Annual Reports Medicinal Chemistry, Volume 07

ANNUAL REPORTS IN MEDICINAL CHEMISTRY Volume 7 CONTRI BUTORS Bach. M . K. . . . . . . . Benet. L . ....... Z . R...

Author: Richard V. Heinzelman

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ANNUAL REPORTS IN MEDICINAL CHEMISTRY Volume 7

CONTRI BUTORS

Bach. M

. K. . . . . . . .

Benet. L

. ....... Z

. R. . . . . . . Bundy. G . L . . . . . . . Burgus. R . . . . . . . . Blohm. T

238

Lienhard. G

259

Morrow. D

169

157 194 99

........ Cheng. . . . . . . . . 129 Colten. H . R . . . . . . . 228 Cronin. T . . . . . . . . 119 Czuba. L . J . . . . . . . 78 Davies. J . . . . . . . . 217 . . . . . . . . 109 Drube. Engelhardt. E . L . . . . . 6 Friis. W. . . . . . . . . 39 G a l l o . D . . . . . . . . . 182 Gandour. R . D . . . . . . 279 . A . . . . . . 47 Harbert. Hitchings. . H. . . . . 1 Hoffer. . . . . . . . . 145 Hudyma. T . W . . . . . . . 208 Juby. P . F . . . . . . . . 208 Kaiser. . . . . . . . . 6. 18 Butler. K C

C

H

C

G

C

G

M

C

. E. . . . . . . .

. F. . . . . . . . . Nagasawa. H . T . . . . . . . . Pachter. 1 . J . . . . . . . . Rachlin. A . I . . . . . . . . Robinson. F . M . . . . . . . . Roe. A . M . . . . . . . . . . Rudzik. A . D . . . . . . . . . Sciavolino. F . C . . . . . . . Schowen. R . L . . . . . . . . Schwender. C . F . . . . . . . Stewart. J . M. . . . . . . . Thomas. R . C . . . . . . . . . Thompson. J . A . . . . . . . . Topliss. J . G. . . . . . . . Tozzi. S. . . . . . . . . . . Waitz. J . A . . . . . . . . . Wechter. W . J . . . . . . . . Weissman. A . . . . . . . . . Wiley. R . A . . . . . . . . . Z i r k l e . C . L. . . . . . . . .

249 182 269

157 145 31

59 39 99 279

69 289 296 269

59

89 109 217

47 259

6. 18

ANNUAL REPORTS IN MEDICINAL CHEMISTRY Volume 7 Sponsored by the Division of Medicinal Chemistry of the American Chemical Society Editor-in- Chief: RICHARD V. HEINZELMAN THE UPJOHN COMPANY KAUMAZOO, MICHIGAN

SECTION EDITORS EDWARD ENGELHARDT IRWIN PACHTER

@ACADEMIC

0

0

JOHN TOPLISS

WILLIAM WECHTER

PRESS

0

0

KENNETH BUTLER

ROBERT WILEY

New York and London 1972

COPYRIGHT 0 1972, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR A N Y OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC P R E S S , INC.

111 Fifth Avenue, New York. New York 10003

Uniied Kingdom Edition published by

ACADEMIC P R E S S , INC. ( L O N D O N ) 24/28 Oval Road, London NW1

LTD.

LIBRARYOF CONGRESS CATALOG CARDNUMBER: 66-26843

PRINTED IN THE UNITED STATeS OF AMERICA

CONTENTS

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

Contributors

ii

Preface

ix

AWARD ADDRESS T h i r d Award Address

-

Medicinal Chemistry

. .- . . .

I n h i b i t o r s o f F o l a t e B i o s y n t h e s i s and U t i l i z a t i o n E v o l u t i o n a r y Changes as a Basis f o r Chemotherapy , George H. H i t c h i n g s , Wellcome Research L a b o r a t o r i e s , Research T r i a n g l e Park, N. C.

1. Section Editor:

1

CNS AGENTS

Edward L . Engelhardt, Merck Sharp and Dohme Research Laboratories, West P o i n t , Pennsylvania 19486

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

6

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

18

1.

A n t i p s y c h o t i c and A n t i - a n x i e t y Agents Charles L. Z i r k l e and C a r l Kaiser, Smith K l i n e & French L a b o r a t o r i e s , Ph i 1ade 1 ph ia, Pa.

2.

Antidepressives and S t i m u l a n t s Carl K a i s e r and Charles L. Z i r k l e , Smith K l i n e & French L a b o r a t o r i e s , Ph i 1 adel ph i a , Pa.

3.

Analgesics and N a r c o t i c Antagonists Frank1 i n M. Robinson, Merck Sharp & Dohme L a b o r a t o r i e s , West P o i n t , Pa.

4.

Sedatives, Hypnotics, A n t i c o n v u l s a n t s and General Anesthetics A. D . Rudzik and W. F r i i s , The Upjohn Company, Kal amazoo, M ich igan

5.

,

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

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

......

Recent Developments R e l a t i n g S e r o t o n i n and Behavior A l b e r t Weissman and Charles A. H a r b e r t , P f i z e r Inc., Groton, Conn.

V

31

39

47

vi

CONTENTS

II. Section Editor:

John G.

PHARMACODYNAMIC AGENTS

Topliss,

S c h e r i n g Corp.,

B l o o m f i e l d , N.

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

6.

A n t i h y p e r t e n s i v e Agents Anthony M. Roe, Smi th K l i n e & French Research I n s t i t u t e , Welwyn Garden C i t y , H e r t f o r d s h i r e , England

7.

A n t i a n g i n a l Agents C h a r l e s F. Schwender, Warner-Lambert Research I n s t i t u t e , M o r r i s P l a i n s , N. J.

8.

A n t i t h r o m b o t i c Agents Leonard J. Czuba, P f i z e r ,

9.

69

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

78

Inc.,

Groton,

Connecticut

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

Pulmonary and A n t i a l l e r g y Agents S . T o z z i , S c h e r i n g C o r p o r a t i o n , B l o o m f i e l d , New J e r s e y

Section Editor:

89

CHEMOTHERAPEUTIC AGENTS

Kenneth B u t l e r , P f i z e r I n c . ,

Groton C o n n e c t i c u t

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

99

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

109

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

119

Antibiotics F r ank C. S c i a v o l i n o ,

P f i z e r Inc.,

Groton, C o n n e c t i c u t

11.

A n t i f u n g a l Agents J. A l l a n Wa i tz , C. G. Drube, S c h e r i n g C o r p o r a t i o n , B l o o m f i e l d , New J e r s e y

12.

A n t i v i r a l Agents T im ot h y H. C r o n i n , P f i z e r In c.,

13.

59

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

Ill.

10.

J.

Groton, C o n n e c t i c u t

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

A n t i n e o p l a s t i c Agents C. C. Cheng, Midwest Research I n s t i t u t e , Kansas C i t y , Missouri

129

vii CONTENTS

14.

A n t i p a r a s i t i c Agents M. H o f f e r and A. 1 . N u t l e y , New J e r s e y

IV. Section Editor:

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

R a c h l i n , Hoffmann-La

METABOLIC D I S E A S E S AND ENDOCRINE FUNCTION

I. J. P a c h t e r , B r i s t o l L a b o r a t o r i e s , New York

Syracuse,

. . . . . . . . . . . . . 157

15.

P r o s t a g l a n d i n s and R e l a t e d Compounds Gordon L. Bundy, The Upjohn Company, Kalamazoo, M i c h i g a n

16.

Atherosclerosis Thomas R. Blohm, M e r r e l l - N a t i o n a l L a b o r a t o r i e s , o f R i c h a r d s o n - M e r r e l l , Inc., C i n c i n n a t i , Ohio

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

169

Division

. . . . . . . . . . 182

17.

S t e r o i d s and B i o l o g i c a l l y R e l a t e d Compounds Duane F. Morrow and Duane G a l l o , Mead Johnson Research Center, E v a n s v i l l e , I n d i a n a

18.

P e p t i d e Hormones o f t h e Hypothalamus and P i t u i t a r y Roger Burgus, The S a l k I n s t i t u t e , La J o l l a , C a l i f .

19.

N o n - s t e r o i d a l A n t i i n f l a m m a t o r y Agents P e t e r F. Juby and Thomas W. Hudyma, B r i s t o l L a b o r a t o r i e s , Syracuse, New York

. . . . . . 194

. . . . . . . . . . . . . 208

V.

Section E d i t o r :

20.

145

Roche I n c . ,

T O P I C S I N BIOLOGY

W i l l i a m J. Wechter, The Upjohn Company, Kalamazoo, Michigan

. . . . . . . . . . . . 217

Mechanisms o f R e s i s t a n c e t o A n t i b i o t i c s J u l i a n Davies, Department o f B i o c h e m i s t r y , U n i v e r s i t y o f Wisconsin, Madison, W i s c o n s i n

viii CONTENTS

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

21.

The Serum Complement System Harvey R. Colten, Harvard Medical School, Boston, Mass.

22.

Immediate H y p e r s e n s i t i v i t y : L a b o r a t o r y Models and Exper imen t a 1 F ind ings Michael K. Bach, The Upjohn Company, Kalamazoo, M i c h i g a n

23.

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

238

........

Trans i t on S t a t e Analogs as Enzyme I n h i b i t o r s G. E. Lienhard, Dept. o f B i o c h e m i s t r y , Dartmouth Medical Schoo , Hanover, N. H.

VI. Section Editor:

228

249

TOPICS I N CHEMISTRY

Robert A. Wiley, U n i v e r s i t y o f Kansas, Lawrence, Kansas

......... . .

24.

Biopharmaceutics and Pharmacokinetics L e s l i e Z. Benet, School o f Pharmacy, U n i v e r s i t y o f C a l i f o r n i a , San F r a n c i s c o , C a l i f .

25.

269 Reactions o f I n t e r e s t i n M e d i c i n a l Chemistry H e r b e r t T. Nagasawa and John A. Thompson, C o l l e g e o f Pharmacy, U n i v e r s i t y o f Minnesota and Veterans Admin s t r a t on H o s p i t a l , M i n n e a p o l i s , Minnesota

26.

I n t r a m o l e c u l a r C a t a l y s i s i n M e d i n i c a l Chemistry R i c h a r d D. Gandour and R i c h a r d L. Schowen, Department o f Chemistry, U n i v e r s i t y o f Kansas, Lawrence, Kansas

27

Peptide Synthesis John Morrow Stewart, U n i v e r s i t y o f Colorado, Medicine, Denver, Colorado

28.

P r e p a r a t i o n o f Radioisotope-Labeled Drugs Richard C. Thomas, The Upjohn Company, Kalamazoo, M i c h i g a n

259

.........

. . . . . ..

. . . . . . . . . . . . . . . . School . . . . o.f

279

.

289

..........

296

PREFACE When a new e d i t o r takes over t h e r e i n s t h e r e i s a n a t u r a l tendency t o make v i s i b l e changes i n t h e new volume o f a s e r i e s . A f t e r consul t a t i o n w i t h many o f our "customers," t h e new e d i t o r concluded t h a t t h e volume i n i t s present form i s admirably s e r v i n g i t s purpose. Consequently the reader w i l l f i n d o n l y minor changes i n format. There i s a growing tendency t o cover c e r t a i n v e r y a c t i v e areas y e a r l y , w h i l e a t t h e same time t r e a t i n g more mature f i e l d s on an everytwo- o r -three-year b a s i s . T h i s leaves room f o r t h e i n t r o d u c t i o n o f new t o p i c s on perhaps a one-time basis. I n f a c t , j u s t h a l f o f t h e present twenty-eight chapters deal w i t h t o p i c s n o t included i n t h e p r e v i o u s v o l urne. The sections, Topics i n Chemistry and Topics i n B i o l o g y c o n t a i n chapters which a r e o f t e n more p r o v o c a t i v e and l e s s drug o r i e n t e d than the rest. I n t h e l a t t e r s e c t i o n t h e emphasis on t h e r a p i d l y maturing f i e l d o f immunology i s d e l i b e r a t e ; some o f these chapter t o p i c s w i l l be developed i n a planned way over t h e n e x t two o r t h r e e years. The many dedicated hours devoted t o t h e volume by authors and s e c t i o n e d i t o r s a r e g r a t e f u l l y acknowledged by t h e e d i t o r and w i l l become apparent t o t h e reader as soon as he begins t o read. Suggestions f o r improvement and f o r new f i e l d s t o cover a r e always welcome.

Richard V. Heinzelman

Ka 1 amazoo, M i c h igan June, 1972

ix

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AWARD ADDRESS

I n h i b i t o r s of F o l a t e B i o s y n t h e s i s and U t i l i z a t i o n E v o l u t i o n a r y Changes As a Basis f o r Chemotherapy

--

George H. H i t c h i n g s Wellcome Research L a b o r a t o r i e s , Research T r i a n g l e P a r k , N. C. Fourth Award i n Medicinal Chemistry, T h i r t e e n t h N a t i o n a l Medicinal Chemistry Symposium of t h e American Chemical S o c i e t y , Iowa C i t y , Iowa, June 18-22, 1972

The e x p l o r a t i o n of enzymes and m e t a b o l i c pathways by means of a n t i m e t a b o l i t e s h a s been p r o d u c t i v e of b o t h new m e d i c i n a l a g e n t s and advances i n fundamental knowledge'. One of t h e i m p l i c a t i o n s of such an approach t o chemotherapy i s t h a t i t i s n e c e s s a r i l y uncommitted w i t h r e s p e c t t o s p e c i f i c t a r g e t s . It feeds on t h e knowledge t h a t i t i t s e l f g e n e r a t e s , and t a k e s f u l l advantage of t h e new d i s c o v e r i e s a r i s i n g from b a s i c work e l s e w h e r e . I t s g o a l i s t h e unv e i l i n g of b i o c h e m i c a l d i f f e r e n c e s t h a t can be e x p l o i t e d f o r t h e development of new m e d i c i n a l a g e n t s . The program w i t h which your M e d a l i s t h a s been a s s o c i a t e d h a s been i n v o l v e d w i t h d i v e r s e c a t e g o r i e s : leukemia, p r o t o z o a l and b a c t e r i a l i n f e c t i o n s , gout and organ t r a n s p l a n t a t i o n . A l l of t h e s e comprise a s i n g l e package of a p p l i c a t i o n s from s t u d i e s of n u c l e i c a c i d metabolism. This e s s a y w i l l a t t e m p t t o d e s c r i b e t h e method of working p r i m a r i l y through one example, s e l e c t i v e i n h i b i t o r s of t h e b i o g e n e s i s and u t i l i z a t i o n Speof f o l a t e s and t h e i r a p p l i c a t i o n t o a n t i - m i c r o b i a l chemotherapy'. c i f i c a l l y , t h e combination of t r i m e t h o p r i m and s u l f a m e t h o x a z o l e i s now a v a i l a b l e i n some 60 c o u n t r i e s of t h e world ( w i t h t h e n o t a b l e e x c e p t i o n of t h e United S t a t e s ) and h a s been used i n some 25 x 10" c o u r s e s of t h e r a p y with highly s a t i s f a c t o r y r e s ~ l t s ~ ' ~ .

'0'

'OCH~ Trimethoprim

Sulfomethoxazole

I t s o r i g i n s can be t r a c e d t o e a r l y f i n d i n g s of t h e p r e s e n t program, t o t h e d e t e c t i o n of a n t i m e t a b o l i t e s t h a t p o s s e s s s e l e c t i v e e f f e c t s ' . Substances were found t h a t e x h i b i t e d a c o n s i s t e n t p a t t e r n of a n t i m e t a b o l i c

-2

AWARD ADDRESS

e f f e c t s i n m i c r o b i a l s y s tens. Some were unusable as chemotherapeutic a g e n t s because of m e t a b o l i c d e s t r u c t i o n o r pharmacodynamic a c t i o n s when they were t e s t e d i n chemotherapeutic t r i a l s . Among them, however, w a s a s i z a b l e group t h a t have " s i m i l a r b u t d i s t i n c t e f f e c t s i n d i f f e r e n t organisms" and i t w a s f e l t t h a t s u b s t a n c e s w i t h such s e l e c t i v e a c t i o n could b e regarded as l e a d s t o chemotherapeutic a g e n t s . S e v e r a l p o s s i b l e modes of selective a c t i o n were recognized: "(1) The compound may p o s s e s s s e v e r a l a c t i v e c e n t e r s which a l l o w i t t o b l o c k , more o r less i n d e p e n d e n t l y , d i f f e r e n t biochemical r e a c t i o n s i n d i f f e r e n t t i s s u e s ; (2) a s i n g l e r e a c t i o n may b e blocked i n a l l organisms b u t t h i s r e a c t i o n may b e of r e l a t i v e l y g r e a t e r i m p o r t a n c e t o one organism t h a n a n o t h e r ; (3) t h e d i s s o c i a t i o n of t h e i n h i b i t o r - c e l l r e c e p t o r complex may v a r y widely from organism t o organism; o r ( 4 ) t h e r e a c t i o n blocked may b e one which o c c u r s only s p o r a d i c a l l y i n n a t u r e as an o b l i g a t e b i o c h e m i c a l me chanism"

'.

Trimethoprim is a n o u t s t a n d i n g example of t h e t h i r d mechanism s t a t e d above. I t i n h i b i t s t h e d&hyd;ofolate r e d u c t a s e of E s c h e r i c h i a c o l i a t a c o n c e n t r a t i o n of ca. 10 9M. w h i l e a c o n c e n t r a t i o n of n e a r l y 10-4M i s r e q u i r e d f o r a comparable i n h i b i t i o n of human l i v e r d i h y d r o f o l a t e reductase'. The r a t i o n a l e f o r i t s use i n combination w i t h a sulfonamide is q u i t e s i m p l y s t a t e d . The t a r g e t is t h e m i c r o b i a l p o o l of t e t r a h y d r o f o l a t e c o f a c t o r s t h a t are e s s e n t i a l t o t h e metabolism and m u l t i p l i c a t i o n of t h e m i c r o b i a l c e l l . Such c o f a c t o r s occur u b i q u i t o u s l y and perform e s s e n t i a l f u n c t i o n s i n a l l cells: i n t h e b i o s y n t h e s i s of p u r i n e s and thymine, t h e s y n t h e s i s of s e r i n e and t h e m e t h y l a t i o n of homocysteine t o form methione. Microorganisms employ similar r e a c t i o n s i n t h e b i o s y n t h e s i s of r i b o f l a v i n , p a n t o t h e n a t e , t h i a m i n and d i h y d r o f o l a t e i t s e l f . A l l of t h e s e l a t t e r m e t a b o l i t e s have become "vitamins," i . e . , t h e mechanisms f o r t h e i r b i o g e n e s i s have been l o s t d u r i n g t h e c o u r s e of e v o l u t i o n , and h i g h e r organisms m u s t have them preformed from food. Perhaps t h i s i n i t s e l f makes t h e microorganism t h e more s e n s i t i v e t o i n t e r f e r e n c e w i t h t h e c a p a b i l i t i e s of t h e t e t r a h y d r o f o l a t e p o o l , A t some p o i n t , f o r example, r e d u c t i o n of t h e t e t r a h y d r o f o l a t e p o o l r e s u l t s i n i n h i b i t i o n of f o l a t e biosynthesis, a vicious cycle effect'

.

COFACTOR ACTIVITY

Cof a c t o r

Precursor

H,,FA

Product

-3

AWARD ADDRESS

The s e l e c t i v e e f f e c t s of sulfonamides rest on a similar b a s e . S u l f a n i l a m i d e , and i t s r e l a t i v e s , act as a n t a g o n i s t s of p-aminobenzoic a c i d , a b u i l d i n g b l o c k of t h e f o l a t e molecule, and t h u s i n h i b i t t h e t h e primary product of t h e b i o s y n t h e t i c b i o g e n e s i s of d i h y d r o f o l a t e r e a c t i o n e . S i n c e man r e q u i r e s preformed f o l a t e , t h i s a n t i m e t a b o l i c e f f e c t is d i r e c t e d a t a r e a c t i o n t h a t is p r e s e n t i n t h e p a r a s i t e and a b s e n t from t h e h o s t . T h i s , however, is only h a l f t h e e x p l a n a t i o n of t h e chemot h e r a p e u t i c a c t i v i t y of s u l f o n a m i d e s . T h e i r a c t i o n i s c o n t i n g e n t a l s o on t h e i n a b i l i t y of p a t h o g e n i c microorganisms t o i n c o r p o r a t e preformed f o l a t e s from t h e environment and s o t o by-pass t h e sulfonamide b l o c k . The a b i l i t y t o use preformed f o l a t e s i n e v o l u t i o n must have preceded t h e d e l e t i o n of t h e b i o s y n t h e t i c r o u t e . Pathogens c o n t i n u e t o s y n t h e s i z e f o l a t e s de novo because they have f a i l e d t o develop t h e a b i l i t y t o take advantage of t h e ample s u p p l i e s of f o l a t e s i n body f l u i d s ' .

--

Biogenesis

Hq FA The e f f e c t produced by a sulfonamide on t h e microorganism's t e t r a h y d r o f o l a t e p o o l s is a d e p l e t i o n c o n t i n g e n t on slow a t t r i t i o n and d i l u t i o n through c e l l u l a r d i v i s i o n . Eventually, the pool f a l l s t o a l e v e l t h a t w i l l no l o n g e r p e r m i t m u l t i p l i c a t i o n , and t h e organism p a s s e s i n t o a r e s t i n g phase, The sulfonamide is t h u s b a c t e r i o s t a t i c i n i t s a c t i o n . Trimethoprim, by b l o c k i n g t h e r e d u c t i o n of d i h y d r o f o l a t e , reduces t h e t e t r a h y d r o f o l a t e pool more r a p i d l y . I n p a r t , t h e a t t r i t i o n of t h e t e t r a h y d r o f o l a t e pool is a s s i s t e d by t h e a c t i v i t y of t h y m i d y l a t e s y n t h e t a s e , s i n c e i n t h i s r e a c t i o n t h e c o f a c t o r n o t only t r a n s f e r s a onecarbon fragment (methylene) b u t c o n t r i b u t e s t o t h e r e d u c t i o n of t h e The methylene group t o methyl, and is i t s e l f o x i d i z e d t o d i h y d r o f o l a t e . e f f e c t of t r i m e t h o p r i m i s t h u s t o t r a p f o l a t e s i n t h e dihydro- s t a t e , and t h e f u n c t i o n a l t e t r a h y d r o f o l a t e pool i s d e p l e t e d .

Dihy d r o f o l a t e Reductase

Thymidylate S y n t h e t ase

-4

AWARD ADDRESS

Trimethoprim, however, i s a c o m p e t i t i v e i n h i b i t o r of d i h y d r o f o l a t e reductase". The accumulation of d i h y d r o f o l a t e , through c o n t i n u i n g b i o s y n t h e s i s and r e o x i d a t i o n of t e t r a h y d r o f o l a t e , t e n d s t o i n c r e a s e t h e m e t a b o l i t e : a n t i m e t a b o l i t e r a t i o and diminishes t h e e f f e c t i v e n e s s of t h e blockade. Conjoint a p p l i c a t i o n of a sulfonamide, however, removes t h e s o u r c e of new d i h y d r o f o l a t e and improves t h e e f f e c t i v e n e s s of t h e i n hibition. I n p r a c t i c e t h e simultaneous u s e of t h e 2 i n h i b i t o r s r e s u l t s i n a 5-10-fold p o t e n t i a t i o n , broadening of t h e spectrum of a c t i o n , a decreased l i a b i l i t y t o t h e development of r e s i s t a n c e , and a c o n v e r s i o n of bacteriostatic to bactericidal effectsg. Trirnethoprim emerged from a group of diaminopyrimidine d e r i v a t i v e s which were viewed "as s t r u c t u r a l analogues of f o l i n i c a c i d of a remote s o r t " t h a t were s e l e c t i v e because they could have a " d i f f e r e n t i a l a f f i n i t y . . . f o r t h e r e c e p t o r s of t h e p a r a s i t e . , . I l l 1 . I n t h e b e g i n n i n g they were c h a r a c t e r i z e d as s u b s t a n c e s t h a t i n t e r f e r e d w i t h t h e u t i l i z a t i o n of f o l i c a c i d by l a c t i c a c i d b a c t e r i a . With t h e i s o l a t i o n and c h a r a c t e r i z a t i o n of d i h y d r o f o l a t e r e d u c t a s e , t h e i r t a r g e t w a s i d e n t i f i e d , The a p p l i c a t i o n of a spectrum of i n h i b i t o r s t o a s e l e c t i o n of p a r t i a l l y p u r i f i e d r e d u c t a s e s 6 documented t h e i r mechanism of a c t i o n , and r e v e a l e d w i t h c l a r i t y t h e magnitude of t h e i r d i f f e r e n t i a l a f f i n i t i e s f o r t h e r e d u c t a s e s of d i f f e r e n t species, Trimethoprim r e p r e s e n t s t h e c u l m i n a t i o n of an e f f o r t t o t a i l o r a molecule f o r maximum b i n d i n g t o b a c t e r i a l r e d u c t a s e s with minimum b i n d i n g t o mammalian r e d u c t a s e s g ' l a .

..

H

Pvr i me t ha mine

Di h y d r o f o l i c

Acid

Trim e t h o p r i m

-5

AWARD ADDRESS

Its s e l e c t i v i t y h a s i m p o r t a n t i m p l i c a t i o n s f o r b o t h chemotherapy and f o r e v o l u t i o n . The most probable i n t e r p r e t a t i o n of i t s a c t i o n is t h a t i t is bound p a r t l y w i t h i n and p a r t l y o u t s i d e t h e a c t i v e c e n t e r of t h e enzyme. It t h u s d i f f e r s i n l o c u s o f b i n d i n g from a s t r u c t u r a l analogue of f o l i c a c i d , such as m e t h o t r e x a t e , which h a s a l l t h e b i n d i n g groups of t h e subs t r a t e and would t h u s b e expected t o b i n d i n p r o p o r t i o n t o t h e s u b s t r a t e binding. The i m p l i c a t i o n of s e l e c t i v e b i n d i n g i n r e g i o n s of t h e enzyme n e a r t o , b u t n o t i n , t h e a c t i v e c e n t e r , i s t h a t e v o l u t i o n a r y changes have been much more a c t i v e i n t h e s e s u p p o r t i n g s t r u c t u r e s t h a n i n t h e a c t i v e c e n t e r i t s e l f . D i h y d r o f o l a t e r e d u c t a s e t h u s f a l l s i n l i n e w i t h hemoglobins and o t h e r p r o t e i n s , now known t o e x i s t i n manifold v a r i a n t s t h a t r e t a i n , i n t h e main, t h e f u n c t i o n of t h e p r i m i t i v e p r o t e i n . It is b e i n g recognized t h a t m u t a t i o n a l changes are o c c u r r i n g a t rates t h a t would have been regarded a s improbably enormous only 5 y e a r s ago, b u t t h a t t h e b u l k o f such mutations are n e u t r a l ' = . An e x c i t i n g c h a p t e r i n t h e h i s t o r y of d i h y d r o f o l a t e r e d u c t a s e s t i l l l i e s ahead when s t u d i e s on t h e sequence and conformation of t h e enzyme from d i f f e r e n t s o u r c e s w i l l permit comparative studies.

For chemotherapy, t r i m e t h o p r i m and i t s r e l a t i v e s c a r r y t h e i m p l i c a t i o n t h a t s e l e c t i v e i n h i b i t i o n of any f u n c t i o n may b e p o s s i b l e , and t h a t t h e most p r o b a b l e access t o s e l e c t i v e e f f e c t s may l i e i n t h e s u p p o r t i v e s t r u c t u r e s of t h e c e l l u l a r r e c e p t o r s .

References G. H. H i t c h i n g s , Cancer Res., 29, 1895 (1969). 417 (1965). G. H. H i t c h i n g s and J. J. B u r c h a l l , Adv. i n Enzymol., L. P. Garrod, Drugs, 1, 3 (1971). E d i t o r i a l , Drugs, 1, 7 (1971). 5. G. H. H i t c h i n g s , G T B . E l i o n , E. A. Falco, P. B . R u s s e l l , M. B. Sherwood and H. VanderWerff, J . B i o l . Chem., 1 (1950). 6 . J. J. B u r c h a l l and G. H. H i t c h i n g s , Mol. Pharmacol., 1, 126 (1965). 444 ( 1 9 7 i ) . 7. G. H. H i t c h i n g s , Ann. N . Y. Acad. sci., 8. G. M. Brown, J. B i o l . Chem., 237, 536 (1962). 9. G. H. H i t c h i n g s , Postgrad. Med. J. s u p p l . , 2, 7 (1969). 10. J. J. B u r c h a l l , Postgrad. Med. J. s u p p l . , 45, 29 (1969) 11. G. H. H i t c h i n g s , E. A. Falco, H. VanderWerff, P. B. R u s s e l l , and G. B. E l i o n , J. B i o l . Chem., 4 3 (1952). 143 (1971). 12. J. J. B u r c h a l l , Ann. N . Y . Acad. S c i . , 13. T. Ohta and M. Kimira, Nature, 233, 118 (1971).

1. 2. 3. 4.

27,

183, 186,

.

199,

186,

Section I Editor:

- CNS Agents

Edward L. Engelhardt

Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486

Chapter 1.

Antipsychotic and Anti-anxiety Agents

Charles L. Z i r k l e and C a r l Kaiser Smith Kline & French Laboratories, Philadelphia, Pa. 19101

- Despite

continuation of the extensive research e f f o r t s i n agents emerged i n 1971. However, t h e e v e r expanding i n v e s t i g a t i o n s of t h e monoaminergic, p a r t i c u l a r l y catecholaminergic, pathways i n t h e b r a i n and of the effects of drugs upon them suggest t h a t this approach, which was so successful i n s e t t i n g t h e stage f o r the discovery o f L-dopa f o r t h e treatment of parkinsonism, w i l l eventually lead t o a b e t t e r understanding of the presently a v a i l a b l e drugs and t o more e f f e c t i v e o r s e l e c t i v e psythe chotropic agents. An important c l i n i c a l development was announced resolution of t h e controversy over the use of L i 2 C 0 3 a s prophylactic treatment of manic-depression.’ Results of an extensive 4 year study t o be reported by NIMH have confirmed the belief long held by s o m e clinicians t h a t d a i l y use of t h e drug i s effective i n preventing broad swings i n mood i n p a t i e n t s w i t h this disorder. A number of books and reviews c l i n i c a l ’ ” and t r e a t i n g t h e biochemical ,2 structure-activity9 ” aspects of t h e antipsychotics and anti-anxiety agents have been pub li shed. Introduction

t h i s f i e l d , no fundamentally new antipsychotics and anti-anxiety

-

-

Tricvclic compounds with a six-membered c e n t r a l r i n q Newer phenothiazine d e r i v a t i v e s with clinical antipsychotic e f f i c a c y included oxaf luma2 p i p o t i a z i n e (2, ,11 a potent cataleptogenic i n animals, zine (la> RP 19,336),‘J which h a s a pharmacological p r o f i l e s i m i l a r t o t h a t of Extensive s t u d i e s fluphenazine, and a r e l a t e d p i p e r i d i n o l SA 124 (g).” indicated c l o s p i r a z i n e ( I d , APY-606) ” had c l i n i c a l antipsychotic a c t i v i t y equivalent t o that of chlorpromazine.’

’

,’*

Several f a t t y a c i d esters of a l c o h o l i c phenothiazine d e r i v a t i v e s , undecyclenate and palmitate, fluphenazine e.g., pipotiazine (2) enanthate’ and decanoate,’ and perphenazine enanthate, as w e l l as t h e r e l a t e d thioxanthene, f lupenthixol decanoate, found c l i n i c a l u t i l i t y i n antipsychotic maintenance therapy.20 These esters can a f f o r d advantages i n absorption t o enable attainment of high drug-levels i n t h e CNS” and to provide clinical e f f i c a c y of 2 to 4 weeks duration following a s i n g l e injection.?O

Chap. 1

Antipsychotics, A n t i - A n x i e t y Agents

Z i r k l e , Kaiser

L

The most e f f e c t i v e of a series of phenothiazines having diazabicyclo[4.4.0Jdecane and -[4.3.03 nonane systems i n t h e s i d e chain w a s 2. I n r a t s l e was s i m i l a r t o perphenazine; i t blocked conditioned avoidance response7CAR) a t 0.5 mg/kg, sc." M c l o p a z i n e (If),5014 (Iq)and 5023 (3) were t h e most potent compounds i n a series of imidazolone and oxazolidone d e r i v a t i v e s of phenothiazine and 1-azaphenothiazine; they w e r e more potent than perphenazine i n tests f o r sedation and antiaggression i n mice and a s antiemetics i n dogs.23

asn NI

d) X=C1; R=

X

mo U

h) X=CF3; R=same a s i n g The most e f f e c t i v e member (&) i n a series of amine-altered congeners of unsaturated thioxanthene antipsychotics was nearly equipotent with chlorpromazine i n blocking CAR i n r a t s and i n depressing spontaneous motor a c t i v i t y (SMA) i n mice.'' Another thioxanthene (2,prothixene) decreased SMA i n mice only a t high doses; however, i t caused marked hypocholest e r e m i a i n rats.25 A dihydroanthracene, SK&F 28175 (2) produced phamacological responses c h a r a c t e r i s t i c of both neuroleptic and antidepressant , only chlorprodrugs whereas a demethyl analog, SK&F 25971 (21caused mazine-like effect ' . s

,

'

H

a

R

X

-2

2

a ) X=CF3; R = @ ( q b) X--ql-+;

a ) R=CH3

OH

R=N(CH3)2

b ) R=H

-

T r i c y c l i c communds with a seven-membered c e n t r a l r i n q A dibenzodiazepine, clozapine (5, W-1854), was e f f e c t i v e i n psychotic patients;'? however, i t produced n e i t h e r neuroleptic-like (e.g., catalepsy, antiemetid) a c t i o n s i n animal$' nor parkinsonism-like symptoms i n man. A d e r i v a t i v e was more potent than chlorpromazine i n of dibenzthiepin, GP 45795 various animal tests f o r neuroleptic a c t i v i t and i t antagonized amphetC l i n i c a l l y , 2 improved amine- and tetrabenazine-induced responses.2

(s),

8

Sect. 1

chronic schizophrenics.’’

-

CNS Agents

Engelhardt, Ed.

Among a series of related s t r u c t u r e s , noroctoand oxyprothepin had neurolepticclothepin (El, oxornetothepin l i k e a c t i v i t y equal t o , o r g r e a t e r than, t h a t of octoclothepin i n mice.” C l i n i c a l antipsychotic p r o p e r t i e s were reported’ for Dehydroclothepin (41, one of a series of dibenzthiepins and dibenzoselenopines, was more potent than perphenazine i n r o t a t i n g rod and c a t a l e p s y tests i n mice; however, t o x i c i t y and i n s t a b i l i t y i n aqueous a c i d are possible d e t e r r e n t s t o i t s c l i n i c a l investigation.” W o unsaturated congeners of peradithepin,” & and 7b, were s u b s t a n t i a l l y more potent than t h e parent i n a rat catalepsy t e s 3 ‘

(x) x.

(z)

H

X=C1; Y-0; R=CH, X=C1; Y = y ; R=H X t o c H s ; Y=%; R=(CH,)SOH x = m , ; y = h ; R=(%),oH

a > R-CH’,

b) R = ( q ) ’ O H

A dibenzazepine, 1f3-chlorocarpipramine1f (8, Y-4153). was e f f e c t i v e i n assays of neuroleptic a c t i v i t y (0.2-1X chlorpromazine) and i t i s claimed t o lack catoleptogenic properties.’*

-

Butyrophenones and r e l a t e d compounds The butyrophenone %was e f f e c t i v e i n t h e therapy of schizophrenics and caused only mild side effects;J6 i t s e l e c t i v e l y blocked CAR (0.U t r i f l u p e r i d o l ) i n mice. AHR-2277 was more potent than chlorpromazine, b u t less potent than haloperidol, i n suppressing CAR i n mice and cats i n doses below those producing obvious A r e l a t e d compound AHR-1900 (&) elicited neurolepticmotor defects.” I n a series l i k e behavioral effects i n mice, r a t s and s q u i r r e l monkeys.ie of butyrophenones bearing unsaturated amino s u b s t i t u e n t s , most potent CNS ~bbott-30360 (2) depressant a c t i v i t y i n mice was noted w i t h 2 . 5 ’ combined neuroleptic p r o p e r t i e s with a n a l g e t i c potency approximately equal The most potent compound (10)i n a series of to t h a t of morphine.‘0 methyl-branched aminobutyrophenones was only weakly depressant i n mice .‘I

(a)

A

Chap. 1

Antipsychotics, Anti-Anxiety Agents

H

10 -

11 -

O t h e r structures with antiDsychotic a c t i v i t y

Z i r k l e , Kaiser

-9

12 -

-

A molindone analog, AL 1612 antiemetic and neuroleptic properties i n animal^;'^ however, clinical antipsychotic a c t i v i t y w a s accompanied by a high i n c i dence of extrapyramidal s i d e effects.' Combined c l i n i c a l antipsychotic and antidepressive actions were noted f o r sulpiride.'s S ~ h - 1 2 ~ 6 7 (g) 9 reduced aggressive behavior i n mice and monkeys; however, a c l i n i c a l study indicated i t i s not a c l a s s i c a l neuroleptic." Chronic schizophrenics were improved by g-penicillamine.'s Prostaglandin like chlorpromazine, decreased CAR i n both naive and trained r a t s f r l A benzothienopyridine, EX 11-349 ( 1 ), decreased SMA i n mice and was e f f e c t i v e i n a f i g h t i n g mouse test ( q o + . 9 mg/kg, po)." Several members of a series of l-acylated-l,2-dihydroquinolines antagonized amphetamine actions, blocked a CAR, and caused neuroleptic-like effects i n mice and rats." The cis-,b u t not the t r a n s , aminoalcohol (g) was e f f e c t i v e i n several pharmacological tests f o r neuroleptic a ~ t i v i t y . ' ~ In mice, the benzothiazinone 16 was the most potent of a series of compounds i n causing depression, catatonia and decreased SMA.50

?s) , demonstrated

14

QQ X

11

14 H ..

4 N - + y N,Bo

16

17

18 -

Benzoquinolizine-like ( short-acting, selective) depletion of c e n t r a l and U-20,057 ,s2 catecholamines w a s produced by RO-8-2580 (17)51 compounds causing neuroleptic-like effects i n animals,

(2)

-

BenzodiazeDines and related compounds The c h d s t r y and s o m e aspects of the relationship between the s t r u c t u r e of 1,kbenzodiazepines and their anti-anxiety a c t i v i t y were reviewed i n 1971.53 In clinical studies, demethdiazepam (*, RO-5-2180, A-101) was more e f f e c t i v e than diazepam for the treatment of anxiety;54 however, Ro-5-3350 w a s less

(a)

10

Sect. 1

-

Engelhardt, Ed,

CNS Agents

e f f e c t i ~ e . ' ~ Several pharmacologicals6 ' s 7 and c l i n i c a l s 8 studies indicated lorazepam wy-4036) i s a potent anti-anxiety agent with a sedativehypnotic component. Sch-12,041 ( 1 d) showed evidence of anti-anxiety Methyloxazepam w a s t h e most a c t i v i t y i n an uncontrolled study+ potent of a series of benzodiazepines i n p o t e n t i a t i n g methamphetamineinduced stereotyped behavior i n rats.60 I n monkeys, Ro-5-4200 ( 1 f > was e f f e c t i v e i n causing EEG and behavioral e f f e c t s (20X diazepam) .6?-Various 7-sulfonamido-1, kbenzodiazepines (3,X = h N S 4 , (CHj l2 N S 0 2 , CHj So, NH; 1-H o r CHj) had a n t i n i c o t i n e a c t i v i t y i n mice, b u t they were much less potent than diazepam.62 Ro-5-6901/3 (3) was t h e most e f f e c t i v e of 8 benzodiazepines t e s t e d f o r anticonvulsant a c t i v i t y i n

(a,

(B)

21 -

X=C1, B r , N 4 20 -

a > x=cl; Y=H b) X = B r ;

&

=2-pyridyl

x=ci; y=ci; 3-OH d) X X 1 ; Y=H; l--CFj e> X=Cl; Y=H; l-CHj; 3 - 0 H f > X = N % ; Y-F; 1-CH5 g> X 4 1 ; Y=F; 1-(C14)2N(C2H5)2

C)

a ) R-4Hj; X=C1; Y=F b) R=CH,; X=Y=Cl C > RrCHj; X=C1; Y=H d) R=Y=H; X=C1 e) X=Y=H; RICHj f > X=H; Y=C1; R=CHj

c1

q$ 0

X=Halogen, CHj

, CHjO

22 I n an extensive series of benzo[6,7]-1, It-diazepino-C5, k-bjoxazoles several 9-methyl d e r i v a t i v e s and t h i a z o l e s r e l a t e d t o oxazolazepam,' (2) were more potent than diazepam a s antagonists of bemigride-induced convulsions i n mice.6' Triazolobenzodiazepines (21)demonstrated potent and s e l e c t i v e a c t i v i t y i n various pharmacological tests generally thought t o i n d i c a t e

Chap.

1

A n t i p s y c h o t i c s , A n t i - A n x i e t y Agents

Z i r k l e , Kaiser

11

anti-anxiety a c t i v i t y . S t r u c t u r e - a c t i v i t y data65 f o r a series o f t h e s e compounds i n d i c a t e s u b s t i t u t i o n of t h e 8-psi t i o n w i t h electron-withdrawi n g groups g e n e r a l l y enhances potency, a s does 1-methyl s u b s t i t u t i o n . S u b s t i t u t i o n of t h e 1-position w i t h a l k y l groups l a r g e r than e t h y l as w e l l a s i n t r o d u c t i o n of a para-methoxyl i n t o t h e 6-phenyl moiety caused a marked decrease of potency.66 S u b s t i t u t i o n of C 1 o r F a t t h e o r t h o posit i o n of t h e C-6 phenyl r i n g gave compounds w i t h d r a m a t i c a l l y enhanced a c t i v i t y i n many tests,65 e.g., & and 2 w e r e very p o t e n t (3OX diazepam) upon S.C. a d m i n i s t r a t i o n t o m i c e i n an a n t i - p e n t y l e n e t e t r a z o l test. Extensive pharmacologicald6'6 and biochemical6 d a t a a r e r e p o r t e d f o r D-65-MI' (3) , D-40-TA , U-31,957 and U-35,005 (21f).

'

(e)

A benzodiazepinone CT 5104 (22)was considerably less p r o t e c t i v e than chlordiazepoxide v e r s u s electroshock-induced convulsions i n mice.6 The benzodiazepindiones 3 were i n e f f e c t i v e a s a n t a g o n i s t s o f pentylenetetrazol-induced convulsions i n r a t s . 7 0 I n humans, O W 8063 e l i c i t e d e l e c t r o p h y s i o l o g i c a l and behavioral changes similar t o those had c l i n i c a l produced by diazepam.71 The pyrazolodiazepine C I 683 (3) anti-anxiety a c t i v i t y . I n r a t s , i t counteracted conflict-induced depression and antagonized pentylenetetrazol-induced convulsion^.^^

(2)

Several miscellaneous s t r u c t u r e s were examined f o r a n t i - a n x i e t y a c t i v i t y . Propranolol w a s e f f e c t i v e i n LSD-induced a n x i e t y i n humans.73 As both ( + > and (-)-isomers produced t r a n q u i l i z a t i o n i n rats, t h e a c t i o n i s n o t a t t r i b u t e d t o P-adrenergic b l ~ c k a d e . ~ ' Although pyridarone (3) was t h e most p o t e n t member of a series s t u d i e d f o r neurodepressant a c t i v i t y i n animals, anti-anxiety a c t i o n could n o t be confirmed i n a clinical study.75 Fenpentadiol (3)displayed anti-anxiety and s t i m u l a n t Meprobamate-like a c t i v i t y w a s noted i n pharmacop r o p e r t i e s i n animals.76 l o g i c a l s t u d i e s o f Insem-54 The pharmacology o f propanediol carbamates w a s reviewed r e ~ e n t l y . ~ '

27

26 -

28 -

bktabolism of benzodiazepine d e r i v a t i v e s - B i o l o g i c a l h a l f - l i f e s t u d i e s of chlordiazepoxide i n man showed desnethylchlordiazepxide (-1 and demoxepam t o be major metabolite^.^^ In dogs, metabolism of i n v o l v e s two o x i d a t i v e pathways. One l e a d s t o two phenolic products: 5-( 4-hydroxyphenyl) and 9-hydroxy d e r i v a t i v e s . The o t h e r i n v o l v e s l o s s of t h e N d x i d e f u n c t i o n t o give N-desoxy-2, oxazepam, and N-desoxy d e r i v a t i v e s of t h e phenolic products. A l s o , h y d r o l y s i s of demoxepam I n man, m e t a b o l i t e s o f demoxepam a f f o r d s an "opened lactam" ( Z > . ' O The pharmacologwere oxazepam conjugates, 30, and phenolic products.'l i c a l effects of diazepam i n mice were r e l a t e d t o b r a i n l e v e l s of i t s metaboli tes, p a r t i c u l a r l y oxazepam .82

a

-

12 -

c1

Sect. 1

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CNS Agents

Engel h a r d t , Ed.

&-

a) X=NH

R

O

N*

a) R = q v ; Y=H

b) X=O

C>

b) R=Y=H

R = C % v ; Y=OH

Metabolism o f prazepam by human and animal l i v e r preparations gave mainly desalkylprazepam p l u s a small amount o f oxazepam, b u t no 3-hydroxyprazepam (jlc) I n c o n t r a s t , t h e p r i n c i p a l urinary metabolites of prazepam i n man were glucuronides of J & and oxazepam and small amounts of

u.''

Diazepam and phenolic diazepam d e r i v a t i v e s were s i g n i f i c a n t m e t a b o l i t e s of medazepam i n dog, r a t and man.e5

-

The r e s u l t s of extensive Hypotheses. Biochemistrv and Pharmacoloqy histochemical mapping by Swedish i n v e s t i g a t o r s 3 ' 5 " " o f catecholaminergic (CAI pathways i n t h e r a t b r a i n suggest t h a t these systems may play c r u c i a l roles i n t h e complex n e u r a l t r a f f i c which courses back and f o r t h between b r a i n s t e m c e n t e r s , hypothalamus, thalamus, basal ganglia (e.g., neostriaturn), limbic system and cortex, and provides the neural b a s i s f o r perception, arousal, motivation and emotional behavior. The most c l e a r l y defined t r a c t i s a n i g r o s t r i a t a l dopaminergic (DA) pathway ascending f r o m the s u b s t a n t i a nigra t o the neostriatum. Another DA system p r o j e c t s from the midbrain t o t h e limbic system. 'Jho noradrenergic (NA) pathways, o r i g i n a t i n g i n the r e t i c u l a r formation and ascending through t h e medial forebrain bundle, together innervate t h e hypothalmus, thalamus, areas of the limbic system and the cortex. Growing evidence i n d i c a t e s t h a t t h e antipsychotics e x e r t many of t h e i r pharmacological and c l i n i c a l e f f e c t s by a c t i o n s on these CA neural systems. The EPS produced by these drugs c l e a r l y i n d i c a t e an a c t i o n on t h e b a s a l ganglia. Lesions i n t h e nigros t r i a t a l pathway, attended by a loss of DA i n the neostriatum, appear t o be i n l a r g e p a r t responsible f o r t h e EPS of parkinsonism, and the EPS produced by reserpine probably a r e t h e consequence of t h e DA-depleting action of t h e drug.5 Although o t h e r types o f n e u r o l e p t i c s such a s the phenothiazines and butyrophenones do n o t have an appreciable e f f e c t on b r a i n l e v e l s of DA they too a f f e c t DA metabolism i n t h e neostriatum. Various biochemical effects of t h e s e agents, e.g., an increased l e v e l o f homovanillic acid, i n d i c a t e t h a t they a c c e l e r a t e t h e turnover of DA i n the neostriatum. It i s postulated t h a t the antipsychotics block postsynatic DA receptors and t h a t t h e r e s u l t i n g functional deficiency of DA a c t i v a t e s a feed-back mechanism t o stimulate synthesis o f t h e transmitter.25 ' The observation t h a t t h e n e u r o l e p t i c s do not appreciably i n c r e a s e turnover of DA u n l e s s nerve impulse flow i s i n t a c t seems t o favor a postsynaptic mechanism of DA blockadee6 r a t h e r than a presynaptic mechanisme7 l i k e t h a t of reserpine. These drugs a l s o i n c r e a s e turnover of DA i n t h e neurons which p r o j e c t t o t h e limbic system and s o m e accelerate turnover However, i n d i v i d u a l drugs of norepinephrine (NE) i n the NA system^.^

Chap, 1

A n tip s y c h o tic s ,

A n ti-A nxiety Agents

Z i r k l e , Kaiser

13

seem t o d i f f e r considerably i n t h e i r effects on t h e two types of neurons. I n an extensive study of a v a r i e t y o f neuroleptics, i t w a s found t h a t perphenazine accelerated turnover of both amines, spiroperidol increased turnover of only DA and pimozide increased turnover o f DA a t low doses and NE a t high doses.” Rats with u n i l a t e r a l l e s i o n s of t h e n i g r o s t r i a t a l pathway have been used a s a model f o r measuring the DA a c t i v i t y of drugs such as amphetamine and t h e DA blocking a c t i v i t y o f a n t i p s y c h o t i c s i n t h e n e o ~ t r i a t u m r. ~ Be70 These animals d i s p l a y motor asymmetries and a tendency t o t u r n toward t h e side of t h e l e s i o n ( i p s i l a t e r a l turning). Amphetamine enhances the turni n g behavior and t h e n e u r o l e p t i c s antagonize this effect. If t h e neostriatum i s lesioned o r removed, t h e n e u r o l e p t i c s cause c o n t r a l a t e r a l asymmetries. A s i m i l a r mouse model h a s been developed and used t o assess t h e a c t i v i t y o f various anti psychotic^.^' The c h a r a c t e r i s t i c cataleptogenic property of t h e antipsychotics has been thought t o be due t o an effect of these drugs i n t h e n e o ~ t r i a t u m . ~ ~ ~ ~ Consistent with this idea i s t h e f i n d i n g of a c o r r e l a t i o n between t h e potencies and t i m e courses of a c t i o n of drugs i n producing c a t a l e p s y and i n producing increased homovanillic a c i d l e v e l s i n t h e neostriatum o f rats.” However, a comparison of the effects of l e s i o n s i n t h e s u b s t a n t i a nigra, neostriatum o r globus p a l l i d u s upon antipsychotic- and cholinergicinduced c a t a l e p s y suggests t h a t i n t e g r i t y of t h e globus p a l l i d u s i s import a n t for t h e a c t i o n o f n e u r o l e p t i c s b u t n o t f o r t h e a c t i o n of choline r g i c s .9 ’9 5

‘

Antagonism of amphetamine- o r apomorphine-induced stereotyped behavior i s another c h a r a c t e r i s t i c effect of the antipsychotics. Fbst d a t a favor t h e hypothesis t h a t drug-induced stereotyped behavior i s the r e s u l t of DA a c t i v i t y i n t h e n e ~ s t r i a t u m . ” ~ ~However, a comparison o f t h e e f f e c t s of l e s i o n s i n the neostriatum o r i n t h e tuberculum olfactorium upon DA- and apomorphine-induced stereotyped behavior suggests t h a t DA a c t i v i t y i n t h e limbic f o r e b r a i n may be important f o r this drug effect i n the rat.96 The antipsychotics are a l s o noted for t h e i r a b i l i t y t o a t t e n u a t e Animals with e l e c t r o d e s implanted i n t h e septum b r a i n self-stimulation. ( i n t h e limbic system) o r t h e medial f o r e b r a i n bundle, through which CA neurons ascend t o t h e limbic system, repeatedly p r e s s a l e v e r to o b t a i n electrical stimulation of these areas. Thus the “reward o r pleasure” centers involved i n this behavior may be located i n areas of t h e limbic f ~ r e b r a i n . ~ ?A study extending earlier work o f SteinP7 s h o w s t h a t selfstimulation i s accompanied by release o f NE f r o m NA terminals i n t h e hypothalamus, p r e o p t l c area, and p a r t s of t h e limbic system.98 Another paper r e p o r t s t h a t self-stimulation also occurs when e l e c t r o d e s a r e placed i n regions near the s u b s t a n t i a n i g r a where DA neurons are located.99 Thus DA a s w e l l as NA pathways may be involved i n t h e effects o f reward on operant behavior and i n t h e a t t e n u a t i n g effects o f t h e n e u r o l e p t i c s on T h i s behavior i s a1 so attenuated when 6-hydroxydopaming self-stimulation. which causes degeneration o f CA neurons and depletion o f t h e amines, i s

14 -

Sect. 1

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E n g e l h a r d t , Ed.

given i n t r a c i s t e r n a l l y t o rats.’” S t e i n and W i s e ’ O ’ have proposed a novel e t i o l o g y of schizophrenia which involves t h e endogenous buildup o f 6-hydroxydopamine and a r e s u l t a n t d e t e r i o r a t i o n of NA pathways t h a t mediate reward. They p o s t u l a t e t h a t t h e a n t i p s y c h o t i c s produce t h e i r c l i n i c a l e f f e c t s by preventing t h e uptake of 6-hydroxydopamine i n t o the NA neurons. Several criticisms’02-’05of this hypothesis and a rebuttala have been published. These p o i n t o u t t h e problems of defining and c h a r a c t e r i z i n g mental disorders as w e l l as some of the experimental and i n t e r p r e t a t i o n a l d i f f i c u l t i e s of research i n this area. Hypotheses regarding a c t i o n o f the antipsychotics a t the c e l l u l a r and molecular l e v e l s range f r o m a view t h a t the drugs i n t e r a c t nons p e c i f i c a l l y with neuronal and s u b c e l l u l a r membranes to a p o s t u l a t e t h a t they s p e c i f i c a l l y block postsynaptic r e c e p t o r s of neurotransmitters, e.g., DA and NE. These drugs a r e potent surface-active agents and s t a b i l i z e c e l l u l a r and s u b c e l l u l a r membranes and i t has been proposed t h a t they act l i k e l o c a l a n e s t h e t i c s by a l t e r i n g membrane permeability and i n t e r f e r i n g with neural transmission.’ O 6 Consistent with t h i s v i e w are observations t h a t procaine, when i n j e c t e d i n t o the neostriatum of cats, produced e f f e c t s s i m i l a r to those of haloperidol’07 and t h a t ouabain, another agent which a l t e r s membrane function, produced many of t h e effects of t h e a n t i A n i n v i t r o study o f the psychotics when given intraventricularly.’O effects of promazine and t h i o r i d a z i n e on membrane preparations suggests t h a t i n h i b i t i o n of Na+-K+-ATPase by these agents i s secondary to more general a l t e r a t i o n s of the c e l l membranes.’ Another hypothesis proposes t h a t t h e a n t i p s y c h o t i c s prevent t h e access of neurotransmitters t o t h e i r r e c e p t o r s by forming a monomolecular f i l m on t h e postsynaptic noting t h a t t h e s o l i d - s t a t e s t r u c t u r e membrane,’ b u t Horn and Snyder,’ of DA i s superimposable upon a portion of the solid-state s t r u c t u r e o f chloropromazine, suggest t h a t t h e antipsychotics might s e l e c t i v e l y i n t e r act with CA receptors. The e f f e c t s of t h e antipsychotics upon b r a i n tissue l e v e l s o f c y c l i c AMP, which could be t h e mediator o f c e n t r a l CA actions, have been studied i n vivo and i n vitro.’ Trifluopera~ind’~ and haloperidol’ were more potent than chlorpromazine i n preventing the rise i n c y c l i c AMP levels produced by norepinephrine o r decapitation. The ” promethazine,’ and protripsulfoxides of t h e two phenothiazines,’ tyline’ were much less potent than chlorpromazine.

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’

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If both antipsychotic and l o c a l a n e s t h e t i c a c t i v i t y are t h e result of nonspecific i n t e r a c t i o n of drugs with membranes, t h e c h a r a c t e r i s t i c CNS a c t i o n s of the antipsychotics must be due t o a p a r t i c u l a r drug d i s t r i b u t i o n i n t o v a r i o u s b r a i n regions, a s e l e c t i v e a f f i n i t y f o r c e r t a i n neurons, o r a d i f f e r e n t i a l s u s c e p t i b i l i t y t o drug a c t i o n by various i n t e r neuronal organizations. It has been observed t h a t d i f f e r e n t regions of t h e b r a i n take up d i f f e r e n t amounts of chlorpromazine, b u t no d i f f e r e n c e in the a f f i n i t y of t h e n e u r o l e p t i c s f o r t i s s u e from various b r a i n regions w a s detected i n i n v i t r o experiments.”‘ A study of phenothiazines showed t h a t the d i f f e r e n t potencies of t h e 1-, 2-, 3- and k h l o r o p r o m a z i n e s i n producing catalepsy i s not due to d i f f e r e n c e s i n t h e a v a i l a b i l i t y o f t h e In c o n t r a s t , a c a r e f u l study of f o u r f o u r isomers i n the brain.”‘ butyrophenones i n d i c a t e d t h a t t h e i r molar concentrations i n t h e b r a i n were

Chap. 1

Antipsychotics, Anti-Anxiety A g e n t s

Z i r k l e , Kaiser

JI

a bout t h e same a f t e r administration of Eq, doses f o r antagonism of Thus, i f amphetamine- and apomorphine-induced stereotyped behavior.’ t o t a l b r a i n l e v e l s of a n t i p s y c h o t i c s are an index of drug a v a i l a b i l i t y a t sites o f a c t i o n , t h e s e l i m i t e d observations suggest t h a t the differences i n t h e potencies of t h e a n t i p s y c h o t i c s can be accounted f o r by differences i n t h e i r a b i l i t i e s t o reach t h e i r d t e s o f a c t i o n i n t h e case of the four butyrophenones b u t not i n the case of t h e isomeric phenothiazines. Much more extensive s t u d i e s c o r r e l a t i n g b i o l o g i c a l a c t i v i t y w i t h t o t a l o r regional b r a i n l e v e l s of a v a r i e t y of a n t i p s y c h o t i c s o f d i v e r s e s t r u c t u r e and potencies must be c a r r i e d o u t before the s i g n i f i c a n c e o f t h e struct u r a l requirements f o r antipsychotic a c t i v i t y can be understood.

’

Although t h e r e a r e s o m e i n d i c a t i o n s t h a t Li,CO, in some way a f f e c t s CA metabolism,”‘ l i t t l e progress has been made i n explaining i t s specific therapeutic effects i n manic-depressive psychosis. Manic o r hypomanic states sometimes develop i n t h e course of L-dopa therapy, p a r t i c u l a r l y i n ” From t h e meager conf licp a t i e n t s w i t h a p r i o r h i s t o r y of mania.’ t i n g d a t a available” e i t cannot be s a i d y e t whether o r n o t Li, COJ i s e f f e c t i v e i n ameliorating Gdopa-induced mania.

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Additional d a t a support t h e i d e a t h a t effects of anti-anxiety agents upon CA pathways may a t least i n p a r t account f o r t h e i r c l i n i c a l action. A study of the effects o f chlordiazepoxide, diazepam and nitrazepam on C4 neurons of non-stressed and stressed r a t s using N s t o chemical and biochemical analyses of amines suggests t h a t these drugs decrease impulse a c t i v i t y i n NA neurons i n n e r v a t i n g t h e c o r t e x and hippocampus and i n DA neurons ascending t o t h e neostriatum and limbic foreThese r e s u l t s are c o n s i s t e n t with e l e c t r o p h y s i o l o g i c a l obserbrain.’ vations t h a t the anti-anxiety agents c o n t r o l abnormal a c t i v i t y i n various components of t h e limbic system a t doses which have l i t t l e o r no effect on the cortex.”’

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2,

2,

2,

14,

2,

26,

fi,

x,

Chap. 1

Antipsychotics,Anti-Anxiety

Agents

17

Z i r k l e , Kaiser

69 M. Julia, N. Hurion and Huynh Dinh T., Chb. Ther., 2, 343 (1970). 70. N. D. Heindel, W. P. Fives, T. F. kmke, J. E. Rowe, H. W. Snady and R. A. Carrano, J. Med. Chem., l4, 1233 (1971). 747 (1971). 71. R. GuerrereFigueroa and D. M. Gallant, Curr. Ther. Res., Clin. Ew., 72. R. J. Santos, C. Bowling, L. Mudgil and J. H. Nodhe, Pharmacologist, 2, 205 (1971). 1039 (1971). 73. A. Linken, Lancet, I1 74. J. G. Bainbridge and D. T. Greenwood, Neurophamacology, l0, 453 (1971). 75. H. J. Ziegler, H. Inion, G. Aussems, A. Christiaens, F. Chaillet and R. Charlier, Chim. Ther., 159 (1971). 76. J. Ginet, J. C. Levy and D. Rolland, Arznek-Forsch., 2, 1 (1971). Bonvarlet, J.-C. C a s h and M. Cazin, Chh. Ther., 6, 14 (1971). 77. C. kspagnol, J.-C. 78. B. J. Ludwig and J. R. Potterfield, Adv. Pharmacol. and Chemotherapy, go 173 (1971). 1500 (19717. 79. M. A. Schwartz, E. Postma, and 2. Gaut, J. Pham. Sci., 80. M. A. Schwartz, E. Postma and S. J. Kolis, ibid., 60, 438 (1971). 81. M. A. Schwartz and E. Postma, ibid., 6 l , 123 ( 1 9 7 2 r 82. F. Marcucci, E. hhssini, A. Guaitani, R. Fanelli and S. Garattini, Eur. J. Pharmacol., l6, 311 (1971). 83. J.-P. Viau, J. E. Epps and F. J. DiCarlo, Fed. ploc., Fed. Amer. SOC. Exp. Biol., 2,225 Abs. (1971). 04. F. J. DiCarlo, J.-P. Viau, J. E. Epps and L. J. Haynes, C l h . Pharmacol. Ther., ll,890 (1970). 85. M. A. Schwftrtz and J. J. Carbone, Biochem. Pharmacol., 2,343 (1970). 86. N.-E. Anden, H. Corrodi, K. Rrxe and U. Ungerstedt, Eur. J. phamcol., l5, 193 (1971). 87. A. Tagliamonte, P. Tagliamonte and G. L. Gessa, J. Neurochem., 2,733 (1970). And&, S. G. h t c h e r , H. Cormdi, K. Fuxe and U. Ungerstedt, Eur. J. Fhamcol., ll,303 88. N.-E. (1970 1. e9. U. Ungerstedt, Acta Physiol. Scand., Suppl. 367, 49 (1971). 90. U. Ungerstedt, ibid., Suppl. 367, 69 (1971). Part I, 781 (1971). 91. V. J. L o t t i , Life Sci., 92. R. Fog, A. Randrup and H. Pakkenberg, Psychophamcologia, l8, 346 (1970). 93. G. S t i l l e and H. Lauener, Arzneim.-Forsch., 2, 252 (1971). 94. B. Costall and J. E. Olley, Neurophamcology, LO, 297 (1971). 95. B. Costall and J. E. Olley, ibid., lo, 581 (1973. 96. G. M. McKenzie, Pharmacologist, l3, 279 (1971). 97. L. Stein and C. D. Wise, Ref. 3, p. 313. 98. G. W. Arbuthnott, K. Fuxe and U. Ungerstedt, Brain Res., 27, 406 (1971). 406P 99. G. M. Anlezark, G. W. Arbuthnott, J. E. Christie and T. J. Crow, B r i t . J. Phamcol.,

2,

(m),

i,

g,

2,

(19711.

loo. G. R. Breese, J. L. Howard and J. P. Leahy, ibid., 101. 102. 103. 104. 105.

106. 107. 108.

log. 110.

111. 112.

113. 114. 115. 116. 117. 118.

119. 120.

5,

255 (1971). L. Stein and C. D. Wise, Science, 1032 (1971). S. M. Antelman, A. S. Lippa and A. E. Fisher, ibid., 175, 919 ( l 9 Z ) . M. B. Bowers, Jr. and M. H. Van Woert, ibid., 175, W?j1972). J. S. Strauss and W. T. Carpenter, Jr., ibid., 175, gU (1972). L. Stein and C. I). Wise, ibid., 175, p 2 (1972). W. 0. Kwant and P. Seeman, Biochem. Pharmacol., 2089 (1971). A. R. Cools, Arch. Int. Pharmacodyn., 259 (1971). N. S. Doggett and P. S. J. Spencer, B r i t . J. Pharmacol., 42, 242 (1971). K. Landmark and I. dye, Acta Pharmacol. Toxicol., 29, 1 (1971). P. A. J. Janssen, Int. J. Neuropsychiat., Suppl. 1, S10 (1967). A. S. Horn and S. H. Snyder, Roc. Nat. Acad. Sci. U.S.A., 63, 2325 (1971). G. C. Palmer, G. A. Robison and F. Sulser, Biochem. Phanoacol., 236 (1971). P. Uzunov and B. Weiss, Neuropharmacoloa, 10 697 (1971). A. L. Green, J. Pham. Phanacol., 2, 207 7i967). P. A. J. Janssen and F. T. N. Allewijn, Arzneb.-bsch., 199 (1969). W. E. Fann, J. M. k v i s , D. S. Janowsky, J. H. Cavanaugh, J. S. KauPmenn, J. D. Gfiffith and J. A. Oates, C l b . Pharmacol. Ther., 2, 71 (1972). F. K. Goodwin, J. h e r . Med. Ass., 2,1915 (1971). C. A. Pearlman, Jr., New Eng. J. Med., 285, 1326 (1971). H. Corrodi, K. Rrxe, P. Lidbrink and L. Olson, B r a i n Res., 1 (1971). S. Margolin and M. Kletzkin, Ref. 3, p. 93.

171,

194,

g,

2

g,

2,

3,

Chapter 2.

Antidepressives and Stimulants

C a r l Kaiser and Charles L. Z i r k l e Smith Kline & French Laboratories, Philadelphia, Pa. 19101

-

Introduction As i n p a s t years, this chapter reviews stimulants together with antidepressives. T h i s arrangement of t o p i c s seems not t o be j u s t i f i e d on c l i n i c a l grounds f o r t h e s p e c t r a of clinical u t i l i t y of t h e two c a t e g o r i e s of drugs are q u i t e d i f f e r e n t . Is t h e r e any reason f o r t r e a t i n g the two s u b j e c t s together o t h e r than one of convenience? Perhaps t h e answer i s yes i f t h e d e f i n i t i o n of *Istimulants" i s l i m i t e d to amphetamine and pharmacologically r e l a t e d agents. Most research reported i n the l a s t year supports t h e long held b e l i e f t h a t the antidepressives and t h e amphetamine-like drugs e x e r t many of t h e i r pharmacological and clinical effects by a c t i o n s on c e n t r a l monoaminergic, e s p e c i a l l y catecholaminergic, pathways. B u t t h e biochemical a c t i o n s of t h e t r i c y c l i c a n t i d e p r e s s i v e s are s t i l l poorly understood and t h e i r relevancy to t h e t h e r a p e u t i c effects has not been established. From the extensive s t u d i e s on amphetamine a more coherent, i f n o t clearer, p i c t u r e of i t s a c t i o n s is emerging. A sign of progress i s t h a t one can now with some assurance describe amphetamine a s a catecholaminergic agent w i t h a pronounced dopaminergic component o f action r a t h e r than a s a c e n t r a l l y - a c t i n g sympathomimetic amine. The 1971 l i t e r a t u r e describes t h e s y n t h e s i s and pharmacological evaluation of numerous p o t e n t i a l antidepressives, b u t p r e s e n t s no evidence t h a t new agents superior t o t h e e s t a b l i s h e d drugs are i n the offing.

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Tricvclic compounds w i t h antidepressive a c t i v i t y An a m i t r i p t y l i n e d e r i v a t i v e , S D 2202-01 (In),exhibited a pharmacological p r o f i l e similar t o t h a t o f the parent.' Antidepressive and anti-anxiety p r o p e r t i e s were Other a m i t r i p t y l i n e r e l a t i v e s , a l s o noted f o r a monomethyl analog (&I.* some 5-methylene-4-substituted dibenzocycloheptenes (21,had l i t t l e a c t i v i t y i n a mouse a n t i r e s e r p i n e test.' A dibenzthiepin, dothiepin (prothiadine), was superior t o a m i t r i p t y l i n e i n a double b l i n d comparison f o r t h e treatment of depression.' Amoxapine (3, cL-67772), a dibenzoxazepine, although devoid of a n t i c h o l i n e r g i c p r o p e r t i e s , produced imipramine-like a c t i v i t y i n animals. C l i n i c a l l y , 3 was an e f f e c t i v e a n t i depressive agent; however, s i d e e f f e c t s (insomnia, d i f f i c u l t u r i n a t i o n ) were e n ~ o u n t e r e d . ~Several related dibenzodiazepines had antidepressive properties. SM-307 (41, an e t h y l s u l f o n y l analog of dibenzepin,6 produced

kCOCH, NRCH'

la:

lb: -

R=CH3 R=H

-2

Antidepressives, S t i m u l a n t s

Chap. 2

19 -

Kaiser, Z i r k l e

imipramine-like e f f e c t s i n animals.’ A r e l a t e d pyridobenzodiazepine, UP-106 (2), a l s o was a c t i v e i n various animal tests i n which most a n t i depressives a r e e f f e c t i v e and i t demonstrated clinical activity.’ The s t r u c t u r a l l y s i m i l a r U-17,660 (6)w a s t h e most potent a n t i a l l e r g i c and antianaphylactic compound i n a series of 5- and 10-aminoalkylated dibenzodiazepines

.’

Antidepressive a c t i v i t y was noted f o r s o m e t r i c y c l i c compounds having a six-rnembered c e n t r a l ring. A series of naphthyridones, C-29 c-42 (2). and C-45 (21, caused imipramine-like a c t i v i t y i n animals.’ The influence of 3 on c e n t r a l biogenic amine levels,’ as w e l l a s i t s absorption, d i s t r i b u t i o n and excretion,’ was investigated i n mice and rats, Several phenothiazine d e r i v a t i v e s had antidepressive a CF, analog of a c t i v i t y , III extensive clinical t r i a l s f l u o r a c i z i n e (&I, chloracizine,’ showed antidepressive e f f i c a c y equivalent to imipramine. It a l s o produced a n t i c h o l i n e r g i c effects,’ ” S t r u c t u r e versus antidepressive a c t i v i t y r e l a t i o n s h i p s were reported f o r a series of phenot h i a z i n e s and r e l a t e d compounds bearing a carbocyclic b a s i c s i d e chain. Che of these compounds (&) was considerably more potent than amitriptyOther l i n e i n preventing reserpine-induced p t o s i s i n mice and rats.” phenothiazines with antidepressive a c t i o n s included &, t h e l-chloroanalog of chlorpromazine, which produced imipramine-like a c t i o n s i n mice and r a t s , ’ and the azaphenothiazine-10-thiolcarboxylate 4 which induced antidepressive-like symptoms (mydriasis, increased s e n s i t i v i t y t o touch) i n mice.‘ The antidepressive e f f e c t s of phenothiazine d e r i v a t i v e s with 1- o r a-substituents may be associated with the steric influence of these groups i n i n t e r f e r i n g with attainment of a nearly f l a t conformation by the t r i c y c l i c system.’ 3

(A),

’

‘

’

0

-7 a> X=H b) X=OCH3

c> x 4 1

-a

2

a ) X=2-CF3 ; R=CO(CY N(C, H, l 2 b) X=Z-Cl; R = t r a n s - T C % N(CH, C > X-1-C1; R = m 3 N(CH3 1 2

Other t r i c y c l i c compounds with antidepressive a c t i v i t y w e r e maprotiline (10, Ciba 34276-Ba), which produced imipramine-like e f f e c t s

20 -

Sec t. I

i n dogs’

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CNS Agents

E n g e l h a r d t , Ed.

,

and blocked r a t synaptoso.na1 uptake of norepinephrine’ (NE) which antagonized tetrabenazineI n mice, t h e phenanthinduced p t o s i s i n mice a t EL+o 20 mgkg, P , o . ~ O rene 12 had no a n t i r e s e r p i n e a c t i v i t g ’ and a 6,g-dihydro d e r i v a t i v e of imipramine f a i l e d t o antagonize tetrabenazine-induced depression .2 and t h e pyridobenzocyclooctane

(z),

7

@cH(q)2N(a3)2

\

(a%)3NHcH3 10 -

(q13 N(CH3 1 2

11 -

((33 1 2

12 -

Several diphenylmethane d e r i v a t i v e s had antidepressive a c t i v i t y . w a s well-tolerated and e f f e c t i v e i n p a t i e n t s with various Tofenacin (3) kinds of d e p r e ~ s i o n ‘.2~4 ~ Like imipramine, s o m e aminopropenes reversed reserpine-induced hypothennia i n mice .2 The (-1 -isomer of 2 caused c l i n i c a l antidepressive a c t i o n s , b u t these were accompanied by unwanted side e f f e c t s . 2 6

14

X=H,j-F,LF; R=H,CH3

14 -

15

Other compounds with antidepressive a c t i v i t y - It has been suggested t h a t rubidium s a l t s ( s e e below) may have antidepressive a c t i v i t y and a remarkable therapeutic response was noted i n an open clinical C l i n i c a l antidepressive a c t i v i t y was produced by A-25794 .2 A molindone r e l a t i v e , EN-3022 ( 9 1 , was e f f e c t i v e i n s e v e r a l tests (antagonism of tetrabenazine-induced p t o s i s i n mice; p o t e n t i a t i o n of pressor response t o NE i n dogs)30 i n which most antidepressives are a c t i v e . Nomifensine w a s more potent than imipramine i n antagonizing reserpineinduced catalepsy i n rats.” Quipazine had a pharmacological p r o f i l e similar t o t h a t of t r i c y c l i c anti depressive^.^^ I n a series of phenacylthioimidazolines, 2 was the most potent antagonist of reserpine-induced hypothermia i n mice (12X imipramine) .l3

(16)

(18)

(a)

Chap. 2

Antidepressives, Stimulants

21 -

Kaiser, Z i r k l e

0

M p r a m i n e - l i ke phannaco logica 1 a c t i v i t y , without a n t i cho l i n e r g ic properties, was noted f o r LG 152 i t also prevented synaptosomal reuptake of NE.35 Potent antitetrabenazine-induced p t o s i s a c t i v i t y , without anticholinergic effects, w a s produced by some e t h e r s of hexanophenone oxime, e.g. 2 3 3 6 The piperidinecarboxamide was equipotent with amitriptyl i n e i n a test f o r dopa-potentiation i n mice.)' A series o f phthalan derivatives having a pharmacological spectrum similar t o t h e tric y c l i c a n t i d e p r e s s i v e s was studied f o r i n h i b i t i o n of mitochondrial activity i n i n t a c t yeast cells a test which c o r r e l a t e s with c l i n i c a l antidepressive potency. Most potent a c t i v i t y was noted f o r Lu 3-04" (2&) , Lu 3-009 (24b) , Lu 3-074 3 X impramine))* and Lu 3-049 4.3X imipramine) C l i n i c a l antidepressive a c t i o n s were noted f o r HT 3261 (3)" and t h e choline d e r i v a t i v e panclar which was an e f f e c t i v e psychostimulant i n p a t i e n t s with schizophrenia, depression and Other compounds with c l i n i c a l antidepressive a c t i v i t y neuroses." included W I N 27,147-2 (271, which w a s devoid of s i d e effects a t e f f e c t i v e which w a s o f doubtful value i n humans dose levels.42 and 8 14.368 (g>,53 and caused untoward s i d e effects although i t produced antidepressive-like a c t i o n s i n animals. Short-lived antidepressive a c t i v i t y w a s observed with ~40pa.'~

(a

(2)

-

(a,

.r

(a,

(261,

(c4L C H 3

ss

c

=NO(CH2)2N% 22 -

24 -

3 HOPO) (% >,N(CH3

26 pcs

a) b) c) d)

X=CH,; R=CH3 X 4 ; R=CH3 X-S; R = C b X=C%; R=H

OH

Several amphetamine d e r i v a t i v e s displayed antidepressive properties. Fenfluramine, an a n o r e c t i c drug which depresses motor a c t i v i t y , showed an imipramine-like p r o f i l e of a c t i v i t i n various phamacological tests i n mice.'5 p3loroamphetamine (*IY6 and i t s N-methyl d e r i v a t i v e ,

22 -

Sect.

1

-

CNS Agents

E n g e l h a r d t , Ed.

RO-4-6861 (29b) ,47 produced c l i n i c a l a c t i v i t y resembling t h a t of the t r i c y c l i c a x d e p r e s s i v e s . BW 65-54 (2) e l i c i t e d antidepressive effects i n animals; i t potentiated responses to amphetamine, apparently by i n t e r f e r i n g with metabolic inactivation,'8 A number of new MA0 i n h i b i t o r s were described i n 1971; however, because of the l i m i t e d c l i n i c a l u t i l i t y of these agents i n t h e treatment of depression, they w i l l not be reviewed i n d e t a i l . Nonetheless, several was included i n a of these substances a r e noteworthy. Sydnophene (2)'9 series of sydnonimines examined f o r MAO-inhibitory a c t i v i t y . It demons t r a t e d m i l d stimulant and antidepressive a c t i o n s i n extensive c l i n i c a l s t u d i e s i n Russia.50 Another MAO-inhibitor, c l o r g i l i n e M&B 9302) had antidepressive a c t i v i t y ; i t s c l i n i c a l effects were s i m i l a r to those of i m i ~ r a m i n e . ~ ' Proportions of two forms of MA0 (Type A, B) were found to vary i n d i f f e r e n t a r e a s of r a t b r a i n . The type A enzyme, predominant i n sympathetic nerves, acted on both 5-HT and tyramine and was inhibited by and acted on tyramine, b u t 32, whereas type B-MA0 was i n s e n s i t i v e t o not 5-HT." I n humans, 32, tranylcypromine and isocarboxazid increased 5HT, NE and dopamine l e v e l s i n various b r a i n a r e a s ; however, t h e effect of tranylcypromine was s i g n i f i c a n t l y g r e a t e r than t h a t of the o t h e r two on dopamine i n t h e caudate nucleus and h y p o t h a l a m u ~ . ~In ~ vitro, Lilly 51641 (21, l i k e harmaline, p r e f e r e n t i a l l y blocked M A 0 oxidation of 5-HT, whereas tranylcypromine and pargyline s e l e c t i v e l y i n h i b i t e d the oxidation of tyramine .5'

(z,

-

Central Stimulants S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s o f c e n t r a l stimul a n t s were reviewed i n 1971.55 '56 The use of central stimulants, notably dextroamphetamine and methylphenidate, i n t h e therapy of hyperkinetic disorders i n children has increased, According t o NIMH estimates more than 200,000, o r about 1 s of hyperactive children, received amphetamine treatment i n 1969?' These agents reduce d i s t r a c t i b i l i t y and promote the c h i l d ' s a b i l i t y to focus on meaningful s t i m u l i and t o organize his bodily movements more purposefully.5 8 ' 5 9 Similar effects w e r e e l i c i t e d by treatment of untrainable hyperkinetic and aggressive dogs with dextroamphetamine."

(21,

N e w e r amphetamine r e l a t i v e s included pyrovalerone which w a s b e n e f i c i a l i n therapy of c h r o n i c a l l y fatigued patients," U-30,405A ( a) and U-28,926~ which caused CNS stimulation i n rhesus monkeys. 6% Amantadine produces amphetamine-like a c t i o n ( see below). An aminot e t r a l i n , ADT ( 6) afforded amphetamine-like e f f e c t s b u t decreased motor a c t i v i t y i n m i c S 3 Adenosine had a psychoanaleptic effect i n a mouse .65 maze test.64 Exploratory a c t i v i t y of mice was enhanced by P1961 (2) Other compounds f o r which CNS-stimulating a c t i v i t y was observed i n mice

(z),

Chap. 2

Ant idepress ives, S t i m u l a n t s

included 4864 ( 8) ,66 a benzocyclobutene % 1-69 and GYI 20238 (j

23 thiolactams,6 *

Kaiser, Z i r k l e

(2) ,6 several

a

c1

2

38

Several s t u d i e s r e l a t e d t o physical p r o p e r t i e s and metabolism of amphetamines. "he b a s i c i t y , t i s s u e d i s t r i b u t i o n and metabolism of p-f luoro-, p, p-dif luoroamphetamine, and amphetamine w e r e s t r i k i n g l y d i f f e r e n t i n rats.70 N m r studies of t h e conformational properties7' of amphetamine and r e l a t e d phenethylamines revealed a preference f o r trans phenylamino rotomers i n aqueous although s u b s t i t u e n t groups, steric and electronic f a c t o r s influence rotomer p o ~ u l a t i o n s . ~A~ quantum mechanical study indicated the folded form of amphetamine i s s l i g h t l y favored.72 Metabolism of amphetamine t o an oxime and subsequent hydrolys i s t o benzyl methyl ketone was shown t o c o n s t i t u t e a route of deaminat i ~ n . ~ ' In a r a b b i t l i v e r oxygenase system this conversion involves an intermediate a-hydroxyamine .75

-

of the various i d e a s about -theses. Biochemistry and Pharmacoloqy biochemical causes of a f f e c t i v e disorders,76 '77 the Vatecholamine (CAI hypothesis" is s t i l l t h e most frequently c i t e d . According to this p o s t u l a t e depression may be r e l a t e d t o a deficiency of CA (usually NE) a t functionally important c e n t r a l CA receptors, w h i l e mania r e s u l t s from excess CA. Perhaps an equally p l a u s i b l e case can be made f o r a "serotonin hypothesis of a f f e c t i v e disorders." Consistent with t h e CA hypothesis a r e the observations t h a t when a-methyltyrosine, an i n h i b i t o r of CA biosynthesis, was given to 7 m a n i c and 3 depressed p a t i e n t s under double b l i n d conditions, 5 of t h e m a n i c p a t i e n t s became less manic and t h e 3 depressed p a t i e n t s became more d e p r e s ~ e d . ~ " ~However, ~ attempts t o overcome t h e postulated deficits of CA o r serotonin (5-HT) by administration of t h e amino acid precursors, Gdopa, tryptophan, o r 5-hydroxytryptophan- a s t r a t e g y successful i n the treatment of parkinsonism have given generEven i n l a r g e a l l y disappointing r e s u l t s i n depressed patient^.^"^^ '13' doses equivalent t o those found t o be e f f e c t i v e i n parkinsonism, M o p a produced an antidepressive effect i n Only 4 of 16 p a t i e n t s . A l l of the p a t i e n t s who responded had retarded depressions; none of the 5 p a t i e n t s with a g i t a t e d depression improved. Five depressed p a t i e n t s who had p r i o r

-

24 -

Sect. 1

-

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Engelhardt, Ed.

h i s t o r i e s o f mania developed hypomania o r mania without a r e v e r s a l o f depression. It i s suggested t h a t CA, p a r t i c u l a r l y dopamine (DA), may be involved i n t h e genesis o f mania i n susceptible i n d i v i d u a l s b u t t h a t These depression and mania are not opposite poles of the same entity." r e s u l t s do not support t h e CA theory of depression b u t n e i t h e r do they rule i t out. Although L-dopa i n c r e a s e s DA i n b r a i n , i t may not i n c r e a s e NE. It also produces o t h e r c e n t r a l biochemical a c t i o n s , e.g., effects suggesting t h a t i t causes a decrease i n turnover o f b r a i n 5-HT i n However, t h e mostly negative clinical r e s u l t s with depressed patients." L a o p a seem t o cast f u r t h e r doubt on the v a l i d i t y of t h e behavioral syndrome and amine depletion induced by r e s e r p i n e (which i s reversed by L-dopa) a s a model of depression. The catechol theory of depression i s based i n p a r t on evidence t h a t the t r i c y c l i c antidepressives and ampheramine a f f e c t CA m e t a b o l i s m i n ways t h a t r e s u l t i n an increased a v a i l a b i l i t y o f t h e t r a n s m i t t e r s a t t h e i r receptor sites. It has been thought f o r s o m e t i m e t h a t t h e w e l l known i n h i b i t o r y a c t i o n o f t h e t r i c y c l i c a n t i d e p r e s s i v e s upon t h e uptake o f NE i n central noradrenergic (NA) neurons r e s u l t s i n an i n c r e a s e of t h e monoarnine a t t h e r e c e p t o r s and may account f o r antidepressive a c t i v i t y . Several observations may not be i n accord with this view. I n d i r e c t evidence has been adduced t h a t some t r i c y c l i c agents with t e r t i a r y amino groups (imipramine, chlorimipramine and a m i t r i p t y l i n e ) p r e f e r e n t i a l l y block 5-HT uptake i n 5-HT neumns w h i l e c e r t a i n secondary amines (desmethylimipramine , n o r t r i p t y l i n e and p r o t r i p t y l i n e ) p r e f e r e n t i a l l y i n h i b i t uptake o f NE. Recent s t u d i e s i n which i n vivo uptake o f NE and 5-HT was d i r e c t l y measured histochemically confirm t h e e a r l i e r data a s d i d funct i o n a l s t u d i e s using the extensor and f l e x o r r e f l e x e s t o assess t h e influence of t h e various drugs on 5-HT and NA receptor a c t i v i t y , respect i ~ e l y . ' ~ I n a d d i t i o n t h e antihistamine drugs, chlorpheniramine and e s p e c i a l l y brompheniramine were found t o be very potent blockers o f 5-HT uptake. However, these drugs, u n l i k e t h e t e r t i a r y amino t r i c y c l i c s , also showed a potent blocking a c t i o n on NE uptake. The questions arise whether t h e c l i n i c a l a c t i v i t y o f these drugs i s more c l o s e l y related t o effects on 5-HT o r on NE neurons o r whether t h e r e are two classes o f antidepressives a c t i n g by d i f f e r e n t mechanisms. Some clinicians feel t h a t t h e c l i n i c a l a c t i o n s of t h e t r i c y c l i c a n t i d e p r e s s i v e s can be divided i n t o a mood-elevating component and a psychomotor-activating component. They b e l i e v e t h e t e r t i a r y amines are more e f f e c t i v e i n brightening of mood, suggesting involvement of 5-HT neurons w h i l e t h e secondary amines are more e f f e c t i v e i n i n c r e a s i n g d r i v e o r producing psychomotor a c t i v a t i o n , suggesting t h a t NE neurons a r e mainly Seemingly a t odds with t h e blockade of amine-uptake hypotheses are the p r o p e r t i e s of one of t h e newer t r i c y c l i c s , iprindole. This drug produces c l i n i c a l effects resembling those of imipramine-like agents y e t i n s o m e pharmacological tests, e.g., r e v e r s a l o f reserpine-induced p t o s i s and hypothermia i n mice, i t i s considerably less potent than i~nipramine.'~ It i s a very weak i n h i b i t o r of uptake of NE and 5-HT &-I v i t r o and i n vivo i n mice and rats.86 Despite i t s weak uptake blocking a c t i v i t y , i t potentiated t h e awakening effect of Gdopa i n r e s e w z e d mice a s

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Antidepressives, Stimulants

Kaiser, Z i r k l e

25

imipramine does. Unlike the a n t i d e p r e s s i v e s which block amine uptake, i p r i n d o l e does not augment the pressor response t o NE o r block t h e blood pressure response t o tyramine i n man,'7 Differences i n t h e e f f e c t s of acute and chronic treatment with imipramine and p r o t r i p t y l i n e on c e n t r a l NE metabolism i n t h e r a t have been observed." The turnover of NE was decreased a f t e r acute administration but increased during chronic administration of these drugs. The i n c r e a s e i n NE turnover occurred sooner when thyroxine was administered with imipramine. It i s suggested t h a t these observations may help t o explain t h e delayed o n s e t of c l i n i c a l antidepressive effects and t h e a c c e l e r a t i n g and enhancing effects o f thyroid hormone on the c l i n i c a l e f f e c t s of imipramine. It has been suggested t h a t thyroxine may e x e r t s o m e o f i t s action by f a c i l i t a t i n g the action of NE i n c e n t r a l NA pathways.e9 Rats t r e a t e d with thyroxine became hyperactive and showed increased s e n s i t i v i t y t o the behaviorally a c t i v a t i n g e f f e c t s o f NE administered i n t r a v e n t r i c u l a r l y . Possibly r e l e v a n t t o the v a r i a t i o n i n effects on NE turnover noted under varied conditions of administration of t h e t r i c y c l i c s i s t h e observation t h a t spontaneous a c t i v i t y i n r a t s w a s decreased s h o r t l y after administration of imipramine but w a s increased over t h a t of c o n t r o l animals one day a f t e r a d m i n i s t r a t i ~ n . ~Much ~ more d a t a o f t h e kinds c i t e d above must be obtained f r o m s t u d i e s o f a v a r i e t y of t r i c y c l i c antidepressives, including i p r i n d o l e , before any conclusions about a r e l a t i o n s h i p between t h e clinical a c t i o n of t h e s e drugs and t h e i r effects on monoamine metabolism can be drawn. A study o f t h e effects of L-dopa and Li,CO, on u r i n a r y excretion o f c y c l i c AMP i n depression and mania has been r e p ~ r t e d . ~ ' Manic p a t i e n t s excreted t h e most c y c l i c AMP and severely depressed p a t i e n t s excreted t h e l e a s t ; c o n t r o l s and moderately depressed p a t i e n t s showed intermediate values. Depressed p a t i e n t s t r e a t e d with L-dopa showed marked i n c r e a s e s i n urinary c y c l i c AMP excretion. P a t i e n t s treated with U 2 C 9 showed changes i n c y c l i c AMP excretion i n t h e d i r e c t i o n o f clinical change; a s depression improved, c y c l i c AMP e x c r e t i o n increased and as mania improved i t diminished. It i s not possible t o a t t r i b u t e these changes i n excretion of c y c l i c AMP t o e i t h e r c e n t r a l o r peripheral mechanisms o r both.

Rubidium i o n seems t o have opposite effects from l i t h i u m i o n on CA metabolism and it has been suggested t h a t i t may have c l i n i c a l antidepressive a c t i v i t y . It produces an i n c r e a s e i n t h e r e l e a s e and turnover o f b r a i n stem NE and, u n l i k e l i t h i u m ion, i t induces an increase i n s t e a d of a decrease i n shock-elicited aggression i n rats.92 Further comparisons of rubidium and l i t h i u m i o n s may shed considerable l i g h t on t h e biochemistry of t h e a f f e c t i v e disorders i f t h e i n i t i a l c l i n i c a l observations t h a t rubidium has antidepressive a c t i v i t y a r e confirmed by c o n t r o l l e d studies?' Although t h e r o l e s o f NE and 5-HT i n t h e antidepressive a c t i o n o f the t r i c y c l i c s have n o t been e s t a b l i s h e d , t h e r e i s l i t t l e doubt b u t t h a t the stimulatory a c t i o n of amphetamine i s t o a l a r g e e x t e n t mediated through CA pathways. Early experiments emphasized effects of t h e drug on NA systems. Now t h e evidence i s stronger f o r a DA mechanism of central

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a c t i o n than f o r a NA mechanism. Although t h e r e is some disagreement, most authors believe t h a t amphetamine produces i t s c e n t r a l e f f e c t s by r e l e a s i n g dopamine and NE a t nerve endings from labile i n t r a n e u r o n a l pools of newlysynthesized amines. Amphetamine i s thought t o induce stereotyped behavior by r e l e a s i n g DA whereas apomorphine produces this behavioral effect by a direct a c t i o n on DA receptors. The increase i n locomotor a c t i v i t y e l i c i t e d by amphetamine appears t o be mediated a t l e a s t i n p a r t by a NA action. Other behavioral effects of amphetamine may also involve NA and/or DA actions. The experimental b a s i s f o r t h e s e statements h a s been A f e w of the observations may be b r i e f l y extensively reviewed.53 summarized a s follows. Depletion of CA i n t h e i n t r a n e u r o n a l storage granules by r e s e r p i n e does not prevent t h e i n c r e a s e i n motor a c t i v i t y o r s t e r e o t y p i c a l behavior produced by amphetamine o r apomorphine. I n h i b i t i o n of CA s y n t h e s i s by a-methyltyrosine blocks the effects o f amphetamine b u t not those of apomorphine. I n h i b i t o r s of dopamine P-hydroxylase (e.g., d i e t h y l dithiocarbamate), which i n h i b i t t h e s y n t h e s i s o f NE but n o t DA, decrease amphetamine-stimulated motor a c t i v i t y , b u t do n o t i n h i b i t and even may enhance amphetamine-induced stereotypies. Recent s t u d i e s confirm these r e s u ~ t s . ~'9'J '95

"'

b s t evidence suggests t h a t amphetamine- and apomorphine-induced s t e r e o t y p i e s a r e t h e r e s u l t of enhanced DA a c t i v i t y i n t h e neostriatum. Consistent with this view i s t h e e f f e c t of amphetamine i n rats with u n i l a t e r a l l e s i o n s of t h e n i g r o s t r i a t a l DA pathway ( s e e Chapter 1, p 19. The drug causes i p s i l a t e r a l turning i n t h e s e animals, presumably as a consequence of release o f DA i n t h e neostriatum from t h e i n t a c t DA neurons of the c o n t r a l a t e r a l (non-lesioned) side. T h i s effect of amphetamine i s attenuated by pretreatment with a m e t h y l t y r o s i n e b u t n o t with reserpine.96 19' I n c o n t r a s t , apomorphine, which i s believed t o be a d i r e c t a c t i n g DA agent, induces c o n t r a l a t e r a l turning behavior, an e f f e c t a t t r i b u t e d t o denervation s u p e r s e n s i t i v i t y o f s t r i a t a l DA r e c e p t o r s i n t h e neostriatum on the lesioned side. T h i s e f f e c t of apomorphine i s not antagonized by e i t h e r r e s e r p i n e o r a-methyltyrosine. I n r a t s with u n i l a t e r a l l e s i o n s i n t h e neostriatum, amphetamine and apomorphine both e l i c i t i p s i l a t e r a l turning. Similar effects are observed i n mice with u n i l a t e r a l l e s i o n s o f t h e caudate nucleus. This preparation provides a simple and rapid model f o r studying effects of drugs on c e n t r a l DA systems.9e Possibly a t odds with t h e hypothesis t h a t drug-induced stereotyped behavior involves a DA mechanism are observations on t h e effects of s o m e stimulants on t h e turnover o f b r a i n CA i n the r a t . cJ-Amphetamine, aminorex and p-chloramphetamine a t doses which increased motor a c t i v i t y , but d i d not induce s t e r e o t y p i e s , caused an i n c r e a s e i n turnover o f DA b u t not of NE. These r e s u l t s a r e d i f f i c u l t to i n t e r p r e t , however, because phenmetrazine did n o t a l t e r turnover of DA o r NE a t a dose t h a t increased motor a c t i v i t y . 9 9 Although t h e c e n t r a l a c t i o n s of amphetamine may r e q u i r e an uninterrupted s y n t h e s i s of CA r a t h e r than reserpine-sensitive s t o r e s of the amines, this does n o t seem t o be true f o r a l l amphetamine-like stimulants.

Chap. 2

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Kaiser, Z i r k l e

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A r e c e n t study, together with e a r l i e r d a t a , suggests that amphetamine-like

stimulants can be divided i n t o two groups according t o t h e ways i n which they i n t e r a c t with r e s e r p i n e and a-methyltyrosine. The e f f e c t s of members of one group - amphetamine, methamphetamine, ephedrine,phenmetrazine and phentermine - are i n h i b i t e d by a-methyltyrosine b u t not by reserpine; those of members of t h e second group benzphetamine, methylphenidate, pipradrol, It-dihydro-bxo-1,acocaine and amfonelic acid ( 7-benzyl-1-ethyl-1, naphthyridine-3-carboxylic acid) are i n h i b i t e d by r e s e r p i n e b u t n o t by m e t h y l t y r o s i n e , a t least i n moderate doses.”’ Although, a s t h e s e results suggest, stimulants may d i f f e r i n t h e i r a c t i o n s on i n t r a n e u r o n a l pools of CA, no d i f f e r e n c e i n t h e i r c l i n i c a l e f f e c t s has been observed. b t h y l p h e n i d a t e produced physiologic, s u b j e c t i v e and behavior effects t h a t w e r e not s i g n i f i c a n t l y d i f f e r e n t from those of amphetamine, methamphetamine, ephedrine and phenmetrazine.‘

-

-

’‘

Further s t u d i e s of t h e metabolism o f amphetamine and o f t h e effects of t h e drug and i t s metabolites on CA metabolism have n o t defined t h e biochemical e f f e c t s underlying t o l e r a n c e development t o t h e drug.102-104 A thorough study was made o f t h e psychomotor and anorexigenic effects of d-amphetamine upon chronic dosing.’ Tolerance development t o t h e drug occurred i n r a t s which w e r e dosed d a i l y s h o r t l y before t e s t i n g . Tolerance d i d not occur i n rats which received t h e same d a i l y doses o f amphetamine but were dosed a f t e r each d a i l y t e s t session. Subsequent dosing of t h e l a t t e r group with amphetamine p r i o r t o t e s t i n g produced psychornotor stimulation and anorexigenic e f f e c t s a s l a r g e a s those i n i t i a l l y observed i n t h e r a t s t h a t had always been dosed d a i l y p r i o r to t e s t i n g . The r e s u l t s suggest t h a t tolerance t o t h e s e effects of amphetamine r e p r e s e n t s a behavioral adaptation of the r a t s t o the. “drugged” state.

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Several reports described effects of t h e methylxanthines and amantadine on b r a i n monoamine m e t a b o l i s m which may be involved i n t h e i r pharmacological actions. Caffeine and theophylline release b r a i n NE i n r a t s and guinea pigs.’o6 The i n c r e a s e i n c y c l i c AMP produced by these drugs, u s u a l l y a t t r i b u t e d t o i n h i b i t i o n of phosphodiesterase, could be I n mice due t o stimulation of adenyl cyclase by t h e released NE. c a f f e i n e and aminophylline appear t o i n c r e a s e turnover of b r a i n DA and NE.lo7 Other d a t a suggest t h a t t h e methylxanthines e i t h e r prevent t h e The antiparkinsonisn release o f b r a i n 5-HT o r i n c r e a s e 5-HT synthesis.”* e f f e c t s of amantadine have stimulated s t u d i e s o f i t s c e n t r a l actions. Extensive biochemical and pharmacological d a t a from s t u d i e s i n r a t s and mice i n d i c a t e t h a t this drug produces i t s effects, e.g. i n c r e a s e i n r o t a t i o n a l behavior i n t h e r a t turning model and i n c r e a s e i n motor a c t i v i t y , by r e l e a s i n g CA, e s p e c i a l l y DA, f r o m e x t r a g r a n u l a r (reserpiner e s i s t a n t ) s t o r e s . Thus amantadine appears t o have an amphetamine-like However, action b u t i t i s much less potent than unlike amphetamine, amantadine-induced i n c r e a s e i n motor a c t i v i t y i n mice was not a l t e r e d by a-rnethyltyrosine.l

’’

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-' 30

206 (1971).

61. G. Gardos and J. 0. Cole, Curr. Ther. Res., C l h . Exp. l3, 631 (1971). 62. A. H. Tang and J. D. Kirch, Rychophannacologia, 2l, 139 (1971). 63. C. F. Barfknecht, D. E. Nichols, B. Olesen and J. A. Engelbrecht, Pharmacologist 2,233 (1971). 64. B. Hach and F. He& Arzneh-Forsch., 2l, 23 (1971). 65. 0. Nieschulz, h i d . , g,1497 (1970). 66. M. Julia and J. deRosnay, Chh. Ther., 2, 337 (1970). .l, 645 (1971). 67. A. A. Siciliano and K. A. Nieforth, J. Med. Chem., 4 68. E. J. Lien, L. L. Lien and G. L. Tong, h i d . , 846 (1971). 69. J. Borsy, I. Elekes, J. I. S&ely and L. Toldy, Acta. Physiol. Acad. Sci. Hung., 39, 275 (1971). 70. R. W. Fuller and B. B. Molloy, Pharmacologist, l3, 294 (1971). 71. G. A. Neville, R. Deslauriers, B. J. Blackburn, and I. C. P. Smith, J. Med. Chem., 717 (1971). 72. B. hllman, J.-L. Coubeils, P. Cwrriere and J.-P. Gervois, ibid., l5, 17 (1972). 73. K. Bailey, A. W. By, K. C. Graham and D. Verner, Can. J. Chem., 3143 (1971). 74. A. H. Beckett, J. P. Van Dyk, H. M. Chissick and J. W. Gorrod, J. Pharm. pharmacol., 23, 560 (1971). 75. C. J. Parli, N. Wang and R. McMahon, Biochem. Biophys. Res. Connnun., 43, 1204 (1971). 76. G. A. Aillon, Psychosomatics, l2, 260 (1971). 77. D. J. McClure, Can. Psychiat. Ass. J., l6, 247 (1971). 872 78. W. E. hnney, Jr., H. K. H. Brodie, D. L. k r p h y and F. K. Goodwin, Amer. J. Psychht., (1971). 79. H. K. H. Brodie, D. L. hrphy, F. K. Goodwin and W. E. Bunney, Jr., C l b . Pharmacol. Ther., 2, 228 (1971 1. 80. F. K. Goodwin, D. L. Murphy, H. K. H. Brodie and W. E. Bunney, Jr., Biol. Psychiat., 2, 341 (1970). 81. B. J. Carroll, C l h . Pharmacol. Ther., l2, 743 (1971). 82. F. K. Goodwin, D. L. h n n e r and E. S. Gershon, Life Sci., lo, Part I, 751 (1971). 83. P. Lidbrink, G. Jonsson and K. Fuxe, Neuropharmacology, 522 (1971). 84. F. Sulser and E. Sanders-Bush, Annu. Rev. Pharmacol., 11, 209 (1971). 85. M. I. Gluckman and T. hum, Psychop8harmacologia,l5, 169 (1969). 86. S. B. Ross, A. L. Renyi and S.-0. Ogren, Life Sci., l0, Part I, 1267 (1971). 87. W. E. Fann, J. Y. Davis, J. S. Kauflpann, J. D. Griffith, D. S. Janmsky and J. A. Oates, F'harmacologist, l3, 207 (1971). 88. J. J. Schildkraut, A. Winokur, P. R. Draskc6zy and J. H. Hensle, h e r . J. Psych&., In, 1032 (1971). 69. W. M e n , D. S. Segal and A. J. Mandell, Science, 2,79 (1972). 90. D. Meltzer and P. A. Fox, Psychophamcologia, 2l, 187 (1971). 91. M. I. Paul, H. Cremer and F. K. Goodwin, Arch. Gen. Psychiat., 24, 327 (1971). 92. J. M. Stolk, R. L. Conner and J. D. Barchas, Psychophamacologia, 2, 250 (1971). 93. T. H. Svensson, Naunyn-Schmiedebergs Arch. Phaxmak. Exp. Pathol., 271, 111 (1971). 94. U. S t h b e r g and T. H. Svensson, Psychopharmacologia, 2,53 ( 1 9 7 1 r 95. J. Maj, 1. Grabonska and E. Mogilnioka, Psychophannecologia,s, 162 (1971). 96. U. Ungerstedt, Acta Physiol. Scand., Suppl., 49 (1971). 97. U. Ungerstedt, ibid., Suppl., 69 (1971). 98. V. J. Lotti, Life Sci., 30., Part I, 781 (1971). 99. E. Costa, K. M. Nabzada and A. Revuelta, B r i t . J. Pharmacol., 43, 570 (1971). 100. J. Scheel-Kzger, Eur. J. Phaxmacol., 47 (1971). 101. W. R. Martin, J.W. Sloan, J. D. Sapira and D. R. Jasinski, C l i n . Phamacol. Ther., l2, 245 (1971). 102. T. Lewander, Naunyn-Sohmeidebergs Arch. Phaxmak. Exp. Pathol., 271, 221 (1971).

2,

2,

2

m,

2,

H,

z,

2,

30

Sect. 1

-

CNS Agents

E n g e l h a r d t , Ed.

103. G. A. Clay, A. K. Cho and M. Roberfroid, Biochem. Pharmacol., g,1821 (1971). 176 (1971). 104. H. C. Rbiger and E. G. McGeer, Eur. J. Phamcol., 105. R. T. H i l l , Abstracts, 7th Middle Atlantic Regional Meeting Amer. Chem. SOC., Feb. 14-17, 1972,

16,

p. B. B. B.

59.

A. Berkowitz, J. H. Tarver and S. Spector, Eur. J. Pharmacol., 2, 64 (1970). Waldeck, J. Pham. hamacol., 23, 824 (1971). A. Berkowitz and S. Spector, Eur. J. h a m c o l . , 322 (1971). U. Stramberg and T. H. Svensson, Acta Phamcol. Todcol., 161 (1971). L.-0. Farnebo, K. Fuxe, M. Goldstein, B. Hamberger and U. Ungerstedt, Eur. J. Fhamacol., (1971). 111. J. E. Thornburg and K. E. Moore, Pharmacologist, 2,202 (1971).

106. 107. 108. 109. 110.

E,

2,

2, 27

Chapter 3 .

Analgesics and Narcotic Antagonists

Franklin M. Robinson Merck Sharp & Dohme Research Laboratories, West Point, Pa. 19486 Literature reports over the last year indicate some decrease in the amount of chemical effort devoted to new analgesic structures, but a continued increase in investigation of biochemical mechanisms of analgesia and dependence. Interest in narcotic antagonists for the treatment and prevention of narcotic abuse has stimulated much current research on new compounds. In neither area was there evidence of a significant advance during the year. An extensive review of the literature on narcotic antagonists and analgesics from July, 1966 to April, 1970 appeared.’ I.

Strong Analgesics

A. New Clinical Studies

-

No clinical studies on new compounds were reported, but new studies on diviminolY2 tilidhe,, and bezitramide4y5 continue to indicate that these are orally effective analgesics in the codeine-morphine potency range which do not produce tolerance.

-

B. Structure-Activity Studies There is an increasing tendency to use the newer tests (inflamed foot, writhing, bradykin) for evaluation of analgesic potency but, unless otherwise specified, potency estimates in the following discussion are based on the mouse hot plate assay.

-

1. Compounds Related to Morphine New reports of the chemistry and activities of ethenothebaines and oripavines include a collated review of la structure-activity relationships containing previously unreported data. The importance of an R configuration at C19 for high analgesic activity has been established6 for the 7-8 as well as the 742 series. The primary ,CH3

~+$,~~ncs~T

7--@

om3

OH

I

IS\ OH

&*#I.. OH

OCH, I1

importance of a lipophilic binding site rather than that previously postulated for the (2-19 hydroxyl was shown by the finding that 11 is as potent (1000 times morphine in the rat tail pressure test) as etorphine (I). Structure-activity relationships in this series vary greatly according to the test used.la

32

Sect. 1

-

CNS Agents

Engelhardt, Ed.

Both natural and synthetic (2) forms of the cholinesterase inhibitor, galanthamine, were reported to be analgesics equipotent with morphine .7 ( 0 )

-

Synthesis and conformation of the 3- (111) and 4-methyl 2. Benzmorphans (IV) benzmorphans were reported.8 IVa (2 times codeine) was about three times as potent an analgesic as IIIa, but IVb, c and d surprisingly were

-

inactive, In the I11 series, all were active the cyclopropylmethyl analog (IIIc) about five times as active as the methyl (IIIa) analog ands without antagonist effect or physical dependence capacity in the monkey.

The benzmorphan analog (V) was comparable to codeine in the acetic acid writing test, but eleven other analogs oxygenated at the carbon 10 bearing the phenyl group were inactive.

-

3. Meperidine Analogs WY-22811 (VI)ll and both of its optical isomers were somewhat more potent analgesics than pentazocine, and both antagonized morphine, the (-) isomer being the more potent. Side effects were minima 1

.

%00C2H5

cH2m3

‘0

VI

VII

(b) n = 2

Chap.

3

Rob i nson

Analgesics, Narcotic Antagonists

33 -

The two dioxolanoalkyl meperidines (VII) shared activity in the meperidine range (VIIb was 3 times VIIa).12 The corresponding dioxane analogs were inactive at 50 mg./kg. but did potentiate meperidine.

VIII

LH2CH2 - @ H C O C H = C H C O O C 2 H j

IX The trans isomer of VIII showed weak analgesic activity while the cis was inactive, although it more closely approximates the structure of morphine than does the trans.13 A series of anileridine derivatives bearing substituents with potential alkylating ability was made in order to investigate the possibility of locating analgesic receptors by specific alkylation.14 The fumaryl derivative (IX) caused apparent inactivation which'could be prevented by naloxone pretreatment. Other analogs of IX were non-selective and toxic. An attempt to label receptors in brain homogenates by photolysis with [H3]-N-B-(p-azidophenyl)-ethylnorlevorphanol also showed insufficient specificity although the compound was a potent narcotic.15

-

New Compounds Extensive pharmacological studies of MZ-144 (X) and 16-19 It was limited data on 20 analogs (of 85 prepared) were reported.

C.

H X

XI

orally active, did not produce tolerance, was not antagonized by nalorphine and strongly potentiated morphine even in tolerant animals. At high doses, MZ-144 was nearly as effective as morphine in animal studies, and it was said to be clinically effective at doses of 0.15-0.3 g. Abbott 30360 (XI) was found to quilizer whose analgesic effect was data on 14 analogs indicated fairly analgesic potency, although the 8-c"

be an orally active analgesic-tranLimited in the morphine range.20 high structural specificity for analog was equally active.

34

Sect. 1

&.J

-

Engelhardt, Ed.

CNS Agents

1 R

XI1 Four quinazolones (XII) of a series of 19 were more potent ana-gesics than codeine. Substitution in the ring or modification of the R-group reduced activity. XIIa has been selected for clinical trial.21 WY-12157 (XIII) showed anal esic activity of the order of morphine as did both of its optical isomers.B2 It was not antagonized by naloxone and produced little or no tolerance, but CNS oxicity and blindness were seen during chronic administration to monkeys. &a

R I

R

t

(a) -H

XIV XIVa was found to be a centrally-acting analgesic more potent than codeine with little respiratory depressant action or physical dependence capacity.24 Twenty of 64 analogs made were also active. XIVb and c antagonized XIVa but potentiated morphine.

PJcH2NHcH3

0%

xv

c$(

IzOOH

0CH,

XVI

XV2’ and XV126 were reported to have significant analgesic activity, but data were limited.

Antiinflammatory Analgesics For complete coverage of this class of compounds refer to the chapter on Antiinflammatory Agents (Section 4). No new compounds with outstanding analgesic activity have appeared. 11.

Reports on new clinical studies27 on clonixin show equivalence of

600 mg. p.0. with 10 mg. of morphine parenterally in post-operative pain.

Chap. 3

Analgesics, Narcotic Antagonists

Rob inson

35

111. Biochemical Mechanisms Many new data concerning the role of neurohumoral agents in the actions of analgesics continue to appear. The conflict between different workers on the effect of morphine on brain serotonin turnover28929 may be due to differeniotime courses of the experiments, since it appears to be an acute effect. Evidence has been reported suggesting that the role of catecholamines in anal esia may depend mainly on dopamine or dopamine/norepinephrine Strong cross tolerance between the analgesic effects of ratios. "pS methamphetamine and morphine develops after 5 days 1 adrninistrati~n.~' Additional studies on the depression of brain acetylcholine release by analgesics34 have shown that morphine also facilitates its action at the neuromuscular junction.35 A review of knowledge concerning the harmacokinetics and sites of action of narcotics in the brain appeared.!36 IV. Narcotic Antagonists Over the past ten years, research on narcotic antagonists has been directed principally toward the discovery of new analgesics, since it was originally thought that such compounds would not have abuse liability. Work leading to thf introduction of pentazocine has been discussed in a , 37-40 Structurally, these antagonists were polynumber of reviews. cyclic potent analgesic molecules with the N-methyl group replaced by an allyl, cyclopropylmethyl or related moiety. The most potent analgesicantagonists did not attain practical utility because of hallucinogenic or other bizarre central nervous system effects that are usually associated with powerful antagonist activity. More recent research on analgesics has produced new types of compounds that will not support morphine dependence, and some of which pharmacologically behave as mild morphine antagonists. These are not related structurally to the antagonists mentioned above. Direct addiction studies on both types of antagonists demonstrated a capacity to produce physical dependence (e .g. pentazocine,*' nalbuphine,& profad01~~ and pro~iram*~) of a type which could lead to drug seeking behavior.44 (The potent antagonists with hallucinogenic effects can also produce a milder type of physical dependence not leading to drug seeking behavior.) Thus, it has been established that a component of antagonist activity does not necessarily eliminate abuse liability. TWO potent antagonists, naloxone (XvIIa) and cyclazocine (XVIII) were administered to selected groups of narcotic addicts in an attempt to Itcure" their addiction by blockin an pleasurable effect of narcotic of the series of preliminary self-administration. The results"*' studies were sufficiently favorable that large-scale controlled studies were begun to better evaluate this type of approach, Research in this

36

Sect. 1

-

Engelhardt, Ed.

CNS Agents

area is being expanded greatly as part of the effort47 now being directed toward alleviation of the drug abuse problem. Although some of the current research in this field has been discussed in newspapers and magazines, few data suitable for this review have been published. Data reported so far indicate that naloxone has too low an oral potency and too short a duration of action to be satisfactory, while the side effects of cyclazocine may be unacceptable.

tg

only reported narcotic antagonist without Naloxone (XVIIa) is other biological activity, This can be demonstrated by an acetylcholine release assay* which measures both agonist and antagonist actions of narcotics, as well as by pharmacological studies. In contrast, analogs

cH3

R

I

1

XVII

XVIII

XVIIb, c and d show both analgesic and antagonist properties. XVIIb (EN-1639) is currently being studied in addicts5' since it appears to be longer acting than naloxone, and shows fewer side effects than cyclazocine. Cyclazocine (XVIII) is a powerful antagonist and analgesic, and its many known analogs show both properties. Diprenorphine (XIXa) is a pure morphine antagonist (3-5 times naloxone) pharmacolo ically, but shows some agonist activity in the acetylcholine assay." In this series, structure around C-19 is critical, e.g. XIXb and c are not antagonists.52

& HO

HO

OCHs XIX

OH

0

xx

Chap.

3

Analgesics, Narcotic Antagonists

Robinson

37

XX shows mild antagonist activity which is unusual for an N-methyl compound la

.

Diphenoxylate and an analog SC-26100 (XXI) will prevent withdrawal symptoms in morphine-addicted animals, and the former has been reported to have the same effect in man. 53

4

0

Haloperidol has been reported to be useful in the treatment of withdrawal symptoms in addicts and also to reduce morphine self-administration in rats at the proper dose.54 REFERENCES

1. J. Lewis, K. Bently and A. Cowan, Ann. Rev. Pharmacol., ll, 241 (1970). (a) P. 249. 2. F. Nobili and G . Bernardi, Europ. J. Clin. Pharmacol., 2, 119 (1971). 3 . J. Klapetek, Med. Welt, 1523 (1971). 4. P. Janssen, C. Niemegeers, K. Schellekens, R. Marsboom, V. Herin, W. Amery, P. Admiraal, J. Bosher, J. Crul, C. Pearce and C. Zegveld, Arzneim. Forsch., 2 l , 862 (1971). 5. H. Knape, Brit. J. Anesthesia, 42, 76 (1971). 6. J. Lewis and M. Readhead, J. Chem. SOC. (C), 2296 (1971). 7. T. Kametani, G., 1043 (1971). 8. R. Parfit and S. Walters, J. Med. Chem., 14,965 (1971). 9. J. Villarreal and M. Seevers, Addendum to the minutes of the 33rd Meeting of the Committee on Problems of Drug Dependence, Nat. Acad. Sci., 1939 (1971). 10. W. Chang, L. Walter and R. Taber, J. Med. Chem., 14, 1011 (1972). 11. P. Goode and A. White, Brit. J. Pharmacol., 9, @P (1971). 12. D. Kriesel and 0. Giavold, J. Pharm. Sci., 60, 1250 (1971). 13. J. Greenhill, J. Hcd. Chem., 14,971 (1971). 14. P. Portoghese, V. Tchng, A . Takemori and G. Hayashi, g., &, 144

(1971).

s,

15. B. Winter and A . Goldstein, Fed. Proc., 528 (1972). 16. J. Knoll, Z. M&z&os, P. Szentmikl6si and S. Furst, Arzneim. Forsch., 21, 717 (1971). 17. 7 Knoll, S. Fursf and Z. Me/szB/ro8, g., 2 l , 719 (1971). 18. J. Knoll, S. Furst and Z. Me/s/ziros,E., 21, 727 (1971). 19. J. ~noll,K. Magyar and D. Banfi, E., 21, 733 (1971).

38

Sect. 1

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CNS

Engelhardt, Ed.

Agents

20. E. Kimura, P. Dodge, P. Young and R. Johnson, Arch. Intern. Pharmacodyn., 190, 124 (1971). 21. M. Vincent, J. Poignant and G. Remond, J. Med. Chem., 714 (1971). 22. J. Malis, Fed. Proc., 2, 563 (1971). 23. Ref. 9, p . 1950. 24. G. Regnier, K. Canevari, J. LeDouarec, S . Holstrop and J. Daussy, J. Med. Chem., Q, 295 (1972). 25. T, Ohgah, N. Hirose, S . Sohda and S . Toyoshima, J. Pharm. SOC. Japan,

2,

603 (1971). 26. H. Hishmat, A. Hashim, M. Shalash and M. Nawito, Arzneim. Forsch., 21, 1026 (1971). 27. Finch and T. DeKornfeld, J. Clin. Pharmacol., 11, 571 (1971). 28. S. Algeri and E. Costa, Biochem. Pharmacol., 2 0 , @' (1971). 29. D. Cheney, A. Goldstein, S. Algeri and E. Costa, Science, IJl, 1169 09-711 30. G. Yarbrough, D. Buxbaum, M. Sanders and E. Bush, Life Sci., 10, 977 (1971) 31. K. Fukui and H. Takagi, Brit. J. Pharmacol., 44,45-51 (1972). 32. A . Takemori and Y. Maruyama, Fed. Proc., 1, 504 (1972). 33. B. Pleuvry, J. Pharm. Pharmacol., a,969 1971). 34. K. Jhamandas, J. Phillis and C. Pinsky, Brit. J. Pharmacol., 4J, 53 (19711 35. R. Frederickson and C. Pinsky, Nature New Biol., 231, 93 (1971). 36. A. Herz and H. Teschemacher, Advances in Drug Research, 6, 79 (1971). 37. W. Martin, Pharmacol. Rev., 2,463 (1967). 38. H. Frazer and L. Harris, Ann. Rev. Pharmacol., 1,277 (1967). 39. S. Archer and L. Harris, Prog. Drug Res., g, 261 (1965). 40. L. Harris and W. Dewey, Ann, Reports in Med. Chem. 1967, 36 (1968). 41. D. Jansinski, W. Martin and R. Hoeldtke, Clin. Pharmacol. Therap., 11, 385 (1970). Jasinski and P. Mansky, E.,Q, 78 (1972). 42. 43. D. Jasinski, W. Martin and R. Hoeldtke, G., 12, 613 (1971). 44. J. Inciardi and C. Chambers, N. Y. State J. Med., 71, 1727 (1971). 45. M. Fink, Science, 1005 (1970). 46. A. Zaks, T. Jones, M. Fink and A. Freedman, J. Am. Med. ASSOC., 3, 2108 (1971). 47'. J. Jaffe, Clin. Res., 2,612 (1971). 48. H. Blumberg, H. Dayton and P. Wolf, Proc. SOC. Exp. Biol. Med., B, 755 (1966) 49, A. Cowie, H. Kosterlitz and A. Watt, Nature, 220, 1040 (1968). 50. A. Hammond, Science, 503 (1971). 51. H. Kosterlitz and A. Watt, Brit. J. Phanaacol., z, 266 (1968).

.

f

.

a,

m,

52. Ref. 1, p. 254. 53. E. Dobrin, D. Krok and W. Potts, Fed, Proc., 2,503 (1972). 54. H. Hanson and C, Cimini-Venema, 503 (1972).

E.,

,

Chapter 4.

Sedatives, Hypnotics, Anticonvulsants and General Anesthetics A. D. Rudzik and W. Friis The Up john Company, Kalamazoo, Michigan 49001

-

Introduction Although an effort has been made in this chapter to exclude depressants classified in other chapters as antipsychotic and antianxiety agents, certain benzodiazepines will be considered here because of the number of compounds in this series which have been found clinically to possess sedative-hypnotic and anticonvulsant activity. In most cases, compounds with more than preliminary pharmacological data o r representatives of new structural types were selected for inclusion in this chapter.

-

Sedatives and Hypnotics Considerable research effort has been expended to elucidate the possible role of serotonin in the neuropharmacology of sleep: Administration of serotonin in proper dosage and by the proper route produces electroencephalographic and ocular signs of (slow wave) sleep. Nonspecific reduction of serotonin levels in the brain by reserpine reduced both rapid eye movement (REM) and non-rapid eye movement (NREM) sleep while sterotaxic destruction of serotonin-containing neurons has produced insomnia in animals.2 With the discovery of a specific inhibitor of serotonin synthesis, para-chlorophenylalanine, it has been possible to deplete brain serotonin without altering the catecholamine content markedly3. A dose dependent reduction in cerebral serotonin, induced by the administration of para-chlorophenylalanine, is followed by a dose-dependent reduction in total sleep lasting as long as 16 days.4 Injection of 5-hydroxytryptophan at the height of the para-chlorophenylalanine action restored slow wave sleep within 10 minutes and after a delay of 5 hours, paradoxical sleep. In man, para-chlorophenylalanine decreased rapid eye movement ( R E M ) sleep, with either no change or a slight increase in non-rapid eye movement (NREM) sleep5. L-Tryptophan, a precursor o f serotonin has also been shown in normal subjects to reduce sleep latency and increase sleep length without altering In a the qualitative characteristics of polygraphically recorded sleep'. double-blind study in hospitalized insomniac patients, doses of 4 to 5 g of L-tryptophan significantly increased sleep time, reduced sleep latency and reduced the number of awakenings

.'

Several reviews have recently appeared on the neurohumoral aspects of sleep control4 and on the chemical families which influence sleep patterns. Many benzodiazepine derivatives have been reported to possess sedative and hypnotic activity in both animals and man. Flurazepam (I) has been found to possess useful hypnotic activity in man without producing In animals flurasignificant "hangover" effects the following morning8". zepam was found to possess activity, in many tests systems, similar in potency and profile to diazepad'.

40

Sect. 1

-

CNS A g e n t s

E n g e l h a r d t , Ed.

I11

IV-X=H Y=H V’X’CH3 Y=H VI-XzCH3 Y 4 1

Flunidazepam (11) was found i n a l l - n i g h t human EEG sleep s t u d i e s and o t h e r e f f i c a c y s t u d i e s t o be a o t e n t h y p n o t i c approximately 15 t o 30 times more a c t i v e t h a n f lurazepam R e c e n t l y a n a n a l o g of oxazepam , lorazepam (111, WY-4036) was r e p o r t e d by Hedges e t a l l 3 t o p o s s e s s p o t e n t s e d a t i v e and d e p r e s s a n t a c t i v i t y i n man and was approximately 5 times more a c t i v e t h a n diazepam i n t h i s s t u d y . Perhaps t h e most s i g n i f i c a n t benzodiazepines r e p o r t e d i n t h e l a s t two y e a r s have been t h e t r i a z o l o b e n z o d i a z e p i n e s ( I V , V , V I ) . These compounds have been found t o p o s s e s s s e d a t i v e and h y p n o t i c a c t i v i t y i n a n i m a l ~ ~ * , ~ 5 , ~ 6 iannman17. d From e x p e r i m e n t a l d a t a i n man, Compound V was p r e d i c t e d t o have l o n g l a s t i n g a n t i a n x i e t y p r o p e r t i e s a t low d o s e s and s e d a t i v e - h y p n o t i c a c t i v i t y a.t h i g h d o s e s 17. Another benzodiazepine d e r i v a t i v e , V I I , has been r e p o r t e d t o possess h y p n o t i c a c t i v i t y i n animals and i s more p o t e n t t h a n diazepam i n t h i s r e g a r d la.

VII

VIII

While t h e most s i g n i f i c a n t s e d a t i v e s and h y p n o t i c s found r e c e n t l y have been benzodiazepine d e r i v a t i v e s , s e v e r a l i n t e r e s t i n g non-benzodiazepine d e r i v a t i v e s have a l s o been r e p o r t e d t o p o s s e s s s e d a t i v e and h y p n o t i c a c t i v i t y . One of t h e s e compounds, p e r l a p i n e ( V I I I ) , has been found t o be a u s e f u l h y p n o t i c i n man.lS

Chap. 4

Rudz i k,

Depressants

Fr i i s

41

c

Janssen et a12' has reported that etomidate (IX, R-16659) is a potent i.v. hypnotic in rats with a rapid onset of action and a duration of hypnosis dependent on the dose administered. The compound has a high safety margin with the toxic dose being approximately 30 times the efficacious dose. Three new pyridazone derivatives (X, XI, XII) were reported by Wermuth et a121 to possess sedative and hypnotic activity. The compounds were synthesized in an analgesic program but were poorly active in analgesic test systems.

--

& &. do X

'

0

-

CH2CH2

XI

I

-

CH2CH2

'- 0

CH2CH2 XI1

An interesting aporphine alkaloid, xylopine (XIII), isolated from the bark of Xylopia brasilienes, possesses both sedative and analgesic activity in mice at moderate doses22. In dogs the compound was found to possess hypotensive activity.

In contrast to many other classes of compounds, the activity of many CNS agents has been found clinical1y.Fenfluramine is an example of such a clinical finding. Fenfluramine (XIV) is presently marketed in 22 countries as an effective and safe anorexigenic drug. Though a phenethylamine derivative, the compound produced sedation and drowsiness in man as a side effect in the anorexigenic studies23. A ureide derivative, capuride (XV, McN-X-94) is a new addition to the nonbarbiturate sedatives. It is especially useful in aiding sleep in preoperative patients2*. Finally, a new derivative of cyclic 3'5' -adenosine monophosphate, HD-233(6-piperidino-purinoribosyl-3 '5'cyclic AMP) has been found to have a sedative effect in rats25. The compound does not alter either serotonin or 5-hydroxy-indoleacetic acid levels in the brain of rats but does increase the brain levels of barbiturates and prolongs barbiturate sleeping time.

