g d e p r e s s i o n of r a b b i t c a r d i a c t i s s u e . 86 FR34235 a l s o d i s p l a y e d s e l e c t i v i t y f o r r e l a x a t i o n o f a r t e r i a l s t r i p s from v a r i o u s v a s c u l a r b e d s . 8 7 Given i . v . t o a n e s t h e t i z e d dogs, BAY K 8644 (41) caused i n c r e a s e s i n m y o c a r d i a l c o n t r a c t i l i t y , blood p r e s s u r e , and p e r i p h e r a l v a s c u l a r resist a n c e . 8 8 , 8 9 These changes were r e v e r s e d by n i f e d i p i n e b u t n o t by a- o r 6a d r e n o c e p t o r blockade. BAY K 8644 i n c r e a s e d c a r d i a c r a t e and c o n t r a c t i l i t y and a l s o caused coronary v a s o c o n s t r i c t i o n i n an i s o l a t e d c a r d i a c p r e p a r a t i o n . T h i s compound may b e u s e f u l a s a t o o l t o a s c e r t a i n t h e f u n c t i o n of CEB i n t i s s u e s . SC-30552 (42) g i v e n o r a l l y t o SHR ( s p o n t a n e o u s l y h y p e r t e n s i v e r a t s ) lowered blood p r e s s u r e and caused b r a d y c a r d i a . Tachycardia caused by i s o p r o t e r e n o l w a s n o t a f f e c t e d by SC-30552, whereas i s o p r o t e r e n o l - i n d u c e d decreases i n blood p r e s s u r e were a t t e n u a t e d . G a n g l i o n i c b l o c k a d e caused by hexamethonium d i d n o t a f f e c t v a s o d i l a t a t i o n caused by t h i s CEB. A pA2 v a l u e a g a i n s t calcium-induced c o n t r a c t i o n s of r a b b i t . a o r t i c s t r i p s of 7.42 w a s c a l c u l a t e d . These e f f e c t s s u g g e s t 42 p o s s e s s e s some $2- b u t n o t $1- o r aa d r e n o c e p t o r a c t i v i t y and may d e p r e s s t h e SA node.

(g),

a new t y p e of CEB, g i v e n t o normotensive rats o r a l l y a t KB-944 10-50 mg/kg produced graded r e d u c t i o n s i n blood p r e s s u r e and i n c r e a s e d c a r d i a c rate.'l The i n c r e a s e d h e a r t r a t e was a p p a r e n t l y due t o t h e b a r o r e c e p t o r r e f l e x as i t was b l u n t e d by concomitant p r o p r a n o l o l t r e a t m e n t . KB-944 w a s i n t e r m e d i a t e i n potency (nifedipine>KB-944>diltiazem) as an a n t i h y p e r t e n s i v e when g i v e n t o SHR, DOCA-NaC1 h y p e r t e n s i v e r a t s , and r e n a l hypert e n s i v e r a t s , and had a l o n g e r d u r a t i o n of e f f e c t . KB-944 g i v e n i n t r a v e n o u s l y t o a n e s t h e t i z e d dogs produced n e g a t i v e d r o m o t r o p i c e f f e c t s r e f l e c t e d by i n c r e a s e d AV n o d a l conduction t i m e and 3' h e a r t b l o c k i n some a n i m a l s and t h u s KB-944 may be u s e f u l as a n a n t i a r r h y t h m i c e g 2

(44)

reduced blood p r e s s u r e i n S e r o t o n i n Receptor A n t a g o n i s t s - K e t a n s e r i n e x p e r i m e n t a l and c l i n i c a l h y p e r t e n s i o n a c t i n g by s p e c i f i c blockade of serot o n i n (5-HT2) r e c e p t o r s . 9 3 However, k e t a n s e r i n h a s been found t o p o s s e s s p o t e n t a-adrenoceptor b l o c k i n g p r o p e r t i e s i n v i t r o and t h e a n t i h y p e r t e n s i v e e f f e c t s of t h i s compound more c l o s e l y c o r r e l a t e w i t h a-adrenoceptor r a t h e r t h a n s e r o t o n i n antagonism. 9 4 LY53857 (45) i s a h i g h l y s p e c i f i c s e r o t o n i n r e c e p t o r a n t a g o n i s t which does n o t lower blood p r e s s u r e i n S H R . 9 5 Thus, i t a p p e a r s t h a t a n t i h y p e r t e n s i v e e f f e c t s of s e r o t o n i n r e c e p t o r a n t a g o n i s t s may be due, a t l e a s t i n p a r t , t o a-adrenoceptor blockade.

66

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Dopamine Receptor Agonists - Compounds which mimicdopamine can lower blood p r e s s u r e by pre- and p o s t j u n c t i o n a l as w e l l as c e n t r a l n e u r a l mechanisms. The R- and S-enantiomers of SKF 82526 (46) are b o t h r e n a l v a s o d i l a t o r s , b u t t h e R-enantiomer o p e r a t e s i n h y p e r t e n s i v e rats by a p o s t j u n c t i o n a l mechis a pergolide anism whereas t h e S-enantiomer does n o t . 9 6 LY141865 (48) d e r i v a t i v e which lowers blood p r e s s u r e a f t e r o r a l a d m i n i s t r a t i o n t o SHR.97 LY141865 i n low doses reduced t h e h y p e r m o t i l i t y observed i n SHR b u t not i n t h e i r corresponding normotensive c o n t r o l s . 9 8 P e r g o l i d e g i v e n i . v . lowers blood p r e s s u r e and reduces p e r i p h e r a l sympathetic n e r v e a c t i v i t y s u g g e s t i n g a c e n t r a l nervous system s i t e of a c t i o n . 9 9

(47)

-

MPV295 (49) lowers blood p r e s s u r e a-Adrenoceptor Agonists and A n t a g o n i s t s bv s t i m u l a t i o n of a-adrenoceptors i n t h e b r a i n s t e m F o o MPV295 s t i m u l a t e s o n l y c a r d i o v a s c u l a r a2-adrenoceptors whereas c l o n i d i n e s t i m u l a t e s b o t h aland a2-adrenoceptors. I n t e r e s t c o n t i n u e s i n t h e area of s e l e c t i v e a l - a d r e n o c e p t o r blockade as a mechanism t o lower blood p r e s s u r e . Compound 50 w a s less t h a n 1 / 1 0 as p o t e n t as p r a z o s i n i n v i t r o as an a-adrenoceptor b l o c k e r b u t w a s n e a r l y e q u i v a l e n t i n lowering 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 rats. l o l Unl i k e p r a z o s i n , t r i m a z o s i n lowered blood p r e s s u r e by some unknown mechanism i n a d d i t i o n t o blockade of a1-adrenoceptors. l o 2 Compound 51 showed pot e n c y as an al-adrenoceptor b l o c k e r i n v i t r o less t h a n t h a t found w i t h p r a z o s i n . l o 3 The compound lowered blood p r e s s u r e i n SHR and may have c e n t r a l a n t i h y p e r t e n s i v e e f f e c t s due t o blockade of b r a i n al-adrenoceptors. Like p r a z o s i n , KF-4942 (52) lowers blood p r e s s u r e due t o blockade of vasT i b a l o s i n e (53) i s a s e l e c t i v e al-adrenoceptor c u l a r a1- adrenoceptors.m a n t a g o n i s t w i t h b o t h c e n t r a l and p e r i p h e r a l a c t i o n s . O 5 T i b a l o s i n e i s about 1000 t i m e s less p o t e n t t h a n p r a z o s i n i n c a u s i n g blockade of v a s c u l a r a l - a d r e n o c e p t o r s and d i s p l a y s a n x i o l y t i c a c t i v i t y i n animals and a t r a n q u i l i z i n g e f f e c t i n man.lo6 The S-enantiomer of WB-4101 (54) w a s more pot e n t t h a n t h e R-enantiomer i n b l o c k i n g a l - and a2-adrenoceptors and w a s a l s o more s e l e c t i v e toward a l - r e c e p t o r s i n p i t h e d rats. l o 7

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P-Adrenoceptor Blocking Agents Compound 55 d i s p l a ed n e a r l y a 9000-fold g r e a t e r a f f i n i t y f o r 61- than f o r $2-adrenoceptors. I C I 141,292 (56) w a s a l s o s e l e c t i v e € o r B 1-adrenoceptor mediated c a r d i o v a s c u l a r and metaboli c responses. I n t r i n s i c sympathomimetic a c t i v i t y w a s found toward c a r d i o v a s c u l a r , b u t n o t m e t a b o l i c 61-adrenoceptors. l o g K-351 (57), an a n t i h y p e r t e n s i v e a g e n t i n SHR, showed n o n - s e l e c t i v e B-adrenoceptor blockade aa d r e n o c e p t o r blockade, and d i r e c t v a s c u l a r r e l a x a t i o n i n v i t r o . l l o y

-

Miscellaneous Agents A t r i o p e p t i n s I and I1 (At1 and A t I I ) , two p e p t i d e s of a t r i a l t i s s u e o r i g i n , have been i s o l a t e d and c h a r a c t e r i z e d . l 1 These s u b s t a n c e s are d i u r e t i c , n a t r i u r e t i c and r e l a x i n t e s t i n a l (At1 and A t I I ) and v a s c u l a r ( A t I I ) t i s s u e . '12 The d i s c o v e r y of t h e s e compounds s u g g e s t s a new endogenous c o n t r o l mechanism i n v o l v i n g t h e h e a r t and kidneys t h a t may b e important f o r maintenance of normal blood p r e s s u r e . The r a t i o of CNS t o a n t i h y p e r t e n s i v e a c t i v i t i e s , t y p i c a l l y from 1 t o

1 0 f o r hypotensive t e t r a h y d r o c a n n a b i n o l a n a l o g s , h a s been i n c r e a s e d t o 100 i n (58). - 113 However, t a c h y p h y l a x i s w a s observed f o r t h e a n t i h y p e r t e n s i v e activity

.

U-54,669F (2)lowered blood p r e s s u r e i n h y p e r t e n s i v e and normotens i v e r a t s and monkeys. '14 The compound i n h i b i t e d sympathetic neuronal funct i o n and d e p l e t e d catecholamines i n c a r d i a c and o t h e r t i s s u e s . However, t h e r e were no e f f e c t s on p o s t j u n c t i o n a l organ responsiveness t o c a t e c h o l a mines n o r e f f e c t s on blood p r e s s u r e d u r i n g p o s t u r a l changes. References 1. M. Moser, Hypertension, 5, 808 (1983). 2. D.S. Goldstein, Hypertension, 5, 86 (1983). 3. B. Folkow, G.F. DiBona, P. Hjemdahl, P.H. Thoren and B.C. Walin, Hypertension, 5 , 339 (1983). 4. B.C. Zimmerman, Circ. Res., 53, 121 (1983). 5. R.F. Furchott, Circ. Res., 2,557 (1983). 6. D.B. Gordon, Hypertension, 5, 353 (1983). 7. M.F. O'Roarke, Aust. NZ J. Med., 3, 84 (1983). 8. T. Inagami, J.J. Chang, C.W. Dykes, Y. T a k i i , M. Kisaragi and K.S. Misono, Fed. Proc. 47, 2729-2734 (1983). Haber, Clin. and Exper. Hyper.-Theory and P r a c t i c e , A5, 1193-1205 (1983). 9. 10. M. Szelke, B. Leckie, A. H a l l e t t , D.M. Jones, J. S u e i r a s , B. Atrash and A.F. Lever, Nature, 555-557 (1982). 11. M.J.A. A m s t e i n , H.B. B e l l , D.P. Clough, J.S. Major, A.A. Oldham and J. Saunders, B r . J. Pharmacol., 2, 253P (1983). 1 2 . J. Boger, N.S. Lohr, E.H. U l m , M. Poe, E.H. Blaine, C.M. F a n e l l i , T.Y. Linn, L.S. Paine, T.W. Schorn, B.I. LaMont, T.C. Vassil, 1.1. S t a b i l i t o , D.F. Veber, D.H. Rich and A.S. Bopari, Nature, 303, 81-84 (1983). 13. European Patent Application 82111359.6. 1 4 . T. Unger, D. Ganten and R.E. Lang, Clin. and Exper. Hyper.-Theory and P r a c t i c e , A5, 1333-1354 (1983). 15. A. Schwab, I. Weinryb, R . Marcerata, W. Rogers, J. Suh and A. Khandwala, Biochem. Pharm., 2,1957-1960 (1983). 16. M. Burnier, G.A. Turini. H.R. Brunner, M. Porchet, D. Kruithof, R.A. Vukovich and H. Gavras. B r . J. Clin. Pharmacol., 12, 893-899 (1981). 1 7 . T.A. Solomon, F.S. Caruso and R.A. Vukovich, Clin. Pharmacol. Ther., 22, 231 a b s t r . c28 (1983). 18. J . R . Powell, B. Rubin, C.W. Cushman. J. Krapcho and M . J . Antonaccio. Abstr. Eur. ~ e ~ t . Hypertens. 1 s t Milan 355 (1983).

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- Pharmacodynamic Agents

Corner, Ed.

19. D.H. K i m , C.J. Guinosso, G.C. Buzby. Jr.. D.R. Herbst, R.J. McCaully, T.C. Wicks and R.L. Wendt, J . Med. Chem., 26, 394-403 (1983). 20. J.L. Dinish, N.K. Metz, R.W. Lappe, D.H. K i m and R.L. Wendt, Pharmacologist, 25, 102 a b s t r . 8 (1983). 21. J.L. Stanton, N . Gruenfeld, J . E . Babiarz, M.H. Ackerman and R.C. Friedmann, J . Med. C h a . , 26, 1267-1277 (1983). 22. K. Hayashi, Y. Ozaki, K. Nunami, T. Uchida, J. Kato, K. Kinashi and N. Yoneda, Chem. Pharm. Bull., 31, 570-576 (1983). 23. T. Unger, T. Yukimura, M. MarinGrez, R.E. Lang, W. Rascher and D. Ganten, Europ. J . Pharmacol., 2, 411-420 (1982). 24. F.J. McEvoy, F.M. L a i and J.D. Albright, J . Med. Chem.. 26, 381-394 (1983). 25. E.D. Thorsett, E.E. Harris, S. Aster, E.R. Peterson, D. Taub, A.A. P a t c h e t t , E.H. Ulm and T.C. Vassil, Biochem. Biophys. Res. Corn., 111,166-171 (1983). 26. C.S. Sweet, Fed. Proc. 42, 167-170 (1983). 27. H.H. Rotmensch, P.H. Vlasses, B.N. Swanson, R.O. Davies, D.D. Merill and R.K. Ferguson. Clin. Pharmacol. Ther., 3, 210 a b s t r . A24 (1983). 28. H.H. Rotmensch, P.H. Vlasses, B.N. Swanson. J . D . I r v l n . K.E. Harris, D.G. Merrill and R.K. Ferguson, Am. J. Card., 53, 116-119 (1984). 29. E.J. Sybertz. T. Baum. H.S. Ahn, S. Nelson, E. Eynon, D.M. Desiderio, K. Pula, F. Becker, c. Sabin, R. Moran, G.V. V l i e t , B. Kastner and E. Smith, J. Cardiovasc. Pharmacol., 2. 643-654 (1983). 30. M.L. Powell, K. Stabins and J.E. P a t r i c k , Fed. Proc., 42, 378 a b s t r . 499 (1983). 31. T. Baum, E . J . Sybertz, R.W. Watkins, H.S. Ahn, S. Nelson, E. Eynon, G.V. Vliet, K.K. Pula, C. Sabin, D.M. Desiderio, F.T. Becker and S. Vermulapalli, J. Cardiovasc. Pharmacol., 2, 655-667 (1983). 32. M. Vincent, G. Remond, B. Portevin, B. Serkiz and M. Laubie, T e t . L e t t . , 23, 1677-1680 (1982). 33. N . Gruenfeld. J.L. Stanton, A.M. Yuan, F.H. Ebetino, L . J . Browne, C. Gude and C.F. Huebner, J. Med. Chem., 26, 1277-1282 (1983). 34. B. J a c o t des Combes, G.A. T u r i n i , H.R. Brunner, M. Porchet, D.S. Chen and Supradip (Ben) Sen, J . Cardiovasc. Phannacol., 5, 511-516 (1983). 35. W. H. Parsons, J . L . Davidson, D. Taub, S.D. Aster, E.D. T h o r s e t t , A.A. P a t c h e t t . E.H. Ulm and B . I . Lamont, Biochem. Biophys. Res. Corn., 117, 108-113 (1983). 36. D.S. Chen, R.A. Dotsen, R.Q. B u r r e l l and B.E. Watkins, Pharmacologist, 25, 240 a b s t r . 715 (1983). 37. R.E. Galardy, V. Kontoyiannfdou-Ostrem and Z.P. Kortylewicz, Biochem., 22, 1990-1995 (1983). 38. R.G. Almquist, P.H. C h r i s t e , W. Chao and H.L. Johnson, J. Pharm. Sci., 72, 63-67 (1983). 39. R. Zelis, Amer. J. Med., 76(6B), 3-12 (1983). 40. M. Konieczkowski, M.J. Dunn, J.E. Stork and A. Hassid, Hypertension, 2, 446-452 (1983). 41. P. Cervoni, P.S. Chan, F.M. Lai and J . E . Birnbaum, Fed. Proc., 2, 157-161 (1983). 42. P.S. Chan, P. Cervoni, M.A. Ronsberg, R.C. Accomando, G . J . Quirk, P.A. Scully and L.M. Lipchuck, J. Phannacol. Exp. Ther., 727-732 (1983). 43. J.B. Bicking, M.G. Bock, E.J. Cragoe, Jr., R.M. DiPardo, N.P. Could, W.J. Holtz, T . J . Lee, C.M. Robb, R.L. Smith, J.P. Springer and E.H. Blaine, J. Med. Chem., 26, 342-348 (1983). 44. J . B . Bfcking. C.M. Robb, E . J . Cragoe, Jr., E.H. Blaine, L.S. Watson and M.C. Dunlay, J. Med. Chem., 26, 335-341 (1983). o ,197-212 45. J . Casals-Stenzel, M. Buse and W. Losert, Prostaglandins Leukotrienes Med., l (1983). 46. B. Muller, J. Schneider, K. Wilsmann, W. Lintz and L. Flohe, Prostaglandins Leukotrienes l , 361-372 (1983). Med., l 47. B.J. Whittle, S. Moncada, K. Mullane and J . R . Vane, Prostaglandins, 2,205-223 (1983). 48. M.A. Orchard, J.M. Ritter, G.L. Shepherd and P . J . L e w i s , B r . J . Clin. Pharmacol., 15. 509-511 (1983). 49. C.W. Howden, H.L. E l l i o t . C.B. Lawrie and J . L . Reid. J. Cardiovasc. Phannacol.. 2. 552556 (1983). 50. M. Catalano, J . P a r i n i and A. L i b r e t t i . Eur. J. Clin. Pharmacol., 2 5 , 157-161 (1983). 51. T. Chiba, M. Hirohashi, I. S u d C f , S. Tanaka, K. Watanabe and A. Akashi, Arzneim.Forsch., 33(1), 112-116 (1983). 52. M.J.M.C. Thoulen, J.C.A. van Meel, B. W i l f f e r t , P.B.M.W.M. Timermans and P.A. vanZweiten, Pharmacology, 27, 245-254 (1983). 53. H. Lape, A. DeFelice and D. Bailey, Pharmacologist, 25, 101 a b s t r . 1 (1983). 54. A. DeFelice and H. Lape, Pharmacologist, 25, 101 a b s t r . 2 (1983). 55. J.M. Evans, C.S. Fake, T.C. Hamilton, R.H. Poyser and E.A. Watts, J. Med. Chem., 26, 1582-1589 (1983). 56. L. Davio, M.N. Agnew, R.C. Effland, J.T. Klein, J.M. Kitzen and M.A. Schwenkler, J . Me& Chem., 26. 1505-1510 (1983). 57. J.M. McCall, J.W. Aiken, C.G. Chidester, D.W. DuCharme and M.G. Wendling, J . Med. Chem.. 26, 1791-1793 (1983). 58. Thastrup, B. Fjalland and J. Lernich, Acta Pharmacol. e t Toxicol., 2,246-253 (1983).

u,

Chap. 7

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

Sprague, Powell

Q

59. A. S a l imb e n i . E. Manghisi. G. Fern1 and G.B. Fregnan. I1 Farmaco.-Ed. S c i . , 38, 904910 ( 1 9 8 3 ) . 60. F.V. DeFeudis, L i f e S c i . , 557 (1983). 61. J.L . Rouleau, W.A. Parmley, J . S t evens. I. Wikrnan-Coffelt, R. S i e v e r s , R.W. Mahley and R.J. Havel, J. Am. C o l l . C a r d i o l . , 1.1453 (1983). 62. P.D. Henry and K . I . B e n t l e y , J. C l i n . I n v e s t . , 68, 1366 (1981). 63. C.M. Heesch. B.M. Miller, M.D. Thames, and F.M. Abboud, Am. J. P h y s i o l . , 245, H653 (1983). 64. W. Kiowski, 0. Bertel, P. E rne, P. B o l l i , U.L. H ulthe n, R. R i t z and F.R. B u h l e r , Hy p e r te n si o n , 5 (S uppl I), 1-70 (1983). 65. R.A.B. McLeay, T . J . S t o l l a r d , R.D.S. Watson, and W.A. L i t t l e r , C i r c u l a t i o n , 67,1084 (1983). 66. G. H a e u s l e r , J. Pharmacol. Exp. T herap., 180,672 (1972). 67. J . R . Powell and L . J . K e w i n , Fed. Proc. 62. 842 (1983). 68. H. Kuriyama, Y. I t o . H. S uzuki , K. Kitamura, T. I t o h , M. Kajwara and S. F u j i w a r a , C i r c . Res., 2 (Suppl I ) , 92 (1983). 69. M. Wibo, I. D e l f o s s e and T . Godfraind. Arch. i n t . Pharmacodyn., 263, 333 (1983). 70. P.A. van Zweiten and P.B.M.W.M. T i mermans. J. Mol. C e l l . C a r d i o l . , 15, 717 (1983). 71. W.G. N a y l e r , J . E . Thompson and B. J a r r o t t , J . Mol. Cell. C a r d i o l . , i 15

ANNUAL REPORTS IN MEDICINAL CHEMISTRY- 19

Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040.519-9

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Effects of kukotrienes on Respiratory Function 1) In Vivo Actions - The in vivo pulmonary response to leukotrienes varies among species. Rats are often resistant to the effects of leukotrienes, but aerosolized LTD causes a bronchospasn in an inbred Basenji-Greyhound dogs , an strain with bronchial hyperresponbiveness. inbred strain with hyperreactive airways, develop an atropine-inhibitable bronchospasm to inhaled LTD while there is no response in mongrel dogs. 2 7 Furthermore, aerosolized LTh4 is 300-900 times more potent than histamine in causing a bronchospasm in Ascaris-sensitive rhesus monkeys, whereas non-allergic monkeys are relatively non-responsive. * The bronchial response in monkeys to aerosol LTD4 is inhibited by inhaled FPL-55712 Administered into the right while intravenous FPL-55712 is inactive. atria of monkeys, LTC4 causes a bronchospasm and a transient increase in atrial pressure followd by a prolonged hypotension.

’

In healthy humans, inhaled LTC4 and LTD4 are more potent than histamine at causing a decrease in maximum expiratory flow rate.31’32 The effect of LTD4 is independent of the production of cyclooxygenase products. In contrast to airway hypersensitivity to histamine among human asthnatics, there is no increased sensitivity to inhaled LTD4. 3 3 ?he importance of cyclooxygenase products, particularly thranboxane A2, in the bronchospastic response to leukotrienes depends on the species and route of administration. The bronchospasn resulting from intravenous LTCq in guinea pigs3’ and LTD4 in cats3 is blocked by cyclooxygenase inhibitors. The response to inhaled LTC in guinea pigs is either not inhibited or is enhanced by cyclooxygenade blockade.

,’

2) In Vitro Actions - Studies using a superfusion technique suggest that a major portion of the contractile response of guinea pig lung parenchymal strips to LTC and LTD is due to the generation of thrcmboxane A2.37 In contrast, rabhit and r& parenchymal strips do not develop a marked contractile response or synthesize thromboxane A2 in response to LTC4 or LTD4. More recent work has shown that under non-flow conditions, the contractile responses of guinea pig parenchyma to LTC and LTD4 are LTB ib 5-10 times more indepedent of thrclmboxane A generati~n.~*’~~ potent than histamine in con&acting guinea pig ludg strip^.^ Intravenous or aerosol administration of LTB4 to guinea pigs produces a These in vitro and in vivo effects of LTB4 are secondary bron~hospasm.~~ to the generation of cyclooxygenase products.

’

Conversion of LTC to LTD and subsequent blockade of the contractile response by FPL-55712 &ring ikxbation with guinea pig trachea has been reported. 4 2 This LTD4-induced contraction may utilize predaninantly intracellular calcium stores because it is inhibited by 1MB-8, an inhibitor of intracellular calcium mobilization, but not by nifedi~ine.~ In contrast, on the guinea pig ileum, the slowness of the contractile response to LTD4, compared to histamine, and the sensitivity to blockade by a slow calcim channel blocker, D.600, suggests that LTD4 utilizes extracellular calcium.4 The Merck-Frosst group prepared the sulfones of LTC4, LTD4 and LTE4 and found that they are almost equipotent to the sulfides on contracting guinea pig trachea and intravenous administration to guinea pigs produces an indanethacin and FPL-55712 inhibitable bronchospasm. 14 The contractile activity of LK4 and LTD analogs has been examined. The rank order of potency is similar on guine4 pig trachea, lung parenchyma and ileum where the 5R, 65 isaners have 0.01-times the potency of the natural

Chap. 24

Biology of Leukotrienes

Kreutner, Siege1

243

5 S , 6R i~aners.~~~48 Eight biosynthetically formed sulfidopeptide leukotrienes (LTC3, 8,9-LTC3, LTC4, 11-trans LIT4, LTC5, LTD4, LTE4 and 11-trans LTE ) were studied for contractile activity on guinea pig lung parenchyma.sif All canpounds are full agonists and 5000-times more potent than histamine. There is no potency difference between 11-cis and 11-trans i s m r s of LTC4 or LTE4. Incubation of LTE4 with glutathione and gm-glutamyltranspeptidase results in the formation of LTF .50 LTF4 is equipotent to LTD on guinea pig trachea, but only 0.01-timed as potent on ileum and in v~vo!’~

Purified human lung mast cells release SRS-A upon stimulation with anti-IgE. 5 2 Eosinophils (horse) release leukotrienes upon stimulation with ionophore A-23187 5 3 and platelets (rabbit) release leukotrienes on stimulation with thranbin or PAF.54 Rat alveolar macrophages 5 5 and human peritoneal macrophages 5 6 stimulated with A-23187 release LTC4 and LTB4, but not LTD4. The production of LTB4 by human alveolar macrophages has also been demon~trated.~~ IgE mediated release of SRS-A occurs with alveolar but not peritoneal rat macrophages.55 Sensitized h w n lung challenged with antigen, releases sufficient amounts of LTC4, D4 and E4 to cause contraction of human bronchi in v i t r ~ . ~ ~ A major finding has been the importance of lipoxygenase products in the secretion of airway mucus. Mucus glycoprotein secretion fran human bronchi in culture is stimulated by l o w concentrations (1-10 nM) of various H!iTEs, with 12- and 15-HETE m s t active.59’60 Antigen-provoked mucus release is inhibited by the lipoxygenase inhibitors ETYA, nordihydrcguiaretic acid and alpha naphthol.59 A recent report claims a significant decrease in mucociliary clearance after oral ingestion of aspirin by healthy volunteers.61 This effect presumably is due to an augmented production of lipoxygenase products after cyclooxygenase inhibition. Other reports have shown increased secretion of mucus after intraarterial injection of LTC4 or LTD4 into dog^,^^-^' intratracheal administration of LTC4 into cats 6 5 or in vitro addition of LTC4 or LTD4 to human bronchial mcosa .6 Leukotriene Actions in the Gastrointestinal System - Large species differences have been found in the response of gastrointestinal tissue to sulfidopeptide leukotrienes. Human gastrointestinal muscle (ileum, stanach, jejunum, colon) does not contract to SRS-A fran guinea pig lung.67 In contrast, both LTCq and LTD contract the rat stomach and colon but not the duodenum or ileum6’ 4LTC4, D4 and E4 produce cyclooxygenase independent-Sontractionsof the guinea pig gall bladder.6 At high concentrations (10 MI, LTC4 and LTD also provoke acid secretion from isolated rabbit gastric parietal c e l l ~ . ~A7 ~role for leukotrienes in inflamnatory bowel disease is suggested by the release of SRS-A fran antigen challenged sensitized colonic m u c o ~ a . ~ ~ Lipoxygenase products may also be essential for insulin release by pancreatic islets. Glucose-induced insulin release is inhibited by lipoxygenase inhibitors, including BW-755CI nordihydrcquairetic acid and 15-HETE.72,73 Also, 5-HETE augments insulin release triggered by low concentrations of glucose.73 Rat pancreatic islets produce 5-, 12- and 15-HETE fran arachidonic acid.73 Actions of Leukotrienes on Cardiovascular Tissue - Purified SRS-A generated fran guinea pig lung has been shown to increase vascular permeability in guinea pig skin, especially if injected together with a vasodilator like EGE2.74 Intradermal LTC4 and LTD4 cause a wheal and

244

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flare75C76and increase microvascular blood flow 77 in human skih. In a hamster cheek pouch preparation, law concentrations of LTC and LTD4 cause intense arteriolar constriction followed by a marked incretse in vascular permeability. 7 8 Airway edema associated with an anaphylactic reaction may be a leukotriene dependent response, since local application of LTC4, D4 or E to guim7$ pig trachea in vivo produces an increase in microvascular perm8ability

.

