Annual Reports Medicinal Chemistry Volume 1

LIST OF CONTRIBUTORS . ..............289 . .................205 . ................... 12 . ................236 . ...

Author: By C.K. Cain

11 downloads 1514 Views 20MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!

Report copyright / DMCA form

DOWNLOAD PDF

LIST OF CONTRIBUTORS

. ..............289 . .................205 . ................... 12 . ................236 . ............... 99 . ................. 99 . .................247 . ................... 30 . ............... 1 . ................. 67 . .................213 ...................247 . ...............136 . .................331 . ..................109 . ..................324 . ................. 40 . .........129 ...................331 . .................331

Anderson. G W Babock. J C Biel. J H Bloom. B M Bolhofer. W A Brodie. D A Buyske. D A Cain. C K Childress. S J Cragoe. E J Diassi. P A Dvornik. D Elslager. E F Finger. K F Flynn. E H Foye. W 0 Harris. L S Herrmann. E C., Jr Higuchi. T Higuchi. W I

. .................... 150 ..................... 277 . ...............191 ............... 78 . ................ 59 . .. .................. 129 ..................213 . ............... 92 ..................... 164 . .................... 51 . ................299 . ................ 224 . ................118 . ............... 314 . ................... 267 . ................. 67 . ................... 85 . ..................311 . ................178 .................... 233

off, D R Horita. A Jorgensen. E C Kennedy. P., Jr Kornfeld. E C Kucera L S Lerner. L J Moreland. W T Pinson. R Poos. G I Schaeffer. H J Scherrer. R A Shepherd. R G Smissman. E E Smith. C G Sprague. J M Tam. R D Taylor. W I Ursprung. J 3 Weiner. M

ANNUAL REPORTS IN MEDICINAL CHEMISTRY, 1965 Sponsored by the Division of Medicinal Chemistry of tbe American Chemical Society Editor-in-Chief: CORNELIUS K, CAIN McNElL LABORATORIES, INC. FORT WASHINGTON, PENNSYLVANIA

SECTION EDITORS JOHN BlEL 0 SYDNEY ARCHER 0 EDWlN FLYNN RICHARD HEINZELMAN 0 IRVING TABACHNICK 0 EDWARD SMISSMAN

@

ACADEMIC PRESS

New York and London 1966

B Y ACADEMIC PRESSI NC. ALL RIGHTS RESERVED. NO PART OF THIS BOOK MAY BE REPRODUCED I N A N Y FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

COPYRIGHT 0 1966,

ACADEMIC PRESS INC. 111 Fifth Avenue, New

York, New York 10003

United Kingdom Edition fiublished by ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House, London W:1

LIBRARY OF

CONGkESS CATALOG CARD

NUMBER: 66-26843

PRINTED I N T H E UNITED STATES OF AMERICA

Preface While excellent reviews in depth covering selected fields of medicinal chemistry a r e available, no publication outlines the c u r r e n t developments and trends in the whole a r e a . The p r e s e n t volume is the f i r s t of a planned annual publication in which m o r e than thirty authors p r e s e n t a c r i t i c a l summary of new and significant contributions appearing in the l i t e r a t u r e of the p a s t year concerning various fields of medicinal chemistry. Its main purpose is to enable r e a d e r s to "catch up" in fields of t h e i r p e r i pheral i n t e r e s t s and, perhaps, to find some different views in fields of their major i n t e r e s t s . The breadth of the subject and limitations of space presented to the authors a very difficult task of selection of m a t e r i a l and condensation of discussion. Recent reviews and leading r e f e r e n c e s a r e cited to guide r e a d e r s to further information if desired. Topics were chosen to achieve reasonable coverage, but a few chapters initially listed had to be postponed to future volumes. Organization of the m a t e r i a l is p r i m a r i l y by pharmacological action, although some chapter titles r e p r e s e n t concepts, chemical c l a s s e s , methods, etc. The manner of presentation v a r i e s , a s is to be expected from the various disciplines of the authors, the nature of the subjects and the lack of a previous volume to s e r v e a s a model.

1 know of no adequate way to e x p r e s s my thanks for the cheerful, enthusiastic and dedicated efforts of a l l who were involved in this undertaking. Especially to be mentioned a r e the section editors and the chapter authors, but s e c r e t a r i e s , proof-readers, a s s i s t a n t s and a l l who supplied m o r a l support and encouragement should not be forgotten. Comments, c r i t i c i s m s , suggestions and, hopefully, p r a i s e will be welcomed by authors and editors.

F o r t Washington, P a . June, 1966

Cornelius K . Cain

V

Section I - CNS Agents Editor: John H. Biel, Aldrich Chemical Co., Milwaukee, Wisconsin Chapter 1. Antipsychotic and Anti-anxiety Agents Scott J. Childress, Wyeth Laboratories, Inc., Radnor, Pennsylvania Most basic biological work concerned with CNS agents that was reported in 1965 falls into the biochemical area. The belief that the antipsychotic drugs function through some interference with adrenergic processes in the brain is widely accepted. Reserpine, for example, is known to deplete the brain of its stores of catecholamines whereas chlorpromazine has a central adrenolytic action. Although the antipsychotic agents can be shown to interfere with the brain amines, the causal relationship between the changes and the behavioral effects is less clear. The mode of action of the anti-anxiety drugs remains unknown.

A symposium on catecholamines held in Milan in 1965 and recently published1 does much to clarify present views of their role in the central nervous system. Other reviews on the biochemical effects of drugs acting on the central nervous system and on the pharmacology of the central nervous system were prepared by Decsi2 and by Bradley.3 More detailed reviews on serotonin4 and y -aminobutyric acid5 appeared. These papers provide excellent background for any fundamental consideration of the CNS drugs. If the brain amines are conceded to play a crucial part in the functioning of the central nervous system, some disorder in the supply, action or disposal of these agents might be responsible for abnormal behavior. ~ellhorn6,for example, has hypothesized that a disturbance in the noradrenalindadrenaline ratio forms a neurophysiological basis for fear and anxiety. The suggestion that toxic substances in the brain might be responsible for schizophrenia is an old one and the search for a faulty metabolic process in schizophrenics has remained a popular pursuit. Bourdillon7 has recently reEmphasized the finding of a "pink spot," probably 3 4-dimethoxyphenethylamine, in the urine of schizophrenics. Work by Ernsd supports the possibility of an abnormal metabolism of dopamine to 3,4-dimethoxyphenethylamine in schizophrenia. He studied the structure-activity relationships of a group of methoxylated phenethylamines in the production of catatonia in cats. He found the presence of a E-methoxy group and the absence of m-hydroxy group to be required with the duration of action determined by the number of methoxy groups in the molecule. Prior treatment with iprpniazid eliminated the requirement for the absence of a m-hydroxy group. The blocking of the aminooxidase resulted in g-methylation by 0-methyltransferase. A behavioral study' of this compound in the dog and cat also indicates some relationship to schizophrenia. Woolley and Gommi'' have detected in the blood of schizophrenics a substance that is synergistic with serotonin in its action on the rat uterus and are studying the possibility of a causal relationship to the mental disorder. An excellent review" on biochemistry and mental function has been published by Kety who has also republished his 1959 review12 criticizing some

2

Sect. 1 - CNS Agents

B i e l , Ed.

of the methodology involved in finding the "needle-in-the-haystack"that might explain the causes of schizophrenia. Six rejoinders from other scientists are included. Most of the compounds cited below were tested by two general methods: 1) the study of animal behavior following drug treatment in experimental models designed to mimic a clinical situation (conflict, avoidance, etc.) and 2) the measurement of classical pharmacological reactions caused or modified by the agent under test (anticonvulsant, antiemetic, etc.). These methods have been collected in a recent Hahnemann symposium volume.13 The testing of the antipsychotic compounds has been reviewed by Janssen, e t &.,I4 who rely strongly upon the production of catalepsy and the antagonism of the emetic effect of apomorphine for predicting the clinical response. In fresh work on test methods, Marriott and Spencer15 have observed that the anti-anxiety agents increase exploratory behavior in inexperienced rats whereas the antipsychotic compounds reduce such activity. The use of a treadmill in approach and avoidance studies of anti-anxiety compounds has been described by Gluckman.16 A criticism of some of the behavioral tests used for evaluating anxiety in animals has been made by Ray.17 The electronencephalographic effects of the psychotropic agents have been reviewed. 18 Methods of clinical study of psychotropic drugs have also been discussed and criticized.l9,zo Phenothiazines and Analogs - Chemical work directed toward modification of the phenothiazine drugs is presently characterized by the preparation of novel tricyclic systems to which the common basic side chains can be attached. Variation of the central ring is most frequent but the peripheral rings are attracting some attention. A group of compounds having a seven-membered central ring has been described by Stille, et a1.21,22 The synthesis of the intermediate lactams was accomplished by ring closure of the appropriate isocyanates.23 The most active of these compounds is the oxazepine (I) which is approximately equipotent with haloperidol in cataleptic and antiapomorphine effects. In a more extensive study of the thiazepine analog (11, clothiapine) an intense antiserotonin effect was measured in the paw-edema test, thus differentiating it from chlorpromazine. It is noteworthy that opening of the piperazine ring as in compound I11 practically eliminates activity.

The Czech group led by Protiva prepared a host of compounds containing seven-membered rings. Two of these having good central depressant effects are IV (octoclothepine) and V.24 Compound IV which has antiserotonin and antihistamine activities as well, is about three times as strong in its central depressant effects as its unchlorinated analog. The compounds were prepared conventionally from the corresponding dibenzothiepinone which, in turn, resulted from ring closure of the requisite 2-phenylthiophenylacetic acid.25 Reports ha e been made on several thioxanthene derivatives: VI (thiothixene), 2& 27 VII (SKF 10812), 28, 29 and VIII (N-7009). 3' Each was indicated to be effective in the treatment of psychotic states. 6iothixene was said to cause only a low incidence of extrapyramidal symptoms.

Chap. 1

Antip sychotic s, Anti -Anxiety Agents

Childres s

3 -

CHC%C%R

XI:

%= - C F 3 j

R10= -(CH

NI

3

II

A

CHCH CH N 2 2

NCH3

u

XI I XI11

\NC%C%OH

v

v

4

Sect. 1 ’

- CNS Agents

Propericiazine (IX)3 1 has been tried in psychopathic patients severe behavioral disorders with fair success and dixyrazine (X)32 found effective in anxiety associated with autonomic disturbances. ~1,33available in Europe, is a homolog of fluphenazine. Although catalepsy it does not have antiemetic properties.

B ’ i e l , .Ed. having has been Compound it produces

Neuroleptic activity has been reported for X d 4 which has a novel ring system. A metabolite of thioridazine has been identified as XIlIand shown to have a much lower neuroleptic potency than its parent.35 Since there was some suggestion of activating properties (suppression of the tetrabenazine syndrome in rabbits, potentiation of serotonin fever and central anticholinergic it was tested clinically as antidepressant agent with negative ~~~~~~~~6 However the activity in schizophrenia seems to be good.

In the continuing study of the metabolism of chlorprmazine, 2-chlorophenothiazine and 2-chlorophenothiazine 5-oxide have now been identified in human urine.37 Butyrophenones - None of the fluorobutyrophenone compounds is at present in use in the United States although they are extensively used in Europe. The recent review by Haase and Janssen is a useful summary of these comp0unds.3~ A n extension from thekr use in ps chiatry to use in surgical procedures for producinf analgesia is underway.3 j40 The appearance of several .clinical studies4 on the psychiatric use of trifluperidol indicates the prospect of its future availability for treatment of schizophrenia. There are some reports of its especial value in paranoid schizophrenia.42

B

Pharmacological and clinical data on compound XI$3 show it to resemble haloperidol except for the addition of an antireserpine effect. A homolog of XIV (XV)44 has also been studied but was found to have troublesome sideeffects. Both of these products are somewhat related to mI45 which, although having an effective antipsychotic action, caused cataracts and disturbances of cholesterol balance. A group of fluorobutyrophenones derived from 4-aminopiperidines has been described.46 The treatment of l-benzyl-4-~-aminopiperidine-4-nitriles with Grignard reagents effected displacement of the cyano group whereas organolithium reagents reacted with the cyano function to afford ketones. Subsequent debenzylation and alkylation gave the corresponding fluorobutyrophenones. These compounds are less active than haloperidol in apomorphine antagonism but are more potent in countering morphine-induced mania in cats. The most potent of these preparations is XVII. A strong neuroleptic of long duration related to the butyrophenones has recently been reported.47 Compound X a I I antagonizes the effect of apomorphine for as long as 100 hours.

Chap. 1

Antipsychotics, Anti -Anxiety Agents

-5

Child re s s

XVIII

Carbamates - A new carbamate, tybamate (XIX)48 closely resembling meprobamate in structure and in activity was marketed during 1965. Its potency is comparable to meprobamate in many tests, yet it has only one-third the potency of meprobamate in the antipentylenetetrazole test. However, in contrast to meprobamate, it antagonises the effect of LSD on the electroencephalogram and has an antiserotonin action. Convulsions are not seen in dogs receiving tybamate upon abrupt withdrawal of the drug, whereas convulsions do result upon withdrawal of meprobamate.49 In the dog tybamate is metabolized by hydroxylation and/or N-dealkylation.50 Hydroxytybamate is the principal metabolite found in the urine. Several clinical studies have appeared demonstrating effectiveness against anxiety. 51,52 One report53 suggested mild stimulant properties, but in a study with alcoholics no separation from placebo could be made.54 A series of meprobamate analogs was prepared containing silicon in place of the quaternary carbon atom.55 Each silicon compound is approximately

equivalent to its carbon analog in the rotarod test and in acute toxicity. monothiol analog (rOr) of meprobamate is also comparable to meprobamate in potency and toxicity.56

A

A chemical review of the carbamates has been written by Adams and Baron57 with some attention to biological activity.

Sect. 1 - CNS Agents

Biel, Ed.

%NOCOC%CC%OCONHC4Hg

I

cH3 XIX

H NOCOCH~CCH~SCONH~ 2

I

CH

3

Benzodiazepines - The introduction of oxazepam in 1965 brought to three the number of 1,4-benzodiazepines comycially available in the United States. ~ particularly the anticonvulsant The animal studies of oxazepam (XXI)' 3 5 8 59 and conflict tests, suggested its use as an anti-anxiety agent and clinical studies60r61 indicated its efficacy for this purpose. Its potency appears to lie between chlordiazepoxide and diazepam. The compound has also been examined as a water-soluble hemisuccinate ester, sodium salt.& In the form of its glucuronide, oxazepam is the principal excretion product in the dog and man of diazep which is also hydroxylated to some extent without N-demethylation. In the circulating serum, the most prominent metabolite of diazepam is the unhydroxylated compound XXIII. There is an indication of the presence of further phenolic metabolites whose precise structures are unknown. The metabolism of chlordiazepoxide in the dog and man results principally in the production of 7-chloro-1,3-dihydro-5-phenyl2g-1,4-benzodiazepin-2-one 4-oxide, but in the rat basic compounds as yet unidentified are produced.65 Some opening of the lactam ring was observed, but no alteration of the aromatic rings or reduction of the N-oxide function was detected. Nitrazepam (XXIV)66,67 has been introduced in Europe and is being promoted as a hypnotic but its activity profile suggests it woul be cffective against anxiety. A clinical report on a related compound (XXV)g8 indicates a resemblance to diazepam. Some additions to the list of benzodiazepinones having functional substituents have been made. French workers succeeded in preparing 3-carboalkoxybenzodiazepinones, e.g. XXVI, 69 by transimidation of the appropriate 2aminobenzophenone imines with a-aminomalonic esters followed by cyclization. The benzodiazepine esters so obtained were converted to amides and also hydrolyzed to salts of the corresponding carboxylic acids. The ease of decarboxylation upon acidification of these salts suggests that the decarboxylation products may be responsible for the high biological activity. Although the esters and amides have typical benzodiazepine profiles they are not so potent as the carboxylic acid salts which compare favorably with diazepam.

Chap. 1

Antipsychotic s, Anti -Anxiety Agents

R

XXI:

R3

-N

9

R5

H

H

CH

H

c6H5 g-FC&

XXVI:

H

C%C%N

C02Et

H

(c@5)2

‘SH5

@ ‘SH5

xxx

02

0

XXXII

c@5

XXV:

c1

‘SH5

H

c6H5

XXIX

K2

c@5

H

‘SH5

c1

R5

OH

H H

XXVII:

XXVIII

3

XXIII: XXIV:

3

I

‘SH5

XXXIII

-7

Child re s s

c6H5 “-FC&

9

c1 c1 c1

N02 C1

c1 C1

8 -

Sect. 1 - CNS Agents

Biel, Ed.

3-Acetamidobenzodiazepines were prepared by cyclization of 2-(2-acetamido2-aminoa~etamido)benzophenones.7~ A novel elimination of acetic acid from the appropriate 2-(N-acetoxyacetamid0)acetamidobenzophenone afforded the intermediate. It was possible to hydrolyze the acetamidobenzodiazepines to the corresponding 3-amino compounds and convert these products into 3-hydroxy compounds by treatment with nitrou acid. The amino and acetamido compounds are merely reported to be active.7

Stempel, et a1.,72 achieved a direct synthesis of a 3-chlorobenzodiazepinone 4-oxide by base treatment of 6-chloro-~-dichloromethyl-4-phenylquinazoline 3-oxide. The course of this ring enlargement, which was responsible for the original discovery of chlordiazepoxide, was clarified by the isolation of an intermediate, ~-dichloroacetamido-5-chlorobenzophenone oxime (ant), which slowly cyclized. Cyclization of the monochloroacetyl analog is too fast to permit its isolation. The 3-chloro substituent of the benzodiazepine product reacted conventionally with nucleophiles following removal of the N-oxide function. A number of benzodiazepinones with functional substituents in the 1position has been published along with test data.73 The general structureactivity requirements already described for the benzodiazepines obtain for the 1-aminoalkyl types. The importance of an 2-fluoro substituent on the 5phenyl ring in increasing the potency of the compounds is clear. Compound XXVII is one of the most potent of the group, but it is slightly less potent than diazepam. The activity of the related 1-aminobenzodiazepinesprepared by use of chloramine was not given.74

Compounds containing a fused naphthalene ring (XXVIII)75 and a fused pyridine ring (xxIX)76 have been disclosed. The former types are inactive and the latter are less active than diazepam. Further examples of compounds related to active benzodiazepines, but for which test data are missing, include XXXR , xw[I,78 XXXII79 and XXXIII.80 Miscellaneous Compounds - Investigations on several compounds that do not fall into the above groups were reported. The pharmacology of trimetozine (XXXIV)81 indicates it to be a tranquillizer without hypnotic or anticonvulsant properties. Compound XXXV82 has sedative properties approximating chlorpromazine but does not produce catalepsy and ataxia. A disruptive effect on conditioned behavior at very low dosage is seen with the adrenolytic compound XXXVr.83 Compound XXXVII84 is less effective than meprobamate against anxiety and it has been shown that poor absorption is not the explanation.85 The tranquillizing potency of XXXVII186 is approximately equivalent to meprobamate. Taborsky, et al.,87 have studied the effect of 1-methylation on a group of psychoactive indoles. In general, the effects on behavior of the methylated and unmethylated pairs are similar. Compound XXXIX, 88 which resembles tetrabenazine in structure, has both stimulant and depressant properties. It blocks conditioned avoidance in rats but the required dose is at least five times that of chlorpromazine. Replacement of the 2-chlorophenyl groups by an alkyl or aralkyl group leads to inactive products. A good clinical response in schizophrenics has been noted. A group of 17-haloyohimbanes89 was examined by observation of the effects on

A n t i p s y c h oti c s , A n t i - A n x i e t y A g e n t s

Chap. 1

Childress

-9

t h e behavior of untamed r h e s u s monkeys. Although 17-CT-bromoyohimbane i s approximately a s p o t e n t a s r e s e r p i n e i n a l t e r i n g behavior i t has u n d e s i r a b l e cardiovascular properties.

XXXVIII

C H -2-Cl 6 4

XXXIX References

1. 2.

3. 4. 5.

6.

7. 8. 9.

10. 11. 12.

13. 14. 15.

16.

G. H. Acheson,

Ed., "Second Symposium on Catecholamines, ' I Pharmacol. 1, P t . 1 (1966). L. Decsi i n E. Jucker, Ed., Progress i n Drug Research, Vol. 8, Birkhauser Verlag, Basel, 1965, p. 53. P.B. Bradley, Ed., "Pharmacology of t h e C e n t r a l Nervous System," Brit. Med. Bull., NO. 1 (1965). s. M r a t t i n i and L. V a l z e l l i , "Serotonin", E l s e v i e r , New York, 1965, p. 199. D.R. C u r t i s and J . C . Watkins, Pharmacol. Rev., E. Gellhorn, P e r s p e c t i v e s Biol. Med., 8, 488 (1 R . E . Bourdillon, C.A. Clarke, A.P. RiGes, P.M. Sheppard, P. Harper and S.A. L e s l i e , Nature, 208 453 (1965). A.M. Ernst, Psychopharmacologia, 383, 391 (1965). Arch. I n t e r n . Pharmacodyn., 12, M.L. Brown, W.J.. Lang and S. 439 (1965). D.W. Woolley and B.W. Gomi, Science, 1 % 670 (1965). S.S. Kety, Nature, 208, 1252 (1965). S.S. Kety, I n t e r n . r P s y c h i a t . , 409 (1965). J . H . Nodine and P.E. S i e g l e r , Eds., "Pharmacologic Techniques i n Drug Evaluation, I' Year Book Medical P u b l i s h e r s , Chicago, 1964. P.A. J. Janssen, C. J . E . Niemegeers, and K.H.L. Schellekens, A r z n e i m i t t e l Forsch., hc' 1 5 104, 1196 (1965); l& 339 (1966). A.S. M a r r i o t t and P.S.J. Spencer, B r i t . J. Pharmacol., 25 432 (1965). wd M . I . Gluckman, Current Therap. Res., 7, 721 (1965).

&., 2, No.

2,

-'

A,

cu,

S e c t . 1 - CNS Agents

10 -

B i e l , Ed.

17. O.S. Ray, Arch. I n t e r n . Pharmacodyn., 1 3 , 49 (1965). 18. 19. 20. 21. 22.

2324. 25. 26. 27. 28. 29. 30.

31.

32-

33-

34. 35.

36. 37. 38

-

39 * 40. 41. 42. 43. 44. 45. 46. 4748. 49. 50.

224 (1965). H.F. Mtfller and A.K. Mtfller, I n t e r n . J. Neuropsychiatry, L.E. H o l l i s t e r and J . E . Overall, J . New Drugs, 286 (1965). M. Ostow, J. New Drugs, 5, 3 (1965). G. S t i l l e , H. Lauener, ET Eichenberger, F. Hunziker and J. Schmutz, 841 (1965). Arzneimittel-Forsch, G. S t i l l e . H. Ackermann, E. Eichenberger and H. Lauener, I n t e r n . J. Neuropharkacol., $, 375- (1965). J. Schmutz, F. K k z l e , F. Hunziker and A . Bcrki, Helv. Chim. Acta, _336 (1965)-. J . O . J i l e k . V. Seidlova, E. Svatek, M. F'rotiva, J. Pomykacek and Z. Sedivy, Mohatsh., 96, 182 (1965). M. P r o t i v a , J.O. J;\iek, J. Metysova, V. Seidlova, I. Jirkovsky, J. Metys, E. Adlerova, I. Ernest, K. Pelz and J. Pomykacek, Farmaco, Ed. Sci., 721 (1965). A.A. Sugerman, H. S t o l b e r g and J. Herrmann, Current Therap. Res., 7, N 310 (1965). G.M. Simpson and J. Iqbal, Current Therap. Res., 7, 697 (1965). D.M. Gallant, M.P. Bishop and W. Shelton, C u r r e n t y h e r a p . Res., 7, h 415 (1965). A.A. Sugerman and F.J. L i c h t i g f e l d , Current Therap. Res., 7, 707 (1965). Y B. Holst, Acta Psychiat. Scand., 40, 415 (1965). D. Turns, H. C. B. Denber and D.N. T e l l e r , J. New Drugs, 5, 90 (1965). T . Fokstuen, I n t e r n . J. Neuropsychiatrp, 1, 294 (1965).* T.M. Itil, Arzneirnittel-Forsch, 2, 817 (y965). Sandoz, Neth. Pat. 6,507,469 (1965). D.M. G a l l a n t , M.P. Bishop and D. Sprehe, Current Therap. R e s . , 7, v 102 (1965). D.M. Gallant, M.P. Bishop and C. S t e e l e , Current Therap. k., 7, N 783 (1965). D . E . Johnson, C.F. Rodriguez and H. P. Burchf i e l d , Biochem. Pharmacol., cvv' 14 1453 (1965). H.J. Haase and P.A.J. Janssen, Eds., "The Action of Neuroleptic Drugs," Year Book Medical Publishers, Chicago, 1965. L. T o r e l l i and F. Schiavi, Aggressologie, 6, 327 (1965). N.W. Shephard, "The A p p l i c a t i o n of Neuro1e;tanalgesia i n Anesthetic and Other P r a c t i c e , Pergamon Press, Oxford, 1965. A.A. Sugerman and B.H. W i l l i a m s , J. New Drugs, 5 318 (1965). L . E . H o l l i s t e r , J . E . Overall, J . L . Bennett, I. 4 K m b e l l , Jr. and J. Shelton, Am. J. Psvchiat., 13, 9 6 (1965). J . A . Christensen, S. Hernestam, J . B . Lassen and N. S t e r n e r , Acts Pharmacol. Toxicol., 23 109 (1965). -' G.M. Simpson, E. Kunz and T.P.S. Watts, Psychopharmacologia, 5 223 (1965). G.M. Simpson, T . Farkas and J . C. Saunders, Psychopharmacologia, 306 ( 1 9 a ) . B. Hermans, P. Van Daele, C . Van De Westeringh, C. Van Der Eycken, J . Boey and P . A . J . Janssen, J . Med. Chem., 5 851 (1965). P.A.J. Janssen, Fr. P a t . 3695M (1965). F.M. Berger, M. Kletzkin and S. Margolin, Med. E x p t l . , fi 327 (1964). S. Margolin, O.J. Plekss and F.M. Berger, Pharmacoloqist, 143 (1965). J.F. Douglas, J. Edelson, A. Schlosser, B. J. Ludwig and F.M. Berger, Federation Proc. No. 1, P t . 1, 489 (1964).

3

9,

45,

a

3

2,

3,

Chap. 1 51. 52. 53 * 54. 55. 56. 57. 58. 59.

Antipsychotics, Anti-Anxiety Agents

11 -

Childress

M. C h i e f f i , Diseases Nervous System, 2 6 369 (1965).

I. Slaughter, J. New Drugs, 177 (lpbs). E . Raab, K. Rickels and E. Moore, Am. J. Psychiat., H.B. Mooney and K.S. Ditman, J. New Drugs, 233 (1 R . J . Fessenden and M.D. Coon, J. Med. Chem., 604 (1965). M.A Leaffer, W.A. Skinner and B . J . Ludwig, J. Med. Chem., 5 208 (1965). P. Adams and F.A. Baron, Chem. Rev., c 5 , 567 (1965). H. Klupp and J. G h l i n g , Arzneimittel-Forsch., 359 (1965). L.O. Randall, C.L. Scheckel and R.F. Banziger, Current Therap. Res., 590 (1965). €3. S c a s s e r r a , Diseases Nervous Sys tem, 2J_6, 511 (1965). 60. 61. W. Janke and K. -D. Stoll, Arzneimittel-Forsch., 15, 366 (1965). 62. S.N. Steen and L.R. Martinez, Anesthesia A n a l g e s g , Current Res., 44, 358 (1965). 63 H.W. -Ruelius, J.M. Lee and H.E. Alburn, Arch. Biochem. Biophys., 1,1-_1, 376 (1965). 64. M.A. Schwartz, B.A. Koechlin, E. Postma, S. Palmer and G. Krol, 2. Phannacol. E x p t l . Therap., lA8, 423 (1965). 65. B.A. Koechlin, M.A. Schwartz, G. Krol and W. Oberhansli, J. Pharmacol. Exptl. Therap., lk8, 399 (1965). 66. L.O. Randall, W. Schallek, C. Scheckel, R . E . Bagdon and J. Rieder, Schweiz. Med. Wochschr., %, 334 (1965). 67- W. F. Borck, Arzneimittel-Forsch., 9, 1155 (1965). 68. H.O. Gerz, Am. J. Psychiat., 121, 495 (1964). 69. Etab. Clin-Byla, Belg. P a t . 6 K 4 0 1 (1965). 70- S . C. Bell, R. J. McCaully and S . J. Childress, Tetrahedron L e t t e r s , 2889 (1965). 71- S. C. Bell, U . S . Pat. 3,198,789 (1965). 4267 (1965). 72- A. Stempel, E. Reeder and L.H. Sternbach, J. Org. Chem., 73. L. H. Sternbach, G.A. Archer, J .V. Earley, R. I. Fryer, E. Reeder, N. Wasilyw, L.O. Randall and R. Banziger, J . Med. Chem., 4, 815 (1965). 74. W. Metlesics, R.F. Tavares and L.H. Sternbach, J. Org. Chem., 30, 1311 (1965). R. L i t t e l l and D.S. Allen, Jr., J. Med. Chem., 5 892 (1965). 75 76. R. L i t t e l l and D . S . Allen, Jr., J. Med. Chem., 5 722 (1965). 77. E. T e s t a and L. Fontanella, Farmaco, Pavia, Ed. Sci., 20, 323 (1965). 78 * P. H.L. Wei, U. S . Pat. 3,185,680 (1965). 79. L.H. Sternbach, H. Lehr, E. Reeder, T . Hayes and N. S t e i g e r , J. Or&. Chem., 0 2812 (1965). icolaus, E. B e l l a s i o , G. Pagani, L. Mariani and E. Testa, 80. B . J . R . a i m . Acta., 1867 (1965). 81. J . - R . B o i s s i e r , P. Simon and J. Fichelle-Pagny, Therapie, 2 3 401 (1965). 82. H. Fujimori and D.P. Cobb, J . Pharmacol. Exptl. Therap., lA8, 151 (1965). V.G. Longo and V. Rosnati, Psychophannacologia, 145 (1965). 83 84. J. Janacek, B . C . Schiele, T.P. B e l l v i l l e , N.D. Vestre and 0. Raths, 2. New Drugs, 5 51 (1965). 85. B.M. P h i l l i p s , C. E. P i l k v i s t and P. J. Kraus, Arch. I n t e r n . Pharmacodyn., 156, 358 (1965). 704 (1965). 86. L.M. Rice and C. H. Grogan, J Med. Chem., 87 * R . G . Taborsky, P. Delvigs, I . H . Page and N. Crawford, J. Med. Chem., fL 460 (1965). 88. C.R. Ganellin and R.G.W. S p i c k e t t , J. Med. Chem., 619 (1965). 89. M. von Strandtmann, G. Bobowski and J. Shavel, Jr., J. Med. Chem., 338 (1965).

3

2,

12,

4.,

-

p

w.

s,

-

3

w1

2,

2,

2,

12 -

Sect. I

- CNS Agents

Biel, Ed.

f

Chapter 2. Antidepressants, Stimulants, Hallucino ens J. H. Biel, Aldrich Chemical Company, Milwaukee, W 8 .

I.

The Antidepressants A. Introduction There were no spectacular break-throughs in the treatment of mental depression durin 1965. However, the development of drugs that displayed a select ve type of antidepressant activity underscored the very complexity of mental depressive illness and afforded a greater understanding of the fact that mental depression is not a single disease entity but requires individualized therapy and the availability of drugs capable of coping with the various facets of this mosaic disease syndrome. Other major developments demonstrated the influence of the antidepressant drugs on catecholamine metabolism, uptake, storage, and intracellular binding. These findings shed new light on the mechanism of action of these drugs pointing to a possible etiology of a breakdown in central chemical homeostasis. Well-controlled clinical investigations brou ht into sharper focus the need for both stimulant and tranquiliz ng antidepressant agents, particularly where depressant symptoms were an overt expression of an underlying psychotic illness which wa exacerbated by the "stimulantT1 type of antidepressant. introduction of monodemethylated imipramine and amitriptyline derivatives was a ste in the direction of achieving greater selectivity of action.g,4,5,6 B. The MA0 Inhibitors 1. New Structures

f

'f

8 , sft;;e

CH3 I $CH2CH(CH3 )N-CH2CECH

ArC O( C H 2)

(IV)

( IVa)

R;R .R -lower '-alkyl groups (VI)

p a H

N-C OR #-R

(V) Ar = phenyl or substituted phenyl R = alkyl, cycloalkyl, heterocyclic amfne

Chap. 2

Stimulants

Biel

13 -

Structures I and I1 are modifications of pargyline (Eutonylm). Knoll et a1.8 d e s c r i b e s t r u c t u r e I1 as being ?'an acute psychostimulant" and ? ? achronic psychic energizer". I t s MA0 i n h i b i t o r y potency i n v i t r o i s s a i d t o be 200 times t h a t of NiamidTM. Unlike pargynn-is agent is a potent c e n t r a l stimulant comparable t o amphetamine i n potency. S t r u c t u r e 111, a phenoxy analog of tranylcypromine w a s o et h i r d a s potent a s the l a t t e r drug and twice a s long-lasting?*g I n r a t s , I11 displayed moderate anorexigenic and increased locomotor a c t i v i t i e s . As an MA0 i n h i b i t o r , compound I V was approximately onehundredth as potent a s pheniprazine (a-methylphenethylhydrazine). As a group, these compounds displayed a v a r i e t y of pharmacologic e f f e c t s : CNS stimulation, tryptamine and nfEotine antagonism The d a t a presented and suppression o f f i g h t i n g mouse behavitvr. do not allow a conclusion concerning t h e i r p o t e n t i a l a s a "lead" i n the a r e a of antidepressants o r CNS s t i m u l a n t s . The s e r i e s is i n t e r e s t i n , however, because of t h e d u a l i s t i c type of psychot r o p i c a c t on ( e x c i t a n t and t r a n q u i l i z i n g ) and bears f u r t h e r watching. Compound V i s another s t r u c t u r e displaying a multitude of pharmacologfc a c t i o n s Both n e u r o l e p t f c and Tfpotenttti n v i t r o MA0 i n h i b i t o r y p r o p e r t i e s a r e ascribed t o t h i s s e r i e s . mec l i n i c a l u t i l i t y of t h i s s t r u c t u r a l group i n mental depression remains i n doubt. Structure i l l u s t r a t e s v a r i a t i o n s on t h e theme of Monasem (etryptam n e ) . Compound V I I has been i n v e s t i g a t e d c l i n i c a l l y by Azirnal3 who found it t o be an e f f e c t i v e a n t i d e p r e s s a n t . The 7-methyl-a-ethyltryptamine ( V I I I ) is s a i d t o be s u p e r i o r a s an MA0 i n h i b i t o r t o etryptamine, both i n v i t r o ( t e n times) and vivo ( t w o t o f o u r t i m e s ) . l 4 The c l s i c a l t i l i t y o f t h i s agent E n t i d e p r e s s a n t therapy has n o t been revealed.

f

B

(VII) MP-809 (VIII) 2 . New Developments on Older MA0 I n h i b i t o r s The hypertensive c r i s e s produced by t r a n lcypromine i n t h e presence of tyramine-rich foods 15915a9 15b, has r e s t r i c t e d the a p p l i c a t i o n of t h i s valuable agent t o h o s p i t a l use. While o t h e r MA0 i n h i b i t o r s evoke s i m i l a r responses i n tyramfnet r e a t e d r a t s , 16 the i n t r i n s i c sympathomimetic a c t i v i t i e s of tranylcypromine presumably enhance i t s potency i n t h i s regard .I7 E f f o r t s t o overcome t h e p e r i p h e r a l s i d e e f f e c t s of t h e MA0 i n h i b i t o r s have taken t w o d i r e c t i o n s : (1) Masking o f t h e f r e e amino group of tranylcypromine by a s u i t a b l e a c y l radical18, 19

Sect. I

14 -

- CNS Agents

B ' i e l , Ed.

(structures I X and X I which would not impede the d r u g ' s passage

( 1x1

(XI across the blood-brain b a r r i e r nor form so s t a b l e an amide linkage as t o preclude enzymatic cleavage t o the f r e e d n e a t the t a r g e t s i t e . ( 2 ) Blockade of the e r i heral c e l l receptors of MA0 by a reversible MA0 i n h i b i t o r w t h greater receptor a f f i n i t y than the therapeutically active MA0 inhibitor and an i n a b i l i t y t o penetrate the blood-brain barrier. I n t h i s way, the therapeutic MA0 i n h i b i t o r Will be forced i n t o the c e n t r a l nervous system r metabolized t o an e a s i l y excretable metabolite. HoritaS8 has succeeded i n demonstrating the u t i l i t y of t h i s concept. Wemedication of the r a t with a reversible MA0 i n h i b i t o r , BW 392C60 ( X I ) , followed by treatment with pheniprazine blocked brain MA0 completely, but maintained h i h MA0 a c t i v i t y i n the l i v e r and other peripheral organs. Phen prazine and pargyline could be e f f e c t i v e l y antagonized, but n o t tranylcypromine o r iproniazid. Compound MO 1255 ( X I 1 1 has been claimed t o be an acgfve antidepressant devoid of cardiovascular side effects. NCH3

?

f

O-CI~~H~NH!!-NHCH~

( X I ) BW 392C60 ( X I I ) MO 1255 (encyprate) neTM (2-piperidino-3-methylpyrazine) has been shown by Rider t o produce g a s t r i c antisecretory e f f e c t s comparable t o atropine. The drug has properties common t o both MA0 inhibit o r s and the imipramine-type agents. A s an MA0 inhibitor, it is f o u r t o s i x t b e s as potent as phenelzine, but unlike the l a t t e r , produces r a t h e r severe o r t h o s t a t i c hypotension i n man.22a 3. Mechanism of Antidepressant Action of MA0 Inhibitors A t the present s t a t e of our knowledge, the mechanism of the antidepressant action of the MA0 i n h i b i t o r s i s thou h t t o be intimately t i e d t o brain catecholamine levels. Sch ldkraut 23 has summarized the evidence favoring the involvement of catecholamine In transmitting the e f f e c t s of the MA0 inhibitors. Spector 28 showed t h a t during selective depletion of norepinephrine and dopamine, the MA0 inhibitor, pargyline, was unable t o reverse reserpine o r tetrabenazine depression u n t i l brain catecholamine levels had stached 50% of pre-dru levels. Recently, Ingvarsson has reported dramat c remissions of long-standing drug r e s i s t a n t depressions following the administ r a t i o n of 50 mg. of DOPA every other day. Marked improvement

a

s9, !da

M

f

f

Chap. 2

Stimulants

Biel

15 -

was seen within a few hours. P a t i e n t s a l s o a f f l i c t e d with asthma and Parkinsonism experienced complete r e l i e f of t h e i r symptoms from the DOPA treatment. Relapse occurred on discontinuance of DOPA therapy. The i n h i b i t i o n of NE biosynthesis by blocking t h e r a t e l i m i t i n g s t e p with a-methyltyrosine r e s u l t e d i airment of motor a c t i v € t y and s e d a t i o n i n animals and man Clinical improvement i n t h e depres9Bd a t i e n t c o r r e l a t e d w e l l with t h e degree of MA0 i n h i b i t i o n . 9 28 Recently, Pletscher30 has presented evidence t h a t the MA0 i n h i b i t o r s may block the uptake of r e l e a s e d NE i n t o s t o r a g e granules, thereby producing an increased concentration of NE around t h e adrenergic synapses. 4. Conclusions a. P o t e n t , i r r e v e r s i b l e i n h i b i t i o n of b r a i n MA0 i s a primary p r e - r e q u i s i t e f o r e f f e c t i v e a n t i d e p r e s s a n t therapy. b. The c l o s e temporal r e l a t i o n s h i p between t h e onset of MA0 i n h i b i t i o n and a n t i d e p r e s s a n t e f f e c t s lends f u r t h e r support t o t h e hypothesis t h a t "free" NE, p r o t e c t e d from o x i d a t i v e metabolism, may be implicated i n mediating t h e a n t i depressant e f f e c t s of the MA0 i n h i b i t o r s . c. C l i n i c a l l y , t h e MA0 i n h i b i t o r s e x e r t optimum a n t i d e p r e s s a n t e f f e c t s i n r e a c t i v e and n e u r o t i c depressions. Activated, psychotic o r endogenous depressions were l e s s s u s c e p t i b l e t o MA0 i n h i b i t o r y therapy.25731 d. The "False Neurochemical Transmitter" theory promulgated by Kopin e t a1.32 t o e x p l a i n t h e sympathetic blocking p r o p e r t i e s of t h e MA0 i n h i b i t o r s , must be s e r i o u s l y considered a l s o w i t h r e s p e c t t o the c e n t r a l p r o p e r t i e s of t h e s e agents. I n essence, t h i s hypothesis proposes t h a t MA0 i n h i b i t i o n w i l l produce an accumulation of sympathomimetic m e t a b o l i t e s which a r e normally n o t present i n t h e body, but have s u f f i c i e n t a f f i n i t y f o r t h e adrenergic r e c e p t o r s i t e s t o d i s p l a c e the r e g u l a r l y p r e s e n t n e u r o t r a n s m i t t e r s (e.g., norepinephrine, dopamine) from sympathetic nerve endings. Furthermore, sympathetic nerve s t i m u l a t i o n w i l l r e l e a s e these agents i n t h e same manner a s it does NE; however, the r e s u l t a n t e f f e c t w i l l be g r e a t l y d i l u t e d , since t h e f a l s e n e u r o t r a n s m i t t e r s have d i s t i n c t l y weaker adrenergic p r o p e r t i e s than NE o r dopamine. Normally, tyramfne is r a p i d l y metabolized by MA0 so t h a t l i t t l e , i f any, octopamine is formed. I n t h e presence of MA0 i n h i b i t o r s , however, s i g n i f i c a n t amounts of octopamine a r e produced.

Ss:B -

H

O

D 2H4NH2

(XIII) Tyramine

H O o C H ( OH )CH2NH2

(XIV) Octo m i n e 0-1

transmitter

5. Review A r t i c l e s t a i l e d information regarding ad For f u r t h s i d e e f f e c t s , 45, comparative c l i n i c a l e f f i c a c y Y$:J!,

!l%

al sb,iysc and

Sect. I - CNS Agents

16 -

B i e l , Ed.

p o s s i b l e mechanism of a c t i o n 3 8 7 3 9 9 4 0 of t h e MA0 i n h i b i t o r s , t h e r e a d e r i s r e f e r r e d t o the c i t e d review a r t i c l e s . S t r u c t u r e- ac t i v i t y , biochemical , pharmacological and c 1i n i c a l pharmacological d a t a on both hydrazine 39, 40 and non-hydrazine MA0 i n h i b i t o r s 41 have been covered extensively i n the respect i v e references. S c h i l d k r a u t ' s paper 38 i s of s p e c i a l i n t e r e s t , s i n c e it provides much of t h e supporting evidence f o r the "Catecholamine Theory" of the antidepressant drug a c t i o n . C. The Thymoleptic Agents ( T r i c y c l i c Antidepressant Drugs) 1. Introduction Listed below a r e t h e s t r u c t u r e s of the t r i c y c l i c antidepress a n t s which a r e e i t h e r commercially a v a i l a b l e o r In advanced

(XV)

I m i ramine

R=CH3: R=H :

h

l ~

(XVI) R=CH3: Amitriptgline R=H : Nortrm

N / e I CH2CH(CH3)CH2N(CH3>2

i

N

(XVII 1

Protriptyline

/

(h,3 N 3 1 C $ € 4 0 H

(XVIII) (XIXI Trimipramine Opipramol Kuhn's discovery42 of t h e antidepressant e f f e c t s of imipramine c o n s t i t u t e d a major break-through i n t h e treatment of mental depression. A e x c e l l e n t and comprehensive review by H'etfliger and BurckhardtQ3 covers t h e chemistry, pharmacology, and c l i n i c a l e f f e c t s of t h i s drug group u n t i l 1963. 2. Evidence f o r Enhancement of Adrenergic Responses by t h e Thymoleptic Drugs The o r i g i n a l pharmacolo i c spectrum of imipramine ave no h i n t of i t s p o t e n t i a l a c t i v t y a s a novel c l i n i c a l a n t depressant; rath the drug behaved l i k e a mild t r a n q u i l i z e r i n animals. S i gEg7w a s t h e f i r s t t o d i s c e r n pharmacologic d i f f e r e n c e s between i m pramine and chlorpromazine on t h e b a s i s of enhancement of c e r t a i n adrenergic responses t o exogenously administered NE by t h e former drug. Sigg ascribed t h i s a c t i o n of the drug t o a I * s e n s i t i z a t i o n of EQe adrenergic r e c e p t o r s Kaumann e t a l . demonstrated an increase i n c a r d i a c r a t e and f o r c e of c o n t r a c t i o n w i t h desipramine which would be blocked by D C I . U r s i l l o and Jacobsen46 working with the i s o l a t e d

f

f

f

Stimulants

Chap. 2

17 -

Biel

v a s deferens achieved marked q u a n t i t a t i v e d i f f e r e n c e s between m x o r and major a n t i d e p r e s s a n t s . Thus, desipramine had 50 100 times t h e potency of methylphenidate. Amitriptyline, chlorprothixene, chlorpromazine produced only i n h i b i t i o n of NE i n t h i s t e s t . Catecholami -induced h erthermia i n r a t s was p o t e n t i a t e d by imipramine.gq Imipram+a-n ne an e s pramhe, i n small doses, enhanced amphetamine stimulant and hyperthermic e f f e c t s . Opipramol produced only a s l i g h t p o t e n t i a t i o n and c l i n i c a l l y , t h i s drug i s l e s s of an a n t i d e p r e s s a n t than an a n x i o l y t i c agent. 3 . Mechanism of NE P o t e n t i a t i o n by t h e Thymoleptic Agents One of t h e major r o u t e s of i n a c t i v a t i o n of f r e s h l y r e l e a s e d (from c e l l u l a r binding s i t e s ) NE i s re-uptake by t h e cell.48a Glowinski and Axelrod 49 devised a technique capable of d e t e r mining b r a i n l e v e l s of exogenously administered (by i n t r a v e n t r i c u l a r i n j e c t i o n ) t r i t i a t e d NE. The exogenous NE i s taken up and r e t a i n e d by the sympathetic nerve endings i n the brain and behaves biochemically l i k e the endogenous neurotransmitter. These i n v e s t i g a t o r s were a b l e t o show t h a t only c l i n i c a l l a c t i v e antidepressant drugs ( h i p r a m i n e , desipramine an am tr p t y m were capable of reducing t h e uptake of t r i t i a t e d NE i n t h e r a t brain. A c l o s e s t r u c t u r a l analog of imipramine, i n a c t i v e a s an antidepressant, was devoid of any a c t i v i t y on NE uptake. The authors suggest t h a t t h e a b i l i t y of c l i n i c a l l y a c t i v e antidepress a n t s t o block t h e re-uptake of f r e s h l y r e l e a s e d "free" ( a c t i v e ) NE by t h e c e r e b r a l t i s s u e s may accoun f o r t h e mechanism of t h e i r antidepressant a c t i o n . Iversent0 working with the i s o l a t e d r a t h e a r t , found desipramine t o be 100 times more potent a s an i n h i b i t o r of NE uptake than chlorpromazine. I n the i s o l a t e d perfused c a t spleen, Thoenen e t &.51 showed t h a t NE output r e s u l t i n g from sympathetic s t f m u l a a o n was increased and t h e i n a c t i v a t i o n of exogenously administered NE delayed i n t h e presence of prev ously administered imipramine o r p r o t r f p t y l i n e . Haefely e t a l . 53 concluded from t h e i r s t u d i e s w i t h t h e , a c t i v e thymoleptic drugs, t h a t such a n t i d e p r e s s a n t s exerted a t l e a s t t h r e e types of a c t i v i t y a t t h e p e r i p h e r a l adrenergfc synapses: (1) a sympathomimetic e f f e c t , presumably due t o r e l e a s e of a c t i v e NE from i t s binding s i t e s , ( 2 ) i n h i b i t i o n of re-uptake of "E by the storage s i t e s of sympathetic nerve endings, and (3) a sympathicolytic or noradrenolytic e f f e c t i n higher doses. The desmethyl d e r i v a t i v e s were uniformly more potent i n i n n s i f y i n g t h e sympathetic e f f e c t s of neuronally r e l e a s e d NE. f

-

4

f

I n t e r a c t i o n of Thymoleptics with Reserpine, Tctrabenazine, o r Benzoquinolizlnes The thymoleptfc agents respond s i m i l a r l y t o t h e bove t h r e e catecholamine "releasing" drugs. S u l s e r and SorokoS3 have shown t h a t the r e v e r s a l of r e s e r p i n e sedation depends on the a v a i l a b i l i t y of NE s t o r e s and t h e r a t e of r e l e a s e of NE from i t s binding 4.

18

Sect. I

-

CNS Agents

B i e l , Ed.

s i t e s . I n animals, s e l e c t i v e l y depleted of t h e i r NE by a - M , the thymoleptic drugs were unable t o r e v e r s e the r e s e r p i n e induced depression. There was a d e f i n i t e temporal r e l a t i o n s h i p between b r a i n l e v e l s of desipramine and r e v e r s a l of the s e d a t i v e response of the benzoquinolizines .54 Hence, t h e authors concluded t h a t t h e r e v e r s a l of r e s e r p i n e s t u p o r by t h e thymolept i c drugs is dependent on r a p i d and copious r e l e a s e of b r a i n catecholamines. 5. A n t i c h o l i n e r g i c E f f e c t s of Thymoleptic Drugs An a l t e r n a t e o r a d d i t i o n a l mechanism of a n t i d e p r e s s a n t drug a c t i o n which would implicate blockade of c e n t r a l cholfgergic responses had been proposed o r i g i n a l l y by B i e l et a l . as imipramine and desipramEe have been Thymoleptic drugs , shown by S u l s e r e t :??6 t o antagonize a l l t h e c e n t r a l parasympathomimetic e f f e c t s of r e s e r p i n e (increased s a l i v a t i o n , muscular r i g i d i t y , hunchback posture and blepharospasms), even i n catecholamine-depleted animals. The c e n t r a l a n t i c h o l i n e r g i c a c t i v i t y of oxygen i s o s t e r e s o g 7 a m i t r i p t y l i n e and n o r t r i p t y l i n e <xX and XXI) w a s demonstrated . by t h e i r antagonism t o t h e c h o l i n e s t e r a s e i n h i b i t o r , d i e t h y l p-nitrophenyl phosphate (Paraoxon) and emetic e f f e c t i n pigeons. Increased p e r i p h e r a l a n t i c h o l i n e r g i c e f f e c t s were displayed by t h e c e n t r a l l y more potent diastereoisomer (B) of s t r u c t u r e X X I I . Diastereofsomer (A) w a s considerabfg l e s s a c t i v e a s an a n t i c h o l i n e r g i c and c e n t r a l stimulant.

Monro e t &.” i n v e s t i g a t e d a s e r i e s of t r i c y c l i c a n t i d e p r e s s a n t a r u g s and found a p o s i t i v e c o r r e l a t i o n between potent CNS p r o p e r t i e s and a n t c h o l i n e r g i c potency i n animals. Giarman and Pepau6& demonstrated t h a t only c e n t r a l l y a c t i v e (animals and man) b a s i c g l y c o l a t e e s t e r s caused a reduction i n b r a i n a c e t y l c h o l i n e (AcCh) l e v e l s of t h e r a t . The authors suggested as a l i k e l y mechanism, i n t e r f e r e n c e of the psychotropic g l y c o l a t e s with s t o r a g e of b r a i n acetylchoF2ne r e s u l t i n g i n reduced uptake of newly synthesized AcGh. Conclusion. On t h e basis of p r e s e n t l y a v a i l a b l e experimental evidence, one would have t o conclude t h a t t h e mechanism of a c t i o n of t h e t r i c y c l i c a n t i d e p r e s s a n t drugs may be due (1) t o an i n t e r ference with c e l l u l a r binding of b r a i n catecholamines, presumably NET thereby producing increased concentrations of catecholamines a t t h e c e n t r a l adrenergic synapses, and (2) a lowering of b r a i n AcCh l e v e l s which would tend t o enhance the o v e r a l l antidepressant effect.

Chap. 2

Stimulants

Biel

19 -

D. Newer Antidepressant Drugs Two drugs which represent structural departures from the currently marketed antidepressants are shown below (XXII1,XXIV):

QcsJ

2H4NMe2

8;

(XXIV) IN 1060

Compound XXIII displayed activity only in "septat;;; rats by suppressing the rage reaction and hyperirritability. Unlike other antidepressants, XXIII did not produce a reserpine reversal. In man, prelim nary data i n E a t e it to be a rapidly acting antidepressant drug. 62 As to compound IN 1060, Sletten et ,1.62 concluded a that the pharmacologic profile of this compous resembles that of imipramine with respect to (1) activation of psychotic processes, ( 2 ) increase in spontaneous motor activity in mice, (3) reversal of reserpine-induced ptos.is and depression in rats, (4) antagoni s m to Ditran-induced behavior in dogs,and ( 5 ) potentiation of epinephrine and NE pressor response of systolic blood pressure in man. E. The Significance of Imipramfne Metabolites K ~ h has n ~ tried ~ to correlate the urinary excretion of imipramine metabolites with clinical improvement. Imipramine Metabolites

(XXVII) desipramine n

(CH ) 3-NHCH3

(XXVIII) While a strict correlation proved difficult, clinical improvement normally became evldent following the maximum urinary excretion of metabolite XXVIII which usually occurred between the sixth and fifteenth day of therapy and was preceded by accumulation of

20 -

Sect. 1

-

CNS Agents

B , i e l , Ed.

metabolite X X V I I , desipramine. Kuhn suggests t h a t t h e formation of metabolite XXVIII i s dependent on t h e presence of s i g n i f i c a n t q u a n t i t i e s of X X V I I whose formation may be t h e r a t e - l i m i t i n g s t e p i n generating metabolite X X V I I I . The p a r a l l e l i s m i n t h e temporal r e l a t i o n s h i p between the o n s e t of t h e a n t i d e p r e s s a n t e f f e c t following imipramine and t h e appearance of maximal amounts of XXVIII i n the u r i n e would e x p l a i n gbe more r a p i d on t of a c t i o n S u l s e r et al.3g have shown (two t o f o u r days) of desipramine. t h a t animal s p e c i e s which a r e unable t o convert s i p r a m i n e t o desipramine a r e a l s o incapable of counteracting r e s e r p i n e o r benzoquinolizine-induced s e d a t i o n w i t h imipramine. F . C l i n i c a l P r o p e r t i e s of Antidepressant Drugs A number of review papers have appeared concerning t h e c l i n i c a l pharmacologic23 1 i 1 r o e i e s of t h e MA0 i n h i b i t o r y and thymoleptic agents. and6g, gy, 68,68,% Ayd has published a compilation of h i s c l i n i c a l experiences w i t h a m i t r i p t y l i n e during a Fix-year p e r i o d . Space permits only a summary of t h e general conclusions which have been reached by t h e s e authors: 1. Mental depression i s a multifaceted d i s e a s e syndrome w i t h varying types of e t i o l o g y . I t may be primary o r secondary ( i . e . , r e s u l t i n g from o t h e r mental disturbances such a s severe a n x i e t y o r an underlying psychosis). It may be r e a c t i v e ( i . e . , i n response t o an environmental s t r e s s ) o r a r i s i n g from within the p a t i e n t due t o a I t i s o f t e n s u b j e c t t o a spontaneous remission. make-up). 2 . W i t h r e s p e c t t o t h e comparative e f f i c a c i e s of t h e a n t i d e p r e s s a n t s , t h e r e c e n t review paper by Wechsler 5 al.69 i s probably most p e r t i n e n t . The a u t h o r s ' conclusion may-6e summarized as follows: a. V a r i a b i l i t Reported e f f e c t i v e n e s s f o r h i p r a m i n e , amit-EST, i p r o n i a z i d , phenelzine, i s o c a r boxazid and nialamide o f t e n ranged from 0 t o 100%. Amitriptyline had t h e l e a s t v a r i a b i l i t y (32 t o 79%). b. Improvement Rate - Average improvement r a t e f o r p a t i e n t s on imipramine, a m i t r i p t y l i n e , and isocarboxazid was 65%, whereas the range f o r i p r o n i a z i d , phenelzine, and nialamide was 40 t o 49%. ECT ranked t h e highest w i t h 72% improvement i n depressive symptoms and placebo therapy the lowest (23%). c . R e l a t i v e Chronicity of Depression Depressions of r e c e n t o r i g i n afforded a much higher improvement r a t e than chronic depressions. For phenelzine, the r a t e decreased from 56 t o 8%, and f o r imipramine and a m i t r i p t y l i n e from 69 t o 45% and 66 t o 32%, r e s p e c t i v e l y . 3. Anergic ( l e t h a r g i c ) depressions responded best t o the "stimulanttt type of a n t i d e p r e s s a n t (imipramine, desipramine, p r o t r i p t y l i n e ) , whereas a g i t a t e d o r anxiety depressions improved most with t h e "sedativet7 type of a n t i d e p r e s s a n t drug ( a m i t r i p t y l i n e , n o r t r l p t y l i n e ) . A l l t h e a n t i d e p r e s s a n t s exacerbated psychotic symptoms; however, a m i t r i p t y l i n e , having a t h e r a p e u t i c spectrum i n between chlorpromazine and imipramine, was t h e s m a l l e s t offender i n t h i s regard. The r e c e n t combination ( T r i a v i l m , E t r a f o n m ) of a potent a n t i d e p r e s s a n t ( a m i t r i p t y l i n e )

kt,

-

-

Chap. 2

Stimulants

Biel

21 -

with a p o t e n t t r a n q u i l i z e r (perphenazine, T r i l a f o n m ) r e p r e s e n t s an attempt t o d e a l more e f f e c t i v e l y with d e p r e s s i o n s accompanied by a n x i e t y , a g i t a t i o n o r schizophrenic s y p t o m a t o l o g y . The p r e l i m i n a r y r e p o r t s appear f a v o r a b l e . 72, 3 ~ 7 4 4. The desmeth 1 d e r i v a t i v e s o f hi ramine (desipramine, , Norpraminm, P e r d ) and a m i t r i p t yl-iptyline A v e n t y l m ) provided a f a s t e r o n s e t of a c t i o n (two t o f o u r days) and a l e s s e n e i n t e n s i t y of parasympatholytic and a d r e n o l y t i c s i d e e f f e c t s . 9 5 , 76977 N o r t r i p t y l i n e proved t o be a valuable adjunct i n t h e treatment of g a s t r o - i n t e s t i n a l disturbances.78 G . C l i n i c a l P r o p e r t i e s of Some Newer Antidepressant Agents More r a d i c a l s t r u c t u r a l d e p a r t u r e s from t h e imipramine and a m i t r i p t g l i n e - t y p e of a n t i d e p r e s s a n t agent a r e shown below: Newer Antidepressant Structures YH2CH2N(CH3l2

(XXX) ID-22

CH3

( X X X I I I ~P-4599

(=I)

(N

HF 1927 o G r m )

(XXXII) SKF 10810

(XXXV) IN 1060

Preliminary c l i n i c a l d a t a i n d i c a t e t h a t ID-22 is a s a t i s f a c t o r y a n t i d e p r e s s a n t endowed w i t h both s t i m u l a n t and t r a n q u i l i z i n g p r o p e r t i e s (dependin on t h e p a t i e n t ) ; s i d e e f f e c t s included o r t h o s t a t i c hypote f o n , dry mouth, c o n f u s i o n a l symptoms, and nocturnal anxiety Compound HF-1927 w a s e f f e c t i v e i n t h e treatment o f i n h i b i t e d and a g i t a t e d endogenous d e p r e s s i o n s ; t h e incidence of psychotomimetic and c a r d i o v a s c u l a r s i d e e f f e c t s w a s high. 807817 82 Freeman e t al.83 have described t h e r a p i d ( t h r e e days) o n s e t of thiazesim, n t e r two weeks' t r e a t m e n t with t h i s agent, 70% of t h e p a t i e n t s could be discharged. Unfortunately, t h e d i s char e r a t e w a s q u i t e comparable i n t h e placebo-treated group. The mipramine-like p r o p e r t i e s of I N 1060 i n animals and man In preliminary c l i n i c a l have been discussed i n S e c t i o n I - D . s t u d i e s , t h e drug e x e r t e d b e n e f i c i a l e f f e c t s i n a n e r g i c schizophrenics and a s m a l l number of depressed p a t i e n t s .84

.

f

22 -

Sect. I

-

CNS Agents

B i e l , Ed.