-

Anticonvulsants During the past two years numerous compounds possessing anticonvulsant activity have been reported. Many of the compounds report-

42 -

Sect. 1

-

CNS Agents

Engelhardt, Ed.

ed were benzodiazepine derivatives and these will be reviewed briefly in this section. Chlorazepate (XVI, A-35616, 4306CB) possesses anticonvulsant activity in mice equipotent to diazepam2e. The monopotassium and the dipotassium salts possessed approximately the same activity and have the advantage of being water soluble. Although the pharmacology of clonazepam (XVII, Ro 5-4023) has been reported previously in this review, more recently numerous papers have appeared reporting the potent anticonvulsant activity in animals27 and man28’29. Clonazepam has been found to be 10 times more active than diazepam.

c 1’ \

XVI

XVII

XvIII-X=C1 XIX- x=C1 XX- X=H XXI- X=H

Y=c1 Y=H Y=H Y X l

A series of triazolobenzodiazepines were found to possess potent anticonvulsant activity in animals. U-33,030 (XVIII) and U-31,889 (XIX) were much more potent than diazepam in mice, rats and cats while U-31,957 (XX) and U-35,005 (XXI) were equipotent to 30. These compounds are extremely active against chemically induced seizures but only weakly active against seizures induced by electroshock. Removal of the chloro-group from position 7 in the diazepam molecule markedly decreased the pharmacologic activity while in the triaaolobenzodiazepine series the compound (XX) still was equipotent to diazepam, This points out the major differences in the triazolobenzodiazepine structure activity relationships compared to the diazepam series. Another benzodiazepine derivative possessing anticonvulsant activity is 0~~?-8063 (XXII). The compound inhibits maximal electroshock, pentylenetetrazol and thiosemicarbazide convulsions in laboratory animals31.

XXII

XXIII

XXIV

Chap.

4

Depressants

Rudzik, Friis

43 -

A series of 22 phenanthridine basic ethers reported by Ashford et a1 were found to possess anticonvulsant activity in mice=. Some of the derivatives, of which compound XXIII is a representative, were as active as diphenylhydantoin in antagonizing pentylenetetrazol and electroshock convulsions but in most cases possessed therapeutic ratios inferior to diphenylhydantoin. Funcke and Zandberg, evaluating a series of tricyclic compounds for anticonvulsant activity, found two compounds, BS7679 ( X X I V ) and BS7029 (XXV), worthy of further studyg3. The compounds were more efficacious than phenobarbital with a low neurotoxicity.

xxv A series of diphenylaminopropanols were evaluated for toxicity, anticonvulsant, anorexigenic and anticholinergic activity34. Several of the derivatives (XXVI , X X V r I ) were potent anticonvulsants against thiosemicarbazide, electroshock and nicotine induced convulsions without possessing anticholinergic activity.

XXVI

XXVI I

Alkoxymethyl derivatives of barbiturates and diphenylhydantoin are The compounds( X X V I I I ,XXIX) possess marked effective anticonv~lsants~~. activity against both maximal electroshock and pentylenetetrazol induced seizures

.

c2:Qi@cH3

CH3O-C H p

-

a CHgCH3

O 0

XXVIII

XXIX

The same authors also reported that acyloxymethyl, bis (acyloxymethyl) and bis (halomethyl) derivatives of 5- e t h y l - 5 - p h e n y l b a r b i t u r i c acid possessed marked anticonvulsant activityg6. Depakine (XXX) has been reported by DeBiolley and Sore1 in uncontrolled studies to be an effective anticonvulsant in the treatment of petit ma1 epilep~y~~’ Some ~ ~ .action was also noted in psychomotor seizures. Side effects in these studies were not a major problem.

44 -

Sect. 1

-

Engel h a r d t , Ed.

CNS Agents

XXX

CH3

XXXII

-

x=NH2 Y e 1

Finally, various compounds related to methaqualone have been found to possess anticonvulsant activity in mice=. Compound XXXI was found to be more active than methaqualone against maximal electroshock seizures. Compound XXXII also possessed potent anticonvulsant activity but was more toxic than Compound XXXI. General Anesthetics - Few new anesthetic agents were reported in the literature in the last two years. The pharmacology of a new dissociative anesthetic, etoxadrol (XXXIII, CL1848C, U-37,862A) in primates and other species= and an evaluation of etoxadrol in normal human volunteers40 has been reported. Etoxadrol has properties similar to ketamine (XXXIV) as an anesthetic. After intravenous administration in monkeys etoxadrol produces complete analgesia with retention of the light and corneal reflexes. In man, etoxadrol appears to be a useful drug in anesthesia and has a longer lasting activity than ketamine. Etoxadrol appears to be devoid of long term psychologic, biochemical or physiologic pathology. Furt er studies have been conducted on compound 347 (XXXV), now called Ethraneb The compound was administered to 250 patients as the major anesthetic41. Its physical properties and clinical characteristics are similar to those of halothane.

HCF&CF2CHFCl 0 C6H5

XXXIII

C2H5

XXXI v

XXXV

REFERENCES 1.

2.

3,

4. 5. 6. 7.

W. P. Koella, Sleep: Its Nature and Physiological Organization, C.C. Thomas, Springfield, Illinois, 1967. M. Jouvet, Advances in Pharmacology, S. Garattini and P. A. Shore; Eds., Academic Press, New York 1968, 265. B. K. Koe and A. Weisman, J. Pharmacol. Exptl. Therap. 499 (1968). W. P. Koella, Biol. Psychiatry 1, 161 (1969). R. J. Wyatt, K, Engelman, D. J. Kupfer, J. Scott, A. Sjoerdsma and F, Snyder, Electroenceph. Clin. Neurophysiol. 3 , 529 (1969). E. Hartmann, R. Chung and C. Chien, Psychopharmacologia 114 (1971). A. J. Mandell, C.E.Spooner and D.Brunet, Biol. Psychiatry 1, 13 (1969).

m,

a,

Chap. 4

45

Rudz i k, Fr i i s

Depressants

A. Kales, J.D. Kales, M.B. Scharf and T. Tan, Arch. Gen. Psychiat. 3, 219 (1970). 9. A.M.Zimmerman, Current Therap. Res. 12, 18 (1971). 10. L. 0. Randall, W. Schallek, C. L. Scheckel, P. L. Stefko, R. F. Banzig e r , W. Pool and R. A. Moe, Arch. I n t . Phannacodyn. 216 (1969). 11. I. Oswald, S. A. L e w i s and J. Tagney, I n t e r n a t i o n a l Symposium on Benzodiazepines, Raven P r e s s , New York, i n press. 12.. A. Kales, I n t e r n a t i o n a l Symposium on Benzodiazepines, Raven P r e s s , New York, i n p r e s s . 13. A. Hedges, P. Turner, T. V. A. Harry, J. Clin. Pharmacol. 11,423 (1971). 14. R. Nakajima, Y. Take, R. Moriya, Y. S a j i , T. Yui and Y. Nagawa, Japan. J. Pharmacol. 2l, 497 (1971). 15. J. B. Hester, A. D. Rudzik and B. V. Kamdar, J. Med. Chem. 1078

8.

m,

16.

14,

( 19711

A. D. Rudzik, J. B. Hester and W.

F r i i s , The Pharmacologist,

12,205

(1971)

17. T. M. I t i l ,

B. S a l e t u , J. Marasa and A. N. Mucciardi, D i s . Nerv. S y s t . , i n press. 18. R. Nakajima, Y. S a j i , S. Chiba and Y. Nagawa, J. Takeda Res. Lab 3, 153 (1970). 19. Chem. Drug. 192,228 (1970). 20. P.A.Janssen, C. J. E. Niemegiers, K. H. L. Schellekens and F. M. L e n a e r t s , Arzneimittel - Forsch. 21, 1234 (1971). 21. C. G. Wermuth, G. LeClerc and J. Schreiber, Chimie Therapeutique lOg

6,

(1971)

22. C. Casagrande and G. M e r o t t i , Farmaco, 3, 799 (1970). 23. B. W. E l l i o t t Current Therap. Res. l2, 502 (1970). 24. J. Katz, S.C. Finestone, M.T.Pappas and J. Fine, Current Therap. Res. 12, 255 (1970 25. L. Vargiu and P. F. Spano, Arch. Pharmakol. 269, 410 (1971). 26. J. Schmitt, P Comoy, M. Suquet, G. C a l l e t , J. LeMeir. T. C l i m , M. 239 Brumaud, J. Mercier, J. S a l l e and G. Siou, Chimie Therapeutique

. Armagnac,

( 1969)

27. J.

4,

F. Boismare, G.

Streichenberger and P. Lechat, Therapie 26,

439 (1971) 28. H. Gastaut, I n t e r n a t i o n a l Sumposium on Benzodiazepines, Raven P r e s s , New York, i n press. 29. G. F. Rossi, C. DiRocco, G. Maira and M. Meglio, I n t e r n a t i o n a l Symposium on Benzodiazepines, Raven P r e s s , New York, i n p r e s s . 30. A. D. Rudzilc, J . B. Hester, A. H. Tang, R. N. Straw and W. F r i i s , Int e r n a t i o n a l Symposium on Benzodiazepines, Raven P r e s s , New York, i n press. 747 31. R. Guerrero Figueroa and D. M. G a l l a n t , Current Therap. Res.

u,

-

(1971).

32. A. R. 33. A. 34. H. 35. C.

Ashford, G. R. Brown, P. J. Palmer, J. W. Ross, R. B. Trigg, and J. Ward, Arzneimittel - Forsch. 21, 937 (1971). B. Funcke and P. Zandberg, Arzneimittel Forsch. 20, 1896 (1970). H. Keasling and R. B. Moffett, J. Med. Chem 1106 (1971). M. Samour, J , F. Reinhard and J. A. Vida, J. Med. Chem. 187

-

(1971)

14,

0

36.

J. A.

Vida, W. R. Wilber and J. F. Reinhard, J. Med. Chem.

14, 14,190

46 -

Sect. 1

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CNS Agents

Engelhardt, Ed.

( 19711

37. D. DeBiolley and L. Sorel, Acta Neurol. Belg. &, 909 (1969). 38. D. DeBiolley and L. Sorel, Acta Neurol. Belg. &, 914 (1969). 39.

J. Hildalgo, R. M. Dileo, M. T. Rikimaru, R. J. Guzman and C. R. Thompson, Anesthesia and Analgesia, 50, 231 (1971). 40. R. D. Wilson, D. L. Traker, E. Barratt, D. L. Creson, R. C. Schmitt, and C. R. Allen, Anesthesia and Analgesia &, 236 (1970). 41. M. H. Lebowitz, C. D. Blitt and J. B. Dillon, Anesthesia and Analgesia &, 1 (1970).

47 Chapter 5.

Recent Developments Relating Serotonin and Behavior

Albert Weissman and Charles A. Harbert, Pfizer Inc., Groton, Conn. 06340 The existing encyclopedic reviews on 5-HT1 make evident that techniques for modifying 5-HT kinetics are almost as varied and voluminously reported as those for studying behavior. As a result, few areas are more difficult to survey briefly than this, relating two such ill-defined yet exhaustively studied variables. We have accordingly chosen to focus on a limited number of issues with particular current importance. Techniques for Manipulating 5-HT Steady State Levels and Receptor Activity

It must be asserted at the outset that there are still no entirely satisfactory means to either elevate or reduce 5-HT steady state levels. Studies purporting to show altered behavior as a function of variations in 5-HT levels are inevitably tainted by factors that covary. For example, the elevation of brain 5-HT levels by administration of the dietary 5-HT precursor, Trp, leads a l s o to the metabolism of excess substrate through other pathways, particularly the kynurenine pathway. Administration of the immediate precursor of 5-HT, 5-HTP, leads to a non-physiological distribution of 5-HT in brain, owing to the ubiquitous distribution of aromatic amino acid decarboxylase. 5-HT derived from either precursor may decrease catecholamine stores by amine displacement, and Trp derivatives also inhibit tyrosine hydroxylase. Conclusions based on high 5-HT levels in selected animal strains, at certain times of day, or at certain ages are confounded by multiple genetic, circadian or ontogenetic effects that covary, Similar problems apply to conclusions drawn from carcinoid victims or other examples of human pathology where 5-HT is elevated. MA0 inhibitors, even those relatively specific for that isoenzyme for which 5-HT is a primary substrate, exert actions other than elevating 5-HT. Comparable lack of specificity plagues known methods of depleting 5-HT from brain: reserpine and similar amine-releasing drugs block the intraneuronal vesicular uptake of catecholamines as well as of 5-HT.4 Lesions in the nucleus Raphe, the medial forebrain bundle and other tracts,5 which deplete 5-HT from brain, deplete NE as well. Prolonged Trp deficient diets6 cause general nutritional deficits and an inefficient decrease in 5-HT levels. Substituted phenethylamine derivatives, such as p-chloromethamphetamine and fenfluramine, have usually been reported to exert relatively small effects on brain 5-HT and the mechanism

______________-__-----------------Abbreviations used in this paper: 5-HT = 5-hydroxytryptamine (serotonin); Trp = tryptophan; 5-HTP = 5-hydroxytryptophan; 5-HIAA = 5-hydroxy-3indoleacetic acid; MA0 = monoamine oxidase; PCPA = p-chlorophenylalanine; NE = norepinephrine

48

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CNS Agents

Engelhardt, Ed.

of their action is unclear. The recent disclosure that intraventricularly administered 5,6-dihydroxytryptamine causes selective degeneration of 5-HT neurons in rat brain7 is exciting, but will require confirmation. The selectivity and activity problems inherent in the above techniques explain why PCPA8 has been seized upon as a tool for studying behavioral and neurophysiological effects after brain 5-HT is depleted. PCPA is perhaps both the most selective and effective depletor of 5-HT known. Nevertheless, it also slightly, but significantly, depletes the brain of catecholamines for a few hours after its administrationY8and several authors have dwelled on this effect in accounting for some of its By blocking phenylalanine hydroxylase ,8,10 PCPA physiological actions. elevates phenylalanine and its metabolitesll and "experimental PKU" may account for certain behavioral effects of PCPA. Other biochemical complications after PCPA include reduced uptake of Trp and phenylalanine.12 Furthermore, 5-HT depletion after PCPA is not "complete", and residual amounts of 5-HT that are synthesized after PCPA may account for its unexpected effects or lack of effects.13 It is unnecessary to alter steady state levels of 5-HT in order to alter 5-HT receptor activity. Blockade of 5-HT reuptake into presynaptic terminals may prolong high extraneuronal concentrations of 5-HT .I4 Agents believed t o exert such an effect -- tertiary tricyclic antidepressants, particularly chlorimipramine -- also have selectivity problems, since all exert at least some effect on NE uptake. Alternatively, it may be possible to stimulate or to block 5-HT receptors specifically. It is suggested that quipazine (2-[l-piperazinyl]quinoline)(III) exerts 5-HT mimetic properties based on its smooth muscle spasmogenic profilei15a its behavioral effects in animal tests also suggest similarities with 5-HT.15b From the plethora of agents exerting peripheral antiserotonin properties some may exert comparable effects in brain; the often-cited anti-5-HT activity of LSD in brain, however , may be complicated by 5-HT mimetic properties as well .I6 Keeping specificity limitations in mind, let us nevertheless focus on the behavioral effects associated with a reduction in brain 5-HT levels caused by the tryptophan hydroxylase inhibitor PCPA or its methyl ester. Where possible the behavioral effects reported to occur after PCPA are related to those produced when 5-HT levels or receptor activity are altered by other means. Behavioral Effects of PCPA-Elicited Serotonin.Depletion in Animals Symptoms - A corollary to the familiar Brodie and Shore h y p ~ t h e s i s ~ ~ that 5-HT mediates a central parasympathetic ("trophotropic") system is that suppression of 5-HT biosynthesis should result in central stimulation because of the dominance of the central sympathetic ("ergotropic") system. Initial symptomatological observations after PCPA, however, included only mild irritability and aggressiveness in isolated rats, without clear evidence of motor stimulation.8 These observations of absent or mild symptomatic effects in rats have frequently been confirmed.18 Quantitative

Chap. 5

Serotonin and Behavior

We i ssman , Ha rbert

49

locomotor activity studies in rodents usually report no elevation, and sometimes decreases in motor activity following PCPA at doses effective in depleting 5-HT.I4 Nevertheless, animals have exhibited elevated activity during prolonged exposure to an activity chamber, as if the novelty of the activity box is sustained.20 In unrestrained larger animals, especially rabbits, cats and rhesus monkeys, irritability and aggressiveness without frank motoric excitement, are observed.21 Consummatory Behavior - Rats treated with moderate doses of PCPA consume laboratory food and water normallyY18b,19c~22 but appear to be more finicky or responsive than controls in their consumption of solutions of alcohol,22~23saccharin,23c dextroselgb or quinine,lgb suggesting that gustatory or olfactory thresholds may be lowered by PCPA. Sleep - Many sleep researchers have found that PCPA causes prolonged EEG activation and sleep disturbances. In rats, there is a significant decrease of both REM and slow wave sleep correspondin to reduced 5-HT levels in brain, but not with PCPA levels.18a,20a,2'f This diminution in REM sleep in rats is most unusual in that no REM rebound occurs.24 Related to the sleep findings is the observation that PCPA inhibits hibernation in squirrels.25 Extensive studies in the laboratories of Jouvet,26 KoellaY2' Dementzla and others have shown that PCPA has the ability to block slow wave sleep and produce prolonged insomnia in cats, effects that are rapidly reversed by 5-HTP. Titration of PCPA and 5-HTP can lead to normal sleep. In rhesus monkeys depleted of brain 5-HT by PCPA, however, REM sleep may be unchanged, and in this respect PCPA effects appear to differ from those seen in rats and cats. The amount of time spent in slow wave (and hence, total) sleep decreases si nificantly, but "total insomnia", as often reported for cats, is not seen.28 Dement et a1,21a however, have been able to produce sleep loss in 2 monkeys resembling the effect in cats. To the extent that conclusions may be drawn from the data available, a different picture occurs in man. In patients seriously ill with carcinoid, Huntington's chorea, migraine, or dystonia, REM sleep is markedly depressed, but slow wave sleep is either unchanged or slightly increased.29 A study of PCPA in normal volunteers revealed no subjective complaints of insomnia.30 The rat and cat sleep results with PCPA, even considered alone, would suggest that 5-HT is functionally involved in normal sleep mechanisms, but these findings are in fact only the latest in a long chain of circumstantial evidence linking 5-HT to sleep.26b Y 27 Exogenous 5-HT, given intraventricularly or in the carotid, and 5-HT precursors -- either Trp or 5-HTP -- given peripherally, have frequently been reported to produce electrophysiological, behavioral and clinical evidence of sedation and sleep. Among other established means of reducing slow wave sleep are administration of reserpine and methysergide. Circadian variations in 5-HT brain levels in animals are positively correlated with circadian variations in sleep. Ablation of the 5-HT-rich Raph6 nuclei also produces insomina. Sensitivity to Pain and Shock Avoidance Behavior - PCPA is hyperalgesic

50

Sect. 1

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CNS A g e n t s

Engel h a r d t , Ed.

i n r a t s , l g c , 3 1 an e f f e c t r e v e r s i b l e by 5-HTP, b u t n o t by 5-HT a d m i n i s t r a t i o n . 5-HT-depleting l e s i o n s of t h e m e d i a l f o r e b r a i n bundle exert a s i m i l a r e f f e c t . 31a Heightened r e s p o n s i v e n e s s t o p a i n may account f o r enhanced avoidance a c q u i s i t i o n a f t e r PCPA.19c,32 I t i s a l s o l i k e l y t h a t e l e v a t i o n s i n avoidance r e s p o n s e r a t e a f t e r PCPA33 d e r i v e i n p a r t from i n c r e a s e d s e n s i t i v i t y t o f o o t shock. PCPA b l o c k s t h e a n a l g e s i c a c t i o n s of morphine, methadone, propoxyCNS phene and m e ~ e r i d i n e . ~ ~ e f f e c t s of morphine o t h e r t h a n a n a l g e s i a are blocked by PCPA,35 b u t t h e f i n d i n g s of Way e t a136 t h a t PCPA b l o c k s symptoms accompanying morphine w i t h d r a w a l have n o t been confirmed. 37

The h y p e r a l g e s i c and a n a l g e s i c - b l o c k i n g e f f e c t s of PCPA are cons i s t e n t w i t h l i t e r a t u r e r e p o r t s showin i n c r e a s e d s e n s i t i v i t y t o p a i n a f t e r lesion-induced d e p l e t i o n of 5-HT3la o r a f t e r r e s e r p i n e - l i k e d r u g s , and an a n a l g e s i c r e s p o n s e f o l l o w i n g MA0 i n h i b i t o r s p l u s 5-HTP.38

-

S e n s i t i v i t y t o Convulsant Treatments PCPA f a c i l i t a t e s c o n v u l s i o n s e l i c i t e d by h y p e r b a r i c oxygen,3Y p e n t y l e n e t e t r a z o l , 4 0 e l e c t r o c o n v u l s i v e shock, 40a,41 a u d i o g e n i c c o n v u l s a n t s t i m u l i , 4 2 f l u r o t h y l 4 3 and w i t h d r a w a l from b a r b i t u r a t e s .44 A s u b s t a n t i a l o l d e r l i t e r a t u r e , o r i g i n a t i n g w i t h t h e work of Chen e t a145 on r e s e r p i n e , d e a l s w i t h t h e r o l e of b i o g e n i c amines i n e x p e r i m e n t a l s e i z u r e s . The preponderance of t h e s e r e p o r t s i n d i c a t e t h a t s t i m u l a t i o n of 5-HT r e c e p t o r a c t i v i t y d e c r e a s e s s e i z u r e s u s c e p t i b i l i t y , and t h a t d e p r e s s i o n of 5-HT r e c e p t o r a c t i v i t y e x e r t s t h e o p p o s i t e e f f e c t , S e n s i t i v i t y t o Other Non-Social E x t e r o c e p t i v e S t i m u l i - S e v e r a l s t u d i e s i n d i c a t e t h a t PCPA i n c r e a s e s v a r i o u s b e h a v i o r a l o r n e u r o l o g i c a l e f f e c t s of e l e c t r i c a l s t i m u l a t i o n . T h r e s h o l d s of c u r r e n t r e q u i r e d t o r e i n f o r c e s e l f s t i m u l a t i o n b e h a v i o r are lowered by PCPA i n r a t s , 4 6 and PCPA h a s been rep o r t e d t o c a u s e s t r o n g e x c i t a t o r y e f f e c t s on i n t r a c r a n i a l s e l f - s t i m u l a t i o n rates i n rats 3 days a f t e r t r e a t m e n t . 4 7 5-HT-depleted animals have b e e n r e p o r t e d t o b e more a c t i v e t h a n c o n t r o l s under c o n d i t i o n s of e x t r a environmental s t i m u l a t i o n , p a r t i c u l a r l y “novel” s t i m u l a t i o n . 1 8 b 9 1 9 ~ Cats “overr e a c t t o s l i g h t n o i s e s ,’I48 s e n s o r y t h r e s h o l d s are lowered,29 t h e r e s p o n s i v e n e s s t o a s t a r t l e s t i m u l u s of rats w i t h low b a s a l r e s p o n s i v e n e s s i s i n c r e a s e d , 4 9 and h a b i t u a t i o n t o an a u d i t o r y - e l i c i t e d s t a r t l e r e s p o n s e , as measured by c o r t i c a l e l e c t r i c a l r e s p o n s e s , is reduced.50 S e x u a l i t y - D e s p i t e o c c a s i o n a l d i s s e n t 5 I t h e r e can now b e l i t t l e doubt t h a t PCPA d r a m a t i c a l l y enhances s e x u a l i t y i n s u i t a b l e animal p r o t o c o l s . 52 L i k e o t h e r e f f e c t s of PCPA, h y p e r s e x u a l i t y i s a b r u p t l y r e v e r s i b l e by low doses of 5-HTP.52a PCPA h a s b e e n observed t o a c t i v a t e l o r d o s i s , a prime i n d i c a t o r of e s t r u s b e h a v i o r , i n o v a r i e c t o m i z e d r a t s t r e a t e d w i t h e s t r o gen and exposed t o a v i g o r o u s male a r t n e r . 5 3 PCPA d r a m a t i c a l l y i n c r e a s e s s e x u a l b e h a v i o r i n cats21a,E4 and r a b b i t s ; 5 5 i n t h e s e s p e c i e s , a l s o , t h e e f f e c t of PCPA i s r e v e r s e d by 5-HTP. Aggression and Other Non-sexual S o c i a l Behavior - Mouse k i l l i n g (muricide) i n rats i s enhanced by PCPA.22b “Cats who normally i g n o r e l a b o r a t o r y rats b e g i n t o k i l l them w i t h a r a p i d i t y and c o n c e n t r a t e d s a v a g e r y t h a t

Chap. 5

Serotonin and Behavior

Weissman, Harbert

51 -

evoke images of the jungle".2la Mice or rats can be extremely aggressive to one another.18a~22b~56 Much of this social behavior is reversed by administration of 5-HTP.22b,52c Summary of Behavioral Effects of Depleting 5-HT with PCPA in Animals - Although isolated animals treated with PCPA show few symptomatic effects, exposure of animals to novel exteroceptive stimulation evokes behavioral hyperresponsiveness. Animals overreact to painful, electrical, auditory, visual, gustatory, olfactory and especially social stimuli. The sustained wakefulness of animals treated with PCPA may feasibly be considered one manifestation of this generalized hyperresponsiveness. The animal behavioral data from PCPA, taken as a whole, may be loosely interpreted as suggesting that 5-HT subserves behavioral inhibitory functions, and that a relative absence of 5-HT produces hypersensitivity to environmental cues. Both the rapid reversal of electrophysiological and behavioral changes following smalldoses of 5-HTP, and the parallel time course of PCPA actions with brain 5-HT levels clearly associate 5-HT with its effects. The PCPA data are also consistent with findings from less selective means of modifying brain 5-HT, such as reserpine or MA0 inhibitor treatment, treatment with 5-HT precursors, lesion-elicited 5-HT depletion, and the use of animal strains, ages, or times of day associated with variations in 5-HT. Implications of PCPA-Elicited Clinical Symptomatology Many clinical investigators who have studied altered attention, sensory and perceptual characteristics in man believe that schizophrenics respond to sensory inputs that do not affect normals.57 In view of this, and the preceding analysis of the role of 5-HT in behavior, one might expect aberrant clinical behavior after selective depletion of 5-HT. Limited reports of such aberrations after PCPA have appeared. In the course of successful trials of PCPA in patients with carcinoid tumor, pronounced behavioral aberrations, ranging from depression to florid hallucinations and paranoia occurred.58 A comparison of sleep patterns in both schizophrenics and PCPA-treated cats revealed several similarities.21a It thus appears at least feasible that the animal behavioral findings after PCPA are manifested in humans as depression and paranoia. This speculation is provocative since abnormalities of 5-HT synthesis and metabolism have been implicated in several psychopathological conditions. We shall accordingly now focus on the role of 5-HT in depression, mania, and schizophrenia and shall emphasize experimental therapeutic approaches current and speculative - which involve an attempt to manipulate brain 5-HT. Indoleamine Dysfunction in Human Behavior Depression - The controversy surrounding the relative importance of catecholamines and indoleamines in depression has culminated in divergent

52

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CNS Agents

Engelhardt, Ed.

"catecholamine"59 and "5-HT"60 hypotheses. However, recent reviews on the role of brain monoamines in psychopathological disorders emphasize the need for understanding their complex interaction.61 Evidence cited to support 5-HT dysfunction in depression includes 1) decreased levels of 5-HT and 5-HIM in the brains of depressed patients who commit suicide,62 2) sub-normal concentrations of 5-HT metabolites in the cerebrospinal fluid (CSF) of depressives,63 and 3) diminished rates of 5-HT turnover, manifested as deficient 5-HIM accumulation in the CSF following treatment with the efflux blocker, probenecid.64 The 5-HT precursors Trp and 5-HTP have been explored most extensively by proponents of the 5-HT deficiency hypothesis, in efforts to increase brain 5-HT levels.65 The earliest trials of Trp in depression found it to potentiate the beneficial effects of MA0 inhibitors.66,67 Ensuing reports have claimed that D L-Trp is as effective as electroconvulsive shock therapy68 or imipraminebg and it has been successfully used "as a last res0rt".7~ In other studies, however, L-Trp has been less impre~sive.7~ Early studies of 5-HTP were limited to low doses given over short periods of time.72 Recent, more prolonged trials, have yielded conflicting results.73 Several recent findings may have an important bearing on the future use of 5-HT precursors in depression. Evidence from CSF experiments suggests an abnormally low rate of 5-HT synthesis in the CNS of some depressed patients,63 perhaps due to impaired Trp-5-hydroxylase activity.74 If this were true, L-Trp would inefficiently elevate brain 5-HT in this population, explaining the divergent clinical results obtained with L-Trp. The use of L-5-HTP, which enters the 5-HT pathway subsequent to the rate-limiting Trp hydroxylation step, might seem a logical alternative. Recent reports, however, indicate that this precursor leads to indiscriminate and non-physiologic elevations of brain 5-HT. 2 75 To what extent these findings apply to the chronic administration of this amino acid to man is not known, but van Praag and Korf73b recently reported L-5-HTP to be effective in depressives who showed evidence of a 5-HT synthesis defect. The continued viability of the 5-HT precursor approach to the treatAdditional definitive research ment of depression is thus questionable. concerning 1) the relationship of change in levels of 5-HIAA in CSF to brain 5-HT turnover, 2) the biochemical classifications of depression, and 3 ) the effects of chronic L-5-HTP on levels of physiologically active 5-HT are needed to determine the future direction of this approachOther potentially significant approaches to the treatment of depression based on the 5-HT hypothesis have emerged during the past few years. These include: 1) selective 5-HT reuptake blockade, 2) selective inhibition of MAO, and 3) 5-HT mimetic activity. Although these approaches may not suffer from the obvious disadvantages of the precursor approach, each possesses unique and challenging problems of its own.

Chap. 5

Serotonin and Behavior

We i ssman , Ha rbert

53

The concept of selective blockade of monoamine reuptake has been studied extensively by Carlsson and associates76 who have shown that tricylic mines such as imipramine and chlorimipramine are selective inhibitors of 5-HT uptake. Furthermore, they have suggested that blockade of 5-HT reuptake is associated with the "mood-elevating" action of the tricyclic antidepressants and that blockade of NE reuptake promotes "drive" in depressed patients. This is consistent with the fact that chlorimipramine, the most potent and selective inhibitor of 5-HT reuptake in this series, has encouraging antidepressant activity in humans77 with at least one report of a selective mood-elevating effect .78

&

At least four different molecular forms of MAO, with different substrate specificities, occur in human brain79 suggesting that depression might be treated by selective inhibition of 5-HT oxidation .80 Several c1

c1 I

I1

I11

selective inhibitors have been reported to date81 and at least two, clorgyline82 (I) and Lilly 51641 (11),83 have been reported to possess antidepressant activity. It is not clear whether they possess sufficient selectivity to obviate the autonomic side effects commonly seen with other non-selective MA0 inhibitors. Another conceivable approach to the problem of 5-HT and depression is the development of an agent that selectively stimulates 5-HT receptors. Such a 5-HT mimetic would require a high degree of CNS selectivity, but this problem does not seem insuperable. Quipazine (111), a smooth muscle stimulant with 5-HT-mimetic properties ,15a possesses a characteristic "antidepressant" profile in animals15b and, depending on clinical activity, may be a significant development toward this end. Mania - Mania has been viewed historically as "the other affective disorder," and emphasis has tended to focus on the polarity of certain manic depressive symptoms. Kety6lc has recently hypothesized that a central indoleamine deficiency permits the occurrence of either mania or depression, and that an accompanying change in adrenergic activity determines the polarity of the affective disorder, adrenergic activity being intensified in mania and diminished in depression. Thus, within the context of this discussion, Kety ' s hypothesis suggests a similar "pro-indole" therapeutic approach to both depression and mania. Clinical trials of 5-HT precursors in mania have been rare, but two recent reports indicate a moderately beneficial effect from L-Trp. 71b ,84 Methysergide has a deleterious effect in manic patients .85

54

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CNS Agents

Engel hardt, Ed.

Schizophrenia - Schizophrenia is almost certainly a class of behavioral disorders and not a single disease entity, which perhaps explains in part the numerous theories and conflicting clinical findings,61 Several theorists invoke defective indoleamine metabolism and transport as key etiological factors in schizophrenia.6la,b Woolley86 suggested that 5-HT concentrations might be elevated in schizophrenics, consistent with observations that L-Trp in combination with an MA0 inhibitor exacerbates psychoses.87 Similar exacerbations and recurrences of individual psychotic patterns are seen following administration of the methyl donors, methioninegg and betaine.89 These findings, together with the known psychotomimetic effects of dimethyltryptaminego and bufotenin91 led several authors88992 to suggest that abnormal methylation of indoleamines, leading t o the endogenous formation of methylated indole(ethyl)amines, may be a primary causative factor in the pathogenesis of schizophrenia. This has been supported by the recent isolation of a specific indoleamine Nmethyltransferase from rat and human brain,93 and the demonstration that tryptamine is converted to dimethyltryptamine in rat and human brain.94 This theory is also compatible with a recent a-2 globulin theoryYg5which postulates an enzyme abnormality that permits excess Trp to enter brain. Clinical attempts to elevate CNS 5-HT levels in schizophrenics have generally had deleterious effects, particularly when L-Trp is combined with an MA0 inhibitor.87 Reports of mild beneficial effects of L-Trp74 and L-5-HTP96 in schizophrenics have appeared recently. Other Behavioral States - Elucidation of the role of 5-HT in sleep26b,27 has prompted numerous clinical studies with L-Trp and L-5-HTP. L-Trp appears t o sedate normals and insomniac patients97 and has recently been described as a "natural sedative".98 Clinical studies of L-5-HTP in sleep have not been as impressive.99 Several behavioral disturbances including psychoses frequently accompany Hartnup's disease, Wilson's disease, porphyria and pellagra, diseases in which indoleamine abnormalities have been identified.100 These behavioral aberrations generally recede during clinical remission of the primary metabolic abnormality. References 1. a) 5-Hydroxytryptamine and Related Indolealkylamines, ed. V. Erspamer, Springer-Verlag, Berlin (1966); b) Advances in Pharmacology, ed. E. Costa and M. Sandler, Academic Press, New York (1968); c) S. Garattini and L. Valzelli, Serotonin, Elsevier, Amsterdam (1965) 2. A. T. B. Moir and D. Eccleston, J. Neurochem., 15, 1093 (1968). 3 . D. K. Zhelzaskov, M. Levitt, and S. Udenfriend, Molec. Pharmacol., 4, 445 (1968).

Chap.

4. 5. 6.

7. 8. 9.

10. 11. 12. 13.

14. 15.

5

S e r o t o n i n and Behavior

We i s sma n , Ha r be r t

55

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367,

.

154,

177,

12,

2,

16. 17. 18.

z,,

188,

19.

20. 21.

181,

47,

22. 23.

15,

15,

24. 25.

10,

56 26. 27. 28. 29. 30. 31.

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a) M. Jouvet, Adv. Pharmacol.,

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12,

169,

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58.

59. 60. 61.

62.

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

71.

57

15,

122,

225,

12,

s,

127,

Sect. 72. 73.

74. 75. 76.

1

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CNS Agents

E n g e l h a r d t , Ed.

S a c k s , and G. M. Simpson, Am. J . P s y c h i a t r y , 129, 379 ( 1 9 6 4 ) . a) H . K . H . B r o d i e , R . S a c k , and L . S i e v e r , C o n f e r e n c e on S e r o t o n i n and B e h a v i o r , P a l o A l t o , C a l . , J a n . 1972 , i n p r e s s ; b ) H . M. van P r a a g and J . K o r f , 3. M. B . Bowers, Neuropharmacology, 9 , 599 ( 1 9 7 0 ) . G. K . A g h a j a n i a n , A. Graham, and E. S h e a r d , S c i e n c e , 169, 1100 ( 1 9 7 0 ) . a) A. C a r l s s o n , H . C o r r o d i , K . Fuxe, and T . H s k f e l t , Europ. J . Pharma c o l . , 5 , 357 ( 1 9 6 9 ) ; b ) A. C a r l s s o n , J . J o n a s o n , and M. L i n q v i s t , J . Pha;. Pharmacol., 769 ( 1 9 6 9 ) . Drug T h e r a p . B u l l . , 2, 99 ( 1 9 7 1 ) . M. H . Synies, S n t . J . N e u r o p s y c h i a t r y , 3 , 60 ( 1 9 6 7 ) . G. G. S . C o l l i n s , M. S a n d l e r , D. E . W i l l i a m s , a n d M . B . H . Yondim, Nature, 817 ( 1 9 7 0 ) . R . W. F u l l e r , Arch. I n t . Pharmacodyn., 1 2 , 32 ( 1 9 6 8 ) . a ) I). W . R. H a l l , B . W . Logan, and G . H . P a r s o n s , Biochem. P h a r m a c o l . , _18, _ 1447 ( 1 9 6 9 ) ; b ) R. W . F u l l e r , B . J . Warren, and B . B . M o l l o y , Riochem. Pharmacol. , 19,2934 ( 1 9 7 0 ) . J . A . H e a r d , C l i n . T r i a l s J . , 6 , 219 ( 1 9 6 9 ) . G . M. Simpson, C . J . I q b a l , a n d F. l q b a l , C u r r . T h e r a p . R e s . , 8, 400 ( 1 9 6 6 ) . A. J . P r a n g e , J . L. S i s k , I . C . W i l s o n , C . E . M o r r i s , C . D . H a l l , and J . S . Carman, C o n f e r e n c e on S e r o t o n i n and B e h a v i o r , P a l o A l t o , J a n . 1972, i n p r e s s . A . Coppen, A. J . P r a n g e , P . C . Whybrow, R. Noguera, and J . M. P a e z , L a n c e t 2, 338 ( 1 9 6 9 ) . D . W . Woolley, The B i o c h e m i c a l Bases of Psycho=, J o h n Wiley and S o n s , Tnc., New York, 1 9 6 2 , p . 1 4 . W . P o l l i n , P . Cardon, J r . , and S . K e t y , S c i e n c e , 122,104 ( 1 9 6 1 ) . G . G . Brune and H . E . Himwich, J . Nerv. Ment. D i s . , 134,447 ( 1 9 6 2 ) . G . G. Brune and H . E . Hirnwich i n R e c e n t Advances i n B A o l o g i c a l P s y c h i a t r y , e d . J . W o r t i s , Vol. 5 , Plenum Press, New York, 1963, 1 4 4 . S . S z a r a , E x p e r i e n t i a , 12, 441 ( 1 9 5 6 ) . H . D. Fabing and J . R. Hawkins, S c i e n c e , 123,886 ( 1 9 5 6 ) . J . A x e l r o d , S c i e n c e , 134,343 ( 1 9 6 1 ) . A . J . Mandell and M. Morgan, N a t u r e , 230, 85 ( 1 9 7 1 ) . J . M. S a a v e d r a and J . A x e l r o d , S c i e n c e , 175,1 3 6 5 ( 1 9 7 2 ) . C . E . Frohman, K. A. Warner, C . T . B a r r y , and R . E . A r t h u r , B i o l . P s y c h i a t . , 1,201 ( 1 9 6 9 ) . R . J . W y a t t , T . Vaughan, J . K a p l a n , M. G a l a n t e r , and R . G r e e n , i n Conf e r e n c e on S e r o t o n i n and B e h a v i o r , P a l o A l t o , J a n . 1 9 7 2 , i n p r e s s . E . Hartmann, R. Chung, and C h i e n , P s y c h o p h a r m a c o l o g i a , 19, 114 ( 1 9 7 1 ) . R. J . Wyatt, K . Engleman, D . J . K u p f e r , D . H . Fram, A. S j o e r d s m a , and F. S n y d e r , L a n c e t 2 , 842 ( 1 9 7 0 ) . I. Oswald, G . W . A s h c r o f t , R. J . B e r g e r , D . E c c l e s t o n , J . I. E v a n s , 391 ( 1 9 6 6 ) . and V . R . T h a c o r e , B r . J . P s y c h i a t . , G. G . Brune i n B i o c h e m i s t r y , S c h i z o p h r e n i a s and A f f e c t i v e I l l n e s s e s , e d . H . H i m i c h , The W i l l i a m s & W i l k i n s Co., B a l t i m o r e , 1 9 7 0 , p . 43. N . S . K l i n e , W.

z,

77.

78. 79. 80. 81.

82. 83. 84.

85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100.

?c,

112,

Section I I - Pharmacodynamic Agents Editor:

John G. Topliss, Schering Corp., Bloomfield, New Jersey Chapter 6 .

Antihypertensive Agents

Anthony M. Roe, Smith Kline and French Research Institute, Welwyn Garden City Hertfordshi re, England Introduction - No major advance n our understanding of the nature of hypertension has been made this year, and it remains true that "until the mysteries of the etiology of essential hypertension are unravelled, treatment must remain empiric and pall iativel'.l Several review^^-^ have discussed various aspects of the clinical conditions which are associated with an elevated blood pressure, and which are the target for so much research. Other review^^-^ have described the cl inical benefits achieved by lowering elevated blood pressure; it is possible that this benefit is obtained by the reversal of the increased ratio of the vascular wall thickness to the diameter of the lumen which is a characteristic of essential hypertension. l o The spontaneously hypertensive rat (SHR) continues to gain attention as a rode1 with which to assess antihypertensives.10-12 The extent to which different anesthetics reduce cardiovascular responses of cats has been studied,13 and the danger of assuming that drug solvents such as polyethylene glycol have no effect on these resporises has been pointed out. l4 Developments in clinically useful antihypertensives - There has been a great deal of discussion15 on work carried out to evaluate the therapeutic potential of combining a vasodilator with a B-adrenoreceptor blocker in order to counteract the tachycardia associated with the former. The combination of hydralazine with propranolol (p.0.) is effective in hypertensive patients at doses which separately do not give a satisfactory hypotensive effect;16 a similar situation holds for dih~dra1azine.l~ Alprenolol (i .v.) also reduced the cardiac stimulation observed in normotensive and hypertensive patients after dihydratazine (i .v.) .18 Minoxidi 1 (PDP), in combination with propranolol and hydrochlorothiazide, appears to be more effective in refractory hypertensive patients than hydralazine. l 9 Practolol effectively blocks the tachycardia in normotensive dogs treated with hydralazine (i.v.) without potentiating the hypotensive effects, which suggests that tachycardia does not diminish the hypotens i ve effect of hydra 1 az i ne . 2o Guancydine has been examined in further cl inical tria1s,21,22 and shown to have no effect on the pressor response of angiotensin in hypertensives.22 The mode of action of guancydine has been studied in vitro leading to the conclusion that its hypotensive properties result

60

Sect. I I

-

T o p l i s s , Ed.

Pharmacodynamic Agents

from i t s n o n - s p e c i f i c i n h i b i t o r y e f f e c t on smooth muscle.23 The c a r d i o v a s c u l a r responses o f p a t i e n t s r e c e i v i n g p r o p r a n o l o l have been compared t o those r e c e i v i n g b e t h a n i d i n e , guanethidine and u - m e t h y l d ~ p a ; no ~ ~ advantage i s gained by combining p r o p r a n o l o l w i t h these sympathetic inhibitor^.^^ Propranolol i s thought t o a c t as an a n t i h y p e r t e n s i v e by decreasing the c a r d i a c o u t p u t and c o n d i t i o n i n g the baroreceptors t o f u n c t i o n a t a lower i n addition t o cardiac B-blockade, t h e r e may be a component o f adrenergic neurone blockade i n t h i s a c t i o n . 2 6 I n c o n t r a s t , t h e a n t i h y p e r t e n s i v e a c t i o n o f p r a c t o l o l may be due t o v a ~ o d i l a t i o n . ~ The ~ problems i n v o l v e d i n t h e c l i n i c a l e v a l u a t i o n of these drugs have been discussed.28 There has been a succession o f r e p o r t s t h i s year on t h e c l i n i c a l e f f e c t s o f c l o n i d i n e ( I ) , which can be used w i t h advantage i n c o n j u n c t i o n with c h l o r t h a l i d ~ n e . ~ ~ ~unique ~~ The and complex mechanism o f a c t i o n o f t h i s drug i s now becoming c l e a r . 3 1 When a l l sympathetic transmission i s blocked, a p e r i p h e r a l vasodi l a t i n g a c t i v i t y has been uncovered,32 i n a d d i t i o n t o i t s p e r i p h e r a l a-receptor s t i m u l a n t a c t i o n which r e s u l t s i n a b r i e f hypertensive response a f t e r i . v . a d m i n i s t r a t i o n a t low doses. The a c t i o n which predominates and makes c l o n i d i n e such a u s e f u l drug i s i t s a b i l i t y t o s t i m u l a t e c e n t r a l a-receptors l e a d i n g t o an i n h i b i t i o n o f sympathetic An e f f e c t on the c e n t r a l c o n t r o l o f vagal a c t i v i t y , a t l e a s t i n dogs, has a l s o been ~ u g g e s t e d . ~ ~

I

Ill

II

Studies on the h e m ~ d y n a m i c sand ~ ~ central s i t e s o f action37 o f several a-receptor s t i m u l a n t s , i n c l u d i n g c l o n i d i n e ( I ) , t h e oxazol i n e ( 1 1 , LD2855), and x y l a z i n e ( I l l , BAY 1470) have been reported. Adrenergic neurone blockers - Several m e t h y l p y r r o l i d i n e analogs ( I V , R = (CH3)2 o r (CH3)3) o f guanethidine have a c t i v i t i e s comparable t o t h a t o f guanethidine when t e s t e d p.0. and S . C . i n renal hypertensive r a t s . ( I V , R = cis-2,4-(CH3)2) i s l e s s a c t i v e than guanethidine as a sympathomirnetic i n dogs and c a t s ( i . v . ) . 3 8

N-CH2CH2NHC //NH NH2

'

IV

\

m N - C C N H

NH2 V

VI

Chap. 6

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

61 -

Roe

The N-carbamidoxime ( V ) lowers the blood pressure o f renal hypertensive dogs a t 2 and 8 mg/kg and i s a l s o r e p o r t e d t o be an e f f e c t i v e a n t i h y p e r t e n s i v e i n neurogenic dogs, as w e l l as r e n a l and spontaneously hypertensive r a t s . T h i s compound resembles guanethidine and debrisoquine ( V I ) i n i t s adrenergic neurone b l o c k i n g and catecholamined e p l e t i n g a c t i v i t y , but i t may have an a d d i t i o n a l c e n t r a l e f f e c t . l l ~ ~ ~ Debrisoquine has now been shown t o d e p l e t e t h a t small p a r t o f the norepinephrine s t o r e which has been p o s t u l a t e d as e s s e n t i a l f o r t h e proper f u n c t i o n i n g o f adrenergic neurones , 4 0 A comparison o f the a n t i h y p e r t e n s i v e e f f e c t o f guanethidine w i t h t h r e e o f i t s m e t a b o l i t e s i n renal h y p e r t e n s i v e r a t s leads t o t h e conclusion t h a t guanethidine p e r se, and not i t s m e t a b o l i t e s , i s responsible f o r i t s hypotensive e f f e c t s . 4 1 Gang1 i o n b l o c k e r s - A t 10 mg/kg, d e r i v a t i v e s o f 4-azahomoadamantane ( V I I ) and (VIII) lower the blood pressure o f anesthetized dogs by a t l e a s t 15% f o r two hours o r more, a p p a r e n t l y by g a n g l i o n blockade.42 The monomethoxymecamylamines ( I X , R1, R2, o r R 3 CH,O) a r e about as a c t i v e as mecamylamine i n l o w e r i n g t h e blood pressure o f r e n a l h y p e r t e n s i v e r a t s , t h e corresponding hydroxy d e r i v a t i v e s a r e l e s s a c t i v e . 4 3

-

R = H = C2H4NHC (NH)NH2

R = NH2 = NHaryl = C H N(alkyll2

= C H COC H F - 4 2 4 6 4

2 4

viI

IX

VI I I

X

H3

-

XI

F u r t h e r d e t a i 1s have now appeared44 on t h e or-Adrenoreceptor b 1ocke r s c a r d i o v a s c u l a r e f f e c t s o f the a-blocker (X, NC 7197) mentioned l a s t year.45 The t r i c y c l i c guanidine ( X I ) , which s i g n i f i c a n t l y lowers t h e blood pressure o f m e t a c o r t i c o i d r a t s a t 5 mg/kg, and conscious normotensive o r neurogenic hypertensive dogs a t 2.5 mg/kg, probably has or-blockade as i t s main mechanism o f a c t i o n . However, s i n c e t h e r e i s no o r t h o s t a t i c hypotension i n the r a b b i t t i l t t e s t , i t s mechanism o f a c t i o n must be complex.46

62 -

Sect.

II

-

Pharmacodynamic Agents

Top1 i s s , Ed.

lndoramine ( X I I , Wy 21901) a b o l i s h e s e p i n e p h r i n e - and ouabai ninduced c a r d i a c a r r y t h m i a s by a combinat i o n o f a - a d r e n o r e c e p t o r b l o c k a d e and membrane s t a b i l i ~ a t i o n . These ~ ~ e f f e c t s have a l s o been s t u d i e d electrophysiological iy.48 l n d o ra m i n e has a PA, o f 7.4 as a c o m p e t i t i v e a - b l o c k e r on a o r t i c s t r i p , and a PA, o f 8.2 as an a n t i h i s t a m i n e o n guinea p i g ileum.49 The b l o o d p r e s s u r e o f c a t s and consci ous r e n a l h y p e r t e n s i v e r a t s i s lowered b y i n d o rami ne a t 20-40 mg/kg (p.0.) ; s e v e r a l analogs a r e a l s o a c t i v e b u t r a t h e r t o x i c . 5 0 A d o s e - r e l a t e d r e d u c t i o n i n b l o o d p r e s s u r e accompanied by an i n c r e a s e i n h e a r t r a t e a f t e r a d m i n i s t r a t i o n o f i n d o ra mi n e has been observed i n man.51 The new compound ( X I 1 1 , AH 5158A) i s p a r t i c u l a r l y i n t e r e s t i n g because i t combines s i g n i f i c a n t B -a d r enoreceptor b l o c k i n g a c t i v i t y w i t h a - b l o c k i n g a c t i v i t y b o t h i n a n i m a l s and i n man.52

IN

H

XI I

XI II

Compounds a f f e c t i n g a d r e n e r g i c t r a n s m i t t e r s - F u s a r i c a c i d amide ( X I V , Sch 10595) i s a v e r y a c t i v e i n h i b i t o r o f doparnine-6-hydroxylase i n v i v o ( b u t n o t i n v i t r o ) , and i t l o w e rs t h e b l o o d p r e s s u r e o f DOCA h y p e r t e n s i v e r a t s f o r many h o u rs i n a d o s e - r e l a t e d manner.53 The c y c l o h e x y l a m i n o o x a z o l i n e ( X V , BAY a6781) p r o b a b l y e x e r t s i t s h y p o t e n s i v e e f f e c t by p r e v e n t i n g t h e r e l e a s e o f c a te c h o l a mi nes f r o m t h e a d r e n a l gl and.54 3-Ethyl-a-methyltyrosine (XVI) i s a competitive i n h i b i t o r o f t y r o s i n e h y d r o x y l a s e and e x h i b i t s s i g n i f i c a n t a n t i h y p e r t e n s i v e a c t i v i t y i n DOCA r a t s a t 25 mg/kg. I t may be p r e f e r a b l e t o a - m e t h y l t y r o s i n e because i t s h o u l d n o t be m e t a b o l i s e d i n t o a ~ a t e c h o l a m i n e . ~ ~

--

CH

[

N ), 3 - H C2H5

XIV

xv

CH

FH3 1 3

1

I 2'NH22

xv I

The mechanism o f t h e c e n t r a l h y p o t e n s i v e e f f e c t s of L-dopa, a-methyldopa and m - t y r o s i n e has been s t u d i e d w i t h t h e a i d o f p e r i p h e r a l dec ar box y las e i n h i b i t o r s . 5 6 3 7 The e f f e c t s o f L-dopa on t h e c a r d i o v a s c u l a r system have been re v i ew ed.5* A c o m p a r a t i v e s t u d y o f t h e dose-dependent a n t i h y p e r t e n s i v e a c t i o n s , and t h e n o r e p i n e p h r i n e d e p l e t i n g a c t i v i t i e s , o f a - m e t h y l t y r a m i n e and a-methyl octopami ne i n r e n a l r a t s has been pub1 i shed. 5 9

Chap. 6

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

63

Roe

A s e r i e s o f hydrazones has been shown t o have a n t i h y p e r t e n s i v e and monoamine o x i d a s e i n h i b i t o r y a c t i v i t y , a l t h o u g h t h e r e was no c o r r e l a t i o n between th e s e a c t i o n s . The most a c t i v e compound ( X V I I ) caused a p r o l o n g e d f a l l o f 40-60 mn Hg i n t h e b l o o d p r e s s u r e o f a n e s t h e t i z e d dogs and c a t s a t 1 mg/kg ( i . v . 1 , and a l s o i n G o l d b l a t t h y p e r t e n s i v e r a t s a t 30 mg/kg (p.0.) when g i v e n f o r 10 d a y s . 6 0 I n a n o t h e r s t u d y , t h e compounds ( X V I I l a and X V l l I b ) were t h e most a c t i v e o f a s e r i e s o f a n t i h y p e r t e n s i v e monoamine o x i d a s e i n h i b i t o r s . 6 1 CH

' 3

XVI I I a) X = -CH=Nb) X = -CH2NHOZN

0 0

0

XVI I

-

N a t u r a l l y o c c u r r i n g and r e l a t e d compounds The s t r u c t u r e and s y n t h e s i s o f oudenone ( X I X ) , t h e h y p o t e n s i v e menti oned l a s t y e a r , 4 5 has now been r e p o r t e d . 6 2 A c e ty l a n d ro me d o l ( X X , g r a y a n o t o x i n I ) , o b t a i n e d from v a r i o u s s p e c i e s o f Rhododendron, i s h y p o t e n s i v e i n c a t s a t 0.04 mg/kg ( i . v . ) ; a r t e f a c t s w i t h r e l a t e d s t r u c t u r e s are a l s o hypotensive i n cats o r r a t s . 6 3 R =

ATS) CH3

-OH

xx

x IX

xx I

i;)

R = , ,

H

Analogs o f n i c o t i n e and anabasine i n w hi ch t h e p y r i d i n e r i n g has been r e p l a c e d by q u i n u c l i d i n e (XXla and b o t h d i a s t e r e o i s o m e r s o f XXlb) show h y p o t e n s i v e e f f e c t s when g i v e n i . v . t o dogs.64 The h y d r a z i d e o f yo him boic a c i d , w h i c h i s c o n s i d e r a b l y l e s s t o x i c t h a n t h e m e t h y l e s t e r ( y o h i m b i n e ) , causes p r o l o n g e d h y p o t e n s i o n a t 1 mg/kg ( i.v.) i n chloralosed dogs.65

-

Prostaglandins The e f f e c t s o f p r o s t a g l a n d i n s on c a r d i o v a s c u l a r responses would appear t o be c r i t i c a l l y dependent on dose l e v e l s : i n f u s e d i n t o humans a t t h e a p p r o p r i a t e dose, PGA, and P G E l cause a f a l l i n b l o o d p r e s s u r e and r e n a l d i l a t a t i o n w i t h o u t t h e r e l e a s e o f r e n i r ~ . ~ However, ~ , ~ ~ th e s e e f f e c t s a r e n o t s i m p l y dose r e l a t e d . 6 8 Ex per im ent s on t h e c a t s p l e e n have l e d t o t h e s u g g e s t i o n t h a t t h e r e i s a continuous basal s e c r e t i o n o f vasodi l a t i n g p r o ~ t a g 1 a n d i n s ; ~ g a t very low i n f u s i o n r a t e s o f PGA, and PGEl i n t h e dog, e f f e c t s due t o d i r e c t v a s o d i l a t i o n can be s e p a ra te d from s y m p a t h e t i c i n h i b i t i ~ n . ~ ~

64 -

-

Sect, I I

Pharmacodynamic Agents

T o p l i s s , Ed.

Compounds a c t i n g d i r e c t l y on vascular smooth muscle o r by unknown The 1,3,4-oxadiazole ( X X 1 1 ) 7 1 and t h e a c e t o n i t r i l e mechanisms ( X X 1 1 1 ) 7 2 cause b r i e f v a s o d i l a t i o n i n c a t s and i n dogs r e s p e c t i v e l y .

-

u1- c Hi-(d) 0

O

0

\ / I

XXI I

q - Nc H2c NO

XXlJ I

NHNH2

XXIV

C H CON-Ph 2 5

CH2CH20H

The benzoquinol i z i n e ( X X I V ) i s a hypotensive v a s o d i l a t o r i n i t lowers the blood pressure of conscious anesthetized dogs and c a t s ; renal hypertensive dogs a t 10 mg/kg/day (p.0.) . 7 3 s 7 4 The hypotensive e f f e c t o f the hydrazine ( X X V , L6150) i n conscious o r a n e s t h e t i z e d dogs (0.5 o r 0.01 mg/kg, i . v . ) appears t o be due t o p e r i p h e r a l v a s o d i l a t i o n . 7 5 The pyrazole ( X X V I ) i s a potent v a s o d i l a t o r which reduces t h e blood pressure o f c a t s and dogs by 40-50 mm Hg f o r 2 hours a t 0.25 mg/kg (i.v.), I t i s a l s o a c t i v e i n renal hypertensive rats.76

a

CH2NH (CH2) TX-NHn

CH3

XXVI I a)

CH3

b) c) d)

Ar

xxv I I

I

4-m-Chlorophenyl-1-piperazino

c6H5 C6H5 c6H5

n 1 2 2

2,6-Me2C6H3

2

11

L c o 2 c pH

R = N - 1 ,2,3,4-Tetrahydroisoquinol i n o 4-Benzyl-1-piperazino

I

Ar

RCH CH C 2 2-

1

X Cti

CH N

CH CH XXlX

A s e r i e s o f twenty-nine 3-pyrrolemethylamines ( X X V I I ) show e) hypotensive a c t i v i t y i n conscious and anesthetized dogs; ( X X V I I , a reduce t h e blood pressure o f t h e l a t t e r by 20 mm Hg a t 1 mg/kg ( i . v , ) o r less.77 Other compounds r e p o r t e d t o be hypotensive i n anesthetized

-

Chap. 6

Antihypertensive A g e n t s

65

Roe

c a t s o r d o g s a r e t h e piperazines ( X X V 1 1 1 ) , 7 8 t h e prenylamine analog ( X X I X , HOE 674), 7 9 t h e hydroxamic acid ( X X X ) e O and t h e amidinium salt ( X X X I ) . a 1

NHOH

xxx

xxx I

XXXI I

T h e highly substituted tetrahydroisoquinol ines ( X X X I I , R = PhCH, and CH3) lower t h e blood pressure o f anesthetized d o g s b y 30 mm Hg for 1 hour at 10 and 4 mg/kg (i.v.) respectively;82 a reduction o f 40% or more for a t least 4.5 hours occurs a f t e r giving several ‘+-substituted * aminoquinol ines ( X X X I I I ) .83 T h e benzocycloheptenes ( X X X I V , a and b) s h o w hypotensive activity in rats a t 10 and 20 mg/kg (i.v.) respectively. 8 4 R HR I XXXI I I

QQ

R = e.g. H, al kyl, furfuryl

m

xxx I v

a) R = 4-(l-methylpiperazinyl.) b) R = N(Ph)CH2CH2N(CH3)2

CHJ09cH2<1 N

xxxv

CH30’

O

H

xxxv I

T h e amidine analog (XXXV, A r = 2,6-C1,C6H3) o f clonidine, and several related compounds (e.g. X X X V , A r = C6H , 2-CH3C6H4, 3’CH3C6~4, are active 2,3-(CH3) ,C,H?, 2,4- ( C H ~ ) Z C ~ H and ~ , 2,2-C12!6H3CH,) antihypertensives in DOCA rats at 10 mg/kg intragastrical ly.85 A detailed study has been made o f t h e unique combination of properties exhibited by t h e hypotensive imidazol idine ( X X X V I , R o 7-2956). In dogs, a direct peripheral vasodilation i s combined w i t h a nonadrenergic myocardial stimulant activity reminiscent o f theophyll ine.86

66

Sect.

I 1 - Pharmacodynamic Agents

T o p l i s s , Ed.

RE FERENC E S

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36,

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Chapter 7.

Antianginal Agents

Charles F. Schwender , Warner-Lambert Research I n s t i t u t e Morris Plains, N.J. Angina pectoris i s a painful symptom of myocardial ischemia occurring when the oxygen supply cannot meet the requirements of the heart. During ischemia, pain and abnormalities of heart metabolism, l e f t ventricular function and electrocardiograms are observed. Blood flow through the myocardium i s the main determinant of oxygen supply.2 The heart extracts oxygen from i t s blood supply a t near maximal efficiency so t h a t any i n crease in oxygen requirements must be met by an increase i n blood flow. Ischemia caused by an i n s u f f i c i e n t oxygen supply may be improved by agents such as n i t r a t e s which increase myocardial perfusion and reduce oxygen utilization. 3 Early work wi t h n i troglyceri n i ndi cated a coronary vasodi 1 a t o r action which led t o a concerted e f f o r t t o find more potent, longer acting vasodi 1 ators s e l e c t i v e f o r the coronary vascul a r bed. These newer agents have been disappointing since l i t t l e r e l i e f of ischemia or anginal pain has been observed. Another more recent approach t o treatment of angina4 includes the reduction of myocardi a1 oxygen requi remen ts through reduced cardi ac work. Also, improved circulation of blood t o ischemic areas by s e l e c t i v e dilation of certain l a r g e r supply vessels appears t o restore normal oxygen and nutrient supply. Circulation through the l e f t ventricle involves epicardial (superficial regions) and endocardial (deeper regions) pathways which compete f o r available blood flow from larger intramural supply vessels. Further branching of the supply vessels leads t o p r e a r t e r i o l a r , a r t e r i o l a r and capillary beds which have a n u t r i t i v e function i n the t i s s u e s . I t has been suggested t h a t circulation in the endocardium i s probably inadequate, thus establishing a r e l a t i v e l y ischemic r e g i ~ n . ~ , ~ Compared w i t h the epicardium, the endocardial region has a lower oxygen tension and a lower level of circulating nutrients. When coronary artery disease r e s t r i c t s blood flow, the endocardial region may be prone t o wocardial insufficiency. Since the endocardium i s r e l a t i v e l y ischemic, autoregulatory responses cause an average a r t e r i o l a r and precapi 1 l a r y sphincter d i l a t i o n of about 91%. The vessels of the better perfused epicardium are only opened about 68% and can b e t t e r t o l e r a t e a decreased flow through compensatory mechanisms. Nitroglycerin - Nitroglycerin i s the oldest and most e f f e c t i v e antianginal agent i n use today. Used prophylactically o r f o r immediate r e l i e f of anginal pain, nitroglycerin acts by reducing heart work and by redist r i b u t i n g blood flow t o the ischemic areas of the myocardium. The beneficial e f f e c t of nitroglycerin on the myocardial circulation of dogs i s the r e s u l t of a number of a c t i o n s . 6 ~ 7 ~ 8The ~ 9 reduced ischemia i s probably the r e s u l t of a decreased oxygen requirement due t o a reduced heart work, f i l l i n g pressure and stroke volume. The major mechanism of action appears

70

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I I - Pharrnacodynarnic A g e n t s

Top1 i s s , Ed.

t o be an i n c r e a s e d oxygen t e n s i o n i n t h e endocardium due t o a r e d i s t r i b u blood flow t i o n o f b l o o d from t h e e p i c a r d i ~ m . ' + ~ While ~ , ~ ~myocardial ~ ~ ~ i s decreased by n i t r o g l y c e r i n , endocardia1 p e r f u s i o n i s m a i n t a i n e d a t p r e t r e a t m e n t l e v e l s b y t h e r e d i s t r i b u t i o n o f b l o o d f r o m t h e epicardium. Endocardia1 oxygen t e n s i o n r i s e s as a r e s u l t o f t h e r e d i s t r i b u t e d b l o o d f l o w and decreased oxygen requirement. N i t r o g l y c e r i n r e d i s t r i b u t e s b l o o d f l o w t o t h e endocardium b y d i l a t i n g t h e l a r g e r s u p p l y o r i n t r a m u r a l vessels c o n t r o l l i n g t h e f l o w d i s t r i b u t i o n between t h e i n n e r and o u t e r r e g i o n s o f t h e myocardium. The i n t r a m u r a l vessels do n o t appear t o be c o n t r o l l e d by t h e a u t o r e g u l a t o r y mechanism s o t h a t t h e y respond d i f f e r e n t l y t o drugs than a r t e r i o l e s . Measurements o f changes i n l a r g e vessel r e s i s t a n c e and oxygen t e n s i o n c o r r e l a t e we1 1 w i t h t h e e f f e c t s caused by n i t r o g l y c e r i n . Microsphere d i s t r i b u t i o n s t u d i e s i n t h e dog a l s o i n d i c a t e d a r e d i s t r i b u t i o n o f f l o w t o t h e endocardium due t o n i t r o g l y c e r i n a c t i o n . I n c o n t r a s t t o n i t r o g l y c e r i n and p e n t a e r y t h r i t o 1 t e t r a n i t r a t e coronary v a s o d i l a t o r s such as d i p y r i d a m o l e 4 and chromonar12 i n c r e a s e d t o t a l coronary b l o o d f l o w i n dogs w i t h o u t r e d i s t r i b u t i n g b l o o d f l o w o r improvi ng endocardi a1 oxygen tens ion. The coronary vasodi 1a t o r s , d i p y r i damole, chromonar, l i d o f l a z i n e , i p r o v e r a t r i l , papaverine and prenylamine were a l l shown t o e x e r t a s e l e c t i v e d i l a t o r a c t i o n on s m a l l coronary a r t e r i e s and a r t e r i o l e s . l 3 I t has been suggested1'+ t h a t t h e smooth muscle r e l a x a n t a c t i o n o f n i t r o g l y c e r i n may be r e l a t e d t o i t s i n i t i a l b l o o d c o n c e n t r a t i o n s r a t h e r than a c e r t a i n optimum b l o o d l e v e l . N i t r o g l y c e r i n appears t o be most e f f e c t i v e i n a b o l i s h i n g e x i s t i n g c o n t r a c t i o n s o f v a s c u l a r smooth muscles r a t h e r than p r e v e n t i n g t h e i r c o n t r a c t i o n . This may i n d i c a t e why n i t r o g l y c e r i n i s t h e r a p e u t i c a l l y more e f f e c t i v e sub1 i n g u a l l y o r by subcutaneous i n j e c t i o n than o r a l l y .

Blum15 was unable t o demonstrate a r e l a t i o n s h i p between reduced oxygen u t i l i z a t i o n , i n h i b i t i o n o f m i t o c h o n d r i a 1 r e s p i r a t i o n and d i l a t a t i o n o f l a r g e coronary a r t e r i e s u s i n g a s e r i e s o f o r g a n i c n i t r a t e s . B i o chemical e v a l u a t i o n d i d n o t c o n s i s t e n t l y p r e d i c t which agents would a f f e c t b l o o d f l o w t o t h e endomyocardium. Coronary V a s o d i l a t o r s - Continued i n t e r e s t i n t h i s approach t o a n t i a n g i n a l agents has been r e f l e c t e d by t h e numerous r e p o r t s o f agents i n c r e a s i n g coronary b l o o d f l o w and oxygen s u p p l y . These agents p r o b a b l y a c t b y i n t e r f e r i n g w i t h m e t a b o l i c a u t o r e g u l a t i o n o f coronary b l o o d f l o w . l6 3 1 7 A u t o r e g u l a t i o n i s a mechanism by which t h e h e a r t m a i n t a i n s s u f f i c i e n t b l o o d f l o w and n u t r i e n t supply f o r i t s m e t a b o l i c a c t i v i t y . Some vasod i l a t o r agents appear t o be s p e c i f i c d i l a t o r s o f s m a l l e r coronary b l o o d vessels by an adenosine-sparing mechanism. The end p r o d u c t o f adenosine t r i p h o s p h a t e (ATP) breakdown i n t h e h e a r t appears t o be adenosine w h i l e i n o t h e r t i s s u e s such as s k e l e t a l muscle, i n o s i n e i s t h e p r o d u c t . Adenosine, a p o t e n t v a s o d i l a t o r , has been shown t o be p r e s e n t i n t h e dog h e a r t i n n u c l e o s i d i c and n u c l e o t i d i c forms. D u r i n g ischemic c o n d i t i o n s , b o t h myocardial and b l o o d l e v e l s o f adenosine a r e increased.

Chap.

7

Ant iang ina 1 A g e n t s

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71

A1 though n o t proven c o n c l u s i v e l y , adenosine has been i m p l i c a t e d as a t l e a s t one m e d i a t o r o f a u t o r e g u l a t o r y c o n t r o l o f myocardial b l o o d f l o w . Oxygen t e n s i o n , potassium i o n c o n c e n t r a t i o n , b l o o d pH and a myogenic response a l s o may be i n v o l v e d i n a u t o r e g u l a t i o n . However, a change i n adenosine c o n c e n t r a t i o n as a r e s u l t o f ischemia can f u l l y account f o r vasodi 1 a t o r responses through a u t o r e g u l a t o r y c o n t r o l . l7 Any t h i n g which may h e l p c r e a t e an oxygen d e f i c i e n c y such as a c t i v e t r a n s p o r t processes, i n c r e a s e d h e a r t work o r i m p a i r e d coronary c i r c u l a t i o n due t o o c c l u s i o n or c a r d i o v a s c u l a r disease a l s o decreases t h e ATP s u p p l y i n t h e myocardium. D u r i n g ischemia, t h e e q u i l i b r i u m between ATP and adenosine diphosphate (ADP) f a v o r s ADP f o r m a t i o n and adenosine. The h i g h e r i n t r a c e l l u l a r adenosine c o n c e n t r a t i o n promotes d i f f u s i o n o f adenosine through c e l l u l a r membranes i n t o t h e b l o o d o f t h e myocardium. A r e s u l t i n g l o c a l i z e d v a s o d i l a t i o n i n c r e a s e s t h e b l o o d f l o w and oxygen s u p p l y t o t h e ischemic area. The i n c r e a s e d ATP/ADP r a t i o p e r m i t s more work.and m e t a b o l i c processes t o be supported. B l o o d f l o w i s m a i n t a i n e d a t a g i v e n l e v e l o f m e t a b o l i c a c t i v i t y and adenosine c o n c e n t r a t i o n . Coronary v a s o d i l a t o r s i n t e r a c t w i t h t h e adenosine-mediated vasod i l a t i o n response by p r e v e n t i n g t h e deamination o r r e a b s o r p t i o n o f adenosine p r e s e n t i n t h e blood. Such agents a r e d i p y r i d a m ~ l e , ~ ~ , ~ ~ hexobendine,2 l i d o f l a z i n e , l 8 2 - a l k y l t h i o a d e n o s i n e s ,2 and o t h e r subs t i t u t e d - a d e n o s i n e s 2 2 i n dogs and p t e r i d i n e r ' analogs i n k i t t e n s . A l though these agents a r e i n h i b i t o r s o f i n t r a c e l l u l a r adenosine deaminase, i t i s b e l i e v e d t h a t t h e i r p r i m a r y a c t i o n i s t h e i n h i b i t i o n o f adenosine d i f f u s i o n across c e l l u l a r membranes where i t i s deaminated.16 Adenosine uptake may i n v o l v e a membrane-bound k i n a s e enzyme which r e l e a s e s adenosine as i t s monophosphate w i t h i n t h e c e l l . 2 4 Dipyridamole i n h i b i t s t h i s membranal k i n a s e i n guinea p i g s t o a g r e a t e r degree than s o l u b l e i n t r a c e l l u l a r adenosine deaminase o r k i n a s e enzymes. Dipyridamole, i n r a t s , does n o t p r e v e n t uptake o r p o t e n t i a t e t h e v a s o d i l a t o r e f f e c t s o f adenosine due t o a l a c k o f t h e membranal k i n a s e t r a n s p o r t mechanism. Thus, species v a r i a t i o n i n adenosine uptake mechanism may t e n d t o confuse s t u d i e s i n mechanisms o f a c t i o n w i t h v a s o d i l a t o r s i n t e r f e r i n g w i t h aden 0s in e

.

Ischemia may cause d e t e r i o r a t i o n o f myocardial f u n c t i o n through excessive l o s s o f ATP and t h e myocardial pool o f p u r i n e n u c l e o s i d e s . The enzyme, x a n t h i ne oxidase, ir r e v e r s i b l y c o n v e r t s p u r i n e s t o u r i c a c i d. A f t e r p e r i o d s o f asphyxia, an i n f u s i o n o f adenosine caused a markedly a c c e l e r a t e d r e s t o r a t i o n o f normal myocardial n u c l e o t i d e s . 2 5 I n coronaryl i g a t e d dogs and sheep, t h e x a n t h i n e oxidase i n h i b i t o r a l l o p u r i n o l p r e vented o r r e v e r s e d n u c l e o t i d e l o s s from t h e myocardium, ST-depression and c a r d i a c arrhythm; 35 due t o myocardial ischemia.26 Hexobendine2 causes a m e t a b o l i c a c i d o s i s which may c o n t r i b u t e t o i t s v a s o d i l a t o r a c t i o n i n humans. Other acje:l?t,z such as amiodarone ,2

bencycl an ,z

chromonar,

72

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Topliss, Ed.

d i a z o x i d e Y 3 l i p r o v e r a t r i l , 3 0 perhexiline,32 and diazepam33 have been shown i n dogs t o e x e r t a v a s o d i l a t o r y a c t i o n on small coronary blood vessels. The mechanisms o f a c t i o n have n o t been f u r t h e r e l u c i d a t e d . These agents a l s o possess a p e r i p h e r a l v a s o d i l a t o r a c t i o n which may be b e n e f i c i a l i n anginal p a t i e n t s by decreasing blood pressure, venous r e t u r n and h e a r t work. An understanding o f t h e m i c r o c i r c u l a t o r y responses o f t h e h e a r t t o n i t r o g l y c e r i n and coronary v a s o d i l a t o r s c l e a r l y i n d i c a t e s why n i t r o g l y c e r i n i s an e f f e c t i v e a n t i a n g i n a l agent, w h i l e v a s o d i l a t o r s are g e n e r a l l y i n e f f e c t i v e o r d e l e t e r i o u s i n ischemic c o n d i t i o n s . 4 ~ 5 N i t r o g l y c e r i n d i l a t e s l a r g e r coronary a r t e r i e s n o t c o n t r o l l e d by a u t o r e g u l a t i o n mechanisms. A r e d i s t r i b u t i o n o f blood f l o w r e s u l t s which d i v e r t s b l o o d from the normal epicardium t o the ischemic endocardium. L i t t l e change i n t o t a l coronary f l o w i s observed. Since coronary v a s o d i l a t o r s a c t by i n t e r ference o f a u t o r e g u l a t i o n mechanisms i n s m a l l e r coronary vessels, v a s o d i l a t i o n o f t h e normal e p i c a r d i a l vessels probably predominates. The smal l e r vessels i n t h e ischemic endocardium are probably maximally d i l a t e d due t o anoxia. Thus, an enhanced e p i c a r d i a l f l o w must d i v e r t b l o o d f l o w from ischemic areas since they compete f o r a v a i l a b l e flow. Coronary v a s o d i l a t o r s could n o t be expected t o be b e n e f i c i a l b u t may be harmful when used t o t r e a t nlyocardial ischemia and angina p e c t o r i s . I n very severe ischemia, h i g h doses o f dipyridamole a c t u a l l y induced anginal a t t a c k s i n man. 3 4

ami odarone

d

bencyclan

H

perhexi lene

hexobendi ne

-

Treatment o f angina p e c t o r i s w i t h BBeta-Adrenergic B l o c k i n q Agents b l o c k i n g agents i s a more r e c e n t !approach which appears t o be o f therap e u t i c value. Exercise and s t r e s s tend t o increase catecholamine l e v e l s and c a r d i a c sympathetic d r i v e i n anginal p a t i e n t s . Excessive sympathetic s t i m u l a t i o n creates an oxygen wasting e f f e c t by i n c r e a s i n g oxygen use i n the myocardium more than i s r e q u i r e d t o perform a d d i t i o n a l work. BBlockade prevents t h i s excessive oxygen consumpti on and reduces c a r d i a c The lower c a r d i a c work and oxygen requirement allows t h e h e a r t t o perform more e f f i c i e n t l y d u r i n g moderate exercise a t a lower h e a r t r a t e .

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A number o f 8-adrenergic blockers have r e c e n t l y been r e p o r t e d i n the l i t e r a t u r e . Two previous reviews have covered a l l b u t t h e most c u r r e n t analogs. 36 , 3 7 Ring s u b s t i t u t i o n o r t h o t o the oxypropanolamine s i d e chain by cyclopropyl (SD-2124-01) has 1a n o n s e l e c t i v e 8-blocker o f potency s i m i l a r t o p r i n d o l o l and g r e a t e r than p r o p r a n o l o l i n dogs. This analog 1acks s igni f icant B-sympathomi meti c and c a r d i odepress ant a c t i v i ty b u t is capable o f r e v e r s i n g ouabain-induced cardiac arrhythmias p o s s i b l y by f a c i l i t a t i n g a t r i o - v e n t r i c u l a r conduction. 38 Two n i t r i l e - s u b s t i t u t e d analogs, Ko-1313 and Ko-1366, have been reO as nonselective 8-blocking agents which were more p o t e n t than ported39,'+ propranolol i n dogs and a l s o reversed ouabain-induced c a r d i a c arrhythmias. However, s i g n i f i c a n t depression o f a t r i o - v e n t r i c u l a r conduction was observed a t a n t i a r r h y t h m i c dosage l e v e l s . This Another analog s i m i l a r i n potency t o propranolol i s D-69-12. agent i s a c t i v e i n the guinea p i g . 4 1 The compound D563 o r 1-3-methoxy-w(l-hydroxy-l-phenyl isopropy1amino)-propiophenone i s a @-blocker i n dogs.42 I n a d d i t i o n t o p r a c t o l o l , o t h e r p a c y l a m i n o analogs43 were shown t o be c a r d i o s e l e c t i v e 8-blockers and more p o t e n t than t h e parent compound. Para-a1 l y l and -a1 l y l o x y s u b s t i t u t e d analogs o f a1 r e n o l o l and oxprenolol have been reported t o be c a r d i o s e l e c t i v e i n cats. !4 RI

Rz

t-butyl

SD-2 124-01

KO-1 313

2-CN

isopropyl

KO-1 366

2 -CN

-t - b u t y l

D-69-12

2-c1

t -b u t y 1 -

0 , C H Z CHOHCHzNHRz

practolol

4-NHCOCH3

isopropyl

alprenolol

isopropyl

oxp reno l o 1

isopropyl

KL-255

2-C1,

5-CH3

t-butyl

B-Adrenergic blockers remain c o n t r a i n d i c a t e d i n those p a t i e n t s s u f f e r i n g from some n y o c a r d i a l decompensation o r h e a r t f a i l u r e . 4 5 I n those cases, B-blockade o f normal sympathetic support o f h e a r t f u n c t i o n may p r e c i p i t a t e c a r d i a c f a i l u r e . Several studies concerning the cause o f tqyocardial depression and i n d u c t i o n o f h e a r t f a i l u r e have been r e ported. % - 4 9 6-61 ockers whi ch possessed s i g n i f ic a n t c a r d i ac depress a n t p r o p e r t i e s may have i n t e r f e r e d w i t h calcium t r a n s p o r t across membranes o f the mitochondria and sarcoplasmic r e t i c u l u m i n dog h e a r t muscle. This r e s u l t e d i n a depression o f the c o n t r a c t i l e s t a t e o f t h e myocardium.

74

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T o p l i s s , Ed.

N a ~ l e rproposed ~ ~ t h a t agents which have s i g n i f i c a n t 8-sympathomimetic a c t i o n d i d n o t i n t e r f e r e w i t h c a l c i u m t r a n s p o r t and t h e c o n t r a c t i l e s t a t e . They were l e s s depressant on t h e myocardium t-han 6 - b l o c k e r s n o t h a v i n g as t i m u l a n t a c t i o n . The depressant e f f e c t was n o t r e l a t e d t o a - b l o c k i n g potency. A t h i g h c o n c e n t r a t i o n s , Noack and G r e e f f 4 7 demonstrated t h a t ab l o c k a d e was n o t i n v o l v e d i n i n h i b i t i o n o f c a l c i u m t r a n s p o r t . Using an v i t r o model, d- and 1-a1 p r e n o l o l were equal l y e f f e c t i v e i n h i b i t o r s o f calcium transport. A f t e r a s t u d y o f t h e c a r d i a c depressant a c t i o n o f KL-255, i t was suggested t h a t m y o c d r d i a l depression may be a r e s u l t of @-blockade o f t h e basal t o n e e s t a b l i s h e d b y endogenously r e l e a s e d catecholamines. 4 8 Catecholamine d e p l e t i o n b y g u a n e t h i d i n e r e s u l t e d i n a l o s s o f t h e dep r e s s i o n o f c o n t r a c t i l e f o r c e n o r m a l l y observed w i t h KL-255. The depress a n t e f f e c t s d i d n o t appear t o he d o s e - r e l a t e d t o l o c a l a n e s t h e t i c a c t i o n . I n t r i n s i c a-sympathomimetic a c t i v i t y o f some B - b l o c k e r s d i d n o t appear t o p r e v e n t t h e onset o f c a r d i a c f a i l u r e i n h e a r t s dependant upon sympathetic d r i v e . I t was suggested t h a t r;-blockers w i t h s i g n i f i c a n t 6sympathomimetic a c t i o n , such as DCI, m i g h t be l e s s l i k e l y t o p r e c i p a t a t e h e a r t f a i l u r e . The more depressant p r o p r a i i o l o l l a c k s 6-sympathomimetic a c t i v i t y . No d i f f e r e n c e was observed between DCI and p r o p r a n o l o l when i n t r o d u c e d d u r i n g t h e sympathomimetic-dependant phase o f e x p e r i m e n t a l h e a r t f a i l u r e i n guinea p i g s . 4 9 Although u - b l o c k e r s t e n d t o reduce coronary b l o o d f l o w , t h e y may n o t be harmful t o p a t i e n t s w i t h myocardial ischemia. I n t h e dog, p r o p r a n o l o l reduces b l o o d f l o w t o normal b u t n o t t o ischemic areas o f t h e h e a r t . Using RbE6 uptake, Xe133 c l e a r a n c e and l a b e l l e d microsphere d i s t r i b u t i o n s t u d i e s , experiments show t h a t p r o p r a n o l o l , l i k e n i t r o g l y c e r i n , r e d i s t r i butes b l o o d f l o w i n t o ischemic r e g i o n s o f t h e h e a r t . 1 0 , 5 0 - 5 2 WinburyS3 used oxygen t e n s i o n t o r e f l e c t i n c r e a s e d r e g i o n d l p e r f u s i o n and decreased m e t a b o l i c requirement o f oxygen. The 6 - b l o c k e r s , b u n o l o l and propranolo'l , i n c r e a s e d oxygen OCHzCHOHCHzNHC(CH3 )3 t e n s i o n i n ischemic areas o f t h e dog endocardium. R e d i s t r i b u t i o n o f b l o o d f l o w i n t o deeper areas of t h e endocardium appears t o be a common a c t i o n o f b o t h n i t r o g l y c e r i n 0 bunolol and a t l e a s t some @ - b l o c k e r s .

Q3

C l i n i c a l Therapy - The v a r i a b l e s i n c l i n i c a l e v a l u a t i o n agents a r e w e l l documented. Unless t h e d r u g s t u d i e d i s judgement o f t h e r a p e u t i c e f f i c a c y i s l i k e l y t o be mixed s u b j e c t t o v a r i e d i n t e r p r e t a t i o n s among i n v e s t i g a t o r s . see agents e f f e c t i v e i n some s t u d i e s b u t no b e t t e r than

o f antianginal 100% e f f e c t i v e , with bias or I t i s common t o placebo i n others.

V a r i a t i o n s i n dosages s t u d i e d , as w e l l as d u r a t i o n o f a c t i o n , o f t e n l e a d t o i n c o n s i s t e n t r e s u l t s . I s o s o r b i d e d i n i t r a t e i s an a c t i v e a n t i a n g i n a l agent w i t h a d u r a t i o n o f a c t i o n s i m i l a r t o n i t r o g l y c e r i n . Some r e p o r t s o f i n e f f e c t i v e n e s s may have been due t o t h e i n e f f e c t i v e dosage

Chap.

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-75

s t u d i e d o r t h e a g e n t ' s s h o r t d u r a t i o n of a ~ t i o n . ~ ' Chronic ~ - ~ ~ treatment w i t h i s o s o r b i d e d i n i t r a t e d i d n o t cause a c r o s s t o l e r a n c e w i t h n i t r o g l y c e r i n t o develop.57 N i t r o g l y c e r i n c o n t i n u e s t o p r o v e u s e f u l i n angina t r e a t m e n t . Using reduced ST-depression as a c r i t e r i o n f o r agent e f f e c t i v e n e s s i n ischemia, i t was shown t h a t n i t r o g l y c e r i n was b e n e f i c i a1 w h i l e s e v e r a l c o r o n a r y v a s o d i l a t o r s i n c l u d i n g chromonar, d i p y r i d a m o l e and hexobendine were i n e f f e c t i v e i n l o n g t e r m the rap^.^^,^^ 6-Adrenergic b l o c k i n g agents c o n t i n u e t o show u t i l i t y i n t h e t r e a t ment o f angina i f a d m i n i s t e r e d t o c a r e f u l l y s e l e c t e d p a t i e n t s . 3 5 , 5 8 , 6 0 ~ 6 1 Anginal p a i n induced by i s o p r o t e r e n o l o r e x e r c i s e was p r e v e n t e d o r e l i m i n a t e d upon t r e a t m e n t w i t h p r o p r a n o l o l o r o x p r e n o l o l . Ischemic changes n o t e d as ST-depressions were reduced by B - b l o c k e r t h e r a p y . A l p r e n o l o l , however, was r e p o r t e d i n e f f e c t i v e i n a s e p a r a t e study.62 I n a t r i a l w i t h p a t i e n t s h a v i n g m y o c a r d i a l i n f a r ~ t i o n ,t h~ e~ c a r d i o s e l e c t i v e B - b l o c k e r p r a c t o l o l showed p o s s i b l e v a l u e i n t h e p r e v e n t i o n o f arrhythmias a s s o c i a t e d w i t h severe ischemia w h i l e i t d i d n o t induce c l i n i c a l d e t e r i o r a t i o n o r f a i 1u r e o f t h e myocardi um. P r o p r a n o l o l , however, d i d cause d e t e r i o r a t i o n i n some p a t i e n t s . Combination therapy w i t h B - b l o c k e r s and n i t r a t e s has c o n t i n u e d . 3 5 3 , h 4 Combined t h e r a p e u t i c e f f e c t s have reduced t h e p o s i t i v e i n o t r o p i c and c h r o n o t r o p i c e f f e c t s o f n i t r o g l y c e r i n and t h e v a s o c o n s t r i c t i v e a c t i o n o f 6 - b l ockers . Benefi c i a1 s y n e r g i s t i c e f f e c t s upon reduced c a r d i ac work , oxygen requirements, and improved f l o w t o ischemic areas o f t h e myocardium have been observed. Combinations o f a l p r e n o l o l o r p r o p r a n o l o l and i s o s o r b i d e d i n i t r a t e s i g n i f i c a n t l y i n c r e a s e d work t o l e r a n c e and delayed p a i n and ST-depression i n some angina p a t i e n t s . The i n d i v i d u a l drugs were l e s s e f f e c t i v e . However, i n another s t u d y , i s o s o r b i d e d i n i t r a t e was i n e f f e c t i v e alone o r i n combination w i t h p r o p r a n ~ l o l . ~I ~n t h e same s t u d y , p r o p r a n o l o l induced h e a r t f a i l u r e i n some p a t i e n t s where no c l i n i c a l d e t e r i o r a t i o n had been d e t e c t e d e a r l i e r .

A s m a l l synergism was n o t e d when e r y t h r i t o l t e t r a n i t r a t e was coinb i n e d w i t h p r o p r a n o l o l o r a l p r e n o l o l . I t was suggested t h a t f u r t h e r i n v e s t i g a t i o n s i n v o l v i n g l a r g e r numbers o f p a t i e n t s a r e needed t o e v a l u a t e t h e p o s s i b l e s i g n i f i c a n c e of combined t h e r a p y . 6 6 References

1. 2.

3.

R. S. ROSS, Myocardial Ischemia, R. S. Ross and F. Hoffman, eds., pp. 1-6, Excerpta Medica, P r i n c e t o n , New Jersey, 1971. R. Kadatz,' Problems i n L a b o r a t o r y E v a l u a t i o n of A n t i a n g i n a l Agents, M. M. Winbury, ed., p. 3, North-Holland, Amsterdam, The Netherlands, 1967. M. M. Winbury, B . B. Howe and H. R. Weiss, J . Pharmacol. Exp. Ther. , 176, 184 (1971).

76 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.

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Pharmacodynamic Agents

Topliss, Ed.

M. M. Winbury, Problems i n Laboratory E v a l u a t i o n of A n t i a n g i n a l Agents, M. M. Winbury, ed. , p. 1 , North-Holland, Amsterdam, The Ne t h e r l ands , 1967. M. M. Winbury, C i r c . Res., 28, (Suppl. I.) 140 (1971). J. K. Vyden, M. Carvalko, E. Boszormenyi, T.-W. Lang, H. B e r n s t e i n and E. Corday, Amer. J. C a r d i o l . ,*25-, 53 (1970). R. A. G i l l i s and K. I. M e l v i l l e , i b i d . , 28, 38 (1971). R. A. O'Rourke, V. S. Bishop, P. A. K o t and J. P. Fernandez, J. Pharmacol. Exp. Ther., 177,426 (1971). R. A. G i l l i s and K. I. M e l v i l l e , Eur. J. Pharmacol., 13,15 (1970). N. J. F o r t u n i , S. Kaihara, L. C. Becker and B. P i t t , Cardiovasc. Res., 5, 331 (1971). P. Mathes and J. R i v a l , i b i d . , 5, 54 (1971). H. R. Weiss and M. M. Winbury, Fed. Proc., 30, ( 2 ) 631 (1971). M. M. Winbury, B. B. Howe and M. A. Hefner, J. Pharmacol. Exp. Ther. , 168, 70 (1969). M. G. Bogaert, A. G. Herman and A. F. De Schaepdryver, Eur. J. Pharmacol , 215 (1970). S. W. Blum, J. B. Quinn, B. B. HOwe, M. A. H e f n e r and M. M. Winbury, J. Pharmacol. Exp. Ther., 176,684 (1971). W. Kubler, P. G. Spieckermann and H. J. B r e t s c h n e i d e r , J. Mol. C e l l . C a r d i o l , 1,23 (1970). R. M. Berne, R. Rubio and B . R. Duling, Myocardial Ischemia, R. S. Ross and F. Hoffman, eds., pp. 28-43, Excerpta Medica, P r i n c e t o n , New Jersey, 1971. N. B i t t a r and T. J. Pauly, E x p e r i e n t i a , 27, 153 (1971). N. Kolassa, K. P f l e g e r and W. Rummel , Eur. J. Pharmacol. , 2, 265 (1970). G. Raberger and 0. Kraupp, i b i d . , 13,312 (1971). M. H. Maguire, D. M. Nobbs, R. E i n s t e i n and J. C. M i d d l e t o n , J. Med. Chem., 415 (1971). J. A. Angus, L. B. Cobbin, R. E i n s t e i n and M. H. Maguire, B r i t . J. Pharmacol., 41, 592 (1971). M. W. N o t t , B r i t . J. Pharmacol., 39, 287 (1970). S. V. Hopkins and R. G. Goldie, Bzchem. Pharmacol., 8, 3359 (1971). W . I s s e l h a r d , D. H. H i n z i n , E. Geppert, W . Maurer, D. Ammermann and I.Sunder-Plassmann, P f l u g e r s Arch. , 320, 195 (1970). R. A. DeWall, K. A. Vasko, E. L. S t a n l e y and P. Kezdi, Amer. H e a r t J., 82, 362 (1971). W. Rudolph and I . Marko, Arzneim.-Forsch., 20, 1190 (1970). J. M. P e t t a and V . J. Zacchio, J. Pharmacol. Exp. Ther. , 176,328 (1971). 1370 (1970). G. Molnar and L. K e l l e r , Arzneim.-Forsch., K. Seta, Jap. C i r c . J., 34, 499 (1970). J. C. S c o t t and A. W. Cowley, Amer. J. C a r d i o l , 24, 865 (1969). G. G. Rowe, D. A. S p r i n g and S. Afonso, Arch. n t . Pharmacodyn. , 187, 377 (1970). R. M. Abel, R. L. Reis and R. N. S t a r o s i c k , B r t. J. Pharmacol , 38, 620 (1970). 0. Mantero and F. C o n t i , C i r c u l a t o r y Drugs, A. B e r t e l li , ed. , pp. 118123, North-Holland, Amsterdam, The Netherlands 1969.

. 12,

.

14,

a,

.

Chap.

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.

7

Ant iang i na 1 Agents

Schwender

77

D. C. Harrison, Myocardial Ischemia, R. S. Ross and F. Hoffman, eds., p. 97, Excerpta Medica, P r i n c e t o n , New Jersey, 1971. W. M. McLamore, Ann. Rep. Med. Chem., 1969, C. K. Cain, ed. , p. 63, Academic Press, New York, 1970. C. F. Schwender, i b i d . , 1970, p. 80, 1971. J. R. B o i s s i e r , J . F. G u i d i c e l l i , P. V i a r o , C. Advenier, P. M o u i l l e and S. Larno, Eur. J . Pharmacol., 15,151 (1971). T. Baum, G. Rowles, A. T. Shropshire and M. I.Gluckman, J. Pharmacol. Exp. Ther., 176,339 (1971). T. Baum, G. Rowles and A. T. Shropshire, ibid., 176, 350 (1971). K. Takagi, I.Takayanogi , Y . Kaziwara, Y . Hiramatsu and A. Izumi , Jap. J. Pharmacol., 20, 504 (1970). K. Sakai, i b i d . , 20, 306 (1970). A. F. Crowther, RFHowe and L. H. Smith, J. Med. Chem., 14, 511 (1971). B. Ablad, M. Brogard, E. Carlsson and L. Ek, Eur. J. P h a z a c o l . , 13, 59 (1970). P. G. Lund-Larsen and E. S i v e r t s s e n , Acta Med. Scand., 186, 187 11969). W. G. Nayler, Postgrad. Med. J., 46, (Nov. Suppl.) 90 (1970). E. Noack and K. G r e e f f , E x p e r i e n t i a , 27, 810 (1971). t R. G. Pendleton, J. M. P e t t a and R. ArHahn, Arch. I n t . Pharmacodyn. , 187, 75 (1970). A . M . B a r r e t t and B. Nunn, i b i d . , 189, 168 (1971). B. P i t t and P. Craven, Cardiovasc. Res., 4, 176 (1970). L. C. Becker, N. J. F o r t u n i and B. P i t t , c i r c . Res. , 28, 263 (1971). P. Mathes and J. R i v a l , i b i d . , 5, 54 (1971). M. M. Winbury, H. R. Weiss and B. B. Howe, Eur. J. Pharmacol., 16, 271 (1971). R. E. Goldstein, D. R. Rosing, D. R. Redwood, G. D. B e i s e r and S. E. Epstein, C i r c u l a t i o n , 43, 629 (1971). H. J. Semler and H. H. O r v i s , C u r r e n t Ther. Res., C l i n . Exp., 13, 523 (1971). W. S. Aronow and H. M. Chesluk, C i r c u l a t i o n , 42, 61 (1970). W. S. Aronow, Myocardial Ischemia, R. S. Ross and F. Hoffman, eds., p. 73, Excerpta Medica, Princeton, New Jersey, 1971. M. Kal tenback, Arzneim.-Forsch. , 20 , 1304 (1970). J.-M. R. D e t r y and R. A. B r u i c e , T i r c u l a t i o n 43, 155 (1971). K. Ushiyama, E. Kinwia, H. K i k u c h i and G. Mabuchi, I s r . J. Med. Sci., -5, 736 (1969). B. Sharma and S. H. Taylor, B r i t . J. Pharmacol., 43, 470P (1971). A. J. Wasserman, J. D. P r o c t o r , F. J. A1 l e n and E. V. Kemp, J. C l i n . Pharmacol. J. New Drugs, 10,37 (1970). D. E. J e w i t t , P. A. Burgess and J. P. S h i l l i n g f o r d , Cardiovasc. Res., 4, 188 (1970). L. Adolfsson, N.-H Areskog and T. Rosmunson, Eur. J. Clin. Pharmacol., 3, 68 (1971). A. N. Goldberg, J. F. Moran, T. K. B u t t e r f i e l d , R. Nemickes and G. A. Bermudez , C i r c u l a t on, 40, 847 (1969). G. A. Dogenais, B. P i t t T n d R. S. ROSS, Amer. J. C a r d i o l . , 28, 10 (1971).

Chapter 8.

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

Leonard J . Czuba, P f i z e r , I n c . , Groton C o n n e c t i c u t

06340

A s u r v e y of r e s e a r c h on p l a t e l e t a g g r e g a t i o n i n h i b i t o r s 1 was p r e s e n t e d i n t h i s s e r i e s i n 1971. A more comprehensive review of a g e n t s a f f e c t i n g thrombosis2 a p p e a r e d d u r i n g t h e p r e c e d i n g y e a r . The p r e s e n t r e p o r t i s i n t e n d e d a s a c o n t i n u a t i o n of t h e s e e a r l i e r works and a t t e m p t s t o summarize t h e s i g n i f i c a n t r e c e n t r e s e a r c h on a l l t y p e s of d r u g s f o r t h e management of thromboembolic d i s e a s e . Rue t o s p a c e limi t a t i o n s , i m p o r t a n t r e l a t e d r e s e a r c h on d i a g n o s t i c t e c h n i q u e s and e x p e r i m e n t a l models of thrumbosis w i l l n o t be reviewed. A roaches t o _Eg---.--..--.

A n t i t h r o m b o t i c Therapy ___(

A s Schor2 p o i n t e d o u t , t h e r e are t h r e e t h e r a p e u t i c a p p r o a c h e s t o t h e t r e a t m e n t of thrombosis : i n h i b i t i o n of p l a t e l e t a g g r e g a t i o n , i n h i b i t i o n of b l o o d c o a g u l a t i o n , and enhancement of t h e f i b r i n o l y t i c p r o c e s s . The a g e n t s i n v o l v e d a f f e c t p l a t e l e t f u n c t i o n , o r a c t on t h e b l o o d c o a g u l a t i o n mechanisms e i t h e r by i n h i b i t i n g t h e f o r m a t i o n o r e n h a n c i n g t h e breakdown of f i b r i n . I n t h i s r e g a r d , i t i s i m p o r t a n t t o d i f f e r e n t i a t e between a c l o t and a thrombus.3-5 A c l o t is a r e l a t i v e l y d is o r g an ized , dark-red s t r u c t u r e t h a t r e s u l t s from t h e c o a g u l a t i o n of whole b l o o d and i s u s u a l l y a s s o c i a t e d w i t h venous c i r c u l a t i o n . The s t r u c t u r a l i n t e g r i t y of t h e c l o t i s m a i n t a i n e d by a f i b r i n network and t h e p r i n c i p a l e v e n t i n c l o t f o r m a t i o n i s t h e a c t i v a t i o n of f i b r i n o g e n t o f i b r i n . I t c o n t a i n s r e d c e l l s , w h i t e c e l l s and p l a t e l e t s i n r o u g h l y t h e same p r o p o r t i o n s as they o c c u r i n whole b l o o d . Thrombi a r e s i m p l e r , more h i g h l y o r g a n i z e d s t r u c t u r e s t h a t o c c u r w i t h v a r y i n g d e g r e e s of ~ o m p l e x i t y ,b~u t t h e u s u a l form on t h e a r t e r i a l s i d e of t h e c i r c u l a t i o n i s a w h i t e h e a d composed a l m o s t e n t i r e l y of a g g r e g a t e d p l a t e l e t s , t o which is a t t a c h e d a r e d t a i l of f i b r i n c o n t a i n i n g r e d c e l l s . The p r i n c i p a l e v e n t s i n thrombus f o r m a t i o n a r e i n i t i a l a d h e r e n c e and t h e p r o p a g a t i o n of p l a t e l e t a g g r e g a t i o n by ADP r e s u l t i n g from t h e p l a t e l e t release r e a c t i o n . 6

A v a r i e t y of c l i n i c a l s t a t e s are a s s o c i a t e d w i t h t h r o m b o s i s . The p r e s e n t d i s c u s s i o n i s l i m i t e d t o a few of t h e more prominent thromboembolic d i s e a s e s ; f u r t h e r i n f o r m a t i o n on t h i s s u b j e c t may b e found i n several r e v i e w s . 3 97-9 The most common c l i n i c a l s t a t e s which i n v o l v e c l o t - l i k e , venous thrombi a r e deep v e i n t h r o m b o s i s , p a r t i c u l a r l y as a p o s t s u r g i c a l c o m p l i c a t i o n , and pulmonary embolism. A n t i c o a g u l a n t s s u c h as h e p a r i n and t h e coumarins have been known f o r y e a r s t o b e e f f e c t i v e i n t h e p r e v e n t i o n of t h e s e t y p e s of thrombi and more r e c e n t e x p e r i e n c e h a s d e m o n s t r a t e d t h e e f f i c a c y of f i b r i n o l y t i c a g e n t s such as s t r e p t o k i n ase f o r t h e i r d i s s o l u t i o n . 3 , 9 P l a t e l e t a g g r e g a t i o n i n h i b i t o r s h a v e o n l y r e c e n t l y been e v a l u a t e d c l i n i c a l l y i n t h e p r e v e n t i o n of venous t h r o m b o s i s . These s t u d i e s a r e c r u c i a l t o t h e r e s o l u t i o n of t h e c o n t r o v e r s y as t o whether p l a t e l e t s p l a y a v i t a l r o l e i n t h e i n i t i a t i o n of venous thrombi.10 There i s p e r s i s t e n t h i s t o l o g i c a l e v i d e n c e t h a t i n d i c a t e s venous thrombi b e g i n as p l a t e l e t a g g r e g a t e s .I1 Myocardial i n f a r c t i o n and s t r o k e are

Chap. 8

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

Czuba

79

t h e major c l i n i c a l m a n i f e s t a t i o n s of a r t e r i a l t h r o m b o s i s . A n t i c o a g u l a n t s have b e e n found t o o f f e r l i t t l e , i f a n y , b e n e f i t t o t h e s u r v i v o r s of m y o c a r d i a l i n f a r c t i o n , and p r o p h y l a c t i c t r e a t m e n t w i t h t h e s e a g e n t s i s overshadowed by t h e d a n g e r of hemorrhage. 3 L a r g e s c a l e c l i n i c a l s t u d i e s of t h e f i b r i n o l y t i c a g e n t s t r e p t o k i n a s e i n m y o c a r d i a l i n f a r c t i o n h a v e b e e n u n d e r t a k e n i n r e c e n t y e a r s . 1 2 Although a s t a t i s t i c a l l y s i g n i f i c a n t improvement is i n d i c a t e d by t h e s e t r i a l s , t h e commitment t o f u r t h e r s t u d i e s t o v e r i f y e f f i c a c y h a s b e e n c r i t i c i z e d on t h e b a s i s of t h e l i m i t e d p o s s i b l e b e n e f i t s of f i b r i n o l y s i s t o t h e p a t i e n t due t o t h e c r i t i c a l t i m e element involved i n myocardial i n f a r c t i o n , S i n c e t i s s u e n e c r o s i s d i s t a l t o t h e c o r o n a r y o c c l u s i o n o c c u r s w i t h i n a few h o u r s , i t h a s b e e n a r g u e d t h a t l i t t l e i s t o b e g a i n e d by r e s t o r a t i o n of b l o o d f l o w a f t e r t h i s i r r e v e r s i b l e damage i s done, and t h a t c l i n i c a l e f f o r t s would b e b e t t e r expended on p r o p h y l a c t i c a g e n t s . 1 3 The r e s u l t s of s t r e p t o k i n a s e trials currently i n progress should resolve t h e controversy and e s t a b l i s h a n i m p o r t a n t p r e c e d e n t i n r e g a r d t o t h e f e a s i b i l i t y o f f i b r i n o l y t i c therapy i n arterial thrombosis. I n g e n e r a l , t h e r e appears t o b e agreement t h a t p l a t e l e t a g g r e g a t i o n i n h i b i t o r s are t h e most r a t i o n a l t y p e of a g e n t f o r t h e p r o p h y l a x i s of a r t e r i a l t h r o m b o s i s . A s p i r i n , t h e p y r i m i d o - p y r i m i d i n e s r e l a t e d t o d i p y r i d a m o l e ( P e r s a n t i n e ) and d e x t r a n are t h e most n o t a b l e a g e n t s f o r which a n t i - p l a t e l e t a c t i v i t y h a s b e e n d e m o n s t r a t e d i n v i v o i n man. However, no m a j o r c l i n i c a l t r i a l s of p l a t e l e t a g g r e g a t i o n i n h i b i t o r s i n p a t h o l o g i c s t a t e s s u c h as m y o c a r d i a l i n f a r c t i o n h a v e b e e n r e p o r t e d . Moreover, s e l e c t i v e p l a t e l e t a g g r e g a t i o n i n h i b i t o r s t h a t a r e l a c k i n g o t h e r m a j o r d r u g e f f e c t s , s u c h as a n t i i n f l a m matory o r h y p o t e n s i v e a c t i v i t y , h a v e n o t undergone c l i n i c a l e v a l u a t i o n . I t i s t h i s l a t t e r c l a s s of a g e n t s t h a t a p p e a r s t o h o l d t h e most p r o m i s e f o r t h e p r e v e n t i o n of a r t e r i a l t h r o m b o s i s . P l a t e l et A g g r e g a t i o n . I n h i b it o r s

P r o s t a g l a n d i n s and C y c l i c AMP - The e f f e c t s of n a t u r a l p r o s t a g l a n d i n s on p l a t e l e t f u n c t i o n h a v e b e e n s t u d i e d e x t e n s i v e l y . A s a g r o u p , t h e y are gene r a l l y i n h i b i t o r s of ADP-induced p l a t e l e t a g g r e g a t i o n . P G E l i s by f a r t h e most p o t e n t b y a t l e a s t two o r d e r s of m a g n i t u d e . PGE2 d i f f e r s i n t h a t i t e x h i b i t s a b i p h a s i c a c t i v i t y ; a t c o n c e n t r a t i o n s less t h a n t h o s e r e q u i r e d f o r i n h i b i t i o n , i t p o t e n t i a t e s p l a t e l e t a g g r e g a t i o n . 1 4 PGEl a l s o i n h i b i t s c l o t r e t r a c t i o n . 1 5 T h i s p o t e n t e f f e c t may b e a t t r i b u t e d t o i n h i b i t i o n of t h e p l a t e l e t release r e a c t i o n . 1 5 The p o s s i b l e c l i n i c a l u t i l i t y of P G E l h a s been s u g g e s t e d , a l t h o u g h s h o r t d u r a t i o n of a c t i o n The l e t h a l e f f e c t of i n t r a v e n appears t o b e an important limitation.16 o u s l y a d m i n i s t e r e d ADP t o r a t s , which a p p e a r s t o c a u s e d e a t h by o c c l u s i o n of t h e c e r e b r a l a r t e r i e s w i t h p l a t e l e t t h r o m b i , w a s p r e v e n t e d by PGE1. T r a n s i e n t t h r o m b o c y t o p e n i a i n d u c e d by ADP i n j e c t i o n w a s a l s o i n h i b i t e d . I 7 The growth of a p a r t i c u l a r t y p e of p l a t e l e t thrombus formed b y e l e c t r i c s t i m u l a t i o n i n c o r t i c a l v e i n s w a s i n h i b i t e d by l o c a l l y a d m i n i s t e r e d PGE1.18 PGEl i s s t a b l e and r e t a i n s p l a t e l e t a c t i v i t y on i n c u b a t i o n w i t h whole human b l o o d . 1 4 R e c e n t l y , a p r o c e d u r e h a s b e e n d e v e l o p e d f o r s t a b i l i z i n g p l a t e l e t f r a c t i o n s w i t h PGEl i n b l o o d b a n k s . P l a t e l e t s t r e a t e d i n t h i s manner e x h i b i t e d n o r m a l h e m o s t a t i c f u n c t i o n f o l l o w i n g t r a n s f u s i o n i n man.19

80 -

Sect.

II

-

Pharmacodynamic A g e n t s

T o p l i s s , Ed.

Two r e p o r t s d e s c r i b e a n a l o g s more p o t e n t t h a n PGE1. The w-homolog of P G E l h a s been shown t o b e about 4 times more p o t e n t t h a n P G E l i n t h e i n h i b i t i o n of p l a t e l e t a g g r e g a t i o n i n v i t r o . 1 7 S i m i l a r l y , an a n a l o g of PGEl w i t h a trans double bond a t t h e 2 - p o s i t i o n was found t o b e 2 1 1 2 times more p o t e n t t h a n PGE1. I t i s i n t e r e s t i n g t h a t a number of isomers of t h i s compound w i t h t h e double bond a t d i f f e r e n t p o s i t i o n s i n t h e s i d e c h a i n s , i n c l u d i n g n a t u r a l PGE2, were a l l much less p o t e n t t h a n PGE1.20 Perhaps t h e most s i g n i f i c a n t event i n p r o s t a g l a n d i n r e s e a r c h d u r i n g t h i s p a s t y e a r was t h e d i s c o v e r y t h a t a s p i r i n i n h i b i t s t h e s y n t h e s i s of p r o s t a g l a n d i n s i n a v a r i e t y of t y p e s of c e l l s , and t h a t t h i s e f f e c t may account f o r many of t h e f a m i l i a r a c t i o n s of a s p i r i n . 2 1 The p r o s t a g l a n d i n s PGE2 and PGF2, are p r o d u c t s of t h e p l a t e l e t release r e a c t i o n induced by thrombin. Although a s p i r i n i n h i b i t s t h e release r e a c t i o n , r e l a t i v e l y high c o n c e n t r a t i o n s of thrombin caused unimpaired release of p l a t e l e t c o n s t i t u e n t s i n t h e p r e s e n c e of a s p i r i n w i t h o u t t h e concomiT h i s i n h i b i t i o n of p r o s t a g l a n d i n product a n t release of PGE2 and PGF2,. t i o n was a l s o demonstrated w i t h p l a t e l e t s from donors dosed w i t h a s p i r i n 1 hour b e f o r e blood was taken.22 Presumably, i n h i b i t i o n of p r o s t a g l a n d i n s y n t h e s i s does n o t c o n t r i b u t e s u b s t a n t i a l l y t o t h e i n h i b i t i o n of p l a t e l e t a g g r e g a t i o n by a s p i r i n . Evidence c o n t i n u e s t o s u p p o r t t h e h y p o t h e s i s t h a t p l a t e l e t aggregat i o n and t h e release r e a c t i o n are i n h i b i t e d by r a i s e d l e v e l s of i n t r a c e l l u l a r c y c l i c AMP.1 The i n h i b i t o r y e f f e c t s on p l a t e l e t f u n c t i o n of such d i v e r s e a g e n t s as P G E l , d i p y r i d a m o l e , t h e m e t h y l x a n t h i n e s and p a p a v e r i n e a l l appear t o b e mediated by t h i s c y c l i c n u c l e o t i d e . 2 3 - 2 8 A s p i r i n - S e v e r a l n o n - s t e r o i d a l anti-inflammatory a g e n t s are known t o i n h i b i t p l a t e l e t a g g r e g a t i o n by p r e v e n t i n g t h e release r e a c t i o n . T h e r e f o r e , a g g r e g a t i o n induced by a g e n t s such as c o l l a g e n , e p i n e p h r i n e and low conc e n t r a t i o n s of thrombin i s i n h i b i t e d , b u t ADP-induced a g g r e g a t i o n i s n o t a f f e c t e d . Among known i n h i b i t o r s of t h i s k i n d , which i n c l u d e s a l i c y l a t e , p y r a z o l e and i n d o l e a c e t i c a c i d t y p e anti-inflammatory a g e n t s , t h e most n o t a b l e are indomethacin and a s p i r i n . 1 9 3 Although r e p o r t s on t h e p l a t e l e t e f f e c t s of indomethacin29 and a n a l o g s such a s "BLR-43"30 c o n t i n u e t o app e a r , t h e v a s t m a j o r i t y of t h e l i t e r a t u r e i n t h i s area i s devoted t o s t u d i e s of a s p i r i n . Although a s p i r i n and s a l i c y c l i c a c i d e x h i b i t h a l f - l i v e s i n man of only 1 3 t o 1 9 min. and 3.5 t o 4.5 h r s . , r e s p e c t i v e l y , 3 1 t h e e f f e c t of a s p i r i n on p l a t e l e t s i n v i v o h a s a d u r a t i o n which approximately p a r a l l e l s p l a t e l e t s u r v i v a l t i m e of 2-7 days. The c u r r e n t l y a c c e p t e d h y p o t h e s i s t o e x p l a i n t h i s a c t i o n h o l d s t h a t a s p i r i n i r r e v e r s i b l y alters p l a t e l e t funct i o n by a c e t y l a t i o n of a p r o t e i n component of p l a t e l e t membrane. A subs t a n t i a l amount of e x p e r i m e n t a l evidence c o n s i s t e n t w i t h t h i s h y p o t h e s i s h a s accumulated. 1 9 3 2 Although a s p i r i n does n o t i n h i b i t ADP-induced a g g r e g a t i o n i t s e f f e c t on t h e release r e a c t i o n d e c r e a s e s p l a t e l e t r e s p o n s e and a b o l i s h e s t h e "second wave'' of a g g r e g a t i o n i n t h e p r e s e n c e of ADP. I t a p p e a r s t h a t a n o t h e r component of a s p i r i n a c t i o n is an e f f e c t on a plasma c o f a c t o r of ADP-induced a g g r e g a t i o n . 3 3 , 3 4 Evidence h a s been

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p r e s e n t e d t h a t Hageman f a c t o r i s n e c e s s a r y f o r p l a t e l e t - a g g r e g a t e s t a b i l i t y . I n a d d i t i o n , t h e a g g r e g a t i n g p l a t e l e t s u r f a c e h a s been proposed as a s i t e of Hageman f a c t o r a c t i v a t i o n . A s p i r i n may i n h i b i t t h e s e i n t e r a c t i o n s by b l o c k i n g t h e a p p r o p r i a t e s i t e on t h e p l a t e l e t s u r f a c e . 35 ,36 The a n t i t h r o m b o t i c e f f e c t of a s p i r i n h a s been demonstrated i n a numb e r of e x p e r i m e n t a l animal models. The d e a t h r a t e due t o pulmonary thromboembolism induced i n mice by i n f u s i o n of a c o l l a g e n s u s p e n s i o n w a s d e c r e a s e d r e p r o d u c i b l y by a s p i r i n . 3 7 A s p i r i n provided s i g n i f i c a n t prot e c t i o n a g a i n s t t h e v a s o c o n s t r i c t i v e e f f e c t s and m o r t a l i t y of pulmonary embolism caused by i n j e c t i o n of f r e s h , a u t o l o g o u s c l o t s i n t o r a b b i t s 3 8 o r dogs .39 Presumably, s u p p r e s s i o n of t h e v e n o c o n s t r i c t i v e component of pulmonary embolism by a s p i r i n w a s due t o t h e i n h i b i t i o n of s e r o t o n i n release from p l a t e l e t s .38 O c c l u s i v e thrombus f o r m a t i o n induced i n i s o l a t e d a r t e r i e s of dogs by mechanical o r chemical i n j u r y w a s s u b s t a n t i a l l y reduced by a s p i r i n i n g e s t i o n . I n t h i s model dipyridamole had no e f f e c t . 4 0 Myocardial n e c r o s i s induced i n dogs by i n f u s i o n of e p i n e p h r i n e i s b e l i e v e d t o b e caused by t h e f o r m a t i o n of p l a t e l e t thrombi. Both a s p i r i n and dipyridamole have a s i g n i f i c a n t p r o t e c t i v e e f f e c t a g a i n s t t h i s phenomenon.41 A new m i c r o i o n t o p h o r e t i c t e c h n i q u e h a s been developed f o r q u a n t i t a t i n g p l a t e l e t a g g r e g a t i o n i n v i v o . T h i s p r o c e d u r e , w a s used t o demonstrate t h a t a s p i r i n w a s s e v e r a l times more e f f e c t i v e i n t h e i n h i b i t i o n of hamster p l a t e l e t a g g r e g a t i o n i n v i v o than i n v i t r o . 4 2 A number of c l i n i c a l t r i a l s w i t h a s p i r i n as an a n t i t h r o m b o t i c a g e n t were r e p o r t e d d u r i n g t h e p a s t y e a r . Some of t h e s t u d i e s have i n v o l v e d venous thrombotic s t a t e s i n which t h e importance of p l a t e l e t a g g r e g a t i o n i s uncertain a t present. The outcome of t h e s t u d i e s r e p o r t e d t h u s f a r

does n o t f u r n i s h an adequate answer t o t h i s i m p o r t a n t q u e s t i o n . I n e f f o r t s t o determine e f f e c t i v e d o s e l e v e l s f o r t h e p r e v e n t i o n of p o s t o p e r a t i v e deep v e i n thrombosis , no a n t i t h r o m b o t i c e f f e c t w a s observed f o l l o w i n g t h e i n g e s t i o n of a s h i g h as 2.4 g of a s p i r i n d a i l y . 4 3 A comparative s t u d y of a s p i r i n , dipyridamole , d e x t r a n and w a r f a r i n w a s c a r r i e d o u t i n 169 p a t i e n t s undergoing h i p r e c o n s t r u c t i v e s u r g e r y . The high rate of o c c u r r e n c e of deep v e i n thrombosis which accompanies t h i s t y p e of s u r g e r y w a s s i g n i f i c a n t l y reduced by a s p i r i n , d e x t r a n and w a r f a r i n , b u t n o t by dipyridamole.44 A c o n t r o l l e d s t u d y of m o r b i d i t y and m o r t a l i t y among 430 aged p e o p l e i n d i c a t e d t h a t a s p i r i n had no e f f e c t on thromboemb o l i c d i s e a s e i n t h i s p a r t i c u l a r group.45 I n hemodialysed p a t i e n t s asp i r i n w a s found t o p r e v e n t f i s t u l a c l o t t i n g which i s c h a r a c t e r i z e d by i n c r e a s e d p l a t e l e t a d h e s i v e n e s s and thrombin p r o d u c t i o n . 4 6 P l a t e l e t consumption a s s o c i a t e d w i t h thromboembolic c o m p l i c a t i o n s of p r o s t h e t i c h e a r t v a l v e s w a s found t o b e uniformly p r e v e n t e d by d i p y r i d a m o l e , b u t a s p i r i n had l i t t l e o r no e f f e c t . 4 7 Some f u r t h e r examples of c l i n i c a l s t a t e s i n which a s p i r i n w a s found t o have s i g n i f i c a n t a n t i t h r o m b o t i c e f f e c t are spontaneous p l a t e l e t a g g r e g a t i o n as a c a u s e of i d i o p a t h i c thrombosis and r e c u r r e n t p a i n f u l t o e s and f i n g e r s , 4 8 t h e r e t i n a l - a r t e r y embolism of amaurosis f ~ g a x and , ~ ~thrombotic thrombocytopenic p ~ r p u r a . ~ ~ The p r e s e n t c l i n i c a l e v i d e n c e f o r t h e a n t i t h r o m b o t i c e f f e c t of asp i r i n i s e q u i v o c a l . Much f u r t h e r s t u d y w i l l b e r e q u i r e d t o d e m o n s t r a t e

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e f f i c a c y , and once e s t a b l i s h e d , any a n t i t h r o m b o t i c b e n e f i t w i l l have t o b e s u f f i c i e n t t o outweigh t h e r i s k of g a s t r i c b l e e d i n g 5 1 7 5 2 on c h r o n i c a d m i n i s t r a t i o n t o p a t i e n t s w i t h thromboembolic disease.

-P l r L m i d o p y r i m i d i n e s -

D i p y r i d a m o l e and two a n a l o g s , RA233 and RA433, h a v e been s t u d i e d e x t e n s i v e l y as p l a t e l e t a g g r e g a t i o n i n h i b i t o r s i n v i t r o and i n v a r i o u s a n i m a l models of t h r o m b o s i s . 5 3 ~ 5 ~ tThe mechanism of a c t i o n of t h e s e compounds on p l a t e l e t s a p p e a r s t o i n v o l v e i n h i b i t o r y e f f e c t s o n g l u c o s e metabolism53155 and c y c l i c AMP p h o s p h o d i e s t e r a s e . 2 7 C l i n i c a l t r i a l s w i t h d i p y r i d a m o l e f o r t h e r e d u c t i o n of thromboembol i c c o m p l i c a t i o n s a f t e r p r o s t h e t i c c a r d i a c v a l v e replacement c o n t i n u e t o show e n c o u r a g i n g r e s u l t s . 4 7 I n a c o n t r o l l e d s t u d y o f 1 6 3 p a t i e n t s , d i pyridaniole ( 4 0 0 mg d a i l y ) s i g n i f i c a n t l y r e d u c e d t h e f r e q u e n c y of a r t e r i a l emboli.56 O n t h e o t h e r h a n d , d i p y r i d a m o l e was found t o have no s i g n i f i c a n t e f f e c t on p o s t o p e r a t i v e d e e p v e i n t h r o m b o s i s .44 New Agents - R e p o r t s of new s t r u c t u r a l t y p e s of p l a t e l e t a g g r e g a t i o n i n h i b i t o r s have i n c r e a s e d d u r i n g t h e p a s t y e a r . The 5 ,SO-dihydro-3-phenylt h i a z o l o [ 3 , 2 - b ] [ 2 , 4 ] b e n z o d i a z e p i n e 1. and f i v e p h e n y l - s u b s t i t u t e d a n a l o g s i n h i b i t e d ADP- i n d u c e d human p l a t e l e t a g g r e g a t i o n i n v i t r o a t c o n c e n t r a to rabbits resulted i n I n t r a v e n o u s a d m i n i s t r a t i o n of t i o n s n e a r lo-5%. i n h i b i t i o n of ADP-induced a g g r e g a t i o n i n s u b s e q u e n t l y drawn b l o o d s a m p l e s , and an o r a l d o s e of i n mice c a u s e d a s i . g n i f i c a n t i n c r e a s e i n b l e e d i n g t i m e . 5 7 Compound 2- w a s t h e most p r o m i s i n g member of a l a r g e g r o u p of a n a l o g o u s a c r i.d i n e de r i v a t i v e s t h a t i n h i b i t e d ADP - i n d u c e d p 1.a t e 1e t a g g r e g a t i o n . T h i s a g e n t was a c t i v e on human p l a t e l e t s i n v i t r o a t conc e n t r a t i o n s of 10-5M. JiIi v z a c t i v i t y was d e m o n s t r a t e d i n r a b b i t s . 5 8 E x t e n s i v e s y n t h e t i c m a n i p u l a t i o n of t h e r i n g s u b s t i t u e n t s and t h e a c r i d i n e n u c l e u s gave a number of a c t i v e a n a l o g s , b u t none w a s found t o b e s u p e r i o r t o 1 . 5 9 The anti.throriibotic a c t i v i t y of a new b e n z o [ c ] [ 1 , 6 ] n a p h t h y r i d i n e 2 w a s d e s c r i - b e d , T h i s compound i n h i b i t e d ADP-induced agThe e f f e c t was n o t g r e g a t i o n i n v i t r q a t c o n c e n t r a t i o n s of 10-51. Rat carotid r e v e r s e d , b u t was s i g n i f i c a n t l y p o t e n t i a t e d by e p i n e p h r i n e . a r t e r y thrombus fozmati.on i n d u c e d by i n s e r t i o n of s y n t h e t i c t u b i n g w a s 2 1 . ~ 0j nlii1,ite.d 60

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A c o m p a r a t i v e s t u d y o€ t h e i n h i b i t o r y a c t i v i t y of 5 and 5 on b i p h a s i c ADP-induced a g g r e g a t i o r t of human p l a t e l e t s i n v i t r o h a s b e e n d e s cribed. Compound 4 i n h i b i t e d b o t h p h a s e s of a g g r e g a t i o n b u t was most e f f e c t i v e on t h e s e c o n d p h a s e . I n c o n t r a s t , i n h i b i t e d o n l y t h e second

Chap.