Low concentrations of LTD (1-lOnM) cause a reversible cessation of beating of rat heart cells in hlture. Evidence was presented by Burke -et al. for a role of leukotrienes in cardiac anaphylaxis. The cardiac dysfunction associated with anaphylaxis is partially duplicated by addition of LTC , D4 or E4. A l l leukotrienes produce a negative inotropic effect, decreasi coronary flow of isolated guinea pig heart, and decrease the contractile force of pectinate muscle f m n human heart. LTD4 also potentiates the tachycardia and arrhythnias caused by histamine. agents on Sulfidopeptide leukotrienes also appear to be potent contra:\ile canine renal arteries8* and rat and cat coronary arteries. These effects are independent of thronboxane A2 generation.

The in vivo effects of leukotrienes on blood pressure and heart rate are quite canplex. Generally, there is a biphasic response consisting of an initial transient increase in systemic blood pressure followed by a sustained systemic hypotension after a bolus of 1-10 pg/kg of LTCq or LTD4. 84-86 Intravenous LTC4 decreases cardiac output and rate while Pretreatment with indmthacin increasing total peripheral resistance. potentiates the initial increase in blood pressure and attenuates the Low sustained hypotension and changes in cardiac function due to LTC doses of LTD4 injected into the left circmflex coronary artery 8f sheep result in coronary vasoconstriction and impaired ventricular function.a7

’

.

Leukotrienes may also play a pathogenic role in hypxic pulmonary hypertension. Hypoxic pulmonary vasoconstriction in sheep is prevented or LTC and LTD are present ‘inlung lavage fluid reversed by FPL-55712. obtained from human newborns &th a clfnical diagnosis of persistent pulmonary hypertension and hypoxemia. Leukotrienes in Inflamnatory Disorders - Leukotrienes are produced by cells ( W s and macrophages) that are present in large numbers at inflmtory sites. If stimulated with the calcium ionophore A-23187, PMNs produce LTB4, which has potent chemokinetic activity and is also an aggregating agent for neutrophilsPl The potency of LTB as a chemotactic agent is similar to that of the canplement ccmponent C5a40r the synthetic peptide F-met-leu-phe. 2In a hamster cheek pouch preparation, LTB4 causes adhesion of leukocytes to vascular endothelim. 76 ’78 Evidence has also been presented that LTB4 is eosinophil chemotactic factor (ECF).95 However, ECF stimulates the migration of eosinophils, but not neutrophils while LTB4 is equally chemotactic for both cell types. 9 6 The neutrophil chanotactic activity of LTB is blocked canpetitively by acetyl LTB 9 7 Intradermal injection of Ld4 (0.2-1.5 m l e ) into human skin caused an imnediate erythema and wheal followed by a delayed reaction of erythema and induration at 1-4 hr with perivascular neutrophil infiltrates. 76

.

LTB has been shown to cause the release of enzymes f m n human 98.4 9 9 Canpared to C and F-met-leu-phe, LTB is a weak secretogcgue and is active’8nly at concentrations 14-fold greater t h a n pMB.

Biology of Leukotrienes

Chap. 24

Kreutner, Si ege1

2115

needed to stimulate chemotaxis or adherence of neutrophils. Neutrophil degranulation caused by LTB4 is inhibited by LTB4-dimethylamide (KD = 0.2 In addition, it has recently been reported that LTB4 may have pM). h u n d u l a t o r y activity For example, LTJ34 appears to cause specific suppression of human lymphocyte function, possibly by inducing suppressor cells. Moreover, LTB4 augments natural cytotoxic cell activity. These latter activities may play important roles in chronic inflamnatory disease. Injection of LTJ3 into rabbit skin,lo6 rabbit eye,l07 guinea pig peritoneal cavityg4 4and human skin results in leukocyte accumulation at the injection site. In a hamster cheek pouch preparation, the movement of leukocytes through the vascular endothelium as a result of the local application of LTB has been directly observed.lo6 The increase in vascular permeability due t8 LTB4 is augmented by the co-administration of a vasdilator prostaglandin and is dependent upon the presence of neutrophils

.

.

The role of LTB4 as an inflammatory mediator is supported by studies showing high amounts of LTB4 in the skin c h e fluid of involved skin from psoriatics and in gouty effusions. In contrast, LTC4 and LTD4, but not LTB4, are detected by bioassay and HPLC in carrageenaninduced pleurisy in rats. 1 1 2 Leukotrienes may also be involved in the . modulation of pain in inflamnatory lesions. In the rat paw, LTB4 causes However, the mechanism of action of this response slight hyperalgesia. is not understood. Leukotrienes C4, D4 and E4 may also be important mediators of inflamnatory processes. LTD4 and LTE4 influence vasopermeability in guinea pig skin and their activities are potentiated by the presence of prostaglandin vasodilators. In addition, LTC4 and LTD increase neutrophil adherance and may also affect release reaction4 of macrophages

.

Elevated levels of both LTBq and LTC4 are found in psoriatic lesions in man.114’11 Ulcerative colitis has among its characteristic features an accumulation of neutrophils in mucosal sites. Samples of tissue f m In inflamed sites in such patients contain elevated levels of LTB4. addition, these samples have an increased ability to synthesize 5-lipoxygenase products ccmpared to normal tissue. Synovial fluids from rheumatoid arthritic or gouty patients also contain large numbers of neutrophils and elevated levels of LTB4. Receptor Binding of Leukotrienes - Initial reports suggested the presence of tissue specific heterogeneity of LTD4 receptors.ll* This is supported by more recent experiments on guinea pig trachea utilizing FPL-55712.119 The availability of high specific activity radiolabeled LTC4 and LTD4 has permitted t p detection of specific binding sites 1~lung for these ligands. [ Hl-LTDq exhibits high affinity (K,,= 10 OM), saturable binding to membrane3+of gy)nea @g lung.lz0 Binding i$ enhanced by divalent cations (Ca , Mg , Mn 1 and inhibited by Na LTE , but not LTC , has high affinity for the binding site suggesting q a t L d 4 and LTD4 ineeract at different sites. High affinity binding of [ HI-LTC was also and guinea pig 122,923 and on an intact demonstrated on lungs from rat snooth muscle cell line, DITl MF-2, derived fran Syrian hamster vas deferens.lZ4 The LTC4 binding is biochemically distinct from LTD binding sites.120’122 The KD for FPL-55712 is greater against LTC than &D4. Binding of LTD4, but not L K 4 is altered in the presence o# guanine nucleotides.

.

24 6

Sect. V

- Topics

i n Biology

Egan, Ed.

Evidence has also been presented that LTB interacts with a specific and secretogogue cell receptor. 9 3 - 1 2 5 The difference in chemot&ic activity of LTB4 has been explained in tern of the former being associated with high affinity receptors and the latter with lcw affinity receptors. Pharmacology of Leukotriene Biosynthesis Inhibitors and Antagonists 1) Biosynthesis Inhibitors - BW-75% (3-1 and phenidone ( 4 ) are inhibitors in of the lipoxygenase and cyclooxygenase pathways 127,128 aEd inhibit vitro anaphylactic contractions of airway smooth muscle fran guinea 7127c129 P1gs’ W 7 5 % inhibits SRS-A, but not histamine, release fran antigen challenged sensitized human lung 1 3 0 and when given by inhalation, attenuates antigen-induced bronchospasm in Ascaris-sensitive monkeys. 1 3 1 Bw-755C inhibits prostaglandin synthesis in inflamnatory exudates but does not inhibit PGI synthesis by gastric mucosa.132 BW-75% also reduces the concentr&ion of Lm4, thrclmboxane B2 and PGE2 in exudates derived fran the subcutaneous implantation of carrageenan impregnated PMN migration into the inflamnatory exudate is also sponges in rats. decreased. BW-75%, but not indanethacin, reduces the size of a myocardial infarct produced in dogs by coronary occlusion followed by reperfusion. The mechanism may involve inhibition of lipoxygenase to reduce the production of LTC and LTDq which constrict coronary arteries and of LTB4 which is c h m t a h for inflamnatory cells.

Me

r

3

-5

At concentrations lower than those needed to inhibit either lipoxygenase or cyclooxygenase, BW-75% enhances lymphocyte activation by BW-755C and phenidone, applied topically to mouse skin, mitogens. prevent the induction of epidermal ornithine decarhxylase caused by application of the tumor p m t e r 12-0-tetra-decanoyl phorphol-13acetate. Nafazatrm (2) inhibits 5- and 12-lipoxygenases but not the cyclooxygenase of B16a tumor cells and also inhibits tumor Other studies have shown nafazatran increases FG12 proliferation. synthesis by aortic strips and has antithrclmbotic activity.138

’

Benoxaprofen (5) has antiinflmtory activity and also inhibits the release of SRS-A.139 At a concentration of 100 pM, benoxaprofen inhibits a 5-lipoxygenase in guinea pig peritoneal cells and HL-60 cells with no effect on the 12-lipoxygenase of h m n platelets or a soybean 15-lipoxygenase.l’+O In rat Rulhis, benoxaprofen is a better inhibitor of cyclooxygenase than 5-lipoxygenase. Its in vivo activity in animal models is consistent with these results, because inhibition of edema occurs at lower doses than inhibition of cellular influx to inflamed sites. Benoxaprofen is claimed to be ineffective clinically to inhibit aspirin-induced bronchospasm 1 4 1 but has shown beneficial activity in the treatment of psoriasis,142c14 a disease associated with the presence of increased levels of lipoxygenase products in epidermal lesions.

Biology of Leukotrienes

Chap. 24

Kreutner, Siege1

-

-

247

OH

7

6

U-60,257 (7) is an inhibitor of glutathione-S-transferase 1 4 4 but its principal action seems to be inhibition of a 5-lipoxygenase. 1 4 5 U-60,257 inhibits SRS, but not histamine, release from human lung, competitively antagonizes the contactile effects of LTC4 and LTD on guinea pig ileum and inhibits lysosamal enzyme release frcm human F d N s . In Ascaris sensitive mnkeys, U-60,257 given by aerosol (0.05-1%) or intravenously (0.01-5 q/kg) inhibits antigen-induced bronchoconstriction. l 4 2) Leukotriene Antaqonists - FPL-55712 (8)consistently inhibits contractile responses to LTD4, but its effect on responses to LTC4 are et al. claim that FPL-55712 does not antagonize LTC4 on less clear. Krell -guinea pig parenchymal strips. llr7 On trachea and bronchus, FPL-55712 antagonizes responses to low concentrations of LTC , but potentiates responses to high concentrations of LIT4. Krell afso showed that antigen-induced bronchospasn in dogs is not inhibited by i.v. or aerosol FPL-55712. 0

0

-8 Limited studies with FPL-55712 have been conducted in humans. Inhaled FPL-55712 in 4 allergic astbtics gave inconclusive results. 1 4 9 'Jho of the four patients s h m d improved FEV1. Tracheal mucous velocity is decreased in asthtics challenged with antigen. 5 0 Inhalation of 0.5-1% FPL-55712 prevents the decrease in mucous velocity but does not inhibit the bronchospasn. Inhalation of LTC4 or LTD by normal subjects induces coughing which is blocked by aerosol FPL-557f2. 1 5 1 In BasenjiGreyhound dogs, the bronchospasn to inhaled citric acid is associated with increased plasma levels of SRS (but not histamine) and is partly blocked by FPL-55712. 1 5 2 Much of the pharmacology of FPL-55712 is ascribed to its antagonism of SRS-A; however, at concentrations only slightly higher than those needed to inhibit SRS-A, FPL-55712 also inhibits thrmboxane synthetase' and lipoxygenase in broken cell, but not intact cell preparations. FPL-55712 also inhibits the extensive necrosis of liver parenchymal cells and death in animals injected with endotoxin and D-galactosamine. 4R,%,62-2-nor LTD (4R-hydroxyl-5S-cysteinylglycyl-6Z-nonadecenoic acid) is an analog of L h 4 which at a concentration of 100 ~ J M , antagonizes

Sect. V

248 -

- Topics

in Biology

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119. R.D. Krell, B.S. Tsai, A. Berdoulay, M. Barone and R.E. Giles, P r o s t a g k d i n s , 171 (1983). 120. S.S. Wng and R.N. DeHaven, Froc. Natl. Acad. Sci. USA, 80, 7415 (1983). 121. S.S. brig, R.N. CeHaven, F.A. KueN and R.W. Egan, J. B i o l . Chen., 258, 9616 (1983) 122. G.K. H o g d x x m , S. Mong, H.L. An and S.T. Crooke, Bicchem. Biophys. Res. C m u n . , 116, 1136 (1983). 123. E. Bruns, W.J. 'Ihomsen and T.A. h g s l e y , L i f e Sci., 3,645 (1983). 124. S. Krilis, R.A. L e w i s , E.J. Corey and K.F. Austen, J. Clin. Invest., 72, 1516 (1983 1. 125. R.A. Kreisle and C.W. Parker, J. Exp. Ned., 157, 628 (1983). 126. D.W. Golchan, L.A. Gifford and E.J. Goetzl, Fed. Proc., 43, 847 (1984). x (1980). 127. F.P. Nijamp and A.G.M. Ramakers, Eur. J. Phannac., 62, 1 128. J. B a m n n , F.V. Bruchhausen and G. Wunn, Prostaglandins, 20, 627 (1980). 129. S.S. Yen and W. Kreutner, Agents and k t i o n s , 10, 274 ( 1 9 8 n . 130. C.L. Anmur, J . M . Hughes, J.P. Seale and D.M. T s l e , Clin. Exp. Phannac. Physiol., 8, 654 (1981). 131. F. Patterson, J.J. hruzansky and K.E. Harris, J. Allergy Clin. Irmolnol., 67, 444 (1981). 132. N.K. Myghton-Smith and B.J.R. Whittle, Proc. B r i t . P h m c . Soc., Sept. 1981, p. 920 P. 808 (1983). 133. J.A. Salmon, P.M. Sinm~nsand S. Moncada, J. Fhann. P h a m c . , 134. K.M. Mullane and S. Moncada, Prostaglandins, 24, 255 (1982). 135. A.T.M. Mobarok and J. Morley, Agents and Actions, 12, 645 (1982). 136. T. Nakadate, S. Yamamto, M. I s h i i and R. Kato, C a r c i n q e n e s i s , 3, 1411 (1982). 65 (1982). 137. K.V. Horn and J.R. Rinn, FEBS L e t t e r s , 138. T.E. Eling, Prostaglandins and Related Lipids, 2, 783 (1982). 139. J.R. Walker, J.R. B o o t , B. Cox and W. Wwson, J. Fhann. Phannac., 32, 866 (1980). 140. J. Harvey, H. Parish, P.P.K. Ho, J.R. Boot and W. h s o n , J. Phann~Pharmac., 35, 44 (1983). 141. W.R. Lumry, J.G. Curd, W.E. Brocklehurst, R.A. S i m n and D.D. Stevenson, J. Allergy Clin. Imunol., 69, 93 (19821. 142. K. Kragballe and T. Herlin, Arch. Dermatol., 119, 548 (1983). 143. J.J. Vorhees, Arch. &nnatol., 541 (1983). 144. M.K. Each, J.R. Brashler, F.A. F i t z p a t r i c k , R.L. G r i f f i n , S.S. Iden, H.G. Johnson, M.L. McNee, J.C. MccUire, H.W. Smith, R.J. S m i t h , F.F. Sun and M.A. Wassennan i n "Mvances i n Imnunopharmacolcgy 2". J.W.'Hadden, L. Chedid, P. D-lkor, F. Spreafico and D. Willoughby, Ed., Pergamon Press, Oxford, 1983, p. 549. 145. S.S. Sun and J.C. McChire, Prostaglandins 2,211 (1983). 146. H.G. Johnson, M.L. McNee, M.K. Back and H.W. Smith, Int. Arch. Allergy Appl. hmunol., 70, 169 (1983). 147. R.D. KrellTM. O'Donnell, R. @born, K. Falcone, L. Vickery, M. Gmm, C. Kinzig, D. B r y a n and J. Gleason, Fed., Proc., g,2586 (1981). 148. R.D. Krell, J. McCoy, R. @born and L.W. Chakrin, I n t . J. I n m m p h a m c . , 2, 55 (1980). 149. T.H. Lee, M . J . Walport, A.H. Wilkinson, M. TurnerWarwick and A.B. Kay, The Lancet, Aug. 8, 1981, p. 304. 150. T. Ahmed, D.W. Greenblatt, S. Birch, 8. Marchette and A. Wanner, Am. Rev. Resp. Dis., 110 (1981). 151. M.C. Holroyde, R.E.C. Altounyan, M. Cole, M. Dixon and E.V. Elliott, Agents and Actions, 11, 573 (1981). 152. C.A. H i r s k , J.E. Peters, J. B u t l e r , J. Hanifin, H. Downes and R.K. Lynn, Am. Rev. Resp. Dis., 125, 65 (1982). 153. A.F. Welton, W.C. Hope, L.D. Tobias and J.G. Hamilton, Biochem. Fhannac., 30, 1378 (1981). 154. F.B. Casey, B.J. Appleby and D.O. Buck, Prostaglandins, g , 1 (1983). 155. w. H~CJMM and De. Keppler, Naturwissenschaften, 69, 594 (1982). 156. B.M. Weictmm, The Pharmacologist, 2, 114 (1983). 157. J.G. Gleason, Bicchem. Biophys. Res. Camnun. 732 (1983).

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Chapter 25.

Endogenous N a t r i u r e t i c Agents

Mary Anna N a p i e r and Edward H. B l a i n e Merck Sharp and Dohme Research L a b o r a t o r i e s Rahway, NJ 07065 and West P o i n t , PA 19486

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Introduction Endogenous n a t r i u r e t i c f a c t o r s , which a r e i m p o r t a n t i n t h e maintenance of t h e e x t r a c e l l u l a r f l u i d volume, have been d e t e c t e d i n plasma and u r i n e and have been e x t r a c t e d from b r a i n t i s s u e and f r o m c a r d i a c a t r i a . Expansion o f t h e e x t r a c e l l u l a r f l u i d volume l e a d s t o i n c r e a s e d sodium i o n e x c r e t i o n ( n a t r i u r e s i s ) by t h e kidney, independent o f changes i n g l o m e r u l a r f i l t r a t i o n r a t e (GFR) and o f t h e r e n i n - a n g i o t e n s i n - a l d o s t e r o n e system.l Considerable evidence i n d i c a t e s t h a t volume expansion (VE) i n l a b o r a t o r y animals and i n man causes t h e r e l e a s e o f a humoral n a t r i u r e t i c f a c t o r , which i s e x c r e t e d i n t h e u r i n e . One such agent t h a t appears t o be an endogenous i n h i b i t o r o f t h e sodium t r a n s p o r t system has been r e f e r r e d t o as n a t r i u r e t i c hormone (NH). Many s t u d i e s have i m p l i c a t e d NH i n t h e pathogenesis o f h y p e r t e n s i o n i n man and e x p e r i mental animals.2-6 A second substance, i s o l a t e d from c a r d i a c a t r i a l t i s s u e and d e s i g n a t e d a t r i a l n a t r i u r e t i c f a c t o r (ANF), does n o t a f f e c t Na+,K+-ATPase and i s s t r u c t u r a l l y d i f f e r e n t f r o m NH. The r o l e o f ANF i n b l o o d p r e s s u r e c o n t r o l o r h y p e r t e n s i o n may r e l a t e t o c a r d i a c a t r i a l r e c e p t o r s t h a t a r e presumed t o be s e n s i t i v e t o changes i n i n t r a a t r i a l p r e s s u r e , p o s s i b l y r e s u l t i n g i n r e l e a s e o f ANF.' This chapter w i l l r e v i e w t h e i s o l a t i o n , t h e c h a r a c t e r i z a t i o n o f t h e chemical and b i o l o g i c a l p r o p e r t i e s , and t h e p o s s i b l e r o l e s o f t h e s e agents i n t h e c o n t r o l o f e x t r a c e l l u l a r f l u i d volume and development o f h y p e r t e n s i o n . More d e t a i l e d reviews o f t h e n a t r i u r e t i c hormone have appeared r e c e n t l y . 2 - 6 N a t r i u r e t i c Hormone - A humoral f a c t o r which causes n a t r i u r e s i s when c r o s s c i r c u l a t e d t o r e c i p i e n t animals has been demonstrated i.n VE donor a n i r n a l ~ . ~ , E ~ x~t r ~a c*t s~ o~f b l o o d o r u r i n e f r o m VE s u b j e c t s a l s o causes n a t r i u r e s i s i n assay animals, i n d i c a t i n g a t r a n s f e r a b l e n a t r i u r e t i c sub~ t a n c e . ~T h i s agent i s e i t h e r l e s s a c t i v e o r u n d e t e c t a b l e i n nonexpanded s u b j e c t s . 4 A n a t r i u r e t i c substance has been e x t r a c t e d f r o m k i d n e t i s s u e o f VE animals, b u t n o t from k i d n e y s o f hydropenic a n i m a l s . l l The evidence suggests t h a t NH i n c r e a s e s sodium i o n e x c r e t i o n by i n h i b i t i n g r e a b s o r p t i o n as a r e s u l t o f d i r e c t i n h i b i t i o n o f t h e r e n a l Nat,K+-ATPase, t h e e n e r g y - r e q u i r i n g sodium i o n t r a n s p o r t pump.12 A h i g h c o n c e n t r a t i o n o f t h e Na+,Kt-ATPase i n h i b i t o r , d i g o x i n , produces n a t r i u r e s i s b y i m p a i r i n g t h e c e l l u l a r pump o f t h e r e n a l t u b u l e . 1 2 An unknown p r e s s o r agent has been observed i n t h e b l o o d o f animals and man w i t h VE, l o w r e n i n , h y p e r t e n ~ i 0 n . l ~Suppressed Na+,K+-ATPase a c t i v i t y i n c a r d i o v a s c u l a r t i s s u e s o f a n i m a l s w i t h e i t h e r one-kidney r e n a l , d e o x y c o r t i c o s t e r o n e a c e t a t e o r reduced r e n a l mass h y p e r t e n s i o n suggests t h a t t h i s p r e s s o r substance i s an i n h i b i t o r o f t h e Na+,Kt-ATPase. By i n h i b i t i n g t h e Nat,K+-ATPase o f v a s c u l a r smooth muscle, i t i s p o s s i b l e t h a t such an agent c o u l d c o n t r i b u t e t o i n c r e a s e d v a s c u l a r r e s i s t a n c e o r

ANNUAL REPORTS IN MEDICINAL CHEMISTRY-I9

Copyright 0 1984 by Academic Press, Inc All rights of reproduction in any form reserved. ISBN 0-12-040519-9'

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Egan, Ed.

r e a c t i v i t y a s s o c i a t e d w i t h hypertension.14 A s u b s t a n t i a l i n c r e a s e i n a r t e r i a1 p r e s s u r e has been observed f o l l owi ng in j e c t i o n o f d i g i t a l is p r e p a r a t i o n s i n i n t a c t animals15 as w e l l as i n e x c i s e d a r t e r i a l 1 6 , 1 7 and venous16 t i ssues , A t h e o r e t i c a l r o l e f o r a s a l t - e x c r e t i n g hormone i n h y p e r t e n s i o n was proposed by Dahl e t a1.l8,lg and expanded by Blaustein,20 Haddy e t a1.,14 and deWardener.21-B=60d pressure i s a f u n c t i o n o f both t o t a l v o l u m e o f t h e b l o o d and t h e r e s i s t a n c e t o i t s f l o w t h r o u g h t h e c i r c u l a t o r y system. The r e s i s t a n c e t o f l o w i s determined by t h e degree of c o n s t r i c t i o n o f t h e a r t e r i o l e s . A p r i m a r y d e f e c t i n t h e k i d n e y t h a t reduces i t s a b i l i t y t o e x c r e t e sodium r e s u l t s i n i n c r e a s e d b l o o d volume, r e l e a s e o f n a t r i u r e t i c hormone, t h e i n h i b i t i o n o f t h e r e n a l Nat,Kt-ATPase and i n c r e a s e d u r i n a r y sodium e x c r e t i o n . A t t h e same t i m e , however, t h e v a s c u l a r t i s s u e s would be exposed t o t h e same Nat,Kt-ATPase i n h i b i t o r , causing increased nons p e c i f i c s e n s i t i v i t y t o c o n s t r i c t o r agents such as a n g i o t e n s i n , vasop r e s s i n o r n 0 r e p i n e p h r i n e . 2 2 9 2 ~ I n h i b i t i o n o f t h e v a s c u l a r Nat,Kt-ATPase leads t o i n c r e a s e d l e v e l s o f i n t r a c e l l u l a r Cat' i n b l o o d vessels, e i t h e r by a Nat-Cat+ exchange mechanism20 o r by p a r t i a l d e p o l a r i z a t i o n o f t h e c e l l membranes i n c r e a s i n g t h e p e r m e a b i l i t y t o ~ a l c i u r n . 1 ~T h i s increases t h e s e n s i t i v i t y t o c i r c u l a t i n g p r e s s o r agents and r e s u l t s i n enhanced c o n t r a c t i o n and increased b l o o d pressure. Another mechanism proposed by Buckalew and Gruber i n v o l v e s a more d i r e c t r o l e o f t h e sympathetic nervous ~ y s t e m . ~I n nonhypertensive i n d i v i d u a l s , NH i n c o n j u n c t i o n w i t h o t h e r n a t r i u r e t i c f o r c e s , i n c r e a s e s r e n a l sodium e x c r e t i o n and m a i n t a i n s normal plasma volume. Since NH can cause n a t r i u r e s i s w i t h no e f f e c t on b l o o d pressure, volume can be regul a t e d i n normal i n d i v i d u a l s w i t h o u t causing hypertension24. I n hypert e n s i v e s u b j e c t s , r e n a l response t o NH i s b l u n t e d , causing NH l e v e l s t o become h i g h e r t h a n i n normotensive i n d i v i d u a l s . P a t h o l o g i c a l l y e l e v a t e d NH l e v e l s l e a d t o i n c r e a s e d b l o o d p r e s s u r e e i t h e r by a c t i v a t i n g t h e sympathetic nervous system, by i n c r e a s i n g v a s c u l a r r e a c t i v i t y , o r both. The i n c r e a s e d b l o o d pressure adds another n a t r i u r e t i c f o r c e h e l p i n g t o overcome t h e d e f e c t i n r e n a l sodium e x c r e t i o n . Thus, h y p e r t e n s i o n i s a r e s u l t o f t h e need t o r e g u l a t e volume i n t h e presence o f a d e f e c t i n r e n a l sodium e x c r e t i o n . C o n t r o l o f NH r e l e a s e by a c e n t r a l s i t e was suggested by i m p a i r e d n a t r i u r e s i s and i m p a i r e d s e c r e t i o n o f NH i n r a t s w i t h l e s i o n s o f t h e a n t e r o v e n t r a l t h i r d v e n t r i c l e (AV3V) r e g i o n o f t h e brain.25 Evidence t h a t NH may be an endogenous Nat ,Kt-ATPase i n h i b i t o r i n c l u d e s t h e i n h i b i t i o n o f sodium t r a n s p o r t i n an anuran membrane, a model o f t h e r e n a l d i s t a l t ~ b u l e ,and ~ i n isolated rabbit collecting tubule.Z6 N a t r i u r e t i c u r i n e e x t r a c t s a l s o d i s p l a c e 3H-ouabain from r e n a l N ~ ' , K + - A T P ~ s ~ . * ~ D i r e c t i n h i b i t i o n o f Nat,Kt-ATPase i n v i t r o has a l s o been r e p o r t e d w i t h e x t r a c t s o f p l a ~ m a and ~ ~ u, r ~i n e~. 4 6 m y n e t a l . show a s i g n i f i c a n t c o r r e l a t i o n between t h e Nat,Kt-ATPase i n h i b i t o r y a c t i v i t y i n t h e plasma and t h e mean a r t e r i a l p r e s s u r e o f t h e donor p a t i e n t . 2 9 A cytochemical assay f o r i n h i b i t i o n o f guinea p i g r e n a l Nat,Kt-ATPase30 has been used t o show t h e presence o f a c i r c u l a t i n g i n h i b i t o r y a c t i v i t y t h a t i s 25-times g r e a t e r i n t h e plasma o f s u b j e c t s on h i g h s a l t t h a n t h o s e on l o w s a l t diets.31 Plasma from h y p e r t e n s i v e p a t i e n t s , plasma w i t h r e n i n values below normal, and plasma from o l d e r s u b j e c t s e x h i b i t e d h i g h e r l e v e l s o f Nat,Kt-ATPase i n h i b i t o r y a c t i v i t y . 3 2 A n a t r i u r e t i c e x t r a c t o f u r i n e from normal o r ~ a l t - l o a d e d 3 3 9 3 and ~ uremic35 humans was a l s o shown t o i n h i b i t t h e Nat,Kt-ATPase i n p i g r e n a l