H. The Anticholinergic Antidepressants The clinical psychotomimetic and Rytidepressant properties A more recent study by of Ditran have been reviewed by Biel. Davis et a1.86 found this drug a "worthwhile, safe, and effective" antidepressant when tested in 78 patients over a period of two and one half years. Improvement became apparent after fi.ve to six treatments spaced two to three days apart. Tachyphylaxis to the psychotomimetic effects were quite pronounced in all patients. Prolonged remissions of illness appeared possible. Reactive, psychotic, and schizo-affective depressions responded best to Ditran, whereas schizophrenic and psychoneurotic depressions proved quite resistant to this type of therap In a well-controlled study by Fink et a1.87 the addition of an anticho1inergic-antiparkinsonism agent, procyclidine, to chlorpromazine produced an antidepressant effect comparable to imipramine . Centrally active anticholinergic agents, either alone or in combination with a tranquilizer, de$erve further evaluation as potential antidepressant drugs. Final Conclusions The principal developments in the field of antidepressant drugs during 1965 may be summed up as follows: 1. An increased understanding of the possible mechanisms of antidepressant drug action was gained through the study of their effects on central adrenergic and cholinergic neurotransmitters. 2. The availability of several drugs with differential types of useful antidepressant activity served to demonstrate that "mental depression'' is a "catch-allf7phrase which encompasses several disease syndromes of varying etiologies and requiring antidepressant drugs with either stimulant, tranquilizing, or antipsychotic properties. 3 . Some progress was made toward achieving a faster onset (two to four days) of action and a lessening of side effects with the desmethyl derivatives of active thymoleptics. 4. Advances in methodology of animal testing for antidepressant activity revealed definite qualitative differences between the antidepressant and tranquilizing agents. 5. On the basis of the mechanism of action of various effective antidepressant drugs, one could conceive of a biochemical etiology for mental depressive illness founded on the inability of the central nervous system to (a) elaborate sufficient amounts of neurotransmitter substance (possibly norepinephrine) (b) release adequate quantities of "active" neurotransmitters from cellular binding sites to the adrenergic synapses or (c) prevent a too rapid metabolic destruction of the critical neurotransmitter substance(s). 6. Clinical evaluation of antidepressant drugs still poses a major problem due to the complex nature of the disease, the hi h incidence of favorable placebo responses and spontaneous remiss ons.

f

Chap. 2

Stimulants

Biel

23 -

C e n t r a l Stimulants The c e n t r a l mechanism of a c t i o n of amphetamine s t i l l remains i n d i s p u t e . Since c e n t r a l catecholamine d e p l e t o r s ( r e s e r p i n e , a - W ) f a i l e d t o block amphetamine-induced motor s t i m u l a t i o n , f e l t t h a t amphetamine a c t s i r e c t l y on t h e CNS. s e v a r ~ 8 8 ~ ~ ~ $ o bO ~na t8h e~ ~ o t~ h e~r dhand, Stein8$ p o i n t s o u t t h a t amphetamine's f a c i l i t a t i n g e f f e c t on i n t r a c r a n i a l s e l f stimulation i n rats blocked by r e s e r p i n e and p o t e n t i a t e d by MA0 i n h i b i t o r s . Hence, he concluded t h a t t h e c e n t r a l stimulant e f f e c t s of amphetamine were mediated by catecholamine r e l e a s e . A novel mechanism h a s now been proposed by Weissman e t al.89a which is based on t h e experimental f i n d i n g t h a t t y r o s i n e T y c o x y l a s e i n h i b i t o r s ( e . g . , a-methyltyrosine) which block t h e biosynt h e s i s of dopamine and NE, a l s o i n h i b i t a l l t h e CNS e f f e c t s e x e r t e d by a m hetamine and amphetamine-like drugs (methamphetamine, Hence, t h e authors conclude t h a t t h e c e n t r a l phenmetrazine? responses induced by amphetamine r e q u i r e a ' ' c r i t i c a l l e v e l of NE a t t h e receptor" and " t h a t t h i s l e v e l d e r i v e s from a f u n c t i o n a l pool of norepinephrine i n t h e CNS h i g h l y s u s c e p t i b l e t o blockade of norepinephrine b i o s y n t h e s i s a t t h e t y r o s i n e hydroxylase s t e which is t h e r a t e - l i m i t i n g s t e p i n catecholamine b i o s y n t h e s i s . %b On t h e o t h e r hand, a-MMT which is a p o t e n t catecholamine-depleting agent, but does n o t i n t e r f e r e with NE b i o s y n t h e s i s , f a i l s t o antagonize t h e c e n t r a l e f f e c t s of amphetamine. I n essence, amphetamine may r e q u i r e t h e presence of f r e s h l y synthesized, r e a d i l y a v a i l a b l e NE t o produce i t s c h a r a c t e r i s t i c CNS e f f e c t s . C l i n i c a l s t u d i e s on a novel sympathomimetic ketone (F-1983) revealed t h a t t h i s substance was capable of i n c r e a s i n g d r i v e and spontaneous a c t h z i t y i n a group of c h r o n i c a l l y depressed and psychotic p a t i e n t s . 11.

=

.

CH3

yHC2H4CH3

("7

(XXXVI) F-1983 C e n t r a l s t i m u l a n t and a n t i t r e m o r i n e e f f e t s were d i s p l a y e d i n animals by a diphenylthiocarbamate (XXXVII). 81 A mild c e n t r a l stimulant (XXXVIII) was shown by Glasky and Simong2 romo t e ~ ~ ' t h e b i o s y n t h e s i s of RNA i n r a t b r a i n and by P l ~ t n i k o ~t o f a c i l i t a t e l e a r n i n g by i n c r e a s i n g memory and r e t e n t i o n of l e a r n e d behavior. This p r o p e r t y was n o t shared by o t h e r c e n t r a l s t i m u l a n t s . jd2NC OSC2H4NEt2

(XXXVII) (XXXVIII 1 (XXXIX) A s t r u c t u r a l l y s i m i l a r cgtpound (XXXIX) has been i n v e s t i g a t e d by G r e e n b l a t t and Osterberg. The pharmacologic spectrum of XXXIX l i e s between amphetamine and imipramine. The compound is

Sect. I

24 -

-

B i e l , Ed.

CNS Agents

a c e n t r a l e x c i t a n t with anorexigenic p r o p e r t i e s . The l a t t e r a r e more pronounced and longer l a s t i n g than those of amphetamine. Tolerance did n o t develop t o e i t h e r e f f e c t . The drug displayed only minimal cardiovascular e f f e c t s and w a s devoid of a n a l e p t i c p r o p e r t i e s . Like imipramine, it delayed t h e onset of r e s e r p i n e depression and i n h i b i t e d tetrabenazine depression. I t protected mice from maximum s e i z u r e s and prolonged b a r b i t u r a t e hypnosis. The Hallucinogens Most d r u s capable of p e n e t r a t i n g t h e CNS have s t r u c t u r a l r e l a t i v e s wh ch w i l l produce psychotomimetic e f f e c t s . T h i s a p p l i e s t o the a n a l g e t i c , antidepressant, a n t i c h o l i n e r g i c , a n e s t h e t i c , sympathomimetic, serotonergic and t r a n q u i l i z i n g drugs. It i l l u s t r a t e s perhaps more than anything e l s e the f i n e l i n e which we a r e s t r a d d l i n g between normal and pathologic emotional behavior and underscores t h e s e n s i t i v e chemical balance necessary f o r t h e maintenance of c e n t r a l homeostasis and presumably ttnormallt behavior. For back round materi reader is r e f e r r e d t o t h e following r e v ew a r t i c l e s . 9gy7 A m t comprehensive review on LSD has been published by Hoffer The s o c i a l and t h e r a p e u t i c fmplications conGQrning the use of LSD2 have been discussed by Cole an Savage and S t o l a r o f f , 200 and McGlothlin and S. Cohen. f 0 f a t z , 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 with regard t o the c e n t r a l stimulant and psychotomimetic p r o p e r t i e s of the basic g l y c o l a t e e s t e r have been presented by B i e l e t a1.102 and Abood and B i e l . 803 A . Analgetics Psychotomimetic p r o p e r t i e s have been reported f o r dexoxadrol Io4 and cyclazocine 05 i n man. In animals, both drugs were devoid of a n a l g e t i c a c t i v i t y but displayed morphine antagonism. I1I .

f

‘si

.!%;

.88

(XL) Dexoxadrol

( X L I ) Cyclazocine

Anesthetics A s an a n e s t h e t i c , phencyclidine (SernylTM) is q u i t e unique from t h e standpoint of high potency, lack of r e s p i r a t o r y and circulafBgy depresfhqn o r disturbance of c a r d i a c rhythm both i n animals . I t s ps chotomimetic p r o p e r t i e s have been and man reviewed by Davles and Beech. 15s The s i m i l a r i t y i n sympathomimetic p r o p e r t i e s phencyclidine, desoxyephedrine and cocaine prompted Chen e t al. t o speculate on a c e n t r a l adrenergic mechanism of a c t i o n , even though the i n s e r t i o n of a methylene group between t h e phenyl and cyclohexyl B.

188

Chap. 2

Stimulants

Biel

25 -

r i n g t o simulate a P-phenethylamine abolished a l l a c t i v i t y . A simple, s p e c i f i c and q u a n t i t a t i v e t e s t f o r t h e assessment of c a t a l e p t i c a c t i v i t y of phen l i d i n e - t y p e compounds i n pigeons The c a t a l e p t i c e f f e c t i s has been developed by Chen. a s c e r t a i n e d by t h e loss of r i g h t i n g r e f l e x without "head drop" over a wide range of dosage A s t r u c t u r e - a c t i v i t y s t u d y was conducted by Maddox e t a l . The most p o t e n t c a t a l e p t i c a g e n t s were those where R = H , ? i i e t h y l o r methoxy and N ( R , R ' ) was p i p e r i d i n o , p y r r o l i d i n o , 3-methylpiperidino, P-methoxyethylamino, methylamino o r ethylamino.

ErB

(XLII

Phenc c l i d i n e

(s e e +

(XLIII) Phencyclidine Derivat i v e s

Adrenergic Agents The proponents of an abnormal m i n e metabolism i n schizophrenic p a t i e n t s received f u r t h e r support f o r t h e i r s p e c u l a t i o n from t h e experimental f i n d i n g of Friedhoff and Van Winkle 11* t h a t 3,4-dimethoxyphenethylamine (DMPEA) was excreted i n t h e u r i n e of schizophrenic p a t i e n t s , but could never be i s o l a t e d t h e u r i n e of normal p a t i e n t s . Bourdillon e t &.I were a b l e t o demonstrate t h a t when t h e schizophrenic population was f u r t h e r subdivided, c e r t a i n groups showed high c o n c e n t r a t i o n s o f t h i s amine i n t h e u r i n e . For an up-to-date l i s t of r e f e r e n c e s on t h i s c o n t r o v e r s i a l s u b j e c t , t h e r e a d e r i s r e f e r r e d t o an E d i t o r i a l . 1 l 4 Friedhoff and Van Winkle115 f u r t h e r found t h a t l i v e r homogenates obtained from b i o p s i e s of schizophrenic p a t i e n t s were capable of 0-methylating both 'OH' groups of dopamine, whereas those obtained from normal s u b j e c t s were unable t o convert dopamine t o DMPEA. I n c a t s , DMPEA produced a c a t a t o n i c e f f e c t s i m i l a r t o t h a t of mescaline. 116,117 D . The C e n t r a l A n t i c h o l i n e r g i c s I t had p r e v i o u s l y been concluded by Bie1118 t h a t p o t e n t a n t i c h o l i n e r g i c a c t i v i t y w a s a p r i m a r y p r e - r e q u i s i t e f o r inducing psychotomimetic e f f e c t s both i n animals and man, but t h a t n o t every potent a n t i c h o l i n e r g i c d r need n e c e s s a r i l y be a psychotogenic agent. Giarman and PepeuBg r e p o r t e d t h a t r a t b r a i n l e v e l s o f a c e t y l c h o l i n e were reduced by t h e psychotomimetic g l y c o l a t e e s t e r s , but n o t by non-psychotogenic a n t i c h o l i n e r g i c s of s i m i l a r s t r u c t u r e . A s i n g l e i n j e c t i o n of 0.5 mg/kg of scopolamine t o r a t s which had been t r a i n e d f o r a s h o r t p e r i o d , produced an amnesic e f f e c t of learned performance which elated well with Both e f f e c t s t h e decrease i n t o t a l r a t b r a i n AcCh l e v e l s . were abolished by e s e r i n e and amphetamine. On t h e o t h e r hand, C.

€30"

€SF

26

Sect.

I

-

CNS Agents

Bi

e l , Ed.

overtrained r a t s experienced no amnesic e f f e c t s , even though b r a i n AcCh l e v e l s were decreased t o t h e same e x t e n t . The authors concluded t h a t only r e c e n t memory may be c h o l i n e r g i c a l l y mediated. I n adequate doses, scopolamine disrupted s e v e r e l y both the s i t i o n and r e t e n t i o n of passive avoidance response in r a t s . I n a r e c e n t paper by Deutsch e t a1.122 t h e i n j e c t i o n of t h e a n t i c h o l i n e s t e r a s e drug, diisopropyl fluorophosphate, i n t o t h e hippocampi of r a t s , 30 minutes a f t e r escape l e a r n i n g , produced a p a r t i a l amnesia l a s t i n g f o r f i v e days. C e n t r a l l y , scopolamine had t e n times t h e a c t i v i t y of a t r o p i n e , whereas i n t h periphery, t h e r a t i o was c l o s e r t o t w o t o one, r e s e c t i v e l y . The compound (Ro 3-1172) was reported by Parkes lq3t o have 50 times t h e CNS a c t i v i t y of a t r o p i n e .

88Yui-

123

3I

Id2C(OH)C02C2H,N

(XLIV) RO 3-1172 S h o r t - l a s t i n g psychotomimetic e f f e c t s (two hours) were induced i n humans by N-allylnoratropine ( 5 mg/man). I n the periphery compound only had 1/16 the autonomic a c t i v i t y o f a t r o p i n e . i2thiS The i n t e r a c t i o n between phenothiazine t r a n q u i l i z e r s i n small doses and the c e n t r a l a n t i c h o l i n e r g i c s , a t r o p i n e , scopolamine, and Ditran (JB-329) was studied by Gershon e t al.125 Chlorpromazine, a t a dose of 0.1 mg/kgtpotentiated t h e e f f e c t s of 0.05 mg/kg of Ditran producing a comatose-like s t a t e i n human s u b j e c t s . Imipramine d i d not p o t e n t i a t e t h e a c t i o n of Ditran. Hence, t h i s t e s t could be applied i n dogs t o d i f f e r e n t i a t e an a n t i d e p r e s s a n t from a t r a n q u i l i z i n g drug. Buehler et a l . l 2 6 synthesized a number of t h i o l analogs of basic g l v c o l a t e e s t e r s which were considerablv weaker a s psychotomimet i c agents than t h e corresponding- oxygen analogs.

= $, cycloalkyl Rl= CH3, C2H5 R

The influence of stereochemical f a c t o r s on a n t i c h o l i n e r g i c p i p e r i d y l g l y c o l a t e s have been psychotomimetic a c t i v i t y of so d i s c w s e d by Gabel and Abood. 137 Summary Present evidence suggests t h a t mental depression may be the cauee o r consequence of a breakdown i n c e n t r a l chemical homeos t a s i s , i . e . , an imbalance between two mutually a n t a g o n i s t i c c e n t r a l neurotransmitter systems, which may possibly be adrenerg i c - l i k e and cholitlergic-like In c h a r a c t e r . Chemical c o r r e c t i o n of t h i s imbalance through drug treatment has afforded a means of accornplisAing remissions i n acute, nonpsychotic depressions.

Chap. 2

Stimulants

27 -

Biel

References L. Klerman and J . 0. Cole, Pharmacol. Rev., lJ-, 101 (1965). L. E . H o l l i s t e r , C l i n . Pharmacol. Therap., 555 (1965). 3 . J. R . Markette and Y. H. H a r r i s , Dis. Nerv. S y s t . , 3.42 (1965). 4. D. M. G a l l a n t , M. P. Bishop, W. Nesselhof and T. E. Fulmer, Curr. Therap. R e s . , 6, 69 ( 1 9 6 4 ) . 5. H. B r i c k , W. H. Doub, and W. C . Perdue, I n t e r n . J. Neuropsychiat., 1,325 ( 1 9 6 5 ) . 6. A. M. Kasich, Cum. Therap. R e s . , 1,542 (1965). 7. B r i t i s h P a t e n t 1,003,686. 8. J. Knoll, Z. E c s e r i , K. Kelcnen, J . Nievel and B. K n o l l , Arch. i n t . Pharmacodyn., 154 (19651. 9. J. F i n k e l s t e i n , E. Chang and J. Lee, J. Med. Chem., 8. 432 (1965). 10. G. A. Youngdale, D. G. Anger, W. C. Anthony, J. P. DaVanzo, M. E. G r e i g , R . V. Heinzelman, H. H. Keasling and J. Szmuszkovicz, J . Med. Chem., 1,,415 (1964). 11. P. A. M. J a n s s e n , U. S. P a t e n t 3,161,637 (1964). 1 2 . The Upjohn Co., French P a t e n t 329lM. 573 (19621. 13. H. Azima, D . A r t h u r s , A. S i l v e r and F. J. Azima, Am. J. P s y c h i a t . , 2. 14. J . B. H e s t e r , M. E. Greig, W. C. Anthony, R . V. Heinzelman and J. Szmuszkovicz, J. Med. Chern., 1, 274 ( 1 9 6 4 ) . 456 (1964). 15. L. I. Goldberg, J. Am. Med. Assoc., 15a. J. M. C u t h i l l , A. B. G r i f f i t h s and D. E. B. Powell, L a n c e t , L. 1076 ( 1 9 6 4 ) . 763 ( 1 9 6 4 ) . 15b. Council on Drugs, J. Am. Med. Assoc., 945 , (1965). 1 5 ~ . E d i t o r i a l , L a n c e t , I16. D. M. Tedeschi and E. J. Fel-lows, S c i e n c e , %, 1225 ( 1 9 6 4 ) . 456 (1964). 17 * L. I . Goldberg, J. Am. Med. Assoc., 18. J. H. B i e l , J. S p o e r l and E. S p r e n g e l e r , B r i t i s h P a t e n t 961,313 ( 1 9 6 4 ) . 19- C. L. Z i r k l e and C. K a i s e r , i n Psychopharmacological Agents, Vol. I , e d . M. Gordon, Academic Press, New York-London, 1964, 445. 20. A . H o r i t a , I n t . J. Neuropharmacol.,.lC, 337 (1965). G. M. E v e r e t t , Second Catecholamine Symposium, Milan, I t a l y , J u l y , 1965, p u b l i s h e d i n 21. (1966). Pharm. Revs., 22. J. A. R i d e r , H. C. Moeller and E . A. DeFelice. T o x i c o l . & Appl. Pharmacol., 1,438 ( 1 9 6 5 ) . 22a. H. Freeman, I . Karacan, W . E. Waterman, A. U. Khan, F. L. Zimmer, A . F e l d s t e i n , C . 0. Tedeschi and E. A. DeFelice. I n t e r n . J. N e u r o p s y c h i a t r y , 1 c5], 513 (19651. J. J. S c h i l d k r a u t , Am. J. P s y c h i a t . . 112, 509 (1965). 23. 856 (1963). 24. S. S p e c t o r , N . Y. Acad. S c i . , 25. C . G. I n g v a r s s o n , A r z n e i m i t t e l F o r s c h . , 3,849 (1965). 86 (1965). 26. S. S p e c t o r , A . Sjoerdsma and S. Udenfriend, J . Pharmacol. E x p t l . Therap.. A. Sjoerdsma, Pharm. Revs., 673 ( 1 9 6 6 ) . 27. '28. A. F e l d s t e i n , H. IIoagland. M. R. Oktem and H. Freeman, I n t . J. N e u r o p s y c h i a t . , L, 384 ( 1 9 6 5 ) . 29 * A. F e l d s t e i n and H. Freeman, C l i n . Pharmacol. T h e r a p . , 6 , 470 (19651. A. P l e t s c h e r , Pharm. Revs., 1 2 1 (1966). 30. 31. R . M. Atkinson and K. S. Ditman, C l i n . Pharmacol. Therap.. 6. 631 (1965). 32. I . J . Kopin, J. E . F i s c h e r , J. M. Musacchio, W. D. Horst and V. K. Weise, J . Pharmacol. E x p t l . Therap., 186 (1965). 33. L. I. Goldberg, J. Am. Med. Assoc., E, 456 (1964). 448 (1964). 34. J . 0. Cole, J . Am. Med. Assoc., 35. G. L. Klerman and J. 0. Cole, Pharmacol. Rev., Q, 101 (1965). 36. L. E. H o l l i s t e r , C l i n . Pharmacol. Therap., 6. 555 (1965). 37 * N. K l i n e , J. Am. Med. Assoc.. E,732 (1964). 38. J. J. S c h i l d k r a n t , Am. J. P s y c h i a t . . 112, 509 (1965). A. P l e t s c h e r . K. F. Gey and P. Z e l l e r , Progr. Drug R e s . , 2, 417 (1960) I I n t e r s c i e n c e ] . 39. J. H. B i e l , A. H o r i t a and A. E. Drukker, i n Psychopharmacological Agents, Vol. I , e d . M. Gordon, 40. Academic P r e s s , New York-London. 1964, 359. 41. C. L. Z i r k l e and C. K a i s e r , i n Psychopharmacological Agents, Vol. I , ed. M. Gordon, Academic P r e s s , New York-London, 1964, 445. 42. R . Kuhn, Schweiz. Med. Wochsch., Q, 1135 (1957). 43. F. H P f l i g e r and V. Burckhardt, i n Psychopharmacological Agents, Vol. I , ed. M. Gordon, Academic P r e s s , New York-London, 1964, 35. 1. 2.

G.

6,

a,

=,

.

a,

s,

2

m,

9.

9,

m,

z,

m,

Sect. I

28 -

-

CNS Agents

B i e l , Ed.

48a. 49. 50 * 51. 52: 53. 54. 55.

E. B. Sigg, Can. Psychiat. Assoc. J., 4. Spec. Suppl.. 7 5 (1959). A. Kaumann. N. Basso and P. Armendia, J. Pharmacol. Exptl. Therap., 54 (1965). R. C. Ursillo and J. Jacobsen, J. Pharmacol. Exptl. Therap.. 247 (1965). A. Jori and S. Garattini, J. Pharm. Pharmacol., lJ, 480 (1565). W. Theobald, 0. &ch, H. A. Kunz, C. Morpurgo, E. G. Stenger and G. Wilhelml, Arch. int. Pharmacodyn., 560 (1964). J. Axelrod. G. Hertting and L. Potter, Nature, Lond.. 297 (1962). J. Glowinski and J. Axelrod, Nature, 1318 (1964). L. L. Iversen, J. Pharm. Pharmacol., 3, 62 (1965). H. Thoener:, A. Huerlimann and W. Haefely, J. Pharmacol. Exptl. Therap., %, 405 ( 1 9 6 4 ) . W. Haefely, A . Huerlimann and H. Thoenen, Helv. Physiol. Acta, 22, 15 (1964). F. Sulser and F. Soroko. Psychopharmacologia, 8. 191 (1965). F. Sulser, M. H. Bickel and B. B. Brodie, J. Pharmacol. Exptl. Therap., 144, 321 (1964). J. H. Biel. P. A. Nuhfer, W. H. Haya and H. A . Leiser, Ann. N. Y. Acad. Sci., $, 251

5657 * 58 * 59. 60. 61.

F. Sulser, M. H. Bickel and B. B. Brodie, J. Pharmacol. Exptl. Therap., 321 (1964). A. Ribbentrop and W. Schaumann, Arzneimittel-Forsch., 3, 863 (1965). A. Ribbentrop and W. Schaumann, Arzneimittel-Forsch., B,863 (1965). A. M. Monro. R. M. Quinton and T. I. Wrigley, J. Med. Chem., 6, 255 (1963). N. J. Giarman and G. Pepeu, Brit. J. Pharmacol., 9, 123 (1964). Z. P. Horovitz. A. R. Furgiuele, L. J. Brannick, 3. C. Burke and B. N. Craver, Nature,

44. 45. 46. 47 * 48.

62. 62a. 63. 64. 6566. 67. 68. 69 * 70.

-

71.

72 73. 74. 75.

76. 77. 78 * 79. 80. 81. 82. 83.

84. 85. 86. 87 88. 88a. 88b. 88c. 886. 8989a. 89b.

-

148.

B,

a,

N,

z,

(1962).

w,

200, 369 (19631. p.1. S. Mapp. R. Dykyj, C. K. Gorby and J. H. Nodlne, Pharmacologist, 5,

234 (1963). I. W. Sletten, X. Pichardo and S. Gershon, Curr. Therap. Res.. 1. 609 (1965). R. Kuhn. Psychopharmaeologia. 8, 201 (1965). A. Ribbentrop and W. Schaumann, Arzneimittel-Forsch., 863 (1965). J. 0. Cole, J. Am. Med. Assoc.. 448 (1964). J. E. Overall, L. E. Hollister. F. Meyer. I. Kimbell, Jr. and J. Shelton, J. Am. Med. Assoc., 605 (1964). G. L. Klerman and J. 0. Cole, Pharmacol. Rev., lJ-, 101 (1965). L. E. Hollister. Clin. Pharmacol. Therap., 6 . 555 (1965). H. Wechsler. G. H. Grosser and M. Greenblatt, J. Nerv. Ment. Dis., 141,231 (1965). N. Kline, J. Am. Med. Assoc., 732 (1964). F. J. Ayd. Dis. Nerv. Syst., 26. 719 (1965). V. #. Pennington, Am. J . Psychiat.. 120. 115 (1964). T. E. Hanlon. J . New Drugs, k, 52 (1964). E. Incas Smith, J. National Med. Assoc., 284 (1965).

x,

u.

m,

*,

z,

J. H. Nodine, P. E. Siegler. T. Bodi. Y. Mapp and R. Dykyj, Am. J. Med. Sci.,

a,

443 (1965). D. M. Gallant, M. P. Bishop, W. Nesselhof and T. E. Fulmer. Curr. Therap. Res., 6 . 69 (196%). J. R. Markette and Y. H. Harris, Dis. Nerv. Syst., 42 (1964). A. M. Kasich. Curr. Therap. Res., 2 , 542 (1965). E. DePerrot, Psychiat. Neurol., 146,341 (1963b.

a,

D. Bente, M. P. Engelmeier, K. Heinrich, H. Hippius and W. Schmitt. Arzneimlttel-Forsch.,

14,538

(1964). J. Angst acd H. Kind, Schweiz. Med. Wochschr., 2, 759 (1964). 0. H. Arnold, H. Bauer and K. Cmyral. Arzneimittel-Forsch.. 14. 510 (1964). H. Freeman, M. R . Otkem, M. W. Kristofferson and C. K. Gorby, Curr. Therap. R e s . , 1, 655 (1965) * I. W. Sletten, X. Pichardo and S. Gershon, Cum. Therap. Res., 1, 609 (1965). J. H. Biel, in Advances in Chemistry Series, No. 45, ed. F. W. Schueler, American Chemcal Society, Baston, Pa., 1964, 114. H. K. Davis, H. F. Ford, J. P. Tupin and!A; Colvin, Dis. Nerv. Syst., 179 (1964). M. Fink, D. F. Klein and J. C. Kramer, Psychopharmacologia, 5, 27 (1964). H. Brick, W. H. Doub. and W. C. Perdue, Intern. J. Neuropsychiat., 1,325 (1965). A. M. Kasich. Cum. Therap. Res., 1, 542 (1965). J. M. VanRossum. J. B. VanDerSchoot and J. A. Th. M. Hurkmans, Experientia, 18,229 (1962). A . Randrup, I. Munkvad and P. Udsen. Acta Pharm. Tox., 20, 145 (1963). R. M. Quinton and G. Halliwell, Nature, 200, 178 (1963). L. Stein. Fed. Proc.. 836 (1964). A. Weissman, B. K. Koe and S. S. Tenen, J. Pharmacol. Exptl. Therap. s,339 (1966). M. Levit, S. Spector, A. Sjoerdsma and S. Udenfriend, J. Pharmacol. Exptl. Therap., 148, 1 (1965).

a,

a,

Chap. 2 90. 91. 9293. 9&. 95. 96 * 97. 98 * 99.

100. 101. 102.

B iel

Stimulants

a. z,

H. Heimann and K. Vetter, Schweiz. Med. Wochschr., 306 (1965). W. Wirth and R. Gosswald, Arch. int. Pharmacodyn.. 393 (1965). A. J. Glasky and L. N. Simon, Science, 702 (1966). N. Plotnikoff. Science, 703 (1966). E. N. Greenblatt and A. C. Osterberg. Toxicol. Appl. Pharmacol., 1, 566 (1965). Symposium on "Some Biological Aspects of Schizophrenic Behavior.'' ed. D. V. Siva Sankar, Art. 1, 1962, 1. Ann. N. Y. Acad. Sci., 3. F. Downing, Quart. Rev., X V I , 133 (1962). D. F. Downing, in Psychopharmacological Agents, Vol. I. ed. W. Gordon, Academic Press, New York-London. 1964. 555. A. Hoffer, Clin. Pharmacol. Therap., 6 , 183 (1965). 3. 0. Cole and M. M. Katz. J. Am. Mad. Assoc., 8 lJ , 758 (1964). C. Savage and M. J. Stolaroff. J. Nerv. Ment. D i s . , 140, 218 (1965). W. H. McGlothlin and S. Cohen. Am. J. Pkychiat.. 572 (1956). J. H. Biel. P. A. Nuhfer, W. K. Hoya and H. A. Leiser, Ann. N. Y. Acad. Sci., &,

z,

s,

s,

x,

251 (1962).

L. 0. Abood and 3. H. Biel, Intern. Rev. Neurobiol., 4. 217 (1962). 104. L. Lasagna and J. W. Pearson, Proc. SOC. Bxptl. Biol. Med., 118,352 (1965). 105. L. Lasagna, Pharmacol. Rev., 16,47 (1964). 106. G. Chen, Fed. Proc., lJ, 358 (1958). 107. F. E. Greifenstein, M. DeVault. J. Yoshitake and J. E. Gajewski, Anesthesiz Analgesia, Current Res., 283 (1958). 108. B. M. Davies and H. R. Beech, J. Ment. Sci., 912 (1960). 1353 (1965). log. G. Chen, C. R. Ensor and B. Bohner, J. Pharmacol. Exptl. Therap., 110. G. Chen, Arch. int. Pharmacodyn., 193 (1965). 111. V. H. Maddox, E. F. Oodefroi and R. F. Parcell. J. Med. Chem., 8_, 230 (1965). li2. A. J. Friedhoff and E. Vanwinkle. Nature, 897 (1962). 113. R. E. Bourdillon, C. A. Clarke, A. P. Ridges, P. M. Sheppard, P. Harper and S. A. Leslie, Nature, @. 453 (1965). 114. Editorial, Brit. Med. J., 1966, I, 119. 115. A. J. Friedhoff and E. Vanwinkle, Nature, 1pe. 1271 (19631. 178 (1962). 116. A. M. Ernest, Nature, 117. R. Michaux and W. 0 . Verly, Life Sci., 1, 175 (1963). 118. J. H. Biel. in "Advances in Chemistry Series," No. 45, ed. F. W. Schueler. American Chemical Society, Easton, Pa., 1964, 129. 119. N. J. Giarman and G. Pepeu, Brit. J. Pharmacol., 2.123 (1964). 120. A. Pazzagli and G. Pepeu, Int. J. Neuropharmacol., k, 291 (1964). 121. B. Meyers. Psychopharmacologia. g. 111 (1965). 122. J. A. Deutsch. M. D. Hamburg and H. Dahl, Science, 2 2 1 (1966). 123. K. W. Parkes, Psychopharmacologia. 1, 1 (1965). 12&. L. F. Soyka and K. R. Unna. Psychopharmacologia. 6 , 453 (1964). 125. S. Gershon, H. Neubauer and D. M. Sundland, Clin. Pharmacol. Therap., 6, 749 (1965). 126. C. A. Buehler. S. F. Thames, L. G. Abood and J. H. Biel, J. Med. Chem., g, 643 (1965). 127. N. W. Gabel and L. G. Abood, J. Med. Chem., 8. 616 (1965). 103.

x,

106.

x,

m,

m.

x,

z,

30 -

Sect. I Chapter 3 .

-

CNS Agents

B i e l , Ed.

Sedatives, Hypnotics, Anticonvulsants, Muscle Relaxants, Gene r a1 Anesthetics

Cornelius K. Cain, McNeil Laboratories, Inc., F o r t Washington, P a .

-

The classification of a given drug according to the pharmacoIntroduction logical actions listed i n the title (plus tranquilizers discussed in Chapter 1) is recognized as a r b i t r a r y and dependent upon dosage being used. The following discussion is divided m o r e on the basis of established o r projected clinical utility than on the basis of widely different pharmacological proper ties o r chemical s t r u c t u r e .

1 Some y e a r s ago, Chen and P o r t m a n undertook a quantitative approach to the problem. They determined the C D 5 0 1 of ~ a convulsant in mice p r e treated with graded doses of a depressant. Plotting these values gave a graph showing two points of inflection, and t h r e e corresponding straight lines were constructed. The slopes of these lines were interpreted a s r e p resenting the sedative, hypnotic and anesthetic potencies of the depressant. The i n t e r s e c t s represented minimal hypnotic and anesthetic doses. The authors concluded that this approach was useful in indicating that a given d e p r e s s a n t drug might be m o r e useful as a hypnotic, an anesthetic o r as a sedative.

;"8

Sedatives and Hypnotics - Several a r t i c l e s d e s c r i b e the favorable r e s u l t s obtained with nitrazepam, Mogadona, RO 4-5360 (I). The pharmacology was thoroug y investigated by Randall and coThey postulate that the sleepworkers inducing effect is due not to a d i r e c t effect on the a r o u s a l system, but to a reduction of stimuli acting on it. Metabolic studies O2 showed the unchanged drug as well a s i t s 7-amino and 7-acetylamino derivative in human plasma and urine. \ I 3 Clinical studies by Wyss and Mgder in 200 patients gave good r e s u l t s in onset and duration of sleep a t doses of 5 to 10 mg. At higher doses (up to 200 mg.), tolerance was excellent. They state h a t the mechanism of action differs f r o m known hypnotics. Lanoir, Dolce and Chirinos4 found that in a battery of neurophysiological t e s t s in cats, nitrazepam and diazepam showed the s a m e type of activity in many respects, such as action on spontaneous rhythm, evoked responses, etc. Both are potent anti-convulsants. The m a j o r difference is that nitrazepam induces a lasting and profound slee while diazepam a c t s essentially as a relaxant and not as a hypnotic. Borck

7

9.

!?

Chap. 3

Depressants

Gain

31 -

reported that the drug in doses of 5 to 15 mg. was a highly suitable hypnotic in psychiatric patients. E g e r t and Jahn6 found it gave v e r y good to good results in 75% of a wide variety of clinical c a s e s . P a r t i c u l a r l y noteworthy were i t s new type of mechanism of action when compared with b a r biturates, i t s low toxicity and i t s u s e l e s s n e s s f o r suicidal pur o s e s . In 40 children ranging in age f r o m a few days to 11 y e a r s , Matthes 7p induced daytime sleep in 50% of the c a s e s . B e s t results were obtained in infants up to 12 months of age. About 10 y e a r s ago, the hypnotic, anticonvulsant and sedative p r o p e r ties of a s e r i e s of 2-alkyl-3-aryl-4(3H)quinazoloneswere reported by Gujral and co-workers 8~ 9 and confirmed by B o i s s i e r . l 0 Recently, a s e r i e s of 79 compounds of this type was examined by Leszkovszky, Erdely and Tardos" who reported that compound I1 showed the highest o r a l activity. Swift, Dickens and Becker12 published a thorough pharmacological study of this compound in 1960. Clinical studies were published by s e v e r a l investigators 13, 14J15J16,17 who 0 compared methaqualone to glutethimide, chloral hydrate and cyclobarbital. A r e cent l e t t e r to the editor cites four I1 patients who consumed l a r g e amounts of the drug daily f o r extended periods, raising the question of physical dependence. In 1965, compound 11 was marketed a s a hypnotic and sedative in the f o r m of 150 m g . tablets as methaqualone (Quaaludem).

qJ$

Fenimide i s the non-proprietary name recently adopted19 for 3-ethyl-

2-methyl-2-phenylsuccinimide(111) which was described by Chen and BassZO

a s a sedative agent comparable to m e p r o bamate and phenobarbital. It protects against s t r e s s -induced u l c e r s while m e p r o bamate and phenobarbital do not.

Q$ . . f . 2Hc3Hc 111

At the same time, the non-proprietary name trimetozine was adopted for 4-(3, 4, 5-trimethoxybenzoyl)morpholine. This compound (IV) was r e o r t ed e a r l i e r by Vargha and co-workers 8 , 2 2

d3

as being selected f r o m a l a r g e s e r i e s of alkoxybenzamides f o r clinical t r i a l as a neurosedative in Hungary. Of particular 6 7 - C OCH3 i n t e r e s t in their p a p e r s is the r e p o r t of OCH3 the marked influence on pharmacological activity of changes in position and/ o r IV nature of the afkoxy groups. B o i s s i e r and c o - w o r l ~ e r sstated ~ ~ that tests in animals show the compound to be a sedative but not a hypnotic even a t toxic doses.

r

/ \

32

Sect. I - CNS Agents

B i e l , Ed.

Metabolic studies of the optical antipodes of glutethimide (V) (Doriden@)by Keberle, Riess and HoffmannZ3 a r e of especial interest since the authors showed stereospecific metabolism in dogs. The dextrorotatory form of V was hydroxylated in the ring to give mainly V I (isolated a s the glucuronide) and VII, resulting from dehydration. The levorotatory f o r m of V was hydroxylated in the ethyl group to give mainly VIII (isolated a s the glucuronide) and IX, resulting from l o s s of acetaldehyde. Total r e covery of these products was 96% of theory. No products resulting f r o m ring opening were detected. CH2CH3

p CH2CH3 I

k

v

VIII

IX

The sedative and hypnotic properties of thalidomide have been almost forgotten in view of i t s reported embryotoxic effects. Recent metabolic studies of thalidomide in abbits, r a t s , mice and guinea pigs by SchuL macher, Smith and Williams 2 4 showed that up to 12 hydrolysis products appear in the urine. These products a r e the same a s those obtained from thalidomide in aqueous solutions a t pH values above 6, 25 and can be accounted for by initial hydrolysis of the phthalimide ring followed by hydrolys i s of the glutarimide ring. E a r l i e r , the same group of investigatorsz6 had studied the relationship of embryotoxic activity and chemical structure in a s e r i e s of compounds related to thalidomide and had concluded that a phthalimide group was important in determining teratogenic activity; and, in a recent article, 27 suggest that the reactivity of the drug towards certain natural diamines such a s spermidine, putresciae, etc., may be of significance in relation to its biological properties. Anti-convulsants - A s in previous years, many compounds were tested and reported to be active a s anti-convulsants during 1965. No new drugs for this use were marketed in the United States, and none marketed in other countries appear to offer considerable advantages over those in general use. A recent review by Millichap28 discusses clinical and EEG indications, efficacy and toxicity of some 2 0 drugs in current use in this field.

Chap. 3

Depressants

Gain

33 -

Central Muscle Relaxants - No new drugs in this field were introduced in the domestic market during 1965. Several reports were published of clinical t r i a l s of various compounds in severe spastic conditions. Bhargava and S r i ~ a s t a v astudied ~~ the anti-tetanus activity in cats of central muscle relaxants and found 3210220, 3-(q-chloropheny1)-3-hydroxy-2,2-dichloropropyl carbamate (X) to be the most potent and longest-acting of 11 compounds tested. Hendrickse and Sherman30 found CHOHCCSLCHZOCONHZdiazepam (Valium@)an effective muscle relaxant in children suffering from X tetanus, although it was not very effective in controlling convulsive spasms. Marsh31 obtained excellent results in 10 of 26 severely cerebralpalsied children using o r a l doses of diazepam in a controlled study, confirming e a r l i e r reports of effectiveness in adults similarly affected. Encouraged by the results of Berman, Noe and Goodfield3’ using chlorzoxazone in cerebral-palsied patients abandoned to supportive care, Darienzo 33 used a combination of chlorzoxazone and acetaminophen and obtained a change from severe to mild symptoms in 12 of 15 children.

A metabolic study of chlormezanone (Trancopala) in rat, dog and man by McChesney and co-workers34 showed that the compound was excreted as such. A review of benzazoles; chemistry, pharmacology and clinical application a ~ p e a r e d ?SKF ~ 13,436, 3 -amino5-trifluoromethyl- 1H -indazole (XI) was reported by Santella and c o - ~ o r k e r to s~~ be a muscle relaxant in r a t s , cats, dogs, F3C&NH2 rabbits and monkeys.

XI Anesthetics (a) - D a t a from thousands of patients were examined for possible relation between post-operative liver damage and anesthesia. Herber and S p e ~ k reviewed t ~ ~ findings in about 20,000 cases and estimated that one case of hepatic necrosis may be expected in about 800 halothane anesthetics. They suggest that careful selection of patients should minimize liver necrosis in the continued use of this valuable anesthetic. Gingrich and Virtue38 reviewed records of nearly 3800 cases using fluorinated hydrocarbons and over 20,000 cases using other inhalation agents a s well a s 48, 000 non-surgical patients. They doubt that either halogenated hydrocarbons o r other anesthetic agents p e r s e have been of significance in the production of hepatic necrosis.

Sect. I

34 -

-

CNS Agents

B i e l , Ed.

OH3 g

(b) Injection Anesthetics

XI1

XI11

-

McCarthy, Chen, Kaump and E n ~ o studied r ~ ~ the general anesthetic and o t h e r pharmacological p r o p e r t i e s of CI 581 (XI), an analog of phencyclidine (Se rnyl@) (XIII) in eight species of animals and concluded that XI1 showed a l e s s e r d e g r e e of CNS XIII. stimulation However, and of Domino, b r i e f e r Chodoff durationand than Corssen40 found that in humans, XIII, like XI, was hallucinatory and that skeletal muscle tone was increased. They sugg e s t the t e r m "dissociative anesthetic" for this c l a s s of agents.

s ~ ~by G. The earlier r e p o r t s by H. Laborit and c o - ~ o r k e r and Laborit and co-workers4' on the clinical u s e of sodium y -hydroxybutyrate f o r sedation, hypnosis and general anesthesia stimulated s e v e r a l other authors to investi4gte the pharmacology of this compound. 43s44,45 B e s s m a n and Skolnick found that the d e p r e s s a n t action paralleled the concentration in brain of the lactone and not of the anion. Recently, Winters and S p ~ o n e suggest r ~ ~ that a r e - a p p r a i s a l of the clinical usefulness of this drug m a y be in o r d e r , based on the epileptiform EEG patterns and grand ma1 s e i z u r e s in cats after intraperitoneal administration of 0,7 to 1.0 g/kg. Propanidid (XIV) and a few other substituted phenoxyacetamides have been investigated f u r t h e r as very short-acting intravenous anesthetics. Gunner and ~ o - w o r k e r sfound ~ ~ propanidid useful in electroconvulsive therapy in 70 patients. They found the OCHZCON(CzH5)z duration of unconsciousness averaged four minutes a t a dose of 5 mg/Kg. and s i x minutes a t a dose of 7 mg/Kg. They a s c r i b e the brevity of action to hydrolysis of the e s t e r group by e s t e r a s e s . E i s t e r e r and c o - ~ o r k e r found s ~ ~ the drug CH2C OOC 3H very suitable in 96 ambulatory patients r e XIV quiring anesthesia of up to five minutes' duration. Hewitt, Hamilton, O'Donnell and Dundee50 reported that, while propanidid showed a slightly higher incidence of venous thrombosis than thiopentone o r methohexitone, the incidence was not enough to influence clini cal acceptability

6"'". .

Adjuncts to Anesthesia (a) - Lunsford and coworkers5l synthesized a s e r i e s of substituted pyrrolidinones (XV) and

Chap. 3

Depressants

Cain

35 -

studied their pharmacological properties. Some were stimulants of r e s p i ration and the CNS, and m o s t of these showed p r e s s o r activity; o t h e r s were depressants. One of the stimulants (XVI) was selected f o r f u r t h e r study.

xv

R = alkyl, cycloalkyl, benzyl B = basic residue

o & c H 2 cR H2B

XVI B R = morpholino ethyl

Dopram@Doxepram

52 E v e r s , Malik and Dobkin found doxepram to be a s effective a s d-amphetamine in shortening the recovery time in dogs given a standardized dose of thiopental; no untoward effects were observed. They a l s o used the drugs a s an intravenous infusion in 50 healthy female patients and found it effective for stimulating respiration (both tidal volume and r e s p i ration r a t e ) without producing any appreciable circulatory changes.

53

Mauro and co-workers studied 52 patients to whom doxepram was given and 23 controls; all were anesthetized with thiopental. A few seconds after a single injection of 0 . 3 mg/Kg. of doxepram, t h e r e was a marked i n c r e a s e in tidal volume to levels approaching control values. Noe, Borrillo and G r e i f e n ~ t e i n injected ~~ 0. 5 mg/Kg. doses into 20 patients anesthetized with (1) pentobarbital-pentothal, (2) nitrous oxideoxygen o r ( 3 ) halothane -nitrous oxide and found r e s p i r a t o r y stimulation with m o r e o r l e s s arousal in a l l t h r e e groups. Brief E E G changes were observed in five subjects and m o s t c a s e s showed moderate hypertension; otherwise, there were no side effects. (b) - Combinations of neuroleptic agents and narcotic analgesics have been widely used in surgical procedures with o r without inhalation o r injection anesthetics. Such combinations a r e r e f e r r e d to a s neuroleptanalgesics and a r e usually considered a s adjuncts to anesthesia. Proceedings of a Symposium held a t Edinburgh were published. 6 8 During 1965, some 20 to 30 a r t i c l e s appeared concerning clinical studies with Innova@, a combination of the neuroleptic droperidol (XVII) and the analgesic fentanyl (XVIII). Contributions by the m o s t active workers

0

XVII

XVIII

36

Sect. I

-

CNS Agents

B i e l , Ed.

in the field a r e : Israel, Jansen and Dobkin;” Corssen, Chodoff, Domino and Kahn;56 DeCastro, Mundeleer and B a ~ d u i n :Gorodetzky ~~ and Martin; 58 Aubry, Denis, Keeri-Szanto and Parent;59 and Gemperle and Buhler. 60 Pharmacological studies using the combination of the two drugs were reported by Dobkin and co-workers;61, 62 Chodoff and D o m i n 0 ; 6 ~and Canellas, Roquebert and Courtois. 64 A combination of the two d r u s was marketed during 1965 as Innova@-Vet. Yelnosky and Fieldg5 investigated i t s u s e i n s i x species of animals and found it m o s t useful in dogs. Franklin and Reid66 obtained good to excellent r e s u l t s in 564 out of 601 surgical procedures usin various b r e e d s of dogs. Mortelmans, Marsboom and Vercruyssee’ reported highly successful r e s u l t s in 174 t r i a l s using p r i m a t e s and lower monkeys.

Chap. 3

D e p r e s s a n ts

Cain

37 -

References (1) G. Chen and R. P o r t m a n , A.M.A. A r c h . Neurol. P s y c h i a t . 68, 498 (1952). L. 0. Randall, W. Schallek, C. Scheckel, R. E. Bagdon and J. (2) Rieder, Schweiz. Med. Wochschr. 95, 334 (1965). S. W w .zy -ie Med. Wochschr. 2, 338 (1965). (3) J. Lanoir, G. Dolce and E. Chirinos, Compt. Rend. SOC.Biol. (4) 431 (1965). W . F. Borck, A r z n e i m i t t e l - F o r s c h . 1155 (1965). (5) (6) H. E g e r t and 0. Jahn, A r z n e i m i t t e l - F o r s c h . l5, 1159 (1965). (7) A. Matthes, A r z n e i m i t t e l - F o r s c h . l5, 1157 (1965). (8) M. L. Gujral, P . N . Saxena and R. S. Tiwari, Indian J. Med. R e s e a r c h 43, 637 (1955), Chem. A b s t r . E, 6662b (1956). (9) M. L. Gujral, K. N. S a r e e n and R. P. Kohle, Indian J. Med. 51 15787h (1957). R e s e a r c h 45, 20? (1957), C h e m . A b s t r . -’ (10) J. R. B c i s s i e r , T h e r a p i e 2, 30 (1958). (11) C. Leszkovszky, I. E r d e l y and L. T a r d o s , A c t a P h y s i o l . Acad. Sci. Hung. 27, 81 (1965), Chem. A b s t r . 62, 16782g (1965). (12) J. G. Swift, E. A. Dickens and B. A. B e c k e r , A r c h . I n t e r n . 112 (1960). Pharmacodyn. (13) A. Ravina, P r e s s e Med. 3, 891 (1959). (14) R. Neubauer, Med. T i m e s s , 61 (1963). (15) N. Asbell, J. Am. G e r i a t . SOC. lo, 1032 (1962). (16) V , Matthews, H. E. Lehmann and T . A. Bau, Appl. T h e r . 6, 806 (1964). (17) T. W . P a r s o n s and T . J. Thomson, B r i t . Med. J. L, 171 (1961). (18) J. S. Madden, B r i t . Med. J. 1 , 676 (1966). 208 (1966). (19) J. Am. Med. A s s o c . *, (20) G. Chen and P. B a s s , A r c h . I n t e r n . P h a r m a c o d y n . E , 115 (1964). (21) L. Vargha, E . K a s z t r e i n e r , J. B o r s y , L. F a r k a s , J. Kuszmann and B . Dumbovich, Biochem. P h a r m a c o l . g, 639 (1962). (22) E. K a s z t r e i n e r , J. B o r s y and L. Vargha, Biochem. P h a r m a c o l . 651 (1962). (23) H. K e b e r l e , W . R i e s s and K. Hoffmann, A r c h . I n t e r n . P h a r m a c o d y n . 142, 117 (1963). (24) H. Schumacher, R. L. Smith and R. T. W i l l i a m s , B r i t . J. P h a r m a c o l . 25, 338 (1965). (25) H. Schurnacher, R. L. S m i t h and R. T . W i l l i a m s , B r i t . J. P h a r m a c o l . 25, 324 (1965). (26) S. F a b r o , H. Schumacher, R. L. Smith and R. T. W i l l i a m s , Life Sci. 2, 987 (1964). 1208 (1965). (27) S. F a b r o , R. L. Smith and R. T . W i l l i a m s , N a t u r e (28) J. G. Millichap, P o s t g r a d . Med. 37, 22 (1965).

m,

2,

z,

z,

-

m,

38 -

Sect. I

- CNS Agents

B i e l , Ed.