8

A n t i thrornbot i c Agents

Czuba

83

p h a s e which i s a s s o c i a t e d w j t h t h e reLease r e a c t i o n . Compound 5 was a b o u t 400 t i m e s as e f f e c t i v e as oxyphcnburazone . 6 1 M o n o d a n s y l c a d a v e r i n e (6), which i s an e f f e c t i v e i n h i b i t o r of t h e f i b r i n c r o s s - l i n k i n g enzyme f i b r i n o l i g a s e , 5 7 ~ s f o u n d t o i n h i b i t ; t h e s e r o n d p h a s e of ADP-induced p l a t e l e t aggregation. A l k y l a t i o r , of t h e p r i m a r y aiuirie f u n c t i o n r e s u l t e d i n analogs t h a t r e t a i n e d v l a t e l e t a c t i v i t y but f i b r i n n l i g a s e i n h i b i t o r y a c t i v i t y was abo 1iThed .5 2

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The a n i l i n e d e r i v a t i v e w a s found t o b e a p o t e n t i n h i b i t o r of ADP-, c o l l a g e n - and t h r o m b i n - i n d u c e d a g g r e g a t i o n of human p l a t e l e t s i n v i t r o . O r a l a d m i n i s t r a t i o n of 2 t o g u i n e a p i g s p r o d u c e d a n i n h i b i t o r y e f f e c t on p l a t e l e t a g g r e g a b i l i t y . I n v i t r o c o n c e n t r a t i o n s of L c o m p a r a b l e t o & v i v o p l a t e l e t i n h i b i t o r y c o n c e n t r a t j o n c h a d no e f f e c t on p l a s m a c o a g u l a t i o n f a c t o r s , b u t h i g h e r c o n c e n t r a t i o n s were f o u n d t o i n h i b i t p r o t h r o m b i n and p a r t i a l t h r o m b o p l a s t i n t i m e s . 6 3 ~ 6 4 A c l o s e a n a l o g o f p r e v i o u s l y d i s c l o s e d a n t i c o a g u l a n t s , 2 compound S , has r e c e n t l y b e e n r e p o r t e d a s a n i n h i b i t o r o f ADP- and c o l l a g e n - i n d u c r d p l a t e l e t a g g r e g a t i o n and c l o t retraction. P l a t e l e t i n h i h i t o i y c o 1 i i e n t r a t i o n s of 8 d i d n o t a f f e c t b l o o d c o a g u l a t i o n . 65

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I t h a s b e e n shown r e c e n t l y t h a t c o n c e n t r a t i o n s of c o l l a g e n , i n s u f f i c i e n t t o i n d u c e a g g r e g a t i o n , p o t e n t i a t e ADP-induced a g g r e g a t i o n of r a b b i t p l a t e l e t s . The noveL t e t r a h y d r o t h i e n o p y r i d i n e d e r i v a t i v e 2, which w a s d e s c r i b e d p r e v i o u s l y ac; an i n h i b i t o r of ADP-induced a g g r e g a t i o n , l i n T h i s o b s e r v a t i o n was i n t e r p r e t e d as e v i d e n c e hibited this potentiation. t h a t 9 a c t s by i n h i b j t i o n of tlie r e l e a s e r e a c t i o n . 6 6 B r i e f m e n t i o n h a s b e e n made of t h e t a c t t h a t some d e r i v d t i v e s of t h e n o v e l t h i a z o l o (3,2-a)pyrrolo(2,3-d) p y r i m i d i n e r i n g s y s t e m are h i g h l y a c t i v e i n h i b i t o r s of ADP-induced p l a t e l e t a g g r e g a t j o n , 67

(10)

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Pharmacodynamic A g e n t s

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NH2

9 -

10 Anticoagulants

The physiology and b i o c h e m i s t r y of blood c o a g u l a t i o n have been t h e Anticoagulants s u b j e c t s of s e v e r a l reviews d u r i n g t h e p a s t year.68-70 i n t e r r u p t i n some manner t h e complex mechanisms c o n t r o l l i n g t h e enzymatic conversion of a s o l u b l e blood p r o t e i n f i b r i n o g e n i n t o i n s o l u b l e f i b r i n . Direct a n t i c o a g u l a n t s , such as h e p a r i n , i n h i b i t t h e conversion of prothrombin t o thrombin and t h e thrombin-fibrinogen r e a c t i o n . Because of e f f i c a c y only by p a r e n t e r a l a d m i n i s t r a t i o n , h e p a r i n i s used i n t r a v e n o u s l y u n t i l o r a l a n t i c o a g u l a n t s b e g i n .to t a k e e f f e c t . 9 The o r a l l y e f f e c t i v e coumarin and indandione t y p e a n t i c o a g u l a n t s a c t i n d i r e c t l y by i n h i b i t i n g s y n t h e s i s i n t h e l i v e r of blood c l o t t i n g f a c t o r s of t h e prothrombin comp l e x . Vitamin K i s e s s e n t i a l f o r t h e s y n t h e s i s of c l o t t i n g f a c t o r s i n t h e l i v e r , and a d e f i c i e n c y produces an a n t i c o a g u l a n t e f f e c t s i m i l a r t o t h a t of t h e coumarin d r u g s . The s i t e of a c t i o n of v i t a m i n K and o r a l a n t i c o a g u l a n t s i n v o l v e s one of t h e f i n a l s t e p s i n t h e p r o d u c t i o n of c l o t t i n g f a c t o r subsequent t o p o l y p e p t i d e c h a i n s y n t h e s i s on t h e ribosomal l e v e l . I n q u i r y i n t o t h e n a t u r e of t h e s e s t e p s and t h e p r e c u r s o r s of t h e rothrombin complex i s c u r r e n t l y an a c t i v e area of i n v e s t i g a t i o n . 71-7t The comprehensive review by Douglas9 p r o v i d e s a r e c e n t d i s c u s s i o n of t h e c l i n i c a l l y u s e f u l a n t i c o a g u l a n t s . Recent s t u d i e s have shown h e p a r i n t o b e c l e a r l y e f f e c t i v e i n c l i n i c a l s t a t e s i n which d i s s e m i n a t e d i n t r a v a s c u l a r c o a g u l a t i o n was i n d i c a t e d to b e a p a t h o l o g i c f a c t o r .75 The most commonly employed o r a l a n t i c o a g u l a n t s are of t h e coumarin t y p e such as w a r f a r i n and nicoumalone. The o r a l a n t i c o a g u l a n t s a p p e a r t o b e of v a l u e i n t h e p r e v e n t i o n of thromboembolic c o m p l i c a t i o n s a f t e r myocardial i n f a r c t i o n . g p 7 6 However, a n t i c o a g u l a t i o n t h e r a p y h a s been found t o have no e f f e c t on d e a t h - r a t e i n t h e s e p a t i e n t s . 7 7 W a r f a r i n h a s been shown t o b e e f f e c t i v e i n t h e p r e v e n t i o n of p o s t o p e r a t i v e venous thrombosis .44 The e f f e c t i v e n e s s of o r a l a n t i c o a g u l a n t s i n t h e p r e v e n t i o n of thromboembolism from p r o s t h e t i c h e a r t v a l v e s i s s t i l l open t o q u e s t i o n . Recent e x p e r i e n c e w i t h acenocoumarol and sodium w a r f a r i n i n h e a r t v a l v e p r o s t h e s e s i n d i c a t e d a low i n c i d e n c e of thromboembolic c o m p l i c a t i o n s .78 P a t i e n t s on o r a l a n t i c o a g u l a n t s are a t c o n s i d e r a b l e r i s k b e c a u s e of p o s s i b l e hemorrhage and t h e dangerous i n t e r a c t i o n s between t h e s e a g e n t s and common drugs s u c h as a s p i r i n and t h e b a r b i t u r a t e s . The complex subj e c t of drug i n t e r a c t i o n s w i t h coumarin a n t i c o a g u l a n t s h a s been e x t e n s i v e l y i n v e s t i g a t e d . 79,80

Chap. 8

Antithrombotic Agents

Czuba

85 -

Reports bf new a n t i c o a g u l a n t s have been r e l a t i v e l y rare. A new s t r u c t u r a l t y p e found t o have a n t i c o a g u l a n t a c t i v i t y i s t h e sodium s a l t of 2,3,5,6-tetrachloro-4-pyridinol. A number of analogs were found t o b e a c t i v e , b u t less p o t e n t . 81 Some novel 4-hydroxycoumarin d e r i v a t i v e s r e l a t e d t o dicoumarol have been r e p o r t e d t o have a n t i c o a g u l a n t a c t i v i t y . The most p o t e n t was about h a l f as a c t i v e a s dicoumarol.82 Aromatic d i a m i d i n e s , which a r e known p o t e n t i n h i b i t o r s of t r y p s i n , i n h i b i t e d human thrombin c l o t t i n g a c t i v i t y and markedly i n c r e a s e d prothrombin and p a r t i a l thromboplastin times of human plasma. These compounds are a l s o much more p o t e n t a n t i - f i b r i n o l y t i c a g e n t s t h a n E-aminocaproic a c i d . 83 F i b r i n o l y t i c Agents A g e n e r a l d i s c u s s i o n of f i b r i n o l y s i s i s a v a i l a b l e i n r e c e n t reviews.9,68,84 The o l d e s t and most thoroughly s t u d i e d f i b r i n o l y t i c agent i s s t r e p t o k i n a s e , a p r o t e i n i s o l a t e d from hemolytic s t r e p t o c o c c i . S i m i l a r t o human a c t i v a t o r , i t d i r e c t l y a c t i v a t e s plasminogen t o plasmin. Although s t r e p t o k i n a s e is a n t i g e n i c and f r e q u e n t l y pyrogenic, t h e s e e f f e c t s have n o t been s u f f i c i e n t l y d e l e t e r i o u s t o p r e c l u d e c l i n i c a l u t i l i t y . 3 9 9 C l i n i c a l e x p e r i e n c e w i t h t h i s agent has been gained now i n most t y p e s of thromboembolic d i s e a s e s , 8 5 w i t h g e n e r a l l y f a v o r a b l e r e s u l t s i n t h e t r e a t m e n t of deep v e i n thrombosis ( w i t h i n 96 hours of o n s e t of A major e f f o r t h a s been expended symptoms) and pulmonary embolism.9,85,86 i n r e c e n t y e a r s t o i n v e s t i g a t e t h e u s e f u l n e s s of s t r e p t o k i n a s e i n myoc a r d i a l i n f a r c t i o n , b u t r e s u l t s i n t h i s a r e a a r e s t i l l open t o ques t i o n . 13 3 85S87-90 Urokinase i s a plasminogen a c t i v a t o r p r e p a r e d from human u r i n e . I t i s produced by kidney c e l l s and h a s been shown t o d i f f e r from v a s c u l a r plasminogen a c t i v a t o r i n terms of immunologic r e a c t i o n , enzymatic a c t i v i t y , and molecular ~ e i g h t . 9 ~Although u r o k i n a s e does n o t e x h i b i t t h e a n t i g e n i c s i d e e f f e c t s a s s o c i a t e d w i t h s t r e p t o k i n a s e , development of t h i s agent h a s been h i n d e r e d by t h e expensive p r o c e s s r e q u i r e d f o r e x t r a c t i o n from u r i n e . However, i t a p p e a r s t o have s u p e r i o r t h e r a p e u t i c potent i a l , and t h e c l a i m h a s been made t h a t c o s t could b e reduced s h a r p l y i f a l a r g e - s c a l e demand were t o develop.90 A c l i n i c a l t r i a l t o s t u d y t h e e f f e c t s of u r o k i n a s e on pulmonary embolism i s c u r r e n t l y i n p r o g r e s s under t h e a u s p i c e s of t h e N a t i o n a l Heart and Lung I n s t i t u t e . 9 2 A r e c e n t s t u d y has demonstrated t h e f e a s i b i l i t y of e v a l u a t i n g u r o k i n a s e i n myocardial infarction.93 S e v e r a l o t h e r h i g h molecular w e i g h t n a t u r a l p r o d u c t s have been i n v e s t i g a t e d a s f i b r i n o l y t i c a g e n t s .2 A t e r o i d i s a n a t u r a l mucopolysacc h a r i d e w i t h f i b r i n o l y t i c a c t i v i t y . The a c t i v a t i o n of f i b r i n o l y t i c processes w i t h t h i s agent i n man h a s been demonstrated i n c l i n i c a l s t u d i e s . I n a d d i t i o n , experiments i n r a t s and r a b b i t s have s u g g e s t e d t h a t i t may a c t by t h e i n h i b i t i o n of endogenous a n t i - a c t i v a t o r s . Recent evidence i n d i c a t e s one important component of t h e f i b r i n o l y t i c a c t i o n may b e due t o n e u t r a l i z a t i o n of antiplasmin.94 A r e l a t i v e l y n o n - s p e c i f i c p r o t e o l y t i c enzyme from A s p e r g i l l u s oryzae, b r i n a s e , c a u s e s l y s i s of e x p e r i m e n t a l thrombi f a s t e r t h a n o t h e r c l i n i c a l t h r o m b o l y t i c a g e n t s . Reduction of

86

Sect.

II

-

Pharrnacodynamic Agents

Topliss,

Ed.

serum a n t i p l a s m i n a c t i v i t y by b r i n a s e may a c c o u n t f o r much of t h e i n v i v o f i b r i n o l y t i c e f f e c t . F i b r i n o l y t i c e f f e c t i v e n e s s and a l a c k of untoward s i d e e f f e c t s h a s b e e n d e m o n s t r a t e d w i t h b r i n a s e i n a number of c l i n i c a l s t a t e s .95 A r v i n and r e p t i l a s e are enzymes w i t h similar a c t i o n i s o l a t e d from t h e venom of Malayan p i t v i p e r , A g k i s t r o d o n rhodostoma, and a S o u t h American s n a k e , B o t h r o p s a t r o x , r e s p e c t i v e l y . T h e s e a g e n t s c o n v e r t f i b r i n o g e n t o an abnormal f i b r i n - l i k e s u b s t a n c e , which i s more r e a d i l y b r o k e n down t o f i b r i n o p e p t i d e s t h a n f i b r i n and d o e s n o t form i n t r a v a s c u l a r c l o t s . For t h e most p a r t , c l i n i c a l s t u d i e s h a v e d e m o n s t r a t e d p r o p h y l a c t i c e f f e c t i v e n e s s , b u t l y s i s of venous o c c l u s i o n s h a s a l s o b e e n c l a i m e d w i t h a r v i n . Both a r v i n and r e p t i l a s e i n h i b i t t h e s e c o n d p h a s e of ADP-induced p l a t e l e t a g g r e g a t i o n . 3996 A c o m p i l a t i o n of s y n t h e t i c d r u g a p p r o a c h e s t o f i b r i n o l y s i s h a s b e e n p r o v i d e d by Schor.97 A v a r i e t y of s i m p l e s y n t h e t i c compounds a r e known t o c a u s e l y s i s of p r e f o r m e d c l o t s . The m a j o r i t y of t h e s e compounds a r e a n t i i n f l a m m a t o r y a g e n t s and s t r u c t u r a l l y s i m i l a r o r g a n i c a c i d s . They do n o t e f f e c t f i b r i n d i r e c t l y , b u t r e q u i r e t h e p r e s e n c e of serum p r o t e i n s f o r c l o t l y s i s . C u r r e n t e v i d e n c e is c o n s i s t e n t w i t h a mechanism of a c t i o n t h a t i n v o l v e s i n h i b i t i o n of a n t i a c t i v a t o r o r a n t i p l a s m i n a c t i v i t y ; b u t t h e p o s s i b l e d i r e c t a c t i v a t i o n of plasminogen h a s n o t b e e n e x c l u d e d . 9 7 398 T h i s g r o u p of compounds a l s o i n h i b i t s p l a t e l e t a g g r e g a t i o n w i t h v a r y i n g d e g r e e s of e f f e c t i v e n e s s . A c o m p a r a t i v e s t u d y of t h e f i b r i n o l y t i c and p l a t e l e t i n h i b i t o r y e f f e c t s h a s been c a r r i e d o u t t o t r y t o o p t i m i z e b o t h a c t i v i t i e s i n t h e d e s i g n of one h i g h l y e f f e c t i v e a n t i t h r o m b o t i c a g e n t .99 B i s o b r i n (EN-1661) i s a & t e t r a h y d r o i s o q u i n o l i n e d e r i v a t i v e t h a t h a s promising f i b r i n o l y t i c a c t i v i t y i n animals;2,97 e x t e n s i v e s t r u c t u r e a c t i v i t y s t u d i e s h a v e b e e n r e p o r t e d r e c e n t l y .loo A p e r i p h e r a l v a s o d i l a t o r , bencyclane, h a s r e c e n t l y been r e p o r t e d t o have p o t e n t f i b r i n o l y t i c a c t i v i t y and t o i n h i b i t p l a t e l e t a g g r e g a t i o n . Io1

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Top1 i s s , Ed.

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R . L u n d h , A. H a r t k o o r n , J . L . G. N i l s s o n , P . S t e n b e r g , J. Bruner-Lorand and L . Lora nd,

J. Med. Chem., 63.

II

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z,

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c,

a,

Chapter 9. S.

Pulmonary and A n t i a l l e r g y A g e n t s

Tozzi, Schering Corporation,

B l o o m f i e l d , New J e r s e y

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I n a r e v e r s i b l e airway o b s t r u c t i v e disease, Introduction s u c h as asthma, r e s p i r a t i o n i s i m p a i r e d b y bronchospasm, vasod i l a t i o n , e x c e s s i v e mucous and edema. V a r i o u s t h e o r i e s have been proposed t o e x p l a i n t h e e t i o l o g y and p a t h o g e n e s i s o f asthma. I t has b e e n c a t e g o r i z e d a s a n i m m e d i a t e t y p e " h y p e r s e n s i t i v i t y " r e s u l t i n g from a n a n t i g e n - a n t i b o d y ( r e a g i n ) r e a c t i o n w i t h s u b s e q u e n t r e l e a s e o f h i s t a m i n e , s e r o t o n i n , bradyk i n i n , slow- r e a c t i n g s u b s t a n c e (S R S - A ) a n d p o s s i b l e o t h e r bronchoconstrictor substances. S i n c e s t r e s s and b r a i n les i o n s a l t e r immunological r e a c t i v i t y and s i n c e e m o t i o n a l fact o r s can e x a c e r b a t e t h e c l i n i c a l s i t u a t i o n , t h e c e n t r a l nerv o u s s y s t e m and c o n d i t i o n i n g may p l a y a r o l e i n asthma.. V a g a l s t i m u l a t i o n a n d c h o l i n e r g i c d r u g s c a n i n d u c e b r o n c h i a l cons t r i c t i o n by e x c e s s i v e p a r a s y m p a t h o m i m e t i c t o n e a n d b l o c k a d e by a n t i c h o l i n e r g i c d r u g s h a s b e e n u s e f u l i n t h e t r e a t m e n t o f asthma. S z e n t i v a n y i ' s l work h a s l e d t o h i s t h e o r y o f " b l o c k ade o f t h e a d r e n e r g i c I3 r e c e p t o r s ' ' i n t h e l u n g s as a p o s s i b l e c a u s e o f b r o n c h i a l asthma. He p o s t u l a t e s t h a t t h e a s t h m a t i c h a s a d e f e c t a t t h e I3 a d r e n e r g i c r e c e p t o r s i t e w h i c h i n d u c e 6 a s t a t e of hyperreactivity. T h i s t h e o r y has b e e n q u e s t i o n e d because i f a s t h m a t i c s have a n i n t r i n s i c B r e c e p t o r blockade, t h e y s h o u l d f i n d no r e l i e f w i t h B a d r e n e r g i c b r o n c h o d i l a t o r s . A new h y p o t h e s i s a b o u t b r o n c h i a l asthma h a s b e e n i n t r o duced which i s s u p p l e m e n t a r y t o t h e c l a s s i c a l t h e o r i e s o f t h e I t v i e w s t h e d i s e a s e a s t h e end r e s u l t o f i n t e r disease. a c t i o n b e t w e e n decreased c i r c u l a t i n g e p i n e p h r i n e and a p a t h o l o g i c a l l y a l t e r e d h y p e r a c t i v e b r o n c h i a l t r e e 2 . Some c l i n i c a l data3'4 i n b r o n c h i a l a s t h m a d o e s i n d i c a t e t h a t t h e r e i s a dec r e a s e i n c i r c u l a t i n g e p i n e p h r i n e and t h i s may be s e c o n d a r y t o a l t e r e d u p t a k e o r enzyme a c t i v i t y (COMT a n d MAO), o r dec r e a s e d r e l e a s e o f e p i n e p h r i n e from t h e a d r e n a l m e d u l l a . The d e c r e a s e d r e l e a s e o f e p i n e p h r i n e from t h e a d r e n a l m e d u l l a may i n v o l v e t h e hypothalamus t h e r e b y l i n k i n g t h e c e n t r a l nervous s y s t e m w i t h asthma. Drugs commonly u s e d f o r s y m p t o m a t i c management a c t by a v a r i e t y o f mechanisms. Sympathomimetic a g e n t s ( i s o p r o t e r e n o l ) m a i n l y a c t by s t i m u l a t i n g b e t a a d r e n e r g i c r e c e p t o r s t o c a u s e b r o n c h o d i l a t i o n a n d some ( e p h e d r i n e and e p i n e p h r i n e ) a l s o s t i m u l a t e a l p h a a d r e n e r g i c r e c e p t o r s t o cause vasoconstriction. The e f f e c t i v e n e s s o f x a n t h i n e derivat i v e s ( a m i n o p h y l l i n e , t h e o p h y l l i n e ) i s b a s e d on t h e i r a b i l i t y t o r e l a x t h e smooth m u s c l e o f t h e t r a c h e o b r o n c h i a l t r e e a n d r e d u c e edema o f t h e b r o n c h i a l mucosa. Their bronchodilator a c t i v i t y may i n v o l v e t h e i n h i b i t i o n o f l u n g c y c l i c AMP phosphodiesterase. Corticosteroids a c t through t h e i r a b i l i t y t o r e d u c e i n f l a m m a t i o n and edema a n d a l t e r t i s s u e r e a c t i v i t y t o

90

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allergens. I n a n a n t i g e n - a n t i b o d y r e a c t i o n , h i s t a m i n e i s rel e a s e d and w h i l e d r u g s t h a t b l o c k h i s t a m i n e a r e s u c c e s s f u l i n t r e a t i n g o t h e r a l l e r g i c d i s o r d e r s , they have been i n e f f e c t i v e i n t h e t r e a t m e n t o f asthma. T h i s i n e f f e c t i v e n e s s c o u l d b e rel a t e d t o t h e i n h e r e n t a b i l i t y of a n t i h i s t a m i n e s t o c o n s t r i c t smooth m u s c l e s s . R e c e n t l y , t h e development o f d i s o d i u m cromog l y c a t e has d i r e c t e d a t t e n t i o n t o w a r d a n o t h e r p o t e n t i a l l y usef u l mechanism f o r t h e t r e a t m e n t o f r e v e r s i b l e a i r w a y o b s t r u c t i v e disease. Disodium c r o m o g l y c a t e i s n o t a b r o n c h o d i l a t o r , antihistamine o r antiinflammatory agent. I n s t e a d , it i s thought t o a c t by blocking t h e release of a l l e r g y mediating s u b s t a n c e s s u c h as h i s t a m i n e , and SRS-A r e s u l t i n g f r o m a n t i gen- a n t i b o d y r e a c t i o n . The p r o s t a g l a n d i n s , a s e r i e s o f natur a l l y o c c u r r i n g hydroxy u n s a t u r a t e d f a t t y a c i d s , have b e e n reported t o possess potent stimulatory o r i n h i b i t o r y properties on r e s p i r a t o r y smooth m u s c l e and i n d e e d may o f f e r a new c l a s s of d r u g t o t h e e x i s t i n g g r o u p o f b r o n c h o d i l a t o r s .

-

The d i v e r s i t y Bronchodilators: Beta Adrenergic Stimulants of a c t i o n s of catecholamines a t 8 adrenergic receptors led Landse t o p o s t u l a t e t h a t t h e r e a r e two B r e c e p t o r p o p u l a t i o n s ; f31 m e d i a t i n g l i p o l y s i s a n d c a r d i a c s t i m u l a t i o n , a n d 82 media t i n g b r o n c h o d i l a t i o n and v a s o d i l a t a t i o n . Isoproterenol, a p o t e n t b r o n c h o d i l a t o r , a c t i v a t e s Be a d r e n e r g i c r e c e p t o r s b u t it a l s o s t i m u l a t e s c a r d i a c adrenergic r e c e p t o r s t o cause tachycardia. P r o b a b l y as a d i r e c t r e s u l t o f t h e c a r d i a c s t i m u l a t i o n , t h e hypoxaemia o f t h e a s t h m a t i c p a t i e n t c o u l d become worse when t h e d r u g i s a b u s e d a n d v e n t r i c u l a r f i b r i l l a t i o n o r T h i s c a r d i a c s t i m u l a t i o n has b e e n i m p l i a s y s t o l e may o c c u r . c a t e d i n t h e r e c e n t r i s e i n asthma m o r t a l i t y t h u s i n d i c a t i n g a n e e d f o r a d r u g t h a t has l i t t l e 191 a c t i v i t y . I n a d d i t i o n , isoproterenol i s a comparatively short a c t i n g bronchodilator b e c a u s e i t i s r a p i d l y m e t a b o l i z e d b y t h e enzyme c a t e c h o l - 0 S u b s t i t u t i o n o f o t h e r groups o r m e t h y l t r a n s f e r a s e (COMT). r e a r r a n g e m e n t s o f e x i s t i n g g r o u p s i n t h e b e n z e n e r i n g have res u l t e d i n compounds more r e s i s t a n t t o COMT w i t h g r e a t e r d u r a t i o n o f a c t i o n , a n d c h a n g e s i n t h e s i d e c h a i n have i n c r e a s e d t h e i r s p e c i f i c i t y f o r Be, o r b r o n c h i a l , as o p p o s e d t o t h e 81, o r cardiac, receptors. The c l i n i c a l , c h e m i c a l and pharmacol o g i c a l c o n s i d e r a t i o n s o f t h e newer B a d r e n e r g i c s t i m u l a n t s h a v e r e c e n t l y b e e n reviewed7. Examples o f d r u g s c l a i m e d t o h a v e more s e l e c t i v e 82 s t i m u l a n t a c t i o n s t h a n i s o p r o t e r e n o l a r e ; salbutamo18, t e r b u t a l i n e s , T h 1 1 6 5 a l 0 , hexo r e n a l i n e ” , clorprenalinel2, t r i m e t h o q u i n o l l 3 , q u i n t e r e n o l l q , WG 2 5 p and s o t e r e n o l l e . S t r u c t u r e a c t i v i t y r e l a t i o n s h i p s of salig e n i n a n a l o g s o f sympathomimetic c a t e c h o l a m i n e s a n d t h e r e s o l u t i o n o f i s o m e r s o f t h e ‘most p r o m i n e n t of t h e s e a n a l o g s , salb u t a m o l , have r e c e n t l y b e e n r e p o r t e d ’ 7r1

,

C l i n i c a l l y some o f t h e s e s e l e c t i v e a d r e n e r g i c bronchod i l a t o r s have s u c c e e d e d i n d e m o n s t r a t i n g l e s s c a r d i o v a s c u l a r

Chap. 9

Pulmonary, A n t i a l l e r g y A g e n t s

Isoproterenol S a l b u t amo 1 Terbutaline Sote r eno1 Clorprenaline Metaproterenol Th 1165a H e x o p r e n a l i ne

3- OH 3- C H 2 O H 3- OH 3- N H S 0 2 C H 3

4-OH

2- C 1

H

3- OH 3- O H 3- OH

5- OH

91 -

Tozz i

4- OH 5- OH 4- OH 5- O H

4- OH

OH

Q u i n te r e n o l 0C H 3

WG 253

T r i m e t hoqui no1

e f f e c t s than isoproterenol. T h e s e d r u g s a r e e f f e c t i v e as a e r o s o l s and u n l i k e i s o p r o t e r e n o l , t h e y a r e a l s o e f f e c t i v e orally. SalbutamolLs'2s has b e e n compared i n d o u b l e b l i n d ~ ~ e t, a p r o t e r e n ~ l ~ ~ r ~ ~ ' ~ ~ . s t u d i e s w i t h i s o p r ~ t e r e n o l ~o r~ m A t 0.2 mg, it i s e q u i a c t i v e t o i s o p r o t e r e n o l and has a l o n g e r d u r a t i o n of a c t i v i t y . T e r b u t a l i n e has demonstrated o t e n t b r o n c h o d i l a t o r a c t i ~ i t y ~ ~when , ~ ' g i v e n o r a l l y ( 5 mgp o r as a n a e r o s o l ( 0 . 2 mg). I t has b e e n compared t o p r o x y p h y l l i n e 4 5 i ~ o p r o t e r e n o la n ~ d~ m e t a p r o t e r e n o l * 4 - 4 7 . Th 1165a a e r o s o l a t 0.2 mg i s f o u r t i m e s as p o t e n t a s i t s a n a l o g u e m e t a p r o t e r e n o l Hexoprenaline and i s a r e l a t i v e l y s a f e b r o n c h o d i l a t o r 4 8 ' s o . i s e f f e c t i v e a s a n a e r o s o l (0.4$) o r o r a l l y (0.5 mg), has a l o n g d u r a t i o n o f a c t i ~ i t y ' ' ~ ~ ~ -a~n d? ,a l l o w s f o r t h e r e d u c t i o n o f c o r t i c o s t e r o i d r e q u i r e m e n t s 4 9 5 5 . Oral d o s e s of t r i m e t h o q u i n o l (1 rng)se,57 and q u i n t e r e n o l (0.4 m g ) s 8 a n d a e r o s o l d o s e s o f WG 253 ( 1 . 5 m g ) 1 5 , 5s a n d s o t e r e n o l ( 0 . 2 2 5 mg)eo h a v e bronchodilator efficacy equivalent t o isoproterenol w i t h a g r e a t e r d u r a t i o n o f a c t i v i t y and lessB1 s t i m u l a t i o n . Clorp r e n a l i n e has f32 b r o n c h o d i l a t o r p r o p e r t i e s i n d o g s 1 2 a n d ratse1. Sympathomimetic amines c a n a l s o i n h i b i t t h e r e l e a s e

92

Sect.

II

-

Pharrnacodynarnic Agents

Topliss, Ed.

o f p h a r m a c o l o g i c a l m e d i a t o r s of t h e immediate t y p e a l l e r g i c reactionse2re3. I n a s e r i e s o f t e s t s y s t e m s i n c l u d i n g pass i v e c u t a n e o u s a n a p h y l a x i s (PCA) a n d d e x t r a n r e s p o n s e i n t h e r a t , i s o p r o t e r e n o l w a s a t l e a s t 1000 times as p o t e n t a s d i s o dim c r o m o g l y c a t e (DSCG)@4. T h e r e f o r e , B a d r e n e r g i c stimul a n t s are a l s o p o t e n t p r o t e c t i v e a g e n t s a g a i n s t a n a p h y l a c t i c r e a c t i o n s and s h o u l d n o t b e c o n s i d e r e d o n l y a g e n t s p r o d u c i n g sympathomimetic r e l i e f o f bronchospasm i n asthma.

Non-Adrenergic B r o n c h o d i l a t o r s - S e v e r a l new b r o n c h o d i l a t o r s have b e e n r e p o r t e d which do n o t d e p e n d on a d r e n e r g i c s t i m u l a t i o n as t h e i r mode o f a c t i o n . P i q u i z i l , 4- (6,7-dimethoxyq u i n a z o 1 i n ) - 4- p i p e r a z i n e - 1-c a r b o x y l i c a c i d i s o b u t y l e s t e r , a new o r a l b r o n c h o d i l a t o r , has b e e n shown t o be e q u a l i n a c t i v i t y t o a c o m b i n a t i o n o f t h e o p h y l l i n e , e p h e d r i n e and phenobarbitale5. Sch 1000, a n a t r o p i n e d e r i v a t i v e , d e c r e a s e s pulmona r y r e s i s t a n c e i n o b s t r u c t i v e pulmonary d i s e a s e when i n h a l e d as a n a e r o s o l (0.025$)saree. D e c l o x i z i n e , a d e c h l o r i n a t e d d e r i v a t i v e of h y d r o x y z i n e , h a s marked a n t i s e r o t o n i n a n d papav e r i n e - l i k e p r o p e r t i e s a n d i s c l a i m e d t o be a n e f f e c t i v e o r a l Fenspiride, an alpha adrenerb r o n c h o d i l a t o r a t 50 mge7-ee g i c b l o c k i n g d r u g , has b e e n c l a i m e d t o b e of v a l u e i n t h e treatment of a ~ t h m a ~ ~ - 7 ~ .

.

S c h 1000

I

C02CH2CH ( C H a ) 2 Piquiz i1

(CH2)20(CH2)20H

Fenspiride

Decloxizine

-

PGEl a n d PGE2 a r e e f f e c t i v e b r o n c h o d i l a t o r s , Prostaglandins c a p a b l e o f r e v e r s i n g t h e i n c r e a s e d a i r w a y r e s i s t a n c e and dec r e a s e d a i r w a y c o m p l i a n c e i n d u c e d by c h o l i n e r g i c s t i m u l a t i o n i n t h e c a t . A d m i n i s t r a t i o n by a e r o s o l produces t h e g r e a t e s t b r o n c h o d i l a t i n g r e s p o n s e w i t h l i t t l e o r no c a r d i o v a s c u l a r The c a r d i o v a s c u l a r e f f e c t s of hypotension and tachycardia. e f f e c t s were most p r o n o u n c e d a f t e r i n t r a v e n o u s a d m i n i s t r a t i o n .

Chap.

9

Pulmonary, Antiallergy Agents

93 -

Tozz i

PGE2 b r o n c h o d i l a t o r e f f e c t s were n o t b l o c k e d b y b i l a t e r a l vagotomy, a d r e n a l e c t o m y , b e t a a d r e n e r g i c o r g a n g l i o n i c b l o c k ade. T h e s e a g e n t s may r e p r e s e n t a new a n d n o v e l c l a s s o f pot e n t b r o n c h o d i l a t o r agents74.

-

Phosphodiesterase I n h i b i t o r s Catecholamines i n c r e a s e t h e c o n c e n t r a t i o n o f c y c l i c AMP b y s t i m u l a t i n g t h e enzyme a d e n y l cyclase. M e t h y l x a n t h i n e s i n c r e a s e t h e c o n c e n t r a t i o n o f cyc l i c AMP b y i n t e r f e r i n g w i t h i t s i n a c t i v a t i o n by p h o s p h o d i e s t e r a s e and t h i s i s t h o u g h t t o b e t h e b i o c h e m i c a l b a s i s f o r t h e bronchodilator a c t i o n of theophylline.

AT’

++

---------------> Mg

Bronchodilation 4 c y c l i c $‘,5’ AMP

adenyl cyclase

Mg

----------------> 5’ ++

AMP

phosphodiesterase

T

T

I n h i b i t e d by t h e op hy 11i ne

S t i m u l a t e d by c a t e c h o l a m i ne s

I C I 58 3 0 1 (3- a c e t a m i d o - & m e t h y l - 8- n - p r o p y l - s- t r i a z o l o [ & , + a ] p y r a z i n e ) i s a new compound t h a t i s a g r e a t e r i n h i b i I n animals, I C I t o r of phosphodiesterase t h a n theophylline. 58 301 i s f a r more p o w e r f u l fn p r e v e n t i n g b r o n c h o s p a s m t h a n i n acting as a bronchodilator. T h i s a c t i v i t y h a s b e e n confirmed i n asthmatic patients7s. P r o x y p h y l l i n e , 7- (2- hydroxyp r o p y l ) t h e o p h y l l i n e , a d m i n i s t e r e d a s a s u p p o s i t o r y ( 5 0 0 mg) i s e f f e c t i v e i n e n h a n c i n g pulmonary f u n c t i o n i n c h r o n i c asthmatic patients76. OH NHCO C H 3

+I

P r o x y p h y l l i ne

ICI

-

58 3 0 1

Disodium C r o m o g l y c a t e (DSCG) T h i s compound a p p e a r s t o b l o c k a s t e p i n t h e c h a i n o f e v e n t s t r i g g e r e d by t h e u n i o n o f a n t i g e n w i t h r e a g i n i c a n t i b o d y which l e a d s t o t h e r e l e a s e o f s p a s mogens and o t h e r m e d i a t o r s o f t h e a n t i p h y l a c t i c and h a s been found u s e f u l i n t r e a t i n g asthma and a l l e r g i c r h i n i t i s . I t d i f f e r s from e x i s t i n g t h e r a p y i n t h a t i t h a s no sympathomimetic, a n t i i n f l a m m a t o r y o r a n t i h i s t a m i n i c a c t i v i t y . DSCG i s a d m i n i s t e r e d by i n h a l a t i o n as a d r y powder i n a spec i a l i n s u f f l a t o r which p e r m i t s c o n t r o l l e d d o s a g e . I n a dose r a n g e o f 20-80 mg d a i l y , i t h a s b e e n u s e d i n t h e t r e a t m e n t o f

94

Sect. I I

-

Pharmacodynamic Agents

T o p l i s s , Ed.

OH I

NaOgC

Disodium c r o m o g i y c a t e ( D S C G ) a l l e r g i c r h i n i t i s and has b e e n f o u n d t o be e f f e c t i v e b y I n e x t r i n s i c asths o m e 7 8 0 0 and i n e f f e c t i v e by o t h e r s a l - 8 3 . m a i n c h i l d r e n , i t has b e e n f o u n d t o be e f f e c t i v e by most i n v e s t i g a t 0 r s ~ 4 -a~n d~ a l l o w s f o r a d e c r e a s e i n t h e r e q u i r e m e n t a n d b r o n c h o d i l a t o r s 8 4 98 D S C G c a n preof s t e r o i d s 93' v e n t a s t h m a t i c a t t a c k s 94 p S T , r e d u c e h i s t a m i n e s e n s i t i v i t y 8 8 3 e@ and e x e r c i s e - i n d u c e d a i r w a y o b s t r u c t i o n l o o i n a s t h matics. The o n l y s i d e e f f e c t r e p o r t e d has b e e n a pharyngeal a n d t r a c h e a l i r r i t a t i o n l o L r L o e a n d a l t h o u g h t h e r e i s no evid e n c e o f d r u g - d e p e n d e n c e t h e r e may b e a loss o f e f f i c a c y a f t e r some months o f u s e L O8.

,

.

-

Antihistamines A n t i h i s t a m i n e s b l o c k h i s t a m i n e a t t h e recept o r s i t e and a r e u s e f u l i n t h e t r e a t m e n t o f u r t i c a r i a and rhinitis. U n f o r t u n a t e l y , t h e m a j o r i t y o f a n t i h i s t a m i n e s produce a n i n c r e a s i n g degree of s e d a t i o n w i t h i n c r e a s e d t h e r a peutic activity. Two new a n t i h i s t a m i n e s , m e c l a s t i n e l o 4 and a z a t a d i n e l o s , have b e e n r e p o r t e d t o be c l i n i c a l l y e f f e c t i v e . Clinical s t u d i e s show t h a t a z a t a d i n e i s a p o t e n t , l o n g a c t i n g a n t i h i s t a m i n e w i t h l o w s e d a t i v e l i a b i l i t y a t d o s e s o f 1 - 2 mg t w i c e daily10er107. A z a t a d i n e , a t 0.009-0.0024 mg/kg p.o., has b e e n r e p o r t e d t o b e more p o t e n t t h a n c y p r o h e p t a d i n e as a n a n t i h i s It t a m i n e and a n t i a n a p h y l a c t i c a g e n t i n m i c e a n d g u i n e a p i g s . i s g e n e r a l l y l e s s t o x i c t h a n c y p r o h e p t a d i n e and p o s s e s s e s s i g n i f ic a n t a n t i c h o l i n e r g i c and a n t i s e r o t o n i n a c t i v i t y 1 05.

n '

I

CH3

0- CH2- C H s - [ z

Meclas t i ne

A z a t a d i ne I t has l o n g b e e n a c c e p t e d t h a t h i s t a m i n e i s formed by t h e enzymatic decarboxylation of h i s t i d i n e . A drug t h a t inh i b i t s h i s t i d i n e - d e c a r b o x y l a s e would d e c r e a s e t h e amount o f

Chap. 9

95

Tozz i

Pulmonary, A n t i a l l e r g y Agents

h i s t a m i n e a v a i l a b l e and t h u s b e o f u s e i n t h e t r e a t m e n t o f allergic disorders. T r i t o q u a l i n e h a s b e e n r e p o r t e d t o have non- s p e c i f i c a n t i s p a s m o t i c and a n t i i n f l a m m a t o r y a c t i v i t y beIt h a s b e e n s i d e s i n h i b i t i n g histidine-decarboxy1aselo8. shown t o b e e f f e c t i v e i n t h e t r e a t m e n t o f hay f e v e r and unl i k e most a n t i h i s t a m i n e s , i t h a s no s e d a t i v e e f f e c t ’ 09. References 1. 2.

3. 4. 5. 6. 7.

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A.A. Mathe and P.H. Knapp, Am. P s y c h o s o m a t i c S o c i e t y , C i n c i n n a t i , 31 March, 1969. D.F. Hawkins, B r . Med. J. 10, 230 (1967). A.M. Lands, A. A r n o l d , J.P. M c A u l i f f , F.P. Luduena, a n d T.O. Brown, N a t u r e , 597 (1967). R.T. B r i t t a i n , D. J a c k a n d A.C. R i t c h i e , Ad. D r . Res., 3, 197 ( 1 9 7 0 ) . R.T. B r i t t a i n , J . B . Farmer, D. J a c k , L.E. M a r t i n a n d W.T. Simpson, Nature, 9,862 (1968). H. P e r s s o n and T. O l l s s o n , A c t a Med. Scand. s u p p l . , 512, 11 ( 1 9 7 0 ) . S . R . O’Donnell, Eur. J. Pharm., 12, 3 5 ( 1 9 7 0 ) . F. Witek, Wein. K l i n . Wschr., 8 -3, 1 1 4 (1971). T. I g a r a s h i , T. Wakabayashi, T. S h o j i and T. Tomiugam, Arzneim. F o r s c h . , 20, 1738 ( 1 9 7 0 ) . F. Fogelman and H.F. Grundy, B r i t . J. Pharm., 2, 416 (1970)Lab. F i l e s of C h a r l e s P f i z e r a n d Co., Groton, Conn. J . P . G r i f f i n a n d P. T u r n e r , J. C l i n . Pharm., ll, 280

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149,

2,

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9, , $, 9,

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28.

S.S.

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C h a t t e r j e e and A.E.

Perry,

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47, 53 (1971).

7

S t r e e t o n and E.B. Morgan, P o s t g r a d . Med. J. suppl., 1 2 5 (1971). K.N. Palmer, J.S. Legge, W.F. H a m i l t o n and M.L. Diament, B r i t . Med. J., 2, 2 3 ( 1 9 7 0 ) . J.W. P a t e r s o n , P o s t g r a d . Med. J. s u p p l . , 47, 38 (1971). A.D. M i l n e r and D. Ingram, Arch. Dis. C h i l d . , 46, 502

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31 32.

(1971).

Hambleton and E . A . S h i n e b o u r n e , Arch. D i s . C h i l d . , 766 ( 1 9 7 0 ) . F. Lustman, P r a c t i t i o n e r , 205, 213 ( 1 9 7 0 ) . D.A. W a r r e l l , D.G. R o b e r t s o n , J . N . Howes, M.E. Conolly, J.W. P a t e r s o n , L . J . B e i l i n and C.T. D o l l e r y , B r i t . Med. J., 2, 65 ( 1 9 7 0 ) . A.E. T a t t e r s f i e l d and M.W. McNicol, N e w Eng. J. Med., 281, 1323 (1969). T.V. O'Donnell, G.M. B u t l e r and M.D. Tocker, P o s t g r a d . Med. J. s u p p l . , 1 1 5 (1971). C. Racoveanu, e t a l . , P o s t g r a d . Med. J. s u p p l . , 83 G.

33

%,

34. 35. 36

37. 38.

-

9,

9,

(1971).

39

0

40.

41. 42.

43

6

44. 45 46. 47. 48. 49 50

51 52.

53

9,

P. Dano, P o s t g r a d . Med. J. s u p p l . , 86 (1971). B. Arner, A. B e r t l e r , T. K a r l e f o r s and H. W e s t l i n g , A c t a Med. Scand. s u p p l , , 512, 4 1 ( 1 9 7 0 ) . U. H e d s t r a n d , Scand. J. Resp. D i s . , 51, 188 ( 1 9 7 0 ) . C.H. Hagelund, Nord. Med., 728 (1971). R. E s p i n o z a , Nord. Med., 729 (1971). H. Formgren, Scand. J. Resp. D i s . , 203 ( 1 9 7 0 ) . H. Formgren, Scand. J. Resp. D i s . , 2, 195 ( 1 9 7 0 ) . B, A r n e r , A c t a Med. Scand. s u p p l . , 512, 45 ( 1 9 7 0 ) . M.J. M a t t i l a and A . M u i t t a r i , Ann. Med. Exp. B i o l . Fenn., 47, 298 (1969). H.K. Tweel, Ann. A l l e r g . , 3, 1 4 2 (1971). J. Hamm and H. Wusthofen, K l i n . Wschr., 48, 415 ( 1 9 7 0 ) . H.P. S c h u s t e r and P. Baum, Arzneim. F o r s c h . , 1p, 1905

a,

3,

-

(1969 1

3,

98 (1971). F. Wilk. Wein. K l i n . Wschr., D. T h i e d e , G. S t e m p e l and W.T. Ulmer, Arzneim. F o r s c h . , 21, 416 (1971). H. P a l l and W. S c h l i c k , Wein. K l i n . Wschr., &, 117

-

(1971

54 55. 56 57. 58.

59. 60.

z,

a,

B. S c h r e i n e r , Wein. K l i n . W ~ c h r . , 80 (1971). R. T i t a c h e r , Wein. K l i n . Wschr., 121, 146 (1971). I. Ando e t a l . , J a p a n Med. News, & ' 1 2 (1971). A. Kiyomoto, Y. Iwasawa and S. H a r i g a y a , Arzneim. F o r s c h , 1, 46 ( 1 9 7 0 ) . E. B l e e c k e r , W. McKinney, H. Lyons and S . N . S t e e n , Anesth. Analg., 48, 7 (1969). I. Carney, M . J . Daly, L.E. L i g h t t o w l e r and R.W. Pickering, Arch. Int. Pharmacodyn., 334 (1971). B.M. Cohen, Curr. T h e r . Res., 11, 713 (1969).

-

194,

Chap. 9

61.

Pulmonary, Antiallergy Agents

Tozz i

97 -

T.

I g a r a s h i , Y. S u s u k i and K. Miyao, Arzneim. F o r s c h . , 234 ( 1 9 7 1 ) . E.S.K. Assem and H.O. S c h i l d , N a t u r e , 3,1 0 2 8 ( 1 9 6 9 ) . E.S.K. Assem and H.O. S c h i l d , I n t . Arch. A l l e r g y , 40,

21, 62.

63

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

a, J.

553 (1971).

Pernod and J. B a t i m e ,

J. Fr.

Med.

C h i r . Thorac.,

24,

783 (1970).

Rev. Lyon Med., 395 ( 1 9 7 0 ) . J. D u h a u l t and M. L a u b i e , Arzneim. F o r s c h . , 4, 1263 (1969). M.E. R o s e n t h a l e , A. D e r v i n i s and J. K a s s a r i c h , J. Pharm. Exp. Therp., 541 (1971). A. J. R i c h a r d s , S.R. Walker and J . W . P a t e r s o n , B r i t . J. D i s . Chest., 247 (1971). P. I n d a , S.S. C h a t t e r j e e , W.D. R i d i n and T. S i n g h , B r i t . J. C l i n . P r a c t . , 3, 5 0 5 (19697. J.S.G. Cox, J . E . Beach and P. S h e a r d , Ad. D r . Res., 3, 197 ( 1 9 7 0 ) . J. C e n t n e r , A r s Medici, 26, 715 (1971). E. H o l o p a i n e n , A. Backman and O.P. S a l o , L a n c e t , 1, 5 5 P. Galy e t a l . , J. C. LeDouaree,

72

73 74

178,

75.

76. 77. 78 79

3

(1971).

80.

81. 82.

83 84.

85 86. 87. 88.

89

-

-4,

71

90

576 (1971).

E.S.K. A s s e m a n d A.W. R i c h t e r , Immun., 21, 729 (1971). K . J . B e r e n y i and S . N . S t e e n , Canad. A n a e s t h . S o c . J., 17, 522 ( 1 9 7 0 ) . W.T. U l m e r , Med. K l i n . , 66, 326 (1971). A. M u i t t a r i , M . J . M a t t i l a and J. P a t i a l a , Ann. A l l e r g . , 274 (1969). J. Van Mechelen, A c t a T u b e r c . Belg., 2, 667 (1968). W.D. L i n e h a n , I b i d . , 3, 549 (1968). M.J. C h a r p e n t i e r , K. B r e u i l and G. Beauchant, Bord. Med.,

.

91. 92.

Pelikan, W.J. Snoek, H. Booij-Noord, N. O r i e and K. d e V r i e s , Ann. A l l e r g . , 28, 548 ( 1 9 7 0 ) . L.H. Cape1 and P. McKelvie, L a n c e t , 1, 575 (1971). R.H. Champion, L a n c e t , 1, 187 (1971): J.M. Smith, L a n c e t , 2, 1 9 5 (1971). I.L. B e r n s t e i n e t al., J. A l l e r g . , 95 (1971). H e Booij-Noord, N.G.M. O r i o , C. Berg and K. d e V r i e s , J. A l l e r g . , 46, ( 1 9 7 0 ) . S. B e d f o r d and J . A . Kuzemko, C l i n . P e d i a t . , 2, 719 (1970 A. Meyer, N. Donat a n d A.M. C l a u z e l , Presse. Med., D, Z.

2,

519 (1971).

D.R. McCourtie, N.M. L e f c o e and J . H . Toogood, Therap e u t i c s , 2, 40 (1971). R.M. S l y , Ann. A l l e r g . , 28, 299 ( 1 9 7 0 ) . T.E. Van Metre, J.Y.L. Chen, S.M. Nagy, P.S. Norman, M. S c h w a r t z and A. Turk, J. A l l e r g - , 131 ( 1 9 7 0 ) . J.S. Hyde, B. B u r a n a k u l and V i t h a y a s a i , Ann. A l l e r g . , 28, 449 ( 1 9 7 0 ) . W.F. Hughes, K.S. Tse and C.E. Arbesman, J. A l l e r g . , Q,

3,

-

98 93

94 95. 96 97 98.

99. 100. 101. 102. 103. 104. 105. 106. 107.

108. 109.

Sect.

II

-

Pharrnacodynarnic Agents

Topliss, Ed.

1 3 2 (1970). H. Chai, L. Molk a n d C . J . F a l l i e r s , A m e r . Med. Ass. 1 1 9 t h . Meeting, Chicago ( 1 9 7 0 ) . 8, 2 8 1 ( 1 9 7 0 ) . J.S. Hyde and B. B u r a n a k u l , C h e s t , O.R. McCarthy, L a n c e t , 2 , 9 8 0 (1970% I,. Molk, H. Chai and C . J . F a l l i e r s , J. A l l e r g . , 132 (1970). 141 (1971). S.C.L. S h o r e , S. A f r . Med. J., J . W . Kerr, M. G o v i n d a r a s and K.R. P a t e l , B r i t . Med. J., 2, 139 ( 1 9 7 0 ) U.Y. Ryo, B. Kang and R.G. Townky, J. A l l e r g . , 96

3,

9,

-

9,

(1971)

a,

S l y , Ann. A l l e r g . , 362 (1971). Mathison, J. J. Condemli, F.W. Lovejoy, Jr. and J.H. Vaughan, J A M , 216, 1 4 5 4 (1971). C.W.M. Cooper, P r a c t i t i o n e r , 205, 230 ( 1 9 7 0 ) . R. Munro-Ford, Ann. A l l e r g . , 2,8 (1971). A. Roncevich, Curr. Ther. Res., 11, 6 2 5 (1969). S. T o z z i , M. d e l Prado, R. L u t z , F.E. Roth and I . I . A . T a b a c h n i c k , Fed. P r o c e e d i n g s , 1972. A. Sabbah, L a V i e M e d i c a l e , 5401 (1969). B. S i g a l and M. H e r b l o t , Gaz. Med. F r a n c e , 77, 364 (1970). F. Hahn a n d H.J. T e s c h e n d o r f , Arzneim. F o r s c h . , 10, 1490 ( 1 9 7 0 ) . C. E b e r h a r t i n g e r a n d H. Ebner, Wrn. Med. Wsche., 119, R.M. D.A.

3,

783 (1969 1

.

Section I11 Editor:

-

Chemotherapeutic Agents

Kenneth Butler, Pfizer Inc., Groton, Connecticut Chapter 10. Antibiotics

Frank C. Sciavolino, Pfizer Inc., Groton, Connecticut

-

General A symposium commemorating thirty years of penicillin therapy was held in May surveyin presently active areas of sc entific research in the penicillin field Commercial 6-APA processes ,la chemical relationships between penicillins and cephalosporins,Ib mechanism of action,lc bacterial resistance,ld and immunological propertiesle of penicillins were discussed, as were current clinical aspects of penicillin therapy .If The physicochemical properties and biological activities of clinically significant 6-lactam antibiotics were reviewed with particular commentary on 8-lactamases.2 A chapter on the chemistry of cephalosporin antibiotics appeared. The Second International Symposium on Gentamicin brought up to date scientific and clinical information relating to this antibiotic, placed gentamicin in perspective among other aminoglycosides and defined areas in which further investigative efforts are needed.4 Structure-activity relationships among chemically and microbially modified lincomycin antibiotics were reviewed. A treatise on bacterial resistance to the macrolide antibiotics and lincomycins was published. The chemistry, biological activity and biosynthesis of the ansamycins were r e ~ i e w e d . ~Detailed accounts of totally synthetic approaches to the tetracycline antibiotics appeared. A monograph on the biochemistry of antimicrobial agents has united significant developments in this area and orients readers to major literature reviews u to 1971.9 A theory on the formation of antibiotics was A symposium11 held in March surveyed recent developments proposed in many areas of the vast field of antibiotics.

.

Aminoglycosides - Comparative in vitro evaluations of tobramycin (nebramycin factor 6) continue to indicate it is two to four times more active than entamicin against clinical isolates of Pseudomonas aeruginosa;l5-I5 mean MIC values of 0.42 and 3.54 y/ml, respectively, were reported.12 Conversely, gentamicin proved four times as potent as tobramycin against isolates of Serratia marcescens ;I4 both antibiotics demonstrated compargble-activity against the majority of other gramnegative pathogens .lz-l' Detailed accounts of the structure elucidations of the gentamicin C components16-18 and sisomicin19s20 were published. Chemical syntheses of 3'-d xykanamycin,21 3' ,4'-dideoxykanamycin B22 and 3 ' ,4'-dideoxyneamine" were announced along with preliminary biological data indicating their in vitro activity against Escherichia and 3. aeruginosa strains which inactivate the parent antibiotics by 3'-0-

100

Sect. I l l

-

Chemotherapeutic Agents

Butler, Ed.

phosphorylation. Among new naturally occurring aminoglycosides, structures were proposed for b u t i r ~ s i n s ~A(k) ~ - ~ ~and B(1b) and lividomycins A(2a)27928 and B(2b) .29 The butirosins, which were isolated from a bacterial fermentation, represent the first reported examples of aminoglycosides containing an N-acylated-2-deoxystreptamine moiety; an account of their biological profiles has not been reported. The lividomycin structures revealed their chemical immunity to enzymatic inactivation by 3'-O-phosphorylation, but 0-phosphorylative inactivation of lividomycin A by strains of g.aeruginosa has been observed3' and apparently occurs at a site other than the 3'-position.

L

HOCHp

Y Rl

NH

'4

OH

k0

I

R, OH

Enzymatic inactivation of gentam' in by cell-free extracts from clinical isolates of g. aeruginosa31,35 and R-factor carrying Klebsiella pneumoniae33~34was described. The mechanism of this pseudomonas resistance to gentamicin was established by showing that acetylation occurs on the +amino group of the 2-deoxystreptamine ring.32 All gentamicin C components and sisomicin were reported to be excellent substrates €or the inactivating enzyme gentamicin acetyltransferase, whereas tobramycin and kanamycin B proved to be poor substrates. A number of gentamicin-resistant aeruginosa from various parts tobramycin-sensitive clinical isolates of of the world have been studied and claimed to contain gentamicin acetyltransferase.32 The klebsiella resistance to gentamicin was

a.

Chap. 10

Antibiotics

Sciavol ino

101 -

reported to occur adenylylation and further the presence of an Rfactor which mediates gentamicin resistance in these klebsiella strains was demonstrated by transfer of this resistance in vitro to E. coli recipients subsequently designated E. & JR66lW677 and M L l ~ l O ~33o,34 A tentative structure for gentamicin C1 adenylate has been pr~posed.~' E. coli JR661W677 has also been shown to adenylylate 3',4'-dideoxykanam y c D 6 and the structure of the inactivated product established as 3 ' ,4'-dideoxykanamycin B-2"-adenylate. 37 A report presenting clinical and laboratory evid nce for the inactivation o.fOgentamicin by carbenicillin appeared.38 However, chemical ,39 kinetic and clinica141 data was published indicating that although gentamicin and carbenicillin can mutually inactivate each other, the reaction requires conditions that are not present in most patients requiring these agents.

Spectinomycin became available in the U.S. for single dose treatment of acute gonorrheal infections; clinical studies indicate IM administration of 2.0 or 4.0 g of spectin m in results in cure rates of over 90% in males and 96% in females.49J3 Ansamycins - Rifampicin became available in the U.S. during 1971 for use in the treatment of tuberculosis. Reviews containing discussions of clinical findings ntimicrobial activity and pharmacology of rifampicin were pubThrombocytopenia, apparently arising an immunological lished.44* mechanism, has been reported as a potentially serious side ef ct resulting from high-dose (1200 mg) twice-weekly rifampicin therapy .Eg The structure of streptovaricin C wt~,igvisedin accord with results of new 49 periodate oxidation experiments and X-ray crystallographic studies. Gross structures for six other naturally occurring streptovaricins were proposed.50 The antiviral properties of the ansamycins are discussed in Chapter 12.

''

+

6-Lactams - Announcements of the discoveries of four novel naturally occurring cephalossprf,i C variants highlighted the year's developments in B-lactam research. The new cephalosporins are uniquely substituted at positions C3 and/or C7 (cf. Table I) and were isolated from Streptomyces fermentations. In general, biological profiles suggest 7methoxycephalosporins are more active than cephalosporin C against most gram-negative bacteria, but show poor activity against g.aeruginosa; they are essentially inactive against penicillin-resistant staphylococci. An elegant tranacylation process for cleaving the aminoadipoyl moiety of 5 with simultaneous introduction of new acyl side chains was reported.55 Synthetic routes to 7-methoxycephalosporins and 6-methoxypenicillins which depart from benzhydryl 7-diazocephalos oranate and benzyl 6-diazopenicillinate, respectively, were described.g6 In related work, 7amethyl-7-phenoxyacetamido cephalosporanic acid and methyl 6amethylbenzylpenicillinate were synthesized as close mimics of the 2-alanyl-g-alanine end of acetylmuramylpentapeptide, but were reported to be substantiall less active than the parent compounds against gram-postive organisms.57

102 -

Sect. I l l

-

Chemotherapeutic Agents

TABLE

-3

Butler, Ed.

I

= Cephamycin A

I

OCH3

4 =

Cephomycin 6

5 =

Cephomycin C (A168868) =

-NH2

& = A16884 Cephalexin (KeflexR ) has proven to be safe and effective in treating commonly encountered in general practice.58 It a variety is reported that successful therapy of urinary tract infections with cephaloglycin (KafocinR) must be mainly attributed to the antibacterial activity of its metabolite desacetylcephaloglycin which is 4-8 times leas active against gram-negative bacilli than cephaloglycin itself. Orally administered acetoxymethyl and pivaloyloxymethyl esters of cephaloglycin were reported to be enzymatically cleaved following absorption from the gastro-intestinal tract and to affor higher blood and urine levels than an equivalent dose of cephaloglycin." Cephapirin (BL-Pl322) ( I ) ,a parenteral ce halosporin, has antibacterial activity equivalent to of 1 g of cephapirin sodium gave serum c e p h a l ~ t h i n . ~ ~IM - ~injection ~ ;61 an 88% levels of 7.2-25 y/ml and urine concentra ions of 1-2.5 mg cure rate was reported for urinary tract6' and respiratory6"infections caused by susceptible organisms. Less pain and phlebitis following i.v. administration is reported to be the mt4or advantage of cephapirin over cephalothin.61 BL-S217 (8) is claimed to be 20 times more effective than cephalothin against Streptococcus pyogenes and Diplococcus pneumoniae infection. infections in mice and 3-4 times more efficacious in an E. ubstitutedStructure-activity relationships among a group of 7-[a-(N,N' amidinothi0)-acetamido cephalosporanic acids were described.3

offinfections

Chap, 10

Antibiotics

S c i a v o l ino

103

Among semi-synthetic penicillins, BL-Pl597 (2)and BL-P1654 were reported to be more active than carbenicillin against Pseudomonas, inhibiting 90% of 150 isolates at 12.5 y/ml; conversely, carbenicillin was more active than both compounds against indole-positive p r ~ t e u s . ~Sulfo~ cillin was reported to have essentially the same in vitro spectrum and potency as carbenicillin.68

-8 . R

Orally administered indanyl carbenicillin (10)is absorbed and hydrolyzed in animals and man to provide carbenicillin activity generally equivalent to parenteral ~arbenicillin;~~ the metabolism of 10 in rats, dogs and humans was reported.70 Satisfactory clinical results have been reported for indanyl carbenicillin therapy f rinary tract infections caused by Pseudomonas and Proteus species.7p-7y Results of a multi-center clinical evaluation of epicillin indicate this orally active ampicillin congener was generally effective in 84% of 683 patients with respiratory, gastro-intestinal or soft-tissue infections caused by a variet ampicillin-sensitive gram-positive and gram-negative bacteria.Y40f Chemical properties of penicillin polymers derived from ampicillin, 6-APA and benzylpenicillin on standing in aqueous solution were described.75 These polymers have been s own to combine covalently with An enzymatic synthesis of protein and to elicit immune ampicillin from 6-APA and various phenylglq5ine esters in the presence of Kluyvera citrophila KY-3641 was described. Evidence has been published be suggesting that the physiological effects of penicillin on E. &may due to interaction with at least two specific targets, both of which are

response^.^'

104 -

Sect. I l l

-

Chemotherapeutic Agents

Butler, Ed.

enzymes. 78 The application of multiparameter regression analysis techniques to the design of semisynthetic penicillins was discussed at length.79 A monograph concerned mainly with chemical reactions and physiologi681 properties of natural and synthetic 6-lactams was published. LincomycinlClindamycin - The clinical efficacy of clindamycin81 and lincomycinBL against infections caused by nonspore-forming anaerobic bacteria was announced. Clindamycin was reported to inhibit 96% of 257 Bacteroides clinical isolates at 3.1 y/ml or less.83 A quantitative structure-activity study among lincomycin/clindamycin analogs suggests that the relative potency of members of these two sets of compounds depends only on the lipophilic nature of the pyrrolidine ring and not that of the molecule as a whole.84 Relative enzymatic hydrolysis rates for different positional and structural esters of lincomycin in dog serum and simulated intestinal fluid were determined as part of a search for a lincomycin congener with superior pharmaceutical and kinetic properties; -in vivo activity correlated with hydrolysis rates but no gross differences in esterase specificity were observed between dog serum and s 8:8.1' and ed intestinal fluid.85 Microbial transformations of clindamycin& N-demethylclindamycin88 by Streptomyces species, in general, lead to prodrug forms of the parent antibiotics.

-

Macrolides Stereochemical problems in the macrolide area were reviewed in depth.-Structures and interrelationships between the leucomycins, maridomycins, espinomycins,SF-837 and YL-704 macrolides were summarized.90 Two reports on syntheses of erythromycylamine indicate the 9-aminoerythromycin con ener can now be conveniently prepared in kilogram quantiA series of chalcomycin analogs were described several of which demonstrated in vitro and in viva activity against staphylococci comparable to the parent antibioticT3 Narbomycin aglycone, narbonolide , was isolated from a Streptomyces fermentation and its biogenetic relationship to narbomycin and picromycin established .94

tie^.^^,'^

Peptide antibiotics were reviewed with respect to biosynthesisg5 and the relationships between primary structure, conformation and antibacterial activity.96 The elusive structure of viomycin was further resolved.97 However, some doubt concerning the peptide sequence remains owing to an X-ray structure determination on closely related antibiotic tuberactinomycin 0 . 9 8 The 65lationship between viomycin and capreomycins 1 A and 1B was established. Tuberactinomycin N was reported to be as active as viomycin against experimental tuberculosis infections in mice and less ototoxic than both viomycin and capreomycin in guinea-pigs. loo The structure ofOfegamycin, a novel broad-spectrum hydrazide antibiotic, was solid-phase determined. An improved synthesis of gramicidin S synthesis and azide cyclization was carried out in 43-51% overall yield.Io2

-

Tetracyclines The disposition of doxycycline by man, dog and rats was investigated.103~104 Results in dog and rats indicate doxycycline passes relatively easily through the intestinal wall and is less

Chap. 10

Antibiotics

105

Sciavol ino

dependent upon normal r e n a l f u n c t i o n € o r i t s e l i m i n a t i o n t h a n o t h e r t e t r a c y c l i n e s of lesser l i p o p h i l i c i t y ; t h e s e f i n d i n g s are c i t e d t o h e l p explain t h e c l i n i c a l observation t h a t doxycycline u n l i k e o t h e r tetrac y c l i n e s does n o t accumulate i n p a t i e n t s w i t h r e n a l i n s u f f i c i e n c y ,105*106 Reports on s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s among t e t r a c y c l i n e a n t i b i d e r i v e d by quantum mechanical c a l c u l a t i o n s 1 0 7 and m i c r o b i a l k i n e t i c s were d e s c r i b e d . An improved s y n t h e s i s of minocycline 4MinocinR) u t i l i z i n g 9-nitro-6-demethyl-6-deoxytetracycline w a s r e p o r t e d , 10

18biCs

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A D i s c u s s i o n of P e n i c i l l i n and R e l a t e d A n t i b i o t i c s P a s t , Present and F u t u r e , E. Chain and J . S t o k e s , O r g a n i z e r s , London, May 20-21, 1971. P r o c . R. Soc. Lond. B., a) T. R. C a r r i n g t o n , 321; b ) D. H. R. B a r t o n and P. G. Sammes, 345; c ) J . L. S t r o m i n g e r , P. M. Blumberg, H. Suginaka, J. Umbreit and G. G. Wickus, 369; d ) G. N . R o l i n s o n , 403; e ) S . S h a l t i e l , R. M i z r a h i and M. S e l a . 411 (1971); f ) C l i n i c a l p a p e r s , J . Roy: C o l l . Phys. Lond. NO, 2

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124,

5. 6.

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10. 11 * 12. 13.

14. 15. 16. 17. 18.

14,

w.,

m.,

106 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.

Sect. I I I

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B u t l e r , Ed.

Chemotherapeutic Agents

D. J . Cooper, R. S. Jaret and H. Reimann, Chem. Comm., 285 (1971). H . Reimann, R. S . Jaret and D. J . Cooper, 924 (1971). S. Umezawa, T . Tsuchiya, R . Muto, Y. Nishimura and H . Umezawa, J . A n t i b i o t i c s (Tokyo), 274 (1971). H. Umezawa, S. Umezawa, T , Tsuchiya and Y. Okazaki, 485 (197l). 711 S, Umezawa, T . Tsuchiya, T . J i k i h a r a , and H. Umezawa, (1971). P . W. K. Woo, H. W. Dion and Q. R. B a r t z , T e t r a h e d r o n L e t t . 2617 (1971). P. W . K. Woo, 2621 (1971). P. W. K. Woo, H. W. Dion and Q. R. B a r t z , 2625 (1971). T. Oda, T. Mori and Y. Kyotani, J . A n t i b i o t i c s (Tokyo), 3, 503 (1971) T. Oda, T , Mori, Y. Kyotani and M. Nakayama, 511 (1971). T. Mori, Y. Kyotani, I. Watanabe and T. Oda, 25, 149 (1972). F. Kobayashi, M. Yamaguchi and S. M i t s u h a s h i , Antimicrob. Ag. Chemother., 1 7 (1972). S. M i t s u h a s h i , F. Kobayashi and M. Yamaguchi, J . A n t i b i o t i c s (Tokyo), 24, 400 (1971) M. B r z e z i n s k a , R. B e n v e n i s t e , J . Davies, P . J . L. D a n i e l s and J. W e i n s t e i n , Biochemistry, 761 (1972). F. Kobayashi, M. Yamaguchi, J . Eda, F. H i g a s h i and S . M i t s u h a s h i , J . A n t i b i o t i c s (Tokyo), 3, 719 (1971). R. B e n v e n i s t e and J . Davies, Febs L e t t . 293 (1971). F. LeGoffic, M. Chevereau and J . TrbfouEl, Compt. Rend. (C) , 274 535 (1972). M. Yagisawa, H. Naganawa, S. Kondo, N. Hamada, T. Takeuchi and H. Umezawa, J . A n t i b i o t i c s (Tokyo), 24, 911 (1971). H . Naganawa, M. Yagisawa, S . Kondo, T. Takeuchi and H. U m e z a w a , w . , 914 (1971). J. E. McLaughlin and D. S. Reeves, Lancet, I , 261 (1971). J . A. Waitz, C. G. Drube, E . L. Moss, J r . , E . M. Oden, J. B a i l e y and G. H . Wagman (Ref. 12) p. 66. L. R i f f and G. G. J a c k s o n , p . 65. P. Noone and J . R. P a t t i s o n , Lancet, 11, 575 (1971). W . R. Holder, D. P. R o b e r t s , W. C. Duncan and J , M. Knox (Ref. 12) p. 48. K. K. Holmes, A. H . B. Pedersen, P . J . Wiesner, L . T . Gutman and D. M. Bear, M.,p . 49. G. Binda, E. Domenichini, A. G o t t a r d i , B. O r l a n d i , E . O r t e l l i , B. P a c i n i , and G. Fowst, Arzneim. F o r s c h . , 21, 1907 (1971). 354 (1971). Anonymous, Drugs, G. P o o l e , P. S t r a d l i n g and S . Worlledge, B r i t . Med. J . 2, 343 (1971). K. S a s a k i . K. L. R i n e h a r t , J r . , F. J. h t o s z , J . A n t i b i o t i c s (Tokyo), 25, 68 (1972). K. L . R i n e h a r t , J r . , and F. J . Antosz, 7 1 (1972). A. H . J . Wang, I . C. P a u l , K. L. R i n e h a r t , J r . , and F . J . Antosz, J . Am. Chem. SOC., 93, 6275 (1971). K. L. R i n e h a r t , J r . , M. L. Maheshwari, F. J . Antosz, H. H . Mathur, K. S a s a k i and R . J . S c h a c h t , 6273 (1971).

u.,

24,

u., w,

-.,

w.,

m., m.,

L,

-

11,

14,

u.,

L,

-

u.,

u.,

Chap. 10

51. 52. 53. 54.

Antibiotics

Sciavol i n o

107

R. Nagarajan, L. D. Boeck, M. Gorman, R. L. H a m i l l , C . E . Higgens, M. M. Hoehn, W. M. S t a r k and J . G . Whitney, 2308 (1971). R. L. H a m i l l , C . B. Carrell, M. M. Hoehn, W. M. S t a r k , L . D. Boeck and M. Gorman (Ref. 1 2 ) p. 7. M. Gorman, M. M. Hoehn, R. Nagarajan, L. D. Boeck, E. A. P r e s t i , J . G. Whitney and R. L. H a m i l l , p. 7. E. 0. S t a p l e y , D . Hendlin, S . Hernandez, M. Jackson, J . M. Mata, A. K. Miller, H. B. Woodruff, T . W . Miller, R. T . Goegelman, R. G. Weston, I . P u t t e r , F. J . Wolf, G . Albers-Schonberg, B . H . Arison and J . L . Smith, p . 8. S. Karady, S. H . P i n e s , L. M. Weinstock, F. E . R o b e r t s , G. S . Brenner, A. M. Hoinowski, T. Y . Cheng and M. S l e t z i n g e r , J . Am. Soc., 2, 1410 (1972). L. D. Cama, W . J . Leanza, T . R. Beattie and B . G . C h r i s t e n s e n , -*i b i d ' 1408 (1972). E. H. W. Bohme, H. E. Applegate, B. T o e p l i t z , J . E . D o l f i n i and J . Z. Gougoutas, 93, 4324 (1971). J. E. Murphy, P r a c t i t i o n e r , 392 (1971). W. E. Wick, W. E. Wright and H . V . Kuder, Appl. M i c r o b i o l . , 21,

s.,

w.,

w.,

55. 56. 57. 58. 59.

w.,

206,

426 (1971). E. Binderup, W. 0. G o d t f r e d s e n , and K , R o h o l t , J . A n t i b i o t i c s (Tokyo), 24, 767 (1971). 61. J. L. Bran, M. E. Levison and D. Kaye, Antimicrob. Ag. Chemother., -1 35 (1972). 62. R. C. Gordon, F. F. Barrett, D. J . C l a r k and M. D . Yow, C u r r . Ther. R e s . , 13,398 (1971). 63. J, Axelrod, B. R. Meyers and S . Z . Hirschman, Appl. M i c r o b i o l . , 22, 904 (1971). 64. C. E. Cox and W. W. King (Ref. 12) p. 33. 65. R. E. Buck, F. L e i t n e r and K . E. P r i c e , Antimicrob. Ag. Chemother. -1, 67 (1972). 66. M. M i s i e k , T. A . P u r s i a n o , L. B. Crast, F. L e i t n e r and K . E . P r i c e , i b i d ' 54 (1972). 67. G. P . Bodey and D. S t e w a r t , Appl. M i c r o b i o l . , 2, 710 (1971). 68. K. T s u c h i y a , T . O i s h l , C. I w a g i s h i and T . I w a h i , J . A n t i b i o t i c s (Tokyo), 24, 607 (1971). 69. ( a ) A. R. E n g l i s h , J . A. Retsema, V . A. Ray and J . E . Lynch, (Ref. 12), p. 84; (b) K. B u t l e r , A. R . E n g l i s h , A. K. Knirsch and J . J . K o r s t , Delaware Med. J . , 43, 366 (1971). p . 43. 70. D. C. Hobbs, 71. G. R. Hodges and R. L . P e r k i n s , p . 32. 72. C. E. Cox, p . 33, 73. J. L. Bran, D. M. Karl and D. Kaye, C l i n . Pharmacol. T h e r . , 12, 525 (1971). 74. J. E. Beck, J . A. Hubsher and D. L . Caloza, C u r r . Thera. R e s . , 13, 530 (1971). 75. H. Smith and A. C. M a r s h a l l , N a t u r e , 232, 45 (1971). 76. A. C. Munro, J . M. Dewdney, H . Smith and A. W. Wheeler, J . Immun. 1971; i n p r e s s . 77. T. Nara, R. Okachi and M. Misawa, J . A n t i b i o t i c (Tokyo), 24, 321 (1971). 60.

w.,

m.,

w.,

108 78.

79. 80

81. 82. 83. 84. 85.

86. 87. 88. 89. 90.

91. 92. 93. 94.

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Chemotherapeutic A g e n t s

235,

R. Hartmann, J . V. Haltje and U. Schwarz, Nature, 426 (1972). A. E. Bird and J . H. C. Nayler i n "Drug Design," Vol. 11, E. J . AriOns, Ed., Academic P r e s s , New York, N. Y . , 1 9 7 1 , Chap. 8. M. S. Manhas and A. K. Bose, "$-Lactams N a t u r a l and S y n t h e t i c , ' ' P a r t I , Wiley-Interscience, New York, N. Y., 1971, R. M. Swenson, T. Michaelson and E . H . Spaulding, (Ref. 12) p. 50. J . G. B a r t l e t t and S. M. Finegold, p. 51. W. J . Martin, M. Gardner and J . A. Washington 11, Antimicrob. Ag. Chemother., 1,148 (1972). Y. C. Martin and K . R. Lynn, J . Med. Chem., 2,1162 (1971). M. J . Taraezka, J . Pharm. S c i . , 60, 1414 (1971). J. H. Coats and A. D. Argoudelis, J . B a c t e r i o l . , 459 (1971). A. D. Argoudelis and J . H. Coats, .I.Am. Chem. SOC., 93, 534 (1971). A. D. Argoudelis, R. D. Birkenmeyer, J. H. Coats and B. J . Magerlein, (Ref. 1 2 1 , p. 1 0 . W. D. C e l m e r , P u r e Appl. Chem., 413 (1971). Y . Yagieawa, J a p . Med. Gaz., 9, No. 2 (1972). G. H. T i m and E. Wildsmith, Tetrahedron L e t t . , 195 (1971). E. Wildsmith, M., 29 (1972). R. D. Westland and T . H. H a s k e l l , A b s t r a c t , 162nd N a t i o n a l Meeting of t h e American Chemical S o c i e t y , Washington, D . C . , S e p t . 13-17, 1971; MEDI-48. T. H o r i , I . Maezawa, N. Nagahama and M. Suzuki, Chem. Conrm., 304

u.,

108,

2,

(19 71)

.

D. Perlman and M. Bodanszky, Ann, Rev. Biochem., 40, 449 (1971). C. Toniolo, Famaco. Ed. S c i , , 26, 741 (1971). B. W. B y c r o f t , D. Cameron, L. R. C r o f t , A. Hassanali-Walji, A. W . Johnson and T. Webb, E x p e r i e n t i a , 27, 501 (1971). 98. H. Yoshioka, T. Aoki, H. Goko, K. Nakatsu, T. Noda, H . Sakakibara, T. Take, A. Nagata, J. Abe, T . Wakamiya, T . Shiba and T . Kaneko, Tetrahedron L e t t . , 2043 (1971). 99. B. W. B y c r o f t , D. Cameron, L. R. C r o f t , A. Hassanali-Walji, A. W. Johnson and T. Webb, Nature, 231, 301 (1971). 100. T. Ando, K, Matsuura, R. Izumi, T. Noda, T . Take, A. Nagata and J . Abe, J . A n t i b i o t i c s (Tokyo), 24, 680 (1971). 101. S. Kondo, S . S h i b a h a r a , S. Takahashi, K. Maeda, H . Umezawa and M. Ohno, J , Am. Chem. SOC., 93, 6305 (1971). 102. M. Ohno, K. Kuromizu, H. Ogawa and N . Izumiya, 5251 (1971). 103. M, Schach von Wittenau and T. M. Twomey, Chemotherapy, l6, 217 (1971). 104. M. Schach von Wittenau, T. M. Twomey and A. C. S w i n d e l l , 26 (1971). 105. C. Revaz, B. Goldenberg and H. A c h t a r i , Schweiz. Med. Wschr., 101, 1 2 7 (1971). 106. R. B. S c o t t , J. S. Cameron, C. S . Ogg and P . Toseland, Lancet, 11, 95. 96. 97.

w.,

E. 17, ,

-

1378 (1971). 107. 108, 109.

F. P e r a d e j o r d i , A. N. Martin and A. Cammarata, J . Pharm. S c i . , 60, 576 (1971). G. H. M i l l e r , S . A. K h a l i l and A. N . Martin, 60, 33 (1971). R. F. R. Church, R. E. Schaub and M. J . Weiss, J . Org. Chem., 36, 723 (1971).

m.,

Chapter 11.

Antifungal Agents

J. Allan Waitz, C. 0 . Drube Schering Corporation, Bloomfield, New Jersey

-

A review of the treatment of mycoses by use of Reviews antibiotics including griseofulvin, nystatin, amphotericin B, pimaricin, 5 -fluoro-cytosine, trichomycin, cycloheximide, clotrimazole and miconazole, also covers the source of the antibiotic, chemistry, in vitro effects, clinical dosages, A recent text,2 together with indications and side effects.l while not specifically related to antifungal agents presents aspects of mycology, of preservation techniques and detailed descriptions of methods related t o diagnosis and culture of dermatophytes and subcutaneous and deep seated mycoses.

-

A new animal model for acute oral candidiasis i n Methods rats may provide an additional system for the preclinical evaluation of new antifungal agents.= Experimentally induced blastomycosis and histoplasmosis tend to mimic natural infecUse of tions and are susceptible t o amphotericin B therapy.* analogue-resistant mutants t o increase levels of end products, has been directed towards biosynthesis of pyrrolnitrins Strains were utilized which were resistant t o 5-fluorotryptophane and 6-fluoro-tryptophane. These studies suggest a possible biosynthetic pathway far pyrrolnitrin i n which D-tryptophane and reduced pyrrolnitrin are intermediates; 3-chloroindole appears to be a by-product since it markedly inhibits pyrrolnitrin synthesis.5 The currently used procedure of cutting out antibiotic producing colonies from the agar i n plugs, and placing them onto bioassay plates has been modified for kasugamycin producing strains. In the "agar piece method" the colonies are cut out prior t o heavy growth, preventing extensive antibiotic diffusion.6

.'

f3-Alanine has been shown to increase the yield of antifungal antibiotic AYF produced by Streptomyces aureofaciens. 7 W light and X-ray generate low-producing mutants as well as high producing mutants of Streptomyces nigrifaciens, with reference t o antifungal production.8 mystatin has been successfully used in the selection of amino acid auxotrophs of Saccharomyces.s Neutron activation analysis for determination of chlorine has been utilized for an assay of griseofulvin i n capsules and in tablet f o r m a l o

-

Intravenous administration of amphoterClinical Experience icin B continues t o be the mpjor therapy for the treatment of systemic mycoses. nebulization of amphotericin B was used t o avoid some aspects of toxicity associated with intravenous use. Nebulization produced an aerosol with 80-90$ of the drug

110

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Chemotherapeutic Agents

particles smaller than 8 microns, a diameter which should be retained in the peripheral bronchi. The successful treatment of 4 patients with coccidioidomycosis by this method, without toxicity, is reported.11 Extracorporeal dialysis has been successful in extracting amphotericin B after intravenous administration in dogs, and in 3 patients with acute renal insufficiency.12 The successful treatment of a case of primary aspergilloma of the cheek due to Aspergillus flavus with intravenous amphotericin B has been reported.13 Rapid intravenous infusion of amphotericin B has been shown to produce higher peak serum levels of amphotericin than slow intravenous infusion, without greater t0xi~ity.l~ The enhancement of fungal keratitis seen with steroid anti-inflammatory agents was not observed in experimental Aspergillus keratitis of the cornea in rabbits when low levels of dexamethasone were combined with pimaricin. It appears that this topical combination may be effective and also avoid the aggravation of fungal keratitis observed with the steroids Pimaricin topically, in combination with the antitrichomonal agent metronidazole orally, appears useful in the elimination of Trichomonas and prevention of the Candida overgrowth which frequently follows antitrichomonal therapy.ls Topical pimaricin therapy of Candida vaginal infections and mixed Candida and Trichomonas infections yielded satisfactory results in approximately 805 of 286 women.17 Several additional clinical reports have appeared on the use of 5-fluorocytosine. The successful therapy of Cryptococcus meningoencephalitis when the drug was given at a level -rams per day for 45 days has been reported. Relapse occurred after withdrawal of the drug; cultures became positive and additional therapy was judged unsuccessful.1* Several other documented relapses while patients are under therapy with 5-fluoro-cytosine for cryptococcal meningitis have been described,” and, in some cases have been attributed to development of resistance during therapy. Oral doses of 2 to 5 grams a day of 5-fluorocytosine have been successful in reducing urinary counts of Candida in some instances, and have apparently eliminated urinary candidiasis in other cases.20 Serum binding of 5-fluorocytosine w a s calculated to be approximately 50%; however the fungicidal effect of 5-fluorocytosine on Candida was enhanced in vitro by the addition of serum to the media. Urine appeared to inhibit the in vitro action of 5 -fluorocytosine.2 O

--

--

Clotrimazole (Bay b 5097) has been the object of additional clinical and laboratory evaluation. In children with C. albicans pyelonephritis who were dosed at a level of 70-200 m g / m o r 10 t o 40 days, good response was noted in all patients although relapses frequently occurred after the

Chap. 1 1

Waitz, Drube

A n t i f u n g a l Agents

111 -

cessation of therapy.21 Contrary to initial reports, a recent study found a number o f z . albicans strains which were not sensitive to obtainable serum levels of clotrimazole.22 Further investigations have shown Candida to be more sensitive -in vitro than Cryptococcus.23 The in vitro activity of clotrimazole against Blastomyces, Histoplasma, Sporothrix, Cryptococcus and Coccidioides was shown to be comparable to amphotericin B, while its activity against Candida and Aspergillus was less than that of amphotericin B, Against dermatophytes clotrimazole had activity comparable to pyrrolnitrin2’ but less than tolnaftate2’ and greater than that of griseofulvin.24’25 Some in vitro activity against In gram-positive bacteria has also been demonstrated.25 guinea pigs experimentally infected with Trichophyton mentagrophytes, topical therapy with clotrimazole wa8 less successful than with tolnaftate; oral therapy gave results similar to that seen with gri~eofulvin,~’ In experimental infections in mice infected with Aspergillus, Blastomyces or Candida, therapy with clotrimazole was unsuccessful, however some protection was obtained in Cryptococcus and Coccidioides infections at high oral dose regimens.26 A drug induced mechanism for in vivo drug inactivation has been

--

--

Thiabendazole, at 25 mg/kg/day orally for 1.5 -22 months, appeared successful in 1/3 of 14 patients with chromomycosis. However, hypersensitivity reactions manifested by elevated serum bilirubin and transaminase levels developed in several patients.27 Griseofulvin continues to be the main oral therapy for a number of dermatophyte infections, and attempts to improve the speed and degree of absorption of griseofulvin following oral administration have continued. Dispersions of griseofulvin in a solid matrix of polyethylene glycol 6000, was demonstrated to lead to higher serum levels in man than those obtained with commercial micronized griseofulvin.2B The vasodilator effect of griseofulvin has been utilized in the treatment of Raynaud’s Phenomenon.29930 The use of dimethyl sulfoxide as a vehicle for griseofulvin has been shown to increase the penetration of griseofulvin into the subkeratinous tissue. Topical therapy with griseofulvin in DMSO was successful in 10 cats with Microsporum canise3’ Miconazole nitrate. previously shown to be effective in topical treatment of Trikhophyton infections, was effective when used topically for 1 to 3 weeks as a 2 % cream in 44 of 46 cases of Candida vaginitis, bbt it was without effect in 2 cases of vaginal Torulopsis glabrata.32

-

Tetranactin, a new macrotetrolid antiNew Antifungal Agents biotic similar to nonactin and produced by a strain of Strep-

112 -

Sect. I l l

-

Chemotherapeutic Agents

B u t l e r , Ed.

tomyces aureus shows i n vitro activity against some fungi and gram-positive bacteria as well as insecticidal activity. The Flavomycoin acute toxicity of tetranactin i n mice is is a new polyene antifungal, isolated from Streptomyces roseoflavus, containing a pentaene system in conjugation with a lactone carbonyl group. It has the partial formula, 1.34'35

'\CH-

I

Tetramycin variety of 34HS 3O14N yeasts and tetramycin

-

CH-CHs I

CHz -CH

I

is another new polyene antifungal produced by a Streptomyces noursei; it has an empirical formula and good in vitro activity against a variety of filamentous fungi. The acute intravenous L D 5 0 of in mice is 50 mg/kge3'

Three new antibiotics related t o phleomycin and bleomyc i n have been isolated from fermentations of' S t r e o t o m m e s bikiniensis variety zorbonensis These products hav; in-vitro activity against gram-positive and gram-negative bacteria as well as-various fungi.- The empirical formulae for the antibiotics are: zorbomycin, CS6H1 00N28024S2C~; zorbonomycin B, C HB 8O S2 C u ; z orb onomyc in C has not b ee n c omple t e ly A new antibiotic, characterized but also contains 24010, possible related t o tunicamycin has activity against a variety of phytopathogenic fungi, Candid8 and Saccharomyces.~8

.

,

Prumycin, has an empirical formula C ~ H l s N ~ 0 5it ; is a new water soluble, basic antibiotic active against some bacteria and fungi and it is produced by a strain of Streptornyces.3s Tunicamycin is a new antibiotic isolated from Streptomyces lysosuperificus which has activity against animal and plant viruses and gram-positive bacteria as well as yeasts and fungi.40 Hikizimycin, produced by a new Streptomyces strain has some antibacterial as well as antifungal activity and appears to be the first example of an antibiotic with cytosine and kanosamine as s ~ b s t i t u e n t s . 4 ~ Cladosporin, 2, is a new antifungal metabolite produced by Cladosporium cladosporioides; it has modest in vitro activity against a variety of' derma-

Chap. 1 1

tophytes

Antifungal Agents

OH

Waitz, Drube

0

HO Amongst a series of semi-synthetic $-lactam compounds, the cephalosporin derivative, 2, and the penicillin derivatives, k,z, showed unexpected antifungal activity i n vitro; compound 3 was ineffective, however, in an experimental systemic Cryptococcus neoformans infection i n S

4 R'

= CsHsOCH(CH3)CO;R"

-5 R'

= C6HsOCH2CO; R" = CHO

= COSK

k, 5 A series of substituted carbamate esters (6-14), have in vitro

R

-6 7

RS02

NHC02Ar

CH3 CH3 2HS

Ar

2,4,6 -tricholorophenyl pent ac hlor op henyl pentachlorophenyl pentachlorophenyl pentachlorophenyl pentachlorophenyl pentachlor ophenyl pent ac hlor op henyl pentachlorophenyl

1 I4 -

Sect.

Ill

-

B u t l e r , Ed.

Chemotherapeutic Agents

activity against C. albicans and Aspergillus niger. The most actibe were 9 and &. The corresponding trichlorophenyl A series of derivatives of 8, 10, 3 2 , 2 were inactive.44 griseofulvin analozes, derived from transformation of 5'formyl griseofulvin, 15 through 24, have good in vitro activity against a number of dermatophytes; 24 was more active 0

CH30

OCH3

R =

cH3

c1 24 -

-in vitro

than griseofulvin. All were without activity in considerably 1 ~ 9 sactive than griseofulvin a g a i n z M. canis in guinea pigs. A series of unsymetrical caTbonyl disulfides, RC (O)SSR', were tested vitro against Histoplasma capsulatum; the most active were those where both R and R' were short unbranched alkyl or unsubstituted phenyl moieties. No meaningful in vivo activity was observed in the series.*' In a large series of thiosemicarbazones and 4-substituted thiosemicarbazones made from through 30, have interesting heterocyclic aldehydes, vitro activity.47 X Y R

vivo except 20 which was

--

a

H

kx

CHoNNHCSNHR

I

Y

-

Chemical and Physical Studies of Antifungal Agents The previous total synthesis of antimycin A3 provided a diasteriomeric mixture. A total synthesis of natural antimycin A2 has

Chap. 1 1

A n t i f u n g a l Agents

W a i t z , Drube

115

now b e e n r e p o r t e d . 4 8 The s t r u c t u r e s of t h e a u t o o x i d a t i o n p r o d u c t s of f i l i p i n and l a g o s i n have been shown t o b e t h e . 4 9 The p o l y e n e s c o r r e s p o n d i n g t e t r a e n e e p o x i d e s 31 and x

OH OH R

OH

OH

OH

OH

OH

OH

t e t r i n A and t e t r i n B have been shown t o have s t r u c t u r e s 2 and 2 r e s p e c t i ~ e l y . ~ ~The ' ~ s~t r u c t u r e of l i p o x a m y c i n h a s

"1; OH R

HO

'OH

OH

CH3

0

CH3 HO

OH

2

R=H

2

R-OH

NH2 b e e n shown t o have s t r i i c t u r e 35.'* Versico lin , an a n t i f u n g a l a n t i b i o t i c i s 2, 3, 6 - t r i h y d r o x y - t o l u e n e , and t h i s h a s b e e n c o n f i r m e d by s y n t h e s i s a 5 3 0 0 OH 0

L i n e a r f r e e e n e r g y r e l a t i o n s , c o r r e l a t i n g d a t a from more t h a n 30 s t r u c t u r a l t y p e s of a n t i f u n g a l a g e n t s w i t h h y d r o p h o b i c and e l e c t r o n i c p a r a m e t e r s have b e e n f o r m u l a t e d . I n a number of t h e s e examples, t h e a n t i f u n g a l a c t i v i t y c l o s e l y p a r a l l e l e d a n t i b a c t e r i a l and h e m o l y t i c a c t i v i t y , s u g g e s t i n g t h a t t h e s e f u n g i c i d e s may b r i n g a b o u t t h e i r a c t i o n by membrane e f f e c t a . 5 4

116

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Chemotherapeutic Agents

Butler, Ed.

-

BiOlOgiCal Studies of Antifungal Agents The primary site of mentagrophytes appears to be the action of siccanin on succinate dehydrogenase system of terminal electron transport. This suggests that siccanin inhibits fungal growth by inhibiThe tion of the respiratory electron transport system.55 primary action of pyrrolnltrin against Microsporum gypseum has been shown to be blockage of the electron transfer between the flavo protein of NADH dehydrogenase and the cytochrome B of the respiratory chain.56 The mode of action of yemenimycin against 2. albicans is suggested to be interference with DNA synthesis. 5 7

r.

The polyenic antibiotic dermostatin which possesses i n vitro activity against dermatophytes and Candida has been re-examined with a view towards its in vitro and in vivo activity against Cryptococcus, Blastomyces and Histoplasma in a variety of test systems. Dermostatin was less active 2 vivo than amphotericin B against C. albicans and go capsulatum while the activities of the 2 antibiotics were comparable against C. neoformans and Blastomyces dermatitidis infections in mice.58

-

Saramycetin has been shown to retard the clearance of sulphobromophthalein (BSP) in man. This may be due to inhibition of the hepatic enzyme which conjugates BSP to reduced glutathione, or to the anticholeretic activity of saramyB has been shown to increase the perme~ e t i n . Amphotericin ~~ ability of frog skin to potassium, chloride and urea.60 It has been suggested that the differential activity of griseofulvin in vitro may against 2. mentagrophytes and Trichophyton rubrum -be useful in the differential diagnosis of these two specieso61 _In vitro studies with Saccharomyces, Candida and several strains of Cryptococcus have shown synergistic activity with a combination of amphotericin B and 5-fluorocytosineo~2 Natural resistance of Candida to nystatin and amphotericin B may not occur;however resistance can be developed in vitro to these agents as well as candicidin, pimaricin and filipin.63 REFERENCES 1. 2.

3. 4,

5. 60

R. Vanbreuseghem, Maroc. Med., 51, 156 (1971). C. Booth, Ed., "Methods in Microbiology", Academic Press, New York (1971). D. Adams and J. H. Jones, J. Dent. Res., 5, 643 (1971). J. W. Ebert, V. Jones, R. D. Jones, R. J. Weeks and F. E. Tosh, Mycopathol. Mycol. Applicata, 45, 285 (1971). R. P. Elander, J. A. Mabe, R e L. Hamill, and M. Gorman, Folia Microbiol., l6, 156 (1971). T. Ichikawa, M. Date, T. Ishikura and A. Ozaki, ibid., 16 218 (1971).

-,

Chap. 1 1

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117

Waitz, Drube

A. A. Abou-Zeid and A. E. A. Jousef, Zentralbl. Bakteriol., 126, 14 (1971). K. C. Basu Chaudhary and S o Gupta, Experientia, 3, 706

7. 8.

(1971) 9. 10.

. .

11

D. R. Thouvenot and C. M. Bourgeois, Ann. Inst. Pasteur, -9 120 617 (1971). M. Margosis, J. T. Tanner, and J. P. F. Lambert, J. Pharm. Sci., &, 1550 (1971). R. S. Eisenber and W. H. Oatway, Amer. Rev. Resp. Dis., 103

-9

f.

289 (1971

16 17o 18.

A. P. Barousse, C. DeBercovich, J. Firmat, and E. E. Malamud, Medecina (Buenas Aires), 26 (1971). V. Rodriguez, J. M. Bardwil, and G o P. Bodey, So. Med. J., 64, 396 (1971). B. T. Fields, Jr., J. H. Bates, and R. S . Abernathy, Appl. Microbiol., 22. 615 (1971). E. Newmark, A. C. Ellison, and H. E. Kaufman, Amer. J. Ophthalmol., 71, 718 (1971). I. Alteras, Brux. Med., 51, 501 (1971). U. Heynen, Z. Allgemeinmed., 47, 829 (1971). J. P. Beine, M. Lontie and J. Vandepitte, Brit. Med.

19

R. J. Fass and R. L. Perkins, Ann. Intern. Med., 74,

20.

Rb R. Davies and D. S. Reeves, Brit. Med. J.,

12

13

0

14

.

15

s,

J.,

1971, 2, 107 (1971).

535 (1971). 21. 22

.

-1,

557 (1971).

W. Marget and D. Adam, Acta Pediatr. Scand.,

1971

3,341

(1971) J. Bodenhoff, Acta Pathol. Microbiol. Scand. Section B.,

79, 345 (1971).

26.

W. Ritzerfeld and M. L. Venschott, Into Z. Klin. Pharmacol., lt, 204 (1971). S. Shadomy, Infect. Immun., 4, 143 (1971). J. A. Waitz, E. L, Moss, and M. J. Weinstein, Appl. Microbiol., 22, 891 (1971). S. Shadomy, Antimicrob. Agents Chemother. 1970, 169

27. 28.

M. A. H. Bayles, Arch. Dermatol., 476 (1971). W. L. Chiou and S. Riegelman, J. Pharm. Sci., 60, 1376

29

B. R. Allen, Lancet, 2, 840 (1971).

23.

24. 25 o

(1971)

-

30 0

31.

104,

(1971)

P. Naidoo, ibid, 2, 1590 (1971). H. B. Levine, J. M. Cobb, and R. H. Friedman, Sabouraudie,

8, 43 (1971).

32

33

34 35

0

P. Godts, P. Vermylen, and J. van Cutsem, Arzneim.Forsch., 21, 256 (1971). K. Ando, H. Oishi, S. Hirano, T. Okutomi, K. Suzuki, H. Okazaki, M. Sawada, and T. Sagawa, J. Antibiotics (Tokyo) 24, 347 (1971). R. Schlegel and H. Thrum, ibid, 24, 360 (1971). R. Schlegel and H. Thrum, ibid, 368 (1971).

z,

118 -

36

-

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'f . M. E.

K. D o r n b e r g e r , R. F h n e r , G. B r a d l e r ,

24,

a n d H.

Thrum,

172 (1971

37

ibid A. D .

38

M. Mizuno, Y. Shimojima, T. Sugawara, and I. Takeda,

-0,

Ed.

Butler,

Argoudelis,

Bergy,

and T.

R.

i b i d , 24,

Pyke,

543 (1971). i b i d . , 24, 896 (1971). S. H i g a s h i k a w a , K. Y a s u i ,

39 40.

i b i d , 3, 900 (197110 A. T a k a t s u k i , K. A r i m a ,

H. T e r a d a , a n d S. Kuyama,

a n d G.

Tamura,

&,

ibid.,

215

(1971)

0

41.

K. Uchida, T. I c h i k a w a , Y. Shimauchi, T. I s h i k u r a , a n d A.

42.

-, 24

M. MacLean,

ibid.,

747 (1971).

W. J. G o t t s t e i n , A . H. Eachus, P. F. Misco, L. C. Cheney,

43 44

Ozaki, i b i d . , 24, 259 (1971). S c o t t , W. van Walbeck, a n d W.

P. M.

.

45

. 47 48 . 46

a

M.

a n d K.

Misiek,

E.

Price,,

J. Med. Chem.,

(1971)

.

I. L a l e z a r i ,

H e G o l g o l a b , and M. Emami,

Fields,

H. Newman,

a n d R.

770

2,1123 i b i d . , 14,

ibid.,

(1971) T. L.

&,

B. A n g i e r ,

767 (1971) L. F i e l d , W.

5. Hanley,

I. McVeigh,

a n d 2.

14 202 (1971).

-¶

14, 252

D. M. Wiles a n d T. Suprunchuk, i b i d . , M. K i n o s h i t a , M. Wada, S. A b u r a g i , and J. A n t i b i o t i c s ,

Evans,

(Tokyo),

S.

ibid.,

(1971).

Umezawa,

24, 724 (1971).

R. W. R i c k a r d s , R. M. S m i z a n d B. T. G o l d i n g ,

3, 603 (1970).

ibid.,

R e C . Pandey, V. F. German, Y. Nishikawa, and K. L. R i n e h a r t , Jr., J. Amer. Chem. SOC., 2, 3738 (1971). K. L. R i n e h a r t , Jr., W. P. T u c k e r , a n d R. C . Pandey, ibid., 3747 (1971). H e A. Whaley, i b i d . , 91, 3767 (1971). R . W. R i c k a r d s , J. A n t i b i o t . , (Tokyo) 715 (1971). C . Hansch and E . J. L i e n , J. Med. Chem., 653 (1971). K. Nose a n d A. Endo, J. B a c t e r i o l . , 105, 176 (1971). D. T. Won J-S. Horng, and R. S. Gordee, i b i d , ,

a,

52

.

53. 54 55 56.

24, 14,

,

106,

168 (19717. 57. 58

I. R. S h i m i , J. A n t i b i o t . , (Tokyo), 24, 593 (1971). R. S . Gordee, T. F. B u t l e r , a n d N . N a r a s i m h a c h a r i , ibid

-09

24,

561 (1971).

D. A. Cooney, R e D. Davis, E. R . Homan, N. R a k i e t e n , U.

.

60. 61

62. 63.

V a n a t t a , B r i t . J. P . N i e l s e n , A c t a P h y s i o l , Scand., B, S c h a e p i, a n d G.

140 (1971 . _. -

R.

Pharmacol.,

&,

-

106 (1971).

M. O t c e n a s e k a n d J. Dvorak, Z e n t r a l b l . B a k t e r i o l . ,

217,

96 (1971)G. Medoff, M. Comfort, and G o S. Kobayashi, P r o c . Exp. B i o l . Med., 571 (1971). M. A. A t h a r a n d H. I. Winner, J. Med. M i c r o b i o l . ,

138,

(1971)

SOC.

2,

505

Chapter 12. Antiviral Agents Timothy H. Cronin, Pfizer, Inc., Groton, Connecticut Introduction - Interest was high in several areas of antiviral research during the past year. A s research continues it becomes clear that interferon is a complex story and most likely just one facet of a much larger immunological picture. No new non-polymeric interferon inducing drugs were discovered. A single human study with tilorone was reported. The role of RNA directed DNA-polymerase with regard to oncogenic viruses has been discussed' and a search for inhibitors of this enzyme as potential antiviral agents has focused on the ansamacrolides. Isoprinosine is claimed to have a range of antiviral activity in experimental laboratory infections and uncontrolled human trials claiming efficacy have been reported from Argentina. A notable advance is the discovery of a drug for the prevention of Marek's disease in poultry. Several reviews2-6 discussed the state-of-the-art during 1971. Interferon - Human interferon was shown to have a 1 mited but measurable activity against a number of strains of aden0virus.j The absence of interferon8 in serial serum specimens collected prior to and during the incubation and acute phase of transfusion associated hepatitis may in part be related to the chronicity of the disease. Human leukocytal interferon has been utilized in at least two controlled clinical trials. In the first trial9 it was administered to patients with confirmed influenza and found to rapidly and dramatically reduce the systemic symptoms (headache, dizziness, malaise) a to a lesser degree the respiratory tract symptoms. The second study" utilized interferon prophylactically and was conducted with children in Moscow during the "flu" season. There was a 45-60% reduction in the incidence of influenza and respiratory infections. Production of interferonll in volunteers infected with either partly attenuated influenza A2/Leningrad/65 or B/England/62 is greatest in those who develop most antibody. There was no correlation between amount of interferon produced and disease severity. The importance of antibody in an untreated infection has recently been demonstrated.12 Normal mice given a sublethal dose of vaccinia virus produce interferon and antibody. Mice immunosuppressed by cyclophosphamide treatment or whole body X-irradiation do not produce antibody and exhibit 100% mortality after vaccinia challenge even though interferon is still formed. Administration of antibody causes a marked reduction in mortality. On the other hand immunosuppressed mice are protected13 against lethal vaccinia infection when treated early enough with the interferon inducers poly I:C or Newcastle disease virus (NDV) Other "factors" which resemble or are identical with interferon have also been identified. A serum protective factor (SPF) has been isolated14 from mice infected with Langat virus. It was shown that SPF is not an immunoglobulin, but it is found in the globulin fraction, and it also differs from interferon in that it does not protect mice under 3 weeks of

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age or neonatally thymectomized mice up to 6 weeks of age against Langat virus infection. The species specificity of SPF was not examined nor was its effect against other viruses tested. Poly IC was found t o cause the formation of a colony inhibitor which appeared to be identical to interferon.15 The interaction of sensitized mouse peritoneal or splenic leukocytes with L-cells was found to release inteferon,16 An experiment conducted with mouse-monkey hybrid cells17 indicated that there were different genetic sites for the synthesis of interferon and the protein responsible for the antiviral state. This data is consistent with recent in vitro experiments18 conducted with actinomycin D. In these experiments cells were incubated with interferon for different time intervals, the interferon removed, and the cells incubated with actinomycin for 3 hours and then challenged with vesicular stomatitis virus (VSV) and plaques counted. There was no antiviral state produced when actinomycin was added prior to interferon, but the antiviral state was achieved and potentiated some 30-fold when actinomycin was added at 4-5 hours and became maximal (100-fold) when actinomycin was added at 5 112-6 hours. These data are consistent with the following scheme: Interferon

hrs*>

Antiviral Protep

4-6 hrs.

y 4-6 hrs >

Control Protein

\1

Antiviral State Similar experiments have also been carried out using poly 1C.l’ In further recent experiments2° a tissue antagonist of interferon was isolated which may be the control protein, because when it was added at 4-6 hours under the same experimental conditions described above, it decreased by 40 fold the antiviral state induced by interferon. Its activity is blocked by actinomycin D. The protection against MM virus afforded by NDV, statalon, endotoxin, pyran copolymer, poly IC or interferon is also antagonized by estrone.21 Interferon Inducers

1) Polynucleotides - Low titres of interferon were seen in 14 out of 20 human subjects given intravenous poly IC in doses up to 4 mg/kg. The chief clinical feature was a febrile response and there were no abnormalities reported in any of the clinical parameters studied. Subjects given a second injection of poly IC gave an interferon response, whereas repeated daily in ections caused a refractory state.22 Experiments with mice indicatei3 that the refractory state can be overcome to some degree by following a strict dosage regimen of either 25 mcg. or 200 mcg. of poly IC every 12 hours for 6 days. This practice was shown to give sustained levels (hundreds of units) of interferon. With rabbits,24 it was shown

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that there was no correlation between interferon response and febrile response to poly IC. In addition, prior administration of indomethacin markedly reduced the elevation of body temperature but was without effect on the interferon stimulation by poly IC. In animal protection studies has been poly IC at 1 mg/kg either intravenously or intramu~cularly~~ demonstrated to protect rabbits against up to 625 LD50 of rabies street virus, and to protect mice against Semliki Forest virus (SFV) infection26 when treatment (100 mg intraperitoneally) was begun as late as 5 days after infection when high titers of virus were already in the brain. It did not prevent infection in calves challenged with infectious bovine rhinotracheitis (IBR) but it did ameliorate the clinical systems of respiratory illness, when adminis ered at doses of 1 mg/kg intravenously and provided complete protection2! from the pneumonic lung lesions of IBR. An attempted cure of a single infant with herpes type 1 encephalitis using a dosage of 1 mg/kg of poly IC for 11 days, was discussed.28 Further toxicological studies with poly IC have shown it to have no adverse effect on HFL-1B cells.29 -In vivo experiments indicate a depression of zinc and iron concentrations in the serum of rats and rabbits but not monkeys;30 in acute and subchronic (42 days) studies using 2 mg/kg/day, poly IC was well tolerated by rodents but not by dogs;3I in calves it caused32 CNS depression and respiratory symptoms at doses of 0.1-1.2 mg/kg intravenously although at these doses very low titers of interferon were detected in the serum. Effects of poly IC on viral onco enesis have been widely studied and a review on the subject has a~peared.~' A recent study34 using a double-stranded RNA of fungal origin dramatically indicates the types of responses that are seen in such experiments. Innoculation of mice with Friend virus results in a progressive increase in spleen size, followed by eventual rupture and death. Administration of double-stranded RNA prior to or in the early stages of infection increases the severity of the disease, whereas treatment with the RNA beginning 5 days after infection causes a marked reduction in spleen size and a more normal histological appearance of the spleen. The treated mice remained in remission for the 6 week duration of the experiment. The beginning of treatment at day 5 corresponds to the time of rapid proliferation of cells which cause the spleens to increase in size. There have been some interesting probes into the structural requirements for activity with double-stranded polynucleotides. Greater in vitro antiviral activity than is found with poly IC can be obtained by the sequential addition of the single-stranded homopolymer constituents;35 e.g. addition of poly I followed at 10 min or even 1 day by poly C causes enhanced antiviral activity as does the addition of poly C followed at 1 min or 10 min by poly I. In vivo the homopolymers must be injected intravenously within 1 minute of each other to achieve interferon levels even comparable with poly IC. This may well be a reflection upon the metabolic stability of single double-stranded RNA. Incubation of poly IC or even the alternating ribonucleotide copolymer poly r (A-U) at 37" in

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Eagles minimal essential medium causes an enhancement of interferon ~ poly IC in which the inducing activity.36 A systematic ~ t u d y 3of molecular weight of only one of the homopolymers was varied at a time leaving the other component of the duplex intact showed that antiviral activity and toxicity (mouse LD50) decreased in parallel as the molecular weight of the variable molecular weight component was decreased. A convenient method for molecular weight determination of polynucleotides is also described.37 A later study with poly C (Ip5)I confirms these results.38 Complexes of poly IC and DEAE-dextran are known to enhance the potency of the former. It was concluded39 from a study of this effect using various concentrations of DEAE-dextran that optimal activity was achieved when the complex aggregates, and that this facilitates uptake by cells and protects against endonucleases. These same authors showed40 that poly I-poly (1-vinylcytosine) is equivalent to poly IC in inducing interferon on human skin fibroblasts and postulated that its reduced chargelmass ratio and tendency to aggregate causes enhanced cellular uptake leading to activity. Additional double-stranded RNAs were discovered to be interferon inducers. The activity of a DNA-RNA hybrid synthesized from singlestranded DNA of fl phage was demonstrated by complement f ation techniques to be entirely due to contaminanting double-stranded RNA." Other ctive double-stranded RNAs were isolated from normal mammalian cells.42,43 Finally, it is claimed that a double-stranded RNA equivalent in potency to poly IC, could be produced on large scale using a restrictive E. & strain and a mutant f2 phage.44 S tat01on~~ administered intranasally protected mice against influenza, and was shown to produce interferon in the trachea and lungs. It was non-antigenic, and its protective effect lasted for at least 1 week.

-

was found to 2) Other Polymers Chlorite oxidized oxyamylose protect mice against foot and mouth disease virus but did not protect swine against the natural infection. In a hog cholera infection COAM provided no increase in survival time but did delay the onset of disease symptoms. Circulating interferon was not detected in swine treated with COAM. Although COAM given prophylactically to ce afforded some protection against influenza A/PR8, it was concluded'' that it acts by triggering host response & interferon. and not y Phosphomannans isolated from several species of yeast48 are a new class of relatively non-toxic (acute LD50) interferon inducers. They cause formation of lower amounts of interferon in the rabbit than poly IC, but have similar induction kinetks. The materials retained their activity after digestion with beef pancreatic ribonuclease, thus ruling out the presence of RNA. 3 ) Tilorone - The bulk of the biological studies reported in preliminary form last year have now been confirmed by others.49 Additional analogs possessing activity similar to tilorone were also described.50 The first clinical experiments were described5I this year. This study examined the

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interferon response after administration of tilorone to the eyes of 10 subjects (1 drop of 200 mg/ml solution), and 1.0 g orally to 3 subjects; no interferon was detected by either route, and it was observed that the drug was deposited in the corneal epithelium. This deposit required up to 2 months after drug administration for complete disappearance. With the exception of some gastrointestinal irritation, no other adverse effects were noted. Tilorone did not induce circulating interferon in the chick, nor did it afford protection against Avian influenza.52 In this test statolon was effective but poly IC was not. Natural Products Rifampicin when applied as a 10% ointment or 15% cream to the vaccination sites of human subjects prevented a positive reaction in about 50% of the cases.53 With the use of the cream formulation a decrease in immune response was noted in all subjects as measured by seroconversion. There was no antiviral activity by the oral route. In animal experiments, oral rifampicin had no effect against herpes simplex, Mengo virus, intranasal nfluenza A/PR 8 or against intracerebral vaccinia infections of mice. 5i However, a significant dose-related reduction of tail lesions in vaccinia infected mice was demonstrated; the optimum regimen was 250 mg/kg of rifampicin beginning 24 hours prior to infection and continuing for 4 days.

-In vitro studies55 of anti-vaccinia activity have been carried out with a series of rifamycin derivatives and with their corresponding aminopiperazine side chains. Several of the side chains per se were claimed to be more active than the rifamycin. These effects could not be demonstrated by other workers.56 It was shown by plaque assays and by morphological changes in cells, that the side-chain of rifampicin had no effect on the course of virus infection. Several mechanisms of antiviral action have been proposed57 ,58 for rifamycin derivatives but the greatest interest centers around the inhibition of RNA-directed DNA polymerase. Several rifamycin derivatives but not rifampicin itself exhibit greater than 50% inhibition5’ of the EWA-directed DNA polymerase of Moloney sarcoma virus (MSV) at concentrations of 50-100 mcg/ml. Rifampicin inhibits60 focus formation by Rous sarcoma virus in chick embryo fibroblasts, but it was postulated that this effect was due primarily to the inhibition of cell proliferation that was also seen. A similar situation was described6I for the l-amino-2,6-dimethyl-benzylpiperazine derivative of rifamycin which, in addition to totally inhibiting focus formation and virus replication of MSV, was also found to reduce cell proliferation by 60%. With a more highly purified sample of drug, however, there was inhibition of viral replication and cell transformation, but there was no decrease in cell proliferation. The streptovaricins, which are also ansamacrolides, appear to elicit effects similar to the rifamycins. Inhibition of focus formation by MSV was demonstrated62 with streptovaricins at doses 4 times lower than the cytotoxic dose. It is speculated that the inhibition of cell transforma-

124

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tion is due to inhibition of the viral DNA polymerase. Streptovaricin when administered at a dose of 1.5 g/kg/day in feed to leukemia virus infected mice, caused a 50% reduction in spleen weight.63 Virus replication in the spleen was unaffected. Since there was no reduction of spleen weight in mice with a non-viral transplantable tumor (L1210) strain, it was reasoned that streptovaricin did not inhibit malignant cell proliferation and therefore presumably inhibited a virus specific event in cell transformation such as the reverse transcriptase. Some miscellaneous natural products which have exhibited limited in~ ~vitro vs. NDV; fusidin65 in vitro,vs. antiviral activity are f i l i ~ i n--influenza A/PR 8; melanacidins66 (from the 3,6-epidithiadiketopiperazine vivo vs. herpes virus; tunicamycin67 in vitro ~6 NDV; antibiotics group) from starf*?n vitro vs influenza; L - a s p a r a z n a in vitro and in viva (rabbit eye) ~6 vaccinia and myxoma; colchicine, demecolcine, vinblastine and vincristine70 vivo (rabbit eye) ~6 herpes simplex. A clinical trial with vitamin C 7 m 40 subjects challenged with a cold virus has been carried out but the results have not been published in the scientific literature. Nucleosides - It appears that there is increasing use of cytosine arabinoside in varicella infections in leukemic children. A dramatic improvement in a disseminated varicella infection in a leukemic patient was achieved72 with cytosine arabinoside administered daily by the intravenous route at doses of 100 mg/meter2. Most recently, it was reported73 that best results are obtained by continuous intravenous infusion at doses of 10-40 mg./ meter2. It was noted that if the patient survives for 48-72 hours there is a good response to the drug. High doses of cytosine arabinoside (100 mg/kg intraperitoneally) were demonstrated74 in a toxicology study to cause malformation of embryos in pregnant rats. Reviews on the use of idoxuridine in herpes simplex75 and herpes h ~ m i n i svirus ~ ~ infections have been published.

-

Isoprinosine (NPT-10381) It is proposed’17 that the anti-influenza activity of isoprinosine (the p-acetamidobenzoate salt of the 1:3 complex of inosine and dimethylaminoisopropanol) is due to an alteration of ribosomes such that they are more selective for host mRNA and less selective for viral mRNA. Isoprinosine administered to mice 24 hours after infection with influenza virus provided protection against mortality78 and with sublethal virus innocula it decreased the respiratory morbidity. In experiments79 with herpes, vaccinia, polio and echovirus there was no reduction in cytopathic effect but there was a reduction in virus yield. -In vivo experiments79 indicated a decrease in mortality from herpes corneal infection in hamsters and a reduction in tail lesions in vaccinia infected mice. No protection was afforded against encephalomyocarditis in the mouse. A controlled study80 of the effect of isoprinosine (1 g q.i.d.) in subjects with viral hepatitis indicated that hepatitis-associated antigen (HAA) positive patients do not respond to the drug, but HAA negative patients improved considerably (liver function parameters) relative to

Chap. 12

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125

the placebo group. Isoprinosine (4-6 glday) was shown81 to be of no value in the treatment of amyotropic lateral sclerosis. A large uncontrolled study82 of isoprinosine (40 mglkglday in children; 50 mg/kg/day in adults) in influenza, rhinovirus, mumps and measles was claimed to ameliorate the systemic symptoms (fever, headache, malaise) and to reduce the time and intensity of the specific disease symptoms.

-

when administered continuously in feed at a level of 0.0002% (1) was effective83 in preH 2 N ~ S 0 2 ~ ~ C O N venting H 2 the mortality and tumor development in birds infected with Marek's 1 disease.

Marek's Disease

p-Ureido-p'-aminodiphenylsulfone

-

Amines Double-blind trials were conducted with cyclooctylamine hydrochloride administered intranasally for 2 days prior to and for 7 days following nasopharyngeal challenge with 64,000 TC ID50 of influenza, A2IHK. The drug was effective in reducing the severity of the disease as measured by reduced febrile responses, lower virus recovery and reduced serum antibody titer. Structure-activity relationships within the amantadine series have been discussed.84 Miscellaneous Synthetics - Florenal (undisclosed structure) was shown to have in vitro and in vivo activity against herpes virus.85 An uncontrolled clinical trial in a large number of patients claimed85 dramatic results in the treatment of herpetic keratitis, adenovirus conjunctivitis and epidemic keratoconjunctivitis. 5-Fluoro-2-(a-hydroxybenzyl)-l-propylbenzimidazole was reported86 to be 10 times more potent than the unfluorinated parent against polio and coxsackie virus in human cell monolayers. Derivatives of the related benzoxazole and benzothiazole series are inactive.a7 N-Methyl and N-e thy1 isatin 6-thiosemicarbazone were shown88 to directly inactivate the Rous sarcoma virus in vitro. Methisazone was inactive against chickenpox and infectious mononucleosis in a controlled clinical trial.89 5-Cyanothiophene-2-aldehyde thiosemicarbazone is reported90 to be more active than methisazone. The L-enantiomer of cytosine arabinoside was inactiveg1 in vitro vs herpes virus. The 02, 02' anhydroderivative of c tosine arabinoside did have anti-herpes activity in vitro and in vivo .81 1-(3,5-Dihydroxy-4-nitrobenzy1)-morpholine was reportedg2 to be active in the mouse against encephalomyocarditis. References

1. D. W. Allen and P. Cole, N. Eng. J. Med., 286, 70 (1972). 2. S. Z. Hirschman, Am. J. Med., 51, 699 (1971). 3 . D. L. Swallow, Progress in Medicinal Chemistry, 8, 119 (1971). . 0 , 210 (1971). 4. D. C. Brown, Invest. Ophthalmology, l

126 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.

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C. Stuart-Harris, Brit. Med. J., I, 387 (1971). K. H. Schmidt-Ruppin, Chemotherapy, 16,130 (1971). J. G. Gallagher and N. Khoobyarian, Proc. SOC. Exptl. Biol. Med., 136 920 (1971). D. A. Hill, J. H. Walsh and R. H. Purcell, Proc. SOC. Exptl. Biol. Med., 136 853 (1971). L. A. Kursinova, E. A. Bukharina, G. I. Makarova and L. I. Neklyudova, Vopr. Virusol, 194 (1971). G. P. Busuek, I. N. Gailonskaya, T. M. Lozinskaya, V. P. Banina, N. I. Zhurnikova and M. A. Tsiprina, Vopr. Virusol, 226 (1971). A. A. Smorodintsev, A. S . Beare, M. L. Byone, B. Head and D. A. J. Tyrrell, Archiv Ges. Virus Forsch., 33, 9 (1971). Federation of American Societies for Experimental Biology, Chicago, Illinois (April 12-17) Abstracts, M. Worthington and S. Baron, p 241 (1971). M. Worthington and S . Baron, Proc. SOC. Exptl. Biol. Med., 136 349 (1971). I. S . Thind and W. H. Price, J. Immunol., 106 1559 (1971). T. A. McNeill and W. A. Fleming, Immunology, 2 761 (1971). L. Borecky, N. Fuchsberger, J. Zemla and V. Lackovic, Europ. J. Immunol., 1, 213 (1971). R. Cassingzna, C. Chany, M. Vignal, H. Suarez, S. Estrade and P. Lazar, Proc. Natl. Acad. Sci., 68 580 (1971). C. Chany, F. Fournier and S . Rousset, Nature New Biology, 230, 113 (1971). Y. H. Ke and M. Ho, Proc. SOC. Exptl. Biol. Med., 136 365 (1971). F. Fournier, C. Chany and M. Sarragne, Nature New Biology, 235, 47 (1972). D. J. Giron, P. T. Allen, F. F. Pindak and J. P. Schmidt, Infect. and Immunity, 3, 318 (1971). A. K. Field, C. W. Young, I. H. Krakoff, A. A. Tytell, G. P. Lampson, M. M. Nemes and M. R. Hilleman, Proc. SOC. Exptl. Biol. Med., 136, 1180 (1971). C. E. Buckler, H. G. DuBuy, M. L. Johnson and S. Baron, Proc. SOC. Exptl. Biol. Med., 136,394 (1971). E. Piperno, A. K. Field and A. A. Tytell (ref. 12) p 242. P. Fenje and B. Postic, J. Infect. D i s . , 123,426 (1971). M. Worthington and S . Baron, Proc. SOC. Exptl. Biol. Med., 136 323 (1971). K. W. Theil, S . B. Moharty and F. M. Hetrick, Proc. SOC. Exptl. Biol. Med., 137 1176 (1971). J. A. Bellanti, L. W. Catalano, Jr., and R. W. Chambers, J. Pediatrics, 78 136 (1971). C. L. Baugh, A. A. Tytell and M. R. Hilleman, Proc. Soc. Exptl. Biol. Med., 137 1194 (1971). R. S. Pekarek, Proc. SOC. Exptl. Biol. Med., 136 584 (1971). B. M. Phillips, R. E. Hartnagel, P. J. Kraus, R. P. Tamayo, E. H. Fonseca and R. L. Kowalski, Toxicology and Applied Pharmacology, 18 220 (1971). B. D. Rosenquist, Am. J. Vet. Res., 32, 35 (1971). C. W. Young, Med. Clin. of North America, 55, 721 (1971).

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34. D. J. F. Pilch and D. N. Planterose, J . Gen. Virol., 10 155 (1971). 35. E. De Clercq and P. De Somer, Science, 173 260 (1971)T 36. E. De Clercq, R. D. Wells, R. C. Grant and T. C. Merigan, J . Mol. Biol. , 56 83 (1971). 37. J . F. Niblack and M. B. McCreary, Nature New Biology, 233, 52 (1971). 38. P. M. Pitha and W. A. Carter, Nature New Biology, 234 105 (1971). 39. P. M. Pitha and W. A. Carter, Virology, 45 777 (1971). 40. J. Pitha and P. M. Pitha, Science, 172 1146 (1971). 41. C. Colby, B. D. Stollar and M. I. Simon, Nature New Biology, 229 172 (1971). 42. E. De Maeyer, J. De Maeyer-Guignard and L. Montagnier, Nature New Biology, 229 109 (1971). 43. R. Stern and R. M. Friedman, Biochemistry, 10 3635 (1971). 44. J . Doskocil, N. Fuchsberger, J. Vetrak, V. Lackovic and L. Borecky, Acta virol. , 15 523 (1971). 45. W. J. Kleinschmidt and F. Streightoff, Infect. and Immunity, 4 738 (1971). 46. J. Leunen, J. Desmyter and P. De Somer, Applied Microbiology, 21 203 (1971). 47. A. Billiau, J . J . Muyembe and P. De Somer, Applied Microbiology, 21 580 (1971). 48. S. Suzuki, M. Suzuki and M. Imaya, Japan J . Microbiol., IS, 485 (1971). 49. E. De Clercq and T. C. Merigan, J . Infect. Dis., 123 190 (1971). 50. Eleventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlantic City, N.J. (Oct. 19-22) Abstracts, R. F. Krueger, G. D. Mayer, K. P. Camyre and S. Yoshimura, p. 26 (1971). 51. H. E. Kaufman, Y. M. Centifanto, E. D. Ellison and D. C. Brown, Proc. SOC. Exptl. Biol. Med., 137 357 (1971). 52. J. Portnoy and T. C. Merigan, J. Infect. Dis., 124 545 (1971). 53. A. Moshkowitz, N. Goldblum and E. Heller, Nature, 229 422 (1971). 54. H. MUckter, H. Saus, G. Poszich and J. Nijssen, Arzneim-Forsch., 21 1571 (1971). 55. G. Lancini, R. Cricchio and L. Thiry, J. Antibiotics, 24, 64 (1971). 56. E. A. C. Follet and T. H. Pennington, Nature, 230, 117 (1971). 57. B. Mass, E. N. Rosenblum and P. M. Grimley, Virology, 45 123 (1971). 58. B. G. T. Pogo, Virology, 44 576 (1971). 59. C. Gurgo, R. K. Ray, L. Thiry and M. Green, Nature New Biology, 229 111 (1971). 60. H. L. Robinson and W. S. Robinson, J . Natl. Cancer Inst., 46 785 (1971). 61. M. Calvin, V. R. Jass, A. J . Hackett and R. B. Owens, Proc. Natl. Acad. Sci., 68 1441 (1971). 62. W. A. Carter, W. W. Brockman and E. C. Borden, Nature New Biology, 232 212 (1971). 63. E. C. Borden, W. W. Brockman and W. A. Carter, Nature New Biology, 232 214 (1971). 64. R. J . Ash, Virology, 43 536 (1971). 65. T. Ya Goncharskaya and S. M. Navashin, Antibiotiki, 16 57 (1971). 66. A. D. Argaudelis and F. Reusser, J. Antibiotics, 24 383 (1971). 67. A. Takatsuki and G. Tamura, J . Antibiotics, 24 232 (1971). 68. Y. Shimizu, Experientia, 27, 1188 (1971).