Chap. 25

Endogenous Natriuretic Factors

Napier, Blaine

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t u b u l a r membranes. The i n h i b i t i o n i s g r e a t e s t i n t h e p r o x i m a l and d i s t a l t u b u l e s and i n t h e t h i c k ascending l i m b o f t h e l o o p o f Henle,30 and t h e e x t r a c t i s a n t i n a t r i f e r i c on t h e s e r o s a l s u r f a c e o f f r o g s k i n . 3 1 Plasma induced i n h i b i t i o n i s s i m i l a r l y d i s t r i b u t e d w i t h i n t h e renal tubule. U r i n e f r o m VE dogs and uremic p a t i e n t s has a l s o been r e p o r t e d t o c o n t a i n an N H - l i k e a ~ t i v i t y , 3 ~ - 3 8and a n a t r i u r e t i c f a c t o r has been d e s c r i b e d i n t h e serum o f p a t i e n t s w i t h c h r o n i c ~ r e m i a . ~ ~ - ~ ~ Buckalew and Gruber h y p o t h e s i z e d t h a t a n t i b o d i e s a g a i n s t a Na+,K+ATPase i n h i b i t o r , such as d i g o x i n , c o u l d be used as probes f o r n a t r i u r e t i c hormone on t h e b a s i s t h a t a n t i b o d i e s t o drugs t h a t b i n d t o s e c i f i c r e c e p t o r s m i g h t r e c o g n i z e and b i n d t o t h e endogenous l i g a n d . $ 8 I n two o f t h e i r s t u d i e s , c r o s s r e a c t i v i t y o f t h e i n h i b i t o r o f Nat,Kt-ATPase w i t h a l . showed t h a t t h e r e a n t i - d i g o x i n a n t i b o d i e s was o b ~ e r v e d . ~ Rudd ~ , ~ _ ~e t _ was s i g n i f i c a n t l y more Na+,K+-ATPase i n h i b i t i o n and d i g o x i n immunor e a c t i v i t y i n t h e plasma o f VE dogs compared t o hydropenic dogs.42 The plasma e x t r a c t w i t h d i g o x i n i m m u n o r e a c t i v i t y f r o m VE dogs i s a l s o n a t r i u r e t i c i n r a t s . D i g o x i n i m m u n o r e a c t i v i t y i s found i n t h e plasma o f Rhesus and A f r i c a n Green monkeys w i t h 2 - k i d n e y Y 1 - c l i p G o l d b l a t t hypert e n s i o n . The h y p e r t e n s i v e monkeys have two- t o t h r e e - f o l d h i g h e r l e v e l s o f d i g o x i n i m m u n o r e a c t i v i t y t h a n t h e normotensive c o n t r o l s . 4 3 Increased e x c r e t i o n o f a d i g o x i n - l i k e hormone i n r a t s d u r i n g s a l t - l o a d i n g , which i s f u r t h e r enhanced d u r i n g t h e development o f h y p e r t e n s i o n and adaptat i o n t o c h r o n i c r e n a l f a i l u r e , has been r e p o r t e d r e c e n t l y . 4 4 I n c o n t r a s t t o t h e s e s t u d i e s , Hamlyn _ et _ a1 d i d n o t observe any a n t i - d i g o x i n immunor e a c t i v i t y i n plasma samples from normal o r h y p e r t e n s i v e p a t i e n t s . 2 9

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I m p a i r e d sodium e f f l u x was measured i n l e u k o c y t e s from p a t i e n t s w i t h e s s e n t i a l h y p e r t e n s i o n . I n c u b a t i o n o f normal l e u k o c y t e s w i t h plasma from h y p e r t e n s i v e p a t i e n t s caused i m p a i r e d sodium t r a n s p o r t . 4 5 F u r t h e r more, p a r t i a l l y p u r i f i e d f r a c t i o n s from plasma, as w e l l as u r i n e , i n h i b i t sodium e f f l u x f r o m p e r i p h e r a l b l o o d l e ~ k o c y t e s . ~The ~ severity o f the d e f e c t i n l e u k o c y t e c a t i o n t r a n s p o r t i s i n v e r s e l y r e l a t e d t o t h e plasma r e n i n a c t i v i t y and i s g r e a t e s t i n p a t i e n t s w i t h e s s e n t i a l h e r t e n s i o n i n whom t h e r e n i n response t o sodium r e s t r i c t i o n was a t y p i c a l Although t h e s e s t u d i e s s u p p o r t t h e h y p o t h e s i s t h a t NH i s a d i g i t a l i s - l i k e substance which i n h i b i t s Na+,K+-ATPase i n numerous t i s s u e s i n c l u d i n g t h e k i d n e y , i t remains t o be determined whether t h e n a t r i u r e t i c e f f e c t o f NH i s due e x c l u s i v e l y t o i n h i b i t i o n o f r e n a l Na+,K+-ATPase.

.$?

It has been proposed t h a t t h e hypothalamus m i g h t s e c r e t e a subs t a n c e which c o n t r o l s sodium e x c r e t i o n . 4 8 The AV3V r e g i o n o f t h e b r a i n i s important f o r r e g u l a t i n g blood pressure, since i t s d e s t r u c t i o n prevents s e v e r a l forms o f h y p e r t e n ~ i o n . ~Furthermore, ~ l e s s Na+ i s e x c r e t e d , t h e b l o o d l e v e l o f Na+ i s e l e v a t e d , and NH i s absent i n animals w i t h AV3V 1e s i o n s Consequently, s e v e r a l groups have prepared hypothalamic o r b r a i n e x t r a c t s and s t u d i e d t h e i r a n t i n a t r i f e r i c o r Na+,Kt-ATPase i n h i b i t o r y p r o p e r t i e s , b u t n a t r i u r e t i c a c t i v i t y was n o t r e p o r t e d . On t h e o t h e r hand, an acetone e x t r a c t o f t h e r a t hypothalamus ( b u t n o t c e r e b r a l c o r t e x , p i t u i t a r y , o r o t h e r t i s s u e s ) possessed v e r y p o t e n t Na',K+-ATPase inhibit o r y a c t i v i t y which was i n c r e a s e d 1 5 0 - f o l d i n animals on a h i g h sodium d i e t . 5 2 The plasma from t h e s e animals a l s o c o n t a i n e d Na+,K+-ATPase i n h i b i t o r y a c t i v i t y , w i t h s e v e n - f o l d more a c t i v i t y i n t h e plasma o f t h e r a t s on t h e h i g h sodium d i e t . Aqueous-acetone e x t r a c t i o n o f r a t b r a i n , i n t h e presence o f n i t r o g e n , r e s u l t e d i n a s p e c i f i c Na+,K+-ATPase i n h i b i t o r . 5 3 A l o w m o l e c u l a r w e i g h t " o u a b a i n - l i k e " f a c t o r , which i n h i b i t s i n v i t r o Na+,Kt-ATPase a c t i v i t y , b l o c k s 3H-ouabain b i n d i n g t o b r a i n m i c r o soma1 Na+,Kt-ATPase o r i n h i b i t s 86Rb u p t a k e i n human e r y t h r o c y t e s , has

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been p a r t i a l l y p u r i f i e d from b o v i n e h y p ~ t h a l a m u s , ~ guinea ~ , ~ ~ p i g 5 6 and rat5' brain. The chemical s t r u c t u r e o f NH has n o t been determined and t h e r e i s no assurance t h a t t h e a c t i v i t i e s a s c r i b e d t o t h e v a r i o u s e x t r a c t s a r e caused by t h e same substance. Furthermore, t h e r e i s no s t a n d a r d i z e d assay which a l l t h e i n v e s t i g a t o r s agree i s b e s t f o r d e t e c t i n g NH. However, t h e r e i s general agreement t h a t t h e substance i s low m o l e c u l a r w e i g h t (< 1000 d a l t o n s ) , a c i d i c , w a t e r s o l u b l e and h e a t s t a b l e . 4 The presence o f two n a t r i u r e t i c f a c t o r s i n t h e plasma and u r i n e o f volume expanded s u b j e c t s has been r e p o r t e d ,58 One f a c t o r causes n a t r i u r e s i s i n r a t s a f t e r a 20 min d e l a y and appears t o be l a r g e r t h a n t h e second f a c t o r which produces immediate n a t r i u r e s i s . The l o w m o l e c u l a r w e i g h t f a c t o r i s a n t i n a t r i f e r i c ( i n h i b i t s i o n f l u x i n i s o l a t e d f r o g s k i n o r toad b l a d d e r membrane p r e p a r a t i o n s ) w h i l e t h e h i g h e r m o l e c u l a r w e i g h t f a c t o r i s n o t . These r e s u l t s and t h e f i n d i n g t h a t NH a c t i v i t y i n c r e a s e s i n plasma f o l l o w i n g i n c u b a t i o n f o r 30 minutes a t room temperature supports t h e presence o f an NH precursor.59 Evidence f o r t h e pe t i d e n a t u r e o f NH i s i n d i c a t e d by i t s s e n s i t i v i t y t o enzymatic d i g e s t i o n 3 9 ~ 3 9 , 4 0 ~ 6 0 , 6 1 and i t s b e h a v i o r d u r i n chromatography by t e c h n i q u e s used f o r t h e i s o l a t i o n o f small peptides.t2 Buckalew and coworkers have s t u d i e d a h e p t a p e p t i d e w i t h sequence homology t o a fragment o f ACTH/a MSH (Met-G1 u-Hi s-Phe-ArgTrp-Gly [ o r Asp]), which may be r e l a t e d t o an endogenous NH s i n c e i t s b i o l o g i c a l e f f e c t s mimic t h o s e o f NH.63-66 The p e p t i d e i n h i b i t s Na+,K+ATPase i s n a t r i u r e t i c a t low doses, and i s h y p e r t e n s i v e a t h i g h d 0 s e s . 6 2 , ~ ~ Others, however, do n o t b e l i e v e t h e f a c t o r t h e y i s o l a t e from u r i n e o f salt-expanded dogs i s a p e p t i d e . 4 ~ ~ 2 ~ 6I 0n s p i t e o f t h e r e p o r t e d c r o s s r e a c t i v i t y o f NH with a n t i - d i g o x i n a n t i b o d i e s , NH does n o t appear t o be a s t e r o i d , s i n c e i t i s i s o l a t e d i n aqueous s o l u t i o n s and i s s u s c e p t i b l e t o a c i d h y d r o l y s i s .4 Expansion o f t h e e x t r a c e l l u l a r f l u i d volume, r e s u l t i n i n a n a t r i u r e t i c response a f f e c t s proximal t u b u l e sodium resorption.6yP68 However, i n h i b i t i o n o f sodium r e s o r p t i o n a t a more d i s t a l nephron s i t e such as t h e c o l l e c t i n g d u c t , a l s o occurs d u r i n g expansion o f t h e e x t r a c e l l u l a r f l u i d volume and i s a major determinant o f t h e magnitude o f n a t r i u r e ~ i s . ~ g ,R~e c~o l l e c t i o n m i c r o p u n c t u r e t e c h n i q u e s were used t o show a s i g n i f i c a n t decrease i n f r a c t i o n a l sodium r e s o r p t i o n i n t h e p r o x i m a l t u b u l e s o f t h e s u p e r f i c i a l nephrons o f uremic r a t s when a n a t r i u r e t i c response occurred f o l l o w i n g t h e a d m i n i s t r a t i o n o f a serum d e r i v e d NH.71 I n r a b b i t , t h e r e n a l c o l l e c t i n g d u c t ( t u b u l e ) has been i m p l i c a t e d as an i m p o r t a n t s i t e f o r t h e r e g u l a t i o n o f a c t i v e sodium t r a n s p o r t by t h e n a t r i u r e t i c f a c t o r . A n a t r i u r e t i c sample a p p l i e d t o t h e p e r i t u b u l a r surface o f t h e i s o l a t e d perfused c o r t i c a l c o l l e c t i n g tubule i n h i b i t e d t h e p o t e n t i a l d i f f e r e n c e and decreased t h e n e t sodium f l u x from t h e lumen t o t h e p e r i t u b u l a r surface.26 T h i s i s c o n s i s t e n t w i t h r e s u l t s from s t u d i e s on t h e i s o l a t e d t o a d b l a d d e r ( a s t r u c t u r e w i t h many s i m i l a r i t i e s t o t h e d i s t a l nephron) which have shown t h a t NH a c t s a t t h e s e r o s a l s u r f a c e and i n h i b i t s t r a n s e p i t h e l i a l t r a n s p o r t by r e d u c i n g sodium movement across t h e s e r o s a l b a r r i e r . 44

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A t r i a l N a t r i u r e t i c Factor Evidence has accumulated which suggests t h a t t h e c a r d i a c a t r i a m i g h t f u n c t i o n as a sensor f o r d e t e c t i n g changes i n Indeed , numerous experimental procedures e x t r a c e l 1u l a r f 1u i d v o l ume which a l t e r i n t r a a t r i a l pressure o r s t r e t c h t h e a t r i a l w a l l r e s u l t i n s i g n i f i c a n t changes i n water and e l e c t r o l y t e e x c r e t i o n . Such a l t e r a t i o n s i n i n t r a a t r i a l p r e s s u r e should r e f l e c t changes i n and p r o v i d e a means f o r r e g u l a t i ng e x t r a c e l 1u l a r f 1u i d v o l ume . 7 ,72

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Changes i n w a t e r e x c r e t i o n t h a t a r e secondary t o a l t e r e d i n t r a a t r i a l p r e s s u r e can be a t t r i b u t e d l a r g e l y t o changes i n t h e s e c r e t i o n o f v a s o p r e s s i n by t h e p o s t e r i o r p i t u i t a r y gland. T h i s r e f l e x has been documented b o t h i n e x p e r i m e n t a l animals and i n man, and t h e r e l e v a n t d a t a has been reviewed r e c e n t l y by Bie.73 On t h e o t h e r hand, i t has n o t been p o s s i b l e , u n t i l r e c e n t l y , t o account f o r t h e i n c r e a s e d u r i n a r y Nat e x c r e t i o n a s s o c i a t e d w i t h changes i n a t r i a l pressure. et a l . have e x t r a c t e d a substance from t h e c a r d i a c a t r i a de B o l d o f r a t s which, when i n j e c t e d i n t o o t h e r r a t s , r e s u l t s i n a 3 0 - f o l d i n c r e a s e i n u r i n a r y Na+ e x c r e t i o n and a 1 0 - f o l d i n c r e a s e i n u r i n e S i m i l a r e x t r a c t s o f c a r d i a c v e n t r i c l e were w i t h o u t e f f e c t . volume.74 Numerous independent l a b o r a t o r i e s have now c o n f i r m e d t h e i n i t i a l observ a t i o n o f a t r i a l n a t r i u r e t i c f a c t o r (ANF).75-88 T h i s a c t i v i t y has been observed i n t h e a t r i u m o f a l l mammalian s p e c i e s s t u d i e d , i n c l u d i n g man.83~89,90 The s p e c i f i c a c t i v i t y appears t o be h i g h e s t i n r a t a t r i a . The d i s t i n c t i o n between t h i s m a t e r i a l and NH r e s t s on s e v e r a l c r i t e r i a : 1 ) t h e minimum m o l e c u l a r w e i g h t f o r ANF i s a t l e a s t 3000 d a l t 0 n s 8 ~ w h i l e NH i s p r o p o r t e d t o be l e s s t h a n 1000,12 2 NH appears t o i n h i b i t Nat,Kt-ATPase w h i l e ANF has no such a c t i v i t y , 4 ~ and 3) NH i s t h o u g h t t o be h y p e r t e n s i v e w h i l e ANF c l e a r l y l o w e r s b l o o d p r e s ~ u r e . ~ , 7 4

I

" S p e c i f i c granules"ql have been d e s c r i b e d i n t h e myocytes o f t h e c a r d i a c a t r i a which c l o s e l y resemble t h e s t o r a g e g r a n u l e s o f o t h e r p e p t i d e hormone s e c r e t i n g t i s s u e s . These g r a n u l e s c o - p u r i f y w i t h ANF92393 and immunocytochemical s t u d i e s c o n f i r m t h e a s s o c i a t i o n between t h e s p e c i f i c a t r i a l g r a n u l e s and ANF.94995 Furthermore, t h e highest concentrations o f antigenic material are l o c a l i z e d i n t h e s u b p e r i c a r d i a l a r e a o f t h e a t r i a l a ~ p e n d a g e s . 9 ~ D i r e c t bioassay o f d i s s e c t e d a t r i a c o n f i r m t h e s e h i g h l e v e l s o f ANF a ~ t i v i t y . 9 ~ It i s n o t understood what f a c t o r s c o n t r o l t h e s y n t h e s i s , s t o r a g e and s e c r e t i o n o f ANF, b u t a l t e r a t i o n s i n body f l u i d volume appear t o change g r a n u l e d e n s i t y and e x t r a c t a b l e a c t i v i t y . G r a n u l a r i t y i n c r e a s e s markedly i n response t o s a l t o r w a t e r d e p r i v a t i o n and decreases when e x t r a c e l l u l a r f l u i d volume i s expanded secondary t o d e s o x y c o r t i c o s t e r o n e However, t h e r e i s no c o r r e l a t i o n between t h e g r a n u l a r i t y treatment.97 It i s p o s s i b l e t h a t o f t h e a t r i a and t h e amount o f e x t r a c t a b l e ANF.g8 t h e s t o r a g e f o r m o f ANF under t h e s e c o n d i t i o n s may n o t be a c t i v e , and f u r t h e r p r o c e s s i n g i s r e q u i r e d b e f o r e b i o l o g i c a l a c t i v i t y can be expressed. Although e x t r a c t a b l e ANF i s reduced i n t h e a t r i a o f spontane o u s l y h y p e r t e n s i v e r a t s , i t i s n o t known whether t h i s change i s r e l a t e d t o t h e e l e v a t e d b l o o d pressure.99

P o t e n t n a t r i u r e t i c a c t i v i t y was e v i d e n t i n a s i m p l e phosphate b u f f e r e d s a l i n e e x t r a c t o f whole a t r i a . 7 4 Subsequently, t h e m a t e r i a l was While t h e e x t r a c t i s b o t h a c i d found t o be a c i d stable.75,87,88,100,1*1 and heat759101 s t a b l e , t r y p ~ i n , ~ ~ , 8 9 , 9as 3 w e l l as o t h e r p r o t e o l y t i c e n z y m e ~ 7such ~ as chymotrypsi n, ami nopept i d a s e A and carboxypept i d a s e s However, t r e a t m e n t w i t h carboxyB and C d e s t r o y n a t r i u r e t i c a c t i v i t p e p t i d a s e A o n l y b l u n t e d a c t i v i t y , 7 g and c o n c a n a v a l i n A had no e f f e ~ t . 7 ~ These o b s e r v a t i o n s a r e c o n s i s t e n t w i t h ANF b e i n g a s m a l l p e p t i d e .

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P u r i f i c a t i o n o f ANF from r a t a t r i a by s e v e r a l independent l a b o r a t o r i e s u t i l i z i n d i f f e r e n t i s o l a t i o n procedures c o n f i r m e d i t s p e p t i d i c n a t u r e . 8 5 ~ 1 0 2 ~ 1 ~ 3 , 1 0 4An amino a c i d c o m p o s i t i o n w i t h o u t sequence was r e p o r t e d from two l a b o r a t o r i e s 8 5 ~ 1 0 2 b u t t h e t o t a l number o f r e s i d u e s

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d i f f e r e d c o n s i d e r a b l y (49 and 36) and t h e r e were d i f f e r e n c e s i n composit i o n . An amino a c i d sequenc f o r r a t ANF was r e p o r t e d e s s e n t i a l l y s i m u l C u r r i e e t a1 ,lo5 Kangawa and Matsuo,1o6 t a n e o u s l y by F1 nn e t a l . , loS et a1 .loJ and Napier e t a1 .87,* T h e l o n g e s t p e p t i d e r e p o r t e d has Seidah t h e 33 amino a c i d sequence shownbelow, 1, which c o n t a i n s t h e C s12-Cys28 d i s u l f i d e b r i d g e , and i s t h e Tyr a c i d r a F h e r t h a n t h e amide.87,{8,107

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H- Leu -A1 a- G1y- Pro-Arg5- Se r-Leu- A r g Arg Se r 10- Se r-Cys- Phe- 61yGly15-Arg- Ile-Asp-Arg-Ile20-Gly-Al a-G1 n-Ser-Gly25-Leu-Gly-CysA~n-Ser3~-Phe-Arg-Tyr-OH

-1 Other sequences a r e N-terminal t r u n c a t e d v e r s i o n s o f t h i s p e p t i d e (22,28, 31,32 residues).87,88,103,107 C u r r i e e t a l . have p u r i f i e d two a t r i a l p e p t i d e s ( a t r i o p e p t i n s I and 11) o f 2 1 a n T 2 3 amino a c i d s . l o 5 The l o n g e r o f t h e s e i s i d e n t i c a l t o t h e 10-32 fragment o f Kangawa and Matsuo have i s o l a t e d ANF from human a t r i a and determined t h e amino a c i d sequence o f a 28 amino a c i d p e p t i d e which d i f f e r s from t h e r a t ANF sequence o n l y a t p o s i t i o n 17 where I l e i s r e l a c e d by Met.1o6 Based on t h e sequence d i s c o v e r e d by Seidah e t a1.,lug s y n t h e t i c m a t e r i a l was made and t h e i m p o r t a n t c o n f i r m a t i o n o f t h e n a t u r a l m a t e r i a l was obtained. The synt h e t i c p e p t i d e composed o f t h e 8-33 sequence o f Seidah e t a l . has f u l l b i o l o g i c a l a c t iv i ty.lU7

1.

I n i t i a l l y , t h e p r i n c i p a l b i o l o g i c a l a c t i v i t y o f ANF was t h o u g h t t o be n a t r i u r e s i s . Most i n v e s t i g a t o r s r e p o r t e d 30 t o 4 0 - f o l d i n c r e a s e s i n u r i n a r y sodium e x c r e t i o n w i t h l e s s e r e f f e c t s on u r i n a r y K+ e x c r e t i o n . Although t h e mechanism o f t h e n a t r i u r e s i s has n o t been s t u d i e d e x t e n s i v e l y , r e p o r t s u s i n g m i c r o p u n c t u r e and m i c r o c a t h e t e r i z a t i on t e c h n i q u e s suggest t h a t i n h i b i t i o n o f Na+ t r a n s p o r t i n t h e f a r d i s t a l nephron ( i . e . c o l l e c t i n g d u c t ) i s t h e p r i n c i p a l mechanism o f a c t i o n . 7 7 ~ 1 0 8 These d a t a must be i n t e r p r e t e d c a u t i o u s l y , however, because t h e magnitude o f t h e n a t r i u r e t i c response would argue f o r a more p r o x i m a l s i t e o f a c t i o n . Indeed, a r e c e n t r e p o r t by Seymour e t al., i n which s y n t h e t i c ANF was i n f u s e d d i r e c t l y i n t o t h e r e n a l a r t e r y o f a n e s t h e t i z e d dogs, i n d i c a t e d t h a t f r a c t i o n a l sodium e x c r e t i o n exceeded 10% d u r i n g maximal n a t r i u r e sis.109 Based on what i s c u r r e n t l y known about sodium d e l i v e r y t o t h e v a r i o u s nephron segments, t h i s would argue f o r a s i t e o f a c t i o n a t l e a s t i n t h e c o r t i c a l d i l u t i n g segment (a s i t e s i m i l a r t o t h i a z i d e d i u r e t i c s ) . Nevertheless, t h e d a t a do not exclude m u l t i p l e s i t e s o f a c t i o n , o r changes i n r e n a l hemodynamics o r nephron h e t e r o g e n e i t y . Some evidence a l s o suggests t h a t changes i n g l o m e r u l a r f i l t r a t i o n a r e i m p o r t a n t i n t h e n a t r i u r e t i c r e s onse, b u t t h e s e s t u d i e s were conducted i n i s o l a t e d p e r Other s t u d i e s i n i n t a c t animals have n o t demonstrated fused k i dneys lP0 m a j o r changes i n e i t h e r g l o m e r u l a r f i l t r a t i o n o r r e n a l b l o o d flow.74,78~79

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I n a d d i t i o n t o t h e p o t e n t n a t r i u r e t i c a c t i v i t y , ANF has s i g n i f i c a n t r e l a x a n t e f f e c t s on v a s c u l a r and perhaps o t h e r smooth muscle t i s ~ u e s . C8u r r i e~ ~a l . observed ~ ~ et r~e l a x a~ t i o n~ o f r~a b b i~t a o ~r t a ~ and c h i c k rectum, which had r e v i o u s l y been c o n t r a c t e d w i t h e p i n e p h r i n e and carbachol, r e s p e ~ t i v e l y . ~They ~ use t h i s s p a s m o l y t i c a c t i o n t o m o n i t o r ANF a c t i v i t y and a r e t h e f i r s t group t o separate smooth muscle r e l a x a n t a c t i v i t y o f s i m i l a r p e p t i d e s i s o l a t e d from c a r d i a c a t r i a , a l though b o t h p e p t i d e s a r e n a t r i u r e t i c . l o 5 A r e c e n t r e p o r t by Winquist -e t a l . has d e t a i l e d t h e v a s c u l a r smooth muscle r e l a x i n g e f f e c t o f synthet i c ANF u s i n g a v a r i e t y o f a g o n i s t s and d i f f e r e n t v a s c u l a r smooth muscle p r e p a r a t i o n s . l 1 4 Based on t h e s e o b s e r v a t i o n s , ANF appears t o have spasmolytic p r o p e r t i e s s i m i l a r t o sodium n i t r o p r u s s i d e .