References

P

g,

(29) K . P. B h a r g a v a and R. K. S r i v a s t a v a , B r i t . J. P h a r m a c o l . 74 (1965). (30) R. G. H e n d r i c k s e and P. M. S h e r m a n , Lancet 1965, p. 737. (31) H. 0. M a r s h , J. Am. Med. A s s o c . &, 797 (1965). (32) H. H. B e r m a n , 0. Noe and F. Goodfield, D i s e a s e s Nervous S y s t e m 2!5, 430 (1964). (33) C. Darienzo, D i s e a s e s Nervous S y s t e m 27, 189 (1965). (34) E. W. McChesney, W . F. Banks, J r . , G. A. P o r t m a n a n d A . V . R . Grain, F e d e r a t i o n P r o c . (2), 418 (1966). (35) C. K . C a i n and A. P. Roszkowski in”Psychopharmacologica1 Agents:‘ Vol. I, M. Gordon, E d . , A c a d e m i c Press, New York, N. Y. 1964, Chap. 10. (36) P. J. Santella, D. H. Tedeschi, J. J. Lafferty, C. L. Z i r k l e and E . J. Fellows, P h a r m a c o l o g i s t ‘7, 164 (1965). 266 (1965). (37) R. H e r b e r and N. W. Specht, A r c h . I n t e r n a l Med. (38) T . F. Gingrich and R. W. Virtue, S u r g e r y = , 241 (1965). (39) D. W . McCarthy, G. Chen, D. H. K a u m p a n d C. E n s o r , J. New D r u g s 5, 21 (1965). (40) E. F. Domino, P. Chodoff and G. C o r s s e n , Clin. P h a r m a c o l . T h e r a p h , 279 (1965). (41) H. Laborit, J. M. Jouany, J. G e r a r d and P. Fabiani, P r e s s e Med. 68, 1867 (1960). 1216 (1961). (42) G. Laborit, A. Kind and C. d e L. Regil, Presse Med. (43) A. B. Drakontides, J. A. Schneider and W. H. F u n d e r b u r k , J. P h a r m a c o l . Exp. T h e r a p . 135, 275 (1965). (44) E. H. Jenny, H. B. M u r p h r e e , L. Goldstein and C. C. Pfeiffer, P h a r m a c o l o g i s t 4, 166 (1962). (45) M. J. HOsko, Jr. and M. I. Gluckman, P h a r m a c o l o g i s t ? , 254 (1963). (46) S . P. B e s s m a n and S . J. Skolnick, Science E,1045 (1964). (47) W. D. W i n t e r s and C. F.Spooner, Intern. J. Neuropharmacol. 4, 197 (1965). (48) B. W. Gunner, G. A. H a r r i s o n , W. D. W a l k e r and I. S. Jenkinson, (11), 327 (1965). Med. J. A u s t r a l i a (49) H. Eisterer, G. F r a u n d o r f e r , P. F o r g e s , H. Seidl and K. Stein638 (1965). b e r e i t h n e r , Wien. Med. Wochschr. (50) J. C. Hewitt, R. C. Hamilton, J. F. O’Donnell and J. W. Dundee, B r i t . J. A n a e s t h e s i a 2 , 115 (1966). (51) c. D. Lunsford, A. D. Cale, J r . , J. W. Ward, B. V. F r a n k o and H. Jenkins, J. Med. Chem. 7 ‘ , 302 (1964). (52) W. E v e r s , K. Malik and A. B. Dobkin, Canad. Anaesth. SOC. J. l2, 281 (1965). (53) A. L. Mauro, L. Labartino, E . Mojdehi and B. Reynolds, Am. J. Med. Sci. 269 (1965).

c,

3,

u,

z,

Chap. 3

Depressants

Cain

39 -

References

(54) F. W. Noe, N. B o r r i l l o and F. E. Greifenstein, A n e s t h e s i a Analgesia, C u r r e n t R e s . 44, 206 (1965). (55) J. s. Israel, G. T. J a n s e n and A. B. Dobkin, Anesthesiology 26, 253 (1965). (56) G. C o r s s e n , P. Chodoff, E. F. Domino and D. R. Kahn, J. T h o r a c . 901 (1965). Cardiov. Surg. 2, (57) J. DeCastro, P. Mundeleer and T. Bauduin, S u r v e y of A n e s t h e s i ology (3, 348 (1965). (58) C. W . Gorodetzky and W. R. Martin, Clin. P h a r m a c o l . T h e r a p . 6, 731 (1965). (59) U. Aubry, R. Denis, M. Keeri-Szanto and M. P a r e n t , Canad. Anaesth. SOC. J. l2, 510 (1965). (60) M. G e m p e r l e and J. C. Buhler, Schweiz. Med. Wochschr. 158 (1965). (61) A. B. Dobkin and P . K . Y . Lee, Canad. Anaesth. SOC. J. l2, 34 (1965). (62) A. B. Dobkin, P . K . Y . L e e and P. H. Byles, Canad. Anaesth. SOC. JI, 39 (1965). (63) P. Chodoff and E . F . Domino, A n e s t h e s i a Analgesia 44, 558 (1965) (64) J. Canellas, J. Roquebert and P. Courtois, Compt. Rend. SOC. Biol. 159 1538 (1965). (65) J. Yelnosky and W. E. Field, Am. J. V e t . R e s . 25, 1751 (1964). (66) I. I. F r a n k l i n and J. S. Reid, V e t . Med. / S m a l l Animal Clinician 60, 927 (1965). (67) J. M o r t e l m a n s , R. M a r s b o o m and J. V e r c r u y s s e e , Bull. SOC. Roy. 2001. d ' A n v e r s 36, 13 (1965). (68) N. W. Shephard, Ed., "The Application of Neuroleptanalgesia in Anesthetic and O t h e r Practice", P e r g a m o n Press, Oxford, 1965. (69) J. R. B o i s s i e r , P. Simon and J. Fichelle-Pagny, TherapieAO, 401 (1965). I

s,

-

-J

Sect. I

40 -

- CNS Agents

B i e l , Ed.

Chapter 4. A n a l g e t i c s --Stronf and Weak Louis S . H a r r i s , U n i v e r s i t y of North Carol na, Chapel H i l l , N.C. Introduction The p a s t few y e a r s have witnessed a ferment i n t h e f i e l d of a n a l g e t i c s . S e r i o u s q u e s t i o n s have a r i s e n t o challenge o u r concept of p a i n as a s p e c i a l i z e d sensory system. Indeed we now take a more A r i s t o t e l i a n view of p a i n and speak of t h e " p a i n experience" which encompasses n o t o n l y t h e s e n s a t i o n of p a i n but a s s o c i a t e d s e n s a t i o n s such as touch and h e a t and f e e l i n g s t a t e s such a s f e a r and p l e a s u r e . I t i s n o t s u r p r i s i n g t h e n t h a t more and more drugs a r e becoming a v a i l a b l e f o r t h e t r e a t m e n t of p a i n which appear t o have mechanisms which d i f f e r , sometimes q u i t e markedly, from those of t h e c l a s s i c a l a g e n t s . We now have s t r o n g a n a l g e s i c s which a r e devoid of morphine-like a d d i c t i o n p o t e n t i a l and mild a n a l g e s i c s which a r e i n c r e a s i n g i n t h e i r p a i n r e l i e v i n g potency. I t would appear t h a t we a r e approaching c l s e r and c l o s e r t o t h e i d e a l a n a l g e s i c agent. A r e c e n t monograph P p r e s e n t s an e x c e l l e n t summary of much of t h i s work.

I.

Strong Analgesics Morphine and Morphinans There was l i t t l e a c t i v i t y i n t h i s a r e a i n 1965. P i r k l e and Gates2 prepared t h e hydroaromatic analogs ( I and 11) of t h e p o t e n t a n a l g e s i c s &-3-hydroxy-N-methylmorphinan and t h e c o r r e s ponding isomorphinan. The morphinan ( I ) w i t h a c i s - r i n g f u s i o n was e s s e n t i a l l y i n a c t i v e while t h e isomorphinan
A.

I B/C I1 B/C

cis -

111 B/C

trans HO

cis

I V B/C t r a n s 6 -OF

CH3 6 only a t n e a r t o x i c doses. The secondary a l c o h o l of I showed a c t i v i t y . Thus, r e d u c t i o n of t h e aromatic r i n g depresses a n a l g e s i c a c t i v i t y and lends evidence t o t h e importance o f t h e p l a n a r r i n g f o r bindin$ t o o r f i t on t h e r e c e p t o r . A n i n t e r e s t i n g m o d i f i c a t i o n of t h e morphinan s t r u c t u r e was described by Sawa and h i s colleague^.^^^ They synthesized (-13-hydroxy-6,N-dimethyl-c-nor-morphinan 111 and t h e corresponding 6-oxo-isomorphan I V , t h e compounds w i t h a 5-membered C-ring. The &-fused compound was 1 9 x morphine and t h e trans-compound 1 2 x morphine i n r a t s u s i n g t h e D'Amour-Smith t e s t . I n t h e way of mechanisms, Way5 and h i s co-workers have shown t h a t h e r o i n ( H ) i s r a p i d l y metabolized t o monoacetylmorphine (MAM) and morphine (M). They p o s t u l a t e t h a t t h e major e f f e c t s of H a r e dependent on t h e formation of MAM. Unchanged H e x e r t s only minor e f f e c t s . H i s biotransformed t o MAM which t h e n a c t s

-

Chap. 4

Analg e ti c s

Harris

41 -

a s a c a r r i e r t o f a c i l i t a t e t h e a c c e s s of M t o r e c e p t o r s i t e s i n t h e C.N.S. Krivoy and h i s group have continued i n t h e i r a t t e m p t s t o r e l a t e t h e development of t o l e r a n c e t o morphine with t h e formation of p r o t e i n s o r p e p t i d e s i n t h e C.N.S. which c o u n t e r a c t t h e drug. Cohen e t a l . 6 r e p o r t some i n t r i g u i n g experiments i n which t h e y a r e a b l e t o d e l a y t h e development of t o l e r a n c e t o morphine by p r e t r e a t i n g t h e animals w i t h actinomycin D . Whether t h e i n h i b i t i o n of t o l e r a n c e development i s r e l a t e d t o d e p r e s s i o n of RNA s y n t h e s i s and consequent changes i n p r o t e i n s y n t h e s i s remains t o be shown a s does t h e s p e c i f i c n a t u r e of t h e p r o t e i n o r p e p t i d e involved. The r o l e of p h y s i c a l and psychological s t r e s s on a n a l g e s i c s was a s s e s s e d by Frome17 who found t h a t t h e s e s t r e s s e s prolonged t h e a c t i o n of morphine i n guinea p i g s . B. Benzomorphans May and h i s c o l l e a g u e s continued t h e i r e l e g a n t s t u d i e s i n t h i s a r e a . C h i g n e l l e t a1.8 r e p o r t e d t h e p r e p a r a t i o n a - ( c i s ) and ~-(trans)-2-hydro~-~9-dimethyl-5-propyl-6,7-benzomorphan. Again, a s h a s been p r e v i o u s l y r e p o r t e d f o r o t h e r members of t h i s s e r i e s , t h e trans-isomer i s c o n s i d e r a b l y more p o t e n t t h a n t h e cis-isomer T h e e - c o m p o u n d , l i k e o t h e r cis- and 5-monoalkyl d e r i v a t i v e s i n t h e benzomorphan s e r i e s , has l i t t l e o r no a b i l i t y t o s u b s t i t u t e f o r morphine i n a d d i c t e d monkeys. The t r a n s compound, however, does s y p r e s s withdrawal symptoms i n d i c t e d monkeys. C h i g n e l l and May prepared t h e 9-methylenedesoxy compound (V) which t h e y t h e n reduced s t e r e o s p e c i f i c a l l y t o t h e c i s - ( V I ) and t r a n s - i s o m e r s ( V I I ) . The methylene compound proved

.

-

VI

V

C3H7

VII

t o have about h a l f t h e a n a l g e s i c a c t i v i t y of morphine a s d i d t h e Jacobson and Maylo prepared a v a r i e t y of 2' - n i t r o - , trans-ieomer. 2' -amino-, and 2 ' -halo-5,9-dialkyl-2-methyl-6,7-benzomorphans. Replacement of t h e p h e n o l i c hydroxyl by t h e s e o t h e r f u n c t i o n a l groups l e d t o a diminution of a n a l g e s i c potency and an i n c r e a s e i n t o x i c i t y . As an i n t e r e s t i n g s i d e l i g h t , it was r e p o r t e d t h a t a n a l g e s i c (Hot P l a t e ) ED50 values i n a new type o f Caesarean d e r i v e d g e n e r a l purpose mlce were approximately 1/2 t h o s e It p r e v i o u s l y o b t a i n e d w i t h o t h e r mice a t t h i s i n s t i t u t i o n . would appear t h a t t h e s e h e a l t h i e r , f a s t e r growing mice a r e more s e n s i t i v e t o a n a l g e s i c s . J o s h i e t a l . 1 1 prepared a homologous s e r i e s of 5-alkyl-2~-hydroxy-6,7-benzomorphans,and have compared t h e s e w i t h a s e r i e s of &-2-alkyl-2'-hydroxy-5,9-

Sect. I

42 -

-

CNS Agents

B i e 1, Ed.

I n t h e 5-alkyl s e r i e s t h e r e l a t i v e I n t h e N-alkyl s e r i e s a c t i v i t y was Me<Et = P r = Bu>Am>Hex. they found Hex>Am>Me with t h e E t , P r and B u being i n a c t i v e . I t had e a r l i e r been reported12 t h a t t h e N-propyl compound i s a potent analgesic antagonist. Kametani and h i s colleaguesL3t l4 have r e p o r t e d an i n t e r e s t i n g m o d i f i c a t i o n of t h e benzomorphan nucleus. They prepared 1 , 2 3,4-tetrahydro-6H-l, 5 - ( e ) ( 1 , 4 ) diazocine and 1 , 2 , 3 , 4 , 5 , 6 - h e x a hydro-2,6-methanobenzo ( e ) ( 1 , 4 ) d i a z o c i n e , two azabenzomorphans I t w i l l be i n t e r e s t i n g t o a s c e r t a i n t o what e x t e n t t h e approp r i a t e N-alkyl d e r i v a t i v e s of t h e s e compounds e x h i b i t a n a l g e s i c activity. C Meperidine D e r i v a t i v e s Despite t h e f a c t t h a t over 4000 meperidine d e r i v a t i v e s have been prepared, work i n t h i s a r e a continues unabated. Hardy e t a1 .I5 described s e v e r a l homologous s e r i e s of nor-meperidines z c l u d i n g N-alkyl, a l k a n o l , a l k y l e t h e r , e t c . where t h e N-side chain v a r i e d i n l e n g t h from 3 - 7 atoms. Their r e s u l t s i n d i c a t e t h a t t h e o v e r a l l l e n g t h of t h e s i d e c h a i n i s q u i t e important f o r a n a l g e s i c a c t i v i t y . Peak a c t i v i t y c o n s i s t e n t l y appeared when t h e s i d e chain s k e l e t o n had an o v e r a l l l e n g t h of 7-9 A O . Their r e s u l t s i n d i c a t e t h a t w i t h i n d i f f e r e n t s e r i e s of compounds i n c r e a s i n g t h e Len t h of t h e N-side chain produces t h e same changes i n analges c potency. A s they p o i n t o u t , t h i s may be t h e r e s u l t of e q u i v a l e n t e f f e c t s on r a t e s of t r a n s p o r t t o t h e a c t i v e s i t e o r t o s i m i l a r changes i n a f f i n i t y f o r t h a t s i t e . Casy and Armstrongl6 prepared a s e r i e s of 4-alkoxy- and 4acyloxy-4-arylpiperidines o f t h e type V I I I and I X f o r comparison

dimethyl-6,7-benzomorphans.

.

.

?!

ArGf

VIIIa:

A

VIII

Ar=$ R =@O(CH2)2 R'=H R" =C 2H5

OCOR"

B

IX

IXa:

Ar=$ R =P((CH2I2 R ' =H R" =CH3

w i t h t h e p r e v i o u s l y r e p o r t e d a n a l g e s i c 4-ethoxy-4-(2-furyl)-3me t h y l - 1 -phene t h y l p i p e r i d i n e . They found t h e 4-phenyl-4-alkoxy d e r i v a t i v e s t o be g e n e r a l l y more a c t i v e t h a n t h e corresponding 4-(2-furyl)-4-alkoxy compounds. The h i g h e s t a c t i v i t y i n the alkoxy s e r i e s w a s found w i t h t h e benzoylethyl d e r i v a t i v e ( V I I I a ) ( 4 x meperidine) while among t h e acyloxy compounds t h e phenethyl d e r i v a t i v e ( I X a ) w a s t h e most a c t i v e (5.7 x meperidine). Kugita and h i s colleagues17 continuing t h e i r e a r l i e r work w i t h 3-alkyl-3-phenyl-piperidines prepared a s e r i e s of 3-alkyl-2methyl-3-arylpiperidines of t h e g e n e r a l s t r u c t u r e X . The Nphenacyl d e r i v a t i v e (Xa) was found t o be about as p o t e n t a s CH3 Xa: R=CH3, R'=@OCH2, R"=OH R" &N-R 1 Xb: R=CH3, R'=CH2=CHCH2, R"=OH

U UX

Chap. 4

Analgetics

Harris

43 -

morphine a s an a n a l g e s i c i n mice. The N-ally1 d e r i v a t i v e (Xb) proved t o be an a n t a g o n i s t and was devoid of a n a l g e s i c a c t i v i t y . T h i s is of i n t e r e s t s i n c e t h i s d i f f e r s markedly from t h e meperidine s e r i e s where t h e N-ally1 compound i s a s t r a i g h t forward a n a l g e s i c . A s was p o s t u l a t e d by Archer and H a r r i s , 1 8 a n t a g o n i s t i c a c t i v i t y may be r e l a t e d t o t h e phenethylamine fragment of molecule, a s t r u c t u r a l f e a t u r e which Xb s h a r e s with t h e o t h e r n a r c o t i c a n t a g o n i s t s and which i s l a c k i n g i n Nallylnormeperidine

.

D.

Miscellaneous

Casy and Meyerslg have prepared a s e r i e s of 1 , 2 - d i a r y l - 4 dimethylamino-3-methylbutan-2-ols. S u b s t i t u e n t s i n t h e 1-phenyl r i n g reduced o r a b o l i s h e d a n a l g e s i c a c t i v i t y . Among t h e a c y l e s t e r s , X I was 1.3 x meperidine. An a d d i t i o n a l methylene group CH3 )CH2N( CH3

I2

)CH2N(CH3 )2 C OCH3 X II

XI

XI11

between Cp and t h e C 2 phenyl ( X I I ) d i d n o t a f f e c t a c t i v i t y . However, a methylene group between C 1 and t h e C phenyl ( X I I I ) e s s e n t i a l l y a b o l i s h e d a c t i v i t y . These changes i n a c t i v i t y a r e r e l a t e d t o t h e r e l a t i v e l y r i g i d molecular s t r u c t u r e of X I and X I 1 a s opposed o t h e l e s s crowded more mobile s t r u c t u r e ( X I I I ) . Portoghese2S, I n h i s c o n t i n u i n g s t u d i e s of t h e r e l a t i o n s h i p of s t e r i c f a c t o r s t o b i o l o g i c a l a c t i v i t y has determined t h e a b s o l u t e c o n f i g u r a t i o n of (-1-phenampromide t o be (R)-N-(1-methyl2 - p i p e r i d h o e thy1)propioanilide ( X I V ) . ( R ) -Phenampromide , ( S ) isomethadone, and (2S:3R)-propoxyphene a r e s t e r e o c h e m i c a l l y This r e l a t e d and have g r e a t e r a c t i v i t y t h a n t h e i r enantiomorphs.

xI V i n d i c a t e s a s t e r e o s e l e c t i v e attachment t o a common r e c e p t o r s i t e . I n a l a t e r p a p e r 2 I Portoghese has p u t f o r t h a concept o f t h e mode of i n t e r a c t i o n of n a r c o t i c a n a l g e s i c s w i t h r e c e p t o r s . He p o s t u l a t e s d i f f e r e n t modes of a n a l g e s i c - r e c e p t o r i n t e r a c t i o n s depending on t h e type of s e r i e s of a g e n t . When p a r a l l e l changes i n a c t i v i t y a r e s e e n i n a s e r i e s of compounds w i t h i d e n t i c a l changes i n t h e N - s u b s t i t u e n t , t h i s would suggest s i m i l a r binding

44 -

Sect.

I

-

CNS Agents

B i e l , Ed.

modes. Nonparallel r e l a t i o n s h i p s would then be i n d i c a t i v e of d i s s i m i l a r i n t e r a c t i o n s . He has demonstrated q u a n t i t a t i v e l y , i n a s e r i e s where t h e mode of binding i s s i m i l a r , t h a t a l i n e a r free-energy r e l a t i o n s h i p e x i s t s . He conceives of t h e r e c e p t a r a s having a charged anionic s i t e a s a common p i v o t p o i n t around which the analgesic may be more o r l e s s t i g h t l y bound depending upon i t s s t r u c t u r a l and conformational c h a r a c t e r i s t i c s . I n t h e l i g h t o f t h i s , and o u r t h i ing about t h i s f i e l d over concerning t h e potent the years, t h e note by Axelrod e t ,1.% a n a l g e s i c p r o p e r t i e s of a non-nErogenous s t e r o i d 2CV i s even more s u r p r i s i n g . This compound was r e p o r t e d t o be 40 x morphine i n a modification of the r a t t a i l - f l i c k method when given by the intravenous r o u t e . I n man, t h e compound a d m i n h t e r e d intravenously, was s t a t e d t o r e l i e v e p o s t o p e r a t i v e and chronic cancer p a i n . F'urther d e t a i l s of t h i s work a r e awaited with g r e a t i n t e r e s t . E. Narcotic Antagonists I t i s , perhaps, among t h e group of compounds known a s analgesica n t a g o n i s t s t h a t t h e g r e a t e s t progress has been made i n r e c e n t years i n t h e f i e l d of a n a l g e t i c s . The t o i c has r e c e n t l y been c r i t i c a l l y reviewed by Archer and Harris.y8 The development of pentazocine (XVIa), cyclazocine (XVIb), and cyclorphan (XVII)

has helped t o change our concepts o f drug development i n t h i s a r e a . These agents antagonize t h e a c t i o n s of morphine, a r e g e n e r a l l y i n a c t i v e o r poorly a c t i v e a s analgesics i n animals, yet a r e potent a n a l g e s i c s i n man and have l i t t l e o r no morphinel i k e addiction p o t e n t i a l . 1. Pentazocine I n t h e p a s t year s e v e r a l s t u d i e s 23-27 have confirmed t h e a n a l g e s i c e f f i c a c y of pentazocine. The consensus of opinion i s t h a t 30-40 mg/70 kg is equlvalent t o 10 mg/70 kg of morphine. Side e f f e c t s resembled those seen with morphine and t h e drug was remarkably f r e e of nalorphine-like psychotomimetic p r o p e r t i e s . Like morphine entazocine produces ome degree of r e s p i r a t o r y depression.28'3g Telford and KeatsP8 have shown t h a t t h i s r e s p i r a t o r y depression can r e a d i l y be reversed by t h e a n a l e p t i c , methyl henidate. I n concomitant tilt t a b l e s t u d i e s , I s r a e l e t a1 .so noted hypotensian only r a r e l y with pentazocine. 2 . Cyclazocine This compound i s bout 40 times more potent than morphine a s an a n a l g e s i c i n man.38 A t equianal e s i c doses it produces about the same degree r e s p i r a t o r y depress on as morphine but, l i k e o t h e r analgesic a n t a g o n i s t s , t h e r e is l i t t l e i n c r e a s e i n t h i s

--

f

Chap. 4

Analgetic s

Harris

45 -

depression as t h e dose i s i n c r e a s e d . Cyclazocine produces a high enough incidence of n a l o r p h i n e - l i k e s u b j e c t i v e e f f e c t s t o make it unacc3qtable as a u s e f u l a n a l g e s i c . Martin and h i s group have r e p o r t e d t h a t t o l e r a n c e develops t o t h e s u b j e c t i v e e f f e c t s of t h e drug and t h a t when p a t i e n t s a r e k e p t on high doses f o r prolonged p e r i o d s and a b r u p t l y withdrawn, abstinence s i g n s appear. These withdrawal symptoms a r e r e l a t i v e l y mild and n o t t y p i c a l of those seen w i t h morphiyj. O n t h e b a s i s of t h e s e and o t h e r s t u d i e s , Martin e t a l . have proposed t h e use of cyclazocine i n maintaining n a r c o t i c a d d i c t s f r e e from n a r c o t i c abuse. Under t h e i n f l u e n c e of cyclazocine t h e euphorogenic e f f e c t s of n a r c o t i c s a r e antagonized and t h e development o f p h y s i c a l dependence i s prevented. An o u t p a t i e n t c l i n i c t r i a l of t h i s t h e r a p y i s being c a r r i e d o u t by J a f f e 3 4 and Freedman.35 The i n i t i a l r e p o r t s a r e q u i t e encouraging. 3 . Cyclorphan T h i s drug i s a l s o a p o t e n t a n a l g e s i c i n man. 36 Like cyclazocine, it produces too high an incidence of n a l o r p h i n e - l i k e s u b j e c t i v e e f f e c t s t o be a c l i n i c a l l y u s e i u l a n a l g e s i c . 4. Oripavine D e r i v a t i v e s One of t h e most i n t e r e s t i n g f i n d i n g s i n r s y e n t years has been that ring the o b s e r v a t i o n by Bentley and h i s c o l l e a g u e s expansion of morphine t o t h e s i x - r i n g 6,14-endoethenotetrahydrothebaine o r o r i p a v i n e s t r u c t u r e l e a d s t o agents of s u r p a s s i n g potency. Thus XVIIIa was found t o be n e a r l y 8,000 times more XVII I a: R=CH3,

AN-"

R' =( CH3 ) y2HCH2CH2

XVIIIb: R= v C H 2 , RT=CH3 X V I I I C : R= y 7 C H 2 ,

HO

RT=(CH3)2CHCH2CH2

J

p o t e n t than morphine.38 These compounds have been used extens i v e l y t o immobilize wild animals s i n c e t h e i r high potency makes d a r t i n g q u i t e f e a s i b l e and t h e i r s a f e t y r a t i o i n four-legged animals i s high. Recently Bentley e t G.39 have described t h e s e r i e s . T h i s s i d e chain N-cyclopropylmethyl d e r i v a t i v e s i n has led t o p o t e n t a n t a g o n i s t s i n t h e morphine, morphinan and benzomorphan s e r i e s . I n t h e 6,14-endoethenothebaine s e r i e s when t h e C-alkanol s i d e chain i s isopropanol (XVIIIb) p o t e n t antagonism (35 x n a l o r p h i n e ) i s produced. When R?=isoamyl (XVIIIc) t h e 1000 x compound behaves more l i k e a s t r o n g a n a l g e s i c (250 morphine). There were, however, some q u a l i t a t i v e d i f f e r e n c e s bet een XVIIIc and morphine. Compound XVIIIb has been given t o mango and has been found t o produce a high incidence of n a l o r phine-like s i d e e f f e c t s .

mis

-

46 -

Sect.

I

- CNS Agents

B i e l , Ed.

5. Miscellaneous There has been some q u e s t i o n a s t o whether t h e n a r c o t i c a n t a g o n i s t s a r e competing w i t h t h e n a r c o t i c s f o r a common r e c e p t o r site i n t h e C.N.S. Evidence f a v o r i n g t h i s view f o r nalorphine and l e v a l l o r p h a n w a s p u t f o r t h by Grumbach and Chernov4I in' a c a r e f u l l y c o n t r o l l e d study. They c a r r i e d o u t Gaddum Drug Ratio s t u d i e s i n r a t s w i t h t h e two a n t a g o n i s t s and a v a r i e t y of a n a l g e s i c s . The r a t i o s f o r f o u r p a i r s of drugs were c o n s t a n t over a wide dose range i n d i c a t i n g t h a t t h e antagonism produced i s a tTsurmountable'vone and t h u s t h e drugs a r e competing f o r a common r e c e p t o r s i t e . The s e a r c h f o r a s p e c i f i c animal &qalgesic t e s t f o r t h i s c l a s s of agent has continued. Ward e t a l . compared nalorphine and pentazocine with morphine i n a m o d i m c z i o n of t h e R a n d a l l - S e l l i t o e T e s t . Winter and F l a t a k e r 4 3 described a more complete study encompassing s e v e r a l a n t a g o n i s t s . Nalorphine, pentazocine, cyclazocine, and N-cyclopropylmethylnormorphine r a i s e d t h e t h r e s h o l d of t h e y e a s t inflamed f o o t but had no e f f e c t on t h e c o n t r o l paw. (-)-3-Hydroxy-N-cyclopropylmethylnorisomorphinan was i n a c t i v e . There was l i t t l e r e l a t i o n s h i p between t h e dose necessary t o r a i s e e t h r e s h o l d and t h e g i i n i c a l e f f i c a c y of t h e drugs. Taber e t al.6k and Blumberg et &. report the effect i v e n e s s of t h e a n t a g o n i s t s i n t h e mouse phenylquinone-writhing t e s t . This has been expanded by P e a r l and H a r r i s . 4 6 To d a t e , a c t i v i t y i n t h i s t e s t g i v e s t h e b e s t c o r r e l a t i o n with c l i n i c a l efficacy. 111. Weak Analgesics Much of t h e l a b o r a t o r y work i n t h i s f i e l d has r e c e n t l y been reviewedl a s has t h e c l i n i c a l pharmacology.47 C e r t a i n a s p e c t s o f t h i s drug c l a s s a r e covered i n g r e a t e r d e t a i l i n t h e Chapter on Non-Steroidal Anti-Inflammatory Agents i n t h e p r e s e n t volume. A. New Agents Takamura and h i s colleagues48 have r e p o r t e d a s e r i e s of a n i l i n e d e r i v a t i v e s of t h e type X I X and XX. The compounds X I X a and b were t h e most p o t e n t a n a l g e s i c s . I n an i n t e r e s t i n g study,

--

CH2CH2C0RXIXa: R=N(CH3)2, XIX

--

XIXb: R=N(CH312,

R'=CH2=CHCH20 R'=p-Br

E h r h a r t e t a1.49 have prepared a s e r i e s of P-hydroxybutyrates and a-dimethylamino-P-hydroxybutyrates o f p-phenetidine and N-methyl-p-phenetidine. These compounds e x h i b i t e d a n a l g e s i c a c t i v i t y of about t h e same l e v e l a s phenacetin but appeared t o produce much l e s s methemoglobin formation. This w a s a t t r i b u t e d t o a metabolic breakdown d i f f e r e n t from t h a t of phenacetin. T h a t i s , t h e r e w a s no formation of p-phenetidine. Casadio e t a l . 50 described a s e r i e s of a,a - d i s u b s t i t u t e d p h e n y l a c e t o n i t r n e s , l-naphthylacetonftriles, phenylacetamides, and l-naphthylacetamides, many of which had a n a l g e s i c and a n t i -

Chap. 4

47 -

Harris

Analge tic s

inflammatory p r o p e r t i e s . Most a c t i v e were t h e l-naphthylacetamfdes, i n p a r t i c u l a r , naphthypramide XXgl which is c u r r e n t l y undergoing c l i n i c a l t r i a l . P a l a e t a l . suggested, from s t r u c -

--

c-c-c

\

N-C-C-C

'i""

XXI ' A corn2 XXII t u r e - a c t i v i t y s t u d i e s of t h e whole s e r i e s , t h a t X X I I r e p r e s e n t s t h e optimal s t r u c t u r e f o r a n a l g e s i c a c t i v i t y . Nishino and h i s group,52 i n a continuing study, prepared a s e r i e s of 1 , 4 - b i s ( s u b s t i t u t e d phenoxyacety1)piperazines XXIII, which showed a n a l g e s i c a c t i v i t y . These compounds a l s o appear t o have C .N.S. 0

R-~OCH20C-N7-com20~-R

W

XXIII

XXIV

Olin and Cashinfi3 r e p o r t e d a s e r i e s of cycloalkanespiro-5-hydantoins (XXIV), which were a c t i v e i n t h e mouse w r i t h i n g t e s t when R o r R' was a s m a l l a l i p h a t i c group o r when R ' was a c e t y l . These compounds a l s o had anti-inflammatory p r o p e r t i e s . Meyer e t a1.54 prepared a group of 3-amfno-2,lb e n z i s o t h i a z o l e s X c a l a r g e number of which showed a n t i n o c i c e p t i v e a c t i v i t y i n mice which was n o t c o r r e l a t e d w i t h t h e i r a n t i b r a d y k i n i n a c t i v i t y . Some resembled aminopyrine i n being Rll I XXVIa: R=p-S02CH3, R ' =Rfl=H depressant p r o p e r t i e s .

as

R l e k T R 1 '

R"

RON. R ' XXV

" er-R

XXVIb: R=p-S02CH3, R ' = H , XXVIc: R=p-S02CH3, R1=H Rii= ( CH3 2

~

R1'=NH2

~

~

2

et a1.55 r e p o r t e d a s e r i e s of a c t i v e i n both t e s t s . Almirante imidazol-[ 1 , P - a I p y r i d i n e s of t h e g e n e r a l s t r u c t u r e XXVI. The most i n t e r e s t i n g a c t i v i t y w a s found i n t h e methylsulfonyl analogs (XXVIa, b, and c ) . There have been r e p o r t s s 6 t h a t t h e dimethylaminoethanol s a l t of 2-(4-biphenyl) b u t y r i c a c i d (namoxyrate) i s an e f f e c t i v e a n a l g e s i c . T h drug appears t o be r a p i d l y absorbed a f t e r o r a l a d m i n i ~ t r a t i o nreaching ~~ a m a x i m u m plasma l e v e l a t one hour. Namoxyrate and/or i t s m e t a b o l i t e s a r e e x c r e t e d slowly over s e v e r a l days. The animal pharmacology has n o t , as y e t , been reported. Mefenamic a c i d X X V I I continues i n a c t i v e c l i n i c a l r e s e a r c h while another compound i n t h i s s e r i e s M N I I I has been r e p o r t e d t o be 150 x more p o t e n t than a s p i r i n (1). Bfsse158 r e p o r t e d

48 -

Sect. I

- CNS Agents

B i e l , Ed.

mefenamic acid t o be equivalent t o codeine compound and superior t o partgetamol i n a double blind crossover study. Williams c a r r i e d out a double blind e v a l u a t i o n of a s p i r i n and et al. X X V I I f o r t h e i r a b i l i t y t o a l t e r experimental ischemic arm pain. Both s i g n i f i c a n t l y increased pain threshold w i t h no s i g n i f i c a n t d i f f e r e n c e between medications. No placebo c o n t r o l was run. An Indomethacin XXIX i s now being market d i n t h e U.S.A. e d i t o r i a l i n t h e B r i t i s h Medical .Journal6g sums up r e c e n t experience with t h i s drug. I n general, it has proven t o be an e f f e c t i v e m i l d analgesic, a l b e i t with a r e l a t i v e l y high incidence of s i d e e f f e c t s . These s i d e e f f e c t s , however, a r e considerably lower when t h e drug i s given by capsule and the dose c a r e f u l l y adjusted. Indomethacin i s p a r t i c u l a r l y u s e f u l i n t h e treatment of many of the rheumatic d i s e a s e s . B. Miscellaneous Portoghese61, continuing h i s s t u d i e s of t h e r e l a t i o n s h i p of s t r u c t u r a l conformation tQ a c t i v i t y , prepared t h e enantiomers of N-2 - (p-me thylbenzylme thy1amino)propyl propioanilide and i t s Npropyl analog. The S-(-)-isomers had g r e a t e r a c t i v i t y . There was a l e s s e r potency r a t i o between the isomers of the N-methyl and t h e N-propyl compound. This was a t t r i b u t e d t o a diminution i n t h e s t e r e o s e l e c t i v i t y of the analgesic r e c e p t o r s and might be r e l a t e d t o d i f f e r i n g modes of analgesic-receptor bindin I t has become apparent from the s t u d i e s of GilfoildfS;t h a t inflammation and hyperesthesia a r e n o t a s i n g l e phenomenon. Many agents produce swelling without a t t e n d a n t increase i n pain s e n s i t i v i t y . A f a c t o r o r f a c t o r s which a r e r e l e a s e d by c e r t a i n agents which produce inflammation may be r e l a t e d t o hyperesthesi This f a c t o r i s n o t s e r o t o n i n o r bradykinin. Winter and F l a t a k e r$3 using a v a r i e t y of hyperesthesia-producing agents, have shown t h a t indomethacin and t h e narcotic-antagonists can reduce hyperesthesia a t doses which do not a f f e c t swelling and which do n o t a f f e c t t h e pain threshold of uninflamed t i s s u e . This might r e v e a l a c e n t r a l analgesic a c t i o n i n c e r t a i n mild analgesics unrelated t o t h e i r p e r i p h e r a l a c t i o n . This work appears t o be leading t o a g r e a t e r understanding of inflammatory pain and how the mild analgesics exert t h e i r effects.

.

Analge tic s

Chap. 4

Harris

49 -

References

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

7. 8. 9.

10. 11* 12.

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

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

G. de Stevens, e d . , A n a l g e t f c s , A c a d e m i c P r e s s , N.Y. and Lond.. 1965. W.H. P i r k l e and M. G a t e s , J. Org. C h e m . , 30, 1769 (1965). Y.K. S a w a , N. T s u j i , K. O k a b e and T. M i y a z t o , Tetrahedron, 21, 1 1 2 1 (1965). T K . S a w a , M. H o r i u c h i and K. Tanaka, i b i d . , 2 1 , 1133 (1965). E . L . Way, J . M . Y o u n g and J . W . Kemp, B u r N a r G t f c s , 27, 25 (1965). M. C o h e n , A . S . K e a t s , W. K r i v o y and G . U n g a r , Proc. SOC. E x p . B i o l . Med., 119, 3 8 1 (1965). E . F r o m m e l , A r z n e i m i t t e l - F o r s c h . , IS, 80 (1965). C . F . C h i g n e l l , J . H . A g e r and E . L . M a y , J. Med. C h e m . , 8, 235 (1965). C . F . C h i g n e l l and E . L . May, i b i d . , 8, 385 (1965). A . E . Jacobson and E . L . M a y , ibid., 8 , 563 (1965). B . C . J o s h i , C . F . C h i g n e l l a n m L . Ray, i b i d . , 8, 694 (1965). S . A r c h e r , N.F. A l b e r t s o n , L.S. H a r r i s , A.K. Pierson and J . G . B i r d , -.' %bid 7, 1 2 5 (1964). T . K a m e t a n i , K. K i g a s a w a , M. H f i r a g i and H . I s h i m a r u , C h e m . Pharm. B u l l . Tokyo, 13, 295 (1965). T . K a m e t a n l , K. K i g a G w a and T. H a y a s a k a , i b i d . , 13, 300 (196 5). D.G. H a r d y , R . E . L i s t e r and E . S . S t e r m , J.m.C h e m . , S, 847 (1965). A . F . C a s y and N.A. A r m s t r o n g , $ b i d . , 8, 57 (1965). H. K u g i t a , 0. T o y a n a r i , H. Inoue and G. H a y a s h i , ibid., 8, 313 (1965). S . A r c h e r and L.S. H a r r i s , Progress i n Drug Research, ed E . Jucker, B k k h g u s e r V e r l a g , B a s e l , V o l . 8 (1965). A . F . C a s y and J . L . Meyers, J. C h e m . S O C . , 4092 (1965). P.S. Portoghese, J. M e d . C h e m . , (1965 1. P . S . Portoghese, P b i d . , 8, 609 (Fb65L1;: L . R . Axelrod, P.N. Rao and D.H. B a e d e r , J. Am. C h e m . SOC -788 856 (1966). M. E n d e , J. Am. G e r . SOC., l3, 775 (1965). R.A. G o r d o n and J . H . Moran, J. C a n . A n e s . SOC., ,2.l 3 3 1 (1956 J . R . H i n s h a w , K . E . K o b l e r , and A.R. B o r j a , Am J. Med. Sci., 251, 57 (1966). M . S . Sadove and R . C . B a l a g o t , J. Am. Med. A s s o c . , 193, 887 (1965). V.K. S t o e l t i n g , A n e s t h e s i a 81 A n a l g e s i a , 44, 769 (1965). J. T e l f o r d and A . S . K e a t s , C l i n . P h a r m . T h e r a p . , 6, 12 (1965) J . W . B e l l v i l l e and J. G r e e n , i b 1965 J . S . I s r a e l , G.T. Jansen and ~ . ~ o ~ i l t ~ ' A ~ e s t h e s i o l o26, p;y, 253 (1965). - - -L. Lasagna, T . J . D e K o r n f e l d and J . W . Pearson, J. Pharmacol.

.

-

.,

@, 12 (1964).

H . F . Fraser, H.C. G o r o d e t z k y and D.E. R o s e n b e r g , J. Pharmacol., 150, 426 (1965). W.R. M a r t i n , C.W. G o r o d e t z k y and T . K . M c C l a n e , Pharmacologist, 7. 163 (1965). ~-~ J. J a f f e and-L. B r i l l , I n t . J. A d d i c t i o n s , 1, 99 (1966). -7

~

-

50 -

Sect. I

- CNS Agents

B i e l , Ed.

35. A.M. Freedman, P e r s o n a l communication. 58, 978 (1965). 36. L. Lasagna, F'roc. Roy. SOC. Med 37. K.W. Bentley, Endeavor, 23, 91 38. R.E. L i s t e r , J. Pharm. Pharmacol., l6, 364 (1964). 39. K.W. B e n t l e y , A.L.A. Boura, A . E . F i t z g e r a l d , D . G . Hardy,

ii9m.

A . McCoubrey, M.L. Aikman and R . E .

(1965).

L i s t e r , Nature,

9,102

40. A . S . Keats, P e r s o n a l communication. 41. L . Grumbach and H . J . Chernov, J. Pharmacol. Exp. Therap., 150, 385 (1965). 42. J . W . Ward, M. Foxwell and W.H. Funderburk, Pharmacologist, 1, 163 (1965). 43. C . A . Winter and L . F l a t a k e r , J . Pharmacol. Exp. Therap., 150, 165 (1965). 44* R.I. Taber, D.D. Greenhouse and S . I r w i n , Nature, 204, 189 (1964). 45. H. Blumberg, P.S. Wolf and D.B. Hyman, P r o c . SOC. Em. B i o l . Med --, 118, 763 (1965). 46. J. P e a r l and L.S. H a r r i s , J. Pharmacol. Exp . Therap., i n p r e s s . 47. W.T. Beaver, Am. J. Med. S c i . , 250, 5//=963). N . Asada, S . Takaku, 48. K. Takamura, A . S h i o y a , i n a m o t o , A . Yoshimitsu and Y . N i t t a , Chem. Pharm. B u l l . Tokyo, 13, 198 and 205 (1965). 49. G . E h r h a r t , E. L i n d e r , and A. KSussler, A r z n e i m i t t e l - F o r s c h . , 15, 727 (1965). 50. S. Casadio, P. Gianfranco, E . C r e s c e n z i , T . Bruzzese, E . Marazzi-Uberti, and G . Coppi, J. Med. Chem., 8, 589 and 594 (1965). 51. G . P a l a , S. Casadio, T . Bruzzese, E. C r e s c e n z i , and V.E. Marazzi, -* i b i d ' 8, 698 (1965). 52. K. NishTno, N. I c h i n o s e k i , K. S h l s h i d o , and T . I r i k u r a , 2. Pharm. S O C . Japan, 85, 715 (1965). 53. W. O l d f i e l d and C.H. Cashin, J. Med. Chem., 8, 239 (1965). 54. R.F. Meyer, B.L. Cummings, P. Bass and H.0.J: C o l l i e r , %bid. 8, 515 (1965). 55. L. Almirante, L. Polo, A. Mugnaini, E . P r o v i n c i a l $ , P. Rugarli, A . B i a n c o t t i , A. Gamba and M . Murmann, i b i d . , 8, 305 (1965). 56. A. Cohen and E.A. DeFelice, J. New Drug-, 153 (1965). 57. 0. Resnick, E.A. D e F e l h e , J . R . Bergen and H . Freeman, C u r r . T H . , 7, 443 (1965). s s e 1, P r a c t i t i o n e r , 194, 817 (1965). 58. 59. M.W. W i l l i a m s , C.S. W i l l i a m s , and M. J. Kartchner, T o x i c o l . A 1. Pharmacol., 7, 45 (1965). 60. Mzd. J., 1329 (1965). 61. T.M. G i l f o i m I. K l a v i n s , Am. J. P h y s i o l , 208, 867 (1965). 62. P.S. Portoghese and T.N. R i l e y , J. Pharm. S c i y z , 1831 (1965).

.-

51 Chapter 5.

Anorexigenic Agents

George I. Poos, McNeil Laboratories, Inc., F o r t Washington, P a .

Introduction - Because of its efficacy and widespread clinical usage, amphetamine has been the standard anorexigenic drug for some time. L 2 , 3 Efforts to reduce the central stimulant and cardiovascular effects of a m phetamine have led to the development o v e r the y e a r s of a number of new products which, hopefully, a r e f r e e of undesirable side effects. Excellent s u m m a r i e s of these developments a r e available. 4, 5* 6~ Along with these new drugs t h e r e has also developed a controversy. A r e t r u e anorexigenic and central stimulant effects s e p a r a b l e ? 6 y Can drug control of a "hunger center" in the l a t e r a l hypothalamus9*"8 11, l2 o r ~ 6 of a "satiety center" i n the ventromedialhypothalamus 13, l4, 1 5 ~ selectively overcome powerful psychological factors that may be associated with obesity? If appetite and hunger a r e effectively drug-controlled in a completely selective manner, w i l l the obese patient be m i s e r a b l e f r o m deprivation without substitution ? Although investigations of anorexigenic agents in 1965 do not s e e m to have provided c l e a r - c u t answers to a l l of these questions, we may well be getting c l o s e r to the truth. Newer Drugs - Both the laboratory and clinical investigation of amphetamine congeners h v e received considerable attention over the p a s t s e v e r a l y e a r s . Of greatest i n t e r e s t s e e m to have been the chloro, trifluoromethyl and alkyl-substituted amphetamine analogs.

-a,a-dimethylphenethylamine)(I), which Chlorphentermine (p-chloro h a s been known f o r some time as an anorectic i n animals' with little CNS stimulant effect, l8 was introduced into g e n e r a l clinical use in April, 1965. At effective anorexigenic dosa e s chlorphentermine seems t o p r o duce little o r no central stimulation 18*2 b , 21 and h a s been reported to c

~

c

I

''

FH3

H - N ~H ~ -

~

CH3 22

produce mild sedation in the f o r m of drowsiness and relaxation. Since chlorphentermine has been shown t inhibit l a t e r a l hypothalamic stimulus induced eating in the satiated c a t as well a s altering the evoked

52 -

Sect. I - CNS Agents

B i e l , Ed.

23

potentials of hypothalamic "appetite centers", answers to the questions mentioned e a r l i e r m a y come f r o m observation of patient and physician acceptance of this new drug. T h e r e a r e a number of r e p o r t s on the investigation of ring-substituted amphetamine analogs (11). A s e r i e s of 4-chloro, 2,4-dichloro and 3,4-dichloro compounds were only of modest i n t e r e s t . 24 An a s s o r t m e n t of analogs monosubstituted at the 2 , 3 and 4 position with methylthio, bensyl-

I1 thio , me thyl s ulf on yl, d imethylami no, isop r opyl, amino s ulf onyl, am in0 , benzoylamino, methylsulfonylamino and trifluoromethyl were examined for anorectic activity in the rat and dog. 25 Only the p-dimethylamino and m - and p-trifluoromethyl derivatives showed m a r g e d activity. The trifluoromGthy1 compounds appear to have little CNS stimulant effect and have been investigated extensively. 2 6 , 2 7 9 289 29* 303 31 The t h r e e i s o m e r i c monofluoro amphetamines a r e a l s o reported to have substantial anorectic activity. 31

-

Recent studies on N-substituted amphetamines have included the n was varied f r o m 2 to 5. In this s e r i e s , N-alicyclic compounds I11 where -

111

IV

V

only the N-cyclop pyl compound was effective in depressing the food consumption of r a t s . 3% Phentermine I V l 7 and Z-phenyl-3-methyl-3-aminobutane (V) 33 a r e recent examples of active amphetamine analogs substituted on the side chain. Multiple substitution such as with the N-methyl-ubenzyl-P-phenethylamine (VI), 34 the N-benzyl-p-chloro compound VII, 35 N -me thyl -E-pentylamphetamine VIII, 6 , and 5 -ethyl -m -trifluoro de riva tive I X (fenfluramine) 38,39,40,41, 42343 has given active compounds of interest. Compounds I, I V , V and I X have been compared with d-amphetamine as to potency and duration of the inhibition of food consumption in r a t s . 4 4 Both chlorphentermine I and fenfluramine IX were l e s s potent d-amphetaa t two hours but of about the same potency a t twelve h o u r s as mine. In a t h r e e day, multiple-dosing experiment, they were clearly superior. 44 Metaboiic studies on chlorphentermine 45 and fenfluramine 42

-

Chap. 5

Anorexigens

53 -

Poos

showed a relatively slow catabolism which undoubtedly contributes to their longer duration of action.

VI

VII

VIII

IX

Research activity with anorexigenic compounds more remotely related in structure to amphetamine continues. In most cases, one can detect a basic nitrogen atom approximately two carbons distant from a benzene ring.

A group of 4 - a m i n o r n o r p h o l i n e ~related ~~ in structure to phenmetrazine were examined f o r their CNS effects.47 The most interesting compound proved to be the N-( 1 -phenyl-2 -propylamino) derivative X, NH II

0-NH - C -NH

A

2

XI

which is an N - N combination of amphetamine and phenmetrazine. Compound X was shown to be an active anorexigenic agent with a behavioral profile different from that of amphetamine. 47 Aralko guanidines have recently been reported to have anorexigenic activity. 4T ' he most active compound, benzyloxyguanidine ( XI), i s about 1/ 5th a s active in inhibiting food consumption in dogs as d-amphetamine. The discovery of anorexigenic activity for certain 2, 5-benzodiazocines 49 provides another new class of potentially useful compounds. The pharmacological 50, 51 reports on 1-(e-chlorophenyl)-l,Z, 3,4, 5,6hexahydro-2,5-benzodiazocine (XII) describe potency in the range of amphetamine with reduced central stimulant and cardiovascular actions. The potent anorexigen Z-amino-5-phenyl-2-oxazoline(MII, aminorex), which was described several y e a r s ago along with a s e r i e s of analogs, 5 2 3 53

54 -

Sect. I

-

CNS Agents

XI11

B i e l , Ed.

XIV

has been reported to be a n effective drug in obese diabetics. 54 In both r a t s and man, aminorex was shown to be rapidly metabolised and eliminated in the urine. 55 The structural1 s i m i l a r 2-dimethylamino-4-phenyl-2-oxazolin-4-one (XIV, thozalinone)x6 is a l s o reported to be an active anorexigen with l e s s acute toxicity and a longer duration of action than amphetamine when compared in mice. 57 Large doses of a-methyl-m-tyrosine have been found to d e c r e a s e the consumption of a sweetened m i l k s o l u t i o n by r a t s . 58 The long-lasting3 dosedependent effect is thought to be due to metabolites of a-methyl-m-tyrosine. 58 Anorexia has been attributed to imipramine. 59 Iproniazid h a s been shown to potentiate the anorexigenic effects of L-E-cocaine in' rats. 6o The anorectic effect of L-n-cocaine is attributed to an amphetamine-like effect i n the CNS r a t h e r t h a n t o i t s local anesthetic activity. 6o Pharmacological Testing Methods - Classical procedures have measured drug effects on food consumption, weight gain o r both. 61, 6 2 * 63 Many r e finements of these methods have been described. 64 One of the m o r e i n t e r e s t ing variations has been the use of obese animals, obtained either by destruction of the ventromedial nuclei 12365 o r by chemical methods such a s the aurothioglucose -obese mou.se. 66s 67 The inhibition of drinking behavior h a s also been used as an index of anorexigenic activity. Effects on the controlled, s h o r t - t e r m consumption of beef broth i n rats have been suggested to m o r e clearly reflect appetite suppression than feeding studies. 53 The u s e of sweetened milk solutions with rats also s e e m s to give satisfactory test r e s u l t s . 58 P e r s p e c t i v e s - Most r e s e a r c h i n the anorexigenic a r e a in the p a s t has concentrated around compounds that a r e either chemically a n d / o r biologically similar to amphetamine. As a result, many drugs a r e known which inhibit food consumption and concommitant weight i n c r e a s e s in a manner s i m i l a r to amphetamine. Available evidence suggests that s e v e r a l of these compounds e x e r t their effect by a r a t h e r specific action in the hypothalamus without producing the central stimulant effects which a r e also associated with amphetamine. We now m u s t wonder i f such a drug alone will provide

Chap. 5

Anorexigens

Po0 s

55 -

adequate and reasonable therapy in obese humans. P e r h a p s a second p h a r macological component will be n e c e s s a r y . Possibly this would be a "psychic stimulant", "psychic energizer" o r anti-anxiety effect n e c e s s a r y to substitute f o r reduced food intake o r relieve the underlying cause of overeating. It has been stated that over 90% of obesity i n humans is psychogenic in origin. 6 Although only centrally acting anorexigenic agents have been considered thus f a r in this review, the possibility should not be overlooked that metabolic, biochemical o r hormonal factors might be drug controlled to combat obesity. Both d-amphetamine68 as well as chlorphentermine, methylphenidate, pipr adol, i m i p r amine, desipramine and a m i t r iptyline show a marked lipid mobilizing activity i n r a t s as shown by i n c r e a s e s in plasma f r e e fatty acids. 69

-

An anorexigenic and faf mobilizing substance (FMS)has been i s o lated f r o m the urine of fasting r a t s . 70 It has been partially fractionated and i s thought to be polypeptide in nature. 71s 72 The function and mechanism of action of F M S remain obscure. Summary - Even though 1965 did not produce any obvious breakthroughs in the a r e a of anorexigenic agents, t h e r e was one significant development. Chlorphentermine was introduced into widespread clinical usage in the United States during 1965. This drug appears to have a d i r e c t amphetaminelike anorectic effect in the hypothalamus with a much lower incidence of CNS stimulation than previous anorexients. Thus, an evaluation of the a c ceptance of chlorphentermine m a y provide an answer a s to whether a "pure anorexigen" is d e s i r a b l e in the t r e a t m e n t of obesity. If i t turns out that some other c e n t r a l effect is a d e s i r a b l e attribute of an anorexigenic agent, then a s u p e r i o r drug may be available f r o m a variety of active compounds that a r e in various stages of development. These v a r y f r o m amphetamine analogs to somewhat different s t r u c t u r e s with a range of both quantitative and qualitative pharmacological differ ences f r o m amphetamine. Research effort continues to be concentrated on amphetamine -like anorexigenic action. The possibility of effective drug control of obesity by a completely different mechanism should not be overlooked.

56 -

Sect. I

- CNS Agents

B i e l , Ed.

References

(1)

E. Davidoff and E. L. Reifenstein, J r . , J. Am. Med. AS'SOC.,

108, 1770 (1937).

H. Nathanson, J. A m . Med. A s s o c . , W 528 (1937) Ulrich, New Eng. J. Med. 696 (1937). A. N u s s b a u m e r and P. B u r i , S-=,, (1965). (5) L. S. Goodman and A. Gilman, "The P h a r m a c o l o g i c a l B a s i s of Therapeutics", T h e Macmillan Company, New York, N. Y . , 1965, pp. 501, 516. (6) W. Modell, "Drugs of Choice", The C. V. Mosby Company, St. Louis, M i s s o u r i , 1964, pp. 313-323. (7) J. R. B o i s s i e r , Therapie,& 803 (1962). (8) S. K. F i n e b e r g , J. Am. Med. A s s o c . , E , 680 (1961). B. K. Anand and J. R. Brobeck, -Y a l e J. Biol. Med., a 1 2 3 (1951). (9) 143 (1955). (10) B. K. Anand, S. Dua and K . Shoenberg, J. Physiol. 248 (1957). (11) S. D. M o r r i s o n and J. M a y e r s , J. P h y s i o l . , e 13,114 (1957). (12) F. R. Stowe, Jr. and A. T. Miller, J r . , E (13) J. R. Brobeck, J. T e p p e r m a n and C.N.H. Long, Y a l e J. Biol. Med., l5, 831 (1943). 358 (14) J. R. Brobeck, S. L a r s s o n and E. Reyes, J. P h y s i o l . , (1956). 89 (1943). (15) A. W . Hetherington, A m e r . J. P h y s i o l . , (16) A. W. Hetherington and S. W. Ranson, A m e r . J. P h y s i o l . , 36, 609 ( 1942). (17) T. Hohn, I. Huus,R. Kopf, I. Moller-Nielsen and P. V. P e t e r s e n , Acta. P h a r m a c o l . Toxicol., l7, 121 (1960). (18) J. A. Gylys, J . J . D . H a r t and M. R. W a r r e n , J. P h a r m a c o l . Exp. T h e r a p . , E,365 (1962). (19) E. I. B o x e r , P r a c t i t i o n e r , 18_7, 214 (1961). (20) L. A. Rudolph and I. MacLachlan, C u r r e n t T h e r a p . R e s . , 4, 629 ( 1 962). (21) H. I. Russek, A m . J. Med. S c i . , 9, 305 (1965). (22) A. Di M a s c i o and D. H. Buie, Clin. P h a r m a c o l . T h e r a p . , 5, 174 (1964). (23) J. F. Emele and M. M. Winbury, Symp. Obesity, Roy. Coll. S u r g . , 22 (1963). (24) J. E. Owen, J r . , J. P h a r m . Sci., 52, 679 (1963). (25) G. F. Holland, C. J. Buck and A . Weissman, J. Med. Chem., 519 (1963). (26) A . W e i s s m a n , F e d e r a t i o n P r o c . , I & 168 (1959). (27) A. W e i s s m a n , J. Exp , g, 271 (1959). (28) A. W e i s s m a n and J. A. Schneider, P h a r m a c o l o g i s t , 2, 71 (1960). (2) N. H. (3) P. (4) 286, 365

217,

,E, E,

-

z,

-

a,

-

Chap. 5

Ano rexig e n s

57 -

Poos

Ref e r ence 8 (29) C. A. Leonard, T. Fujita, D. H. Tedeschi and E. J. Fellows, Pharmacologist, 2, 79 (1961). (30) A. Kandel, R. B. Miller, C. A. Leonard and E. J. Fellows, Pharmacologist, 2, 78 (1961). (31) J. C. LeDouarec and L. Beregi, Conf. Hung. Therap. Invest. P h a r macol. , 2, Budapest (1962), pp. 115-125 [Chem. Abs., 63, 227113 (1965)l. w (32) M. F r e i f e l d e r , J. Med. Chem., &, 813 (1963). (33) W. A. Behrendt and R. Deininger, Arxneimittel-Forsch, 7x1 (1963). (34) V. 0. Nieschutz and G. Schneider, Arxneimittel-Forsch, ,l4, 104 (1964). (35) M. F r e i f e l d e r , Y. H. Ng and F. N. P r e s t o n , J. Med. Chem., 1, 381 (1964). (36) M. Morin-Jomain, J. A b r a h a m and R. Jacquot, Arch. Sci. Physiol., 17, 261 (1963). (37) F r e n c h P a t e n t M2563, June 29, 1964 [Chem. Abs., 6l, P 16014d (1964)); Unlisted Drugs, 17, 62a (1965); Unlisted Drugs, l8, 27c (1966). (38) J. Vague and J. L. Codaccioni, Rev. P r a t i c i e n , l2, 3533 (1962). (39) P. Lambusier, Presse Med., 7 l , 181 (1963). (40) R. S. Alphin, W. H. Funderburk and J. W. Ward, Tox. Appl. P h a r macol. , p, 340 (1964). (41) B. V. Franko, L. J. Honkomp and J. W. W a r d , J. P h a r m . P h a r m a col., l7, 222 (1965). (42) J. Duhault and S. F e n a r d , A r c h . i n t e r . Pharmacodyn., 251 (1965). (43) H. Schmitt and J. C. LeDouarec, Therapie, 20, 111 (1965). (44) V. K. Opitz, F. K e m p e r and A. L o e s e r , A r z n e i m i t t e l - F o r s c h . , s , 278 (1965). (45) B. Dubnick, G. H. Leeson, R. Leverett, D. F. M o r g a n a n d G . E. Phillips, J. P h a r m a c o l . Exp. Therap., E, 85 (1963). 427 (1964). (46) M. J. Kaln, J. Med. Chem., 433 (1964). (47) D. J. Knapp, J. Med. Chem., (48) D. G. Martin, E. L. Schumann, W. Veldkamp and H. Keasling, J. Med. Chem., 8, 456 (1965). (49) T. S . Sulkowski and M. H. W i l l e , A b s t r a c t s 150th A.C.S. Meeting, Atlantic City, N. J., September 13-16, 1965, p . 16P. (50) M. I. Gluckman, Pharmacologist, 1, 146 (1965). (51) T . Baum, Pharmacologist, 1, 147 (1965). (52) G. I. Poos, J. R. C a r s o n , J. D. Rosenau, A. P. Rosxkowski, N. M. Kelley and J. McGowin, J. Med. C h e m . , 9, 266 (1963). (53) A. P. Roszkowski and N. M. Kelley, J. P h a r m a c o l . Exp. Therap. , 367 (1963).