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83.

84. 85. 86. 87. 88. 89. 90 91. 92.

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R. Maral and G. H. Werner, Nature New Biology, 232 187 (1971). T. Tokumaru and A. Avitabite, Proc. SOC. Exptl. Biol. Med., 137 29 (1971). Chemical Week, February 10, 1971, p. 50. D. Prager, M. Bruder and A. Sawitsky, J. Pediatrics, 78 321 (1971). Medical World News, January 7, 1972, p 51 E. J. Ritter, W. J. Scott and J. G. Wilson, Teratology, 4, 7 (1971). H. Ashton, E. Frenk and C. J.: Stevenson Brit. J. Dermat., 84 496 (1971). A. Luger, Z. Haut-Geschl. Kr., 46 399 (1971). P. Gordon, E. R. Brown and B. Rorsen (ref 12.) p. 242. E. R. Brown and P. Gordon (ref. 12) p. 242. T. W. Chang and L. Weinstein, (ref. 50) p 29. E. A. Dainko (ref. 50) p. 30. A. K. Percy, L. E. Davis, D. M. Johnston and D. B. Drachman, N. Eng. J. Med., 285 689 (1971). J. Ink, F. J. Andres, G. M. Antonini, V. Scarpiello, J. C. Stefano, N. Coronel, M. G. Genoves, R. Spataro, C. Natelli, 0. Buey, R. L . Ink, I. Goijman, N. Pernas, R. J. Andres, 0. R. Sumary, I. Casol and 0. Cudemo, Prensa Medica Argentina, 57 2050 (1971). 162nd American Chemical Society Meeting, Washington, D.C. (Sept. 12-17), Abstracts, T. Y. Chen, D. B. R. Johnston, N. P. Jensen, W. V. Ruyle, J. J. Friedman, M. W. Fordice, J. F. McPherson, K. H. Boswell, T. A. Maag, R. W. Burg, R. M. Pellegrino, M. E. Jewell, C. A. Morris, H. L. Easterbrooks and B. J. Skelly, P MEDI-44 (1971). P. E. Aldrich, E. C. Hermann, W. E. Meier, M. Paulshock, W. W. Prichard, J. A. Snyder and J. C. Watts, J. Med. Chem., 14, 535 (1971). Y. F. Maichuk, Investigative Ophthalmology, 10, 408 (1971). D. G. O'Sullivan, C. M. Ludlow and A. K. Wallis, Experientia, 27, 1025 (1971). F. Gualtiere, G. Brody, A. H. Fieldsteel and W. A. Skinner, J. Med. Chem., 14 546 (1971). W. Levinson, B. Woodson and J. Jackson, Nature New Biology, 232, 116 (1971). A. K. Sinha, Brit. J. Clinical Practice, 25, 177 (1971). Phannascope, Investigational Drugs, Dec. 1971; from Seventh Int. Cong. Chemother. Prague, Czechosl. Aug. 71, abst. A-5/13. D. T. Gish, G. L. Neil and W. J. Wechter, J. Med. Chem., 14,882 (1971). Unlisted Drugs, 24, 3 (1972).

Chapter 13.

A n t i n e o p l a s t i c Agents

C . C. Cheng, Midwest Research I n s t i t u t e , Kansas C i t y , M i s s o u r i 64110

I n t r o d u c t i o n - An account of a conference on t h e N a t i o n a l Cancer I n s t i t u t e chemotherapy program has been p u b l i s h e d . Topics i n c l u d e d s c r e e n i n g , sel e c t i o n of a g e n t s f o r s c r e e n i n g , c e l l k i n e t i c s and chemotherapy o f c a n c e r , t r e a t m e n t schedule dependency o f experimental a n t i l e u k e m i c (L1210) d r u g s , l a b o r a t o r y of t o x i c o l o g y , drug development programs o f t h e N a t i o n a l Cancer I n s t i t u t e , c l i n i c a l t r i a l s and combination chemotherapy, and s u p p o r t i v e c a r e i n cancer therapy. I n a d d i t i o n t o t h e s u b j e c t s which a r e p r e s e n t e d i n t h i s r e v i e w , o t h e r s t u d i e s beginning t o have an impact on t h e approaches t o cancer chemotherapy i n c l u d e molecular o r b i t a l c a l c u l a t i o n s and mathematical c o r r e l a t i o n s of l i p o p h i l i c parameters and p a r t i t i o n c o e f f i c i e n t measurements of a g e n t s of b i o l o g i c a l importance. Human p i t u i t a r y growth hormone, p r o s t a g l a n d i n s , c y c l i c AMP, RNA-dependent DNA polymerase--their f u n c t i o n , r e l a t i o n s h i p , immunological a s p e c t s , and i n f l u e n c e on o t h e r hormones, Poly A:U, and tumor growth--also show promise. n

A l k y l a t i n g Agents - 5-Aziridinyl-2,4-dinitroben~amide~ ( I ) has e x c e p t i o n a l a c t i v i t y a g a i n s t t h e Walker carcinoma. I t s a n t i t u m o r a c t i v i t y i s r e v e r s e d by 4-amino-5 -imidazolecarboxamide. Compound 1 i s s i m i l a r t o melphalan ( L - s a r c o l y s i n ) i n i t s e f f e c t s on DNA and RNA s y n t h e s i s . An azo compound 2 , formed i n vivo from two molecules o f 1, h a s been proposed a s a p o s s i b l e a c t i v e form.

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Suppression of c e l l membrane t r a n s p o r t p l a y s a r o l e i n t h e r e s i s t a n c e t o n i t r o g e n mustard (HN2) by t h e Yoshida sarcoma c e l l s 3 . Metabolism of cyclophosphamide (?a, cytoxan) by r a t h e p a t i c microsomes has been r e p o r t ed4. The a c t i v e m e t a b o l i t e & i s formed by a t h r e e - s t e p enzymatic r e a c t i o n .

0 II,NH2 N-P, C 1 CH2 CH2’ 6 O-CH2 -CH2 -C02H C1-CH2 -CH2,

-3a. b.

X = CH2 X = CO

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Butler, Ed.

4-Ketocyclophosphamide (2b) is an inactive metabolite.5 Significant synergistic action of potassium iodide on the therapeutic effect of 2 was not confirmed.6 Busulfan appears to be the treatment of choice for chronic mye locytic leukemia. 9

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5-Azacvtidine 5-Azacytidine inhibits the incorporation of tritiated thymidine or deoxyadenosine into DNA to a greater extent than it does tritiated uridine into RNA. The inhibition can be nullified by cytidine, or by uridine, but not by deoxycytidine or deoxyuridine. 5-Azacytidine inhibits the S phase (DNA synthesis) of the cell cycle.’ The activity of uridine kinase in mouse leukemia cells resistant to 5-azauridine is reduced to ca. 50% of that in the sensitive cells.lO,ll This enzyme is responsible for the initial phosphorylation step of uridine, cytidine, and 5-azacytidine

.’

Cytosine Arabinoside (1-B-D-arabinofuranosylcytosine, ara-C, cytarabine) Enzymatic phosphorylation studies confirm that ara-C and its phosphorylated derivatives are deoxycytidine antagonists rather than cytidine antagonists.I2 Inhibition of DNA polymerase activity is considered more significantl.3 to antineoplastic properties than either blocking the uptake of thymidinel4 or the inhibition of the conversion of cytidine diphosphate to deoxycytidine diphosphate. Ara-C rapidly kills S phase cells. It affects the passage rate of cells from S to G2 phase more than from G1 to s15.

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Ara-C is effective against acute lymphocytic and myeloblastic leukemia,l6 and also in the palliation of advanced epidermoid carcinoma of the head and neck but duration of therapeutic benefit is brief.17 Unlike 5-FU and MTX, ara-C does not cause mucositis. Recovery of cellular colony-forming unit is much faster after ara-C treatment than with BCNU.la A sensitive test for detecting ara-C in plasma has been developed.19 The 5’-adamantoate of ara-C has been studied as a depot form of ara-C.20 Many 5’-esters of ara-C possess immunosuppressive and antileukemic activities .zl The 3-N-oxide derivative retains antileukemic action against leukemia L-1210 ,22 but 1-B-L-arabinofuranosylcytosine, the enantiomer of ara-C, has no antineoplastic a ~ t i o n . ~ ~ , ~ ~

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5-Fluorouracil and Related Pyrimidines 5-Fluorouracil (5-FU) might be more effective in patients with liver metastases when given orally, due to high drug concentrations in the portal system, Oral dosage with 5-FU is safe and effective in metastatic colorectal carcinoma.25 5-FU enhances the antitumor effect of irradiation in bronchogenic carcinoma.26 It supports hepatic tyrosine aminotransferase (TAT) induction by altering in vivo turnover of glucocorticoid-induced TAT .27 Catabolism of 5-FU is delayed by the presence of 5-cyano~racil.~~ 5-FU

Chap.

13

A n t i n e o p l a s t i c Agents

Cheng

131 -

i n combination with an a n t i c o a g u l a n t (warfarin) i n c r e a s e s t h e antitumor e f f e c t .29 2 '-Deoxy-5-(trifluoromethyl)uridine (F3TDR) induces remissions i n a c u t e leukemias. Doses of F3TDR causing leukopenia i n p a t i e n t s a r e n o t accompanied by g a s t r o i n t e s t i n a l t o x i c i t y . 3 0 Prolonged bone marrow depress i o n has hampered the good i n h i b i t o r y a c t i v i t y of F3TDR i n p a t i e n t s with b r e a s t and colon cancer .31 5-Bromo-2 I-deoxyuridine i s incorporated i n t o c e l l u l a r DNA, b u t i n h i b i t s t h e t r a n s c r i p t i o n of only c e r t a i n genes i n t o Z-RNA. 32

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5-Formamide 1-B - r i b 0 furanosy 1t h i o imidazo le-46-Mercap topurine carboxamide 2 ' , 3 ' , 5 ' - t r i f o r m a t e , i s comparable with 6-mercaptopurine (6-MP) f o r the treatment of leukemias.33 It i s c r o s s - r e s i s t a n t with 6-MP. Conv e r s i o n of 6-MP ribonucleoside t o 6-MeMP r i b o n u c l e o t i d e has been shown i n L-1210 leukemia c e l l s , adenocarcinoma 755 c e l l s , H. Ep. No. 2 c e l l s and o t h e r systems. 6-MeMP r i b o n u c l e o t i d e i s r e s p o n s i b l e f o r t h e i n h i b i t i o n of purine s y n t h e s i s produced by 6-MP.34 Metabolism of 6-MeMP i n E h r l i c h asc i t e s c e l l s has been studied.35 Breakdown of the a c t i v e n u c l e o t i d e form of the 6-thiopurines by a l k a l i n e phosphohydrolase i s probably r e s p o n s i b l e f o r the r e s i s t a n c e of n u c l e o p l a s t i c c e l l s t o these agents. 36

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F o l i c Acid Antagonists In c e r t a i n c e l l s f o l i c acid antagonists, such a s methotrexate (M'M) , homofolic a c i d o r tetrahydrohomofolic a c i d , i n h i b i t the i n c o r p o r a t i o n of t h e C-8 carbon i n t o 4-aminoimidazole-5-carboxamide r i b o t i d e . I n h i b i t i o n of DNA s y n t h e s i s by MTX and r e l a t e d antago n i s t s is not s e l e c t i v e , but i s balanced by concurrent i n h i b i t i o n of RNA and/or p r o t e i n s y n t h e s i s .37-39 A t h e r a p e u t i c advantage with reduced t o x i c i t y i s achieved by t h e a d m i n i s t r a t i o n of high doses of MTX, followed by "rescue" with f o l i n i c a c i d a s an a n t i d o t e . 37,40,41 Dosage schedules and t o x i c o l o g i c a l s t u d i e s of methasquin, a p o t e n t i n h i b i t o r of d i h y d r o f o l a t e MTX, t h i s quinazoline compound is reductase , were r e p 0 r t e d . 3 ~943 ~ ~ Unlike ~ poorly absorbed from t h e g a s t r o i n t e s t i n a l t r a c t . The N-10 n i t r o g e n atom of f o l i c a c i d i s b i o l o g i c a l l y ~ i g n i f i c a n ts i~n~c e N-10-deazafolic a c i d has no antineop l a s t i c a c t i v i t y .45 A study o f i n d u c t i o n of c y t o t o x i c i t y by MTX has shown t h a t MTXt r e a t e d lymphocytes added t o a l l o g e n e i c primary c u l t u r e s of embryo c e l l s produced a c y t o t o x i c e f f e c t ; no e f f e c t w a s observed when they were added t o syngeneic c e l l s of p r i m a r y embryo c u l t u r e . Normal syngeneic lymphoc y t e s when t r e a t e d with MTX, suppressed tumor c e l l growth i n mice.46 MTX i n h i b i t s the immune response t o i n s u l i n . The suppression i s r e versed by f o l i n i c a ~ i d . 4 ~Vitamin B12 promotes DNA-thymidine s y n t h e s i s i n C3H mouse c e l l s by r a i s i n g t h e l e v e l of f o l a t e coenzymes.48

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B u t l e r , Ed.

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[ N , N ' - ( 4 , 4 ' - D i a c e t y l ) d i p h e n y l l u r e a Bisguanylhydrazone (DDUG) DDUG (5) i n h i b i t s a number o f leukemias and some mammary tumors i n mice. R e s i s t a n c e t o DDUG i s a s s o c i a t e d w i t h a reduced uptake of t h e compound, b u t t h e i n t r a c e l l u l a r d i s t r i b u t i o n and drug b i n d i n g was unchanged. S t r a i n s r e s i s t a n t t o DDUG a r e c r o s s - r e s i s t a n t t o 2-chloro-4' ,4"-bis (2imidazolin-2yl)terephthalanilide, but remain s e n s i t i v e t o methylglyoxal b i s (guanylhydrazone) and v i n c r i s t i n e . The s i t e s o f i n h i b i t o r y a c t i o n of a l l four agents a r e different.49

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The c y t o t o x i c i t y of hydroxyurea was compared i n n o r Hydroxyurea mal and i n r a p i d l y p r o l i f e r a t i n g epidermis ;5O deoxyribonucleoside i n c o r p o r a t i o n was i n v e s t i g a t e d i n a l k y l a t e d , non-dividing human lymphocytes .51 The a b s o r p t i o n , d i s t r i b u t i o n , and e x c r e t i o n of hydroxyurea i n p a t i e n t s was reported.52 The c y t o t o x i c i t y o f hydroxyurea i s due t o i n h i b i t i o n of r i b o n u c l e o t i d e r e d u c t a s e i n DNA s y n t h e s i s . Hydroxyurea i n h i b i t s s k i n c a r c i n o g e n e s i s ;53 a c t i v i t y a g a i n s t nonr e s e c t a b l e cancer of t h e lung i s comparable w i t h t h a t of cyclophosphamide.54 It i s a n e f f e c t i v e agent i n epidermoid carcinoma of t h e head.55 However, c l i n i c a l t r i a l s a g a i n s t malignant melanoma56 and a c u t e myelocyt i c leukemia57 a r e d i s c o u r a g i n g .

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1,3-Bis (2-chloroethyl) - 1 - n i t r o s o u r e a (BCNU) , 1- (2Nitrosoureas c h l o r o e t h y l ) -3-cyclohexyl-1-nitrosourea (CCNU) a r e extremely l i p i d s o l u b l e and r e a d i l y c r o s s t h e blood-brain b a r r i e r . 5 8 CCNU i s more e f f i c a c i o u s than BCNU a g a i n s t implanted gliomas and leukemia L-1210. Clinically, BCNU and CCNU a r e e f f e c t i v e i n p a t i e n t s w i t h bronchogenic carcinoma, mal i g n a n t lymphoma and a c u t e leukemia i n children.59 T h e i r most important use i s f o r t h e t r e a t m e n t o f p a t i e n t s with malignant b r a i n tumors.59-62 Because of t h e i r l i p i d s o l u b i l i t y and r e l a t i v e nonionic c h a r a c t e r , n i t r o s o u r e a s a r e d i s t r i b u t e d widely i n plasma, c e r e b r a l s p i n a l f l u i d , b r a i n , muscle and o t h e r t i s s u e s . They a r e r e a d i l y metabolized; o n l y a small amount of p a r e n t m a t e r i a l can be i s o l a t e d from t i s s u e s t o g e t h e r w i t h l a r g e amounts of metabolite^.^^,^^ The a n t i n e o p l a s t i c a c t i v i t y of CCNU may be due t o m o d i f i c a t i o n o f c e l l u l a r p r o t e i n s and t o a l k y l a t i o n o f n u c l e i c a c i d s . 6 3 N i t r o s o u r e a s a c t on marrow s t e m c e l l s , cause a double f a l l i n the WBC count, and induce a more prolonged r e d u c t i o n i n colonyforming u n i t s t h a n does c y t o s i n e a r a b i n o s i d e . 18 N i t r o s o u r e a s o f t e n cause delayed bone marrow t o x i c i t y , t r a n s i e n t h e m a t o c r i t d e p r e s s i o n and hematop o i e t i c s u p p r e s s i o n , which may be r e l a t e d t o t h e drug-induced a l t e r a t i o n s i n t h e c e l l cycle.64 This e f f e c t may be r e l i e v e d by a change i n dosage regimen .62 S t r u c t u r a l m o d i f i c a t i o n s of N-nitrosoureas a r e r e p o r t e d .65

Chap. 13

Ant i neopl a s t i c Agents

Cheng

I33

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"Pseudourea" 2,2 '-(9,lO-Anthrylenedimethylene)bis-(2-thiop~eudourea) dihydrochloride (6), has shown activity against mouse leukemia L-1210. It complexes with DNA and inhibits both DNA synthesis and DNA-dependent RNA polymerase activity.66 Initial clinical studies revealed undesired phototoxicity in most patients with metastatic cancer.67 NH II

-6

CH2-S-C-NH2 II NH

-

5-(3,3-Disubstituted 1-triazeno)imidazole-4-carboxamide(7 DIC) DIC is used for treating disseminated malignant melanoma;68,fi it is reported to have activity against carcinoma of the lung70,71 and primary brain tumors.71 However, it is of no use in malignant melanoma in Uganda.72 Bone marrow depression, vomiting, diarrhea, oral ulceration, hepatocellular damage, CNS effects, fever, and a delayed flu-like syndrome are reported side effecgs. DIC is transformed to 9 via 5-diazoimidazole-4-carboxamide (8) .73 DIC undergoes s tepwise demethylation to 5-aminoimidazole-4-carboxamide (AIC) .74975 The monomethylated intermediate (w75or diazomethane76 may be the active metabolites of DIC The imidazole-4-carboxamide 11 (BIC) is transformed into an ionic product 12 77 in vivo,which may explain the differences in antileukemic action and resistance development of DIC and BIC.75 Cytotoxicity study78 and preliminary clinical trials76 of BIC are reported. The action of 5-diazoimidazole-4-carboxamide (8) was studied in rabbit platelets ; the sulfhydryl group and pyrophosphate structures may be involved in the mechanism of release of 5-hydroxytryptamine by &,79 Two isomeric compounds of DIC were synthesized. 5-(3,3-Dimethyl-lstable toward light and heat; it has good activity against leukemia L-1210 and less toxicity than 1.80 triazeno)pyrazole-4-carboxamide. (13)is

The isomeric 5carboxamide 14 lacks antileukemic activity.80 H

-7

R = N=N-N(CH3)2 R = (N2)+ 10 R = N=N-NH(CH3)

-8

11 R

= N=N-N(CH~CH~C~Z)

1

N- N-CHzCH2-Cl

134

Sect, I l l

-

Chemotherapeutic Agents

Butler, Ed.

-

Steroids Large doses of estrogen inhibit prolactin stimulation of mammary tumor growth as demonstrated by the effect of estradiol benzoate to DMBA-induced mammary adenocarcinomas in female rats Estrogen may be involved in transport of RNA from nucleus to cytoplasm.82 Specific binding of estradiol within the nucleus is confined to hormone -dependent tumors. 83 6a-Methylpregn-4-ene-3,llY2O-trione produces favorable responses against malignant melanoma, carcinoma of the breast and prostate with only mild toxicity when given orally .84 Hydroxyprogesterone caproate gave excellent responses in adenocarcinoma of the uterine corpus .85 Preliminary clinical studies with phenesterin in patients with advanced breast cancer were disappointing.86 A spectrophotometric analysis for diethylstilbestrol diphosphate has been deve loped.87 Andros terone defi ciency is associated with poor prognosis of lung cancer.88

-

l-(g-Chlorophenyl) -1-@-chlorophenyl) -2,2-dichloroethane (0,p I DDD) o ,p '-DDD produces favorable clinical responses of adrenocortical carcinoma in man is effective in an adrenal preparation in vitro but not in the adrenal slice. ~,E'-DDD does not affect base-line steroid production when ACTH-induced steroidogenesis is completely blocked, which indicates that ~,E'-DDD interferes with the mechanism by which ACTH stimulates steroidogenesis.89

-

-

Actinomycin D Kinetic studies with actinomycin D (act-D) in transplanted leukemic mice showed that a single dose provided cytotoxic concentrations for up to 24 hours." The characteristics of act-D resistance in L5178Y cells show that alterations in membrane composition and conformation in the drug-resistant subline accounts for the observed changes in the permeability of this drug.91 Act-D enhances the toxicity of morphine by increasing brain ~ermeability.~~ Exposure of leukemia P388 to act-D causes respiratory depression, a decrease in malic and lactic dehydrogenase and an increase in glucose6-phosphate dehydrogenase. These effects are similar to those caused by nitrogen mustard.93 Act-D stimulates isoperoxidase activity at low concentration and represses it at higher ~oncentration.~4 It inhibits the activity of DNA polymerase .95

Chap. 13

Antineoplastic Agents

Cheng

135

Vitamin A acid causes mucous metaplasia in the skin tumor keratoa ~ a n t h o m a . ~Mucus ~ was not observed after topical application of act-D to the skin tumor prior to the usage of Vitamin A acid.97 Adriamycin and Daunorubicin - The structure and absolute stereochemistry of daunorubicin (& daunomycin, rubidomycin) were determined.98 0

OH

15a. b. C.

R = CO-CH3 R = CHOH-CH3 R = CO-CH20H

Clinical trials of in children with acute lymphoblastic leukemia were compared and reviewed.99 This antibiotic has some value in children with advanced disseminated neuroblastoma,loo but may ultimately find its use in combination chemotherapy.lol, lo2 Daunorubicin is rapidly metabolized in rats to daunorubicinol and the aglycones of 15a and m . 1 0 3 Compound 15b was isolated from human urine and enzymatic reactions.104 and adriamycin complex strongly to DNA, inhibit Daunorubicin (*) mitotic activity, DNA s y n t h e ~ i s ~ 0 5and ~ ~ proliferation ~6 of HeLa cells. Daunorubicinol is only slightly active in inhibiting DNA synthesis and cellular proliferation.106 When the amino sugar of these compounds is masked or exchanged €or a-D-glucosamine, the resulting derivatives lack biological activity. The ability of these compounds to bind DNA is associated with the structure of the amino sugar.lo5 This observation supports the antileukemic triangulation pharmacophore hypothesis .44

(z), (u)

The pharmacokinetics, pharmacology, chemotherapeutic effects, toxicity, chromosome aberations and mutagenic potency of adriamycin were reported.105-109 Adriamycin is useful for acute lymphoblastic and chronic myelogenous leukemias, transitional cell carcinoma, liposarcoma, squamous cell carcinoma, and adenocarcinoma of the breast. It is less toxic than daunorubicin. Resistance to daunorubicin is due to changes in the cellular membrane.110 Both antibiotics possess immunosuppressive properties.111

-

Complete structures of the bleomycins have not been Bleomycins elucidated. Bleomycin A2 is active against the ascites type of Ehrlich carcinoma and sarcoma 180 in mice, and is clinically useful in the treatment of human epidermoid cancer, squamous cell carcinoma of the head and neck, lymphosarcoma, Hodgkin's disease, mycosis funoides, Kaposi's sarcoma, carcinoma of the thyroid, and brain tum0rs.~~2-114It exhibits very

I36

Sect. I I I

-

Butler, Ed.

Chemotherapeutic Agents

low renal toxicity and rarely causes leukopenia, thrombocytopenia or hematopoiesis in patients. Bleomycin A2 frequently causes pulmonary toxicity. It inhibits DNA synthesis in E. Ehrlich carcinoma and HeLa cells, inhibits ATP-dependent DNA ligase activity, causes DNA strand scission -in vivo and in vitro, and decreases the melting temperature of DNA in the It prevents presence of hydrogen peroxide or sulfhydryl compounds.115 3 cells from entering visible mitosis. 117 The activity of DNA polymerase from rat ascites hepatoma AH-130 cells is reduced by bleomycin but -in vitro addition of this antibiotic to the DNA polymerase assay system does not produce noticeable inhibition. An increase in the number of chromosomal aberrations was observed in cancer patients following administration of the antibiotic.l19

a,

The sulfur-containing chromophore (E),which is the "core" of all bleomycins , was revealed through x-ray analysis and confirmed by synthesis.l2O Partial structure of bleomycins A2, Ap', A5, 4 , B1 and B2 are represented by 17. In bleomycin A2 , X is 3-aminopropyldimethylsulfonium halide; in other bleomycins X is a straight chain polyamine.

17 -

16 -

Bleomycin A2 (M.W. ca 1400) contains two sugars (1-glucose and 3-0carbamoyl-D-mannose) and the peptide and amino acids 18-20.121 (L) p 2 YH2 H2N -CH2-CH-C02H I H02C-CH2-kH C02H N,?

T-cH-To

y,..

NH2 18

19 -

N\

N '

OH

CH3-FH

NH2

~H-OH

20

-

NH I

CH3

CH3-CH-C02H

Mitomycin C Human sarcoma cells exposed to mitomycin C stimulated DNA synthesis in autochthonous lymphocytes. Preexposure of the tumor cells to autochthonous serum abrogated lymphocyte stimulation.122 The use of mitomycin C in combination with cycle-phase specific drugs that may block cell repair.or with agents having a different mode of action has been suggested,123 but preliminary combinations with phenylalanine mustard, and vincristine in the treatment of osteogenic sarcoma was without much effect.124

Chap. 13

Antineoplastic Agents

Cheng

137 -

-

Camptothecin An alkaloid from the tree Camptotheca acuminata, camptothecin (21) exhibits antineoplastic activity against the mouse leukemias, the plasma cell tumor YPC-1, Walker 256 rat carcinosarcoma and has cytotoxic activity against several cell lines.125-127 Total synthesis128,129 and preliminary structure-activity relationship studies130 are reported. Camptothecin is not crossresistant to leukemias made refractory to the antifols, BCNU, cytosine arabinoside, 6-mercaptopurine, L-asparaginase, or vin0 cristine.127 The primary effect of this 21 alkaloid is on DNA synthesis. It also causes a G2 "lesion" which prevents subsequent mitosis. The major effect of 21 is to affect cells in S phase and the capacity of these cells to go through Go phase.127 Inhibition of DNA synthesis in L-1210 cells by camptothecin is initially reversible, but becomes irreversible when exposure to the drug is prolonged. 131 Camptothecin resistant L-1210 subline is not characterized by a drug permeability barrier nor by altered levels of cell surface glycoprotein, which differ from characteristics of actinomycin D-resistance cell lines.131

pJJyq

Camptothecin inhibits ribosomal RNA synthesis without affecting the RNA synthesis inhibition transfer RNA or protein synthesis;132 is rapidly reversible upon removal of the drug. 133 Initial claims of clinical activity of camptothecin in gastrointestinal cancer134,135 have not yet been confirmed.

& -

Among other antineoplastic agents of plant origin, the narcissus alkaloid pseudo lycorine (22) is active against Rauscher leukemia.136 Taxol , Ho from Taxus brevifolia, is active against a number of experimental neoplastic systems; its structure CH~O (23)was reported. 137 The sesquiterpene lactones138 l.39 and bufadien~lidesl~~ are cytotoxic. 0

I

C6H5 -Co

I

COCH3

I

OCH~

24 -

22 -

138 -

Sect. I l l

-

Chemotherapeutic A g e n t s

B u t l e r , Ed.

The f a c t o r r e s p o n s i b l e f o r c y t o t o x i c i t y of t h e s e s q u i t e r p e n e l a c t o n e s i s t h e O=k-k=CH* moiety.l38 T o t a l s y n t h e s i s of t h e t u m o r - i n h i b i t o r y aporphine a l k a l o i d s , t h a l i c a r p i n e (24) and h e r n a n d a l i n e was accomplished. 141 Emetine - A s an i n h i b i t o r of p r o t e i n s y n t h e s i s a t t h e t r a n s c r i p t i o n l e v e l , emetine i s a l s o a c t i v e a g a i n s t leukemias L-1210 and P-388, B16 melanomas, E h r l i c h a s c i t e s carcinoma and Yoshida sarcoma. Pharmacologic s t u d i e s showed t h a t i t e f f e c t s a sympathetic blockade, a n t a g o n i z e s h y a l u r o n i d a s e , i n h i b i t s o x y d a t i v e N-demethylation of aminopyrine and N-ethylmorphine a s w e l l a s t h e S-demethylation of 6-MP r i b o s i d e i n v i t r o . 14*9 143

P r e l i m i n a r y c l i n i c a l t r i a l s i n d i c a t e d t h a t emetine reduced lung tumor s i z e and p u r u l e n t bloody v a g i n a l d i s c h a r g e ; s e v e r e muscle weakness i s t h e most important d o s e - l i m i t i n g t o x i c e f f e c t . Emetine i s n o t myelos u p p r e s s i v e and may be u s e f u l i n p a t i e n t s w i t h poor marrow r e s e r v e . l 4 4 C a r d i o t o x i c e f f e c t s o f t h i s a l k a l o i d and i t s 2-dehydro analog were noted i n r a b b i t h e a r t . The observed c a r d i o p a t h y compares w i t h t h o s e n o t accompanied by v a s c u l a r symptoms b u t a s s o c i a t e d w i t h n e c r o s e s due t o e l e c t r o l y t e and s t e r o i d d i s t u r b a n c e . 145 N-Substituted a-emetine d e r i v a t i v e s have a l s o been found t o i n h i b i t growth of E h r l i c h a s c i t e s c a r c inoma . I 4 6 Vitamin A Acid - Vitamin A a c i d i s c a p a b l e o f inducing r e g r e s s i o n s i n a benign as w e l l a s malignant e p i t h e l i a l tumor. It has a t h e r a p e u t i c e f f e c t on e s t a b l i s h e d s k i n papillomas and s k i n carcinomas induced by 7,12-dimethybenz[g]anthracene and c r o t o n o i l . 147 I n i t i a l c l i n i c a l t r i a l has e s t a b l i s h e d t h e e f f e c t o f t o p i c a l l y a p p l i e d v i t a m i n A a c i d on b a s a l c e l l carcinomas on t h e s k i n i n man.148

-

Two L-glutamine a n t a g o n i s t s (6-diazo-5-0x0-L-norL-Asparaginase l e u c i n e and a z a s e r i n e ) produce a d d i t i v e a n t i l e u k e m i c e f f e c t s w i t h La s p a r a g i n a s e i n mouse leukemia L5178y.149 Rapid development of L-aspara g i n a s e r e s i s t a n c e w a s observed. 149 L-Asparaginase from d i f f e r e n t s o u r c e s p o s s e s s e s v a r i e d tumor i n h i b i t o r y p r o p e r t i e s . The enzyme from S e r r a t i a marcescens i s more i n h i b i t i v e a g a i n s t 6C3HED lymphoma t h a n t h a t from E. c o l i . 1 5 0 L-Asparaginase h a s a weak a c t i v i t y i n a d u l t a c u t e myelocytic leukemia; i t produces about 50% r e m i s s i o n s i n a d u l t s w i t h a c u t e lymphoc y t i c leukemia151 and h a s some a c t i v i t y i n c h i l d r e n w i t h advanced l e u It w i l l have t o be combined w i t h o t h e r drugs i f i t i s t o kernia.152,153 s i g n i f i c a n t l y a f f e c t s u r v i v a l of leukemia p a t i e n t s . 1 5 4 A c r o l e i n i n t o b a c co smoke155 and hormonal a n t i b o d i e s 1 5 6 reduce t h e t h e r a p e u t i c a c t i v i t y of L- a,sparaginase

.

Chap. 13

Antineoplastic Agents

Cheng

139

L-Asparaginase is a potent immunosuppressive agent. 157-159 This effect was not decreased by heat treatment or simultaneous administration of L-asparagine.lG0 L-Asparaginase from E. coli has a tetrameric structure with identical units, each possessing a single catalytic site.161,162 Extensive cross-linkage and alterations of substrate specificity can occur when the enzyme is treated with tetranitromethane.163 The native enzyme has 10% b-helices and 45% @-structure; the rest are o f the unordered types. Lyophilization increases the content of B-structure.164 Production by tumor cells and the effect of asparagine analogs were studied.165 Its action on mitotic activity during induced hepatocarcinogenesis was reported. 166 t-RNA Methyltransferase - Abnormally high levels of methyltransferase enzymes methyltransferase activity were found in neoplastic tissues including virally induced, chemically induced, and spontaneous tumors.167-171 The t-RNAs of many tumors contain highly elevated amounts of N-methylated, C-methylated, and the 2'-0-methylated nucleosides .I68 t-RNA from solid human tumors with t-RNAs from normal tissues was compared chromatographically.172 t-RNAs are associated with the regulation of protein synthesis at the translation level and since alkylating carcinogens were found to alkylate t-RNA in vivo, the aberrancy of methyltransferase could be involved in the initiation of tumor induction and neoplasia. 168 Inhibitors of methyltransferase are postulated to be of potential value in cancer chemotherapy. 168 Ricin - A phytotoxic protein from Ricinus communis is inhibitory towards protein synthesis in experimental tumor cells. It has a moderate inhibitory effect on DNA synthesis without affecting RNA synthesis. Its mode of action is not known, but is not due to impairment of glucose metabolism nor amino acid uptake in tumor cells.173 Gallium Salts - With a special affinity for malignant tumors, gallium salts are used as diagnostic tools for detection of neoplastic bone metabolism and allied neoplastic diseases. 174,175 Gallium bromide, chloride, citrate, iodide, and nitrate as well as ammonium gallium chloride have antitumor activity against Walker carcinosarcoma 256 and P-1798 lymphosarcoma. Gallium 5-fluoroorotate is active against leukemia L-1210 in mice. Autoradiographic studies of gallium-67 salts in rodent tissue show more than 70% of the 67Ga activity was localized in the cytoplasm. High concentrations of 67Ga were observed in the epithelium of renal convoluted tubules. 176 Four Group 111-A elements (aluminum, gallium, indium, and thallium) had some antitumor activity but only Ga+3 and, to a lesser extent, In+3 inhibited tumor growth when the tumor was innoculated by a

140

Sect. I l l

-

Butler, Ed.

Chemotherapeutic Agents

route different from that of the salts. The decreasing order of toxicity was In(NO3)3 2 TlCl3 > Ga(N03)3 > A1(N03)3. Some gallium salts may be useful for treatment of solid tumors in man.177j178

-

exhibited Platinum Complexes cis-Diamminedichloroplatinum (g), potent antitumor activity against sarcoma 180, Ehrlich ascites carcinoma, leukemia L-1210, Dunning ascites leukemia, Walker 256 carcinoma, DMBAinduced mammary carcinoma, and virus-induced reticulum cell sarcoma in experimental animals.179 2 180

- - - -Nr3 :\ ' Pt4 2 ; C1-

These platinum complexes cause a consistent inhibition of DNA synthesis in vivo.181 At very low concentrations, 25 selectively inhibited the

#

/

\ '

C1.- ---NH3

25

incorporation of th~idine-CHg-~H, uridine-5-3H, L-leucine-lk and Lleucine-3H into mammalian cells.181,182 It may undergo sequential transformations with loss of chloride, and the resultant platinum species may act bifunctionally to crosslink adjacent nuclephilic centers of DNA through covalent binding. 183 The acute toxic and pathogenic effects of 25 in rats include pronounced effects on many of the rapidly proliferating normal tissues (i.e., in the intestines , the marrow, and the renal tubules). 184,185 Hematologic studies show a decrease in circulating reticulocytes and lymphocytes after administration of 25, and histologic studies showed striking changes in thymus and spleen.186 Deafness was noted in preclinic trials, An aryl congener of 25, cis-dichlorodipyridineplatinum, possessed actions similar to those of the inorganic species, albeit l e s s potent.187 References 1. 2.

3. 4.

5.

6. 7. 8.

C . G . Zubrod, e t a l . , " F i r s t J o i n t Working Conference, N a t i o n a l Cancer I n s t i t u t e Chemotherapy Program," Cancer Chemother R e p t s . , P a r t 3,2, 1 (1971). T. A. Connors and D. H . Melzack, I n t . J . C a n c e r , 1,86 ( 1 9 7 1 ) . M . I n a b a and Y. S a k u r a i , I n t . J . Cancer, l,430 (1971). N . E. S l a d e k , Cancer Res., 1, 901 (1971). H . - J . H o h o r s t , A. Ziemann, and N . Brock, A r z n e i m . - F o r s c h . , 25, 1254 (1971). A. Primack, Proc. SOC. E x p t l . B i o l . Med., 604 ( 1 9 7 1 ) . M. P. G o l l e r k e r i and G. B. Shah, Cancer, 3, 596 ( 1 9 7 1 ) . D. T. Kaung, H . P . C l o s e , R. M. W h i t t i n g t o n , and M. E. P a t n o , Cancer, 3, 608

x,

(1971).

c(".

L. H. Ll. E. J . O l i n , T. F r a s e r , and 3. K. Bhuyan, Cancer R e s . , 1, 2770 (1971). 10. J . Vesely; M . gvo:ik, A. i h s k , a n d F. Sorm, I n t . J . Cancer, S, 310 (1971). 11. J. Vesely', A . Cihak, and F. $ o m , Eur. J. Biochem., 2,551 ( 1 9 7 1 ) .

9.

12. Y . Kozai and Y . Sugino. Cancer R e s . , 2, 1376 (1971). 13. F. L. Graham and G. F. Whitmore, i b i d . , ;So, 2627, 2636 ( 1 9 7 0 ) . 14. F. D . B e r t a l a n f f y and M . H. L. Gibson, i b i d . , 1, 66 (1971). 861 ( 1 9 7 0 ) . 15. M. Karon and S. S h i r a k a w a , J . Nat. C:ncer I n s t . , 9, 16. D. G . T r a g g i s , A . D o h l w i t z , L. Das, N . J a f f e , W. C . Moloney, and T . C . H a l l , Cancer, 28, 815 (1971).

Chap. 17. 18. 19. 20. 21.

13

A n t i n e o p l a s t i c Agents

25.

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 * 6c. 61. 62.

63. 64.

I41 -

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5,

14,

1159 22. R . P. 23. D. T . 24. R. L. 26.

Cheng

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a,

18,

186,

a,

5,

D,

a,

5,

s,

z,

z,

142 65.

66. 67. 68. 69. 70.

71. 72.

73. 74. 75.

76. 77. 78. 79. 80.

81.

Sect.

Ill

- Chemotherapeutic Agents

B u t l e r , Ed.

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46,

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44,

s,

68,

u,

6.

m,

Chapter 14.

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

M. H o f f e r and A . I.Rachlin, Hoffmann-La Roche Inc., Nutley, N.J. 07110

I n t r o d u c t i o n - The r e c e n t 1 i t e r a t u r e contains many c i t a t i o n s t o p a r a s i t e r e l a t e d diseases and problems p e r t a i n i n g thereto. I n a r e c e n t paper' several areas r e q u i r i n g i n t e n s i f i e d research and the development o f new o r improved drugs were i d e n t i f i e d . I n t h e past year, a s i n g l e p e r i o d i c a l has c a r r i e d papers on amebicides,2 ant helm in tic^,^ m a l a r i a , 4 y 5 amebic and b a c t e r i a l dysentery,6 i n t e s t i n a l helminths and f i l a r i a s i s , 7 trypanosomia s i s and leishmaniasis,8 schistosomiasis ,9 and immunodiagnosis o f paras i t i c diseases. 0 Considerable i n f o r m a t i o n r e l a t i n g t o c u r r e n t treatment of these a f f l i c t i o n s may be found i n these papers as w e l l as i n a r e p o r t c a l l ed "Drugs f o r P a r a s i t i c I n f e c t i o n s " . The proceedings of two r e c e n t symposia have been published, i . e . , on methodology f o r t h e development, s e l e c t i o n and t e s t i n g o f a n t i c o c c i d i a l drugs12 and on t h e development of drug r e s i s t a n c e i n malaria.13 Evidence i s accumulating t h a t n a t u r a l o r acquired immunity t o patho l o g i c p a r a s i t e i n f e c t i o n s i s abol ished by immunosuppression, p a r t i c u l a r l y by betamethasone, a c o r t i c o i d . 14-16 Consequently l a t e n t p a r a s i t i c i n f e c t i o n s may f l a r e up i n p a t i e n t s being t r e a t e d w i t h c o r t i s o n e o r i t s '8 derivatives

.

3

Antimalarials General - A p p l i c a t i o n o f an automated method o f mass drug t e s t i n g t o a n t i m a l a r i a l screening has been proposed.19 I t has been suggested t h a t CMLR1066, a commercial c u l t u r e medium, i s u s e f u l f o r t e s t i n g s u s c e p t i b i l i t y t o a n t i m a l a r i a l drugs.20 I d e n t i c a l d i h y d r o f o l a t e reductase enzymes, i s o l a t e d from P. berghei and p. knowlesi, have been found t o be s e n s i t i v e t o pyrimethamine. There i s no e m t h a t i n h i b i t i o n of the enzymes leads t o i n h i b i t i o n o f DNA synthesis . 2 1 , 2 2 Other d i hydrofol i c a c i d reduc tase i n h i b i t o r s , c y c l oguan i l , W.R. 38839 ( I ) , trimethoprim, methotrexate and W.R. 40070 (11), the l a t t e r t h r e e a t h i g h e r concentrations, acted i n a manner s i m i l a r t o p y r i methami ne . 2 3 NH,

I

I1

-+

Adenosine, t r i t i a t e d i n t h e 8 - p o s i t i o n , i s incorporated i n t o the RNA and, t o a l e s s e r e x t e n t , i n t o t h e DNA o f P. ber h e i . I n d i c a t i o n s a r e t h a t adenosine monophosphate i s degraded t o adenosine y e r y t h r o c y t e s before i t

146

Sect.

Ill

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can e n t e r the p a r a ~ i t e . 2 4 ~ 2 5 Labeling experiments show t h a t blood c e l l s i n f e c t e d w i t h P. knowlesi and P. f a l c i p a r u m synthesize coenzyme Q (ubiquinone) from p-hydroxybenzoic a c i d a n d t h a t coenzyme Q8 i s a p p a r e n t l y t h e dominant coenzyme Q f o r p. knowlesi p. lophurae and P. cynomolgi.26 Consequently t h e synthesis o f p o t e n t i a l r e v e r s i b l e i n h i b n o r s of t h e biosynthesis o f f u n c t i o n of coenzyme Q8 has been ~ n d e r t a k e n . ~ ~ An attempt has been made t o q u a n t i t a t e what appear t o be q u a l i t a t i v e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i ps f o r c hloroquine analogs .28 Resistance t o chloroquine and o t h e r antiplasmodials

- The development o f

h a l t s degradation o f hemoglobin by t h e m a l a r i a l p a r a s i t e s which then succumb t o amino a c i d s t a r v a t i o n . Experiments i n d i c a t e 2 9 t h a t chloroqu i n e r e s i s t a n c e develops by a s w i t c h i n t h e t r o p h o z o i t e stage t o t h e normally l a c k i n g c i t r i c a c i d c y c l e from which amino a c i d s a r e produced. A l t e r n a t i v e s t o overcome drug r e s i s t a n c e were reviewed i n 1 96830 and a d d i t i o n a l suggestions have appeared i n the most r e c e n t l i t e r a t ~ r e . ~ l - ~ ~ The search f o r new a n t i m a l a r i a l s - A new cinchona a l k a l o i d synthesis paved t h e way t o various q u i n i n e analogs35 of which t h e 6'-desmethoxy-7'-chlorodihydroquinines were found t o be 4 times as p o t e n t as n a t u r a l q u i n i n e a g a i n s t P. ber h e i i n mice. The n a t u r a l and unnatural antipodes and racemic f o r m s o d k l k a l o i d s showed no appreciable differences i n a c t i v ity.36 Increased a c t i v i t y was found among some 2'-a1 k y l - and a r y l - q u i n i n e d e r i v a t i v e s t h e most a c t i v e of which a r e useless, however, because o f phot o t 0 x i c i t y . 3 ~ Compounds I I I a and I I I b a r e new q u i n o l i n e a n t i m a l a r i a l s comparable t o , o r s u p e r i o r i n a c t i v i t y t o chloroquine and primaquine against P. v i n c k e i (mice) and p. cathemerium ( c a n a r i e s ) . S i q n i f i c a n t cross r e s i s t a n c e t o chloroquine was found i n p. ber h e i w i t h - I I I b b u t none w i t h I I I a . 3 8 The weak a n t i m a l a r i a l menoctone30 naphthoquinone analog o f IVa o r I V c ) exerted some i n h i b i t i o n o f coenzyme Q f u n c t i o n . Compounds

F-

H

OCH-R

a: R=CH3 b: R=OCH3

IVa-d were b e t t e r coenzyme Q i n h i b i t o r - s and compared f a v o r a b l y w i t h menoctone as antiplasmodial s . Hydrogenation of t h e h e t e r o c y c l i c r i n g destroyed the a c t i v i t y . 2 6 Compound (Va) and analogs,notably Vb, s t r u c t u r a: Rl=cyclohexyloctyl , R2=OH b: Rl=n-pentadecyl , R2=OH

J+1R2' R

0

IV

c: R1=OH, R2=cyclohexyloctyl d : R'=OH , R2=n-pentadecyl

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a l l y r e m i n i s c e n t of coenzyme Q, have s i g n i f i c a n t a n t i p l a s m o d i a l a c t i v i t y . S t r u c t u r a l v a r i a t i o n s have l i t t l e e f f e c t on a c t i v i t y . 3 9 The b e s t o f r e -

a: R=H b: R=Br c e n t l y prepared s i d e c h a i n analogs of c h l o r o q u i n e a r e comparable i n act i v i t y t o c h l o r o q u i n e . ~ o , ~ l Analogs of t h e c o c c i d i o s t a t benzoquate ( V I a ) have been recognized a s a n t i m a l a r i a l s ; I C I 56780 (VIb) and I C I 60128 ( V I c ) supress P. berghei asexual e r y t h r o c y t i c i n f e c t i o n s i n mice a t doses o f 1 mg/kg sca.nd b o t h a r e p o t e n t i a t e d by sulfadoxine.42 I C I 56780 a c t s p r o p h y l a c t i c a l l y a g a i n s t P. berghei i n mice and p. cynomolgi i n rhesus monkeys. B-663, an a n t i l e p r o s y and a n t i t u b e r c u l a r drug,43 has been C02CH3

H

VI found t o be a c t i v e i n c h l o r o q u i n e r e s i s t a n t s t r a i n s o f Plasmodium.44 Novel s t r u c t u r e BA-41799 ( V I I ) i s a c t i v e a g a i n s t P. chabaudi45 and s t r a i n s of P berghei r e s i s t a n t t o c h l oroquine , mepacrineTprimaqui n e , c y c l oguani 1 and d i a m i n o d i p h e n y l s u l f o n e (DDS) . 4 6 Sulfamonomethoxine monohydrate (DJ-1550) i s r e p o r t e d t o be p a r t i c u l a r l y a c t i v e a g a i n s t p. f a l c i p a r u m and P. vivax.47 New a n t i f o l a t e s and a n t i f o l a t e combinations have become i n c r e a s i n g 1y impor0 ~9 i t t a n t as a n t i p l a s m o d i a l s 2 3 , 3 2 , 3 ~ , ~ ~ ,and i s a l o n g these l i n e s t h a t f u t u r e progress can VII be expected.

.

A H

A c a u t i o n was r e c e n t l y sounded w i t h r e s p e c t t o t h e use of DDS which

is p a r t l y m e t a b o l i z e d t o a t o x i c , methemoglobin-producing hydroxylamine derivative.

50

Nitroimidazoles The use o f n i t r o i m i d a z o l e s f o r t h e t r e a t m e n t o f protozoan and, t o some e x t e n t , metazoan i n f e c t i o n s i s becoming i n c r e a s i n g l y more i m p o r t a n t . Furthermore a g i v e n member o f t h i s c l a s s o f compounds i s f r e q u e n t l y eff e c t i v e a g a i n s t a v a r i e t y o f p a r a s i t e s . The 5 - n i t r o i m i d a z o l e s have gene r a l l y proven t o be s u p e r i o r t o t h e 4- and 2 - n i t r o i m i d a z o l e s 5 1 as a n t i p a r a s i t i c agents b u t t h e l a t t e r seem t o have b e t t e r a n t i b a c t e r i a l properties.

148 -

Sect.

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-

&LRl

02N

;1

B u t l e r , Ed.

Chemotherapeut c Agents

i: R=CI

VIII

j : R=CH2CH20H, R1=

eF

a: R=CH3, R1=CH3 b: R=CH2CH20H, R1=CH3

k: R=CH3, R1= a C O N H ,

c : R=CH3 , R1=CH20CONH2 d: R=CH3, Rl=CH(CH3)2 e: R=CH2CHOHCH2C1, R1=CH3 f : R=(CH2)2S02C2H5, R1=CH3

n

+

D i m e t r i d a z o l e ( V I I I a ) c o n t i n u e s t o be used i n v e t e r i n a r y medicine a g a i n s t trichomonas i n cows52 and Histomonas melea r i d i s i n f e c t i o n s i n fowl. 5 3 M e t r o n i d a z o l e ( V I I I b ) , t h e standard r u g f o r Trichomonas v a g i n a l i s and T. f o e t u s i n f e c t i o n s , has gained some prominence a s an i n t e s t i n a l and h e p a t i c amebicide,54-56 b u t more r e c e n t l y a t t e n t i o n has been c a l l e d t o t h e p o t e n t i a l development o f trichomonal r e s i s t a n c e . 57 Ronida z o l e ( V I I I c ) 5 8 and i p r o n i d a z o l e ( V I I I d ) 5 9 - 6 1 a r e p o t e n t histomonastats. The l a t t e r substance i s n o t o n l y more a c t i v e a g a i n s t H. m e l e a g r i d i s than c l o s e l y r e l a t e d n i t r o i m i d a z o l e s i n c l u d i n g dimetridazo%,62 b u t i t a l s o has s u p e r i o r a n t i trichomonal p r o p e r t i e s . 6 3 Compound V I I I e (Ro 7-0207) has been found t o be h i g h l y a c t i v e a g a i n s t Entamaeba h i s t o l y t i c a , i n t e s t i n a l i n r a t s and h e p a t i c i n hamsters, and e f f e c t i v e a g a i n s t pinworms.64 T i n i dazole ( V I I I f ) , 6 5 compared w i t h m e t r o n i d a z o l e , has been found t o be 4-16 v a g i n a l i s and T . f o e t u s and about equal a g a i n s t times as a c t i v e a g a i n s t E. h i s t o l y t i c a . I t was a l s o found t o be a c t i v e a g a i n s t Eimeria t e n e l l a and H. m e l e a g r i d i s b u t i n e f f e c t i v e toward Trypanosoma b r u c e i , T. c o n g s e n s e , T. c r u z i and P. b e r g h e i . 6 6 N i t r i m i d a z o l e (VIIIS) has been t r i e d c l i n i c a l l y as a trichomonastat67,6* b u t i t was r e p o r t e d t o be i n f e r i o r t o m e t r o n i d i z o l e . 6 9 Panidazole ( V I I I h ) , when t e s t e d c l i n i c a l l y as an amebicide, was found t o be e f f e c t i v e a t doses o f 100 mg, 2 times d a i l y f o r 4 days. 7 0 M F - n i t r o i m i d a z o l e ( V I I I i ) was r e p o r t e d t o be p a r t i c u l a r l y e f f e c t i v e i n amebiasis, f l u n i d a z o l e (MK-915) ( V I I I j ) a g a i n s t vaginalis and T . f o e t u s and MCA-nitroimidazole ( V I I I k ) a g a i n s t T. b r u c e i i n mice. 7 1 C l i n E a e f f i c a c y f o r V I I I i was demonstrated. 7 2 C o m E u V I I I l was r e p o r t e d t o be e q u i v a l e n t t o m e t r o n i d a z o l e a g a i n s t trichomonas. 7 3 CL64855 ( h y d r o c h l o r i d e , CL-75805) ( V I I I m ) , p r e v i o u s l y c i t e d f o r i t s a n t i b a c t e r i a l a c t i v i t y 7 4 has now been r e p o r t e d t o be 7-8 t i m e s as p o t e n t as

T.

--

T.

Chap.

I4

A n t i paras i t i c Agents

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149

metronidazole a g a i n s t trich0monas.~5 I t has been found t o be an e f f e c t i v e i n t e s t i n a l and hepatic amebicide76 as w e l l as being a c t i v e a g a i n s t T. equiperdum i n mice a t doses of 20-40 mg/kg and a g a i n s t Leishmania donovani i n mice when f e d a t l e v e l s o f 0.04% i n feed f o r 7 days.77 I t i s , however, 4 times as t o x i c as m e t r o n i d a ~ o l e . ~ 5Compound V I I I n has been r e p o r t e d t o be an e f f e c t i v e t r i c h o m o n a ~ i d e . ~C1 ~ i n i c a l e f f i c a c y as an amebicide has been r e p o r t e d f o r BT-985, a n i t r o i m i d a z o l e o f undisclosed s t r u c t u r e ; amebic h e p a t i t i s 7 9 and a case o f lupus erythematosus a l s o r e sponded .a0 A second n i t r o i m i d a z o l e o f undisclosed s t r u c t u r e , MK-910, was a l s o r e p o r t e d t o be e f f e c t i v e Y a 1 b u t i n f e r i o r 8 2 t o metronidazole, as an amebicide. Coccidiostats

A method o f e v a l u a t i n g c o c c i d i o s t a t s under simulated f i e l d condit i o n s has been r e p 0 r t e d . ~ 3 I n a d d i t i o n t o t h e a n t i b i o t i c m o n e n ~ i n , ~ ' + ~ ~ ~ t h e r e l a t e d dianemycin86 and a n t i b i o t i c X-20687 a r e r e p o r t e d t o be e f f e c t i v e i n t h e c o n t r o l o f c o c c i d i o s i s . The a , a , a - t r i f l u o r o t o l u a m i d e s I X a r e r e p o r t e d t o be a c t i v e a t doses o f 0.0065% i n feeda8 and t h e p i p e r a z i n e s u b s t i t u t e d dithiosemicarbazones X a r e claimed i n t h e p a t e n t 1 i t e r a t u r e a 9

I

CR2-NUN-

\

( CH2),-NH-CS-NH-N=CR1f2

IX X RI,R~=H, CH3, C2H5 t o be c o c c i d i o s t a t s . N i t r o n e X I and analogs a r e r e p o r t e d t o be a c t i v e m e l e a g r i d i ~ . ~ ORofenaide [Ro 5-0013, s u l a g a i n s t E. t e n e l l a and a l s o

H.

1! 04 . CH=N-CH2CH20H

02N

XI

6

fadimethoxine t ormetoprim ( X I I ) 9 1 i n a r a t i o o f 5:3] i s completely e f f e c t i v e a g a i n s t a v a r i e t y o f Eimeria s t r a i n s a t t h e 0.02% l e v e l i n feed.92 Another treatment f o r c o c c i d i o s i s i s t h e use a t 0.0125% i n feed o f bec l o t i a m i n e ( X I I I ) , a d e r i v a t i v e o f thiamine. T h i s substance has l i t t l e o r no a n t i t h i a m i n e a c t i v i t y i n chickens o r r a t s . 9 3

X II

=

XI11

150

Sect.

Ill

-

Chemotherapeutic Agents

B u t l e r , Ed.

Histomonastats

A comparative study o f several establ ished histomonastats i n c l u d i n g 4-ni trophenylarsonic a c i d , p-ureidobenzenearsonic a c i d , 3,5-dini t r o s a l ic y l i c acid-5-ni t r o f u r f u r y l idene hydrazide94 and d i m e t r i d a z o l e was publ i s h e d . 5 3 The 5 - n i t r o i m i d a z o l e s ronidazole58 and ipronidazole59-61 a r e superior new histomonastats. A n t i trypanosomal Agents The need f o r new antitrypanosomal agents was pointed o u t i n a j o i n t r e p o r t o f t h e FA0 and WHO e x p e r t committee on A f r i c a n trypanosomiasis. 9 5 It has a l r e a d y been noted t h a t some o f t h e n i t r o i m i d a z o l e s a r e a c t i v e i n t h i s area, i . e . CL-64855 ( V I I I m ) a g a i n s t T. e u i erdum i n mice and T. brucei i n donovani i n mice68 and MCA-nitroimidazoleTV*nst mice. 71 The antimal a r i a l s 2,4-d iami no-6- (3,4-d i c h l or0 benzgamino)$u inazol i n e and i t s nitrosamine d e r i v a t i v e CI-679 were found t o supress, b u t n o t eradicate, T. c r u z i i n mice a t doses o f 60 mg/kg/day and 46 mg/kg/day f o r 6 t o 8 d a y s ~ ~ ~ i e t h y l a m i n o e t h y l - 5 , 6 - d i h y d r o c a r b a z o lt eh ,e b e s t o f a s e r i e s , has been found t o be u s e f u l as an a d d i t i v e t o donor blood t o prevent t h e spread o f Chagas-Mazza disease. 97 5'-O-Sulfamoyladenosine (XIVa), an analog o f t h e a n t i b i o t i c XIVb i s reported t o be 100% c u r a t i v e a t s i n g l e i . p . doses o f 2 mg/kg o r a t 0.1 mg/kg/day i . p . f o r 3 days.98 Trypanomycin (A-l0091/A) , a a: X=H new pigment a n t i b i o t i c i s r e p o r t e d t o be a c t i v e b: X=F a g a i n s t trypanosomes as w e l l as several bacteria.99 XIV OH OH A n t i sc his t o soma 1 Aqen t s General - Schistosomiasis and i t s treatment have r e c e n t l y been r e viewed. 1 0 0 - 1 0 2 Moreover , t h e development o f screening t e s t s f o r p o t e n t i a l drugs and assays f o r t h e disease have received considerable a t t e n t i o n . One proposed i n v i t r o screen f o r schistosomacides uses t h e c e r c a r i a l o r schistosomular form o f S. mansoni and a f i x e d concentration o f t h e t e s t substances. l o 3 An assayf-stosomiasis uses a f r a c t i o n of S. mansoni a n t i g e n l a b e l e d w i t h r a d i o a c t i v e iodine.104 A group o f 420 compounds, among them being 27 chemical enzyme i n h i b i t o r s , 4 a n t i s c h i s t o s o ma1 drugs and 2 s p e c i f i c i n h i b i t o r s f o r t r y p s i n and chymotrypsin, were t e s t e d f o r schistosome hemoglobin protease i n h i b i t i o n 1 0 5 on t h e theory t h a t ingested host hemoglobin i s r e q u i r e d f o r n u t r i t i o n o f t h e parasite.106 A comparison o f t h e e s t a b l i s h e d schistosomacides mirasan, lucanthone, n i r i d a z o l e and hycanthone has been published.107 Hycanthone i s s t i l l w i d e l y used i n s i n g l e i . m . doses o f 3 mg d e s p i t e t h e unpleasant s i d e e f f e c t s , mainly emesis, anorexia and abdominal pain.108 I t has been reported t h a t thiosinamine (a1 l y l t h i o u r e a ) blocks normal egg formation i n S. mansoni by i n h i b i t i n g t h e a c t i v i t y o f polyphenoloxidase.lo9

Chap. 14

Antiparasitic Agents

H o f f e r , Rachl in

151 -

New Chemotherapeutic Agents - A s i n g l e 50 mg/kg p.0. dose o f MK-3883 (XVa)llo-111 cures i n f e c t e d monkeys. Sinclle 5-7.5 ma doses o f a metabol i t e . ( X V b ) a r e a c t i v e . Compounds-XVI a,b'lclln o f a group o f naphthoa: R=H

0 2N ' RCH

m

xv

C H 2NHCH (CH 3) H

b: R=OH

quinone mono-oximes, and X V I I ,113 o f a group o f nitrosophenols, were found t o be p a r t i c u l a r l y a c t i v e . A r e c e n t patent114 claims antischistosomal ac-

t i v i t y f o r t h e 1,4-diarninonaphthalene d e r i v a t i v e XVIIIa and i t s quinol i n e analog XVIIIb. Antimonyl-dimethylcysteino-tartrate (NAP) gave 94% cures

i n humans i n f e c t e d w i t h S. mansoni w i t h 5 d a i l y doses o f 8% NAP s o l u t i o n , e q u i v a l e n t t o a t o t a l do= o f 2 g o r 290 mg o f antimony per person.'15 SQ-18506 ( X I X ) l 1 6 i s v e r y a c t i v e i n mice, hamsters,l17 and rhesus monkeys''' i n f e c t e d w i t h & mansoni. A t t e n t i o n has been d i r e c t e d t o t h e conformational analogy between SQ-18506 and n i r i d a z o l e. 1 1 9 S-2- ([ 2- (2-Thiat

NHCO( CH2) 2NH2( CH2)2SSO;

xx

XIX

zoylcarbamoyl )ethyl]amino)ethyl a c i d t h i o s u l f a t e (XX), i t s t h i o l hydroc h l o r i d e and d i s u l f i d e have been r e p o r t e d t o be h i g h l y e f f e c t i v e a g a i n s t S . mansoni i n mice and monkeys.120 2-(2-Methyl-4-amino-5-pyrimidinylcarbarn-nitrothiazole, an a n t i m e t a b o l i t e o f Vitamin B i , i s a p o t e n t schistosomacide t h e a c t i v i t y o f which i s destroyed, however, by simul t a neous a d m i n i s t r a t i o n o f h i g h doses o f v i t a m i n B1 . I 2 1

-

Anthel m i n ti cs Three weeks a f t e r i n o c u l a t i o n o f r a t s w i t h metacercariae, glutamico x a l o a c e t i c transaminase appears t o be g r e a t l y elevated. Since treatment w i t h v a r i o u s f a s c i o l a c i d a l agents caused normal i z a t i o n o f t h e enzyme

152

Sect.

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- C h e m o th e rapeuti c Agents

B u t l e r , Ed.

l e v e l s w i t h i n one week, i t was suggested t h a t t h i s o b s e r v a t i o n be used a s a means of e v a l u a t i n g p o t e n t i a l drugs f o r l i v e r f l u k e i n f e s t a t i o n s . The f o l l o w i n g substances were found t o be a c t i v e a t t h e i n d i c a t e d l e v e l s (mg/ k g ) : hexachlorophene (50) , b i t h i o n o l (300) , menichlopholan ( 5 ) , 3,4' ,5tri bromosal i c y 1 a n i 1 i d e (1 00) , 3 I - c h l o r o - 4 I - (p-chlorophenoxy)-3,5-di bromos a l i c y l a n i l i d e (300) , 5-bromo-3'-chloro-4'-( p-chlorophenoxy)-3-nitrosal i c y l a n i l i d e (50), o x y c l o z a n i d e (XXIa) (300).'22 The v a r i o u s s a l i c y l a n i l i d e s , XXIa,123 r a f o x a n i d e (XXIb)124 and n i c l o s a m i d e ( X X I C ) were ~~~ e v a l u a t e d i n v a r i o u s c l i n i c a l and f i e l d t r i a l s a g a i n s t i n t e s t i n a l worms, l u n g worms and l i v e r f l u k e s .

&a

C a p i l l a r i a s i s i n man, caused by t h e round worm C a p i l l a r i a h i l i inensis was c o n t r o l l e d R1 'ONH R .P-%T3 e t t e r by t iabendazole O (XXIIa) xx I than by b i t h i o n o l and bephenium.126 Thiabendazole has been a: R1=3,5,6-Cl3, R2=2-OH-3,5-C12 compared w i t h 1evamisol e t h e 1e v o - r o t a t o r y f o r m o f t e t r a m i s o l e ( X X I I I ) , and parbendazole (XXIVa) b: R1=3,5-12, R,=3-C1-4-(-O-@l) against natural helminthic infect i o n s i n sheep.l27 Thiabendazole has been found u s e f u l i n t r e a t i n g C : R1=5-CI, R2=2-C1-4-N02 i n f e s t a t i o n s o f 1agoc h i 1asc a r i s . 1 2 0 Levamisole was found t o be e f f e c t i v e a g a i n s t l u n g worms i n pigs,129 i t c o n t r o l l e d nematodes i n c a t t l e w i t h 94-99% e f f i c a c y a g a i n s t a d u l t worms and 68-81% a g a i n s t l a r vae,l30 and i n angora goats, 8 mg/kg was a s a f e dose, 16 m /kg caused f r e quent drug i n t o x i c a t i o n and 64 mg/kg was g e n e r a l l y f a t a 1 . l I t i s useful i n worm i n f e c t i o n s i n h 0 r ~ e s . l ~Parbendazole ~ which i s p r i m a r i l y e f f e c t i v e a g a i n s t nematodes, was a l s o found t o be a c t i v e a g a i n s t c e ~ t o d e s l ~ ~ and T r i c h i n e l l a s p i r a l is i n m i c e . l 3 4 Mebendazole (XXIVb) i s a c t i v e a g a i n s t nematodes; i t e r a d i c a t e s E n t e r o b i u s v e r m i c u l a r i s t o t h e e x t e n t of

9

a : R=C4H9 b: R=NHCO,CH(CH3)2

C6H5

XXIII

XXII

H

xxv

a : R=CH3 b: R=H

Chap.

14

An t ipa r a s i t i c Ag en t s

H o f f e r , Rachl i n

153 -

90% w i t h a s i n g l e dose of 100 mg/kg and i s a c t i v e a g a i n s t Syphacia muris i n r a t s . l 3 5 Carbendazole ( X X I I b ) i s approximately 3 times as e f f e c t i v e as thiabendazole a g a i n s t nematodes. I t i s a l s o e f f e c t i v e a t 5-15 mq/kg doses, p.0. , i n sheep a g a i n s t Haemonchus , O s t e r t a i a , T r i c h o s t r o n g u l u s , -Cooperia , Nematodirus, S t r o n g y l o i d e s , Chabertia an Oesophagostomum. 1 3 6 - 1 3 8 Morant e l (XXVa), a t 6.25 mg/kg, e l i m i n a t e d 96-100% o f N i o s t r o n l u s and Nematosplrioides compared w i t h 76-89% e f f i c a c y f o w b ) i n para l l e l t r i a l s . 1 3 9 The l a t t e r compound was 96.7% e f f e c t i v e a t 5 mu/lb a g a i n s t A s c a r a i s lumbricoides; i t was a c t i v e a g a i n s t Necator americanus, i n a c t i v e a g a i n s t T r i c h i n e l l a l 4 0 b u t a c t i v e i n s i n g l e 10 mg/kg doses i n ent e r o b ias is 1 1 a nd a scar ias is . 2 [2- ( 4 - Hydr o x y p heny 1 ) -6 benz im ida zolyl l-6-( 1-methyl - 4 - p i p e r a z y l )benzimidazol e (Hoechst 33258) i s e f f e c t i v e against m i c r o f i l a r i a e i n cotton r a t s infected with Litomsoides c a r i n i i . 1 4 3 Quaternary ammonium s a l t s of c e r t a i n palmitoylaminoal k y l amides a r e h i g h l y e f f e c t i v e a g a i n s t t h e nematode Penagrell us r e d i v i v u s . 144 Some 1 -carbony1 -3-methyl -2-pyrazol ine-4,5-dione-4-aryl hydrazones a r e a c t i v e a g a i n s t T r i c h i n e l l a s p i r a l i s . 1 4 5 A g a i n s t m i c r o f i l a r i a l i n f e c t i o n s , 2e t h y l -6-methyl -2,3,4,4a ,5,6,7,8-octah dro-1 H-pyrazino( 1 , 2 - c ) p y r i m i d i n e - l one seems t o be v e r y promising.146y14T 0,0-Dimethyl-2,2,2-trichloro-lhydroxyethylphosphonate, a chol i n e e s t e r a s e i n h i b i t o r , immobilizes t h e a d u l t Onchocerca v o l v u l u s and consequently i s u s e f u l i n t r e a t i n g onchocerciasis.148

__$_

-

-

The a n t i b i o t i c paromomycin ( s u l f a t e ) cured 12 o f 13 p a t i e n t s i n f e c t e d w i t h t h e cestode Hymenolepis w . 1 4 9 Cephamycin C (A-11884), an a n t i b i o t i c i s o l a t e d f r o m Ltreptomyces 1 i m a n i i i s a c t i v e a g a i n s t A s p i c u l a r i s t e t r a p t e r a , Syphacia o b v e l a t a and A s c a r i s l u m b r i c o i d e s . 1 5 0 REFERENCES P. E. Thompson, J. Parasitol.. 67,3 (1971). D. R. Seaton. Practitioner. 16 (1971). A. C. E. Cole, ibid., 206. 20 (1917). L. J. Bruce Chwatt, ibid., 207, 143 (1971). J. H. Walters. ibid.. 207. 147 (1971). P. E. C. Manson Bahr and W. E. Ormerod, i b i d , 207,154 (1971). A. W. Woodruff and G. S. Nelson, ibid.. 207, 173 (1971). 8. W. H. R. Lumsden and P. D. Marsden. ibid.. 207, 181 (1971). 9. H. A. K. Rowland and G. Webbe. ibid, 27.186 (1971). 10. D. A. Denham. D. S. Ridley. and A. Voller, ibid., 207, 191 (1971). 11. Medical Letter. 12 (6). Issue 266. 1969. 12. W. M. Reid. Exp. Parasitol., 21, 1 (1970). 13. K. M. Cahill. Bull. N. Y. Acad. Med.. g, 997 (1969). 14. R. C. Bagai and D. Subrahmanyam. Nature. 228, 682 (1970). 447 (1971). 15. A. S. Young and F. E. G. Cox. Parasitology. 16. M. E. Rose, ibid, 137(1970). 17. J. H. Seabury. S. Abadie. and F. Savoy. Jr.. Amer. J. Trop. M e d Hyg., 3209 (1971). 245 (1970). 18. Y. Yim, Y. Kikkawa. H. Tanowitz, and A. Wittner. J. Trop. Med. Hyg.. 19. R. J. Cenedella L. H. Saxe. and K. Van Dyke, Chemotherapy, 9 , 1 5 8 (1970). 188 (1970). 20. W. A. Siddiqi. J. V. Schnell, and Q. N. Geiman. J. Parasitol.. 9. 21. W. E. Gutteridge and P. 1. Trig& Parasitol.. L2. 431 (1971). 22. W. E. Gutteridge and P. 1. Trigg. J. Protozool.. E.89 (1970). 23. G. J. McCormick. C. J. Canfield. and G. P. Willet. Exp. Parasitol.. 33, 88 (1971). 24. C. H. Lantz. K. Van Dyke. and G. Carter. ibid.. 29,402 (1971).

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

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Butler, Ed.

25. C. H. Lantz. K. Van Dyke, Biochem. Pharmacol.. 2.1157 (1971). 26. J. V. Schnell, W. A. Siddiqui. Q. M. Geiman, F. S. Skelton. K. D. Lunan. and K. Folkers. J. Med. Chem.. 14,1026 (1971). 27. F. S. Skelton. C. H. Bowman. T. H. Porter, K. Folkers. and R. S. Pardini, Biochem. Biophys. Res. Commun., 43,102 (1971). 28. G. E. Bass, D. R. Hudson, J. E. Parker, and W. P. Purcell, J. M e d Chem. 1 3 275 (1971). 29. R. E. Howells, W. Peters. C. A. Homwood. and D. C. Warhurst. Nature, 228.625 (1970). 30. A. R. Surrey and A. Yarinsky. Ann. Rep. Med. Chem., 1968 (1969)P. 126 . 402 (1971). @ 31. W. H. Hiser, B. S. MacDonald. C. J. Cranfield, a n d J. J. Kane, Amer. J. Trop. Med. Hyg.. 2 804 (1971). 32. D. F. Clyde, R. M. Miller, A. R. Schwartz, and M. M. Levine, ibid. 33. D. F. Clyde, G. T. Shute. V. C. McCarthy. and R. P. Sangalang. J. Trop. M e d HYR. 74, 101 (1971). 34. C. J. Canfield. E. G. Whiting. W. H. Hall. and B. S. MacDonald, Amer. J. Trop. Med. Hyg.. g.524 (1971). 35. G. Grethe. H. L. Lee, T. Mitt, and M. Uskokovic, J. Amer. Chem. Soc.. 93, 5904 (1971). 36. A. Bros& M. Uskokovic. J. Gutzwiller, A. K. Krettli. and 2. Brener, Experientia 21.1100 (1971). 62 (1971). 37. R. W. A. Rees. P. B. Russell, J. P. Yardley, Z. E. Bright, L. Rane. a n d H. Smith. J. Med. Chem., g+ 38. E. Fink. P. Nickel, and 0. Dann, Anneim.-Forsch.. 20.1775 (1970). 39. E. L. Stogryn. J. Med. Chem.. 14,171 (1971)., l3. 864.1106 (1970). 40. T. Singh, J. F. Hoops. J. H. Biel, W. K. Hoya. R. G. Stein, and D. R. CNZ. J. M e d Chem.. l4,532 (1971). . D. R. Cmz. ihid., l4, 283 (1971). 41. T. Singh, R. G. Stein, J. F. Hoops. J. H. Biel, W. K. H o Y ~and 4 2 J. F. Ryley and W. Peters, Ann. Trop. Med. ParasitoL, 64, 209 (1970). 43. L. Doub. Ann. Rep. Med Chem., 1967 (1968). p. 105. 44. J. N. Sheagren, J. ParasitoL, 54,1260(1968). 45. W. Peters, An% Trop. Med ParasitoL. 64. 189 (1970). 46. W. Peters, Trans. Roy. SOC.Trop. M e d Hyg., 66.11 (1971). 47. T. Yoshinaga, Y. Tsutsumi, K. Tsunoda. and Y. Yamamura, Anneim.-Folsch, 20,1206 (1970). 48. P. 0.Fasan. Ann. Trop. Med ParasitoL. 2.117 (1971). 49. L. Donno and V. Sanguineti, Chemotherapy, l5,118 (1970). 50. S. A. Cucinell, Z. Israili, J. Vaught. and P. G. Dayton. Intersci. Conf. Antimicrob. Ag. Chemother., 11th. Atlantic City. N. J.. 1971. Abstracts. p.45. 51. G. C. Lancini, E. Lazzari, V. Arioli. a n d P. Bellai, J. Med. Chem. 12, 775 (1969). 52. D. K. McLoughliR J. ParasitoL. 66.39 (1970). 53. P. W. Waldroup. J. F. Maxey, R. E. Ivy. D. E. Greene, and E. L. Stephenson, Poultry Sci., 48.978 (1969). 54. D. R. O'Holohan and J. Hugoe-Mathews, Ann. Trop. Med ParasitoL. 64,475 (1970). 55. J. Antani and H. V. Srinivas. Amer. Trop. M e d Hyg.. 12, 762 (1970). 56. J. Antani H. V. Srinivas K. R. Krishnamurthy, and B. R. Jahagirdar. ibid, l9, 821 (1970). 377 (1971). 67. I. de Carnieri and F. Trane. Arzneim.-Forsch., 58. G. C. Shelton and H. C. McDougle, Poultry Sci.. 49,1077 (1970). 69. M. Mitrovic and E. G. Schildknecht, ibid., 49, 86 (1970). 60. W. L. Marusich, E. F. Ogrinz. and M. Mitrovic. ibid., ,3!4 92 (1970). 61. R. E. Messersmith. L. P. Hambly, and C. T. Orton, ibid., a.960 (1971). 62. M. Hoifer, M. Mitrovic. A. Beaman. and A. Brossi. J. Med.Chem., 14.993 (1971). 63. K. Butler, H. L. Howes, J. E. Lynch. and D. K. Pirie. J. Med. Chem., lo.891 (1967). 64. E. Grunberg. R. Cleeland. H. N. Prince, and E. Titsworth, Proc. SOC.Exp. Biol. Med.. 133, (Z),490 (1970). 65. M. W. Miller. H. L. Howes, Jr., R. V. Kasubick. and A. R. English, J. Med. Chem., 849 (1970). 66. M. W. Miller. H. L. Howes, Jr.. and A. R. English, Antimicrob. Ag. Chemother., 1969. p. 257 (1970). 67. M. Moffett. M. I. McGill. C. B. S. Schofield. and G. Masterton. Brit. J. Vener. nis., 47. 173 (1971). 68. L. Cohen, ibid., 9.177 (1971). 69. R. D. Catteral, Practitioner, 207, 516 (1971). 70. S. Pickholz. H. Shapero. D. M. Ryan. H. S. Jefferies. and B. T. Warren, Experientia, 26. 1025 (1970). , J. Trop. Med. Hyg.. 12. 916 (1970). 71. A. C. Cuckler. C. M. Malanga. and J. C o n r o ~Amer. 7 2 R. Liechti. Schweiz. M e d Wochenschr.. 100.2117 (1971). 73. C. Rufer, H.-J. Kessler. and E. Schroeder. J. Med. Chem.. 1 3 94 (1971). 74. L. Doub. Ann. Rep. Med Chem.. 1968 (1969).p. 108. 75. E. J. Burden, E. Schumacher. and M. Kelly, Antimicrob. Ag Chemother.. 1968.p. 538 (1969). 76. S. Carvajal. S. Pidacks. H. Steinmann, H. Maxon, and A. Wozniak. i b i d , 641. 77. E. J. Burden and E. Racette. ihid.. 545. 78. C. Rufer. H.-J. Kessler. and E. Schroeder, Chim. Ther.. l6. (4), 286 (1971). 79. H. Abd-Rabbo, G. Hillal. Y. El Gohary, J. Trop. M e d Hyg., 74.62 (1971). 80. H. Abd-Rabbo, ibid.. 73. 47 (1970). 81. M. V. Chari and B. N. Gadiyar. Amer. mop. Med Hyg., 19,926 (1970). 82. S. J. Powell and R. Elsdon Dew, ibid. 20, 839 (1971). 83. D. 1. Gard. D. C. Young, and M. E. Callender. Poultry Sci.. 31811 (1969).

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84. R. F. Shumard and M. E. Cdlender. ibid.. 48,1870 (1969). 86. M. E. Callender and L. V. Tonkinson, ibid., 60. 1660 (1971). 86. Eli Lilly. U S 3,677,631 (1971). 87. Ell LillY. US 3,827,883 (1971). 88. D. E. Welch, R. R. Baron, and B. A. Burton. J. Med. Chem.. 12, 299 (1969). 89. Burroughs-Wellcome, U S 3.677.421 (1971). 301 (1971). 90. H. K. Kim, R. E. Bamburu. a n d H. K. Yaktin, J. Med. Chem., 2, 91. M. Hoffer. E. Grunberg, M. Mitrovic, and A. Brossi. J. Med. Chem.. 14.462 (1971). 9 2 M. Mitrovic. E. G. Schfldknecht, and G. Fuaiek. Poultry Sci.. 48, 210 (1969). 93. M. Yamazaki. M. Sasaki, T. Matauzawa K. Kawada, and T. Yusa, ibid.. 60.1415 (1971). 94. E. W. Bemdt, H. Van Essen, 8. G. Held, and R. D. Vatne. J. Med. Chem., l2,371 (1969). 96. World Health Organization, Tech. Rep. Ser.. No. 434 (1969). 96. P. E. Thompson and A. Baules. J. Parasitol.. 66.616 (1970). 448 (1971). 97. J. Gallo Pecca and S. M. Albonico, J. Med. Chem., g, 636 (1970). 98. J. J. Jaffe, J. J. McCormack. and E. Meymerian. Exp. Parasitol., 99. W. Fleck, D. Strauss, Ch. Schoenfeld, W. Jungstand. and Chr. Seeber, Intersci. Conf. Antimicrob. Ag. Chemother.. 11th. Atlantic City, N. J.. 1971. Abstracts. P. 22. 100. P. Jordan and G. Webbe, “Human Schistosomiasis”, Charles C. Thomas, Springfield, Ill., 1969. 101. L. M. Werbel. Top. Med. Chem.. & 125 (1970). 236 (1970). 102. B. A. Southgate. J. Trop. Me& Hyg.. 3. 103. M. Muftic, i b i d , L3, 232 (1970). 104. J. S. Williams, E. H. Sadun, a n d R. W. Gore. J. Parasitol., 57, 220 (1971). 106. R. A. Zussman and P. M. Bauman, ibid.. 57, 233 (1971). 106. R. A. Zuisman, P. M. Bauman, and J. C. Petruska, ibid.. 66, 76 (1970). 107. R. Foster, 8. L. Cheetham. E. T. Mesmer. and D. F. King, Ann. Trop. Med. Parasitol.. 6&, 46 (1971). 108. J. A. Cook and P. Jordan, Amer. J. Trop. Med. Hyg., 22. 84 (1971). 109. A. B. M. Machado, M. L. Henriquez da Sflva, and J. Pellegrho, J. Parsaitol.. 56,392 (1970). 110. C. A. R. Baxter and H. C. Richards, J. M e d Chem., 14.1033 (1971). 111. R. Foster, E. T. Mwner. B. L. C b e e t h m and D. F. King. Ann. Trop. Me& Parasitol. L6. 221 (1971). 1 1 2 E. F. Elslager and D. F. Worth. J. Med. Chem., 13,370 (1970). 113. E. F. Eldager, L. M. Werbel. and D. F. Worth, ibid., 19.104 (1970). 114. Parke Daviaand Co.. US 3,667,718 (1971). 115. M. R. Pedrique, S. Barbera, and N. Ercoli. Ann. Trop. Med. Parasitol.. 64. 256 (1970). 1 708 (1969). 116. H. Breuer, J. Med. Chem., 2 117. E. Bueding, C. Naquira, S. Bouwman,and G. Roae, J. Pharm. Exp. Ther., 178.402 (1971). 118. D. G. Erlckson. J. G. Bourgeois. E. H. Sadun. and E. Bueding, i b i 6 . m 411 (1971). 119. E. Buedin& J. Parasitol., 66.663 (1970). 120. R. D. Westland. L. M. Werbel, J. R. Dice, J. L. Holmes and B. G. Zahm, J. M e d Chem., l4. 916 (1971). 121. I. Nabih and H. Zoroob, Experientia 27.143 (1971). 122. W. C. Campbell and T. A. Borry. J. Panuitol.. 56, 326 (1970). 1583 (1971). 123. S. E. Knapp, M. W. Schlegel, P. J. A. Presidente. and J. N. Armstrong. Amer. J. V e t Rea, G, 1289 (1971). 124. 5. E. Knapp and P. J. A. Presidente, ibid., g, a n d G. P. Scbeinesson. Amer. J. Trop. Med. Hyg., 20,206 (1971). 126. H. Most, M. Yoeli. J. H-ond, 126. G. E. Whalen. E. B. Rosenbeq, R. A. Gutman, J. Cross, J. W. Fresh. T. Strickland, and C. Uylangco. Amer. J. Trop. M e d HYS, 22, 96 (1971). 127. K. C. Kateg M. L. Colglazier. F. D. Enzie. I. L. L i n d a and G. Samuelson, J. Paradtol.. 67. 366 (1971). 128. B. F. J. Oostburg, Amer. J. Trop. Med. Hyg.. 22,680 (1971). 129. W. D. Linguist. S. E. Leland. Jr.. and R. K. Ridley, Amer. J. Vet. Res, B,1301 (1971). 646 (1971). 130. H. Ciordia and H. C. McCampbell. ibid, g, 131. D. G. I n a m and W. R. Mitchell. ibid., g.876 (1971). 132 M. J. Clarkson and M. K. Be& Ann. Trop. M e d Paradtol.. g6,87 (1971). 133. D. W. Kunkle and V. J. Theodorides J. Parasitol., S l . 319 (1971). 134. V. J. Theodorides and M. Laderman. ibid.. 65.678 (1969). 136. J. P. Brugmang D. C. Thienpont. I. van Wijngaarden. 0.F. Vanpariis. V. L. Schuermana and H. L. Lauwera J. Amer. Med. Ass., 217, 313 (1971). 186. D. R. Hoff, M. H. Fisher. R. J. Bochio, A. Lusl. F. Waksmunokl. J. R. Egerton, J. J. Yakstia W. C. Campbell, a n d 660 (1970). A. C. Cuckler. Experientia, 3. 137. G. W. Benz, J. Parasitol.. 57. 286 (1971). 138. N. F. Baker and G. T. Waltels, Amer. J. V e t Rea, 92, 29 (1971). Med. Parasitol., f34.626 (1970). 139. R. L. Cornwell and M. A. Blore, Ann. ROP. 140. V. M. Vlllarejos. J. A. Arguedm Gamboa E. Eduarte, and J. C. Schwartzwelder, Amer. J. Trop. M e d Hyg.. 20. 842 (1971).

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A. Sanati and E. G. Ghadirian, J. ROP. Med. Hyg..

B u t l e r , Ed.

Iff, 160 (1971).

W. A. Cervoni and S. 0. Gonzalez. Amer. J. Trop. Med. Hyg., 22, 589 (1971).

W. Raether and G. Laemmler. Ann. Trop. M e d Parasitol.. 66.107 (1971). R. R. M o d F. C. Magne. E. L. Skau, and G. Sumrell. J. Med. Chem., l4. 558 (1971). H. G. Garg. ibid, L4, 266 (1971). Council of Scientific and Industrial Research, New Delhi, India, US 3,560,503 (1971). R. Bindra. R. N. Iyer and N. Anand, XXlII IUPAC Cong., Boston, Mass., 1971. Abstract No. 180. p.75. M. Salazar Mallen. D. G. Barranco. and J. J. Mendoza, Ann. Trop. Med. Parasitol., 65. 393 (1971). M. Wittner and Tanowitz, Amer. J. Trop. Me& Hyg.. 20,433 (1971). R. L. Hamill C. B. Carre& M. M. Hoehn, W. M. Stark, L. D. Boeck, and M. Gorman, Intersci. Conf. Antimitrob. Ag. Chemother.. 11th. Atlantic City, N. J., 1971. Abstracts, p.7.

Section I V - Metabolic Diseases and Endocrine Function Editor:

I . J. Pachter, B r i s t o l Laboratories, Syracuse, New York Chapter 15.

Prostaglandins and Related Compounds

Gordon L . Bundy, The Upjohn Company, Kalamazoo, Michigan

I n reviewing a f i e l d which i s growing a s r a p i d l y and i n a s many d i r e c t i o n s a s prostaglandins, one must e i t h e r l i m i t t h e scope of t h e review or make it s o long a s t o be unwieldy. I n keeping with t h e former choice, t h i s review w i l l emphasize chemical developments i n t h e prostaglandin area i n 1971 ( s i n c e the l a s t review i n t h i s series’). A r e c e n t general review of prostaglandins i s available2, a s well a s comprehensive reviews of t h e i r chemistry3, biochemistry4, and pharmacology5 6. Vergroesen, e t a17 have summarized recent progress i n c l i n i c a l a p p l i c a t i o n s of prostaglandins while other reviews have focused on t h e r e l a t i o n s h i p of prostaglandins with human reproductiona-9 and f e r t i l i t y control’’. J

I . Syntheses of n a t u r a l prostaglandins - J . Fried and h i s associates’’ r e c e n t l y described a new synthesis of prostaglandins which u t i l i z e s metala l k y l epoxide openings f o r t h e introduction of both s i d e chains. Using d i a l l y l copper lithium, epoxide &” was converted i n high y i e l d t o 3

2 -

1

2

4 1. C o l l i n s 2. Wittig 3. KOH

AC 20 HOA c

b

bA c

OA c

6

L \=M C 0;1H

&

DH

7

which via t h e 4-step process indicated above afforded d i o l epoxide 2. An important f e a t u r e of F r i e d ’ s synthesiz i s t h e r e g i o s p e c i f i c opening of epoxide 2 with dimethyl ( S ) -( -) - 3 - ~ - b ~ t y l o x y - l - o c t y n y l a l a n e ’ ~leading

158

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&

Pachter, Ed.

Endocrine

.

t o a pair of diastereomers k i n 65% y i e l d (no a t t a c k a t C-11) This r e g i o s p e c i f i c i t y i s made p o s s i b l e by complexation of t h e alane with t h e ethanol side chain of 1 followed by i n t e r n a l d e l i v e r y of t h e reagent t o C-12. Chromatographic separation of 2 from t h e corresponding 15-epienantiomeric compound completed t h e resolution. Exclusive of t h e preparat i o n and r e s o l u t i o n of t h e o p t i c a l l y a c t i v e alane reagent, F r i e d ’ s r o u t e a f f o r d s PGF2a i n 3% o v e r a l l y i e l d from 1 and i s stereo- and regios p e c i f i c throughout.

(I)

The discovery by Upjohn researchers13, l4 t h a t n a t u r a l [i .e. 15 (S) ] PGA,, PGE2 and e s t e r i f i e d d e r i v a t i v e s a r e found i n t h e gorgonian c o r a l , Plexaura homomalla (from various l o c a t i o n s i n t h e Caribbean a r e a ) has l e d t o a short, e f f i c i e n t p a r t i a l synthesis of PGE2 and PGF2a13>15.15(S)-PGA2, acetate, methyl e s t e r (-1.5% of the frozen wet weight of c o r a l ) was con-

-

1. H202, OH2. A ~ ( H R ) 3. hydrolysis

-8

OA c

CO2H

6H

-OH

2

verted t o an epimeric mixture of a,B-epoxyketones, reduced with aluminum amalgam, and following chromatographic separation of C - 1 1 epimers, t h e PGE2, 15-acetate, methyl e s t e r was hydrolyzed enzymatically, thereby affording c r y s t a l l i n e PGE2 (2)i n 50% o v e r a l l y i e l d from 8. Reduction of 4 gave pure PGF$ i n varying y i e l d depending on t h e reducing agent. This represents improvement i n both number of s t e p s and o v e r a l l y i e l d over a s i m i l a r process reported e a r l i e r 1 6 which u t i l i z e d coral-derived prostaglandins w i t h 15 ( R ) configuration. E. J . Corey has r e c e n t l y outlined a number of modifications of h i s 16-step synthetic r o u t e reported e a r l i e r 1 7 . Corey and h i s a s s o c i a t e s found t h a t t h a l l o u s cyclopentadienide 10 offered s e v e r a l advantages over

t h e commonly used a l k a l i metal salts i n t h e i n i t i a l a l k y l a t i o n of cyclopentadiene’’. Using t h i s modification (and w i t h X=Br, R=CH3 above), 2 (R=CH3) was i s o l a t e d i n 55% y i e l d from cyclopentadiene. The use of a i n s t e a d of a methyl ether offered a n advanbenzyl ether (11,R=CH&)19 tage f o r scale-up s i n c e i t s eventual removal by c a t a l y t i c hydrogenation was experimentally simpler than t h e boron tribromide s t e p required with t h e methyl ether17. Corey a l s o found t h a t reduction of 12 (R=pphenylbenzoyl) with hydride reagent Q, gave a b e t t e r r a t i o of 15a/15B reduction products than a l a r g e number of a l t e r n a t i v e s ( d i f f e r e n t R, d i f f e r e n t hydrides)”. I n a d d i t i o n t o promoting a good 15a/15B r a t i o t h e 1-

Chap. 15

Pros t a g 1 and i n s

159

Bundy

phenylbenzoyl group gave c r y s t a l l i n e , e a s i l y separable reduction products 03914). Hydride reagent 2 and l i t h i u m perhydroborophenalyl hydride2O, 21 were both found t o be e f f e c t i v e f o r t h e s t e r e o s p e c i f i c reduction of t h e PGE prostaglandins t o t h e PGFa series21. Use of these reagents obviates t h e necessity of an PGFcl/PGFP chromatographic separation. Improvement i n t h e s e l e c t i v i t y of hydrogenation of t h e PG2 t o t h e PG1 s e r i e s 2 1 r e s u l t e d from t h e use of hindered s i l y l e t h e r s ( a t C-11 and C-15 i n PGE2) which render t h e C-13,14 double bond even l e s s a c c e s s i b l e than t r i m e t h y l s i l y l o r tetrahydropyranyl ethers22. reported t h e synthesis of g-PGE2 methyl e s t e r Ferdinandi and and t h e corresponding 5,6-dehydro compound v i a a bismesylate s o l v ~ l y s i s ~ ~ route. The key s t e p , t h e rearrangement of a cyclopropyl c a r b i n y l system, was investigated i n d e t a i l . Miyano and h J s coworker^^^^^^ have elaborated on t h e i r e a r l i e r synt h e t i c route27, s t a r t i n g w i t h 3-oxo-undecan-l,ll-dioic acid and s t y r y l glyoxal and have made G-PGE1, G-PGFla: and g-13,14-dihydro-PGE1 by t h i s approach. While this r o u t e i s f a i r l y d i r e c t , it does r e q u i r e a number of complex separations of isomers. Taub25728 has reported s e v e r a l improvements i n t h e Merck prostaglandin synthesis2’ which allowed higher y i e l d s , a savings of s e v e r a l s t e p s and incorporated a r e s o l u t i o n procedure. 11. Syntheses of s t r u c t u r a l l y modified prostaglandins - I n t h e course of synthesizing prostaglandin analogs with a six-membered r i n g i n place of t h e usual five-membered ring, N.S. Crossley3’ developed a new s y n t h e t i c approach which i s outlined below. Reduction of r e a d i l y available31 d i ketone gave a mixture of products from which g c r y s t a l l i z e d i n 24% y i e l d . Conversion of t h e l a t t e r t o c r y s t a l l i n e aldehyde 20 was performed a s i n d i c a t e d . (Epoxide i s t h e major product of an epimeric p a i r . )

g

18

NaBH4 0

16 -

1. HBr 2 . oxid.

,

3. OAC

18 -

-mr)

160 -

Sect,

IV

- M e t a b o l i c t Endocrine

Pachter, Ed.

20

re

21 OH

OH

OH

22

22

The remaining s t e p s p a r a l l e l e d very c l o s e l y those of t h e Corey synthesis17 and f i n a l l y afforded 22 and 2 which were " . . . l e s s potent than t h e n a t u r a l prostaglandins. i n s e v e r a l b i o l o g i c a l assays. '"O

..

..

A new n a t u r a l l y o c c u r r i prostaglandin isomer, 5-trans-PGA2 & was i s o l a t e d by Upjohn chemists 13% from t h e gorgonian Plexaura homomalla. The t r a n s isomer content usually ranged between 5 ant h e =A2

OH

24

&

6H

OH

26 -

3

6H

~5~ 5-transpresent. Using chemical t r a n s f o m a t i o n s described e a r 1 i e 1 - l 1 PGA2 was converted i n t o 5-trans-PGE2 333 (map. 76-77") and 5-trans-FGFfl 26 (m.p. 95-96').

-

J . Fried34 has reported t h e synthesis of (+)-7-oxa-PGF$l and (+)-7oxa-15-epi-PGFla and t h e i r enantiomers by a general route described earlier=. O f these f o u r isomers, o n l y 7-oxa-PGFp i t s e l f , i n which a l l c h i r a l centers possess t h e absolute configuration of n a t u r a l prostaglandins, e x h i b i t s t y p i c a l prostaglandin a c t i v i t y , while t h e others are e i t h e r i n a c t i v e o r a c t as antagonists. A l s o o n l y 7-oxa-PGFla was a substrate f o r prostaglandin 1 5 - d e h y d r o g e n a ~ ewhile ~~ the other t h r e e isomers were competitive i n h i b i t o r s of t h i s enzyme.

Another r e c e n t l y disclosed 15-dehydrogenase i n h i b i t o r i s t h e thiaalkyne 3''. This competitive i n h i b i t o r e x h i b i t s a K1=5.5 polar, while t h e corresponding oxa-analog i s a non-competitive i n h i b i t o r with 50% i n h i b i t i o n a t 200-400 p o l a r .

28

~

x-x

C =

\

O

Z

v 30, x-x

{CH=CH$;

=

H

40-09

Chap.

I5

Pros t a g l a n d ins

161 -

Bundy

a

The bicyclo[2.2.l]heptene d e r i v a t i v e prepared v i a a Diels-Alder r e a c t i o n by Samuelsson and C ~ r e y ~ i s~a, s e l e c t i v e i n h i b i t o r of t h e biosynthesis of PGEl ( b u t not PGFla) from 8,11,14-eicosatrienoic a c i d . This d e r i v a t i v e bears obvious resemblance t o t h e endoperoxide a proposed intermediate i n prostaglandin E l and Fla b i o ~ y n t h e s i s ~ ~ .

a

Microbial transformations of dione

& y i e l d i n g 15-epi-a8(12)-PGE1

(z) were ,described by Miyano and coworkers38. F l a v o b a c t e r i m 2.NR€U,B-3874 reduced t h e (-1 form of (+>-31 y i e l d i n g ( -1-32 (wok),while Pseudomonas 2.NRRL-B-3875 reduced t h e (+> form of t h e racemate stereos e l e c t i v e l y producing (+)-z. The enantiomorph not used up i n each reduction was o p t i c a l l y a c t i v e and could be separated chromatographically. I n n e i t h e r case was t h e corresponding 15-epimer formed.

&

R=O; 32, R+OH

22

A number of analogs have been reported which were made by modificat i o n s of t h e Corey route17. Using t h e o p t i c a l l y a c t i v e W i t t i g reagent

11.

( a v a i l a b l e i n 7 s t e p s from (S)-malic a c i d ) , Corey's group3' prepared PGE3 and PGF3a, i d e n t i c a l with the same compounds of n a t u r a l o r i g i n . 11-Deoxy prostaglandins have been synthesized by two groups. E. J. Corey4' genera t e d aldehyde 2 u t i l i z i n g a modification of a thallium n i t r a t e r i n g conprocedure t o t r a c t i o n r e a c t i o n 4 1 (&+,.), and converted 2 by t h e ll-deoyy-PGE2 and ll-deoxy-PGF2a. Crabbe and Guzm&ne dehydrated Corey I B then by hydrogenation and oxidation obtained t h e same iodohydrin 36 t o

____.7b

34

11-deoyy-PGs

0

POC13 o r

3 stem

-,12

ZI.

o ~ ~ t h e conversion of 36-i.. aldehyde 25. Corey and G r i e ~ accomplished using simply methanesulfonyl chloride and pyridine and then transformed 37 f u r t h e r without reducing t h e double bond, thereby a f f o r d i n g a d i r e c t synthetic r o u t e t o P G A ' s ( i . e . avoiding t h e n e c e s s i t y of dehydrating PGE's) The b i o l o g i c a l a c t i v i t y of ll-deoxy-PGEl was reported s e v e r a l years

.

Crabbe' and h i s coworkers45 have synthesized analogs with an a l l e n y l moiety i n place of t h e 5,6-double bond ( e . g . ki) a s indicated below. The

162 -

Sect.

key s t e p

IV

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M e t a b o l i c & Endocrine

P a c h t e r , Ed.

(S-+Q)involved a reductive process followed by elimination OAc

l.LICfC(C&) & .2-

L

OAC

i

AcCl

OTHP

DTHP

9

OAc

amf? 1.K2co3 2.oxid.3. HOAc

6THP

OTW

a

-

-om

c I MedhLi

2

0

'\= OH

40

e

0 -,2H

-

OH

41 -

and rearrangement. Several a d d i t i o n a l c l a s s e s of analogs were made by Crabb&45 involving modifications of the five-membered r i n g . These include 9,ll-bisdeoxy-PG's ( e . g . Q), 10ahydroxy-PG's ( e . g . 46) and t h e acid l a b i l e d e r i v a t i v e G, prepared a s indicated below: 1.H2, RaNi

-C02Me Carey: \CHO route 42 ~

4. Collins

ZI

-

,o

0'-

CO2H

-OH

4. H2 ,PdC 5. C o l l i n s

44

L

-OH

OH

,

1.AgOAc HOAc 2 . K2CO3 OAC

42

3

v -OH 46 -

n

Liz

48

9

No b i o l o g i c a l data were disclosed f o r these analogs.

Upjohn c h e m i s t ~ ' ~ , *r~e c e n t l y disclosed an improved synthesis of 15methyl-PGE2 methyl e s t e r . 15-Methyl-PGF2a methyl esterL6 was converted t o t h e C - 1 1 mono-trimethylsilyl d e r i v a t i v e , which on C o l l i n s oxidation and subsequent removal of t h e s i l y l group afforded 15-methyl-PGE2 methyl e s t e r i n about 45% y i e l d . K a r i ~ nhas ~ ~found t h a t 15-methyl-PGE2 methyl e s t e r i s a t l e a s t 100 times more a c t i v e than PGE2 a s a u t e r i n e stimulant i n humans and i t s e f f e c t s l a s t a t l e a s t t h r e e times a s long a s PGEz.

Chap.

15

Prostaglandins

163 -

Bundy

Corey4* has reported a f a i r l y e f f i c i e n t method f o r e f f e c t i n g invers i o n of configuration a t C-11, thus making possible t h e s y n t h e s i s of 11epi-prostaglandins.

,&

LzA

0

p-5

1.T s C l 2.Bu4N+OR-

-

-

OH

OR

50

+

z

o.;dd 52

Treatment of t h e t o s y l a t e of 50 with tetrabutylammonium forma-ce gave a 3 : l mixture of 2 (R=CHO) and 52. With a c e t a t e instead of formate, t h e r a t i o was 1.2:1and with oxalate, only 52 was formed. Merck chemists4’ reported t h e s t r u c t u r e s of s e v e r a l by-products formed i n transformations of PGE1. Hydrogenation of PGE1 with 10% Pd/C i n e t h y l a c e t a t e gave, i n a d d i t i o n t o t h e expected 13,lb-dihydro-PGEl (511, about 15% of the diketone implies t h a t double Formation of

54.

OH

54

54

0

bond migration (A134A14) i s competitive with hydrogenation under t h e s e conditions. Acidic dehydration of 2 gave dihydro-PGA1 and -35% of t h e cyclic ether (from i n t e r n a l a d d i t i o n of t h e C-15 OH a t C - 1 1 of dihydroPGAl), while base treatment of gave the Michael product 56.

2

A paper has appeared r e c e n t l y from t h e Unilever Laboratories5’ desc r i b i n g prostaglandin analog synthesis by bioconversion of a wide v a r i e t y of s y n t h e t i c polyunsaturated f a t t y a c i d s . Prostaglandins formed i n t h i s 5 -trans-PGE2 18-cismanner included 2 - t r ans-PGE 3-trans-PGE b-&pPGE, F‘GE. This work confirms t h e idea t h a t not only t h e p o s i t i o n of t h e double bonds, b u t a l s o t h e chain length i s an important f a c t o r f o r e s s e n t i a l f a t t y acid a c t i v i t y . Also a close c o r r e l a t i o n i s evident between EFAa c t i v i t y and r a t e of prostaglandin formation i n v i t r o .

,

,

,

Pace-Asciak and Wolfe have e l a b ~ r a t e d ~ ’ ,on ~ ~the s t r u c t u r e proofs

164

Sect.

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P a c h t e r , Ed.

of s e v e r a l novel prostaglandin d e r i v a t i v e s formed from arachidonic acid by r a t stomach homogenates51 and sheep seminal vesicles52. Preliminary accounts of t h i s work appeared e a r l i e r s 3 . Two approaches t o t h e synthesis of t h e PGFlcl carbon skeleton, which merge i n t h e i r f i n a l stages, have been reported by Katsube and cosynw o r k e r ~ ~ ~The , ~ key ~ . intermediate i n t h e s e routes i s t h e n i t r i l e thesized i n one case5* from t r i o n e Zp and a l t e r n a t i v e l T 5 from the DielsAlder product These r o u t e s have t h e advantage of being short, but t h e

a.

dA c A

bH

57

OH

58

overriding disadvantage of being nonstereoselective a t C-9, 11 and 1 5 . Sih, e t a15' r e c e n t l y disclosed t h e preparation of dl-15-deoxy-FGE1 by a six-step sequence, t h e key f e a t u r e s of which include t h e 1,4-

cycloaddition of chemically generated singlet oxygen ( 61j62) and addision of 1-lithio-trans-oct-1-ene (with Bug-CuI, e t h e r ) t o - t o e s t a b l i s h t h e l5-deoyy a l k y l chain. 111. Assay, a n a l y s i s - Chemical i o n i z a t i o n mass spectrometry i s a powerf u l a n a l y t i c a l t o o l which has only r e c e n t p 7 been applied t o prostaglandin s t r u c t u r e problems. The b a s i s o f t h i s technique i s t h e i n t e r a c t i o n between prostaglandin molecules and ions ( CHsf, C2H5+, C&+) produced by e l e c t r o n impact on methane--a process t h a t involves much lower energy than d i r e c t e l e c t r o n impact on t h e prostaglandins and hence r e s u l t s i n l e s s complex fragmentation p a t t e r n s .

I n t h e area of gas chromatographic analysis o f prostaglandins, Axen, e t a15' prepared 3,3,4,4-tetradeutero-PGE~ and -PGF@ and used these a s c a r r i e r s i n t h e glc-mass spec determination of PGE2 and PGF$ i n picomole amounts. (Measure r a t i o of . This represents approximately a 10f o l d increase i n s e n s i t i v i t y over t h e glc-mass spec method reported earlier5'. Pace-Asciak and Wolfe" i l l u s t r a t e d t h e usefulness of c y c l i c nbutylboronates a s g l c d e r i v a t i v e s of the PGF s e r i e s . Using t h i s derivat i v e a mixture containing PGE1, pGE2, PGFla and PGFg may be analyzed

h.)

Chap. 15

Pros t a g 1 and ins

Bundy

165

without p r i o r c l a s s separation. Analysis of sub-nanogram l e v e l s of PGFla and PGF@ (heptafluorobutyrate e s t e r s ) was accomplished by g l c w i t h triti u m e l e c t r o n capture detection61. A review of the various d e r i v a t i v e s used f o r g l c a n a l y s i s of prostaglandins has appearede2. For t h e measurement of physiological l e v e l s of prostaglandins, radioimmunoassay remains t h e most s e n s i t i v e technique. I n a d d i t i o n t o s e v e r a l such assays reviewed l a s t year', K.T. K i r t ~ n ' and ~ H . R . Behrmans4 reported new immunoassays s e n s i t i v e t o picogram q u a n t i t i e s of prostaglandins. Levine, e t a165 examined t h e s p e c i f i c i t i e s of antibodies against PGB1, PGFla and PGFS and found t h a t t h e cyclopentane ring appears t o be immunodomlnant. One f e a t u r e of t h e b i o l o g i c a l a r e a t h a t deserves s p e c i a l I V . Biology4-'mention here i s t h e recent establishment of t h e connection between prostaglandins and anti-inflammatory drugs such as a s p i r i n and indomethacin. Two B r i t i s h research groups have independently presented evidence t h a t some of t h e therapeutic e f f e c t s of a s p i r i n and r e l a t e d drugs are due t o t h e i n h i b i t i o n of prostaglandin synthesis. Vane" found t h a t i n c e l l - f r e e homogenates of guinea p i g lung, which w i l l normally synthesize PGE2 and PGF& on incubation with arachidonic acid, prostaglandin synthesis was inh i b i t e d by sodium a s p i r i n , indomethacin, and sodium s a l i c y l a t e t o an ext e n t varying w i t h t h e dose of t h e anti-inflammatory drug added. The conc e n t r a t i o n s necessary t o p r d u c e t h i s i n h i b i t i o n were c l e a r l y lower than t h e average therapeutic concentration i n human blood. Smith and Willise7 reported t h a t these same drugs when added t o resuspended human p l a t e l e t s i n h i b i t e d thrombin-induced production of prostaglandins without impairing t h e " r e l e a s e r e a c t i o n . " A similar l e v e l of i n h i b i t i o n (80-954&) of prostaglandin synthesis was found i n t h e blood from normal volunteers taken one hour a f t e r an o r a l dose of 600 mg of a s p i r i n o r 100 mg of indomethac i n . Vane, e t als8 added f u r t h e r evidence i n showing t h a t indomethacin and a s p i r i n block PGE2 and PGFg p r d u c t i o n by i s o l a t e d dog spleen. Prostaglandins have been shown t o induce fever and t o be involved i n inflammation--two conditions f o r which a s p i r i n treatment i s e f f e c t i v e . (Vane has found no evidence f o r a l i n k between i n h i b i t i o n of prostaglandin synthesis and a s p i r i n ' s analgesic e f f e c t s , however. ) This work represents a start a t unravelling the mechanism of a c t i o n of a s p i r i n and hence has extremely far-reaching impllcations. Vane6' suggests t h a t a s p i r i n and indomethacin might b e u s e f u l i n the treatment of other conditions thought t o be t h e result of prostaglandin r e l e a s e . Such conditions might include thrombosis, threatened abortion, rheumatism and I U D failures". Tothil17' found t h a t s e v e r a l of t h e sedative drugs ( e . g . desipramine, chlorpromazine, e t c . ) commonly used t o prevent threatened miscarriage a l s o prevent PGE2 formation i n i s o l a t e d r a t u t e r i n e s t r i p s , and he suggests t h a t more e f f e c t i v e therapy might be possible by giving a s p i r i n i n conjunction with t h e psychotropic agent. Colller7' disclosed t h a t t h e PGE and PGF content of human semen i s reduced i n s u b j e c t s who a r e taking a s p i r i n ( p o s s i b l e cause of t e m p o r a r i b reduced f e r t i l i t y ) . V.

Clinical

-

No e n t i r e l y new c l i n i c a l a p p l i c a t i o n s of prostaglandins

166 -

Sect. I V

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6 Endocrine

Pachter, Ed.

have appeared i n t h e last year. PGAl continues t o be of i n t e r e s t as an i n h i b i t o r of g a s t r i c secretion72 and f o r treatment of e s s e n t i a l hy-pertension73,74. The hypotensive e f f e c t s of PGEl have a l s o been investigated i n c l i n i c a l situation^^^. A t doses where t h e blood gressure e f f e c t s of PGEl a r e minimal i n healthy subjects, p a t i e n t s with e s s e n t i a l hypertension exh i b i t e d decreases i n blood pressure which s t a r t e d soon a f t e r i n f u s i o n of PGEl and l a s t e d 20-30 minutes. The cardiovascular e f f e c t s of PGE2 and PGF2a76,77,7a as well as PGA1, PGFlCl and PGF1B7' were studied i n man with s p e c i a l emphasis on t h e incidence of side e f f e c t s or cardiovascular eff e c t s a s a function of dose and mode of administration.

The g r e a t e s t volume of c l i n i c a l prostaglandin research s t i l l appears t o be d i r e c t e d toward t h e a r e a s of reproduction and f e r t l l i t y - - l a b o r induction, therapeutic abortion and contraception. Several groups have subs t a n t i a t e d and extended t h e r e p o r t s reviewed l a s t year' t h a t intravenous administration of e i t h e r PGE2 o r PGF$ i s e f f e c t i v e f o r terminating prege f f e c t i v e , therapeutic abortions by t h i s r o u t e a r e n a n ~ y ~ ~ - 'While ~. accompanied by a high incidence of vomiting and diarrhea. The s i d e e f f e c t s found i n the intravenous route can be minimized using i n t r a - u t e r i n e administrationa4 (lower doses required). Intra-amniotic i n j e c t i o n of P G E ~ or PGFza: a l s o overcomes these problems and has been used e f f e ~ t i v e l y ' ~ - ~ ~ . I n t r a v a g i n a l administration i s u s e f u l both f o r therapeutic abortion and labor induction8849. Several groupsao, 827 a7 have suggested t h a t t h e p r i mary use of prostaglandins f o r therapeutic abortion may be i n t h e second t r i m e s t e r where a l t e r n a t i v e methods a r e d i f f i c u l t or hazardous ( e . g. hypot o n i c s a l i n e o r glucose, hysterotomy) . For labor induction, a l l of t h e aforementioned modes of administrat i o n of prostaglandins have been used successfully', a8,a0,a2. I n addit i o n o r a l l y administered PGE2 and P G F s a r e e f f e c t i v e f o r labor induction", whereas t h e l a r g e r doses required f o r o r a l l y induced therapeutic abortion produce marked l e v e l s of g a s t r o i n t e s t i n a l s i d e effectsa3. K a r i m showed t h a t t h e a c t i o n of prostaglandins on the human uterus i s not mediated through the r e l e a s e of oxytocina2. References

1. G.L.Bundy, Annual Reports in Medicinal Chemistry, 1970, C.K.Czin, Ed., Academic Press, New York, 1971, p. 137. 2 . J.E.Pike, Scientific American 225 NO.^), 84 (1971). 3. U.Axen, J.E.Pike and W.P.Schneider in "The Total Synthesis of Natural Products", V01.111, J.ApSimon, Ed., Wiley, New York, New York, in press. 4. J.W.Hinman, Ann.Rev.Biochem.Vg1. 41 (1972), in press. 5. J.R.Weeks, Ann.Rev.Pharmacol.c, 317 (1972). 6. J. R. Weeks, Naunyn-Schmieaebergs Arch. exp.Pharmak. 347 (1971). 7. A.J.Vergroesen, P.Gans, J.J.Gottenbos and F.tenHoor, Klin.Wschr. 3 , 889 (1971). 8. V.R.Pickles, Biblphy.Reprod. 1,155 (1971). 9. T.H.Lippert, Dt.med.Wschr. $, 916 (1971). 10. S.M.M.Karim, "The Prostaglandins: Progress in Research," Medical and Technical Publ. Co. Ltd., Oxford and Lancaster, pp. 71-164 ( 1 9 ' 7 2 ) . 11. J.Fried, "7th Middle Atlantic Regional Meeting, American Chemical Society," Philadelphia, Pennsylvania, Feb. 16, 1972. 12. J.Fried, C.H.Lin, M.M.Mehra, W.L.Kao and D.Dalven, Ann.N.Y.Acad.Sci.s,38 (1971). 13. G. L.Bundy, "7th Middle Atlantic Regional Meeting, America1 Chemical Society,"

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a.,

m.

z,

a.

=.

z,

z,

m.

m. w.

u.

s,

x,

s,

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E

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Endocrine

64. H.R.Behrman, Physiologist 2, 110 (1971). 65. L.Levine ,R.M. Gutierrez Cernosek and H. vanvunakis , J.Biol. Chem. &, 06. J.R.Vane, Nature New Biol. G, 232 (1971).

z,

6782 (1971).

235 (1971). 67. J.B.Smith and A.L.Willis, Nature New Biol. 68. S.M.Ferreira,S.Moncada and J.R.Vane, Nature New Biol. 237 (1971). s9 * Evidence that IUD’s may function via inducing prostaglandin release is found in

z,

G. Chaudhuri, Lancet (l), 480 (1971). A.Tothill,D.Bamford and J.Draper, Lancet (2), 381 (1971). J.G.Collier,R.J.Flower, Lancet (Z),852 (1971). D.E.Wilson, C.Phillips and R.A.Levine, Gastroenterology &, 201 (1971). J. B. Lee, J. C. McGiff , H. Kannegiesser , Y. Y. Aykent ,J.G. Mudd and T.F.Frawley , Ann. 703 (1971). Internal Med. 830 (1971). 74. A. A. Carr, Ann. Internal Med. 1370 (1970). 75. F.Okada, Shinryc 76. S.M.M.Karim,K.Somers and K.Hillier, Cardiov.Res. 2 , 255 (1971). 77. S.M.M.Karim,K.Hillier and K. Somer, J. Obstet. Gynaec.Brit. Comm. g , 172 (1971). 78. L.A.Carlson,L.G.Ekelund and L.Oro, Acta Med.Scand. 188,553 (1970). 79. C.H.Hendricks,W.E.Brenner and L-Ekbladh, Amer.J.0bstet.Gynec. 111,564 (1971). 80. K.Kinoshita ,T.Wagatsuma and M.Hogaki , Arner. J.Obste t. Gynec. 111,855 (1971). 81. G . M. Filshie , J. Obstet. Gynaec.Brit. Comm. g , 87 (1971). 82. R.G.Kaufrnan, R.K.Freeman, and D.R.Mishel1, Contraception 2. 121 (1971). 83. M. P.Ernbrey , J.Reprod.Med. 6, 256 ( 1971). 84. M.P.Embrey and K.Hillier, Brit.Med.J. (l), 588 (1971). 85. S.M.M.Karim and S.D.Sharma, Lancet ( 2 ) , 47 (1971). 85. M.Toppozada, M.Bygdeman and N.Wiqvist, Contraception i,293 (1971). 87. M.Bygdeman,M.Toppozada, and N.Wiqvist, Acta.Physiol.ScLnd. 82, 415 (1971). 88. S.M.M.Karim and S.D.Sharma, J.0bstet.Gynaec.Brit.Comm. 294 (1971). 89. S.M.M.Karim, Contraception 2, 173 (1971). 90* M.P.Embrey, Proc.Roy.Soc.Med. 64,1018 (1971). 546 (1971). 91. N.S.Rangarajan,G.E.LaCroix and K.S.Moghissi, Obstet.Gyneco1. 92. S.M.M.Karim and S.D.Sharma, J.Obstet.Gynaec.Brit.Comm. @, 251 (1971). 93. S.M.M.Karim and S.D.Sharma, Brit.Med.J. (l), 260 (1971).

B,

a,

a,

E,

s,

Chapter 16.

Atherosclerosis

Thomas R. Blohm, MerreIl-Nationa1 L a b o r a t o r i e s , D i v i s i o n of Richardson-Merrell I n c . , C i n c i n n a t i , Ohio INTRODUCTION A t h e r o s c l e r o s i s i s a n extremely common d i s e a s e of t h e l a r g e a r t e r i e s , which c o n s t i t u t e s t h e p a t h o l o g i c b a s i s f o r most coronary h e a r t d i s e a s e , s t r o k e , aneurysm and o c c l u s i v e a r t e r i a l disease.' A r e p o r t has been published2which conveys t h e magnitude of t h e h e a l t h problem a s s o c i a t e d w i t h a t h e r o s c l e r o s i s , and reviews t h e evidence f o r i d e n t i f i c a t i o n of r i s k f a c t o r s (major r i s k f a c t o r s : h y p e r c h o l e s t e r o l e m i a , h y p e r t e n s i o n and c i g a r e t t e smoking). A program i s o u t l i n e d f o r c o n t r o l of r i s k f a c t o r s u t i l i z i n g a l l a v a i l a b l e procedures. The s t a t e m e n t on drugs i n d i c a t e s a need f o r c o n t i n u e d e v a l u a t i o n of c u r r e n t l y a v a i l a b l e drugs and f o r development of new ones, w i t h emphasis on s a f e t y . B a s i c pathology of a t h e r o s c l e r o s i s i s w e l l d e s c r i b e d i n a t e x t , l a n d a n important m o n o g r a p P p r w i d e s a broad c w e r a g e of t h e d i s e a s e , from e t i o l o g y t o c l i n i c a l management. An o l d e r , though s t i l l v a l u a b l e , monograph4deals w i t h t h e e v o l u t i o n of t h e a t h e r o s c l e r o t i c plaque. A comprehensive p i c t u r e of c u r r e n t r e s e a r c h i n a t h e r o s c l e r o s i s can be o b t a i n e d from t h e r e c e n t l y p u b l i s h e d Proceedings of t h e Second I n t e r n a t i o n a l Symposium on A t h e r o s c l e r o s i s .5 Much of t h e material c o n t a i n e d i n t h i s book has n o t y e t appeared i n j o u r n a l s . BIOLOGICAL RESEARCH L i p o p r o t e i n Metabolism - Previous reviews have i n d i c a t e d that major r e s e a r c h e f f o r t h a s been d i r e c t e d toward development of d i e t s , s u r g i c a l procedures and drugs which lower e l e v a t e d blood l i p i d s . The r e a s o n s f o r Blood t h i s emphasis on blood l i p i d c o n t r o l a r e d e t a i l e d elsewhere.2 9 3 J~ c o a g u l a t i o n and p l a t e l e t a g g r e g a t i o n , areas w i t h i m p o r t a n t re l a t i o n s h i p s t o a t h e r o s c l e r o s i s , w i l l be d i s c u s s e d i n c h a p t e r 8. The l i p i d s of plasma e x i s t as components of l i p o p r o t e i n s , and i t i s t h e s e which must be c o n s i d e r e d as p o t e n t i a l pathogenic a g e n t s , s i n c e u l t i mately i t i s t h e physicochemical and b i o l o g i c a l p r o p e r t i e s of t h e s e comp l e x molecules which determines whether o r n o t a g i v e n l i p i d w i l l r e a c h t h e a r t e r i a l subintima and be d e p o s i t e d t h e r e . On t h e b a s i s of t h i s reasoning, s t u d i e s have been conducted t o determine t h e a t h e r o g e n i c pot e n t i a l of t h e v a r i o u s l i p o p r o t e i n classes, s t a r t i n g w i t h t h e work of Gofman and coworkers." For r e c e n t concepts t h e r e a d e r i s r e f e r r e d t o t h e c l a s s i c series of papers by F r e d r i c k s o n and caworkers,7and t o r e c e n t reviews.8 79 F a m i l i a r i t y w i t h t h e F r e d r i c k s o n c l a s s i f i c a t i o n of hyperl i p o p r o t e i n e m i a s w i l l be assumed i n t h e following d i s c u s s i o n . Large e l e v a t i o n s of low-density l i p o p r o t e i n s (LDL), such as occur i n homozygous t y p e I1 hyperlipidemia a r e profoundly a t h e r o g e n i c ; l O t h e s i t u a t i o n i s less c l e a r f o r s l i g h t o r moderate e l e v a t i o n s of LDL. A

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proposed r e l a t i o n s h i p of plasma t r i g l y c e r i d e e l e v a t i o n t o c o r o n a r y h e a r t d i s e a s e l l implies a t h e r o g e n i c i t y of t h e t r i g l y c e r i d e - r i c h v er y low-density l i p o p r o t e i n s (VLDL). VLDL a l s o c o n t a i n c h o l e s t e r o l esters and f r e e c h o l e s t e r o l , a s w e l l a s LDL a p o p r o t e i n ( s e e below). An a n a l y s i s h a s b e e n r e p o r t e d l z o f r e l a t i o n s h i p o f l i p i d s a n d l i p o p r o t e i n s t o development of c o r o n a r y h e a r t d i s e a s e ( 0 ) o v e r 14 y e a r s i n 5127 men and women i n t h e Framingham S t u d y . Serum c h o l e s t e r o l , LDL a n d VLDL showed s t r o n g s t a t i s t i c a l d i s c r i m i n a t o r y power f o r development of CHD, VLDL somewhat less t h a n t h e o t h e r two. I n men of a l l a g e s a n d i n young women VLDL d i d n o t p r e d i c t CHD when i t s c h o l e s t e r o l r i s k f a c t o r was s t a t i s t i c a l l y removed. I n women of 5 4 - 6 9 y e a r s , however, VLDL a p p e a r e d t o b e t h e i m p o r t a n t r i s k f a c t o r , r a t h e r t h a n c h o l e s t e r o l . With t h e e x c e p t i o n of t h e s e o l d e r women, t h e d a t a seem t o i n d i c a t e t h a t c h o l e s t e r o l , p r e s e n t e d t o t h e a r t e r y e i t h e r as LDL o r VLDL, i s t h e p a t h o g e n i c a g e n t . No p r o t e c t i v e r o l e c o u l d b e imputed t o serum t o t a l p h o s p h o l i p i d , s i n c e t h e cholesterol/phospholipid r a t i o showed no a b i l i t y t o d i s c r i m i n a t e f o r o r a g a i n s t CHD. The a n a l y s i s h a s been c r i t i c i z e d on t h e b a s i s of t h e b a s i c data.13

Two groups have a t t e m p t e d t o c o r r e l a t e CHD, documented by c o r o n a r y a r t e r i o g r a p h y , with hyperlipoproteinemia and o t h e r r i s k f a c t o r s . B e m i s e t a114measured l e s i o n p r o g r e s s i o n by r e p e a t e d a r t e r i o g r a p h y a n d found p r o g r e s s i o n t o b e s t r o n g l y c o r r e l a t e d w i t h h y p e r l i p i d e m i a ( c h o l e s t e r o l and /or triglyceride). Although t h e numbers a r e small, t y p e s I11 a n d IV hyp e r l i p o p r o t e i n e m i a were b e t t e r c o r r e l a t e d t o l e s i o n p r o g r e s s i o n t h a n t y p e 11. I n a s t u d y o f 50 c o n s e c u t i v e h o s p i t a l a d m i s s i o n s f o r CHD, S a l e 1 e t a115determined t h e p r e v a l e n c e of v a r i o u s r i s k f a c t o r s . The h i g h e s t p r e v a l e n c e by f a r was f o r male sex; t y p e I V h y p e r l i p o p r o t e i n e m i a was n e x t , a n d t y p e I1 a poor t h i r d . P a r t i c u l a r l y i m p o r t a n t was t h e f i n d i n g t h a t p r e v a l e n c e of t y p e I V was s i g n i f i c a n t l y g r e a t e r i n t h e CHD g r o u p t h a n i n a c o n t r o l p o p u l a t i o n , w h i l e p r e v a l e n c e of t y p e I1 was n o t s i g n i f i c a n t l y d i f f e r e n t f o r t h e two p o p u l a t i o n s . The lower p r e v a l e n c e of t y p e I1 i n t h e t o t a l p o p u l a t i o n may r e q u i r e a l a r g e r sample t h a n was used. These s t u d i e s r e p r e s e n t e a r l y e f f o r t s t o d e t e r m i n e a t h e r o g e n i c i t y o f LDL ( t y p e 11) a n d VLDL ( t y p e IV) e l e v a t i o n s . Much more needs t o be done. It a p p e a r s a t t h i s p o i n t t h a t b o t h LDL and VLDL s h o u l d b e r e g a r d e d a s a t h e r o g e n i c , b u t t h a t q u a n t i t a t i v e comparisons c a n n o t b e made. A g e n e r a l p i c t u r e of l i p o p r o t e i n metabolism has nuw e v o l v e d , w i t h several i m p o r t a n t d e t a i l s a d d e d d u r i n g 1971. T r i g l y c e r i d e (TG) i s s y n t h e s i z e d i n t h e l i v e r when f a t t y a c i d s become a v a i l a b l e , e i t h e r from l o c a l s y n t h e s i s o r from t h e b l o o d ( m o b i l i z a t i o n from a d i p o s e t i s s u e ) . Formation of VLDL i s a n immediate consequence of TG p r o d u c t i o n i n t h e hepatocyte.lsr1e B e f o r e e n t e r i n g t h e c i r c u l a t i o n t h e n a s c e n t VLDL d i f f e r s i n p r o t e i n c o m p o s i t i o n from VLDL i s o l a t e d from t h e c i r c u l a t i o n , 1 7 cont a i n i n g r e l a t i v e l y more of t h e p e p t i d e c h a r a c t e r i s t i c of LDL (apo-LDL) and less of p e p t i d e s found i n h i g h - d e n s i t y l i p o p r o t e i n (HDL). Evidence has been o b t a i n e d t h a t p e p t i d e s a r e t r a n s f e r r e d r e v e r s i b l y from HDL t o n a s c e n t VLDL i n t h e c i r c u l a t i o n . l S 9 2 0 Two such t r a n s f e r r a b l e p e p t i d e s have been i d e n t i f i e d , d e s i g n a t e d a c c o r d i n g t o C - t e r m i n a l amino a c i d a s

Chap.