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ANF has p o t e n t e f f e c t s on c y c l i c GMP (cGMP) l e v e l s . I n v i t r o , a d d i t i o n o f a t r i a l e x t r a c t s t o minced k i d n e y t i s s u e o r t o p r i m a r y i d n e y c e l l c u l t u r e s r e s u l t s i n i n c r e a s e d l e v e l s o f ~ G M p . 1 1 ~I n j e c t i o n i n t o a n e s t h e t i z e d r a t s r e s u l t e d i n a 4 - f o l d i n c r e a s e i n plasma cGMP l e v e l s and a 2 8 - f o l d i n c r e a s e i n u r i n a r y e ~ c r e t i 0 n . l ~Sodium ~ nitroprusside also causes i n c r e a s e d cGMP l e v e l s i n t r e a t e d t i s s u e s , b u t p r o b a b l y a c t s d i r e c t l y t h r o u g h s t i m u l a t i o n o f g u a n y l a t e c y c l a s e . F u r t h e r s t u d i e s must be done t o c l a r i f y t h e r o l e o f cGMP i n t h e v a s o r e l a x a n t and t h e n a t r i u r e t i c a c t i o n s o f ANF. The p o t e n t n a t r i u r e t i c a c t i v i t y o f ANF has i n e v i t a b l y l e d t o comp a r i s o n s w i t h known d i u r e t i c s . One r e p o r t by Sonnenberg e t a l . i n d i c a t e d t h a t probenecid b l u n t e d t h e n a t r i u r e t i c a c t i v i t y o f ANF.lm B e c a u s e p r o b e n e c i d i n t e r f e r e s w i t h t h e n a t r i u r e t i c a c t i v i t y o f most o t h e r d i u r e t i c s b y competing w i t h t h e i r s e c r e t o r y t r a n s p o r t i n t o t h e nephron lumen, t h e s e a u t h o r s suggest t h a t ANF m i g h t be a c t i n g as an endogenous d i u r e t i c . Furosemide i s t h e d i u r e t i c most f r e q u e n t l y c i t e d as s i m i l a r t o ANF, w i t h r e g a r d t o e l e c t r o l y t e e x c r e t i o n . ANF promotes sodium, potassium and c a l c i u m e x c r e t i o n w i t h c h l o r i d e as t h e p r i n c i p a l anion. Although f u r o s e mide has been a r e f e r e n c e s t a n d a r d i n a t l e a s t one paper,89 i t s h o u l d be p o i n t e d o u t t h a t r i g o r o u s dose-response s t u d i e s comparing ANF t o o t h e r d i u r e t i c s have n o t been conducted. The most d e f i n i t i v e s t u d i e s suggested a c e i l i n g s i m i l a r t o t h e t h i a z i d e d i u r e t i c s , a maximum f r a c t i o n a l sodium Also, l o o p d i u r e t i c s such as furosemide e x c r e t i o n o f about lU%.109 i n h i b i t NaCl t r a n s p o r t i n b u l l f r o g cornea, an analog o f t h e m e d u l l a r y t h i c k ascending l i m b o f t h e l o o o f Henle whereas a t r i a l e x t r a c t s a r e i n a c t i v e i n t h i s preparation.ll! Because o f t h e p o t e n t i a l f o r ANF t o be i n v o l v e d i n b l o o d p r e s s u r e c o n t r o l , s e v e r a l i n v e s t i g a t o r s have s t u d i e d i t s e f f e c t s on t h e c a r d i o v a s c u l a r system o f normal and h y p e r t e n s i v e animals. I n a n e s t h e t i z e d normotensi ve and spontaneously h y p e r t e n s i v e r a t s , a t r i a l e x t r a c t decreased a r t e r i a l b l o o d p r e s s u r e i n a s s o c i a t i o n w i t h a decrease i n b o t h 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 and c a r d i a c c o n t r a c t i 1 i t y . l l 6 Also, a n e g a t i v e c h r o n o t r o p i c e f f e c t has been r e p o r t e d i n r a t s . l 1 7 Dahl s a l t s e n s i t i v e r a t s appear t o have g r e a t e r amounts o f ANF i n t h e i r a t r i a , b u t a r e hyporesponsive when exogenous m a t e r i a l i s i n j e c t e d . 9 0 R e s u l t s from t h i s s t u d y a l s o suggest an i n c r e a s e i n r e n a l p a p i l l a r y plasma f l o w and a washout o f t h e m e d u l l a r y osmotic g r a d i e n t , f a c t o r s which c o u l d c o n t r i b u t e s i g n i f i c a n t l y t o t h e a s s o c i a t e d n a t r i u r e s i s and d i u r e s i s . Increased m e d u l l a r y and i n n e r c o r t i c a l b l o o d f l o w a l s o were r e p o r t e d i n normotensive r a t s .I18

___ Summary - I n t h e case o f n a t r i u r e t i c hormone, t w e n t y - f i v e y e a r s o f r e search have f a i l e d t o c h a r a c t e r i z e a p u r e substance t h a t has n a t r i u r e t i c a c t i v i t y and i s an i n h i b i t o r o f Nat,Kt-ATPase, although, a v a s t body o f c i r c u m s t a n t i a l evidence does f a v o r t h e e x i s t e n c e o f such a substance. A t r i a l n a t r i u r e t i c f a c t o r , on t h e o t h e r hand, i s a r e c e n t d i s c o v e r y which y i e l d e d r e a d i l y t o chemical c h a r a c t e r i z a t i o n and s y n t h e s i s . I t s b i o l o g i c a l p r o f i l e suggests t h a t i t may be a component o f t h e v a r i o u s c o n t r o l systems s u b s e r v i n g f l u i d volume and p r e s s u r e r e g u l a t i o n . However, no c o n v i n c i n g evidence has y e t been o f f e r e d t h a t a t r i a l n a t r i u r e t i c f a c t o r p l a y s an i m p o r t a n t p h y s i o l o g i c a l r o l e i n p r e s s u r e o r volume homeostasis.

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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.

78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88.

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G.A. MacGregor and H.E. dewardener, B r i t . Med. J., 3,847 (1981). L. Poston, S. W i l k i n s o n , R.B. Sewell and R. W i l l i a m s , C l i n . S c i . , 63, 243 ( 1 9 8 2 ) . R.P.S. Edmondson and G.A. MacGregor, B r i t . Med. J., 282, 1267 ( 1 9 8 n . H.W. Smith, Amer. J. Med., 23, 623 (1957). M. Brody and A.K. Johnson i n " F r o n t i e r s i n Neuroendocrinology," L. M a r t i n i and W.F. Ganong, Eds., Raven Press, New York, Vol. 6, 1980. A.K. Johnson i n " P e r s p e c t i v e s i n Nephrology and Hypertension," H. S c h m i t t and P. Meyer, Eds., Wiley, New York, 1976, p 106. S . B e a l e r , J.R. Haywood, A.K. Johnson, K.A. Gruber, V.M. Buckalew and M.J. Brody, Fed. Proc., 38, 1232 (1979). J. Alaghband-Zadeh, S. Fenton, K . Hancock, J . M i l l e t t and H.E. dewardener, J. E n d o c r i n o l . , 98, 221 (1983). K.R. Whitmer, E.T. W a l l i c k , D.E. Epps, L.K. Lane, J.H. C o l l i n s and A. Schwartz, L i f e Sci 30, 2261 (1982). G.T. H a u p e r c Jr., L. Kormos, L. Anderson, S. Ray and L.C. Cantley, A b s t r a c t s , 1 6 t h Ann. Mtg. Amer. SOC. Nephrology, p 192A (1983). G.T. Haupert, Jr. and J.M. Sancho, Proc. N a t l . Acad. Sci., 4658 (1979). M.C. Fishman, Proc. N a t l . Acad. Sci., 2, 4661 (1979). D. L i c h t s t e i n and S. Samuelov, Biochem. Biophys. Res. Comm., 96, 1518 ( 1 9 8 0 ) . H.E. dewardener, C l i n . Sci. Mol. Med., 53, 1 (1977). K.A. Gruber and V.M. Buckalew, Jr., Proc. SOC. Exp. B i o l . Med., 463 ( 1 9 7 8 ) . V.M. Buckalew, Jr. and K.A. Gruber i n "The Kidney i n L i v e r Disease," 2nd ed., M. E p s t e i n , Ed., E l s e v i e r , New York, N.Y., 1983. p 479. K.A. Gruber and V.M. Buckalew, Jr. i n "Hormonal R e g u l a t i o n o f Sodium E x c r e t i o n , " 6. L i c h a r d u s , R.W. S c h r i e r and J. Ponec, Eds., E l s e v i e r , New York, N.Y., 1980, p 349. A.N. Radhakrishnan. 5 . S t e i n . A. L i c h t . K.A. Gruber and S. Udenfriend. J. Chromatography, 5 5 2 - ( 1977). . J.F. Hennessy, V.M. Buckalew, Jr., J.R. Lymanqrover and K.A. Gruber, C l i n . Res., 30, 848A (1982). M.C. K l e i n , P.M. Hutchins, V.M. Buckalew, J r . , J.F. Hennessy, J.R. Lymangrover and K.A. Gruber, Fed. Proc., 42, 1020 (1983). K.A. Gruber, J.F. Hennessy, J.R. Lymangrover, M.C. K l e i n , P.M. H u t c h i n s and V.M. Buckalew, Jr., Fed. Proc., 42, 1174 (1983). J.R. Lymangrover, J.F. Hennessy, M.C. K l e i n , P.M. H u t c h i n s , V.M. Buckalew, Jr. and K.A. Gruber, Fed. Proc., 42, 1174 (1983). B.M. Brenner, K.H. Falchuk, R . I . Keimowitz and R.W. B e r l i n e r , J. C l i n . I n v e s t . , 48, 1519 (1969). J.H. D i r k s , W.J. Cirksena and R.W. B e r l i n e r , J. C l i n . I n v e s t . , 1160 ( 1 9 6 5 ) . H. Sonnenberg, Amer. J. Physiol., 223, 916 (1972). J.H. S t e i n , R.W. Osgood, 5 . B o o n j a r e r n and T.F. F e r r i s , J. C l i n . Invest., 52, 2313 (1973). H. Weber, J.J. B o u r g o i g n i e and N.S. B r i c k e r , Amer. J . P h y s i o l . , 226, 419 ( 1 9 7 4 ) . K.L. Goetz, G.C. Bond and D.D. Bloxham, P h y s i o l . Rev., 55. 157 (1975). P. B i e , P h y s i o l . Rev., 60, 961 (1980). A.J. de Bold, H.B. B o r e n s t e i n , A.T. Veress and H. Sonnenberg, L i f e Sci., 89 (1981). N.C. T r i m o d o . A.A. MacPhee. F.E. Cole and H.L. B l a k e s l e -v .. Proc. SOC. EXD. B i o l . Med., 502 (1982). G. T h i b a u l t , R. Garcia, M. C a n t i n and J. Genest. H y p e r t e n s i o n 5 (Suppl. I ) , 1-75 (1983). ,J.P. B r i g g s , B. S t e i p e , G. Schubert and J. Schnermann, P f l u e g e r s Arch., 395, 271 (1982). R. K e e l e r , Can. J. P h y s i o l . Pharm., 3, 1078 (1982). D.M. P o l l o c k and R.O. Banks, C l i n . Sci., 65, 47 (1983). A. Bonham, K. Barron, M. A l l e n , 0. P o l l o c k , R. Banks and M.J. Brody, Fed. Proc., 42, 494 (1983). Hathaway and S. Solomon, Fed. Proc., 42, 475 ( 1 9 8 3 ) . M.J.F. Camargo, H.D. K l e i n e r t , J.E. S e a l z , J.H. Laragh and T. Maack, Fed. Proc., 42, 475 ( 1 9 8 3 ) . M.N. Nemeh and J.P. Gilmore, C i r c . Res., 53, 420 ( 1 9 8 3 ) . M.G. C u r r i e , D.M. G e l l e r , B.R. Cole, J.G.Boylan, W. YuSheng, S.W. Holmberg and P. Needleman, Science, 221, 7 1 (1983). R.T. Grammer, H. Fukumi, T. Inagami and K.S. Misono, Biochem. Biophys. Res. Commun., 696 (1983). I. K i t o r , D.J. Levenson, O.C. Ohuoha and V.J. Dzau, A b s t r a c t s , 1 6 t h Ann. Mtg. Amer. SOC. Nephrology, p 167A (1983). M.A. N a p i e r , R.S. Dewey, G. Albers-Schonberg, C.D. Bennett, J.A. Rodkey, E.A. Marsh, M. Whinnery, A.A. Seymour and E.H. B l a i n e , Fed. Proc., 43, 501 (1984). M.A. N a p i e r , R.S. Dewey, G. Albers-Schonberg, C.D. Bennett, 5 . K Rodkey, E.A. Marsh, M. Whinnery, A.A. Seymour and E.H. B l a i n e , Biochem. Biophys. Res. Commun. ( i n press).

.,

76,

159,

132,

2,

28,

a,

> -

77.

Endogenous N a t r i u r e t i c Factors

116,

262 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.

Sect. V

- Topics in Biology

Egan, Ed.

5

N.C. Trippodo, A.A. MacPhee and F.E. Cole, Hypertension (Supp. I ) , 1-81 (1983). M. Ganquli, Y. H i r a t a and L. Tobian, A b s t r a c t s , 1 6 t h Ann. Mtg. h e r . SOC. Nephro-

l o g y , p 191A (1983). Jamieson and G.E. Palade, J. C e l l B i o l . , 23, 151 (1964). A.J. de Bold, Can. J. P h y s i o l . Pharm., 60, 32471982). R. Garcia, M. Cantin, G. T h i b a u l t , H. Ong and J. Genest, E x p e r i e n t i a , 1071 (1982). M. Cantin, J. Gutkowski, G. T h i b a u l t , R.W. M i l n e , S. Ledoux. S. M i n l i , R. Garcia, P . Hamet and J. Genest, Histochem. ( i n press). M. Cantin, J. Gutkowski, R.W. M i l n e , G. T h i b a u l t , R. Garcia and J. Genest, 37th Ann. Mtg. Council f o r High Blood Pressure Research, 1983, abst. 39 (1983). J. B r i g g s , W. K r i z and J. Schnermann, C i r c u l a t i o n 68 (Suppl. 1 1 ) , abst. 172 (1983). A.J. de Bold, Proc. SOC. Exp. B i o l . Med., 50871979). G. T h i b a u l t , R. Garcia, M. C a n t i n and J. Genest, Fed. Proc., 42, 611 (1983). H. Sonnenberg, S . M i l o j e v i c , C.K. Chong and A.T. Veress, Hypertension, 5. 672 (1983). A.J. de Bold, Fed. Proc., 40, 554 (1981). A.J. de Bold, Proc. SOC. Exp. B i o l . Med.. 133 (1982). A.J. de B o l d and T.G. Flynn, L i f e Sci., 2,297 (1983). T.J. Flynn, M.L. de Bold and A.J. de Bold, Biochem. Biophys. Res. Comm., 859 (1983). G. T h i b a u l t , R. Garcia, N.G. Seidah, C. Lazure, M. Cantin, M. C h r e t i e n and J. Genest, FEBS L e t t . , 286 (1983). M.G. C u r r i e , D.M. G e l l e r , B.R. Cole. N.R. S i e g e l , K.F. Fok, S.P. Adams, S.R. Eubanks, G.R. G a l l u p p i and P. Needleman, Science, 223, 67 (1984). K. Kangawa and H. Matsuo, Biochem. Biophys. Res. Comm., 131 (1984). N.G. Seidah, C. Lazure. M. C h r e t i e n , G. T h i b a u l t , R. Garcia, M. Cantin, J . Genest, R.F. N u t t , S.F. Brady, T.A. L y l e , W.J. Paleveda, C.D. Colton, T.M. Ciccarone and D.F. Veber, Proc. N a t l . Acad. Sci. ( i n press). H. Sonnenberg, W.A. Cupples, A.J. de B o l d and A.T. Veress, Can. J. P h y s i o l . Pharm., 60, 1149 (1982). A.A. Seymour, R. N u t t , E. Mazack and E.H. B l a i n e , Fed. Proc., 43, 502 (1984). H.D. K l e i n e r t , J.E. Sealey, J.H. Laragh and T. Maack, P h y s i o l o g s t , 26, A-59 (1983). H. Sonnenberg, C.K. Chong and A.T. Veress, Can. J. P h y s i o l . Pharm., 59. 1278 (1981). D.C. Throckmorton and J.P. Gilmore, Fed. Proc., 42, 475 (1983). R.C. Deth, K. Wong, S. Fukozawa, R . Rocco, J.L. Smart, C.J. Lynch and R. Awad, Fed. Proc., 3, 983 (1982). R.J. Winquist, E.P. Faison and R.F. N u t t , Europ. J. Pharmacol. ( i n p r e s s ) . P. Hamet, J. Tremblay, G. T h i b a u l t , R. Garcia, M. C a n t i n and J. Genest, A b s t r a c t s , 6 5 t h Ann. Mtg. Endocrine S o c i e t y , p 289 (1983). U. Ackermann, T. I r i z a w a and H. Sonnenberg, Fed. Proc., 3, 1353 (1982). U. Ackermann, T.G. I r i z a w a , S . M i l o j e v i c and H. Sonnenberg, Fed. Proc., 2, 1096 (1983). H.B. Borenstein, W.A. Cupples, H. Sonnenberg and A.T. Veress, J. Physiol., 133 (1983). J.D.

38,

161, 170,

117,

164,

118,

334,

Section VI

- Topics in Chemistry and

Drug Design

Editor: Richard C. Allen, Hoechst-Roussel Pharmaceuticals Inc., Somerville, New Jersey 08876 Chapter 26. Enzymic Methods in Organic Synthesis Mark A. Findeis and George M. Whitesides, Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138 Introduction - Enzyme-based synthetic chemistry has continued to grow rapidly in recent Technical problems,which have inhibited the widespread use of enzymes as catalysts, have been much reduced by the introduction of new procedures for enzyme immobilization and stabilization and for in situ cofactor recycling. A widespread interest in asymmetric synthesis has focussed attention on the demonstrated utility of enzymic catalysis in producing chiral fragments. In fact, the major hindrance to the widespread use of enzymic catalysis is the residual unfamiliarity of many classically trained synthetic chemists in the techniques of enzyme isolation, manipulation, and assay. This last barrier is disappearing as biochemistry and enzymology become an accepted part of the education of an organic chemist. This review emphasizes procedures which use partially or highly purified enzymes to catalyze organic reactions potentially useful in medicinal chemistry. Biochemical procedures using microbiological transformations or cell culture are not discussed. General Techniques - More than two thousand enzymes are known,' and several hundred can be obtained commercially. Many other enzymic activities are available through straightforward isolations or small-scale fermentations. Increasing attention is being paid to large-scale production of enzymes;8 Kula has developed efficient methods based on liquid-liquid extractions. Given sufficient demand, many enzymes can be produced in quantity by recombinant DNA techniques. We note that highly purified enzyme preparations are not always necessary in synthetic applications since contaminating enzymes may have no effect on the reactants and products present in the reaction mixture. If an enzyme to be used in synthesis has intrinsically low specific activity (units of catalytic activity per weight of protein), crude preparations can cause practical problems by requiring large volumes of immobilization medium and correspondingly large reactor volumes. The use of microbial cells in enzyme-catalyzed synthesis represents a limiting case in purification; since no purification is involved, their manipulation is straightforward. The activity of these preparations may be low. In favorable cases, however, they represent

ANNUAL REPORTS IN MEDICINAL CHEMISTRY- 19

Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any forni reserved. ISBN 0- 12-040519-5,

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the simplest basis for enzyme-catalyzed reactions, and several successful industrial processes have been developed using immobilized whole cells. 12 Enzyme Immobilization - Enzymes used in synthetic applications are commonly immobilized in or on insoluble materials because immobilization enhances their stability and allows their recovery and reuse. While bench-scale experiments are most conveniently carried out as batch processes, industrial processes often depend on long-lived immobilized enzymes in continuous processes. Many immobilization methods have been developed. 12-14 Only a few of these techniques deserve explicit comment. Glutaraldehyde is the most commonly used bifunctional reagent in immobilization,l2 forming covalent linkages of still incompletely defined nature between amino groups. It is used to bind enzymes to solid supports or cross-link enzymes adsorbed on a support, and to cross-link enzymes with carrier proteins or with themselves to form insoluble aggregates. Immobilization procedures for industrial applications based on functionalized ceramics cross-linked with enzyme via glutaraldehyde have been developed. 1 4 , l5 Wood et al. have developed an immobilization procedure using Whitesides et al. have developed an polyurethane-based membranes. l6 immobilization method based on polyacrylamide-E-N-acryloxysuccinimide cross-linked with triethylene tetramine in the presence of enzyme. 17 This method is particularly useful with the relatively delicate enzymes useful in‘ organic synthesis. Kula et al. have used membrane reactors containing soluble enzymes. l8 The enzymes are not immobilized, but reactor performance has been good.

-

Enzyme Stabilization Enzyme stabilization is a concern before, during, and after immobilization. Immobilization often greatly increases the stability of enzyme^.^'-^^ Reasons for this stabilization are not clearly understood. Enzyme deactivation during the immobilization process is often troublesome. Addition of substrates or inhibitors of the enzyme during immobilization helps to occupy and protect the active site, and increases yields on immobi1i~ation.l~ A number of strategies are useful in maintaining activity in soluble and immobilized enzymes during use. 23-26 Thiol reagents (dithiothreitol, D-mercaptoethanol, 1,3-dithiopropan-2-01) maintain the reduced state of catalytically essential thiols in the enzyme. Chelating agents inhibit metal ion catalyzed oxidations of en~ymes.~’ The stability of soluble enzymes can be enhanced by the addition of 01 ols, salts, and certain polymers, and by chemical modification. 23,2zp-3H Thermally inactivated immobilized enzymes have been reactivated by thiol reagents and reversible heat treatment. 33

-

Cofactor Regeneration About 70% of enzymes use nucleoside triphosphates, nicotinamide derivatives [NAD(P)(H)], or CoA as cofactors. These enzymes include many of those of greatest interest in the synthesis of fine chemicals. Since these cofactors are too expensive to be used stoichiometrically, it has been necessary to develop recycling systems for them. The problem of recycling the nucleoside triphosphates

Chap. 26

Enzymic Methods

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265

is essentially solved at the level of laboratory-scale synthesis,34-38 and that of recycling NAD derivatives is well-advanced toward solution. 39-46 None of these schemes has been tested on a production scale, although they work satisfactorily for syntheses of several moles of products. Recycling of ATP from ADP or AMP rests on the development of practical syntheses of the phosphate donors acetyl phosphate37 and phosphoen~lpyruvate~~ to be used with the enzymes acetate kinase and pyruvate kinase, respectively. Acetate kinase is also applicable to recycling of GTP, UTP, CTP and the corresponding 2’-deoxy-nucleoside triphosphates.’ ,34 Regeneration of these species from nucleoside monophosphates is not truly practical since adenylate kinase is specific for AMP, but relatively few reactions generate nucleoside monophosphate. CoA recycling has not been explored. Recycling of S-adenosyl-l-methionine, a cofactor in enzyme-catalyzed transmethylations, is currently difficult.47 The conversion of reduced nicotinamide cofactors [NAD(P)H] to the oxidized form [NAD(P)], developed by Jones et al., is the most widely used procedure for this transformation, but suffers from the need for large amounts of flavin and from slow reaction rates.39 Oxidative regeneration based on the conversion of a-ketoglutarate to lutamic acid works well and does not require the presence of oxygen. The best system for reductive regeneration of NADH from NAD is based on formate dehydrogenase. 18,46*48 This procedure works very well, although the enzyme does not accept NADP as a substrate and is relatively expensive. The most practical procedure for regeneration of NADPH uses glucose-6-phosphate dehydrogenase. 42

“

Equilibrium Manipulation - Enzymes are catalysts and therefore serve only to accelerate attainment of equilibrium. In many instances the equilibrium constant for a given reaction in water does not adequately favor the desired product or water acts as an undesired reactant. Sometimes product can be favored using excess starting material, or by reacting the product in an irreversible manner.49 Occasionally precipitation of product will drive an unfavorable reaction, but precipitation can foul immobilized enzymes. Kinetic control of a reaction can sometimes be used to maximize yields.49 In cases where less polar materials are being manipulated, water miscible organic cosolvents have been used, but this technique can reduce enzyme activity. 50-53 Careful selection of cosolvent can, however, maintain enzyme activity and dramatically shift equilibrium.54 A more general approach for working with less polar compounds is the use of a two-phase system incorporating a waterimmiscible organic solvent. This approach has been discussed extensively by Martinek and coworkers. 55-59 Enzyme Mediated Processes

-

Simple Hydrolases and Isomerases These are enzymes requiring no added cofactors, and which catalyze hydrolyses, isomerizations, some condensations, and related reactions. Such enzymes are among the simplest to use and are the most widely used in industry (Table 1). A valuable introduction to processes using these enzymes has been published. 12

266 -

Sect. VI Table 1.

- Topics in Chemistry and Drug Design

Allen, Ed.

Selected Industrial Applications of Enzymes.

-

ref.

6-aminopenicillanic acid penicillin-G fumaric acid -L-aspartic acid ~ L - m a l i acid c fumaric acid starch glucose glucose fructose N-acetyl-D,L-amino acids L-amino acids L-arginine L-citrulline

12,60,61 12,62 12,63 30,64 30,65,66 12 12

Amidases - Acylase is used in continuous production of L-amino It has been used to resolve D,L-phenylalanine6' and a-formylacids." e -acyl-D ,L-lysine. Penicillins have been synthesized from 6-aminopenicillanic acid with penicillinase. 69

-

Esterases Esterases from several sources have been used in stereoselective and regioselective hydrolysis of esters in the preparation of chiral esters, acids, and alcohols. Sih and coworkers have developed a valuable theoretical treatment for quantitative analysis of such biochemical kinetic resolutions relating the extent of conversion (c) of racemic substrate, the optical purity (ee) of the starting material and products, and the enantiomeric selectivity of the enzyme.70 Recycling of enantiomerically enriched substrate can greatly increase ee. Racemic threo ester L w a s hydrolyzed with pig liver esterase (PLE) to (-)-acid 1 (64% ee). Reesterification and incubation to 80% c resulted in 1_ with >90% ee. Erythro ester 2 was treated with Gliocladium roseum to hydrolyze the 2&,32isomer. At 70% c the remaining ester had 95% ee.

/l\rC02R OH

(4 I , R=CH, 1-1 2 , R = H

/+

(4 3

CO,CH,

OH

PLE was used in the enantiotopically selective hydrolysis of diethyl-@. Reduction, derivatization and methylglutarate to the half-ester 2 resolution raised the ee of 2 from 69% to 91%. 70

R02C

C02Et

4,RzEt; 5 , R = H

6(69 %eel

6 (91% eel

Substituent effects have been studied in PLE-catalyzed hydrolysis of a range of symmetrical dicarboxylic acids and a model has been developed to determine absolute configuration in the monoester product^.^' Esterases have successfully resolved compounds 7-2. Imidazolone Dimeth 1-2,4-dimethylglutarates 2 and r~seum.~' Dimethyl-b-aminoglutarate 2 was used as starting material for syntheses of &- and S=4-[(methoxycarbonyl)methy1]-2-azetidinone~,~~useful nuclei for carbapenem p-lactam antibiotic synthesis. &-Esters of 3-hydroxy-3methylalkanoic acids 11

-7 was converted to (+)-biotin.72 -9 were resolved with PLE and 5.