,a

3

-

-

E,

z, z,

s,

58 -

Sect. I

- CNS Agents

B i e l , Ed.

References (54) L. C. Wood and J. H. Owen, J r . , J. New Drugs, 53, 181 (1965). The name "aminoxaphen" was used instead of the c o r r e c t adopted name "aminorex" f o r compound XIII in this article. (55) G. A. Braun, G. I. P o o s and P. C. Johnson, Fed. Proc., 546 (1965). (56) C. F. Howell, N. Q . Quinones and R. A. Hardy, J r . , J. Org. Chem., 27, 1679 (1962). (57) E. N. Greenblatt and H. C. Osterberg, Toxicol. Appl. P h a r m a c o l . , 7, 566 (1965). (58) P. L. Carlton and A. R. Furgiuele, Life Sci., 4, 1099 (1965). (59) H. Chimenes, P r e s s e Med., 72, 1131 (1964). (60) G. Schmidt, Arch. i n t e r . Pharmacodyn., %, 87 (1965). (61) M. L. Tainter, J. Nutr., 27, 89 (1944). (62) W . Sangster, M. I. G r o s s m a n and A. C. Ivy, Am. J. Physiol., 152, 259 (1948). (63) H. Wendel, F. M. Sturtevant and I. Shemano, Pharmacologist, 2, 71 (1960). (64) B. Andersson and S. Larsson, Pharmacol. Rev., l3, 1 (1961). (65) See, f o r example, H. J. Adams and L. C. Greene, J. P h a r m . Sci., 22, 1405 (1964), and G. Stille and H. Lauener, Helv. Physiol. Pharmacol. Acta, 22, C 46 (1964). (66) G. Friedman, L. A. Weingarten and H. D. Janowitz, A m . J. Clin. Nutr., lo, 225 (1962). (67) P. D. Cullen and H. A. Swartz, Canad. P h a r m . J., 97, 33 (1964). (68) R. Santi and G. F a s s i n a , J. P h a r m . Pharmacol., 131 (1964). (69) R. Santi and G. Fassina, J. P h a r m . P h a r m a c o l . , l7, 596 (1965). ( 7 0 ) J. A. F. Stevenson, B. M. B o x a n d A. J. Szlavko, P r o c . SOC. Exp. Biol. Med., 9, 424 (1964). (71) J. R. Beaton, A. 3. Szlavko and J. A. F. Stevenson, Can. J. Physiol. P h a r m a c o l . , 42, 647 (1964). (72) J. R. Beaton, A. J. Szlavko, B. M. Box and J. A. F. Stevenson, Can. J. Physiol. P h a r m a c o l . , 42, 657 (1964). ,

a,

u

-

16,

S e c t i o n I1 Editor:

- Pharmacodynamic A g e n t s

S , Archer, Sterling-Winthrop Research I n s t i t u t e R e n s s e l a e r . N e w York

Chapter 6. E,

C.

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

K o r n f e l d , E l i L i l l y and Company, I n d i a n a p o l i s , I n d .

Of t h e t w e n t y - t h r e e new c h e m i c a l e n t i t i e s i n t r o d u c e d i n t o m e d i c i n e i n t h e U n i t e d S t a t e s i n 1965, none were c o n c e r n e d The y e a r w a s c h a r primarily w i t h antihypertensive therapy,' a c t e r i z e d , t h e r e f o r e , by t h e e x t e n s i o n of e s t a b l i s h e d l e a d s r a t h e r t h a n by r a d i c a l i n n o v a t i o n . The f o l l o w i n g g r o u p s of drugs received a t t e n t i o n :

Diuret i c s The o r a l d i u r e t i c s of t h e t h i a z i d e or r e l a t e d c l a s s c o n t i n u e d t o be t h e one most i m p o r t a n t g r o u p of a g e n t s for u s e e i t h e r a l o n e or, more o f t e n , i n c o m b i n a t i o n w i t h o t h e r hypot e n s i v e drugs. T h i s a r e a i s r e v i e w e d i n d e t a i l i n C h a p t e r 7. Use of t h e t h i a z i d e s w i t h p o t a s s i u m c h l o r i d e t o m a i n t a i n p o t a s s i u m b a l a n c e h a s l e d t o small bowel u l c e r a t i o n p r o b l e m s ; 2 h o w e v e r , a d m i n i s t r a t i o n of p o t a s s i u m i n r e a d i l y d i s p e r s i b l e or slow-release forms a p p e a r s t o o b v i a t e t h i s d i f f i c u l t y O 3 R e s e r p i n e AnaloRs A p p l i c a t i o n of t h e method d e v e l o p e d by R, B. Woodward' f o r t h e t o t a l s y n t h e s i s of r e s e r p i n e h a s been e x t e n d e d t o t h e p r e -

p a r a t i o n of u n n a t u r a l a n a l o g s i n F r a n c e , 5 and o t h e r d e r i v a t i v e s h a v e been s t u d i e d i n t e n s i v e l y i n C z e c h o s l o v a k i a , ' Reearpines w i t h s u b s t i t u e n t s i n r i n g s A , C , and E have been d e v e l o p e d ; however, i t i s u n c e r t a i n a s y e t w h e t h e r t h e s e o f f e r a s i g n i f i cant advantage over t h e n a t u r a l alkaloids. G a n g l i o n i c BlockinR D r u g s The a p p l i c a t i o n of t h e s t e r i c a l l y h i n d e r e d amine g a n g l i o n i c b l o c k i n g d r u g s , s u c h as mecamylamine and p e m p i d i n e , has diminiehed i n favor of a g e n t s w i t h fewer a i d e e f f e c t s . N e v e r t h e l e s s , work on a few r e l a t e d d r u g s h a s been p u b l i s h e d , Workers a t Parke+-Davis l a b o r a t o r i e s h a v e r e p o r t e d on D i b u t a d i a m i n (1)' w h i l e pempidine d e r i v a t i v e s s u c h a s I1 and I11 have been s t u d i e d a t Lakeside.'

60 -

Sect. II

- Pharmacodynamics

A r c h e r , Ed.

I1

I

I11

Amino Acid Enzyme Inhibitors a-Methyl dihydroxyphenylalanine (a-methyl-DOPA) continued to find growing application a s a moderately potent hypotensive agent. Although it i8 still the only drug of this class in wide use, a few related colapounds have been studied. The a-ethyl-DOPA (IV) and the tropone derivative ( V > appear to be

T

hu

IV

.. V

H

decarboxylase inhibitors,"'lo while a,p,@-trimethyl-DOPA is reported to inhibit tryptophane hydroxy1ase.l'

HGrZ

CH3 N TH 2 Cp O O H

(VI)

Ho-Q-cHzEL

80

P

VI

VI I

Still another member o f this group. a-methyl-L-p-tyronine (a-MPT) (VII), inhibits tyrosine hydroxylase. a-MPT has been shown to be effective in the treatment of hypertension caused by pheochromocytoma,l* Hydralizine Analons Although it is one of the oldest hypotensives, hydralizine (VIII) is stiL*l widely used. usually in combination with other agents, A n analogous compound IX, reported from the Lakeside l a b ~ r a t o r i e s .has ~ ~ been shown to lower blood preseure by decreasing peripheral resistance.

Chap. 6

Antihypertensives

Kornfeld

61 -

NkNH2

VIII W.A.O.

I X

Inhibitors

I n t e r e s t i n t h i s g r o u p h a s waned b e c a u s e o f p r o b l e m s a s s o c i a t e d w i t h s i d e e f f e c t s and w i t h h y p e r t e n s i v e c r i s e s f o l l o w i n g c o n c o m i t a n t i n g e s t i o n of t g r a m i n e - c o n t a i n i n g f o o d s i 4 P a r g y l i n e ( X I i s t h e b e s t known of t h e s e a g e n t s .

J3-Receptor

X

Blockers

which was d e v e l o p e d f o l l o w i n g t h e l e a d

Pronethalol ( X I ) ,

XI1

XI

o r i g i n a l l y discovered i n t h e drug D O C - I . ( X I I ) , 1 5 h a s been SUSs t u d i e d i n t h e t r e a t m e n t of a n g i n a and a r r h y t h m i a s . t a i n e d f a l l s i n blood p r e s s u r e have been n o t e d w i t h t h i s d r u g a f t e r o r a l administration f o r several ronths,le Pronethalol was s u b s e q u e n t l y shown t o be c a r c i n o g e n i c i n r o d e n t s , s o t r i a l s have been s h i f t e d t o p r o p r a n o l o l ( X I I I ) , a r e l a t e d member of t h e s e r i e s . l 7 & ~ ! ~ C € I 2 N H C H ~ C H ~ ~ 2 eHC1 \

/

XIII

62

Sect. I1

- Pharmacodynamics

A r c h e r , Ed.

G u a n e t h i d i n e Analogs B y f a r t h e most i n t e n s i v e s y n t h e t i c e f f o r t d u r i n g t h e p e r i o d has been d i r e c t e d t o w a r d t h e p r e p a r a t i o n of r e l a t i v e s of g u a n e t h i d i n e ( X I V ) . T h i s d r u g h a s been d e s c r i b e d

XIV

by I. H. Page ae “ t h e most s a t i s f a c t o r y a n t i h y p e r t e n s i v e , . . t o date”18 f o r f a i r l y severe hypertension. Consequently, t h e q u e s t f o r even s u p e r i o r v a r i a n t s h a s been a n a c t i v e one. G u a n e t h i d i n e i e composed of a h e p t a m e t h y l e n i m i n o p o r t i o n ( A ) l i n k e d t h r o u g h a two-carbon c h a i n ( B ) t o a q u a n i d i n e m o i e t y (C), The f o l l o w i n g new a n a l o g s h a v e been d e r i v e d from g u a n e t h i d i n e by v a r i a t i o n of any o r a l l of t h e s e t h r e e structural features, C l i n i c a l r e s u l t s a r e l a c k i n g w i t h some of t h e new compounds; w i t h 0 t h e r 8 , i t a p p e a r s t o o e a r l y t o a s s e s s t h e c l i n i c a l u t i l i t y r e l a t i v e t o t h a t of g u a n e t h i d i n e . I t a p p e a r s l i k e l y t h a t a number of t h e new d r u g 8 w i l l be available. D e b r i s o q u i n (XV)l* and B e t h a n i d i n e (XVI)eo h a v e

xv

XVI

a s h o r t e r d u r a t i o n of a c t i o n t h a n g u a n e t h i d i n e , a f e a t u r e which may make d o s a g e more f l e x i b l e , Each i s l e s s l i k e l y t o produce t h e d i a r r h e a s e e n o f t e n w i t h g u a n e t h i d i n e , Envacar (Guanoxan) ( X V I I ) * l and V a t e n e o l ( X V I I I ) e P h a v e e a c h been

c1

c1 XVII

XVIII

Chap. 6

Antihypertensives

Kornfeld

found t o be e f f e c t i v e i n c e r t a i n g u a n e t h i d i n e - r e s i s t a n t cases. A n o t h e r c l i n i c a l l y e f f e c t i v e a g e n t i s t h e German d r u g , G u a n a c l i n e ( L e r o n ) (XIX);p3 and a f u r t h e r a n a l o g w i t h b o t h h y p o t e n a i v e and a n t i d e p r e s s a n t p r o p e r t i e s is Parke, Davis and Company's Guanoxyfen (XX)." The

XIX g u a n e t h i d i n e i s o m e r s X X I and X X I I h a v e a l s o been p r e p a r e d 2 5 ' 2 e and may o f f e r some q u a n t i t a t i v e a d v a n t a g e o v e r

xx I

XXII

t h e p a r e n t drug, O f i n t e r e s t a l s o from a s t e r e o c h e m i c a l p o i n t of view is t h e o b s e r v a t i o n t h a t t h e o l e f i n i c a n a l o X X I I I i 8 more a c t i v e i n t h e Cis form t h a n i n t h e t r a n s ,

XXIII The o r a l l y a c t i v e Cis form i s more a c t i v e and a l s o of e h o r t e r duration than guanethidine. F i n a l l y , the foilowing a r e l i s t e d t o i l l u s t r a t e o t h e r m o d i f i c a t i o n s which h a v e been reported:

R

P

8 , CHa

(ref. 28)

Guanoctine ( r e f , 29)

63 -

Sect. I1

64 -

CH3

(ref.

30)

(ref.

31)

- Pharmacodynamics

A r c h e r , Ed.

(ref.

24)

32)

(ref.

Miscellaneous AntihjRertensives The p r o s t a g l a n d i n s h a v e shown some h y p o t e n e i v e p r ~ p e r t i e s ,but ~ ~ t h e i r r o l e i n t h i s a r e a is n o t y e t f u l l y elucidated. Among new s y n t h e t i c t y p e s t o ehow a n t i - h y p e r t e n e i v e q r o p e r t i e s a r e t h e Bulgarian d r u g Depreton (XXIV),3 which i s c l a i m e d t o be e f f e c t i v e i n human h y p e r t e n s i o n , and d i a l l y l m e l a m i n e (XXV),35 which i s h y p o t e n s i v e /CH p CH t C H p

“y

f

b

H

2CH r CH 2

by v i r t u e of d i r e c t a c t i o n on v a s c u l a r smooth m u s c l e . REFERENCES d e H a e n , D r U R a n d Cosmet.

Ind.,

s, 27

(1966).

1.

P.

2.

W. B. Ashby, J. Humphreye, and S. J. S m i t h , B r i t . Med. J., 11, 1409.

3.

V.

4.

R. B. Woodward, F. E. B a d e r , H . B i c k e l , A. J. F r e y , and R. W. K i e r s t e a d , J. Am. Cher. SOC., 7A, 2023 (1956).

5.

R o u s s e l l UCLAF, F r . P a t e n t s 1366721, 1366723, 1369956, 1369957 (Derwent 1 3 6 9 0 , 1 3 6 9 1 , 14065, 14066 1.

6.

I. J i r k o v e k y , I. E r n e s t , and M. Chem. C o m m . , 3355 (1956).

Wynn, L a n c e t , l S 5 11, 1241.

x,

P r o t i v a , Coll.

Czech.

Chap. 6

7.

D. K.

Antihypertensives

65 -

Kornf eld

McCarthy, G. M. Chen, D. H. Kaump, D. P o t t e r , K. H o l a p p a and C. E n s o n , Arch. I n t . Pharmacodyn., 154, 263 L1965). A.

4 1 ) 5 .

8.

W. B. S e v e r s , W. Pharm. S c i . ,

9.

R.

10

J. K i n n a r d , and J. P.

2. 859

.

.

J. L e v i n e and A.

lL6, 42 (1964).

Sankyo, Jap.

and 1 0 2 6 ( 1 9 6 5 ) .

S j o e r d e m a , J.

B u c k l e y , J.

Pharm.

EXR. T h e r . ,

P a t e n t 12930/64. Roche, Neth.

P a t e n t 6414247 (Derwent 17316).

11

Hoffmann-La

12.

A. S j o e r d a m a , K. Engelman, S. S p e c t o r , and S. U d e n f r i e n d , L a n c e t I1 ( 7 4 2 2 1 , 1 0 9 2 ( 1 9 6 5 ) . A. I. E d l i n , W. J. B u c k l e y , J. Pharm.

14.

a,

K i n n a r d , E. E. V o g i n , and J. Sci., 2 0 . (1965).

P.

S i m p s o n , and a, 754 ( 1 9 6 4 ) .

F. S. G l a z e n e r , W. M. Morgan, J. M . P. K . J o h n s o n , J. Am. Med. A B B . , C.

E. P o w e l l and I. H. S l a t e r , J. Pharm. EXR. T h e r . ,

E,480

(1958).

16

B. N.

Prichard, B r i t .

17.

J. Hamer, T. G r a n d j e a n , L. M e l a n d d z , and G O Sowton, Brit. Med. J., - 1 720.

18.

A. C. C o r c o r a n , D r u R s of C h o i c e 1966-1967, C. V. Woeby Co., S t . Louis (1966).

19.

M. H. L u r i a and E. 289 (1965).

20

.

21 22 23

24

.

C.

M.

W.

G i f f o r d , J.

W.

S.

P e a r t and M .

398 ( 1 9 6 5 ) .

J.,

The

D. Freis, C u r r e n t Ther. Ree., Am.

Med.

ABB.,

x,901

HcWahon, B r i t .

T.

T. D. V. L a w r i e , A. R. 8. R e i n e r t , B r i t . Med.

Med.

-

(1965).

J.,

1.

L o r i m e r , S. 0 . H c A l p i n e , and 402 ( 1 9 6 4 ) .

J.,A

K. Kuehna, C. J a n o c h a , V. Muench. Med. Wschr.,

--

Hed.

J a n o c h a , and D. (1965).

3, 2599

P a r k e , D a v i s and Co. , U.S. A s s , , 1 9 4 , 1376 ( 1 9 6 5 g .

P a t e n t 3203988

Hagnus,

17.

Am.

Med.

Sect. I1

66 25,

R.

26.

Sandoz Co.,

27.

W. E. B a r r e t t , R. 2 1 , 506 (1965).

G i u d i c e l l i , H.

- Pharmacodynamics

Najer, s, 726 (1965).

23,

a n d 2’.

A r c h e r , Ed.

L e f e v r e , ComVt. Rend.,

Fr. P a t e n t 1413924 ( D e r w e n t 18888). M u l l , a n d A.

P.

J.

Plummer, Exver,,

/ch.

28.

Bager. U . S .

29.

H. J.

P a t e n t 3236859.

G. S c h o e p k e , H. L.

z,

D. BrondyK, L. H. Weimeter, and I n t . Pharmacodyn., 185

S c h m i d t , Arch.

(1965).

30.

D. I. B a r r o n , I. L. N a t o f f , a n d D. J. Pharm., 534 (1965).

31.

C i b a , U.S.

32.

Lab

33.

E. W. H o r t o n , E x p e r , , $ ( 3 ) ,

34.

D. P a s k o v , A. S p a s o v , I. K r u s h k o v , and E. Arch. I n t . P h a r m a c o d y n . , x 7 . 1 3 2 (1965).

35.

W. A . Arch.

2.

K.

V a l l a n c e , Brit.

P a t e n t 3178443.

Lumiere, B r i t .

P a t e n t 973533 ( D e r w e n t 1 4 2 3 7 ) .

113 (1965).

F r e i b u r g e r , J. R. Weeks, a n d D. W. Exa. P a t h . Pharm., 39 (1965).

3,

Golovinsky, Ducharme,

Chapter 7.

Diuretic Agents

Edward J. Cragoe, Jr. and James M. Sprague Merck Sharp & Dohme Research laboratories, West Point, pa.

A perusal of the current l i t e r a t u r e , including the patent literature, leads one t o believe t h a t compounds with d i u r e t i c a c t i v i t y a r e most abundant. However, many of these reports a r e undocumented o r the supporting biological data a r e meager. This review i s r e s t r i c t e d t o selected reports that contain credible biological support o r present chemical novelty. The l i t e r a t u r e coverage includes p a r t of 1964 and extends through April 1966.

Organomercurials. The advent of newer d i u r e t i c s has diminished the ckinical importance of the organomercurial d i u r e t i c s and few new compounds have been r - m r t e d i n recent years. Nevertheless, an active i n t e r e s t continues in the locus of action of t h i s c l a s s of agents i n the kidney nephron and the definition of t h e molecular events involved i n the action of these substances on nephron function, p a r t i c u l a r l y a s it relates t o sodium transport, potassium excretion and e f f e c t s on uric acid and glucose metabolism. Additional important evidence has been reported i n support of the mercuric ion hypothesis1 over the i n t a c t molecule hypothesis* f o r the action of mercurials. The mercuric ion theory1 implies t h e presence i n the kidney of mercuric ion during the period of active diuresis following organomercurial administration; solid evidence of t h i s has been lacking. Using a novel isotope exchange diffusion analysis f o r @3, evidence has been presented t h a t the l e v e l of mercuric ion i n the rat kidney correlated w e l l w i t h the onset and duration of diuresis a f t e r Hgm3-chlormerodrin. Mercuric ion was released a l s o i n kidney t i s s u e of the r a b b i t and the chicken.3 Similar results were obtained i n the rat w i t h p-chloromercuribenzoic acid, 4,5 an organomercurial t h a t i s not d i u r e t i c i n the dog and does not release mercuric ion i n a model i n v i t r o system where other active mercurial d i u r e t i c s readily yield mercuric ion'lTaowever, t h i s substance has been shown t o be rapidly d i u r e t i c i n t h dog when administered by retrograde infusion i n t o t h e renal tubular lumen.

-

%

Important studies on t h e locus of action i n the nephron7 and the e f f e c t of the sodium reabsorption and oxygen consumption8 of these mercurials have been reported. Recently reported data support the concept that mercurial d i u r e t i c s in-

h i b i t active sodium reabsorption & vivo by inhibition of the Na+-@-activated component of the membrane ATPaSe SYSThus, an enzyme system has been

identified t h a t can be correlated w i t h active sodium and potassiumtransport and that i s inhibited i n v i t r o and i n vivo by mercurial diuretics.

--

--

Acylphenoxyacetic Acids. Compounds of t h i s type containing an tY,&unsaturated-acyl group were designed t o react w i t h functionally important sulfhydryl gro s in a manner similar t o t h a t believed t o occur w i t h mercurial diuretics.% Biological data have now appeared indicating t h a t such a react i o n indeed does occur i n v i m as well as i n v i t m . Ethacrynic acid (I) i s

68 -

Sect. I1

c1

- Pharmacodynamics

A r c h e r , Ed.

c1

excreted, in part in t h e dog as the cysteine -uct;d mercurials are known t o b e p r e t e d as the cysteine 5-11 conjugates. The ethacrynic acidc'4! cysteine adduct was shown to be an I active diuretic i n man.13 In the dog, ethacrynic acid caused a decrease in protein-bound sulfhydryl It had no such effec in the groups in kidney c e l l s as do t h e mercurials. rat, a species i n which it is not d i ~ r e ! t i c . l ~ , ~The 5 uptake of G 3 - c h l o r merodrin by s l i c e of renal cortex of t h e dog and the rat was reduced by ethacrynic acid.

-

C H C-!-&-c~~2~

2

Using thirteen cosnpounds of t h i s class ranging from highly active to inactive in the dog diuretic assay, l i t t l e correlation was found between diuret i c a c t i v i t y and reactivity toward sulfhyaryl (cysteine) in an 2 v i t r o model system. Better correlation was found with the a b i l i t y to inhibit m t r o Na+-K?-(membrane)-ATPase from guinea pig kidney. However, the enzyme derived f r o m t h e kidney of the dog and the t was inhibited in v i t r o t o approximately the same extent by ethacrynic 8 c i d . e Also, ATPase from the kidenys of rats t h a t had received e t crynic acid shoved substantial inhibition without resulting in diuresis.

--

3

Substitution on the ammatic nucleus o r on the unsaturated-acyl group markedly influences diuretic a c t i v i t y as measured in the dog. Compounds without nuclear substituents showed only weak activity. Substitution in the 2-position increased a c t i v i t y modestly while substitution in the 3-position o r in t h e 2,3-positions, especially by chlorine o r methyl, gave highly active compounds. Additional substitution in the 5 and/or 6 positions resulted in l o w activity. However, the 1,kaaphthalene analog was quite active. Substireduced activity; the tution on the methylene of the unsaturated-acyl -up mono-methyl campounds showed reduced a c t i v i t y and the dimethyl compounds had only l o w activity. Saturation of t h e double bond of the acyl group yielded compounds w i t h marginal activity.lOil? Detailed documentation of the pharmacology of ethacrynic acid and some of i t s congeners has appeared. I n dogs, they caused a prompt and profound water diuresis and the excretion of nearly equivalent amounts of sodium and chloride when administered either parenterally or orally. There was some increase i n potassium excretion, especially following intravenous administration. They have a steeper dose response curve and a substantially greater maximal saluretic effect than the thiazides, and a t least as great an effect as the organomercurials. Results w i t h combinations indicate a mechanism of action f o r ethacrynic acid different from t h a t of the thiazides, acetazolamide and t h e mercurials. The ascending loop of Henle is a major s i t e of action i n the nephron.ll,lg Numerous reports on ethacrynic acid support i t s c l i n i c a l efficacy.

Aromatic Sulfonamides. Interest continues in derivatives and structural variants of the m-benzenedisuli'oaamides. A number of 4-chloro-5-( substitut e d - a m i n o ) - m - b e n z e n e d i s u d e s and some khydroxy analogs were effective

Chap. 7

Diuretics

Cragoe and Sprague

69 -

diuretics Bd the 4-chloro-5-(2-guanidinoethylamino) compound was weakly hypotensive. Several 5- and 6-hydrazino derivatives were less effective in dogs and rats than dichlorphenamide.21 However, the major recent i n t e r e s t in compounds of t h i s class l i e s i n analogs where one sulf'amyl group has been replaced by other electmn a t t r a c t i n g gmups, such as, alkylsulfonyl, acyl o r carboxyl and i t s functional derivatives. Diuretic a c t i v i t y i n rats mice has been reported f o r several 3-nethylsulfonylbenzenesulfonamides.8Bt23 The carboxylic acid analogs have yielded highly active and useful drugs of current interest. Four campounds of this category have received considerable study. The chemistry and pharmacology of clopamide (11) has appeared earlier, but evidence of i t s c l i n i c a l u t i l i t y continues t o appear.

V

111, R=H; R'=N(CH3)2

Iv, R=R'=CH3 The related acylhydrazine (III)(CI-546) was an effective diuretic, natriuretic and cbloruretic in rats, dogs, monkeys and man. Results in the dog indicated that it exerts its effect on t h e renal tubule and that carbonic anhydrase inhibition becomes significant only a t high doses. In man, kaluresis did not occur t o any marked extent in the effective daily o r a l dose of 20-320 mg. resulting in a favorable sodium-potassium excretion r a t i o . Metabolism studies in several laboratory species, following o r a l administration, indicated rapid absorption and excretion principally i n the urine with recoveries of 80-100$ of the dose. The major urinary excretion products were unchanged drug and a minor amount of the corresponding benzoic The related c a r b p m i d e derivative (IV)$cI-456) was similarly effecacid* The methyl substitu ion on the sulfamoyl group t i v e in t h e same species. of t h i s compound i s noteworthy. The f a t e of t h e methyl group is not reported but, from r e s u l t s with other methylated sulfonamides i n vim, demethylation t o yield an unsubstituted sulfamoyl compound would be anticipated. The action of these compounds and the thiazides on active ion transport and m o t h muscle contractility has been studied i n relation t o the hypotensive a c t i v i t y of the benmthiadiazines .27

ange

Remarkable a c t i v i t y has been found i n a series acids appropriately substituted i n the amino group. was achieved when t h e amlno substituent was arylmethyl o r heterocyclic-methyl.

Sect. I1

70 -

- Pharmacodynamics

A r c h e r , Ed.

One member of t h i s series, furosemide (V)(also designated, f u r e d e , frusemide) had strikingly different properties from t h e sulfonamides of a l l other types. Eowever, the isomeric compounds, the 2 - c h l o r o - 4 - s u b s t i t ~ e d ~ i n o - 5 sulfamoylbenzoic acids, were not saluretic.28 The meximum naturesis, chloruresis and diuresis obtainable with furosemide, following o r a l o r parentera1 administration t o man and t o several species of animals, was several-fold greater than that produced by the most active thiazides.30 &tassim excret i o n increased but, due to the massive sodium excretion, the sodim/potassim r a t i o is favorable. R e n a l studies indicated a s i t e of action in the renal tubule different from the thiazides and mercurials; furosemide a f f e c t s not only proximal and istal tubule sites but a l s o t h e ascending limb of the loop of Henle.3l-3 These properties of furosemide are shared 'by ethacrynic acid, a non-sulfonamide, of entirely unrelated structure. Furthermore, interaction of furosemide w i t h other diuretics i n the dog indicated that the mechaniam of furosemlde is different from that of the t h i a z i es and mercuria l s and i s similar o r identical t o that of ethacrynic acid. 39

P;

Furosemide was excreted largely as unchanged drug i n the urine36 but 2-chloro-5-sulfamoylanthranilic acid, a r i s i n g from loss of the furfury1 group, was identified as a metabolite.37,38 The c l i n i c a l efficacy of t h i s drug i s w e l l s ~ p p o r t e d . 3 9 9 ~m s e m i d e i s reported t o produce t h e same stde e f f e c t s c o m n t o other s onamide diuretics, i e hypokalemia, hyperglycemia and hyperuricemia. 393

.

3

Benzothiadiazines and Analogs. Many new publications on thiazide d i u r e t i c s continue to appear, but no strikingly novel structural variants have been disclosed. Variations of t h e heterocyclic ring have given active compounds. The 3-aza analog of chlorothiazide, 6-chlo1-0-2~-1,2,3, kbenzothiatriazine-7-sulfonamide 1 , l - d i xide, showed a c t i v i t y comparable to hydrochlomthiazide i n rats and rabbits.& 6-chloro thiochrcnnan-7-sulfonamide 1,ldioxide i s reported to be active in r a t ~ . ~ 6-Chloro-3,4-dihydro-2~-1,23 benzothiazine-7-sulfonamide 1,l-dioxide (the isostere of pprochlorothiazide i n which the b N H I s replaced by C%) wa8 active i n dogs. Detailed attention has been given t o the ieoindoline analogs, i.e. the phthalimides ( V I ) and the corresponding 1-oxoisoindolines. h e compound of the latter series, chlorexolone (VII) was suff i tly active t o j u s t i f y c l i n i c a l trial and has received further study.

43-fa

VI

VII

In each series, the nature of the substituent on the ring nitrogen had pronounced e f f e c t s on a c t i v i t y a s measured in the rat. Cycloa-1 groups were

Chap. 7

Diuretic s

Cragoe and Sprague

71 -

most favorable i n each series, and maximum a c t i v i t y wa6 found w i t h cyclohexyl o r substituted-cyclohexyl although, i n the phthalimide series, cyclopentyl, cycloheptyl and cyclo-octyl showed comparable a c t i v i t y . These derivatives were 3-6 times a s a c t i v e as chlomthiazide. Reduction of one carbonyl group t o CHOH and t o Cf4! (VII) increased a c t i v i t y some 10 and 50-fold respectively. Chlorexolone i s 300-450 times as active as chlorothiazide i n the rat and 50-fold i n t h e dog. Conversion of the other carbony1 group t o C+ to give the chlorexolone isomer abolished a c t i v i t y . The sodium, potass um and chloride excretion i n t h i s s e r i e s resembled t h a t of the thiazides.'5 Metabolism studies i n rats and dogs showed chlor olone t o be excreted as monohydroxy derivatives of t h e cyclohexane ring.@ C l i n i c a l r e s u l t s showed c rexolone t o resemble hydroflumethiazide i n its action and t o be e q u i p o t e n t Y t 4 7 O f i n t e r e s t are the 3-(ketoalkyl) derivatives of t h e hydrothiazide series.49,w These a r e formed from the reaction of ketoaldehydes, RCO(CH )nCHO, with t h e appropriate disulfamoyl aniline. With pketoaldehydes fn=l), intermediate a n i l s were isolated when the ortho-sulfamy1 group carried a t least one substituent. These were assigned t h e anil-enol struct u r e (VIII). On f u r t h e r reaction, under acid conditions, IX, R - R k H zides (M)were fomed. Certain of these compounds (e.g. the X=CF3, n-1 or 2; V I I I , R 2 = a l l y l , R3=CH3 o r C6H5, X=C1) exhibited diure&

Yrothia-

VIII

Ix

a c t i v i t y i n the dog comparable t o trichlormethiazide. P a r t i c u l a r i n t e r e s t l i e s i n the hydrazones derived from M and 1-hydrazinophthalazine (hydralazine), an antihypertensivs agent. Some properties two such compo ds have X 4 8 p fi 4 H 3 , R31CH3, n=l, X=CF3)?' and EX-5004 (R !? =HI been reported, E R k H , n=l, ~ 4 1 ) . 59 5 These hydrazones were not active d i u r e t i c s but t ey showea a unique blocking action on t h e d i u r e t i c a c t i v i t y of the thiazides. k9 T h i s blocking action of E X - 4 8 7 has been employed t o analyze the mechanism of action of t h e t h i a ~ i d e s . 5 ~W-48n did not a l t e r the d i u r e t i c response t o acetazolamide but it blocked the d i u r e t i c a c t i v i t y of hydrochlorothiazide. The a c t i v i t y of chlorothiazide was only p a r t i a l l y blocked. Thus, it was concluded t h a t EX-487" i n h i b i t s the "B" (benzothiadiazine) e f f e c t of the thiazides, i.e. both hydrochlorothiazide and chlorothiazide, but leaves the carbonic a drase inhibitor "A" (acetazolamide) response of chlorothiazide unaffect&T1 EX-5004 was reported to be hypotensive in t h e rat and dog, probably by the lowering of eriphexal resistance through a d i r e c t action on vascular smooth muscle.~3

72 -

Sect. I1

- Pharmacodynamics

A r c h e r , Ed.

y r i d i n e s (xy. The diuretic properties of triamterene, 2,4,7-triamino- -phenylpteridine, have been known f o r m e time. Some structurea c t i v i t y results have now been published. The nature of the substituents not odiy influence the potency of the compounds, but the qualitative charact e r i s t i c s of water and electrolyte excretion. ~n rats, 2,1C-diaminO-6,7dimetbylpteridine (SKF-371), like

4 X

2-anilino- 4,7-diamino-6-phenyl derivat i v e caused saluresis with lraluresis and no diuresis.55

The 6-cartmmyl derivatives were g e n e m l u more natriuretic than triamterene, especially in dogs. Although these derivatives prpduced l i t t l e kaliuresis, they did not display the antikaliuretic e f f e c t s exhibited by triamterene Thus, 2-phenyl- 4,7-diaminopteridine-6-carboxmide (SKF-6874) in the adrenalectomized rat receiving aldosterone produced natriuresis w i t h no e f f e c t on potassium excretion. In contrast triemterene increased sodium excretion and depressed potassium excreti0n.5~ Substitution on the carbarmy1 nitrogen with o r without simultaneous substitution on the 7-amino nitmgen can lead t o increased a c t i v i t y in rats. In a series of N-R-2phenyl-lcamim-7-NMc-6-pteridinecarboxamides, the most active compound was ~y-5256, where R=2-methoxyethyl. Replacing the 2-phenyl w i t h p-methoxyo r p-chloro-phenyl, 2-thienyl, methylmercapto-, diethylaminoethylamino or hydrogen gave l e k s active compounds as did replacing 7-NHEi w i t h hydroxyl. 56 ~n more detailed studies in rats and dogs, ~y-5256was shown t o have about the same o r a l diuretic a c t i v i t y as hydrochlorothiazide. About equal amounts of sodium and chloride were excreted along w i t h some potassium and, when combined w i t h hydrochlomthiazide, t h e e f f e c t s of t h e two drugs were additive. 57

.

Another substituted-amide, N- (2-mrpholinoethyl) -2-phenyl-4,7-diaminopteridinecarbommide (Vy-3654), i n t h e dog produced marked sodium and chloride excretion w i t h some potassium excretion. From studies on combinations of Wy-3654 w i t h other diuretic agents, it has been concluded that the mode and/or s i t e of action of ~y-3654differs from that of hydrochlorothiazide, acetazolmide and ethacrynic acid but are similar to t h a t of mercaptomerin .58 The metabolism of triamterene in humans results in five products i n unchanged drug, 2,4,7-triamino-6-p-hydroxyphenylpteridine, the corresponding s ic acid ester, a N-glucuronide of triamterene and an unknown m e t e b o l 3 9 From the kidney ti sue of triamterene-treated rats, a pteridine nucleotide has been isolated.& Clinical studies using a combination of triamterene and a thiazide indicated t h a t no levels could be maintained without potassium supplements. t h e urine:

T1

-fk-!N%

Chap. 7

Diuretics

73 -

Cragoe and Sprague

zincs. N - A m i d i n o - 3 - ~ p ~ z i n e c a r b o x a m i d e s(XI) constitute a new class o diuretic agents. They are unique both in structure and in their strong basic character. In norma1 rets, they produced marked diuresis while leaving unaffected o r repressing potassium excretion. They antagonized 6 the renal actions of exogenously administered aldostemne, DOCA o r hydmcorti5 sone in the adrenalectamized rat. N 3 Although the potency in dogs YBS lower, t h e relative a c t i v i t i e s of the various members f the series paralleled those XI in rats. 83

Halogen in the 6-position enhanced activity; the potency was in the order C l > B r > I . The 6-chloro derivative (XII) was more t e n t than triamterene and considerably more active than spironohctone. 630 Introduction of alkyl, substituted-alkyl, alkylideneamino o r a r y l substituents on one o r both of the terminal nitrogens of t h e guanidino moiety gave compounds that generally were l e s s active than XII; however, derivatives of XI1 bearing N-benzyl, N-(2-hydroxyethyl) o r N, N' -dimethyl substituents were equipotent with XII. Derivatives of XI1 having one acyl group on t h e 3-amino o r on one o r both of the termina nitrogens of the guanidine moiety were considerably less active than XII. k2 The introduction of a second amino group, a t position-5, further improved activity. N-Amidino-3-amino-5-dimethylamino-6-chloropyrazinecarboxamide (XIII) was more active than XI1 in rats and dogs with a slower onset and a longer duration of action. When XI11 was administered t o rats dogs In o r humans, it was demethylated stepwise t o the 5-amino analog (XIV).'4 rats, X N was as natriuretic a s hydrochlorothiazide with no kaliuretic effect. S m a l l doses augmented the natriuresis produced by large doses of acetazolamide while decreasing potassium excretion. A synergistic increase i n sodium excretion occurred when X N and hydrochlorothiazi e were coadministered while potassium excretion was considerably reduced. 85

In dogs, XIV was qualitatively similar but less potent than in rats. Small doses of XIV in combination w i t h either hydrochlomthiazide o r ethacrynic acid produced enhanced natriuresis and depressed kaliuresis. Although the mode of action is different from spironolactone and organomercurials, stop-flow studies in icated that X I V i n h i b i t s d i s t a l potassium secretion and sodium r e a b ~ o r p t i o n . ~ 5

I n patients w i t h cirrhosis and ascites, XIV, in doses of 8 to 30 mg./day, produced diuresis and saluresis with some bicarbonate excretion with reduced kaliuresis. In combination w i t h hydrochlorothiazide o r ethacrynic acid, sed t h e excretion of potasXIV enhanced the excretion of sodium and d slum mre effectively than spironolactone.

878

g-Triazines. A recent c r i t i c a l study in rats has confirmed and extended earlier diuretic and natriuretic structure-activity r e l a t ons in a large series of 2-mono-6ubstituted-amino-4-amino-e-triazines.55r L

b

74

Sect. I1

- Pharmacodynamics

A r c h e r , Ed.

2-(p-Chlorophenylamino)-4-amino-~-triazhe (Chlorazanil) has received considerable study in man, However, a more recent study indicated that the m-chloro isomer may be significantly more active in rats and dogs and i n I U R In comparison with the present day potent diuretics, campounds of t h i s c l a s s have l i t t l e p r a c t i c a l importance. The principle remaining interest l i e s i n t h e i r modest e f f e c t on potassium excretion and i n the modification of the action of several adrenalocorticoid steroids such as the antagonism of minerslocorticoids and of t h e deposition of hepatic glycogen induced by the g l u c o c o r t i c o i d s . ~ ~A detailed study has shown the influence on potassium excretion i n the rat; a t l e a s t , it is complex and strongly dose related. A t non-toxic doses, most of the compounds can produce kaluresis. Miscellaneous Heterocyclic Compounds. From a s e r i e s of 2-methylene-4thiazolidones ,.(O the d i u r e t i c a c t i v i t y of one member, 2-ethoxycarbonylmethylene-3-methyl-5-piperidino-bthiazolidone(XV), has been re r t e d under the name Etozolin. It was effective in both the rat and dog.7l-B I n the

xv

XVI

XVII

E

rat, it was reported to give a grea er response than chlorothiazide, hydrochlorothiazide and acetazolamide.7 r75 I n t h e dog, however, it was l e s s active than hydrochlorothiazide and meralluride.75 When given t o dogs i n clearance type experiments, it enhanced the maximal diureses of the mercuria l s and of chlorothiazide, indicating a probable mechanism different from these d i u r e t i ~ s . 7 ~ Results i n man a r e not available. Substituted sydnones (XVI) have been shown to increase the excretion of sodium and chloride i n rats.77~78 O f the three compounds reported i n d e t a i l , the 3-butyl-Lethyl derivative showed a c t i v i t y a t lower doses than the 3-sec. -butyl compound and t h e 3-isopropyl-4-carboxylic acid; however, t h e a c t i v i t y was not great, and t h e i r p o t e n t i a l usefulness is limited by t h e i r convulsive properties. A series of 3-(aminoaUryl)-sydnones were not a c t i v e .79

I n a s e r i e s of thirty-nine s-triazoles, only the lead member, 5-(paminophenyl)-5-triazole-3-thiol 7XV I ) showed d i u r e t i c and n a t r i u r e t i c act i v i t y i n rats a t reasonable doses.

80

The intense diuresis produced derivatives was related to i n vivo polyuria was not accompanied by an t h e action bears no resemblance t o

--

i n the rat by a number of ethyleneimine l i b e r a t i o n of ethyleneimine. This marked increase i n electrolyte excretion, hence that of the d i u r e t i c s i n c l i n i c a l use.

~ . ~ ~

Chap. 7

Diuretics

Cragoe and Sprague

75 -

An extrarenal mechanism may be involved, perhaps a t the p i t u i t a r y , since t h e polyuria was similar t o t h a t of diabetes insipidus.81

Aldosterone Antagonists. Although many 17-spirolactones of t h e s t e r o i d type have proven t o be p o t e n t aldosterone antagonists in animals and i n r a m , few new compounds of t h i s type have been reported. However, t h e c l i n i c a l u t i l i t y of these compounds have stimulated the examination of other s t e r o i d s f o r e f f e c t s on e l e c t r o l y t e t i o n i n animals and i n man. Oxygenated derivatives of progesterone@" and testosterone85 showed augmented act i v i t y . The a c i d s corresponding t o c e r t a i n s t e r o d spirolactones exhibited antagonistic a c t i v i t y comparable t o the lactone.& Diuretic a c t i v i t y of some adrenocortical steroids in t h e rat have been compared. Cortisone, hydrocortisone and some of t h e i r 1,2-dehydro and fluorinated analogs have a c t i v i t y exceeding t h a t of chlorothiazide .87 Compounds that are possibly non-steroid aldosterone antagonists have turned up i n a series of 3-substituted-coumarins (e.g. 3-carboq1, 3-acety1, The a c t i v i t y is said t o be confirmed i n man.@ An extensive 3-glyoxyloyl). review of aldosterone antagonists has appeared.@

REFERENCES

2,

1. I.M.

Weiner, R . I . Uvy and G.H. Mudge, J. P h a m c o l . Fxptl. Therap., (1962). 2. R.H. Kessler, R. Zozano and R.F. P i t t s , J. C l i n . Invest., 656 (1957). 3. T.W. Clarkson, A. Rothstein and R. Sutherland, B r i t . J. Pharmacol., 24, 1 (1965 . 1. 4. T.W. Clarkson and M. Greenwood, B r i t . J. Pharmacol., :5O (1966). 5. T.W. Clarkson, Federation P m c . , ' w 6. E.J. Cafruny, N. Y. Acad. Sci. Symp., Mar. 3-5, 1966. 7. E. Littman, M. H. Goldstein, I,. Kasen, M.F. Levitt and R . P . Wedeen, J. Pharmacol. Exptl. Therap., 152, 130 (1966); N. Y. Acad. Sci. Symp., Mar. 3-5, 1966. ,221 8. R.H. Kessler, S.W. Weinstein, F.D. Nash and M. FuJimto, Nephron, 1, (1964); R.H. Kessler, N. Y. Acad. Sci. Symp., Mar. 3-5, 1966. 9. V.D. Jones, G. Zockett a n d h a x m a c o l . Exptl. Therap. 23 - (1965). . _- _ 10. E.M. Schultz, E.J. Cragoe, Jr., J.B. Bicking, U.A. Bolhofer and J.M. Sprague, J. Med. Pharm. Chem., 5, 660 (1962). 11. K.H. Beyer, J.E. Baer, 7.K. Michaelson and H.F. Russo, J. Phaxmacol. Exptl. them^*, 1 (1965). 12. I . M . Weiner and O.H. Muller, J. Pharmacol. Ekptl. Therap., 113, 241 (1955). 13- Y.H. Kong, G.V. Irons, Jr., W.M. G i n n , Jr., G.E. Garrison and E.S. Orgain, Circulation, 32, Suppl. 11, 128 (1965). 14. R.M. Komorn anhE.J. Cafruny, Science, 133 (1964). 15 R.M. Komorn and E.J. Cafruny, J. P h a m c o l . Exptl. Therap., 148, 367 (1965). 16. R.Z. Gussin and E.J. Cafruny, J. Pharmacol. Exptl. Therap., 1 (1965). 17. D.E. Duggan and R.M. Noll, Arch. Biochem. Biophys., log, 3-965). 18. J.B. Hook and H.F. Williamson, Proc. Soc. Exptl. B i o m e d . , 120, 358 (1965). SOC. Exptl. 19- J.E. Baer, J.K. Michaelson, D.N. McKinstry and K.B. Beyer, Biol. Med., 3, 87 (1964).

96

&,

-

26,

-

,

147,

143,

m,

a,

76 -

Sect. I1

- Pharmacodynamics

A r c h e r , Ed.

20.

A. Lespagml, D. Bar, C. I&spagnol and A. Anouwtr, Bull. 8oc. chim. France,

21.

A. u-gml,

22.

HFKugita, M. Zbkeda, T. Oine and K. Higaki, Chem. Phaxm. Bull., 10, 1001 to 1008 (1964) J.R. Boissier, C. Dumont, J. Usbms and C. %lea, Thempie, 20, 1 3 5 (1965)D. H. KaW, R.L. F m ~ m y ,LIT. Blouin and D. Williams, J. New Drugs, 21

23. 24. 25. 26. 27.

28.

29. 30.

800 (1965).

D. Bar, A. Robclet, C. Mizon-Capron and J. B i z a r d , Thempie,

-

19, 1423 (1964).

5,

(1964)

R.E. Ober, S.F. Chang, L.T. B l o u i n and G.W. Gymn, Federation Proc., 532 (1966). L.T. B l O ~ i n , D.H. Kaumip, R.L. Fm8-y and D. WiUiamS, J* New D W S ,

302 (1963).

25, 2,

Daniel and C.W. Nash, Arch. Intern. Pharmacodyhamie, 158, 139 (1965). K. Sturm, U. Siedel, R. Weyer and H. Ruschig, Chem. Ber., 99, 328 (1966). W. Siedel, K. Stuxm and W. Scheurich, Chem. B6 3-45 n 9 6 6 ) . R.J. Timuexman, F.R. Springman and R.ii. T h c S9 Current Therap. Res., 88 (1964). J.B. Hook and H.E. Williamson, Pmc. SOC.Exptl. Biol. Med., ll8, 372 (1965). W. Suki, F.C. Rector, Jr. and D.Y. Seldin, J. C l i n . Invest., 1458 (1965). G. Malnic, F.L. Vieria and H. Enokibara, Nature, E.E.

5,

31. 32. 33. 34. A. small and E.J. Cafruny, ~ h a x m a c o l o g i s t - , ~ 6 5 ~ ~ ~ . ~ 35. J.B. Hook and H.E. Yilliemson, J. Pharmacol.Exptl. Therap., 3,88 (1965). 36. J. Gayer, Klin Yochschr., 2,$ 1 5 . 37. A. $ussler and P. J3ajd6, Arznezidz-iorsch., 14, 710 (1964). 38. A. EIaussler and H. Wicha, Arzneimittel-Forsch., 15, 81 (1965). 39. D.E. Hutcheon, D. Mehta and A. Romano, A.M.A. A r x . Internal Med., 9, 542 (1965). b. R.G. Mu*, J. Am. M ed. Assoc., 195, 1066 (1965). 41. L. Bencomo, J. Fyvolent, S. a and L. Knhnlul, Current Therap. Res., 2, 339 (1965). 42. A. Bose, S.K. Bose and D. Chakravarty, Indian J. Pharm., 27, 3 (1965). 43. J.R. Boissier, C. DuWnt, J. Lesbros and C. Malen, Theraps 20, 1305 (1965). 44. Merck & co., Inc., French Pat. M-3136 (g,64, 1 1 2 i , 4 ) 7 450 E.J. Cornish, G.E. Lee and U.R. Wragg, J. PGm. Phannacol., 18, 65 (1966). 46. A.F. Lant, W.I. Baba and G.M. Wilson, Ciin. Phanuacol. Therap: 7, 196 (1966). 47. W.I. Baba, A.F. Iant and G.M. Yilson, Clin. Pharmacol. Therap., 1, 212 (1966). 48. K. Corbett, S.A. Edwards, G.E. Lee and T.L. Threlfa l l , Nature, 208, 286 (1965). 49. J.E. Robertson, D.A. Dusterhoft and T.F. Mitchell, Jr.,'nMed.chem., 8, 9 (1965). 50. H. Inoue, M. Take&, K. H i g s k i and H. Kugita, J. Pham. Soc. Japan, 85, 95

1m.

-

51. 52. 53. 54.

55.

(1965)

C.R.

Ross and E.J.

R.A. Yea-,

C.A.

Cafruny, J. Pharmacol. Exptl. Therap., 140, 125 (1963). Brahm and D.L. Miller, Toxicol. Appl Fhai5=col., 1, 598

.

-

(1965) A.I. E d l i n , W.J. Kinnard, E.E. Vogin and J.P. Buckley, J. Pharm. Sci., 54 20 (196). V.D. Wiebelhaus, J. Weinstock, A.R. Maass, F.T. Breanan, G. Sosnowski and T. larsen, J. Phaxm. Gxptl. Therap., 397 (1965). T.U. S h l a n and F.E. Shidemm, J. Phazm. Exptl. Therap., 148, 356 (1965).

149,

-

Chap. 7

Diuretics

Cragoe and Sprague

77 -

56.

A.A. S a n t i l l i , L.E. McCardle and T.S. Osdene, Abstr. papers, 150th Am. Chem. Soc., Meeting Abstracts, 16~-17~+, September, 1965. 57- M.E. Rosenthale and T.S. Osdene, Pharmacologist, 7, 165 (1965). 58. M.E. Rosenthale, L. Datko, J. Kassarich and F. Sckeider, J. Pham. Exptl. ThemPo, 147, 399 (1965)15 812 (1965). 59- K ~ m > z n e i m i t t e l - F o r s c h . , 60. H. Herken, V - Neuhoff and G. Senft7ki.n. Wochschr., 43, $0 (1965). 61. R.A. Thompson and M.F. Cmwley, Postgrad. Med. J., 41, 706 (1965). 62. J.B. Bicking, J.W. Mason, O.W. Woltersdorf, Jr., J.H. Jones, S.F. Kwong, C.M. Robb and 6. J. Cragoe, Jr., J. M e d . Chem., 8, 638 (1965). 63 M.S. Glitzer and S.L. Steelman, PIVC. Soc. ExptlT Biol. Med., 1x), 364 (1965) 64. C.B. Jones, H.F. RUESO and A. Zacchei, Federation Proc., 25, 197 (1966). 65 J.E. Baer, C.M. Mucha, S.A. Spitzer and H.W. Yee, Federatsn Proc., 25, 197

-

(1966)-

66. N.W. 67. T.B. 68. W.B. 69. K. N. 70.

71 72

73

9

74. 75 76. 77. 7879.

Moulcheibir and W.M. Kirkendall, C l i n . Research, 13, 425 (1965). Repold8 and H.C. Pelle, C l i n . Research, 2, 184719661 McKeon, Jr., Arch. Intern-mie, 151, 225 (1964). Modi, C.V. D e r I n . Pharmacodynamie, 151, 13 - (1964). . - G. Satzinger, Ann. Chem. Uebigs, 150 (1963). 0. Heidenreich and L. Baumeister, Arch. Exptl. Pathol. Pharmakol., 247,

-

665,

-

347,,(1964)*

G. Fulgraff, L. Bauueister and 0. Heidenreich, Arch. Exptl. Pathol. Pharmakol. 3% (1964). 0. Heidenreich, G. Fulgraff, L. Baumeister and K. Schmiz, Arch. Exptl. Pathol. Pharmakol., 249 432 (1964). 0. Heidenreich, G. Gharemani, P. Keller, Y 1 Kook and K. Schmiz, Arzneimittel-Forsch., 1242 (1964). R.J. Girerd, C.L. Rsssaert, G. D i P a s q u a l e and C.R. Smith, Rev. Can Biol., 23, 197 (1964). OFHeidenreich and L. Baumeister, Klin. Wochschr., 42, 1236 (1964). -L.B. K i e r , D. D h a m and M.J. Fregly, J. Pham. S c i z 53, 677 (1964). M.J. Fregly, L.B. Kier and D. Dhawan, Toxicol. Appl. P&mcol., 6, 529

, 247,

-’ 14,

-

(1964)

T. Bruzzese, S. Casadio, E. Marazziuberti and C. Turba, J. Pham. Sci., 54 1041 (1965). 80. H d Yale and J. J. Prala, J. Med. Chem., 2, 42 (1966). 81. R.M.V. James and H. Jackson, Blochem. Pharmacol., 14, 915, 1847 (1965); B r i t . J. Pharmacol., 223 (1965). 82. R.C. %it and C.M. Kagawa, J. Med. Chem., 7, 524 (1964). 83 C.M. Kagawa, Endocrinology, 2, 724 (1964184. J.M. George, G. Saucier and F.C. Bartter, J. Clin. Endocrinol. Metabolism, 25 621 (1965)999 (1964). 85 C.M. Kagaua and R. Pappo, Endocrinology, 86. C.M. Kagawa, D.J. Bouska, M.L. Anderson and W.F. Krol., Arch. Intern. Pharm a c o F , 8 (1964). ’ J 368 , (1965). 87 P.F. D Arcy and E.M. Howard, J. Wsm. Pharmacol., l 88. R. S e l l e r i , 0. Caldini, R. Spano’, G. Cascio and M. Palazzoadriano, Arzneimittel-Forsch., 15, 910 (1965). 89. E.J. Ross, C l i n . ~ r m c o l .Therap., 6, 65 (1965).