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171 -

Blohm

apoLP-gluzO (R-glu21 ) and apoLP-ala (R-ala). F u n c t i o n a l r o l e s f o r t h e s e p e p t i d e s a r e s t r o n g l y s u g g e s t e d by t h e f i n d i n g s t h a t apoLP-glu a c t i v a t e s t h e enzyme l i p o p r o t e i n l i p a s e , and that apoLP-ala can a c t i v a t e , o r , d e Lipoprotein lipase i s t h e pending on c o n c e n t r a t i o n , i n h i b i t it."'-24 enzyme which removes TG from VLDL by h y d r o l y s i s a t s i t e s of t i s s u e TG u t i l i z a t i o n . During h y d r o l y s i s of VLDL-TG, LDL, apoLP-glu and apoLP-ala a r e released.20 LDL, l a b e l e d i n t h e p r o t e i n moiety w i t P 2 5 1 , was not r e u t i l i z e d f o r VLDL s y n t h e s i s . 2 5 An o v e r a l l scheme f o r t r i g l y c e r i d e t r a n s p o r t c a n now be v i s u a l i z e d l g w i t h r o l e s a s s i g n e d t o a l l of t h e l i p o p r o t e i n classes. While o t h e r f u n c t i o n s of t h e plasma l i p o p r o t e i n s c e r t a i n l y e x i s t , t h e r e i s a good p r o b a b i l i t y t h a t t h e i r t r i g l y c e r i d e t r a n s p o r t f u n c t i o n i s a major d e t e r minant of t h e i r plasma c o n c e n t r a t i o n s .

Carbohydrate

I

-%.

F

nVLDL peptide

Ir a c i d s l

'I

amino a c i d s

choles t e r o l

II

1

trans

I

apo L D L 1

I

'

fat mobili?c

I

peptides

f

(adipose I

I

nVLDbnascent VLDL; HDL-p=HDL w i t h t r a n s f e r r a b l e p e p t i d e s a t t a c h e d ; FFA= f r e e f a t t y a c i d s . Symbols and arrows o u t s i d e o f boxes r e p r e s e n t t r a n s p o r t i n blood plasma.

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

According t o t h i s scheme , t y p e I V h y p e r l i p i d e m i a ( e l e v a t e d VLDL) could be t h e r e s u l t o f h e p a t i c w e r p r o d u c t i o n of TG o r of d e c r e a s e d p e r i p h e r a l u t i l i z a t i o n of VLDL-TG. Type I1 could be due t o a c c e l e r a t e d TG t u r n o v e r , exceeding a normal c a p a c i t y t o degrade LDL formed by h y d r o l y s i s of VLDL-TG, o r t o d e c r e a s e d a b i l i t y t o degrade LDL. Recently a subtype 11, type I I b , has been r e c o g n i z e d , 2 6 i n which both LDL and VLDL a r e e l e v a t e d . T h i s subtype commonly i s accompanied by o b e ~ i t y , ~ ~ aint dmay be t h a t t r i g l y c e r i d e w e r p r o d u c t i o n has exceeded t h e capacities of b o t h t h e TG removal system and t h e LDL d e g r a d a t i o n system i n t h e s e p a t i e n t s . Levy25 has shown t h a t c h o l e s t y r a m i n e i n c r e a s e s t h e f r a c t i o n a l c a t a b o l i c rate

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& Endocrine

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o f 1 2 6 I - l a b e l e d LDL, w i t h o u t a f f e c t i n g i t s endogenous p r o d u c t i o n rate. S i n c e t h i s drug i s t h e most e f f e c t i v e f o r t r e a t m e n t of c l a s s i c a l t y p e I1 (type I I a ) , i t may be i n f e r r e d t h a t LDL d e g r a d a t i o n i s f a u l t y i n t h i s d i s o r d e r . Levy a l s o found that n i c o t i n i c a c i d , which i s a l s o e f f e c t i v e i n type 11, d e c r e a s e d s y n t h e s i s of LDL w i t h o u t a f f e c t i n g t h e rate of i t s catabolism. Combination o f c h o l e s t y r a m i n e w i t h n i c o t i n i c a c i d produced synergism i n r e d u c t i o n of c h o l e s t e r 0 1 . ~ 5,aa ,as Using a s i m i l a r r a t i o n a l e , Howard and Hyams30found that a combin a t i o n of c l o f i b r a t e and DEAE-Sephadex s y n e r g i z e d i n reducing plasma c h o l e s t e r o l of type I1 p a t i e n t s . These p a t i e n t s had f a i l e d t o respond t o c l o f i b r a t e a l o n e , and responded modestly (13% c h o l e s t e r o l r e d u c t i o n ) t o DEAE-Sephadex a l o n e , b u t e x p e r i e n c e d a n a v e r a g e c h o l e s t e r o l r e d u c t i o n of 33% on t h e combination. By i n c r e a s i n g t h e combination dose of DEAE-Sephadex from 12 t o 18 gm/day, c h o l e s t e r o l r e d u c t i o n could be i n c r e a s e d t o 40%. S u r p r i s i n g l y , combination of cholestyramine w i t h c l o f i b r a t e was n o t as e f f e c t i v e as t h e c l o f i b ra t e -DEAE -Sepha dex combination. W h i te3 has shown t h a t c l o f i b r a t e cannot p r e v e n t t h e i n c r e a s e i n h e p a t i c c h o l e s t e r o l synt h e s i s o c c u r r i n g d u r i n g cholestyramine a d m i n i s t r a t i o n t o t h e rat. S t u d i e s a t t h e NHLI have c l e a r e d up some of t h e mystery surrounding t y p e I11 hyperlipidemia (broad-B d i s e a s e ) . I n t y p e I11 p a t i e n t s , VLDL i s p a r t i a l l y degraded a t a normal r a t e t o a n i n t e r m e d i a t e l i p o p r o t e i n 3 2 w i t h B-mobility. This l i p o p r o t e i n accumulates and i s only slowly degraded i n t h e s e i n d i v i d u a l s , due t o a d e f i c i e n c y of one component of l i p o p r o t e i n l i p a ~ e . 3 ~This r e s u l t i m p l i e s t h a t VLDL d e g r a d a t i o n occurs i n a t l e a s t two s t a g e s , one of which i s d e f e c t i v e i n t y p e I11 hyperlipidemia. Other evidence s u g g e s t i v e of t h i s canes from s t u d i e s of l i p o p r o t e i n l i p a s e (LPL) a c t i v a t i o n by t h e HDL p e p t i d e ~ . ~ 4

,

C o n s i d e r a t i o n of t h e TG t r a n s p o r t scheme o f f e r s a n e x p l a n a t i o n f o r t h e o b s e r v a t i o n t h a t h e p a r i n t r e a t m e n t , w h i l e reducing c i r c u l a t i n g VLDL, o f t e n i n c r e a s e s plasma 23)L.35 J 3 = Heparin releases LPL i n t o t h e c i r c u l a t i o n , where i t hydrolyzes TG from VLDL and chylomicrons. The r e l e a s e d f a t t y a c i d s must be p a r t i a l l y r e u t i l i z e d by t h e l i v e r f o r s y n t h e s i s of TG, and consequently VLDL. Turnover of VLDL i s t h u s i n c r e a s e d , w i t h a n o b l i g a t o r y i n c r e a s e i n LDL s y n t h e s i s . It seems b e t t e r t o a t t e m p t t o i n c r e a s e t h e TG-clearing mechanism a t i t s p h y s i o l o g i c a l s i t e , t h e t i s s u e c a p i l l a r y endothelium, where t h e r e l e a s e d FFA are t r a n s f e r r e d i n t o t i ~ s u e . 3 ~Glueck3e has r e p o r t e d t h a t oxandrolone , a n a n a b o l i c - a n d r o g e n i c s t e r o i d , reduced plasma VLDL and chylomicrons i n t y p e s 111, N and V, and produced s t r i k i n g i n c r e a s e s i n LPL enzymes which could be r e l e a s e d i n t o t h e c i r c u l a t i o n by h e p a r i n . Such i n c r e a s e s probably r e f l e c t i n c r e a s e d amounts of enzyme a t t h e t i s s u e c a p i l l a r y s i t e . LDL v a l u e s were n o t rep o r t e d i n t h i s s t u d y , b u t c h o l e s t e r o l levels, most of which were o r i g i n a l l y i n t h e normal r a n g e , showed l i t t l e change. S i m i l a r e f f e c t s a r e reported38 f o r t h e p r o g e s t a t i o n a l s t e r o i d n o r e t h i n d r o n e a c e t a t e . Obesity & C h o l e s t e r o l Metabolism - A r e l a t i o n s h i p between o b e s i t y and a t h e r o s c l e r o s i s has been s u s p e c t e d and proposed40on a n e m p i r i c a l b a s i s , b u t l i t t l e understanding has evolved, r e l a t i v e t o t h e p o t e n t i a l importance

Chap. 16

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o f such a r e l a t i o n s h i p . I n p a r t i c u l a r , no c o n s i s t e n t r e l a t i o n s h i p has been found between plasma c h o l e s t e r o l and o b e s i t y .

-

Schreibman e t a141 s t u d i e d c h o l e s t e r o l k i n e t i c s i n v e r y obese Pa t i e n t s w i t h normal c h o l e s t e r o l levels, and found c h o l e s t e r o l t u r n o v e r r a t e s 2 112 times normal. E x c r e t i o n s o f b o t h n e u t r a l s t e r o l s and b i l e a c i d s were i n c r e a s e d . A n a l y s i s of plasma l a b e l e d - c h o l e s t e r o l decay curves showed normal s i z e of t h e r e a d i l y exchangeable c h o l e s t e r o l pool (pool A), b u t t h e slowly exchanging pool (pool B ) was doubled. The e x t r a mass of c h o l e s t e r o l i n pool B could be accounted f o r by t h e c h o l e s t e r o l c o n t e n t of excess adipose t i s s u e . I n t h e rat, Angel and F a r k a ~ 'r~e p o r t e d t h a t c h o l e s t e r o l s y n t h e s i s from g l u c o s e occurs a t about o n e - f i f t h t h e rate i n a d i p o s e t i s s u e a s i n l i v e r , p e r u n i t weight of t i s s u e . Because of t h e g r e a t e r mass of a d i p o s e t i s s u e , i t ranked w i t h l i v e r a s a c h o l e s t e r o g e n i c organ. Under c o n d i t i o n s of d i e t a r y c h o l e s t e r o l i n t a k e , c h o l e s t e r o l s y n t h e s i s i n a d i p o s e t i s s u e was suppressed much less t h a n i n l i v e r , and a d i p o s e t i s s u e became a p r i n c i p a l endogenous s o u r c e of c h o l e s t e r o l . These s t u d i e s show that a d i p o s e t i s s u e serves as a p o t e n t i a l l y i m p o r t a n t s i t e of c h o l e s t e r o l p r o d u c t i o n and as a r e s e r v o i r of slowly mob i l i z a b l e c h o l e s t e r o l . I n s p i t e of t h i s , t h e v e r y obese p a t i e n t s of Schreibman e t a1 m a i n t a i n e d normal plasma levels of c h o l e s t e r o l . It i s h a r d t o see how o b e s i t y posed a t h r e a t t o t h e a r t e r i e s of t h e s e p a t i e n t s , a t least v i a c h o l e s t e r o l , i n a s t e a d y s t a t e of body w e i g h t . Conceivably, r a p i d l o s s of body weight c o u l d m o b i l i z e l a r g e q u a n t i t i e s of c h o l e s t e r o l and r a i s e t h e plasma c o n c e n t r a t i o n u n t i l a new s t e a d y s t a t e was e s t a b l i s h e d . Obese persons might e x h i b i t r e s i s t a n c e t o hypocholesteremic drugs by v i r t u e o f a n a b i l i t y t o compensate, out of a d i p o s e t i s s u e s t o r e s , f o r d e c r e a s e d c h o l e s t e r o l s y n t h e s i s o r i n c r e a s e d e x c r e t i o n . Ahrens43 has obs e r v e d maintenance of e l e v a t e d plasma c h o l e s t e r o l i n t h e f a c e of n e t l o s s of body c h o l e s t e r o l d u r i n g c l o f i b r a t e t h e r a p y , b u t d i d n o t r e p o r t t h a t t h i s was r e l a t e d t o o b e s i t y .

-

I n s p i t e of t h e high p r o b a b i l i t y t h a t i n f i l t r a t i o n of liAtherogenesis p o p r o t e i n s i n t o t h e a r t e r i a l wall i s a n e s s e n t i a l e v e n t i n a t h e r o g e n e s i s , t h e r e i s r e l a t i v e l y l i t t l e understanding of t h e mechanisms of subsequent e v e n t s : l i p i d d e p o s i t i o n , i n t i m a l p r o l i f e r a t i o n , smooth muscle c e l l mig r a t i o n t o t h e s u b i n t i m a , foam c e l l formation, etc. It i s known t h a t low-density and v e r y low-density l i p o p r o t e i n s can form complexes w i t h a c i d mucopolysaccharides (AMPS), prominent components of e a r l y l e s i o n s . SJch complexes have now been i s o l a t e d from e a r l y human l e s i o n s by Berenson e t a l , 4 4 a n d shown t o i n c l u d e b o t h LDL and VLDL comp l e x e s , w i t h predominance of LDL. The AMPS i n v o l v e d were c h o n d r o i t i n s u l f a t e and h e p a r i t i n s u l f a t e .

The e l a s t i c lamellae, which provide e l a s t i c i t y t o t h e a r t e r i a l w a l l , f i g u r e prominently i n t h e e a r l y phases of development of t h e a t h e r o s c l e r o t i c l e s i o n . They a r e s i t e s f o r m i c r o s c o p i c a l l y v i s i b l e l i p i d

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d e p o s i t i o n and undergo f r a g m e n t a t i o n and d i s r u p t i o n . Kramsch e t a145 have p u b l i s h e d a s t u d y of e l a s t i n composition i n normal a n d a t h e r o s c l e r o t i c human a o r t a s . S t r i k i n g i n c r e a s e s i n l i p i d c o n t e n t o f e l a s t i n were c o r r e l a t e d w i t h s e v e r i t y of a t h e r o s c l e r o s i s . E i g h t y p e r c e n t of t h e l i p i d i n e l a s t i n from normal t i s s u e and from plaques was c h o l e s t e r o l , p r e s e n t c h i e f l y a s e s t e r s . Concomitant w i t h t h e i n c r e a s e i n l i p i d s i n a t h e r o s c l e r o t i c samples, t h e r e were a n i n c r e a s e i n p o l a r amino a c i d s i n e l a s t i n and a d e c r e a s e i n c r o s s q i n k i n g amino a c i d s . Using a d i f f e r e n t i s o l a t i o n procedure, Y ~ ~ ~ a found l s o a n i n c r e a s e d c o n t e n t of p o l a r amlno a c i d s i n e l a s t i n from a t h e r o s c l e r o t i c a o r t a s , b u t e s s e n t i a l l y no change i n c r o s s l i n k i n g amino a c i d s . Robert e t a14?have d i s c u s s e d t h e b i o c h e m i s t r y of e l a s t i n as r e l a t e d t o a g i n g and a t h e r o s c l e r o s i s . The high e l a s t i n c o n t e n t of a r t e r i e s may a c c o u n t i n p a r t f o r t h e i r e x t r a o r d i n a r y a b i l i t y t o t a k e up c h o l e s t e r o l from t h e c i r c ~ l a t i o n . ~ *Uptake of l i p i d s by e l a s t i n may p a r t i a l l y d e n a t u r e t h e p r o t e i n , r e n d e r i n g i t more s u s c e p t i b l e t o hydrol y s i s by elastase a n d o t h e r p r o t e a s e s d e r i v e d from p l a t e l e t s o r phagoc y t e s .47 L i p i d - f i l l e d c e l l s , "foam c e l l s , " a r e a prominent f e a t u r e of t h e a t h e r o s c l e r o t i c l e s i o n , and c o n t r i b u t e t o t h e c o r e of c e l l u l a r d e b r i s , t h e s o - c a l l e d " g r u e l " of advanced l e s i o n s . T h i s material can embolize d i r e c t l y from r u p t u r e d plaques o r s e r v e a s a thrombogenic focus. The o r i g i n of t h e s e c e l l s has been d i s p u t e d , a l t h o u g h c o n s i d e r a b l e e v i d e n c e has accumulated r e c e n t l y t h a t t h e y d e r i v e from smooth muscle c e l l s . The c o n t r i b u t i o n , i f a n y , of c i r c u l a t i n g macrophages t o t h i s pool of c e l l s has remained u n s e t t l e d . Cookson,49in a n e l e c t r o n m i c r o s c o p i c s t u d y of l e s i o n s i n c h o l e s t e r o l - f e d r a b b i t s , confirmed t h e c o n t r i b u t i o n o f smooth muscle c e l l s , and o b t a i n e d e v i d e n c e t h a t c i r c u l a t i n g macrophages p l a y a prominent r o l e i n p h a g o c y t o s i s of l i p i d i n t h e l e s i o n , and i n f o r m a t i o n of d e b r i s by r a p i d d e g e n e r a t i o n . Wurster and Z i l v e r s m i t , 6 0 i n a s t u d y of phagocytic a c t i v i t y of p e r i t o n e a l macrophages from c h o l e s t e r o l - f e d r a b b i t s , concluded that t h i s t y p e of c e l l probably does n o t c o n t r i b u t e i m p o r t a n t l y t o l i p i d accumulation i n t h e a o r t a . The two s t u d i e s are n o t n e c e s s a r i l y i n c o m p a t i b l e , b u t f u r t h e r work w i l l b e r e q u i r e d t o s e t t l e t h i s problem. Cookson s u g g e s t s t h a t t h e a c t i v i t y of t h e smooth muscle c e l l i s e s s e n t i a l l y r e p a r a t i v e , and s h o u l d be s t i m u l a t e d t o promote h e a l i n g of t h e l e s i o n . Dzoga e t a l s l h a v e r e p o r t e d t h a t h y p e r l i p e m i c serum, o r LDL i s o l a t e d t h e r e f r o m , from c h o l e s t e r o l - f e d r a b b i t s o r monkeys s t i m u l a t e s growth of homologous a r t e r i a l smooth muscle c e l l s i n t i s s u e culture. LDL from normal a n i m a l s d i d n o t have t h i s e f f e c t a t t h e same concentration. C h o l e s t e r o l accumulated i n atheromatous l e s i o n s i s l a r g e l y esteri f i e d w i t h f a t t y a c i d s , e s p e c i a l l y o l e i c a c i d . The change i n f a t t y a c i d composition of c h o l e s t e r o l esters between c i r c u l a t i n g l i p o p r o t e i n s and atheromas i n d i c a t e s a n e s t e r i f i c a t i o n p r o c e s s o p e r a t i v e i n t h e developing l e s i o n . K o t h a r i e t a F 2 have demonstrated enzymes i n normal artery which c a t a l y z e b o t h s y n t h e s i s a n d h y d r o l y s i s of c h o l e s t e r o l esters. R e a c t i o n rates of e s t e r i f i c a t i o n and h y d r o l y s i s were found t o be s t r o n g l y dependent on t h e p h y s i c a l s t a t e of t h e r e a c t a n t s : a n e m u l s i f i e d s t a t e promoted e s t e r i f i c a t i o n , whereas a m i c e l l a r s t a t e promoted h y d r o l y s i s .

Chap. 16

Blohrn

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A t h e r o s c l e r o t i c t i s s u e i s r e p o r t e d t o have a h i g h e r c h o l e s t e r o l esteri f i c a t i o n r a t e t h a n normal a r t e r i a l t i s s u e , 5 3 , 5 4 a s w e l l as a g r e a t e r p e r m e a b i l i t y t o c h o l e s t e r o l e s t e r s bound t o l i p o p r o t e i n . 5 4 , 5 5 Caro e t a156have p u b l i s h e d a new h y p o t h e s i s r e l a t i n g s h e a r a t t h e blood-wall i n t e r f a c e t o remwal of c h o l e s t e r o l from t h e a r t e r y w a l l . An assumption t h a t c h o l e s t e r o l s y n t h e s i s i n t h e w a l l i s a n important s o u r c e of l e s i o n c h o l e s t e r o l i s probably i n v a l i d , b u t c o u l d be r e p l a c e d by a n assumption r e g a r d i n g c h o l e s t e r o l e s t e r i f i c a t i o n . The h y p o t h e s i s i s i n tended t o account f o r t h e p a t t e r n of d i s t r i b u t i o n of human l e s i o n s . E s t r o g e n s and o t h e r Hormones - Evidence from c l i n i c a l t r i a l s i n d i c a t e s but not e t h i n y l e s t r a d i 0 1 ~ , 5~ 90r t h a t conjugated e q u i n e e s t r o g e n s ,E7 A n ~ e n e F0 , ~ p~r o t e c t a g a i n s t d e a t h from coronary h e a r t d i s e a s e i n s u r v i v o r s of myocardial i n f a r c t i o n . D i s s o c i a t i o n of t h e p r o t e c t i v e e f f e c t from changes i n blood c h o l e s t e r o l was observed,58 ,59and l a r g e doses o f e q u i n e e s t r o g e n s , s u f f i c i e n t t o a l t e r blood l i p i d s , r e s u l t e d i n excess c a r d i o vascular mortality,57 e s p e c i a l l y during t h e e a r l y p o s t - i n f a r c t i o n p e r i o d .5 Wolinskf" has shown t h a t h y p e r t e n s i o n produces i n c r e a s e s i n a o r t i c t h i c k n e s s , c e l l u l a r p r o t e i n , e l a s t i n and c o l l a g e n , and t h a t t h e s e changes reverse i n female rats when t h e blood p r e s s u r e i s r e t u r n e d t o normal. The changes d i d n o t r e v e r s e i n males, b u t e s t r a d i o l t r e a t m e n t of males d u r i n g Progestin t h e h y p e r t e n s i v e phase prevented t h e h y p e r t r o p h i c changes .63 t r e a t m e n t s l i g h t l y enhanced t h e e f f e c t s of h y p e r t e n s i o n . F i s c h e r has rep o r t e 8 t h a t e s t r a d i o l replacement i n w a r i e c t o m i z e d r a t s i n c r e a s e d t u r n wer of e l a s t i n and c o l l a g e n i n a o r t a , d e c r e a s e d t o t a l a o r t i c c o l l a g e n and d e c r e a s e d c o l l a g e n - t o -e l a s t i n r a t i o

.

These s t u d i e s s t r o n g l y s u g g e s t t h a t e s t r o g e n s reduce a r t e r i a l responses t o i n j u r y , which a r e important i n development of t h e a t h e r o s c l e r o t i c l e s i o n , s p e c i f i c a l l y c e l l u l a r p r o l i f e r a t i o n and l a y i n g down of conn e c t i v e t i s s u e f i b e r s . Such e f f e c t s , i f l i m i t e d , could r e t a r d t h e c h r o n i c a t h e r o g e n i c p r o c e s s , b u t t h e y could be harmful i n circumstances r e q u i r i n g s t r e n g t h e n i n g and r e p a i r of s e v e r e l y d i s e a s e d t i s s u e , e s p e c i a l l y weakened walls of arteries and h e a r t . The p o t e n t i a l importance of t h e s e f i n d i n g s r e q u i r e s t h e i r c o n f i r m a t i o n , p r e f e r a b l y i n a n o t h e r s p e c i e s i n which wera l l c a t a b o l i c e f f e c t s of e s t r o g e n s a r e less pronounced. Marked hypocholesterolemic e f f e c t s of c h r o n i c glucagon a d m i n i s t r a t i o n have been d e s c r i b e d f o r r a t s i n a v a r i e t y of d i e t a r y and e n d o c r i n e situation^.^^ The d a t a i n d i c a t e that t h i s hormone must be t a k e n i n t o account i n e v a l u a t i n g e f f e c t s of e n d o c r i n e m a n i p u l a t i o n on blood cholesterol.

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Dayto$6 has d e l i n e a t e d t h e need f o r h y p o l i p i d e T r i a l s of Druq E f f i c a c y m i c drugs a s p a r t of a t o t a l program f o r l i p i d c o n t r o l i n a t h e r o s c l e r o s i s . Although t h e r a t i o n a l e f o r use of such drugs i s s t r o n g , f i n a l proof of

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e f f i c a c y must come from unequivocal evidence of p r e v e n t i o n o r r e v e r s a l of human a t h e r o s c l e r o s i s a n d l o r i t s c o m p l i c a t i o n s . w i t h t h e advent of c l o f i b r a t e as a n a c t i v e hypolipidemic drug, s e v e r a l s t u d i e s were i n i t i a t e d . R e s u l t s from two o f t h e s e have now been r e p o r t e d . Both s t u d i e s ( d e s i g n a t e d t h e N e ~ c a s t l e ~ ~ aSnc do t t i s h G 8 s t u d i e s ) f a l l i n t h e c a t e g o r y of secondary p r e v e n t i o n t r i a l s , i . e . t h e y were designed t o determine t h e e f f e c t s of t r e a t m e n t on p r o g r e s s of a t h e r o s c l e r o t i c d i s e a s e m a n i f e s t a t i o n s i n p a t i e n t s e s t a b l i s h e d as having t h e d i s e a s e a t t h e o n s e t . Although d i f f e r e n c e s between t h e s t u d i e s e x i s t , l e a d i n g t o some d i f f e r e n c e s i n f i n d i n g s , c e r t a i n important and unexpected r e s u l t s were common t o b o t h s t u d i e s . C l o f i b r a t e was found t o p r o t e c t a g a i n s t d e a t h and new myocardial i n f a r c t i o n i n t h o s e p a t i e n t s e x h i b i t i n g a n g i n a as a m a n i f e s t a t i o n of t h e i r d l s e a s e , b u t s i g n i f i c a n t p r o t e c t i o n was n o t a f f o r d e d t o p a t i e n t s whose m a n i f e s t a t i o n was p r e v i o u s myocardial i n f a r c t i o n w i t h o u t a n g i n a . Among t h e c l o f i b r a t e - t r e a t e d subgroups i n t h e Newcastle s t u d y , t h e r e was no rel a t i o n s h i p between c h o l e s t e r o l r e d u c t i o n and p r o t e c t i o n a g a i n s t c a r d i o v a s c u l a r d e a t h o r myocardial i n f a r c t i o n , a l t h o u g h such a r e l a t i o n s h i p obt a i n e d i n p a r t of t h e S c o t t i s h s t u d y . These r e s u l t s s u g g e s t t h a t t h e m j o r p r o t e c t i v e e f f e c t of c l o f i b r a t e i n t h e s e groups of p a t i e n t s may n o t have been mediated by r e d u c t i o n of blood l i p i d s . Although t h e s u b j e c t s were chosen f o r t h e i r m a n i f e s t (and t h e r e f o r e advanced) d i s e a s e and might be expected t o respond r e l a t i v e l y poorly t o l i p i d r e d u c t i o n , t h i s c o n c l u s i o n i s d i s t u r b i n g . The a u t h o r s of t h e Newcastle s t u d y s u g g e s t t h a t c l o f i b r a t e somehow p r o t e c t s a g a i n s t dysrhythmia, a common cause of d e a t h i n a n g i n a p a t i e n t s . C l o f i b r a t e - Space does n o t p e r m i t d i s c u s s i o n of t h e many p u b l i c a t i o n s on t h i s drug. Some of t h e more important ones d e a l t w i t h e f f e c t s on chol e s t e r o l s y n t h e s i s ,69 j 7 0 c h o l e s t e r o l and b i l e a c i d e ~ c r e t i o nt ,r ~ i g~l y c e r i d e ~ y n t h e s i s , ~j73and ' e f f e c t s on c e l l u l t r a s t r ~ c t u r e . ~F o~r g e n e r a l i n f o r mation, two s o u r c e s a r e r e c ~ m m e n d e d ,76 .~~

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I n v e s t i g a t i o n a l Drugs A number of c l i n i c a l p u b l i c a t i o n s on nafenopin (SU-13,437, Melipan) e.ppeared d u r i n g t h e p a s t year.77-83 I n general, r e s u l t s confirm p r e v i o u s r e p o r t s of g r e a t e r r e d u c t i o n s of serum t r i g l y c e r i d e s t h a n of serum c h o l e s t e r o l . E a r l y claims f o r a n advantage over c l o f i b r a t e i n t r e a t m e n t of t y p e I1 hyperlipidemia have n o t been confirmed. I n a comparative s t u d y of t h e two d r u g s , Dujovne e t a177found nafenopin (600 mg p e r day) s l i g h t l y , b u t n o t s i g n i f i c a n t l y s u p e r i o r t o c l o f i b r a t e (2 gm p e r day) i n overall r e d u c t i o n of serum l i p i d s , b u t c h o l e s t e r o l red u c t i o n was n o t s u p e r i o r i n t y p e 11, and t h e r e may have been a n "escape" of serum TG i n t h e t y p e IV p a t i e n t s w i t h nafenopin. I n c i d e n c e of l i v e r f u n c t i o n a b n o r m a l i t y was h i g h e r on nafenopin t h a n on c l o f i b r a t e , b u t c l o f i b r a t e produced a s i g n i f i c a n t e l e v a t i o n of serum c r e a t i n e k i n a s e ( u s u a l l y r e l a t e d t o muscle damage), whereas nafenopin d i d n o t . Hartmann,78Manucci e t a179and F e l l i n e t a l e o f o u n d Type I1 h y p e r l i p i d e m i a less r e s p o n s i v e t h a n t h e h y p e r t r i g l y c e r i d e m i a s t o n a f e n o p i n ; a l l r e p o r t e d no, o r minimal, e f f e c t s on l i v e r f u n c t i o n , b u t deGennes and coworkerse1 r e p o r t e d f o u r cases of j a u n d i c e "of unknown mechanism" i n a s e r i e s of 126 p a t i e n t s . Manucci e t a1 found nafenopin t o lower e l e v a t e d plasma f i b r i n o g e n l e v e l s i n hy-

Chap. 16

Atherosclerosis

Blohm

177 -

p e r l i p i d e m i c p a t i e n t s , b u t t o i n c r e a s e plasma plasminogen and d e c r e a s e e u g l o b u l i n l y s i s t i m e ( t h e l a t t e r two i n d i c a t i n g a p o s s i b l e r e d u c t i o n i n a b i l i t y t o l y s e c l o t s ) . No e f f e c t was s e e n on p l a t e l e t a d h e s i v e n e s s t o g l a s s . Nafenopin i s r e p o r t e d t o i n c r e a s e h e p a t i c f a t t y a c i d s y n t h e s i s i n t h e mouse w i t h l i t t l e e f f e c t on c h o l e s t e r o l s y n t h e s i ~ . ~ *A f i n d i n g of l i v e r pathology i n long-term, high-dose s t u d i e s i n rats has r e s u l t e d i n withdrawal of t h e IND f o r nafenopin.82 H a l o f e n a t e (MK -185, 2-acetamidoethyl (p-chlorophenyl) ( m - t r i fluoromethylphenoxy) a c e t a t e ) i s a hypolipidemic compound w i t h a cons i s t e n t hypouricemic e f f e ~ t . ~ I~n -r a~ t s~, h a l o f e n a t e reduced b o t h chol e s t e r o l and TG, w i t h a potency 5.7 t i m e s t h a t of c l ~ f i b r a t e , ~ ~ bi nu tman i t has demonstrated l i t t l e a b i l i t y t o reduce serum c h o l e s t e r o l , a l t h o u g h i t i s about twice as p o t e n t as c l o f i b r a t e , b u t no more e f f e c t i v e , i n r e ducing serum TG.B6-88 H a l o f e n a t e is r e p o r t e d t o d i s p l a c e t h y r o x i n e from plasma t h y r o x i n e b i n d i n g g l o b u l i n , 8 5 t h e r e b y reducing protein-bound i o d i n e ; t h e r e i s no a p p a r e n t e f f e c t on c l i n i c a l t h y r o i d s t a t u s . Serum u r i c a c i d ~ t h e coml e v e l s a r e lowered through a u r i c o s u r i c m e c h a n i ~ m . ~Although pound, l i k e c l o f i b r a t e , causes l i v e r enlargement i n r a t ~ , ~ ~a bnn o r m a l i t i e s of l i v e r f u n c t i o n were r e p o r t e d . Serum c r e a t i n e k i n a s e was e l e v a t e d , and t h e g r e a t e s t e l e v a t i o n s were a s s o c i a t e d w i t h g a s t r o i n t e s t i n a l symptoms.85 Removal of b i l e a c i d s v i a i n c r e a s e d f e c a l e x c r e t i o n has been accompl i s h e d through use of b i l e - a c i d b i n d i n g r e s i n s , and by i l e a l by-pass o p e r a t i o n s . Both procedures have been e f f e c t i v e i n reducing serum chol e s t e r o l l e v e l s i n p a t i e n t s w i t h t h e heterozygous form of type I1 hyperl i p i d e m i a , b u t much less s o i n t h e homozygous Grundy e t a l g 2 have examined t h e dynamics of c h o l e s t e r o l and b i l e a c i d s i n t y p e I1 p a t i e n t s r e c e i v i n g cholestyramine o r undergoing i l e a l e x c l u s i o n . They found t h e two t r e a t m e n t s e q u i v a l e n t , even q u a n t i t a t i v e l y . G e n e r a l l y , t h e Grundy e t a1 found t h a t o p e r a t i v e procedure has been more e f f e c t i v e . 9 o b i l e acid removal l e d t o i n c r e a s e d c h o l e s t e r o l s y n t h e s i s , probably b o t h i n l i v e r and i n t e s t i n e , and t h a t t h i s compensated f o r t h e b i l e a c i d l o s s i n some c a s e s , i n which plasma c h o l e s t e r o l d i d n o t d e c r e a s e , o r even i n c r e a s e d . KuS3 has r e p o r t e d t h a t t h e f r e q u e n t l y observed e l e v a t i o n of plasma VLDL d u r i n g c h o l e s t y r a m i n e t h e r a p y can be c o n t r o l l e d by carbohyd r a t e and c a l o r i e r e s t r i c t i o n . C o l e s t i p o l (U-26,597A), a copolymer of t e t r a e t h y l e n e p e n t a m i n e and e p i c h l o r h y d r i n , has been r e p o r t e d t o be s i m i l a r t o cholestyramine i n p o t e n c F 4 jg5and t o l e r a n c e . 9 5 Combinations of b i l e a c i d - b i n d i n g a g e n t s w i t h o t h e r drugs a r e d i s c u s s e d under l i p o p r o t e i n metabolism i n t h i s review. The mechanism of t h e hypocholesteremic e f f e c t of neomycin has been reviewed and i n v e s t i g a t e d by Thompson and coworkers .96 They found t h a t t h i s b a s i c a n t i b i o t i c caused p r e c i p i t a t i o n of c h o l e s t e r o l , f a t t y a c i d s and b i l e a c i d s from t h e micellar phase of a s p i r a t e from t h e small i n t e s t i n e , f o l l o w i n g a d m i n i s t r a t i o n w i t h a t e s t m e a l . D i s r u p t i o n of mixed m i c e l l e s of these s u b s t a n c e s could be expected t o i n t e r f e r e w i t h a b s o r p t i o n , e s p e c i a l l y of c h o l e s t e r o l . Lipase a c t i v i t y was n o t a f f e c t e d . Samuelg7 has rep o r t e d a s t r o n g c o r r e l a t i o n between t h e a b i l i t y o f neomycin t o p r e v e n t f e c a l c o n v e r s i o n of c h o l i c a c i d t o deoxycholic a c i d and i t s a b i l i t y t o

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lcwer plasma c h o l e s t e r o l i n hypercholesteremic c h i l d r e n .

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Miscellaneous and New Drugs Kritchevsky9* has reviewed r e c e n t l y developed hypolipidemic drugs. Cardiac t o l e r a n c e t o D-thyroxine ( d e x t r o - t h y r o x i n e , D-T4) i s r e p o r t e d t o be improved by concomitant a d m i n i s t r a t i o n of t h e @-blocking a g e n t , p r o p a n o l ~ l . ~A ~c l i n i c a l r e p o r t l o o i n d i c a t e s t h a t D - t r i i o d o t h v r o n i n e (D-T3 ,dextro-triiodothyronine) i s a n e f f e c t i v e hypol i p i d e m i c a g e n t w i t h minimal c a r d i a c s i d e e f f e c t s . A new t h y r o x i n e a n a l o g , D,L-a-methylthyroxine e t h y l ester (CG-635) has been s t u d i e d i n Germany.lol lo: The compound i s r e p o r t e d t o have improved r a t i o s of hypocholesteremic e f f e c t t o e f f e c t s on C&-consumption, h e a r t weight and h e a r t rate i n t h e r a t , i n comparison t o t h e T, a n d T4 isomers.102 The c l i n i c a l dose is r e l a t i v e l y l a r g e ( 4 0 mg/day) and P B I i n c r e a s e s markedly.lo3 CG-635 lowered plasma c h o l e s t e r o l moderately i n t y p e s I1 and IV p a t i e n t s , and t r i g l y c e r i d e s i n type IV.101-103 Some e f f e c t s on l i v e r f u n c t i o n were noted.lo1

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(DH-581, Biphenabid) was shown t o produce moderate reProbucol d u c t i o n s i n plasma c h o l e s t e r o l i n s e v e r a l c l i n i c a l s t u d i e s ; l o 4 - l o 7 t r i g l y c e r i d e s were n o t reduced, and i n some cases were i n ~ r e a s e d . 1 ~ ~ Mechanism of t h e hypocholesteremic e f f e c t remains i n doubt, b u t may i n v o l v e i n h i b i t i o n of r e l e a s e of l i p o p r o t e i n c h o l e s t e r o l from t h e 1 i v e r . l 0 * Kritchevsky e t a l l o g found probucol t o reduce plasma c h o l e s t e r o l and a t h e r o n a h r m a t i o n i n c h o l e s t e r o l - f e d r a b b i t s . Probucol a p p a r e n t l y was w e l l t o l e r a t e d i n t h e human s t u d i e s .

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(methyl 2,10-dichloro-12H_-dibenzo[d,g][1,3]dioxocinTreloxinate 6 - c a r b o x y l a t e ) i s a new hypolipidemic a g e n t of t h e g e n e r a l c l o f i b r a t e class.ll0 y l l l The p-chlorophenyl s u b s t i t u e n t s a r e h e l d i n a f i x e d conformation a s p a r t of t h e d i o x o c i n r i n g s t r u c t u r e . I n r a t s t r e l o x i n a t e i s 8 t i m e s a s p o t e n t as c l o f i b r a t e i n reducing plasma c h o l e s t e r o l , and 30 t i m e s a s p o t e n t i n reducing t r i g l y c e r i d e s . T r e l o x i n a t e i n h i b i t e d h e p a t i c s y n t h e s i s of c h o l e s t e r o l from a c e t a t e , b u t not from rnevalonate i n t h e rat, and i n c r e a s e d o x i d a t i o n of c h o l e s t e r o l - 2 6 ' 4 C t o 1 4 C 4 . In hypothyroid dogs, i t markedly reduced LDL c h o l e s t e r o l , b u t n o t HDL c h o l e s t e r o l .

Chap.

I6

Atherosclerosis

I79

B lohm

D-N-(a-methylbenzyl) l i n o l e a m i d e i s t h e most p o t e n t o f a s e r i e s o f N - s u b s t i t u t e d l i n o l e a m i d e s w h i c h p r e v e n t h y p e r c h o l e s t e r a m i a and a t h e r ogenes is i n c h o l e s t e r o l - f e d r a b b i t s . ” 2 The compound does n o t a f f e c t c h o l e s t e r o l s y n t h e s i s o r s i d e - c h a i n o x i d a t i o n i n v i t r o , b u t appears t o i n h i b i t c h o l e s t e r o l a b s o r p t i o n . l l 3 T h i s e f f e c t may be r e l a t e d t o i t s a b i l i t y t o f o r m a complex w i t h c h o l e s t e r o l . l l 4 The D-isomer i s more s l o w l y m e t a b o l i z e d t h a n t h e L -i s o me r, w h i c h may a c c o u n t f o r i t s g r e a t e r a c t i v i t y . l l 5 Some s i m p l e mono- and d i - s u b s t i t u t e d n i c o t i n i c a c i d s a r e r e p o r t e d l ’ s t o be more p o t e n t t han t h e p a r e n t compound i n i n h i b i t i n g c h o l e s t e r o l and f a t t y a c i d synthesis i n r a t l i v e r . References

1. 2.

3. 4.

5. 6.

7.

P a t h o l o g y o f t h e H e a r t and Blood V e s s e l s , 3 r d e d i t i o n , S . E . Gould, e d . , C h a r l e s C . Thomas, S p r i n g f i e l d , Ill. 1968. R e p o r t Of I n t e r - S o c i e t y Commission f o r H e a r t D i s e a s e R e s o u r c e s , C i r c u l a t i o n 4 2 , A-55 11970). A t h e r o s c l e r o s i s - P a t h o l o g y , P h y s i o l o g y , A e t i o l o g y and C l i n i c a l Management,. F.G. S c h e t t l e r and G.S. Boyd, e d s . , E l s e v i e r , New York 1969. E v o l u t i o n o f t h e A t h e r o s c l e r o t i c P l a q u e , R . J . J o n e s , e d . , U n i v e r s i t y of Chicago P r e s s 1963. A t h e r o s c l e r o s i s - P r o c e e d i n g s of t h e Second I n t e r n a t i o n a l Symposium on A t h e r o s c l e r o s i s , R . J . J o n e s , e d . , S p r i n g e r , New York 1970. J . W . Gofman, F. L i n d g r e n , H . E l l i o t t , W . Mantz, J . H e w i t t , B. S t r i s o w e r , V . H e r r i n g and T . P . Lyon, S c i e n c e 111, 166 ( 1 9 5 0 ) . 3 4 , 9 4 , 1 4 8 , 215, D.S. F r e d r i c k s o n , R.I. Levy and R . S . L e e s , New Eng. J . Med.

s,

273 (19671.

8,

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V . N . Schumaker and G.H. Adams, Ann. Rev. Biochem. 113 ( 1 9 6 9 ) . 9 . P . A l a u p o v i c , A t h e r o s c l e r o s i s 2, 1 4 1 ( 1 9 7 1 ) . 10. D . S . F r e d r i c k s o n , R . I . Levy and R.S. L e e s , N e w Eng. J . Med. 215 (1967). 11. M.J. A l b r i n k , J . W . Meigs and E.B. Man, Am. J . Med. 1 . 4 (1961). 1 2 . W.B. K , n n e l , W.P. C a s t e l l i , T . Gordon and P.M. McNamara, Ann. I n t . Med. 1 (1971). 796 (1971). 13. M. T z a g o u r n i s and P.E. S t e f f e n s , Ann. I n t . Med. 14. C.E. Bemis, L.M. E b e r , H . G . KemF and R. G o r l i n , C i r c u l a t i o n S u p p l . 11, 47 (19’11). S u p p l . 11, 47 15. A.F. S a l e l , E.A. Amsterdam, D.T. Mason and R . Z e l i s , C i r c u l a t i o n

a,

z,

B,

44,

44,

(1971). 1 6 . A.L. J o n e s , N.B. Ruderman and M.G. H e r r e r a , P r o c . SOC. Exp. B i o l . (1966).

& Med.

123, 4

1 7 . R.L. H a m i l t o n , F o u r t h I n t e r n a t i o n a l Symposium on Drugs A f f e c t i n g L i p i d Metabolism, W.L. Holmes, Lankenau H o s p i t a l , P h i l a . , Secy. A b s t r a c t s p. 11, 1971. I n p r e s s a s P h a r m a c o l o g i c a l C o n t r o l o f L i p i d Metabolism, Adv. Exp. Med. B i o l . , Plenum P u b l . C o r p . , New York. Given a s DALM i n s u b s e q u e n t r e f e r e n c e s . 18. 0. S t e i n and Y. S t e i n , J . C e l l B i o l . 319 (1967). S u p p l . 11, 9 ( 1 9 7 1 ) . 1 9 . R.J. H a v e l , M.L. Kashyap and J . P . Kane, C i r c u l a t i o n 20. S . E i s e n b e r g , D.W. B i l h e i m e r , F.T. L i n d g r e n and R . I . Levy, C i r c u l a t i o n $, S u p p l .

z,

44,

r r , 9 (1971). 21. B. S h o r e and V . S h o r e , i n A t h e r o s c l e r o s i s , P r o c e e d i n g s of t h e Second I n t e r n a t i o n a l Symposium, R . J . J o n e s , e d i t o r , S p r i n g e r , New York, p. 144 ( 1 9 7 0 ) . 22. J . C. L:Rosa, R . I . Levy, P. H e r b e r t , S.E. Lux and D. S . F r e d r i c k s o n , Biochem. B i o p h y s . Res. Communications 57 ( 1 9 7 0 ) . 23. R . J . H a v e l , V . G . S h o r e , B. S h o r e and D . M . B i e r , C i r c . Res. 11,595 ( 1 9 7 0 ) . S u p p l . 11, 9 ( 1 9 7 1 ) . 24. W.V. Brown and M.L. B a g i n s k y , C i r c u l a t i o n 25. R . I . Levy, DALM ( s e e r e f . 1 7 ) A b s t r a c t s p. 11 ( 1 9 7 1 ) . 26. J . L . Beaumont, L.A. C a r l s o n , G . R . Cooper, Z . F e j f a r , D.S. F r e d r i c k s o n , T . S t r a s s e r , 891 ( 1 9 7 0 ) . B u l l . WHO 9, S u p p l . 11, 58 (1971). 27. S.L. Nunn and J . Roubion, C i r c u l a t i o n 28. C . D . M o u t a f i s , N.B. Myant, M. Mancini and P. O r i e n t e , A t h e r o s c l e r o s i s 3 , 247 ( 1 9 7 1 ) .

5,

44,

9,

180 -

Sect. I V

-

Metabolic & Endocrine

Pachter, Ed.

29. H . R . Casdorph, DALM A b s t r a c t s ( s e e r e f . 17) 37 (1971). 30. A . N . Howard and D.E. Hyams, B r i t . Med. J . 3, 2 5 ( 1 9 7 1 ) . 31. S. B u a r f o r t , R . I . Levy, and D.S. F r e d r i c k s o n , J . C l i n . I n v e s t . 2, 744 ( 1 9 7 1 ) . 32. L.W. W h i t e , DALM A b s t r a c t s ( s e e r e f . 17). 1 2 1 ( 1 9 7 1 ) . 33. D.W. B i l h e i m e r , S. E i s e n b e r g and R . I . Levy, C i r c u l a t i o n 4 4 , S u p p l . 11, 56 ( 1 9 7 1 ) . 3 4 . D. Ganesan, R . H . B r a d f o r d , P . Alaupovic and W . McConathy, FEBS L e t t e r s 3. 205

(1971).

35. R . H . Furman, R . P . Howard and P. A l a u p o v i c , Metabolism 11,879 ( 1 9 6 2 ) . 36. A . V . N i c h o l s , E . H . S t r i s o w e r , F . T . L i n d g r e n , G . L . Adamson and E.L. C o g g i o l a , C l i n . Chim. Acta 20, 277 (1968). 37. R . J . H a v e l , i n HTndbook o f P h y s i o l o g y , v o l . 5 , Adipose T i s s u e , A.E. Renold and G.F. C a h i l l , e d . W i l l i a m s and W i l k i n s , B a l t i m o r e , p. 499 ( 1 9 6 5 ) . 38. C . J . G l u e c k , Metabolism 20, 691 ( 1 9 7 1 ) . 39. C . J . Glueck, F. Swanson, and T. H u t s e l l , C i r c u l a t i o n 158 ( 1 9 7 0 ) . 40. M.J. A l b r i n k and J . W . Meigs, i n Conference on Adipose T i s s u e Metabolism and O b e s i t y , Ann. N.Y. Acad. S c i . 673 ( 1 9 6 5 ) . 41. P . H . Schreibman, P. N e s t e l and E.H. Ahrens, C i r c u l a t i o n S u p p l . 11, 1 ( 1 9 7 1 ) . 42. A. Angel and J . F a r k a s , C i r c u l a t i o n S u p p l . 11, 1 (1971). 43. E . H . Ahrens, DALM A b s t r a c t s ( s e e r e f . 17) 2 ( 1 9 7 1 ) . 44. G . S . B e r e n s o n , S . R . S r i n i v a s a n , P . J . Dolan and B. Radhakrishnamurthy, C i r c u l a t i o n 4 4 , S u p p l . 11, 6 (1971). 45. D.M. Kramsch, C. F r a n z b l a u and W . H o l l a n d e r , J . C l i n . I n v e s t . 1666 ( 1 9 7 1 ) . 46. S.Y. Yu, Lab. I n v e s t . 3,1 2 1 ( 1 9 7 1 ) . 47. L. R o b e r t , B. R o b e r t and A . M . R o b e r t , Exp. G e r o n t . 5, 339 ( 1 9 7 0 ) . 249 ( 1 9 7 1 ) . 48. K . J . Ho and C.B. T a y l o r , P r o c . SOC. Exp. B i o l . & Med. 49. F.B. Cookson, B r i t . J . Exp. P a t h o l . 2,62 (1971). 50. N.B. W u r s t e r and D.B. Z i l v e r s m i t , A t h e r o s c l e r o s i s 309 (1971). 51. K. Dzoga, R.W. W i s s l e r and D. V e s s e l i n o v i t c h , C i r c u l a t i o n S u p p l . I1 6 ( 1 9 7 1 ) . 5 2 . II. K o t h a r i , B.F. M i l l e r and D. K r i t c h e v s k y , C i r c u l a t i o n S u p p l . 11, 5 ( 1 9 7 1 ) . 53. R.W. S t . C l a i r , C i r c u l a t i o n S u p p l . 11, 5 ( 1 9 7 1 ) . 5 4 . H . B . L o f l a n d , J r . , R . W . S t . C l a i r and T.B. Ckarkson, DALM A b s t r a c t s ( s e e r e f . 1 7 ) 12 (1971). 55. K.G. McCullagh and A.L. R o b e r t s o n , C i r c u l a t i o n S u p p l . 11, 5 ( 1 9 7 1 ) . 56. C . G . Caro, J . M . F i t z - G e r a l d and R . C . S c h r o t e r , P r o c . Roy. S O C . (London) B. 109 ( 1 9 7 1 ) . 57. J . S t a m l e r , R . P i c k , L.N. K a t z , A . P i c k , B.M. Kaplan, D.M. Berkson and D. C e n t u r y , J A M A ,&I 632 ( 1 9 6 3 ) . 58. J . Marmorston, F . J . Moore, C.E. Hopkins, O.T. Kuzma and J . Weiner, P r o c . S O C . Exp. B i o l . & Med. 110, 400 ( 1 9 6 2 ) . 59. M.F. O l i v e r and G.S. Boyd, L a n c e t 2, 499 ( 1 9 6 1 ) . 6 0 . V . A . D r i l l , D . L . Cook and R . A . E d g r e n , i n Hormones and A t h e r o s c l e r o s i s , G . P i n c u s , e d . , Academic P r e s s , New York 1958. 61. R e p o r t o f Coronary Drug P r o j e c t R e s e a r c h Group, JAMA 2,1303 ( 1 9 7 0 ) . 62. H . Wolinsky, C i r c . R e s . 622 ( 1 9 7 1 ) . 63. H. Wolinsky, C i r c u l a t i o n S u p p l . 11, 7 (1971). 64. G . M . F i s c h e r , C i r c u l a t i o n 3, S u p p l . 11, 7 (1971). 6 5 . M . Friedman, S . O . Byers and R . H . Rosenman, L a n c e t 2, 464 ( 1 9 7 1 ) . 66. S. Dayton, Fed. P r o c . 0, no. 3 , P a r t I , 849 (1971). 67. R e p o r t of Newcastle P h y s i c i a n s Group, B r . Med. J . 767 (1971). 68. R e p o r t Of R e s e a r c h Committee of S c o t t i s h S o c i e t y o f P h y s i c i a n s , B r . Med. J . 4, 775 ( 1 9 7 1 ) . 69. H.S. S o d h i , B . J . Kudchodkar, L. H o r l i c k and C . H . Weder, Metabolism 20, 348 (1971). 7 0 . L.W. W h i t e , J . Pharmacol. @, 3 6 1 ( 1 9 7 1 ) . 71. L. H o r l i c k , B . J . Kudchodkar and H.S. S o d h i , C i r c u l a t i o n 299 (1971). 7 2 . J . N . P e r e i r a , G.A. Mears and G.F. H o l l a n d , DALM A b s t r a c t s ( s e e r e f . 1 7 ) 9 4 (1971). 73. L . L . Adams, W . W . Webb and H . J . F a l l o n , J . C l i n . I n v e s t . 2, 2339 (1971). 74. D . J . Svoboda and D.L. A z a r n o f f , Fed. P r o c . 0, no. 3 , P a r t I , 8 4 1 (1971). 75. Symposium on Pharmacology of Hypolipidemic D r u g s , Fed. P r o c . no. 3 , P a r t I ,

g,

m,

w,

44,

2,

G,

9,

9,

w,

3,

9,

x,

*,

18,

2,

5,

2,

827 (1971).

Chap.

16

181 -

Blohm

Atherosclerosis

76. A t h e r o s c l e r o s i s , P r o c e e d i n g s of t h e Second I n t e r n a t i o n a l Symposium, R . J . J o n e s , e d . , S p r i n g e r , New York, S e c t i o n s XV and XVI, ( 1 9 7 0 ) .

77. C . A . DuJovne, P. Weiss and J . R . B i a n c h i n e , C l i n . Pharm. Therap. 12, 117 ( 1 9 7 1 ) . 78. G. Hartmann, DALM A b s t r a c t s ( s e e r e f . 1 7 ) 64 (1971). 79. P.M. Manucci, F. P a r e t i , C.A. Maggi and M. D i a g u a r d i , A t h e r o s c l e r o s i s 2, 1 (1971). 8 0 . R. F e l l i n , D . F e d e l e , G . B a g n a r o l and G . C r e F a l d i , DALM A b s t r a c t s ( s e e r e f . 1 7 ) , 5 4 (1971). 8 1 . J . L . deGennes, G . T u r F i n and 3 . T r u f f e r t , DALM A b s t r a c t s ( s e e r e f . 1 7 ) 43 (1971). 8 2 . C Russo and M. Mendlowitz, C l i n . Pharm. Therap. 12, 676 ( 1 9 7 1 ) . 83. A.S. T r u s w e l l , G. Watermeyer and J . I . Mann, DALM A b s t r a c t s ( s e e r e f . 1 7 ) 116 ( 1 9 7 1 ) .

8 4 . R . J . C e n e d e l l a , L i p i d s 5, 475 ( 1 9 7 1 ) . 85. J . P . Morgan, J.R. Bi a n c h i n e , T . H . Hsu, and S. M a r g o l i s , C l i n . Pharm. Therap. 12, 517 (1971). 86. J . R . Ryan, A . J a i n , G . E . Maha and F.G. McMahon, C l i n . Pharm. T h e r a p . 12, 464 (1971). 87. C.R. S i r t o r i and D.L. A z a r n o f f , J . Am. Oil Chem. S o c . , 3,92A ( 1 9 7 1 ) . 88. D. B e r k o w i t z , DALM A b s t r a c t s ( s e e r e f . 1 7 ) 2'7 (1571). 89. J . L . G i l f i l l a n , V.M. Hunt and J . W . Huff, P r o c . S O C . E X F . B i o l . & Med. lj6, 1274 (19711.

9,

S u p p l . 11, 9 0 . R.B. Moore, I . D . F r a n t z , R . L . Varco and H. Buchwald, C i r c u l a t i o n 47 (1971). 9 1 . C . D . M o u t a f i s , N.B. Myant, M. Mancini and P. O r i e n t e , A t h e r o s c l e r o s i s 247 (1971). 92. S.M. Grundy, E.H. Ahrens and G. S a l e n , J . Lab. C l i n . Med. @, 94 (1971). 93. P . T . Kuo, DALM A b s t r a c t s ( s e e r e f . 1 7 ) 77 (1971). 94. K. Gundersen, DALM A b s t r a c t s (see r e f . 17) 63 (1971). 95. C . J . Glueck, P.M. S t e i n e r , D. S c h e e l and S. F o r d , C i r c u l a t i o n $, S u p p l . 11, 59

a,

(1971).

96. G . R . Thompson, J . Barrowman, L. G u t t i e r e z and R.H. Dowling, J . C l i n . I n v e s t . 2, 319 (1971). 97. P. Samuel, A . Schussheim and S. Liebermann, C i r c u l a t i o n $, S u p p l . 11, 8 (1971). 98. D. K r i t c h e v s k y , Fed. Proc. ,O, no. 3 , P a r t I , 835 ( 1 9 7 1 ) . 99. D.M. K r i k l e r , D. L e f e v r e and B. Lewis, Lancet 1,934 (1971). 100. H.J. Meyer, DALM A b s t r a c t s ( s e e r e f . 1 7 ) 81 ( 1 9 7 1 ) . 101. F.A. Gries, H . D . Miss, H . C a n z l e r , T . Koschinsky, K.H. Vogelberg and K. Jahnke, DALM A b s t r a c t s (see r e f . 17) 62 (1971). 102. R. Beckmann, G . Hillmann, W . L i n t z and W. V o l l e n b e r g , DALM A b s t r a c t s (see r e f . 1 7 ) 26

(1971).

103. H. D i t s c h u n e i t , H . H . 104. 105. 106. 107. 108. 109.

D l t s c h u n e i t , H . U . K l o r , D. Rakow and P. Schwandt, DALM A b s t r a c t s ( s e e r e f . 17) 45 (1971). T . A . M i e t t i n e n , Scand. J. C l i n . Lab. I n v e s t . 3, S u p p l . 116, 17 (1971). 2. Kalamis, M. D a c q u i s t o and G.S. K o r n e t t , Curr. Therap. Res. g,692 (1971). D.T. Nash, DALM A b s t r a c t s (see r e f . 1 7 ) 86 (1971). A . A . P o l a c h e k , H.M. Katz and M. L i t t m a n , DALM A b s t r a c t s ( s e e r e f . 1 7 ) 95 ( 1 9 7 1 ) . J . W . B a r n h a r t , J.D. Johnson, D . J . R y t t e r and R.B. F a i l e y , DALM A b s t r a c t s (see r e f . 1 7 ) 25 (1971). D. K r i t c h e v s k y , H.K. K i m and S.A. T e p p e r , Proc. SOC. Exp. B i o l . & Med. 1216

m,

(1971)110. T. K a r i y a , T.R. Blohm. J . M . G r i s a r , R . A . P a r k e r and J.R. M a r t i n , DALM A b s t r a c t s (see r e f . 17) 72 (1971). 111. D.L. Wenstrup, R . G . Johnson, R . A . P a r k e r , J . M . G r i s a r , T. K a r i y a , T.R. Blohm, R.W. Fleming and V. Petrow, A b s t r a c t s 162nd Meeting, Am. Chem. SOC. A b s t r a c t MEDI 066 (1971). 112. K. Toki and H. N a k a t a n i , Adv. Exp. Med. B i o l . 491 (1969). 113. D. K r i t c h e v s k y and S.A. T e p p e r , A r z n e i m i t t e l f o r s c h . 3,1024 (1971). 114. Y.H. Abdulla and C.W. M. Adams, A t h e r o s c l e r o s i s y. 6 1 (1971). 115. K. Toki and H. N a k a t a n i . DALM A b s t r a c t s (see r e f . 1 7 ) 114 (1971). 116. J . G . Hamilton, A. C. S u l l i v a n , M. G u t t i e r e z and O.N. Miller, Fed. Proc. No. 2 ,

i,

s,

519 (1971).

Chapter 17.

S t e r o i d s and B i o l o g i c a l l y Related Compounds

Duane F. Morrow and Duane G a l l o , Mead Johnson Research Center, E v a n s v i l l e , Indiana

-

Introduction The f l o w o f p u b l i c a t i o n s i n t h e area o f s t e r o i d research continued unabated i n 1971. However, i n much o f t h i s the r i g i d s t e r o i d s k e l e t o n served s o l e l y as a convenient framework f o r i n v e s t i g a t i o n o f the stereochemistry of new r e a c t i o n s . Many o t h e r papers d e a l t w i t h t h e i s o l a t i o n and i d e n t i f i c a t i o n of new s t e r o l s from n a t u r a l sources, w h i l e o t h e r s were r e p o r t s o f new d e r i v a t i v e s which seemed t o us t o have l i t t l e prospect for practical u t i l i t y . I n t h i s review we have s e l e c t e d p r i m a r i l y those a r t i c l e s which appeared t o have p a r t i c u l a r i n t e r e s t f o r the m e d i c i n a l chemi s t

.

T o t a l Synthesis - Several new procedures f o r the t o t a l s y n t h e s i s o f s t e r o i d s have been developed. A novel s y n t h e s i s o f a 19-nor s t e r o i d was r e p o r t e d i n which =-butyl a c e t o n e d i c a r b o x y l a t e was a l k y l a t e d t o g i v e which was condensed w i t h t h e d i s u b s t i t u t e d cyclopentanedione 2 to g i v e t h e i n 34% t r i c y c l i c i n t e r m e d i a t e 3 . H y d r o l y s i s and r i n g c l o s u r e a f f o r d F d The u t r l i t y and convenience of t h i s procedure may p e r m i t overall yield.’ i t t o be adapted t o the syntheses o f a l a r g e v a r i e t y o f u s e f u l s t e r o i d s and s t e r o i d intermediates.

1,

k

0

-

COntBu

+ c1

-

CH 2C02 t BU

0

Two new t o t a l syntheses of c - e s t r o n e u t i l i z e t h e b l c y c l i c aldeas key intermediates. hyde z2’and the v i n y l k e t o n e These a r e condensed r e s p e c t i v e l y w i t h fi-methoxybenzyltriphenylphosphonium c h l o r i d e and 2methylcyclopentanedione t o g i v e t h e a p p r o p r i a t e intermediates f o r r i n g c l osu r e .

a3

Chap.

17

S t e r o i ds

183 -

Morrow, Gal l o

-5

6 -

An i n t e r e s t i n g a p p l i c a t i o n o f asymmetric s y n t h e s i s a f f o r d e d t h e i n from the t r i k e t o n e & danedione The use o f aminoacids o f t h e (S)-conf i g u r a t i o n such as L - p r o l i n e and L - a l a n i n e as c a t a l y s t s f o r t h e condensat i o n gave o p t i c a l l y a c t i v e b i c y c l i c i n t e r m e d i a t e s o f the (S)-configurat i o n i n y i e l d s o f 60-85% w i t h o p t i c a l p u r i t y i n t h e same range. 4

I

I

R

-7

R R = H, Me

-8

R ' = Me, E t A t o t a l s y n t h e s i s o f n o r g e s t r e l was d e s c r i b e d , i n w h i c h t h e key s t e p i s the condensation o f ethylcyclopentanedione w i t h the tetrahydropyran d e r i v a t i v e 9.5 A s i m i l a r method was used f o r t h e t o t a l s y n t h e s i s o f r e t r o t e s t o s t e r o n e and i t s derivative^.^'^'^ A new method was d e v i s e d f o r i s converted t o the c l o s i n g the D-ring, i n which t h e a c e t y l e n i c a l c o h o l k e t o n e 1 1 i n 95% y i e l d t o g i v e an 80:20 m i x t u r e o f t r a n s : c i s r i n g j u n c tures.

10

'-

py2 p Et

1

+

&-t

-9

OtBu

10 -

11 -

184 -

Sect.

- Metabolic

IV

& Endocrine

Pachter, Ed.

The a p p l i c a t i o n o f t h e s t a n d a r d Torgov procedure f o r t h e p r e p a r a t i o n o f 7,7-dimethyl-6-oxasteroids l e d s u r p r i s i n g l y t o t h e f o r m a t i o n o f t h e 8a,I46-isomer.’O The a u t h o r s p o s t u l a t e t h a t a s t e r i c i n t e r a c t i o n between t h e =-dimethyl group and t h e 15-proton caused i s o m e r i z a t i o n o f t h e A 1 4 Subsequent r e d u c t i o n o f t h e A’-double i n t e r m e d i a t e t o a 146-A’5-isomer. bond was r e p o r t e d l y i n f l u e n c e d by t h e 146-center t o produce an 8 a , 9 a - r i n g j u n c t i o n . However, when employing a s i m i l a r s y n t h e s i s t o p r e p a r e a 7amonomethyl-6-oxa system, t h e normal 86,9a, 1 4 a - s t e r o i d was produced, a l though a g a i n some d i f f i c u l t y was encountered i n t h e r e d u c t i o n o f t h e A 1 4 bond t o t h e 14a-i somer. l1 The Torgov p r o c e d u r e was a l s o used t o g r e p a r e 2,4-dimethoxyestrone and t h e c o r r e s p o n d i n g 3-desoxy d e r i v a t i v e . ’ ’ 1 3 The former compound was o f i n t e r e s t a t one t i m e because o f r e p o r t e d a n a l g e s i c a c t i v i t y . A method f o r c o n v e r t i n g 19-nor i n t e r m e d i a t s uch as 4 t o 106-methyl produced t h e s t e r o i d s was r e p o r t e d . l 4E p o x i d a t i o n o f t h e A 5 ? ” 7 - 3 - k e t a f 5a,10a-epoxide w h i c h was c o n v e r t e d t o t h e 10B-methyl-A4-3-ketone by t r e a t ment w i t h methyl magnesium bromide.

-

Other S y n t h e t i c Procedures A new s y n t h e t i c r o u t e , of p o s s i b l e commercial i n t e r e s t , t o 6-dehydroretroprogesterone was r e p o r t e d . Progesterone, as e i t h e r t h e 3 2 0 - b i s k e t a l o r t h e 3-monoketal, was c o n v e r t e d t o t h e c o r r e sponding A5”-diene w h i c h was i s o m e r i z e d t o t h e 96,lOa-isomer by p h o t o l y sis. H y d r o l y s i s a f f o r d e d t h e d e s i r e d 6-dehydroretroprogesterone. Also formed i n t h i s r e a c t i o n was t h e b y p r o d u c t 6-dehydro-8a,lOa-progesterone. A new p r e p a r a t i o n o f 19-norprogesterone was d e s c r i b e d i n w h i c h d i o s g e n i n was c o n v e r t e d t o 19-nordiosgenin v i a t h e 6 , l g - o x i d e and t h e 19m e t h y l o l d e r i v a t i v e s . Standard d e g r a d a t i o n o f t h e s i d e c h a i n a f f o r d e d 19norpregnenolone i n 30% o v e r a l l y i e l d . l 6

New methods f o r t h e s y n t h e s i s o f 17- and 17,21-hydroxylated s i d e C a t a l y t i c reducchains have been t h e s u b j e c t o f s e v e r a l i n v e s t i g a t i o n s . t i o n o f 16-methyl-16-dehydropregnenolone produced t h e 20-eno1, w h i c h r e a c t e d w i t h a i r t o f o r m t h e 17a-hydroperoxide. Reduction w i t h t r i e t h y l Howphosphi t e a f f o r d e d 166-methyl-17a-hydroxypregnenolone i n 70% y i e l d . e v e r , a t t e m p t s t o p r e p a r e s i m i l a r l y t h e 16-unsubsti t u t e d a n a l o g r e s u l t e d i n much p o o r e r y i e l d s . ” Treatment w i t h m e t h y l l i t h i u m o f t h e 176-cyano17a-alcohol p r e p a r e d from t h e 17-ketone a f f o r d e d t h e 17a-hydroxypregnan20-one system i n good y i e l d . 1 4 The l a t t e r c o u l d a l s o be o b t a i n e d f r o m 17v i n y l i d i n e s t e r o i d s ( 1 2 ) , which a r e e a s i l y p r e p a r e d f r o m t h e c o r r e s p o n d i n g 1 7 B - a c e t o x y - 1 7 a - e t h i n ~ s t e r o i d s by t r e a t m e n t w i t h z i n c d u s t . P e r a c i d y i e l d e d t h e 17a-hydroxy-20-ketone, whereas o x i d a t i o n w i t h oxidation o f osmium t e t r o x i d e a f f o r d e d t h e 17a,21-dihydroxy-20-keto s i d e c h a i n i n 53% yield. 1 8

12

Some p o t e n t addition o f n i t r o n l y 16a-carbon, produce a l s o t h e

a l l y u s e f u l new s y n t h e t i c methods were r e p o r t e d , The l e o x i d e s t o A16-20-ketones, p r e v i o u s l y thought t o g i v e 17a-oxygen-linked i s o x a z o l i n e s , has now been shown t o i s o m e r i c 17a-carbon l i n k e d compounds I f the

(u).”

Chap.

17

St e r o ids

Morrow, Gal l o

185

y i e l d s c o u l d be improved, th e s e i s o x a z o l i n e s would be u s e f u l i n t e r m e d i a t e s f o r t h e s y n t h e s i s o f p r o g e s t e r o n e s c o n t a i n i n g n o v e l 17a-carbon l i n k e d f u n c t i o n a l i zed s u b s t i t u e n t s

.

H

An i n n o v a t i v e method f o r c o n v e r t i n g an a r o m a t i c A r i n g t o a A 4 ” - 3 k e t o system was r e p o r t e d , ” a r a t h e r remarkabl e r e v e r s a l o f t h e u s u a l a r o m a t i z a t i o n p ro c e s s . The “remote p h o t o l y t i c o x i d a t i o n ” o f s t e r o l d s has now been ex t end e d t o i n c l u d e o x i d a t i o n a t C 9 o r c 1 4 and c 1 5 by attachment o f t h e benzophenone r e a g e n t t o 17B- o r 2 4 - a l ~ o h o l s . ~The ~ oxidation a t C g , which c o u l d lead t o a v a r i e t y of C - r i n g s u b s t i t u e n t s i n c l u d i n g 1 1 oxygenated d e r i v a t i v e s , appears t o be a p a r t i c u l a r l y v a l u a b l e a p p l i c a t i o n o f t h i s new method. The s y n t h e s i s o f A-nor s t e r o i d s c o n t i n u e s t o be o f i n t e r e s t , and s e v e r a l new r o u t e s have been d e v i s e d . P i n a c o l rearrangements o f t h e d i o l s pre par ed by h y d r o x y l a t i o n o f 3-methyl-A2-5a- and 3 - m e t h y l - A 3 - 5 B - s t e r o i d s l e d t o 2- ac et y l -A-n o r-5 a and 3 - a c e t y l - A - n o r - 5 B - s t e r o i d s respectively. 22 For m ic a c i d - c a t a l y z e d rearrangement o f a l-methyl-1a,2a-epoxy-3-ketoa ndr os t ane l e d i n 70% y i e l d t o an e p i m e r i c m i x t u r e o f I - f o r m y l - l - m e t h y l - 2 ke t o- A - nor andr o s ta n e s , w h i c h was c o n v e r t e d t o t h e c o r r e s p o n d i n g l - m e t h y l 2-ketone i n good y i e l d . 2 3 Oth e r r e c e n t developments i n c l u d e t h e s y n t h e s i s o f 3a-methyl-2-keto-A-n0randrostanes,~~and t h e p r e p a r a t i o n o f a 5ame t hy l- 3- k et o- A -n o r s t e r o i d by t h e p i n a c o l rearrangement o f 3a-methyI-SB, 5B-d i hydroxy-A-norpregnan-20-one.

-

Heterocyclic Steroids Much s y n t h e t i c work c o n t i n u e s t o be p u b l i s h e d i n t h i s a r e a . Two n o n -b a s i c aza s t e r o i d s , 13-aza-17-ketones, were p r e p a r e d w i t h t h e s t a t e d purpose o f t e s t i n g a h y p o t h e s i s t h a t t h e u s u a l l a c k o f hormonal a c t i v i t y o f a z a s t e r o i d s i s due t o an i n a b i l i t y o f b a s i c s t e r o i d s t o p e n e t r a t e c e l l membranes. However, no b i o l o g i c a l i n f o r m a t i o n was 1 5 , 1 6 - d i a ~ a - , ~and ~ 11,15,16-triaza~teroids~~ g i v e n . 2 6 ’ 2 7 Some lS-aza-,” were s y n t h e s i z e d . The f o r m e r two t y p e s a r e r e p o r t e d t o have a n t i - b a c t e r i a l a c t i v i t y , whereas 17a-aza-D-homosteroids a r e i n a c t i v e . 3 1 The a c e t y l a t i o n o f one i n a c t i v e 17B-hydroxy-4-azasteroid was shown t o c o n f e r a n t i - b a c t e r i a l a c t i v i t y t o t h e compound, b u t t h i s e f f e c t was n o t gene r a 1 . 3 2 The s y n th e s e s o f a 5 , l O - d i a z a ~ t e r o I d ~and ~ o f an A ,B -i ndol os t e r o i d (14) 3 4 were a l s o r e p o r t e d . The s u c c e s s f u l p r e p a r a t i o n s o f 2-oxa,

-

186 -

IV

S e c t.

-

M e t a b o l i c & E ndocri ne

P a c h t e r , Ed.

2 - t h i a , and 2-aza s t e r o i d s were n o te d , b u t a t t e m p t s t o i n t r o d u c e a phoso f t h e A - r i n g were unphorus atom i n t o e i t h e r t h e 2 - 3 5 o r s u c c e s s f u l . An i n t e r e s t i n g s e r i e s o f r e a c t i o n s o f 2a,5a-oxido and 2a,5at h i o s t e r o i d s l e d t o a v a r i e t y o f A-nor-3-oxa and 3 - t h i a s t e r o i d s . 3 7 The s y n t h e s i s o f 5 - a z a c h o l e s t e r o 1 3 * was r e p o r t e d , as w e l l as f u r t h e r work on t h e syntheses o f 1 1 - , 4 0 ' 4 1 and 12-aza4* systems. P r e p a r a t i o n s o f t h e unusual s t r u c t u r e s , A-b i snor-B-homo-5-azapregnane-2,2O-d i o n e 4 and a s e r i e s o f 5 a , 7 ~ - d i s u l f i d e s , ~were ~ announced. H

-

M i s c e l l a n e o u s C h e m i s try Se v e ra l o t h e r new s t e r o i d s t r u c t u r e s were p r e par ed b a v a r i e t y o f s y n t h e t i c r o u t e s . Among these a r e 8,19-oxi do-steroids," 9a-amino-11-ox enated s t e r o i d s , 4 6 a v a r i e t o f 21-substituted17-vi n y l i d i ne s t e r o i d s ,"' 4 8 a 17B-, 1 8 - c y c l o s t e r o i d and ga-methy 1 B (ga) -homo-A-norpregna-3 ( 5 ) ,9a-di ene-2,20-dione. 5 0

,Ig

-

-

Seco S t e r o i d s The i mp o rta n c e o f t h e r i g i d s t e r o i d s k e l e t o n f o r hormonal a c t i v i t y has been f a i r l y we1 1 e s t a b l i s h e d , b u t f u r t h e r i n v e s t i g a t i o n s cont i n u e . The s l i g h t p r o g e s t a t i o n a l a c t i v i t y o f 15,16-secoprogesterone i s n o t s u r p r i s i n g , b u t t h e r e l a t i v e l y h i g h potency (ca. 1/5 x p r o g e s t e r o n e ) o f t h e i s o m e r i c 15,16-seco-17-isoprogesterone i s r a t h e r unexpected.51 Both 9 , l l - s e c o p r o g e s t e r ~ n e ~ ~ ~5,6-secoproges a n d t e r ~ n e ~ ~ ~ i n aa cr tei v e as p r o g e s h i n s . Some weak a n d r o g e n i c a c t i v i t y was shown b 5 , 6 - s e c o t e s t o s t e r one,52 b u t a 3,5-seco-5-keto d e r i v a t i v e was i n a c t i v e . 5 1

A s e r i e s o f 9,11-seco-11-hydroxy and 1 1 - c a r b o x y l i c a c i d d e r i v a t i v e s o f e q u i l e n i n were s y n t h e s i z e d . I t i s n o t e w o r t h y t h a t o f t h e f o u r isomers t e s t e d , o n l y t h a t one w h i c h c o rre s p o n ds t o t h e l & e p i m e r o f t h e n a t u r a l A v e r y f l e x i b l e B,Chormone has any s i g n i f i c a n t e s t r o g e n i c a c t i v i t y . bis-seco estrone d e r i v a t i v e was p r e p a r e d and found t o have o n l y v e r y weak e s t r o g e n i c a c t i v i t y (ca. 1/250 x e s t r o n e ) . 5 5

(5)

An i n t e r e s t i n g s t u d y o f t h e i mp ortance o f shape on e s t r o g e n i c a c t i v i in t y was accompl i s h e d by t h e s y n t h e s i s o f t h e two compounds whic h t h e B and D r i n g s o f e s t r a d i o l were " f l i p p e d o v e r ' ' t o t h e "North" s i d e o f the molecule. Since and I J, a r e v e r y weak e s t r o g e n s , and s i n c e they r e t a i n b o t h t h e r i g i d i t y and t h e same a c c e s s a b i l i t y t o t h e a- and 6f a c e s as t h e normal e s t r o g e n s , t h e i r low a c t i v i t y i s most l i k e l y a s s o c i a t e d w i t h changes i n t h e "edge p r o f i l e " o f t h e w hol e m o l e c u l e . 5 6 An i n c o n t a i n i n g a s p i r o C,D-ring j u n c t u r e was t r i g u i n g analo g o f e s t r o n e

(16,2)

16

(18)

Chap.

I7

Steroids

Morrow, Gal l o

187 -

pre par ed, 57 and 5,7-secomestranol was ~ y n t h e s i z e d ,b~u~t no b i o l o g i c a l i n f o r m a t i o n was p r o v i d e d f o r e i t h e r o f t hese compounds. 0

OH

0

-

Progestins S e v e r a l v e r y p o t e n t new p r o g e s t a t i o n a l s t e r o i d s were des c r i b e d . The 1 7 a -p ro p a d i e n y l d e r i v a t i v e s o f n o r e t h i n d r o n e and n o r g e s t r e l have o r a l p r o g e s t a t i o n a l p o t e n c i e s 10 and 12 ti mes, r e s p e c t i v e l y , t h a t o f n o r e t h i ndrone. 59,60 The c o rre s p o n d i ng 17a-vi n y l e t h i n y l d e r i v a t i v e i n t h e 19-methyl s e r i e s a l s o has g e s t a g e n i c a c t i v i t y . 6 1 The 6 , 6 - d i f l u o r o a n a l o g o f n o r e t h i n d r o n e i s two o r t h r e e ti me s more p o t e n t than n o r e t h i n d r o n e i t ~ e l f , ~ * , 6w 3 h i l e t h e 1 8 - v i n y l analog o f noret hi ndrone i s e q u i p o t e n t t o norges t r e 1 . 6 4

19

The d i h a l o p r o g e s t e r o n e d e r i v a t i v e i s about 100 ti mes more p o t e n t t h a n pr oges t er o n e ,6 5 ,6 6 whereas a v a r i e t y o f s u b s t i t u t e d 3-desoxy-17aac et ox y pr oges t e ro n e s a r e a l l l e s s a c t i v e t h a n t h e p a r e n t 17a-acetoxy-20oxopregna-3,5-diene. 67 The i n t r o d u c t i o n o f a 4 - c h l o r o s u b s t i t u e n t enhances p r o g e s t a t i o n a l a c t i v i t y , 6 8 b u t m e t a b o l i s m s t u d i e s i n d i c a t e t h e 4c h l o r o gr oup i s v e r y l a b i l e , and, i n f a c t , i n v i t r o s t u d i e s have demons t r a t e d removal o f t h i s c h l o r i n e atom v i a nonenzymatic r e a c t i o n w i t h merca pt ans . The proposed mechanism i s d i s c u s s e d . 69,70 The i n t r o d u c t i o n o f s e v e r a l o t h e r s u b s t i t u e n t s a t t h e 4 - p o s i t i o n (e.g. cyano, f o r m y l , aminomethylene) d e s t r o y s hormona 1 a c t iv i t y 7 1 9 7 2 a'72bThe proges t a t i ona 1 a c t i v i t y o f 17a- ac et o x y p ro g e s te ro n e i s enhanced f i v e t o t e n ti mes by t h e a d d i t i o n o f a 7 a - t h i o e t h e r mo i e ty ,7 3 b u t h y d r o x y l s u b s t i t u t i o n a t C 7 reduces t h e u s u a l p o t e n t i a t i n g e f f e c t o f a 16-methylene group.74

--

.

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188 -

Sect.

IV

-

Metabolic & Endocrine

rF

P a c h t e r , Ed. H

c1

I n a s e a r c h f o r agents w h i c h would s e l e c t i v e l y a l k y l a t e t h e Clauberg " r e c e p t o r , " t h e s y n t h e s i s and t e s t i n g o f l 6 a - s u b s t i t u t e d p r o g e s t e r o n e derivative^^^ and o f w - s u b s t i t u t e d e s t e r s o f 1 7 a - h y d r o x y p r o g e ~ t e r o n e ~ ~ was r e p o r t e d . A l t h o u g h s e v e r a l compounds d i s p l a y e d weak p r o g e s t a t i o n a l a c t i v i t y , no e v i d e n c e i n d i c a t i n g r e c e p t o r a l k y l a t i o n was o b t a i n e d .

Estrogens and Androgens - I n t h e e s t r o g e n s e r i e s , t h e e p i m e r i c 17-carboxye s t r a d i o l - 3 - m e t h y l e t h e r s a r e r e p o r t e d and, i n t e r e s t i n g l y , t h e "wrong" isomer, t h e 17B-carboxy-17a-estradiol e t h e r i s weakly e s t r o g e n i c whereas t h e 17a-carboxy-17f3-estradiol e t h e r i s i n a c t i v e . 7 7 Thus t h e r e a r e t h r e e r e p o r t e d cases i n 1971 ( n o t e t h e o t h e r two on p.186)of e p i m e r i c analogs o f s t e r o i d s i n which t h a t epimer c o r r e s p o n d i n g t o t h e active natural steroid i s the p o t e n t analog. The reasons f o r t h i s a r e u n c l e a r and o b v i o u s l y much remains t o be l e a r n e d about s t r u c t u r e - a c t i v i t y r e l a t i o n ships i n the s t e r o i d f i e l d . I n c o n t r a s t t o t h e p r o g e s t i n s e r i e s , 18m e t h y l e s t r o g e n s a r e l e s s a c t i v e than t h e p a r e n t compounds.78 F u r t h e r s t u d i e s w i t h 4-mercur i - 1 76-es t r a d i o l demonstrated t h a t t h i s compound e f f e c t i v e l y competes w i t h 8 - e s t r a d i o l f o r t h e 8s r a t u t e r i n e c y t o s o l e s t r o g e n r e c e p t o r i n an i r r e v e r s i b l e manner. Unexpectedly, t h e 8s-mercury s t e r o i d complex i s n o t t r a n s f e r r e d t o t h e p a r t i c u l a t e f r a c t i o n under c o n d i t i o n s i n w h i c h t h e n u c l e a r u p t a k e o f B - e s t r a d i o l t o a 4-5s complex c o u l d be demonstrated. S i n c e t h e mercury s t e r o i d was p r e v i o u s l y shown t o have b i o l o g i c a l a c t i o n s t y p i c a l o f e s t r o g e n s , t h e p r e s e n t o b s e r v a t i o n s a r e n o t i n a c c o r d w i t h c u r r e n t concepts t h a t o n l y i n t r a n u c l e a r Be s t r a d i o l can t r i g g e r s p e c i f i c c e l l u l a r responses.79 F u r t h e r e v i d e n c e t h a t t h e 4-mercury group undergoes m e r c a p t i d e bond f o r m a t i o n w i t h t h e u t e r i n e e s t r o g e n r e c e p t o r was o b t a i n e d i n s t u d i e s w i t h 4-mercuri-17ae s t r a d i o l . T h i s compound has l e s s e s t r o g e n i c p o t e n c y than t h e correspond i n g 1 7 6 - d e r i v a t i v e and d i s p l a y e d p e r s i s t e n t a n t i e s t r o g e n i c a c t i v i t y e q u a l i n potency t o 1 7 a - e s t r a d i o l b u t o f much l o n g e r duration.*O I n t h e androgen s e r i e s , some 16a, 17a-cyclopropano d e r i v a t i v e s o f 19n o r t e s t o s t e r o n e were s y n t h e s i z e d i n t h e hope t h a t t h e s m a l l e r s t e r i c b u l k o f t h e methylene group, i n comparison w i t h t h a t o f a 17a-methyl group, would enhance o r a l a n d r o g e n i c a c t i v i t y ; however, no b i o l o g i c a l d a t a were

Chap.

17

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189

reported.'l A 7 - a c e t y l d e r i v a t i v e o f t e s t o s t e r o n e i s being i n v e s t i g a t e d as an antiandrogen, b u t again no b i o l o g i c a l data a r e a v a i l a b l e . 8 2 Some 8a-methyl t e s t o s t e r o n e d e r i v a t i v e s were prepared b u t were n e a r l y devoid o f androgenic and a n a b o l i c a c t i v i t y . 8 3 Since 8a-testosterone r e t a i n s most o f the a c t i v i t y o f the n a t u r a l 8B-epimer, and s i n c e the i n t r a m o l e c u l a r d i s tances between the 3- and 17-oxygen atoms a r e about equal, the d e a c t i v a t i n g e f f e c t o f t h e 8a-methyl group i s r a t h e r unexpected, f o r i t i s c u r r e n t l y h e l i e v e d t h a t a n a b o l i c and androgenic agents approach t h e i r res p e c t i v e r e c e p t o r s i t e s from the B-side o f the molecule.

-

Anti-fertility The p o t e n t i a l o f s t e r o i d s i n t h e a n t i - f e r t i l i t y area i s probably s t i l l t h e l e a d i n g m o t i v a t i o n f o r research i n t h i s f i e l d , b u t l i t t l e new i n f o r m a t i o n has appeared. Two s t e r o i d s , oxymetholone and nandrolone phenpropionate, b o t h w e l l e s t a b l i s h e d a n a b o l i c agents, were shown t o cause r e s o r p t i o n of fetuses a f t e r i m p l a n t a t i o n . The term " i n t e r c e p t i v e s " was proposed f o r t h i s type o f e f f e c t . T h i s a c t i o n o f b o t h compounds could be reversed by progesterone. I t was p o s t u l a t e d t h a t oxymetholone suppresses p i t u i t a r y gonadotrophin release, w h i l e nandrolone phenpropionate e i t h e r i n h i b i t s l u t e a l synthesis o f progesterone o r compet t i v e l y blocks a u t e r i n e r e c e p t o r s i t e necessary f o r pregnancy maintenance .a4 A pyrazoloandros tane der i v a t l ve (20) has ant1 - imp1a n t a t i o n a c t i v and 2a-aminodihydrotestosterone has moderate a n t i - u t e r o t r o p h i c a c t i v i Papers c o n t i n u e t o appear on the a n t i - f e r t i l i t y a c t i v i t y o f a v a r i e t y t r i a r y l e t h y l e n e derivatives.87-90 Extension o f the D o s t - c o i t a l a n t i f e r t i i it y e f f e c t o f the methyl e s t e r of dl-cis-bisdekydrodoisynol i c a c i d i n monkeys t o s t u d i e s i n humans would be o f considerable i n t e r e s t . 9 1 H CH3

(a)

CH 30 22 -

21 -

The c l i n i c a l e f f i c a c y o f d i e t h y l s t i l b e s t r o l as a p o s t - c o i t a l c o n t r a c e p t i v e i n women was documented.92 No pregnancies occurred i n 1000 women given, w i t h i n 72 hours o f exposure, 25 mg o f DES t w i c e d a i l y f o r f i v e days. No s e r i o u s s i d e e f f e c t s were r e p o r t e d and the m a j o r i t y o f p a t i e n t s e x h i b i t e d e s s e n t i a l l y normal menses f o l l o w i n g treatment. The use of DES as an emergency c o n t r a c e p t i v e i s perhaps more tenable w i t h t h e c u r r e n t l y g r e a t e r avai l a b i 1 i t y o f backup a b o r t i o n i n case of f a i l u r e . 9 3

-

I n t h e c o r t i c o i d area, several C 2 1 d e r i v a t i v e s o f Miscellaneous A c t i v i t y known a c t i v e c o r t i c o i d s were DreDared and e v a l ~ a t e d . ~ ~ A - ~new ~ c non. . halogenated g l u c o c o r t i c o i d possessing " e x t r a o r d i n a r y a n t i - i n f l a m m a t o r y potency" was r e p o r t e d , 9 9 and the pharmacology o f 9a-f l u o r o h y d r o c o r t i s o n e was e x t e n s i v e l y reviewed.100 The r e l a t i o n s h i p s between s t r u c t u r e and

190

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IV

-

M e t a b o l i c & Endocrine

P a c h t e r , Ed.

b i o l o g i c a l a c t i v i t y and potency i n t h e c o r t i c o s t e r o i d s e r i e s were d i s cussed,101,102 S t u d i e s o f t h e i n v i t r o i n t e r a c t i o n between c o r t i s o n e - 2 1 i o d o a c e t a t e and 2 0 B - h y d r o x y s t e r ~ d ~ d r o g e n a s el e d t o t h e c o n c l u s i o n t h a t t h e enzyme was i r r e v e r s i b l y i n a c t i v a t e d by a l k y l a t i o n o f a h i s t i d i n e res due l o c a t e d a t i t s a c t i v e s i t e . l o 3

(22,23)

Two new types o f s t e r o i d s possessing a n t i - a l d o s t e r o n e act i v t y were r e p o r t e d . A h i g h l e v e l o f a c t i v i t y was c l a i m e d f o r G,lo4 b u t 23 was o n l y weakly a c t i v e . l o 5

-

23

24 -

I n t h e h y p o c h o l e s t e r o l e m i c area, 2 5 - a z a l a n o s t e r o l i s s u p e r i o r t o 2 0 , 2 5 - d i a z a c h o l e s t e r o l , l o 6 whereas t h e 170,-epimer o f 2OY25-diazacholeshas t e r o l i s inactive.107 An unusual cobal t - c o n t a i n i n g d l a z a s t e r o i d a h i g h degree o f i n h i b i t i o n o f c h o l e s t e r o l s y n t h e s i s . l o 8 S e v e r a l d e r i v a t i v e s o f 5a-cholestane-3B,5a,6B-triol were t e s t e d f o r h y p o c h o l e s t e r o l e m i c a c t i v i t y i n r a b b i t s . l o 9 None were as a c t i v e as t h e p a r e n t compound. Members o f t h i s s e r i e s do, however, i n h i b i t t h e i n v i t r o s y n t h e s i s o f c h o l e s t e r o l .109,110

(24)

--

Several 17f3-aminosteroid d e r i v a t i v e s were r e p o r t e d t o have a n t i microbial activity.l" Others were found t o r a t h e r s p e c i f i c a l I y i n h i b i t t h e b i o s y n t h e s i s o f androgens f r o m [21 1 4 C ] 17a-hydroxyprogesterone by a r a t t e s t i c u l a r microsomal p r e p a r a t i o n . The s t r u c t u r a l analogy between t h e i n h i b i t o r s and t h e n a t u r a l s u b s t r a t e o f t h e 17,20-lyase enzyme was p o i n t e d o u t , and i t was p o s t u l a t e d t h a t t h e i n h i b i t o r s resemble a n a t u r a l enzym a t i c i n t e r m e d i a t e o r t r a n s i t i o n s t a t e b u t , l a c k i n g a 17a-hydroxyl, cann o t proceed t o p r o d u c t s . l 1 2 The 10a-epimer o f n o r e t h i n d r o n e has no p r o gestational a c t i v i t y but i s a strong estrogen.ll3 Presumably t h i s i s due t o a f a c i l e e l i m i n a t i o n o f t h e 9 , l O - c i s hydrogen atoms t o f o r m t h e a r o matic A-ring.l14 Recent r e p o r t s have s t i m u l a t e d renewed i n t e r e s t i n t h e p o s s i b i l i t y o f a medical t r e a t m e n t f o r g a l l s t o n e s . Chenodesoxycholic a c i d a d m i n i s t r a t i o n presumably reduces t h e "1 i t h o g e n i c p o t e n t i a l " o f b i l e by i ncreas i ng t h e c o n c e n t r a t i o n o f c h o l e s t e r o l - s o l ub i 1 i z i ng components ,1 l5 and f u r t h e r i t has been r e p o r t e d t o reduce t h e s i z e o f g a l l s t o n e s , as v i s u a l i z e d by c h o l e c y s t o g r a p h y , i n 4 o f 7 p a t i e n t ~ . l l ~ A, ~m~i x ~t u r e o f 3a-hydroxy-5a-pregnane-lly20-dione and i t s 21-acetoxy d e r i v a t i v e i s r e p o r t e d t o be a u s e f u l i n t r a v e n o u s anesthetic.118'120

Chap.

17

S t e r o i ds

191 -

Morrow, Gal l o

Considerable progress was made i n e l u c i d a t i n g the b i o t r a n s f o r m a t i o n s o f c h o l e c a l c i f e r o (Vitamin D 3 ) t o b i o l o g i c a l l y a c t i v e forms. The f i r s t o f these has been i d e n t i f i e d as 25-hydroxy c h o l e c a l c i f e r o l , which i n t u r n i s f u r t h e r metabo i z e d t o the 1,25- and perhaps a l s o t o the 21,25-dihydroxy d e r i v a t i v e s . C o n t r o l over calcium t r a n s p o r t i s assured, s i n c e the i n i t i a l h y d r o x y l a t i o n i s o b l i g a t o r y i n c o n f e r r i n g b i o l o g i c a l a c t i v i t y and the b i o s y n t h e s i s o f the a c t i v e m e t a b o l i t e s i s c o n t r o l l e d by feedback regul a t i o n . While these h y d r o x y l a t e d d e r i v a t i v e s , u n l i k e Vitamin D 3 i t s e l f , a r e a l l d i r e c t l y e f f e c t i v e a t t h e c e l l u l a r l e v e l , t h e r e i s some evidence o f organ s p e c i f i c i t y . l 2 1 , ’ 2 2

-

Reviews A number o f review a r t i c l e s appeared, i n c l u d i n g two on t h e t o t a l s y n t h e s i s o f h e t e r o c y c l i c steroids.123s124 Other reviews covered the search f o r c a t a t o x i c s t e r o i d s , 1 2 5 r e t r o s t e r o i d s , 1 2 6 and 1 6 - s u b s t i t u t e d s t e r o i d s . 12’ References 1.

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J.M. M o r r i s , N . E n g l . J . Med., 286, 265 1 9 7 2 ) . E.T. O r d o G z , Arzneim. - F o r s c h . , 21, 248 1 9 7 1 ) . E . Diamanti and G.E. B i a n c h i , 21, 251 ( 1 9 7 1 ) . R. Nonhoff and K.F. Z e t h , 21, 382 1 9 7 1 ) . R. Yamamoto, S. F u j i s a w a and M. Kawamura Yakugaku Z a s s h i , 91, 8 5 5 ( 1 9 7 1 ) . M. Kawamura, R . Yamamoto and S. F u j i s a w a a) 863 ( 1 9 7 1 ) ; b ) 871 ( 1 9 7 1 ) ; c ) 41. 879 ( 1 9 7 1 ) . 99. K . - H . Bork, F . von Werder, H. Metz, K. BrGckner and M. Baumgarth, J u s t u s L i e b i g s Ann. Chem., 123 ( 1 9 7 1 ) . 100. A r z n e i m . - F o r s h . , 1, 1105-1157 ( 1 9 7 1 ) . 101. T . Uete and N . Shimano, J . Biochem. ( T o k y o ) , iO ' -, 723 ( 1 9 7 1 ) . 1 0 2 . C . H . E g e r , M . J . G r e i n e r and D . A . N o r t o n , S t e r o i d s , 18, 231 ( 1 9 7 1 ) . 103. M. Ganguly and J . C . Warren, J . B i o l . Chem., 3 6 z (1971). 104. W.F. J o h n s , J . Org. Chem., 711 (1971). 105. R . K . Sharma, N . J . Doorenbos and N.S. Bhacca, J . Pharm. S c i . , 60, 1677 ( 1 9 7 1 ) . 106. M. C . Lu, F. Kohen and R.E. C o u n s e l l , J . Med. Chem., 136 (1971). 107. V.V. Ranade, F. Kohen and R.E. C o u n s e l l , 38 ( 1 9 7 1 ) . 108. L.G. Donaruma and P . U . F l a t h , 999 ( 1 9 7 1 ) . 109. D . T . W i t i a k , R . A . P a r k e r , D . R . B r a n n , M.E. Dempsey, M . C . R i t t e r , W.E. Connor and D . M . Brahmankar, 216 ( 1 9 7 1 ) . 110. D . T . W i t i a k . R . A . P a r k e r , M.E. Dempsey and M . C . R i t t e r , 684 ( 1 9 7 1 ) . 111. K1 Y a g i s h i t a , Nihon DRigaku Nojuigakubu G a k u j u t s u Kenkyu Hokoku, 1; LChem. A b s t s . . 36456r (197111. 1 1 2 . G.E. A r t h , A . A . P a t c h e t t , T. J e f o p o u l u s , R . L . B u g i a n e s i , L.H. P e t e r s o n , E.A. Ham, F.A. Kuehl, Jr. and N . G . B r i n k , J. Med. Chem., 675 ( 1 9 7 1 ) . 113. E. F a r k a s and D . J . O ' T o o l e , S t e r o i d s , 9, 317 ( 1 9 7 1 ) . 114. E. F a r k a s and N . J . Bach, J. Org. Chem., 2715 ( 1 9 7 1 ) . 115. J . L . T h i s t l e and L . J . S c h o e n f i e l d , G a s t r o e n t e r o l o g y , 488 ( 1 9 7 1 ) . 116. R.D. D a n z i n g e r , A.F. Hofmann, L . J . S c h o e n f i e l d and J . L . T h i s t l e , N.Engl.J.Med., 286, 1 ( 1 9 7 2 ) . ll7.K.J. Isselbacher, 40 ( 1 9 7 2 ) . 118. D . Campbell, A . C . F o r r e s t e r , D. C. M i l l e r , I . H u t t o n , J . A . Kennedy, T.D.V. L a w r i e , A . R . Lorimer and D. McCall, B r i t . J . A n a e s t h . , %, 1 4 ( 1 9 7 1 ) . 119. E . M . W . B r a d f o r d , D. C. M i l l e r , D. Campbell and W.L.M. B a i r d , 940 (1971). 120. M. Swerdlow, S.K. Chakraborty and M . A . H . M . Zahangir, Q, 1075 ( 1 9 7 1 ) . 121. J . Omdahl, M . H o l i c h , T. S u d a , Y. Tanaka and H.F. DeLuca, B i o c h e m . g , 2935 ( 1 9 7 1 ) . 122. L . G . R a i s z , C.L. Trummel, M.F. H o l i c h and H.F. DeLuca, S c i e n c e , D, 768 ( 1 9 7 2 ) . 5ll ( 1 9 7 1 ) . 123. H.O. Huisman, Angew. Chem., 1 2 4 . H. S i n g h , J . S c i . I n d . R e s . , 0, 176 ( 1 9 7 1 ) . 1 (1971). 125. H . S e l y e , J . Pharm. S c i . , 126. M.P. R a p p o l d t , Chem. Weekbl.,,i'6 S18 ( 1 9 7 1 ) . 127. I . Weisz-Vincze, G . S c h n e i d e r , M. Halmos, J . A . Szabo and K. Kovacs, Acta Phys. Chem., 3, 67 ( 1 9 7 1 ) .

93.

94. 95. 96. 9'7. 98.

m.,

m,

s,

w., g,

747,

246,

s,

e,

w.,14,

s., fi, u., 14,

w.,14,

m,

E,

14,

6,

61,

m.,286,

u., 9,

m.,

8,

60,

Chapter 18. P e p t i d e Hormones of t h e Hypothalamus and P i t u i t a r y . Roger Burgus, The S a l k I n s t i t u t e , La J o l l a , C a l i f .

T h i s survey w i l l p l a c e emphasis on t h e more r e c e n t s i g n i f i c a n t d e v e l opments which have occurred d u r i n g t h e two y e a r s s i n c e t h i s s u b j e c t was reviewed 1 w i t h t h e a i m of l e t t i n g p a p e r s r e c e n t l y p u b l i s h e d i n a g i v e n a r e a l e a d t h e reader t o e a r l i e r r e p o r t s . S y n t h e s i s of hormones per se i s covered i n a n o t h e r survey (S.M. S t e w a r t , t h i s volume). Hypothalamic P e p t i d e s . S e v e r a l g e n e r a l reviews and proceedings of c o n f e r e n c e s on hypothalamic p e p t i d e hormones have appeared s i n c e t h e l a s t survey2-6. A comprehensive r e v i e w on t h e c h e m i s t r y of t h e hypothalamic r e l e a s i n g f a c t o r s i s f o r t h coming7. Methods of Bioassav - One of t h e d i f f i c u l t i e s i n t h e p a s t i n t h e i s o l a t i o n and e l u c i d a t i o n of t h e s t r u c t u r e of t h e v a r i o u s hypothalamic r e l e a s i n g f a c t o r s h a s been t h e problem o f o b t a i n i n g s p e c i f i c and r e l i a b l e b i o a s s a y s f o r t h e s e f a c t o r s . The d i s c u s s i o n of t h e l i m i t a t i o n s of t h e v a r i o u s b i o a s s a y s i s f a r beyond t h e scope of t h i s survey and c r i t i c a l d i s c u s s i o n s can b e found i n some o f t h e g e n e r a l review^^-^. Radioimmunoassay of plasma l e v e l s of t h e v a r i o u s p i t u i t a r y hormones h a s provided a more r e l i a b l e endp o i n t f o r t h e d e t e r m i n a t i o n of hypothalamic r e l e a s i n g f a c t o r s . The u s e of d i s p e r s e d p i t u i t a r y c e l l c u l t u r e s h a s g r e a t l y i n c r e a s e d p r e c i s i o n and quant i t a t i v e a b i l i t y o f t h e r e l e a s i n g f a c t o r b i o a s s a y 8 , as w e l l a s b e i n g more amenable t o m u l t i p l e t r e a t m e n t experiments t h a n o t h e r a s s a y methods. Ano t h e r b i o a s s a y t e c h n i q u e i n v o l v e s t h e u s e of r a d i o - l i g a n d b i n d i n g t o t a r g e t c e l l r e c e p t o r ~ i t e s 9 - 1 A~ radiaimmunoassay f o r t h e d i r e c t d e t e r m i n a t i o n of TRF h a s been d e s c r i b e d ii

.

Thyroid S t i m u l a t i n g Hormone (TSH) R e l e a s i n g F a c t o r , TRF - The c h e m i s t r y and physiology of TRF h a s r e c e n t l y been r e v i e w e d 3 , L L , n . The s t r u c t u r e of o v i n e TRF'was c o n c l u s i v e l y e s t a b l i s h e d by h i g h r e s o l u t i o n mass s e c t r o m e t r y and a d d i t i o n a l chemical c h a r a c t e r i z a t i o n t o b e alu-His-Pro-NH2 If: Subseq u e n t l y , p o r c i n e TRF vas shown c o n c l u s i v e l y t o have t h e same s t r u ~ t u r e l ~ , 1 ~ .

.

The f i r s t chemical s y n t h e s i s of TRF involved t h e ammonolysis of t h e ~. methyl ester prepared from t h e f r e e a c i d , e l u - H i ~ - P r o - O H ~ Subsequently, many d i f f e r e n t k i n d s of t o t a l s y n t h e s e s of TRF have appeared, employing c l a s s i c a l 1 8 - 2 ° and s o l i d - p h a s e methods19 3 21s 22 u s i n g chloromethyl r e s i n o r a benzhydrylamine r e s i n 1 2 . The l a t t e r method was used t o s y n t h e s i z e TRF l a b e l l e d a s [3€I]-Pro ( c a . 50 Ci/mM)7. Well over 50 s t r u c t u r a l analogues of TRF have now been s y n t h e s i z e d and t e s t e d f o r b i o l o g i c a l a c t i v i t y . Many of t h e r e s u l t s of t h e s e s t u d i e s have been t a b l u l a t e d and d i s c u s s e d e l ~ e w h e r e 7 , 1 2 , ~ 3 . Most of t h e m o d i f i c a t i o n s , which have included t h e a l t e r a t i o n o r replacement of e v e r y amino a c i d , have r e s u l t e d i n s u b s t a n t i a l r e d u c t i o n of t h e b i o l o g i c a l a c t i v i t y ,

Chap. 18

Peptide Hormones

Burgus

195

indicating that every part of the molecule is an essential requirement for biological activity. Substitution of the histidine by more basic amino acids such as arginine, l y ~ i n e 2 3 , 2 ~ m,y-diaminobutyric , acid23, or otnithine24, yields compounds with little activity. alu-His-(nMe)-Pro-NH has .04% of the TRF biological activity, but @lu-His(TMe)-Pro-NH2 has 800% activityz4. Interestingly, the dipeptide alu-His(~Me)-NH2 exhibits some biological a ~ t i v i t y 2 ~ .It is unclear whether or not the biological effects observed result from inductive effects of the methyl group on the aromatic ring or are due to steric effects. If the -position of TRF is substituted by Phe25, or by P-(pyrazolyl-3)-alanine ,'2y3 a considerable amount of biological activity is retained; apparently, the aromaticity of the imidazole ring is important. alu-Tyr-Pro-NH2[ Tyr2]TRF, has much less activi y than [PheZlTRF, probably because of the influence of the hydroxyl group". The stereoisomer "L-D-L"-TRF (L-Elu-D-His-L-Pro-NHZ)exhibited 3% of the biological potency of the native compound , the "L-L-D" and 'ID-L-L"analogues were much less active, and "D-D-D"-TRF was inactive at the concentrations studied27. No structural analogues of TRF have been reported to act as inhibitors. The binding of TRF to whole pituitary cells in culture or plasma cell membrane fractions in competition with r3H]TRF has been s t u d i e d 7 ~ 9 , ~ ~ ~ ~ ~ . The binding affinities of the TRF analogues have shown a close correlation with the observed biological activity, suggesting that the limiting factor in biological activity is the affinity for the receptor. Initial studies on the biosynthesis of TRF indicate that it may be n o n - r i b o ~ o m a l ~ ~ -TRF 3 ~ .is rapidly inactivated in vitro by plasma, serum or whole blood. The evidence best supports enzymatic i n a ~ t i v a t i o n ~ ~ One ~~~. of the major products appears to be the free acid, Ci?lu-His-Pro-OH33. The dipeptide Rlu-His-OMe, which has little or no TRF activity, apparently acts as a competitive inhibitor of plasma inactivation of TRF34. TRF has The clinical use of TRF has been r e v i e w e d l l ~ ~ ~Synthetic . been extensively tested in patients to test pituitary TSH reserve or pituitary response to TRF. Doses have ranged from 250 kg to 1 mg i.v. and 2 to 10 mg orally with no serious adverse effects. TRF has been shown to be highly specific in the release of TSH, having no effect on the secretion of luteinizing hormone (LH), follicle stimulatin hormone (FSH), adrenocorticotropic hormone (ACTH) in vivo or Recently, Tashjian et al. have reported that TRF stimulates in vitro3?'. the release of prolactin (PRL) in ~ i t r o ~ These ~. results have been confirmed by other laboratories7. A s in the case with TSH, the effect of TRF on the release of prolactin is inhibited by thyroxin. Although large amounts of TRF are required to achieve an effect in rats, it a pears to be relatively more potent for the release of prolactin in man 37,3/i* Luteinizing Hormone Releasing mone Releasing Factor (FRF) identified as having the same Leu-Arg-Pro-Gly-NH2.A review

Factor (LRF) and Follicle Stimulating HorPorcine3Y and ovine4" LRF have now been primary structure, a l u - H i s - T r p - S e r - T y r - G l y of the work on the porcine material by

196 -

Sect, I V

-

P a c h t e r , Ed.

Metabolic & Endocrine

S c h a l l y ' s group h a s appeared41. The r e s u l t s from F o l k e r s ' l a b o r a t o r y on p a r t i a l c h a r a c t e r i z a t i o n of bovine and p o r c i n e LRF have been reviewed by ~ i e v etrs s o n l 3 . The f i r s t d e s c r i p t i o n of t h e t o t a l s y n t h e s i s of t h e LRF d e c a p e p t i d e of

was t h a t of Monahan e t a l . 4 2 . The p e p t i d e had t h e b i o l o g i c a l a c t i v i t o v i n e LRF i n v i v o and i n v i t r o ( r a t s ) and caused o v u l a t i o n i n

rabbit^^^^^^.

Subsequent t o t h a t r e p o r t , s e v e r a l o t h e r d e s c r i p t i o n s of s y n t h e s e s have a peared which employed c l a s s i c a l m e t h o d s l 3 ~o ~r ~s o l i d - p h a s e m e t h o d ~ ~ 3 ~ ~

Judging from programs o f forthcoming meetings and c o n f e r e n c e s i n J u n e , 1972 ( e . g . , IVth I n t e r n . Congr. of Endocrinology, Washington; Serono Fdn. Conf., Acapulco, Mexico), t h e r e i s a c o n s i d e r a b l e amount of a c t i v i t y now i n t h e area of s y n t h e s i s and b i o l o g i c a l t e s t i n g of a n a l o g u e s of LRF. However, a t t h i s w r i t i n g , on1 a s m a l l p o r t i o n of t h i s h a s appeared i n p r i n t . In o u r own l a b o r a t o r y 5 4 7 , we have t e s t e d , among o t h e r s , s y n t h e t i c LRF p e p t i d e a m i d e s s h o r t e n e d from t h e C-terminus (G., @lu-NH2 through des-GlylO-LRF). The nonapeptide des-GlylO-LRF h a s a b o u t 10% o f t h e potency of LRF i n t h e p i t u i t a r c e l l c u l t u r e a s s a y , and t h e o t h e r s h o r t e r p e p t i d e s , w i t h t h e e x c e p t i o n of hlu-His-Trp-NH2 which h a s a .13% a c t i v i t y , have It i s i n t e r e s t i n g , however, t h a t some o f l i t t l e o r no a c t i v i t y , (60.02%). t h e s h o r t e n e d p e p t i d e s , i n c l u d i n g Elu-His-NH2, have some i n t r i n s i c b i o l o g i c a l a c t i v i t y . The g r e a t e r a c t i v i t y of t h e t r i p e p t i d e s u g g e s t s t h a t t h e C-terminal p o r t i o n s of t h e l a r g e r p e p t i d e analogues may b e a c t u a l l y i n t e r f e r i n g w i t h t h e b i o l o g i c a l a c t i v i t y of t h e t r i p e p t i d e p o r t i o n of t h e molec u l e . F o l k e r s ' l a b o r a t o r y l 3 , i n c o l l a b o r a t i o n w i t h Bowers, h a s t e s t e d seve r a l d i - and t r i p e p t i d e analogues of LRF. None of t h e s e , i n c l u d i n g R l u His-NH2,(see below) were found by them t o have LRJ? a c t i v i t y a t t h e d o s e s t e s t e d . I n a s e r i e s of 6 t e t r a p e p t i d e - a m i d e s h a v i n g a R l u N-terminus i n combination w i t h Tyr, Arg, and Trp, o n l y one, Ci?lu-Tyr-Arg-Trp-NH2, w a s rep o r t e d t o have some LH r e l e a s i n g a c t i v i t y 1 3 I n o u r hands, t h e t e t r a p e p t i d e Blu-Tyr-Arg-Trp-NH2 h a s l e s s t h a n .001% of t h e a c t i v i t of LRF, which i s less t h a n w e f i n d f o r t h e d i p e p t i d e a l u - H i s - N H 2 ( . 0 2 % 4 ~ ~ ' ~ The . disregarding c r e p a n c i e s i n t h e o b s e r v a t i o n s of t h e two l a b o r a t o r i e s 7 , t h e r a t i o s of LRF a c t i v i t y of a l u - H i s - N H and Rlu-Tyr-Arg-Trp-NH2 may n o t 2 b e s i g n i f i c a n t s i n c e t h e l e v e l s of b i o l o g l c a l a c t i v i t y a r e extremely low A tetrapeptide, and t h e methods of b i o a s s a y a r e d i f f i c u l t t o compare. @lu-His-Pro-Gly-Mi2, which i s 35% a c t i v e as a TRF, a l s o h a s d i s t i n c t LRF a c t i v i t y i n d i c a t i n g t h a t t h e r e c e p t o r s , a l t h o u g h n o t i d e n t i c a l , might have some d e g r e e of s i m i l a r i t y ? We have a l s o t e s t e d 7 a s e r i e s of a n a l o g u e s i n which s u b s t i t u t i o n s i n t h e v a r i o u s amino a c i d r e s i d u e s have been made. P l a c i n g t h e methyl group on t h e 7 - o r I--imidazole p o s i t i o n of h i s t i d i n e r e s u l t s i n analogues w i t h 3%and 6% LRF a c t i v i t y , r e s p e c t i v e l y . These e f f e c t s a r e i n c o n t r a s t t o t h o s e observed f o r TRF i n which t h e .r-methyl s u b s t i t u t i o n g r e a t l y enhances b i o l o g i c a l a c t i v i t y . I n a s e r i e s of a n a l o g u e s i n which g l y c i n e h a s r e p l a c e d each of t h e v a r i o u s amino a c i d s , a l l of t h e analogues show c o n s i d e r a b l y reduced b i o l o g i c a l a c t i v i t y . I n t h e i n v i t r o c e l l c u l t u r e system, one of t h e s e a n a l o g u e s , [Gly2]LRF, i n a d d i t i o n t o a s l i g h t a g o n i s t a c t i v i t y , showed c o m p e t i t i v e i n h i b i t i o n of LRF a c t i v i t y . The nonapeptide analogue i n which h i s t i d i n e had been d e l e t e d , des-His2-LRF, showed v e r y l i t t l e a c t i v i t y as an a g o n i s t and w a s a l s o a b l e t o a n t a g o n i z e

.

J~~

Chap. 18

P e p t i d e Hormones

197

B u rgus

c o m p e t i t i v e l y t h e a c t i o n of LRF48,49. Claims have appeared i n t h e l i t e r a t u r e of t h e s e p a r a t i o n from hypothalamic e x t r a c t s of a s e p a r a t e FRF from LRF a c t i v i t y , b u t t h e s e conclus i o n s have been d i s p u t e d on t h e b a s i s of t h e t e c h n i q u e s of b i o a s s a y 2 , 3 * 5 . It i s now w e l l e s t a b l i s h e d t h a t t h e n a t u r a l product i s o l a t e d a s LRF a l s o o s s e s s e s c o n s i d e r a b l e FSH r e l e a s i n g a c t i v i t y i n a l i t h e systems t e s t e d 7 9 Furthermore, t h e s y n t h e t i c LRF d e c a p e p t i d e , l i k e t h e n a t u r a l product from p o r c i n e and ovine m a t e r i a l , r e l e a s e s FSH. The analogues of LRF which we have t e s t e d s o f a r a l s o have FSH r e l e a s i n g a c t i v i t y i n about t h e same p r o p o r t i o n , b u t t h e FSH r e l e a s i n g potency i s much l e s s t h a n t h a t of LH and, although i t i s s t a t i s t i c a l l y s i g n i f i c a n t , i t i s much more d i f f i c u l t t o q u a n t i t a t e . I n c o n t r a s t t o t h e c l a i m of S i e v e r t s s o n e t a l 1 3 , t h a t t h e t e t r a p e p t i d e Glu-Tyr-Arg-Trp-NH2 i s i n a c t i v e a s an FRF a t h i g h d o s e s , we have found i t t o have about t h e same r a t i o of FRF/LRF a c t i v i t y a s o t h e r analogu e s t e s t e d 7 . Although t h e LRF d e c a p e p t i d e i s c a p a b l e of r e l e a s i n g ’ b o t h LH and FSH, t h e q u e s t i o n of whether o r not t h e r e e x i s t s a s e p a r a t e chemical e n t i t y which r e l e a s e s FSH, remains t o b e e s t a b l i s h e d . P o r c i n e 4 I , ovine50 s t i m u l a t e t h e r e l e a s e of LH and FSH i n human b e i n g s . and s y n t h e t i c LRF41,51, D i s c u s s i o n s of p o s s i b l e c l i n i c a l u s e of LRF i n f e r t i l i t y c o n t r o l have appeared4I 52 53. J

Growth Hormone (Somatotropin) R e l e a s i n g F a c t o r , SRF (GRF) Considerable orts the existevidence from p h y s i o l o g i c a l and c l i n i c a l areas s t r o n g 1 The p u r i f i ence of hypothalamic c o n t r o l of growth hormone r e l e a s e c a t i o n of hypothalamic f a c t o r s i n f l u e n c i n g t h e c o n t e n t of p i t u i t a r y growth hormone has been r e p o r t e d by s e v e r a l l a b o r a t o r i e s , b u t t h e s i n i f i c a n c e of assays5)ased on p i t u i t a r y hormone c o n t e n t h a s been questionedF4. S c h a l l y et a l . have r e p o r t e d t h e p u r i f i c a t i o n and sequencing of t h e p e p t i d e ValHis-Leu-Ser-Ala-Glu-Glu-Lys-Glu-Ala from p o r c i n e hypothalami on t h e b a s i s have s y n t h e s i z e d t h e p e p t i d e . Both t h e of t h i s a s s a y , and Veber e t a l ? n a t i v e p e p t i d e and i t s s y n t h e t i c r e p l i c a t e were a c t i v e i n t h e p i t u i t a r y d e p l e t i o n a s s a y b u t f a i l e d t o r e l e a s e radioimrnunoassayable growth hormone i n t h e plasma of normal animals o r humans. Moreover, Veber e t a l . 5 6 have noted t h e s t r u c t u r a l s i m i l a r i t y of “GRH” t o t h a t of t h e amino t e r m i n a l sequence of t h e p - c h a i n of p o r c i n e hemoglobin. The p h y s i o l o g i c a l s i g n i f i cance of “GRH” t h u s remains t o be e s t a b l i s h e d . S e v e r a l l a b ~ r a t o r i e s ~ ~ - ~ ’ have r e p o r t e d t h a t e x t r a c t s of hypothalami ( p o r c i n e , ovine, r a t ) and p a r t i a l l y p u r i f i e d m a t e r i a l s from t h e s e e x t r a c t s s t i m u l a t e r e l e a s e of r a d i o immunoassayable growth hormone. Others have r e p o r t e d growth hormone reThe chemil e a s e i n h i b i t i n g a c t i v i t y i n crude hypothalamic e x t r a c t s 6 0 , 6 1 . c a l n a t u r e of any of t h e s e s u b s t a n c e s h a s n o t been c h a r a c t e r i z e d .

3 i?iy.

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C o r t i c o t r o p i n R e l e a s i n g F a c t o r , CRF Although CRF was one of t h e f i r s t of t h e r e l e a s i n g f a c t o r s t o b e s t u d i e d , t h e chemical n a t u r e of t h i s f a c t o r s t i l l remains e l u s i v e , p a r t l y because of t h e d i f f i c u l t i e s i n t h e b i o a s s a y t e c h n i q g ~ s ~and - ~ p o s s i b l y a l s o because of chemical i n s t a b i l i t y of t h i s It i s now t h e consensus of most of t h o s e working i n t h e f i e l d hormone t h a t a hypothalamic CRF does e x i s t and t h a t a l t h o u g h v a s o p r e s s i n i s c a p a b l e of r e l e a s i n g ACTH i n t h e v a r i o u s a s s a y systems, i t i s probably not t h e physThe e x a c t r e l a t i o n s h i p of hypothai o l o g i c a l m e d i a t o r of ACTH release2-’.

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lamic CRF t o e i t h e r v a s o p r e s s i n o r a-MSH o r b o t h , a s w e l l a s i t s r e l a t i o n s h i p t o CRF of neurohypophysial o r i g i n , remains t o be e s t a b l i s h e d 3 , 5 . Chan e t a 1 . 6 2 have suggested t h a t t h e r e may b e two CRF's i n t h e r a t hypothalamus. P r o l a c t i n Release I n h i b i t i n g F a c t o r , PIF, and P r o l a c t i n R e l e a s i n g F a c t o r , There i s p h y s i o l o g i c a l evidence t h a t t h e s e c r e t i o n of p r o l a c t i n i s under some s o r t of t o n i c i n h i b i t i o n by t h e h y p 0 t h a l a m u s 2 - ~ . D e s c r i p t i o n s of a t t e m p t s t o p u r i f y t h i s f a c t o r have placed i t i n o r near t h e zone of m a t e r i a l having LRF r e l e a s i n g a c t i v i t y i n g e l f i l t r a t i o n experiments. LRF does n o t s t i m u l a t e o r i n h i b i t t h e r e l e a s e of p r o l a c t i n i n v i v o o r i n v i t r o 63. No meaningful chemistry on t h e n a t u r e of t h i s f a c t o r i s a v a i l a b l e .