Chap. 26

Enzymic Methods

Findeis Whitesides

267

were prepared for testing as inhibitors of 3-hydroxy-3lnethylglutarylCoA reductase and in compactin analogue syntheses.75 Schneider and coworkers have used PLE-catalyzed hydrolyses in asymmetric syntheses of (1R 3&)-chrysanthemic 12,permethrinic 13 and caronic acid 1 4 derivatives,7 6 and in syntheses of disubstituted monoalkyl malonates7/nd kane dicarboxylate monomethyl esters. 78 0

yy

'

PhbNKNAPh

MeO,C

H++i nPrOzC

Me0,C

C0,Me

8

C0,nPr

CO,Me

1 2-cycloal-

i;'

Me0,C

Cope

10

9

7 Me OH

R

&Cop II

R'

12, R = H , R ' s CH=CMe2 13, R = H, R'= CH=CCI, 14, R = Me, R'. CO,H

6% cOzR

Cambou and Klibanov7' have used transesterifications catalyzed by PLE and yeast lipase to resolve a variety of alcohols with great effectiveness. Their novel approach employed a biphasic system of aqueous enzyme solution absorbed in a porous solid phase placed in a mixture of "matrix ester'' (methyl propionate or tributyrin) and racemic alcohol (Scheme I). 0 Scheme I. 0 &O-(-)-R chiral ester t esterase

matrix ester

r a cemic alcohol

chiral alcohol

+ Me OH

The low water content of this system simplified subsequent purification and effectively suppressed ester hydrolysis. Lipases have also been applied to resolutions of chloroglycerol derivatives,80181 3-acetylthiocycloheptene,82 and 2-chloromethyl-1-propyl propionate .83 Cholinesterases have been used to resolve D,L-carnitine. 84 Proteases ~-

-

Proteases have been applied to several synthetic purposes the most important of which are peptide bond synthesis and protein semisynthesis. Recent extensive reviews cover this area.85,86 Proteases have been used in ester synthesis87 and resolution.88 Semisynthesis of human insulin has been achieved by enzymic removal and replacement of one amino acid in porcine i n ~ u l i n . ~ ~ , ~ 'All peptide bonds in the N-terminal octapeptide of dynorphin [H-Tyr-Gly-Gly-Phe-LeuArg-Arg-Ile-OH] have been formed using proteases. 91 A precursor of aspartame has been made by themolysin-catalyzed condensation of benzyloxycarbonyl-L-aspartic acid and L-phenylalanine methyl ester. 92 Aldolases - Aldolases catalyze reversible aldol condensations of sugars.y' A well-studied enzyme is fructose-lY6-diphosphate aldolase from rabbit muscle. This enzyme exhibits a high specificity for dihydroxyacetone phosphate as the nucleophile, but tolerates a range of aldehydes as electrophiles (Scheme 11) .94 This broad specificity allows synthesis of sugars such as 6-deoxyfructoseg5 and isotopically labeled glucose

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Allen, Ed.

derivatives.94 Other aldolases exist with different substrate specificities for possible application to preparative sugar synthesis. Scheme 11.

Other - Farnesylpyrophos hate synthetase has been used in asymmetric synthesis of isoprenoids.g6 Potato acid phosphatase has been applied to mild hydrolysis of polyprenyl pyrophosphates. 97 Sulfatase-catalyzed hydrolysis of &naptho1 sulfate has been used to separate a- and B-napthol~.~~ NADg9 and flavin adenine dinucleotide“’ have been made by enzymic coupling reactions. Cofactor Requiring Enzymes Phosphorylation - Enzymic phosphorylation with coupled in situ ATP regeneration has been used to prepare g l u c o s e - 6 - p h o s p h a t e ~ ~ - g l y cerol-3-phosphate,lo* creatine-phosphate,lo3 and 5-phosphoribosyl-lpyrophosphate. 104

-

Chiral Redox Chemistry Nicotinamide cofactor dependent oxidoreductases have been applied in chiral synthesis. Enantioselectivity is often only moderate, but in certain cases excellent results have been obtained. Horse liver alcohol dehydrogenase (HLADH) is the most widely explored enzyme for these purposes with most of the work in this area having been done by Jones and coworkers. lo5 HLADH has been studied sufficiently Cis-3-( 2-hydroxythoroughly to allow modeling of the active site. ethy1)cyclopentanol 2 was oxidized by HLADH to (+)35-(2-hydroxyethyl) cyclopentanone 16with 97% ee; the remaining diol had 70% ee.lo7

-

2-Substituted tetrahydropyran-4-ones were reduced to (-)trans alcohols with 100% ee. lo8 Remaining (+)ketones had 51-86% ee. HLADH-catalyzed oxidation of monocyclic mesodiols x p r o c e e d s to bicyclic lactones g i n

high yield with complete stereotopic selectivity. log Essentially no selectivity is seen in oxidation of the trans diols. =-decalin-2,719 is reduced specifically to (-)(7S-,92,10&)-7-hydroxy-&-decdione alin-2-one 20llo*lll which can be converted to (+)4g-twistanone 21 in 51% yield from the dione. D- and L-lactic dehydrogenases reduce chloropyruvic acid Z t o D- and L-chforolactic acids 23; these can be

Enzymic Methods

Chap. 26

3

H

H

0

Findeis, Whitesides

OH

converted to epoxyacrylic acids 24. 'I2 Progesterone has been reduced to ZO-B-hydroxy-pregn-4-ene-3-one with 20-P-hydroxysteroid dehydrogenase using reversed micelles in organic solvent and H2 as ultimate reductant Microbial reductions have been used in intermediate steps in syntheses of natural brefeldin-A, ' 1 4 (+)compactin 'I5 and L-carniL-leucine dehydrogenase has been used in the reductive tine amination of a-ketoisocaproate to L-leucine18 in a membrane reactor. NAD was covalently modified by attachment of polyethylene glycol to make it unable to cross the membrane.

. .

Multi-Enzyme Cofactor Requiring Processes - A more complex level of applied enzymology is reached in the use of multi-enzyme schemes to synthesize complex molecules. Examples of such syntheses include ribu10se-l,5-diphosphate,~~ im ortant in the study of ribulose-diphosphate carboxylase; lacto~amine,'~~ from the first use of the Leloir pathway enzymes in synthesis; and S-adenosyl-L-methionine. 47 Oxidations - Enzymes which functionalize inactivated carbon are often difficult to obtain and handle. Many examples exist of oxidations using microbial e.g. for steroids, and recently in olefin oxidation. Such preparative transformations have not been achieved with purified enzymes and are unlikely to be amenable to large-scale 2 vitro approaches because of instability and complexity of the enzyme systems. Klibanov and coworkers, however, have developed systems with horseradish peroxida~e'~~,~~' and xanthine oxidaset2' for oxidation of aromatic alcohols and amines, for use in syntheses and waste water treatment. Hydroxyphenyl compounds can be oxidized to dihydroxy derivatives. L-DOPA has been made from L-tyrosine in this manner. l Z 3 c clohexanone was oxidized to t-caprolactone with a bacterial oxygenase. 1 2 4 The Future - Enzymic synthetic methods will see increased use in research and industry. Numerous examples exist of preparations of useful quantities of chiral compounds for use in synthesis, and the use of hydrolytic enzymes for simple synthesis. More importantly, enzymes will allow the facile synthesis of complex molecules important in biological research. Immunology, neurobiology, endocrinology, molecular genetics, membrane biology, and plant and insect biology are areas becoming more molecular in scope. Research in such fields will increasingly depend on biologically active compounds not readily accessible by more conventional chemistry. Molecules that are water soluble, or highly functional-

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ized such as carbohydrates, nucleic acids, lipids, and proteins may prove available by enzymic synthetic methods. Enzymology will be useful in modifications of poly- and oligosaccharides and proteins. Enzymes will also see growth in applications in medical diagnostics and treatment, and in food chemistry. 125 Recombinant DNA and RNA methods rely on enzymes, and as these methods are developed,the op ortunity for enzyme engineering of synthetic catalysts will grow. 126*p27 Enzyme-based synthetic methods will be an important part of future organic synthesis, especially in synthesis of new pharmaceutical products. Acknowledgement - We acknowledge financial support from the National Institutes of Health, Grant GM 30367. 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.

G.M. Whitesides and C.-H. Wong, Aldrichimica Acta, g, 27 (1983). J.B. Jones in "Asymmetric Synthesis," J.D. Morrison, Ed., Academic Press, New York, N.Y., 1983. C.J. Suckling and H.C.S. Wood, Chem. Brit., 2,243 (1979). J.B. Jones, C.J. Sih and D. Perlman, Eds., "Applications of Biochemical Systems in Organic Chemistry," Tech. Chem., lo,Wiley, New York, N.Y., 1976. J.B. Jones, Method. Enzymol., 831 (1976). C.J. Sih, E. Abushanab and J.B. Jones, Annu. Repts. Med. Chem., 12, 298 (1977). International Union of Biochemistry. Nomenclature Committee. "Fzyme Nomenclature, 1978," Academic Press, New York, N.Y. (1979). B.S. Hartley, T. Atkinson and M.D. Lilly, Eds., Phil. Trans. R. SOC. Lond., 237 (1983). M.R. Kula, K.H. Kroner and H. Hustedt. Adv. Biochem. Eng., 2, 73 (1982). P.-A. Albertsson, Adv. Protein Chem., 2, 309 (1970). P.-A. Albertsson, "Partition of Cell Particles and Macromolecules," 2nd ed., 1971. Almquist and Wiksell, Stockholm, Wiley-Interscience, N.Y., I. Chibata, "Immobilized Enzymes--Research and Development." Halsted Press, New York, N.Y., 1978. K. Mosbach, Ed., "Immobilized Enzymes," Method. Enzymol., 4ft (1976). A.M. Klibanov, Science, 219, 722 (1983). R.P. Rohrbach, U.S. 4,268,419 (1979). L.L. Wood, F.J. Hartdegen, P.A. Hahn, U.S. 4,312,946 (1982). A. Pollak, H. Blumenfeld, M. Wax, R.L. Baughn and G.M. Whitesides, J. Am. Chem. SOC., 102, 6324 (1980). C. Wandrey, A.F. Buclanann and M.R. Kula, Biotechnol. Bioeng., 2,2789 (1981). A.M. Klibanov, Anal. Biochem., 1 (1979). K. Martinek, A.M. Klibanov, V.S. Goldmacher and I.V. Berezin, Biochim. Biophys. Acta, 485, 1 (1977). K. Martinek, A.M. Klibanov, V.S. Goldmacher, A.V. Tchernysheva, V.V. Mozhaev, I.V. Berezin and B.O. Glotov, Biochim. Biophys. Acta, 485, 13 (1977). A.M. Klibanov, N.O. Kaplan and M.D. Kamen, Arch. Biochem. Biophys., 199, 545 (1980). R.D. Schmid, Adv. Biochem. Eng., &, 41 (1979). A.M. Klibanov, Biochem. SOC. Trans., 2. 19 (1983). A.M. Klibanov, N.O. Kaplan and M.D. Kamen, Enzyme Eng., 2, 135 (1980). A.M. Klibanov, N.O. Kaplan and M.D. Kamen, Proc. Nat. Acad. Sci. U.S.A., 15, 3640 (1978). A.M. Klibanov, N.O. Kaplan and M.D. Kamen, Biochim. Biophys. Acta, 547, 411 (1979). J.B. Jones and D.R. Dodds, Can. J. Chem., 2533 (1979). S. Branner-Jdrgensen, Biochem. SOC. Trans., 2, 20 (1983). C. Bucke, Biochem. SOC. Trans., &, 13 (1983). K. Gekko and S.N. Timasheff, Biochem., 2,4667; 4677 (1981). T. Arakawa and S.N. Timasheff, Biochem., 2, 6536; 6545 (1982). A.M. Klibanov and V.V. Mozhaev, Biochem. Biophys. Res. Comm., 2, 1012 (1978). C.-H. Wong, S.L. Haynie and G.M. Whitesides, J. Am. Chem. SOC., 105, 115 (1983). B.L. Hirschbein, F.P. Mazenod and G.M. Whitesides, J. Org. Chem., 2, 3765 (1982). C.-H. Wong, A. Pollak, S.D. McCurry, 'J.M. Sue, J.R. Knowles and G.M. Whitesides, Method. Enzymol., g,I08 (1982). D. Crans and G.M. Whitesides, J. Org. Chem., 3130 (1983). R.L. Baugh, 0. Adalsteinsson, and G.M. Whitesides, J. Am Chem. SOC., 100, 304 (1978).

*,

a,

z,

x,

z,

Chap. 26 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. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92.

Enzymic Methods

F i n d e i s , Whitesides

271

J.B. Jones and K.E. Taylor, Can. J. Chem., 15, 2969 (1976). C.-H. Wong and G.M. Whitesides, J. Am. Chem. S O C . , 104,3542 (1982). C.-H. Wong and G.M. Whitesides, J. Org. Chem., 2816 (1982). C.-H. Wong and G.M. Whitesides, J. Am. Chem. SOC., 103,4890 (1981). C. Burstein, H. Ounissi, M.D. Legoy, G. Gellf and D. Thomas, Appl. Biochem. Biotechnol., L, 329 (1981). E. Chave, E. Adamowicz and C. Burstein, Appl. Biochem. Biotechnol., L. 431 (1982). C.-H. Wong and G.M. Whitesides, J. Am. Chem. SOC., 105,5012 (1983). Z. Shaked and G.M. Whitesides, J. Am. Chem. SOC., 102, 7104 (1980). A. Gross, S. Geresh and G.M. Whitesides, Appl. Biochem. Biotechnol., g, 415 (1982). H. Schutte, M.R. Kula and H. Sahm, Method. Enzymol., 2,527 (1982). K. Martinek and A.N. Semenov, J. Appl. Biochem., 2, 93 (1981). L.G. Butler, Enzyme Microb. Technol., L, 253 (1979). J.B. Jones and H.M. Schwartz, Can. J. Chem., g,335 (1982). J.B. Jones and D.H. Pliura, Can. J. Chem., 2, 2921 (1981). J.B. Jones and M.M. Mehes, Can. J. Chem., 2245 (1979). G.A. Homandberg, J.A. Mattis and M. Laskowski, Jr., Biochem., 17,5220 (1978). K. Martinek, A.N. Semenov and I.V. Berezin, Biochim. Biophys. Acta, 658, 76 (1981). K. Martinek and A.N. Semenov, Biochim. Biophys. Acta, 658, 90 : ) 1 8 9 ( K. Martinek, A.N. Semenov and I.V. Berezin, Dokl. Akad. Nauk. SSSR, 253, 358 (1980). K. Martinek, A.N. Semenov and I.V. Berezin, Dokl. Akad. Nauk. SSSR, 252, 394 (1980). A.M. Klibanov, G.P. Samokhin, K. Martinek and I.V. Berezin, Biotechnol. Bioeng., E, 1351 (1977). B.J. Abbott, Adv. Appl. Microbiol., 20, 203 (1976). E. Lagerlof, L. Nathorst-Westfelt, B. Ekstrom and B. Sjoberg, Method. Enzpol., 3, 759 (1976). I. Chibata, T. Tosa and T. Sato, Method. Enzymol., 739 (1976). I. Chibata, T. Tosa and I. Takata, Trends. Biotechnol., 1 ,9 (1983). M.K. Weibel, W.H. McMullen and C.A. Starace, Dev. Ind. Microbiol., 19, 103 (1977). N.B. Havewala and W.H. Pitcher, Jr., Enzyme Eng., L, 315 (1974). N.H. Mermelstein, Food Technol., 2,20 (1975). T. Kitahara and S. Asai, Agric. Biol. Chem., 7, 991 (1983). Y. Minematsu. Y. Shimohigashi, M. Waki and N. Izumiya, Bull. Chem. SOC. Jap., 1899 (1978). A.N. Semenov, K. Martinek, V. Yu.-K. Shvyadas. A.L. Margolin and I.V. Berezin, Dokl. Akad. Nauk. SSSR, 258, 1124 (1981). C.-S. Chen, Y. Fujimoto, G. Girdaukas and C.J. Sih, J. Am. Chem. SOC., 104 7294 (1982). P. Mohr. N. Waesue-Sarcevic, C. Tamm, K. Gawronska and J.K. Gawronska, Helv. Chim. Acta, 2501 (i983). S . Iriuchijima, K. Hasegawa and G. Tsuchihashi, Agric. Biol. Chem., 66, 1907 ( 982). C.-S. Chen, Y . Fujimoto, C.J. Sih, J. Am. Chem. SOC., 103,3580 (1981). M. Ohno, S. Kobayashi, T. Iimori, Y.-F. Wang and T. Izawa, J. Am. Chem. SOC., 103. 2405 (1981). W.K. Wilson, S.B. Baca, Y.J. Barber, T.J. Scallen and C.J. Morrow, J. Org. Chem., 48, 3960 (1983). M. Schneider, N. Engel, and H. Boensmann, Angew. Chem. Int. Ed. Engl., G,64 (1984). M. Schneider, N. Engel, and H. Boensmann, Angew. Chem. Int. Ed. Engl., 2, 66 (1984). M. Schneider, N. Engel, P. Honicke, G. Heineman and H. Gorisch, Angew. Chem. Int. Ed. Engl., 2, 67 (1984). B. Cambou and A.M. Klibanov, J. Am. Chem. SOC., 106,in press (1984). S . Iriuchijima and N. Kojima, Agric. Biol. Chem., 1153 (1982). 1593 (1982). S . Iriuchijima, A. Keiyu and N. Kojima, Agric. B i o l . Chem., 3, S. Iriuchijima and N. Kojima, J.C.S. Chem. Corn., 185 (1981). J. Lavayre, J. Verrier and J. Baratti, Biotechnol. Bioeng., 24 2175 (1982). E.P. Dropsy and A.M. Klibanov, Biotechnol. Bioeng. 2, in press (1984). J.S. Fruton, Adv. Enzymol., 2, 239 (1982). I.M. Chaiken, A. Komoriya, M. Ohno and F. Widmer, Appl. Biochem. Biotechnol. 1,385 (1982). D. Tarquis, P. Monsan and C. Durand, Bull. SOC. Chim. Fr. II., 76 (1980). Y. Nishida, H. Ohrui and H. Meguro, Agric. Biol. Chem., 7, 2123 (1983). K. MOKihaKa, T. Oka, H. Tsuzuki, Y. Tochino and T. Kanaya, Biochem. Biophys. Res. Comn., 396 (1980). K. Inouye, K. Watanabe, K. Morihara, Y. Tochino, T. Kanaya, J. Emura and S . Sakakibara, J. Am. Chem. SOC., 101,751 (1979). W. Kullman, J. Org. Chem., 67,5300 (1982). K. Oyama, S. Nishimura, Y. Nonaka, K. Kihara and T. Hashimoto, J. Org. Chem., 5, 5241 (1981).

x,

x,

c,

x,

&,

s,

z,

272 93. 94. 95. 96. 97. 98. 99. 100.

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- Topics

i n Chemistry and Drug Design

P.D. Boyer, Ed., "The Enzymes," 7-, Chapters 6-9 (1972). C.-H. Wong and G.M. Whitesides, J. Org. Chem., 2, 3199 (1983). C.-H. Wong, F.P. Mazenod and G.M. Whitesides, J. Org. Chem. 3493 (1983). T. Koyama, A. Saito, K. Ogura and S. Seto, J. Am. Chem. Soc., 102, 3614 (1980). H. F u j i i , T. Koyama, K. Ogura, Biochem. Biophys. Acta, 712. 716 (1982). G. Pelsy, A.M. Klibanov, Biotechnol. Bioeng., 3. 919 (1983). D.R. Walt, M.A. Findeis. V.M. Rios-Mercadillo, J. Auge and G.M. Whitesides, J. Am. Chen. SOC., 234 (1984). S. Shimizu, K. Yamane, Y. Tan1 and H. Yamada, Appl. Biochem, Biotechnol., 237

ss

106,

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(1983). 101. 102. 103. 104. 105. 106. 107. 108. 109.

A l l e n , Ed.

z,

A. Pollak, R.L. Baughn and G.M. Whitesides, J. Am. Chem. SOC., 2366 (1977). V.M. Rios-Mercadillo and G.M. Whitesides, J. Am. Chem. SOC., 5828 (1979). Y.-S. Shih and G.M. Whitesides, J. Org. Chem., 2, 4165 (1977). A. Gross, 0. Abril, J.M. Lewis, S. Geresh and G.M. Whitesides, J. Am. Chem. SOC.,

101,

105, 7428 (1983). T. Takemura and J.B. J.B. A.J. J.A.

Jones, J. Org. Chem., 5. 791 (1983). and previous papers. Jakovac, Can. J. Chem., 9 19 (1982). Jones, J. Am. Chem. SOC., 1625 (1977). Haslegrave and J.B. Jones, J. Am. Chem. SOC., 106. 4666 (1982). Jacovac, H.B. Goodbrand, K.P. Lok and J.B. Jones, J. Am. Chem. SOC., 106, 4659 Jones and I.J. Irwin and J.B.

z,

I.J. (1982).

110. D.R. Dodds and J.B. Jones, J.C.S. Chem. C m . , 1080 (1982). 111. M. Nakazaki, 8. Chikamatsu and M. Taniguchi, Chem. L e t t . , J l 1761 (1982). 112. B.L. Hirschbein and G.M. Whitesides, J. Am. Chem. SOC., 104,4458 (1982). 113. R. Hilhorst, C. Laane and C. Veeger, FEBS Lett., 159, 225 (1983). 114. C. LeDrian and A.E. Greene, J. Am. Chem. SOC., 2,5473 (1982). 115. C.T. Hsu, N.Y. Wang, L.H. Latimer and C.J. Sih, J. Am. Chem. SOC., 105, 593 (1983). 116. B. Zhou, A.S. Gopalan, F. Vanmiddlesworth, W.-R. Shieh and C.J. Sih, J. Am. Chem. SOC., 105,5925 (1983). 117. C.-H. Wong, S.L. Haynie and G.M. Whitesides, J. Org. Chem., 5416 (1982). 118. K. Kieslich, "Microbial Transformations of Non-Steroid Cyclic Compounds," John Wiley h Sons, New York, N.Y., 1976. Chem. Corn., 849 (1978). 119. H. Ohta and H. Tetsukawa, J.C.S. 120. A.M. Klibanov, B.N. A l b e r t i , E.D. Morris and L.M. Felshin, J. Appl. Biochem., 1,414 (1980). 121. A.M. Klibanov and E.D. Morris, Enzyme Microb. Technol., 2, 119 (1981). 122. G. Pelsy and A.M. Klibanov, Biochim. Biophys. Acta, 762, 352 (1983). 123. A.M. Klibanov, 2. Berman and B.N. A l b e r t i , J. Am. Chem. SOC., 103,6263 (1981). 124. J.M. Schwab, J. Am. Chem. SOC., 103,1876 (1981). 125. H. Samejima, K. Kimura and Y. Ado, Biochemie, g , 299 (1980). 126. W.H. Rastetter, Appl. Blochem. Biotechnol., 3 423 (1983). 127. T.H. Maugh, 11, Science, 223, 154 (1984).

zs

27 3 -

Chapter 27.

Stable Isotopes in Drug Metabolism and Disposition

Thomas A . Baillie and Albert W. Rettenmeier, Department of Medicinal Chemistry, University of Washington, Seattle, WA 93195 Lisa A . Peterson and Neal Castagnoli, Jr., Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 Since the 1977 review in Annual Reports,' the applications of stableisotope-labeled compounds to biomedical research have increased dramatically. Three international conferences devoted to the use of stable isotopes in the life and one to synthesis and general applicat i o n ~ have ~ been held. Review articles have focused on mass spectrometric-based analytical methods, 13'' on applications of stable isotopes in clinical8-'' and pharmacological research , "-' and on more specific studies concerning groblems in drug metabolism, l r '15 pharrnacokinetics16' and synthesis. "' The present review covers mainly the literature published since 1981 and focuses on recent developments in the use of stable isotopes for investigations of drug metabolism and disposition.

'

Metabolite Structure Elucidation Isotope Clusters - The isotope cluster (isotope doublet, twin ion) technique has proved to be a powerful method for the detection and structural identification of drug metabolites in complex biological matrices. An equimolar mixture of [8-13C;1,3-15N~]caffeineand unlabeled caffeine was used to establish that the acetyl group of the metabolite, 4-acetyl-6amino-3-methyluracil, did not contain the original C-8 carbon atom since it was labeled with two 15N atoms only." In this and a related studyYZL [ 2-14C]caffeine was co-administered to aid in monitoring the excretion of drug metabolites. Although a useful adjunct to the stable isotope cluster technique, the administration of radiotracers in human research is limited on ethical grounds, particularly in pediatric and obstetric populations. Studies on the biotransformation of theophylline in premature newbornsz2 and on the placental transfer and fetal metabolism of this compound in humansz3 relied exclusively on the use of [ 2-1 3C;1,3-15N2 Itheophylline as tracer. The stable isotopes of carbon and nitrogen also have been successfully applied to ion cluster metabolism studies of [ 13C, "N~]hexobarbital. " Although the use of deuterium in drug metabolism involving the isotope cluster technique may be complicated by isotope effects,2 5 both cost and convenience have led most workers t o rely on this isotope. Recent examphencyclidine, ples include reports on arninopyrine ,z 6 pencycuron , fentanyl, 3 0 and diethylstilbestrol. 3 1 In the case of steroids and their derivatives, ion cluster studies have been performed exclusively with deuterium-labeled substrates. Examples include investigations with [ 'H5 Iethynylestradiol,3 z [ 'H2 ]4-hydroxyandrost-4-ene-3, 17-dione, [ 'H2 ]ursodeoxycholic acid, 3 y and [ 'H~lbudesonide.3 5

''

ANNUAL REPORTS IN MEDICINAL CHEMISTRY-19

''"'

Copyright 0 1984 by Academic Prebs, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040519-9

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Allen, Ed.

The isotope cluster technique has proved valuable in the identification of metabolites similar or identical to endogenous compounds present in the biological extracts. Use of a hexadeutero variant of valproic acid, a short-chain fatty acid, led to the identification of a new biotransformation product, 2-g-propyl-4-oxopentanoic acid. 3 6 A similar approach was used to reveal a novel pathway of benzoic acid metabolism in the horse. 3 7 - 3 9 Metabolites of rutin, a flavinol glycoside, were identified in the urine of rats" and humans'l given [2',5',6'-2H3]rutin. Deuteriumlabeled phenylephrineW2 has been used in a similar fashion. Derivatization of samples with an equimolar mixture of labeled and unlabeled reagents to produce ion clusters has been applied recently to the characterization of the bleomycins by FD and FAB mass ~pectrometry.'~ A variation of this technique utilizes a mixture of labeled and unlabeled reagent gases { pyridine/[ 2Hs]pyridine ," trimethylchlorosilane (TMCS)/ [ 2H9]TMCS, tetramethylsilane (TMS)/[ 2H12 ]TMS," and NH3/N2H3" ' r 7 1 in conjunction with CI mass spectral analyses. 'lhe isotope cluster technique also may be exploited with the newer ionization methods of SIMS48 and FAB" through the addition of appropriate counter-ions (for example ,

''

Ag'"),

Work continues on the development of computer software for automatic screening of large amounts of mass spectral data for specified isotope clusters. O ' ' The Shift Technique - The stable isotope shift technique refers to a procedure in which the mass spectrum of an unlabeled compound is compared with that of a stable-isotope-labeled counterpart. The observed differences in masses between corresponding ions in the two spectra provide valuable information on ion composition and hence on molecular structure. This technique may be used in two distinct ways. The drug may be labeled at a specific site(s) and the mass spectra of metabolites derived from the labeled compound compared with those obtained from the unlabeled drug. Recent examples of this application include reports on the metabolism of oxybutynin,5 2 tocainide, and ketamine. 54 Alternatively , a heavy isotope (usually 'H) is introduced into the metabolite of interest by reaction with a labeled derivatizing reagent. For example, perdeuteromethylation results in a mass difference of 3 daltons between labeled and unlabeled derivatives per methyl group. Structures of metabolites obtained from caffeine" and phenytoin5' have been confirmed by this technique. Tri(deuteromethy1)silylation has been employed in the identification of metabolites of carbama~epine~'and afloqualone.