-

-

-

25,

-’

2,

149,

-

78 -

Sect. I1

- Pharmacodynamics

A r c h e r , Ed.

Chapter &A. Angina P e c t o r i s and Antianginal Agents Paul Kennedy, Jr., Pharmaceutical Division, Geigy Chemical Corp.

, Ardsley,

N.Y.

During t h e last few y e a r s t h e symptom complex of angina p e c t o r i s h a s been t h e s u b j e c t of an i n t e n s e renewal of i n t e r e s t . Angina i t s e l f , by d e f i n i t i o n is pain, t h e e a s i l y described y e t poorly understood c l i n i c a l s i g n of a complex patho-physiological problem which occurs when t h e need €or oxygen and p o s s i b l y o t h e r n u t r i e n t s by myocardial t i s s u e exceeds t h e a v a i l a b l e supply. The need of t h e h e a r t i s governed by t h e work t h e h e a r t must perform and t h e supply i s dependent on an adequate coronary a r t e r i a l system and flow of blood through t h i s system. The oxygen s a t u r a t i o n of blood, t h e s t a t e of t h e c a p i l l a r y bed, t h e a b i l i t y o f myocardial t i s s u e t o u t i l i z e oxygen, and t h e metabolic s t a t u s of t h e h e a r t a r e r e l a t e d f a c t o r s . A t h e r o s c l e r o s i s of t h e coronary vessels i s t h e major e t i o l o g i c f a c t o r y e t i t should be noted t h a t angina p e c t o r i s can o c c a s i o n a l l y occur without evidence of coronary d i s e a s e , a s i n s e v e r e anemia, c o n g e n i t a l h e a r t d i s e a s e and advanced pulmonary d i s e a s e . I n c o n t r a s t , some p a t i e n t s w i t h evidence of coronary a r t e r y d i s e a s e never experience angina. Antianginal drugs have as t h e i r goal t h e a l l e v i a t i o n , a b o l i t i o n o r prevention of t h e symptom, angina p e c t o r i s , without a d v e r s e l y a f f e c t i n g c a r d i a c performance. I n a d d i t i o n , most drugs base t h e i r claim f o r a n t i a n g i n a l e f f i c a c y on p r o p e r t i e s which hopefully w i l l improve t h e b a s i c coronary i n s u f f i c i e n c y . No compound a v a i l a b l e today alters t h e fundamental a t h e r o s c l e r o t i c process. N i t r o g l y c e r i n has been and s t i l l i s t h e most important agent f o r a b o r t i n g t h e a c u t e a t t a c k of angina p e c t o r i s , though as w e s h a l l soon see, i t s e f f i c a c y has been challenged by some very thought-provoking experiments. I n animals n i t r o g l y c e r i n a c t s a s a pure d i l a t o r by i n c r e a s i n g coronary flow, decreasing coronary a r t e r i o v e n o u s d i f f e r e n c e and by e f f e c t i n g no s i g n i f i c a n t change i n c a r d i a c oxygen consumption. The same w a s thought t o be t r u e f o r man. I n 1959, Brachfeldl s t u d i e d f o u r normal and s i x mild c a r d i a c p a t i e n t s b e f o r e and a f t e r n i t r o g l y c e r i n . Following n i t r o g l y c e r i n , myocardial oxygen consumption increased as w e l l a s coronary flow. There w a s no change i n c a r d i a c work and a f a l l i n myocardial e f f i c i e n c y w a s observed. Also noted w e r e decreases i n pulmonary a r t e r y , pulmonary wedge and r i g h t a t r i a l p r e s s u r e s . A t t h e same time, Gorlin2 reported on seventeen p a t i e n t s , f o u r t e e n of whom had angina p e c t o r i s . Following n i t r o g l y c e r i n t h e r e was a decrease i n c a r d i a c output, blood pressure, and l e f t v e n t r i c u l a r work accompanied by f a l l s i n t h e systemic and pulmonary venous pressures. Coronary flow as measured by t h e n i t r o u s oxide flow method w a s e s s e n t i a l l y unchanged and even decreased i n some p a t i e n t s . Gorlin concluded t h a t n i t r o g l y c e r i n a c t e d by temporarily reducing t h e work of t h e h e a r t r a t h e r than by r e l a x i n g t h e coronary v a s c u l a r tree. The time-honored conclusion t h a t n i t r o g l y c e r i n increased coronary flow i n angin a l p a t i e n t s had been challenged using t h e technique of c a r d i a c c a t h e t e r i z a t i o n . Gorlin2 confirmed h i s f i n d i n g s using I 1 3 l serum albumin t o measure transcoronary c i r c u l a t i o n time with a n e x t e r n a l counting technique. Coronary c i r c u l a t i o n time was decreased i n normal i n d i v i d u a l s a f t e r n i t r o g l y c e r i n b u t remained t h e same o r increased i n t h e abnormal group. I n c o n t r a s t , Johnson and Sevelius,3 using r a d i o a c t i v e iodopyrocet, demonstrated i n c r e a s e s i n myocardial blood flow i n

Chap. 8A

Antiang inal

Kennedy

79 -

arteriosclerotic patients followin sublingual nitroglycerin and oral pentaerythritol tetranitrate. While the substances seem to have fallen from favor, a variety of external countin! techniques have been developed for the study of the coronary circulation. Rb 6, Rb84, Na24, Kr42, Kr85 and ~e133,among others, have been employed to measure myocardial blood flow.

,bl

Bing4 has worked with a coincidence counting technique using RbB4, a positron emitter, to determine coronary flow equivalent. With this method, nitroglycerin increased the clearance equivalent of Rb84 in patients without coronary heart disease and caused a decrease in clearance equivalent in patients with coronary disease. Ross5 has measured human myocardial blood flow by determining precordial radioactivity following the injection of solutions of radioactive Kr85 or Xe133 directly into coronary arteries after coronary arteriography. The right and left coronary circulations could be studied separately and comparisons made with anatomical findings. No significant differences in flow at rest were found between normal and coronary artery disease patients. Bernstein6 using Xe133 has studied the effect of nitroglycerin in thirty patients undergoing selective coronary arteriography for the evaluation of chest pain. Nitroglycerin (0.4 mg) sublingually caused a decrease in myocardial blood flow, left ventricular work and no significant decrease in coronary vascular resistance in patients with normal as well as diseased coronaries. Nitroglycerin (0.1 to 0.2 mg) injected directly into the coronary vessel caused an immediate though transient increase in myocardial blood flow and a decrease in coronary vascular resistance in the same patients. This blood level, attained by intracoronary administration, could not be achieved by the sublingual route using accepted dosages. However in attempting to explain the apparent conflict between arteriographic dilation caused by nitroglycerin and other nitrates7 and the failure of these compounds to increase blood flow, Bernstein has presented a hypothesis based on a biphasic action. He suggests that during the first phase there is coronary vasodilation, a drop in coronary vascular resistance and increase in myocardial blood flow. During the second phase of the drug's action, the effects on the systemic circulation predominate and there is a decrease in arterial pressure and cardiac output. The decrease in coronary vascular resistance persists into the second phase, but because of the drop in arterial pressure, the myocardial blood flow falls below control levels. Winbury8 and Conng have presented detailed reviews and critiques of the use of radioactive tracers to which the reader is referred. Ross5 feels that the accurate location and discreet measurement of flow in unperfused areas of heart muscle which result from disease, may prove difficult using external counting techniques. The advent of selective coronary arteriographylo has made it possible to study the pathological anatomy of the diseased coronary artery. Sones7 and Likoffll have demonstrated an increase in the calibre of the main coronary arteries and those branches large enough to be visualized following sublingual administration of nitroglycerin and other nitrates. Not only have presumably sclerotic vessels been seen to dilate, but also previously non-visualized collaterals were visualized following drug administration. However an increase in the calibre of a vessel, "vasodilation", does not imply an increase in myocardial blood flow. Winbury8 has pointed out that smaller vessels which regulate flow cannot be visualized by the approach and questions whether or not the results obtained by

80 -

Sect. I1

- Pharmacodynamics

A r c h e r , Ed.

these techniques have any greater significance than those obtained by the measurement of blood flow. Their value in anatomic appraisal of the larger visible vessels cannot be challenged. Nitroglycerin exerts harmacological effects on both the venous and arterial circulation. Mason, 15 using normal patients, demonstrated that nitroglycerin dilated capacitance vessels of the forearm and caused significant pooling of blood. This finding represents additional evidence for the fall in cardiac output following nitroglycerin. W illiamsl3 demonstrated with cineradiograms a decrease in ventricular dimensions after nitroglycerin. These observations further suggest that nitroglycerin may relieve angina by reducing the work and oxygen requirements of the heart. Cohenl4 has attempted to correlate in sixty patients the findings of selective coronary cinearteriography with clinical history, physical examination, electrocardiogram, Kr85 coronary blood flow studies, and myocardial oxygen and lactate extraction at rest and during stress. Some predictions could be made from the patient's history which were not previously possible. Disease of a single coronary vessel usually indicated pain less than one year's duration. Increasing duration in years revealed more extensive pathology. Pain limited to the chest usually indicated milder anatomic pathology than when the patient had pain in two or more sites or experienced prandial or nocturnal angina. Disease of three vessels was more common in this group. Seventy percent of the resting electrocardiograms were abnormal and two-thirds of the remainder were abnormal with exercise. The measurement of coronary flow at rest or after stress could not distinguish patients with coronary disease from those without, and no consistent correlation could be made with anatomic pathology. MyQcardial lactate production or decreased extraction of lactate by the myocardik could be correla ted with the severity of disease. Raab15 feels that interest in the metabolic and neuro-regulatory factors affecting the myocardium has been lagging due to a "one-sided preoccupation" with vascular anatomic factors. According to Raab, data on total coronary flow, arteriography and coronary sinus oxygen saturation do not locate circumscribed areas of anoxia or define the causative mechanism. In addition to the atherosclerotic process he feels that environmental, emotional and nicotine-induced over-stimulation of the sympatho-adrenal system and the deterioration of counter-regulatory mechanisms secondary to physical inactivity should receive more attention. Since catecholamines augment the uptake and consumptior.of oxygen far beyond energy requirements, factors such as stress, smoking, emotion and unusual physical exercise must be controlled by measures which can cushion this sympathetic activity. Emotional relaxation, nicotine abstinence and systematic physical training are recommended. Thus far we have limited our review to a few of the current techniques used to study the coronary circulation and in so doing have shown some of the controversy surrounding the mechanism of action of nitroglycerin. The problem of angina pectoris and its relief is equally complex. Angina cannot be shown by the arteriogram or measured by coronary sinus oxygen or lactate extraction studies, or identified by the varied techniques used to calculate coronary flow. Vasodilation is not synonymous with increased coronary blood flow and neither expression means relief of angina. Several factors, the exact inter-relationship of which are not understood are probably etiologic, and relief of angina

Chap. 8A

Antianginal

Kennedy

81 -

can probably be attributed to several mechanisms which are also not fully understood as yet. Sublingual nitroglycerin has long been considered the most useful agent for the relief of acute anginal attacks and for the prevention of an attack if taken prior to a stressful situation known to precipitate angina. While evidence has been mainly subjective, various exercise tests have been developed to achieve objectivity. Nitroglycerin and other compounds are studied for their effect on prevention, delay or decrease of anginal pain associated with a standard amount of exercise. An evaluation of simultaneous or post-exercise electrocardiographic changes may be included in the evaluation. Bernsteinld has criticized these approaches because the patients studied are often limited to those whose anginal attacks are reproducible after a given amount of exercise or to those with reproducible electrocardiographic abnormalities. These patients represent but a small fraction of the total group of patients with angina pectoris. Sandler, l7 using a double-blind technique has reported that nitroglycerin does not alter the duration of an established anginal attack when compared to placebo, but that the drug increased work performance and shortened the duration of ensuin angina when administered prophylactically three minutes before exercise. RussekfB corroborated this later phase of Sandler's experiment giving nitroglycerin five minutes prior to exercise. Fischlg has been unable to demonstrate any effect of nitroglycerin on the duration of the actual attack, and in addition, found the drug no better than placebo when given three, five and ten minutes before exercise. Because of the difficulty in matching nitroglycerin with a perfect placebo Fisch feels that the responses of patients in the previously mentioned studies may have been influenced by drug recognition. This controversy involving nitroglycerin accentuates the host of difficulties inherent in the evaluation of drug efficacy in angina pectoris. If the attacks are of very short duration, for example, under ninety seconds, it is conceivable that the effects of nitroglycerin might not have time to be distinguished from those of placebo. It should also be pointed out that Fisch's patients were carefully picked for reproducibility of symptoms and are not necessarily representative of the majority of patients with angina pectoris. While angina is frequently related to effort, it also occurs at rest, during or after eating and at the time of emotional stress. Nitroglycerin, despite controversy, remains the drug of choice for immediate relief from an acute anginal attack and for very short-term prophylaxis. Amy1 nitrite though faster in'onset of action, has a shorter duration of action and must be inhaled. Isosorbide dinitrate and erythrol tetranitrate are used sublingually, and by this route have been shown to afford clinical protection in anginal patients for periods up to two hours. These agents'have little or no value in aborting the acute anginal episode. Newer sublingual nitrates, such as clonitrate20 and stereo-isomers of pentanitrates21 do not seem to possess any advantages over nitroglycerin. Several long-acting nitrates are available for long-term prophylaxis of anginal attacks. This concept of therapy is far different from treatment of acute attacks or the relatively short prophylaxis mentioned previously in relation to the sublingual agents. The efficacy of the organic nitrates available for oral administration such as pentaerythritol tetranitrate, erythrol tetranitrate,

82 -

Sect. I1 - Pharmacodynamics

A r c h e r, Ed.

isosorbide dinitrate and trolnitrate phosphate 'must be established by long term investigation. As a class they seem to have pharmacological activity similar to nitrogl~cerin~~-~4 but the results of long term treatment are difficult to evaluate in terms of their effect on anginal pain. Investigations conducted without placebo control are generally enthusiastic, while those employing double-blind methods have frequently not shown a statistical difference from placebo. The stresses of every day living involve a myriad of difficult-tomeasure variables which influence the number and severity of anginal attacks, and therefore, a high degree of placebo responses can be expected. Bernstein16 in considering this problem, suggests that treatment periods continue at least one year for each drug under evaluation. Unfortunately, longer studies of this type are extremely difficult to undertake from a practical standpoint. Dipyridamole, a relatively new entry in the antianginal field, and quite unrelated to the nitrates, has a chemical nucleus of pyrimido-pyrimidine rings. When administered intravenously or orally to laboratory animals, the drug increases coronary blood flow and coronary sinus oxygen content. Normal individuals, as well as patients with impaired coronary circulation, have shown similar increases in these parameters following intravenous administration.25 Laboratory studies with heart preparations have shown dipyridamole to protect against disturbances in function and fall in high energy phosphate levels induced by hypoxia.26 Heart mitochondria of animals pretreated with the drug and exposed to hypoxia demonstrate preservation of anatomic structure. 27 Vineberg28 employed long-term oral administration of dipyridamole in animals with experimental coronary occlusion, resulting in enhanced survival and increased coronary collateral circulation as compared to untreated controls. B ~ n a g has ~ ~shown that dipyridamole, in the presence of intact red blood cells, prevents the deamination of exogenous adenosine suggesting this mechanism may account for effects on the coronary blood flow. Recently Emmons30 and co-workers demonstrated a favorable effect by dipyridamole on human platelet aggregation. Dipyridamole is not intended for the relief of an acute attack of angina pectoris but for long-term administration. In some instances several months of continuous therapy may be required to achieve a satisfactory response. Clinical observations without placebo controls in long-term treatment of angina pectoris have been quite enthusiastic and investigations have run as long as 24 months.31 The results of studies using placebo controls and the double blind technique have been equivocal. Chromonar hydr~chloride~~ and phenylamine,33 not yet available commercially in the United States, are agents that increase coronary blood flow. In addition, phenylamine is reported to have sympatholytic, adrenolytic and central sedative actions. Clinical reports from abroad have been, for the most part, encouraging. The monoamine oxidase inhibitors have been used in the therapy of angina pectoris as well as in hypertension and certain psychiatric disorders. While experimental evidence for this effect on angina may be scant, several theories regarding mechanisms of action have been proposed.34 These include central nervous system stimulation, mood elevation, central analgesic effect, increase in coronary flow (though this seems unlikely in man), a blocking effect on pain transmission, and decreased oxygen requirement of the tissues. However, there is no clear pharmacological basis for beneficial effects of these compounds on the coronary circulation or the myocardium.

Chap. 8A

Antianginal

Kennedy

83 -

According to a recent review35 most clinical studies indicate that the monomine oxidase inhibitors cause a subjective increase in exercise tolerance and patients can do more work during standardized exercise tests than during control periods. Nevertheless, ischemic ECG changes are not altered or delayed and may be more severe. Controlled and uncontrolled studies, while not unanimous, do seem to suggest some effect on anginal pain or the patient's reaction to it. This group of drugs can produce serious toxic reactions which have limited their use, by and large, to cases resistant to other forms of therapy. The use of beta-adrenergic blocking agents in the therapy of angina pectoris has been the subject of recent interest. Since the catecholamines increase oxygen utilization by the heart as previously discussed,l5 compounds that may block this effect present an interesting approach to the problem. Pronethalol has been studied fairly extensively abroad. The drug reduces heart rate at rest and after exercise. While there appears to be a normal exertional increase in stroke volume, overall cardiac output after exercise may be red~ced.3~9~7 The drug should not be used in patients with cardiac failure or impending failure because of its myocardial depressant action. There were several favorable reports on the use of this agent in angina pectoris. More extensive use has been partly limited by frequent CNS side effects and by a report of its tumor-producing activity in mice.38 Propanalol has a similar mode of action, and has been reported to have a therapeutic effect ten times greater than pronethalol without carcenogenic otential. Initial impressions of this agent have been generally favorable.39-11 No attempt has been made to consider all possible approaches to the problem. The use of sedatives and tranquilizers in angina pectoris can be quite useful for those individuals in whom anxiety, excitement and tension are precipitating factors. Alcohol in moderation, should not be overlooked in this regard. Papaverine, xanthines and anticoagulants have their advocates. Treatment of coexisting cardiac decompensation, arrythmias and hypertension benefits the anginal patient, as does reduction of weight and correction of anemia. Chemical or surgical thyroidectomy has been successful in some severe cases. Interest in surgical revasculization seems to be gaining some momentum. Though many if not all approaches are probably beneficial to certain individual patients or groups of patients, none as yet represent the ideal. None cure or halt the atherosclerotic process. The search must continue. References

1. 2. 3.

4.

5. 6. 7.

Brachfeld, N.; Bozer, J, and Gorlin, R.: Circulation 19, 697 (1959). Gorlin, R., Brachfeld, N., MacLeod, C. and Bopp, P.: Circulation 3 705 (1959). Johnson, P.C. and Sevelius, G.: JAMA 173, 1231 (1960). Bing, R.J., Bennish, A., Bluemchen, G., Cohen, A., Gallagher, J.P. and Zaleski, E. J. : Circulation 29. 833 (1964). Ross, R.S., Ueda, K., Lichtlen, P.R. and Rees, J.R.: Circulat. Res. 15, 28 (1964). Bernstein, L., Friesinger, G.C., Lichtlen, P.R. and Ross, R.S.: Circulation -9 33 107 (1966). Sones, F.M., Jr.: presented at 14th Annual Convention of the American College of Cardiology, Boston, Mass., February 18, 1965.

a4 -

Sect. 11

- Pharmacodynamics

A r c h e r, Ed.

Winbury, M.M. : Advances . i n Pharmacology, S. G a r a t t i n i ; P.A. Shore (ed.), New York: Academic Press, 1964, vol. 3, pp 1-78. 9. Conn, H.L., Jr. : C i r c u l a t . Res. 10, 505 (1962). C i r c u l a t . Res. 527 (1959). 10. West, J.W. and Guman, S.V.: C i r c u l a t i o n 26, Likoff, W., Lehman, J.S., Kasparian, H. and Segal, B.I.: 11. 751 (1962). 12. Mason, D.T. and Braunwald, E. : C i r c u l a t i o n 32. 755 (1965). C i r c u l a t i o n 32. 767 (1965). 13. W i l l i a m s , J.F., Jr., Glick, G. and Braunwald, E.: h e r . J. Cardiol. 14. Cohen, L.S., E l l i o t t , W.C., Klein, M.D. and Gorlin, R.: 17, 153 (1966). 15. Raab, W.: Amer. Heart J. 66. 685 (1963). Vasc. D i s . 2, 357 (1965). 16. Bernstein, A. and Simon, F.: Lancet #7415, 747 (1965). 17. Sandler, G.: 18. Russek, H. I. and Howard, J.C., Jr, : JAMA 189, 108 (1964). 19. Fisch, S.: presented a t t h e American Therapeutic Society, 66th Annual Meeting, June 1965. 20. Warshaw, L.J., Shafer, N., Caginalp, N. and Stein, I.: Med. Times 93, 614 (1965). Koretsky, S. and Altman, G.E.: h e r . J. Cardiol. 15, 220 21. Riseman, J.E.F., (1965). J. New Drugs 5, 143 (1965). 22. Scriabine, A. and McShane, W.K.: Amer. J. Med. S c i . 23. Afonso, S., Rowe, G.G., Lowe, W.C. and Crumpton, C.W.: 246, 584 (1963). Chelius, C.J., Afonso, S., Gurtner, H.P. and Crumpton, C.W.: 24. Rowe, G.G., J. Clin. Invest. 40, 1217 (1961). Amer. Heart J. 63, 146 (1962). 25. Kinsella, D., Troup, W. and McGregor, M.: 26. Kunz, W., Schmid, W. and Siess, M.: Arzne 12, 1098 (1962). Rev. Argent. Cardiol. 27. Lozada, B.B., Laguens, R.P. and Riuz Beramendi, A,: 32(5), (1965). Canad. Med. Ass. 28. Vineberg, A., Chari, R.S., P i f a r r e , R. and Mercier, C.: J. 87, 336 (1962). Douglas, C.R., I m a i , S. and Berne, R.M. : C i r c u l a t . Res. 15. 29. Bunag, R.D., 83 (1964). 30. Enrmons, P.R., Harrison, M.J.G., Honour, A.J. and Mitchell, J.R.A.: Lancet 603 (1965). 31. Griep, A.H. : Vasc. D i s . 1, 299 (1964). 32. Lochner, W. and Hirche, H j . : Arzneimittelforsch. 13, 251 (1963). Deutsch. Med. Wschr. 85. 1405 (1960). 33. Bohm, C.: 34. Zbinden, G.: Amer. J. Cardiol. 5, 1121 (1960). "Vasodilator Drum". i n Goodman, L.S.: and Gilman. A.: 35. Nickerson. M.: The Pharmacological Basis of Therapeutics, ed. 5, New-York: The k c m i l l a n Co., 1965, chap. 34, p. 749. 36. Dornhorst; A. C: and. Robinson, B.F. : Lancet #7251, 314 (1962). 37. Schr'dder, G. and Werk'd, L.: Amer. J. Cardiol. 15, 58 (1965). B r i t . Med. J. 2, 1266 (1963). 38. Paget, G.E.: 39. Srivastava, S.C., Dewar, H.A. and N e w e l l , D.J.: B r i t . Med. J. 2, 724 (1964) B r i t . Med. J. 2, 337 (1965). 40. G i l l a m , P.M.S. and Prichard, B.N.C.: 41. Keelan, P.: B r i t . Med. J. 1, 897 (1965). 8.

L

.

~

-

+u ~

-

I

Chapter 8 B.

Antiarrhythmics

Ralph D. Tanz, Department of Pharmacology, Geigy Chemical Corp., ArdsleY, IJ. Y.

From the therapeutic approach, despite ~rmerousinvestigations, the degree of advance in the area of antiarrhythmic therapy has not been as mked 88 one might desire. Every year many new dn;lgs are reported, but few ever reach clinical evaluation and none have thus far surpassed the traditional agents. A systematic synthesis of antiarrhythmic and antifibrillatory compounds is rendered difficult by the fact that most antiarrhythmic agents are quite different in chemical structure. A rnrmber of substances with entirely different chemical structures are known to possess antiarrhythmic activity and it is extremely difficult to attribute this action to a basic structure commn to a l l agents in this field. Diverse drugs m y abolish or prevent arrhythnias by influencing different cardiac parameters (e.g., refmctoriness, excitability, rhythmicity and conductivity). Cardiac stimulants or depressW, ,synpathamimetics, sympatholytics, agents enhancing catecholanine depletion or storage, cholinergics, anticholinergics, local anesthetics, sntihistaminics, autonomic and central neural depressants all have offered ~lurmhrougpathways for pharnracological investigation. Thus a clear demonstration of structure-activityrelationships is relatively impossible and meaningless.

-

The basis for this relative lack of success may be in the anatomical a d physiological nature of the heart itself, which, from the standpoint of arrhythmias, can really be considered to possess five distinct structures. The epecialized conduction system, which allows the heart to contract in a synchronous manner, consists of the sino-atrial node, the atrioventricular node, and the Bundle of His and diffuse Purkinje fiber system. In addition, the auriculssr and ventricular musculature also conducts impulses, and m y , under certain conditions, initiate impulse formation resulting in ectopic beats. Disturbances in either the conduction system or musculature, can lead to a ,disorganizedbeat and inefficient cardiac contraction which in turn m y diminish cardiac output. Such disturbances in iqpulse formation and/or conduction are referred to as cardiac arrhythdas, though a preferable term might be dysrhythmias. Since most serious arrhythmias result in a faster than normal ventricular rate, it is not surprising that the two principal agents, qlinidine and procaine amide have, as a camon property, the ability to depress impulse formation and, therefore, rate. As mentioned, there are msny chemical substances that can accompUsh the same end, but quinldine and procaine amide also possess pharmacological specificity for cardiac tissue that other agents often lack. Cardiac glycosides (alone or in combination), although usually used to improve the contractile force of the heart, are often employed to depress specific areas of the arrhythmic heart with the hope of restoring a more normal conduction time and efficient ventricular rate. The complex nature of the problem ha8 of necessity forced pharmacologists to resort to a number of different laboratory models in order to screen potential antiarrhythmic compounds, for no one model is suf'f'iciently useful by itself. The phsnaacologist must test for action upon rate production, excitability and conduction time. Deraagenents of impulse formtion due to chemical

86 -

Sect. I1

- Pharmacodynamics

A r c h e r , Ed.

agents (e .g. aconitine, cardiac glycosides, acetylcholine, catecholamines, etc.) o r physical events ( e l e c t r i c a l l y o r surgically induced arrhythmias) must be measured, and the degree of protection afforded by a test substance t o a standard agent, usually quinidine And, he must be ever c-ed mindm of the toxic properties of a new compound. These d i f f i c u l t laboratory limitations have t o date, yielded no chemlcal agent possessing greater therapautic efficacy than the three time-tested stalwarts, quinidine, procaine amide and cardiac glycosidides. What is equally disturbing i s the fact that the original observations of the antiemrhythmic properties of these substances resulted, not Avlm laboratory study, but rather from astute c l i n i c a l observation.

.

The presence of acetylcholine and norepinephrine i n proper ratio i n the f'unctional heart is Important i n the maintenance of cardiac rhythmicity. The ideal antiarrhythmic should therefore support a balanced r a t i o bsttreen cardiac norepinephrine and acetylcholine, yet at the same time it should ensure a diminution of myocardial e x c i t a b i l i t y t o circulating catecholamines. I n addition, the ideal agent should depress the i n t r i n s i c rhythmicity of the conducting pathways, and regulate the flux of intracellular sodium and potassium, thereby maintaining ionic balance. It seems obvious from the outset that no one drug will be found t o possess all the properties inherent i n the definition o f ' t h e ideal antiaxrhythmic. More likely, the i d e a l agent becomes many drugs capable of interfering with, or reversing, the numerous abnormal physiologic patterns of the heart. 1. Beta Adrenergic Blockers. With the a v a i l a b i l i t y of beta adrenergic blocking agents, pharmacologists have been handed a most important research tool. The agents of greatest current i n t e r e s t are dichloroisoproterenol ( X I ) , M J 1999,pronethala 1, propranolol and N-isopropyl-p-NitrophenylethamUznine (INPW). A l l have been shown t o protect against the i n i t i a t i o n of various, laboratory induced arrhythmias and t o terminate SQPY! existing arrhythmias Pronethalol and DCI have been reporged t o afford protection against u b a i n induced f i b r i l l a t i o n i n guinea pigs and t o reduce hythmias resulting from a combination of ouabain plus e l e c t r i c a l s t i m u l a t i o r Doubt has been cast, however, as t o whether the antierrhythmic properties of these agents are solely attributable t o t h e i r beta adrenergic blocking action? ye Recent evidence obtained by comparing the beta adrenergic blocking and antiarrrhythmic a c t i v i t y of the dextro-isomer and the racemic mixture of propranolol has resulted i n the current view that antiarrhythmic a c t i v i t y i s i n part due t o a non-sqcific (depressant) "quinidine-lib'' action, as w e l l as beta blocactivity. This conclusion i s based in p a r t on the f a c t that the racemic mixture of propranolol reversed the arrhythmias resulting from coronary ligation plus epinephrine, but the d-isomer did not; yet both agents reversed ouabain-induced arrhythmias and neither was effective against ventricular arrhybhmias due t o coronary l i g a t i o n alone. Since the d-is-r possesses only 1/50 the beta adrenergic blocking action of the racemic mixture, it appears that i n those arrhythmias associated with catecholamine release, beta blockade i s of importance.

,

.

Chap. 8B

Antiarrhythmics

Tanz

87 -

A number of studies have been performed comparing the antiarrhythmic activities of these agents with quinidine?"The results obtained appear to depend on the laboratory models employed, but, in general, propranolol is more potent than pronethalol insofar as beta adrenergic blocking and antiarrhythmic activity are concerned. Several British clinicians have also geported success in the treatment of ventricular arrhythmias with propranolol.l ' Pronethalol is somewhat different than its newest derivative, propranolol, in that it possesses both sympathom5metic activity and an antivagal action, both of which result in increased heart rate. Moreover, pronethalol lengthenes the . reflractory period, diminishes excitability and is a potent local anesthetic equa to procaine .1',13 One of the newer ents, INPEA, was compared to pronethalol for antiarrhythmic activlty ? '% Both agents antagonized catecholamine-induced arrhythmias (epinephrine and methylchloroform plus epinephrine), but were ineffective against the arrhythmias resulting f'rom coronary artery ligation. Furthermore, only pronethalol was effective in reversing ouabain-induced ventricular tachpxixdia, indicating once again, that pronethalol also possesses a nonspecific depressent action, similar to quinidine.

MJ 19% is a beta adrenergic blocker which also has the ability to antagonize cardiac irregularities produced by (large doses o f ) catecholamines, but affords no protection against the arrhythmias resulting f'rom ouabain or coronary 1igation.l' Insofar as protective potency against arrhythmias is concerned it appears that propranolol > pronethalol > MJ 1999.

.

2. Catecholamine Depletors It follows that since beta adrenergic blocking agents are able to antagonize certain types of arrhythmias that catecholaminedepletion might also afford protection against some arrhythmias. In 1963 Roberts et al.17 reported that following reserpinization the ability of digitalis to induce ventricular arrhythmias was diminished. Moreover, $TM 10, an agent which prevents the release of catecholamines, markedly reduced the response of the ventricular pacemaker to digitalis. The abilit of reserpine to antagonize but it should be noted arrhythmias has been confirmed by several workers,al'* that the degree of success is much higher if an adrenalectomized preparation is employed so as to reduce the circulating catecholamines. In addition, guanethidine and bretylium have been reported to antagonize the arrhythmias pro21 duced by acetylcholine and electrical stimulation

-

.'"

3 . Sympathomimtic and Pressor Agents. Certain types of arrhythmias (especially

supraventricular tachycardias) may be converted to normal sinus rhythm by raising the blood pressure, thereby eliciting reflex vagal stimulation. E l l i s a2 has reported that metho-ne and some closely related substituted phenylisopropylamines can effectively convert ventricular tachycardias to sinus m h m . Special mention was made of a-methyl-$-hydroxy-$-( 2,5 diethoxyphenyl)-N-isopropyl ethylamine, which was found to be effective in restoring sinus rhythm under a number of experimentally produced ventricular tachycardias (e .g ouabain, epinephrine with or without hydrocarbon anesthesia, DMPP and coronary artery ligation). Similarly, isopropyl methoxamine has been found to be effective in converting digitalis-induced ventricular tachycardia to sinus rhythm, though the duration was rather transient.'' In their review on the use of vasopressor d r u g s in the therapy of cardiac arrhythmias, Corday and co-workerg4note that in addition

.

88

Sect. I1

- Pharmacodynamics

A r c h e r , Ed.

to methoxmuine, such agents as norepinephrine, mephentermine, m e t a r a m l , phenylephrine and angiotensin have been used successAiLly. However, such therapy is only u s e m when the arrhythmia is associated wlth hypotension. A possible exception lies in the usage of angiotensin which has been found to protect against a variety of experimentally induced arrhythmias both in vitro and in vivo ?,' aeAccordingly, these investigators also ascribed a "quinidine-like" action to angiotensin. In the presence of heart block, ventricular tachycardia and quinidine The mechanism inintoxication, isoproterenol has been used success~ully.~~ volved is apparently based on the fact that isoproterenolaccelerates the basic idioventricular pacemaker thereby suppressing ectopic ventricular activity. The increase in heart rate, coronsry f l o w and cardiac output may also be responsible for terminating arrhythmias. O f course, driving the heart at a more rapid rate could achieve the s8me result as drug therapy. The inherent danger in the use of isoproterenol is that it is almost a pure beta adrenergic stimulator and may precipitate ventricular tachycssdia or fibrillation.a7

4. Local Anesthetics. Since the observation that procaine was effective in suppressing arrhythmias, and the subsequent developnt of procaine amide, numerous local anesthetics psVe been employed in antiarrhythmic drug therapy. Lidocaine, a synthetic local anesthetic, has recently been reported to be of value in the treatment of cardiac arrhythmias.asThe mechanism of action appears to be similar to that of procaine. The conduction time is slowed, excitability depressed and the refractory period prolonged. In therapeutic doses mlyocardial contractility does not appear to be depressed nor is the blood pressure depressed. It ha8 a brief duration, is relatively safe and may be given i.v., all of which may mske it useful in arrhyt,hmias which sometimes occur during cardiac surgery. Pruss and Hidalgo2@have reported on a new substance kvoxadrol!, which is a local anesthetic and a ganglionic blocking agent. The authors claim that it is slightly less potent than quinidine but more potent than either procaine amide or lidocaine in reversing atrial fibrillation caused by the topical application of acetylcholine. Moreover, it is very effective in restoring sinus rhythm in dogs with coronary artery ligation ventricular arrhythmias.

5. htihistaminics. In 1948 it was demonstrated that a number of antihistamines possessed sane pharmacologic properties in common with atropine, procaine amide and quinidine. By 1952 the suppression of ventricular premature

systoles by antazoline had been described, and in 1959 a report appeared noting that antazoline was more effective than quinidine in the suppression of spontaneous and surgically induced ventricular fibrillation in the hypothermic dog?' In conrmDn with quinidine, antazoline is a local anesthetic, possesses sane anticholinergic properties and causes mild adrenergic blockade. However, as opposed to quinidine, It increases peripheral vascular resistance but decreases both stroke volume and cardiac output without altering mean blood pressure. Thus, pressor agents are never required?* Like quinidine it produces its antiarrhythmic effects primarily by depressing conduction velocity. Apparently it is effective in atrial, nodal and

Chap. 8B

Antiarrhythmics

T anz

89 -

ventricular tachycardias, as well as arrhythmias associated with d i g i t a l i s intoxication. Diphenhydramine has also proven t o be an effective31antif l b r i l l a t o r y agent at low doses both i n the laboratory and c l i n i c .

6. Central Nervous System (CNS) Agents. Diphenylhydantoin (Dilantifl) is a potent antiepileptic which produces generalized depression of the CNS. It has been used successfldly i n abolishing numerous arrhythmias both i n the laboratory and i n the c l i n i c ?1 j a 4 * 7 Its duration of action i s however, relatively short (2 30 mins .) I n the heart Dilantin i s said t o diminish conduction velocity, may depress c o n t r a c t i l i t y and produces a net efflux of i n t r a c e u u l a r sodium and an influx of potassium, which tends t o r a i s e the resting membrane potential thereby reducing the likelihood of cardiac arrhythmias. The general impression i s that it should not be used as an antiarrhythmic u n t i l procaine amide and quinidine have been tried f i r s t .

-

.

38

Fekete and Borsy have shown that certain thymoleptics, such as imipramine, desmethylimipramine, amitriptyline and trimepropimine display mild antiarrhythmic a c t i v i t y i n rats and dogs.

7. Miscellaneous Agents. Space limitations preclude the inclusion of the

numerous substances that have been tested for antiarrhythmic a c t i v i t y . The antimalarial substance chloroquine, depresses myocardial e x c i t a b i l i t y and exerts a n t i f i b r i l l a t o r y a c t i v i t y as does the o r a l hypoglycemic phenformin (DBfl).33 Sparteine is a potent, short acting a n t i f i b r i l l a t o r y substance which, i n contrast t o quinidine, increases myocardial conduction velocity. 33 Much i n t e r e s t has been generated i n the antiarrhythmic properties of synthetic oxytocin (Syntocino#) especially by Canadian workers. Syntocinon has been demonstrated t o suppress a variety of laboratory and c l i n i c a l srrythmias, including those related t o catecholamines as w e l l as arrhythmlas induced by injecting metrazol into the fourth ventricle. 39-43 It should be noted however, that 0.55 chlorobutanol is used i n the vehicle and that it a l s o displays antiarrhythmic a c t i v i t y (un ublished observations). The cholinergic drug edrophonium chloride (Tensilo& has been reported t o display transient antiarrhythmic a c t i v i t y , eliminating paroxysmal auricuLar tachycardia i n about 505 of treated patients .a4 Russian workers have recently described the use of cocarboxylase as being effective against a variety of c l i n i c a l l y observed arrthythmias .45 Iproveratril (Isoptin@) i s a new beta adrenergic blocker that has been claimed t o possess antiarrhythmic a c t i v i t y greater than e i t h e r pronethalol or MJ l ~ f ~ ~ 4 7 H o w e v it e r i, s also a toxic substance with a ra.t;her n a r r o w margin of safety. Chelating agents, such as EUI!A, have been employed with various degrees of success i n the past. A new substance in t h i s group i s 10-phenanthroline which was reported t o be as effective as quinidine i n reversing auricular f i b r i l l a t i o n of isolated rabbit hearts. Apparently it accomplishes t h i s by lengthening the refractory period and the depolarization p e r i ~ d f * r ~ ~ Newer chemicalmieties which have been reported t o possess antiarrhythmic activity, but which cannot be c r i t i c a l l y evaluated a t t h i s t i m e , are:

90 -

Sect. I1

-

Pharmacodynamics

A r c h e r , Ed.

,

a s e r i e s of N,N-diisopropyl-N' -isoamyl-N' -diethylaminourea (P-286)s0 N-(~aminoalkyl)phthalimidese~ *5 the procaine amide analogs of p-&no-N[2-( substituted amino) ethyl) benzamides,66 derivatives of morphanthridineB7 2-diethylmino-l-phenylethy1, 2-ethoxy HC1 and i t s 4-ethoxy analoff*, and the l o c a l anesthetic N,N-bis (phenykarbamoyl-nethyl) dimethylaamnium C 1 (QX$ 7 ~ ? ) ' ~More . work w i l l have t o be done on these substances before a fair pharmacologic appraisal can be made.

,

,

References 1. b e , R. A. and Abrams, W. B.: i n "Cardiovasculaz Drug Therapy", ed. A. N. w e s t and J. H. Moyer, G r u n e and Stratton, I?. Y. p.455 (1965). 97 ( 1 9 ~ 1 2. E r l i j , D. and Mendez, R.: J. m c o l . , 21, 462 (1963). 3. Selciya, A. and E. M. Vaughan W i l l i a m s : 4. Wyte, S. R. and R. E. Tan Eick: Fed. Proc., 5. Lucchesi, B. R.: J. Pharmacol., 6. Stickney, J. L., B. R. Lucchesi and G, D. Abrams : Fed. Proc a , 6 2 1 (1966.. 7. Lucchesi, B. R., L. S. Whitsitt and J. L. Stickney: Confr. on New Adrenergic Blocking Drugs; N. Y. Acad. Sci., Feb. 24-26, 1966. 8. Benfey, B. G. : Fed. Proc 24, 234, (1965) 9. Benfey, B. G. and D. R. Varma: Brit. J. P h w c o l . , 26, 3 (1966) 10. Rowlands, D. J,, G. Howitt and P, Ihrkman: Brit. Med. J., 1,891 (1965) ll. Sloman, G., J. S. Robinson and K. Mchan: B r i t . Med. J., 1, 895 (1965). 12. G i l l , E. W. and E. M. Vaughan W i l l i a m s : Nature, 201, 199 T1964) 13. Wallace, A . G., W. G. Troyer, M, A. Lesage and Zotti: 140 (1965) 14. Somani, P. and B. K. B. Lua: J. Pharmacol., 147, 194 (1964). Fed. Proc., &, 712 (1965). 15 Somani, P. and B. K. B. h: 16. Somani, P., J. G. Fleming, G, K. Chan and B. K. B. Lum: J. Phe;rmacol., 151, 32 (1966) 149 (1963). 17. Roberts, J., R . Ho, J. Reilly and V. J. Cairoli: Circ. Res., 325 (1964) 18. Lu, G. G.: Fed. Proc. 19 Ievitt, B., F. Ciofalo and J. Roberts: Fed. 20. Leveque, P. E. and R. C. Grubbs: Fed. Proc. M.: ~ m .J. cardioi., 21. -caner, (1964). 22. E l l i s , C H. : Arch. i n t Phamcodyn., JJO, 23 Paradise, R. R. and V. K. Stoelting: Proc. SOC. Exp , Biol. Med., U6,72 (1964) 24. Corday, E., F. F. Barbieri and T-W. lang: i n Cardiovascular Drug Therapy, ed. A. N. Brest and J. H. Moyer, Grune and Stratton, N. Y., p. 485, 1965. 25 8 Beltrami, E. and A. Beaulnes: Rev. Can. B i o l . , a , 191 (1964). 121 (196; Beaulnes, A., G. Gariepy, J. Brodeur and E. Beltrami : Fed. Proc 25 26. Dreifus, L. S., M. D. Rabbino and Y. Watanabe: i n Cardiovascular Drug Them y, ed. A. N. Brest and J. H. Moyer, G r u n e and Stratton, N. Y., p.491,

144,

145,

.

.,

0

E. x., 18,

a,

a,

.

.

IJ,=~

h.4

a,

0

(196523. . - _ _ 27. Dresel, P. E., M. 28. 29

30 31 32

.

140, 67

C Hart and B. C Stromblad: J. Pharmcol., Frieden, J. : Am. H e a r t J., 713 (1965). pruss, T. P. and J. Hidalgo: Fed. Proc., 32'7 (1964). Angelakos, E. T. and A. R. Hegnauer : J. Pharmacol., 12, 37 (1959) Dreifus, L. S., M. D. Rabbino and Y. batanabe: Med. m n . N. Amer., 371 (1964). Bellet, S : B r i t . Pract 19 (1961).

.

s,

., 186,

a,

(1963).

. 48,

Chap. 8B

Tanz

Antiarrhythmic s

x,

33 34 35 36

9

37 38 39

91 ,-

Lamla, M.: Arch. int. Pharmacodyn., 442 (1965). Mixter, C. G., J. M. &ran and W. Austen: Am. J. Cardiol., 332 (1966). Ruthen, G. C.: Am. Heart Jln., 70, 275 (&). Bernstein, H., H. Gold, T-W. Law, S. Pappelbaum, V. Bazika and E. Corday: JAA-A., 695 (1965)Conn, R. D.: New Engl. J. Med., 2J2, 277 (1965). Fekete, M. and J. Borsy: _Med. exp. l0, 93 (1964). Klassen, G. A.,J. W. Rubin and M. McGregor: Am. J. Cardiol., l2, 523 (1963). Varma, D. R., K. I . Melville and M. D. S i l v e r : Arch. i n t . Pharmacodyn., 4-40(1963). M e l v i l l e , K. I. and D. R. Varma: Biochem. Pharmacol., 8, 136 (1961). Panesset, J. C. and A. Baulnes: Rv. Can. Biol., 20, 47-(1961).

x,

a,

40.

41.

.

42.

43 Bircher, R. P. : Fed. Proc 3, 560 (1964). 44. Moss, A. J. and L. M. Aledort: Am. J. Cardiol., JJ, 62 (1966). 45 Bakumenka, M. S.: Kardioloaiya (U.S.S .R.), abstracted i n Excerpta Med. 2, 1965. 46. Melville, K. I., Shister, H. E. and S. Huq: C a n . Med- Jln., g,761 (1964). 47. Schmid, J. R . and C. Hanna: Fed. Proc., 3, 382 (1966). 48. Yaw, W. C., S. Rothman and A. Gimeno: Proc. SOC. EXP Biol. Med., 2, 136 (1963). 49 Rothmsn, S. and W. C. Yaw: Am. J. Physiol., 206, 283 (1964) 50 Garrett, J., M. Goncalves Moreira, W. Osswald and S. GUimaraeS: 9

s.,

.

143,

51 52

53

54. 55

-

56 57

.,

J. Pharmacol., 243 (1964). Kempen, R. R . : Fed. Proc 3, 327 (1964). Hideg, K and H. 0. Hankovszky: Acta Chima. Acad. Sci. Hun@;., 3, 391 (1963) Hideg, K., L. Szekeres, H. 0. Hankovszky and J. Pam: Biochem. pharnaacol. (Suppl.), 12, 171 (1963). Hideg, K. and H. 0. Hankovszky: J. Med. Chem., 8 257 (1965). Szekeres, L., K. Hideg, H. 0. Hankovoszky and G. Papp: Acta phxsiol. Hung., 26, 287 (1965). Thyrum, P and A . R. Day: J. Med. Chem., 107 (1965) Werner, L. H., S . Ricca, E. Mohacsi, A , Rossi and V. P o Arya: J. Med. Chem.,

6

-8 74 (1965). 58. Grelak, R. P. and T. R. Sherrod: Fed. Proc. 24, 234,(1965). 59 Covino, B. G. and P. Rachwell: J. New Dru@;6 , Jan-Feb., 30 (1964). 0

92 -

Sect. I1 Chapter 9 .

- Pharmacodynamics

A r c h e r, Ed.

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

Walter T. Moreland, Chas. P f i z e r ti Company, Inc.,

Groton, Conn.

Medicinal chemical p r o g r e s s i n t h e f i e l d s of pulmonary and a n t i a l l e r g y a g e n t s * h a s been slow i n r e c e n t y e a r s . A t least t h r e e f a c t o r s a p p e a r t o have been r e s p o n s i b l e , i n v a r y i n g d e g r e e s , f o r t h i s h i a t u s : ( 1 ) t h e r e have been r e l a t i v e l y few advances i n i n s i g h t i n t o t h e n a t u r e of u n d e r l y i n g mechanisms, SO t h a t s c r e e n i n g methods have tended t o remain u n s p e c i f i c o r i n a p p r o p r i a t e ; ( 2 ) t h e p u r s u i t of e x i s t i n g s t r u c t u r a l l e a d s h a s f a i l e d t o p r o v i d e a g e n t s s u p e r i o r to t h o s e a l r e a d y i n u s e ; and ( 3 ) i n some i n s t a n c e s t h e r e h a s n o t been a n obvious need t o improve upon e x i s t i n g d r u g s , e s p e c i a l l y i n t h e f a c e of more u r g e n t probl e m s i n o t h e r areas, which h a s tended to d i s c o u r a g e s p e c i f i c r e s e a r c h c a l c u l a t e d t o produce new l e a d s and new methodology. R e t r o s p e c t i v e l y , t h i s p a t t e r n was s u b s t a n t i a l l y unchanged i n 1965, and a d e q u a t e summaries of c u r r e n t s t a t u s are provided i n a p p r o p r i a t e s e c t i o n s of newly r e v i s e d e d i t i o n s of s t a n d a r d reference t e x t s. 192 The f i e l d of pulmonary a g e n t s seems c e r t a i n t o become c o n s i d e r a b l y more a c t i v e i n t h e f u t u r e , however, i n t h e l i g h t of c l i n i c a l developments t h a t have r e c e i v e d widespread a t t e n t i o n i n 1965. The N a t i o n a l Heart I n s t i t u t e , i n a r e p o r t t o t h e S e n a t e A p p r o p r i a t i o n s Subcommittee,3 s t a t e d t h a t "emphysema h a s mushroomed i n t o a major h e a l t h problem, seemingly o v e r n i g h t d i s a b i l i t y benef i t s f o r emphysema are (now) exceeded o n l y by d i s a b i l i t y b e n e f i t s f o r a t h e r o s c l e r o s i s and coronary h e a r t d i s e a s e . 'I Many c l i n i c a l d i s c u s s i o n s of c h r o n i c o b s t r u c t i v e lung d i s e a s e p r e s e n t e d similar c o n c l u s i o n s .4 I n s p e c t i o n of t h e a g e n t s p r e s e n t l y a v a i l a b l e t o t h e p h y s i c i a n shows t h e l i s t 5 t o be dominated by sympathomimetic and x a n t h i n e b r o n c h o d i l a t o r s , e x p e c t o r a n t s and mucolytic a g e n t s and none of t h e s e i s p a r t i c u l a r l y new. ( C e r t a i n r e s p i r a t o r y s t i m u l a n t s 3 , 6 and a n t i i n f l a m a t o r y a g e n t s 3 are a l s o r e p o r t e d , b u t t h e i r u s e i s n o t u n i v e r s a l l y f a v o r e d . 4 ~ 7 ) The e n t i r e f i e l d was t h o r o u g h l y reviewed by Aviado i n two volumes c i t i n g ' s e v e r a l thousand r e f e r e n c e s . 8 It i s clear t h a t t h e r e i s room f o r improvement i n a l l classes, and i t may be e x p e c t e d t h a t o t h e r approaches t o complement o r d i s p l a c e t h o s e i n c u r r e n t use w i l l emerge as r e s e a r c h i n t e r e s t i n c r e a s e s .

...

****** *For t h e purpose of t h i s review, t h e c a t e g o r y "pulmonary and a n t i a l l e r g y a g e n t s " is c o n s i d e r e d t o i n c l u d e b r o n c h o d i l a t o r s , a n t i t u s s i v e s , e x p e c t o r a n t s , m u c o l y t i c s , n a s a l d e c o n g e s t a n t s and a n t i p r u r i t i c s ( a n t i h i s t a m i n e s ) . A n t i b i o t i c s , which occupy a n important t h e r a p e u t i c p o s i t i o n i n t h e r e s p i r a t o r y area, and a n t i i n f l a m m a t o r y a g e n t s are excluded because they u s u a l l y would n o t be developed s p e c i f i c a l l y f o r these indications (although antiinflammatory s t e r o i d s d e s i g n e d f o r t o p i c a l u s e might c o n s t i t u t e a n i m p o r t a n t e x c e p t i o n ) . D e s e n s i t i z a t i o n p r o c e d u r e s are c o n s i d e r e d t o be o u t s i d e t h e scope of medicinal chemistry.

Chap. 9

Mo reland

Pulmonary, Antiallergy

93 -

There can be l i t t l e doubt t h a t b r o n c h o d i l a t i o n by sympathom i m e t i c a g e n t s i s mediated through p - r e c e p t o r s , so t h a t i t i s n o t s u r p r i s i n g t h a t s i d e e f f e c t s such as t a c h y c a r d i a , r e s u l t i n g from e x c e s s i v e s t i m u l a t i o n o f p - r e c e p t o r s a t o t h e r s i t e s , should be a common o c c u r r e n c e . l a , 2a Poor o r e r r a t i c o r a l a b s o r p t i o n o f t e n p r e s e n t s an a d d i t i o n a l problem. I s o p r o t e r e n o l (I) , t h e p r o t o t y p e p-sympathomimetic, dominates !he group. 3Y4 Most o f t h e newer a g e n t s are c l o s e l y r e l a t e d t o i s o p r o t e r e n o l , and a l l are most o f t e n a d m i n i s t e r e d l o c a l l y i n some form o f a e r o s o l t o minimize s i d e e f f e c t s . Two a d d i t i o n a l a n a l o g s , c l o r p r e n a l i n e ( I n 9 and metaprot e r e n o l ( o r c i p r e n a l i n e , 111) ,lo r e c e i v e d c o n s i d e r a b l e c l i n i c a l a t t e n t i o n , e s p e c i a l l y i n Europe. The former i s s u i t a b l e f o r o r a l a d m i n i s t r a t i o n , b u t

I

i t a p p e a r s t o e l i c i t s u f f i c i e n t CNS s t i m u l a t i o n t o r e q u i r e concomitant a d m i n i s t r a t i o n o f a CNS d e p r e s s a n t . The l a t t e r , which i s r e p o r t e d t o have a minimal e f f e c t on h e a r t r a t e , h a s been most o f t e n a d m i n i s t e r e d by a e r o s o l . A v e r y r e c e n t report'' d e s c r i b e d a s e r i e s o f catecholamine a n a l o g s i n which t h e p h e n o l i c hydroxyls a r e r e p l a c e d by a l k y l - o r a r y l sulfonamido s u b s t i t u e n t s o f comparable a c i d i t y . When t h i s s u b s t i t u t i o n i s meta t o t h e ethanolamine s i d e c h a i n , p o t e n t a - o r p - r e c e p t o r s t i m u l a n t s r e s u l t ; t h e most a c t i v e p - a g o n i s t s o f t h e s e r i e s ( I V Y V and VI) approach t h e potency of e p i n e p h r i n e o r i s o p r o t e r e n o l i n smooth muscle p r e p a r a t i o n s . B r o n c h o d i l a t o r s t u d i e s w i t h t h e s e a n a l o g s have n o t been r e p o r t e d , and l i t t l e can be d i s c e r n e d from p r e s e n t e v i d e n c e r e g a r d i n g s p e c i f i c i t y o f a c t i o n . I n t e r e s t i n g l y , comparable s u b s t i t u t i o n i n t h e para p o s i t i o n

R1 IV V

R2

H

H

OH

H

VI a f f o r d s predominantly p-blocking a g e n t s . The problem o f s p e c i f i c i t y of a c t i o n among $-sympathomimetics remains a s u b s t a n t i a l c h a l l e n g e , which many would a r g u e t o be e s s e n t i a l l y u n r e s o l v a b l e because of t h e wide a n a t o m i c a l d i s t r i b u t i o n o f p - r e c e p t o r s . However, t h e work o f Lands and Brown," showing t h a t a - e t h y l s u b s t i t u t i o n i n c e r t a i n 6 - a g o n i s t s markedly f a v o r s p - r e c e p t o r s i n t h e b r o n c h i o l e s o v e r t h o s e i n t h e h e a r t ( T a b l e I ) , s u g g e s t s t h a t d i f f e r e n t i a t i o n among p - r e c e p t o r s i s p o s s i b l e and that c a r e f u l s t u d y may r e v e a l c o n s i d e r a b l e o p p o r t u n i t y t o achieve s p e c i f i c i t y f o r a particular site.

Sect. I1

94 -

- Pharmacodynamics

A r c h e r , Ed.