PRF -

The a b i l i t y of TRF, a l u - H i s - P r o - N H 2 , t o r e l e a s e p r o l a c t i n h a s a l ready been mentioned. The r e a l t i o n s h i p of TRF t o a p o s s i b l e n a t u r a l PRF i n hypothalamic t i s s u e remains t o be e s t a b l i s h e d . Melanocyte S t i m u l a t i n g Hormone (Release) I n h i b i t i n g F a c t o r , MIF, and R e l e a s i n g F a c t o r , MRF L i k e p r o l a c t i n , t h e release of MSH a p e a r s t o b e under a t o n i c i n h i b i t i o n by t h e h y p o t h a l a m ~ s ~ - ~C.e l i s e t alet: have proposed t h a t t h e C-terminal fragment of o x y t o c i n , Pro-Leu-Gly-NH2, i s an MSH release i n h i b i t i n g hormone, MIF; t h i s s u g g e s t i o n was confirmed by Nair et a165, who sequenced m a t e r i a l o b t a i n e d from t h e hypothalamus. Both groups supported t h e i r o b s e r v a t i o n s u s i n g s y n t h e t i c m a t e r i a l . The M I a c t i v i t y of t h i s p e p t i d e could n o t be confirmed by o t h e r l a b o r a t o r i e s 7,%6 On t h e o t h e r hand, t h e r i n g s t r u c t u r e of o x y t o c i n ( t o c i n o i c a c i d 6ys-Tyr-IleGln-Asn-Cy's-OH, was r e p o r t e d t o e x h i b i t some MIF a c t i v i t y 26 Both of t h e s e

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p e p t i d e s may a r i s e from enzymatic d e g r a d a t i o n o f o x y t o c i n i n t h e h y p o t h a l amus64. The p e n t a p e p t i d e Cys-Tyr-Ile-Gln-Asn-OH h a s been r e p o r t e d t o exh i b i t MSH r e l e a s i n g a c t i v i t y 6 7 . These r e s u l t s remain t o be confirmed. The s i g n i f i c a n c e of t h e s e p e p t i d e s a s m e d i a t o r s of MSH s e c r e t i o n remains t o b e e s t a b l i s h e d i n view of t h e q u e s t i o n s which a r i s e concerning t h e v a l i d i t y of t h e a s s a y systems. Mechanism of A c t i o n of Hypothalamic R e l e a s i n g F a c t o r s The mechanisms of a c t i o n of hypothalamic f a c t o r s have been reviewed3ybi12,68. E f f e c t s of [K*] , [Ca+] , o t h e r c a t i o n s and m e t a b o l i c i n h i b i t o r s , a s w e l l a s p o s s i b l e r o l e s of cyclic-AMP and p r o s t a g l a n d i n s i n t h e r e l e a s e of p i t u i t a r y hormones a r e d i s c u s s e d . The a c u t e e f f e c t s of r e l e a s e of TSH and LH by TRF and LRF r e s p e c t i v e l y appear n o t t o b e a f f e c t e d by p r e t r e a t m e n t w i t h i n h i b i t o r s of p r o t e i n s y n t h e s i s , such a s cycloheximide, puromycin o r actinomycin D , b u t c o n f l i c t i n g r e p o r t s occur r e g a r d i n g t h e e f f e c t of t h e s e i n h i b i t o r s on t h e r e l e a s e of o t h e r p i t u i t a r y hormones. The a c u t e o r c h r o n i c e f f e c t of t h e r e l e a s i n g f a c t o r s on t h e s y n t h e s i s of p i t u i t a r y hormones has been s t u d i e d u s i n g c r u d e hypothalamic e x t r a c t s and measuring l e v e l s of t h e p i t u i t a r y hormone i n v a r i o u s t i s s u e s and f l u i d s . The e f f e c t s observed a r e minimal w i t h r e s p e c t t o t h e c o n f i d e n c e l i m i t s of t h e b i o a s s a y s i n v o l v e d ; s t u d i e s u s i n g h i g h 1 r e l i a b l e measurements of t h e p i t u i t a r y hormones a r e q u i t e l i m i t e d . l&-glucosamine and 14C-alanine had been r e p o r t e d t o be i n c o r p o r a t e d i n t o immunologically p r e c i p i t a b l e t h y r o t r o p i n . A s i m i l a r r e p o r t of t h e i n c o r p o r a t i o n of 3H-glucosamine i n t o LH by h i g h l y p u r i f i e d p o r c i n e

Chap. I8

P e p t i d e Hormones

Burgus

LRF h a s r e c e n t l y appeared69. P i t u i t a r y Hormones. The t e c h n i q u e o f radioimmunoassay of p i t u i t a r y hormones i s now w e l l established1y70. Assays of human a n t e r i o r p i t u i t a r y hormones a s t e s t s of p i t u i t a r y f u n c t i o n have been reviewed71. Reports have appeared d e s c r i b i n g r a d i o - l i g a n d b i n d i n g of p i t u i t a r y hormones t o t a r e t t i s s u e s f o r mechani s t i c s t u d i e s o r a s a p o s s i b l e means of a ~ s a y 7 2 , 7 ~ The . biological action of ACTH74 and bovine growth hormone (BGH)75 using hormones bound t o l a r g e molecules, h a s a l s o been s t u d i e d . Growth Hormone - The s t r u c t u r e of human growth hormone (HGH) a s o r i g i n a l l y proposed by L i e t a1?6y h a s been revised77378. The p e n t a d e c a p e p t i d e fragment c o n t a i n i n g Trp o r i g i n a l l y a s s i g n e d t o p o s i t i o n 17-31 was found t o occupy p o s i t i o n s 77-91; a m i s s i n g d i p e p t i d e fragment, Leu-Arg, w a s found i n p o s i t i o n s 92-93 and t h e sequence Gly-Ser-Pro i n 130-132 was c o r r e c t e d t o P r o - S e r - G l ~ ~ ~ I,n~ a~d.d i t i o n , t h e Asp r e s i d u e a t p o s i t i o n 47 and Glu a t p o s i t i o n s 4 9 , 9 0 and 121, were r e a s s i g n e d a s Asn and G l n , r e s p e c t i v e l y 7 8 . A s y n t h e t i c p r e p a r a t i o n of a p e p t i d e o b t a i n e d by s o l i d - p h a s e methods based on t h e o r i g i n a l l y proposed growth hormone sequence had 10% growth promoting potency and 5% l a c t o g e n i c a c t i v i t y i n comparison t o t h e n a t i v e hormone and i t a l s o r e a c t e d immunologically w i t h rat a n t i s e r u m t o HGH79. L i e t a 1 8 O y 8 l have a l s o r e p o r t e d t h e s y n t h e s i z e d p r o t e c t e d p e p t i d e fragmenbg by c l a s s i c a l methods based on t h e o r i g i n a l s t r u c t u r e . C h i l l e m i and P e c i l e have r e p o r t e d t h a t p e p t i d e s corresponding t o t h e sequence 81 t o 121 and t h e sequence 122 t o 153 of HGH76 prepared by s o l i d - p h a s e s y n t h e s i s , were a c t i v e i n t h e t i b i a t e s t . A s noted above, t h e sequence upon which t h e s e s y n t h e s e s were based h a s been revised c o n s i d e r a b l y i n t h e 81-121 r e g i o n , a l t h o u g h t h e sequence of 30 of t h e 4 1 amino a c i d r e s i d u e s a r e i n segments homologous t o t h e r e v i s e d sequence. It i s , n e v e r t h e l e s s , of i n t e r e s t t h a t such small segments of the GH molecule would have a c t i v i t y . These c o n c l u s i o n s a r e i n agreement w i t h e a r l i e r r e p o r t s t h a t growth hormone fragments o b t a i n e d f r p r o t e o l y t i c o r cyanogen bromide c l e a v a g e r e t a i n some b i o l o g i c a l a c t i v i t y Reports from v a r i o u s l a b o r a t o r i e s have appeared on sequence s t u d i e s of growth hormones of o t h e r s p e c i e s . Fellows e t a l . 8 3 have provided e v i d e n c e t h a t t h e m u l t i p l e c h a i n s of BGH a r i s e from enzymatic d e g r a d a t i o n of t h e Nt e r m i n a l p o r t i o n . D’ff r e n c e s may o c c u r , however, i n a d d i t i o n t o t h e Nterminal sequences Sequence s t u d i e s show t h a t BGH86-88 , o v i n e GH8’, and p o r c i n e GHgl, have a high d e g r e e of homology w i t h HGH, e s p e c i a l l y i n t h e C-terminal p o s i t i o n .

E.

P r o l a c t i n , PRL - Bovine p r o l a c t i n (BPRL) h a s been r e p o r t e d ” t o d i f f e r from t h e sequence of o v i n e p r o l a c t i n (OPRL)93 by o n l y two amino a c i d r e sid u e s . The p r o l a c t i n s show c o n s i d e r a b l e s t r u c t u r a l homology w i t h growth hormone and w i t h human p l a c e n t a l l a c t o g e n (more r e c e n t l y c a l l e d human c h o r i o n i c somatotropin, HCS), HCS shows a h i g h d e g r e e of homology w i t h GH; over 80% of t h e amino a c i d s a r e i d e n t i c a l and t h e remaining p a i r s are rel a t e d by h i g h l y favored codon s u b ~ t i t u t i o ny sg 4 .~ ~ N i c o l l and L i c h t g 5 have compared t h e e l c t r o p h o r e t i c m o b i l i t i e s of p r o l a c t i n s and somatotropins from a v a r i e t y of s p e c i e s and have reviewed e x t e n s i v e l y t h e f u n c t i o n a l

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r e l a t e d n e s s of t h e s e m o l e c u l e s . I n a l l t h e t e t r a p o d s p e c i e s examined, p r o l a c t i n s and growth hormones appeared t o b e s e p a r a t e m o l e c u l a r e n t i t i e s bu b o t h hormones shared p r o l a c t i n and s o m a t o t r o p i c a c t i v i t i e s . N i a l l et alS7 have observed t h a t , i n a d d i t i o n t o t h e " e x t e r n a l homologies" of prol a c t i n s and growth hormones, a c o n s i d e r a b l e d e g r e e of " i n t e r n a l homology'' e x i s t s , t h e r e a p p e a r i n g t o b e f o u r r e g i o n s i n which sequences of a p p r o x i m a t e l y 20 amino a c i d s a r e c l e a r l y r e l a t e d w i t h i n t h e i n d i v i d u a l molecules. These f i n d i n g s had l e d them t o s u g g e s t t h a t t h e s e hormones have a l l a r i s e n from a s h o r t e r p r i m o r d i a l p e p t i d e by gene r e d u p l i c a t i o n . Evidence i s mounting, p r i m a r i l y on t h e b a s i s of immunological s t u i t h a t a human p r o l a c t i n s e p a r a t e from human growth hormone, existsgg-": Continued rep o r t s have ap e a r e d concerning t h e p h y s i c a l - c h e m i c a l p r o p e r t i e s of HGH99lol, BGH, OGHPa2, and 0PRL1Oo. G l y c o p r o t e i n Hormones; Thyroid S t i m u l a t i n g Hormone, TSH, L u t e i n i z i n g Hormone, LH, F o l l i c l e S t i m u l a t i n g Hormone, FSH - The complete primary s t r u c t u r e s of bovine TSHlU3 and o v i n e LH104-106 have now been r e p o r t e d . The s t r u c t u r a l s t u d i e s on TSH and comparisons of TSH w i t h o t h e r glycoprot e i n hormones have been reviewed by P i e r c e l 0 7 . These two hormones103-107 a s w e l l as FSHlo8-ll1, d i s s o c i a t e i n t o two s e p a r a t e c h a i n s of approximately 15,000 m o l e c u l a r w e i g h t each. The hormones c o n t a i n about 15-30% carbohyd r a t e , a l t h o u g h t h e s u g a r c o n t e n t s v a r y . Bovine and o v i n e TSH and LH cont a i n l i t t l e o r no s i a l i c a c i d whereas human TSH and LH and FSH a r e r e p o r t e d t o c o n t a i n more s i a l i c a c i d l o 7 which a p p a r e n t l y i s n e c e s s a r y f o r f u l l h o r monal a c t i v i t y 1 1 2 , l13. However, d e s i a l y l a t i o n of human LH i n c r e a s e s b i n d i n g a f f i n i t y t o s u b c e l l u l a r t e s t i s o r o v a r i a n f r a c t i o n s 1 1 4 and a l s o seems t o enhance immunologic a c t i v i t y 1 l 5 . The s t r u c t u r e work on TSH and LH r e v e a l e d t h a t one s u b u n i t , TSH-a and L H e , i s e s s e n t i a l l y t h e same i n b o t h hormones and t h e o t h e r s u b u n i t s , TSH-p and LH-P, a r e t h e hormone s p e c i f i c u n i t s ; n o t o n l y can t h e cy and p s u b u n i t s of a g i v e n hormone b e recombined t o g i v e a r e c o n s t i t u t e d complex w i t h 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 of t h e p a r e n t hormone, b u t a l s o e i t h e r TSH-a o r LH-CY can b e comb i n e d w i t h TSH-P t o g i v e TSH and e i t h e r a - c h a i n can b e combined w i t h LH-P t o g i v e a molecule w i t h t h e p r o p e r t i e s of LH. A s i m i l a r r e l a t i o n s h i p e x i s t s between t h e s u b u n i t s of human c h o r i o n i c gonadotropin (HCG) and t h e s u b u n i t s of TSH and LH. The s u b u n i t s of human LH, FSH and human HCG116, c a n a l s o b e s u b s t i t u t e d f o r each o t h e r i n combination w i t h t h e p subu n i t of t h e o t h e r hormones116. The sequence of h e o v i n e LH-p s u b u n i t rep o r t e d by Ward's l a b o r a t o r y l o 5 and b o v i n e LH-pllfj a r e now i n complete agreement; t h e sequence proposed by L i ' s grouplo6 f o r o v i n e LH-P d i f f e r s from t h e s e p r o p o s a l s by t h e placement of 1 7 amino a c i d s , most of which rep r e s e n t r e v e r s a l sequences. A l s o , Ward e t al1'5 propose an a c y l a t e d amino t e r m i n u s i n c o n t r a s t t o t h e f i n d i n g s of Papkoff e t a 1 . I o 6 . There i s a s t r i k i n d e g r e e of homology (around 50%) between t h e TSH-P and LH-P seq u e n c e ~ ~ ' ~ .Hennen e t a l .l19 have noted an " i n t e r n a l homology" i n t h e a - c h a i n of bovine LH i n a s i t u a t i o n analogous t o t h a t observed by N i a l l et a1.77 f o r growth hormone and t h e l a c t o g e n s . Chemical and b i o l o g i c a l c h a r a c t e r i z a t i o n of t h e g l y c o p r o t e i n s u b u n i t s i s c o n t i n u i n g . There a p p e a r s t o b e agreement t h a t t h e c i r c u l a r d i c h r o i s m (CD)of i n t a c t o v i n e LH i n d i c a t e s l i t t l e o r no h e l i c a l c h a r a c t e r b u t t h e r e is some disagreement r e g a r d i n g t h e CD c h a r a c t e r of t h e subunits120-122. Yang e t a l . 1 2 3 r e p o r t t h a t t h e

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20 1 -

LH-P s u b u n i t i n d u c e s o v u l a t i o n i n t h e hamster. The o v i n e L H w s u b u n i t h a s l i p o l y t i c a c t i v i t y a s does t h e i n t a c t hormone, whereas t h e LH-fl i s i n a c t i v e t o r e l e a s e f r e e f a t t y a c i d s from mouse a d i p o s e t i s s u e 1 2 4 . The p s u b u n i t a p ears t o p r o v i d e most of t h e a n t i g e n i c i t y of o v i n e TSH107 and ~ ~ 1,124 0 7- P26

.

A d r e n o c o r t i c o t r o p i c Hormone, ACTH - Reports on b i o l o g i c a l a c t i v i t i e s of s y n t h e t i c ACTH and i t s analogues a r e c o n t i n u i n g . Fragments as s m a l l as ACTH4-10 ( p o r c i n e c o r t i c o t r o p i n (4-10) h e p t a p e p t i d e ) and ACTH5-10 hexap e p t de e x h i b i t s t e r o i d o g e n i c a c t i v i t y l 2 7 . ACTHlli s a competitive a n t a g o n i s t of ACTHISgg ( p o r c i n e ) , s u g g e s t i n g t h a t tEe r e g i o n 11-24 i s n o t involved i n a c t i v a t i o n b u t p r o v i d e s a f f i n i t y f o r t h e r e c e p t o r l 2 8 . ACTHI-24 d i a z o t i z e d t o polyacrylamide s t i m u l a t e s s t e r o i d o g e n e s i s i n r a t adrenal c e l l s without entering t h e c e l l , suggesting t h a t t h e r e i s a possib l e i n t e r a c t i o n of t h e ACTH a t t h e c e l l s u r f a c e r e c e p t o r s l 2 9 . ACTH d i a z o t i z e d t o a g a r o s e induced s t e r o i d o g e n e s i s w i t h o u t e n t e r i n g t h e c e l l b u t , a p p a r e n t l y d i d n o t a d h e r e t o t h e c e l l surface13'. Reports from s e v e r a l l a b o r a t o r i e s have now appeared, i m p l i c a t i n g t h e involvement of ACTH o r ACTH analogues i n memory o r b e h a v i o r a l r e s p o n s e s 131-133~135. A p e p t i d e "BC" h a s been o b t a i n e d from p i t u i t a r y which s t i m u l a t e s avoidance a c q u i s i t i o n i n r a t s b u t h a s no ACTH, MSH o r p r e s s o r a c t i v i t y l 3 4 . The z i n c phosphate v e h i c l e used i n some of t h e s e s t u d i e s i s a l s o a b l e t o mimic some of t h e b e h a v i o r a l e f f e c t s i n t h e cond i t i o n e d avoidance r e s p 0 n s e l 3 ~ . P r o t o n N M R s t u d i e s have l e d P a t e l l 3 6 t o conclude t h a t no i n t e r a c t i o n Structural relationo c c u r s between sequences 1-10 and 11-24 i n ACTHl-24. s h i p of ACTH t o b i o l o g i c a l a c t i v i t y h a s been d i s c u s s e d by Toniolol'l. Melanocyte S t i m u l a t i n g Hormone, MSH - S h a p i r o e t al.137 have s t u d i e d MSH a c t i v i t i e s i n a v a r i e t y of v e r t e b r a t e s p e c i e s , comparing t h e s e t o known CYand B-MSH's, and have concluded on t h e b a s i s of g e l f i l t r a t i o n b e h a v i o r , radioinununlogical a s s a y s and b i o a s s a y s t h a t t h e s t r u c t u r e s of t h e major MSH hormones i n s e v e r a l of t h e species a r e d i f f e r e n t from t h o s e which have been c h a r a c t e r i z e d p r e v i o u s l y . Sandman e t a l .138 have claimed i n v o l v e ment of MSH i n t h e p a s s i v e avoidance r e s p o n s e . The involvement of p i t u i t a r y hormones i n reL i p o t r o p i c Hormone, LPH l e a s i n g f r e e f a t t y a c i d s from a d i p o s e t i s s u e ( l i p o l y s i s ) h a s been d i s c u s s e d a t a symposiuml39. The amino a c i d sequence (58 amino a c i d s ) of p o r c i n e a - l i p o t r o p i c hormone (a-LPH) i s c l o s e l y homologous t o t h a t p r e v i o u s l y r e p o r t e d f o r o v i n e 7-LPH. The p o r c i n e Y-LPH sequence a p p e a r s t o b e i d e n t i c a l t o t h e N-terminal 1-58 sequence of p o r c i n e P-LPH i n a r e l a t i o n s h i p s i m i l a r t o t h a t p r e v i o u s l y observed f o r p - and a - L P H of bovine o r i g i n . A l s o , as h a s been t h e c a s e w i t h t h e o v i n e hormone, t h e C-terminal sequence of porc i n e y-LPH i s i d e n t i c a l t o t h e e n t i r e 18 amino a c i d sequence of P-MSH from t h e same s p e c i e s . P a r t i a l c h a r a c t e r i z a t i o n of human l i p o t r o p i n s was d e s cribed.

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Posterior Pituitary Hormones; Oxytocin, Vasopressin - The chemistry, physiology, and assay of oxytocin and vasopressin have been extensively reviewedl40. Investigation of the properties o f synthetic neurohypophysial hormones continues to be a very active area. [Thr4]oxytocin exhibits enhanced oxytocin-like activities and diminished vasopressin-like activities; [ Thr4]mesotocin exhibits similar p r ~ p e r t i e s l ~ ~Recent . studies142 have revealed the somewhat surprising result that removal of the N-terminal amino group from these derivatives results in reduction of biological activity rather than enhancement as might have been expected in the light of previous observations concerning ox tocin and other 4-substituted analogues of o ~ y t o c i n l ~ ~Manning . et al.K42 have suggested that the solubility differences of these various analogues offer one possible explanation for the differences in biological properties. The laboratory o f du Vigneaud has described a series of analogues of oxytocin having the Cys residue in position 1 replaced with L-penicillamine (L-p-P-dimethylcysteine) derivatives which are devoid o f avian vasodepressor, oxytocic, and pressor activities but which inhibit the oxytocic activity of oxytocinl44. [ Leu2, Leu4] oxytocin, which has natriuretic activit and weak diuretic effects, is also an inhibitor of oxytocic activitylX5 in contrast to [Leu41 oxytocin which has considerable diuretic and natriuret ic effects as well as anti-ADH activity (inhibiting the antidiuretic effect of vasopressin); other 4-substituted oxytocins have been reported to have anti-ADH effects146. Conformations of oxytocin, vasopressin and their derivatives have been studied by NMR147-149. The sequence of porcine neurophysin I has been determined 150. References. 1. 2. 3.

4. 5. 6.

7. 8.

9.

10. 11. 12. 13. 14.

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107. 108. 109. 110. 111. 112. 113. 114.

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Burgus

207 -

145. V.J. Hruby and W.Y. Chan, J.Med.Chem., 14,1050 (1971). 146. H. Datta and R.R. Chaudhury, J.Endocrinol., 4 6 , 117 (1970). 147. D.W. Urry and R. Walter, Proc.Nat.Acad.Sci.(USA), 68, 956 (1971). 148. J. Feeney, G.C.K. Roberts, J.H. Rockey and A.S.V. Burgen, Nature, New Biology, 232, 108 (1971). 149. P.H. Von Dreele, A.I. Brewster, F.A. Bovey, H.A. Scheraga, M.F. Ferger and V. du Vigneaud, Proc.Nat.Acad.Sci.(USA), 68, 3088 (1971). 150. F.C. Wuu, S. C r u m and M. Saffran, J.Biol.Chem., 246, 6043 (1971).

Chapter 19.

Non-steroidal Antiinflammatory Agents

Peter F. Juby and Thomas W. Hudyma Bristol Laboratories, Syracuse, New York 13201

-

Introduction Research in the field of non-steroidal antiinflammatory agents (NAA) encompasses too wide an area to be completely covered in a review of this size. As was the case in last year’s Report,1 we have had to confine our survey mainly to the work relating to the chronic arthritic diseases. For those readers interested in the broader aspects of the subject, several excellent texts29 3 and reviews4, are now available. Reports have appeared on three important symposia. 6-8

-

Etiolow and Pathogenesis The widely held belief that infectious organisms play some part in the initiation of rheumatoid arthritis (RA) is proving difficult to substantiate. New investigationsg, 10 have indicated that mycoplasma may be implicated in the pathogenesis of RA, and skeletal lesions have appeared in micell and chicks12 which have received injections of human rheumatoid synovial tissue. A recent study,13 however, has demonstrated no significant benefit from long term tetracycline therapy for patients with RA.

-

Animal Test Models In vitro screens for N U have been largely neglected, with continuing reliance being placed mainly on the adjuvant arthritis and carrageenin foot edema models in the rat. By the use of both non-established and established adjuvant diseases it has been possible to differen tlate be tween immunosuppress ive agent s and pure 1y anti inflammatory l5 Di Rosa and Willoughby have delineated16 the sequence of agents. events in the carrageenin-induced foot edema model. The initial mediators, histamine, serotonin, and kinins, are followed by a prostaglandin, the release of which is associated with the migration of leukocytes into the inflamed site. NAA suppress this migration. Experimental rabbit models of osteoarthritis have been reported. 1 7 ~ 1 8

-

To add to the many existing proposed modes of action of Modes of Action NAA, several new hypotheses have been advanced. It has been suggestedlg-22 that acidic NAA owe their effectiveness to their demonstrated ability to inhibit the synthesis or release of prostaglandins such as PGE and =Fa. thin the The prostaglandin blockade occurs at drug concentrations well range attained clinically in plasma. 19 Prostaglandins have been i licated as mediators in certain types of inflauunatory conditions in the ra36923-26 and in man,2’ but their role in RA has not been established. Perhaps the prime action of the NAA is to inhibit the release of damaging lysosomal enzymes from invading leukocytes, since these enzymes are envisioned28 as being responsible for the ultimate production of the prostaglandins.

8i

It has been proposed29 that antirheumatic drugs act by displacing certain small peptides from their binding sites on serum proteins. These free peptides can then protect connective tissues against the effects of

Chap.

-

19

A n t i inf 1 arnrna to r y Ag en t s

chronic inflamnatory reactions. cy of free peptides.

Juby, Hudyrna

209 -

Patients with RA have a relative deficien-

Several acidic NAA have been shown3O to inhibit nicotinate phosphoribosyl transferase in human platelet lysate by reversible competition with nicotinic acid. The resulting suppression of nicotinamide adenine dinucleotide biosynthesis may inhibit mucopolysaccharide biosynthesis and reduce the inflammatory response. Still a matter of some controversy is whether NAA either stablize or labilize lysosomes. The answer seems to depend on the source of the lyaosomes and the experimental conditions used in the assay. 31932

-

Shock Considerable interest has been shown of late in the use of NAA against shock due to various causes. Treatment of dogs with NAA abolished some of the symptoms of shock caused by injection of living g. &organi s m ~ .Doses ~ ~ of NAA required for activity against cholera toxin in the rat were in the same range as those re uired for antiinflannnatory activity. 34 Membrane stabilization effectsfZ and the inhibition of prostaglandin release or synthesis35 have been su gested as possible modes of action for the drugs, although a brief report38 has appeared on the beneficial effect of PGEl and PGFa in endotoxin shock. Agents under Investination

*

-

General A working draft of clinical testing guidelines for NAA has been introduced by the FDA.37 Of particular interest is the proposal that compounds are needed with less side effects than aspirin without necessarily any greater effectiveness than aspirin. An excellent survey of the probleas associated with the clinical assessment of drugs for use against RA has been published. 38

-

Arylalkanoic Acids and Related Compounds Without doubt this area has been the most widely explored for potential NAA. In a double blind crossover trial, 1.5 g/day of alclofenac (r)was judged equivalent to 75 %/day of indomethacin in patients with osteoarthritis of the hip.39 In a separate study, 3.0 g/day of h was effective in 60% of patients with RA, ankylosing spondylitis, and osteoarthritis.40 Clinical improvement with the absence of side effects was noted in rheumatoid arthritics receiving the ethanolamine salt of 1 at 0.5-1.5 g/day, i.rn.41 About 707. of 983 patients with RA, osteoarthritis, and allied conditions have shown improvement on longIn a review on 2, however, it was conterm therapy with ibuprofen (2J.42 cluded that 2 was an effective analgetic agent but has not so far been shown to have antiinflammatory (AI) effects in man.43 Fenoprofen (3, an antiinflarmnatory-analgetic agent, was reported to inhibit swelling in the carrageenin rat foot edema test (CE), inhibit adjuvant arthritis, and to have an ED5o of 0.5-1.0 mg/kg in the W erythema assay.44 The (+)- and (-)-isomers appeared equipotent. The major urinary metabolites of 2 in the rat and rabbit were free and conjugated 4'-hydroxy derivatives. 45 Patients

* Unless

stated otherwise, agents were administered orally.

210

Sect. I V

- Metabolic

with RA who were t r e a t e d with

-1

&

2 obtained

Endocrine

Pachter, Ed,

symptomatic r e l i e f a f t e r 4 days.46

z

5 (9

(9

A c t i v i t y comparable t o indomethacin i n the carrageenin abscess assay i n the r a t was reported f o r 4 (19 583 R.P.) which is apparently undergoing c l i n i c a l evaluation.47 A comprehensive phannacolo ical paper on the A 1 and a n a l g e t i c p r o p e r t i e s of naproxen ( 5 ) has appeared,'f8 together with a claim t h a t the compound s i g n i f i c a n t l y reduced hind paw inflammation i n the established adjuvant a r t h r i t i s assay a f t e r 14 d a i l y doses of 0.2 mg/rat.49 A c l i n i c a l t r i a l has indicated 5 t o be e f f e c t i v e a g a i n s t RA a t doses of 300500 mg/day.s0 Tolmetin (6J had 0.38 times the potency of indomethacin (CE) and w a s claimed t o be a c t i v e i n the adjuvant a r t h r i t i s assay.51

&.,

702H

0 \

e

\

CH3

(9

-6

Wy 23205 (I) had twice t h e potency (CE) of phenylbutazone and was s l i g h t l y less potent than indomethacin i n the adjuvant a r t h r i t i s assay. 52 Leo-1028 (8J had a minimal e f f e c t i v e dose of 13 mg/kg (CE).s3 Comparable potency t o indomethacin was claimed f o r the racemic a c i d 2 (TAI-284) i n a wide v a r i e t y of a c u t e and chronic A 1 animal s c r e e n ~ . ~ Most 4 of t h e a c t i v i t y resided i n the (9-(+)-isomer. 55 Evidence has been presented56 which does n o t support t h e proposal55 t h a t the A 1 a c t i v i t y of S-cyclohexylindan1-carboxylic a c i d s is due t o a s t e r o i d - l i k e mechanism. COH

N

P 2

c 2 H 5 ) ( n 1 2 p2z"

H

C02H

c1 CH3

-7

Cl

-8

Compound 10. with the usual aromatic r i n g p a r t l y saturated, 0.05 t i m e s the potency o f indomethacin i n the adjuvant a r t h r i t i s in Compound 11 w a s equipotent t o indomethacin (CE), with an LD between 50 and 136 ~ n g / k g . Metiazinic ~~ a c i d (12)had a pro&ent e f f e c t a t d a i l y doses o f 0.75-1.5 grams i n p a t i e n t s with v a r i o u s

had only assay.57 rats of analgetic rheumatic

Chap.

19

A n t i - i n f l a m m a t o r y Agents

21 1 -

Juby, Hudyma

a f f l i c t i o n s . 59 7°2H

The indol zine -3omers of indamethacin (15).60

'i02H

YH3

13 and & ha-

about

t i m e s the potency (CE CO H 1 2

c1

13 -

c1"

d" 14 -

-

c1

15 -

Fenamic Acids and Aza Analogs The N-nitroso d e r i v a t i v e 16 (ITF 611) was reported t o have twice the potency (CE) of flufenamic acid, with l e s s toxicity. 61,62 Clonixin (17)had 0.5 times the potency (CE) of flufenamic acid, 63 and i n a c l i n i c a l t r i a l 600 mg of 17 was found comparable t o 12 mg of morphine s u l f a t e (i.m.1 i n the r e l i e f of postsurgical pain.& A t d a i l y doses of 0.75-1.0 g, niflumic acid (18) w a s shown t o be of some b e n e f i t i n a wide v a r i e t y of rheumatic a f f l i c t i o n s . 65

&3

-

-

16

17

-

c1

b

18

-

S a l i c v l i c Acids Three days a f t e r s u b s t i t u t i o n of a s p i r i n (19. 3.6 g/day) by placebo, exacerbations were evident i n approximately two t h i r d s of pat i e n t s with RA.66 Benorylate (20) a t 4 g/day6' w a s found t o be as e f f e c t i v e a s a s p i r i n (3 glday) i n RAY and a t 6 g/day68 lowered the erythrocyte eedimentation r a t e more s i g n i f i c a n t l y than phenylbutazone (0.32 glday), a l though the p a t i e n t s preferred the phenylbutazone. F l u f e n i s a l (21)may not possess s i g n i f i c a n t advantages over a s p i r i n t o j u s t i f y continued c l i n i c a l evaluation.69

21 2

Sect. I V

- Metabolic t Endocrine

Pachter, Ed.

-

Benzothiazines The acidic 4-hydroxy-2methyl-2H_..l, 2-benzothiazine-3carboxanilide 1 1-dioxide (22. R e ) had about twice the potency (CE) of phenylbutazone.50 The major urinary metabolite i n man (22 R 4 H ) had nearly twice the plasma h a l f - l i f e of the parent com ound? but w a s reported to have weak anti-edema a c t i v i t y i n the rat.y2 Compound 23. a member of a potent series of isomeric 1,2-benzothiazine 1,l-dioxide A 1 agents had 1.5 t i m e s the potency (CE) of indomethacin.73

-

The parenteral use of gold salts i n RA has received prominent a t tention i n the past year, and i t has been concluded t h a t chrysotherapy is beneficial i n active rheumatoid disease. 74 New impetus for research i n t h i s area may have been provided by the discovery of an o r a l l y e f f e c t i v e agent i n the adjuvant a r t h r i t i s assay. Triethylphosphinogold chloride (p.0.) w a s as e f f e c t i v e as gold sodium thiomalate (i.m.) a t equal gold doses and produced no overt kidney damage following chronic administration.55 Gold salts may exert t h e i r beneficial e f f e c t s by preventing prot e i n denaturation76 and/or modifying the immune response. 9977

-

I n s p i t e of the accumulating evidence for the pro-inflamProstanlandins matory nature of prostaglandins (see Modes of Action), =El and PGE2 have been shown t o have a n t i a r t h r i t i c roperties when given S . C . to rats a t d a i l y doses of about 1 mg/rat.78,99 Glenn and Rohloff have suggested79 t h a t the a n t i a r t h r i t i c e f f e c t s of the prostaglandins may be r e l a t e d to the concomitant adrenal hyperplasia, prostration, and diarrhea caused by the high pharmacologic doses used.

-

Imrmnosuvpressives I t has been enerally accepted t h a t RA is an example of an imune complex disease,80,8f and so c l i n i c a l t r i a l s of cytotoxic immunosuppressive a e n t s such as chlorarnbucil, 82, 83 azathioprine, 84, cyclophosphamide ( a , B 6 and isophosphamide (25),87 have continued. The r i s k of severe s i d e effects, however, has resulted i n a recommendation88 that these drugs be used only i n closely supervised treatment of p a t i e n t s who a r e unresponsive t o conventional therapy. The p o s s i b i l i t y t h a t the cytotoxic agents may a c t by predominantly antiinflanrmatory rather than immunosuppressive mechanisms has been discussed. 89

Chap.

19

Anti-inflammatory

Agents

Juby, Hudyma

/CH CH C 1 2

2

O=P-#

24

25 -

-

Miscellaneous C l i n i c a l experiences with apazone (26) have been sumaarized.90 Good A 1 a c t i v i t y , with l i t t l e incidence of s i d e e f f e c t s , w a s noted i n p a t i e n t s with a v a r i e t y of inflammatory d i s e a s e s receiving 26 a t 0.4-1.6 glday. Tinoridine HC1 (27) was introduced on the market i n Japan a s an antiinflammatory-analgetic agent. 91 The benzophenone (28) had about twice the potency (CE) of phenylbutazone. 92

Compound 2 proved comparable t o phenylbutazone (CE). g3 Additional r e p o r t s have appeared on the A1 a c t i v i t i e s of diflumidone sodium ( 9 9 4 and t r i f l u m i d a t e ( m 9 5 i n acute and chronic rodent screens.

R

0 + C 6 H II 5 C y y ' 'S02CHF2

c 6 H0 II5 c ~ ~ N S7°2c2H5 02CF.,

c6H5PNHCOCH20C2H5

22

30

31

Benefit has been claimed f o r p a t i e n t s with RA who were t r e a t e d with g-penicillamine (3JJ96 and &-histidine ( m . 9 7 The t r i a z o l e 34 had comparable A1 a c t i v i t y (CE) to phenylbutazone.98 ( a 3 ) 27-fI-C02H SH NH2

r=7/4 CH2F-co2H N NH2

C 2 H s 0 L -NL S H

I

34 32 33 Conanent - Our o v e r a l l impression is t h a t t h e r e have been no r e a l l y e i g n i f i cant advances i n t h e p a s t year, An encouraging sign, however, ie t h e i n -

m2

creasing mount of research which is being d i r e c t e d toward a b e t t e r understanding of the inflananatory processes. We hope t h a t from t h i s work w i l l come the breakthrough so badly needed i n t h i s f i e l d .

21 4 -

Sect.

IV

-

M e t a b o l i c 6 Endocrine

P a c h t e r , Ed.

References 1. 2. 3.

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

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

P. F. Juby and T. W. Hudyma, Annu. Rep. Med. Chem., 1970, 182 (1971). A. G. S. Hill, Ed., "Modern Trends in Rheunatology", Vol. 2, AppletonCentury-Crofts, New York, N. Y., 1971. H. 2. Movat, Ed., "Inflanrmation, Imrmnity and Hypersensitivity", Harper and Row, New York, N. Y., 1971. J. H. Bland, Seminars in Drug Treatment, L( 93 (1971). 263 (1971). B. Unterhalt, Deut. Apoth.-Ztg., &l B. K. Forscher and J. C. Houck, Eds., "Imnunopathology of Inflanmation", Excerpta Medica, Amsterdam, 1971. Arzneim. -Forsch. & 1757 (1971). Abstracts of the 7th European Rheumatology Congress, Brighton, England, June, 1971. M. H. Williams and F. E. Bruckner, Ann. Rheum. Dis., 30. 271 (1971). E. Jansson, P. Gkisara, K. Vainio, U. Vainio, 0. Snellman, and S. Tuuri, 30. 506 (1971). S. L. Warren, L. Mannor, and D. M. Liebes, Arch. Intern. Med., &l 614 (1971). S. L,- Warren, L. Mannor, R. Boak, and D. M. Liebes, 128. 619 (1971). M. Skinner, E. S. Cathcart, J. A. Mills, and R. S. Pinals, Arthritis Rheum., & 727 (1971). R. J. Perper, B. Alvarez, C. Colombo, and H. Schroder, Proc. SOC. Exp. Biol. Med., 137. 506 (1971). D. T. Walz, M. J. DiMartino, and A. Misher, J. Phannacol. Exp. Ther., && 223 (1971). M. Di Rosa and D. A. Willoughby, J. Pham. Phannacol., 23. 297 (1971). A. Hulth, L. Lindberg, and H. Telhag, Acta Orthop. Scand., 41. 522 (19 70). R. W. Moskowftz, W. Davis, J. Samnarco, M. Martens, and S. Chase, Arthritis Rheum., 14( 176 (1971). J. R. Vane, Nature (London), New. Biol., && 232 (1971). J. B. Smith and A. L. Willis, 231. 235 (1971). S. H. Fcrreira, S. Moncada, and J. R, Vane, 231. 237 (1971). R. V. Tomlinson, H. J. Ringold, M. C. Qureshi, and E. Forchielli, Biochem. Biophys. Res. Comnun., 46. 552 (1972). A. L. Willis, Pharmacol. Res. Ccnmnun., 5 297 (1970). G. Kaley and R. Weiner, Ann. N. Y. Acad. Sci., J 8 l 338 (1971). G. Kaley and R. Weiner, Nature (London), New Biol., &2 114 (1971). P. Crunkhorn and A. L. Willis, Brit. J. Phannacol., 41. 49 (1971). M. W. Greaves, J. Sdndergaard, and W. McDonald-Gibson, Brit. Med. J. 2, 258 (1971). A. J. Anderson, W. E. Brocklehurst, and A. L. Willis, Phannacol. Res. Canmun., 2( 13 (1971). J. N. McArthur, P. D. Dawkins, M. J. H. Smith, and E. B. D. Hamilton, Brit. Med. J., 2, 677 (1971). 2. N. Gaut and H. M. Solomon, J. Pharm. Sci., 60. 1887 (1971). L. J. Ignarro, Biochem. Fharmacol., 20. 2847 (1971). L. J. Ignarro, 20. 2861 (1971). J. R. Culp, E. G. Erdos, L. B. Hinshaw, and D. D. Holmes, Proc. SOC.

,

w.,

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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.

. 19

-

A n t i in f 1 ammatory Agents

Juby, Hudyma

21 5 -

Exp. Biol. Med., 137. 219 (1971). H. I. Jacoby and C. H. Marshall, Nature (London), 235. 163 (1972). A. Bennett, 231. 536 (1971). T. M. Glenn, G. T. Raflo, S. L. Wangensteen, and A. M. Lefer, Pharmacologist, 13. 293 (1971). Drug Research Reports, (25), S-2 (1971). 28 (1972). F. D. Hart and E. C. Huskisson, Lancet, R. DesprogesGotteron, V. Leroy, and B. Comte, Curr. Ther. Res., Clin. Exp., &I 393 (1971). C. Klemm, R. Fricke, M. Schattenkirchner, W. Treiber, and H. Mathies, 2. Rheumaforsch., 30. 17 (1971). K. Pavelka, D. KaFikova', F. Wagenhzuser, and B k i , _ibid., 30. 50 (1971). A. A. J. Goldberg, J. E. Hall, J. W. Buckler, P. G. Dodsworth, and J. Agar, Practitioner, 207. 343 (1971). E. C. Huskisson, F. D. Hart, G. M. Shenfield, and R. T. Taylor, 207. 639 (1971). R. C. Nickander, R. J. Kraay, and W. S. Marshall, Fed. Proc., Fed. her. SOC. Exp. Biol., 30. 563 (1971). 30. 564 (1971). H. W. Culp, A. S. Ridolfo, C. M. Gruber, and W. M. Mikulascheck, Clin. Pharmacol. Ther., 12. 300 (1971). L. Julou, J. 4.Guyonnet, R. Ducrot, C. Garret, M. -C. Bardone, G. Maignan, and J. Pasquet, J. Phannacol., 2, 259 (1971). A. P. Roszkowski, W. H. Rooks 11, A. J. Tomolonis, and L. M. Miller, J. Pharmacol. Exp. Ther., 179. 114 (1971). W. H. Rooks 11, Fed. Proc., Fed. Amer. SOC. Exp. Biol., 30, 386 (1971). A. Lussier, K. L. MacCannell, S. J. Alexander, C. V. Multz, G. Boost, and E. J. Segre, Clin. Phannacol. Ther., 13. 146 (1972). J. R. Carson, D. N. McKinstry, and S. Wong, J. Med. Chem., 14. 646 (1971). A. Y. Green and D. Green, Brit. 3. Pharmacol., 4 3 638P (1971). 0. Rohte, Acta Pharmacol. Toxicol., 2 (Suppl. 4), 47 (1971). K. Kawai, S. Kuzuna, S. Morimoto, H. Ishii, and N. Matsumoto, Jap. J. Phannacol., 621 (1971). S. Noguchi, S. Kishimoto, I. Minamida, M. Obayashi, and K. Kawakita, Chem. Pharm. Bull., 646 (1971). P. F. Juby, T. W. Hudyma, and R. A. Partyka, J. Med. Chem., 15. 120 (1972). M. Vincent, G. Remond, and J. -C. Poignant, 15. 75 (1972). J. Redel, H. Brouilhet, F. Delbarre, N. Bazely, and M. Jouanneau, C. R. Acad. Sci., Ser. D, 273, 911 (1971). D. Gross, Praxis, 60. 1334 (1971). C. Casagrande, A. Invernizzi, R. Ferrini, and G. Miragoli, Farmaco, Ed. Sci., 26. 1059 (1971). G. Zani, M. L. Molinari, and S. Banfi, 26. 191 (1971). G. Zoni, M. L. Molinari, and S. Banfi, 26. 525 (1971). A. S. Watnick, R. I. Taber, and I. I. A. Tabachnick, Arch. I n t . Phannacodyn. Ther., 190. 78 (1971). J. S. Finch and T. J. DeKornfeld, J. Clin. Pharmacol. New Drugs,. 371 (1971). E. Colinet, Famaey, and Bauduin, J. Belge Rheumatol. Med. Phys., 26.

u.,

w.,

u.,

w.,

w.,

u.,

21 6 -

Sect.

IV

- Metabolic E

Endocrine

Pachter, Ed.

21 (1971). 66. C. V. Multz, G. C. Bernhard, W. J. Blechman, S. Zane, and R. A. Restifo, Arthritis Rheum., 14. 406 (1971). 67. K. Pavelka, R. Wagenhzuser, and R. Geiregat, 2. Rheumaforsch., 30, 45 (1971). 68. D. I. Haslock, P. A. Nicholson, and V. Wright, Clin. Trials J., 5 43 (1971). 69. L. H. Sarett, Arzneim. -Forsch., 21. 1759 (1971). 70. J. G. Lombardino, E. H. Wiseman, and W. M. McLamore, J. Med. Chem., 14. 1171 (1971). 14. 1175 (1971). 71. J. Chiaini, E. H. Wiseman, and J. G. Lombardino, 72. E. H. Wiseman and J. Chiaini, Pharmacologist, 13. 284 (1971). 73. J. G. Lombardino and E. H. Wiseman, J. Med. Chem., 14. 973 (1971). 74. A. R. Myers, Mod. Treat., 12, 761 (1971). 75. D. T. Walz, M. DiMartino, B. Sutton, and A. Misher, Fed. Proc., Fed. her. SOC. Exp. Biol., 30. 386 (1971). 76. D. T. Walz, M. J. DiMartino, J. Flagiello, Jr., S. Morgan, and S. Davidheiser, Pharmacologist, &I 237 (1971). 77. H. W. Baenkler and F. Scheiffarth, Z. Rheumaforsch., 30. 236 (1971). 78. R. B. Zurier and F. Quagliata, Nature (London), 234. 304 (1971). 79. E. M. Glenn and N. Rohloff, Proc. SOC. Exp. Biol. Med., 139. 290 (1972). 80. N. J. Zvaifler, J. Exp. Med., 134. 276 S (1971). 81. W. H. Dodson, Mod. Treat., 5 778 (1971). 82. D. Bontoux, A. Kahan, H. Brouilhet, B. Amor, F. Delbarre, M. Jouanneau, and E. Gniazdowska, Rev. Eur. Etud. Clin. Biol., 16. 166 (1971). 83. P. Thorpe, J. Hassall, and J. York, Med. J. Aust., 2, 796 (1971). 84. J. Harris, J. D. Jessop, and D. M. Chaput de Saintonge, Brit. Med. J., 4, 463 (1971). 85. M. B. Urowitz, D. A. Gordon, H. A. Smythe, W. Pruzanski, and M. A. Ogryzlo, Arthritis Rheum., 14. 419 (1971). 86. N. V. Jarlqh and K. Sgrensen, Ugeskr. Laeg., 133. 587 (1971). 87. W. Kunz, R. Rau, and A. Ski, Schweiz. Med. Wochenschr., 101. 1151 (1971). 88. R. S. Schwartz and J. D. C. Gowans, Arthritis Rheum., 14. 134 (1971). 879 (1971). 89. H. L. F. Currey, Clin. Exp. Imunol., 90. li. Sausgruber, Arzneim. -Forsch., &2 1230 (1971). 91. Jap. Med. Gaz., S (81, 7 (1971). 92. R. Coornbs, W. J. Houlihan, J. Nadelson, and E. I. Takesue, J. Med. Chem., 14. 1072 (1971). 93. D. Artini, A. Buttinoni, E. Dradi, W. Logemann, V. Mandelli, P. Melloni, R. Tonmasini, G. Tosolini, and G. Vita, Arzneim. -Forsch., 2L( 30 (1971). 94. K. F. Swingle, R. R. Hamilton, J. K. Harrington, and D. C. Kvam, Arch. Int. Phannacodyn. Ther., &I 129 (1971). 95. K. F. Swingle, J. K. Harrington, R. R. Hamilton, and D. C. Kvam, *-ibid 9 192. 16 (1971). 1815 (1971). 96. K. Miehlke, Arzneim. -Forsch., &2 . ( 4 ) , 18 (1971). 97. D. A. Gerber, Drug Therapy, l 98. T. George, D. V. Mehta, R. Tahilramni, J. David, and P. K. Talwalker, J. Med. Chem., & 335 (1971).

u.,

Section V

-

Topics i n Biology

E d i t o r : W i l l i a m J. Wechter, The Upjohn Company, Kalamazoo, Michigan Chapter 20.

Mechanisms of Resistance t o A n t i b i o t i c s

J u l i a n Davies, Department of Biochemistry, University of Wisconsin Madison , Wisconsin Introduction - The search f o r new antimicrobial agents, e i t h e r from natur a l sources or by t h e chemical or biochemical modification of e x i s t i n g compounds i s predicated by t h e d e s i r e t o o b t a i n agents with a ) broad s p e c t r a of b a c t e r i a l e f f e c t i v e n e s s , b ) low t o x i c i t y , c ) a c t i v i t y a g a i n s t n a t u r a l l y occurring r e s i s t a n t organisms. Thousands of a n t i b i o t i c derivat i v e s have been prepared using numerous approaches t o s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s , which are s t i l l l a r g e l y empirical. Comparatively f e w a n t i b i o t i c s have been modified s u c c e s s f u l l y as t h e r e s u l t of r a t i o n a l biochemical p r e d i c t i o n s . I t i s now clear t h a t n a t u r a l r e s i s t a n c e mechanisms, f o r a l l t h e i r c l i n i c a l problems, do provide some c l u e s about s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s and one can d e r i v e u s e f u l conclusions about funct i o n a l groups important t o b i o l o g i c a l a c t i v i t y . The occurrence of r e s i s t a n c e s t r a i n s - I t i s easy t o generate b a c t e r i a l mutants which are r e s i s t a n t t o a n t i b i o t i c s ; biochemical s t u d i e s of such mutants have provided much information on c e l l s t r u c t u r e and metabolism1. These mutants arise by s i n g l e or multiple-changes i n chromosomes and t h e p r o p e r t i e s are i n h e r i t e d i n Mendelian fashion. However, t h e r e s i s t a n t s t r a i n s which are i s o l a t e d from n a t u r a l sources (during a n t i b i o t i c therapy i n humans and animals r a r e l y correspond t o t h e types mentioned above2 s3. C l i n i c a l i s o l a t e s of both gram-positive and gram-negative b a c t e r i a are often r e s i s t a n t t o unrelated groups of a n t i b i o t i c s , are not produced by mut a t i o n of t h e parent s e n s i t i v e s t r a i n s , and t h e r e s i s t a n c e c h a r a c t e r s are i n h e r i t e d i n non-Mendelian fashion4. The r e s i s t a n t s t r a i n s c a r r y blocks of genes which determine r e s i s t a n c e t o a v a r i e t y of a n t i b i o t i c s ; t h e s e blocks of genes can be t r a n s f e r r e d from s t r a i n t o s t r a i n by t h e processes of conjugation or transduction. The complete extrachromosomal u n i t is u s u a l l y r e f e r r e d t o as a r e s i s t a n c e f a c t o r (R-factor) i n gram-negative organisms and as a plasmid i n gram-positive organisms. I t i s most l i k e l y t h a t t h e s e r e s i s t a n c e mechanisms existed within t h e b a c t e r i a l population before t h e advent of a n t i b i o t i c s , and were s e l e c t e d when a n t i b i o t i c s were used i n quantity5. Since "naturally-occurring" r e s i s t a n c e is of g r e a t e s t s i g n i f i cance i n terms of t h e medical uses of a n t i m i c r o b i a l agents t h i s review w i l l concentrate on t h i s form of r e s i s t a n c e .

General mechanisms of r e s i s t a n c e - In 1 9 5 2 , Davis and Maas discussed t h e ways by which an organism might become r e s i s t a n t t o an a n t i b i o t i c 6 . These mechanisms are outlined i n Table 1, together with examples of each type.

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Table 1 Biochemical mechanisms of drug r e s i s t a n c e Examples Mechanism I n a c t i v a t i o n of drug by bond cleavage or modification

Modification of drugsensitive site

Loss of permeability t o drug Increased l e v e l s of enzyme i n h i b i t e d by drug

Clinical" Isolates R-factor or plasmid determined i n a c t i v a t i o n of B-lactam or aminoglycoside a n t i b i o t i c s

Labor a t ory it Jc Isolates

?

erythromycin r e si s tance i n - 5 . aureus ( a l t e r e d ribosomes

streptomycin, spectinomycin r e s i s t a n c e ( a l t e r e d ribosomes)

sulfonamide r e s i s t a n c e i n Pneumococci ( a l t e r e d enzyme )

rifamycin ( a l t e r e d RNA polymerase sulfonamide r e s i s t a n c e i n Pneumococci.

R-factor determined resistance t o tetrac y c l i n e or sulfonamides

r e s i s t a n t s t r a i n s produced by s e r i a l transfer ? 5-methyltryptophan r e s i s t a n c e , Psicofuranine r e s i s t a n c e

New biochemical pathway which bypasses i n hibited reaction Increased concentrat i o n of a metabolite t h a t antagonizes t h e inhibitor Decreased r e q u i r e ment f o r product of inhibited reaction

sulfonamide r e s i s t a n c e ?

s u l f onamide r e s i s t a n c e ?

J(

refers t o mechanisms of r e s i s t a n c e i n b a c t e r i a which appear during t h e r a p e u t i c use of an a n t i b i o t i c .

itit

refers t o mechanisms of r e s i s t a n c e i n b a c t e r i a l s t r a i n s i s o l a t e d under laboratory conditions. e.g. d e l i b e r a t e s e l e c t i o n of a n t i b i o t i c resistance.

A n t i b i o t i c Resistance

Chap. 20

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The mechanisms favoured by " n a t u r a l l y - r e s i s t a n t " organisms involve drug i n a c t i v a t i o n ( e i t h e r by bond cleavage o r d e r i v a t i z a t i o n ) , an a l t e r e d drug receptor s i t e , o r reduced permeability t o t h e drug. Studies of laboratory-derived ( s i n g l e - s t e p ) r e s i s t a n t mutants have proved t o be of considerable biochemical u t i l i t y . For example, s t r e p t o mycin-resistant mutants have an a l t e r e d receptor s i t e due t o a change i n a p r o t e i n of t h e small ribosomal subunit, while erythromycin-resistant mutants have a change i n a p r o t e i n of t h e l a r g e ribosomal subunit7. Altered RNA polymerases are found i n mutants resistant t o rifampicin and s t r e p t o l ydigin8,g. Fusidic a c i d r e s i s t a n c e occurs as a r e s u l t of mutations which a l t e r a f a c t o r required i n p r o t e i n s y n t h e s i s l o . Such forms of a n t i b i o t i c r e s i s t a n c e have been u s e f u l i n determining t h e mode of a c t i o n of t h e a n t i b i o t i c and i n s t u d i e s of macromolecular s t r u c t u r e and function. By cont r a s t , i n "natural" resistance, t h e r e s i s t a n c e mechanism i s , i n most cases, unrelated t o t h e biochemical mode of a c t i o n of t h e drug. The a n t i b i o t i c s with known plasmid-determined r e s i s t a n c e are l i s t e d i n Table 2. Table 2 R-Factgr o r plasmid-determined resistance t o a n t i m i c r o b i a l agents tetracycline chloramphenicol s u l f onamides erythromycin lincomycin penicillins cephalosporins 3%

*

neomycin kanamycin st r ep t omyc i n spectinomycin gent amicin tobramycin ? f u s i d i c acid

Plasmid-determined r e s i s t a n c e has not been found t o n a l i d i x i c a c i d , n i t r o f u r a n s , polymixin, b a c i t r a c i n , o r rif ampicin.

S p e c i f i c mechanisms of r e s i s t a n c e A. Sulfonamide resistance is widespread among c l i n i c a l i s o l a t e s of bacter i a l s t r a i n s , and it was t h e appearance of sulfonamide resistance i n Japan i n t h e 1950's, which provided t h e f i r s t example of an "epidemic" of resist a n c e l l . Within 5 years a f t e r t h e i n t r o d u c t i o n of sulfonamides f o r t h e treatment of b a c i l l a r y dysentery, 80-90% of t h e S h i g e l l a d y s e n t e r i a e isolates were found t o be r e s i s t a n t t o t h e s e drugs. A l a r g e proportion of t h e s e s t r a i n s was found t o be resistant t o o t h e r unrelated a n t i b i o t i c s such as streptomycin and chloramphenicol, and g e n e t i c s t u d i e s l e d t o t h e discovery of t r a n s m i s s i b l e drug r e s i s t a n c e characterized by t h e presence of R-factors o r plasmids. R-factor o r plasmid-determined sulfonamide res i s t a n c e i s now common among b a c t e r i a l pathogens. The biochemical mechanism of sulfonamide resistance in t h e s e s t r a i n s has been l i t t l e s t u d i e d ; it i s c u r r e n t l y accepted t h a t t h e R+ s t r a i n s ( s t r a i n carrying t h e R-factor) have a decreased permeability f o r t h e drug, although t h e biochemical evidence f o r such a mechanism is f a r from convincingll. Not a l l sulfonamide r e s i s t a n c e i s determined by R-factors o r

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plasmids. Genetic a l t e r a t i o n s i n t h e dihydropteroic acid-forming enzymes have been found i n some r e s i s t a n t s t r a i n s of Pneumococci12; t h e sulfonamide drugs do not act as competitive i n h i b i t o r s of t e t r a h y d r o f o l a t e format i o n i n such s t r a i n s . B. Tetracycline r e s i s t a n c e i s probably t h e most common form of R-factordetermined r e s i s t a n c e . As far as can be ascertained a l l t e t r a c y c l i n e r e s i s t a n t s t r a i n s obtained from c l i n i c a l s i t u a t i o n s are r e s i s t a n t because they c a r r y an R-factor or a plasmid. I t has been concluded t h a t t e t r a c y c l i n e r e s i s t a n t s t r a i n s are impermeable t o t h e drug, although t h e evidence is not convincing13. Biochemical s t u d i e s i n d i c a t e t h a t t e t r a c y c l i n e i s a c t i v e l y transported i n t o s e n s i t i v e c e l l s which implies t h a t r e s i s t a n c e t o t h e drug is due t o i n a c t i v a t i o n of a s p e c i f i c t r a n s p o r t system for t e t r a cycline. ( I t seems s t r a n g e t h a t b a c t e r i a would want t o take up such a drug!) Rt s t r a i n s possess a b a s a l l e v e l of r e s i s t a n c e t o t e t r a c y c l i n e which is enhanced when t h e c e l l s are grown i n t h e presence of t h e drug; some evidence f o r an inducible genetic system has been provided but det a i l e d g e n e t i c and biochemical a n a l y s i s w i l l be necessary t o e s t a b l i s h t h i s mechanism14. The t e t r a c y c l i n e s have been used extensively f o r a g r i c u l t u r a l purposes as animal feed a d d i t i v e s ; however, t h e i r use has been discontinued i n England on evidence of t h e r e p o r t of t h e Swann committeel5. This recommendation was based on findings t h a t t h e b a c t e r i a l f l o r a of animals fed t e t r a c y c l i n e s (and o t h e r a n t i b i o t i c s ) on a continuous b a s i s contained a high proportion of s t r a i n s carrying R-factors. Such animals c o n s t i t u t e a r e s e r v o i r of resistant s t r a i n s which may be passed t o human s u b j e c t s and possibly transmit t h e i r r e s i s t a n c e c h a r a c t e r s t o a n t i b i o t i c s e n s i t i v e pathogens. There i s no d i r e c t evidence t h a t such t r a n s f e r occurs, but t h e r e i s l i t t l e doubt t h a t t h e extensive use of a n t i b i o t i c s s e l e c t s f o r an a n t i b i o t i c - r e s i s t a n t population of b a c t e r i a among c a t t l e , p o u l t r swine and f i s h which have been maintained on a n t i b i o t i c feed a d d i t i v e s

16 .

There i s s t r o n g genetic and biochemical evidence t h a t t h e gene f o r t e t r a c y c l i n e r e s i s t a n c e is c l o s e l y linked t o t h e r e s i s t a n c e t r a n s f e r fact o r (RTF, t h e portion of t h e R-factor which determines t r a n s m i s s i b i l i t y ) . Indeed, when t h e R-factor d i s s o c i a t e s , t h e gene f o r t e t r a c y c l i n e r e s i s t a n c e a s s o c i a t e s with t h e RTF and not t h e r - d e t e r m i n a n t ~ l ~ . This would imply t h a t s e l e c t i o n f o r t e t r a c y c l i n e r e s i s t a n c e must automatically s e l e c t f o r t h e a b i l i t y t o t r a n s f e r . If t r a n s f e r a b i l i t y i s considered t o play an i m portant r o l e i n t h e development of a n t i b i o t i c r e s i s t a n c e i n human pathogens, t h e use of t e t r a c y c l i n e must c e r t a i n l y be r e s t r i c t e d . However, t h e present s t a t e of our knowledge cannot allow us t o say whether transmissib i l i t y i s a f a c t o r i n t h e increase i n occurrence of a n t i b i o t i c - r e s i s t a n t pathogens. Cross-resistance t o a l l t h e t e t r a c y c l i n e a n t i b i o t i c s i s common; one possible exception i s minocycline, a semisynthetic t e t r a c y c l i n e which has some a c t i v i t y a g a i n s t t e t r a c y c l i n e r e s i s t a n t s t r a i n s l 8 . C. Resistance to.+lactam a n t i b i o t i c s ( p e n i c i l 1 i n s and CeDha~osDorins)has been extensively s t u d i e d , since it has s i g n i f i c a n t l y d i c t a t e d t h e applicat i o n and use of a n t i b i o t i c s of t h i s class. With one notable exception ( m e t h i c i l l i n r e s i s t a n c e i n Staphylococcus) r e s i s t a n c e t o t h e s e a n t i b i o t i c s

Chap. 20

A n t i b i o t i c Resistance

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22 1 -

i s due t o t h e production of a 6-lactamse ( p e n i c i l l i n a s e ) which i n a c t i v a t e s t h e a n t i b i o t i c s by opening t h e B-lactam r i n g l g . There appear t o be a number of B-lactamases which show a broad spectrum of a c t i v i t y on t h e B-lactam a n t i b i o t i c s i n c l i n i c a l use; none of t h e s e a n t i b i o t i c s are comp l e t e l y i n e r t 2 0 . Resistance t o t h e B-lactams i n Staphylococcus i s d e t e r mined by a plasmid; s u b s t a n t i a l g e n e t i c and biochemical d a t a have been accumulated i n t h i s system, notably by Novick21, and by Richmond22, and t h e i r c o l l a b o r a t o r s . B-Lactamase production i n Staphylococcus i s induci b l e ( r e q u i r e s t h e presence of a n t i b i o t i c f o r expression) and very low ( s u b i n h i b i t o r y ) concentrations of a @-lactam a n t i b i o t i c are s u f f i c i e n t t o induce maximal expression of t h e gene(s) f o r t h e B-lactamase.

The problem of r e s i s t a n c e t o t h e B-lactam a n t i b i o t i c s was o r i g i n a l l y r e s t r i c t e d t o gram-positive s t r a i n s . The discovery and use of B-lactam a n t i b i o t i c s with a c t i v i t y a g a i n s t gram-negative s t r a i n s ( a m p i c i l l i n , carb e n i c i l l i n , cephalosporins) has l e d t o t h e appearance of s t r a i n s o f E . c o l-i , P. aeruginosa and K. pneumoniae, etc. which are r e s i s t a n t t o -tre B-lactam a n t i b i o t i c s . In almost a l l gram-negative s t r a i n s , r e s i s t a n c e is due t o t h e R-factor determined production of a B - l a ~ t a m a s e ~ ~Unlike . t h e 8-lactamase i n gram-positive s t r a i n s t h e enzyme i s produced c o n s t i t u t i v e l y (does not r e q u i r e t h e presence of a n t i b i o t i c t o induce formation of t h e enzyme). In Staphylococcus t h e f3-lactamase i s released i n t o t h e medium, but t h e f3-lactamase of e n t e r i c b a c t e r i a appears t o belong t o t h e class of p e r i plasmic enzymes, which a r e s i t u a t e d between t h e c e l l wall and t h e c e l l memixane24. The existence of c a r b e n i c i l l i n - r e s i s t a n t s t r a i n s of Pseudomonas aeruginosa, although not widespread, is of considerable concern i n burn c l i n i c s s i n c e c a r b e n i c i l l i n has proved t o be an e f f e c t i v e agent i n t h e treatment of Pseudomonas i n f e c t i b n s . Studies have shown t h a t drug resistance i s t r a n s f e r a b l e (R-factor-determined) i n manv of t h e s e instances and t h e r e is c i r c u m s t a n t i a l evidence t h a t t h e Pseudomonas s t r a i n s acquired t h i s r e s i s t a n c e by accepting R-factors from s t r a i n s of Proteus or K l e b s i e l l a which w e r e present i n t h e f l o r a of burns25. Lowbury and coworkers have demonstrated R-factor t r a n s f e r i n infected burns i n mice; t h i s i s t h e f i r s t evidence which emphasizes t h e importance of both t r a n s f e r and s e l e c t i o n i n t h e development of resistance26. Not a l l s t r a i n s of Pseudomonas c a r r y R-factors, and an inducible 6-lactamase ( s i m i l a r t o t h a t i n gram-positives?) has been reported i n some i s o l a t e s . The B-lactamase which is R-factor determined and which i s responsible f o r c a r b e n i c i l l i n r e s i s t a n c e i n burn c l i n i c s i s , l i k e o t h e r R-factor-determined enzymes, produced c o n ~ t i t u t i v e l y ~ ~ . Large numbers of p e n i c i l l i n and cephalosporin a n t i b i o t i c s have been made i n e f f o r t s to.expand t h e spectrum of t h e s e drugs and t o produce a n t i b i o t i c s r e s i s t a n t t o t h e a c t i o n of 6-lactamases. It seems u n l i k e l y t h a t a B-lactam a n t i b i o t i c which i s completely r e f r a c t o r y t o t h e a c t i o n of B-lactamases w i l l be found. This pessimism is based on t h e following argument: t h e B-lactam r i n g is c r i t i c a l t o a n t i b i o t i c a c t i v i t y and cannot

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Substances introduced adjacent t o t h e 8-lactam r i n g can be modified. change t h e effectiveness of t h e molecule as a s u b s t r a t e f o r 8-lactamase, but cannot completely eliminate t h i s a c t i v i t y . The range of known 8-lactamases makes it probable t h a t any modified 8-lactam is going t o be a substrate f o r one of t h e enzymes present i n nature and t h e subsequent use of t h i s a n t i b i o t i c w i l l s e l e c t f o r r e s i s t a n t s t r a i n s producing t h e most e f f e c t i v e enzyme, Methicillin-resistance i n Staphylococcus aureus is not associated with 8-lactamase production. I t seems most l i k e l y , i n view of t h e r a t h e r unusual p r o p e r t i e s of t h e s e s t r a i n s (co-resistance t o cephalos p o r i n ) , t h a t r e s i s t a n c e is due t o impermeability t o t h e a n t i b i o t i c 2 8 .

Chloramphenicol r e s i s t a n c e has been extensively studied by S h a ~ ~ ~ , and by Okamoto and Suzukiau, and is due t o i n a c t i v a t i o n of t h e drug by O - a c e t y l a t i ~ ni~n~e n t e r i c b a c t e r i a and Staphylococcus. The enzyme, chloramphenicol a c e t y l t r a n s f e r a s e is determined by an R-factor i n t h e Enterobacteriacae and by a plasmid i n Staphylococcus; it is produced cons t i t u t i v e l y i n E. c o l i and i t s production is induced by chloramphenicol i n Staphylococcus a u r a . Chloramphenicol usage i s t i g h t l y c o n t r o l l e d and it i s s u r p r i s i n g , perhaps, t h a t a l a r g e proportion of multiply r e s i s t a n t organisms i s o l a t e d from n a t u r e a r e chloramphenicol r e s i s t a n t . U n t i l rec e n t l y , no o t h e r t y p e of chloramphenicol r e s i s t a n c e had been recognized although Nagai and Mitsuhashi have reported t h e i s o l a t i o n of a number of chloramphenicol-resistant R+ s t r a i n s which seem t o be impermeable t o t h e antibiotic32.

D.

E. Aminoglycoside a n t i b i o t i c s have s e v e r a l well-characterized modes of enzymatic i n a c t i v a t i o n and a l l c l i n i c a l i s o l a t e s of b a c t e r i a r e s i s t a n t t o t h e s e a n t i b i o t i c s have been shown t o possess t h e s e i n a c t i v a t i o n mechan i s m ~ ~Some ~ . seven d i f f e r e n t enzymes have been characterized which ina c t i v a t e one o r more of t h e aminoglycoside a n t i b i o t i c s (Table 3 ) . Streptomycin (and spectinomycin) adenylylation was apparently t h e f i r s t mechanism t o be recognized ( s e l e c t e d ) and most multiply r e s i s t a n t s t r a i n s , i s o l a t e d after 1956, c a r r y t h i s form of r e s i s t a n c e . This r e s i s t a n c e determinant has been detected i n s t r a i n s lyophilized i n 1945, which suggests t h a t R-factors were present before t h e extensive c l i n i c a l use of a n t i b i o t i c ~ ~Shortly . after t h e introduction o f kanamycin as a broad-spectrum a n t i b i o t i c , a number of s t r a i n s carrying neomycin and kanamycin r e s i s t a n c e were isolated34. Such r e s i s t a n c e is now q u i t e widespread and i n a c t i v a t i o n of neomycin and kanamycin by phosphorylation is a common form of aminoE. c ol i , P. aeruginosa and K. pneumoniae. I n a c t i glycoside r e s i s t a n c e i n vation of kanamycin by a c e t y l a t i o n of t h e 6-amino group of t h e glucosamine r i n g has been demonstrated i n some s t r a i n s . This r e s i s t a n c e mechanism gives a low l e v e l of r e s i s t a n c e and is rare among c l i n i c a l i s o l a t e s .

Gentamicin, t h e newest o f . t h e aminoglycoside a n t i b i o t i c s t o be used t h e r a p e u t i c a l l y , has proved t o be a successful drug35; f o r one r e a s o n , it is e f f e c t i v e a g a i n s t neomycin and kanamycin-resistant s t r a i n s , such as many Pseudomonads. However, two forms of gentamicin i n a c t i v a t i o n have been found i n gentamicin-resistant s t r a i n s i s o l a t e d i n h o s p i t a l s and c l i n i c s using t h e drug.

Chap. 20

Ant i b io t ic streptomycin phosphotrans f e r a s e

A n t i b i o t i c Resistance

Dav i es

Table 3 Inactivation Substrates Mechanism phosphorylation streptomycin, of L-glucosamine ring

223

Resistance Spectrum streptomycin

streptomycin

adenylylation of L- gluc 0 s amine ring

streptomycin, spectinomycin ,

streptomycin spectinomycin

kanamycin acetyltransferase

a c e t y l a t i o n of D-glucosamine ring

kanamycin A , B, neomycin, gentamicin

kanamycin A, (low l e v e l resistance t o other s u b s t r a t e s)

neomycin-kanamycin phosphotransferase I

phosphorylation of D-glucosamine ring

neomycin, kanamycin, paromomycin

neomycin , kanamycin , paromomycin

neomycin-kanamycin phosphotransferase I1

phosphorylation of D-glucosamine ring

neomycin, kanamycin, paromomycin, ambutyrosine

neomycin, kanamycin , paromomycin , ambutyrosine

gentamicin adenylate synthetase

adenylylation of garosamine r i n g

gentamicin , kanamycin

gentamicin, kanamycin ,tobramycin

gentamicin a c e t y l t r a n s f e r as e

a c e t y l a t i o n of deoxystreptamine ring

gentamicin C

gentamicin C

Gentamicin r e s i s t a n t s t r a i n s of E. c o l i and K l e b s i e l l a pneumoniae have been reported i n h o s p i t a l s i n t h e U.S.A.36,3/ and France38. The a n t i b i o t i c i s i n a c t i v a t e d by adenylylation; kanamycin and tobramycin can be i n a c t i v a t e d by t h e same mechanism39. The enzyme , gentamicin adenylyl a t e synthetase, i s determined by an R-factor gene. S t r u c t u r a l s t u d i e s with dideoxykanamycin which had been i n a c t i v a t e d by t h i s enzyme have shown t h a t t h e adenyl r e s i d u e is on t h e 2”OH of t h e kanosamine ring40. I n vivo t r a n s f e r of t h e R-factor t o s p e c i e s o t h e r than E. c o l i and K l e b s i e l l a has not been reported. This adenylylating enzyme has provided t h e b a s i s f o r a r a p i d and s e n s i t i v e assay f o r gentamicin i n b i o l o g i c a l f l ~ i d s ~ l ~ ~ ~ .

--

Gentamicin r e s i s t a n c e i n Pseudomonas aeruginosa occurs as a r e s u l t of a reaction a c e t y l a t i o n of t h e 3-amino group of t h e deoxystreptamine r i n which seems t o be s p e c i f i c t o t h e gentamicin C a n t i b i o t i c s 4f:44. This i s t h e f i r s t r e p o r t of modification of t h e deoxystreptamine moiety i n any a n t i b i o t i c , which is somewhat s u r p r i s i n g since t h i s sugar i s common t o t h e aminoglycoside a n t i b i o t i c s . This r e s i s t a n c e mechanism has not been found i n s t r a i n s o t h e r than P. aeruginosa and it has not been demonstrated t o be transmissible. By c o n g a s t , c a r b e n i c i l l i n r e s i s t a n c e , and neomycinkanamycin r e s i s t a n c e i n P. aeruginosa can be conjugally t r a n s f e r r e d t o E. c o l i .

--

224 -

Sect. V

- Topics

i n Biology

Wechter, Ed.

An attractive feature of gentamicin and tobramycin45 (3-deoxykanamycin B) is that they are structurally incapable of inactivation by the ubiquitous neomycin-kanamycin phosphorylating enzymes, which modify the 3'-OH group of the D-glucosamine ring. The presence of these enzymes is probably the reason why most clinical isolates of Pseudomonas aeruginosa are resistant to neomycin-kanamycin. Gentamicin and tobramycin contain a 3'-deoxy sugar that lacks the site modified by the phosphorylating enzymes; as an additional bonus they can inhibit the phosphorylation of other antibiotic^^^. The occurrence of such aminoglycoside antibiotics has indicated the possibility of structural modifications of aminoglycosides with the group modified by the inactivation removed without reducing the potency of the antibiotic. The aminoglycosides offer great potential in this respect and may have an advantage over the $-lactam antibiotics, where the site of enzymatic inactivation cannot be removed. The production of synthetic and semisynthetic aminoglycoside antibiotics may allow one to keep ahead of nature's ability to produce inactivating enzymes. Marked success has been reported by Umezawa and his co-workers47 who have synthesized 3,4-dideoxykanamycin B, a close derivative of gentamicin and tobramycin, which is effective against a number of kanamycin resistant organisms including Pseudomonas. It is to be hoped that similar developments in this area will be forthcoming. Would an aminoglycoside still be an antibiotic, when all of its enzymatically modifiable functions are removed? -.F Macrolide and lincosamide resistance in Staphylococcus is the only naturally-occurring resistance mechanism which appears to involve an alteration in the target site of the antibiotic, rather than a change in the antibiotic itself. Considering erythromycin and lincomycin as typical antibiotics, two types of resistance can be considered: a) inducible resistance, in which the presence of sub-inhibitory concentrations of erythromycin can induce resistance to both erythromycin and lincomycin and b) constitutive resistance, in which strains are resistant to both classes of drug without requiring previous exposure to erythromycin o r lincomycin. Lincomycin resistance without concomitant erythromycin resistance is rare in clinical isolates. The work of Weisblum48, and M i t s ~ h a s h i ~and ~ , their colleagues has shown that inducible and constitutive erythromycin resistance result from a change in the ribosome, such that the drugs are incapable of binding at their target site. Recent work by Lai and Weisblum50 has shown that erythromycin resistant strains of S. -aureus contain an enzyme which methylates specific sites on the RNA of the 50s ribosomal subunit. Ribosomes containing the unmethylated RNA can bind the antibiotic, but ribosomes with methylated RNA are refractory to the antibiotic. Erythromycin resistance in-Streptococcus pyogenes may also be the result of methylation of ribosomal RNAsl. The evidence suggests that erythromycin-lincomycin resistance in Staphylococcus is determined by plasmid genes52.

Conclusion - Mechanisms of resistance to the common, clinically-used antibiotics are summarized in Table 4.

Chap. 20

A n t i b i o t i c Resistance

Dav i es

Table 4 Mechanisms of r e s i s t a n c e i n b a c t e r i a Antimicrobial Agent Penicillins

1

B-lactamase

r i n g cleavage

kinase

phosphorylation

Spectinomycin

adenylyltransferase

adenylylation

Kanamycin

acetyltransferase

a c e t y l a t ion

kinase

phosphorylation

Gentamicin Gentamicin Kanamycin

acetyltransferase

acetylation

adenylyltransf eras e

adenylylation

Chlmamphenicol

acetyltransferase

acetylation

Ebythromycin Lincomycin

methylas e

methylation of ribosomal RNA

Cephalos por i n s Streptomycin Streptomycin

*

Kanamycin Neomycin Paramomycin

A Acetylation of neomycin and gentamicin a l s o occur, but t h e s e a n t i b i o t i c s are not completely i n a c t i v a t e d by t h i s form of modification.

The i n f e c t i o u s d i s e a s e s c l i n i c i a n wants a n t i b i o t i c s of broad spectrum arid low t o x i c i t y . Unfortunately, l i t t l e i s known or can be done about t h e l a t t e r . With r e s p e c t t o spectrum, it i s i n t h e hands of t h e pharmaceutic a l houses and physicians t o decide whether broad spectrum a n t i b i o t i c s remain as such, or i f they develop reduced s p e c t r a as a r e s u l t of indiscrimi n a t e use with consequent s e l e c t i o n of r e s i s t a n t organisms. R-factors are probably t h e most common form of r e s i s t a n c e determinant i n n a t u r a l isol a t e s , and t h e promiscuity of t h i s agent i s undoubtedly r e l a t e d t o i t s widespread occurrence i n c l i n i c a l l y - i s o l a t e d s t r a i n s 5 3 . A l a r g e number of s t r a i n s are known t o be capable of carrying R-factors (Table 5 ) . Table 5 Bacterial s p e c i e s known t o c a r r y R-factors

S e r r a t i a marcescens Aerobacter a e r o enes & Klebsiella Dneumoniae Vibrio cholerae Pasteurella sp. Citrobacter

226 -

Sect. V

-

Wechter, Ed.

T o p i c s i n Biology

There seems t o b e l i t t l e hope t h a t a g e n t s w i l l be developed t o p r e v e n t t h e s t r a i n - t o - s t r a i n t r a n s f e r , o r promote t h e s e g r e g a t i o n ( c u r i n g ) o f R-factors. T h e r e f o r e , t h e p r o p o r t i o n o f r e s i s t a n t bacterial s t r a i n s i n n a t u r e must be k e p t t o a minimum by t h e c a r e f u l l y c o n t r o l l e d u s e of a n t i b i o t i c s , which might i n c l u d e r o t a t i n g u s e o f a n t i b i o t i c s with q u i t e d i f f e r e n t r e s i s t a n c e mechanisms. There is good evidence t h a t Rt r e s i s t a n t s t r a i n s are outgrown by t h e s e n s i t i v e , wild-type b a c t e r i a l f l o r a when t h e s e l e c t i n g a n t i b i o t i c is withdrawn from use53. REFERENCES 1. D. G o t t l i e b and P. D . Shaw, " A n t i b i o t i c s " , Springer-Verlag, New York, (1967). 2. J . Davies, J . Infect. D i s . 124, S7 (1971). 3. M. J. Weinstein, C. G. D r u b C E . L. MOSS, J r . , and J . A. Waitz, J . Infect. D i s . 124, S11 (1971). 4. T. Watanabe, B a c z i o l . Rev. 27, 87 (1963). 5. D. H. Smith, J. Bact. 94, 2 0 7 1 ( 1 9 6 7 ) . 6. B. D. Davis and W. K. G a s , F'roc. Nat. Acad. S c i . U.S. 38, 775 (19521, see also, H. S. Moyed, Ann. Rev. Microbiol. 1 8 , 347 (1964). 7. J. Davies and M. Nomura, Ann. Rev. Genetics, i n p r e s s (1972). 8. "RNA polymerase and t r a n s c r i p t i o n " . L e p e t i t Colloquia on Biology and Medicine, 1. North Holland P u b l i s h i n g Co., Amsterdam, 1970. 9. R. S c h l e i f , Nature 223, 1068 (1969). 1 0 . G. Kuwano, D. S c h l e z n g e r , G. R i n a l d i , L. F e l i c e t t i and G. P. Tocch i n i - V a l e n t i n i , Biochem. Biophys. Res. Commun. 42, 441 (1971). 11. S. Mitsuhashi " T r a n s f e r a b l e drug resistance factor R" , U n i v e r s i t y Park Press , Baltimore , 1971. 1 2 . P. J . O r t i z , Biochemistry, 9 , 355 (1970). 371 (1967). 13. T. J. F r a n k l i n , Biochem. J. , 14. T. J. F r a n k l i n and J . M. Cook., Nature 229, 273 (1971). 15. Report o f t h e J o i n t Committee on t h e u s e f a n t i b i o t i c s i n Animal Husbandry and V e t e r i n a r y Medicine. H.M.S.O. London, England. 1969. 16. See numerous articles i n Ann. N.Y. Acad. S c i . 182, 1971; also H. D. Mercer, D. P o c u r u l l , S. Gaines, S. Wilson and J . V . Bennett, Appl. Microbiol. 22, 700 (1971). 17. T. Watanabe, Ann.3.Y. Acad. S c i . 182, 126 (1971). 18. H. Jarolman, D. H e w e l , and E. K a i n x n f e c t . Immunity 1, 321 (1970). 19. E. P. Abraham and E. Chain, Nature 146, 837 (1940). 20. G. W. Jack and M. H. Richmond, J. G Z M i c r o b i o l . 6 1 , 43 (1970). 21. R. P. Novick, Fed. Proc. 26, 29 (1967). 22. M. H. Richmond, Biochem. J. 94, 584 (1965). 23. N . D a t t a and P. K o n t o m i c h a l o c Nature 208, 239 (1965). 1 7 , 783 (1969). 24. H. C. Neu, Appl. Microbiol. 25. E. J. L. Lowbury, A. Kidson, H. A. L i l l y , G. A. J . A y l i f f e , and R. J. J o n e s , Lancet, 1969, 448. 26. E. Roe, R . J. J o n e s , and E . J . L. Lowbury, Lancet, 1971, 149. 27. R. B. Sykes and M. H. Richmond, Lancet, 1971, 342. 28. F. H. Kayser, E . J. Benner, R. Troy, and P. D. Hoeprich, Ann. N.Y. Acad. S c i . 182, 106 (1971).

-

105,

-

-

-

Chap. 20

29. 30. 31. 32. 33.

W. Y. W. Y. J.

Antibiotic Resistance

Dav i es

V. Shaw, J. B i o l . Chem. 242, 687 (1967). Suzuki and S. Okamoto, oiJ.l Chem. 242, 4722 (1967). V. Shaw, Ann. N.Y. Acad. S c i . 182, 2 3 4 7 9 7 1 ) . Nagai and S. Mitsuhashi, J. B a z r i o l . 109, 1 (1972). Davies, M. Brzezinska, and R. B e n v e n i s t e , Ann. N.Y. Acad. S c i .

182, 226 (1971). 34. C L e b e k , Zentr. B a k t e r i o l . P a r a s i t e n k , Abt.1. b i g . , 188, 494 (1963). 35. J . L. Adler and M. F i n l a n d , Appl. Micro. 22, 870 (1971). 36. C. M . M a r t i n , N.S. S k a r i , J . Zimmerman a n d J . A. Waitz, J . I n f e c t . Dis. 124, S24 (1971). 37. S. A . K a b i n s , C. R. Nathan, and S. Cohen, i b i d , S65. 38. J. L. W i t c h i t z and Y. A. Chabbert, J . A n t i b i o t i c s 24, 137 (1971). 39. R. Benveniste and J . Davies, FEBS L e t t , 1 4 , 293 (1-1). 40. H. Naganawa, M . Yagisawa, S. Kondo, T . Takeuchi and H. Umezawa, J . A n t i b i o t i c s 24, 913 (1971). 41. D. H. Smith, B . T a n O t t o , and A . L. Smith, New England J . Med., 286, 583 (1972). 42. T S . Lietman, J. J . Lipsky, R. S. Johannes and J . E. Davies, B a c t e r i o l . Proc. 1972, i n p r e s s . 43. S. Mitsuhashi, F. Kobayashi and M . Tamaguchi, J. A n t i b i o t i c s 24, 400 (1971). 44. M. Brzezinska, R . Benveniste, J . Davies, P. J . L. Daniels and J. Weinstein, Biochemistry, 11, 7 6 1 (1972). 45. J . Dienstag and H. C. Neu, A z i m i c r o b . Ag. Chemother. 1, 4 1 (1972). 46. J . Davies and R. H. Rownd, S c i e n c e , i n p r e s s (1972). 47. H. Umezawa, S. Umezawa, T. Tsuchiya, and Y. Okasaki, J. A n t i b i o t i c s 24, 485 (1971). 48. B. Weisblum and V. Demohn, J . Bact. 98, 447 (1969). 49. "Drug a c t i o n and d r u g r e s i s t a n c e i n bacteria; Macrolide a n t i b i o t i c s and lincomycin". Ed. S. Mitsuhashi, U n i v e r s i t y Park F'ress, Baltimore , Maryland. 1972. 50. C. J. La: and B. Weisblum, Proc. Nat. Acad. S c i . U.S. 68, 856 (1971). 51. C. J. La; and B. Weisblum, p e r s o n a l communication. 52. R. P. Novick and D. Bouanchaud, Ann. N.Y. Acad. S c i . 182, 279 (1971). 53. S. Cohen, Am. J. Clin. Nutr. 23, 1480 (1970).

-

-

-

-

-

Chapter 21

- The

Serum Complement System

Harvey R. Colten, Harvard Medical School, Boston, Mass.

-

Introduction Complement was discovered in the late nineteenth century when it was recognized that the bactericidal activity of fresh serum required the participation of at least two factors: a heat-stable factor, specific for the particular organism (antibody); and a second, non-specific, heat-labile factor designated complement. During the two decades following the discovery of complement, it became apparent that cmplement did, in fact, consist of several components which act in a definite sequence to effect lysis and death of the bacteria. In the 1950'8, the development of sophisticated methods in protein chemistry and suitable functional assays for individual complement components facilitated the elucidation of the chemical, physical, and functional properties of the complement system. As a result, the serum complement system is now known to consist of nine distinct proteins that interact to mediate and amplify many of the biological effects of immune reactions (1,2). It should be noted that a discussion of the complement system as an entity separate from the balance of the plasma proteins is somewhat artificial, albeit convenient. Plasma contains several multicomponent enzymatic systems which interdigitate to form a complex functional unit. This functional unit, in turn, interacts with cellular elements and/or with exogenous agents to effect a multiplicity of homeostatic, protective, and occasionally injurious events. For instance, recent studies have demonstrated the intimate connections between components of the coagulation mechanism, the kinin-generating system, and complement (3,4). Furthermore, it has been recognized that other non-immunologic systems, such as bacterial proteases and lysosomal enzymes, can initiate complement activation (5). The purpose of this report is to review briefly some of the pathophysiological and homeostatic functions of the complement system and to indicate some of the clinical conditions in which pharmacologic regulation of complement activity might offer a rational approach to the therapy of allergic and immunologic disease.

-

Nomenclature For a number of years, a great deal of confusion had been generated by the lack of agreement on a consistent language for describing the complement system. In 1968, a standardized nomenclature was agreed upon (6). According to this nomenclature, the individual complement components are designated numerically in the order of their reaction sequence as follows: C1, C4, C2, C3, C5, C6, C7, C8, C9. Since initially an understanding of the functional properties of the complement components derived from studies of immune hemolysis, a convention was established for designating the reaction sequence and intermediate complexes on cell surfaces. E represents erythrocytes; A, antibody directed against the erythrocytes; and the intermediates indicated by EAC, followed by the numbers corresponding to the individual components that have reacted with EA. For

Chap. 21

Serum Complement System

Col ten

229

example, EAC142 describes an intermediate consisting of erythrocytes sensitized with antibody and containing the first, fourth, and second components of complement. Activated forms of the componznts are designated by a bar above the symbol; e.g., activation of C1 to C1. Fragments of the ccnnponents are indicated by a lower case letter; e.g., C3a, C3b, C ~ C ,and C3d.

-

Physicochemical Properties of Complement The first component of com lement (Cl) is a macromolecule of molecular weight approximately 1 x 10 which can be dissociated in the presence of sodium EDTA into three distinct protein subunits designated Clq, Clr, and Cls (7). Clq, the largest of the three with a molecular weight of about 400,000, electrophoretic mobility in the g a m a region, and a sedimentation constant of lls, has been visualized in the electronmicroscope as a coiled chain of six to eight globular subunits ( 8 ) . Clq is the portion of the C1 macromolecule that possesses the binding site capable of interacting immunoglobulin molecules; namely, IgG (principally of the gaxnnal, gamma2, and gamma3 subclasses) and most IgM molecules; but not with IgA, IgD, or I g E . The amino acid composition and carbohydrate content of Clq are similar to those of basement membrane proteins (9). Clq can be quantitated in serum by functional and innnunochemical methods. The concentration in normal human serum is approximately 20 mgl. Clr, a 7s protein with beta mobility, normally exists in serum non-covalently bound to Clq. Clq probably activates Clr by a mechanism as yet poorly understood (lo), and the Clr, in turn, activates Cls (111, altering its electrophoretic mobility and converting the proesterase to an active esterolytic enzyme (12). The esterolytic activity of Cls can be inhibited by phosphous esters (131, as well as by the serum protein inhibitor of C1. Cls, an alpha-2 protein, molecular weight 80,000, present in normal serum at a concentration of approximately 12 mgt, possess the active enzymatic site(s) of the C1 molecule which facilitate the cleavage of the natural substrates of this enzyme, C4 and C2 (14).

g

The binding and cleavgge of C4, a beta globulin of molecular weight approximately 250,000, by C1 apparently uncovers another enzymatic site on the Cls fragment which effects the cleavage of the C2 molecule into at least two fragments (15). One of the C2 fragments (C2a) can m i n e with a fra-nt of C4 (C4b) to form a new enzyme, C3 convertase (C42) (15). This C42 enzyme is extremely unstable under physiologic conditions; but if C3 is available, it splits the C3 into two fragments, C3a and C3b. C3, a genetically polymorphic protein, is the complement component present in highest concentration in normal serum ( 150 2 50 mgt). Immunochemical quantitation of this protein is relatively simple. Hence, most clinical laboratories generally assess abnormalities of complement in disease s g t e s by measurements of C3. As a consequence of cleavage of C3 by the C42 enzyme, a fragment of the molecule (C3b) acquires proteolytic enzymatic activity that probably is involved in cleavage of the next component in the series, C5 (5). C5, another beta globulin, has a molecular weight of about 180,000, and a sedimentation rate of 9s (16). This protein normally is present in serum at a concentration of about 8 mg%. C5 canbines with C6 and C7 to form an active complex capable of interacting with C8 and C9 (17).

230 -

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-

Topics i n Biology

Wechter, Ed.

The characteristics of several other proteins intimately associated with the Complement system should also be mentioned. These include the naturally occurring inhibitors of the complement system, the C1 inhibitor and the C3 inhibitor. The C1 inhibitor is an alpha-2 protein normally present in serum at a concentration of 18 5 4 mg%. The protein has a molecular weight of approximately 100,000 and a sedimentation rate of 35. It is capable of inhibiting the esterolytic and hemolytic activities of C1 stoichiometrically, but apparently does not interact with the precursor form of the C1 molecule. Interestingly, the C1 inhibitor is also capable of blocking the activity of several other proteins; namely, plasmin, kallikrein, and a permeability factor designated PF/dil (18). The C3 inhibitor is a 5s heat-stable beta-globulin that is capable of blocking the activity of C3b (19). Some of the physicochemical properties and the concentrations of each of the complement proteins in normal human serum are given in Table I:

Complement Component c1 Clq Clr Cls c2 c3 c4 c5 C6 c7 C8 c9

Properties of the Complement Components Normal Serum Conc. Relative Estimated Sedimentation Electrophoretic Hemol. Eff, ~ ~ ( ~ 1 0 - Coefficient 3) (S) Mobility 1000 400 150 80 117 185 240 180 95 110 150 79

19.0 11.0 7 .O 4.0 5.5 9.5 10.0 9.0 5.6 5.6 8.0 4.5

v2

P

6 2

A2 $1

5;

$:

Y1 OL

-

There are several pathways by which all or part of the Reaction Sequence complement system can be activated. The nexus of these various activation pathways, the C3 molecule, and the later-acting complement components, yield the bulk of the biologically active components of the complement system. However, as will be described shortly, some of the biological effects of complement are the consequence of the action of C1, C4, and C2 alone. The classical complement activation sequence is initiated by the binding of C1 to an antigen-antibody complex. A single IgM in complex *Serum proteins have been separated according to their electrophoretic mobilities in a standard buffer at p H 8.6. In this procedure, albumin is the most rapidlymigrating, and gamma globulin the slowest serum protein. **Hemolytically effective molecules assayed in whole serum using standard functional assays for the individual complement components ( 2 ) . Normal values for some of the components have not yet been established.

Chap. 21

Serum Complement System

Col t e n

23 1 -

with its corresponding antigen is capable of binding one C1 molecule; whereas at least two IgG molecules of the appropriate subclass are required for C1 fixation (20). As a consequence of binding, the C1 undergoes a conformational change and an autocatalytic activaten. The activated C1 is then capable of cleaving C4 and C2, forming the C42 complex. Interact k o f the active C423 with native C5 generates an unstable intermediate C4235 which then acts on C6 and C7. Subsequently, C8 and C9 are bound and activated. It should be noted that this pathway can be activated by non-imunologic mechanisms as well. For example, it has been demonstrated that a protein active in the clotting mechanism, plaein (the activated form of plasminogen), is capable of activating C1 to C1, thus initiating the classical complement sequence(3). Likewise, precipitation of C1 with highly charged molecules, such as carrageenin, also results in C1 activation (21). In addition, trypsin, lysosomal enzymes, and bacterial proteases can directly activate the complement system without the participation of specific immune complexes (5). A number of years ago, a major alternate pathway of complement activation was described. This pathway, initiated by incubation of endotoxin or yeast with fresh serum, is mediated by the properdin system, bypassing the early Complement components (Cl, C4, and C2) to effect the activation of C3 directly (22). Properdin and at least two proteins designated Factors A and B constitute the principal components of this bypass-activating system. Two of these proteins have recently been isolated: properdin, a euglobulin with a molecular weight of 280,000 (23); and Factor B (previously designated GBG) , a genetically polymorphic pseudoglobulin (24) Recently, it has been shown that certain immunoglobulins (guinea pig gamnalr human IgA, and possibly IgE) that do not fix C1 are capable of initiating the "bypass" sequence of complement activation (25,26). It is not clear at the present time whether these antibodies activate the classical properdin system or activate still another alternate pathway.

.

-

Biological Effects of Complement Activation of the complement system under appropriate conditions leads to the generation of a number of biologically active fragments that participate in the inflammatory response. Activation of all nine of the components results in membrane damage (i.e., cytolysis, bacteriolysis, and possibly the immunopathogenic lesions of immune complex disease). Anaphylatoxins and Chemotactic Factors (cf, reference 5 for extensive reThere are two complement-associated anaphylatoxic facview of this area) tors which effect smooth muscle contraction and increase vascular penneability. The first of these, a polypeptide of molecular weight approximately 7,000, C3a, arises from the cleavage of C3 in the following reactions:

-

C3

*

p C3a

+

C3b

t alternate pathway

232

Sect. V

-

Topics i n Biology

Wechter, Ed.

The second anaphylatoxic factor is generated from C5 by the following react ion :

-

c

C423 5 -

A C5a + C5b

C5a is a fragment of the parent molecule with a molecular weight of about 10,000. The actions of both anaphylatoxins can be blocked by antihistamines, but apparently the biological effects of each operate via separate mechanisms since smooth muscle no longer sensitive to C3a can be stimulated by C5a. It should be noted that response of smooth muscle to anaphylatoxin exhibits tachyphylaxis; whereas the response to histamine does not. It is clear, however, that activation of the complement system at least through C3 leads to complement-dependent histamine release. This phenomenon is mediated by immunoglobulins other than IgE. Both C3a and C5a have chemotactic activities as well ( 2 7 , 2 8 ) . That is, they are capable of promoting migration of white blood cells to a site of inflammation. It is not clear whether the C5a and/or C3a fragments responsible for chemotaxis are chemically identical with the corresponding anaphylatoxin molecules. The precise mechanism by which chemotaxis is accomplished is only poorly understood at the present time, but it does appear that at least two serine esterases supplied by the polymorphonuclear leukocytes are required for this response to chemotactic factors.

-

Immune Adherence and Opsonization It is known that, although phaqocytosis can proceed in the absence of complement, this process is greatly accelerated by an intact complement system (29). Phagocytosis of IgM antibody-antigen complexes requires complement. IgG, on the other hand, by itself promotes phagocytosis, but this effect is augmented by the fixation and activation of complement. Based on experimental evidence, as well as clinical observations, it is now clear that i m u n e adherence and opsonization require activation and uptake of complement (at least through C3b). It has been suggested that receptors on phagocytes capable of recognizing C3 in effect cement a particle to the white cell, thereby facilitating both phenomena. There is evidence, however, that the fixation of C5 may also be required for phagocytosis of certain microorganisms, such as monilia (30).

-

Membrane Damage Activation of the complement system at discrete sites on cell surfaces results in irreversible damage to a cell membrane, leading to lysis of the cell. Red blood cells, most if not all nucleated cells, and many bacteria are susceptible to the lytic action of complement. The action of complement on biological membranes results in characteristic electronmicroscopically identifiable lesions. These lesiogs, demonstrated with negative-staining techniques, are approximately 80 A in diameter (31). Recent evidence suggests that the lesions appear after the complement reaction sequence has proceeded through C5 (321, although there are claims that all nine components are required. In any case, a functional lesion in the membrane is not detectable until at least the first eight components have been activated. This functional lesion leads to a disruption of the

Chap. 21

Serum Complement System

Col t e n

233 -

osmotic integrity of the cell and a sequential loss of small ions, amino acids, polypeptides, and macromolecules (33). In nucleated cells, there is, in addition, a profound enzymatic cleavage of DNA following immune cytolysis (34).

-

Coagulation Recent evidence derived from studies of rabbits deficient in C6 indicated that the clotting mechanism is facilitated by this component, since C6-deficient rabbit plasma showed a defect in coagulation that could be restored to normal by the addition of C6 (35).

-

Virus Neutralization The neutralization of viruses under some conditions requires the participation of anti-viral antibody and at least some of the complement components. For example, it has been shown that neutralization of herpes virus by IgM anti-herpes antibody requires the activation and binding of C1 and C4 to the virus particle. Smaller amounts of C4 will suffice if C3 is available (36).

-

Ontogeny, Phylogeny, and Biosynthesis of Complement Fluid phase, heatlabile hemolytic factors have been identified in primative invertebrates (37). However, a clearly defined complement system appears phylogenetically first in cartilagenous fish. So far, of the vertebrates that have been studied, all have complement, although the amount as detected in a standard hemolytic assay varies widely from one species to another. Many of the components are immunochemically and physicochemically species specific. Nonetheless, several components are sufficiently similar functionally to permit interaction of a component of one species with that of another. In short, phylogenetic studies, while only fragmentary at the present time, do indicate that the complement system arose early in evolution and that the basic structural and functional properties of many of the components have undergone relatively little genetic change. A significant exception to this general rule can be found in the genetic polymorphism of a few of the components, the principal example being C3 (38). By means of electrophoretic analysis, C3 has been shown to exist in two common genetically determined forms, designated S for the slower or cathodal type and F for the faster or anodal type. A number of rare variants of S and F alleles have been described, These variant molecules, while functionally indistinguishable, must have resulted from minor amino acid substitutions and, therefore, are reflections of some degree of evolutionary variability. A similar polymorphism of C4 has been described, but the exact genetic basis for this has not been completely clarified (39). The discovery of polymorphic form of the complement proteins has been of considerable importance in one approach to the study of the ontogeny and biosynthesis of complement. According to a number of independent lines o f investigation, complement biosynthesis by the fetus is initiated early in gestation (40,41). It has been shown that C3 and C4, as well as whole Complement activity, are present in fetal serum and, furthermore, that the electrophoretic variants of C3 in fetal sera may differ from the maternal type ( 4 2 1 , suggesting a lack of transplacental passage and thus active fetal synthesis of this component. In addition, human fetal tissues are capable of synthesizing & vitro biologically active C1 through C5 (the components that have been

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been studied thus far). Recently, the sites of synthesis of most of the individual complement components have been identified in adult, as well as fetal tissues. C1 is synthesized primarily, if not exclusively, in the gastrointestinal tract (large and small bowel) (43); C2 and C4 by macrophages derived from a variety of organs, including liver, spleen, lunq, and bone marrow (44,451; C3 and the C1 inhibitor, by the liver (probably in parenchymal cells) (41,461; and C5 in the liver, the gastrointestinal tract, and possible bone marrow (47,481. The sites of synthesis of C6 to C9 are less firmly established.

-

Complement Fixation Probably the most important practical applicatiw of complement research has been the development of complement fixation tests for the quantitation of antigens and antibodies. Standard complement fixation tests are performed by mixing dilutions of antigen and antibody in the presence of a known amount of complement. After a suitable incubation period, erythrocytes sensitized with antibody (EA) are added. If complement has not been fixed, the indicator cells (EA) will lyse; fixation by the antigen-antibody complex will lead to a loss of hemolytic complement activity; hence, no lysis of the jrldicator cells (details given in ref. 4 9 ) . Antigen (Ag) Antibody (Ab) Ag + Ab

+C

+

+

C C

-

__c. _I+

+ +

-

EA EA AgAbC + EA -No

Lysis Lysis lysis

Recently, a more sensitive Complement fixation test that permits detection on a molecular basis of nanogram quantities of antigens or antibodies has been developed (50). This method, the C1 fixation and transfer test, has had useful research applications in the estimation of cell surface antigens and their corresponding antibodies (51). Future applications of this method to clinical problems should be fruitful.

-

Complement and Human Disease The complement system plays a central role in the pathogenesis of several human diseases. These clinical conditions can be divided into two general groups: those in which a normal complement system, activated by immune or non-immune mechanisms, leads to damage of the host tissues; and a second group, due to genetic deficiencies of complement biosynthesis. Abundant evidence implicates activation of the complement system in development of the pathological features of systemic lupus erythematosus, rheumatoid arthritis, acute and some chronic forms of glomerulonephritis, serum sickness, and certain autoimmune or drug-induced hemolytic anemias (reviewed in ref. 9). In each case, increased consumption of complement components and/or deposition of complement at sites of tissue damage have been demonstrated. In addition, numerous animal models of these have substantiated the role of complement in the development of characteristic lesions. It should be noted that participation of the complement system is not always accompanied by a change in the concentration of complement in serum. For example, serum complement is normal or even elevated in rheumatoid arthritis, whereas in the joint space consumption of complement and generation of biologically active complement fragments have been clearly demonstrated (52). In lupus erythematosus and acute glomerulonephritis, on the other hand, serum complement is profoundly depressed (53);

Chap. 21

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23 5 -

and, in fact, chanaes in complement levels can be used as an index of exacerbation and remission of the disease. A number of inherited abnormalities of the complement system have been described (54). While generally these abnormalities are qualitatively less severe than most of the other immunodeficiency diseases, several, such as hereditary angioneurotic edema (HANE) (551, and Clr (56) and C 5 aeficiency disease (30), may have dramatic, even life-threatening, clinical consequences. Hereditary angioneurotic edema (HANE) is a disease in which a genetic deficiency of the natural inhibitor of the first companent of complement (C1 INH) leads to attacks of angioedema. Two variants of this disease have been recognized. In the more common form of the disease, the concentration of C1 INH in patients' sera is markedly reduced although the specific activity of the C1 INH that is present is normal. The second and less common form of HANE is characterized by the presence of antigenically intact, but functionally inactive, C1 INH molecules (54). These two forms of the disease cannot be distinguished clinically but are easily identified by the distinctive laboratory findings. In both forms of the disease, during an attack, C1 is activated and the patient develops angioedema probably due to the uninhibited generation of a vasoactive fragment of CZ. This disease, therefore, is the result of abnormal regulation of complement activation. A similar circumstance leads to the clinical features of a condition known as congenital hypercatabolism of C3 (57). In this disease, characterized by recurrent severe infections and allergic manifestations, the lack of a naturally occurring enzyme inhibitor (GBGase) leads to increased catabolism of C3. As a result, C3 is not available for normal defense against bacterial infection.

-

Summary The chemical isolation and identification of individual complement proteins and the development of sensitive and specific functional measures of complement-dependent biological activities has made it possible over the past few years to advance studies of complement into areas of clinical, genetic, biochemical, and fundamental pathophysiological importance. These advances now permit a logical search for pharmacologic agents that will control complement-dependent disease factors. References 1. 2.

3. 4. 5. 6. 7. 8.

9.

H.J. MUller-Eberhard, Adv. Imunol., g, 2 (1968). H. J. Rapp and T. Borsos in "Molecular Basis of Complement Action, I' Appleton-Century-Crofts, New York, 1970. O.D. Ratnoff and G.B. Naff, J. Imunol., 125, 337 (1967). I. Gigli, A.P. Kaplan, and K.F. Austen, J. Exp. Med., 134, 1466 (1971) I.H. Lepow in "Progress in Immunology,'' B. Amos Ed., Academic Press, New York, 1971, p. 579. Bull. Wld. Hlth. Org., 2, 935 (1968). I.H. Lepow, G.B. Naff, E.W. Todd, J. Pensky, and C.F. Hinz, J. Exp. Med., 117, 983 (1963). M.J. Polley in "Progress in Immunology," B. Amos Ed., Academic Press, New York, 1971, p. 593. H.J. MUller-Eberhard in "Progress in Imunology," B. Amos Ed., Academic Press, New York, 1971, p. 553.

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M. Uos, T. Borsos, and H . J . Rapp, J. Immunol., 108, 683 (1972). 11. G.B. Naff and O.D. Ratnoff, J. Exp. Med., 128, 571 (1968). 1 2 . K. Nagaki and R.M. S t r o u d , J . Immunol., 103, 1 4 1 (1969). 13. E.L. Becker, Biochim. Biophys. Acta, 147, 289 (1967). 14. I. G i g l i and K.F. Austen, J. Exp. Med., 130, 833 (1969). Muller-Eberhard, M . J . P o l l e y , and M.A. Calcott, J. Exp. Med., 15. H.J.

10.

125, 359 (1967).

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.

Milller-Eberhard, J. Exp. Med., 122, 277 (1965). Haxby, C.M. Arroyave, and H.J. Mtiller-Eberhard, J. w,P. Kolb, J.A. Exp. Med., E, 549 (1972). O.D. Ratnoff, J. Pensky, D. Ogston, and G.B. Naff, J. Exp. Med., 129, 315 (1969). S. Ruddy, L.G. Hunsicker, and K.F. Austen, J. Immunol. 657 (1972) T. Borsos and H . J . Rapp, S c i e n c e , 150, 505 (1965). T. Borsos, H . J . Rapp, and C. C r i s l e r , J . Immunol., 94, 662 (1965). L. P i l l e m e r , L. B l u m , I . H . Lepow, O.A. Ross, E.W. Todd, and A.C. Wardlaw, S c i e n c e , 120, 279 (1954). J. Pensky, C.F. Hinz, E.W. Todd, R.J. Wedgewood, and I . H . Lepow, J. I m u n o l . , 100, 142 (1968) T. Boenisch and C.A. Alper, Biochim. Biophys. A c t a , 221, 529 (1970). A.G. O s l e r , B. O l i v e i r a , H . J . Shin, and A.L. S a n d b e r c J . Immunol., 102, 269 (1969). 0. Goetze and H.J. MCiller-Eberhard, J. Exp. Med. ( S u p p l . ) , 134, 90 (1971). P.A. Ward i n " B i o l o g i c a l A c t i v i t i e s o f Complement," S . Karger, Basel, 1972. R. Snyderman and S.E. Mergenhagen i n " B i o l o g i c a l A c t i v i t i e s of Complement," S . Karger, Basel, 1972. H.K. Ward and J.F. Enders, J. Exp. Med., 57, 527 (1933). 354 (1970). M.E. Miller and U.R. N i l s s o n , New Engl. 3. Med.,=, J.H. Humphrey and R.R. Dourmashkin i n "Complement: C i b a Symposium," C h u r c h i l l , London, 1965. M . J . P o l l e y , H . J . Mflller-Eberhard, and J . D . Feldman i n "Biological A c t i v i t i e s of Complement," S. Karger, Basel, 1972. ti. Green, R.A. F l e i s c h e r , P. B a r r o w , and B . Goldberg, 3. Exp. Med., 109, 505 (1959). E.S h i p l e y , A.R. Baker, and H.R. C o l t e n , J. Immunol., 576 (1971). T.S. Zimmerman, C.M. Arroyave, and H . J . Milller-Eberhard, J. Exp. Med., 134, 1591 (1971). G. Daniels, T. Borsos, H . J . Rapp, R. Snydennan, and A.L. Notkins, 508 (1969). Science, H. Gewurz, J. F i n s t a d , L.H. Muschel, and R.A. Good i n "Phyloqeny of Immunity," U n i v e r s i t y o f F l o r i d a P r e s s , G a i n e s v i l l e , 1966, p. 105. C.A. A l p r and R.P. Propp, J. C l i n . Invest., 47, 2181 (1968). S.E. Rosenfeld, S . Ruddy, and K.F. Austen, J. C l i n . I n v e s t . , 48, 2283 (1969). D. G i t l i n and A. B i a s u c c i , J. C l i n . I n v e s t . , 48, 1433 (1969). H.R. Colten, J. C l i n . I n v e s t . , 51, 725 (1972). C.A. A l p e r , A.M. Johnson, A.G. B i r t c h , and F.D. Moore, S c i e n c e , E,

r N i l s s o n and H . J .

108,

.

.

-

106,

165,

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Col ten

237

286 (1969). 43.

H.R.

D.J.

Colten, J . M .

Gordon, T. Borsos, and H . J .

Rapp, J. Exp. Med.,

128 (1968).

44.

Rubin, T. Borsos, H . J .

Rapp, and H.R.

Colten, J. I I N n U n O l . ,

106,

295 (1971). Wyatt, H.R. Colten, and T. Borsos, Fed. P r o c . , E , 355 (1971). Johnson, C.A. A l p e r , F.S. Rosen, and J . M . Craig, Science, 173,

45. 46.

H.V. A.M.

47.

M.E. P h i l l i p s , U.A. Rother, K.O. Rother, and G . J . Thorbecke, J . Immunol., 315 (1969). H.R. Colten, Unpublished observations. M.M. Mayer i n “Mayer’s and Kabat’s Experimental Immunochernirtry,” C. C. Thomas, S p r i n g f i e l d ( I l l i n o i s ) , 1961. T. Borsos and H . J . Rapp, J . Imnunol., 95, 559 (1965). T. Borsos, H.R. Colten, J.S. S p a l t e r , N. Rogentine, and H . J . Rapp, J. Immunol. 392 (1968) G. Fostinopoulos, K.F. Austen, and K . J . Bloch, Arth. hi Rheum., 8,

553 (1971).

17,

48. 49. 50. 51.

52.

,=,

.

219 (1965). 53. 54. 55. 56.

57.

Cooper, M . J . Polley, and H.J. MUler-Eberhard i n “InUnUnOlOgiCal Diseases,” 2nd Ed., L i t t l e Brown, Boston, 1971, p. 289. C.A. Alper and F.S. Rosen, Adv. Inmunol., 14, 251 (1971). V.H. Donaldson and R.R. Evans, Amer. J. Med., 35, 37 (1963). N.R.

R.J.

131,

Pickering, G.B.

Naff, R.M.

Stroud, and R.A.

803 (1970). C.A. A l p e r , N. Abramson, R.B. Johnston, Jr., J . H . Rosen, New Engl. J. Med., 282, 349 (1970).

Good, J . Exp. Med., J a n d l , and F.S.

Chapter 22. Immediate H y p e r s e n s i t i v i t y : L a b o r a t o r y Models and Experimental Findings Michael K. Bach, The Upjohn Company, Kalamazoo, Michigan 49001, USA The general concept of the ability of an animal to fight infection with its immune system is nearly 100 years old, and the role of blood leukocytes and the various cells in lymphoid tissues in this process is well established. It was only in the last 10 years, however, that progress in molecular biology, genetics, cellular biochemistry, and protein chemistry contributed to the unraveling of the multisystem jigsaw puzzle which is immunology. It has been traditional to refer to two major types of responses as comprising the totality of the immune response. These are the immediate type response and the delayed type resp0nse.l The names refer to the speed with which an immunologic reaction to a foreign substance is mounted in an animal which had been previously sensitized to that substance. The immediate type response generally occurs within minutes, and the delayed type response takes one to two days to reach its peak. Furthermore, the immediate type response is initiated primarily by circulating antibody molecules (humoral response) while the delayed response involves the infiltration of the affected site by a variety of leukocytes which, in the apparent absence of any demonstrable circulating antibody, are capable of mounting an attack on the offending foreign substance (cellular response). This review is concerned only with reactions of the immediate type. Furthermore, as will be defined below, it is primarily concerned with only one subgroup of the immediate type reactions, namely the models for atopy or "allergy" which are initiated by reagin-like antibodies.

-

Definitions To facilitate the discussion, a few of the commonly used terms are listed and defined below:

-

Adjuvant A substance which is used in conjunction with an immunogen to augment the immune response or to modify the quantitative distribution of the antibodies of the various immunochemical classes which are produced. A n a p h y h i s - The acute and rapid reaction of an animal having an immediate type of allergy to an antigenic challenge. The reaction can be active if the animal was itself the producer of the responsible antibodies or passive if the antibodies had been administered preformed. Anaphylaxis can furthermore be systemic or local, an example of the latter being passive cutaneous anaphylaxis (PCA).

-

Antigen A substance which can combine with an antibody. It is also referred to as an allergen. An immunogen is a substance which induces the production of antibodies. It may, but need not, be an antigen. Antibodies - Serum proteins which can combine specifically with an antigen. Structurally, antibodies are immunoglobulins and belong to several,

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chemically distinguishable classes. These are IgG (Immunoglobulin G), molecular weight 160,000, IgM, molecular weight 900,000, IgA and IgE (or reagin) .z A t o p y - The immunologic disorders caused by IgE antibodies, colloquially referred to as I'allergies." Examples are hay fever, certain forms of asthma and drug hypersensitivities (e .g., "penicillin allergy").

- A relatively low molecular weight substance which, in itself is not immunogenic or antigenic, can be combined with polymeric carriers such as serum proteins and then induce the production of antibodies specific against itself.

Hapten

-

Leukocytes The white cells of the blood. Among these are lymphocytes, monocytes and polymorphonuclear cells (PMN's). Cells in the last two categories are rich in lysosomes (subcellular organelles containing an assortment of hydrolytic enzymes) and are capable of phagocytosis (i.e., the ingestion of particulate foreign matter). The PMN's are further divided into neutrophils, eosinophils and basophils based on the staining properties of the cytoplasmic granules they contain. Basophils and their relatives, the tissue mast cells, contain in their granules the bulk of the animal's stores of pharmacologic mediators of anaphylaxis (notably histamine and serotonin) and, therefore, play a key role in anaphylaxis while eosinophils also appear to be involved in atopic conditions.

The mechanisms of immediate type hypersensitivity - Aside from the temporal considerations mentioned above, the immediate type reactions can be further categorized by the nature of the antigen and the immunoglobulin class of the antibody initiating the response. Responses initiated by IgG and IgM antibodies usually result in the activation of a series of plasma proteins which act in cascade fashion. These are known as the complement system. While this system will be discussed in detail in the next chapter, it is important to stress here that one consequence of its activation is the production of substances which act as chemotactic agents, attracting the various phagocytic cells to the site where the immune complex is located and, secondarily, bringing about the lysis of the cells to which the complex is attached. In addition, the anaphylotoxins, which are also produced upon the activation of the complement system can affect vascular permeability directly and indirectly. The site of all these events, and the tissue damage which ensues are a function of the localization of the antigen-antibody complex. Since the complexes often accumulate in the kidney, kidney damage is a frequent consequence. There is a large group of diseases which, while they appear to be immunologically induced, and generally fit into the immediate type of hypersensitivities, nevertheless differ from the classical pattern in that they do not involve the activation of the complement system nor tissue damage. These are the atopic conditions, and their distinguishing feature is that exposure of the organism to the sensitizing allergen results in the direct release of the pharmacologic mediators of anaphylaxis. In addition to histamine and serotonin which are present preformed

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in the basophils and the tissue mast cells, several other substances are produced by these cells during anaphylaxis. These are certain prostaglandins ,4 slow-reacting substance of anaphylaxis (SRS-A) and the newly described eosinophil chemotactic factor of anaphylaxis (ECF-A) .6 The structu'res of neither of the last two substances are known. They both have molecular weights of less than 1000. SRS-A may be an acidic lipid and ECF-A appears to be a peptide. The release of ECF-A during anaphylaxis explains, at least in part, the eosinophilia which generally accompanies atopic conditions, and SRS-A (rather than histamine) may be the major culprit in bringing about asthmatic attacks. Characteristics of IgE - The physical nature of the immunoglobulin which is responsible for the atopic reactions was an enigma for a long time. It is destroyed by a relatively brief exposure to 56Ocy7 is eluted from sephadex columns at a volume corresponding to a molecular weight of about 190,000, only slightly larger than common IgG. Its concentration in the serum of even highly allergic individuals is so small that it is not possible to demonstrate its presence by the formation of antigen-antibody precipitates. The coincidental discovery of a human patient with a strange myeloma by Johansson and Benniche and the careful separation work of the Ishizaka's7 finally led to the identification of the agent responsible for atopy as a new class of immunoglobulin which has been termed IgE. In the five years since IgE has been first described, immunoglobulins which are immunochemically and physically similar to IgE have been described in various animal species, including the mouse,g rat,1° rabbit,ll guinea pig,12 d0g,13314monkey15 and the cow.16 In all these species, the characteristics which distinguish IgE (reagin) from all other immunoglobulins are its lability to heat and mercaptoethanol treatment, its presare in trace amounts in the serum, its characteristic fast migration (as a fast y or slow B band) in immunoelectrophoresis, its ability to bind to and persist in skin sites for very long periods of time, and the complete non-dependence on the complement system for the release of pharmacologic mediators of anaphylaxis upon the interaction of cell-bound IgE with antigen. The persistence of IgE in the skin has given this immunoglobulin its descriptive name of homocytotropic antibody. It is the basis for its routine assay: Serial dilutions of the antibody solution are injected into the skin of recipient animals. One to three days later, anti en, together with a colloidal dye is injected intravenously. The PCAl reaction which results is due to the diffusion of the antigen out of the vasculature, and the combining of the antigen with the tissue-bound antibody molecules causing the release of the pharmacologic mediators from the tissue mast cells. The mediators cause increased vascular permeability, rendering the capillaries in the vicinity of the reaction more permeable to the colloidal dye so that a colored spot results. The size and intensity of the colored spot can be quantitatively related to the concentration of antigen-specific IgE which was present in the injected serum. While the reaction is generally carried out in homologous animals in keeping with the w c y t o t r o p i c name of the antibody, it has been possible to assay human reagin in monkeys,18*19and

9

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mouse reagin in rats.18,19 Recently, there have been reports21 that it is even possible to assay human reagin with rat mast cells in an in vitro analog of the PCA reaction. In fact, a serologic homology between human and rat IgE has been demonstrated.22 When dealing with a heterocytotropic antibody, however, it is essential to be wary of complications which can be caused by the much tighter binding of certain other immunoglobulins to skin sites in the heterologous species than in the homologous species. While most atopic conditions in man can be explained on the basis of hypersensitivity involving IgE, evidence has been obtained that under certain conditions certain subclasses of IgG may also be homocytotropic .23,24 The analogous situation in rodents is well established. Here one class of IgG in the mouse,25 guinea pig26 and rat27 is capable of causing a PCA reaction and of causing the antigen-dependent release of pharmacologic mediators from mast cells without the involvement of the complement system. The implications of this dual mechanism remain the subject of much interest. Production of reaginic antibodies - A number of principles have evolved in recent years to help achieve conditions f o r evoking an IgE response in laboratory animals. In ordinary immunization schedules IgE, if it is produced at all, is produced in a transient manner early in the immunization. In rats and mice titers are generally low and attempts to boost them by reimmunization fail. An exception is the response of all species tested to infection with live intestinal parasites such as ascaris28 o r the nematode Nippostrongylus brasiliensis (in the rat) .29 Infection with this agent at critical times after immunization with an unrelated protein antigen also can act as an adjuvant for the production of hi h titers of IgE antibody which is specific for the protein antigen.3f The use of the proper adjuvant is critical: The use of aluminum hydroxide gels to adsorb the antigen is useful for the immunization of rabbitsY3lmice32 and guinea pigs,33 while in rats a specially prepared B. pertussis vaccine consisting of heat-killed Bordetella pertussis organisms is apparently essential.34 B. B. Levine and his collaborators have reported that repeated immunization with exceedingly small doses of protein antigens adsorbed to aluminum hydroxide gel produced quite high titers of IgE antibody in mice and guinea pigs.32y Large differences were seen between the responses of various inbred lines of mice to the same haptens when the haptens were coupled to different carrier proteins. The ability to respond to a given protein carrier appears to be linked to the genes controlling histocompatibility (transplantation) antigens .32 In this respect , as well as in the low doses of antigen which are required for immunization, this system appears to be a good model for the study of the conditions which control reagin production in man. Differences in the response to a hapten when it is coupled to different carrier proteins have also been reported in rats.28 Tada and his colleague^^^,^^,^^ as well as others28 have presented evidence that treatments which ordinarily result in inhibiting the immune

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response, such as thymectomy, splenectomy, X-irradiation or treatment with immunosuppressive drugs (cyclophosphamide, actinomycin D, or 5bromodeoxyuridine) all stimulated production of IgE. Similar observations have been made by Dr. G . J. White of these l a b o r a t ~ r i e susing ~ ~ horse anti-rat thymocyte serum as the immunosuppressant. Thus, IgE synthesis appears to be normally inhibited by some other component of the immune response, the production of which is, in turn, inhibited by the immunosuppressive regimens. The thymus derived lymphocytes (T cells) have been shown40 to play this role. Methods for studying sensitization and mediator release - The term "sensitization" is used for convenience to denote the process which renders cells capable of releasing histamine upon challenge with antigen. A number of experimental systems are available in which the release of mediators as a consequence of sensitization with reagin and challenge with antigen can be studied both in vivo and in vitro. In addition to the PCA reaction which has already been described, it is possible to quantitate the release of mediators in a unique "in vivo test tube'' arrangement by injecting the antibody into the peritoneal cavity and some time later injecting the antigen into the same space.41 The interaction of antigen with the antibody which is bound to the numerous mast cells in the peritoneal cavity causes these cells to release their pharmacologic mediators of anaphylaxis. The peritoneal washings can be removed, freed of cells, and the supernatant fluid can be assayed for its content of mediators. Other in vivo systems employ anaphylactic collapse or death as their end point. Most of these require the use of actively immunized animals. Aside from the crudeness of the assay end point, these systems introduce further variation caused by animal to animal differences in the degree of immunologic responsiveness, in addition to the variation in the amount of mediator released, and in the systemic sensitivity to the action of the mediators.

-In vitro methods for studying cell sensitization and release of pharmacologic mediators offer much better control over animal to animal variation. The tendency of mast cells to degranulate non-specifically -in vitro, thus releasing their pharmacologic mediators, requires that a number of experimental variables be meticulously controlled, however. Peritoneal cells from rats42 and mice9 have been used. In agreement with the firm binding of IgE to mast cells in skin in vivo where it may persist for periods of several weeks,43 this antibody binds firmly to the cells in vitro and can cause the release of mediators from the cells even after the cells have been washed free of non-adhering antibody. By contrast, homocytotropic antibodies of the IgG class are much less firmly bound, and do not persist on the cells even after a single washing.44945 It is of interest that despite this difference in the avidity of the attachment of the two classes of homocytotropic antibodies to the cells, they can compete with one another for binding to the This observation supports the hypothesis that there is a specific receptor for these immunoglobulins on the mast cells. The chemical nature of this receptor is of considerable interest, and the elucidation of its structural features may point the way to the formulation of specific inhibitors of

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the attachment of reagins to their receptors. In vitro methods for studying sensitization and mediator release -have also been developed using rabbit leukocytes .47,48y49 Here matters are somewhat more complex in that the bulk of the histamine in rabbit blood is actually found in the platelets and not the basophils. Challenge of sensitized basophils with antigen results in the release of a soluble factor which, in turn, causes the release of histamine from the platelets. Finally, human leukocytes from non-atopic individuals can be sensitized -in vitro with sera from atopic individuals,50 and histamine can be released upon challenge of the sensitized cells with antigen. The in vitro system which is the closest model available for studying mediator release in asthma involves the sensitization of chopped lung fragments (from guinea pigs, monkeys or humans)2o s 5 l with reaginic sera, and collecting the pharmacologic mediators which are released upon challenge of the tissue fragments with antigen, Not only is this the only in vitro system in which the release of SRS-A along with histamine has been described, but it can be shown that it is inhibitable by realistic concentrations of all the drugs which are active in the laboratory and in the clinic in the treatment of asthma. Sensitive methods are available for the quantitation of the amounts of the various pharmacologic mediators of anaphylaxis which are released during anaphylactic challenge: 1. Histamine can be determined by a fluorometric procedure.5 2 ,5 3 Alternatively, it can be estimated by a bioassay which depends on measuring the contraction of a piece of guinea pig ileum.27 The latter procedure is better adapted to handling large numbers of samples although it is not as specific as the fluorometric procedure. 2 . SRS-A is also assayed by measuring the contraction of the guinea pig ileum. For this purpose, the muscle is treated with an antihistamine to block its response to histamine.