- The applications of stable isotopes coupled with NMR analysis have multiplied as higher field magnets and sophisticated computer techniques have increased sensitivity. A recent review which discussed NMR studies employing stable isotopes is available. 5 9 NMR Studies

Natural abundance 13C-NMR spectroscopy has been applied to the structure analysis of several metabolites derived from acetaminophen." The structures of metabolites of prenaltero16 and trans-sobrerol" have been studied. 13C-NMR analyses of metabolites derived from I3C-enriched amitriptyline,6 3 BHT," propachl~r'~ and aminopyrine66'67 have been reported.

Chap. 27

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Bailie et al. 275

Mechanistic Studies Cytochrome P-450 Catalyzed Reactions - Studies with "02 have established that the cvtochrome P-450 mediated hvdroxvlation of camDhor bv the bacterial enzymk68 and the enzyme purified from rat liver6' reshts in the incorporation of atmospheric oxygen in the 5-*-hydroxylation product. Cumene [ "02]hydroperoxide will transfer its peripheral oxygen atom to a variety of compounds which serve as substrates for mammalian cytochrome P450." AS expected, " 0 2 served as the oxygen source for the bacterial cytochrome P-450 catalyzed epoxidation of 5,6-dehydrocamphor .7 1 N-Hydroxymethylcarbazole, formed by the cytochrome P-450 catalyzed oxidation of Nmethylcarbazole, incorporates " 0 exclusively from dioxygen. 7 z ' Under anaerobic conditions cytochrome P-450 may catalyze the intramolecular transfer of oxygen present in tertiary amine N-oxides. a Mechanistic studies on S-dealkylation and S-oxidation reactions also have used " 0 tracer methods.7' The oxidation of 1,2-dideuterocyclohexene by both cytochrome P-450 and model chemical systems gave cyclohexenol with various degrees of allylic rearrangement of the deuterium atoms, suggesting a mechanism involving caged radical intermediates. The bioactivation of terminal olefins by cytochrome P-450 leads to the alkylation of the heme prosthetic group. Mechanistic studies using trans-1-[ 'Hloctene as substrate demonstrated that the trans-stereochemistry is retained in both the heme alkylation product and the epoxide. This result is inconsistent with heme alkylation occurring by reaction with octene oxide itself. 76 The structures of heme adducts produced from several olefins now have been determined and it has been established, using oxygen-18, that the adducts contain an atom of oxygen derived from the atmosphere." Mechanistic studies with deuterium-labeled arenes have provided evidence against a proton abstraction or direct insertion mechanism for the hydroxylation of certain benzenoid systems at the meta p~sition.~' Finally, "N-labeling has been employed in an investigation of the process through which metabolism of 3,5-diethoxycarbonyl-l,4-dihydrocollidine by cytochrome P-450 leads to self-destruction of the enzyme and formation of N-methylprotoporphyrin IX. 7 9 Metabolic Pathways - Compounds labeled at specific sites with stable isotopes have been employed in the identification of short-lived, potentially toxic metabolites. The metabolic formation of reactive episulfonium ions from the conjugation of 1,2-dihaloethanes with glutathione has been studied with deuterium-labeled substrates."' 8 2 The detection of iminocyclophosphamide from cyclophosphamide was aided by deuterium and oxygen18 labeling techniq~es.'~ Quantitative studies on the a-hydroxylation bioactivation pathway of carcinogenic nitrosamines were approached by measuring 'N2 evolution from doubly "N-labeled N-nitrosodimethylamine and N-nitrosomethylaniline from rat liver homogenates. " The evolution of I3CO from l3Ccl4 could be distinguished from unlabeled CO derived from the destruction of heme and lipid per~xidation.'~ The two isotopes of chlorine (35Cl and 37Cl) have been used t o probe the origin of electrophilic chlorine species generated during the metabolism of CClt, in hepatic microsoma1 preparations. 8 6 Reductive dehalogenation of Cc11, to the trichloromethylperoxy radical has been proposed as the initial event which leads to the electrophilic chlorine specie^.'^ In a study using oxygen-18 as a metabolic tracer, the porphyrinogenic agent allylisopropylacetamide was shown to undergo biotransformation to three different chemically reactive intermediates which appear to alkylate different cellular constituents

."

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Allen, Ed.

Using ''02 Anderson et al. showed that the two major phenolic metabolites of N-phenyl-2-naphthylamine produced by liver enzymes are likely to be derived via arene oxide intermediate^.'^ The mechanism by which bromobenzene is converted to 4-bromocatechol in isolated rat hepatocytes was shown with " O z to proceed via dehydrogenation of a dihydrodiol intermediate rather than by two successive hydroxylation reactions of the substrate. Similar studies have been performed on chlorpromazine. 9 1 Using 'H-labeled drugs, arene oxides have been implicated in the metabolism of phenytoing2 and propranolol. Deuterium-labeled variants of propranolol also have been used to study stereoselective aspects of its metabolism and disposition. 9 3 '" Spontaneous decomposition of anti-neoplastic agents such as the 2-haloethylnitrosoureas to give reactive alkylating and carbamoylating intermediates has been examined with deuterium and oxygen-18 labeled compounds.9 5 - 9 7 Studies of the enterohepatic recycling of 3C- and 2H-labeled mercapturic acid conjugates of propachlor have been reported. 6 s '

'

'-'

Deuterium Kinetic Isotope Effects - The analysis of deuterium kinetic isotope effects provides a useful tool for the elucidation of mechanisms Recent studies of deuterium isoof drug metabolism and toxicity. l o '-lo tope effects on cytochrome P-450 catalyzed reactions have led to a general consensus that these types of oxidations proceed by hydrogen radical abstraction, with the transient formation of a carbon radical prior to recombination with a hydroxyl radical to form the final product. '4-1 Cytochrome P-450 catalyzed oxidative N-dealkylations tend to proceed with modest isotope effects at best.107-'og Studies with 5-['H~]phenyl-5phenylhydantoin showed that no isotope effect was observed for both paraand s-hydroxylations with rat liver microsomes. Analogous results were obtained with deuterium-labeled ~arfarin,~'suggesting that these biotransformations occur by an addition-rearrangement mechanism. In vitro kinetic isotope studies have been reported with monoamine oxidas7" "12 and epoxide hydrolase.ll3 The introduction of deuterium at C-6 of penicillanic acid resulted in a significant increase in its a-lactamase inhibiting properties.

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vivo alterations in rates of metabolism and excretion of deuterIn ium-labeled versus unlabeled drugs have been discussed. I' Such changes in metabolic patterns can influence the pharmacological properties of some drugs such as the increased potency of the hexadeuterated analog of the gastric antisecretory agent N,N-dimethyl-"-[ 2-(diisopropylamino)ethyl]N'-(4,6-dimethyl-2-pyridyl)urea. l6 Substitution of deuterium in the Nmethyl groups of imipramine caused a decrease in systemic clearance, increase in half life and, upon oral administration, an increased bioavailability. ' 1 7 The uptake, clearance and brain levels of the hallucinogen N,N-dimethyltryptamine were potentiated upon deuterium substitution at the ct and f3 positions of the side chain. 'I8 The in vivo rate of degradation of the antidepressant phenelzine was found to be retarded in a deuterium-labeled analog,' which apparently was responsible for alterations in the observed biochemical , behavioral , " and spontaneous motor activity effects of the drug."' Kinetic isotope effects also have been used to aid in the elucidation of the in vitro and in viva metabolic pathways of the anticancer agent 6-mercaptopurine. 12' A variety of isotope effect studies have been conducted in an effort to characterize. critical bioactivation steps which may be responsible for the toxicity of xenobiotics. Examples include investi ations on the metabolism of ally1 alcohol,'23 methoxyflurane, BHT,12' 1,2-dibromoethane9l Z 6 chloroform 12 7 9 1 2 8 and carcinogenic nitrosamines. 1 2 9 - 1 3 4

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Chap. 27

Stable Isotopes

B a i l i e , et al. 277

Pharmacokinetic Investigations Quantitative Applications - The use of stable-isotope-labeled compounds as internal standards for the quantitation of drugs and metabolites in biological fluids offers a unique combination of sensitivity and selectivity of detection for pharmacokinetic studies. The principles of the technique have been outlined, and applications up to 1981 have been compiled.135 Specific aspects of the isotope dilution method, e.g. its utility as a reference technique' and associated procedures for handling the data generated from the use of multiple isotope tracers simultaneously,l 3 'have been discussed. Examples of isotope dilution methods used in clinical psychopharmacology have also been reviewed. 3 8 Deuterium continues to be the most widely used isotope for preparation of labeled standards and appears to be well suited for this purpose. However , certain 'H-labeled compounds have been found to become diluted with the protio forms upon storage, presumably due to isotope exchange phenomena. A case in point is [ 3,3,4 ,4-2H4 ]6-oxoprostaglandin Fla,1.59 Stable isotopes other than deuterium have been employed with increasing frequency for internal standards. A sensitive assay for glyceryl trinitrate in human plasma by GC-negative ion CIMS made use of two multiply15N-labeled analogs of the drug. The [l5N3] variant was employed to minimize absorption of glyceryl trinitrate on the GC column and a [2H5,15N3] s ecies served as internal standard for quantitative purposes. l Y 0 [ 3C 15N3]Guanfacine has been used as an internal standard for [ 2H3,13C6]dexamethasone for the corresponding unlaguanfacine14' "'*'and beled corticosteroid. 1 4 3

P

In certain situations, "0 may be suitable for labeling purp o s e ~ ~ ~ ~and ~ ' some " ~ exploratory work has been carried out with polychlorinated compounds enriched at > 90% excess with 37C1.146y127In the latter case, greatly simplified molecular ion clusters were evident in the mass spectra of 37C1-labeled compounds such as heptachlor and related organochlorine insecticides. Although ' S is available commercially , compounds enriched in this isotope do not appear to have been used as yet in the field of drug metab~lism."~ Lithium is important in the treatment of manic depressive illness and a stable isotope dilution assay for 6Li, using 'Li as internal standard , has been published. l 4 Where preparation of stable-isotope-labeled drug metabolites is required, an economical approach is to administer the labeled drug to a suitable animal species and to isolate the corresponding labeled metabolites from urine samples. Deuterium-labeled metabolites of antipyrine were obtained in this manner , while deuterated metabolites of 6-0x0PGFla were prepared by incubation of the labeled parent prostaglandin with Mycobacterium rhodochrous.

''

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The "soft" ionization MS techniques of FD15* and FAB"3-155 have been employed successfully for quantitative applications with stable-isotopelabeled internal standards and interesting developments in the use of microwave plasma discharges, either alone or in combination with mass spectrometic detection, promise to extend the utility of stable isotope labeling methods in quantitative studies of drug dis New methods have been reported for the measurement of lgN enrichment in ammonia, based on conversion into hexamethylenetetramine, 5 8 and for the ' analysis of 3C0 by combustion to "C$.

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Pharmacokinetics Determined under "Steady-State" Conditions - Administration of a single "pulse dose" of a stable-isotope-labeled analog is an effective method for defining the kinetics of a drug which is being given chronically, as there is no need to interrupt the dosage regimen to conduct the study.11'16 The value of this technique for use in clinical pharmacology is illustrated convincingly by work on the changes in valproic acid pharmacokinetics which take place during pregnancy. These parameters were determined by following the disappearance of a bolus dose of [ 1,2-13C2]valproic acid given to a pregnant epileptic patient. 16' For ethical reasons, this type of study could not have been carried out by alternative methodology using radioactive isotopes. A similar investigation of valproic acid pharmacokinetics in non-pregnant humans was performed using a deuterium-labeled variant of the drug. l b 2 It is essential that the labeled species chosen for administration be demonstrated to be pharmacokinetically equivalent to its unlabeled counterpart. The likelihood for non-equivalence is remote when 13C or 15N is employed. Re orts have appeared in which investigators have validated the use of [ 13C,P5NN]phenobarbital'63-16s and [ 13C,'5N2]phenytoin166 for such applications. Concern over apparent differences in vivo between phenytoin and a deuterium-labeled analog led Poupaert et &. to synthesize [ 3C6]phenytoin for pulse dose studies. Nevertheless, when properly validated, drugs labeled with deuterium can be used for administration in Recent examples of the latter have involved studies kinetic experiments. on the influence of long-term infusions of lidocaine on the kinetics of this agent 16' to investigations of methadone kinetics during maintenance treatment,l6 17' and to stereoselective aspects of the disposition of methadone enantiomers in humans given the racemic drug. 17' The singledose kinetics of A'-tetrahydrocannabinol were studied in light and heavy cannibis users with the aid of a 'H3-1abeled variant of the drug.172

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Bioavailability Studies - The use of stable isotopes in studies of absolute or relative bioavailability offers a number of important advantages over the conventional "cross-over" approach in which different (unlabeled) formulations are administered on separate occasions. l 1 " ' l7 "7 4 This is especially true for drugs which are subject to extensive first-pass elimination, since these compounds usually exhibit the greatest variation in bioavailability as a function of time. l7 The absolute bioavailabilities of timol01~'~''~~and bepridil17' have been studied by the simultaneous oral and intravenous administration of unlabeled and 13C-labeled forms, respectively. The bioavailability of a standard formulation of clovoxamine fumarate was compared with that of a slow-release preparation using [ '3C]clovoxamine as the reference dosage form. 17' The absolute bioavailabilities of verapamillso and methadone'" have been investigated with the aid of specifically deuterated analogs, and the results of a ilot study on the oral bioavailability of captopril have been published. 1 P 2 Additional examples of the use of stable isotopes in measurements drug bioavailability include the study of stereoselective increases propranolol bioavailability during chronic dosinglB3 and the influence urinyq: pH and route of administration on meperidine disposition This technique also can be employed to quantify the conversion man. prodrugs into the active species in vivo, as illustrated in a study prodrugs of indomethacin. l E 5

of in of in of

on

Chap. 27

S t a b l e Isotopes

Bailie

e t al.

279

Conclusions S t a b l e i s o t o p e t e c h n i q u e s o f f e r a number o f s i g n i f i c a n t a d v a n t a g e s o v e r a l t e r n a t i v e methods f o r i n v e s t i g a t i o n s of d r u g m e t a b o l i s m and d i s T h e s e a d v a n t a g e s , some of w h i c h h a v e b e e n h i g h l i g h t e d i n t h i s position. b r i e f r e v i e w , a r e becoming more w i d e l y a p p r e c i a t e a a n d w i t h t h e growing a v a i l a b i l i t y of mass s p e c t r o m e t e r s s u i t a b l e f o r t h e measurement of i s o t o p i c a l l y - e n r i c h e d m o l e c u l e s , t h e u s e o f s t a b l e i s o t o p e s i n m e t a b o l i c and p h a r m a c o k i n e t i c s t u d i e s may b e e x p e c t e d t o i n c r e a s e s t e a d i l y i n t h e coming years. Although s t a b l e i s o t o p e s w i l l n e v e r r e p l a c e t h e i r r a d i o a c t i v e c o u n t e r p a r t s f o r many t y p e s of a p p l i c a t i o n , t h e y have become w e l l estab l i s h e d as i n d i s p e n s i b l e t o o l s f o r u s e i n m e t a b o l i c i n v e s t i g a t i o n s .

Aden~icPress, N&r Ymk, 1Y% Smidt. H. F'c;rsbl erd K. HRinzirger, Ms., Wable Isotopes. h c e d h g s of the 4th Internatioral ccnferencs," i&&.€r,k m t d a n , 1982. 5. W.P. Dncm and A.B. susul, Ms., " S y n ~ aand A M c a U m of hu@caUylabaled ' h p U X l s , f f Elsevier,

L. H A .

Apstcndam, 1933. 6. A.L. B~.llngem,T.A. W e , P.J. Denick ard O.S. hizhov, A d . h., 2,214R (193). E-, A. Dell and D.H. b d ,Ad.. o l e m a , 2, 363R (1$82)7. A.L. B 8. D. IktllidayandKJ. Reonie, CUn. sci., 63, 485 (1982). 9. R.M. Capriau and D.M. Ber, in f W & d ApplcatiOnS of hSpsCkxmhy. First &pphrmtmy Voluue," NRJ Ymkp 1W,p. 8%. C.R. WBUar and 0.C. Darnar, M S . 8 10. J, Sjb;all, in flAdvanm in h s Spctraretry," 8B, A. W l e , M., byden and Sen, Ladcn, lW,p. .@l 11. T.A. m e , Pnanmml. REV., 2,81 (1961). 12. T.A. Paillie, H. Ibgbs, ard D.S. &vies, in fqstahleI S O ~ ~H.-L.S , ~W ~ t , H. Fb;gtsl, and K. Heinzirgsr, Ms., Elsevler, Amstsrdam, 1482, p. 187. 13. N.J. Hasldns, fftomed. h s SpsCtrau., 9, 26q (1%?). d g 8 and I.L. Ims, Ms., ?he 14. D.R. Haddns, in %otapjs: Essential Ctmistty ard ApplicationS," I.A. W olenrical Society, kdn,lW,p.232. 15. W.J.A. V m u v d , in "Synthesis and A@icaticm of IsotCpblJy Iateled Ccmpnarls," W.P. Rncan erd A.B. 3Ms., Mw, Amdeadam, 1933, pa 77. 16. P.J. M.u-&v and H.R. Eullivan, Antn~.Rev. €lmmcol. T d m l . , 20, 0 (1933). 17. M. Bcheltaum, G.E. MI bruh and A. Sopngyi, C&. Ruumscaldn., I,4 9 (1982). 18. D.G. Gtt, YiynVeems w i t h Stable Isotopes of Cartcn, Nitrogen and Wgm," J& Mley and Scns, Nev York,

193.

19. A.P. Tulloch, Rog. Lipid Res., 22, 235 (1963). 20. A.R. Branfbn, M.F. b h t i s h , R.J. M, M.M. CaUahm, R. Fbtertmn and D.W. Y e s a i r , h g b h b . DispJs.,

2, 2% (1981). 21. M.M. callahan, R.S. b b e r b n , A.R. hnhn,M.F. IkCudsh a d D.W. Yesair, hg &tab. Disp3s., 2, 2ll (1963). 22. J.L. kdm,B. M h , M. Desage and B. selle, Ebsd. h s SpsCtrcm., 2, 169 (1W). 23. J.L. M, B. ~ibcn,M. ~esageand B. salle, in t1mw-b in MWS spectranew in iaochemistry, Mdicine and FZwhmena Research," 7, A. -0, M., Elsevier, A m s t d a ~ , 1981, p. 27. 2.4. M. Demge, J.L. M e r erd R. Int. J. hss Spectrcm. Ion ms., &, 85 (1583). 25. K. Mmna and S. Rib, @em. W. W. (Tolg.o), 2, P-43(1961). 26. T. C o r a m ~ ~T., ma.uta, S. Rib, A. W a n d s . Iguchi, olem. h. Bull. (T&o),a, 17&(1%1). 27. I. Ileyana, S. h q c c h i , I.Kotcal,T.H&iw,Y. I s h i i a n i I . T a b e , J.Pgric.FwdO1Em.,3l, 1061 (1982). 28. A.K. b,R.C. Ihmmmr and L. Ate, Life sci., 3,1075 (191). 23. G. Fa&&, R.C. lhnmrer, C.H. wan, D.A. W t z , E.W. Di %fen, and A.K. &, hg b b b . DispDs., 3 47 (1987). b h b . Msp3e;., 33. T. Ganmaru, H. ktamm, T. Fbuta, S. Rib, N. Y a , T. M p d i i , T. Semsshimn, 10, 542 (1462). 31. rblddmfeld, P. Carter, V . N . R a i n h o l d , S . B . l s n n e r I V a n d L . L . ~ , ~ . ~ s S F e c t r c m . , 5 , 5 8 7 (1978). 32. B.4. W k g , M. Axalscn, D.J. cdllins and J. Sf&lJ., J. bum-., 277, 453 (1961). 33. D.A. k&, L. Ropamfp, K.I.H. WFWams, H.J. Bndie and A.M.H. M e , Bocbm. hrmml., 2,7M (1982) 2. M.Tahm,Y.N&nta,H. hut&, T.Kurosaka, I.MddmandS.kkagaka,@em. Rrurm. W. TWO),^, 137 (1481).

w,

280 35.

36.

37.

3. 39. 40.

41

.

42. 43. M.

45.

16. L7. @. 49.

50. 51 * 52.

53.

54.

55. 56. 57. 58. 59. 60. 61. 62. 63.

66. 65. 63. 67.

68. CN. 73.

n. n. 73. 74.

75. 76.

n. 78.

79. 8).

01. 82.

83.

86. 85.

ab. 87. 88. €9. 9. 91.

Sect. V I

- T o p i c s i n Chemistry and Drug Design

A l l e n , Ed.

Bailie

Stable Isotopes

Chap. 27

e t al.

281

W t a f a , M. C l a e s a , J. Adline, D. V a n d m o r s t ard J.H. Finpie&, Dng kth D i p . , 2, 574 (1983). ktab. bpx., lo, 122 (1932). 93. T. hhlle, J.E. Oatis, Jr., U.K. W& ard D.R. Krapp, %. T. Wle, M.J. wilscn, U.K. k l l e ard S.A. Bai, h g &tab. Dlspss., 2, 54.4 (1933). (1981). 95. J.W. Lam ard S3.S. &uhn, J. Q'g. m.,&, $6. J.W. Lam ard S.M.S. U , J. (kg. Qlecn., Q, 851 (1982). 9. J.W. Lam, R.R. K c g e n e ard A.V. J C ~ WJ., Org. M.,Q, 2M7 ( 1 9 ) . 9. J.E. W, J. Rafter, G.L. Iarsen, J.A. htafsson ard B.E. htafssan, ktab. D i p . , 2, 525 (1931). 9. G.L. knrsan ard R. ftyhage, Xembiotioa, 2, 855 (1%). 103. G.L. Iarssn ard J.E. W, Xenobiotiar, 13, 115 (1907). 101. J.P. KUnmn, Ariv. byuol., &, L15 (14785. l a . D.B. Northup, Arm. Rev. Biochem., 103 (1981). 103. W.W. clelani, m &it. ~ e v . ~tiochem., 2,385 (1982). l a . J.T. Groves, S. Kriehmn, G.E. A v a r i a , T.E. h, in W a u i m t i c QamLshy,"D. C. Wnnh, Y. k a k a m i , I. 'Igt*lshi, Ms., American G-mical Society, bhingtm, D.C., 1583, p. 277. 105. M.H. Celb, D.C. Heimfnwk, P. &&&nard S.C. sllgar, W s t r y , 21, 370 ( 1 9 ) . 105. T. Sam, Y. Mzu, T. Tcda and N. oshino, Emg &tab. 2, 4$(1$81). 107. G.T. M-a, W.A.carlerd, B.J. Hcdson ard A.Y.H. Lu, J. Ebl. M., a 9 (199). l a . G.T. H-a, J.S. Wsh, G.L. Weris ard P.F. H o l l e n b e r g , J. Blol. W., 258, 1&5 (1983). ( & 2633 , (192). l@. T. &no, T. T d a ard N. Oshjm, J. Am. a m . Soc., I 110. M. CLeesen, M.A.A. h t a f a , J. Adline, D. Vezxlervorst ard J.H. pouFaert, fhvg kth Msps., l o ,667 (1932). 111. M. I6lsain, D.E. EMumdsm ard T.P. singer, Biochemistry, Zl, 595 (1W). 112. P.H. Yu, S. kclay, B. hvis ardA.A. Boultcn, B i h m . p)uurmcol., 30, 339 (191). XB, 195 113. R.B. kstkaempr ard K.P. FanzUk, Arch. B i h m . Bio*s., 114. D.C. and J.R. W l e s , loche em is try, 0, 3683 ( ~ ) 7 115. A.B. Fcater, in "Advances i n I h l g Research," B. T e s t a , Fd., A c a d m L c Press, I.d.m, 1984, in p s s . HofM,C.N. Kakker, A.M. Pietruszkiewicz, W.A. blhofer, E.J. CraCJoe, Jr., M. Lxl T c a r h i a r a ard 116. J.M. R. Kirschnenn J. M b m . , 26, 1650 (1983). 117. I.W. Taylor, C. Icamides, P. %ma, J.C. Tuner ard D.V. parke, Biochem. pharmncol., 32, 6!,1 (1933). 118. S.A. Barker, J.M. h t m , S.T. Qlristian, J.A. hti and P.E. Elcaris, Blcchm. Eharmocal., 31, 2513 (1982). 119. L.E. Q&, D.A. ~sden,P.H. YU, B.A. mvis and A.A. ~oulton,mochem. pharmncol., 3, 151971983). 120. C.T. h r i s h , K.M. W, L.E. Qck ard A.A. Boultcn, PsychopuumLcol.,8l, 122 ( 1 9 3 . 1. Ricchd. W v . , l9, 471 (1583). 121. C.T. M s h , A.J. Cmenshu ard A.A. Bmltcn, 122. M. Jamn, J.H. Kihuis, C.B. E m , V.C. Knick, G. Lamb, D.J. Nelscn ard R.L. Tuttle, in I I S t a h l e Isotqe~,~~ H.-L. Schmidt, H. F'drstel ard K. €k&jngm, Ells., Elsevier, Amsterdam, 1982, p. 217. 123. J.M. patel, W.P. Coldcn,.S.D. N e b ard K.C. Isitam, h g Metab. Disps., 2, 164 (1933). 103 (1982). 124. J.M. hien, S.A. Rice ard R.I. Wze, Anesthesiolag, 125. T. Hzutani, K. yamamoto ard K. Tajim, Tcudcol. A@. Ruumpml., 283 (1983). Ruunacol., 63, 170 (1933). 1%. R.D. Wnite, A.J. cendolfi, G.T. Bxkn ard I.C. Sips, Toxiwl. 127. R.V. hnchfhm ard L.R. Pohl, Taxlwl. A M . Pluumsool, 61, w)7 (1931). 128. M. hmdimkh, C . 4 . Kuo and J.B. h k , J. Toldcol. hvlrca. Wth, 8, 105 (191). 5019 (1981). 129. S.S. Hecht ard R. Yourg, Cancar Res., 1 3 . M.S. %a, D.G. SarpUi ard W. LijinsQ, &rdccgenssis, 2, 731 (1981). 131. W. LijW ard M.D. Reutar, Cancer Lett., & 273 (199). 132. W. Lijinsky, M.D. R m k , T.S. hvies and C Y . Pigps, J. ktl. Cancar Inst., 1127 (192). i j w ,Center Res., &, 2105 (1982). 133. G. Farrally, M.L. S W , J.E. SBaVedra and W. L 134. S.S. Mt, D. Lin ard A. Cas-, carcinogenesis, 4, 305 (1933). h m m w . Sci., 2, 392 (1931). 135. W.A. G s r l a r x i a r d M . L . hell., J. C 136. I. B j & k h , in "stable Isotopes," H.-L. W d t , H. Fmtel and K. Heinzinger, Ells., Elsevier, Ansterdam,

9. M.A.A.

so,

blm,

m.,

255,

k) .

,

2,

9,

m.

g,

9,

19% p.

593.

k b y , K. Nakanua, M.J. Kreek and P.D. &in, in "stable Isotops," €I.-L. S h i d t , H. F k t e l and K. kimirga, Ms., Elsevier, Amsterdnm, 1982, p.235. L. BEo-tilssan, i n V M c a l FhmmcoogY in Psychiaq," E. usdin, Fa., Elsavier, New York, 1981, p. 59. A.I. Millet, Prcateglaniins leuko~enesM., t3, 181 (1982). G. Idzu, M. Ishiard H. Mpmki, J. Qrromstogr., g,327 (1%). ,C.- J C. Lap, D. Iavene, J.R. Kiechel and J.J. Bassaller, Int. J. h s Spectrom. Ian @s.,

137. D.L. 138. 119. 140. 1Ll.

&, m

(1983).

kk ard J s L Raid, J. h. -., 5,114 (1983). Ye J A M t h s , J.P. FreemEln ard R.K. Nt&m, in ''Syntbda arrl A @ h t i ~ of Is~topicaUy IakLed cupnds," W.P. k c a n ard A.B. Susan, Ms., Elsevier, Amsterdam, 1983, p. 369. W.C. Plckett ard R.C. Anal. Bicchem., lll,115 (1931). D.M. anl M. ~ a i ,~ ~ o m e db~ . spcb., 2, Ln (1933). D.H.T. Cnien ard J.R. k y s , J. Agric. F a d &an., 30, 3% (1982). N. H. b h b ard L.A. W f , €Lo&. Miss Spctrcin., 2, l+5 (1%). R.H. White, Anal. Biochem., l & 369 (1931). J.R. Lbyl ard F.H. Field, B l d . Miss Spctmm., a, 19 (1931).