Table I E f f e c t of a - E t h y l S u b s t i t u t i o n on t h e S e c i f i c i t y o f A c t i o n o f P2 Sympat homime t i c A m i ne s

Rl -

H C2H5 H C2H5

R2 isopropyl isopropyl cyc l o pent y 1 cyclopentyl

R e l a t i v e P o t e n c i e s ( I s o p r o t e r e n o l = 1) +Cb Bronchodi l a t i o n +Ia -

1 0.06 .6

.016

1 0.001 .6 .0005

1 0.33

.7

.45

(a) positive inotropic effect (b) positive chronotropic e f f e c t Over tbe y e a r s , a c o n s i d e r a b l e number of x a n t h i n e a n a l o g s and d e r i v a t i v e s have been s t u d i e d . However, none h a s y e t succeeded i n d i s p l a c i n g t h e o p h y l l i n e , which remains t h e predominant non-sympathomimetic bronchol b , 21 d i l a t ~ r , ~ i n- s~p i t e of s u b s t a n t i a l o r a l a b s o r p t i o n and s i d e e f f e c t problems. Because of t h e a p p a r e n t s t r u c t u r a l and pharmacological l i m i t a t i o n s i n h e r e n t i n t h e sympathomimetic approach, t h e f u t u r e seems c e r t a i n t o b r i n g a s u b s t a n t i a l i n c r e a s e i n e f f o r t s t o d e v e l o p a g e n t s more c l o s e l y r e l a t e d t o t h e x a n t h i n e s . A novel workin h y p o t h e s i s , which d e r i v e s from t h e e l e g a n t work of S u t h e r l a n d and h i s g r o u p Y f 3 o f f e r s a p o s s i b l e new approach t o t h e development o f improved a g e n t s i n t h i s class. Although t h e r e i s some evidence t h a t i s not e n t i r e l y c o n s i s t e n t , l b t h e h y p o t h e s i s s u g g e s t s t h a t t h e b r o n c h o d i l a t o r a c t i o n of theop h y l l i n e i s t h e r e s u l t o f i n c r e a s e d l e v e l s of c y c l i c 3',5-AMP brought about by p h o s p h o d i e s t e r a s e i n h i b i t i o n . T h i s would d i r e c t l y r e l a t e t h e o p h y l l i n e t o p - r e c e p t o r s , where c y c l i c 3 ' ,5'-AMP s y n t h e s i s i s thought t o be i n c r e a s e d through s t i m u l a t i o n of a d e n y l c y c l a s e by a p p r o p r i a t e a g o n i s t s . There i s y e t l i t t l e d i r e c t e v i d e n c e , however, t o s u g g e s t t h a t t h i s i s t h e o n l y , o r even t h e major, m e c h a n i s t i c component o f t h e o p h y l l i n e b r o n c h o d i l a t i o n , so i t remains t o be determined how f r u i t f u l t h e approach w i l l be. Mucolytic a g e n t s and e x p e c t o r a n t s l C y 2a' r e used t o d e c r e a s e t h e v i s c o s i t y o f , and a i d i n c l e a r i n g b r o n c h i a l s e c r e t i o n s . A number o f a g e n t s are a v a i l a b l e Y 5 i n c l u d i n g many o l d e r p r e p a r a t i o n s such as i o d i d e s and g u a i a c o l a t e s . The more r e c e n t a d d i t i o n s , N - a c e t y l e y s t e i n e , r i b o n u c l e a s e and v a r i o u s p r o t e o l y t i c enzymes, are u s u a l l y a d m i n i s t e r e d l o c a l l y i n a nebul i z e d form. The c l i n i c a l v a l u e o f t h e s e a g e n t s does n o t a p p e a r t o be unanimously acce t e d Y 4and o b j e c t i v e d e m o n s t r a t i o n of e f f i c a c y i s o f t e n d i f f i c u l t t o o b t a i n . l I T h i s s u g g e s t s a decided need f o r more e f f e c t i v e p r o d u c t s , s i n c e t h e r e seems t o be l i t t l e doubt t h a t t h i s i s a v a l u a b l e t h e r a p e u t i c a p p r ~ a c h . ~ , ~

Chap. 9

Mo reland

Pulmonary, Antiallergy

95 -

Better a g e n t s w i t h l o c a l a c t i o n , designed from chemical knowledge o f s p e c i f i c components i n b r o n c h i a l s e c r e t i o n s , l5 r e p r e s e n t a n obvious avenue o f a t t a c k . The r e p o r t s t h a t p i m e t i n e ( V I I I ) 1 6 and Bisolvon ( I X ) 1 7 reduce sputum v i s c o s i t y a f t e r systemic a d m i n i s t r a t i o n a r e probably of g r e a t e r p o t e n t i a l i n t e r e s t , s i n c e a g e n t s n o t dependent on d e l i v e r y through a i r w a y s t h a t may be s e v e r e l y o b s t r u c t e d would be e x p e c t e d t o have i n h e r e n t advantages.

Br VIII

IX

A n t i t u s s i v e d r u g s l c y Z cwere reviewed by Bucher,18 who c l e a r l y p o i n t e d o u t t h e need f o r more s o p h i s t i c a t e d s c r e e n i n g t o o l s based on a b e t t e r u n d e r s t a n d i n g of t h e cou h r e f l e x . A n t i t u s s i v e a c t i v i t y i n s e v e r a l s t r u c t u r a l t y p e s was r e p o r t e d , f9-25 b u t t h e s e do not a p p e a r t o r e p r e s e n t 26 s u b s t a n t i a l d e p a r t u r e s from t h o s e reviewed e a r l i e r by Chappel and von Seeman. Drug t r e a t m e n t of a l l e r g i c d i s o r d e r s , i f a n t i i n f l a a o r y a g e n t s a r e excluded, i s l i m i t e d almost e x c l u s i v e l y t o antihistamines','d and c e r t a i n sympathomimetic a g e n t s . l a , 2a The l a t t e r class i n c l u d e s b o t h a-mimetics, used as n a s a l d e c o n g e s t a n t s , and p - m i m e t i c s ( d i s c u s s e d a b o v e ) , which are g e n e r a l l y f o r a c u t e use as i n a n a p h y l a c t i c r e a c t i o n s . A new a - m i m e t i c , amidephrine (X) , from t h e s e r i e s of sulfonamido a n a l o g s a l r e a d y 11,2 mentioned, was r e p o r t e d t o be a n e f f e c t i v e and l o n g - a c t i n g n a s a l d e c o n g e s t a n t .

CHCHzNHCH3

I

OH

CH3

X The complexity o f a l l e r g i c phenomena, and t h e need f o r t r u e understanding o f t h e i n t e r p l a y of t h e v a r i o u s c o n t r i b u t i n g f a c t o r s h i s t a m i n e , s e r o t o n i n , k i n i n s and o t h e r s - i s a p p a r e n t i n the m e c h a n i s t i c s t u d i e s 2 8 t h a t c o n t i n u e t o i n v e s t i g a t e v a r i o u s a s p e c t s of h y p e r s e n s i t i v i t y and e s p e c i a l l y i n t h e review o f methods f o r t h e i n d u c t i o n o f e x p e r i m e n t a l h y p e r s e n s i t i v i t y by Spencer and West.29 A symposium c h a i r e d b Ungar e x p l o r e d t h e v a r i e t y of p h y s i o l o g i c a l f u n c t i o n s proposed f o r h i s t a m i n e ," and G a r a t t i n i and V a l z e l l i p r e s e n t e d a n e x t e n s i v e review of ~ e r o t o n i n . ~ ' Cursory reviews o f p r e s e n t a n t i h i s t a m i n e usage32 s e r v e d t o emphasize t h e r e l a t i v e q u i e s c e n c e o f t h e f i e l d i n terms of d r u g development.

96

Sect. I1

- Pharmacodynamics

A r c h e r , Ed.

Various 6-7-6 t r i c y c l i c r i n g systems - s t r u c t u r a l l y t h e most i n t e r e s t i n g class of a n t i h i s t a m i n e s , even though t h e i r impact h a s been f a r g r e a t e r i n o t h e r f i e l d s - continued t o receive a t t e n t i o n . Structurea c t i v i t y s t u d i e s i n dibenzocycloheptene a n a l o g s r e l a t e d t o cyproheptadine were reported.33 Rubin d e s c r i b e d t h e pharmacology of a dibenzoxazepine w i t h 1-10 times t h e potency of p y r i l a ~ n i n e ,and ~ ~ P r o t i v a reviewed r e c e n t I n o t h e r areas, a developments i n s e v e r a l o t h e r , r e l a t e d systems.35 series of t e t r a h y d r o q u i n a z o l i n e s w a s described,36 one of which (XI) was r e p o r t e d t o be comparable t o chlorpheniramine i n t h e c l i n i c . 3 7 CH2CH2N(CH3)2

0

XI References (1)

" D r i l l ' s Pharmacology i n Medicine," 3rd ed., J . R. D i P a l m , Ed., M c G r a w - H i l l Book Company, New York, N. Y . , 1965. ( a ) B. Levy and R. P. A h l q u i s t , Chapter 31; ( b ) E . B. T r u i t t , Chapter 27; ( c ) E. M. Boyd, Chapter 49; ( d ) F. E . Roth and I. I. A. Tabachnick, Chapter 48.

(2)

'The Pharmacological Basis of T h e r a p e u t i c s , ' ' 3rd e d . , L. S. Goodman and A. Gilman, Ed., Macmillan Company, New York, N. Y . , 1965. ( a ) I. R. Innes and M. Nickerson, Chapter 24; ( b ) J . M. R i t c h i e , Chapter 19; ( c > J. H. J a f f e , p. 280; ( d ) W. W. Douglas, C h a p t e r s . 2 9 and 30.

(3)

Anon., Drug Research Reports, 4, No. 34 ( s u p p l . ) , S-2 (August 25, 1965)

(4)

see, f o r example: H. A . Lyons, J . Am. Med. ASSOC., 1 9 4 , 1234 (1965); R. J . Johnston and H. W . Harris, Med. C l i n . N. Am., 49, 1309 ( 1 9 6 5 ) ;

w.,

B. M. Cohen, Geriatrics, 20, 999 ( 1 9 6 5 ) ; F. J . C . M i l l a r d , 20, 854 (1965); R. F. Corpe, S. Grzybowsbi, F . M. MacDonald, M. M. Newman, A. H. Niden, A. B. Organick, and W. Lester, Am. Rev. Respirat. Dis., 92, 513 (1965); R. M . Cherniak, Ann. N. Y. Acad. S c i . , 121,942 (1965). (5)

Anon.

, pharmhdex,

41 ( J a n u a r y 1965).

Chap. 9

Pulmonary, Antiallergy

P. Sadoul, Ann. N. Y . Acad. S c i . ,

121, 836

Mo reland

97

(1965).

M. E . Bader and R. A. Bader, Am. J. Med., 38, 165 ( 1 9 6 5 ) . D. M. Aviado, 'The Lung C i r c u l a t i o n , " Vole. I and XI, Pergamon P r e s s , New York, N. Y . , 1965.

B. K r a f t and J. G. Armstrong, Appl. Ther.,Z,

829 ( 1 9 6 5 ) .

M. E . Coleman and L. A. Howard, Ann. Alergy, 23, 434 ( 1 9 6 5 ) ; examples from t h e European l i t e r a t u r e i n c l u d e : A. Knopp, Med. Welt., 764 (1965); H. I l l i g , Med. Klin.(Munich), 60, 1452 ( 1 9 6 5 ) ; M. F i s c h e r and U. Mielki, g ,1824 ( 1 9 6 5 ) .

a.,

R. H. Uloth, J . R. K i r k , W. A. Gould, and A. A. L a r s e n , J. Med. Chem., 88 (19661, and r e f e r e n c e s t h e r e i n .

-9 ,

Lands and T. G. Brown, Jr., Proc. SOC. E x p t l . B i o l . Med., 116, cf. D. R. Van Deripe and N. C. Moran, E e d e r a t i o n P r o c . , 24, 712 (1965); B. Levy, J. Pharmacol. E x p t l . Therap., 129 (1964) f o r r e l a t e d observations with p-blocking agents. A. M.

331 (1964);

146,

E . W. S u t h e r l a n d , I. $ye, and R. W . B u t c h e r , Recent P r o g r . Hormone Res 21, 623 (1965)

- - 3

Committee on Drugs of t h e Research C o u n c i l , J. A l l e r g y ,

3,:488

(

1965).

P. L. Masson, J. F . Heremans, and J. P r i g n o t , Biochim. Biophys. Acta, (1965).

111, 466

S. Chodosh, A b s t r a c t s , 6 6 t h Annual Meeting of t h e American T h e r a p e u t i c S o c i e t y , June 1965, p . 29. see f o r example, W . Junge, W. Guenthner, E . Haslreiter, K . Hess, and M. Loos, Med. K l i n . (Munich), @, 2058 (1965); cf. J. Hamm and E . Hunekohl, A r z n e i m i t t e l - F o r s c h . , l6, 209 (1966). K . Bucher i n " P h y s i o l o g i c a l Pharmacology," V o l . 11, W. S . Root and F. G. Hofman, Ed., Academic P r e s s , New York, 1965, p . 175. M. C a r i s s i m i , A . C a t t a n e o , R. D'Ambrosio, V. DePascale, E . G r u m e l l i , E. Milla, and F. Ravenna, J . Med. Chem., 2, 542 ( 1 9 6 5 ) . F . P . Doyle, M. D. Mehta, R. Ward, J . B a i n b r i d g e , and D . M. Brown, 571 ( 1 9 6 5 ) .

m.,2,

S. Casadio, G. P a l a , E . C r e s c e n z i , T . Bruzzese, E . Marazzi-Uberti, -8, 589 (1965). and G. Coppi,

w.,

Sect. I1

98 -

- Pharmacodynamics

A r c h e r , Ed.

#

(22)

J . C. LeDouarec, G. Regnier, and R. C a n e v a r i , A r z n e i m i t t e l - F o r s c h . , 1 5 , 1330 (1965).

(23)

E . M a r c h e t t i , G. Mattalia, and F. Samueli, Farmaco, Ed. S c i . , 696 (1965).

(24)

M. Pesson, S. Dupin, M. Antoine, D . Humbert, and M . J o a n n i c , SOC. Chim. F r a n c e , 2262 ( 1 9 6 5 ) .

(25)

K . Takagi, I. Takayanagi, and M. Imamura, J . Pharm. SOC. J a p a n , 85, 550 (1965).

(26)

C.

(27)

(28)

I . Chappel and C . von Seeman, P r o g r . Med. Chem.,

m.

89 (1963).

H. C. S t a n t o n , K . W. Dungan, and P. M. L i s h , I n t e r n . J . Neuropharmacol., 235 ( 1 9 6 5 ) ; K . W. Dungan, H . C . S t a n t o n , and P . M. L i s h , 4, 219 ( 1 9 6 5 ) .

w.,

4,

T y p i c a l examples are:

209, 545, 550 ( 1 9 6 5 ) ; 13, 189 (1965).

B. M. A l t u r a and B. W. Zweifach, Am. J . P h y s i o l . , G. B. Fregnan and A. H . G l b s e r , Med. E x p t l . ,

P. S . J. Spencer and G. B. West, P r o p r . Med. Chem., G. Ungar, F e d e r a t i o n P r o c . ,

24,

1293ff.

4,

1 (1965).

(1965).

S . G a r a t t i n i and L. V a l z e l l i , " S e r o t o n i n , " N e w York, N . Y . , 1965.

(32)

3,

E,

E l s e v i e r P u b l i s h i n g Company,

R. W. M o r r i s , p h a r m h d e x , 4 ( J a n . 1 9 6 6 ) ; J . D. Horan, Can. Med. Assoc. 93, 1031 (1965).

2-9

(33)

E . L. E n g e l h a r d t , H . C . Z e l l , W. S . S a a r i , M. E . C h r i s t y , C. D . C o l t o n , C. A. S t o n e , J . M. S t a v o r s k i , H. C . Wenger, and C. T . Ludden, J . Med. Chem., 2 , 829 ( 1 9 6 5 ) .

(34)

B. Rubin, J . J . P i a l a , R. M i l l o n i g , and B. N . C r a v e r , Arch. I n t e r n . Pharmacodyn., 155, 47 ( 1 9 6 5 ) .

(35)

M. P r o t i v a , Farmaco, Ed. S c i . ,

(36)

G . Muacevig, H .

(37)

H. J. Lange,

J

(1965).

21,

76 ( 1 9 6 6 ) .

S t B t z e r , and H . Wick, A r z n e i m i t t e l - F o r s c f i . ,

m.,l5, 1411 ( 1 9 6 5 ) .

l5, 613

Chapter 10.

Agents Affecting G a s t r o i n t e s t i n a l Functions

W i l l i a m A. Bolhofer and David A. Brodie, Merck Sharp & Dohme Research Laboratories, West Point, Pa. EXPERIMENTAL INVESTIGATIONS Stimulation of Gastric Secretion

A major advance i n t h e biochemistry of g a s t r i c s e c r e t i o n has the determination of s t r u c t u r e and synthesis of g a s t r i n I and I1 from yarious species. Porcine g a s t r i n I has been shown t o be the heptadecapeptide I. Porcine g a s t r i n I1 (11) has t h e same peptide sequence but with n

RL

1-GluGluGluGluGluAlaTyrGlyTryMetAspPheNH2 I

@GluGlyProTry-R

2

R1 = Met, R = H 1 2 11, R = Met, R = SO H I,

3

111,

R

1

= Leu, R~ = H

IV, R~ = Leu, R~

= SO~H

Both were equipotent t h e tyrosine hydroxyl i n t h e form of i t s s u l f a t e ester.' and showed the same b i o l o g i c a l e f f e c t s . The pure h y n g a s t r i n s have been isol a t e d , 2 and degradative analyses3 and t o t a l synthesis showed t h a t human gast r i n I (111) and human g a s t r i n I1 (IV) w e r e heptadecapeptides i d e n t i c a l t o t h e porcine g a s t r i n s except f o r replacement of one of t h e methionine components by leucine. The porcine g a s t r i n s stimulated s e c r e t i o n i n other species including dogs and man. Elucidation of t h e amino acid sequence of t h e g a s t r i n s permitted an exami n a t i o n of the e f f e c t of s t r u c t u r e modification on the b i o l o g i c a l a c t i v i t y and presented an opportunity f o r the synthesis of i n h i b i t o r y analogs of t h e n a t u r a l The Chormone. A series of peptides r e l a t e d to g a s t r i n I has been t e ~ t e d . ~ terminal t e t r a p e p t i d e (V) was the minimum fragment of t h e g a s t r i n molecule which V,

TryMetAspPheNH2

showed a l l t h e physiological a c t i v i t i e s of t h e n a t u r a l hormone. Based on s t i h u l a t i o n of g a s t r i c acid s e c r e t i o n i n dogs, i t was about 20% as potent as gast r i n . The C-terminal amide was e s s e n t i a l f o r a c t i v i t y , whereas acylation of t h e N-terminal amino group had l i t t l e e f f e c t on t h e a c t i v i t y . The e f f e c t on a c t i v i t y o f a numb r of s t r u c t u r a l modifications of the t e t r a p e p t i d e (V) has been determined.' The v a r i a t i o n s i n t h e main group of analogs studied consisted of s i n g l e or multiple s u b s t i t u t i o n of one or more of the amino acid residues, including replacement o f the normal L amino acids by D amino acids. Activity of products with respect t o g a s t r i c s e c r e t i o n i n dogs varied from equipotent with g a s t r i n f o r c e r t a i n acylated d e r i v a t i v e s (VI, VII)

100 -

Sect. 11. - Pharmacodynamics VI9

A r c h e r , Ed.

H2NCOTryMetAspPheNH2

VII, (CH3) 3COCD.&AlaTryMetAspPheNH2 to inactive for those peptides not having a terminal amide group. In addition, the effect of some of the analogs on other physiological functions such as gastric motility was not uniformly consistent with the direction of the effect on gastric secretion, thus indicating a separation of the various gastrin activities. The presence of D amino acids gave peptides of decreased activity, and none of these products, including the inactive all D tetrapeptide, inhibited the action of gastrin. It was concluded that the most important structural feature of the tetrapeptide for activity was the aspartic carboxyl-terminal amide combination.

In humans, gastrin was 80-fold more potent than histamine in stimulating ~ecretion.~At present, the major clinical use of gastrin or active fragments thereof would appear to be for the replacement of histamine and other secretagogues for diagnostic analysis of gastric secretion^.^" Gastrin appeared to exert a stimulatory and inhibitory effect on acid secretion, at l o w and high doses, respectively, and a stimulatory effect on pepsin secretion at high doses regardless of whether the initial stimulation was histamine or However, it has been suggested that timulation was the normal effect and inhibition was the result of overdosage.” The histamine content of the stomach mucosa was decreased by astrin, indicating that it may act as a secretory agent by releasing histamine.f2 Acid secretion stimulated b exogenous gastrin was inhibited by heparin13 and by insulin hypoglycemia. ll Gastrin I1 has been shown o increase oxygen consumption and cause vasodilation in the stomach m u c o ~ a . ~It~ also caused a pronounced increase in gastrointestinal motility which was inhibited by atropine.16 Gastrin I and I1 were approximately 20-fold more active than histamine for the stimulation of intrinsic factor secretion by the human ~tomach.~ 2-Deox~glucose: Study of acid secretion stimulated by 2-deoxyglucose indicated that it functions by foming nonmetabolizable 2-deoxyglucose-6-phosphate, resulting in cytoglucopenia at the vagal secretory center. Thus, in effect, this mechanism of stimulation of gastric secretion was apparently equivalent to the hypoglycemic mechanism of stimulation resulting from insulin administration. It has recently been shown that, after insulin stimulation, the increase in f l o w of gastric contents occurred simultaneously with or followed the lowest point reached by the blood sugar. l8 Since the central regulatory mechanism for gastric secretion acts through the vagus nerve, stimulation of the secretory center by insulin is a method which has been used to study the completeness of vagotomy in the surgical treatment of peptic ulcer. Tests in humans showed that 2-deoxyglucose produced the same hypersecretion as insulin without the attendant side effects. Therefore, it has been suggested that insulin be re laced by 2-deoxyglucose for evaluation of patients who have undergone vagotomy. P9

Chap. 10

Gastrointestinal

Bolhofer and Brodie

101 -

Histamine: Numerous studies devoted to the evaluation of the role of histamine in stimulating gastric secretion have been reported. All results indicated that histamine is the chemical mediator of gastric secretion c m o n to all stimulants. Inhibition of specific histidine decarboxylase by the hydrazino analog of histidine and NSD-1055 4-bromo-3-hydroxybenzyloxyamine), agents which lower tissue histamine levels,‘O in pylorus-esophagus-ligated rats prevented stimulation of acid secretion by reserpine, bethanechol chloride (2-carbamoylpropyltrimethylanrmonium chloride), insulin and gastrin, but not exogenous histamine. These results showed hat acid secretion was dependent on the presence of histamine in the mucosa.“ It has also been shown that vagal stimulation led to mobilization of gastric histamine.22 Gastric secretagogues, such as gastrin, insulin, betazole &(&aminoethyl)pyrazola and bethanechol chloride, lowered gastric tissue histamine levels, but it was not luiown if their stimulatory effect on secretion was a consequence of gastric histamine release. However, the enzyme histaminase prevented gastric acid secretion in dogs under the stimulating effect of a gastrin extract.23 Portacaval shunt, which in man results in an increased incidence of duodenal ulcer, in rats caused an increase in acid secretion. Als6, histidine decarboxylase activity in the fundus, the acid-secreting portion of the stomach, has been shown to undergo a 4-fold increase after this operation. The elevated acid secretion was reduced essentially to normal by treatment with NSD-1055, the previously mentioned inhibitor of histidine decarboxylase. 24 Inhibition of Gastric Secretion Benzothiazoles: Antisecretory activity in a series of 3-amincr2,l-benzisothiazoles (VIII), determined by measuring gastric volume reduction after

VIII,

c , ” > s R 1/

\R2

f

subcutaneous administration to pylorus-li ated rats, varied from an ED50 of 1.5 mg./kg. to greater than 50 mg./kg. R and R2 consist of hydrogen, lower alkyl and phenyl, and some members of the series were also substituted in the benzenoid ring. Representative examples from the series did not show anticholinergic activity.25 Anticholinergics: The effects of anticholinergic agents on gastric acid secretion were compared with pharmacological effects on gastric emptying and pupil diameter, and it was concluded that the relationship of the secondary actions to the desired action’ofgastric acid inhibition was more important in evaluation than the absolute potency. 26 Experimental Ulcer Production The major objectives of the examination in laboratory animals of agents which produced gastrointestinal irritation or ulceration have been the discovery of preventative or counteracting agents and elucidation of the mechanism

102

Sect. I1 - Pharmacodynamics

A r c h e r , Ed.

of ulceration. The ulcerative action of serotonin was significantly diminished by administration of the antiserotonin methysergide (Deseril; 1-methyl-Dlysergic acid butanolamide tartrate) .29, 2g Antienzymes of pancreatic origin (Kunitz inhibitor) 29 or parotid origin (Frey inhibitor)3O markedly reduced the number of lesions resulting from reserpine or phenylbutazone. Aspirin damage to the intestinal mucosa occurred regard1 ss of whether It has been contact with the drug was from the lumen or the cir~ulation.~~ shown that aspirin greatly increased the ionic permeability of the mucosa and broke down its normal barrier to diffusion of sodium, potassium and hydrogen ions, so that absorption of hydrogen ions through the damaged barrier from the stomach led to mucosal bleeding. During active secretion of acid these effects It has also been proposed that aspirin decreased the rate may be enhan~ed.~~t33 of formation of the mucous lining and also lowered its resistance to proteolysis.34 Acetazolamide damage to the mucosa appeared to depend upon the presence of acid and, in addition, it accelerated the l o s s of hydrogen ions from the luminal fluid through the surface muc0sa.~5 Enhancement of gastric secretion was suggested to be a part of the mode of action of caffeine in ul~erogenesis.3~ The polysaccharide carrageenin has been found to have a protective effect from ulceration induced by histamine, cortisone or the Shay t e ~ h n i q u e . ~ ~ , ~ ~ Absorption and Transport Most experimental studies on absorption and transport were carried out to determine mechanism of these actions rather than to search for agents affecting in vivo action. Much of the work was done in in vitro gut sac preparations. Stimulation of glucose and sodium transport by reserpine resulted, at least in part,3$s a result of release of catecholamines from storage sites in the intestine. Similarly, Lepinephrine and &norepinephrine stimulated glucose and sodium absorption, but stimulation was blocked by prior treatment with dichloroisoproterenol or ergotamine, although the latter two agents alone had no effect. Cyclic AMF’ also had no effect.40 It was concluded that calcium binding substances reduced gastrointestinal absorption of drugs mainly by markedly decreasing the rate of gastric emptying.41 Transport of amino acids across the intestinal wall was inhibited by 4deoxypyridoxine, a vitamin B6 antagonist, whereas glucose transport was unaffected. It was concluded that vitamin B6 may be necessary for a metabolic process involving the amino acid carrier systems.42 CLINICAL INVESTIGATIONS Peptic Ulcer Therapy Anticholinergics: Although anticholinergic drugs are prescribed for the inhibition of gastric acidity in peptic ulcer disease, there was no indication that average doses of these agents significantly inhibited gastric acid secre-

Chap. 10

Gastrointestinal

Bolhofer and Brodie

103 -

Thus, for any significant degree of inhibition of gastric secretion, an anticholinergic agent must be administered at the maximally tolerated However, combinations of antacids and anticholinergics were found to be more effective in reducing gastric acidity than either drug given alone. Several new anticholinergics have undergone clinical trial, but none appeared to be any more potent or selective in action than a t r ~ p i n e . ~ ~Glycopyrrolate ,~~ (1-methyl-3-pyrrolidyl aphenylcyclopentaneglycolate methobromide) (IX) has been

CH3

'CH3

Br-

shown to be a clinically useful anticholinergic,47'48 but the drug had no special qualities which distinguished it from the more than 50 anticholinergics now available. The Wphenylcyclohexaneglycolate congener of glycopyrrolate, which was equiactive in animals, was found to be only minimally effective in suppressing gastric acidity in man.49 Antacids: These agents are widely used to reduce gastric acidity and relieve pain in the peptic ulcer patient. The mechanism of pain relief was unclear, since new telemetry techniques for measuring gastric pH50 have confirmed the older reports that the acid-neutralizing action lasted only from 30 to 40 minutes, while pain relief lasted for hours. A new antacid, bismuth aluminate, has been reported to be effective in relief of pain, but this agent had no effect on gastric acidity or peptic a~tivity.~' The effect was postulated to be due to a coating action of the gastric mu~osa.~* As with anticholinergics, the antacids53 gave symptomatic relief, but did not alter the natural course of the disease. Antipeptic Agents: Since acid-pepsin digestion is considered to be essential for the production of peptic ulceration, regardless of the underlying cause of the disease, reduction of pepsin concentration of gastric juice remains a practical aim in ulcer therapy. Sun54 has reviewed this area with particular attention to a new antipeptic agent, Depepsen, a sulfated amylopectin. The place of these agents in peptic ulcer treatment is still controversial. Gastric Ulcer Therapy Gastric ulcer patients differ from those with duodenal ulcer, in that the former have normal or low gastric acidity and -amuch higher incidence of gastric cancer Carbenoxolone (B iogastrone) , the pentacyc 1ic triterpene, glycyrrhetinic acid, has been reported to give symptomatic relief in gastric ulcer patients and to increase the rate of gastric ulcer healing, confirming the original clinical s t ~ d i e s . 5 ~Some ~ ~ investigators, however, have failed to find effects better than placebo,58 and side effects related to salt and water retention were common. The mechanism of the antiulcer action was not clear, but could be related to the drug's antiinflannnatory effects.

.

104

Sect. I1 - P h a r m a c o d y n a m i c s

A r c h e r , Ed.

Functional Gastrointestcnal Disorders Anticholinergics have been used f o r treatment of hypermotility s t a t e s with some success, but with t h e usual s i d e e f f e c t s . A l a r g e series of thioxanthene d e r i v a t i v e s was studied f o r s e l e c t i v e anticholinergic action, and 9-flN-methyl3-piperidy1)methylJ thioxanthene, marketed as methixene (Trest) (X) , has been found t o provide symptomatic r e l i e f w i t h l o w incidence of atropine-like s i d e

H C-

.

3

e f f e c t s 59s60 It i s too e a r l y t o t e l l i f t h i s drug w i l l replace t r a d i t i o n a l anticholinergic treatment i n hypermotility states. I n t h e carcinoid syndrome, i t has been suggested t h a t serotonin was responsible f o r t h e d i a r r h e a seen i n t h i s condition. Treatment with a serotonin a n t a g o n i s t J 6 l methysergide, was e f f e c t i v e i n c o n t r o l l i n g t h e diarrhea and was f u r t h e r evidence f o r t h e r o l e of serotonin i n i n t e s t i n a l m o t i l i t y . The search f o r agents which a l t e r smooth muscle a c t i v i t y yielded several new compounds. Antispasmodic e f f e c t s were reported f o r ipyrrole, 62 acetophenone63 and piperidine derivatives,64 as w e l l as d e h y d r o m e t h y s t i c ~ s nand ~ ~ 2-(2'phenylace tylhydraz ino)-4,6-bisdiethylamino- 1,3 ,5- t r i a z ine (Ciba 28,882-Ba) . 66 The spasmo enic e f f e c t of dihydrocodeine w a s reversed by n i c o t i n i c acid esteri f i ~ a t i o n . Nicotinyldihydrocodeine ~~ was found t o i n h i b i t drug-induced spasm of t h e b i l e duct sphincter. Stimulation of smooth muscle a c t i v i t y w a s reported f o r t et r ahydr oaminoacr i d i n e c ompounds 68

.

Metoclopramide ~-(2-diethylaminoethyl)-2-methoxy-4-amino-5-chlorobenzamide d i h y d r c x h l o r i d g (XI) has been widely used i n Europe as a "digestive C O W C H ~ C H ~ - N(C 2139

I

c1QH3

2 HC1

modifier" t o r e l a x t h e pylorus and increase g a s t r i c m o t i l i t y and as an a i d t o t h e x-ray study@ of the g a s t r o i n t e s t i n a l t r a c t . I t s primary use has been as an antiemetic. A t t h e present t i m e , t h i s d k g i s not a v a i l a b l e i n t h e United States.

Chap. 10

Gastrointestinal

Bolhofer and Brodie

105 -

Ulcerative Colitis Therapy The choice between medical and surgical treatment of this disease remains a problem to the clinician. Reviews of the l i t e r a t ~ r e ~indicated ~ , ~ ~ that steroids were the most commonly used drugs; however, as with anticholinergic therapy for peptic ulcer, they alleviated the symptoms but did not cure the disease. Drug Effects on Gastrointestinal Motility Stimulation of gastrointestinal motility has been studied, usually with isolated tissues, with a wide variety of drugs. Serotonin has been suggested to be a naturally occurring material which stimulates motility, perhaps as a local hormone. However, its role remained unclear, since it has been shown to stimulate human tissues in ~ i t r o but ,~~ up to 90% depletion in the rat did not inhibit peri~talsis.~3 A study on the isolated rat colon indicated that the effect of serotonin was dose-related: small doses had a direct effect on muscle fibers, while higher doses acted by stimulating parasympathetic ganglia in the gut wall.74 Bile Drug-induced jaundice as a side effect of phenothiazine derivatives is well documented. The effect of these drugs and C17~alkyl-substitutedsteroids was studied by in vivo and in vitro t e s t ~ , ~and s it was concluded that these agents inhibited glucuronyl transferase. The steroids prevented excretion of conjugated bilirubin by blocking a stage after conjugation, but the mechanism of phenothiazine jaundice was still unclear. The steatorrhea observed in patients receiving cholestyramine (a basic ion exchange resin which binds bile acids) and a diet containing triglyceride fats of normal chain length was eliminated by replacing the long chain triglycerides with medium chain triglycerides (less than C12). Bile acid absorption by the cholestyramine was comparable for both diets. The medium chain triglycerides may be of value in clinical management of patients with malabsorption due to biliary 0bstruction.7~ Salivary Glands Physalaemin, a polypeptide was found to be the most active sialogogic agent tested in rats and dogs.7f Its action appeared to be a direct one on the salivary gland, since its effect was not blocked by sympathomimetic, parasympathomimetic or ganglionic blockers, but its effect was reduced by serotonin. Drug-Induced Gastrointestinal Lesions The incidence of pathological changes in the gastrointestinal tract due to therapeutic agents has increased as new drugs and formulations appeared on the market. Gastrointestinal ulceration due to steroids and antiinflammatory drugs is well known and continues to be a major side effect. Reduction of ulcerogenic activit was reported with ketophenylbutazone as compared to phenylbutazone itself,78 while the use of indomethacin capsules reduced the incidence of peptic ulcers seen with indomethacin The mechanism of steroid-

tablet^.^'

106

Sect. I1

-

Pharmacodynamics

A r c h e r , Ed.

induced g a s t r o i n t e s t i n a l l e s i o n s i s s t i l l not resolved. Hydrocortisone d i d not a f f e c t g a s t r i c s e c r e t i o n i n t h e r a t , leading t o t h e conclusion t h a t some mechanism o t h e r increased g a s t r i c a c i d i t y was t h e cause of c o r t i c o s e r o i d ulceration.86518' There d i d not s e e m t o be a s p e c i f i c s t e r o i d ulcer8' a s distinguished from u l c e r s due t o o t h e r causes. It has been suggested83 t h a t s t e r o i d s aggravated e x i s t i n g inflammation by loosening i n t e r c e l l u l a r cement and permitting p e n e t r a t i o n of t h e mucosa by t h e g a s t r i c j u i c e . S t e r o i d therapy i n cancer p a t i e n t s appeared t o induce more severe g a s t r i c complications than ,in p a t i e n t s with nonmalignant disease.84

A new i a t r o g e n i c .lesion, small bowel u l c e r a t i o n following a d m i n i s t r a t i o n of enteric-coated t a b l e t s of potassium c h l o r i d e and a t h i a z i d e d i u r e t i c , 8 g 39 described i n l a t e 196485 and r a p i d l y confirmed by numerous c a s e r e p o r t s . Experimental s t u d i e s i n d i c a t e d t h a t t h e l e s i o n was due t o t h e release of a high l o c a l c o n c e n t r a t i o n of potassium from t h e enteric-coa ed t a b l e t over a s h o r t segment of t h e i n t e s t i n e . 9 0 I n one c l i n i c a l report," a p a r t i a l l y digested t a b l e t was found i n a l e s i o n c r a t e r i n t h e i n t e s t i n e . The t h i a z i d e d i u r e t i c d i d n o t appear t o be connected with t h e l e s i o n p r o d u c t i ~ n . ~ ~ , ~ ~ REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16.

17.

18.

19. 20. 21. 22.

23.

H. Gregory, P.M. Hardy, D.S. Jones, G.W. Kenner and R.C. Sheppard, Nature, 204, 931 (1964). R.A. Gregory, H.J. Tracy and M.I. Grossman, Nature, 209, 583 (1966). P.H. Bently, G.W. Kenner and R.C. Sheppard, Nature, 209, 583 (1966). J. Beacham, P.A. Bently, R.A. Gregory, G.W. Kenner, 3.K. MacLeod and R.C. Sheppard, Nature, 209, 585 (1966). H.3. Tracy and R.A. Gregory, Nature, 204, 935 (1964). J.S. Morley, H.J. Tracy and R.A. Gregory, Nature, 207, 1356 (1965). G.M. Makhlouf, J.P.A. McManus and W . I . Card, Lancet, 2, 485 (1964). L.S. Semb and J.A. Myren, Scand. J. Clin. Lab. Invest., 1 7 , 311 (1965). W.J. I r v i n e , Lancet, 1, 736 (1965). S . Konturek and M.I. Grossman, Proc. SOC. Exptl. Biol. Med., 119, 443 (1965). R.B. Quintana, C. D e La Rosa and L.R. Dragstedt, U e s t . D i s . , 10, 745 (1965). B.J. Haverback, L.B. Tecimer, B.J. Dyce, M. Cohen, M . I . S t u b r i n and A.D. Santa Ana, L i f e Sci., 3, 637 (1964). P.H. Jordan and R. Quintana, Gastroenterology, 47, 617 (1964). W.G. Gobbel, Jr., R.H. Edwards and C.F. Zukoski, Southern Med. J., 58, 1044 (1965). J.D. Cunnning, E.H.L. H a r r i e s and P. Holton, J. Physiol. (L0nd.Z 181, 33P (1965) A.N. Smith and D. Hogg, Lancet, 1, 403 (1966). B.I. Hirschowitz and G. Sachs, Am. J. Physiol., 209, 452 (1965). R.A. Davis, F.P. Brooks and C.McN. Robert, Jr., Am. J. Physiol., 208, 6 (1965). W.W. Duke, B.I. Hirschowitz and G. Sachs, Lancet, 2, 871 (1965). R.J. Levine, T.L. S a t 0 and A. Sjoerdsma, Biochem. Pharmacol., 14, 139 (1965). R.J. Levine, Fed. Proc., 24, 1331 (1965). P.A. Shore, Fed. Proc., 24, 1322 (1965). M. S t u b r i n , €3. Dyce, L. T e c i m e r and B.J. Haverback, Clin. Res., 13, 97 (1965).

.

Chap. 10 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

Gastrointestinal

Bolhofer and Brodie

J.E. F i s c h e r and S.H. Snyder, Science, 150, 1034 (1965). C o l l i e r , J. Med. Chem., 8, R.F. Meyer, B.L. Cummings, P. Bass and H.O.J. 515 (1965). D.A. Brodie, Gastroenterology, 50, 45 (1966). R. Lambert, L. Descos, J. Pasquier and M. Martin, Gastroenterologia, 103, 365 (1965). R. Lambert, L. Descos and J. Pasquier, Compt. Rend. SOC. Biol., 158, 1367 (1964). F. Guerrin, A. D e m a i l l e , P. Merveille and C. B e l , Compt. Rend. SOC. Biol., 159. 1172 (1965). F. Guerrin, A. Clay, C. L'Hermine, A. D e m a i l l e and R. Druart, Compt. Rend. SOC. Biol., 158, 1626 (1964). A. Lynch, H. Shaw and G.W. Milton, Gut, 5, 230 (1964). H.W. Davenport, Gastroenterology, 49, 189 (1965). K.W. Anderson, Arch. I n t e r n . Pharmacodyn., 157, 181 (1965). R. Menguy and Y.F. Masters, Surg. Gynecol. Obstet., 120, 92 (1965). J.L. Werther, F. Hollander and M. Altamirano, J. Physiol., 209, 127 (1965). C.J. P f e i f f e r and G.H. Gass, Toxicol. Appl. Pharmacol., 7, 732 (1965). W. Anderson and P.D. Soman, Nature, 206, 101 (1965). G. Lambelin, Med. Exptl., 14, 136 (1966). K.A. Aulsebrook, Proc. SOC. Exptl. Biol. Med., 119, 387 (1965). K.A.,,Aulsebrook, Biochem. Biophys. Res. C m n . , 18, 165 (1965). E. Sognen, Acta Pharmacol. Toxicol., 22, 31 (1965). B.G. Munck, Biochim. Biophys. Acta, 94, 136 (1965). J.B. Kirsner and H. Ford, _Current Therap. Res., 7, 116 (1965). J.E. Berk, Current Therap. Res., 7, 149 (1965). S.M. Chernish and B.D. Rosenak, Am. J. Digest. D i s . , 10, 697 (1965). P.B. Rowe, B. Fenton and D. Beeston, Am. J. Digest. D i s . , 10, 205 (1965). W.P. S i n c l a i r and E.S. Schnier, Am. J. Gastroent., 44, 62 (1965). B.O. Amure, P r a c t i t i o n e r , 195, 335 (1965). M.D. Kamionkowski and B. F l e s h l e r , Current Therap. R e s . , 6, 459 (1964). H.G. Noeller, Therapiewoche, 15, 47 (1965). Swan and J. Kelleher, B r i t . Med. J., 1, 753 (1965). S.G.F. Matts, C.H.J. G.W. Milton, B r i t . Med. J., 1, 1672 (1965). Thomas, Postgrad. Med. J., 41, 48 (1965). P.R.C. Evans, S. N o w e l l and I.A.P. D.C.H. Sun, Current Therap. Res., 7, 790 (1965). R. Doll, I.D. H i l l , C. Hutton and D.J. Underwood, Lancet, 2, 793 (1962). R. Doll, I.D. H i l l , and C.F. Hutton, Gut, 6, 1 9 (1965). L. Horwich and R. Galloway, B r i t . Med. J., 2, 1274 (1965). W.R. Middleton, A.R. Cooke, D. Stephen and A.P. Skyring, Lancet, 1, 1030 (1965). H. Lauener and R.C. Pogge, J. Pharm. Sci., 53, 568 (1964). J.K. Martins, Clin. Med., 72, 1313 (1965). K.L. Melmon, A. Sjoerdsma, J.A. Oates and L. L a s t e r , Gastroenterology, 48, 18 (1965); A. Robelet, F. Erb-Debruyne, N. Bizard-Gregoire and J. Bizard, Therapie, 20, 427 (1965). R. Cahen, A. Boucherle, M. H i c t e r , M. S a u t a i , J. Pessonier and F. Rambert, Compt. Rend. SOC. Biol., 159, 1353 (1965). A. Vaccari, F. Cugurra and M. Ferro, Clin. Terap., 32, 483 (1965).

&I.

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.

107 -

108

Sect. I1

- Pharmacodynamics

A r c h e r , Ed.

65. H.J. Meyer, Arch. Intern. Pharmacodyn., 154, 449 (1965). 66. P.P. Spinnler, Arch. Intern. Pharmacodyn., 156, 217 (1965). 67. R.A. Cahen, A. Boucherle and M. Sautai, Compt. Rend. SOC. Biol., 159, 1123 (1965). 68. M.J. Bleiberg, Life Sci., 4, 449 (1965). 69. R. Van Lerberghe, Acta Gastroent. Belg., 28, 440 (1965). 70. M. Sparberg and J.B. Kirsner, Current Therap. Res., 7, 324 (1965). 71. T.P. Almy and A.G. Plaut, Gastroenterology, 49, 295 (1965). 72. D.J. Fishlock, A.G. Parks and V. Dewell, Gut, 6, 338 (1965). 73. D.J. Boullin, Brit. J. Pharmacol., 23, 14 (1964). 74. K. Ulrich, J. Pharm. Pharmacol., 17, 710 (1965). 75. T. Hargreaves, Nature, 206, 154 (1965). 76. R.B. Zurier, S.A. Hashin and T.B. Van Itallie, Gastroenterology, 49, 490 (1965). 77. G. Bertaccini and C. de Caro, J. Physiol. (Lond.1, 181, 68 (1965). 78 2. Horakova, 3. Metys and V. Cepelak, Therapie, 20, 617 (1965). 79. 0. L%rgren and E. Allander, Brit. Med. J., 1, 996 (1965). 80. P.H. Guth and R. Mendick, Am. J. Gastroenterol., 44, 545 (1965). 81. R.E. Wiederanders, J, Lancet, 85, 315 (1965). 82. A.E. Garb, E.H. Soule, L.G. Bartholmew and J.C. Gain, Arch. Intern. Med., 116, 899 (1965). 83. D.H. Johnston, Gastroenterology, 48, 823 (1965). 84. D.L. Roseman and S.C. Economou, Arch. Surg., 90, 488 (1965). 85. D.R. Baker, W.H. Schrader and C.R. Hitchcock, J. Am. Med. ASSOC., 190, 586 (1964). 86. R.J. Rosen and D.T. Borucki, J. Am. Med. ASSOC., 191, 419 (1965). 87. D.J. Buchan, F. Murphy and C.S. Houston, Gastroenterolopy, 48, 808 (1965). Lancet, 2, 593 (1965). 88. L. 89. T.B. Binns, A.K. Pittman, D.M. Burley and J.M. O'Brien, Brit. Med. J., 1, 248 (1965). 90. S.J. Boley, L. Schultz, A. Krieger, S. Schwartz, A. Elguezabal and A.C. Allen, J. Am. Med. ASSOC., 192, 763 (1965). 91. H. Payan and A. Blaustein, Gastroenterology, 48, 877 (1965). 92. R.M. Diener, D.H. Shoffstall and A.E. Earl, Toxicol. Appl. Pharmacol., 7, 746 (1965). 93. F.D. Lawrason, E. Alpert, F.L. Mohr and F.G. McMahon, J. Am. Med. Assoc., 191, 641 (1965).

sf,

S e c t i o n I11 Editor:

Edwin H.

Chemotherapeutic Agents

Flynn, E l i L i l l y a n d Co.,

C h a p t e r 11. Edwin H.

-

Indianapolis,

Ind.

A n t i b i o t i c s and R e l a t e d Compounds

Flynn, E l i L i l l y and Co.,

Indianapolis,

Ind.

The y e a r 1965 w a s marked by a p p e a r a n c e of a number of v a l u a b l e r e p o r t s b e a r i n g on t h e u s e of a n t i b i o t i c s i n t h e chemotherapy of i n f e c t i o u s d i s e a s e s . Nek advances have been made i n u n d e r s t a n d i n g t h e b i o s y n t h e s i s of some a g e n t s and e x p e r i m e n t s have been r e p o r t e d which were d e s i g n e d t o make t h e mode of a c t i o n u n d e r s t a n d a b l e . Although n o t a l l - i n c l u s i v e , p a p e r s r e f e r r e d t o i n t h e f o l l o w i n g pages s h o u l d g i v e t h e r e a d e r a s t a r t i n g p o i n t f o r f u r t h e r r e a d i n g and may i n d i c a t e t h e d i r e c t i o n s b e i n g t a k e n by r e s e a r c h i n t h i s a r e a . Penicillins new d e r i v a t i v e s of 6 - a m i n o p e n i c i l l a n i c a c i d ( I) c o n t i n u e t o

f i n d a p l a c e b o t h i n e x p e r i m e n t a l work on i n f e c t i o u s d i s e a s e Two d e r i v a t i v e s c l o s e l y r e l a t e d t o and i n m e d i c a l p r a c t i c e . o x a c i l l i n ( 11) a r e c l o x a c i l l i n ( 111) and d i c l o x a c i l l i n ( IV) These compounds, though d e s c r i b e d e a r l i e r , have been s t u d i e d further. It has been said1 t h a t c l o x a c i l l i n i s p r i m a r i l y u s e f u l i n i n f e c t i o n s c a u s e d by p e n i c i l l i n a s e p r o d u c i n g s t a p h y l o c o c c i . Blood l e v e l s a f t e r o r a l and i n t r a m u s c u l a r a d m i n i s t r a t i o n w e r e s l i g h t l y more d e l a y e d when compared w i t h o x a c i l l i n b u t l a s t e d longer. Peak l e v e l s were t h o u g h t t o be s i m i l a r , a l t h o u g h o t h e r workers2 have r e p o r t e d b e t t e r a b s o r p t i o n t h a n t h a t o b t a i n e d w i t h oxacillin. A c l i n i c a l s t u d y 2 of c l o x a c i l l i n showed good r e s u l t s i n s t a p h y l o c o c c a l , pneumococcal and s t r e p t o c o c c a l i n f e c t i o n s and c o n s i s t e n t l y h i g h e r a n t i b a c t e r i a l blood l e v e l s

.

110

Sect. 111

-

Chemotherapeutics

F l y n n , Ed.

a f t e r o r a l administration. It w a s c o n c l u d e d t h a t c l o x a c i l l i n c o u l d s u b s t i t u t e f o r m e t h i c i l l i n when o r a l t h e r a p y i s f e a s i b l e . D i c l o x a c l l l i n a p p e a r s t o be a b s o r b e d e x t r e m e l y w e l l , g i v i n g p e a k blo& l e v e l 8 t w i c e t h u s e of c l o x a c l l l i n and f o u r - f o l d greater than o x a c i l l i n . g It w a s f e l t t h a t serum b i n d i n g w a s comparable t o t h e o t h e r i s o x a z o l y l p e n i c i l l i n s . These blood l e v e l s have been confirmed.4 A m p i c i l l i n ( V ) , known t o be a b s o r b e d w e l l o r a l l y and t o have a b r o a d e r s p e c t r u m of a c t i v i t y t h a n o t h e r p e n i c i l l i n s , has been s t u d i e d a s t h e sodium s a l t f o r p a r e n t e r a l a d m i n i s t r a t i o n . 5 R e s u l t s i n d i c a t e d t h i s form t o be s a t i s f a c t o r y f o r p a r e n t e r a l u s e , t h u s e n h a n c i n g t h e c l i n i c a l v a l u e of a m p i c i l l i n . H e t a c i l l i n ( V I ) , t h e r e a c t i o n p r o d u c t of a m p i c i l l i n and a c e t o n e , has been r e p o r t e d . 6 It is s a i d t o g i v e more p r o l o n g e d b l o o d l e v e l s a f t e r o r a l dosage t h a n does a m p i c i l l i n , and a p p a r e n t l y is a c t i v e i n v i v o by v i r t u e of h y d r o l y s i s t o ampicill~n.~

N a f c i l l i n ( V I I ) has been r e p o r t e d t o be a n e f f e c t i v e and w e l l t o l e r a t e d a n t i s t a p h y l o c o c c a l a g e n t comparable t o o t h e r penicillinase resistant penicillins.* a - a l k y l d e r i v a t i v e s of phenoxymethyl p e n i c i l l i n ( p e n i c i l l i n V) have been d e s c r i b e d and t u r n o u t t o be e s s e n t i a l l y i n a c t i v e as a n t i b a c t e r i a l s u b s t a n c e s when compared w i t h t h e u n s u b s t i t u t e d compound.8 A c t i v e d e r i v a t i v e s of 6 - a m i n o t h i o p e n i c i l l a n i c a c i d ( V I I I ) have been r e p o r t e d . 1 0 An i n t e r e s t i n g s e r i e s i s t h a t r e p r e s e n t e d by I X ; t h i s member showing good gram n e g a t i v e as w e l l as gram p o s i t i v e a c t i v i t y . ' l

(VIII)

A p r o p o s a l c o n c e r n i n g t h e mechanism of a c t i o n of p e n i c i l l i n s on t h e b a c t e r i a l c e l l has been advanced.12 An i n t e r e s t i n g p a p e r c o n c e r n i n g t h e i n i t i a l s t r u c t u r a l l e s i o n s roduced by p e n i c i l l i n a c t i n g on t h e b a c t e r t a l c e l l has appeared. It v e r i f i e s a h i g h l y s p e c i f i c p o i n t o f a t t a c k by t h i s a n t i b i o t i c .

Antibiotics

Chap. 11

Flynn

111 -

A w o r t h w h i l e r e v i e w of p e n i c i l l i n s h a s a p p e a r e d L 4 and a book h a s been p u b l i s h e d x S which is c o n c e r n e d p r i m a r i l y w i t h b i o c h e m i c a l and b i o l o g i c a l f a c t o r s r e l a t e d t o p e n i c i l l i n . Cephalosporins The c e p h a l o s p o r i n g r o u p of a n t i b i o t i c s , whose g e n e s i s may be t r a c e d t o t h e d i s c o v e r y of c e p h a l o s p o r i n C ( X ) , h a s demons t r a t e d t h e i r v a l u e i n t h e t r e a t m e n t of i n f e c t i o u s d i s e a s e . C e p h a l o t h i n ( X I ) , t h e f i r s t of t h e s e s u b s t a n c e s t o find wides p r e a d u s e , w a s f o l l o w e d by c e p h a l o r i d i n e ( X I I ) which is now employed in many c o u n t r i e s f o r t h e r a p y of b a c t e r i a l i n f e c t i o n s . It seems a p p a r e n t t h a t o t h e r v a l u a b l e compounds of t h i s c l a s s may be e x p e c t e d .

X, R = H 0 2 C - C H N H 2 ( C H 2 ) 3 C O XI,

R = O H z C O - , S

XIII,

-

R'

, R'

= CHsC02-;

= CH3C02-;

R = D - C B H ~ C H N H ~ C O - , R'

CH3C02-

C e p h a l o r i d i n e h a s been e v a l u a t e d i n a number of c l i n i c a l and l a b o r a t o r y o t h e r r e p o r t s n o t c i t e d h e r e may be f o u n d in t h e A b s t r a c t s of t h e F i f t h I n t e r s c i e n c e C o n g r e s s on A n t i m i c r o b i a l Agents and Chemotherapy. It c a n be s a i d i n g e n e r a l t h a t t h e a g e n t w a s e f f e c t i v e i n a v a r i e t y of b o t h gram n e g a t i v e and gram p o s i t i v e b a c t e r i a l i n f e c t i o n s . Cephaloridine was ' i n e f f e c t i v e a g a i n s t Pseudomonas, t h e t u b e r c l e b a c i l l u s , a n d fungi. I n a comparison w i t h c e p h a l o t h i n s i g n i f i c a n t d i f f e r e n c e s in t h e r a p e u t i c r e s p o n s e were n o t d e t e c t e d . 1 7 G e n e r a l l y s a t i s f a c t o r y r e s u l t s were s e e n i n a p e d i a t r i c study" of cephaloridine. A s e r i e s of 92 c a s e s w i t h a v a r i e t y of i n f e c t i o n s w a s s t u d i e d with impressive r e s u l t s . l s !Cwo c a s e s of r e n a l f a i l u r e were e n c o u n t e r e d w h i l e u n d e r t r e a t m e n t w i t h 8-12 grams of c e p h a l o r i d i n e d a i l y ; t h e complex h o s p i t a l c o u r s e s d i d n o t I n a n o t h e r s e r i e s of 70 a l l o w d i r e c t i m p l i c a t i o n of t h e d r u g . c a s e s a t what would seem t o be low d a i l y d o s e s , good r e s u l t s were r e p o r t e d . 2 0 The same r e p o r t m e n t i o n s t o x i c c h a n g e s in r a b b i t k i d n e y a t a s i n R l e d o s e of 90 mg./kg. b u t not a t 45 mg./kg. d a i l y f o r e i g h t weeks. A l l of t h e f o r e g o i n g s t u d i e s employed p a r e n t e r a l a d m i n i s t r a t i o n . The b a c t e r i c i d a l a c t i v i t y o f c e p h a l o r i d i n e h a s been commented upon f a v o r a b l y 2 1 a n d i t s s t a b i l i t y t o s t a p h y l o c o c c a l p e n i c i l l i n a s e h a s been ~ t u d i e d . ~ ~ AJ l ~ l e 3r g y t o c e p h a l o r i d i n e h a s r e c e i v e d n o t i c e 2 * as h a s a c a s e of a n a p h y l a x i s a t t r i b u t e d t o c e p h a l o t h i n . 2g

112

S e c t I11

- Chemotherapeutics

F l y n n , Ed.