3. ECF-A is estimated6 by its chemotactic activity for guinea pig eosinophils. The assay depends on determining the relative rate at which eosinophils penetrate a Millipore@ membrane separating a compartment containing mixed leukocytes and a compartment containing a presumed source of ECF-A. In considering the magnitude of the mediator releasing response, there is, unfortunately, no way of estimating what the maximum amount of SRS-A or ECF-A which might be released might be since neither of these materials is demonstrable in the tissue prior to challenge. In the case of histamine, on the other hand, it is possible under optimal conditions to bring about the release of virtually all of the 30-40 picograms of this mediator which are present in an average rat peritoneal mast

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Electron microscopic and fluorescence microscopic studies have shown that the localized application of an antigen to limited regions on the surface of a mast cell result in a similarly localized release of histamine.55,56 This is most readily shown by use of the releasing agent, compound 4 8 / 8 0 , a polymeric amine which can mimic the reagin-mediated histamine release reaction. In both cases the release of mediator occurs within seconds and is not a cytotoxic reaction in that it is not accompanied by the release of other soluble components from the cells, such as potassium ion or soluble enzymes. The antigen concentration which is required for optimal release of histamine is characteristically very low-of the order of a few tenths of a microgram per ml.57 While inhibition can be achieved when excess antigen is used, the dose response curves tend to be much less steep than the usual antigen-antibody precipitation curves. Desensitization - The release of histamine from mast cells is an energyrequiring process. In the case of human leukocytes, 2-deoxyglucose, an inhibitor of glucose respiration, inhibits the release of histamine.58 Similarly, there is no release when the antigen is added to cells in the cold. The first step in the release reaction also requires calcium ions,59 and is dissociable from the second , respiratory energy-requiring step. While histamine release from rat peritoneal cells is also prevented in the cold, it has not been possible to demonstrate a calcium requirement for that reaction.44 In both systems the capacity to release histamine once antigen has been added decays rapidly whether or not the release of histamine actually occurs. Thus, when cells are reacted with antigen under conditions which do not permit the release of histamine (e.g., in the absence of calcium ion, in the cold, or in the presence of an inhibitor) , 6 0 y 6 1 it is found that the reestablishment of conditions which would ordinarily result in the release of histamine no longer brings about this response. The 'lanergicl' state which results from such an abortive antigenic challenge is referred to as a pharmacologic desensitization. While the mechanism of desensitization is not clearly understood, it is apparent that the biochemical sequence of events starting with the reaction of antigen with antibody and culminating with release of histamine must have a time limit built into it such that histamine release stops even when there still is histamine remaining in the cells. Similarly, if something intervenes to prevent histamine release, the timed reaction may proceed nevertheless, and once it has run its course no histamine release is possible anymore until the limiting step in the release mechanism has been "rewound." It is clear that drugs which are capable of causing desensitization are of considerable interest in the control of atopic conditions since their action is independent of the nature of the antigen or pharmacologic mediator causing the disease symptoms. The role of cyclic adenosine monophosphate, prostaglandins, and catecholamines in the regulation of mediator release - In 1936, long before the role of catecholamines in the control of adenyl cyclase was appreciated, Schild62 described the inhibition of the release of histamine from guinea pig lung by epinephrine. It remained for Lichtenstein and M a r g ~ l i s , ~ ~ however, to revive interest in this area. Data have now been accumulated

Chap. 22

Immediate Hypersensitivity

Bath

245

from various laboratories to document the fact that the cyclic AMP system is involved in the control of the release of pharmacologic mediators of anaphylaxis.51 ,58,64 1 6 5 , 6 6 Conditions which increase the level of cyclic AMP in the cells, such as stimulation of adenyl cyclase by catecholamines or inhibition of the cyclic AMP-destroying diesterase by methylated xanthines (such as aminophylline), markedly inhibit the release of mediators. The actions of agents of these two types can be shown to be synergistic. Conversely, the action of the catecholamines can be inhibited by the 6-adrenergic blocking agent, propranolol. Alpha-adrenergic agents (such as norepinephrine) combined with 6-adrenergic blockade increase the release of mediators. These effects have been demonstrated with chopped lung preparations from guinea pig, monkey and man, and with the rat peritoneal mast cell system. In all these systems, the catecholmines are active at concentrations in the range M. In the human leukocyte system, the catecholamines are only involved in the calcium independent, antigen-requiring phase of the release reaction5’. The ability of prostaglandins, and especially PGEl and PGE to relax bronchial smooth muscle has been recognized for some time.g7,68 It was thus of considerable interest when it was found that these compounds, in concentrations a s low as a few nanograms per rat, could inhibit the release of mediators as well.69 Similar effects have been reported for the human leukocyte system as well. 5 8 Drug mode of action studies

-

In view of the regulatory role for cyclic

AMP in the release of pharmacologic mediators, it is of interest to determine if any of the drugs which are being evaluated for the control of atopic disorders might interact with this system. The antifilarial drug , diethylcarbamazine, has been known to inhibit mediator release.57 By studying the effect of combinations of this drug with catecholamines, propranolol, and methylxanthines, it was shown that diethylcarbamazine apparently acts as an inhibitor of phosphodiesterase.64 Disodium cromoglycate, by contrast, could not be shown t o interact with any of the components of the cyclic AMP system, and it is therefore concluded that it must inhibit mediator release at a different step in the biochemical sequence leading to release.64 In fact, it has been suggested70 that this drug inhibits the action of the phospholipase A which has been postulated by HiSgberg and U v n F i ~ , to ~ ~ be involved in the release sequence. Effect of mediators on end organs - As already mentioned, histamine, serotonin, SRS-A and the kinins, all cause the contraction of smooth muscle when they are added to the tissue in vivo or in vitro in minute concentrations. The action of histamine can be inhibited by antihistamines, such as pyrilamine or chlorophenyramine and that of serotonin by serotonin antagonists such as lysergic acid and its derivatives .72 Specific inhibitors of SRS-A or bradykinin have not been described, although the action of these may be inhibited by a variety of non-steroida1 antiinflammatory drugs . 7 3 Caution is essential in interpreting results dealing with the inhibition of the action of mediators on smooth muscle. Not only is there a variation in the sensitivity of various organs of the same species to a given mediator, and a variation in sensitivity of

246 -

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Wechter, Ed.

t h e same organ from d i f f e r e n t s p e c i e s Y 3b u t even t h e i n h i b i t i o n of t h e a c t i o n of a given mediator on d i f f e r e n t smooth muscle p r e p a r a t i o n s can d i f f e r markedly from one system t o t h e n e x t . 7 4 There i s c o n s i d e r a b l e d i f f e r e n c e between s p e c i e s r e g a r d i n g t h e a n a t o m i c a l s i t e of t h e primary anaphylactic l e s i o n . I n the rat i t i s t h e i n t e s t i n e 7 2 while i n t h e guinea p i g and i n man i t i s t h e l u n g s , where t h e c o n t r a c t i o n of t h e b r o n c h i a l smooth muscle ("bronchospasm") causes s e v e r e d i f f i c u l t y i n b r e a t h i n g . Thus, as long a s t h e r e i s v a r i a t i o n i n r e s p o n s e between v a r i o u s muscle p r e p a r a t i o n s , human b r o n c h i a l smooth muscle should be t h e end organ of primary i n t e r e s t . J u s t as t h e r e l e a s e of pharmacologic m e d i a t o r s from m a s t c e l l s , t h e i r a c t i o n on smooth muscles i s c o n t r o l l e d i n p a r t by t h e 6-adrenergic system. It has been found, however, t h a t c e r t a i n B-adrenergic a g e n t s are r e l a t i v e l y more p o t e n t i n t h e i r a c t i o n on c a r d i a c muscle t h a n t h e y a r e on b r o n c h i a l smooth muscle, and v i c e versa, T h i s h a s l e d t o t h e p r o p o s a l t h a t t h e r e a r e two t y p e s of 6-adrenergic r e c e p t o r s , 6 1 and 42, t h e former being r e p r e s e n t a t i v e of t h e r e c e p t o r s on c a r d i a c muscle w h i l e t h e l a t t e r i s t h e r e c e p t o r found predominantly on b r o n c h i a l smooth muscle.75 Y e t o t h e r s u b t y p e s of B r e c e p t o r s may e x i s t , f o r example, on t h e m a s t cells.51 E f f o r t s are underway i n many l a b s t o c a i t a l i z e d on t h e d i f f e r e n c e s i n s p e c i f i c i t y of 81 and B2 but i t remains t o be s e e n i f t h e s e d i f f e r e n c e s a r e s u f f i c i e n t t o be t h e r a p e u t i c a l l y useful.

receptor^,^^^^^,^^

Beta-adrenergic blockade and asthma - Of t h e a t o p i c c o n d i t i o n s , asthma i s by f a r t h e most troublesome. It would be m i s l e a d i n g t o imply, however, t h a t r e a g i n a n t i b o d y i s t h e cause of a l l asthma. A c t u a l l y , many p a t i e n t s s u f f e r i n g from t h i s d i s e a s e do n o t have any demonstrable a t ~ p y . ~ ' Furthermore, even i n t h o s e a s t h m a t i c s where such a h y p e r s e n s i t i v i t y can be shown ( t h e s o - c a l l e d " e x t r i n s i c " a s t h m a t i c s ) , i t i s o f t e n p o s s i b l e t o demonstrate a much h i g h e r s e n s i t i v i t y of t h e b r o n c h i a l muscle t o t h e a c t i o n o f m e d i a t o r s and a lower s e n s i t i v i t y t o t h e i n h i b i t o r y a c t i o n of t h e catecholamines. An a d d i t i o n a l r o l e of c h o l i n e r g i c s t i m u l a t i o n i n t h e e t i o l o g y of asthma i s r e c o g n i z e d , b u t as y e t p o o r l y understood. These c o n s i d e r a t i o n s have l e d S ~ e n t i v a n y it o ~ ~p o s t u l a t e t h a t one of t h e c a u s e s of asthma may be a p a r t i a l 6-blockade. Thus, superimposed on t h e immunol o g i c mechanism of release of pharmacologic mediators and t h e p o s s i b i l i t i e s of s e e k i n g d r u g s t o c o n t r o l t h e s e , a g e n t s are needed which can reverse / t h i s a p p a r e n t B-blockade. A few animal models f o r s t u d y i n g such a g e b t s have been proposed. One of t h e s e stems from t h e o b s e r v a t i o n t h a t mice which have been t r e a t e d w i t h B. p e r t u s s i s v a c c i n e appear t o have a n i n c r e a s e d s e n s i t i v i t y t o h i s t a m i n e and o t h e r w i s e behave as i f they were p a r t l y B-blocked. A v e r y i n t e r e s t i n g new model h a s been desc r i b e d r e c e n t l y , c o n s i s t i n g of i n b r e d l i n e s of guinea p i g s having high and low P r o n c h i a l s e n s i t i v i t y t o pharmacologic m e d i a t o r s .*O The supply of t h e s e animals i s s t i l l v e r y s h o r t , b u t i t is t o be hoped t h a t c a r e f u l s t u d i e s b f t h e s p e c i f i c d i f f e r e n c e s between t h e s e strains w i l l p o i n t t h e way t o a b e t t e r understanding and e v e n t u a l c o n t r o l of t h i s a s p e c t of atopic disease.

Chap. 22

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Immediate H y p e r s e n s i t i v i t y

247

References 1.

2. 3.

4.

5. 6.

7. 8. 9. 10. 11. 12.

13 * 14. 15. 16. 17* 18. 19 9

20. 21. 22. 23 24. 25. 20. 27 28. 29.

-

30 * 31.

32 *

33. 34.

35. 36.

37* 38. 39. 40. 41. 42.

43

1

44. 45. 46.

47. 48.

L.H. Criep, Clinical Immunology and Allergy, 2nd Edition, Grune and Stratton, N.Y., 1969, p. 5. L.H. Criep, Advances in Immunology, g,62 (1971). E.L. Becker, loc. cit., p. 267. P.J. Piper and J.R. Vane, Nature, 3, 29 (1969). W.E. Brocklehurst, J.Physio1. (London), 151, 416 (1960). 602 (1971). A.B. Kay, D.J. Stechschulte and K.F. Austen, J.Exp.Med., 330 (1968). K. Ishizaka and T. Ishizaka, J.Allergy, S.G.O. Johansson and H. Bennich, Immunology, 2. 381 (1967). A. Prouvost-Danon and R. Binaghi, Nature, g , 66 (1970). D.J. Stechschulte, R.P. Orange and K.F. Austen. J.Immunol., 1082 (1970). K. Ishizaka, T. Ishizaka and M.M. Hornbrook, Immunochem., 1,515 (1970). C. Dobson, J.H. Rockey, E. J. L. Soulsby, J.Immuno1. , 1431 (1971). R. Patterson in P. Kallo’s and B.H. Waksman (editors), Progress & I Allergy, Karger, Basle, Volume 13, p. 332 (1969). R.M. Schwartzman, J.H. Rockey and R.E. Halliwell, Clin.Exp.Immunol., 2, 549 (1971) 924 (1969). R. Patterson and C.H. Talbot, J.Lab.Clin.Med., D.K. Hammer, B. Kickhafen and T. Schmid, Europ. J.Immunol., 1,249 (1971). Z. Ovary, Progress Allergy, 5, 459 (1958). N.R. Rose, J.H. Kent, R.E. Reisman and C.E. Arbesman, J.Allergy, 3,520 (1964). T. Ishizaka, K. Ishizaka, R.P. Orange and K.F. Austen, J.Imunol., 104,335 ( 1970 1. M.K. Bach, unpublished results. L. Perelmutter and A. Liakopoulou, Int.Arch.Allergy, g, 481 (1971). B. Kanyerezi, J.-C. Jaton and K.J. Bloch, J.Immunol., 106,1411 (1971). A . I . Terr and J.D. Betz, J.Allergy, 433 (1965). W.E. Parish, Lancet, 1970 11, 591 (1970). N.M. Vaz and Z. Ovary, J.Immunol., 100,1014 (1968). 951 (1963). Z. Ovary, B. Benacerraf and K.J. Bloch, J.Exp.Med., D.J. Stechschulte, K.F. Austen and K.J. Bloch, J.Exp.Med., 3, 127 (1967). G.H. Strejan and D . G . Marsh, J.Immunol., 306 (1971). R.J.M. Wilson and K.J. Bloch, J.Immunol., 100, 622 (1968). T.S. C. Orr, P. Riley and J.E. Doe, Immunol., 0 , 185 (1971). N.J. Zvaifler and J.O. Robinson, J.Exp.Med., Ijo,907 (1969). N.M. Vaz, E.M. Vaz and B.B. Levine, J.Immunol., 104,1572 (1970). B.B. Levine, H. Chang, Jr. and N.M. Vaz, J.Immunol., 106,29 (1971). H.G. Johnson and C.A. VanHout, J.Immuno1. (1972) in press. T. Tada and K. Okumura, J.Immunol., 1137 (1971). T. Tada, M. Taniguchi and K. Okumura, J.Immunol., 106,1012 (1971). K. Okumura and T. Tada, J.Immunol., 106,lOl9 (1971). M. Taniguchi and T. Tada, J.Immunol., 107, 579 (1971). G.J. White, unpublished observations. 1682 (1971). K. Okumura and T. Tada, J.Immunol., R.P. Orange, M.D. Valentine and K.F. Austen, Science, 318 (1967). 752 (1971). M.K. Bach, K.J. Bloch and K.F. Austen, J.Exp.Med., K. Ishizaka and T. Ishizaka, in B. Amos (editor), Advances Immunology, Academic Press, N.Y. (1971), p . 859. M.K. Bach, K.J. Bloch and K.F. Austen, J.Exp.Med., 772 (1971). A. Prouvost-Danon, M. Queiroz Javierre and M. Silva Lima, Life Sciences, 5 , 1751 (1966). M.K. Bach, J.R. Brashler, K.J. Bloch and K.F. Austen in K.F. Austen and E.L. Becker (editor), Biochemistry of the Acute-Allergic Reactions, Blackwell, Oxford (19711, P - 65. C.G. Cochrane in B. Amos (editor) Advances &Immunology, Academic Press, N.Y. (1971). P. 143. R.P. Siraganian and A.G. O s l e r , J.Immunol., 1244 (1971).

z,

z,

x,

x,

z,

6,

x,

m,

x,

x,

z, u, a,

s,

248 49.

Sect. V M. Aikawa, M . J .

-

Wechter, Ed.

Topics i n Biology

Schoenbechler, J.F.

B a r b a r o and E.H.

Sadun, Am.J.Path.,

(1971). 50

A.G.

O s l e r , L.M.

L i c h t e n s t e i n and D.A.

Levey, Advances i n Immunology,

9, 85

8,

183

(1968).

51. 52 *

53. 54.

55. 56 * 57.

E.S.K. Assem and H . O . S c h i l d , B r i t . J . P h a r m a c o l . , 2 , 620 (1971). P.A. S h o r e , A. B u r k h a l t e r and V . H . Cohn, J.Pharm.Exp.Therap., 111,182 (1959). L.T. Kremzner and I . B . W i l s o n , Biochim. Biophys. A c t a , 364 (1961). M.K. Bach, u n p u b l i s h e d o b s e r v a t i o n s . R . H a s t i e , Clin.Exp.Immunol., 8, 45 (1971). B. UvnYis i n K.F. Austen and E.L. Becker ( e d i t o r s ) B i o c h e m i s t r y of t h e AcuteA l l e r g i c R e a c t i o n s , B l a c k w e l l , O x f o r d , 1 9 7 1 , p . 175. R.P. Orange, M.D. V a l e n t i n e and K.F. Austen, Proc.Soc.Exp.Biol.Med., 127

2,

x,

(1958).

58 59. 60.

61. 62.

63. 64.

65. 66.

67. 68.

L.M. L i c h t e n s t e i n and R. DeBernardo, J.Immunol., 107, 1131 (1971). L.M. L i c h t e n s t e i n , J.Immunol., 107, 1122 (1971). J.S.G. Cox, J . E . Beach, A . M . J . N . B l a i r , A . J . C l a r k e , J. King, T.B. L e e , D.E.E. Loveday, G.F. Moss, T.S.C. Orr, J . T . R i t c h i e and P. S h e a r d , Advances i n Drug R e s e a r c h , 2 , 115 ( 1 9 7 0 ) . M. Lopez and K . J . B l o c h , J . I m m u n o l . , l o j , 1428 (1969). H.O. S c h i l d , Q u a r t e r l y J . E x p . P h y s i o l . , 165 ( 1 9 3 6 ) . L.M. L i c h t e n s t e i n and S. M a r g o l i s , S c i e n c e , 902 (1968). W . J . Koopman, R . P . Orange and K.F. A u s t e n , J.Immunol., 1096 ( 1 9 7 0 ) . L . J . L o e f f l e r , W. Lovenberg and A . S j o e r d s m a , Biochem.Pharmacol., 20, 2287 ( 1 9 7 1 ) . M . A . K a l i n e r , R . P . Orange, W.J. Koopman, K.F. Austen and P . J . L a r a i a , Biochim. Biophys. A c t a , 160 ( 1 9 7 1 ) . J . R . Weeks, J . R . S c h u l t z and W.E. Brown, J . A p p l . P h y s i o l . , 3,783 (1968). M.E. R o s e n t h a l e , A. D e r v i n i s , A . J . Begany, M. Lapidus and M . I . Gluckman, E x p e r i e n t i a , &, l l l 9 ( 1 9 7 0 ) . W. J . Koopman, R . P . Orange and K.F. Austen, Proc.Soc.Exp.Biol,Med., 64 (19'71). T.S.C. Orr and J.S.G. Cox, N a t u r e , 223, 197 (1959). B. Hugberg and B. Uvnas, Acta P h y s i o l . S c a n d . , 345 ( 1 9 5 7 ) . K.F. Austen and J . H . Humphrey, Advances i n Immunology, 2. 1 (1963). P.A. B e r r y and H . O . J . C o l l i e r , B r i t . J . P h a r m a c o l . , 3,2 0 1 ( 1 9 6 4 ) . M.E. G r e i g , p e r s o n a l communication. A . M . Lands, A . A r n o l d , J . P . M c A u l i f f , F.P. Luduena and T.G. Brown, J r . , N a t u r e ,

6, 161,

105,

E,

x,

5,

76. 77. 78. 79. 80.

2111, 597 (1967).

S.R. O'Donne11, E u r o p . J . P h a r m a c o l . , 12, 35 ( 1 9 7 0 ) . J . R . P a r r a t t and R.M. Wadsworth, B r i t . J . P h a r m a c o l . , 2 , 296 ( 1 9 7 0 ) . B. Levy, J.Pharmaco1. and E x p - T h e r a p . , 413 ( 1 9 6 6 ) . A. S z e n t i v a n y i and C . W . F i s h e l , i n G . F i l l i p p ( e d i t o r ) P a t h o g e n e s e 5T h e r a p i e A l l e r g i s c h e r R e a k t i o n e n , F e r d . Enke, S t u t t g a r t , 1 9 6 6 , p. 588. Y. T a k i n o , K. S u g a h a r a and I . H o r i n o , J . A l l e r g y , 247 (1971).

a,

a,

249 Chapter 23,

Transition State Analogs a s Enzyme Inhibitors

G. E. Lienhard, Dept. of Biochem., Dartmouth Medical School, Hanover, N. H,

-

Introduction This review describes a general approach for the design of specific, potent reversible enzyme inhibitors, The inhibitors that a r e prepared according to this approach a r e called transition state analogs. A transition state analog for an enzyme is a compound that resembles (or, in certain special cases, is easily capable of resembling) in structure the substrate portion of the transition state of the enzymatic reaction, The review is divided into two sections, a theoretical section in which the basis for the inhibitory potency of transition state analogs is explained and an experimental section in which the types of transition state analogs that have been prepared are described. A review of this subject by Wolfenden has recently been published. Theory Enzymatic Reactions of Single Substrates - Scheme 1 describes a single substrate enzymatic reaction and the corresponding nonenzymatic reaction in terms of the transition state theory of kinetics. E , S, P, ES, E P , ES*, and

KS

___)

Jr ES &

E + P

11 ES*

__$

(Scheme 1)

EP

+I

S represent the free enzyme, the substrate, the product, the enzymesubstrate complex, the enzyme-product complex, the transition state of the enzymatic reaction, and the transition state of the corresponding nonenzymatic reaction, respectively. The transition states a r e defined a s the structures of highest energy in the interconversion of S and P and of ES and EP, I$ and KZ a r e the equilibrium constants for formation of the transition states o the nonenzymatic and enzymatic reactions, respectively; K is the is t t e association constant for formation of ES from E and S; and association constant for the hypothetical reaction, the binding of S+' to E. The constants in scheme 1 a r e related according to the expression Since in transition state theory2 the equilibrium constant for transition state is equal to the rate constant of the reaction times the constant h/kT (k is Boltzmann's constant; h is Planck's constant; T is the absolute temperature), this expression becomes = Ksk where k is the first-order rate constant for the transformation opthe E enzyme-substrate complex to enzyme-product complex and k is the firstorder rate constant for the corresponding nonenzymatic rea8ion. For a

yr

4,

250 -

Sect, V

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i n Biology

Wechter,

Ed.

'.

typical enzymatic reaction the ratio k /k is 1O1O o r more', Thus the transition state, S", is predicted to fin$Jto the enzyme at leasts -1 more tightly than the substrate. This analysis of enzymatic catalysis in terms of transition state theory does not give any information about the degree of similarity between the structure of the substrate portion of E d and the structure of S*', However, it is generally the case that the mechanism of an enzymatic reaction and the mechanism of the corresponding nonenzymatic reaction a r e similiar in so far as the basic bond-making and bond-breaking steps.3 Thus, an analog of the substrate portion of ES* (a transition state analog) is generally also an analog of s*'. Consequently, a transition state analog for such an enzyme should bind to the enzyme much more tightly than the substrate. The fact that the affinity of an enzyme for is so enormous relative to its affinity for S suggests that even a crude transition state analog should be a potent inhibitor. In addition, it can that even if the strucbe shown, by arguments which a r e given elsewhere, ture of the substrate portion of ESS and the structure of S*' a r e not similar, a transition state analog should still bind to the enzyme much more tightly than the substrate. '9

Enzymatic Reactions of Two Substrates That Proceed Via a Ternary Complex Scheme 2 describes, in terms of transition state theory, an enzymatic reaction between two substrates (S1, S2) and the corresponding nonenzymatic reaction. The enzymatic reaction is one that proceeds by a direct reaction between the two substrates that occurs through a ternary complex (ES1S2) of E, S1 and S2.

E+S,+S

4E + P

2-

ES1 + S

(Scheme 2)

2

KS2

Jr ES*

%=

' *

__$

EP

In t h i s case K21Ksa K E / S = Ks1K s';k kF/%. This relationship differs from that ase on cheme 1 only by su stitu ion of a product of association constants for a single association constant and by the dimensions of kN, which is here a second-order rate constant. Since k /k is often 1O1O g o r larger and K and K a r e typically l o 4 M-l (except trfJe case of H 0 as s2 2 a substrate'), K? is immense, and very tighcbinding of an analog of the T substrate portion of ES* is expected.

251 -

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Enzyme I n h i b i t o r s

Chap. 23

It is worth noting here that Scheme 2 shows that potent inhibitors for enzymes that catalyze two substrate reactions a r e single molecules that incorporate in an appropriate arrangement the primary binding determinants of S 1 and S but lack a functional group that is similar to the structure of the atoms 2 undergoing reaction in the transition state. The association constant for the binding of such inhibitors would be expected to be e ual to the product of K times a factor that could be as large as 10 , This factor is due that the binding of the S portion of the inhibitor is an intramolecular reaction within an oriented compfeex, whereas the binding of S itself is inter2 molecular (Scheme 3 ) , 5 , 6 These inhibitors may be more simllar in structure

%

/

E

S

1

+

Ks2 s2

E

&'

/ (Scheme 3)

\ s2

E

E

\

I

,

where S

1

- S2

= inhibitor

*

to the substrate portion of ES S than to that of ES and may be called multiHowever! i%ey a r e also crude transition state analogs, substrate analogs. since the initial step in the progression to the transition state of both the enzymatic and nonenzymatic reactions is the coming together of the substrates, Types of Transition State Analogs The design of a transition state analog for an enzyme requires a knowledge of the mechanism of the enzymatic reaction o r at least an understanding of the possible mechanisms. Fortunately, our knowledge of the mechanisms of enzymatic reactions and of the corresponding nonenzymatic reactions that may be models for the enzymatic one^^,^ is sufficiently detailed so that the main structural features of the transition state of almost any enzymatic reaction a r e either known or can be postulated with some confidence, In the design of a transition state analog, it is useful to focus attention upon the metastable intermediate(s), such a s a carbanion o r carbonium ion, that occurs in the mechanism, An analog of a metastable intermediate is, in fact, a crude transition state analog, since a metastable intermediate and the transition state from which it is formed resemble each other in structure and energye8 Because enzymatic reactions fall into classes of reactions with similar mechanisms, a transition state analog for one particular enzymatic reaction should be able to be variously modified to give analogs for the other enzymes in the same mechanistic class. The substituents attached to the functional group that resembles the reacting atoms in the transition state are simply changed to those that satisfy the substrate specificity requirements

252

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i n Biology

of each enzyme within the mechanistic class. The examples of transition state analogs that a r e described below have been chosen a) because they appear to resemble the postulated transition state and b) because they appear to bind to the enzyme much more tightly than the substrate(s). It should be emphasized that in most of the examples the structures of the transition state and of the corresponding enzyme-transition state analog complex a r e reasonable postulates rather than verities established through extensive experimentation. The inhibitory potency of the analogs (P) has been expressed a s the ratio of the association constant for binding of the analog to the enzyme to that for binding of the substrate (K ). In many cases, the value of K has not been determined; and the reciproca?of the Michaelis S constant, a kinetic parameter whose value is equal to the substrate concentration required for half-maximal velocity of the enzymatic reaction,' has been used in its place.

-

Analogs of tetrahedral transition states Adenosine deaminase catalyzes the hydrolysis of adenosine (1) to inosine (3), probably via a tetrahedral-like transition state formed by the attack of water (2). 1,6-Dihydro-6-hydroxymethylpurine (4) resembles 2 by virtue of the H on N5, the tetrahedral geometry of C6, and, in one conformation the position of hydroxyl group. The value of P for one isomer of 4 is 40.1' The deamination of cytosine

+

HO \

R 1

2

3

4

R = P-D-ribofuranosyl nucleosides (5) by enzymes from various sources 11,l 2 is markedly inhibited by 3,4,5,6-tetrahydrouridine (6), with P for cytidine deaminase from l3. being 830. l2 Although it is reasonable to hypothesize that the transition state for this reaction is similar to 2 and so accounts for the inhibition by 6, the possibility that a nucleophilic group of the enzyme adds reversibly to the dehydrated form of 6, which may form readily from 6 (7,8),l3 should also be considered. If the enzymatic reaction proceeds by a double displacement via 4-pyrimidinyl enzyme intermediate (9), 8 is analogous to the tetrahedral transition states for the formation and hydrolysis of 9 (see the description of chymotrypsin), Chymotrypsin is one of a class of enzymes that catalyze the transfer of acyl groups (R-C = 0) between various acceptors (XR') by way of a covalent

Chap. 23

NH2

HO

H

H ? N D

N>

1 ' 1

0-

0-

I R

5

J5

9>): 33 EX

.+EXH

,

EX

H

0-N

0-N

I

253

L ienha r d

Enzyme I n h i b i t o r s

0-N

I

I

I

R

R

R

R

6

7

8

9

R = P-D-ribofuranosyl,

EXH = enzyme

acyl-enzyme intermediate (10-12). In the case of chymotrypsin, the acylenzyme (12) is formed from the hydroxyl group of a seryl residue (CH OH), with the participation of the imidazole group of a nearby histidine resi8ue (N) in proton transfers.l4 The transition states for the acyl transfer reactions a r e probably very similar to the tetrahedral intermediate represented by 11. CH2-0-C /R R R CH2 CH2 E

/

\

\

I

0 /

C = O I

+ E

XR'

.eH

'

\

'GH

N' 10

-

I 0 - C - 0 I

/

+O

f E

XR' 12

11

XR' = O R , OH, NHR A possible transition state analog for chymotrypsin is 2-phenylethaneboronic 15 acid (13), which is an effective competitive inhibitor of the enzyme (F = 125). Since the trigonal, planar boronic acids a r e known to ionize in dilute base by the addition of hydroxide ion to form anionic tetrahedral adducts (14), the seryl hydroxyl group of chymotrypsin may add to the boron in the same way to yield a structure (15) similar to 11. The 2-phenylethyl group satisfies the

/

R-B

OH

'

OH

13

R

-

OH I B-- OH I OH

14

CH ' 2\ E

'

IfH

R 1-

0-B-OH I

OH

15

R = PhCH CH 2 2specificity of chymotrypsin for an aromatic side chain. 14'16 Other boronic acids are also inhibitors of chymotrypsin, but they are less effective than 13, 16,l7 The structures of the complex that is formed between chymotrypsin and 13 is, by no means, proven to be 15; several other possibilities have been hypothesized.15-17 It should be pointed out that if 15 is correct, this type of transition state analog is unusual because it forms a covalent bond

254

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with the enzyme and so has a different structure in the complex than in solution, A method for predicting the maximal possible affinity of an enzyme for such an analog is described in reference 15. Penicillin has been suggested to be a transition state analog as well as a covalent inhibitor of peptidoglycan transpeptidase, l8 The basis of this suggestion is the fact that the p-lactam function of penicillin is not planar. Thus, it is possible for the spatial relationships among the atoms of the tetrahedral intermediate that is formed from the susceptible planar peptide bond in the substrate to be similar to those among the atoms of penicillin. Since it appears that subsequent to the noncovalent interaction between penicillin and the transpeptidase, virtually irreversible acylation of the active site SH function by the lactam group occurs, it will probably be very difficult to determine the association constant for the initial noncovalent binding of penicillin to transpeptidase. Thus, it may not be possible to determine whether the criterion that a transition state analog binds more tightly than the corresponding substrate is satisfied here, In any event, this hypothesis for the action of penicillin illustrates a useful general approach for the design of very specific and potent irreversible enzyme inhibitors, which is to devise structures that are both transition state analogs and aminoacid-modifying reagents. 1 Other possible analogs, which will only be mentioned here, a r e Lmethionine- (S)-sulfoximine phosphate and L-benzylsuccinate; these compounds bind tightly to glutamine synthetase and carboxypeptidase, respectively and can resemble to some extent the tetrahedral-like transition states. 19, io Analogs of Carbanion-Like Transition States - There is a similarity between the structure of an enolate anion (16) and a carboxylate group (17), and it has been found that several enzymatic reactions in which the transition state probably resembles an enolate anion are strongly inhibited by the analogous carboxylate anion. The most thoroughly studied analog of this type is

\cJ.rc.c-4

/

0

0

-2

OLL'C

\ 16

\ 17

0

li CLIJO

I

C H 2 0 PO;

18

H-C-OH

cr :s- o I CH2OPO;

19

phosphoglycolic acid (18) which resembles the metastable enediolate intermediate (19) that occurs in the interconversion of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate catalyzed by triose phosphate isomerase, 21 The value of P for phosphoglycolate varies with pH; at pH 7 it is 75. Another example of this type may be oxalate (21), which is a potent inhibitor of an Mn++-requiring enzyme (P = 300)22, 23 that decarboxylates oxaloacetate (20). Oxalate is also an effective inhibitor (P = 200) of the Mn-containing enzyme

Enzyme I nh i b i t o r s

Chap. 23

255 -

L ienha rd

24 that catalyzes the transfer of this carboxyl group from oxaloacetate to biotin and may also act here as a transition state analog, although the investigators propose an alternative mechanism for carboxyl transfer. CH2

0

-

C

0

.'.&

c02

/.,

\. 0

I

E 20

*

1

0

0

-

*$

C

,y 0

-c

& -

\ 0

++

Mn I

E

21

The enzyme acetoacetate decarboxylase catalyzes the decarboxylation of acetoacetate by way of a Schiff base formed with the €-amino group of alysyl residue of the enzyme (22). Acetylacetone is a potent inhibitor of the enzyme (l =? 10, 000), probably because it can easily form an enamine with the enzyme (23) that resembles the transition state. 25 Other examples of transition state

22

23

analogs for planar, carbanion-like transition states, which a r e described in detail elsewhere, are pyrrole-2-carboxylate and 17-p-dihydroequilenin; these compounds resemble the transition states that are postulated for the enzymatic racemization of proline and isomerization of A5-3-ketosteroids, respectivelx26

Analogs of Carbonium-Ion Like Transition States - Most glycosyl-transferring enzymes are strongly inhibited by the 4-lactone (25) analogous to the corresponding substrate (P as large as 10,000),27-29 The explanation is probably that the transition states for these reactions are similar to alkoxycarboniwn ions (24), which like the lactones, have a half-chair conformation and positive charge on the ring oxygen atom and carbon 1 of the ring. Norjirimycin, an antibiotic that differs from glucose only by the substituion of an NH group for

24

25

26

27

oxygen in the ring, can exist in a form that is an even better analog of 24 (26) and has been found to be an even better inhibitor of glucosidases than Dglucono-$-lactone. 30 It appears that in certain cases a relatively planar conformation of the ring is a sufficient similarity to 24 to allow tight binding,

256

Sect. V

7

-

Topics i n Biology

Wechter, Ed,

since p-galactosidases are inhibited (P = 3-160) by D-galactal (27).

31

Analogs of Transition States for Phosphoryl Transfer - The transition states for most enzymatic phosphoryl transfer reactions a r e probably very much like a trigonal bipyramid of pentacovalent phosphorus, with the entering and leaving groups 6,Y) in apical positions (28). A complex between oxovanadium ion (IV) and uridine binds very tightly to the enzyme ribonuclease (P = 1700),

-

U

Y HO,

1

-

-

*

U

HoH2c%

I

-

0,I

HOY7-O

X 28

OH2 29

U

L

OH2 30

= uracil

probably because the complex has a structure (29) that mimics the transition state for ribonuclease-catalyzed hydrolysis of uridine-2', 3'-cyclic phosphate (30).4 The enzyme that catalyzes the transfer of a phosphoryl group from adenosine triphosphate to creatine forms a very stable quarternary complex with adenosine diphosphate (ADP), creatine, and nitrate (or similar anions). This case appears to be an example of the organization on the enzyme of a transition-state-like complex from several compounds, which is possible here due to the similarity of NO- and the base of the phosphorus trigonal 3 bipyramid (31). 32 ADP-0

Analogs For Multisubstrate Reactions - N- (Phosphonacety1)-Laspartate (32) incorporates features of both substrates of the enzyme N-creatine aspartate transcarbamylase (33), although it does not contain a 31, Z = P for the transition state tetrahedral functional group analZ = N for the analog complex ogous to the tetrahedral-like transition state that is expected for this carbamyl transfer reaction (34). The association constant for bindin of this compound to aspartate transcarbamylase is very large (4 x 107 g-').53 The virtually irreversible inhibition of the enzyme that catalyzes the transfer

of an acetyl group between the thiol group of coenzyme A and the hydroxyl group of carnitine by a compound in which coenzyme A and carnitine a r e appropriately linked together (35) appears to be another example of this type

Chap. 23

Enzyme I n h i b i t o r s

Lienhard

257

of analog. 34 Multisubstrate analogs haye also been prepared for nicotinamide adenine dinucleotide-dependent enzymes" and for ribulose diphosphate carboxylase. 36 i 0 II C CH2POi

0 II

-

H2N-C-OPOg

+

I

RNH

RNH2 33

32

RNH2 34

R = -CH(CO-)CH co2 2 2

Conclusion - There a r e many other strong inhibitors of 0 2 C C H CHO enzymes that have not been 21 \\ CoA discussed here. The explanaYH2 CH2 tion for the effectiveness of some of these compounds w i l l almost certainly be found in 35, CoA = coenzyme A their resemblance to the transition states of the enzymatic reactions that they inhibit. The application of the transition state theory of kinetics to enzymatic catalysis has provided both an explanation of why many potent inhibitors of enzymes a r e potent inhibitors and a basis for the design of new potent inhibitors. 0

-

-

It C

-

-

The author is indebted to Dr, Kim Collins for his aid in Acknowledgment searching the literature. References 1. 2.

3, 4.

5. 6. 7. 8.

5, 10 (1972). K, J, Laidler, Theories of Chemical Reaction Rates, McGraw-Hill, New York, N. Y., 1969, Chapter 3, W, P, Jencks, Catalysis in Chemistry and Enzymolog& McGraw-Hill, New York, N. Y., 1969. G . E, Lienhard, I. I. Secemski, K, A. Koehler, and R. N. Lindquist, Cold Spring Harbor Symposia on Quantitative Biology, XXXVI, 45 (1971). D, E, Koshland, Jr., J. Theor, Biol., 2, 75 (1962). M. I, Page and W. P, Jencks., Proc, Nat. Acad. Sci. 68, 1678 (1971). T, C, Bruice and S , J, Benkonic, Bioorganic Mechanisms, W. A. Benjamin, New York, N. Y., 1966. G. S, Hammond, J. Amer. Chem. Soc., 77, 334 (1955). R. Wolfenden, Acc, Chem. Res.,

Sect. V

9.

15. 16.

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

36.

- Topics

i n Biology

Wechter, Ed.

S. A. Bernhard, The Structure and Function of Enzymes, W. A. Benjamin, New York, N. Y., 1968, Chapter 4. B, Evans and R, Wolfenden, J. Amer, Chem, Soc., 92, 4751 (1970). G, W. Camiener, Biochem, Pharmacol., 17, 1981 (1968). R, M, Cohen and R, Wolfenden, J. Biol. Chem,, 246, 7561 (1971). A, R. Hanze, J, Amer. Chem. Soc,, 89, 6720 (1967). D, M, Blow in The Enzymes, 3rd e d , , P, D, Boyer, Ed., Academic Press, New York, N. Y. 1971, Volume 111, Chapter 6. K, A. Koehler and G. E. Lienhard, Biochemistry, 10, 2477 (1971). V. K, Antonov, T, V, Ivanina, A. G. Ivanova, I. V. Berezin, A. V, Levashov, and K. Martinek, FEBS Lett,, 20, 37 (1972). M. Philipp and M. L, Bender, Proc, Nat. Acad. Sci., 68, 478 (1971). B. Lee, J. Mol. Biol,, 61, 463 (1971). J. D, Gass and A. Meister, Biochemistry, 9, 1380 (1970). L, D, Byers and R. Wolfenden, J. Biol, Chem., 247, 606 (1972). R, Wolfenden, Biochemistry, 9, 3404 (1970). A. Schmitt, I, Bottke, and G. Siebert, Hoppe-Seyler's Z. Physiol. Chem, , 347, 18 (1966). G, W. Kosicki and F, H. Westheimer, Biochemistry 1, 4303 (1968). A, S. Mildvan, M, C, Scrutton, and M. F, Utter, J. Biol, Chem., 241, 3488 (1966). I. Fridovich, J, Biol, Chem,, 243, 1043 (1968). Reference 3, page 300. D, H. Leaback, Biochem. Biophys. Res, Comm., 32, 1025 (1968). I, I. Secemski andG, E, Lienhard, J. Amer, Chem, SOC,, 93, 3549 (1971). A. M, Gold, E, Legrand, and G. R, S h c h e z , J. Biol. Chem., 246, 5700 Q971). E. T, Reese, F. W. Parrish, and M, Ettlinger, Carbohyd, Res., Is, 381 (1971). Y. C, Lee, Biochem, Biophys, Res, Comm., 35, 161 (1969). E, J, Milner-White and D, C. Watts, Biochem. J., 122, 727 (1971). K, D, Collins and G. R. Stark, J. Biol, Chem,, 246, 6599 (1971). J, F, A, Chase and P, K, Tubbs, Biochem. J., 111, 225 (1969). J, Everse, E. C. Zoll, L, Kahan, and N. 0, Kaplan, Bioorg. Chem., 1, 207 (1971). M, Wishnick, M, D. Lane, and M, C, Scrutton, J, Biol. Chem., 245, 4939 (1970).

-

-

Section 6 Editor:

-

Topics i n Chemistry

Robert A. Wiley, U n i v e r s i t y o f Kansas, Lawrence, Kansas Chapter 24.

Biopharmaceutics and Pharmacokinetics

L e s l i e Z. Benet, School o f Pharmacy U n i v e r s i t y of C a l i f o r n i a , San Francisco Biopharmaceutics - The r e l a t i o n s h i p between t h e physicochemical prop e r t i e s of a drug i n a dosage form and the b i o l o g i c a l response observed f o l l o w i n g i t s a d m i n i s t r a t i o n i s the s u b j e c t o f biopharmaceutical studies. The a b i l i t y t o c a r r y o u t i n v i t r o studies on t h e dosage form, which show i n v i v o c o r r e l a t i o n s w i t m i c a l a v a i l a b i l i t y i s a fundamental and has r e s u l t e d i n an e x c e l l e n t "Guide f o r Biopharmaceutical Studies i n Man" pyepared by a committee o f t h e APhA Academy o f PharmaI n addition, three have been published i n c e u t i c a l Sciences the l a s t year d e a l i n g w i t h biopharmaceutics and r e l a t e d pharmacokinetics. I n v i t r o d i s s o l u t i o n studies were shown t o c o r r e l a t e w i t h i n v i v o a v a i l -measurements f o r commercial acetaminophen t a b l e t s m i f f e r e n t timed release n i t r o l y c e r i n tablet^^'^, d i f f e r e n t formulations o f s u l phadimidine tablets!, and f i v e d i f f e r e n t c r y s t a l l i n e phases o f f l uprednisol oneg.

.

demonstrated a strong c o r r e l a t i o n between i n v i t r o Wagner e t a1 .lo d i s s o l u t i o n studies and i n v i v o blood l e v e l s f o l l o w i n g o r a l dosing o f f o u r d i f f e r e n t c o m n e r c i a l s o f w a r f a r i n t a b l e t s , There were marked i n t e r s u b j e c t and i n t r a s u b j e c t v a r i a t i o n s i n t h e t e r m i n a l h a l f - 1 i v e s o f warfarin, which r e q u i r e d the authors t o make some assumptions concerning e i t h e r constant clearance o r constant volumes o f d i s t r i b u t i o n from study t o study when r e l a t i v e a v a i l a b i l i t i e s were determined, since no I . V . doses were given t h e p a t i e n t s i n t h i s r e p o r t . The authors chose t o assume t h a t t h e volume o f d i s t r i b u t i o n was constant and although r e l a t i v e a v a i l a b i l i t i e s o f t h e f o u r dosage forms would be maintained i f constant clearance were assumed, t h i s problem should receive a t t e n t i o n i n f u t u r e studies when varying h a l f - l i v e s a r e observed. Workers a t t h e Food and Drug D i r e c t o r a t e o f Canada published a s e r i e s o f a r t i c l e s p o i n t i n g o u t t h e inadequacy o f using a s i n g l e d i s s o l u t i o n t e s t t o p r e d i c t r e l a t i v e b i o a v a i l a b i l i t y f o r an e n t i r e s e r i e s o f comnercial products. Studies d e a l t w i t h s u l f a d i a z i n e ' l, phenylbutazone12,13 and nitrofurantoin"+. Chiou and R i e g e l ~ n a nreviewed ~~ t h e pharmaceutical a p p l i c a t i o n s of s o l i d d i s p e r s i o n systems. These authors found complete and r a p i d abs o r p t i o n o f g r i s e o f u l v i n dispersed i n polyethylene g l y c o l 6000 f o r both capsule and t a b l e t dosage forms, i n c o n t r a s t t o i r r e g u l a r and incomplete absorption from commercially a v a i l a b l e micronized drug. l6 Munzel l7 reviewed various f o r m u l a t i o n v a r i a b l e s which can i n f l u e n c e drug a c t i o n . Kakemi and co-workers18 s t u d i e d t h e e f f e c t s o f b u f f e r components, osmotic

260

Sect. V I

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Wiley, Ed.

i n Chemistry

pressure, injection volume, buffercapacity and pH of formulated intramuscular injections. Low osmotic pressure and low pH solutions yielded decreased absorption of nonionized d r u g s due t o morphological changes in the muscle. Miller and Fincherl9 demonstrated an inverse correlation between area under the blood level time curve and the particle size of phenobarbital i n intramuscular suspensions given t o beagle dogs. Cressman and Sumner20have shown t h a t purebred beagle dogs may serve as a good model for evaluating the bioavailability of aminorex fumarate from sustained-release tablets. Hanselmann and VoigtZ1 reviewed the literature on prolonged action drugs. White e t a1.22 showed t h a t oral administrat i o n of digoxin with and without m m a d l i t t l e effect on the bioavailability of the drug. Although the areas under the plasma curves for the. f i r s t eight hours appear different, the d r u g has such a long half-life t h a t when total areas t o infinite time are compared, no difference would be expected, as was confirmed i n a four week multiple dosing study. Lindenbaum e t a l Z 3 found marked variances i n the blood levels of digoxin during the f i r s t five hours a f t e r oral administration of four commercial products. Unfortunately they d i d not take blood samples beyond five hours and therefore i t i s possible t h a t no differences in biologic availability between the products exist, Since the study concerns a d r u g w i t h a long half-life which i s given repeatedly, availability of the drug i s the parameter which i s important clinically, not i n i t i a l blood levels, The study i s poorly designed since availability can only be inferred by assuming absorption i s complete in five hours.

-

Physiologic factors and d r u g interactions may also affect the extent of drug absorption. Nightingale and co-workersZ4 found t h a t stimulation of bile flow in the rat increased the gastrointestinal absorption of sulfadiazine, while Winne25 described two different four compartment models which relate intestinal absorption t o blood flow. The relative amount of drug absorption from the stomach was shown t o be negligible compared t o intestinal absorption for digitoxin26, sul famethoxazoleZ7, and cephalothin30. Kojima e t a1 3 1 found ~ a r f a r i n ~ *cephaloridine *~~, t h a t the presence of food decreased the pharmacological activity of phenobarbital by decreasing the rate of absorption in the rat and t h a t this decreased absorption rate i s due primarily to slowed gastric emptying, Bianchine e t a1.32 correlated the pharmacologic effect of L-dopa t o i t s blood l e v e f i d pointed o u t the marked effects o f stonach cn:Fi;yir,s on the rate and extent of availability o f this drug. In a d d i t i o n , a number of drug-drug interactions may be explained as a result of altered stomach emptying rates which can in turn influence the rate and extent of bioavailability. Hayton and Levy33 showed t h a t SKF 525A decreased the absorption rate of o r a l l y administered sulfacetamide by inhibiting stomach emptying. I t i s suggested t h a t the tricyclic anti-depressants delay oxyphenbutazone absorption34 by the same process, while barbital probably increases the absorption rate of aminopyrine in the rabbit by increasing the stomach emptying rate. 3 5

.

Antacids are a class o f over-the-counter drugs which might markedly affect the bioavailability of another d r u g . For example, Hurwitz and

Chap. 24

B iopharmaceu t i c s ,

Pha rmacoki net i c s

Benet

26 1 -

S h e e h a r ~showed ~~ t h at although co-administration of antacids containing A1 ion slowed the r a t e of pentobarbital absorption and delayed i t s pharmacologic e f f e c t , the t o t al amount of drug absorbed was unaffected by the antacid. Hurwitz3' al s o studied the e f f e c t s of antacids on sulfadiazine and quinine, noting changes i n stomach emptying time and solubility. Barr e t a1 3 8 demonstrated the decreased a v a i l a b i l i t y (approx. 50%) o f t e t r a cyc'line from corrmercial capsules when 2 g . of sodium bicarbonate is given a t the same time. When tetracycline was dissolved prior t o administration, no differences i n a v a i l a b i l i t y were observed w i t h o r without concomitant administration o f sodium bicarbonate, indicating that the dissolution r a t e step i s involved i n the decreased absorption of tetracycline capsules. Robinson e t al!9 found t h a t concomitant administration o f a b u l k forming colloid, m i u m hydrophyllic mucilloid o r an antacid w i t h warfarin, decreased the a v a i l a b i l i t y of the drug, while cholestyramine decreased warf ar i n blood levels apparently by b i n d i n g the d r u g and decreasing absorption.

.

Since the majority of drugs a r e given o r a l l y , much work has been directed toward understanding the mechanisms of intestinal absorption and attemptin to modify the drug o r dosage form so as t o improve absorption. Taraszka 4% found t h at the t ra n s fe r r a t e across the i n v i t r o r a t intestine f o r a clindamycin analogue could be f a i r l y well predicted by assuming that only unionized molecules of the d r u g were transferred and by correcting clindamycin t r a n s fer rat e s f o r the difference i n PKa of the analogue. However, Crouthamel e t a1 . ' + l reported significant transport of ionized sulfaethidole and baFbital from the r a t i n t e s t i n e , and very reduced absorption of drug from the stomach a t pH values ranging from 3.5 t o 8.3. Benet e t a1.42 and Morishita and c o - ~ o r k e r sstudied ~~ the e f f e c t of buffer components on the absorption of drugs, while Mayersohn and co-workers studied the influence of various sugars44, hy otonic and hypertonic solut i o n ~ and ~ ~ h, i g h potassium ion concentratione6 on passive drug t r a n s f e r across the everted r a t intestine. This work and the study of Nayak and Benet47 on d r u g t r a n s f e r across r a t i ntestinal musculature a f t e r EDTA treatment ar e consistent w i t h a proposed mechanism whereby polar compounds a re assumed t o traverse the isolated, everted r a t intestine via i n t e r c e l l u lar channels existing between adjacent mucosal epithelium c e l l s .

Drug transport and a v a i l a b i l i t y have also been measured f o r routes of ~~ administration other than o r a l . Rectal absorption of a c e t a m i n ~ p h e nand ~ a l i c y l a t $ ~buccal ~, absorption o f barbituatesS0 and a series of n-a1 kaooic acidss1, peritoneal d i a l y s i ~ ~ ~ -penetration ~'+, across the vitreous b a r r i e r of the eyes5, and the significance of vehicle composition i n the skin penetration of fluocinolone a c e t ~ n i d were e ~ ~a l~l ~s t ~u d i e d . Lymphatic absorption of digitoxin, d i g 0 x i t - 1 ~and ~ iopanoic acid59 was found t o be insignificant. Lucas e t a1 . 6 0 experimentally confirmed that compounds administered by intraperi toneal injection were absorbed primarily through the portal ci rc u l a t i o n , while Bederka and co-workers61 found that absorption of drug into the circulation following an i . m . injection was much more sensitive t o changes i n blood flow to the muscle than t o molecular weight of the absorbed species.

262

Sect. V I

- Topics

i n Chemistry

Wiley,

Ed.

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Pharmacokinetics While biopharmaceutics i s e s s e n t i a l l y the study o f t h e e f f e c t s o f the dosage form on the i n p u t (absorption) o f a drug i n t o a b i o l o g i c a l system, pharmacokinetics i s concerned w i t h t h e d i s p o s i t i o n o f t h e drug once i t becomes a v a i l a b l e f o r i n p u t . The goal o f a pharmacok i n e t i c study should be t o r e l a t e a c l i n i c a l response t o t h e pharmacok i n e t i c parameters used i n d e s c r i b i n g t h e time course o f a drug and i t s metabolites i n the body, Studies r e l a t i n g pharmacologic e f f e c t t o pharmac o k i n e t i c parameters have appeared f o r ethanol62, f e n c l o z i c acid63, disopyramide phosphate64, and heparin65. G i b a l d i e t a1 . 6 6 presented a very w e l l w r i t t e n a n a l y s i s o f the r e l a t i o n between pharmacologic e f f e c t s and drug d i s t r i b u t i o n i n mu1ticompartment systems. J ~ s k oshowed ~ ~ t h a t the n e c r o b i o t i c response o f chemotherapeutic agents which a t t a c h i r r e v e r s i b l y t o c e l l receptors may be q u a n t i t a t e d by l o g e f f e c t vs. dose p l o t s i f one assumes t h a t very l i t t l e drug i s a c t u a l l y l o s t by i r r e v e r s i b l e binding. I n essence he has approximated t h e i r r e v e r s i b l e nature o f these agents by assuming t h a t they appear t o i n t e r a c t r e v e r s i b l y . Smolen and Schoenwald68 used t h e m y d r i a t i c response o f tropicamide i n r a b b i t s , a pharmacologic measurement, t o measure t h e absorption r a t e o f t h e drug i n t o the c e n t r a l systemic compartment. The v a l i d i t y o f t h e method i s t e s t e d using known i.v. i n f u s i o n r a t e s o f the drug, Transcorneal absorption was a l s o measured69. A1 though t h e method appears impressive, i t i s unfortuna t e t h a t d i r e c t v e r i f i c a t i o n o f t h e r e s u l t s was n o t made, a t l e a s t once, by measuring plasma concentrations, S m ~ l e na l~s o~ used pharmacologic data t o determine t h e b i o a v a i l a b i l i t y and k i n e t i c model f o r t r i d i h e x e t h y l c h l o r i d e f o l l o w i n g ophthalmic and o r a l a d m i n i s t r a t i o n o f the drug. Although t h e r e a r e minor e r r o r s i n modelling and computer f i t t i n g o f the data, t h e p r i n c i p l e s brought f o r t h a r e important c o n t r i b u t i o n s . Most work d u r i n g t h e past year involved use o f t h e more conventional compartment models which have been described i n a number o f books2s3971 and article^.^*'^^ However, a few i n v e s t i a t o r s described s o l u t i o n s f o r W e ~ t l a k ep~o i~n t s o u t t h a t nonlinear pharmacokinetic equations,2,75) even though the 1east-squares estimates o f pharmacoki n e t i c model parameters might be considerably i n e r r o r , they w i l l probably enable reasona b l y accurate p r e d i c t i o n s o f blood l e v e l s t o be made, However, considera b l e d i s p a r i t y w i l l e x i s t between exact t i s s u e drug l e v e l s and p r e d i c t e d l e v e l s based on least-squares estimates. Although t h i s i s t r u e w i t h i n the c o n t e x t presented, t h e author should have a l s o p o i n t e d o u t t h a t t h e r e can be no exact t i s s u e l e v e l s , since t h e amount o f drug i n an unsampled d i s t r i b u t i o n compartment i s model dependent and o n l y r e s u l t s as a f u n c t i o n o f how t h e author defines e l i m i n a t i o n from t h e system. This l a s t comment i s a l s o appropriate t o t h e work o f N o ~ r d h o e kwho ~ ~ failed to f i n d p r e d i c t e d t i s s u e l e v e l s o f hexobarbital i n mice. G i b a l d i and W e i n t r a ~ bpointed ~~ o u t t h a t premature t e r m i n a t i o n o f s i n g l e dose pharmac o k i n e t i c studies due t o poor sampling design o r t o a n a l y t i c a l methods which a r e n o t s u f f i c i e n t l y s e n s i t i v e , may y i e l d erroneous underestimates o f h a l f - l i v e s . T h i s could l e a d t o gross e r r o r s i n t h e estimates o f steady-state blood l e v e l s o r t o t h e i n v a l i d assumption t h a t dose depende n t k i n e t i c s were observed f o l l o w i n g mu1t i p l e dosing.

3

Chap. 24

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During t h e past year more than 100 papers have appeared describing the pharmacokinetics-the absorption, d i s t r i b u t i o n , and metabolism o f various drugs. Although many o f these papers cannot be covered i n t h i s l i m i t e d review, the importance of these studies should n o t be minimized, since over half o f these studies were c a r r i e d out i n man, and serve as a comprehensive data base f o r the use o f pharmacokinetic data i n the development of r a t i o n a l dosing regimensE0. Human studies f o l l o w i n g i.v. administration of phosphonomycinsl and cyclophospkmides2 y i e l d e d good data consistent w i t h a two compartment model. Data on toxogonins3 and p-chlorbenzo1sulfonee4 would probably y i e l d b e t t e r f i t s i f a two compartment model was u t i l i z e d . Mark e t a1.85 used a whole-body counter t o f o l l o w the ptake, d i s t r i b u t i o n x e x c r e t i o n o f a s i n g l e breath o f halothane-8yBr A two-compartment model was used t o f i t the r e s u l t i n g data. B i s c h o f i e t a1 ,86 expanded t h e i r preliminary model o f methotrexate p h a r m a c o k i n e t i c s b y n c l u d i n g multicompartment representations f o r b i l i a r y excretion and movement of the drug through the g.i. t r a c t w i t h p a r t i a l reabsorption. The new model i s consistent w i t h data from t h e moyse, r a t , dog, monkey and man a t several doses. D o l u i s i o e t a1.87 showed t h a t f o l l o w i n g i.m. i n j e c t i o n o f a m p i c i l l i n t r i h y d r a t e suspensions o r a d i c l o x a c i l l i n sodium solution, the slope o f the terminal phase o f the serum time curves corresponded t o the absorption r a t e o f the drug from level the i.m. i n j e c t i o n s i t e r a t h e r than t o the e l i m i n a t i o n r a t e o f the drug from the body. Without i.v. data f o r these compounds erroneous r e s u l t s as t o absorption and e l i m i n a t i o n rates would have resulted. These authors a l s o pointed o u t t h a t i.m. i n j e c t i o n s o f the above drugs as w e l l as ampic i l l i n sodium solutions r e s u l t e d i n decreased a v a i l a b i l i t y o f t h e drug (65-75%) as compared t o i.v. i n j e c t i o n s .

-

-

A number o f i n v e s t i g a t o r s noted decreased a v a i l a b i l i t y o f drugs subject t o hepatic metabolism when these drugs were administered o r a l l y as compared t o i.v. dosing. The influence o f route of administratfoh on a v a i l a b i l i t y was noted f o r o r a l doses o f l i d o ~ a i n e , ~o ~ r a*l ~propran~ , intraperitoneal injections o f 0 1 0 1 9 ~ ' ~ ~inhaled , isoproterenol marihuanag3, and o r a l helveticosal acetonideg4. Gibaldi e t a1 . 9 5 developed equations which would p r e d i c t the f r a c t i o n o f drug metabolized on the f i r s t pass through t h e l i v e r f o l l o w i n g o r a l a d m i n i s t r a t i o n i f i.v. areas under the curve were known. The a p p l i c a t i a n t o propranolol data was incomplete, however, since although they used blood flow rates, the areas under the curve were from plasma data, and would only y i e l d v a l i d estimates i f the p a r t i t i o n c o e f f i c i e n t f o r propranolol between plasma and red blood c e l l s were u n i t y . Alvarezq6 pointed out t h a t although v i v o h a l f - 1 ives o f pentobarbital and thiopental d i f f e r e d from those found i n the i s o l a t e d perfused r a t l i v e r , clearance measurements were s i m i l a r . Ohnhaus e t a L q 7 noted changes i n l i v e r blood f l o w during enzyme induction.

-

in

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The e f f e c t s o f b i l i a r y excretion on drug k i n e t i c s i s r e c e i v i n g i n creasing a t t e n t i o n i n pharmacokinetic studies. Specific studies on the b i l i a r y excretion o f n i t r o f ~ r a n t o i n ~barbituratesg9, ~, a series o f penic i l l i n s ' O0, and demethylchlortetracyclinelO1 have been reported.

264

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W i l ey,

Ed.

Phenobarbital was found t o increase b i l i a r y flow i n r a t s l o 2 . This increase i s due t o enhanced formation o f t h e b i l e salt-iqdependent f r a c t i o n o f c a n a l i c u l a r b i l e production. Clark e t a1 .103showed t h a t l o w molecular weight anionic drugs administered t o r a t s by retrograde b i l i a r y i n f u s i o n a r e excreted p r i m a r i l y i n the u r i n e i n d i c a t i n g absorption from the b i l i a r y t r a c t . High molecular weight anionic drugs, i n contrast, were excreted predominantly i n the b i l e . The p r o t e i n binding o f diphenylhydantoin was found t o decrease i n uremic p a t i e n t s l o 4 . The free metabolite l e v e l s o f t h i s drug, estimated on the basis o f i n v i t r o binding studies, were found t o c o r r e l a t e w i t h the i n vivo incidence o f diphenylhydantoin-induced g i n g i v a l hyperplasia i n m a n x r a t . l oDrug 5 i n t e r a c t i o n s i n v o l v i n g displacement from proteins were investigated f o r phenyl butazone and sul phadoxine’ 06and f o r w a r f a r i n and a series o f a c i d i c drugs.lo7 McArthur e t a l , l o e showed t h a t s a l i c y l i c acid and phenobarbital bind t o human red c a l a s w e l l as plasma proteins and t h a t calculations as t o f r e e drug concentration 9 v i v o w i l l be i n c o r r e c t i f they are based o n l y on i n v i t r o plasma p r o t e r n i n d i n g studies. These same a u t h o r s l o g suggest t h a t t h e antirheumatic drugs may a c t by binding t o serum proteins and thereby increase the proportion o f f r e e peptides i n the blood by competitive p r o t e i n binding o f the drugs, Drug-drug i n t e r a c t i o n s i n man received considerable study during the past year. Levy’ reviewed those aspects o f drug biotransformation i n t e r a c t i o n s i n man t h a t are seen w i t h administration o f the nonnarcotic analgesics,while Dayton and Perel presented a more general review o f the mechanisms involved i n drug i n t e r a c t i o n s . Levy and co-workers found no i n h i b i t i o n i n glucuronide and s u l f a t e formation when acetaminophen and sal icy1 i c acid were given concomitantly as compared t o separate administ r a t i o n o f each drug. These s t u d i e ~ l ~ ~ l, el d~ t3o the conclusion t h a t o f the several combinations of acetaminophen, s a l i c y l a t e , and s a l i cylamide studied, salicylamide i s the major determinant i n the biotransformation i n t e r a c t i o n s encountered. The e f f e c t o f metabolic enzyme i n duction was a l s o studied f o r the f o l l o w i n g inducer-drug pairs: d i s u l furagt-antipyrine 14, phenobarbital l y c e r y l guaiacolate ether’ 5 , imipra, ~ ~ ~ , hydantoin-phenobarmine and d e ~ i m i p r a m i n e - b a r b i t u r a t e s ~ ~ ~diphenyl bital and hexobarbital ’l9, hypnotic drugs-warfarin120, c h l o r a l hydratecoumarin anticoagulants121, phenylbutazone and diphenyl hydantoind i g i t o x i n 1 2 2 , carbamazepine-diphenyl hydantoin and warfarin. 1 2 3 References 115,118 and 120-123 r e p o r t increased metabolic r a t e s f o r the drug as a f u n c t i o n o f the inducer, The remaining reported i n t e r a c t i o n s r e s u l t e d i n decreased metabolic rates. Other drug-drug i n t e r a c t i o n s appeared t o i n fluence d i s t r i b u t i o n and membrane transport. I n s u l i n a l t e r e d the t i s s u e d i s t r i b u t i o n o f to1 butamide, b u t not chlorpropamide. 1 2 4 Ethanol was found t o increase the i n v i v o l i p i d s o l u b i l i t y o f phenobarbital and i t s r a t e o f transport across the peritoneum and uptake i n t o the blood, and therefore a corresponding uptake i n t o the brain.125 The e f f e c t was much l e s s pronounced on pentobarbital uptake because o f i t s inherent high l i p i d solub i l i t y and r a p i d absorption. Increasing i n t e r e s t i n d i f f e r e n t i a t i n g gene t i c and environmental influences on drug disposition, i s r e f l e c t e d i n a

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Chap. 24

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number o f p u b l i c a t i o n s t h i s past year. 1 2 6 S p e c i f i c studies have d e a l t w i t h halothane metabol ism127,128, the pharmacokinetics o f monomethylated tric y c l i c a n t i d e p r e s ~ a n t s l ~and ~ , ethanol metabolism. 130 Davies and c o - w o r k e r ~ ldemonstrated ~~ t h e use o f a n t i p y r i n e h a l f - 1 i f e as a measure o f an i n d i v i d u a l ' s c a p a c i t y t o metabolize phenylbutazone and oxyphenbutazone. Shibasaki e t a1 .l32 f o l l o w e d acetaminophen and i t s g l u curonide m e t a b o l i t e i n t h e b E o T a n d u r i n e o f r a b b i t s , They found t h a t the r a t e l i m i t i n g step f o r appearance o f t h i s drug i n t h e u r i n e was t h e e x c r e t i o n o f t h e m e t a b o l i t e r a t h e r than t h e m e t a b o l i t e formation as had p r e v i o u s l y been assumed. Since a simple A+B+C model was n o t found t o hold, a catenary chain model w i t h an a d d i t i o n a l compartment which may be part i a l l y bipassed was introduced. Although good f i t s o f t h e data a r e obtained, the model has no b i o l o g i c a l meaning s i n c e i t suqgests a s i g n i f i cant amount o f an intermediate metabolite. Fleischmann noted t h a t metampicil l i n administered i n t r a m u s c u l a r l y o r intraveneously does n o t undergo metabolic changes i n t h e blood o r b i l e , b u t r a t h e r i n t h e kidney. Findings o f s i g n i f i c a n t kidney metabolism i n t h i s work and o t h e r prelimi n a r y r e p o r t s suggest t h a t t h i s f a c t o r must be adequately i d e n t i f i e d when comparisons o f dosage form a v a i l a b i l i t y by d i f f e r e n t routes o f administrat i o n are made. S t r u c t u r e a c t i v i t y r e l a t i o n s h i p s between physicochemical p r o p e r t i e s o f a drug and i t s {harmacokinetic d i s p o s i t i o n have been i n v e s t i g a t e d . Seydel and Wempe13 prepared a sequence o f new h i g h l y a c t i v e s u l f a p y r i dines on t h e basis o f p r e d i c t e d c o r r e l a t i o n s o f pharmacokinetic behavior i n physiolo i c a l systems w i t h c e r t a i n physicochemical p r o p e r t i e s . Johnson and M a i b a ~ h ?found ~ ~ t h a t p a r t i t i o n c o e f f i c i e n t , i n a d d i t i o n t o pK, i s important i n determining t h e r a t e o f e x c r e t i o n o f drugs i n human e c c r i n e sweat, Beckett and Brookes136 o r a l l y co-administered a number o f amphetamine type drugs and followed the u r i n a r y e x c r e t i o n o f t h e unchanged drug using g.1 .c. analyses. They found t h a t halogen s u b s t i t u t i o n caused a marked decrease i n t o t a l unchanged drug e x c r e t i o n as w e l l as a delayed peak e x c r e t i o n time. S u b s t i t u t i o n o f a second methyl group on t h e alpha carbon atom caused a s i g n i f i c a n t increase i n t o t a l unchanged drug excret i o n w i t h o u t a f f e c t i n g t h e peak e x c r e t i o n time. REFERENCES 1.

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64. R.E. Ranney, R.R. Dean, A. Karim and F.M. Radzialowski, Arch. Int. Pharmacodyn. Ther.

a, 162 (1971).

65. J.W. Estes, Ann. N. Y. Aczd. Sci. llp, 187 (1971). 66. M. Gibaldi, G. Levy and H. Weintraub, Clin. Pharmacol. Therap. 12, 734 67. W.J. Jusko, J . Pharm. Sci.,

60,

68. V.F. Smolen and R.D. Schoenwald, 69. 70. 71* 72

73 74. 75. 76. 77* 78* 79. 80. 81. 82. 83.

84. 85. 86.

(1971).

892 (1971).

w.60, w.

96 (1971).

R.D. Schoenwald and V.F. Smolen, @, 1039 (1971). V.F. Smolen. %. 60, 354 (1971). E.J. Ariens, ed., “Drug Design.” Vol. 1, Academic Press, New York, M. Mayersohn and M. Gibaldi, Am. J. Phsrm. Ed. $, 19 (1971). L.Z. Benet and J.S. Turi, J. Pharm. Sci. 60, 1593 (1971). C.M. Metzler, J. Am. Statistical Assoc. 66, 49 (1971). J.J. Coffey, F.J. Bullock and P.T. Schoenemann, J. Pharm. Sci. @, J.J. DiStefano 111 and R.F. Chang, Am. J. Physiol. 221, 1529 (1971 W.J. Westlake, J. Pharm. Sci. @, 882 (1971). J. Noordhoek, Arch. Int. Pharmacodyn. Ther. 388 (1971). M. Gibaldi and H. Weintraub, J. Pharm. Sci. &, 624 (1971). D.S. Alberts, N.R. Bachur, J.L. Holtzman, Clin. Pharmacol. Therap. K.C. Kwan, D.A. Wadke and E.L. Foltz, J. Pharm. Sci. @, 678 (1971 J.L. Cohen, J.Y. Jao and W.J. Jusko, Brit. J. Pharmacol. 9, 677 ( F.R. Sidell and W.A. Groff, J. Pharm. Sci. @, 860 (1971). A. Fahndrich. W. Krause and E. Gores, Phtrmazie 6, 173 (1971). L.C. Mark, I.C. Geddes, J.R. Schemer, C.S. Dombrowski and S.H. Cohen, Int. J. App. Rad. 2, 171 (1971). K.B. Bischoff, R.L. Dedrick, D.S. Zaharko and J.A. Longstreth. J. Pharm. Sci. @, 1128 (1971).

z.

60, 715 (1971). 87. J . T . Doluisio, J.C. LaPiana and L.W. Dittert, 88. R.N. Boyes, D.B. Scott, P.J. Jebson, M.J. Goodman and D.G. Jullan, Clin. Pharmacol. 89*

Therap. 12. lo5 (1971). M. Rowland. P.D. Thomson, A. Guichard and K.L. Melmon. Ann. N. Y. Acad. Sci.

383 (1971). 90.

C.T. Dollery, D.S. Davies and M.E. Conolly, Arch. Int. Pharmacodyn. Ther.

93-

214s (1971).

91 Idem., Ann. N . Y . Acad. Sci., 11p, 108 (1971). 92- A. Hayes and R.G. Cooper, J. Pharmacol. Exp. Ther.

G, 302

m,

E,

(1971).

T. HO, G. Fritchie. L.F. Englert, M. McIssac and J.E. Idanpaanheikkila, J. Pharm. Pharmacol. 3,309 (1971). 94. V.F. Zielski, H. Dovidat, G. Betzien, and W. Viogtlander, Arzneimforsch. 21,

566 (1971). 95. M. Gibaldi, R.N. Boyes and S. Feldman, J. Pharm. Sci. @, 1338 (1971). 96. R.A. Alvarez, Arch. Int. Pharmacodyn. Ther. 3,380 (1971). 97 E.E. Ohnhaus, S.S. Thorgelrsson, D.S. Davies and A. Breckenrldge, Biochem. Pharmacol. 20, 2561 (1971). 140 (1971). 98. J.D. Conklin and D.L. Wagner, Brit. J . Pharmacol. 9, 161 (1971). 99* C.D. Klaassen, w. 9,

-

A. Ryrfeldt, J. Pharm. Pharmacol. 2.463 (1971). A.M. Guarino. S.I. Helman and L . S . Schanker, Xenobiotica 1, 257 (1971). C.D. Klaassen, J. Pharmacol. Exp. Ther. 176,743 (1971). A.G. Clark, P.C. Hirom, P. Millburn and R.L. Smith, J. Pharm. Pharmacol. 3, 150 (1971)104. L. Lund, P.K. Lunde, A. Rane, 0. Borga and F. Sj:qvist, Ann. N. Y. Acad. Sci. 2. 1OCI 101. 102. 103.

723 (1971)-

268

Sect. V I

7

- Topics

Wiley, Ed.

i n Chemistry

105. G.J. Conard. C . O . Haavik and K.F. F i n g e r , J. Pharm. S c i . 60, 1642 (1971). 106. E.G. McQueen, W . M . W a r d e l l , B r i t . J . Pharmacol. 9, 312 (1971). 107. E.M. S e l l e r s and D. Koch-Weser. Ann. N. Y. Acad. S o l . 213 (1971). 108. J . N . McArthur, P.D. Dawkins and M . J . H . S m i t h , J. Pharm. Pharmacol. 9. 32 (1971). 109. Idem., 9, 393 (1971). 110. G. Levy, A n n . N.Y. Acad. S c i . 32 (1971). 111. P.G. Dayton and J . M . P e r e l , 67 (1971). 112. 0 . Levy and H. Yamada. J . Pharm. S c i . @, 215 (1971). 113. G. Levy and C.G. Regardh, 60, 608 (1971). 114. E.S. V e s e l l , T. P a s s a n a n t i and C.H. Lee, C l i n . Pharmacol. Therap. 12. 785 (1971). 115. S.N. G i r l , Arch. I n t . Pharmacodyn. Ther. 141,147 (1971). 116. K. Kakemi, H. S e z a k i , R. K o n i s h i and T. Kimura, Chem. Pharm. B u l l . (Tokyo) 9,

x,

m.

za, ,

=.

s.

1395 (1971). 117. H.C. Shah and H. L a l , J. Pharmacol. Exp. Ther.

D, 4U4

(1971).

m,

88 (1971). 119. J.W. Kemp, M. Tannhauser and E.A. Swinyard, Arch. I n t . Pharmacodyn. T h e r . 37 (1971). 120. A. B r e c k e n r i d g e and M. Orme, Ann. N . Y . Acad. Sci. 421 (1971). 1 2 1 . M. Weiner, w. D, 226 (1971). 1 2 2 . H.M. Solomon, S . R e i c h , N. S p i r i t and R . B . Abrams, 362 (1971). 123. J . M . Hansen. K. S i e r s b a e k - N i e l s e n and L. S k o v s t e d , C l i n . Pharmacol. Therap. 2, 539 (1971). 124. S.N. Madsen, F. Fog-Moller and I. P e r s s o n . Eur. J. Pharmacol. 2,374 (1971). 125. B.B. C o l d w e l l , H.L. Trenholm, B.H. Thomas and S. Charbonneau, J. Pharm. PhFrmacol. 23, 947 (1971). 126. Kalow, I n t . J . C l i n . Pharmacol. 5, 38 (1971). 127. H.F. C a s c o r b i , E.S. V e s e l l . D.A. Blake and M. H e l r i c h , C l i n . Pharmacol. Therap. 1 2 , 9 (1971). 128. &. , Ann. N . Y . Acad. Sc;. B, 244 (1971). 129. B. Alexanderson and F. S j o q v i s t , m.D. 739 (1971). 130. E.S. V e s e l l , J . O . Page and G . T . P a s s a n a n t i , C l i n . Pharmacol. Therap. 11,192 (1971). 131. D.S. Davies and S . S . T h o r g e i r s s o n , Ann. N. Y. Acad. S c i . D, 411 (1973). 132. J. S h i b a s a k i , R . K o n i s h i , Y. Takeda and T. Koizumi, Chem. Pharm. B u l l . (Tokyo), 1 2 , 18OC (1971). 133. Fleischmann, B. Gradnik and V. Guzzon, I1 Farmaco, Ed. Pr. 26, 106 (1971). 134. J . K . S e y d e l , E. Wempe, Arzneimforsch. 2, 187 (1971). 135. H.L. Johnson and H . I . Maibach, J . I n v e s t . D e r m . 182 (1971). 136. A.H. . B e c k e t t , L.G. Brookes, J. Pharm. Pharmacol. 3,837 (1971). 118. P.L. M o r s e l l i , M. Rizzo and S . G a r a t t i n i , Ann. N. Y. Acad. S c i .

a, u. m,

s,

z,

Chapter 25.

Reactions of Interest in Medicinal Chemistry

Herbert T. Nagasawa and John A . Thompson College of Pharmacy, University of Minnesota and Veterans Administration Hospital Minneapolis, Minnesota 55417 The stepped up abstracting of useful synthetic procedures from the international chemical literature by the journal Synthesis is noted (1844 abstracts to Dec. 1971) as well as the appearance (in soft cover) of the first of the Annual Reports Organic Synthesis, edited by J. McMurry and R.B. Miller. To avoid possible overlap with material in other chapters, specialized reactions for steroids and amino acids have not been included here; and, as in previous years, the materials actually presented are necessarily highly selective. Useful Reagents (not elsewhere classified) - A stable (several months under N 2 ) , storable hydroborating and reducing agent which is liquid at room temperature is provided by dimethylsulfide-borane. An excellent reagent for the oxidation of a) Schiff bases to oxaziridines, b) nitrogen-containing aromatic heterocycles to N-ocCides, and c) nitrosamines to n i t r d n e s is t-amylhydroperoxide with M0cl5.~ Tl(NO3)3 is useful for a) the degradation of monoalkylacetylenes to carboxylic acids containing one less carbon atom, b) synthesis of acyloins from dialkylacetylenes, and c) synthesis of methyl arylacetates from acetophenones. s 4 An extremely simple procedure for the preparation of e s t e r s , acetals or ketazs at room temperature involves the use of a dehydrated sulfonated On a preparpolystyrene copolymer together with a drying agent (CaS04). ative scale, 1,4-dioxaspiro[4.5]decane was produced in 90% yield from cyclohexanone and ethylene glycol. Somewhat analogously, ketimines and enantines can be prepared in high yield by the reaction of the appropriate ketones and amines in benzene or ether at room temperature in the presence of molecular sieves.6 This general method has been successful even with medium-sized ring ketones and camphor, which are rather hindered. Sodium borohydride on silica gel reduces oximes to the corresponding h y d r o q 2d n e s in benzene at room temperature.

-

Protective Groups and Their Removal The acetylenic linkage in compounds containing both double and triple bonds can be protected as the acetylenedicobalt hexacarbonyl complex by treatment with cobalt carbonyl. After reaction at the non-coordinated olefinic bond is completed, the acetylene may be regenerated swith Fe(N03)3*9H20. Methylene groups adjacent to the carbonyl in carbonyl compounds may be protected as dithiolanes or dithianee by condensation with ethylene or trimethylene dithiotosylate, or for unreactive methylenes, by initial activation to an enamine or a hydroxymethylene derivative followed by condensation. The methylene groups may be regenerated by Raney Ni reduction or may be converted to a carbonyl function by mercuric ion catalyzed hydrolysis. Oxidative hydrolysis appears to be a superior method for removal of the dithioketal groups,

'

270 -

Sect. V I

-

Topics

in Chemistry

especially when oxygenated or olefinic functions are present. bromosuccinimide or N-chlorosuccinimide-AgN03 in aqueous CH3CN readily converts this group to a ketone at room temperature or Chloramine-T in H20 or MeOH has also been used successfully to cleave 1,3-dithiolanes and 1,3-oxathiolanes.

Wiley, Ed. Thus, Nor Me2CO below.'' oxidatively

-

Carbocyclic and Heterocyclic Ring Formation Halogen-substituted carbocyclic three-membered rings formed from olefins and phenyl(trihalomethy1)mercury compounds are the subject of a recent review.13 An improved organomercury route to difluorocarbene has been described.l4 CycZopropyZ compounds (or a z i r i d i n e s ) can be prepared vi a the attack of a carbanion (or amine) on the readily obtainable dimethylaminophenyl-(2-phenylvinyl)oxosulfonium fluoroborate followed by elimination of the sulfur grouping.15

-Diphenylsulfonium cyclopropylidel and the ylide derived from the N ,Ndimethyl salt of cyclopropylphenylsulfoximine17 reacts with a,B-unsaturated and B-dimethylamino ketones (in the latter case) t o give high yields of spiropentane derivatives.

Certain nitriles, sulfones, and sulfonamides can be converted to their a,a-dilithio derivatives and then to three- and five-membered ring A 4+2 cyclo-addition of a -CH2CO- unit compounds with dihaloalkanes. has been accomplished using 2-chloroacrylyl chloride.l9

Fwlmzs are readily obtained according t o the following scheme.20

'

0 EtOH @ Me2SCH2CiCH Br -Me2SCH=C=CH2

R' COCH2R" NaOE t

+

Me2S

A synthesis of 8-Zactam8 involves the addition of one mole of a primary

amine to cyclopropanone (ketone + diazomethane) and treatment of the resulting carbinolamine with t-butyl hypochlorite followed by AgNO3 in CH3CN.21 3-PyrroZid~nones have been prepared by the addition of azo-

Chap. 25

Chemical React ions

Nagasawa, Thompson

271 -

methine oxides to allenes.22 A one-step synthesis of oxazoles involves the reductive cyclocondensation of acyl halides and a-azidocarbonyl compounds in the presence of Ph3P.23

-

Reductions For unsaturated compounds containing oxygen (allylic alcohols and ethers, ynols, epoxides, a,%-unsaturated ketones, aldehydes, acids and epoxides) nickel boride (prepared from Ni acetate and NaBH4) selectively and quantitatively hydrogenates the C-C double bonds without rearrangements, hydrogenolysis or carbonyl reductions. 24 An aryl trifluoromethyl group can be reduced to a methyl group with LiAlH4 if an electron-donatin amino or hydroxyl substituent is present Aromatic diazoniwn fluoroborates are in an ortho or para position.'5 reduced to the corresponding arenes in DMF at <80° when catalyzed by rhodium complexes, RhCl(C0) (PPh3)3 or RhCl(PPh3)c~. 26 Electron attracting substituents appear to favor this reduction. Reductive decyanation of alkyl nitriles may be effected in 58-100% yields by the action of Fe(C5H702) 3 and Na. 27

The remarkable selectivity of sodiwn cyanoborohydride (NaBH3CN) as a reducing agent in aqueous or aqueous MeOH systems has been elaborated.28 Oximes are smoothly reduced to alkylhydroxylamines, and enamines are reduced to amines under acid catalysis. Reductive amination of an aldehyde or ketone with BH CN@ in the presence of NH3, '1 amine or 2O amine at pH 7 leads to lo, 2 or '3 amines, respectively. Reductive amination of substituted p ruvic acids leads to fair yields of the corresponding a-amino acids. PsN-labeling can be accomplished by using I5NH3. Also the hydrogens of BH3CNQ can be readily exchanged for either 2H or 3H, thus permitting the synthesis of 2H or 3H-labeled alcohols, amines and amino acids, Tosyl hydrazones of aliphatic aldehydes and ketones are selectively reduced to the corresponding hydrocarbons with this reagent; hydrazones of aromatic ketones are not reduced.29 Primary and '2 iodo and bromo groups as well as '1 tosylates can also be reductively removed in the presence of practically all other functional groups with sodium cyanoborohydride in hexamethylphosphoramide.(HMPH) 30

a

.

A single-step reductive e l i m h a t i o n of epoxides to olefins with Zn-Cu couple in ethanol has been described. Sulfoxides, including the conformationally restricted thioxanthine sulfoxide, are quantitatively deoxygenated with NaBH4-CoC12 in 95% ethanol. 3 1 Sulfoxide's and sulfilimines are also reduced to sulfides at room temperature by 0,O-diethyldithiophosphoric acid. 32

Conjugate Addition Reactions - The conjugate additions of lithium diaZky Zcopper reagents (LiR2Cu) have been further elaborated. In conjugated dienones, the presence of an alkyl group at one of the substitution sites directs the conjugate addition to the other position.33 For l-alkenylcuprolithium complexes, the cis or t r m integrity of the alkenyl group is preserved during conjugate additions or in alkylations. Thus, L gives 100% 2. 34

272 -

Sect, V I

- Topics

i n Chemistry

Wiley, Ed.

-

cv

Stereospecific B-vinylation has been observed in a special case.35 Acetylenic groups may be added to a,B-unsaturated ketones v i a diethylalkynylalanes.36 PhC3CLi

+

Et2AlC1

-

acocH3 Et2AlCZCPh

-

acocH CECPh

-

Alcohols and Alkyl Halides Controlled oxidation of the hydroboration product of olefins of the type RR'C-CHR" with pure 02 followed by hydrolysis of the trialkylborates gives the corresponding alcohols R(R')CHCH(R")OH. 37 Trialkylboranes are converted readily to t r i a l k y l carbinols by reactions with chlorodifluoromethane under the influence of lithium triethylcarboxide.3 8 T e rt i a ry alcohols are also produced when trialkylboranes are photobrominated in the presence of water and the (rearranged) products subsequently oxidized.39

A one-step four-carbon homologation leading to a variety of 4-alkyl2-buten-1-01s is provided by reaction of 1,3-butadiene monoxide with primary or secondary trialkylboranes in the presence of catalytic amounts of 02 or other free-radical initiator^.^^ R3B

+

CH=CHCH-CH2 ' 0 '

Cat 2' ,RCH2CH=CHCH20H H20

+

R2BOH

Chromic acid oxidation of bicyclo[3.3.l]nonane, bicyclo[3.2.2]nonane, bicyclo[3.3.2]decane and adamantane affords the bridgehead t e r t i a r y carbirfozs which provide the best entry to functionalization at these sites.4 Allylic alcohols can be transformed to their corresponding unrearranged a l l y Zic chlorides by the use of r n e t h a n e s u l f o n y l c h l o r i d e and a mixture of LiC1-DMF and 2-collidine at 0°.42 The reversible 1,3- transposition of sulfoxide and alcohol functions in an allylic system appears t o be of general synthetic utility.43

Solvolysis of oxiranes with dimethyl sulfoxide and 2,4,6-trinitrobenzenesulfonic acid affords threo-glycols from cis-epoxides and

Chap. 25

Chemical Reactions

273 -

Nagasawa, Thompson

erythro-glycols from trans-epoxides .44 A new route to 0-amino alcohols from ketones is especi'ally suitable for ketones which do not form cyanohydrins or condense with n i t r ~ m e t h a n e . ~ ~

A new method for the-conversion of alcohols to alkyl halides involves carbazate intermediates which are oxidized with N-halogenosuccinimides in the presence of ~yridine.~ Gem-dichlopoalkenes have been prepared from the tosylates of aryl-, alkyl- and alkynyl(trichloromethy1)carbinols by reaction with PhMgBr.47 In another anti-Markownikov hydrobronrination of internal olefins, the olefin is condensed with 9-borabicyclo63.3.l]nonane and the intermediate subjected to a-bromination-HBr cleavage.48

Amines and Azides. A wide variety of organic azides has been converted to aZkyZethylamines by reaction with triethyl borane followed by methanolEnamines may be reduced to the corresponding ysis of the inten~ediate.~~

00 R-N-NrN

+

Et3B

"' :

'r'

R-N-BEt2-

MeOH

RMIEt

+

E t2BOMe

t e r t i a r y d n e s with NaBH4 zda the C-mercurated immonium salts. A general method for exhaustively alkylating amines to quarternary m o n i w n compounds at room temperature makes use of the sterically hindered base 1,2,2,6,6-pentamethyl piperidine. 5 1 Cyclic or acylic higher olefins may be aminomethylated with CO, H20, and a secondary amine in the presence of rhodium oxide (andlor iron pentacarbonyl) 5 2

.

Cycloaliphatic aaides can be prepared at room temperature by the reaction of the Li amide of the primary cycloaliphatic amine with tosyl azide. 5 3 Phenyliodosochlorideazide [PhI(Cl) (N3) 3 is a reagent which converts olefins to chloro-azido compounds, generally adding c i s to double bonds, whereas ClN3 adds trans. 54 a-Azido-6-nitratoalkanes have been produced when olefins were treated with sodium azide and ceric ammonium nitrate in acetonitrile. 5 5

-

Aldehydes and Ketones Aldehydes are obtained from terminal acetylenes according to the scheme: 56 RCZCH

+

R3SifI

-

RCWHSiR3

-

RCH-CHSiR3

H2S04

RCH'CHO

'0'

Several labile aldehydes have been prepared from alkyl halides and methylthiomethyl sulfoxide.57

'S-CH3

J

J

274

Sect. V I

- Topics

in Chemistry

Wiley, Ed.

The conversion RX-RCH2CHO ma be arcomplished utilizing commercially available 2,4-dimethylthiazole.3 8 @

1) BuLi

1) Me30 BF4

Mecsc~2~ 2) NaBH4

-

RCH2CHO

S

CH2R

Ferrate(V1) ion (K2Fe04) rapidly oxidizes primary amines and alcohols to aldehydes and secondary alcohols to ketones without affecting double or triple bonds, aldehyde groups, or tertiary alcohol or amine functions.59 Oximes are converted to aldehydes and ketones virtually instantaneously with Tl(N03)3.60 Ketones are conveniently obtained from olefins by oxymercuration, transmetallation with palladium chloride and subsequent decomposition of Ketones can also be prepared from the organopalladium intermediate. the corresponding nitro compounds using aqueous TiC13. 6 2 Symmetrical alkyl or aryl ketones are formed from organomercuric halides and Ni(CO)4.63 Highly-branched carbonyl compounds are obtained via the 1,4-addition of B-alkylboracyclanes with bulky alkyl groups to a,$-unsaturated carbonyl compounds.64 The conversion of aldehydes to ketones can be accomplished by protecting the derived aldehyde cyanohydrin with ethyl vinyl ether and alkylating this species with lithium diisopropylamide and an alkyl halide following by deprotection.6 5 A procedure for the vinylogous homoZogation of aldehydes (RCHORCH CHCHO) has been developed.5 6 The reagent 1,3-bis(methylthio) alkyllithium reacts with carbonyl compounds (or alkyl halides) to afford the corresponding unsaturated trans aldehydes.66 The a-hydrogen of an

-

a,B-unsaturated ketone can be replaced by an alkyl group via a metalloenamine.67 AlZylic oxidation of trisubstituted olefins by Se02 has been established to give selectively trans allylic alcohols, or trans a,Bunsaturated aldehydes in certain case^.^^,^' A new synthesis of a,$unsaturated ketones involves a Wittig type addition to a ketone and hydrolysis of the resulting ketimine.70

Disubstituted alkynes are oxidized to a-diketones by NaOCl (or NaIO4) with a catalytic amount of Ru04. 71 Symmetrically disubstituted acyclic olefins o r large ring alicyclic olefins are directly converted to a-diketones by KMnO4-Ac20 in the cold.

25 Chemical Reactions Nagasawa, Thompson 275 Carboxylic Acid Derivatives - Stable lithium ester enolates, prepared by react.ion of carboxylic esters with lithium N-isopropylcyclohexylamide (LiICA) in THF at low temperatures, may be potentially more versatile than sodiomalonic ester in synthetic applications. The coupling reactions are carried out at room temperatures or below and give good yields of the desired products. Thus, the lithium ester enolates may be a) alkylated to the corresponding aZky Zated e s t e r , 7 3 b) carboxylated with CO to produce malonic e s t e r derivatives,74 c) halogenated to a-iodo and a-homo e s t e r s , 7 5 and d) acylated withacylchlorides to give the corresponding B-keto e s t e r s . 76 The lithium salts of the a-lithiated carboxylic acids have been condensed with esters to give 6-ketoacids, and added in Michael fashion to a ,0-unsaturated esters. 77 8-Hydroq-acids are produced directly when these a-lithiated carboxylic acid salts are condensed with ketones.7 8 Chap.

The chain-lengthening of carboxylic acids by three carbon atoms to y-keto acids (or nitriles) by way of oxazoline-5-one anions have been described. 79 The conversion of aldehydic cyanohydrins to a-hydroxy-N-tbutylcarboxamides followed by oxidation and hydrolysis constitutes a new general procedure for the preparation of a-ketoacids. 8o A mild general procedure for preparing p e r o q acids in 70-85% ields involves the acid catalyzed perhydrolysis of acyldiethylphosphates.8Y

NitriZes have been prepared from a) carboxylic acids in one step by heating with amidosulfuric acid and ureaYB2b) aldoximes by reaction with TIC14 in the presence of pyridineYB3and c) aldehydes v i a a modified Wittig reaction with cyanomethylene-bis(tripheny1phosphonium) dibromide, this reaction giving rise to a , B-unsaturated n i t r i l e s . 84 a, &Unsaturated Br2CHCN

+

2PPh3

-

CDQ

[Ph3P-P-PPh3]28? CN

OH0

RCH=CHCN

+

2Ph3PcO

aldehydes have been converted oxidatively to amides without affecting the olefinic bond by treatment with manganese dioxide in the presence of NaCN and an amine.85 A new isocyanide synthesis involves simply heating a monosubstituted aliphatic or aromatic formamide (readily prepared at '0 from the amine and phenylformateB6) in CHC13 or CH2C12 with triphenylphosphine, Cc14 and Et3N. 87 Miscellaneous - Dialkyl, aralkyl, alicyclic and certain diaryldisulfides undergo facile s t e r e o s p e c i f i c desuzfurization with t r i s (diethylamino)phosphine. Inversion of configuration occurs at one of the carbon atoms a to the disulfide group. Some biologically important disulfides such ROOCGCOOR

s-s

+

(Et2N) 3P--., ROO+&COOR

H S

S

-

R O O C G C O O R

276 -

Sect. V I

-

Topics i n Chemistry

Wiley, Ed.

as derivatives of lipoic acid and cystine have been desulfurized in high yield to the corresponding thioethers.89 A two-step process for preparing N-monoazky Z s u Z f o n d d e s from the alkyl bromide without going through the alkylamine involves the alkylation of the sodio derivative of the eth 1 N-(arylsulfony1)carbamate and subsequent hydrolysis of the carbamate.&J The CZaisen rearrangement has been applied to yrepare 6-allyl-5The reaction is hydroxyuridine from 5-allyloxyuridine in 79% yield. general for the 5-ally1 ethers of hydroxyuracils and methods are available for the eventual removal of the 5-hydroxy group. A new class of d i hydropyrimidinea, v i z . , analogs of 5,6-dihydro-5,6-cyclobutanyluracils and their nucleosides, are obtained when aqueous solutions of 5-alkyl uracils (ethyl, propyl, isopropyl) or their nucleosides are irradiated at 254 nm.92 The conversion of uridine or its analogs or derivatives to cytidines has H L % m 2 m 3 hv been accomplished in one step by heat0 ing the uridine in an autoclave with B B i hexamethyldisilazane and ammonia. The 5’-hydroxy group in purine and pyrimidine ribonucleosides has been selectively replaced with C1 or Br at room temperature with SOC12 (or SOBr2) in HMpA.94 L

A novel synthesis of 2-, 8- and 9-substituted adenines where the substituents are introduced unambiguously has been described.9 5 The reactions start with 4,6-diamino-5-nitrosopyrimidines either substituted or unsubstituted in the 2position and the eventual adenine 8- and 9-substituents are introduced on the intermediate 7-aminofurazano[3,4-d]pyrimidine. A new approach to the synthesis of 8-azapurine nucZeosides involves opening the imidazole ring of the appropriately protected pre-formed purine nucleosides and cyclization of the resultant 5-amino-4-glycosylaminopyrimidine with nitrous acid.96 _ _ ) -

The difficultly accessible 3-substituted pyridines have been synthesized in one step by alkylation (or bromination) of lithium tetrakis(N-dihydropyridyl) aluminate which is readily prepared by the reduction of pyridine with LiA1H4.9J The diazonium fluoroborates prepared i n s i t u from 2- and 4aminoimidazoles have been photolyzed at room temperature or below to give 2- and 4-fZuoroimidazoZes in fair yields. This reaction, which did not take place in the absence of U.V. light even under pyrolytic conditions, has been applied to the synthesis of 4-fluoro-DL-histidine.9 8

Chap. 25

Chemical Reactions

277

Nagasawa, Thompson

References

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Chapter 26. I n t r a m o l e c u l a r C a t a l y s i s i n Medicinal Chemistry Richard D. Gandour and Richard L . Schowen Department of Chemistry, U n i v e r s i t y of Kansas, Lawrence I n t r a m o l e c u l a r c a t a l y s i s i s i m p o r t a n t i n m e d i c i n a l chemistry because (1) i t i s a f e a t u r e of t h e r e a c t i v i t y of m u l t i f u n c t i o n a l molecules, (2) i t p l a y s a s p e c i a l r o l e i n t h e d e s i g n and b e h a v i o r of l a t e n t i a t e d d r u g s , and ( 3 ) i t s e r v e s as a model f o r c a t a l y s i s by a c t i v e - s i t e f u n c t i o n a l i t i e s i n r e a c t i o n s o f enzyme-substrate complexes. During 1 9 7 1 , e x c e l l e n t reviews of t h e s u b j e c t appeared by Bender,' Capon2 and F e r s h t and K i r b y , 3 which d e a l t c r i t i c a l l y and comprehensively w i t h p r e v i o u s work. This a r t i c l e c o n c e n t r a t e s on developments d u r i n g 1971 and i s h i g h l y s e l e c t i v e . I t i s organized a c c o r d i n g t o t h e t y p e of r e a c t i o n b e i n g c a t a l y z e d , i n o r d e r of i n c r e a s i n g complexity: p r o t o n t r a n s f e r from carbon; a c e t a l h y d r o l y s i s ; a c y l t r a n s f e r ; h y d r o l y s i s of phosphate esters. A t t h e end, p a p e r s c o n t r i b u t i n g t o t h e " o r b i t a l s t e e r i n g " c o n t r o v e r s y are reviewed. Throughout, t h e a b b r e v i a t i o n "TS" i s employed f o r " t r a n s i t i o n s t a t e . " Hydrogen T r a n s f e r From Carbon- Deuteron t r a n s f e r t o t h e i n t r a m o l e c u l a r (CH3)zN group i n (CD,)ZC=NH+CHR~CHR~CH~N(CH~)~ l e a d s t o 7x t o lOOx r a t e enhancements i n t h e d e d e u t e r a t i o n of (CD3)2CO, c a t a l y z e d v i a t h e immonium i o n s by t h e corresponding diamines. Only amines c a p a b l e of forming an 8r i n g c y c l i c TS a r e e f f e c t i v e and only t h e n i n c a s e s where t h e c a t a l y t i c f u n c t i o n is syn about t h e immonium c e n t e r t o t h e p r o t o n donor group.4 The 8-ring TS i s n o t g e n e r a l f o r i n t r a m o l e c u l a r p r o t o n t r a n s f e r from carbon. For example, a 6-ring TS i s f a v o r e d f o r p r o t o n t r a n s f e r t o t h e c a r b o x y l a t e c e n t e r i n CH3CHN02CH2CH2C02- t o form t h e n i t r o c a r b a n i o n . Hydride t r a n s f e r is i n t r a m o l e c u l a r l y b a s e - c a t a l y z e d i n t h e bromine o x i d a t i o n of o-formy1 benzoate i o n t o produce a ?Ox a c c e l e r a t i o n ( o t h e r s u b s t i t u e n t s a f f e c t t h e benzaldehyde rate only w i t h i n 3 x ) . N u c l e o p h i l i c a d d i t i o n of -CO2- t o t h e formyl group i s excluded because p h t h a l i c anhydride h y d r o l y z e s t o o s l o w l y t o be an i n t e r m e d i a t e ; presumably t h e h y d r a t e i n t e r v e n e s (L). The subs t r a t e exists a s the l a c t o l i n a c i d i c s o l u t i o n and then o x i d i z e s a t least l o x 1 more s ZowZy than benzaldehyde. Intramolecular a c i d c a t a l y s i s of h y d r i d e t r a n s f e r by Zn*-activation of t h e hy' - Br d r i d e a c c e p t o r g i v e s a rate c o n s t a n t of 19 2 4 M-lsec-l t o t h e alcohol-dehydrogenase model r e a c t i o n 2, w h i l e no react i o n a t a l l i s observed i n t h e absence This i s t h e f i r s t non-enzymatic of Znu. r e d u c t i o n of an aldehyde f u n c t i o n by a dihydropyridine. '"7

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n

A c e t a l Hydrolysis- Acid-base c a t a l y s i s of a c e t a l h y d r o l y s i s i s i m p o r t a n t f o r t h e mechanism of g l y c o s i d a s e a c t i o n .

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Carboxyl groups might s t a b i l i z e t h e h y d r o l y s i s TS by ( a ) p a r t i a l p r o t o n d o n a t i o n ( g e n e r a l a c i d c a t a l y s i s ) ; (b) complete, p r i o r p r o t o n d o n a t i o n t o t h e acetal l i n k a g e , followed by e l e c t r o s t a t i c i n t e r a c t i o n of -C02- and t h e p o s i t i v e a c e t a l l i n k a g e d u r i n g CO bond f i s s i o n ( e l e c t r o s t a t i c c a t a l y s i s ) , o r ( c ) p r i o r p r o t o n d o n a t i o n followed by c o v a l e n t b i n d i n g of -C02- t o t h e i n c i p i e n t carbonium c e n t e r d u r i n g CO f i s s i o n ( n u c l e o p h i l i c c a t a l y s i s ) . Recent work b e a r s on a l l of t h e s e . A c e t a l s were t h o u g h t s u b j e c t o n l y t o s p e c i f i c a c i d c a t a l y s i s u n t i l i t was shown t h a t i f t h e RO- group t o b e p r o t o n a t e d were u n u s u a l l y weakly b a s i c o r i f t h e carbonium i o n l e f t by RO- d e p a r t u r e were u n u s u a l l y s t a b l e t h e n e a r l y c l e a v a g e and g e n e r a l c a t a l y s i s would b e s e e n . I n t r a m o l e c u l a r c a r b o x y l c a t a l y s i s i s promoted by t h e same f e a t u r e s which f a v o r t h e i n t e r molecular r e a c t i o n ; c u r r e n t work has c e n t e r e d on whether t h e mechanism i s g e n e r a l a c i d , e l e c t r o s t a t i c o r n u c l e o p h i l i c c a t a l y s i s . The c h a r g e 6+ i n t h e i n c i p i e n t carbonium i o n of TS 2 H ( i . e . , 2 w i t h Y = H) i s t h e same (p = -3 f o r v a r i a t i o n a t R) whether X = CH3, H o r From t h i s i t w a s concluded t h a t y t h e r e w a s no d i r e c t TS i n t e r a c t i o n of t h e C 0 2 X group 3,v and e i t h e r t h e OH group o r t h e i n c i p i e n t carbonium i o n ( t h u s no g e n e r a l c a t a l y s i s and no n u c l e o p h i l i c HCe+ VOCH c a~ t a lR y s i s , l e a v i n g e l e c t r o s t a t i c c a t a l y s i s as t h e only e x p l a n a t i o n of t h e 500x a c c e l e r a t i o n when X = -.'IThe c o n c l u s i o n i s v a l i d i f hydrogen-bonding of C02- t o t h e OH would a l t e r 6+, as seems r e a s o n a b l e . On t h e o t h e r hand, t h e e l e c t r o s t a t i c hypot h e s i s t o a f i r s t approximation would n o t p l a c e any o r i e n t a t i o n r e q u i r e ment on t h e c a r b o x y l a t e group; b u t 2-methoxymethoxy-l-naphthoic a c i d ( i n which c o p l a n a r i t y of t h e -C02H i s p r e v e n t e d ) i s hydrolyzed 5x more slowly t h a n t h e r e a c t i o n of H , w h i l e s t e r i c p r e s s u r e on t h e a c e t a l group which does n o t i n t e r f e r e w i t h t h e carboxyl o r i e n t a t i o n ( a s i n 3 H 3 , 2N02, and 3C02H, as w e l l as l-methoxymethoxy-2-naphthoic a c i d ) g i v e s a c c e l e r a t i o n s i s e s t i m a t e d , assuming of 4x t o 490x over Furthermore, compound no s t a b i l i z a t i o n by t h e COz- n e g a t i v e c h a r g e , t o b e t o o u n s t a b l e an a c i d t o be an i n t e r m e d i a t e i n t h e f a s t obe r v e d r e a c t i o n . Only i f i t s Ka i s d e c r e a s e d a t l e a s t 4 slOOOx by t h e -C02- is t h e e l e c t r o s t a t i c - c a t a l y s i s mechani s m p o s s i b l e . I f t h e s t a b i l i z a t i o n i n v o l v e s hydrogen CH,OCHCGH, bonding, t h e mechanism i s i n e f f e c t f e n e r a 1 c a t a l y s i s , as f a v o r e d by t h e a u t h o r s . E l e c t r o s t a t i c cat5 a l y s i s i s invoked t o e x p l a i n t h e 30x a c c e l e r a t i o n i n 5 o v e r t h e g l u c o s i d e s , i n which no -C02- group i s p r e s e n t . Here t h e c a r b o x y l a t e \2-Naph i s s t e r e o c h e m i c a l l y i l l - s i t u a t e d f o r e i t h e r OH g e n e r a l o r n u c l e o p h i l i c c a t a l y s i s . l 2 J u s t as s o their t h e benzylidene catechols experience intermolecular c a t a l y s i s , h y d r o l y s i s i s a l s o a c c e l e r a t e d i n t r a m o l e c u l a r l y (6) e i t h e r by e l e c t r o s t a t i c o r g e n e r a l c a t a l y s i s , s i n c e t h e carboxyl group is i n no p o s i t i o n t o e f f e c t n u c l e o p h i l i c s t a b i l i z a t i o n a t t h e i n c i p i e n t p o s i t i v e carbon. l 4 Benzaldehyde d i s a l i c y l a c e t a l (1) h y d r o l y z e s more t h a n 109x f a s t e r t h a n i t s d i methyl ester, w h i l e t h e monoanion forms t h e a c y l a l a n o t h e r 65x f a s t e r . 1 5 While t h e c y c l i z a t i o n does n o t prove T S - p a r t i c i p a t i o n by -C02-, i t i s

g;:: co;

-

m 0 P

*

-.

Chap. 26

I n t ramol e c u l a r Ca t a lys i s

suggestive. A search f o r nucleophilic assF=ta?me by c a r b o x y l a t e and o t h e r centers y i e l d e d c y c l i z e d p r o d u c t s i n some cases, but generally insufficient r a t e HO,C a c c e l e r a t i o n s t o j u s t i f y p o s t u l a t i o n of //C\o’H TS c a r b o x y l a t e a s s i s t a n c e e x c e p t i n 5082% d i o x a n e - w a t e r s o l u t i o n s . For example, 2-carboxybenzaldehyde d i e t h y l ace t a l h y d r o l y z e s 3000x f a s t e r than t h e 4-carboxy isomer i n b a s i c 82% dioxanewater b u t o n l y 3x f a s t e r i n w a t e r . The o r i g i n of t h e dioxane e f f e c t i s u n c e r t a i n . 1 6

o l d

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Acyl T r a n s f e r - I n t r a m o l e c u l a r c a t a l y s i s of t h e s e p r o c e s s e s h a s been a s u b j e c t of i n t e r e s t f o r y e a r s , p z r t l y because of i n t e r e s t i n a c y l - t r a n s f e r enzymes. Among t h e new examples are t h e lox a c c e l e r a t i o n produced by t h e 0-0- group i n t h e b a s i c h y d r o l y s i s of o-hydroxyphenyl e t h y l c a r b o n a t e (over t h e 0-OCH3 and p - 0 compounds) l 7 and >150x enhancement by -C02- i n h y d r o l y s i s of C6H502CCH2CO2-.l8 Both r e a c t i o n s a r e thought t o proceed by g e n e r a l b a s e c a t a l y s i s . I n t r a m o l e c u l a r a c y l s h i f t s (which a r e i m p o r t a n t p e r s e , and which a l s o occur a s s t e p s i n n u c l e o p h i l i c c a t a l y s i s ) i n c l u d e t h e b i o t i n model r e a c t i o n 8 s i m u l a t i n g t h e presumed 1 , 3 - O , N s h i f t of COP i n b i o t i n . 1 9 A t

pH < 9 , where t h e s u b s t r a t e is p r o t o n a t e d , t h e syn-anti i s o m e r i z a t i o n i s f a s t and a c y l t r a n s f e r i n t h e f r e e b a s e i s r a t e determining ( o v e r a l l b a s e c a t a l y s i s ) . Above pH 10 t h e f r e e b a s e i s t h e major s p e c i e s and i s f r o z e n i n t h e u n r e a c t i v e syn form; i t r e p r o t o n a t e s , i s o m e r i z e s t o anti i n a r a t e - d e t e r m i n i n g s t e p and t h e s h i f t f o l l o w s r a p i d l y ( o v e r a l l a c i d c a t a l s i s ) . Thus t h e r e is a rate maximum a t pH 9-10.24; The c l o s u r e of o-hydroxymethylbenzamide t o p h t h a l i d e i s c a t a l y z e d by i m i d a z o l e , y i e l d i n g a c l o s e model f o r a c y l a t i o n of chymotrypsin. An i n t e r e s t i n g c o m p e t i t i o n of general-base and nucleop h i l i c c a t a l y s i s i s found f o r 9. The h y d r o l y s i s rate i n c r e a s e s w i t h t h e d e g r e e of i o n i z a t i o n of t h e carboxyl group below pH 8, w i t h f o r m a t i o n of s u c c i n i c anhydride ( n u c l e o p h i l i c c a t a l y s i s by -C02-). Above pH 8, t h e r e CO(CH,),COOH i s a second sigmoid i n c r e a s e , p a r a l l e l i n g t h a t f o r c a t e c h o l monoacetate ( i n b o t h cases k H 2 0 / k D 0 = 1.8) and owing t o general-base c a t a l y s i s by -O-.32 * 2 Acyl T r a n s f e r : A l k y l E f f e c t - The k i n e t i c and thermodynamic d r i v i n g of r i n g c l o s u r e by a l k y l s u b s t i t u e n t s

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i n t h e s u c c i n y l moiety of compounds l i k e 9 i s well-known. Log k f o r t h e n u c l e o p h i l i c c a t a l y s i s of h y d r o l y s i s of 9 p a r a l l e l s v a l u e s of l o g Ke f o r anhydride formation from a l k y l a t e d s u c c i n i c a c i d s w i t h a s l o p e of 0.23 (TS r e c e i v i n g 53% of t h e e q u i l i b r i u m d r i v i n g f o r c e ) .2 With s u c c i n a n i l i d e s ( i n t r a m o l e c u l a r g e n e r a l acid c a t a l y s i s ) , where e x p u l s i o n of t h e l e a v i n g group should be more d i f f i c u l t , t h e corresponding s l o p e is 0.75, c o n s i s t e n t w i t h t h e expected more p r o d u c t - l i k e TS,24 P r e v i o u s work h a s tended t o emphasize conformational f a c t o r s as t h e o r i g i n of t h e a l k y l e f f e c t i n t h e s e systems, b u t s e m i q u a n t i t a t i v e e s t i m a t e s of t h e e n e r g e t i c s of nonbonded i n t e r a c t i o n s s u g g e s t r e l i e f of i n i t i a l - s t a t e s t r a i n as t h e main c o n t r i b u t o r . 2 4 Compelling evidence f o r t h i s view a p p e a r s i n t h e enormous a c c e l e r a t i o n s experienced by d i a l k y l s u b s t i t u t i o n i n maleamides (10) where no q u e s t i o n of conformational isomerism a r i s e s . 2 5 Intramolecular general-acid RxcONHR3 ' C H R 3 c a t a l y s i s of anhydride formation proceeds 2 w i t h r a t e determining decomposition of 7 t h e c y c l i c adduct (shown by a l t e r n a t i v e g e n e r a t i o n of t h e adduct from t h e isoimi d e , which g i v e s o n l y t h e maleamic acid-thus t h e adduct r e t u r n s t o r e a c t a n t f a s t er than going on t o p r o d u c t ) . Monoalkyla t i o n of t h e double bond produces moderR 1 c : R 3 a t e , apparently e l e c t r o n i c e f f e c t s . Dim e t h y l a t i o n (R1 = R2 = CH3) , however, produces a 2 3 , 5 0 0 ~a c c e l e r a t i o n ! The e f f e c t appears as a 10 kcal/mol r e d u c t i o n i n AH", opposed by a 10 B U d e c r e a s e i n R2 0 R2 0 AS*. I n c l u s i o n of t h e two a l k y l groups i n a 5- o r & r i n g completely r e v e r s e s t h e e f f e c t : t h e h a l f amides of cyclopentene and cyclobutene d i c a r b o x y l i c a c i d s r e a c t , r e s p e c t i v e l y , 104x and 105x more szowzy than t h e p a r e n t compound. S i n c e a l l t h e anhydrides hydrolyze a t very s i m i l a r ra te s , t h e e f f e c t i s presumed t o o r i g i n a t e i n i n i t i a l - s t a t e s t r a i n r e l i e v e d i n t h e t r a n s i t i o n s t a t e and p r o d u c t .25 Int e r e s t i n g l y , Keq f o r dimethylmaleic anhydride formation from t h e a c i d appears t o be only a b o u t 530x l a r g e r than Keq f o r m a l e i c anhydride format i ~ n a, f~a c ~t o r of 45x s m a l l e r than t h e a c c e l e r a t i o n of amide c l o s u r e . Perhaps an e l e c t r o n i c requirement f o r n e a r c o p l a n a r i t y of t h e amide funct i o n and t h e double bond i n t e n s i f i e s t h e i n i t i a l s t a t e s t r a i n , o r perhaps a n e a r - t e t r a h e d r a l c e n t e r i n t h e TS l e a d s t o g r e a t e r s t r a i n r e l i e f t h e r e than i n t h e anhydride. The g e n e r a l l y small e f f e c t s of m o n o s u b s t i t u t i o n a r e confirmed by t h e narrow range of r a t e s of r i n g c l o s u r e of v a r i o u s aand B- s u b s t i t u t e d coumaric a c i d s .26

"G 4

Acyl T r a n s f e r : Other S t r u c t u r a l F a c t o r s - Ring-size p r e f e r e n c e s i n t h e TS are expected (and a r e found) t o vary w i t h t h e c a t a l y t i c mechanism. The a m i n o l y s i s o f a c e t y l i m i d a z o l e by diamines shows lOOx r a t e enhancements w i t h 2 o r 3 methylene groups between t h e n u c l e o p h i l i c and presumed b a s e - c a t a l t i c n i t r o g e n s , w h i l e 4- o r 5- atom b r i d g e s gave only 15x a c c e l e r a t i o n s . 2: I n t h e very l a r g e e f f e c t s produced by i n t e r n a l n u c l e o p h i l i c a t t a c k i n 11 (more t h a n 105x f a s t e r than t h e corresponding i n t e r m o l e c u l a r r e a c t i o n ) , t h e 5-ring TS (x = 1) is p r e f e r r e d by a small f a c t o r , 5x i n rate, o v e r t h e

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6-ring TS ( x = 2 ) . 2 8 The same i s t r u e f o r g e n e r a l b a s e c a t a l y z e d l a c t o n i z a t i o n of w-hydroxyalkanoic a r y l esters, i n which t h e b u t y r a t e s c y c l i z e lox 20x f a s t e r than t h e v a l e r a t e s By c o n t r a s t , t h e X v a l e r a t e s undergo a c i d - c a t a l y z e d h y d r o l y s i s probably v i a t h e l a c t o n e about 2x as f a s t as t h e b u t y r a t e s . 2 8 The hydroxyl group appears n o t t o p a r t i c i p a t e i n t h e a c i d i c h y d r o l y s i s of t h e c o n j u g a t e a c i d of 2. The placement of p o t e n t i a l l y c a t a l y t i c groups i s a matter of obvious importance. Thus l o c a t i o n of a carboxyl group a t R1 i n 12 g i v e s h y d r o l y s i s r a t e s (water a t t a c k a t c a r b o n y l ) l i t t l e d i f f e r e n t from t h o s e f o r c a r b o x y l a t R 2 , w h i l e c a r b o x y l a t R 3 g i v e s an enhancement of a t least l o x . Apparently p r o t o n a t i o n of t h e leaving-group n i t r o g e n i s more i m p o r t a n t t h a n p r o t o n @ N H :z R3 d o n a t i o n t o t h e carbonyl oxygen . 2 9

fl

.

2!

C a t a l y t i c power may be c o n f e r r e d on o t h e r w i s e i n e r t f u n c t i o n a l groups by an a c t i v a t i n g chemical r e a c t i o n . For example, hydroxide i o n adds t o t h e k e t o f u n c t i o n of o-acyl esters t o R produce an i n t r a m o l e c u l a r b a s i c group (13)which i n subsequent r a p i d s t e p s produces h y d r o l y s i s of t h e ester 30 Cis-3-benzoylacrylates e n j oy a similar c a t a l y s i s of h y d r o l y s i s w i t h benzoyl s u b s t i t u e n t s g i v i n g p % 2-2.5, w h i l e i n trans3-benzoylacrylates , no such p o s s i b i l i t y e x i s t s Another example of a c t i v a t i o n i s provided by esters of and p % 0.6-0.7.31 3-hydroxybutyric and cis-2-hydroxycyclopentanecarboxylic a c i d s , i n which e s t e r i f i c a t i o n of t h e hydroxyl groups by b o r a t e l e a d s t o i n t r a m o l e c u l a r b o r a t e c a t a l y s i s of h y d r o l y s i s of t h e ester f u n c t i o n . The hydroxyl groups by themselves prodcce only a s m a l l a c c e l e r a t i o n , perhaps connected w i t h solvent structuring.32

.

Acyl T r a n s f e r : B i f u n c t i d n a l C a t a l y s i s - Many mechanisms € o r enzyme c a t a l y s i s p o s t u l a t e c o o p e r a t i v e ("push-pull") c a t a l y s i s by an acid-base p a i r i n t h e a c t i v e s i t e . The analogous b i f u n c t i o n a l i n t r a m o l e c u l a r c a t a l y s i s has been widely s o u g h t ; t h e evidence p r e s e n t e d g e n e r a l l y c o n s i s t s of a maximum r a t e a t a pH s u f f i c i e n t l y a c i d i c f o r t h e a c i d - c a t a l y t i c f u n c t i o n t o be prot o n a t e d y e t s u f f i c i e n t l y b a s i c f o r t h e b a s e - c a t a l y t i c f u n c t i o n t o be f r e e . The h y d r o l y s i s of hexachlorophene monosuccinate (14), f o r example, e x h i b i t s such a rate maximum a t pH 6 . 8 (between pKa 5.20 f o r t h e carboxyl group and 8.4 f o r t h e p h e n o l i c group). S u c c i n i c anhyd r i d e can be t r a p p e d , d e m o n s t r a t i n g a nucleoThe -OH i s presumed a p h i l i c r o l e f o r -Cop-. g e n e r a l a c i d . Hexachlorophene monoacetate CI hydrolyzes 500x f a s t e r t h a n t h e d i a c e t a t e givi n g a rough measure of t h e -OH c a t a l y s i s al o n e , w h i l e t h e monoanion of 3 reacts 3 x 104x f a s t e r t h a n t h e m n o a c e t a t e , a measure of t h e nucleophi li c-p l u s -cooperat i v e con t r i b u t i ~ n .This ~ ~ system c o n t r a s t s w i t h t h e

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competing n u c l e o p h i l i c and g e n e r a l b a s e c a t a l y s i s by -CO2- and -0- s e e n f o r 2 above.22 A r a t e maximum a t pH 2.5 f o r t h e h y d r o l y s i s of methyl 2,6dicarboxybenzoate was taken t o i n d i c a t e b i f u n c t i o n a l c a t a l y s i s . The monoa n i o n , however, r e a c t e d only 12x f a s t e r t h a n t h e n e u t r a l ester which w a s e x p l a i n e d by t h e g r e a t e r importance of leaving-group p r o t o n a t i o n than of b a s e c a t a l y s i s i n t h e probably r a t e - d e t e r m i n i n g breakdown of t h e t e t r a h e d r a l i n t e r m e d i a t e . 3 4 A v e r y s e v e r e blow was d e a l t t h e use of b e l l - s h a p e d pH-rate p r o f i l e s as evidence f o r b i f u n c t i o n a l c a t a l y s i s by t h e o b s e r v a t i o n of such curves i n t h e h y d r o l y s i s of molecules l i k e 15 and p-H02CC&+0CO(CH2)2C02H, where no d i r e c t p a r t i c i p a t i o n by t h e second f u n c t i o n i s s t e r e o c h e m i c a l l y p o s s i b l e . 35 I n 15, p r o t o n a t i o n of N i n c r e a s e s t h e rate by ~o(CH2)COOHimproving t h e l e a v i n g group; deproton3 a t i o n t h u s reduces t h e r a t e ( " e l e c t r o n i c 0' i n h i b i t i o n " ) . S i m i l a r l y d e p r o t o n a t i o n of t h e p-CO2H group i n p-carboxyphenyl s u c c i n a t e produces " e l e c t r o n i c i n h i b i t i o n . " Even when t h e grou i s s t e r e o c h e m i c a l l y a b l e t o c a t a l y z e , i t s e f f e c t may be e l e ~ t r o n i c . ~ ' T h i s c r i t i c i s m a p p l i e s t o t h e two examples c i t e d above. I n f a c t , t h e p o s t u l a t e d a c i d i c b i f u n c t i o n a l component i n seve r a l c a s e s of t h i s k i n d was shown t o obey a l i n e a r f r e e energy r e l a t i o n d e f i n e d by remote s u b s t i t u e n t s , s u g g e s t i n g a n o n - c a t a l y t i c r o l e . 35 Acyl T r a n s f e r : Complexation- I n t r a m o l e c u l a r c a t a l y s i s w i t h i n a r e v e r s i b l y formed complex o f f e r s a s t r o n g analogy t o r e a c t i o n s w i t h i n t h e enzyme-subs t r a t e complex. Complexing a g e n t s such as c a f f e i n e , v a r i o u s a l k y l ammonium i o n s , p i p e r a z i n e d i o n e and t r i g l y c i n e e x h i b i t e d r e v e r s i b l e b i n d i n g ( w a t e r , pH 4.6) w i t h t h e 0 - a c e t y l , hexanoyl and o c t a n o y l esters of 8-hydroxyquinol i n e b u t t h e a t t a c k of water on t h e ester c a r b o n y l , general-base c a t a l y z e d by t h e q u i n o l i n e n i t r o g e n , was reduced i n r a t e i n t h e complex. I n cont r a s t , d i r e c t complexation ( i n f e r r e d from chain-length e f f e c t s ; no s a t u r a t i o n k i n e t i c s observed) between t h e q u i n o l i n e esters and long-chain a l k y l amines i n c r e a s e d t h e rate of t h e c o r r e s p o n d i n g l y c a t a l y z e d a m i n o l y s i s rea c t i o n s . Decylamine r e a c t s 2Ox-4Ox f a s t e r t h a n ethylamine w i t h t h e quinol i n e esters, and r e a c t s more t h a n 2x f a s t e r w i t h t h e 8 - d e r i v a t i v e (capable of i n t r a m o l e c u l a r c a t a l y s i s ) t h a n w i t h t h e 6 - d e r i v a t i v e . 36 The macroc y c l i c hydroxamic a c i d 16 c o n t a i n s a hydrophobic b i n d i n g s i t e ( t h e c a v i t y CH,CONOH of t h e r i n g ) and two f u n c t i o n s f o r intracomplex r e a c t i o n . S a t u r a t i o n k i n e t i c s i s observed w i t h p - n i t r o hH3 p h e n y l b u t y r a t e (Km = 1 mM) even though t h e medium cont a i n s 11% methanol and 2% a c e t o n i t r i l e . "Burst" k i n e t i c s is s e e n w i t h a l l p - n i t r o p h e n y l esters, t h e s i z e of t h e b u r s t i n d i c a t i n g t h a t only one hydroxamic-acid group i s a c t i v e . The a c c e l e r a t i o n over t h e r e a c t i o n '"3 r a t e w i t h N,N-diisobutylglycine N-methylhydroxamic a c i d , produced by 16,rises from 1 . 7 ~( p - n i t r o p h e n y l a c e t a t e ) C ~ ~I ~ ~ O (p-nitrophenyl H to N 7600x dodecanoate) 37

'

.