142. N.D.

14.3.

14.

145. 146. 147.

148. 1L9.

w,

28 2 1%. 151.

152. 153. 1%.

155. 156.

157.

158. 159. 160. 161.

162. 163. 1 Q.

165. 1%.

167. 163. I@. 170. 171. 172.

173. 174. 175. 1%.

177.

178. 179. 183. 181. 1 8 .

183.

14. 185.

Sect. V I

- Topics i n Chemistry and Drug Design

Allen, Ed.

Chapter 28. Drug Discovery at the Molecular Level: A Decade of Radioligand Binding in Retrospect Michael Williams and David C. U'Prichard Nova Pharmaceutical Corporation P.O. Box 21204, Wade Avenue Baltimore, MD 21228 Introduction - In the design of new therapeutic entities, the medicinal chemist works from information Erom known active compounds or from the structure of the putative neuroeffector agent thought to be involved in the etiology of the disease state under study.' The search for novel chemical entities which have therapeutic potential has become increasingly more complex;2 thus any rational strategy that will increase the probability O E finding a new drug is of considerable interest to those involved in drug research. One such approach is that utilizing radioligand binding assays , 3 which permits the evaluation of compounds for their direct interaction with cell surface recognition sites (receptors) independent of events distal to the actual recognition process. Structure-activity relationships generated using radioligand binding assays theoretically reflect the actual physicochemical properties required for receptor recognition, and thus provide information directly pertinent to the improvement of specificity and potency. In addition, receptor assays require only small amounts of compound and animal tissue, and are relatively rapid to perform. However, like classical pharmacological screens, these assays have certain drawbacks. They take no account of compounds that require metabolic activation, and generally measure only relative potency rather than efficacy, so that it is difficult to assess whether a compound interacting with a ligand binding site is an agonist or antagonist. These issues, and the advances and insights gained through the technique of radioligand binding, are the subject of this review. The Binding Technique - The radioligand binding assays developed to date are based on the fact that compounds known to interact with given receptors, when labeled to high specific radioactivity (10 Ci/mmole or H or 1251,bind to such sites in membrane or intact cell greater) with preparations from mammalian tissue with high affinity (Kd 'lo-' M) in a reversible, saturable manner. The difference between total and nonspecific binding, the specific binding, can then be used to measure compound interactions with the receptor. Ideally, specific binding should represent 70% or greater of the radioactivity bound. However, 50% specific binding, if not ideal, is acceptable. Binding assays where specific binding is only 20-30% of the total, present problems in 'signal-to-noise' reliability for routine screening purposes. In general for screening purposes, compounds with Icso values greater than M can be deemed 'inactive,' especially if at other recognition sites, they have I C s o values in the lo-' M range. The large majority o f radioligand binding assays make use of brain tissue because of its ease in preparation, and i t s richness in receptors

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on a weight per weight basis.3 Other tissues which have known physiological responses to a given agonist tend not to be good targets for radioligand binding because of the low density of receptors present as compared to CNS tissue.3 The ease with which CNS tissue can be utilized should not, however, preclude examination of peripheral tissue radioligand binding, since it is highly likely that subtle pharmacological and biochemical nuances exist among receptors of a given type as a function of their tissue source. For instance, despite considerable effort, the nicotinic antagonist, mecamylamine, which is exceedingly potent in autonomic ganglia, has no demonstrable binding in the CNS. 4 Furthermore, ligands specific for dopaminergic receptor subtypes in the CNS have not proven suitable for labeling the subtypes present in peri phera 1 tissues

.

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Validation of Receptor'Binding Assays There has been considerable concern that because of the non-physiological conditions used to examine radioligand binding, the sites labeled may represent in vitro artifacts. Such conditions include the use of hypoosmotic buffers; long incubation periods, which are inconsistent with the millisecond time courses related to the proposed physiological actions of neurotransmitter analogues and antagonists; and the use of non-physiological assay temperatures. These basic criticisms have some validity, but in general, like many in vitro test paradigms, reflect the interface between the complexity of nature and mankind's necessarily simplistic approaches. Several criteria have been proposed5 to validate a binding assay. These are saturability, indicating a finite number of binding sites; reversibility, consistent with a biological process involved in information transfer; correlation of binding affinity with the demonstrated concentration of the neurotransmitter or drug under physiological conditions; tissue and subcellular distribution of binding consistent with the known localization or target site of the ligand; agonist and antagonist pharmacology of the binding site corresponding to the known properties of the compound from other pharmacological tests; and finally, correlation of binding data with biological dose-response curves in identical tissue preparations. Most binding assays currently used fulfill a good many of these criteria. With these guidelines in mind, the status of the radiollgand binding assays currently in use will be considered. In many cases the initial observations of relatively simple recognition sites has progressed to the delineation of receptor subtypes. While offering exciting possibilities in terms of drug development, many of the assays for receptor subtypes have a weak pharmacological basis and in some instances do not fulfill many, if not all, of the criteria listed above. Binding and Receptor Subtypes

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a) Dopamine receptors Recognition sites for this monoamine, which is implicated i n the etiology of both schizophrenia and Parkinsonism, have been labeled with more than 20 iigands.6 Five or more distinct receptor subtypes have been described; of these, only those designated D-1 and D-2, which activate and inhibit adenylate 7 cyclase activity, respectively, are considered tangible entities. Apomorphine, spipelone, and domperidone label both D-1 and D-2 recognition sites. While newer antagonists such as S$H 23390 (1) appear to be selective for the D-1 receptor in vitro, in vivo this

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distinction is not apparent.” Newer agonists, such as RDS127’l (2) and PHNO (?), appear to be selective D-2 ligands. Putative dopamine autoreceptor agonists, such as 3-PPP and TL-99, have generated considerable interest Unfortunately, neither binding nor i,n viva pharmacological procedures have provided definitive evidence that either of these compounds has absolute selectivity for presynaptic dopamine Whether pre- and post-synaptic receptors represent distinct pharmacological, as opposed to anatomical, entities is a matter f o r further investigation.

I -

2

3 -

b) Alpha-adrenoceptors - A stronger association has been established between radioligand binding sites and functional receptor entities for epinephrine and norepinephrine. Alpha-1 and alpha-2 receptors were originally discriminated by location, being post- and pre-junctional respectively.16 Subsequently, alpha-2 receptors were shown to be negatively coupled to adenyiate cyclase, while alpha-1 receptors stimulate PI turnover and Ca2 entry.17 Among antagonist radioligands, peptide ergots (e.g. dihydroergocryptine) and phentolamine label both receptors with equal affinity,” while prazosin and the are highly potent and selective aminotetralone, HEAT (RE 2254)” alpha-1 ligands; WB-4101 i s partially selective for the alpha-1 receptor,’l while yohimbine and its isomer, rauwolscine, are somewhat Alpha-2 antagonists appear to accelerate alpha-2 receptor selective.” antidepressant- induced down-regulation of beta- and 5HT-2 receptors, and are currently the subject of intense intere~t.’~ The selective alpha-2 antagonist, the imidazoline, RX 781094, has been successfully used as a radi~ligand.’~ Several catecholamines and imidazolines selectively label high affinity states of the alpha-2 receptor.25 Preand postjunctional components of alpha-2 receptor binding have been identified, but no differential pharmacological characteristics have yet been observed.

c) Beta-adrenoceptors - These have been readily labeled with antagonists such as dihydroalprenolol, carazolol, iodohydroxybenzylpindolol ,26,27 cyanopindolol and pindolol.” Beta receptors were originally subdivided into beta-1 and beta-2 types on the basis of differential agonist and antagonist pharmacology and tissue location. Ligand binding studies corroborate the pharmacological subclassification quite precisely. Since the above mentioned antagonists are not subtypeselective, estimates of subtype relative abundance and competitor preference have relied on the generation of biphasic competition curves, with computer-assisted curve-fitting. These have not, however, been without problems; IPS 339 (4-1, a beta-1 selective antagonist as judged by computer assisted binding data3’,has little subtype selectivity in other tissue receptor assays. 3 1

’’

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OH

..

-4 I

5

-

The biochemical characterization of beta receptors and their coupling to adenylate cyclase has been intensively studied 32 using agonist radioligands (epinephrine, hydroxybenzylisoproterenol) to label high affinity receptor conformations, especially of the beta-2 receptori3 more hydrophilic antagonists (e.g. CGP 12177 (5))to label cell surface, but not internal, receptor populations (especially useful for examining regulation of beta receptors in cell culture); 34and photoactivated, irreversibly binding antagonists such as p-azidobenzylcarazolol, to tag the receptor during purification to apparent homogeneity.35

-

d) Serotonin receptors These have been labeled with serotonin itself and LSD,36 spiperoneg7using cortical tissue, and more recently, ketanserin.38 Serotonin receptors in brain tissue appear to exist in two distinct forms termed 5HT-1 and 5HT-2. The former, which can be labeled with serotonin, have been further subdivided Into 5-HT-1A and 5-NT-1B sites.39 Both spiperone and ketanserin label 5-HT-2 receptors in cortical and other brain regions. Interestingly, the pharmacological actions of ketanserin are mediated via alpha-1 receptor blockade. 4 0 The 5-HT-2 receptor has been implicated in depression, chronic treatment with the tricyclic, amitriptyline, reducing the number of 5-HT-2 binding sites in rat frontal cortex.41 The physiological relevance of the receptor has, however, been questioned since atypical antidepressants such as mianserin can reduce the density of serotonin-2 sites when given acutely.42 The relationship of central serotonin binding sites to those designated as M (excitatory; blocked by morphine) and D (inhibitory; blocked by dibenyline) is unclear at the present time.

e) Muscarinic cholinergic receptors - Many highly potent and selective antagonlsts have been used to label muscarinic receptors in central and peripheral tissues, including N-methylscopolamine, 3-quinuclidinyl benzilate (QNB), N-methyl-4-piperidinyl benzilate, and the covalent ligand, benzylcholine mustard. Excellent correlations have been obtained between affinities at these binding sites and pA values in classical organ preparations such as guinea pig ileum.43 Agonist competitors at antagonist binding sites exhibit complex, heterogeneous interactions manifested by very "shallow" competition curves; these can be partitioned into "super- high" (SH), "high" (H) and "low" (L) affinity states. Agonist ligands (*-methyldilvasene, acetylcholine, oxotremorine-M) preferentially label, and improve resolution of, the SH state of the receptor. 4 4 The relative proportion of these three "states" was found to differ markedly in different tissues and brain areas. 4 4 I t has become apparent, however, that there exist two pharmacologically distinct types of muscarinic receptor, 4 5 and antagonist designated M-1 and M-2. The agonist McN-A-343 are selective for M-1 receptors; no M-2 receptor pirenzepine selective agents are known. Given the apparent tissue specificity for the two receptor types (M-1 in most brain regions and autonomic ganglia;

(L),46

(a),

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M-2 in peripheral effector organs such as heart), there is considerable interest in the development of subtype-selective agents.

(CH,),~CH~C=CCH,OCONH

-Q \

6

CI 7

8

-

f ) Nicotinic cholinergic receptors Acetylcholine (ACh) receptors sensitive to the alkaloid nicotine have been labeled in Torpedo tissue and in autonomic and central nervous system using radiolabeled ACh,47 nicotine 4aand alpha-bungarotoxin (BTX).49 BTX is, however, not a good ACh antagonist in mammalian nervous tissue. 5 0 The classical ganglionic blockers, hexamethonium and mecamylamine do not displace these ligands from CNS tissue,5 0 a finding which may indicate that agonist and antagonist binding sites are distinct entities. Mecamylamine shows no speci€ic binding in brain tissue. The neuromuscular blocker, dihydro-beta-erythroidine (DBE; (S)) does label central nicotinic recognition sites. DBE binding is insensitive to ganglionic blockers, suggesting that the nicotine recognition site in mammalian brain is more selective for neuromuscular type than ganglionic receptor blockers.4

g) GABA receptors - These have been labeled with tritiated GABA51 and muscimol.’L Electrophysiological and binding studies in mammalian tissue have led to the discovery of two distinct types of GABA re~eptor:~ the GABA-A site, which is labeled by GABA and muscimol and which is sensitive to blockade by bicuculline; and the GABA-B site,which can be selectively labeled by baclo€en and is insensitive to blockade by bicuculline. The GABA-B receptor may be linked in a modulatory manner to a membrane bound adenylate ~ y c l a s e . ~ ~ h) Excitatory amino acids - As many as four excitatory amino acid receptor subtypes have been described. These are sensitive to the agonists, N-methyl-D-aspartate (NMDA), kainic acid, quisqualic acid and APB (2-amino-4-phosphonobutyric acid) .5 Excitatory amino acids have been termed ‘excitotoxins’and can cause cell death in a manner similar to that seen in Huntington’s disease.55 In general, the affinities of the various ligands used to label excitatory amino acid recognition sites in mammalian brain tissue are low compared with other putative neurotransmitter recognition sites, with Kd values around M.55

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i) Purinergic receptors Receptors for the purines may be divided into P-1 and P-2 subtypes.’b P-1 receptors are adenosine-sensitive and cyclase-linked, while P-2 receptors are ATP-sensitive, affect prostaglandin synthesis, and have no effect on cyclic AMP production. Two subtypes of the P-1 receptor exist; 57’5%he A-1 or Ri and A-2 or Ra, which, respectively, inhibit o r activate adenylate cyclase. Both A-1 and A-2 receptors are sensitive to blockade by xanthines such as caffeine and theophylline. To date, ligand binding assays have only been described for the A-157’5%nd P-2 5 9 receptors. Binding studies have led to the description of further subtypes which show species 60 dependence. Functional differences in A-1 receptor function have

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also been reported.61'62 The 5'-N-ethylcarboxamide. of adenosine, NECA, may show preferential selectivity for A-2 sites. 6 3

-

j) Histamine receptors Mepyramine has been extensively used to label H-1 receptors,ss while tiotidine has been shown to demonstrate specific binding consistent with the labeling of an H-2 receptor. 6 5 The recently described irreversible H-2 blocker, L-643441 may be an ideal ligand for this receptor subclass.66 In view of the profound effects of histaminergics on CNS tissue function and the search for second generation antiulcer agents, i t seems highly appropriate that a reliable H-2 binding assay should be available for drug screening.

(z),

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k) Opiate receptors Binding assays for opiates have contributed significantly to the discovery and characterization of the enkephalins and endorphins. At least four, and perhaps five opiate receptor subtypes are amenable to study by radioligand binding techniques. 6 7 The mu-receptor can be labeled with dihydromorphine (DHM) and by the enkephalin analog, DAGO. The delta receptor, which is involvedsin spinal analgesia, is labeled with the enkephalin analog, DADLE. The kappa receptor, thought to be involved in the psychotomimetic effects of some opiate partial agonists, es6 Becially benzomorphans,70can be labeled with ethylketocyclazocine (EKC) or bremazociyf (10).The sigma opiate receptor can be labeled with SKF 10,047. The epsilon receptor can be labeled with beta-endorphin, although this is controversial at a newly introduced analgesic agent, has this time. Meptazinol been reported to interact with a subtype of the mu receptor, mu-1 which 72 is responsible for the central analgesic actions of the opiates. BY inference, the mu-2 receptor is responsible for the respiratory depression and inhibition of gastric motility associated with opiate action. Radiolabeled meptazinol has not proven to be a satisfactory ligand because of its lipid solubility.

(c),

Hd 9

0

lo

1) Benzodiazepine (BZ) receptors - Using 311-BZs, up to three subclasses of BZ receptors have been described;73 two central receptors, BZ-1 and BZ-2, which have been reported to mediate the anxiolytlc and sedative/ataxic actions of the BZ's, respectively,74 can be labeled with clonazepam; a third peripheral type receptor, which has low affinity for clonaeepam, can be labeled with the BZ, Ro 5-4864.75 Whether the two central receptors are distinct entities or different forms of a single receptor remains to be determined.76 Ro 15-1788, the BZ antagonist or 'inverse agonist' can be used to label central BZ receptors.77 Using the central BZ binding assay, several non-BZs with anxiolytic potential have been reported. These include the pyrazoloquinoline, CGS 9896 78and the triazolopyridine, CL 218872,74 which displace BZ binding; and the pyrazolopyridine, etazolate,79 which enhances binding. The BZ receptor is somewhat unique in that it exists as a receptor complex involving a GABA recognition site and chloride ionophore, the latter being responsible for the physiological actions of the B Z S . ~ ~

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m) Peptide receptors - Many neuromodulatory peptides in addition to the enkephalins and endorphins have been discovered in both the gut and brain which have a common embryological,origin. Radioligands have been used to identify the cell surface recognition sites for peptides. The classical peptides first studied were insulin 5 and glucagon; assays for Substance P, cholecystekinin, neurotensin, bombesin, angiotensin, and arginine vasopressin have been described. 8 0 Although some progress has been made in delineating substance P receptor subtypes using binding criteria, radioligand binding assays for peptides and other related entities such as the leukotrienes 8 2 should at the present time be described as recognition site, rather than receptor assays. Nonetheless they are exceedingly useful screening tools.

!2

!3

-

n) Receptors-for calcium agonists and antagonisAs- While calcium antagonists bind to ion channels and cannot be considered as receptor antagonists per se, the binding of three classes of such agents has been described: the dihydropyridines 8 3 (DHP) (e.g. nifedipine and nicardepine); the nitrilesE4 (e.g. verapamil and D-600); and the benzothiazepines (e.g. diltiazem). Binding assays for the DHPs have been characterized in brain and visceral tissue. Verapamil, D-600 and diltiazem are weak, partial displacers of DHP binding, which may imply the existence of either distinct calcium channels or a 'receptor' complex with different recognition sites. A binding assay for verapamil has been described re~ently.'~ Diltiazem can stimulate DHP binding and can reverse the inhibitior, of binding seen with tiapamil. 8 6 More recently DHPs have been described which facilitate calcium entry including Bay K 8644 8 7 (2) and CGP 28392 (2). 8 8 While calcium antagonist binding sites are present in brain, their pharmacological significance is unclear since they do not appear to affect calcium fluxes in synaptosomal preparation^.^^

-

Potpourri A variety of ligand binding sites are unassociated with defined receptors but are useful in the drug screening process. These include the proteins, labeled by antidepressants, which are associated with reuptake processes for 5-HT ( H-imipramir~e~,~H-paroxetine 9 9 and norepinephrine ( H-desmethylimipramine 1. Other potentially important sites are those for the psychotomimetic phencyclidine (PCP) 9 3 and the atypical antidepressant, trazodone. 9 4 The potential significance and usefulness of these and other such sites must remain in limbo until a relevant biochemical or cellular function has been attributed to them. Specific 'drug' recognition sites need not, however, be invoked for each and every novel compound with therapeutic potential. The search for unique binding sites using radiolabeled

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probes and tee extrapolation to a search for the "endogenous ligand" may lead to unnecessary complications. A potentially useful therapeutic agent may have a distinct 'non-receptor,' but still locus specific, site of action. An example of this in the anticonvulsant/central sympathomimetic/anxiolytic MK 801g5 which has in vivo activity but has no appreciable activity in several ligand binding assays. The pharmacological activity of a compound may also be dependent on activity at more than one recognition site. The example of ketanserin, an in vitro ligand for the 5HT-2 receptor but a alpha-1 antagonist in vivo has been cited.40 The is another instance where the binding serotoninomimetic, MK 212 (g), profile of the compound and its in vivo activity do not c ~ r r e l a t e . ~ ~ ' ~ ~

(E),

Ligand binding methods have facilitated the study of receptor alterations in disease states, whether secondary or presumptively etiological examples incude human postmortem brain tissue obtained from patients with neurodegenerative (Parkinsonism, Huntington's Chorea, Alzheimer's Disease) or psychiatric (Schizophrenia) disorders and in formed blood elements obtained from patients before, or during, drug treatment (e.g. platelet alpha-2 receptors and H-imipramine binding sites; white cell beta receptors). 9 9 Especially in the realm of neuropsychiatric disorders, a variety o f receptor data has been amassed that is at this stage primarily phenomenological and as yet of little value with regard to etiological issues or rational therapy.

,'*

-

Conclusion The future of radioligand binding methods for research on receptors and their function can be predicted to follow several directions: (a) the increased use of receptor autoradiography with computer-assisted quantitative imaging to combine biochemical precision with high morphological and spatial resolution;100 and (b) an increased emphasis on isolation and purification of receptors, facilitated by the use of covalent affinity probes, to allow controlled reconstitution of receptor-effector systems, and to facilitate development of receptor antibodies which in turn will be used as probes to determine specific receptor RNAs and DNAs. This will ultimately lead to a better understanding of receptor expression, and the effects of drugs thereon, and to cloning of receptors in host cells on a scale large enough to provide sufficient material to study in depth the physico-chemical properties of the ligand-binding "active site" of receptor proteins, allowing for truly rational drug design.

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292 56. 57. 58,

59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80.

81. 82. 83. 84. 85.

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A l l e n , Ed.

G. Burnstock in "Regulatory Function of Adenosine," R . H . Berne, T.W. Rall and

R. Rubio, Eds., Nijhoff, Boston, 1983, p. 49. J.W. Daly, J. Med. Chem., 3 . 1 9 7 (1982). M. Williams, Handbook Neurochem., a.1 (1984). R.M. Levin, R. Jacoby and A.J. Wein, Mol. Pharmacol.,23,1 (1982). K.M.M. Murphy and S.H. Snyder, Mol. Pharmacol., 22,250 (1982). T.V. Dunwiddie and T. Worth, J. Pharmacol. Exp. Ther., =,70 (1982). J.J. Katims. 2. Annau and S.H. Snyder. J. Pharmacol. Exp. Ther., 227.167 (1983). S-M. Yeung and R.D. Green, PharmacoloRist, 23.184 (1981). R.S.L. Chang. V. Van Tran and S.H. Snyder. EUK. J. Pharmacol.. 3 . 4 6 3 (1978). G.A. Gajtkowski, D.B. Norris, T.J. Rising and T.P. Wood, Nature. 3 2 , 6 5 (1983). M.L. Torchiana. R.J. Pendleton. P.C. Cook, C.A. Hanson and B.V. Clineschmidt. J. Pharmacol. Exp. Ther., 224,514 (1983). M.C.C. Cillan and H.W. Kosterlitz, Brit. J. Pharmacol., 77,461 (1982). L.E. Robson and H.W. KosteKlitZ, Proc. Roy. SOC. London. Series B. 205.425 (1979). G.W. Pasternak. Proc. Aat. Acad. Sci. U.S.A.. 77. 3691 (1980). R.W. Foote and R. MauKeK. EUK. J. Pharmacol., E.99 (1982). G.W. Pasternak, M. CaKKOll-BUatti and K. Spiegel, J. Pharmacol. Exp. Ther.. 2 , 1 9 2 (1981). K. Spiegel and C.W. Pasternak, J. Pharmacol. Exp. Ther.. 228.414 (1984). M. Williams, J. M&d. Chern.. g.619 (1983). A.S. Lippa, D.J. Critchett, M.C. Sano, C.A. Klepner, D.W. Creenblatt, J. Cooper and B. Beer, Pharmacol. Biochem. Behav., 10.831 (1979). P.J. Marangos, J. Patel. J-P. Boulenger and Clark-Rosenberg, Mol. Pharmacol., 22.26 (1982). 1.L. Martin, C.L. B r o w and A. Doble. Life Sci..L2.1925 (1983). C. Braestrup, R. Schmiechen, G. Neef. M. Nielsen and E.N. Petersen, Science. 216,1241 (1982). =okiyama and T. Glenn in "Abstracts of Papers;' 184th National Meeting of the American Chemical Society, Kansas City, MO.,Sept. 12-17, 1982. Abstr. MEDI 58. M. Willlams and E.A. Risley. Life S c i . , 24,833 (1979). K-J. Chang and P. CuatreCaSaS i n "Brain Peptides," D.T. Krieger. M.J. Brownstein and J.B. Martin, Eds., Wiley Interscience, New York, 1983, p. 565. C-M. Lee, L.L. Iversen. M.R. Hanley and B.E.B. Sandberg. Naunvn- Schmiedeberg's Arch. Pharmacol., 318,281 (1982). R.F. Bruns, W.J. Thomsen and T.A. Pugsley. Life Sci., 33,645 (1983). R.A. Janis and D.J. Triggle, J. Med. Chem., 2 , 7 7 5 (1985). R.J. Miller, Life Sci., in press (1984). J-P. Calizzi, M. Fosset and M. Lazdunskl, Biochem. Biophys. Res. Commun.. 118.239 (1984). R.J. Could, K.M.M. Murphy and S.H. Snyder. Proc. Nat. Acad. Sci. U.S.A.,=3656 (1983). M. Schramm, G. Thomas, R. Towart and C. Franckoniak, Nature, 303.535 (1983). P. Erne, E. Burgissen, P.R. Buhler, B. Dubache, H. Kuhnis, M. Meier and H. Rogg, Biochem. Blophys. Res. Commun., 118,842 (1984). I. Shallaby, S.A. Freedman and R.J. Miller, SOC. Neurosci. Abstr.. 2,565 (1983). R. Raisman, M.S. Briley and S.Z. Langer, Nature, 281,148 (1979). E.T. Mellerup, P. Plenge and M. Engelstoft, Eur. J. Pharmacol., 9 6 , 3 0 3 (1983). C.M. Lee, J.A. Javitch and S.H. Snyder, J. Neurosci., 2.1515 (1982). R. Quirion and C.B. Pert, Eur. J. Pharmacol., E , I 5 5 (1982). D.A. Kendall, D.P. Taylor and S.J. Enna, Mol. Pharmacol., 2 , 5 9 4 (1983). B.V. Clineschmidt, M. Williams, J.J. Witoslawski, P.R. Bunting, E.A. Risley and J.A. Totaro, Drug Development Res., 2,147 (1982). B.V. Clineschmidt, Gen. Pharmacol.. 10.287 (1979). M. Williams and E.A. Risley, Fed. P r z . , 2 . 1 3 3 0 (1982). R.W. Olsen. P. Van Ness. C. Napias, M. Bergman, and W.W. Tourtelotte, Adv. Biochem. Psychopharmacol., 21.451 (1980). M.S. Briley, S.Z. Langer, R. Raisman, D. Sechter and E. Zarifan, Science, =,30(198G) W.S. Young and M.J. Kuhar. Brain Res., 179,255 (1979).

Chapter 29. Forskolin and Adenylate Cyclase: New Opportunities in Drug Design Kenneth B. Seamon, National Center for Drugs and Biologics, FDA Bethesda, MD 20205 Cyclic AMP is synthesized by the membrane-bound enzyme adenylate cyclase which is present in almost all mammalian tissues. Hormonal regulation of adenylate cyclase is initiated by the hormone binding to cell surface receptors. The resulting hormone-receptor complex interacts with either stimulatory regulatory proteins, (Ns), or inhibitory regulatory proteins, (Ni), which bind guanine nucleotides and interact with the catalytic subunit (C) of adenylate cyclase to modulate the enzyme activity. ATP

CAMP

-FO

RS KO LI N

/ /

GTP > 40nm

INHIBITION

STIMULATION

Hormone receptor agonists and antagonists have been used for years as therapeutic agents which affect cyclic AMP levels by acting at receptors. Although there has been much progress in the isolation and purification of the hormone receptors and the regulatory proteins, Ns and Ni, there has been less progress in the purification and characterization of the catalytic subunit of adenylate cyclase. Forskolin (l), the major pharmacologically active diterpene isolated from the roTts of Coleus for~kohlii,l-~ can directly activate the

-1

0

H3C

15

CH3 OH

catalytic subunit of adenylate cyclase, and is offering new insights into the regulation of this complex enzyme system. This review will discuss the unique properties of forskolin as an activator of adenylate cyclase which are of potential interest to the medicinal chemist.