A n i n t e r e s t i n g i n v e s t i g a t i o n of s y n e r g i s m between p e n i c i l l i n s and c e p h a l o s p o r i n s a g a i n s t pS. pyocyanea h a s been p u b l i s h e d show‘ing marked enhancement of a c t i v i t y i n c e r t a i n c a s e s . 26 C e p h a l o g l y c i n ( X I I I ) has been e v a l u a t e d 2 7 I n t h e l a b o r a t o r y b o t h in v i t r o and in v i v o and shows a good l e v e l of a c t i v i t y v e r s u s gram n e g a t i v e organisms. The a r t i c l e p o i n t s o u t some d i f f i c u l t i e s i n h e r e n t in e v a l u a t i n g a s u b s t a n c e which is r e l a t i v e l y u n s t a b l e in s o l u t i o n as is t h e c a s e w i t h c e p h a l o g l y c in. Chemical p a p e r s have a p p e a r e d , one d e s c r i b i n g p h y s i c a l c h e m i c a l d a t a on a s e r i e s of c e p h a l o s p o r i n s P 8 and a n o t h e r m e n t i o n i n g p r e p a r a t i o n of a number of d e r i v a t i v e s . = e Lincomyc in L l n c o m n i n . a n a n t i b i o t i c e f f e c t i v e in t r e a t m e n t of i n f e c t i o n s caus;d by gram p o s i t i v e organisms, is f i n d i n g Earlier w i d e s p r e a d u s e in t h e . t r e a t m e n t of human d i s e a s e . r e p o r t s ( 1 9 6 3 and l a t e r ) d e m o n s t r a t i n g c l i n i c a l e f f i c a c y have been e x t e n d e d . 30-91 T h a t s t r u c t u r a l m o d i f i c a t i o n of l i n c o m y c i n is p o s s i b l e w i t h o u t loss of a n b i b a e t e r i a l a c t i v i t y has been shown in s e v e r a l papers.32’3s*34 Some s t u d i e s o n b i o s y n t h e s i s have been r e p o r t e d . 3 5

\

w r a l r e p o r t s c o n c e r n e d w i t h a new e n t i t y c a l l e d kasugamycin hav; a p p e a r e d . T h i s s u b s t a n c e has been shown t o have t h e s t r u c t u r e , XIV.36 O r i g i n a l l y d i s c o v e r e d b e c a u s e of i t s

H2N-i-iPHs NH

H

~~

( XIV)

H

OH

o

~

o

~

OH

a c t i v i t y i n p. o r y z a e i n f e c t i o n s of t h e r i c e p l a n t , it has a n t i b a c t e r i a l a c t i v i t y of a f a i r l y wide r a n g e , though of a low order.37 O r a l l y a b s o r b e d , and a p p a r e n t l y r e l a t i v e l y n o n - t o x i c , kasugamycin may f i n d a p p l i c a t i o n in t r e a t m e n t of human infections. I t s u t i l i t y in Pseudomonas i n f e c t i o n s of t h e g e n i t o u r i n a r y t r a c t has been s u g g e s t e d in one pa er.36 D e r i v a t i v e s of r i f a m y c i n have been r e p o r t e d e * 4 0 a n d a t l e a s t one compound has been shown t o be e f f e c t i v e in human disease.41 The d i e t h y l a m i d e of r i f a m y c i n B has shown e f f e c t i v e n e s s i n a c u t e c h o l e c y s t i t i s and i n s t a p h y l o c o c c a l i n f e c t i o n s . L e s s favorab1.o r e s u l t s were o b t a i n e d in pneumonia. A revidw has a p p e a r e d on r i f a m y c i n SV.42

P

Chap. 1 1

Antibiotics

Flynn

113 -

A d d i t i o n a l i n f o r m a t i o n on t h e g e n t a m i c i n complex, f i r s t r e p o r t e d i n 1963, h a s been made a v a i l a b l e d u r i n g 1965. T h i s m u l t i p l e f a c t o r a g e n t ( s ) has been shown t o have v a l u e i n t h e t r e a t m e n t of Pseudomonas s e p s i s i n burns4cg and t h e ( r a b b i t ) eye44 and h a s been u s e d i n p e d i a t r i c s t o t r e a t gram n e g a t i v e i n f e c t i o n s . 4 5 Although somewhat t o x i c , g e n t a m i c i n a p p e a r s t o have c l i n i c a l v a l u e . Some c h e m i c a l s t u d i e s have been r e p o r t e d , 4 6 showing t h e p r e s e n c e i n t h e m o l e c u l e of D-glucosamine, 2 - d e o x y s t r e p t a m i n e and a n amino s u g a r named g e n t o s a m i n e A. A c o n f e r e n c e t o d i s c u s s new a n t i t u b e r c u l o u s a g e n t s w a s h e l d by t h e New York Academy of S c i e n c e s September 13-15, 1965, i n N e w York C i t y . The u s e of e t h a m b u t o l and capreomycin i n t h e r a p y was discussed. Aatinospectacin, a n a g e n t w i t h broad spectrum a ~ t i v i t y , 4 ~ showed promise i n a c u t e i n f e c t i o n s of t h e g e n i t o u r i n a r y t r a c t s 4 ' The r e l a t i v e l y low p e r c e n t a g e of "good" r e s p o n s e s w a s p r o b a b l y due t o i n a d e q u a t e dosage. I s o l a t i o n a n d b i o l o g i c a l a s s e s s m e n t of coumermycin has been reported.4a The s t r u c t u r e has been e s t a b l i s h e d 5 O and i t s s y n t h e s i s from a n o v o b i o c i n i n t e r m e d i a t e h a s been mentioned.'l Chemically r e l a t e d t o n o v o b i o c i n , coumermycin A 1 shows c r o s s r e s i s t a n c e w i t h novobiocin; i t a p p e a r s t o be more a c t i v e i n It is i d e n t i c a l t o sugordomycin. Coumermycin A 2 i s general. c l o s e l y r e l a t e d t o A 1 hhemically.s2 Chemistry of t h e polymyxins h a s been discassed.'" S y n t h e s i s of polymyxin E ( c o l i s t i n A) w a s t h e s u b j e c t of a r e c e n t study.s4 S t r u c t u r e s t u d i e s were d e s c r i b e d showing t h e i d e n t i t i e s of polymyxin El and E2 w i t h c o l i s t i n A and B, respectively.' F u s i d i c a c i d , t h e s t e r o i d a l a n t i b i o t i c now i n c l i n i c a l u s e , has been shown t o be i d e n t i c a l w i t h ramycin.56 A comprehensive s t u d y of t h e e f f e c t of s t r u c t u r a l v a r i a t i o n on a n t i b a c t e r i a l The s t r u c t u r a l r e l a t i o n s h i p w i t h a c t i v i t y has been r e p o r t e d . s 7 h e l v o l i c a c i d and c e p h a l o s p o r i n P1 i s mentioned. Erythromycin has been shown t o be e f f e c t i v e c l i n i c a l l y 5 * in pneumonia due t o Mycoplasma pneumoniae, t h e Eaton a g e n t pneumonia. Bios n t h e s i s and metabolism of e r t h r o m y c i n s were s t u d i e d u s i n g 1-f4c p r o p i o n a t e as p r e c u r s o r . sg The a g l y c o n e w a s formed and gave e r y t h r o m y c i n s A, B, and C. A biosynthetic scheme w a s proposed. The s t r u c t u r e s of s p i r a m y c i n and magnamycin were published.6o The s t r u c t u r e of magnamycin r e p r e s e n t s a r e v i s i o n of an e a r l i e r p r o p o s a l . The s t r u c t u r e of viomycin h a s been d e t e r m i n e d . e l Moenomycin, a macromolecule h a v i n g h i g h i n t r i n s i c a n t i b a c t e r i a l a c t i v i t y % v i t r o and i n v i v o when a d m i n i s t e r e d The recommendation p a r e n t e r a l l y , has been r e p o r t e d . 6 2 , 6 3 , 6 4 w a s made t h a t t h i s i n t e r e s t i n g s u b s t a n c e be r e s e r v e d f o r u s e a s a n a n i m a l growth s t i m u l a n t .

114

Sect. III

-

Chemotherapeutics

F l y n n , Ed.

T e t r a c y c l i n e d e r i v a t i v e s have been p r e p a r e d by a l k o x y a l k y l a t i o n of t h e amide f u n c t i o n . 6 5 The mode(s) of a c t i o n of t e t r a c y c l i n e s have been s t u d i e d u s i n g A. a e r o g e n e s . 0 0 They a r e s a i d t o i n t e r f e r e w i t h hydrogen t r a n s f e r r e a c t i o n s . A disturbing r e p o r t of t r a n s d u c t i o n of 2. a u r e u s t o t e t r a c y c l i n e r e s i s t a n c e in v i v o h a s a p p e a r e d . 6 7 Although a similar c i r c u m s t a n c e seems A further remote in humans, t h e p o s s i b i l i t y w a s noted. e l a b o r a t i o n of b i o s y n t h e t i c pathways f o r t e t r a c y c l i n e s h a s been provided.ee The 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 h-hydroxy-6m e t h y l p r e t e t r a m i d , a t e t r a c y c l i n e p r e c u r s o r accumulated by a b l o c k e d mutant, has been r e c o r d e d . e a A new t o t a l s y n t h e s i s of 6-deoxy-6-demethyltetracycline w a s a c h i e v e d by an e l e g a n t p r o c e d u r e .70 The c h e m i s t r y of t h e d e p s i p e p t i d e t y p e a n t i b i o t i c s ( s t r u c t u r e s made up of hydroxy- and amino a c i d m o i e t i e s l i n k e d t h r o u g h e s t e r a n d amide bonds) apd t h e i r mechanism of a c t i o n has been reviewed.71 A d e t a i l e d a c c o u n t of t h e c h e m i s t r y a l s o h a s been p u b l i s h e d . 7 2 It is t h o u g h t t h a t t h e s e s u b s t a n c e s a c t by a l t e r i n g ion t r a n s p o r t t h r o u g h c e l l membranes v i a changes in t h e l i p o p r o t e i n component of t h e c e l l membrane. Some s t r u c t u r a l a s p e c t s of t h e vancomycin m o l e c u l e have been d e f i n e d . 7 3 C l i n i c a l l y , vancomycin has been shown t o be e f f e c t i v e in s t a p h y l o c o c c a l e n t e r o c o l i t i s . 7 4 , 7 5 L y s o s t a p h i n , a l y t i c enzyme p r e p a r a t i o n s p e c i f i c f o r t h e genus S t a p h y l o c o c c u s , h a s been r e p o r t e d t o be a m i x t u r e of two enzymes, a h e x o s a m i n i d a s e and a p e p t i d a s e . 7 6 L y t i c a c t i o n is due t o t h e p e p t i d a s e . I t s t h e r a p e u t i c a c t i v i t y i n animals has been d i s c u s s e d . 7 7 A paper h a s been p u b l i s h e d which r e c o g n i z e s t h e d i f f i c u l t y of d e t e r m i n i n g r e l a t i v e c l i n i c a l e f f i c a c y of a n t i b i o t i c s and proposes p o s s i b l e alternatives.78 The i m p o r t a n c e of o b t a i n i n g a n optimum b a l a n c e of p r o p e r t i e s i n a d d i t i o n t o u s a b l e a n t i b a c t e r i a l a c t i v i t y has been n e g l e c t e d by many e x p e r t s . A comprehensive s t u d y and d i s c u s s i o n of serum b i n d i n g of a n t i b i o t i c s has been p u b l i s h e d . 7 e S t i l l t o be c o r r e l a t e d e x p e r i m e n t a l l y is t h e s u g g e s t e d d e l e t e r i o u s e f f e c t on c l i n i c a l e f f e c t i v e n e s s v i t h t h e n a t u r e and d e g r e e of serum p r o t e i n binding P o s s i b l e involvement of p r o t o p l a s t forms of b a c t e r i a in an i n f e c t i o n have been c o n s i d e r e d . I n a n e x p e r i m e n t a l approach, e v i d e n c e w a s o b t a i n e d t o s u g g e s t t h a t a c o m b i n a t i o n of p e n i c i l l i n a n d kanamycin e l i m i n a t e d b o t h c l a s s i c a n d p r o t o p l a s t forms in e x p e r i m e n t a l p y e l o n e p h r i t i s . 8 0 Major advances in i n f e c t i o u s d i s e a s e t h e r a p y a r e d e s t i n e d t o o r i g i n a t e from s t u d i e s of t h e r e l a t i o n s h i p of a b e r r a n t b a c t e r i a l forms t o disease. The b i o s y n t h e s i s of m a c r o l i d e a n t i b i o t i c s w a s considered.81 A s p e c t s of t h e b i o g e n e s i s of s e v e r a l o t h e r a n t i b i o t i c groups have been c o v e r e d in a c o l l e c t i o n of papers.82 A u s e f u l r e v i e w of t h e c h e m i s t r y of nev a n t i b i o t i c s h a s appeared.89

--

.

Chap. 11

115 -

Flynn

Antibiotics

References

-

1. J. W. Kislak, T. C. Eickhoff and M. W. Finland, Am.' J. Med. Sci., 249,

636 (1965).

2. M. Turck, A. Ronald and R. G. Petersdorf, J. Am. Med. Assoc. , 13, 961.

(19651 3. C. F. Gravenlramper, J. V. Bennett, J. L. Brodie and W. M. M. Kirby, Arch. Int. Med., 15-6, 340 (1965).

4. P. Naumann, "Fifth Interscience Conference on Antimicrobial Agents and 5.

6.

7. 8.

Chemotherapy" Abstracts , American Society for Microbiology, Ann Arbor? Mich., 1965, p. 71. T. Eickhoff, J. W. Kislak, M. W. Finland and C. Wilcox, Am. J. Med. -*sci 9 2 9 , 163 (1965). G. A. Hardcastle, D. A. Johnson, C. A. Panetta, A. I. Scott and S. A. Sutherland, J. Org. Chem. , 31, 897 (1966). P. Bunn, J. Lunn and S. Milicich, "Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy" Abstracts, American Society for Microbiology, Ann Arbor, Mich. , 1965, p. 71. T. C. Eickhoff, J. W. Kislak and M. W. Finland, New EnKl. J. Med. , 272,

699 (1965) 9. T. Leigh, J. Chem.

SOC.,

1965, 3616.

10. W. J. Gottstein, R. B. Babel, L. B. Crast, J. M. Essery, R. R. Fraser,

J. C. Godfrey, C. T. Holdredge, W. F. Minor, M. E. Neubert, C. A.

Panetta and L. C. Cheney, J. Med. Chem., 5 794 (1965). ll. P. Bamberg, B. Ekstrom, K. Undheim and B. Sjoberg, Acta Chem. Scand., 19, 352 (1965)J. Tipper and J. L. Straninger, Proc. Natl. Acad. Sci., 2, 1133 12. 13

14. 15

16.

17

18. 19

20.

21.

22.

23

24.

25

(1965).

P. Fitz-James and R. Hancock, J. Cell. Biol., ,&, 657 (1965). F. P. Doyle and J. H. C. Nayler, Adv. Drug Res. ,A, 1 (1964). G. T. Stewart, "The Penicillin Group of Drugs." Elsevier Publishing Co., New York, N. Y., 1965. P. G. Cohen, M. J. Romansky and A. C. Johnson, "Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy" Abstracts, American Society for Microbiology, Ann Arbor, Mich., 1965, p. 68. E. J. Benner, J. L. Brodie and W. M. M. Kirby, E. p. , 67. G. Flux, A. W. Nunnery and H. D. Riley, Jr., E.,p. 70. M. A. Apicella, R. L. Perkins and S. Saslaw, Am. J. Med. Sci., 2 , 266 (1%). G. Moessner, H. Maurer and M. Flege, Med. Klin., 60, 19b4 (1965). Chem. Abstr., 9,2645 (1966). J. Bernard and S. Lambin, Ann. Inst. Pasteur, 109, 907 (1965). M. Ridley and I. P h i l l i p s , Nature, 208, 1076 (1965). E. J. Benner, J. V. Bennett, J. L. Brodie and W. M. M. Kirby, J. Bacteriol., g, 1599 (1965). J. H. Mitchell, Lancet, I, 125, 915. S. A. Kabins, B. Eisenstein and S. Cohen, J. Am. Med. Assoc., 193, 159

(1965)

e,

-

26. L. D. Sabath and E. P. Abraham, Nature, 1066 (1964). 27 W. E. Wick and W. S. Boniece, Appl. Microbiol., 2 (2), 248 (1965). 28. G. F. H. Green, J. E. Page and S. E. Staniforth, J. Chem. S O C . , lE5,

-

1595

116 -

Sect. I11

- Chemotherapeutics

F l y n n , Ed.

E. Van Heynhgen, J. Med. Chem., 5 22 (1965). W. J. Holloway and E. B. Scott, Am. J. Med. Sci., &9, 691 (1965). A. J. McLeod, H. B. Ross, R. L. Ozere, G, Digout and C. E. van Rooyen, Can. Med. Assoc. J., 2,1056 (1964). B. J. Magerlein, WFifth Interscience Conference on Antimicrobial Agents and Chemotherapy" Abstracts , American Society for Microbiology, AM Arbor, Mich., 1965, p. 17. B. Bannister, p. 17. R. B. Birkenmeyer, p. 18. A. D. Argoudelis, J. A. Fox and D. J. Mason, Biochemistry, 5 710

29 30 31 32

s., s.,

(1965)

Y. Suhara, K. Mae& and H. Ihnezawa, Tetrahedron Letters, l s , 1239. M. Hamada, T. Hashimto, T. Takahashi, M. Miyake, S. Yokoyama, T. Takeuchi, Y. Okami and H. Umezawa, J. Antibiotics (Tokyo), Ser. A, I&,

104 (1965). T. Takeuchi, M. Ishizuka, H. TakaJrema, K. Kineha, M. Hamada and H. Umezawa, E., 107 (1965). 39. N. Maggi, V. Arioli and P. Sensi, J. Med. Chem.,i, 790 (1965). 40. N. Maggi, R. Pn'lza and P. Sensi, "Fif'th Interscience Conference on Antimicrobial Agents and Chemotherapy" Abstracts, American Society for Microbiology, AM Arbor, Mich., 1965, pp. 47, 48. 41. B. C. Stratford, S. Dixon, B. F. Stratford and J. Forbes, ibid., p. U4. 42. N. Bergami and G. Forust, Arzneimittel-Forsch., 2,951 (lm. 43 H. H . Stone, J. D. Martin and L. Kolb, Surg., Gynecol. Obstet., 120,

38

44.

45

g,

351 (1965).

F. P. Furgiuele, R. Kiesel and L. Martyn, Am. J. Opthahol.,

(1965).

&, 818

A. W. Nunnery and H. D. Riley, Jr., "Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy" Abstracts, American Society for Microbiology, Ann Arbor, Mich., 1965, p. 37. 46. C. P. Schaffher and H. Maehr, Abstracts, 149th lational Meeting of the American Chemical Society, Detroit, Mich., April, 1965, p. 9C. 47 C. Lewis and H. Clapp, Antibiot. Chemotherapy, &&, 127 (1961). 48. R. Marsh and F. Ceccarelli, CUm. Med., 82'7 (1965). 49 Hi Kawaguchi, H. Tsukiura, M. Okanishi, T. Miyaki T. Ohmori, K. Fudisawa and H. Koshiyama, J. Antibiotics. (Tokgoj, Ser. A, l8, 1 0

-

50 51

53

54 55

56 57 58

9

(1965)

H. Kawaguchi, T. Naito and H. Tsukiura, g., 18, ll (1965). A. Furlenmeier, J. Berger, A. Batcho, €I Spiegelberg, . H. Schocher, 0. Keller, B. Pecherer and B. Vaterlaus, Chimia, 597 (1965). H. Kawaguchi, T. Miyaki and H. Tsukiura, J. Antibiotics (Tokyo),

a

Ser. A, &l 220 (1965). K. Vogler, h e w . Chem., 77, 974 (1965). R. 0. Studer, Helv. Chim.Tcta, 48, 1371 (1965). T. Suzuki, K. Hayashi, K. FujikaG and K. Tsukamoto, J. Biochem. (Tolgro), 57, 226 (1965). H. VanderGghe, P. VanDijck and P. DeScmer, Nature, 225, 710 (1965). W. 0. Godtfredsen, W. VonDaebne, L. Tybring and S. Vangedsl, J. Med. B., 8, 15 (1966). J. R. Rasch and W. J. Mogabgab, "Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy" Abstracts, American Society for Microbiology, Ann Arbor, Mich. , 1965, p. 36.

Antibiotics

Chap. 1 1

Flynn

59. P. Hung, C. L. Marks and P. Tardrew, J. Biol. Chem., 60. M. Kuehne and B. Benson, J. Am. Chem. S O C . , ~ , 4660 61. J. Dyer, C. Kellogg, R. Nassar and W. Streetman, Tetrahedron Letters, 1965, 585. 62. m e s e m a n n , P. Prave, A. Steigler and K. H. WaUhauser, "Fifth

63. 64. 65. 66.

Interacience Conference on Antimicrobial Agents and Chemotherapy" Abstracts, American Society for Microbiology, Ann Arbor, Mich., 1965, p. 44. E. V. Wasielewski, R. Muschaweck and E. Schutze, E., p. 45. F. Bauer and G. Dost, E.,p. 46. C. R. Temorria and R. C. Esse, J. Med. Chem.,& 870 (1965). J. Benbough and G. A. Morrison, J. Pham. Phannscol., 17 409 (1965). H. Jarolmen, A. Bondi and R. L. Crowell, J. Bacteriol.T&, 1286 (1965). P. A. Miller, J. A. Hash, M. Lincks and N. Bohonos, Biochem. Biophys. Res. Cam., 325 (1965). J. R. D. McCormick and E. R. Jensen, J. Am. Chem. SOC., ,7'8 1794 (1965). H. lvfuxfeldt and W. Rogalshi, 87, 933 (1965). M. M. Shemyakin, "Fifth Interscience2onference on Antimicrobial Agents and Chemotherapy" Abstracts, American Society for Microbiology, Ann Arbor, Mich., 1965, p. 16. M. M. Shemyakin, E. I. Vinogradova, M. Yu. Feigina, N. A. Aldanova, N. F. Loginova, I. Ryabova and I. Pavlenko Experientia, 21, 548 (1965). F, J. Marshall, J. Med. Chem.,A ' , 18 (19651. J. F. Wallace, R. H. Smith and R. G. Petersdorf, New Engl. J. Med., 2 3 , 1014 (1965)W. E. Herrell, Clin. Med., 72, 19U (1965). H. P. Browder, W. A. Zy-7 J. R. Young and P. A. Tavormina, Biochem. &383 (1965). Biophys. Res. Canrm., I E. F. Harrison and C. B. Cropp, "Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy" Abstracts, American Society for Microbiology, Ann Arbor, Mich., 1965, p. 115. B. A. Waisbren, Am. J. Med. Sci., 2 0, 406 (1965). G. N. Rolinson and R. Sutherland, J. E'harmacol., 3, 638 (1965). J. Z. Montgamerie, G. M. Kalmanson, W. L, Hewitt and L. B. Guze, "Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy" Abstracts, American Society for Microbiology, Ann Arbor, Mich., 1965,

E.,

72.

73 74. 75 76

77. 78 79

80.

117 -

At.

P. 78. 81. H. Brisebach, p. 22. 82. Z. Vanek and Z. Hostalek, Eds., "Biogenesis of Antibiotic Substances," Academic Press, New York, N. Y., 1965. 83 E. P. Abraham, Am. J. Med., 39, 692 (1965).

s.,

K.

118 -

Sect. I11

-

Chemotherapeutics

Flynn, Ed.

Chapter 12. Synthetic Antibacterial Agents Robert G. Shepherd, Lederle Laboratories, American cyanamid Co., Pearl River, New York Developments during 1965 involved primarily laboratory and clinical study of analogs of known antibacterial structures and of their metabolism and mechanism of action. The importance of antibacterials is indicated by the large research effort being expended. The difficulty of finding new antibacterial structures is demonstrated by the fact that Only a very few have been reported in this decade in spite of this effort. Most of the 119 y e a r s since the beginning (Semlweiss 1847 and Lister 1865) of modern(1) antibacterial chemotherapy have been spent in stagnation or in significant but slow improvement on the small numbers of different active types so far discovered. Into the attack on the antibacterial frontiers are being brought biochemistry at the enzyme level, molecular vibrations, electronic energy-level calculations and pharmacokinetics via computers but the push-button age of "magic bullets" is still far in the future. In Vivo Actives.--The new compounds first shown by published data in 1965 to be active against infections in animals are discussed here. A laboratory study(2) on 2-sulfanilamido-4,6-diethyl-s-triazine showed it to be the most active of the sulfatriazines in vlvo an3 the first clinical report was promising(3). It is highly soluble, well absorbed, less conjugated than other clinical sulfas, and excreted largely unchanged(3). Its behavior is between short-acting and long-acting and its renal clearance is uniquely acceleratable by adjustment of urinary pH(3). Chemotherapeutic application of 4-sulfa-1,2,3-thiadiazoles is limited by their explosive intermediates (4). 5-Dimethylsulfamyl-antnthranilic hydrazide is only formally a sulfanilamide derivative since it is not antagonized by PAB. The in vivo antibactes ial activity(5) is rather specific structurally and is limited to Staphylococcus @D50 25-5Orng/kg) and Pneumococcus (ED50 250mg/kg) but, curiously, shows up in vitro only against Pneumococcus. Low activity against the expen imental TF3 infection in mice was reported for 2-carbethoxyaminoethylnbutylsulfide(6) and for beta-alanylhydroxamic acid(7). The latter shows some similarities in mechanism of action to cycloserine. Additional data(8) on 1-(aralkoxy and higher alkyloxy)-k, 6-diamino-l,2-dihydro-2,2-dimethyls-triazines show that some are active in vivo against Strep. hemolyticus but only at just below toxic doses. The broad-spectrum in vitro activity and the toxicity appear to be antifolic in nature. New activity (leprosy, below) was reported for ethambutol and for thalidomide as well as for some azo dyes. Antituberculous Agents.-Although major advances in TI3 therapy have been made, treatment is still far from satisfactory and the incidence of new urban cases is increasing, i n some instances drastically(9). Ethambutol (dextro-2,2'-ethylenediimino-di-l-butanol), one of two new active types reported in this decade, has successf'ully undergone clinical trials (10, 11, 12, 13) in several countries. Ethambutol acts clinically (12, 13) and experimentally(l4, 15) against strains of mycobacteria resistant to other agents, and combinations of sub-effective concentrations are inhibitory (16) in vitro. Resistant strains, which develop with difficulty(17, IS), have different biochemical characteristics than isoniazid-resistant strains (19). Human and bovine mycobacterial strains, which were experimentally

-

Chap. 12

Antibacterials

Shepherd

119 -

made resistant to ethambutol, have been fo~na(16) to be attenuated for mice. Using 3513 and 32P to estimate protein and nucleic acid synthesis, it has been concluded(l9, 20) that ethambutol acts by interfering with a function of cellular polyamines and divalent cations in the synthesis or stabilization of RNA. Preliminary reports of its activity against human(21) and mine(22) leprosy have appeared. Quickly following a report drawing attention to the carcinogenic activity of isoniazid was a word of caution(23), much needed in the present, solnewhat hysterical atmosphere'surraundingW s . The production of tumors only in mice may well have prevented introduction of isoniazid in recent years but should not diminish its use until a harmful effect is established and weighed against its proven merits. Extensive investigation of N,N'-diaryl and N-aryl-N'Aeteroaryl thioureas appears to be ending in naught. N,N'-Bis(k-isoamyloxyphenyl)thiourea, the member most studied clinically, is judged to be only slightly active in man, by the usual criteria(24, 25, 26), on the basis of all the published controlled trials(27) in several countries. In addition, there is a high frequency(28) of natural resistance to this agent and cross-resistance with 4acetamidobenzalthiosemicarbazone(29). 2-Ethyl isonicotinic thicamide kthionamide) is undergoing clinical study at decreasing dosage. Although effective(30, 31) in producing clinical improvement and preventing emergence of resistance, high incidence of liver (31, 32, 33) and gastrointestinal(30, 34, 35) toxicity seems likely to restrict its use to retreatment of resistant cases. Mental disturbance and neural effects were cited(31, 36) as reasons for not using it in ambulatory patients. It is converted in man to several metabolites(37), among them the thioamide sulfoxide. It is not cross-resistant with isoniazid but shows B?m cross-resistance with thiocarbamylhydrazides (thiacetazone) and diarylthioureas(38, 39, 40, 41). Reports on the N-morpholino-methylde of pyrazinoic acid, proposed as a form of pyrazinamide with decreased toxicity and maintained activity(froln conversion to pyrazinamide), fail to confirm either of these characteristics(&). Gram-negative Antibacterials. -l-Ethyl-7-methyl-~oxo-l, 8-naphthyridine3-carbo~ylicacid(na1idixic acid) is one of two new active types reported & this decade. Its Gram-negative activity was first published by Lesher in 1962 and a brief structure-activity summary followed in 1964(43). There is no cross-resistance(44, 45) with other agents used in Gram-negative infections and its ED50 is comparable(4.6). The primary antibacterial action of nalidixic acid against E. coli is specific inhibition of DNA synthesis(47, 48). This antibacterial is unusual in being active as such and in rapidly forming an equally active metabolite, the 7-hydroxymethyl anslog(49). About one-third is excreted as this metabolite plus two-thirds as glucuronides of parent and metabolite. Antibacterial activity(50) in urine is predominantly (7:l) due to 7-hydroxymethyl metabolite while in tissue the two agents are about equal in concentration(@). Clinical assessments(51, 52) rate it rapidly effective orally against certain Gram-negative bacteria. Since genitourinary tract infections due to these organisms should be treated for several weeks to avoid relapse, it is an important shortcoming that a high 53) or after a degree of resistance can develop rapidly during therapy(% s m a l l number of transfers in vitro. A small trial(54) against brucellosis was successful. As is common in the early stages of clinical use, reports are conflicting on the breadth of application(55, 56, 57, 58) to urinary

120

Sect. 111 - Ghemotherapeutics

F l y n n , Ed.

infections (reported percentages of sensitive strains: 2-22s) Wb 8% 9%) and on side-reactions( 51, 59). Sulfanilamides.-Work on sulfa drugs is beginning its 4th decade with i& terest in and use of these agents continuing, especially since microbes ap~n apPliparently have not meanwhile progressively developed resistance(@). cation for sulfas (and other antibacterials) now receiving attention(6l) is treatmt of bacteriuria(frequently asymptamatic) of P r e V t W-n, a Condition with serious consequences for the child (higher incidence of' prematurity, neonatal death, congenital abnormalities, cerebral palsy, and lower I&) as well as for later chronic disease of the mother. The various drug-ofchoice uses of sulfas have been sunmarized(62) and chemotherapeutic research with sulfanilamide derivatives reviewed(63). B means of pharmacokinetic equations and computers, various papers(64, 657 attempt to adjust experimental and clinical dosage of sulfas to get equitherapeutic plasma-water concentration-time curves. Several long-acting sulfas introduced in recent y e a r s are in clinical evaluation (in approximate order of increasing persistence): 4-sulfa-3,6dimethoxypyrid~zine(66), 3-sulfa-4-iodo-5-methylisoxazole(67), 2-sulfa-5methoxypyrimidine(68), 2-sulfa-3-methoxypyrazine( 69), and ksdfa-5,6-dimethoxypyrimidine(70). The latter is so slowly excreted that oral, parenteral or rectal doses once per week or per disease episode are being explored(71, 72, 73). In contrast to its 2,&dimethoxy is-r, It yields only about 2$ of glucuronide. In relation to crystalluria potential(68), the l o w solubility of the long-acting sulfa8 is partly compensated for by glucuronide formation, but more significant is the amount of sulfa and/or metabolite excreted per unit time in relation both to actual urine flow rate and to the therapeutic dosage required for the particular sulfanilamide. Considerable study has been made of the binding of sulfas to blood albumin. Although bacterial inhibition varies directly with the percent unbound in a simple experimental mode1(74), the activlty relationship in vivo can not be aseumed to be the same as that of relative binding, and a highly bound compound need not be less desirable than a slightly bound one. Two agents with different degrees o f binding to blood albumin also differ in binding to tissue, in vltro potency, &, lipid solubility, excretion, metabolism, and distribution in body water, all of which directly or indirectly affect in vivo efficacy. In addition, the relationship of blood albumin binding to the degree of binding to the key enzyme site where the sulfa competes with PAB(75) is unknown. In an excellent sympoeium on interaction between drugs, Brodie(76) pointed out that binding is an advantageous and necessary characteristic without which a therapeutic agent would oscillate between toxic and inactive plasma concentrations and require frequent administration. Releasing an agent from binding to protein can be accomplished by means of a broadly similar chemical compound because of the rela tively non-specific nature of the binding but this release can lead to greater toxicity(76) and excretion and metabolism as w e l l as to the desired physiological action. Of interest in laboratory and clinical studies, it has been observed that the mouse, dog, man and other species v a r y 17-fold in extent of albumin binding of sulfas. Variation of binding is 2-fold among normal human sera, and 10-fold in certain illnesses(77) partly due to circulating blood constituents and partly due to albumin variation. The locus of binding of several sulfas to serum albumin has been shown(78) by relaxation-time

Chap. 12

Antibacterials

Shepherd

121 -

measurements on high-resolution pmr spectra to involve the benzene ring and not the heterocycle; in 5-sulfa-1-phenylpyrazole the two benzene rings bind at different sites. Infonoation about sulfa drug metabolism in man has been reviewed(79). &Acetylation has been known to occur enzymatically to an extent dependent on N1-substituent and animal species. -Glucuronides(l-4$) result from nonenzymatic chemical reaction whileI*!J sulfamstestl (about I$) are true metabolites. Formation of N1-glucuronides occurs quite generally; with some N1heterocycles it amounts to only a few percent of dosage(sulfisoxazole, sulfathiazole) but with sulfamethoxyppidazine is substantial(l5-20$) and with sulfadimethoxine is 60-85$ of the substances excreted. 4-Sulfa-6-methoxy-2methylpyrimidine yields a large amount of 2-hydroxymethyl 0-glucuronide. Wfferences in the So;! infrared frequency in amino and imino isomers led to assignment of the sulfadimethoxine metabolite as NL rather than ring-N substltuted(80, 81). 5-Sulfa-1-phenylpyrazole is 70-9srenally excreted as a glucuronide of still uncertain structure( 82 ). 2-Sulf'a-4,5-dimethyloxazole is reported(83) in a study by thin-layer chromatography to be grossly unstable in water as w e l l as urine and blood, but a paper chrumatographic study (84)flatly contradicts this. Nitrof'urans.-Twenty-two years after the first report of their antibacterial activity, this class is receiving renewed clinical and laboratory interest. In spite of several thousand publications cove ng some six hundred analogs and 803116) structure-activity generalizations required: 5-N% o 3- or 4-substituan ring, some kind of 2-substituent and the critical characteristics that govern their activity especial. ly in vivo are far from clearly defined, The chemotherapeutic properties of the earlier nitrofurans have been reviewed(86). 4-(5'-Nitro-2'-furyl)-2-(3' '-pyridy1)thiazole is unusual in being a nitrofurylheterocycle bearing a heterocyclic substituent and in having broadspectrum activity in vivo (about 8 times as active as furaltadone and furadantin). Unfortunately, blood dyscrasias occur at doses just below the therapeutic range(87). An experimental model was reported(88) for the polyneuropathy which occurs with furadantin and other nitrofurans(86, 89). Recent work has involved linking nitrofuran to various heteroaromatic rings either directly or through one or more ethylene units. 3-(5'-Nitro2'-fUryl)-1,2,4oxadiazoles and thiadiazoles and 2-(nitrofury1)thiazoles were found low in activity in vitro and "largely ineffectivev in animals (90)while the 1,3,4-isamers and 4-(nitro 1)thiazoles reported earlier were active in vivo (many at max. tol. doses Hydroxymethyl- and bishydroxymethyl-amino derivatives(g1) are less active and less toxic acutely than the parent known nitrofbylvinyl-aminoheterocycles. It is stated that in vitro activity is increased by insertion of vinyl or substituted vinyl between the 5-nitro-2-furyl unit and a pyridazine or thiadiazole(92), oxadiazole(93, 94), quinoline(g5), pyridine(96) and its l-oxl&?(97), range of minimal inhibitory concentration6 being 0.03 to lOmcg/ml. The insertion of another vinyl group is claimed to increase the in vitro activity still W h e r for oxadiazoles(98) and pyrimidines(gg), range of m.i.c. 0.02 to O.%cg/ml. Several were found active in vivo but only at maximum tolerated doses. 3-( beta-Substituted ethyl)-1-( 5'-nitro-2'-~furylideneamino)-imidazolidin-2-ones are claimed(100) to be more active in vivo but less active in vitro than the 3-H parent(101).

r

E%5T,

7.

122

Sect. I11

-

Chemotherapeutics

F l y n n , Ed.

Antileprotic Agents.--Although the age of chemotherapy has benefitted its treatment, the effect on this wide-spread(lW) disease is incomplete and slaw. The slowness is befitting this, the most ancient infectious disease of man, which has the longest known incubation period (from 3 to 8 or more years) and the longest generation time (30 dws)(l(X?, 103). New agents have come from trying in human leprosy anything active against tuberculosis, hoping for activity on the basis of the taxonomic relationship of the causative organisms. Neither an in vitro test nor an experimental form of the disease has been available but recent advances have been made(lOe, 103, 104, 105). Although some antituberculotics are effective against human leprosy, activity against tuberculosis is an unreliable indicator(l06) of humin antileprotic activity, and vice versa. Three reports of activity have come from clinical use of agents available for widely differing purposes. ~li~de(cCphthalimidoglutarimide),while being employed as a sedative, showed activity against human lepromatous leprosy in 6 consecutive cases by several criteria. It will be interesting to await confirmation of this preliminary report(107). In another preliminary report(108) involving a more extensive trial(50 patients) in a controlled comparison with diaminodiphenylsulfone, ethambutol was reported equally active. Against an experimental Wco. lepraemurium infection(109) in mice it was estimated to be s o m e a t less active than the sulfone. &re active than this standard in the murine infection are Lsulfa-6-methoxypyrimidine and 3-sulfa-6-methoxypyridazine. Several papers on clinical ficacy of the latter have appeared(ll0, 111). Using the murine test, 1$44- ubstituted phenyl or pyridyl)-5,6,7,8-tetrahydro-l-naphthyltuninopropyiJ piperidines were found to have an interesting level of activity(ll2). In Vitro Actives.-Such compounds are potentially useful for bacterial control of body cavities, genitourinary tract, wounds, skin prior to operations, hands of medical personnel, and for preservation of parenteral and topical~dicinals. The significance of many publications can not be judged since tests are not adequately defined or related to standards nor are toxicity, in vivo results and effect of protein on activity given. The following were active against Gram-positive and Gram-negative organisms at the minimal inhibitory concentrations given: 6-( 4t-methoxyphenyl)-Mdazo @, at 8-60, 2-(phenyl and 2' -pyridyl)-isatogen( 114) at 25-200, -2-(dichloroacetyl)guanidine(ll5) at 0.05-25, and 3-(methyl and ethyl)-l-hydroxy-2-oxoquinoline(116) at 0.3-5mcg/ml. In the latter which is 1-10 times as active as aspergillic acid, the hydroxamic acid moiety is necessary but not sufficient for activity. Dibenziodolium(ll7, 118) and dibenzoxaiodinium analogs(119) were claimed to be more stable and more active (m. i. c. 0.3-25mcg/ml) than the diphenyliodonium analogs; acute LD50 10-

1-d

1oow/kg.

Work continues on detergent bases and cationic surfactants, whose activity was first reported in 1935. Antibacterials of this kind generally inhibit both Gram-positive and Gram-negative organisms. 2-(N-Decylpyrrolidinium) ethyl trans-beta-(l-naphthyl)acrylate(l20) was 50-100-fold less active in the presence of serum, an effect common to many agents of this class. Other compounds active were polychloro-2,3-diphenylpropylamines( 121) and 3,3,3tris(4' -chlorophenyl)propyl-N,N-bis(beta-pyridinium ethy1)amine dichloride at l-lOOmCg/ml. Several kinds of phenylenebis (4-amino-6-amidinoquinainoquines (122) were active at the same concentration but were not active in vivo. The activity of N-( 3-phenyl-2-propyl)-3,3-diphenylpropylamine( 123) at l-lOmcg/ml

Chap. 12

Antibacterials

Shepherd

123 -

against Gram-positive organisms is reversed by nucleic acids or by divalent cations. The latter also antagonized the action of steroidal tertiary amines or quaternaries(l24) whose cellular damage is prevented by spermine also. 5-Azacytidine at 0.2mcg/ml inhibits growth of E. coli as a result of spontaneous triazine ring-opening in the nucleic acid into which it is incorporated( 125). Mono and di-chloro Z-benzamido-4,6-dialkoxypyrimidines (126) inhibited Staph. aureus at l-5mcg/ml but the somewhat more active Lsulfa-2, 6-dimethoxypyrimidine standard inhibited Gram-negative organisms also. Their activity may be a variant of the halo-salicylanilide type(l27). 2,bDialkanoyl phloroglucinols( 128) also have narrow spectra(Staph. and Strep. inhibition at 0.1-0.5 mcg/ml); related resacetophenones are inactive(129). A rela tively unstable chelate permits cell penetration by salicylaldehydes whose activity against Pseud. aeruginosa is dependent on the amount of free aldehyde group liberated inside the cells(l29). This organism is subject to bactericidal action (130) by ethylenediaminetetraacetic acid but not other chelators; it produces damage to the protoplast membrane. Research Techniques and Screening &tho&.-Radioactive tracers have been used in studying pharmacology and mechanism of action. The primary antibacterial action of nalidixic acid was shown(47, 48) to b specific inhibition of DNA synthesis by following the incorporation of lEC- and 3H- labeled purines, pyrimidines and amino acids into DNA, RNA and protein of E coli. Ethionamid ' 8 mechanism of action was investigated(38) by me s of j2P-, 35S- and lEC- labeled nucleic acid and protein precursors. lg-Ethambutol was e loyed in human pharmacological(20) and mechani.sm of action(l9) studies. lq-Iabeled quinaldinium compounds were used to determine their mechanism of antibacterial action( 131). Pharmacokinetic equations are being solved by analog computers and used (64, 65, 132, 133) to devise optimal dosage regimens which are important to safe, effective clinical trials and can make laboratory comparisons more incisive by equalizing exposure of infections to drugs. These equations have been applied in a series of papers to sulfa drugs(64, 65, 133) for which extensive pk, protein binding, excretion, and distribution data are available. Adequate screening methods for leprosy have been lacking but tissue-culture(102) and in vitro growth(lO5) have been disclosed. Growth of the human strain in cell-free medium has resulted(105) from use of unisolated growth factors from lysed saprophytic mycobacteria. One method available has been the mouse foot-pad growth(103) of Myco. leprae which does not produce a leprosy infection and might be considered a form of tissue-culture. A mco. lepraemurium infection(109) in mice has several similarities to the human disease but also certain differences. According to a recent report(104), the infection of black mice with "Chatterjee" bacilli is not the hoped-for major step forward in antileprotic screening but seems to be a modified M. lepraemurium infection. A lethal Tl3 infection was developed(134) in guinea pigs by intracardial injection of M. tuberculosis H37Rv but has a more variable survkvaltime than the mouse infection and requires more compound for testing. Various kinds of delayed and intermittent treatment of tuberculous mice have been studied(l35) with half a dozen reference drugs. Correlations of molecular vibrations,7%electron energies and of calculated molecular orbital and bond-hybridization character with antibacterial activity(l36, 137, 1-38>139) provide interesting summaries of where one has

124 -

Sect. III

- Chemotherapeutics

F l y n n , Ed.

been rather than where one is going. Perhaps when we know how to transfer in vitro data successfully to in vivo tests, we will be able to make use of such specialized, "unidirectional" theoretical approaches. Modern physical methods and chromatographic techniques are being applied to problems of separation and identification of synthetic antibacterial structures and their metabolites, which in the case of nalidixic acid makes a major contribution to activity: gas chrmtography (14, 140); thin-layer chromatography of sUlfanilamides(l41, lk),amines(143), and heterocycles (144); mass spectrametry of pyrimidines(145) and d s ( 1 4 6 ) ; nmr spectrometry of phenols(147, 148), peptides(l49), and pyrimidines(l50); X-ray spectrompem of P-, s-, and halogen- containing ccarrpounds(l5l.); and --ray spectrof neutron-activated pharmaceuticals(1%) employed in forensic investigations but broader in potential(l53). enzymic bases of now well-defined mechanisms Mechanism Of Action.-The for bacterial control (cmpetitive inhibition of essential enzymes, feed-back inhibition of biosynthesls of the natural competitor, repression of fonnation of a necessary enzyme, and non-competitive inhibition by reacting with the enzyme active center) have been reviewed(l54). The biochemical basis for differential action of an antibacterial toward host and microorganism is clear for a number of synthetic agents(156). Differences in response to dihydrofolate reductase inhibitor6 reflect differences in the structural limitations of the binding site of the same enzyme from different bacteria(l55). Sulfanilamides are unique in having their mode of action almost completely known at the enzyme level. The step in folic acid synthesis at which they intervene has recently been shown to be the reaction of PAB with the pyrophosphate ester of 2-amin0-4-0~0-6-hydro~thyldihydropteridine (155). The sulfa8 are now known to participate in this reaction and form spurious folic acids, but their significance in growth inhibition is not certain. Some differences in responses of bacteria are due-to relative abilities to transport and assimilate folic acid. Sulfanilic acid is an inhibitor of a cell-free enzyme system but not of bacterial growth, presumably due to difficulty with cellular transport(155). Bactericidal action of sodium tetrapropylenebenzenesulfonate resulted. from lysis of protoplast membranes; in contrast to many other agents, this action decreased as cellular metabolic activity increased( 156). Sodium lauryl sulfate produces inhibition of biosynthesis of S. aureus cell walls similar to that produced by penicillin(157). Prevention of emergence in vitro of drug resistance to a variety of antibacterials by spermine or atabrine was reported(l58) to result f r o m preventing induction of resistance. An antimutagenic action of spermine toward random as well as induced mtations(159) confirms the effect (stated not to be co-inhibition) in vitro but demonstration of a ureful effect in vivo is still lacking. Reviews.-Reviews appeared on tuberculosis(15, 29, 160, 161), leprosy( 106), sulfa antgs(63, 64, 65, "7, 79, 1331, nitrofuram(%), drug interaction(76), protein binding(77, 133), mechanisms of action(154, 155, 162), and combinstion therapy( 163). REFEREmCES 1. The mercurial8 used for syphilis in the 1500's might also be considered as the first antibacterials. 2. W. E. Taft, H. M. Krazinski, F. C. Schaefer, and R. G. Shepherd, J. Med. -* 8, 784 (1965).

-

Chap. 12

Antibacterials

125 -

Shepherd

3. A. R. Frisk and E. Hultman, 4th Internat. Congr. Chemotherapy, 1965, Washington, D. C., Abstracts, p. 33. 4. D. L. Pain and R. Slack, J. Chem. SOC. (Lond. ) 5166 (1965). 5. P. Schmidt and L. Neipp, Proc. 3rd Internat. C o n g r . Chemotherapy, 1963, Stuttgart, 1355 (1964). 6. G. Siefert, Arzneimittel-Forsch. 9, 1359 (1965). 7. G. R. Gale and J. E. Hawkins, Am. Rev. Resp. D i s . E, 64.2 (1965). 8. P. Mmalis, L. J e f f r i e s , S. A. Price, M. J. R i x , and D. J. Outred, J. Med. Chem. 8, 684 (1965). 9. Editors, Mod. Wd. 32, 54 (1964). 10. I. Ksss, Tubercle ( E d . ) 46, 166 (1965). 11. Conference on New Antitubezulous Agents, Sept. 1965, Ann. N. Y. Acad. Sci. 135, 680-1120 (1966). 12. T F . Corpe and F. A. Blalock, MS. Chest 48, 305 (1965). 13. I. D. Bobrowitz and K. S. Gokulanathan, DiK Chest 48, 239 (1965). 14. J. Po !L'honkasy C. 0. Baughn, R. G. Wilkinson, and R . 3 . Shepherd, Am. Rev. Resp. D i s . 5, 891 (1961); R. G. Wilkinson, M. L. Cantrall, and R. G. Shepherd, J. Meed. Chem. 5, 835 (1962). 15. F. Grumbach, Advan. Tuberc. Res. 2, 74 (1965). 16. S. K O Gupta and I. S. Eilathur, Indian J. Exptl. Biol. 2, 81 (1964). 17. M. Forbes, N. A. Kuck, and E. A. Peets, J. ~acteriol.-89, 1299 (1965). 18. F. Grumbach, Ann. Inst. Fasteur 110, 69 (1966). 19. S. K. Gupta and I. S. Mathur, InJ. E x p t l . Biol. 3, 176 (1965).

20. E. A. Peets, W. M. Sweeney, V. A. Place, and D. A. BU%ke, Am. Rev. Reap. D i s . 91, 51 (1965). 21. J. G.Tolentino, R. S. Quint0 and R. M. Abalos, 25th Res. Conf. i n Pulm. Diseases of VA-Armed Forces, Jan. 1966, Cincinnati, Ohio. 22. Y. T. Chang, Antimicrobial Agents Chemotherapy 777 (1965). 23. Editors, B r i t . Med. J. 1(5449), 1508 (1965). 24. M. Olejnicek, M. Weberova, M. Novak, and E. Jancik, Tubercle (Lond.)

1964,

188 (1965).

5,

25. A. S. Moodie, M. Aquinas, and R. D. Foord, Tubercle (Lond.) G , 1% (1964). 26. S. Sunahara, Tubercle (Lond.) 46, 178 (1965). 27. Editors, Tubercle (Lond.) 46, 8 8 (1965). 28. G. Meissner, Praxis h e w r 19, 387 (1965). 29. F. Grumbach, Advan. Tuberc. R G . 14, 70 (1965). 30. M. Boszormenyi, I. Fauszt, I. B ,* and 0. Schweiger, Tubercle (Lond.) 46, 143 (1965). 31.

J.M.

Schless, R. F. Allison, R. M. Inglis, E. F. White, and S. Topperman,

Am. Rev. Resp. Die. 91, 728 (1965). 32. F. I t o and K. HayanoTAm. Rev. Resp. Ms. 91, 796 (1965). 33. A. W. Lees, B r i t . J. D i s . Chest 59, 228 (1'965)34. A. Pines, Tubercle (Lond.) 46, l z (1965). 35. I. Kass, Tubercle (Lond.) 4 c 151 (1965).

36. A. W. Lees, Am. Rev. R e s p . 3 8 . 91, 966 (1965). 37. H. Iwainsky, I. Sehrt, and M. G r u n e r t , Arzneimittel-Forsch. 15, 195 (1965). 38. M. T s ~ ~ k a m r aIgaku , To Seibutsugaku 67, 80 (1963); Chem. Absc. 63, 12024f (1965). 39. H. Hartwlgk, Tuberculosearzt 9, 522 (1965).

40. S. Oka, J. Konno, M. Kudo, K. Oiztani, and S. Yamaguchi, Am. Rev. Resp. D i s .

41.

919 638 (1965). Verbist, 4th Internat. Congr. Chemotherapy, 1965, Washington, D, Abstracts, p. 60.

C.,

126

Sect. I11 - Chemotherapeutics

F l y n n , Ed.

42. L. Tnka, J. K u s k a , and A. Havel, Chemtherapia 9, 158, 168 (1965). 43. G. Y. Lesher, Proc. 3rd Internat. Congr. Chemoth
-

Chap. 12

Antibacterials

127 -

Shepherd

82. W. Riess, K. Schmid, and H. Keberle, KLin. Wochschr. 43, 740 (1965). 83. J. Seydel, H. Buettner, and F. Portwich, Klin. WochscG. 43, 1060 (1965). 84. E. Serick and W. Loop, Chemotherapia 10, 74 (1965). 85. V. N. Solov'ev, G. A. Konyaev. S. S. Xvikov, L. I. Khel'nitskii, and T. S. Novikova, Fbrmakol. i Toksikol. 28, 316 (1965). 86. H. E. Paul and M. F. Paul i n "Experimexal Chemotherapy", R. J. Schnitzer and F. Hawking, Eds., Acad. Press, New York, 1964, V o l . 11, p. 307. 87. J. K, LEtndquist and A. R. Martin, Antimicrobial Agents Chemotherapy 1964,

-

-

591.(1965).

88. A.

Behar, E. Rachmilewitz, R. Rahamimoff, and M. Denman, Arch. Neurol.

13, 160 (1965).

w. Beverungen,

K. W. F r i t z , and J. Ross, Muench. Med. Wochschr. 107, 953 ( 19651 90. W. R. Sherman and A. V. Esch, J. &d. Chem. 8, 25 (1.965). 91. K. Miura, M. Ikeda, T. Oohashi, Y. Igarashi,-and I. Okada, Chem. Pharm.

89.

Bull* ( T O W O ) 13, 529 (1965). 92. W. Voemel, P r o z 3rd Internat. Congr. Chemotherapy, 1963, Stuttgart, 530 (1964). 93. I. Saikawa and A. Takai, Yakugaku Zasshi 85, 948 (1965). 94. I. Saikawa, H. Kodsma, A. Takai, T. Wada,%hd Y. Suzuki, Japanese patent 12,619 (1965). 95. K. Miura, M. Ikeda, T. Oohashi, Y. Igarashi, I. Okada, T. Shikimi, and S. I s h i i , Y a k u g a k u Zasshi 85, 289 (1965). 96. K. H a r a d a and S. Emto, ChG. Pharm. B u l l . (Tokyo) 13, 389 (1965). 97. S. Minami, A. Fujita, and J. Matsumoto, Japanese p a c n t 28,591 (1965). 98. I, Saikawa, Y. Kodama, A: Takai, T. Pllaeda, and I. Takamichi, Japanese patent 12,706 (1965). 99. I. F a i k a w a , Y. Kodama, S. Krtnamori and A. Takai, Japanese patents 19~65% 4 and 26,408 (1964). 100. Norwich Pharm. Co., Belgian patent 653,hl (1965). 101. J. R. O'Connor, H. E. Russell, J. G. Michels, P. V. Newland, and W. F. Carey, Proc. 3rd Internat. Congr. Chemotherapy, 1963, Stuttgart, 558

(1964).

102. Editors, J. Am. Med. Assoc. 192, 32-36 (1965). 103. C. C. Shepard and D. H. M c R & T J . Bacteriol. 2, 365 (1965). 104. H. M. La1 and J. M. Robson, B r i t . J. Exp. Pathol. 46, 318 (1965). 105. A. L Olitzki and Z. Gershon, I s r a e l J. Med. Sci. 1F1004 (1965). 106. R. J. Schnitzer, Antibiotica e t Chemotherapia, Fohschr. 9, 145 (1961). 107. J. Sheskin, Clin. Pharmacol. Therap. 6 , 303 (1965). 108. J. G. Tolentino, R. S. Quinto, and R.-M. Abalos, 25th Res. Conf. i n Pulm. Diseases of VA-Armed Forces, Jan. 1966, Cincinnati, Ohio. log. Y. T. Chang, Antimicrobial Agents Chemotherapy 777 (1965). ll0. J. Schneider and J. Languillon, Internat. J. Leprosy 31, 551 (1963). 111. E. Asshauer, Tropenmed. Parasitol, 16, 73 (1965). 112. Y. T. Chang, 4th Internat. Congr. Chemotherapy, 1965, Washington, D. C., Abstracts, p. 82. 113. W. Weuffen, T. Pyl, W. Gruebner, and W. D. Juelich, F'harmazie 20,

1964,

-

629 (1965).

-

114. M. Hooper, D. A. Wtterson, and D. G. Wibberly, J. pharm. Pharmacol. 17,

734 (1965).

115. A. F. Mcky, U. S. patent 3,169,146

116. R. T. Coutts, 792 (1965).

(1965).

W. N. Pitkethly, and D. G. Wibberly, J. Pharm. Sci.

54,

128

Sect. 111 - Chemotherapeutics

F l y n n , Ed.