Complexation w i t h Ni* t o form 1 7 g i v e s a 56x i n c r e a s e i n t h e esterh y d r o l y s i s rate, w h i l e i o n i z a t i o n of t h e s a l i c y l carboxyl produces a f u r t h e r 1 . 7 ~i n c r e a s e , l e a d i n g t o t h e p o s t u l a t e of b f f u n c t i o n a l ( c a r b o x y l a t e m e t a l i o n ) c a t a l y s i s . The c a r b o x y l a t e e f f e c t i s i n t h e wrong d i r e c t i o n f o r

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an e l e c t r o n i c , n o n - c a t a l y t i c e x p l a n a t i o n . The s m a l l b i f u n c t i o n a l a c c e l e r a t i o n i s termed "semicooperative The r e a c t i o n of 2-cyanopyridine w i t h water t o g i v e yridine-2-carboxamide, catalyzed by Cuu o r N,i' is completely circumvented

0 - - - Ni

t+

a l k o x i d e c o n j u g a t e b a s e w i t h 2-cyanopyridine w h i l e b o t h a r e l i g a n d s on t h e m e t a l i o n . The metal i s bel i e v e d t o a c i d - c a t a l y z e a t t a c k of t h e a l k o x i d e oxygen on t h e n i t r i l e group. Phenomenologically, t h e metal-ion c a t a l y s i s a p p e a r s i n AS* .39 Hydrolysis of Phosphate Esters- I n t r a m o l e c u l a r c a t a l y s i s i n t h e h y d r o l y s i s of second-row d e r i v a t i v e s h a s f i g u r e d l a r g e i n t h e d e s i g n of k i n a s e and phosphatase mechanisms. The dominant theme h a s been t h e s t e r e o c h e m i c a l l i m i t a t i o n s imposed by t h e i n t e r m e d i a t e t r i g o n a l - b i p y r a m i d a l a d d u c t , 40 e x e m p l i f i e d by 19 f o r phosphoenol p y r w a t e . E l e c t r o n - r i c h groups s t r o n g l y p r e f e r t h e e q u a t o r i a l p o s i t i o n s of such s p e c i e s yOR2 w h i l e only r e l a t i v e l y e1ectro.n-poor groups may a t* t a i n the a x i a l positions ("polarity rule"). Ent r a n c e t o and d e p a r t u r e from phosphorus m u s t o c c u r O-pA: I 0 - i n a x i a l p o s i t i o n s . When R2 = - ( i n t e r m e d i a t e d e r i v H2C< e d from CH2=C(C02-)OP03Ri), 2 is t h e only s t r u c t u r e 0 which can form because t h e two e l e c t r o n - r i c h -00 groups are locked e q u a t o r i a l and t h e 5 - r i n g cannot Thus 19 can o n l y r e t u r n t o span t h e e q u a t o r i a l p o s i t i o n s ( " s t r a i n r u l e " ) . r e a c t a n t s o r e x p e l R l O - . When R1 = CgHgCH2, e x p u l s i o n of t h e l a t t e r ( w i t h a c i d c a t a l y s i s ) i s t h e major (85%) r e a c t i o n i n s p i t e of t h e expected g r e a t er leaving-group r e a c t i v i t y of t h e e n o l v s b e n z y l a l c o h o l . Protonation of one of t h e -0- a l s o occurs (13%) t o g i v e OH which can occup an a x i a l p o s i t i o n . Then i s o m e r i z a t i o n of 2 t o g i v e 20 (pseudorotation'O) i s p o s s i b l e . Departure of t h e e n o l 0 i n 20 (R1 = H , OR' OR R2 = CH2CgHg) g i v e s an open-chain a c y l phosphate which hydrolyzes t o t h e observed p y r u v i c a c i d and monobenzyl phosphate. The d i b e n z y l s u b s t r a t e ( y i e l d i n g 9 o r 20, R 1 = R2 = CH2CgHg) e x p e r i e n c e s no h i n d r a n c e t o p s e u d o r o t a t i o n and forms t h e openc h a i n a c y l phosphate from 20 350x f a s t e r t h a n i t h y d r o l y z e s . However, t h e open-chain compound n e a r l y always r e - c l o s e s and t h e c h i e f (85%) o v e r a l l r e a c t i o n i s a g a i n benzyl-alcohol e x p u l s i o n . K i n e t i c a l l y , t h e h y d r o l y s i s r e a c t i o n s appear as pH-independent r e a c t i o n s of t h e phosphoenol p y r u v i c acid d e r i v a t i v e ( i n t r a m o l e c u l a r g e n e r a l a c i d c a t a l y s i s ) . S i m i l a r l y , phenyl s a l i c y l phosphate h y d r o l y s i s g i v e s o n l y phenol and s a l i c y l phosphate ( v i a 21, R1 = C g H g , R2 = -) w h i l e t h e t r i e s t e r formed from s a l i c y l i c a c i d Rp = (cH2)3, which can undergo and propane-1,3-diol y i e l d s 21 w i t h R1 p s e u d o r o t a t i o n e a s i l y . The p r o d u c t above pH 5.3 i s t h u s s a l i c y l i c a c i d and p r o p a n e d i o l phosphate; t h e r e a c t i o n shows k i n e t i c i n t r a m o l e c u l a r general b a s e c a t a l y s i s . The rate d e t e r m i n i n g s t e p i s probably t h e h y d r o l y s i s

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of t h e r e v e r s i b l y formed open-chain a c y l phosphate d e r i v a t i v e . B e l o w pH 2.0, t h e predominant p r o d u c t i s t h a t from h y d r o l y s i s of one of t h e p r o p a n e d i o l P-O bonds and k i n e t i c i n t r a m o l e c u l a r g e n e r a l a c i d c a t a l y s i s i s s e e n . The r a t e - d e t e r m i n i n g s t e p may now b e a c i d - c a t a l y z e d c l e a v a g e of R10-P i n 3, R1 + R2 = (CH2)3. The a c c e l e r a t i o n s by b o t h -Copand -C02H are lo',, r e l a t i v e t o phenyl d i a l k y l

p h o s p h a t e s . 42

A l o n g - s t a n d i n g p o i n t of i n t e r e s t h a s been t h e i n t r a m o l e c u l a r genera l a c i d c a t a l y z e d e x p u l s i o n of PO3- from s a l i c y l phosphate which shows no deuterium s o l v e n t i s o t o p e e f f e c t i n s p i t e of t h e a p p a r e n t TS hydrogen bonding o r p r o t o n t r a n s f e r . 4 3 SO3 e x p u l s i o n from s a l i c y l s u l f a t e i n a presumabl s i m i l a r p r o c e s s a l s o shows no s o l v e n t i s o t o p e e f f e c t . J 4 T h i s l a c k has been a s c r i b e d i n b o t h c a s e s t o a p r o d u c t - l i k e s t r u c t u r e f o r t h e p r o t o n - t r a n s f e r p r o c e s s i n t h e TS (comp l e t e t r a n s f e r t o 0 l e a v i n g -C02-). A c r u c i a l experiment h a s now d i s p r o v e d t h i s h y p o t h e s i s . S u b s t i t u e n t s a t R1 and R2 i n 22 b e a r a p a r t i c u l a r r e l a t i o n s h i p t o t h e p h e n o l i c and c a r b o x y l f u n c t i o n s : R1 is para t o -C02H and meta t o -0PO3- and R2 i s vice ver8a. The rates of hydroR2 l y s i s of s u b s t i t u t e d compounds may t h e r e f o r e b e f i t t e d t o a two-term H a m m e t t e q u a t i o n l o g klk, = p l a l p 2 a 2 where p 1 r e p r e s e n t s t h e s u b s t i t u e n t e f f e c t a t t h e c a r b o x y l and p 2 t h e e f f e c t a t t h e p h e n o l i c tent e r , w i t h a1 and a2 a p p r o p r i a t e l y chosen. The v a l u e s are p 1 = -1 and p 2 = +1.74. The former shows t h e -C02H t o b e i n t a c t as s u c h i n t h e TS Thus t h e l a c k of ( t h e s u b s t r a t e i s i n t h e form R1R2C6H2(CO2-)OPO3H-). i s o t o p e e f f e c t i s from a r e a c t a n t - l i k e TS f o r p r o t o n t r a n s f e r ; t h i s is confirmed by p 2 , which i n d i c a t e s a l a r g e n e g a t i v e charge a t t h e TS p h e n o l i c c e n t e r . The o r i g i n of t h e c a t a l y t i c e f f e c t i s s u g g e s t e d t o b e r o t a t i o n of t h e c a r b o x y l i n t o t h e r i n g p l a n e simultaneous w i t h P-O f i s s i o n , and res u l t i n g d e l o c a l i z a t i o n of t h e p h e n o l i c charge .45

(z),

+

Metal i o n s are g e n e r a l l y r e q u i r e d f o r enzymic phosphoryl t r a n s f e r . T h e i r c a t a l y t i c e f f e c t s on phosphate ester and h y d r o l y s i s i n model systems are, however, q u i t e modest i n t h e absence of an i n t r a m o l e c u l a r b i n d i n g s i t e . Given t h e l a t t e r , l a r g e a c c e l e r a t i o n s are s e e n . An 0i m i d a z o l y l f u n c t i o n b i n d s Cuu as i n 23 t o y i e l d a g r e a t e r than 104x a c c e l e r a t i o n of PO3u l s i o n o v e r t h e r a t e of t h e r e a c t i o n w i t h o u t Cu*e3 The analogy t o 22 above 0 i s i n t e r e s t i n g . Phosphoryl d o n a t i o n from an imidaII 0. p'ozolium t o an oximate c e n t e r i s a c c e l e r a t e d about lo3, ( v s h y d r o l y s i s of phosphorylimidazole r a t h e r t h a n I oximate a t t a c k ) by t h e agency of Znu (24). The 0 N T r e a c t i o n is c o n s i d e r e d a good model f o r n u c l e o s i d e NH diphosphokinase , which r e q u i r e s Mgu and p a s s e s through a p h o s p h o r y l h i s t i d i n e i n t e r m e d i a t e .48

-

,c{+

O r b i t a l S t e e r i n g - The i d e a t h a t enzymes might d e r i v e

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Gandou r , Schowen

287

v e r y l a r g e a c c e l e r a t i o n s from e f f e c t i n g t h e p r e c i s e o r i e n t a t i o n of r e a c t i n g groups ( " o r b i t a l s t e e r i n g " ) a r o s e from t h e o b s e r v a t i o n t h a t t h e a c i d - c a t a l y z e d l a c t o n i z a t i o n of 25 i s 106x f a s t e r t h a n e t h a n o l - a c e t i c a c i d es t e r i f i c a t i o n . An a t t e m p t e d d i s s e c t i o n of t h i s and r e l a t e d accelerations i n t o contribuapproximation, s t r a i n and 24 12 rt ieosnt rs i cfrom t i o n of r o t a t i o n s l e f t a f a c t o r of 2 x l o 4 unaccounted f o r which was OH a s s i g n e d t o o r b i t a l s t e e r i n g . 4 9 However, d i f f e r e n t c o r r e c t i o n s f o r s t r a i n and r o t a t i o n a l r e s t r i c t i o n s u g g e s t t h a t l a c t o n i z a t i o n of 25, r a t h e r t h a n b e i n g a c c e l e r a t e d 45x r e l a t i v e t o y-hydrox b u t y r i c - a c i d l a c t o n i z a t i o n by o r b i t a l s t e e r i n g , is retarded about S t i l l a n o t h e r s e t of c o r r e c t i o n s roduces a s i m i l a r conclusion .51 Both s t a t i s t i c a l - m e c h a n i c a l and rough ent r o p y estimates50 l e a d t o a c c e l e r a t i o n s of up t o 106x-109x, merely f o r i n t r a m o l e c u l a r vs i n t e r m o l e c u l a r r e a c t i o n , w h i l e o n l y 55x w a s o r i g i n a l l y used. The e n e r a l q u e s t i o n of p r o x i m i t y e f f e c t s was c r i t i c a l l y reviewed last year. 58

-

calculation^^^

I f r e a c t i o n s are g r e a t l y speeded o r slowed by s m a l l changes i n t h e o r i e n t a t i o n of groups i n t h e t r a n s i t i o n s t a t e , t h e " i d e a l " t r a n s i t i o n s t a t e must be v e r y r i g i d , a t least n e a r t h e r e a c t i n g c e n t e r . Force-cons t a n t estimates based on an o r b i t a l - s t e e r i n g f a c t o r of lo8 show t h e req u i s i t e f o r c e c o n s t a n t s t o b e f a r t o o l a r g e , about l O O x l a r g e r t h a n t h o s e A c o l l i s i o n - t h e o r y c a l c u l a t i o n a p p e a r s , however, i n s t a b l e molecules. t o a l l o w a c c e l e r a t i o n s of about 3Ox-1OOx p e r r e a c t i n g group ,54 f o r " p r e c i s e " o r i e n t a t i o n w i t h i n 10' of s o l i d a n g l e . 5 5

REFERENCES 1. 2.

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

11. 12.

13. 14. 15. 16.

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M.L. Bender, "Mechanism of Homogeneous C a t a l y s i s from Protons t o P r o t e i n s , " W i l e y - l n t e r s c i e n c e , New York, N . Y . , 1971. 6 . Capon, Essays i n Chemistry, 3, 127 (1972). A.J. Kirby and A . R . Fersht, Prog. Bioorg. Chem. , 1 (1971). J. H i n e , M.S. Cholod and J . J e n s e n , J . Amer. Chern. SOC. 93, 2321 (1971). H . Wilson and E.S. Lewis, i b i d . , 94, 2283 (1972). B . G . Cox, J . Chem. SOC. B , 1704 (1971). D.J. Creighton and D.S. Sigman, J . Amer. Chem. SOC. 2,6314 (1971). B . Capon, Biochimie 53, 145 (1971). B.M. Dunn and T.C. BKice, J . Amer. Chem. SOC. 93, 6314 (1971). 8. Capon, E . A n d e r s o n , N.S. Anderson, R . H . Dahm and M.C. Smith, J . Chem. SOC. B , 1963 (1971). T.H. F i f e and E . Anderson, J. Amer. Chem. SOC. 2,6610 (1971). B . Capon and B . C . Ghosh, J . Chern. SOC. B y 739 (1971). B . Capon and M , I . Page, J . Chem. SOC., Perkin T r a n s . I I , 522 (1972). 8. Capon, M . I . Page and G . H . Sankey, i b i d , 529 (1972). E . Anderson and T. H . F i f e , Chem. Commun., 1470 (1971). E . Anderson and B. Capon, J. Chem. SOC., Perkin Trans 1 1 , 515 (1972). J. T i l l e t and D. E . Wiggins, Tetrahedron L e t t . , 911 (1971).

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Sect. V I T o p i c s i n Chemistry W i l e y , Ed. A.J. K i r b y and G.J. L l o y d , Chem. Commun., 1538 (1971). 19. R.F. P r a t t and T.C. B r u i c e , Biochem. 3178 (1971). 20. T.C. B r u i c e and R.F. P r a t t Chem. Commun., 1259 (1971) and J. Amer. Chem. SOC., 2823 (19723. 21. J.A. Shafer, C.J. B e l k e and S.C.K. Su, J. Amer. Chem. SOC. 93, 4552

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L . Eberson and L.A. Svensson, i b i d . 93, 3827 (1971). L. Eberson and L.A. Svensson, Acta Chem. Scand., i n p r e s s . L. Eberson and H. W e l i n d e r , J. Amer. Chem. SOC. 93, 5821 (1971). A.J. K i r b y and P.W. L a n c a s t e r , J. Chem. SOC., P e x i n Trans. 1 1 , i n press. B.C. L i p p o l d and E.R. G a r r e t t , J. Pharm. S c i . 60, 1019 (1971). M . I . Page and W.P. Jencks, Fed. Proc. 30, 1 2 4 0 7 1 9 7 1 ) . B. Capon, S.T. McDowell and W.V. R a f t e G , Chem. Commun.,389 (1971). A.F. Hegarty, R.F. P r a t t , T. G i u d i c i and T.C. B r u i c e , J. Amer. Chem. SOC. 93, 1428 (1971). K. B o x e n and G.R. T a y l o r , J. Chem. SOC. B, 145, 149 (1971). K. Bowden and M.P. Henry, i b i d . , 156 (1971). B. Capon and M. I . Page, i b i d . , 741 (1971). T. H i g u c h i , H. Takechi, I . H . Pitman and H.L. Fung, J. Amer. Chem. SOC.

93, 539 (1971).

34. 35. 36. 37. 38. 39. 40.

41. 42.

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46. 47. 48. 49. 50.

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K H . H u r s t and M.L. Bender, i b i d . , 93, 704 (1971). T. Maugh I I and T.C. B r u i c e , i b i d . , 3237 (1971). T. Maugh I I and T.C. B r u i c e , i b i d . , 6584 (1971). R. H e r s h f i e l d and M.L. Bender, i b i d . , 1376 (1972). R. Breslow and C. M c A l l i s t e r , J. Amer. Chem. SOC. 93, 7096 (1971). R. Breslow and M. Schmir, i b i d . , 93, 4960 (1971). F.H. Westheimer, Accts. Chem. R 1,es. 70 (1968). K.J. Schray and S.J. Benkovic, J. G e r . Chem. SOC. 93, 2522 (1971). R.H. Bromilow, S.A. Khan and A.J. K i r b y , J . Chem. B, 1091 (1971). M.L. Bender and J.M. L a w l o r , J. Amer. Chem. SOC. 85, 3010 (1963). S.J. Benkovic, Ann. N. Y . Acad. S c i . 172, 563 (19m. R.H. Bromilow and A.J. K i r b y , J. C h e m z o c . , P e r k i n Trans. I I , 149 ( 1972) G.J. L l o y d , C.M. Hsu and B.S. Cooperman, J. Amer. Chem. SOC., 4889 (1971) S.J. Benkovic and L.K. D u n i k o s k i , J r . , i b i d . , 93, 1526 (1971). G.J. L l o y d and B.S. Cooperman, i b i d . , 93, 4 8 8 3 7 1 9 7 1 ) , D.R. Storm and D.E. Koshland, J r . , P r o c N a t . Acad. S c i . U . S . , 445 ( 1970) 8. Capon, J. Chem. SOC. B, 1207 (1971). T.C. B r u i c e , A. Brown and D.O. H a r r i s , Proc. N a t . Acad. S c i . U . S . ,

2, 2, 94,

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68, 658 (1971).

68,

52. KI. Page and W.P. Jencks, i b i d . , 1678 (1971). 53. T.C. B r u i c e , " P r o x i m i t y E f f e c t s i n Enzyme C a t a l y s i s , " i n "The Enzymes,'' 3 r d ed., v o l . I I , P.D. Boyer, ed., Academic Press, New York, N.Y., 1970. 54. G.A. D a f f o r n and D.E. Koshland, J r . , B i o o r g . Chem. 1, 129 (1971). 55. D.E. Koshland, K.W. Carraway, G.A. D a f f o r n , J.D. Gays and D.R. Storm, C o l d S p r i n g Harbor Symp., 3 6 , 13 (1971).

Chapter 27.

Peptide Synthesis

John Morrow Stewart, U n i v e r s i t y o f Colorado School o f Medicine, Denver, Colorado 80220

T h i s review w i l l summarize progress i n methods o f p e p t i d e s y n t h e s i s and t h e s y n t h e s i s o f c e r t a i n s p e c i f i c peptides and p r o t e i n s from the l i t e r a t u r e s i n c e these t o p i c s were l a s t reviewed i n t h nnual Reports , and f i v e u s e f u l f o r 1961. Two u s e f u l books1,2, f i v e symposium r e p o r t s reviews - l 2 have appeared.

5-9

S y n t h e t i c Methods - P r e p a r a t i o n and use o f ena i n e p r o t e c t i n g groups13 and a new p r o t e c t i n g group based on 2 - n i t r o p h e n o l have been described.

4

Procedures f o r removal of Boc groups w i t h mercaptoethanesulfonic a c i d l 5 , which should be u s e f u l f o r p r o t e c t i o n o f tryptophan, as w e l l as f o r removal of r i f l u o r o a c e t y l (TFA) and t r i c h l o r o a c e t y l groups by r e d u c t i o n w i t h NaBH l6have appeared. The imidazole o f h' t i d i n e appears t o be most e f f e c t i v e v y blocked by the t o s y l (Tos) group, 4,'5 although t h e 2,4d i n i t r o p h e n y l (Dnp) group i s goodl8. Both o f these groups i n h i b i t racemization o f h i s t i d i n e 7 . The Tos group i s removed by HF, w h i l e t h e Dnp i s n o t . The Tos group f o r the guanidine o f a r g i n i n e appears t o o f f e r advantages over t h e n i t r o group, s i n c e the former does n o t cause cont a m i n a t i o n o f products w i t h o r n i t h i n e , due t o decomposition o f t h e a r g i n i n e l g . The g u a n i d i n o - t o s y l group i s a l s o cleaved by HF. A new group, d i isopropylmethoxycarbonyl, ("Dipmocl') has been proposed f o r the E-amino group o f l y s i n e , s i n c e i t i s more s t a b l e than the carbobenzoxy group and should a v o i d t h e problem o f branching a t l y s i n e i n s o l i d phase synthesis20. However, t h i s group appears t o be more s t a b l e than would be i d e a l . Simultaneous deblocking and o x i d a t i o n o f c y s t e i n e i n peptides can be accomplished w i t h 12 i f t h e acetamidomethyl group i s used f o r p r o t e c t i o n 2 1 . The S-isobutoxymethyl p r o t e c t i n g group f o r c y s t e i n e 2 2 has a l s o been described. Papers have appeared on the use o f 2 - p y r i d y l e s t e r s 2 3 and 2 - p y r i d y l t h i ~ e s t e r si n~ ~ synthesis. Simultaneous deblocking o f 2 - n i t r o p h e n y l s u l f e n 1 peptides and c o u p l i n g a r e p o s s i b l e u s i n g the method o f Mukaiyama 28 , which has now been shown t o be r a c e m i z a t i o n - f r e e . The Mer k method of s y n t h e s i s w i t h Ncarboxy anhydrides i n aqueous solution2g,27 has been described i n d e t a i 1 . Coupling o f peptides w i t h DCC i n t h e presence o f hydroxybenzotriazole28 has been shown t o reduce racemization s i g n i f i c a n t l y . Cleavage of peptides a t Thr-Pro bonds29 i n Na-NH r u l e s out t h i s reagent as a general one f o r p e p t i d e s y n t h e s i s , a though some peptides a r e remarkably r e s i s t a n t t o damage. A c a r e f u l study o f a f a c i l e N-0 s h i f t i n n s u l i n has appeared30.

7

Racemi z a t ion i n pept ide synthes i s has been r e v i ewed9, and Mann ing has improved h i s method f o r d e t e c t i n g racemization by a c o r r e c t on f o r racemi za t i on dur ing hydro1ys is 31

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S o l i d Phase Synthesis (SPPS) - I n the e i g h t years s i n c e t h e i n t r o d u c t i o n o f t h i s new technique, achievements i n s y n t h e s i s o f peptides and even p r o t e i n s by t h i s method have been impressive. However, much u n c r i t i c a l work and naivete/ o f approach on t h e p a r t o f many i n v e s t i g a t o r s has s e r i o u s l y hampered development o f the method. Although many peptides have been synthesized s u c c e s s f u l l y on t h e conventional p o l y s t y r e n e r e s i n , p a r t i c u l a r l y when t h e 1% c r o s s l i n k e d r e s i n i s used, i t i s apparent t h a t new types o f s o l i d supports a r e needed, e s p e c i a l l y f o r the synthesis o f l a r g e peptides and r o t e i n s . Recent work has r e r t e d t h e use o f m a c r o r e t i c u l a r r e ~ i n s 3 ~ ~ 3r e3s, i n coated glass beads!g, polystyrene grafted onto halocarbon nucleus35, and even peptides bound c o v a l e n t l y t o g l a s s beads36. New types o f l i n k a g e t o a conventional p o l y s t y r e n e r e s i n i n v o l v e t h e use o f h a l o a c y l r e s i n s from which t h e p e p t i d e can be removed readi l y by nucleophi 1 i c reagents37, and t h e use o f p e p t i d e - r e s i n bonds which a r e very s t a b l e d u r i n g t h e s n t h e s i s , b u t can be r e a d i l y m o d i f i e d t o d e r i v a t i v e s which a r e very l a b i l e 38- 40. U n f o r t u n a t e l y , none o f the systems proposed so f a r appear t o be u n i v e r s a l l y a p p l i c a b l e . Attachment o f t h e p e p t i d e t o the r e s i n v i a €-amino group o f l y s i n e was used i n a novel s y n t h e s i s o f vasopressin L o f f e t has proposed t h e use o f a quaternary base i n t h e usual e s t e r ! ic a t i o n procedure f o r attachment o f t h e f i r s t amino a c i d t o t h e r e s i n This should e l i m i n a t e f o r m a t i o n o f quaternary ammonium groups on t h e r e s i n , which may complicate s y n t h e s i s as w e l l as m o n i t o r i n g d u r i n g t h e syn thes i s

$3; . fl.

.

Incomplete removal o f Boc grou s d u r i n g SPPS has long been recognized as a source o f d i f f i c u l t y t 2 . U n f o r t u n a t e l y t h e s o l u t i o n found by M e r r i f i e l d a t t h a t time, t h e use o f 1% c r o s s l l n k e d r e s i n and a d e p r o t e c t i o n reagent which s w e l l s t h e r e s i n maximally, has n o t been w i d e l y adopted. For example, the s y n t h e s i s o f a p e p t i d e r e c e n t l y described as llimpossible"43 was r e a d i l y accomplished by the use o f a 1% c r o s s l i n k e d I t i s evident (1:3) as t h e d e p r o t e c t i o n reagent43a. r e s i n and TFA-CHCl t h a t f o r successfu s y n t h e s i s o f l a r g e peptides o r p r o t e i n s , m o n i t o r i n g w i l l be e s s e n t i a l , both o f d e p r o t e c t i o n 4 3 ~ 4 4 , as w e l l as c o u p l i n g .

7

The c o u p l i n g r e a c t i o n i n SPPS has been s t u d i e d from t h e aspects o f solvent45, n a t u r e o f the amino a c i 46, c h a i n length47, a d s o r p t i o n o f t h e amino a c i d d e r i v a t i v e t o t h e r e s i nf!8 , and e f f e c t o f t r i a z o l e a d d i t i v e s on coup1 i n g o f a c t i v e e s t e r s 4 9 . N-Ethoxycarbonyl-2-ethoxydi h y d r o q u i n o l i n e (EEDQ) has been i n v e s t i g a t e d as a cou l i n g agent50, as w e l l as a reversed SPPS u s i n g the a z i d e c o u p l i n g methods'. The c o u p l i n g o f p e p t i d e fragments i n SPPS ma become extremely important f o r the s y n t h e s i s o f l a r g e molecules~~ e s~p~e c~i a, l l y w i t h t h e use o f a reagent such as hydroxyb e n z o t r i a z o l e t o reduce racemization53, o r o f a system such as t h a t of Wang and M e r r i f i e l d s h which gives p r o t e c t e d p e p t i d e hydrazides d i r e c t l y from SPPS. Hydroxybenzotriazole i s a l s o u s e f u l f o r suppression o f racemization o f benzyl h i s t i d i n e i n SPPS55, although use o f t h e Dnp o r Tos d e r i v a t i v e 5 6 would seem t o be a b e t t e r s o l u t i o n t o t h a t problem. Use o f Na-NH3 o r hydrogenolysis f o r removal o f benzyl groups from h i s t i d i n e cannot be recommended g e n e r a l l y . A n i n h y d r i n t e s t f o r m o n i t o r i n g

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completeness o f c o u p l i n g i n SPPS has proved t o be very popular57. Although t h r e l i a b i l i t y of such m o n i t o r i n g techniques has been s e r i o u s l y q u e ~ t i o n e d s ~some , problems have been revealed by monitoring59. The consequences o f incomplete r e a c t i o n s i n SPPS, the s o - c a l l e d " f a i l u r e sequences", have been discussed60, and reagents have been described f o r t e r m i n a t i n g p e p t i d e chains where complete coup1 i n g cannot be achieved61 962. A r a p i d method f o r cleavage o f amino a c i d residues from SPPS r e s i n s has been p roposed63. Several tr p t o han-containing peptides have been synthesized Mercaptoethanol and 1,2-dimercaptoethane b o t h r e c e n t l y by SPPS appear t o be s a t i s f a c t o r y f o r p r o t e c t i o n o f tryptophan i n a c i d i c reagents. Hydro1 s i s o f peptides by t o l u e n e s u l f o n i c a c i d i n the presence o f i n d o l e 9 a l l o w s d i r e c t ana y s i s o f tryptophan.

t4-6t.

5?-

4

The s y n t h e s i s of p e p t i d e amides i s important f o r endocrinology. Recent r e p o r t s d e s c r i b e cleavage o f peptides from the standard SPPS r e s i n by ammonolysis69, o r by t r a n s e s t e r i f i c a t i o n t o t h e methyl e s t e r followed by ammonol s i s i n s o l u t i o n f o r C-terminal v a l i n e peptides (aAnother s y n t h e s i s o f MSH was achieved by conversion melanotropin, M S H ) 7 )5 o f the standard r e s i n t o a hydroxyphenyl r e s i n d e r i v a t i v e , from which t h e p e p t i d e c o u l d be removed by ammonolysis68. I n t h i s case racemization was a problem i f t h e ammonolysis was done a t room temperature. Benzhydrylamine resins71 o f f e r much promise f o r t h e s n t h e s i s o f amides, and have already been u t i l i z e d i n several syntheses73,73.

.

The s y n t h e s i s o f peptides on s o l u b l e p o l y m e r i c supports continues t o receive some i n v e s t i g a t i o n 7 4 , 7 5 , as w e l l as t h e use o f i n s o l u b l e reagents76,77 f o r s y n t h e s i s o f peptides i n s o l u t i o n . Synthesis o f S p e c i f i c Peptides - By f a r the most s p e c t a c u l a r achievement o f p e p t i d e s y n t h e s i s i s t h a t o f ribonuclease A by Gutte and M e r r i f i e l d , This s y n t h e t i c p r o t e i n has now been p u r i f i e d t o 80% s p e c i f i c a c t i v i t y 7 8 . Progress i n improvement o f the s y n t h e s i s o f ribonuclease S - p r o t e i n by t h e c l a s s i c a l method has been reviewed79. S y n t h e t i c studies80 have revealed a very i n t e r e s t i n g p e p t i d e - p r o t e i n recombination a t t h e carboxyl end of ribonuclease s i m i l a r t o t h a t o f t h e S-peptide a t t h e amino end. synthesis o f a 188-residue chain proposed f o r human g r w t h hormonLkT was marred by the s y n t h e s i s o f the wrong sequence82, and by the use o f , which probably cleaved the chain a t Thr-Pro. The f a c t t h a t some growt hormone and p r o l a c t i n a c t i v i t y was o b t a i n e d probably i n d i c a t e s Na-NHl? t h a t o n l y p o r t i o n s o f the chain a r e necessary f o r these a c t i v i t i e s . T h i s has already been suggested by work on bovine growth hormone83. The synthesis o f E. e a c y l c a r r i e r p r o t e i n 5 8 appears t o be very c a r e f u l work, and h i g F b i o l o g i c a l a c t i v i t y was o b t a i n e d . S i g n i f i c a n t b i o l o g i c a l a c t i v i t y was a l s o o b t a i n e d i n the s y n t h e s i s o f t h e b a s i c p a n c r e a t i c t r y p s i n i n h i b i t o r p e p t i d e , b i i the s y n t h e t i c methods used caused much I n c o n t r a s t , the s y n t h e s i s o f damage t o the n a t i v e p e p t i d e p a r a t h y r o i d hormone residues 1-34 gave a p e p t i d e w i t h very h i g h b i o l o g i c a l a c t i v i ty35. C a r e f u l s y n t h e t i c s t u d i e s on staphylococcal nuclease continue t o appear from the l a b o r a t o r y o f Anfinsen85. D e t a i l s o f

.

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Katsoyannis' c l a s s i c a l work on i n s u l i n have appeareda6 as w e l l as c o n t i n u i n g s t u d i e s by SPPS on i n s u l i n from the l a b o r a t o r y o f Weitze187. Reports o f new o x y t o c i n analogs continue t o appear. P a r t i c u l a r l y i n t e r e s t i n g are a-hydroxy oxytocin88, which i s more than t h r e e times as a c t i v e as t h e parent hormone, and 4-threonine oxytocin89, which has a very h i g h r a t i o o f o x y t o c i c t o a n t i d i u r e t i c a c t i v i t y . Several peptides from t h e C- termi nus o f chol ecys t o k i n i n (CCK) have been synthes i zed, up t o a tridecapeptidego. The s m a l l e s t p e p t i d e possessing CCK a c t i v i t y I s a heptapeptide. [8-lsoleucinel-angiotensin I I seems t o be a good i n h i b i t o r o f a n g i o t e n s i n a c t i o n g l , and t h e new approach o f I n c o r p o r a t i n g a l k y l a t i n g groups i n t o p e p t i d e hormones92 o f f e r s g r e a t promise f o r t h e future. S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s i n the a n g i o t e n s i n and k i n i n f i e l d s have been r e v i e w e d 3 ~ 6 ~ 7 . Several synthe s o f a n t i b i o t i c peptides have appeared, n o t a b l y those o f g r a m i c i d i n S 9 3 ~ 3 ~t y, r o c i d i n s 895, C96 and E97, and actinomycin D and analogs98. The p o t e n t hypotensive p e p t i d e , Substance P, has been c h a r a c t e r i z e d and synthesized73. For i n f o r m a t i o n on synthesis o f hypothalamic and p i t u i t a r y p e p t i d e hormones, t h e reader i s r e f e r r e d t o Chapter 18. Re f e r e nces

1. 2.

3. 4.

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6. 7. 8. 9. 10.

11. 12.

13. 14.

15. 16. 17. 18. 19. 20.

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P.G. Katsoyannis, J . Ginos, C . Z a l u t , M. T i l a k , S . Johnson and A.C. T r a k a t e l l i s , J.Am.Chem.Soc., 93, 5877 (1971). G. W e i t z e l , K. E i s e l e , H. G u g x e l m i , W . Stock and R. Renner, 2. P h y s i o l .Chem., 352, 1735 (1971). D.B. Hope and M. W a l t i , Biochem.J., 125, 909 (1971). M. Manning, E . Coy and W.H. Sawyer, B i o c h e m i s t r y , 2, 3925 (1970). M.A. O n d e t t i , J. Pluscec, E.F. Sabo, J.T. Sheehan and N. W i l l i a m s , J. Am.Chem.Soc. , 92, 195 (1970). F.M. Bumpus, R X . T u r k e r and M. Yamamoto, i n Proceedings, Second I n t l . Symp. P r o t e i n and P o l y p e p t i d e Hormones, L i e g e , 1971. M. Margoulies and F.C. Greenwood, eds., V o l . 2, E x c e r p t a Medica, Amsterdam, 1972. T.B. Paiva, A.C.M. Paiva, R.J. F r e e r and J.M. S t e w a r t , J.Med.Chem., 15, 6 (1972). M. Ohno, K. Kuromizu, H . Ogawa and N. Izumiya, J.Am.Chem.Soc., 93, 5251 (1971). L. Losse and K. Neubert, Tetrahedron L e t t e r s , 1267 (1970). K. Kuromizu and N. Izumiya, Bul1.Chem.Soc. Japan, 43, 2199 (1970). K. Kuromizu and N . Izumiya, Bull.Chem.Soc. Japan, 43, 2944 (1970). N. Mitsuyasu, S . Matsura, M. Waki, M. Ohno, S . Makzumi and N . Izumiya, B u l l .Chem.Soc. Japan, 43, 1829 (1970). J . Meienhofer, J .Am.Chem.Soc. , 92, 3771 (1970). T-Y. L i u , Y.H. Chang, J.Biol.Ch%., 246, 2842 (1971).

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Chapter 28.

P r e p a r a t i o n of Radioisotope-Labeled

Drugs

Richard C. Thomas, The Upjohn Company, Kalamazoo, Michigan T h i s r e p o r t i s concerned w i t h t h e p r e p a r a t i o n o f r a d i o i s o t o p e l a b e l e d d r u g s f o r u s e as drug t r a c e r s , n o t of r a d i o p h a r m a c e u t i c a l a g e n t s f o r u s e i n d i a g n o s i s and t h e r a p y . S i n c e t h i s h a s n o t been t h e s u b j e c t of p r e v i o u s r e p o r t s i n t h i s series, p a r t i c u l a r a t t e n t i o n w i l l b e g i v e n t o general principles, L i t e r a t u r e Sources - D e s c r i p t i o n s of t h e p r e p a r a t i o n of l a b e l e d d r u g s are remarkably few, s c a t t e r e d , and, t o some e x t e n t , hidden i n r e c e n t p e r i o d i c a l l i t e r a t u r e , S i n c e 1965 The J o u r n a l of L a b e l l e d Compounds h a s provided t h e g r e a t e s t c o n c e n t r a t i o n of s u c h p u b l i c a t i o n s , T h i s j o u r n a l a l s o cont a i n s an a b s t r a c t s s e c t i o n which p r o v i d e s a d d i t i o n a l i n f o r m a t i o n on synt h e s i s w i t h i s o t o p e s drawn from 40 p e r i o d i c a l s as w e l l as Chemical abs t r a c t s and Nuclear S c i e n c e A b s t r a c t s . Otherwise, p r o c e d u r e s f o r l a b e l i n g d r u g s a p p e a r i n f r e q u e n t l y as s u c h , o r as p a r t of a more comprehensive s t u d y , i n a number of p e r i o d i c a l s . A number of r e f e r e n c e worksl-10 and proceedings of c ~ n f e r e n c e s l l - ~d~e ,a l i n g i n whole o r i n p a r t w i t h t h e prepa r a t i o n of l a b e l e d compounds, have appeared d u r i n t h e p a s t 10 y e a r s . S e v e r a l i m p o r t a n t works, n o t a b l y t h o s e of Calvin", Murray and W i l l i a m s 1 7 9 l a , Ronizo19, and Kamen20, a l t h o u g h more t h a n 10 y e a r s o l d , are u s e f u l s o u r c e s of i n f o r m a t i o n , Design of S y n t h e s i s - I n u s i n g a r a d i o i s o t o p e - l a b e l e d drug as a tracer one o r d i n a r i l y is concerned w i t h f o l l o w i n g t h e carbon s k e l e t o n o f t h e drug molecule. To accomplish t h i s , u s u a l l y o n l y one of t h e atoms i n t h e molecule is l a b e l e d t o s e r v e as t h e tracer f o r t h e e n t i r e molecule. S i n c e j u s t t h e l a b e l e d atom is r e a l l y b e i n g d e t e c t e d , i t is i m p o r t a n t t h a t t h i s atom t r u l y r e p r e s e n t t h e carbon s k e l e t o n of t h e drug molecule. This concept s h o u l d be c o n s i d e r e d when choosing a r a d i o i s o t o p e and, p a r t i c u l a r l y , when d e c i d i n g on i t s l o c a t i o n i n t h e molecule. Possible changes t h e drug w i l l undergo d u r i n g t h e a n t i c i p a t e d t r a c e r s t u d y should be c o n s i d e r e d c a r e f u l l y , Carbon-14, i n a s u i t a b l e p o s i t i o n , u s u a l l y i s s a t i s f a c t o r y as a tracer f o r t h e carbon s k e l e t o n of a d r u g , b u t a n o t h e r r a d i o i s o t o p e such as t r i t i u m , s u l f u r - 3 5 , o r c h l o r i n e - 3 6 , s e r v i n g as an a u x i l i a r y tracer f o r carbon, may be used t o advantage. The a c t u a l c h o i c e of r a d i o i s o t o p e i n v o l v e s a number of f a c t o r s , i n c l u d i n g t h e m o l e c u l a r s t r u c t u r e and i n t e n d e d u s e of t h e d r u g , t h e d i f f i c u l t y and expense of i n c o r p o r a t i n g t h e i s o t o p e i n t o t h e molecule, and t h e equipment a v a i l a b l e f o r d e t e c t i n g r a d i o a c t i v i t y . The maximum B energy (MeV), h a l f l i f e ( y e a r s ) and h i g h e s t p r a c t i c a l s p e c i f i c a c t i v i t y (mCi/m Atom), r e s p e c t i v e l y , f o r t h e r a d i o i s o t o p e s mentioned are: 0.155, 5570, and 40 f o r 1 4 C ; 0.018, 12.5, and 25000 f o r 3H; 0.167, 0.244, and 1000 f o r 35S; and 0.714, 308000, and 0.3 f o r 3 6 C l . S i n c e carbon and hydrogen are common t o a l l d r u g s , carbon-14 and t r i t i u m u s u a l l y s a t i s f y t h e molecular s t r u c t u r e r e q u i r e m e n t s . Carbon-14 g e n e r a l l y is p r e f e r r e d , s i n c e i t i s a more d i r e c t tracer of t h e d r u g ' s

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carbon s k e l e t o n and i s somewhat e a s i e r and more convenient t o u s e . On t h e o t h e r hand, t h e need f o r h i g h d e t e c t i o n s e n s i t i v i t y , as w i t h c e r t a i n r a d i o immunoassays, may r e q u i r e a s p e c i f i c a c t i v i t y a t t a i n a b l e o n l y w i t h t r i t i u m o r sulfur-35. I n a u t o r a d i o g r a p h y s t u d i e s t h e h i g h e r energy of t h e carbon14 B i s d e s i r a b l e f o r whole-body s e c t i o n work whereas t h e weaker energy of t h e t r i t i u m 6 is n e c e s s a r y f o r high r e s o l u t i o n a t t h e c e l l u l a r l e v e l . Chlorine-36 is n o t used o f t e n , because of i t s low s p e c i f i c a c t i v i t y . A p o t e n t i a l l y l a b i l e l i n k a g e between two p a r t s of a d r u g ' s carbon s k e l e t o n o c c a s i o n a l l y n e c e s s i t a t e s d o u b l e - l a b e l i n g t h e drug. For t h i s purpose i t is d e s i r a b l e t o l a b e l each p a r t of t h e s k e l e t o n w i t h a d i f f e r e n t i s o t o p e , such as carbon-14 and t r i t i u m , s o they c a n b e d i f f e r e n t i a t e d e a s i l y by r a d i o a c t i v i t y a n a l y s i s d u r i n g t h e tracer s t u d y . I n p r a c t i c e each i s o t o p e u s u a l l y i s i n t r o d u c e d i n a completely s e p a r a t e s y n t h e s i s s o t h a t two lab e l e d d r u g s are o b t a i n e d . This g e n e r a l l y r e s u l t s i n h i g h e r y i e l d s of each r a d i o i s o t o p e and g i v e s t h e u s e r f l e x i b i l i t y i n s e l e c t i n g a n i s o t o p e r a t i o f o r h i s double-label s t u d i e s . The t o t a l amount of l a b e l e d drug t o p r e p a r e , i n terms of r a d i o a c t i v i t y , depends on t h e a n t i c i p a t e d s t u d i e s , t h e r a d i o i s o t o p e , and methods f o r d e t e c t i n g t h e r a d i o a c t i v i t y . I n a t y p i c a l drug-metabolism s t u d y i n v o l v i n g 3 animal s p e c i e s and man, u s i n g l i q u i d s c i n t i l l a t i o n c o u n t i n g t e c h n i q u e s , approximately one m C i of a carbon-14 o r s u l f u r - 3 5 l a b e l e d drug w i l l s u f f i c e . S i m i l a r s t u d i e s w i t h t r i t i u m would r e q u i r e 2 t o 4 m C i s i n c e t h e d e t e c t i o n e f f i c i e n c y f o r t r i t i u m by t h e same techn i q u e s is o n l y one-half t o one-fourth t h a t f o r carbon-14 and s u l f u r - 3 5 . On t h e o t h e r hand, t h e d e t e c t i o n e f f i c i e n c y f o r t r i t i u m d u r i n g whole-body a u t o r a d i o g r a p h y is o n l y about o n e - f i f t i e t h t h a t of t h e two more e n e r g e t i c i s o t o p e s , s o , i f such s t u d i e s are a n t i c i p a t e d , even more t r i t i u m - l a b e l e d drug would have t o b e p r e p a r e d . The s p e c i f i c a c t i v i t y r e q u i r e d f o r a l a b e l e d drug depends on t h e same 3 f a c t o r s d e t e r m i n i n g t h e t o t a l r a d i o a c t i v i t y r e q u i r e d , and i n addit i o n depends on t h e a n t i c i p a t e d drug dosage l e v e l i n terms of w e i g h t . The more b i o l o g i c a l l y p o t e n t d r u g s , s u c h as s t e r o i d hormones, g e n e r a l l y r e q u i r e h i g h e r s p e c i f i c a c t i v i t i e s t h a n t h e less p o t e n t o n e s , such as a n t i b i o t i c s , A carbon-14 l a b e l e d drug having a s p e c i f i c a c t i v i t y of 10 m C i p e r mM can be q u a n t i f i e d a t a l e v e l of one nanogram p e r m l i n body and e x c r e t o r y f l u i d s by l i q u i d - s c i n t i l l a t i o n c o u n t i n g . A c o r r e s p o n d i n g l y h i g h e r s p e c i f i c a c t i v i t y would be n e c e s s a r y f o r a t r i t i u m - l a b e l e d drug. Care must be t a k e n t o p r e p a r e a l a b e l e d drug a t a s u f f i c i e n t l y h i g h s p e c i f i c a c t i v i t y f o r i t s i n t e n d e d u s e ; i t can always b e d i l u t e d w i t h n o n r a d i o a c t i v e drug i f n e c e s s a r y . However, t o o h i g h a s p e c i f i c a c t i v i t y may make t h e s y n t h e s i s u n n e c e s s a r i l y d i f f i c u l t and w i l l l e a d t o i n c r e a s e d r a d i a t i o n decomposition of t h e drug d u r i n g s t o r a g e . The t o t a l amount of l a b e l e d drug t o p r e p a r e , i n terms of w e i g h t , w i l l depend on t h e s p e c i f i c a c t i v i t y and t h e t o t a l amount of r a d i o a c t i v i t y r e q u i r e d . This w i l l v a r y widely b u t u s u a l l y does n o t f a l l much below 0 . 5 mM f o r a drug p r e p a r e d by a s y n t h e t i c sequence. I n t h e case of t r i t i u m exchange and r e d u c t i o n r e a c t i o n s i t may b e c o n s i d e r a b l y lower, however.

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Carbon-14 Labeling of Drugs The most d i s t i n c t i v e f e a t u r e s of carbon-14 s y n t h e s i s , as compared t o r e g u l a r o r g a n i c s y n t h e s i s , are: 14C02 , t h e p r i mary form of carbon-14, is t h e common s t a r t i n g m a t e r i a l ; high c o s t of t h e i s o t o p e ; and need t o work on a very small scale. S i n c e 14C02 i s t h e s t a r t i n g material, i t f i r s t i s n e c e s s a r y t o c o n v e r t i t t o a multi-carbon i n t e r m e d i a t e , e i t h e r d i r e c t l y by c a r b o n a t i o n of a Grignard o r l i t h i u m r e a g e n t , o r i n d i r e c t l y f o l l o w i n g i t s conversion t o a n o t h e r s i n g l e - c a r b o n These s i n g l e compound such as K14CN, H14COOH, H 3 1 4 C O H , o r H 2 N l 4 C N . carbon i n t e r m e d i a t e s , a s w e l l as a wide v a r i e t y of multi-carbon i n t e r m e d i a t e s , l a b e l e d w i t h carbon-14 are a v a i l a b l e from s u p p l i e r s . Whether t o s y n t h e s i z e such an i n t e r m e d i a t e o r purchase i t from a s u p p l i e r depends on t h e l o c a l e x p e r t i s e , t h e amount r e q u i r e d , and t h e c o s t . The need f o r 25 t o 50 m C i of a l a b e l e d i n t e r m e d i a t e might d i c t a t e i t s l o c a l s y n t h e s i s , whereas one o r two m C i might b e more j u d i c i o u s l y purchased. Whatever t h e s o u r c e of t h e l a b e l e d i n t e r m e d i a t e i t is c o n v e r t e d t o t h e d e s i r e d drug by r e g u l a r s y n t h e t i c t e c h n i q u e s , u s u a l l y modified f o r improved y i e l d s and h a n d l i n g small amounts of material. Although a well-developed s y n t h e t i c procedure f o r p r e p a r a t i o n of t h e n o n r a d i o a c t i v e drug u s u a l l y is a v a i l a b l e , i t may be of l i m i t e d v a l u e as a r o u t e t o t h e carbon-14 l a b e l e d drug. The s y n t h e t i c r o u t e w i l l b e determined from a c o n s i d e r a t i o n of p o s s i b l e l a b e l ed i n t e r m e d i a t e s , t h e d e s i r e d l o c a t i o n of t h e l a b e l i n t h e carbon s k e l e t o n of t h e d r u g , and t h e u s u a l l y small scale of t h e r e a c t i o n s ,

The carbon-14 l a b e l s h o u l d be i n t r o d u c e d as l a t e as p o s s i b l e i n t h e s y n t h e t i c sequence s i n c e t h e y i e l d based on i s o t o p e u s u a l l y i s of most The y i e l d should b e b o t h good and r e l i a b l e . To h e l p a s s u r e importance. t h i s , n o n r a d i o a c t i v e "cold" r u n s should b e conducted s o t h a t t h e r a d i o a c t i v e "hot" r u n becomes r o u t i n e . These c o l d r u n s s h o u l d b e made u s i n g t h e same s c a l e , r e a g e n t s , and equipment t h a t w i l l b e used i n t h e h o t run. They s h o u l d b e c a r r i e d through t h e e n t i r e s y n t h e t i c sequence, n o t l i m i t ed t o a s t u d y of each s t e p s e p a r a t e l y u s i n g pure s h e l f r e a g e n t s , The j u d i c i o u s a d d i t i o n of n o n r a d i o a c t i v e c a r r i e r a t v a r i o u s s t a g e s i n t h e synt h e t i c sequence is p a r t of t h e s t r a t e g y i n carbon-14 s y n t h e s i s , A d d i t i o n of p u r e c a r r i e r s e r v e s t o i n c r e a s e t h e chemical p u r i t y of a l a b e l e d i n t e r mediate i n t h o s e cases where s u c c e s s of a r e a c t i o n is dependent on a p u r e r e a c t a n t . I t a l s o can be h e l p f u l f o r i n c r e a s i n g r a d i o c h e m i c a l y i e l d s d u r i n g r e c r y s t a l l i z a t i o n s and o t h e r s o l u b i l i t y l i m i t i n g o p e r a t i o n s , Although adding carrier i n c r e a s e s chemical p u r i t y , i t h a s no e f f e c t on radiochemical p u r i t y . B i o s y n t h e t i c i n c o r p o r a t i o n of 14C02 a l t h o u g h v a l u a b l e f o r prep a r a t i o n of carbon-14 l a b e l e d endogenous materials, a p p e a r s of l i m i t e d u t i l i t y f o r p r e p a r a t i o n of l a b e l e d d r u g s . Exceptions a r e t h e n a t u r a l l y o c c u r r i n g drugs s u c h as d i g i t o x i n and n a t u r a l l y o c c u r r i n g compounds such as c a r b o h y d r a t e s and amino a c i d s t h a t would be u s e f u l i n t e r m e d i a t e s f o r chemical o r b i o s y n t h e t i c p r e p a r a t i o n of l a b e l e d d r u g s . The b i o s y n t h e t i c conversion of l a b e l e d multi-carbon i n t e r m e d i a t e s t o l a b e l e d drugs o r drug p r e c u r s o r s by s p e c i f i c enzyme systems o r growing organisms is of much more u t i l i t y . For example, carbon-14 l a b e l e d p r o s t a g l a n d i n E1-2-14C h a s been p r e p a r e d by i n c u b a t i n g 8,11, 1 4 - e i c o ~ a t r i e n o i c - 2 - ~ ~ aCc i d w i t h sheep v e s i c u l a r gland t i s s u e 2 I . The p r e p a r a t i o n s of numerous carbon-14 l a b e l e d a n t i -

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b i o t i c s by f e r m e n t a t i o n of l a b e l e d c a r b o h y d r a t e s 2 2 , amino a c i d s 2 , and f a t t y a c i d s 2 4 have been d e s c r i b e d . A cheap carbon-14 l a b e l e d s u b s t r a t e f o r some f e r m e n t a t i o n s might b e t h e s u g a r s y r u p o b t a i n e d by e x t r a c t i o n of detached canna l e a v e s f o l l o w i n g t h e i r p h o t o s y n t h e t i c i n c o r p o r a t i o n of 14c02. The most u s e f u l r e f e r e n c e works d e a l i n g w i t h t h e g e n e r a l p r o c e d u r e s of carbon-14 s y n t h e s i s are t h o s e of C a l v i n 1 6 , Catch1, and Ronzio19. Murr a y and Williams17, w r i t t e n i n t h e form of Organic S y n t h e s e s , and S c h u t t e 3 , c o n t a i n i n g an e x t r e m e l y comprehensive t a b u l a t i o n of carbon-14 s y n t h e s i s through 1965, are of p a r t i c u l a r v a l u e . Less comprehensive t r e a t m e n t s of carbon-14 s y n t h e s i s are i n c l u d e d i n books by Kamen2' , Wang6 , Wilson7 , Chase and Rabinowitz' , and Raaen9. S e v e r a l c o n f e r e n c e e r o c e e d i n g s c o n t a i n s p e c i f i c p a p e r s d e a l i n g w i t h carbon-14 s y n t h e s i s 1 Y l 2 Y Y 15. T r i t i u m L a b e l i n g of Drugs - T r i t i u m can b e i n c o r p o r a t e d e f f e c t i v e l y i n drugs by t h r e e g e n e r a l methods: chemical s y n t h e s i s , exchange r e a c t i o n s , and b i o s y n t h e s i s . One of t h e p r i n c i p a l d i f f e r e n c e s i n p r e p a r i n g carbon-14 and t r i t i u m l a b e l e d d r u g s is t h a t i n t h e former c a s e t h e i s o t o p e i s i n t r o duced d u r i n g f o r m a t i o n of t h e carbon s k e l e t o n of t h e drug whereas i n t h e l a t t e r case t h e i s o t o p e u s u a l l y i s added t o t h e preformed s k e l e t o n . Cons e q u e n t l y , i n c o r p o r a t i o n of r a d i o a c t i v i t y i n a d r u g i s somewhat easier w i t h t r i t i u m , b u t g e n e r a l l y t h e c a r b o n - 1 4 l a b e l e d drug i s s u p e r i o r as a t r a c e r , The monograph of Evans2 i s t h e s i n g l e most comprehensive and u s e f u l volume d e a l i n g w i t h p r e p a r a t i o n of t r i t i u m - l a b e l e d com ounds. Other genera1 r e f e r e n c e s i n c l u d e books by Feinendegen4, S c h u t t e , and Murray and Williams18, a review by Lee and S ~ h m i d t - B l e e k ~and ~ , a small b r o c h u r e comp i l e d by Evans26. The r e c e n t monograph by Thomaslo, d e a l i n g w i t h d e u t e r i um, s h o u l d p r o v i d e v a l u a b l e i n f o r m a t i o n f o r tritium l a b e l i n g of d r u g s .

!

The method of c h o i c e f o r i n t r o d u c i n g t r i t i u m i n t o a drug w i t h h i g h p r o b a b i l i t y of s u c c e s s , a t t h e h i g h e s t s p e c i f i c a c t i v i t y , and i n s e l e c t e d , o r a t l e a s t somewhat p r e d i c t a b l e , p o s i t i o n s i n t h e molecule i s by chemic a l s y n t h e s i s . T r i t i u m g a s , t h e primary form of t r i t i u m i s t h e u s u a l s t a r t i n g material a l t h o u g h t r i t i a t e d water is i m p o r t a n t a l s o . Although t r i t i u m g a s can b e , and o f t e n i s , used i n t h e c a r r i e r - f r e e s t a t e , t r i t i a t e d water u s u a l l y i s n o t , p r i m a r i l y because of t h e h i g h r a d i a t i o n d e n s i t y i n such a condensed-phase system. Thus, f o r o b t a i n i n g a drug of t h e h i g h e s t s p e c i f i c a c t i v i t y , a method u s u a l l y must b e d e v i s e d t o i n t r o d u c e t r i t i u m gas d i r e c t l y i n t o t h e drug o r a s y n t h e t i c p r e c u r s o r . Although most tracer u s e s of drugs do n o t r e q u i r e such h i g h s p e c i f i c a c t i v i t i e s , i n c r e a s i n g u s e of t h e radioimmuno method € o r drug a n a l y s i s , t h e s e n s i t i v i t y of which depends on t h e s p e c i f i c a c t i v i t y of t h e l a b e l e d d r u g , is i n c r e a s i n g t h e demand f o r t h e h i g h e s t s p e c i f i c a c t i v i t i e s attainable

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Methods f o r d i r e c t i n t r o d u c t i o n of t r i t i u m g a s i n t o a drug by s y n t h e s i s g e n e r a l l y i n v o l v e c a t a l y t i c h y d r o g e n a t i o n , by e i t h e r t r i t i u m a d d i t i o n t o an u n s a t u r a t e d ~ y s t e m ~ , ~o r~ t, r~i t 'i u m replacement of a h a l ~ g e n ~ ~ Such ~ ~ r~e a~c t' i o . n s are u s u a l l y conducted on a micro o r s e m i -

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micro s c a l e , a t room t e m p e r a t u r e , and a t atmospheric p r e s s u r e . Catalysts such as p l a t i n u m and p a l l a d i u q sometimes s u p p o r t e d on c h a r c o a l o r calcium c a r b o n a t e , a r e used. P a r t i c u l a r l y f o r h i g h s p e c i f i c a c t i v i t i e s , nonpolar s o l v e n t s a r e r e q u i r e d s i n c e t r i t i u m g a s undergoes c a t a l y t i c exchange w i t h hydroxyl and o t h e r l a b i l e hydrogens t o d i l u t e t h e t r i t i u m . I f a p o l a r s o l v e n t can n o t be avoided i t s volume s h o u l d be s m a l l and t h e h y d r o g e n a t i o n r a t e should be r a p i d s i n c e t h e exchange i s u s u a l l y slower t h a n hydrogen a t i o n . C a t a l y t i c hydrogenation h a s been used s u c c e s s f u l l y f o r t r i t i u m l a b e l i n g a wide v a r i e t y of compounds, p a r t i c u l a r l y amino a c i d s , s t e r o i d s , f a t t a c i d s , and t h e p u r i n e and pyrimidine b a s e s a t h i g h s p e c i f i c a c t i v i The main d i s a d v a n t a g e s of t h e method are t h e d i f f i c u l t y of o b t a i n ties i n g a s u i t a b l e p r e c u r s o r f o r hydrogenation and t h e p o s s i b i l i t y of a cert a i n amount of n o n s p e c i f i c l a b e l i n g , p a r t i c u l a r l y w i t h u n s a t u r a t e d precursors.

z.

T r i t i u m l a b e l i n g by c a t a l y t i c exchange of a compound's s t a b l e hydrogen w i t h t r i t i u m of a s o l v e n t h a s been widely used f o r i n t r o d u c i n g tritium i n t o a v a r i e t y of drugs and o t h e r compounds2. I n t h i s method t h e compound, c u r i e amounts of a t r i t i a t e d s o l v e n t , and t h e c a t a l y s t are mixed and allowed t o react, u s u a l l y a t an e l e v a t e d t e m p e r a t u r e , f o r s e v e r a l h o u r s t o s e v e r a l days. The system can be homogeneous, as w i t h a c i d i c and b a s i c c a t a l y s t s , o r heterogeneous, as w i t h c e r t a i n t r a n s i t i o n metal c a t a l y s t s . Homogeneous a c i d c a t a l y s i s u s i n g t r i t i a t e d s u l f u r i c 3 1 , p h o ~ p h o r i c ~p ~e r, c h l o r i c 3 , and t r i f l u o r o a c e t i c a c i d s 34 , as w e l l as aluminum c h l o r i d e i n t r i t i a t e d water35 and t r i t i a t e d phosphoric acid-boron t r i f l u o r i d e complex32 have been u s e d 2 . They are g e n e r a l l y s u i t a b l e f o r l a b e l i n g a r o m a t i c compounds, b u t o f t e n t h e c o n d i t i o n s are s o d r a s t i c t h a t e x t e n s i v e decomposition of complex compounds o c c u r s . Homogeneous b a s i c c a t a l y s i s i n l i q u i d t r i t i ~ a m m o n i a and ~ ~ i n s t r o n g l y b a s i c media37 h a s been r e p o r t e d b u t does n o t appear t o be g e n e r a l l y u s e f u l f o r l a b e l i n g drugs. T r i t i a t i o n by heterogeneous c a t a l y s i s u s i n g an a c t i v a t e d t r a n s i t i o n metal c a t a l y s t and a t r i t i a t e d s o l v - e n t , u s u a l l y p l a t i n u m and water o r 70% a c e t i c a c i d , a t t e m p e r a t u r e s t o 200°C h a s been w i d e l y e m p l o y e d 2 ~ 3 8 - 4 0 . I t h a s been s u c c e s s f u l w i t h a v a r i e t y of s t r u c t u r e t y p e s , p a r t i c u l a r l y a r o m a t i c compounds, s t e r o i d s , amino a c i d s , and h e t e r o c y c l i c compounds s u c h as p u r i n e and p y r i m i d i n e b a s e s 2 . T h i s u s u a l l y i s t h e method of c h o i c e f o r exchange l a b e l i n g s i n c e i t i s r a t h e r g e n e r a l l y a p p l i c a b l e , g i v e s p r o d u c t s of h i g h s p e c i f i c a c t i v i t y , r e q u i r e s o n l y s h o r t exchange p e r i o d s , and Its d i s a d v a n t a g e s are h e a t s e n s i t i v causes l i t t l e r a d i a t i o n d e g r a d a t i o n . i t y of some compounds, low exchange rates f o r many a l i p h a t i c compounds, and c a t a l y s t p o i s o n i n g . A s w i t h o t h e r solvent-exchange methods, t h e spec i f i c a c t i v i t y of t h e p r o d u c t i s l i m i t e d by t h a t of t h e t r i t i a t e d s o l v e n t , The t r i t i u m gas-exposure method f o r r a d i a t i o n - i n d u c e d exchange l a b e l i n g of o r g a n i c compounds w a s i n t r o d u c e d by Wilzbach4' i n 1 9 5 7 and h a s come t o b e known as t h e "Wilzbach m e t h ~ d " ~ , ~ I, n~ t~h .i s method t h e compound t o be l a b e l e d i s exposed t o c u r i e amounts of c a r r i e r - f r e e t r i t i u m gas i n a s e a l e d f l a s k f o r a p e r i o d of s e v e r a l days t o s e v e r a l weeks, The a c t i v a t i o n energy n e c e s s a r y f o r exchange of s t a b l e hydrogen atoms i n t h e compound w i t h t r i t i u m gas i s d e r i v e d from t h e &decay of t r i t i u m . T r i t i u m

Chap. 28

Radioisotope-Labeled

Drugs

Thomas

30 1 -

i n c o r p o r a t i o n is d i r e c t l y p r o p o r t i o n a l t o t h e amount of t r i t i u m gas and t h e exposure t i m e . For drug-type compounds i n c o r p o r a t i o n of s t a b l y bound t r i t i u m is o f t e n 10 t o 200 WCi p e r curie-day e x p ~ s u r e ~ ~ By - ~ p~r o. p e r c h o i c e of t h e amount of drug exposed, such t r i t i u m i n c o r p o r a t i o n s p r o v i d e s p e c i f i c a c t i v i t i e s s u f f i c i e n t l y h i g h f o r many, b u t n o t a l l , d r u g - t r a c e r u s e s . The major d e f i c i e n c y w i t h t h e Wilzbach method i s e x t e n s i v e format i o n of t r i t i a t e d by-products of v e r y h i g h s p e c i f i c a c t i v i t i e s . Although t h e s e by-products may r e p r e s e n t only minor chemical i m p u r i t i e s , t h e y usua l l y account f o r most of i n c o r p o r a t e d t r i t i u m . S i n c e t h e s e i m p u r i t i e s o f t e n c l o s e l y resemble t h e d e s i r e d p r o d u c t , h i g h - r e s o l u t i o n p u r i f i c a t i o n methods s u c h as chromatography and c o u n t e r - c u r r e n t d i s t r i b u t i o n s h o u l d be employed. Radiochemical p u r i t y must be e s t a b l i s h e d w i t h g r e a t c a r e f o r d r u g s l a b e l e d by t h e Wilzbach method. M o d i f i c a t i o n s of t h e Wilzbach method have been aimed a t i n c r e a s i n g t h e rate of t r i t i u m i n c o r p o r a t i o n and d e c r e a s i n g t h e f o r m a t i o n of imp u r i t i e s 2 g 5 ,42. Three g e n e r a l areas have been i n v e s t i g a t e d ; a c t i v a t i o n of t h e system by e x t e r n a l r a d i a t i o n s o u r c e s , a d s o r p t i o n of t h e compound and t r i t i u m on c h a r c o a l , and u s e of a noble-metal c a t a l y s t , R a d i a t i o n s o u r c e s have i n c l u d e d an e l e c t r i c d i s c h a r g e 4 3Y ~ u~l t r ,a v i o l e t l i g h t 4 * , microwave^^^ y 5 0 , and Y- and x-rays4'. These methods g e n e r a l l y have been s u c c e s s f u l i n i n c r e a s i n g t h e r a t e of t r i t i u m i n c o r p o r a t i o n b u t have n o t been p a r t i c u l a r l y e f f e c t i v e i n d e c r e a s i n g t h e f o r m a t i o n of h i g h s p e c i f i c a c t i v i t y impurities. Adsorption of t h e compound and tritium gas on charcoa151 a p p e a r s t o b e a worthwhile m o d i f i c a t i o n of t h e Wilzbach method b u t h a s n o t been e x t e n s i v e l y s t u d i e d f o r drug-type compounds. The t h i r d modif i c a t i o n , t h a t of exposing an i n t i m a t e m i x t u r e of t h e com ound and a catal y s t , such as p l a t i n u m o r p a l l a d i u m b l a c k , t o t r i t i u m gast5 Y~~ ,5 2 a p p e a r s t o b e t h e most promising m o d i f i c a t i o n of t h e Wilzbach method. Incorpor a t i o n of t r i t i u m u s u a l l y is g r e a t e r and p r o d u c t i o n of i m p u r i t i e s i s no g r e a t e r and o f t e n less, t h a n by t h e c o n v e n t i o n a l Wilzbach method. B i o s y n t h e t i c methods f o r d i r e c t i n t r o d u c t i o n of t r i t i u m from a t r i t i a t e d - w a t e r medium i n t o a drug o r i n t e r m e d i a t e can i n v o l v e s p e c i f i c i n c o r p o r a t i o n of t r i t i u m i n t h e compound, u s i n g a p a r t i c u l a r enzyme system, o r n o n s p e c i f i c i n c o r p o r a t i o n , i n v o l v i n g growth of an organism. Such methods have n o t been of g r e a t u t i l i t y f o r p r e p a r i n g t r i t i u m l a b e l e d drugs because of t h e l a r g e amounts o f r a d i o a c t i v e s u b s t r a t e which must b e handled and t h e r a t h e r l i m i t e d s p e c i f i c a c t i v i t i e s a t t a i n a b l e . This a p p e a r s t o b e i n h e r e n t i n t h e method s i n c e t h e l a b e l e d subs t r a t e , t r i t i a t e d water, i s t h e growth medium and t h e r e f o r e must b e used i n l a r g e e x c e s s . Also, t h e s p e c i f i c a c t i v i t y of t h e t r i t i a t e d water must be s u f f i c i e n t l y low t o p r e v e n t r a d i a t i o n damage of t h e enzyme system o r organism. N e v e r t h e l e s s an a n t i b i o t i c h a s been The uats e of low b u t a d e q u a t e s p e c i f i c a c t i v i t y f o r metabolism s t u d i e s b i o s y n t h e t i c methods f o r c o n v e r t i n g t r i t i a t e d o r g a n i c i n t e r m e d i a t e s , prepared by chemical o r exchange methods, t o l a b e l e d d r u g s is more u s e f u l . By t h i s method d r u g s of r e l a t i v e l y h i g h s p e c i f i c a c t i v i t i e s can b e o b t a i n ed s i n c e s u b s t r a t e s of h i g h s p e c i f i c a c t i v i t i e s can b e employed i n rel a t i v e l y l a r g e amounts. Other antibiotic^^^ , s e v e r a l s t e r o i d s 5 5 , and p r o s t a g l a n d i n E256 have been p r e p a r e d i n t h i s manner.

Gepared .

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P u r i t y o f Labeled Drugs R e g a r d l e s s of whether t h e l a b e l e d drug i s prepared on t h e premises o r by a s u p p l i e r , t h e u s e r s h o u l d a s s u r e himself t h a t i t is s u f f i c i e n t l y p u r e immediately p r i o r t o i t s use. Radiochemical p u r i t y is of primary importance, b u t chemical, r a d i o i s o t o p i c , " r a d i o i s o m e r i c , " and p h a r m a c e u t i c a l p u r i t i e s may b e i m p o r t a n t a l s o . The d e g r e e of r a d i o c h e m i c a l p u r i t y and t h e r e l e v a n c e of t h e o t h e r c r i t e r i a of p u r i t y depend on t h e i n t e n d e d u s e of t h e l a b e l e d drug. S a t i s f a c t o r y radiochemic a l p u r i t y , t h a t i s , a s s o c i a t i o n of t h e r a d i o a c t i v i t y w i t h t h e d e s i r e d chemical form of t h e d r u g , n e a r l y always must b e g r e a t e r t h a n 95% and o f t e n exceeds 99%. S i n c e most l a b e l e d d r u g s are p r e p a r e d i n r e l a t i v e l y small amounts, e s p e c i a l l y when a high s p e c i f i c a c t i v i t y is n e c e s s a r y , c r i t i c a l e s t a b l i s h m e n t of chemical p u r i t y may be d i f f i c u l t o r even imposs i b l e , However n o n r a d i o a c t i v e i m p u r i t i e s may a c c e l e r a t e decomposition of t h e d r u g 5 7 , 5 Q , i n t e r f e r e w i t h t h e u s u a l b i o l o g i c a l o r chemical a c t i o n of t h e d r u g , and o b s c u r e t h e t r u e s p e c i f i c a c t i v i t y of t h e l a b e l e d d r u g t o t h e p o i n t t h a t c o n v e r s i o n of r a d i o a c t i v i t y l e v e l s t o a b s o l u t e druge q u i v a l e n t l e v e l s i s meaningless. Thus chemical p u r i t y of a l a b e l e d d r u g , a l t h o u g h o f t e n overlooked, can be i m p o r t a n t . R a d i o i s o t o p i c p u r i t y , t h a t i s , t h e a s s o c i a t i o n o f t h e r a d i o a c t i v i t y of t h e l a b e l e d drug w i t h t h e d e s i r e d i s o t o p e , is n o t o f t e n a problem. There h a s been a t l e a s t one r e p o r t 5 9 , however, of a carbon-14 l a b e l e d compound, o b t a i n e d from a supp l i e r , c o n t a i n i n g t r i t i u m , a p p a r e n t l y i n t h e same chemical form. Radioi s o m e r i c p u r i t y , t h a t i s , t h e a s s o c i a t i o n of t h e r a d i o i s o t o p e w i t h t h e s p e c i f i e d l o c a t i o n i n t h e molecule, a l s o i s n o t o f t e n a problem. P a r t i c u l a r l y i n t h e c a s e of t r i t i u m - l a b e l e d d r u g s , however, p o s i t i o n s i n t h e molecule o t h e r t h a n t h o s e s p e c i f i e d may c o n t a i n t h e i s o t o p e . Although t h i s would c a u s e no d i f f i c u l t y i n many cases, such material c o u l d l e a d t o e r r o n e o u s c o n c l u s i o n s i n c e r t a i n s t u d i e s . P h a r m a c e u t i c a l p u r i t y of a r a d i o i s o t o p e - l a b e l e d drug i s a measure of i t s s u i t a b i l i t y and s a f e t y f o r I t s h o u l d p a s s s u b s t a n t i a l l y t h e same chemical p u r i t y , u s e i n humans. s t e r i l i t y , and p y r o g e n i c i t y tests r e q u i r e d f o r t h e n o n r a d i o a c t i v e form of t h e drug. Radiochemical p u r i t y i s b e s t determined d i r e c t l y by h i g h - r e s o l u t i o n s e p a r a t i o n p r o c e s s e s , such as t h i n - l a y e r , p a p e r , g a s - l i q u i d , and column chromatography, e l e c t r o p h o r e s i s , and c o u n t e r c u r r e n t d i s t r i b u t i o n , i n which i t i s convenient t o a n a l y z e f o r s e p a r a t i o n of components by m o n i t o r i n g f o r r a d i o a c t i v i t y ( i . e . , a u t o r a d i o g r a p h y , chromatogram s c a n n i n g , e l u a t e m o n i t o r i n g ) . I n most c a s e s several such systems s h o u l d be employed; two o r t h r e e well-chosen ones may s u f f i c e b u t t h e r e are c a s e s where many more systems d i d n o t r e v e a l a l a t e r - r e c o g n i z e d r a d i o c h e m i c a l i m p u r i t y . Another d i r e c t method f o r d e t e r m i n i n g r a d i o c h e m i c a l p u r i t y is by r e v e r s e i s o t o p e d i l u t i o n a n a l y s i s . I n t h i s method t h e l a b e l e d drug is d i l u t e d w i t h a s u b s t a n t i a l amount of p u r e , n o n r a d i o a c t i v e - c a r r i e r drug and t h e material is p u r i f i e d ( i , e . , c r y s t a l l i z a t i o n , chromatography). A r e s u l t i n g d e c r e a s e i n t h e s p e c i f i c a c t i v i t y ( c o r r e c t e d € o r d i l u t i o n ) can b e q u a n t i f i e d i n terms of t h e r a d i o c h e m i c a l p u r i t y of t h e u n d i l u t e d l a b e l e d d r u g , T h i s method is a v a l u a b l e supplement t o t h e chromatographic methods p r e v i o u s l y mentioned b u t i s n o t n e a r l y as s e n s i t i v e . I n some cases, p a r t i c u l a r l y t h o s e i n v o l v i n g a s y n t h e t i c sequence, r a d i o c h e m i c a l p u r i t y can b e i n f e r r e d i n d i r e c t l y from t h e chemical p u r i t y of t h e l a b e l e d d r u g . This i s t o t a l l y

Chap. 28

Radioisotope-Labeled

Drugs

Thomas

303

u n j u s t i f i e d i n t h e case of Wilzbach t r i t i a t i o n s and where n o n r a d i o a c t i v e carriers have been added a t v a r i o u s s t a g e s of a s y n t h e t i c sequence. Chemical p u r i t y of r a d i o i s o t o p e - l a b e l e d drugs u s u a l l y must b e d e t e r mined u s i n g a n a l y s e s which r e q u i r e v e r y small amounts of m a t e r i a l o r a l l o w r e c o v e r y of t h e a n a l y t i c a l sample. T h i s o f t e n can o n l y b e accomplished by working c l o s e l y w i t h t h e a n a l y s t . For example, t h e UV-absorption spectrum, s p e c i f i c a c t i v i t y , and chromatographic p u r i t y can be determined w i t h a s o l u t i o n c o n t a i n i n g one mg o r less of a drug. Although a r e l a t i v e l y l a r g e sample is r e q u i r e d f o r o p t i c a l r o t a t i o n measurements, most of i t c a n b e recovered. R a d i o i s o t o p i c p u r i t y of d r u g s l a b e l e d w i t h t h e i s o t o p e s ment i o n e d i n t h i s r e p o r t u s u a l l y can b e determined by s c i n t i l l a t i o n s p e c t r o metry and h a l f - l i f e measurement ( i f n e c e s s a r y ) . O r d i n a r i l y t h e p r e s e n c e of a f o r e i g n r a d i o i s o t o p e would b e due t o c o n t a m i n a t i o n ; i t would t h u s c o n s t i t u t e a r a d i o c h e m i c a l i m p u r i t y and b e d e t e c t e d as such. Radioisomeric p u r i t y of a l a b e l e d drug o f t e n can b e i n f e r r e d from t h e method o f p r e p a r a t i o n . This is n o t always s a f e , however, p a r t i c u l a r l y i n t h e case of t r i t i u m . For example, unexpected i n t r a m o l e c u l a r s h i f t s and unexpected exchanges i n t h e p r e s e n c e of h y d r o g e n - t r a n s f e r c a t a l y s t s have been obs e r v e d . Thus, i f t h e p o s i t i o n of t h e l a b e l i s c r i t i c a l t o t h e s t u d y , t h e T h i s may r e q u i r e e x t e n s i v e u s e r s h o u l d a s s u r e himself of i t s l o c a t i o n . d e g r a d a t i o n and d e r i v a t i z a t i o n procedures. A u s e f u l a l t e r n a t i v e , i n t h e c a s e of t r i t i u m , might be t o c a r r y o u t a t r i a l s y n t h e s i s w i t h d e u t e r i u m P h a r m a c e u t i c a l p u r i t y of a and d e t e r m i n e t h e i s o t o p e d i s t r i b u t i o n by NMR. r a d i o i s o t o p e - l a b e l e d drug is determined p r i m a r i l y on t h e b a s i s of i t s chemical p u r i t y , t o g e t h e r w i t h i t s s t e r i l i t y and freedom from pyrogens i f intended f o r p a r e n t e r a l use. The v a r i o u s p u r i t y r e q u i r e m e n t s s h o u l d be c o n s i d e r e d when p l a n n i n g s y n t h e s i s of t h e l a b e l e d drug. The i n t e n d e d u s e r s h o u l d d i s c u s s t h i s d i r e c t l y w i t h t h e p e r s o n o r o r g a n i z a t i o n p r e p a r i n g t h e l a b e l e d drug. For example, chemical and r a d i o i s o m e r i c p u r i t i e s may b e a s s u r e d o n l y by t h e method of s y n t h e s i s , Determination of chemical p u r i t y may b e p o s s i b l e o n l y by t h e s u p p l i e r who h a s access t o t h e b u l k of t h e l a b e l e d drug. Brief d i s c u s s i o n s of p u r i t y r e q u i r e m e n t s f o r l a b e l e d compounds a r e i n c l u d e d i n s e v e r a l of t h e g e n e r a l r e f e r e n ~ e s l - ~ r ~I .n a d d i t i o n , an a r t i c l e by P e y s e r G O , a b o o k l e t by CatchG1, and a p a p e r p r e s e n t e d by Merrill and L e w i s s 9 a r e p a r t i c u l a r l y r e l e v a n t .

-

S t a b i l i t y and S t o r a p e of Labeled Druas Having a s s u r e d himself of t h e p u r i t y of a r a d i o i s o t o p e - l a b e l e d drug t h e u s e r must t r y t o m a i n t a i n t h i s purity. This is n o t an easy t a s k s i n c e decomposition, due t o s e l f i r r a d i a t i o n , o r d i n a r i l y w i l l b e more pronounced t h a n f o r t h e n o n r a d i o a c t i v e form of t h e drug. The f i r s t g e n e r a l o b s e r v a t i o n s of t h i s e f f e c t were p u b l i s h e d by T o l b e r t , et & i n 1 9 5 3 6 2 . S i n c e t h e n s e v e r a l o t h e r e x c e l l e n t reviews and t a b u l a t i o n s on t h e s e l f - d e c o m p o s i t i o n of r a d i o i s o t o p e l a b e l e d compounds have a p p e a r e d G 3 , p a r t i c u l a r l y from t h e group a t The g e n e r a l d i s c u s s i o n s of t h e Radiochemical C e n t r e i n A m e r ~ h a m 7. ~ ~ - Other ~ problem can b e found i n books by Feinendegen4, S c h u t t e 3 , and WangG. Although knowledge of s e l f - i n d u c e d r a d i a t i o n decomposition of r a d i o -

304 -

Sect. V I

- Topics

i n Chemistry

W i l e y , Ed.

a c t i v e compounds is l a r g e l y e m p i r i c a l i t i s recognized t o r e s u l t from f o u r b a s i c f a c t o r s . Bayly and Weigelt4 have c l a s s i f i e d t h e s e as primary ( i n t e r n a l ) , primary ( e x t e r n a l ) , secondary, and chemical e f f e c t s . The primary ( i n t e r n a l ) e f f e c t r e f e r s t o t r a n s f o r m a t i o n of t h e r a d i o a c t i v e atom i n a molecule t o a n o t h e r element r e s u l t i n g i n a c h e m i c a l l y d i f f e r e n t molec u l e . Although t h e r e s u l t i n g molecule c o n s t i t u t e s a chemical i m p u r i t y , u n l e s s t h e daughter atom is r a d i o a c t i v e o r two r a d i o a c t i v e atoms are i n t h e same molecule, a radiochemical impurity w i l l n o t b e formed. Fortun a t e l y , s i n c e i t can n o t be c o n t r o l l e d , t h i s e f f e c t i s of l i t t l e consequence i n t h e c a s e of r a d i o i s o t o p e - l a b e l e d drugs. The primary ( e x t e r n a l ) e f f e c t i n v o l v e s d i r e c t i n t e r a c t i o n of t h e r a d i o a c t i v e p a r t i c l e w i t h molec u l e s of t h e compound t o form i m p u r i t i e s , b o t h chemical and radiochemical. Appreciable decomposition r e s u l t s from t h i s mechanism. The secondary e f f e c t r e f e r s t o decomposition of t h e compound by chemically r e a c t i v e s p e c i e s produced by t h e r a d i o a c t i v e p a r t i c l e . These chemically r e a c t i v e species may arise from any material w i t h i n t h e range of t h e r a d i o a c t i v e p a r t i c l e , n o t simply from t h e compound i t s e l f . This e f f e c t , a l t h o u g h d i f f i c u l t t o d i s t i n g u i s h from t h e primary ( e x t e r n a l ) e f f e c t , seems t o account f o r most s e l f - i n d u c e d r a d i a t i o n decomposition. The chemical e f f e c t r e f e r s t o decomposition u n r e l a t e d t o t h e r a d i a t i o n and would o c c u r d u r i n g s t o r a g e of t h e n o n r a d i o a c t i v e form of t h e compound. Chemical decomposition may be more s e v e r e , however, i n t h e c a s e of a r a d i o a c t i v e compound when i t i s p r e p a r e d i n such a s m a l l q u a n t i t y t h a t chemical is n o t a s s u r e d and s t o r e d a t a very low c o n c e n t r a t i o n i n s o l u t i o n 5 7 ,

rlrity *.

A compound's s u s c e p t i b i l i t y t o r a d i a t i o n is e x p r e s s e d i n terms of a v a l u e , t h a t is, t h e number of molecules d e s t r o y e d p e r 100 e l e c t r o n v o l t s of r a d i a t i o n energy absorbed. Although t h e g e n e r a l r e l a t i o n s h i p between t h e e x t e n t of decomposition and G(-M) i s e x p o n e n t i a l , i t i s n e a r l y l i n e a r when decomposition does n o t exceed lo%, and is d i r e c t l y proport i o n a l t o t h e product of G(-M) and t h e amount of r a d i a t i o n energy absorbed. The l a t t e r v a l u e is t h e product of 4 f a c t o r s : F, t h e f r a c t i o n of t h e r a d i a t i o n absorbed; E , t h e a v e r a g e energy of t h e r a d i a t i o n ; so, t h e s p e c i f i c a c t i v i t y of t h e compound; and t , t h e t i m e (when i t is s h o r t r e l a t i v e t o the isotopes half l i f e ) 6 4 . I n general t h i s relationship holds f o r both primary ( e x t e r n a l ) and secondary e f f e c t s when F r e f e r s t o t h e compound i n t h e former case and t o both compound and i t s immediate environment i n t h e l a t t e r case. G(-M)

C o n t r o l of t h e t h r e e f a c t o r s , t , so, and F is e f f e c t i v e i n l i m i t i n g decomposition due t o both primary ( e x t e r n a l ) and secondary r a d i a t i o n e f f e c t s by minimizing d i r e c t i n t e r a c t i o n of t h e r a d i a t i o n w i t h t h e system. S i n c e decomposition i s t i m e dependent, i t can be minimized by keeping t h e t i m e , t , between p u r i f i c a t i o n of a compound and i t s u s e a s s h o r t as poss i b l e . Keeping t h e s p e c i f i c a c t i v i t y , so, of t h e compound a t t h e lowest r e a s o n a b l e v a l u e a l s o w i l l minimize i t s r a d i a t i o n decomposition, The f r a c t i o n of t h e r a d i a t i o n , F, absorbed by t h e compound can be minimized by d i s p e r s i n g o r d i l u t i n g t h e compound. D i s p e r s i n g t h e compound as a t h i n f i l m on t h e c o n t a i n e r w a l l o r on paper o r s i l i c a g e l may a l l o w t h e r a d i a t i o n t o e s c a p e from t h e compound w i t h minimum i n t e r a c t i o n . Although t h i s is f e a s i b l e € o r carbon-14 and s u l f u r - 3 5 , i t is i m p r a c t i c a b l e f o r t r i t i u m

Chap. 28

Rad i o i so tope-La be 1 ed Drugs

Thomas

305 -

due t o i t s low r a d i a t i o n energy. D i l u t i o n of t h e l a b e l e d compound w i t h n o n r a d i o a c t i v e compound, a s o l v e n t , o r an i n e r t material such as p a p e r o r s i l i c a g e l s e r v e s t o l i m i t i n t e r a c t i o n of r a d i a t i o n w i t h t h e l a b e l e d molec u l e s by i n t e r p o s i n g i n a c t i v e molecules i n t h e p a t h of t h e r a d i a t i o n , Although d i l u t i o n of t h e l a b e l e d compound w i t h a s o l v e n t is an e f f e c t i v e , and o f t e n n e c e s s a r y , means of l i m i t i n g d i r e c t i n t e r a c t i o n of rad i a t i o n w i t h t h e compound, secondary e f f e c t s due t o p r o d u c t i o n of chemic a l l y r e a c t i v e s p e c i e s from t h e s o l v e n t may l e a d t o e x t e n s i v e decomposit i o n . Benzene is a good s o l v e n t s i n c e i t does n o t produce h i g h concent r a t i o n s of r e a c t i v e s p e c i e s . Water i s p a r t i c u l a r l y poor i n t h i s r e s p e c t , a l t h o u g h i n c l u s i o n of a s c a v e n g e r such as e t h a n o l may r e t a r d f o r m a t i o n of r e a c t i v e s p e c i e s . Scavengers have n o t been found p a r t i c u l a r l y u s e f u l i n nonaqueous systems. I n g e n e r a l a l a b e l e d compound, whether i t is n e a t o r d i l u t e d , s h o u l d b e s t o r e d a t a low t e m p e r a t u r e , i n t h e absence of l i g h t , and i n an i n e r t atmosphere t o p r o t e c t a g a i n s t secondary r a d i a t i o n , as w e l l as p u r e l y chemical , decomposition. 1. 2. 3.

4. 5.

6. 7.

8. 9. 10.

11. 12.

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13 14. 15. 16. 17. 18. 19* 20. 21. 22.

References J.R. catch, "Carbon-14 Compounds," Butterworth, Washington, D.C., 1961. E.A. Evans, "Tritium and Its Compounds," D. Van Nostrand, N.Y., 1966. H.R. Schutte. "Radioaktive Isotope in der Organischen Chemie und Biochemie ,I' Verlag Chemie. Weinheim, 1966. L.E. Feinendegen. "Tritium-Labeled Molecules in Biology and Medicine." Acedemic Press. N.Y., 1967. M. Wenzel and P.E. Schulze, "Tritium-Markierung," Welter de Gruyter and Co., Berlin, 1962. C.H. Wang and D.L. Willis. "Radiotracer Methodology in Biological Science," Prentice-Hall, Englewood Cliffs, 1965. "The Radiochemical Manual," 2nd ed. ,B.J. Wilson (Ed.), The Radiochemical Centre, Amersham, 1966. G.D. Chase and J . L . Rabinowitz, "Principles of Radioisotope Methodology," 3rd ed., Burgess, Minneapolis, 1967. V.F. Raaen, G.A. Ro~p.and H.P. Raaen, "Carbon-14," McGraw-Hill, N.Y., 1968. A.F. Thomas, "Deuterium Labeling in Organic Chemistry,'I Appleton-Century-Crofts, N.Y.. 1971. "Advances in Tracer Methodology." Vols. 1-4, S. Rothchild (Ed.) , Plenum Press, N.Y., 1963, 1965, 1966, 1968. "Radioisotopes in the Physical Sciences and Industry," Vol. 111, IAEA, Vienna, 1962. "Tritium in the Physical and Biological Sciences," Vol. 11, IAEA, Vienna, 1962. "Proceedings of the Conference on Methods of Preparing and Storing Marked Molecules." J . Sirchis (Ed.) , Euratom ReLort EUR-1625.e. Brussels, 1964. "Methods of Preparing and Storing Labelled Compounds," J. Sirchis (Ed.), Euratom Report EUR-3746. Brussels, 1968. M. Calvin. C. Heidelberger, J . C . Reid, B.M. Tolbert and P.F. Yankwich, "Isotopic Carbon," John Wiley and Sons, N.Y. , 1949. A. Murray, I11 and D.L. Williams, "Organic Syntheses with Isotopes," Part I. Interscience, N.Y., 1958. A. Murray, I11 and D.L. Williams, "Organic Syntheses with Isotopes." Part 11, Interscience, N.Y., 1958. A.R. Ronzio. "Microsynthesis with Tracer Elements," Chptr. XI11 in "Technique of Organic Chemistry." Vol. VI, A. Weissberger (Ed.1, Interscience, N.Y., 1954. M.D. Kamen. "Isotopic Tracers in Biology," 3rd ed., Academic Press, N.Y., 1957. M. Hamberg, Eur. J. Blocher.. 6 , 135 (1968). G.D. .Hunter and D.J. Hockenhall, Biochem. J . , Zp. 268 (1955).

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47.

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Wiley, Ed.

in Chemistry

0. C i f e r r i , A. A l b e r t i n o , and G. C a s s a n i , "Proceedings of t h e Conference on Methods of P r e p a r i n g and S t o r i n g Marked Molecules," J. S i r c h i s ( E d . ) , Euratom Report EUR-1625.e, B r u s s e l s , 1964. p. 961. Y. Komatsu, J. A n t i b i o t . , 3, 566 (1971). J . K . Lee and F. Schmidt-Bleek, "Advances i n A n a l y t i c a l Chemistry and I n s t r u m e n t a t i o n , " Vol. 7 , C . N . R e i l l e y and F.M. McLafferty ( E d s . ) , I n t e r s c i e n c e , N . Y . , 1968. P. 67. E.A. Evans, " A Guide t o T r i t i u m L a b e l l i n g S e r v i c e s . " Radiochemical I n f o r m a t i o n Booklet from A m e r s h a d S e a r l e , Chicago. W.H. Pearlman. Biochem. J . , 17 (1957). P.N. R r o , S t e r o i d s . Is, 219 (1971). M. Gut and M. Uskokovic, J . Org. Chem., 3, 673 (1959). A.S. F a i r h u r s t , J. Label. Compounds, 1,133 (1971). P. Avinur and A. N i r . B u l l . Res. Coun. I s r . , G , 74 (1958). P.M. Yavorsky and E. G o r i n , J . Am. Chem. S o c . , &, 1071 (1962). B. A l i p r a n d i and F. Cacace, Ann. Chim. (Rome), 2011 (1959). B. A l i p r a n d i and F. Cacace, 397 (1961). C. Mantescu, A. Genunche, J . Label. Compounds, 1, 178 (1965). K. Bloss, 5, 355 (1969). J.R. J o n e s , 2, 197 (1968). J.L. G a r n e t t , N u c l e o n i c s , 0, ( 1 2 ) , 86 (1962). J.L. G a r n e t t and W . S o l l i c h , J . Phys. Chem., 436 (1964). I. P r i - B a r and 0. Buchman, J. Chem. S o c . ( D ) , 1631 (1970). K.E. Wilzbach, J . Am. Chem. SOC.. 2, 1013 (1957). M.L. Whisman and B.H. E c c l e s t o n . Nucleonics, 3, 98 (1962). R.C. Thomas and G . J . I k e d a . J . Pharm. S c i . , 112 (1966). R . C . Thomas, D.R. B u h l e r , and G. J . I k e d a . *, $, 507 (1967). R . C . Thomas, 0. J . I k e d a , and H. H a r p o o t l i a n , 862 (1967). R . C . Thomas, G . J . I k e d a , and L.M. Reineke, J. Label. Compounds, 517 (1971). R.M. L e m o n , B.M. T o l b e r t , W. S t r o h m e i e r , and I . M . Whittemore, S c i e n c e , 1740

66,

w ,s,

*,

w,

u,

E,

z,

w, s,

(1959) *

z,

2,

D . A . Ghenem and T . Westermark, J . Am. Chem. S o c . , &, 4432 ( 1 9 6 0 ) . T. Westermark, H. L i n d r a t h , and B. Enander, I n t . J . A p p l . R a d i a t . 1 s o t o p . , 1 , 3 3 1 (1960) 50 . T. Gosztonyi, J. Label. Compounds, 2 , 196 (1969). 51. M. Wenzel. P.E. S c h u l z e , and H. Wollenberg, N a t u r w i s s e n s c h a f t e n , 3 , 515 ( 1 9 6 2 ) . 1510 (1962). 52 * T. Mesh1 and T. T a k a h a s h i , B u l l . Chem. S o c . , J a p . , 53. R . E . Ober, S.A. F u s a r i , G.L. Coffey, G . W . Gwynn and A . J . Glazko, "Advances i n T r a c e r Methodology," V o l . 1, S. R o t h c h i l d ( E d . ) , Plenum P r e s s , N . Y . , 1963, p. 56. 54. E. K a t z , A.B. Mauger, and H . Weissbach. Mol. Pharmacol., 1,lo7 ( 1 9 6 5 ) . 55. P . J . Ayres, " T r i t i u m i n t h e P h y s i c a l and B i o l o g i c a l S c i e n c e s , " Vol. 11. IAEA, Vienna, 1962, p. 131. 56. S. Bergstrom, H. D a n i e l s s o n , and B. Sammuelsson, Biochim. Biophys. Acta, E, 207 (1964) 57. L.E. G e l l e r and N. Silberman, J. Label. Compounds, 2. 66 (1969). 749 (1966). 58 * A . I . F r a n k e l and A . V . Nalbandov, S t e r o i d s , 59. E . J . M e r r i l l and A.D. Lewis, Chem. Eng. News, 3 (18), 24 (1971). 60. P. P e y s e r , "Advances i n T r a c e r Methodology," Vol. 4, S . R o t h c h i l d ( E d . ) , Plenum P r e s s , N . Y . , 1968, p. 41. 61. J.R. Catch, " P u r i t y and A n a l y s i s o f L a b e l l e d Compounds," Radiochemical Informat i o n Booklet from Amersham/Searle, Chicago. 62. B.M. T o l b e r t , P.T. Adams, E.L. B e n n e t t , A . H . Hughes, M . R . K i r k , R.M. L e m o n , R.M. N o l l e r , R . Ostwald, and M. C a l v i n , J. Am. Chem. S o c . , E , 1867 (1953). 685 (1965). 63. P. R o c h l i n , Chem. Rev., 64. R . J . Bayly and H. Weigel, Nature (London), 384 ( 1 9 6 0 ) . 65. R.J. Bayly and E.A. Evans, J. Label. Compounds, 2 , 1 (1966). 66. R . J . Bayly and E.A. Evans, 2 (Suppl. l), Jc9 (1967). 67. R . J . Bayly and E.A. Evans, " S t o r a g e and S t a b i l i t y o f Compounds L a b e l l e d w i t h r a d i o i s o t o p e s , " Radiochemical I n f o r m a t i o n Booklet from Amersham/Searle, Chicago.

48. 49.

B,

-

5,

w,

188,

COMPOUND NAME AND CODE NUMBER INDEX

4864, 23 48/80, 5014, 5023,

l-amino-2,6-dimethyl-benzyl 244

7 7

19 583 R.P., 210 24010, 112 34276-Ba (maprot i 1 ine) , 19 A- 1009 1 /A ( t rypanomyc i n) , 150 A- 1 1884 (cephamyc in C ) , 153 A-25794, 20 A-30360, 8 A-30360, 33 A-35616 ( c h l o r a z e p a t e ) , 42 acenocuma r o l , 84 acetaminophen, 259, 261, 264, 265 17a-acetoxy-6-chloro-21 - f l u o r o - 1 6 m e t h y l enepregna-4,6-d i ene-3,20dione, 187 21 -acetoxy-3a-hyd roxy-5a-pregnane11,20-dione, 190 a c e t o x y m e t h y l cephalog l y c i n , 102 acetylacetone, 255 a c e t y l and romedo 1 (g rayanotoxin I ) , 63 acetylcholine, 35, 36 ACTH ( a d r e n o c o r t i c o t r o p i c hormone), 195 actinomycin D (act-D), 120, 134, 242 adenosine, 22, 70, 71, 145 adenosine analogs, 71 adenos ine d iphosphate (ADP) , 71 adenos ine monophosphate (AMP) , 145 adenos i ne t r i phosphate (ATP) , 70, 71 ad renoco r t icoco t r o p ic ho r m n e ( A C T H ) , 195 adriamycin, 135 ADT, 22 AH 5158A, 62 8 AHR-1900, AHR-2277, 8 AL 1612, 9 alclofenac, 209 l-alkenyl-4-(l-H)-quinazolones, 2-a1 k y l t h i o a d e n o s i ne, 71 al lopurinol, 71 alprenolol, 59, 73, 74, 75 amantadine, 22, 26, 125 amfonelic acid, 27

34

piperazine, 123 4-am i n o - 5 - e t h y l - 3 - m e r c a p t o - i triazole, 213

2-aminomethyl-7-methoxy-2,3dihydrobenzofuran, 34 aminophylline, 27, 89, 245 aminopyrine, 260 aminorex, 26 aminorex fumarate, 260 amiodarone, 71, 72 a m i t r i t y l ine, 24 amoxapine (CL-677721, I8 amphetamine, 26, 265 103, 263 a m p i c i l 1 in, a m p h o t e r i c i n B, 109, 110, 116 androsterone, 134 ang i o t e n s i n , 292 02, O2 ' - a n h y d r o c y t o s i ne a r a b inos i d e , 125 a n i l e r i d i n e , N-acyl d e r i v a t i v e s ,

33 a n t i b i o t i c peptides, 292 a n t imonyl -d i m e t h y l c y s t e i no151 t a r t r a t e (NAP), a n t i m y c i n A2, 114 antipyrine, 264 anvene ( m y t a t r i e n e d i o l ) , 175 6-APA, 99, 103 apazone, 213 abomorphine, 26 APY-606 ( c l o s p i r a z i n e ) , 6 arvin, 86 L-asparaginase, 124, 138 a s p i r i n , 79-82, 165, 166, 211 ateroid, 85 AYF, 109 5-azacytidine, 130 25-azalanosterol, 190 azatadine, 94 a z a t h i o p r i ne, 21 2 5-az i r i d i nyl-2,4-d i n i t robenzami de (CB-1954), 129

B-663, 147 BA-41799, 147 bacitracin, 219 barbital, 260, 261 BAY 1470 ( x y l a z i n e ) , BAY 4503 ( p r o p i r a m ) ,

60

35

3 08 -

COMPOUND NAME AND CODE N U M B E R INDEX camptothecin, 137 carbamazepine, 264 ca rbendazo 1 e , 1 53 carbenicillin, 101, 103, 221, 223 capreomycin lA, 104 capreomycin lB, 104 capur ide (McN-X-941, 41 CB- 1954 (5-azi r id i nyl-2,4-d i n i t robenzami de) , 129 C C N U (1-(2-chloroethyl)-3-cyclohexyl- 1 -n i t rosourea) , 132 cephalexin, 102 cephaloglycin, 102 cepha 1 or id i ne, 260 cephalosporins, 219, 220, 221, 225 cephalothin, 102, 260 cephamyc i n C (A- 1 1884), 1 53 cephap i r i n (BL-P 1322) , 102

BAY a6781, 62 BAY b5097 (clotrimazole), 110 BCNU ( 1 ,3-b i s (2-chlo roethy 1 ) -1

-

nitrosourea), 132 beclotiamine, 149 betamethasone, 145 bencyclan, 71, 72, 86 benorylate, 211 benzmorphans, 32, 36 benzoquate, 147 benzphetamine, 27 benzylpenicillin, 103 L-benzylsuccinate, 254 bephenium, 152 betaine, 54 bethan id i ne, 60 bez i tram i de (R4845), 3 1 dl-cis-bisdehydrodoisynolic

acid, 189 bisobrin (EN-1661), 86 bithionol, 152 bleomycin A2, 135 BL-P1322 (cephapirin), 102 BL-P 1597, 103 BL-P 1654, 103 BL-S217, 102 brinase, 85 5-brow-3 -ch 1 or0-4~ (p-chlorophenoxy)-3-nitrosal icylanilide, 152 5-brow deoxyuridine, 242 4 I -brom0-3,4 I -d i hyd ro-2-methy 1-3-

-

oxo-2H-1,2-benzothiazine-4-

carboyanil ide 1 ,I-dioxide, bromopheniramine, 24 BS 7029, 43 BS 7679, 43

149

BT-985,

bufotenin, 54 bunolol, 74 busulfan, I30 butadienolides, 9 butirosin A, 100 butirosin B, 100 BW 65-54, 22 C-29, 19 C-42, 19

C-45,

19

caffeine,

27

212

CG-635, 178 cha 1 comyc i n , 104 chenodesoxycholic acid, 190 chloral hydrate, 264 chlorambucil, 212 chloramphenicol, 219, 222, 225 chlorazepate (A-356161, 42 p-chlorbenzol sul fone, 263 chlordiazepoxide, 15 chlorimipramine, 24, 48, 53 chlorite oxidized oxyamylose, 122 p-chloroamphetamine, 22, 27 3-~-chlorobenzoyl-7-methoxy-2methyl-1-indolizineacetic acid, 21 1 l-~-chlorobenzoyl-6-methoxy-2methyl-3-indolizineacetic acid, 21 1 3-chlorocar ipramine (Y-4153), 8 3l-chl oro-by-(p-ch lorophenoxy)3,5-dibrowsalicylanilide, 152 5-chloro-4-cyclohexylphenylacetic acid, 210 chlorophenirarnine, 24 p-chlorophenylalanine (PCPA), 39,

48-5 1 chloroquine, 146, 147 chlorpromazine, 166 ch 1 orpropam ide, 264 chlorthal idone, 60 cholecalciferol (Vitamin D 3 ) ,

191

COMPOUND NAME AND CODE NUMBER INDEX c h o l e c y s t o k i n i n , 292 cholestyramine, 171, 172, chromonar, 70, 71, 75 C I -572 ( p r o f adol) , 35 Cl-679, 150

Cl-683, 11 CL-1848C ( e t o x a d r o l ) , CL-64855, 148, 150

D-65-MT,

177,

261

44

CL-67772 (amoxapine), I8 CL-75805, 148 cladosporin, 112 c l indamycin, 104, 261 clofibrate, 172, I 7 7 clonazepam (Ro-5-4023), 42 clonidine, 60 c l o n i x i n , 34, 211 22, 53 c l o r g y l i n e (MGB 9302), c l o r p r e n a l ine, 90, 91 c l o s p i r a z i n e (APY-6061, 6 c l o t r i m a z o l e (BAY-b 50971, 110 c l o z a p i n e (HF-18541, 7 CMLR- 1066, 1 45 cocaine, 27 coenzyme Q, 146, 147 coenzyme Q8, 146 colchicine, 124 c o l e s t i p o l (U-26,597A), I77 colony i n h i b i t o r , 120 conjugated equine estrogens, 175 c o r t i c o t r o p in r e l e a s i n g f a c t o r (CRF), 197 CP 14.368, 21 CT 5104, 11

5-cyanothiophene-2-aldehyde thiosemicarbozone, 125 5-cyanourac i 1 , 130 cyclazocine, 35, 36 c y c l i c AMP, 80, 129, 198, 245 cycloguanil, 145, 147

(+) -2- [4-~ycIohexyl)cyclohexenI - y l l p r o p i o n i c acid, 210 cyclooctylamine, 125 cyclophosphamide, 212, 242, 263 cyproheptadine, 94 c y t a r a b i n e (ara-C, c y t o s i n e a r a b i noside, I-6-D-arabinofuranosylcytosine), 124, 125, 130 cytoxan (cyclophosphamide) , 129

D-bO-TA,

11

11

D-69-12, 73 0563 [ 1-3-methoxy-w- ( 1 -hydroxy-1phenyl isopropylamino) p r o p i o phenonel , 73 daunorub i c i n (daunomyci n, rub idomycin, 135 DCI, 74 0 , ~-DDD ' ( 1 (0-c h 1 o ropheny 1 ) 1 c h 1 o ropEeny 1 ) 2,2 d i c h 1 o r o ethane), 134 DDUG ( [ N , N ' - ( 4 , 4 ' - d i a c e t y l ) d i pheny 1 ] urea b i sbuany 1 hydrazone), 132 DEAE dextran, 122 DEAE Sephadex, 172 61 debr i soqu i ne, decloxizine, 92 dehydroclothepi n, 8 6-dehydroret roproges terone, 184 demecolcine, 124 demethdiazepam (Ro-5-2180), 9 104 N-demethylclindamycin, 263 deme thy1 ch 1 o r t e t racyc 1 ne, demethy 1 d iazepam (Ro- 5 2180) 9 9 demoxepam, 11 31-deoxykanamycin, 99 F3TDR ( 2 I - d e o x y - 5 - t r i f uoro13 methylur i d i n e ) , depakine, 43 dermostatin, 1I 6 12 desa 1 ky 1 prazepam, desipramine, 24, 166, 264 11 desmethy 1 c h i o r d iazepox ide, desmethyl imiprarnine, 24, 166, 264 N-deson/demoxepam, 11 dextran, 79, 81 dextroamphetamine, 22, 27 DH-581 (probucol, B i p h e n a b i a ) , 178 2,4-d i ami no-6- (3,4-d i c h l o r o b e n z y l amino) quinazol ine, 150 d iam inod iphenyl su 1 fone (DDS) , 147 c i s -d iamm ined i c h 1o rop 1 a t inum , 1 40 149 dianemycin, diazepam, 1 1 , 12, 15, 72 71 diazoxide, D I C (5- (3,3-d imet hy 1 1 t r iazeno) i m i d a z o I e - 4 - c a r b o ~ a m i d e ) ~133

(e-

-

- -

-

-

- -

--

-

310 COMPOUND NAME AND CODE NUMBER INDEX c is-d ich 1 o r o d ip y r i d in e p l a t i num, d i c l o x a c i l 1 i n sodium, 263

140

3',4'-dideoxykanamycin B, 99 3',4'-dideoxykanamycin, 99, 223, 224 3 I ,4 I -d ideoxyneami ne , 99 N-d ie t h y l arni noet hyl-5,6-dehyd r o carbazole, 150 d i e t h y l c a r b a m a z i n e (Hetrazana), 245 d i e t h y l d i thiocarbamate, 26 189 diethylstilbestrol, d i e t h y l s t i l b e s t r o l diphospha e, 134 d i f l u m i d o n e sodium, 213 B,B-difluoroamphetamine, 23 6,6-d i f 1 u o r o n o r e t h i ndrone, 87 digitoxin, 260, 261, 264 digoxin, 260, 261 d i hydra 1 az ine, 59 1 7 - @ - d i h y d r o e q u i l e n i n , 255 4 d i h y d r o f o l i c acid, 1 ,6-d i hyd ro-6- hyd roxymet hy 1 purine, 252 6 . 9 - d i h v d r o i m i ~ r a r n i n e ~ 20 1 ;25-d ihydroxychol e c a l c i f e r o l , 191 21,25-dihydroxycholecalciferol, 191 1 (3,s-d ihydroxy-4-n it robenzyl ) morphol ine, 125 5,6-dihydroxytryptami ne, 48 4,7-dimethoxybenzofuran-5-carboxam ide-6-0-acet ic ac i d, 34

-

-

-

0,0-dimethyl-2,2,2-trichloro-lhydroxyethylphosphonate, 153 dimethyl tryptamine, 54 dimetridazole, 148, 150 d i phenyl hydan t o i n, 264 d i p h e n o x y l a t e , 37 d i prenorphf ne (M-5050), 36 dipyridamole (Persantine), 70, 71, . 7 2 , 75, 79, 81, 8 2 disodium chromoglycate (DSCG, I n t a p ) 90, 92, 93, 94 disopyramide phosphate, 262 disulfiram, 264 d i v i m i n o l (Z 424), 31 DJ 1 550 ( su 1 f amonome t hox i ne monohydrate), 147 DNA RNA h y b r i d , 122 L-dopa, 21, 24, 25, 62, 260 dopamine, 3 5

-

-

dothiepin (prothiadine), 18 doxycycl ine, 104 DPC (3- ( 3 , 3 - d i m e t h y l - l - t r iazeno) p y r a z o l e-4-ca rboxam ide) , 133 D S CG ( d isod ium c h romog 1 y Ca t e , I n t a m , 90, 92, 93, 94, 245 emetine, 138

EN-1639, 36 EN-1661 ( b i s o b r i n ) ,

-

86

EN-2234A ( n a l b u p h i n e ) , 35 EN-3022, 20 endotox i n , 120 ephedrine, 27, 89, 92 epicillin, 103 89 epinephrine, erythritol tetranitrate, 75 erythromycin, 218, 219, 224, 225 erythromycylami ne, 104 espinomycins, 104 estradiol, 175 e s t r a d i o l benzoate, 134 estrogens, c o n-~ j u g a t e d equine, 175 estrone, 120 ethanol, 262, 264 265 e t h a n o o r i p a v i nes, 31 ethinyl estradiol, 175 4- ( e t hoxyace t y 1 ) am no-3-methyl benzophenone, 2 3 e t h r a n e , 44 N - e t h y l i s a t i n B-thiosemicarbazone, 125 5,6,7,82 - e t h y l -6-methy 1-2,3,4,4a, o c t a h y d r o - 1H-pyraz ino ( 1 ,2-c) pyrimidine-1-one, 153 3-ethyl-a-methyl tyrosine, 62 41 e t o m i d a t e (R-166591, etorphine, 31 e t o x a d r o l (cL-l848C), 44 EX 11-349, 9 f e n c l o z i c acid, 262 f e n f luramine, 41 f enop r o f en, 209 11 fenpentadiol, f e n s p i r i d e , 92 filipin, 124 flavomycoin, 112 f lorenal, 125 flufenisal, 211 f lunidazepam, 40

-

31 1 -

COMPOUND NAME AND CODE NUMBER INDEX f l u n i d a z o l e (MK-915), 148 f l u o c i n o l o n e acetonide, 261 fluoracizine, 19 B-fluoroamphetamine, 23 5-fluorocytosine, 110, 116 9 a - f luorohydrocortisone, 189 5 - f luoro-2- (a-hydroxyl benzyl) - 1 propylbenzimidazole, 125 f l u p e n t h i x o l decanoate, 6 f 1 uphenaz ine decanoa t e, 6 fluphenazine enanthate, 6 fluprednisolone, 259 f lurazepam, 39 f o l l i c l e s t i m u l a t i n g hormone (FSH), 195 f o l l i c l e s t i m u l a t i n g hormone r e l e a s i n g f a c t o r (FRF), 195 5-FU ( 5 - f l u o r o u r a c i l ) , 130 f u s a r i c a c i d amide (Sch 10595), 62 f u s i d i c acid, 219 124 fusidin, D-galactal, 256 galanthamine, 32 139 gallium salts, gentamicin, 99, 100, 101, 219, 222, 223, 224, 225 g 1 ucagon, 1 75 D-glucono-6-lactone, 255 glyceryl guaiacolate ether, 264 gold salts, 212 GP 45795, 7 gramicidin S , 104 g r a yanotox in I (ace t y 1 and romedo 1 )

-

,

63

(SRF) (GRF),

197

177

haloperidol, 14, 37 halothane, 263, 265 41 HD-233, h e l v e t i c o s a l acetonide, heparin, 84, 172, 262

263

FOE 674,

65

Hoechst 33258 (2-[2-(4-hydroxypheny 1 ) -6-benzimi dazol y 1 ] -6( 1 -me thy1 -4-p i perazy 1 ) benz i m i dazole), 153 HT 3261, 21 5-HTP (5-hydroxytryptophan), 52,

,

-

54 human chor i o n i c soma t o t rop i n (HCS), 199 hycanthone, 150 h y d r a l a z i n e , 59 hyd roch 1 o r o t h i a z i de, 59 25- hydroxyc ho 1eca 1 c if e r o 1 , 19 1 6-hydroxydopamine, 14 4-hydroxy-2-methyl-2l- 1 ,2-benzot h i a z ine-3-ca rboxan i 1 ide 1,l-dioxide, 212 2- [2-(4-hydroxyphenyl) -6benz imidazol y 1 ] -6- ( 1 -methy 1 4-pi perazy 1) benz imidazol e 153 (Hoechst 33258), 3-hydroxyprazepan1, 12 3a-hydroxy-5a-pregnane-l1,20dione, 190 5-hydroxytryptophan (5-HTP) , 52, hyd roxyurea 132 92 hyd roxyz i ne, ibuDrofen, 209 C I ' 56780; 47 C I 58 301, 93 C I 60128, 47 doxuridine, 124 7 mic lopaz ine rnipramine, 24; 25, 53, 264 103 ndanyl c a r b - n i c i l 1 in, ndornethacin, 80, 165, 166, 211 ndoramine (Wy 219011, 62 nserm-54, 11 nsulin, 264 nterferon, 119 opanoic acid, 261

-

,

griseofulvin, 109, 1 1 1, 259 growth hormone ( G H ) , 197, 291 growth hormone r e l e a s i n g f a c t o r guancyd ine, 59 guanethidine, 60, 61 G Y I 20238, 23 ha1o f ena t e (MK-185, 2-acetamidoe t h y l (p-chlorophenyl) ( m - t r i f 1 uo rome t hy 1 -phenoxy) a c e t a t e )

hernandaline, 138 hexachl orophene, 152 hexobarbital, 262, 264 hexobendine, 71, 72, 75 hexoprenaline, 90, 91 HF-1854 ( c l o z a p i n e ) , 7 hikizimycin, 112 L-histidine, 213

54

COMPOUND NAME AND CODE NUMBER INDEX iprindole, 25 ipronidazole, 148, 150 i p r o v e r a t r i 1 , 70, 72 i s o c a r b o x a z i d , 22 isophosphamide 212 i s o p r inos i ne, 124 i s o p r o t e r e n o l , 75, 89, 90, 91, 92, 263 isosorbide d i n t r a t e , 74, 75 ITF -9611 211 kanamycin, 219, 222, 223, 224, 225 109 kasugamyc i n, ketamine, 44 KL-255, 73, 74

KO-1313,

KO-1366,

73 73

L6150, 64 LD2855, 60 L ~ O - 1 0 2 8 210 1 eucomyc i ns , 104 levamisole, 152 l e v o r p h a n o l , [ H 3 ] -N-B-(p-azidopheny1)-ethyl, 33 LG 152, 21 1 i d o c a i n e , 263 70, 71 lidoflazine, L i l l y 51641, 22, 53 lincomycin, 104, 219, 224, 225 1 i p o t r o p i c hormone (LPH), 201 lipoxamycin, 115 l i t h i u m carbonate, 6, 15, 25 l i v i d o m y c i n A, 100 l i v i d o m y c i n 8, 100 lorazepam (Wy-4036)) 10, 40 LU 3-009, 21 LU 3-047, 21 LU 3-049, 21 LU 3-074, 21 1 ucan thone , 1 50 l u t e i n i z i n g hormone ( L H ) , 195 1 u t e i n i z i ng hormone r e 1 eas i ng f a c t o r (LRF), 195 M-5050 (d i p r e n o r p h ine) , 36 m a p r o t i 1 i n e (34276-Ba), 19 mar idomyc ins, 104 mar i huana, 263 MGB 9302 ( c l o r g y l i n e ) , 22, 53 MCA-nitroimidazole, 148, 150 McN-X-94 (capur i d e ) 41 mebendazole, 152

-’

,

m e c l a s t i n e , 94 medazepam, 12 me1anac i d i ns, 124 me a n o c y t e s t i m u l a t i n g hormone (MSH), 198 me a n o c y t e s t i m u l a t i n g hormone (release) i n h i b i t i n g f a c t o r (MIF), 198 me a n o c y t e s t i m u l a t i n g hormone 198 releasing - f a c t o r (MRF). a-me 1 a n o t r o p i n, 291 menichlopholan, 152 menoctone, 146 meperidine analogs, 33 6-MP (6-mercaptopurine) , 131 4 - m e r c u r i - 1 7 f 3 - e s t r a d i o l , 188 metampicill in, 265 m e t a p r o t e r e n o l , 91 methamphetamine, 35 1,5-methano-3-methyl1 ,2,3,4,5,6hexahydro-3-benzazocines, 32 methasquin, 131 m e t h i c i l 1 in, 220, 222 methionine, 54 L-meth i o n ine- ( S ) -su 1 f o x i m i ne phosphate, 254 meth isazone, 125 m e t h o t rexa t e (MTX, amet h o p t e r in) , 131,145, 263 7-methoxycephalosporins, 101 6-methoxypenicill ins, 101 2- (2-methy 1 -4-am ino-5-py r i m i d iny 1 c a r barn i d e ) -5-n i t r o t h ia z o l e, I

151 D-N- (a-methy 1 benzy 1

-

-

1 i no 1 eami de ,

178 N-methyl-p-chloroamphetamine ( Ro-4-6861 ) , 22 a-methyldopa, 60, 62 a-methyloctopamine, 62 methyloxazepam, 10 6 - m e t h y l p e n i c i l l in, 101 methylphenldate, 22, 27 6a-methyl pregn-4-ene-3,11,20trione, 134 7a-methyl t h i o - l 7 a - a c e t o x y progesterone, 187 D,L,-a-methylthyroxine ethyl ester

(CG-6351, 5-methyl

178

tryptophan,

218

COMPOUND NAME AND CODE NUMBER INDEX a-methyl t y r a m i n e , 62 a-methyltyrosine, 23, 27 methylsergide, 53 m e t i a z i n i c acid, 210 metronidazole, 110, 148 MF-ni t r o i m i d a z o l e , 148 miconazole n i t r a t e , 111 minocycline, 105, 220 m i n o x i d i l (PDP), 59 mirasan, 150 136 m i t o m y c i n C, MK-185 ( h a l o f e n a t e ) , 177 MK-910, 149 MK-915 ( f l u n i d a z o l e ) , 148

MK-3883, monensin, morantel, morphine,

151

149 153 31, 32, 34, 35, 37

2-morpholinomethylbenzophenone h y d r o c h 1o r ide, 2 13 m y t a t r i e n e d i o l (anvene),

33 n a f e n o p i n (SU-l3,437, 176

175

MZ-144,

Me1 i p a n a ) ,

35 n a l b u p h i n e (EN-2234A), n a l i d i x i c acid, 219 naloxone, 33, 34, 35, 3 6 nandrolone phenpropionate, 189 NAP ( a n t imony 1 -d i m e t h y l c y s t e i notartrate), 151 naproxen, 210 narbomycin, 104 n a r b o n o l ide, I04 35 n a r c o t i c antagonists, NC 7197, 61 nebramycin f a c t o r 6, 99 negamyc i n, 104 177, 219, 222, 223, neomycin, 224, 225 neurophysin I , 202 niclosamide, 152 n i c o t i n i c acid, 172, 178 niflumic acid, 211 niridazole, 150, 151 nitrate, 256 n i t razepam, I5 nitrimidazole, 148 nitrofurans, 219 nitrofurantoin, 259, 263

nitroglycerin, 69, 70, 72, 74, 75, 259 4-nitrophenylarsonic acid, 150 3,5-nitrosalicylic acid-5-nitrof u r f u r y l i d e n e hydrazide, 150 nomifens i n e , 20 norepinephrine, 245 noreth indrone acetate, 172 norgestrel, 183 n o r j i r imyc i n, 255 no r o c t o c 1o t h e p in, 8 184 19-norprogesterone, n o r t r i p t y l ine, 24 nystatin, 109 ORF-8063, 1 1 , 42 o r m e t o p r im, 149 ouabain, 14, 73 oudenone, 63 o x a f lumazine, 6 o x a l i c acid, 254 oxandrolone, 172 oxazepam, 1 1 , 12 oxazolazepam, 10 oxometothepin, 8 oxovanadium ( I V ) i o n , 256 oxprenolol, 73, 75 oxyclozanide, 152 oxymetholone, I89 oxyphenbutazone, 260, 265 oxyprothepin, 8 oxytocin, 198, 292 P1961, 22 panclar, 21 panidazole, I48 papaverine, 70, 80 p a r a t h y r o i d hormone, 291 parbendazole, 152 153 paromomycin s u l f a t e , PCPA ( p - c h l o r o p h e n y l a l a n i n e ) ,

48- 5 1 PDP ( m i n o x i d i l ) , 59 D-penicillamine, 9, 213 p e n i c i l l in, 254 penicillins, 219, 220, 221, 225 p e n t a e r y t h r i t o l t e t r a n i t r a t e , 70 pentazocine, 32, 3 5 p e n t o b a r b i t a l , 261, 263, 264 peradithepin, 8

314 COMPOUND NAME AND CODE NUMBER INDEX perhexi 1 ine, 72 40 p e r 1 ap ine, 6 perphenazine enanthate, p e r s a n t ine (see d ip y r idamole) phenmet r a z ine, 27 phenoba r b it a 1 , 92, 260, 264 phentermine, 27 phenyl butazone, 259, 264, 265 2-phenylethaneboronic acid, 253 phosphoglycol i c acid, 254 phosphomannans, 122 N- ( phosphonacety 1 -L-aspa r t a t e , 2 56 phosphonomyc i n, 263 picromycin, 104 pimaricin, 110 pipotiazine, 6 pipotiazine palmitate, 6 p i p o t i a z i n e undecylenate, 6 pipradrol, 27 p i q u i z i l , 92 102 polymixin, 219 121 p o l y r (A-U), 121 p o l y r C, p o l y r c : ( I ) I, 122 p o ~ yr I, 121P5 119, 120, 121, p o l y r I : poly r C, 122, 123 poly r I : poly (I-vinylcytosine), 122 59, 60, 73, 75 practolol, prazepam, 12 p r e n y l a m i n e , 70 73 prindolol, p r o b u c o l (DH-581, B i p h e n a b i d a ) ,

pivaloyloxymethylcephaloglycin,

178 p r o f a d o l (C I - 5 7 2 ) , 35 progestin, 175 p r o l a c t i n (PRL) 95 p r o l a c t i n release n h i b i t i n g f a c t o r (PIF), 198 p r o l a c t i n r e l e a s i n , f a c t o r (PRF),

,

198

promazine, I4 14 promethaz i ne su 1 f o x ide, p r o p a n o l o l ( p r o p r a n o i o l ) , 1 1 , 59, 60, 73, 74, 75, 178, 245, 263 35 p r o p i ram (BAY-4503),

p r o p r a n o l o l ( p r o p a n o l o l ) , 1 1 , 59, 60, 73, 74, 75, 178, 245, 263 63, 166 p r o s t a g l a n d i n A1 ( P G A ] ) , p r o s t a g l a n d i n E l (PGEI), 63, 92, 79, 80, 166, 212, 245 p r o s t a g l a n d i n E2, 9, 79, 80, 92, 93, 166, 167, 212, 245 p r o s t a g l a n d i n E2, 15-methyl, methyl e s t e r , 162 p r o s t a g l a n d i n F l a , 7-oxa, 160 prostaglandin F (PGF2a), 80, 2a'

166, 167

prostaglandins, 129, 198 prothiadine (dothiepin), 18 prothixene, 7 p r o t r i p t y l ine, 24, 25 14 p r o t r i p t y l ine sulfoxide, proxyphylline, 93 prumycin, 112 pseudolycorine, 137 pseudourea ( 2 , 2 ' - ( 9 , l O - a n t h r y l e n e ) -d i m e t h y l ene) b i s- ( 2 - t h i o pseudourea)d i h y d r o c h l o r i d e ) ,

133 ps i c o f u r a n i n e , 218 71 pteridines, p y r a n copolymer, 120 pyrantel 153 p y r idarone, 11 pyrimethamine, 4, 145 pyrrole-2-carboxyl i c acid, 255 pyrrolnitrin, 109, 1 I 6 p y r o v a l e r o n e , 22 quinine, 261 quinterenol, 90, 91 quipazine, 20, 48, 53 R4845 ( b e z i t r a m i d e ) , 31 R-16659 ( e t o m i d a t e ) , 41 rafoxanide, 152 r e p t i I ase, 86 retrotestosterone, 183 r i b o n u c l e a s e A, 291 ricin, 139 rifampicin, 99, 123, 219 rifamycin, 218, 219 rifamycins, 123 t-RNA methyl transferase, 139 Ro 4-6861 ( N - m e t h y l - p - c h l o r o amphetamine), 22

,

COMPOUND NAME AND CODE NUMBER INDEX

Ro 5-0013 ( R o f e n a i d n ) , 149 Ro 5-2180 (demethydiazepam) , 9 Ro 5-3350, 9 Ro 5-4023 (clonazepam), 42 RO 5-4200, 10 RO 5-6901/3, 10 RO 7-0207, 148 Ro 7-2956, 65 RO 8-2580 9 R o f e n a i d Q (Ro 5-00131, 149 ronidazole, 148, 150 rubidium s a l t s , 20, 25 SA 124, 6 salbutamol, 90, 91 salicylamide, 264 s a l i c y l i c acid, 261, 264 saramycetin, 116 SC-26100, 37 Sch 1000, 92 Sch 10595 ( f u s a r i c a c i d amide), 62 Sch 12041, 10 Sch 12679, 9 SD 2124-01, 73 SD 2202-01, 18 serotonin, 35, 39 serum p r o t e c t i v e f a c t o r , 119 sesquiterpine lactones, 137 SF-837, 104 116 siccanin, sisomicin, 99 SKdF 525A, 260 SKEF 25971, 7 SKSF 28175, 7 SM-307, 18 soterenol, 90, 91 spectinomycin, 101, 218, 219, 222, 223, 225 SQ- 1 8506, 151 statalon, 120, 122 78, 79, 85 streptokinase, streptolydigin, 219 streptomycin, 218, 219, 222, 223, 225 s t r e p t o v a r i c i n C, 101 streptovaricins, 123 Su-13,437 ( n a f e n o p i n , Me1 i p a n a ) ,

176 292 substance P, 260 su 1 facetamide, sulfadiazine, 259, 260, 261

315 -

sulfadimethoxine, 149 s u l f a d i m i d i ne, 259 su l f a d o x ine, 147 sulfaethidole, 261 1, 260 sulfamethoxazole, sulfamonomethoxine monohydrate (DJ- 1550) , 147 5 1 -0-sulfamoyladenosine, 150 s u l f a p y r i d ine, 265 sulfocillin, 103 sulfonamide, 218, 219, 220 sulfadoxine, 264 9 sulpiride, sydnophene, 22 TAI-284, 210 t e r b u t a l ine, 90 tetracycline, 208, 218, 219, 220, 26 1 3,4,5,6-tetrahydrouridine, 252 tetramisole, 152 tetramycin, 112 111 tetranactin, t e t r i n A, 115 t e t r i n B, 115 Th 1165a, 90, 91 thal icarpine, 138 t h e o p h y l l i n e , 27, 89, 92, 93 thiabendazole, 1 1 1 , 152, 153 S-2- ( [ 2 - ( 2 - t h i a z o y l carbamoyl) e t hy 1 ] am ino) e t hy 1 ac i d t h i0sulfate, 151 thiopental, 263 t h i o r i d a z ine, 14 thiosinamine, 150 t h y r o i d s t i m u l a t i n g hormone (TSH),

194 t h y r o i d s t imu 1a t ing hormone re1 ease f a c t o r (TRF), 194 thyroxine, 25, 195 D- t h y r o x i n e ( d e x t r o - t h y r o x ine, D-TG), 178 tilidine, 31 tilorone, 122 t inidazole, 148 t in o r i d ine hydroch l o r ide, 21 3 tobramycin, 99, 219, 223, 224 tofenacin, 20 tolbutamide, 264 tolmetin, 210 111 tolnaftate,

316 -

COMPOUND NAME AND CODE NUMBER INDEX

toxogon in, 263 t r a n y l cyprom i ne, 22 I78 treloxinate, 3,4' ,5-t r ibromosal ic y l an i 1 ide, 152 t r i d ihexethyl c h l o r ide, 262 t r i e t h y l phosph inogol d c h l o r i d e , 21 2 triflumidate, 213 t r i f 1 uoperazi ne, 14 D - t r i i o d o t h y r o n i n e ( d e x t r o - t r i iodothy178 ronine, D-T3), trimethoprim, 1-5, 145 t r i m e t h o q u i n o l , 90, 91 1 (3,3,3-t r i phenyl p r o p y l ) -4- (2p y r i m idy 1 ) p i peraz ine, 3 4 95 tritoqualine, t rop icami de, 262 t r p ( t r y p t o p h a n ) , 52, 53, 54 trypanomycin ( A - ~ o o ~ ~ / A 150 ), tryptophan ( t r p ) , 52, 53, 54 L- t ryptophan, 39 tuberact i nomyc in-N, 104 t u b e r a c t i nomyc i n-0, 104 112, 124 tunicamycin, m- t y r o s ine, 62 U-17,660, 19 U-20,057, 9 U-26,597A ( c o l e s t i p o l ) , 177 U-28, 926A, 22 U-30,405A, 22 U-31,889, 42

-

U-31,957,

1 1 , 42

U-33,030, 42 11, 42 U-35,005, 19 UP-106,

p-ureido-p'-aminodiphenylsulfone, 125 p-ureidobentenearsonic a c i d , 150 urokinase, 85 vasopressin, 197 versicol in, 115 vinblastine, 124 vincristine, 124 viomycin, I04 v i t a m i n A acid, 135, 138 v i t a m i n C, 124 81, 84, 259, 260, 261, warfarin, 264 WG 253, 90, 91 W I N 27,147-2, 21 W.R. 38839, 145

W.R. 40070, 145 Wy-4036 (lorazepam), 10, 40 My-12157, 34 Wy-21901 ( i ndorami ne 9 62 Wy-22811, 32 Wy-23205, 210 X-206, 149 x y l az ine (BAY 1470), 60 xylopine, 41 Y-4 153 (3-ch 1oroca r p pramine), yemenimycin, 116 YL-704, 104 yohimboic a c i d hydrazide, 63 2 424 ( d i v i m i n o l ) , 31 zorbomycin, 112 zorbonomycin B, 112 zorbonomycin C, 112

8