ANNUAL REPORTS IN MEDICINAL CHEMISTKY- 19

Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-0405 19-9

29 4

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Drug Design

Allen, Ed.

Activation of Adenylate Cyclase - Metzger and Lindner first demonstrated that forskolin activates adenylate cyclase in rabbit heart Subsequent studies demonstrated that forskolin and liver membranes .4 9 could activate adenylate cyclase in rat brain membranes,6 and in membranes from most mammalian tissue^.^ The EC50 for forskolin activation is generally between 211M and 101~M. The maximal extent of activation varies among different tissues and cells. Adenylate cyclase in membranes from rat liver is activated 20-f0ld,~while adenylate cyclase in turkey erythrocyte membranes is activated only 3-fold over basal level^.^ Forskolin activation of adenylate cyclase is observed in the presence of cyclic AMP phosphodiesterase inhibitors and does not require the presence of calcium or calmodulin.6 Forskolin activates solubilized adenylate cyclase with similar EC50's and maximal activations as observed for membrane-bound enzyme .l0-12 Forskolin increases the Vmax of adenylate cyclase with little or no effect on the Km for the substrate, MgATP or MIIATP.~~Forskolin has no effect on the Ka for MgC12 in activating liver or platelet adenylate cyclase.*r13 Forskolin can protect adenylate cyclase in membranes from human platelets, 549 lymphoma cells, and rat brain against thermal denaturation, proteolytic inactivation, and inactivation with N-ethylmaleimide (NEM) .I3 Solubilized preparations of adenylate cyclase are also protected against thermal denaturation by forskolin.l1 These include preparations which do not contain functional Ns protein. Aden late cyclase from sperm or testis is not stimulated by f~ r s k o l i n . ~ ~ These enzymes are not hormonally responsive and are not associated with Ns or Ni proteins. Forskolin can activate sperm membrane adenylate cyclase when extracts from human erythrocyte membranes are added to sperm membranes; however, it is not clear if the reconstitution is revealing adenylate cyclase activity in the human erythrocyte membrane or adding back a factor required for forskolin stimulation of the sperm membrane adenylate ~yclase.~5,16 Aden late cyclase from invertebrates such as mollusks 17-20 and insects21sH2 is stimulated by forskolin. Adenylate cyclases from 5. Pertussis,23 E. C~liN , e~u r o ~ p o r a ,and ~~ D. Discoidium7 are not stimulated by forskolin or Ns. Interaction with the Catalytic Subunit - Forskolin stimulates adenylate cyclase in membranes from the S49 c c- 1 phoma cell about 20-fold with an EC50 of about 25 micromolar.10~23,25-~ As this mutant of the S49 lymphoma cell line does not contain a functional Ns subunit, it was proposed that forskolin is activating adenylate cyclase by an interaction at the catalytic subunit or other closely associated protein.1° Forskolin can activate other adenylate cyclase preparations which are unresponsive to hormones or guanine nucleotides and do not contain functional Ns protein. These include detergent solubilized preparations of aden late cyclase from rat striatum,ll cortex,12 heart,12 and liver.3B In the absence of a functional Ns protein, it is necessary to include Mn2+ in the assay to measure the activity of the catalytic subunit. Forskolin does not require Mn2+ to stimulate the catalytic subunit.

Chap. 29

Forskolin and Adenylate Cyclase

Seamon

295

The 7-0-hemisuccinate-7-desacetyl derivative of forskolin has been coupled to Sepharose to form an affinity resin.12 Solubilized adenylate cyclase from rat brain and heart, but not pigeon erythrocytes, binds to this resin and can be eluted from the resin when forskolin is included in the buffer. Preparations of adenylate cyclase devoid of Ns activity are recovered from this column consistent with the proposai that forskolin binds to the catalytic subunit(s). Adenylate cyclase preactivated with guanosine-5',3,y-imidodiphosphate (GppNHp) also binds to the forskolin-Sepharose column and elutes from the column as a complex of the catalytic subunit(s) and the activated Ns protein.31

-

Potentiation of Forskolin Stimulation by the Ns Protein Forskolin stimulation of adenylate cyclase can be potentiated by guanine nucleotides and the Ns protein. Forskolin stimulation of adenylate cyclase in wild type S49 lymphoma cell membranes exhibits a time lag and biphasic kinetics indicative of low (Ka=22.OllM) and high (Ka=0.35uM) affinity sites of a ~ t i o n . ~ Forskolin ~,~~ stimulation of adenylate cyclase in S49 cyc- membranes, which do not contain Ns protein, has kinetics consistent with one site of action with a Ka of 244pM. The high affinity component for activation of the adenylate cyclase in S49 membranes by forskolin requires the Ns protein. The lag time in activation of the wild type S49 adenylate cyclase by forskolin is also associated with the presence of the Ns protein and is not observed in the presence of isoproterenol. Preparations of the catalytic subunit which do not have functional Ns protein can be stimulated by the addition of Ns and GppNHp and forskolin can potentiate this stimulation.ll This potentiation requires that forskolin, Ns, GppNHp, and the catalytic subunit be present at the same time. Preactivated Ns (Ns-GppNHp) and forskolin are only additive in their stimulation of the catalytic subunit when they are present in the assay at the same time. Forskolin can potentiate GppNHp stimulation of adenylate cyclase in membranes from some tissues. This potentiation can be observed when the effect of the Ni protein is suppressed by treatment with pertussis toxin,33 or by including manganese chloride in the assay medium.34 Activation of Cyclic AMP Generation in Intact Cells - Forskolin activates adenylate cyclase in intact cells and tissues with similar characteristics as those observed for activation of the enzyme in membranes and solubilized preparations. These include preparations of rat35 and human36 adipocytes, human latelets,37 tissue slices from brain and other peripheral tissues,315 and various endocrine and secretory tissues.39 Forskolin stimulates adenylate cyclase in S49 lymphoma cells,25p32 rat astrocytomas 40341 rat pheochromoc toma cells,42 cultured pituitary cells ,43-28 cardiomyocytes,49 9 51; cultured leydig cells,51 and cultured kidney cells. 52 * 53 Forskolin increases intracellular cyclic AMP rapidly with an EC50 of about 10uM, and results in elevations of cyclic AMP over basal levels which range between two and fifty-fold, depending on cell type and tissue. Increases in intracellular*cyclic AMP by forskolin occur rapidly and are reversible. Increased levels of cyclic AMP are maintained in tissue culture over long periods of time when forskolin is kept in the culture media. Cyclic AMP phosphodiesterase inhibitors augment the increases in intracellular cyclic AMP elicited by forskolin.

29 6

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Allen, Ed.

Paralleling membrane studies, forskolin elicits increases in intracellular cyclic AMP in the cyc- mutants of the S49 lymphoma cell line which are about ten-fold less than the increase elicited in wild type S49 lymphoma cells.25 Similar results are seen in the H21a mutant which contains a lesion in Ns impairing its coupling to the catalytic subunit. A maximal stimulation of cyclic AMP synthesis by forskolin in S49 cells requires the presence of a functional Ns protein. Forskolin stimulation of cyclic AMP synthesis in C6-2B rat astrocytoma cells is decreased after the cells are grown in the presence of cycloheximide, a protein synthesis inhibitor.4O This occurs without the loss of isoproterenol or cholera toxin stimulated adenylate cyclase. It was suggested that the cycloheximide blocks the synthesis of a protein which has a high turnover rate and is required for forskolin stimulation of adenylate cy~lase.~o

-

Potentiation of Hormone Action Low concentrations of forskolin (50uM) inhibits basal, hormone-stimulated GppNHp stimulated, and forskolin stimulated adenylate c y ~ l a s e . ~ ~ This inhibition is not competitive with forskolin and has been suggested to be due to the interaction of the metal ion at a cation binding site on the catalytic subunit. Forskolin activation of adenylate cyclase can be inhibited b high concentrations of detergents and low molecular weight alcohols.74 ~~5 Inhibition of forskolin stimulated adenylate cyclase by ethanol can be observed at 0.2M and results in 50% inhibition at 1.OM. Butanol and propanol are more effective at inhibiting forskolin stimulated adenylate cyclase than ethanol. Dimethyl sulfoxide inhibits the stimulation of adenylate cyclase by forskolin much less than ethanol .75 Physiological Effects of Forskolin - Forskolin elicits physiological effects consonant with its ability to stimulate adenylate cyclase and increase intracellular cyclic AMP-. This has been studied in many different tissues and cells and a complete description of all systems is beyond the scope of this review. Secretory responses which are elicited by forskolin include: chloride-secretion across rat colon descendens,76 acid and pepsinogen secretion from astric gland ,77 ~ 7 8prolactin secretion from pituitary cells y43 *4f luteinizing hormone secretion from pituitary,79 renin secretion from perfused kidney,80 insulin release from pancreatic cells,81 ACTH release from pituitary cell^,^^,^^ and amylase secretion from pancreatic acinor cells .82 Other responses which are elicited by forskolin and have been associated with increased intracellular cyclic AMP include li 01 sis in adipocytes,35 ~ 3 6 ~ 5 4 inhibition of platelet a g g r e g a t i ~ npT2 ,~~ ,83 relaxation of smooth muscle,2,3,84 and stimulation of steroidogenesis.57 ,85 Forskolin increases cyclic AMP content in oocytes and inhibits progesterone induced meiosis. 86-92 Forskolin increases sodium transport in cultured epitheliag3 and bicarbonate transport in choroid plexus.94 Reduction of intraocular pressure b forskolin has been observed in rabbit, human, and monkey eye.95,9g Forskolin stimulates transcription of the prolactin gene in pituitaryg7 and increases the biosynthesis of enkephalin in bovine chromafin cells .98 Forskolin elicited physiological responses are usually potentiated by the natural hormone consistent with the hormonal potentiation of forskolin elicited increases in cyclic AMP. Forskolin has been shown to affect enzymes other than adenylate cyclase indirectly. Treatment of rabbit cardiac slices with forskolin results in the inhibition of the membrane bound Na+ K+ ~ ~ p ~ ~ ~ . 9 9

29 8

Sect. VI

- Topics in Chemistry and Drug Design

Allen, Ed.

The inhibition of the ATPase is probably due to a cyclic AMP mediated phosphorylation. Tyrosine hydroxylase is activated in striatal slices treated with forskolin,lOO also due to a cyclic AMP mediated phosphorylation. Structure Activity Relationships for Forskolin Analogs - Forskolin makes up 1% of the dry weight of the roots of Coleus forskohlii and is the major diterpene that is isolated from the plant. Other structurally similar diterpenes that occur naturally in the plant include 6-acetyl-7-desacetylforskolin (k), 7-desacetylforskolin (5), 1,9-dideoxyforskolin (7), and 9-deoxyforskolin @). A numEer of semisynthetic derivatiTes of forskolin have been prepared.101,102 These and the naturally occurring analogs of forskolin have been tested for their ability to activate adenylate cyclase.103

2 3

4

OH OH OH

5 OH OH Z H OH

OH OH OH OH OH

OH

OCOEt OC0,Et OSOZ-&Me-Ph OH OH OCOMe OCOMe

OH

OCOMe

OH

OH 'H

OH OH

OCOMe OCOMe

OH OH

0 0

OH OH OH OH

OCOMe OH

CH-=H, CWH, CHXH, cn=H, CH=CH2 CH=CH, CMH, CH=CH,

H H

9 o\

The 1-and 9-hydroxyl groups define an important area for forskolin's action. The 1,9-dideoxyderivative (L) is totally inactive and the 1,9,6,7-dicarbonate (9), and 1,9 sulfonate are very weak at activating adenylate cyclase. OtFer structurally related compounds which are unable to activate adenylate cyclase are 1,6-diketoforskolin, the 14,15-oxide (12), virescenol-B, abietic acid, podocarpic acid, retene, gibberellin, and other diterpenes(l3-15) - - 023983 None of the inactive diterpenes antagonize forskolin stimulation of adenylate cyclase. Derivatives of

Chap. 29

Forskolin and Adenylate Cyclase

Seamon 299

forskolin which are partially active include a number of 7-acyl 3) derivatives. The most active of these include the 7-ethylcarbonate ( and the 7-propionate (2) which are almost equipotent with forskolin. The 7-formyl, 7-desacetyl and 7-hemicsuccinate are less potent than forskolin with EC50's that range from 50pM to 150pM. The 7-tosyl derivative (4) is much less potent than the other 7-acyl derivatives. The 14,15-diKydroforskolin (11) and llphydroxy-forskolin (10) are less potent than forskolin w z h EC50's of about 50 pM. The activity of forskolin analogs does not correlate with lipophilicity.

(z),

12-t3H] forskolin (sp. act. 27 Ci/mmol) has been s nthesized and used to determine forskolin binding sites in membranes 12-[3H] forskolin binds to rat brain membranes with high affinity, Kd = 27nM, Bmax = 275 fmol/mg protein. The ability of forskolin analogs to compete for these binding sites correlates with their ability to activate adenylate cyclase.

.Io4

Therapeutic Potential - Forskolin is a potent hypotensive agent due to its peripheral vasodilatory properties. Reduction in blood pressure is observed in dogs (20ug/kg i.v.), renal hypertensive rats (50uglkg i.v.), and spontaneous hypertensive rats (10 mg/kg, p.0.) .2s105 Forskolin is also a potent positive inotropic agent as observed in isolated guinea pig heart and atrial preparations, and in dog and cat hearts ~ i t r o ,Io5 . ~ Cardiac output in anesthesized dog was increased at doses ranging from 5ug/kg to 100ug/kg, i.vm2 Forskolin antagonizes histamine or acetylcholine induced bronchoconstriction making it a potent bronchodilator.lo6 ,Io7 Forskolin inhibits platelet aggre ation in vitro and -in vivo, making it a possible anti-thrombotic agent.3$,72,83Forskolin produces a rapid and long lasting reduction in intraocular pressure upon topical application to the eye, and would appear to be a valuable agent to treat glaucoma .959 96 Forskolin has been shown to inhibit pulmonary tumor colonization in mice b B16 murine melanoma cells and is a potential antimetastatic agent .log Summary - Forskolin activates hormone sensitive adenylate cyclase in a completely unique manner. Its ability to potentiate hormonally induced increases in cyclic AMP provides a new mechanism to potentiate hormonal responses -in vitro and -in vivo; The exact site of action of forskolin still remains unclear, and it is possible that forskolin acts directly at the catalytic subunit of adenylate cyclase or at another subunit which has not yet been identified. The possibility still remains that an endogenous forskolin compound might exist, which would have the ability to either stimulate adenylate cyclase directly or to put the enzyme in a potentiated state which would be exquisitely sensitive to hormonal stimulation. It also remains to be determined if -in vivo effects of forskolin are due to the direct stimulation of adenylate cyclase by forskolin or to the potentiation of the action of natural hormones which are in the circulation. Binding sites for forskolin in membranes can now be studied using the radiolabelled forskolin, providing an easy assay to search for forskolin agonists and antagonists. The radiolabelled forskolin should also provide a tool to study the metabolism and disposition of forskolin after -in vivo administration.

300

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Allen, Ed.

In over 300 articles in the past three years forskolin has been established as a powerful tool to study adenylate cyclase in vitro and in vivo. It is also anticipated that potential new uses of forskolin as -a therapeutic agent will continue to be investigated. Studies with forskolin have thus provided medicinal chemists with a new target in the design of therapeutic agents, the adenylate cyclase enzyme. Acknowledgement - I would like to acknowledge the cooperation and support of Dr. Noel J. deSouza, Dr. Jurgen Reden, and their colleagues at Hoechst Pharmaceuticals, Bombay during our investigations on forskolin. This review is dedicated to the memory of Dr. Balbir S. Ba jwa who made such significant contributions to our understanding of the medicinal chemistry of forskolin.

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Chap. 29

F o r s k o l i n and A d e n y l a t e C y c l a s e

-

Seamon 301

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302,

302

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- Topics in Chemistry and Drug Design

Allen, Ed.

85. L. Hedin and S. Rosberg, M o l . Cell. Endocrinology, 33, 69 (1983). 86. N. Dekel and I. S h e r i z l y , FEBS Lett., 151,153 (198g. 87. S.B. Oldham, C.T. Molloy. L.G. Lipson, and T.T. Boggs, Endocrinology, 114, 207 (1984). 88. P.J. O l sie wski and W.H. Beers, Dev. Bi ol ., 100. 287 (1983). 89. S. S c h o r d e r e t s l a t k i n e and E.M. Baul i eu, Endocrinology 111, 1385 (1982). 90. R.M. S c h u l t z , R.R. Montgomery, and J.R. B e la noff, Dev. Biol. , 97, 264 (1983). 91. R.M. S c h u l t z , R.R. Montgomery, P.F. Wardbailey, and J.J. Eppig, Dev. B i o l . , 294 (1983). 92. F. Urner, W.L. Herrmann, E.E. Baulieu, and S. S c h o r d e r e t s l a t k i n e , Endoc rinology, 113, 1170 (1983). 93. A.W. C u t h b e r t , and J.A. Spayne, Br. J. Pharmacol., 2, 33 (1982). 94. Y. S a i t o and E.M. Wright. J. P h y s i o l . (Lond.), 336, 635 (1983). 95. S.P. Bartels, S.R. Lee, and A.H. Neufeld, Curr. Eye Res., 2, 673 1983). 96. J. C a p r i o l i and M. S e a r s , L ancet , 1, 958 (1983). 97. G.H. Murdoch, M.G. Rosenfeld, and E.M. Evans, Sc ie nc e , 2,1315 1982). 98. L.E. Eiden, and A.J. Hot chki ss, Neuropept ide s , 4, 1 (1983). 99. F. Spah, J. Cardiovasc. Pharmacol., 6, 99 (19847. 100. I.R. Katz, D. Smith, and M.H. MakmanT Brain Res., 264, 173 (1983). 101. S.V. Bhat, A.N. Dohadwalla, B.S. B a j w a , N.K. Dadkar, H. Dornauer, and N.J. Desouza, J. Med. Chem., 2, 486 (1983). 102. S.V. Bhat, B.S. Bajwa, H. Dornauer, and N.J. de Souza, J. Chem. Soc., P e r k i n Tra ns . I, 767 (1982). 103. K.B. Seamon, J.W. Daly, H. Metzger, N . J . de Souza, and J. Reden, J. Med. Chem., 26, 436 (1983). 104. KB. Seamon, R. V a l l a n c o u r t , M. Edwards, and J.W. Daly, Proc. Nat. Acad. S c i . U.S.A., in p r e s s (1984). 105. N . J . de Souza, A.H. Dohadwalla, and J. Reden, Med. Res. Rev., 2, 201 (1983). 106. J.F. Burka, J. Pharmacol. Exp. "her., 225, 427 (1983). 107. J.F. Burka, Can. J. P h y s i o l . Pharmacol., 61, 581 (1983). 108. K.C. Agarwal and R.E. P arks, I n t . J. Cancer, 32, 801 (1983).

Chapter 30.

Recent P r o g r e s s i n t h e R a t i o n a l Design of P e p t i d e Hormones and N e u r o t r a n s m i t t e r s

V i c t o r J. Hruby, John L. Krstenansky, and Wayne L. Cody Department of Chemistry, U n i v e r s i t y of Arizona, Tucson, AZ 85721

- Any e f f o r t t o d i s c u s s p r o g r e s s i n " r a t i o n a l design" of pept i d e n e u r o t r a n s m i t t e r s and hormones ( h e r e a f t e r r e f e r r e d t o as p e p t i d e hormones) poses c e r t a i n d i f f i c u l t i e s . P e p t i d e hormones g e n e r a l l y produce t h e i r b i o l o g i c a l e f f e c t s by i n t e r a c t i o n w i t h membrane bound r e c e p t o r s , but w e know l i t t l e about t h e s p e c i f i c chemical and p h y s i c a l ( t h r e e dimens i o n a l ) p r o p e r t i e s of t h e s e binding sites. Thus any r a t i o n a l d e s i g n must u t i l i z e o u r a b i l i t y t o deduce t h e p r o p e r t i e s of t h e r e c e p t o r system ( h o s t ) by u s i n g w e l l designed p e p t i d e hormone analogs ( g u e s t s ) , and c a r e f u l chemi c a l and p h y s i c a l a n a l y s i s of t h e s t r u c t u r e , conformation and dynamic prop e r t i e s of t h e s e analogs i n r e l a t i o n t o b i o l o g i c a l a c t i v i t i e s . -I-n--t r o d u c t i o n

U l t i m a t e l y hormone-receptor i n t e r a c t i o n s are t h r e e dimensional i n n a t u r e , i n which two complementary t o p o l o g i c a l rsurfaces i n t e r a c t t o produce a b i o l o g i c a l response. These t h r e e dimensional f e a t u r e s o f t e n can be examined by conformational r e s t r i c t i o n of t h e p e p t i d e hormones. 1-8 For l a r g e r p e p t i d e hormones 0 2 0 r e s i d u e s ) , design of e x t e n s i v e secondary s t r u c t u r e s such as amphiphilic h e l i c e s can provide important i n s i g h t s i n t o membrane binding, r e c e p t o r binding, and o t h e r a s p e c t s of b i o l o g i c a l recogn i t i o n . This approach has r e c e n t l y been reviewed9-11 and w i l l n o t be d i s c u s s e d here. Rather we w i l l c o n c e n t r a t e on smaller p e p t i d e s (0*co \ TI

(CH2) 2OCH3

Hb-.

Enocitabine is an antileukemic agent closely related to cytarabine. It appears more resistant to deamination than cytarabine, thus allowing greater in vivo phosphorylation into an active cytotoxic metabolite.

EperisomeHydmchbn'de (muscle r e l a ~ a n t ) 3 ~ , 3 9 Country of Origin: Japan Originator: Eisai First Introduction: Japan

. C2Hg-*'

Introduced Trade Name: by: MYONAL E M

/-*\ 'o=o'

k . 0 HCH2

ZH3

.--.

'0-0':~~~

Eperisone hydrochloride is a centrally acting muscle relaxant useful in the management of various spastic conditions including cervical spondylosis and cerebral palsy. It is structurally related to tolperisone. Epoprostenol sodium (platelet aggregation i n h i b i t ~ r ) ~ O , ~ l Country of Origin: Originator: First Introduction: Introduced by: Trade Names:

United Kingdom Burroughs Wellcome United Kingdom Burroughs Wellcome; Upjohn FtoLAN;CYCLO-PROSTIN

8H

Epoprostenol sodium (prostacyclin) is a naturally occurring prostaglandin indicated for the preservation of platelet function during cardiopulmonary bypass, prevention of platelet aggregation during charcoal hemoperfusion of patients in hepatic failure, and as an alternative to heparin during renal dialysis.

Fenbuprol (choleretic)4&43 Country of Origin: Originator: First Introduction: Introduced by: Trade Name:

W. Germany Klinge Pharma W. Germany Rhom Pharma VALBIL

,.=. l H

,8"8/.-O-CHz

HCliflC4Hg

Fenbuprol produces an increase in the volume of bile secretion. It is useful in patients having symptomatology associated with biliary tract dysfunction. Flutamide (antineoplastic)%45 Country of Origin: Originator: First Introduction: Introduced by: Trade Name:

USA scheting Chile Schering DROGENIL

( CH 3 2

Flutamide is an orally active, non-steroidal antiandrogen indicated for the treatment of prostatic cancer in both castrates and noncastrates.

Allen

To Market, To Market

Chap. 31

Fluvoxamine Maleate (serontonergic antidepressant)46-48 Country of Origin: Netherlands Originator: First Introduction: Introduced by: Trade Name:

Duphar Switzerland Kali-Duphar FLOXYFRAL

F3C-0,

.=.

p*-*\,

/ 3i-CH2-CH2-CH2-CH2-0-CH3 N-O-CH 2-CH 2-NH 2 ‘C4H404

Fluvoxamine maleate is the most recent of the serotonin-specific antidepressants to reach the market. In vitro and in vivo animal experiments have shown fluvoxamine t o have a marked effect on 5-HT mediated processes and little effect on norepinephrine. Clinical trials suggest similar efficacy t o imipramine and clomipramine with a somewhat lower incidence of side effects, especially anticholinergic effects. Fluvoxamine, in contrast to the tricyclic antidepressants, does not appear to produce heart rate increase, postural hypotension or prolongation of the intraventricular conduction t i m e and QT interval.

Gallopamil Hydrochloride (antianginal)49 Country of Origin: Originator: First Introduction: Introduced by:

W. Germany

Knoll W. Germany Chem. Werke Minden Trade Name: PROCORUM

CH 30

Gallopamil hydrochloride is a somewhat more potent methoxy analog of calcium channel blocker verapamil with a similar profile. It is useful in the treatment of angina and auricular arrhythmia.

Gemeprost ( a b ~ r t i f a c i e n t ) ~ ~ , ~ ~ Country of Origin: Originator: First Introduction: Introduced by:

Japan On0

MalaysiaJSingapore May & Baker

Trade Name: CERVAGEM

*/*\ */’\*/

o\*-.P+*\

CO 2CH 3

/

'a-•

Ho\+’

1.4

\*/

\*/ \*/

dH

Gemeprost, a metabolically stabilized analog of PGE1,has been demonstrated t o reliably induce termination of early pregnancy when administered as a vaginal suppository. Its effect is presumably due to both uterine contraction and rapid decline of steroid hormone levels. Minimal side effects have been reported.

Guanadrel Sulfate (antihypert ensive)%53 Country of Origin: Originator: First Introduction: Introduced by: Trade Name:

USA Cutter USA Penuwalt WLOREL

Guanadrel sulfate is an antihypertensive belonging t o the class of adrenergic neuron blocking drugs. It diminishes sympathetic vasoconstriction by inhibiting norepinephrine storage and release from neuronal storage sites. It appears similar in effectiveness and side effect profile to its structural relative guanethidine.

319

320

Sect. VII

- Worldwide Market

Introductions

a

Halometasone (topical antiinflammatory)%55 Country of Origin: Originator: First Introduction: Introduced by: Trade Name:

Allen, Ed.

Switzerland Ciba-igy Switzerland Ciba-Geigy SICORTEN

Halometasone is a Dotent,. topical - steroid useful in a variety of acute and chronic eczematous dermaioses and psoriasis. It is reportedly devoid of skin toxicity and systemic effects. Hydrocortisone Butyrate Propionate (topical antiinflammat0ry)5~ GH flCOCH 2CH 3 Country of Origin: Originator: First Introduction: Introduced by: Trade Name:

Japan Taiaho Japan Taisho

PANDEL

Hydrocortisone butyrate propionate is a potent, topical steroid reported to possess minimal systemic side effects. It is indicated in the treatment of various acute and chronic contact, eczematous, and atopic dermatoses and psoriasis. Jndalpine (serotonergic a n t i d e p r e ~ s a n t ) ~ ~ - ~ g

Country of Origin: Originator: First Introduction: Introduced by: Trade Name:

France Pharmuka France Labs. Fournier Freres UPSTENE

.’! /.B c r ‘0-.

\*/

yH

Indalpine is a non-tricyclic antidepressant with a serotonin selective profile. It is 6-7 times more potent than fluoxetine and clomipramine in inhibiting serotonin reuptake in vitro in rat brain synaptosomes. Statistically significant clinical effects within one week of onset of treatment have been reported. An anxiolytic effect may accompany the antidepressant effect. Indalpine appears devoid of anticholinergic and cardiovascular side effects and does not promote weight gain or affect appetite. sox xi cam (antiinflammatory)6&62 Country of Origin: Originator: First Introduction: Introduced by: Trade Name:

USA Warner-Lambert W. Germany Warner-Lambert

PAC=

Isoxicam is a non-steroidal antiinflammatory agent useful in the treatment of various forms of rheumatoid arthritis, osteoarthritis and musculoskeletal disorders. It is about one-tenth as potent as its structural relative sudoxicam; its similar long TQ (>30hrs.) allows once-daily dosing.

Chap. 31

To Market, To Market

Allen

Loprazolam Mesylate (hypnotic/sedative)63,64 Country of Origin: United Kingdom Originator: R o w e l First Introduction: United Kingdom Introduced by: Roussel Trade Name: DORMONOCT

*-a

p