117. W. N. Cannon, U. S. patent 3,207,660 (1965). 118. H. T. Openshaw and E. Walton, B r i t i s h patent L,013,571 (1965). 119. E. L i l l y and Co., Belgian patent 652,071 (1965). 120. G. Coppi and C. C. Bonardi, Farmaco (Pavla), Ed. Scl. 20, 203 (1965). 121. M. Schorr, Proc. 3rd Internat. Congr. Chem. 1963, Stuttgart, 1396 (1964). 122. H. Loewe and J. Urbanietz, Arzneimittel-Forsch. 15, 604 (1965). 123. H. M. Rauen, Arzneimittel-Forsch. 2,1318 (1965T l24. R. F. Smith and D. E. Shay, Appl. Microbiol. &I 706 (1965). 125. A. Cihak and F. Sorm, Collection Czech. Chem. .C2,2091 (195). 126. Y. N i t t a , T. Miyamoto, T. Nebashi, and>F. Yoneda, Chem. Pharm. B u l l . ( T O e O ) 13, 901 (1965). 127. A. &aushaar, Arzneimittel-Forsch. 4, 548 (1954). 128. K. Bowden and W. J. Ross, J. l?harm.-F'harmacol. 3, 239 (1965). 129. D. E. Barton, K. C l a r k e , and G. W. Gray, J. Chem. SOC. 438 (1965). 130. G. W. Gray and S. G. Wilkinson, J. Appl. Bacteriol. 28, 153 (1965). 131. W. A. Cox, Appl. Mlcrobiol. 956 (1965). 132. W. E. Moore, G. A. portmann, H. Stander, and E. W. McChesney, J. Pharm. Sci. 2, 36 (1965). 133. E. Ihteger-Thiemer, W. Diller, L. D e t t l i , P. Buenger, and J. Seydel, Antibiotica e t Chemotherapia, Fortschr. 12, 171 (1964). 134. S. K. Gupta and I. S. Elathur, Indian J. %d. Res. 52, 973 (1964). 135. B, Kreis and S. Fournaud, Ann. Inst. Pasteur 108, 113 (1965). 136. F. Yoneda and Y. N l t t a , Chem. Pharm. B u l l . ( T x o ) 12, 1264 (1964). 137. J. Seydel, Antlbiotica e t Chemotherapia, Fortschr. 12, 135 (1964). 138. R. S. Schnaare and A. N. Martin, J. Pharm. Sci. 54,1707 (1965). 139. J. Seydel, 4th Inter. Congr. Chemo. 1965, Washinzon, D. C., Abstr. p. 88. 140. E. Brochmann-Hanssen and A. B. Svendsen, Pharmazie 20, 591 (1965). 141. G. Ritschel-Beurlln, Arzneimittel-Forsch. 15, 1247 7i965). 142. J. Pastor and R. Raimondi, B u l l . Soc. Chim. France 1965, 24.26. 143. H. Grasshof, J. Chromatog. 20, 165 (1965). 144. H. Bieling, W. A l m s , and A.Pogadl, Z. Chem. 5, 376 (1965). 145. J. M. Rice, G. 0. Dudek, and M. Barber, J. Am7 Chem. SOC. 87, 4569 (1965). 146. R. G r l g g , M. V. Sargent and J. A. Knight, Tetrahedron 21, 3441 (1965). 147. R. J. Highet and P. F. Highet, J. Org. Chem. 30, 902 (1965). 148. M. -do, Bull. Chem. SOC. Japan 38, I271 (19bj). 149. B. J. Millard, Tetrahedron ~ e t t e r T l 9 6 5 ,3041. 150. L. Bauer, G. E. Wright, B. A. Milcrut and C. L. B e l l , J. Heterocyclic Chem. 2, 447 (1965). 151. S. Natelson, cline chem. 290 (1965). J. Assoc. Offic. Agr. 152. H. L. Schlesinger, M. J. Pro and C. M. Hof-, chemists 48, -U39 (1965). 153. J. M. A. Lenand S. J. Thomson, Eds., "Activation Analysis", Acad. Press, New York, 1965. 154. M. H. Richmond, Blol. Rev. Cambridge Phllos. Soc. 40, 93 (1965). 155. G. H. Hitch* and J. J. Burchall, Advan. EnzymolTg, 418 (1965). 156. A. C. Schwartz, Z. Allgem. Mikrobiol. 2, 147 (1965). 157. T. B e r t 1 and M. Trocca, Giorn. Microblol. 2, 129 (1963). 158. M. G. Sevag and B. Ashton, Antimicrobial Agents Chemo. I & + 410 , (1965). 159. H. G. Johnson and M. K. Bach, Nature 208, 408 (1965). 160. D. A. Mltchison, B r i t . Med. J. 1(5446), 1333 (1965). 161. F. Trendelenburg, Progress i n Drug Research 7, 196 (1964). 162. A. Albert, "Selective Toxicity", Wiley and Sons, New York, 1965. , 163. M. Barber, Proc. Roy. SOC. Med. 2, R. 2, 990 (1965).

a,

-

-

s,

136 -

Sect.

III - Chemotherapeutics

Chapter 14a.

F l y n n , Ed.

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

Edward F. Elslager Parke, Davis and ComFny, Ann Arbor, Mich.

The g r e a t advances made i n chemotherapy during t h i s century have enabled some measure of control of t h e major k i l l i n g diseases of t h e t r o p i c s . Notwithstanding p a s t progress, man today i s confronted by a catalog of challenging problems, and t h e incentive t o develop novel and improved a n t i p a r a s i t i c agents has not diminished. Adequate drugs a r e s t i l l lacking for t h e treatment of many chronic and d e b i l i t a t i n g d i s eases including leishmaniasis, Chagas' disease, f i l a r i a s i s , schistosomiasis, dmmcchiasis, t r i c h u r i a s i s , and s t r o n g y l o i d i a s i s . Moreover, r e p o r t s of d r u g r e s i s t a n c e have o f t e n followed advances i n chemotherapy l i k e a " f a i t h f u l shadow", and t h i s shadow w i l l no doubt lengthen i n t h e f u t u r e . The philosophy of global eradication of communicable diseases' and t h e evolution of techniques for i t s achievement have added a new dimension t o research i n p a r a s i t e chemotherapy, namely t h e urgent need f o r drugs with protracted a c t i o n . While t h e c l i n i c i a n i n t e r e s t e d i n t r e a t i n g an individual o f t e n has a v a r i e t y of good drugs a t h i s disposal and may f e e l l i t t l e need f o r new agents, t h e public h e a l t h worker, whose aim i s t h e ultimate eradication of a disease from an e n t i r e community, f a c e s problems of a magnitude and type not encountered by t h e c l i n i c i a n and i s frequently unable t o achieve h i s objective of t o t a l coverage with currently a v a i l a b l e drugs. Future progress i n p a r a s i t e chemotherapy w i l l depend mainly on an increased awareness of such c r i t i c a l problems and needs and on appropria t e recognition of biochemical processes within both p a r a s i t e and host. Intensive e f f o r t s t o develop useful agents for t h e treatment of p a r a s i t i c diseases of livestock, poultry, and o t h e r domestic animals have a l s o y i e l d ed a v a r i e t y of promising new substances (see Chapter 14b), and t h e b e n e f i t s derived from i n t e r p l a y between human and veterinary research a r e already apparent. 11.

MALARIA

I n s p i t e of t h e impressive gains made i n global malaria eradication, malaria s t i l l d i s a b l e s more people and imposes a heavier economic burden than any o t h e r disease. Today more than one b i l l i o n people l i v e i n malarious regions: 723 m i l l i o n i n a r e a s where e r a d i c a t i o n programs a r e i n progress and 393 m i l l i o n i n a r e a s where programs have not y e t s t a r t e d O 2 The ready a v a i l a b i l i t y of a v a r i e t y of useful synthetic a n t i m a l a r i a l drugs i n t h e post-World War I1 e r a contributed t o a f e e l i n g t h a t malaria would soon cease t o be a world problem, and i n t e r e s t i n t h e development of

Chap. 14a.

Antipa r as i tic s

Elslager

137 -

new drugs waned. Moreover, only r e c e n t l y has t h e p o t e n t i a l usefulness of A chemotherapy i n a l l phases of malaria e r a d i c a t i o n been f u l l y recognized.' r e a p p r a i s a l indicated: (1)t h e need f o r long-acting drugs i n malaria eradica(2) t h e seriousness of t h e s i t u a t i o n created by t h e possition b i l i t y of widespread r e s i s t a n c e of Plasmodium falciparum t o t h e 4-aminoquinol i n e s and t h e r e s u l t i n g urgent need f o r new types of f a s t - a c t i n g suppressive drugs;'J7 and ( 3 ) t h e importance of developing s a f e and e f f e c t i v e a n t i - r e l a p s e drugs capable of e f f e c t i n g a r a d i c a l cure of p. vivax and p. malariae, p r e f e r ably i n a s i n g l e dose o r a t most i n a three-day regimen.' Several excellent, a u t h o r i t a t i v e reviews on malaria chemotherapy have recently been p u b l i ~ h e d . ' , ~ ~

-

A. Repository Rrugs. The development of repository a n t i m a l a r i a l drugs required t h e synthesis of substances t h a t would prolong t h e r e l e a s e of a c t i v e moieties from depot s i t e s or, l i k e 6,8-dichloro-2-phenyl-a-2-piperidyl-4-quinolinemethanol (SN-10,275), be firmly bound by host t i s s u e s and then slowly released. lo

-

1. Dihydrotriazine and Pyrimethamine S a l t s . The synthesis of cycloguanil (DRP)pamoate (Camolas) ( I)lO and o t h e r dihydrotriazinelo~ll and p y r i methaminel', l2 s a l t s t h a t e x h i b i t remarkable repository a n t i m a l a r i a l properties5J1' has been reported. A s i n g l e intramuscular dose of cycloguanil pamoate has t h e unusual capacity t o p r o t e c t man f o r many months against challenges with susce t i b l e s t r a i n s of Plasmodib ~ i v a x , ~ "P., ~f a~l ~ i p a r u m , ~ ~P., ~ ' malariae,lg and 2. ova1e.l' Although cycloguanil g m o a t e has so f a r noF shown a l i a b i l i t y t o induce rapid r e s i s t a n c e , p a r a s i t e s known t o be r e s i s t a n t t o chlorguanide, DRT, o r pyrimethamine are less susceptible t o it .14,17-1s

NHR

I

I1

-

2. Sulfones and Sulfonamides. The sulfones and sulfonamides (vide i n f r a ) a c t a t a d i f f e r e n t s i t e than DHT o r pyrimethamine, presumably by preventing t h e incorporation of E-aminobenzoic a c i d i n t o f o l i c acid.20 P o t e n t i a l long-acting sulfone and sulfonamide d e r i v a t i v e s were synthesized t o provide substances t h a t , i n combination with cycloguanil pamoate o r r e l a t e d compounds,11,12 might enable a sequential block i n t h e metabolic synthesis of nucleotides and a f f o r d broader repository a c t i o n a g a i n s t drug-resistant l i n e s than e i t h e r drug alone .21 Various 4-acylaminodiphenylsulfones exhibited

138

Sect. 111

-

F l y n n , Ed.

Chemotherapeutics

i.

promising repository a’ction i n mice. Against trophozoite-induced berghei i n f e c t i o n s i n mice, a s i n g l e subcutaneous 100-400 mg./kg. dose of N,N’-diacetyl-4,4’ -diaminodiphenylsulfone (WE) (IIb) almost invariably prevented or strongly suppressed patent i n f e c t i o n s through 6 t o 14 weeks. A single 50 mg./kg. intramuscular dose of DADDS prevented patent g. cynomolgi i n f e c t i o n s i n monkeys f o r 63-268 (average 158) days and g r e a t l y suppressed t h e p a r a s i t e mia f o r many weeks longer. A comparison of DADDS, cycloguanil pamoate, and a 1:l mixture a g a i n s t l i n e s of 2. berghei highly r e s i s t a n t t o e i t h e r D I E or DBT demonstrated t h a t t h e mixture had broader repository a c t i o n against t h e drugr e s i s t a n t l i n e s than e i t h e r d r u g alone.21J22 Further evaluation of t h e DAD%cycloguanil p m o a t e mixture i n connection with t h e prevention and e r a d i c a t i o n of malaria i s i n progress.

-

3 . 4-Aminoquinolines . Several novel 4aminoquinolines have a l s o been t e s t e d f o r therapeutic and repository a c t i v i t y against trophozoite~ * most i n t e r e s t i n g of these, 4,4’-[1,4induced 2. b e r h e i i n m i ~ e . ~ ~ ,The piperazined&l-methylethyleneimino) 1b i s [ 7-chloroquinoline] (III)? prot e c t e d mice against intervening challenge with 2. berghei f o r 8 weeks followi n g a s i n g l e o r a l dose of 500 mg./kg. The compound has a marked a f f i n i t y f o r l i v e r and kidney, and a delay i n t h e r e l e a s e of t h e drug from l i v e r parenchym a l and Kupffer c e l l s i s apparently responsible f o r t h e s t r i k i n g repository e f f e c t s . No r e p o r t s have y e t been published t o i n d i c a t e whether or not t h e drug has s i g n i f i c a n t repository a c t i o n i n o t h e r laboratory animals or i n man. The synthesis of I11 and many r e l a t e d compounds has been reported.25

-

4. Silicone Rubber Implants. S i l a s t i c capsules have been used successfully i n experimental animals f o r t h e sustained r e l e a s e of antimalarial and antischistosomal agents .26

-

B. Suppressive Agents. From t h e dawn of chemotherapy progress has been shackled by t h e appearance of drug r e s i s t a n c e . Sporadic observations of l e s s ened s e n s i t i v i t y of malarial p a r a s i t e s t o quinine, quinacrine, pamaquine, chlorguanide, and pyrimethamine appeared p r i o r t o 1960, but t h e impact of t h i s phenomenon on malaria eradication was considered i n s i g n i f i c a n t .? Recent i n v e s t i g a t i o n s i n d i c a t e t h a t c e r t a i n s t r a i n s of 2. falciparum from South America t h e most widely and Southeast Asia are r e s i s t a n t t o t h e 4aminoquinolines used drugs i n malaria chemotherapy and t h a t some of t h e s e strains a r e a l s o l e s s susceptible t o many, o r i n a f e w instances widely used a n t i m a l a r i a l The possible i m p a c t of these findings on t h e prevention, treatment, and e r a d i c a t i o n of malaria i s obvious and underscores t h e need for new a n t i m a l a r i a l d r u g s . 3, 6-8,27,30

-

-

s,

-

1. Sulfones and Sulfonamides. It has been known for several decades t h a t sulfones and sulfonamides have a n t i m a l a r i a l action, but none has been used extensively i n t h e treatment of human malarias .3,8,21 Recent observations, however, have revived i n t e r e s t i n t h e possible u t i l i t y of t h e s e substances both a s repository (vide supra )21,22 and s u p p r e s ~ i v e ~ ~ -agents. ”5

Certain l i n e s of 2. berghei, p. cynomolgi, and p. allinaceum made r e s i s t a n t t o 4,4’-diaminodiphenylsulfone (diaphenylsulfone, ? DDS7 7 IIa - or t o DIIT or pyrimethamine were s t i l l susceptible t o t h e heterologous drug with only a l o w order of cross-resistance.31 Further, a 1:l mixture of DIEI) and DDS proved

Chap. 14a.

Antiparasitic s

El slager

139 -

highly e f f e c t i v e against t h e parent, DHT-resistant, and DDS-resistant l i n e s of 2. berghei, and t h e r a t e of emergence of r e s i s t a n c e i n t h e parent s t r a i n was s i g n i f i c a n t l y l e s s with t h e mixture than with e i t h e r drug alone.31 In man d a i l y o r a l doses of 25 t o 50 mg. of D B protected 22 of 26 volunteers from i n f e c t i o n by s t r a i n s of r e s i s t a n t 2. falciparum i s o l a t e d i n Southeast Asia.30 Pyrimethamine ( 5 0 mg. d a i l y f o r 3 days) o r sulfadiazine (2.0 g. d a i l y f o r 5 days) given alone did not cure i n f e c t i o n s with t h e m l a y a (Camp.) s t r a i n of

chloroquine-resistant 2. falciparum; these doses of pyrimethamine and s u l f a diazine administered concurrentlgb although not rapidly effe t i v e , cured i n f e c t i o n s i n 5 of 6 volunteers. Sulforthomidine (Fanasi& (IV) given weekl y i n 500 mg. doses alone o r i n combination with 25 mg. of pyrimethamine cleared symptomless asexual parasitemia i n East African schoolchildren i n f e c t e d with pyrimethamine-resistant 2. falciparum. 32 Although t h i s study indicated t h a t sulforthomidine acted t o o slowly f o r use a g a i n s t acute a t t a c k s , 25 semiimmune p a t i e n t s were subsequently t r e a t e d with s i n g l e 1 . 0 g. doses, and a l l but 3 were cured within 3 days.33 Sulforthomidine appears t o have no sporontocidal a c t i o n against 2. falciparum.34 The above findings, together with i n d i c a t i o n s t h a t t h e combined o r a l use of a sulfone or su1fonami.de with pyrimethamine o r chlorguanide may l e a d t o p o t e n t i a t i o n of a n t i m a l a r i a l effects,8,24.t31.t35 pinpoint an area worthy of extensive i n v e s t i g a t i v e e f f o r t s .

Iv

I11

-

2. 4-Aminoquinolines. In therapeutic s t u d i e s a g a i n s t a normal 2. berghei s t r a i n i n mice, I11 had an o r a l CD50 of 1 0 mg./kg. d a i l y x 4.23 A l though chloroquine-resistant s t r a i n s studied t o date have proved uniformly r e s i s t a n t t o other 4 - a m i n o q ~ i n o l i n e s , ~ a chloroquine-resistant s t r a i n of 2. berghei s u r p r i s i n g l y was reported t o be cross-resistant t o III.24

3 . New S t r u c t u r e s . - Several new a c t i v e chemical moieties have been reported. 3-Piperonylsydnone ( V ) was a c t i v e a g a i n s t 2. berahei a t a dose of 1 0 mg./kg." The glyoxal dithiosemicarbazones VIa and b showed high a c t i v i t y a g a i n s t P. pallinaceum i n t h e chick; t h e median e f f e c t i v e doses per 0s were 0.7 rug./&. x 7 and 0.5 mg./kg. x 7, respectively.37 It i s not y e t c l e a r whether these or r e l a t e d compounds will have p r a c t i c a l application.

-

C. Radical Curative Agents. The 8-aminoquinolines are t h e only substances having s u f f i c i e n t a c t i v i t y against secondaq t i s s u e schizonts t o be of p r a c t i c a l value i n achieving r a d i c a l cure of r e l a p s i n g malarias. However, primaquine and c e r t a i n o t h e r 8-aminoquino~inesproduce acute hemolytic anemia

140

Sect. I11

- Chemotherapeutics HC=N-NBCSNHX

a,X=H b, X = C&OCHs

I

HCd-NHCSNBX

V

F l y n n , Ed.

VI

i n persons whose red c e l l s are unusally susceptible t o drug-induced hemolysis. The rapid degradation and elimination of primaquine necessitates d a i l y @&inist r a t i o n , which c o n s t i t u t e s - a f u r t h e r drawback l i m i t i n g i t s use. The discovery that 4- (2-bromo-4,5-dimethoxyphenyl)-1,1,7,7-tetraethyldiethylenetriamine (RC-12) ( V I I ) has a pronounced effect on t h e exo-erythrocytic stages of 2. cathemerium i n canaries3 stimulated an appraisal of the a c t i v i t i e s of RC-12 against 2. cynomolRi i n t h e monkey.38 Although t h e drug per se had l i t t l e promise as a schizonticidal or suppressive drug, it showed s i g n i f i cant promise as a prophylactic or r a d i c a l curative agent and m i g h t f i n d use when: (1)t h e cambination of chloroquine-primaquine i s not e f f e c t i v e i n causal prophylaxis, as appears t o be t h e case where there i s chloroguine resistance; ( 2 ) there i s a need f o r a curative agent which can produce benefits i n l e s s than 14 days; and (3) there a r e fears of enhanced susce t i b i l i t y t o the hematot o x i c i t y of primaquine. Clinical studies are planned. Si:

m

S

VIII

VII

6- [ [4-(Diethylamino)-l-methylbutyl]amino]-5,8-dimethoxyquinaldine (B-505) (VIII)39 a l s o produced r a d i c a l cures of 12. cathemerium infections i n canaries

but the relative t o x i c i t y of t h i s compound precluded c l i n i c a l studies i n man. ?i

-

D. Test Methods. The demonstration of multiple drug resistance i n 2. falciparum (vide supra) spurred research t o develop better experimental models for studying t h i s phenomenon. Strains of 2. bernheihave been induced t o acaquire resistance t o representatives of a l l the major classes of suppressive drugs, and the r e s i s t a n t l i n e s have been employed i n cross-resistance tests t o determine the i n t e r r e l a t i o n s h i p among t h e various chemical types,20,31,40-44 Anopheles ste&ensi has been shown t o be a suitable experimental vector of 2. berghei t o serve as a source of sporozoites and f o r cyclic transmissions and the existence of primary t i s s u e stages of 2. berRhei has been c o n f i r ~ n e d . ~ ~ , ~ ~ The r e l a t i v e ease of infecting the splenectomized gibbon with blood-induced infections of 2.S c i m r u m indicates that t h i s species may be a suitable l a b oratory model f o r studies of fa3.cimm.m malaria. 47

Chap. 14a.

Elslag e r

Antipa r a s itic s

141 -

Leishmania i s responsible f o r millions of smoldering u l c e r s among inhabi t a n t s of endemic regions i n Africa, t h e Americas, and t h e Near and Far East. Antimonials a r e usually e f f e c t i v e i n leishmaniasis, but t h e necessity f o r r e peated (10-30)i n j e c t i o n s and t h e well-known contraindications of t h e s e drugs impose serious r e s t r i c t i o n s on t h e i r widespread use. Inasmuch a s some regression of e a r l y l e s i o n s of dermal leishmaniasis was observed i n malaria p a t i e n t s t r e a t e d with c h l o r g ~ a n i d e ,it ~ ~was of i n t e r e s t t o i n v e s t i g a t e t h e p o t e n t i a l usefulness of t h e repository drug cycloguanil pamoate ( I ) (vide supra). Early r e s u l t s from Costa Rica and Mexico a r e encouraging; a s i n d e intramuscular i n j e c t i o n of cycloguanil pamoake (280-350 mg. base) cured 26 of 30 p a t i e n t s with b r a ~ i l i e n s i s ~and ' 31 of 40 percutaneous and mucocutaneous i n f e c t i o n s of sons with cutaneous leishmaniasis produced by &. nexicana .49

L.

IV. CHAGAS' DISEASE A t l e a s t 7 m i l l i o n persons i n South and Centrql America a r e i n f e c t e d with Trypanosoma cruzi, t h e causative agent of Chagas' disease. In c o n t r a s t t o t h e success attending t h e development of e f f e c t i v e drugs f o r t h e African trypanosomiases, t h e treatment of cruzi i n f e c t i o n s has been generally unsatisfactory. There i s some evidence t h a t n i t r o f u r a n s may be e f f e c t i v e a g a i n s t acute i n f e c t i o n s , but no curative agent has y e t been found.50 The discovery t h a t strong chemotherapeutic i n t e r e s t . 51 The nature modifications of t h e N1-

N1- (3-dimethylaminopropyl )piperazines (IX) e x h i b i t a c t i v i t y against 2. c r u z i i n mice i s of p a r t i c u l a r of t h e R s u b s t i t u e n t i s r e l a t i v e l y non-specific, but (3-dimethylaminopropy1)piperazine moiety d r a s t i c a l l y

X reduce a c t i v i t y . In comparative s t u d i e s with 2. cruzi i n mice, piperamide maleate (X), given o r a l l y i n s i x t e e n 2.5 or 10 m g x doses over 7 days, prolonged t h e median survival t i m e of t r e a t e d mice s i g n i f i c a n t l y longer than mice t r e a t e d with these doses of furaltadone or primaquine.sl However, t h e curat i v e value and t h e s a f e t y of piperamide maleate and r e l a t e d substances52 have y e t t o be established.

v.

IS

The i d e a l antiamebic agent must be capable of e r a d i c a t i n g Ehtamoeba h i s t o l y t i c a from both t h e bowel and from e x t r a - i n t e s t i n a l s i t e s and should b y v i r t u a l l y non-toxic. Agents with sustained, prophylactic antiamebic p r o p e r t i e s a r e a l s o needed. Although many s a t i s f a c t o r y antiamebic agents a r e available, no s i n g l e preparation i s capable of exerting a l l t h e a c t i o n s required. Theref o r e , t h e search f o r new agents continues.=

Sect. I11

142 -

- Chemotherapeutics

F l y n n , Ed.

-

A. 2-Amino-5-nitrothiazoles. Over a decade ago it was reported t h a t 2-acetamido-5-nitrothiazole (aminitrozole ) ( X I ) possessed curative a c t i o n

against i n t e s t i n a l amebiasis i n r a t s and dogs and k i l l e d a d u l t Schistosoma mansoni i n mice a t well-tolerated 0 s e s . ~ 4 More r e c e n t l y a new 2-amino-5n i t r o t h i a z o l e d e r i v a t i v e ( f m b i l h a h , 32, 644-Ba ) ( X I I ) having strong therapeut i c e f f e c t s against amebiasis and schistosomiasis (vide i n f r a ) was discovered.55 !The minimum i n h i b i t o r y concentration of 32,644-& against g. h i s t o l y t i -

XI

XI1

ca i n v i t r o w a s 10 pg./ml.

G emetine

This i s approximately t h e same potency exhibited

or 4,7-phenanthroline-5,6-quinone under comparable conditions. 56

!Phe compound strongly suppresses amebic l i v e r abscess i n hamsters following d a i l y o r a l doses of 60-120 mg./kg. f o r 7 days (chloroquine was e f f e c t i v e a t 100-200 mg./kg. ). I n t e s t i n a l amebic i n f e c t i o n s i n rats and guinea-pigs were markedly reduced following d a i l y o r a l doses of 30-60 mg./kg. f o r 4 and 5 days, respectively .56 Results of preliminary human t r i a l s i n d i c a t e t h a t 32,644-&, administered i n d a i l y doses of 20-25 mg./kg. f o r 5 t o 10 days, i s e f f e c t i v e against both amebic dysentery and amebic l i v e r abscess, although s i d e e f f e c t s including frequent ECG changes and occasional neuropsychic episodes were noted.57 More extensive t r i a l s w i l l be required t o a s s e s s t h e r e l a t i v e s a f e t y and e f f i c a c y of t h e drug compared with o t h e r forms of treatment. Rel a t e d heterocyclic compounds a r e a l s o reported t o have antiamebic and a n t i schistosomal p r o p e r t i e s .58

-

B. 7-Quinolinols. Another new amebicide of p o t e n t i a l i n t e r e s t i s 5chloro-7- 3-diethylaminopropylaminomethyl ) -8-quinolinol (clamoxyquin PAA-3854 CI-433) ( X I I I ) s9 The r e l a t i v e amebicidal concentrations of clamoxyquin hydrochloride and emetine hydrochloride a r e 20-40 pg./ml. and 2.5 pg./ml. respect i v e l y . 6o The e f f e c t s of clamoxyquin hydrochloride and chloroquine against amebic h e p a t i t i s i n hamsters were roughly comparablej clamoxyquin hydrochloride,

.

,

,

OH

c1 XI11

XIV

pamoate, and s a l i c y l a t e w e r e a c t i v e against i n t e s t i n a l amebiasis i n r a t s i n gavage doses ranging from 75 t o 600 mg./kg. d a i l y f o r 4 days." Clamoxyquin hydrochloride and pamoate cured amebic dysentery in do s i n d a i l y doses of 3.13 t o 50 mg./kg. and 12.5 t o 25 mg./kg. respectively.6' Relative t o t h e 8-quinol i n o l s i n c l i n i c a l use t h e clamoxyquin s a l t s were deemed worthy of c l i n i c a l

Chap. 14a.

Antipar as itic s

Elslager

143 -

t r i a l . Clamoxyquin hydrochloride was t o l e r a t e d w e l l i n man following d a i l y 1 5 mg./kg. doses f o r 5-10 days and was highly e f f e c t i v e against various forms of Additional s t u d i e s w i l l be required before the r e l a i n t e s t i n a l amebiasis."' t i v e importance of the clamoxyquin salts i n amebiasis chemotherapy can be assessed

.

C. Anisomycin. - The determination of t h e s t r u c t u r e and stereochemistry of the potent antiprotozoal a n t i b i o t i c anisamycin (XIV)62 may serve t o stbiul a t e f u t u r e research on t h e synthesis and biological properties of r e l a t e d substances.

VI,

SCHISTOSOMIASIS

Nearly 200 m i l l i o n people a r e infected with schistosomiasis, and on a world b a s i s these i n f e c t i o n s a r e ~ p r e a d i n g . ~The degree of prevalence, t h e seriousness of t h e infection, and t h e paucity of adequate drugs make schistosomiasis one of t h e g r e a t e s t challenges i n p a r a s i t e chemotherapy today. Seve r a l promising new agents have been discovered, but t h e i r usefulness i n man has yet t o be adequately evaluated. Therefore, one s t i l l must r e l y on the a n t i monials with some assistance from lucanthone hydrochloride (ma) against Schistosoma haematobium and c e r t a i n s t r a i n s of S. mansoni. Research on schistosomiasis6Y'64 and other trematode infections63 has r e c e n t l y been reviewed.

-

A. 2-Amino-5-nitrothiazoles. To date 1-(5-nitro-2-thiazolyl)-2-imidazolidinone ( A m b i l h d 32 644-E!a) ( X I I ) has been evaluated more extensively i n ~ c h i ~ t o s a m i a s i than s ~ ~ i n ~amebiasis ~ ~ ~ ~(vide ~ ~ supra). " ~ ~ ~ When ~ adult S . mansoni were exposed t o 32,644-Ba i n v i t r o for prolonged periods (100 hr.), oviposition was i n h i b i t e d a t concentrations of 1 pg./ml. and worms were k i l l e d a t 100 p g . / m l . 5 5 ~ 6 5 The destruction of the v i t e l l o g e n i c gland i n the female coincides with a reduction i n body-length i n both male and female worms and i n t h e s i z e of t h e ovary i n t h e female.57 The drug.was highly a c t i v e against S . manSOn155,65 and 2. j a ~ o n i c u mi ~ n ~mice i n o r a l doses of 100 mg./kg. d a i l y f o r 10 days and had a strong prophylactic and chemotherapeutic e f f e c t on experimental 2. mansoni infections i n monkeys .57 Temporary, r e v e r s i b l e impairment of sljermatogenesis occurred i n mice- the drug apparently a c t s d i r e c t l y on t h e germinal epithelium of t h e t e s t e s . 66 Results of preliminary c l i n i c a l t r i a l s i n man i n d i c a t e t h a t 72 644-h has considerable e f f i c a c y against 2. h a e m a t ~ b i u m ~ ~and , " ~2. mansoni5 j Q 8 i n o r a l doses of 20-40 mg./kg. d a i l y f o r 5-15 days; S . japonicum i n f e c t i o n s appear t o be more refractory.57 Three types of side e f f e c t s were encountered which merit s p e c i a l a t t e n t i o n : (1)frequent ECG changes, involving mainly t h e T waves; ( 2 ) neuropsychic e f f e c t s , ranging from severe headaches t o convulsive a t t a c k s with infrequent hallucinations; (3) t r a n s i t o r y impairment of spermatogenesis .57 The ultimate place of 32,&4-& i n schistosomiasis chemotherapy w i l l depend l a r g e l y on t h e incidence and s e v e r i t y of these side e f f e c t s a t e f f e c t i v e dose l e v e l s when compared with t h e side e f f e c t s encountered i n t h e c l a s s i c a l treatments. The drug has a l s o been reported t o be e f f e c t i v e i n t h e treatment of Dracunculus medinensis (guinea worm) i n f e s t a t i o n i n humans .69

144

Sect. III

-

Chemotherapeutics

F l y n n , Ed.

-

B. Thiaxanthenones. Incubation of lucanthone (XVa) with sclerotiorum produces t h e a c i d - l a b i l e hydroxymethyl d e r i v a t i v e Surprisingly, t h e (Xvb) together with smaller amounts of t h e aldehyde five-day ED50 f o r lucanthone and hycanthone a g a i n s t S . mansoni i n hamsters were 8.0 mg./kg. and 0.78 mg./kg., respectively.70 The unusual potency of hycanthone l e d t o t h e hypothesis t h a t it might indeed be t h e e l u s i v e a c t i v e metabol i t e of lucanthone. The a c i d s e n s i t i v i t y of Xvb could explain why it had

escaped detection by previous i n v e s t i g a t o r s . Accordingly, monkeys were medicated with lucanthone, t h e w i n e s were c a r e f u l l y e x t r a c t e d avoiding a c i d treatment, and t h e e x t r a c t s were examined by thin-layer chromatography. In agreement with e a r l i e r work,71 t h e sulfoxide (XVd) was t h e major metabolite, and only t r a c e s of XVb w e r e detected. When t h e urine e x t r a c t s were incubated with glucuronidase p r i o r t o chromatography, however, hycanthone appeared as a major component.7o Idthough t h e sulfone XVe i s reported t o be t h e major urinary metabolite i n hycanthone was a l s o found i n mouse urine a f t e r glucuronidase incubation but t o a much l e s s e r extent t h a n i n t h e monkey.7o The results of t h e s e elegant s t u d i e s support t h e hypothesis t h a t hycanthone i s an important a c t i v e metabolite of lucanthone; it now becomes apparent why a 2-toluidine function i s an absolute requirement f o r a c t i v i t y among all lucanthone r e l a t i v e s . Other s t u d i e s u t i l i z i n g s u b t i l i s suggest t h a t t h e major i n vivo a c t i o n of lucanthone i s t o complex with DNA, thereby blocki n g DNA-dependent RNA synthesis .73 Various o t h e r schistosomicides r e l a t e d t o hycanthone and t h e aldehyde XVc have been prepared.74 It has a l s o been disclosed t h a t lucanthone N-oxide (XVI) i s considerably l e s s t o x i c than lucanthone .75

-

Tris (paminophenyl )carbonium Salts. Tris (paminopheny1)carbonium C. (TAC) pamoate (XVII)76 and c e r t a i n antimonials have broad antischistosome a c t i v i t y i n experimental animalsT7 and i n man.78 However, both groups have important l i m i t a t i o n s . Thus t h e slow-acting TAC pamoate must be administered for s e v e r a l weeks t o achieve a strong degree of e f f e c t while t h e f a s t - a c t i n g antimonials have a r e l a t i v e l y narrow therapeutic index.

In a recent study of t h e j o i n t a c t i o n of TAC s a l t s and t a r t a r emetic a marked degree of synergism was observed against S. mansoni i n v i t r o and i n mice, Moreover, n e i t h e r drug had p a r t i c u l a r l y a f t e r pretreatment with TAC pamoate?” an appreciable a f f e c t on t h e gross t o x i c i t y of t h e other f o r normal mice. An encouraging degree of j o i n t antischistosomal a c t i o n by TAC pamoate and t a r t a r

Chap. 14a.

Antiparasitics

-OOC

.OH

145

Elslager

H(

XVIII

XVII

emetic was also observed in monkeys. The therapeutic advantage of such a combination hinges largely upon whether o r not the co-administration of TAC pamoate produces higher o r more sustained antimony levels in the blood or tissues. A subsequent study utilizing 124Sb-labeled tartar emetic demonstrated that co-administration or pretreatment with TAC pamoate had no significant effect on the disposition of antimony in mice o r monkeys." Available evidence suggests that the mode of action of TAC pamoate and the trivalent antimonials is dissimilar. Briefly, the antimonials interfere with schistosome glycolysis by inhibiting phosphofructokinase, which catalyzes the reaction: fiuctose-6-phosphate

f

ATP

F'ructose-1,6-diphosphate

+ ADP

Recent studies with 14C-labeled glucose indicate that tartar emetic blocks both the Ehbden-Meyerhof and the hexose-monophosphate pathways of glycolysis Organic antimonials have also been reported to inhibit schistosome glutamicpyruvic transaminase

.

.

The TAC salts apparently act by an entirely different mechanism.e2 In mice subcurative doses of TAC chloride have no demonstrable effect on the carbohydrate and protein metabolism of the worms but produce a localized paralysis of the acetabulum, pharynx, and oral sucker which histochemically has been correlated with a pronounced decrease of cholinesterase activity in the central ganglia. This paralysis is completely reversed in vitro by the cholinergic blocking agent mecamylamine, suggesting an accumulation of acetylcholine in these structures. Simultaneously, glycogen depletion is observed in the papillae located below the cuticle of the worm. Either one or a combination of these changes could account for the antischistosomal effects observed.82

-

D. Cucurbitine. For several years reports emanating from mainland China have claimed that the dried, ripe seeds of the pmpkin varieties Cucurbita pep0 L. and C. moschata Duch. are effective in the treatment of schistosomiasis. The amino acid cucurbitine (XVIII) has recently been isolated from C. moschata Duch." Cucurbitine reportedly inhibits the growth of immature 2. japonicum, produces degenerative changes in the genital organs of the worms resulting in depressed egg-producing capacity, and is virtually nontoxic.s3 It would appear worthwhile to confirm the structure of cucurbitine and study its effects on 2. haematobium and 2. mansoni. VII.

CLONORCHIASIS

Clonorchiasis is caused by the human liver fluke Clonorchis sinensis (syn.

Sect. 111 - Chemotherapeutics

146 -

F l y n n , Ed.

Opisthorchis s i n e n s i s ) and i s widely d i s t r i b u t e d i n t h e Far East, especially i n Indochina, Central and South China, Formosa, Korea, and Japan. Approximatel y 19 million people are a f f l i c t e d with t h i s disease, y e t no r e l i a b l e treatment i s known. Therefore, preliminary r e p o r t s t h a t t h e f a s c i o l i c i d e a,a,a,a',@,a'hexachloro-2-xylene (XIX) i s highly e f f e c t i v e against C. s i n e n s i s i n experimen-

t a l animalss4"' and i n a t well-tolerated doses a r e most encouraging. Expanded human t r i a l s a r e indicated.

The world-wide incidence of i n t e s t i n a l helminthic i n f e c t i o n s i n man approaches t h e staggering f i g u r e of two b i l l i o n . * The prevalence of these i n f e c t i o n s and t h e i r complex and far-reaching e f f e c t s upon body function cause them t o rank among t h e most important disease problems of man. F i r s t reports" on tetramisole ( X X ) , a potent new anthelmintic with broad a c t i o n against nematodes, a r e highly encouraging. The drug i s reported t o be e f f e c t i v e against Ascaris lumbricoides and Enterobius vermicularis i n man" and against a v a r i e t y of adult and immature g a s t r o i n t e s t i n a l and pulmonary nematodes i n laboratory animals, poultry, and livestock. ,l' s2 Although devoid

xx

XXI

XXII

of antihistaminic, anticholinergic, adrenolytic o r other c l a s s i c a l pharmacol o g i c a l properties, tetramisole e x e r t s a rapid paralysing a c t i o n on a variety of nematodes i n v i t r o a t low concentrations.

''

The evolution of tetramisole points once again t o t h e importance of strong biochemical support i n drug development. Thus, t h e observation t h a t t h i a z o t h i en01 (XXI)'~ was a c t i v e against nematodes during a routine screening program i n chickens stimulated a search for a c t i v e metabolites of XXI and l e d t o t h e isol a t i o n and i d e n t i f i c a t i o n of XXII a s t h e major transformation product i n t h e feces. The c y c l i c metabolite XXII was a c t i v e a t s i g n i f i c a n t l y lower dose l e v e l s t h a t t h e parent compound (XXIX and these results prompted the s n t h e s i s ?' . and b i o l o g i c a l evaluation of an extensive s e r i e s of r e l a t e d compounds Tetramisole emerged a s t h e most promising substance f o r expanded s t u d i e s .

Antipa rasitics

Chap. 14a.

147 -

Els lage r

IX. BIBLIOGRAPHY

&I.

m., a.s.

(1) E. H. Hinman, 2. Tro Med. 2, 125 (1966). (2) Chronicle, 3 3 d a (3) L. J. Bruce-Chwatt, Trans. Trop. g . 2, 105 (1965). (4) E. F. Elslager and P. E. Thompson, Pharmacol.,& 193 (1962). ( 5 ) P. E. Thompson, B. J. Olszewski, E. F. Elslager, and D. F. Worth, &.' J& 481 (1963 -J. Tro Med. (eRexution, JJ, 361 (1964).

.

&. &.

m.,

m.,

w.,

1965,

S. Goodman and A. Gilman, Ed., t h e Mamillan Co., New York, N.Y.,

1087-1117.

ed., L. pp.

(10)E. F. Elslager and P. E. Thompson, Abstracts, 9th National Eledicinal Chemistry Symposium of t h e American Chemical Society, Minneapolis, Minn., June 1964, p. 6A. (11)E. F. Elslager and D. F. Worth, U. S. Patents 3,223,705 (1965) and 3,248 422 (1966). (12) E. F. Elslager and D. F. Worth, U. S. Patents 3,161,641 (1964); 3,236,849 (1966); 3,244,716 (1966)j and 3,248,422 (1966). (13) G. R. Coatney, P. G. Contacos, and J. S. Lunn, J. Trop. &d. &g.,

&I.

-

13 383 (1964)")(14) R. D. Powell, R. L. DeGowin, and R. B. Eppes, M.,&, 913 (1965). (15) P. G. Contacos, G. R. Coatney, J. S. Lunn, and J. W. Kilpatrick,

L;zI 386

u.,

(u@+)*

(16) 1. A. McGregor, K. Williams, G. H.'Walker, and A. K. Rahman, Brit. E .

2 . , 3 695 (1966).

- m.,

(17 P. E. Thompson, B. Olszewski, and J. A. Waitz, &. 2. m.Med. 343 (1%5)* (18)W. Chin, J. S. Lunn, J. Buxbaum, and P. G. Contacos, w., &I 922

&

(1965)

(20) A.

(19) P. G.

(1965

Contacos, G. R. Coatney, J. S. Lunn, and W. Chin,

a

w.,

925

Bishop, ParasitolopV, 53, 10 (1963); 407 (1965). (21) E. F. Elslager and D. F. S r t h , Nature, 206 630 (1965). (22) P. E. Thompson, B. Olszewski, and J. A. &z, &. 2.

r14 U1

343 (1965)-

&. Med. a.,

(23) J. Schneider, M. Bouvry, and J. Le Quellec, Abstracts, I n t e r n a t i o n a l Colloquium on , P a berghei, I n s t i t u t e of Tropical Medicine "Prince Leopold," Antwerp, Belgium, December 1964. (24) J. Hill i n "mperimental Chemotherapy," V o l N, R. J. Schnitzer and F. Hawking, Ed., Academic Press, New York, N.Y., 1966, p. 448. (25) Rhone-Poulenc S. A., B r i t i s h Patent 973,903 (1964); Eire Patents 461/62, 1097/62, 591/63, and 620/63 (1963). (26) K. G. Powers, J. Parasitol., 2, 53 (1965). (27) C. G. H u f f , 'Itrop. m e , & 339 (1965). (28) R. L. De Gowin and R. D. Powell, 519 (1965). (29) R. L. De Gowin and R. D. Powell, J. &&.B. 851 (30) 3. % (131, 38 ( i s ) . (31) P. E. Thompson, A. Bayles, B. Olszewski, and J. A. Waitz 198 (1965).

&.-z.

m.

m,

- a*,*,

@.

w.,

s., a,

, Am-

148

Sect. IU

- Chemotherapeutics

'Flynn, Ed.

(35) P. C. Basu, N. N. Singh, and N. Singh, B u l l . World Health Qrgan., & 699 -_ ( .1-964.). (36) W. H. Nyberg and C. C. Cheng, 2. B., 531 (1965). (37) P. A. Barrett, E. Beveridge, P. L. Bradley, C. G. D. Brown, S. R. M. Bushby, M. L. Clarke, R. A. Neal, R. Smith, and J. K. H. Wilde, Nature, 1340 (1965). (38) L. H. Schmidt, R. N. Rossan, R. Fradkin, J. Woods, W. Schulemann, and L. Kratz, presented a t t h e 14th Annual Meeting of t h e American Society of Tropical Medicine and Hygiene, New Orleans, L a . , Nov 1965. (39) F. Schonhofer and W. Schulemann, B r i t i s h Patent 970,949 (1964). (40) R. L. Jacobs, 2. Parasitol., 481 (1965). (41) W. Peters, 9. Parasitol., 80, 90, 97 (1%5)(42) W. Peters, Nature, (43) P. E. Thompson, B. %ze:ski, A. Bayles, and J. A. Waitz, 2. P a r a s i t o l . , /s 51 (21, 54 (1965). (44) P. E. Thompson, A. Bayles, B. Olszewski, and J. A. Waitz, Science, 1240 (1965). (45) M. Yoeli and H. Most, &. 2. Trop. €&., 700 (1965). (46) M. Yoeli, J. Vanderberg, R. Nawrot, and H. Most, & 927 (1965). (47) R. A. Ward, J. H. Morris, D. J. Gould, A. T. C. B u r k e , and F. C. Cadigan, Jr., Science, 1604 (1965 ) (48) A. Pel& Chavarria, E. Kotcher, and C. Lizano, 2. Assoc., 1142 (1965). (49) F. Beltrh, F. Biagi, and S. Gonza'lez, presented a t t h e 14th Annual hketing of t h e American Society of Tropical Medicine and Hygiene, New Orleans, La., NOV 1965. H&., 211 (1964). (50) F. Hawking, 2. Trop. (51) A. S. Tomcufcik, R. I. Hewitt, P. F. Fabio, A. M. Hoffman, and J. Entwistle, Nature, 2ez, 605 (1965). , (52) American Cyanamid Co., South African Patent 3833/63 (1964) and Belgian Fatent 654,017 (1965). (53) G. Woolfe i n "Drug Research," Vol VIII, E. Jucker, Ed., Birkhauser AG, Basel, Switzerland, 1965, pp. 13-52. (54) A. C. Cuckler, A. B. Kupferberg, and N. Millman, Antibiot. Chemother540 (1955). C. R. Lambert, M. Wilhelm, H. S t r i e b e l , F. Kradolfer, and P. Schmidt, Ek e r i e n t i a , 452 (1964). %adolfer and R. Jarumilinta, Trop. E.Parasitol., 210

@.

&

a

.

e.

s. & u., &. w.

a

2

&.

(19651* (57) "Th6rapeutique Nouvelle de l a Bilharzioae e t de 1'Amibiase, Symposium de Lisbonne, June, 1965, Acta WOE., SuppL. 9, 1-314 (1966). ( 5 8 ) Ciba Ltd., South African Patents 3781/64 (1965) and 4336/65 (1966); Neth. Patents 6,505,225 and 6,505,226 (1965). (59) J. H. Burckhalter, W. S. Brinigar, and P. E. Thompson, 2. & 4070 (1961). I'

a. e., Meisenhelder, J. e-

(60) P. E. Thompson, A. Bayles, P. McClay, and J. E. sitol., 817 (1965). (61)R. Hugonot, E. Granotier, and J. P. Farges, Therapie, & 329 (1965). (62) J. J. Beereboom, K. Butler, F. C. Pennington, and I. A. Solomons, 2. B., 2334 (1965).

a-

Chap. 14a.

Antiparas iti cs

Elslager

149 -

s., &,

(63) G. Uinrmler, 2. Tropenmed. Parasitol., 95, 164, 337 (1964). (64) World Health Organ. Tech. Rep. 317. (65) C. R. Lambert, Trop. Parasitol., 292 (1964). (66) C. R. Lambert, V. S. P. S i n a r i , and J. Tripod," Acta T r o ~ . , & , 155 (1965). (67) C. R. Lambert and F. S. I& C r u z F e r r e i r a , B u l l . World Health Organ.,& 73 (1965)(68) P. Jordan, Brit. w .z., A 276 (1966). (69) G. Raffier, Acta T r o ~ . ,.& 350 (1965).

a., s.

(70) D. Rosi, G. Peruzzotti, E. W. Dennis, D. A. Berberian, H. Freele, and

S. Archer, Nature,%

(71) R. Strufe,

1006 (1965).

w. Chem.

%.--.

Abhandl. Forschungsstaetten Farbenfabriken Bayer, & 337 (1963). (72).Gijnnert, B u l l . World Health O r g a n . , a 702 (1961). (73) I. B. Weinstein, R. Chernoff, I. Finkelstein, and E. Hirschberg, Pharmacol. 1 297 (1965). m e r h 'n4g Drug Inc., South African Patent 4081/64 (1965). (75) Farbenfab. Bayer A. G., Belgian Patent 663,790 (1965). (76) F. W. Short and E. F. Elslager, U. S. Patent 3,218,339 (1965). (77) E. F. Elslager, F. W. Short, D. F. Worth, J. E. Meisenhelder, H. Najarian, and P. E. Thompson, Nature, 628 (1961). (78) J. R. da Silva, A. Prata, C. A. Argento, H. American0 do B r a s i l , and L. F. Ferreira, Hospital (Rio de Janeiro), & 25 (1964). (79) P. E. Thompson, J. E. Meisenhelder, A. K. Moore, and J. A. Waitz, B u l l . World Health Organ., 2 517 (1965). (80) J. A. Waitz, R. E. Ober, J. E. Meisenhelder, and P. E. Thompson,

s.

z,

w.,

81) J. A. Waitz, Life Sci., 3 377 (1964). (82) E. L. S c h i l l e r and E. Buding, 2. P a r a s i t o l . Supplement, 51, 43 (1965). (83) J. H. Zhou, F. Y. Fu, and H. P. Lei i n "Proceedings of t h n e c o n d International Pharmacological Meeting, Prague, 20-23 August, 1963," Vol V I I , K. K. Chen, B. Mukerji, and L. Volicer, Ed., t h e Macmillan Co., New York, N. Y., 1965, p. 25. N. N. Plotnikov and L. I. Sinovich, Meed. Parazitol. & Parazitarn. Bole301 (1964); Diseases g., l m ) . 9 6 5 % . Yokogawa, H. Koyama, H. Yoshimura, and C. S. Tsai, J. Chiba Med. SOC., & 286 (1965). ( 8 6 ) H. L. Chung, Chinese Med. J., & 232 (1965). (87) J. Liu, (19657. ( 8 8 ) N. N. Plotnikov, No N. Ozeretskovskaya, V. K. Karnaukhov, N. S. Zalnova, G. M. Faibusovich, G. I. Kukhta, and M. I. Alekseeva, Med. Parazitol. i Egmzitarn. Bolezni, 676 (1964); IIl.op. Diseases B u l l . , & 32771965). Tv. N. mozdov, Diseases & 59 (1966). (90) M. Yokogawa, M. Tsuji, K. Araki, T. Nomoto, M. mtsumoto, M. Koyama, R. Yoshiba, S. Fukuchi, A. Hiratsuka, and M. Kitamura, Japan. 3. P a r a s i t o l . , & 526 (1965)(91) D. Thienpont, 0. F. J. Vanparijs, A. H. M. Raeymaekers, J. Vandenberk, P. A. Demoen, F. T. N. Allewijn, R. P. H. Marsboom, C. J. E. Niemegeers, K. H. L. Schellekens, and P. A. J. Janssen, Nature, 3 & 1084 (1966). (9) J. K. Walley, Vet. Record, J&, 4 0 6 m ) . (93) Janssen &arm., N. V., Belgian Patent 653,087 (1965). (94)Janssen Pharm., N. V., Neth. Patent 6,505,806 (1965).

(a)

-

m.

=.-

a

J.

us,

m.

u.,

136 -

Sect.

III - Chemotherapeutics

Chapter 14a.

F l y n n , Ed.

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

Edward F. Elslager Parke, Davis and ComFny, Ann Arbor, Mich.

The g r e a t advances made i n chemotherapy during t h i s century have enabled some measure of control of t h e major k i l l i n g diseases of t h e t r o p i c s . Notwithstanding p a s t progress, man today i s confronted by a catalog of challenging problems, and t h e incentive t o develop novel and improved a n t i p a r a s i t i c agents has not diminished. Adequate drugs a r e s t i l l lacking for t h e treatment of many chronic and d e b i l i t a t i n g d i s eases including leishmaniasis, Chagas' disease, f i l a r i a s i s , schistosomiasis, dmmcchiasis, t r i c h u r i a s i s , and s t r o n g y l o i d i a s i s . Moreover, r e p o r t s of d r u g r e s i s t a n c e have o f t e n followed advances i n chemotherapy l i k e a " f a i t h f u l shadow", and t h i s shadow w i l l no doubt lengthen i n t h e f u t u r e . The philosophy of global eradication of communicable diseases' and t h e evolution of techniques for i t s achievement have added a new dimension t o research i n p a r a s i t e chemotherapy, namely t h e urgent need f o r drugs with protracted a c t i o n . While t h e c l i n i c i a n i n t e r e s t e d i n t r e a t i n g an individual o f t e n has a v a r i e t y of good drugs a t h i s disposal and may f e e l l i t t l e need f o r new agents, t h e public h e a l t h worker, whose aim i s t h e ultimate eradication of a disease from an e n t i r e community, f a c e s problems of a magnitude and type not encountered by t h e c l i n i c i a n and i s frequently unable t o achieve h i s objective of t o t a l coverage with currently a v a i l a b l e drugs. Future progress i n p a r a s i t e chemotherapy w i l l depend mainly on an increased awareness of such c r i t i c a l problems and needs and on appropria t e recognition of biochemical processes within both p a r a s i t e and host. Intensive e f f o r t s t o develop useful agents for t h e treatment of p a r a s i t i c diseases of livestock, poultry, and o t h e r domestic animals have a l s o y i e l d ed a v a r i e t y of promising new substances (see Chapter 14b), and t h e b e n e f i t s derived from i n t e r p l a y between human and veterinary research a r e already apparent. 11.

MALARIA

I n s p i t e of t h e impressive gains made i n global malaria eradication, malaria s t i l l d i s a b l e s more people and imposes a heavier economic burden than any o t h e r disease. Today more than one b i l l i o n people l i v e i n malarious regions: 723 m i l l i o n i n a r e a s where e r a d i c a t i o n programs a r e i n progress and 393 m i l l i o n i n a r e a s where programs have not y e t s t a r t e d O 2 The ready a v a i l a b i l i t y of a v a r i e t y of useful synthetic a n t i m a l a r i a l drugs i n t h e post-World War I1 e r a contributed t o a f e e l i n g t h a t malaria would soon cease t o be a world problem, and i n t e r e s t i n t h e development of

Chap. 14a.

Antipa r as i tic s

Elslager

137 -

new drugs waned. Moreover, only r e c e n t l y has t h e p o t e n t i a l usefulness of A chemotherapy i n a l l phases of malaria e r a d i c a t i o n been f u l l y recognized.' r e a p p r a i s a l indicated: (1)t h e need f o r long-acting drugs i n malaria eradica(2) t h e seriousness of t h e s i t u a t i o n created by t h e possition b i l i t y of widespread r e s i s t a n c e of Plasmodium falciparum t o t h e 4-aminoquinol i n e s and t h e r e s u l t i n g urgent need f o r new types of f a s t - a c t i n g suppressive drugs;'J7 and ( 3 ) t h e importance of developing s a f e and e f f e c t i v e a n t i - r e l a p s e drugs capable of e f f e c t i n g a r a d i c a l cure of p. vivax and p. malariae, p r e f e r ably i n a s i n g l e dose o r a t most i n a three-day regimen.' Several excellent, a u t h o r i t a t i v e reviews on malaria chemotherapy have recently been p u b l i ~ h e d . ' , ~ ~

-

A. Repository Rrugs. The development of repository a n t i m a l a r i a l drugs required t h e synthesis of substances t h a t would prolong t h e r e l e a s e of a c t i v e moieties from depot s i t e s or, l i k e 6,8-dichloro-2-phenyl-a-2-piperidyl-4-quinolinemethanol (SN-10,275), be firmly bound by host t i s s u e s and then slowly released. lo

-

1. Dihydrotriazine and Pyrimethamine S a l t s . The synthesis of cycloguanil (DRP)pamoate (Camolas) ( I)lO and o t h e r dihydrotriazinelo~ll and p y r i methaminel', l2 s a l t s t h a t e x h i b i t remarkable repository a n t i m a l a r i a l properties5J1' has been reported. A s i n g l e intramuscular dose of cycloguanil pamoate has t h e unusual capacity t o p r o t e c t man f o r many months against challenges with susce t i b l e s t r a i n s of Plasmodib ~ i v a x , ~ "P., ~f a~l ~ i p a r u m , ~ ~P., ~ ' malariae,lg and 2. ova1e.l' Although cycloguanil g m o a t e has so f a r noF shown a l i a b i l i t y t o induce rapid r e s i s t a n c e , p a r a s i t e s known t o be r e s i s t a n t t o chlorguanide, DRT, o r pyrimethamine are less susceptible t o it .14,17-1s

NHR

I

I1

-

2. Sulfones and Sulfonamides. The sulfones and sulfonamides (vide i n f r a ) a c t a t a d i f f e r e n t s i t e than DHT o r pyrimethamine, presumably by preventing t h e incorporation of E-aminobenzoic a c i d i n t o f o l i c acid.20 P o t e n t i a l long-acting sulfone and sulfonamide d e r i v a t i v e s were synthesized t o provide substances t h a t , i n combination with cycloguanil pamoate o r r e l a t e d compounds,11,12 might enable a sequential block i n t h e metabolic synthesis of nucleotides and a f f o r d broader repository a c t i o n a g a i n s t drug-resistant l i n e s than e i t h e r drug alone .21 Various 4-acylaminodiphenylsulfones exhibited

138

Sect. 111

-

F l y n n , Ed.

Chemotherapeutics

i.

promising repository a’ction i n mice. Against trophozoite-induced berghei i n f e c t i o n s i n mice, a s i n g l e subcutaneous 100-400 mg./kg. dose of N,N’-diacetyl-4,4’ -diaminodiphenylsulfone (WE) (IIb) almost invariably prevented or strongly suppressed patent i n f e c t i o n s through 6 t o 14 weeks. A single 50 mg./kg. intramuscular dose of DADDS prevented patent g. cynomolgi i n f e c t i o n s i n monkeys f o r 63-268 (average 158) days and g r e a t l y suppressed t h e p a r a s i t e mia f o r many weeks longer. A comparison of DADDS, cycloguanil pamoate, and a 1:l mixture a g a i n s t l i n e s of 2. berghei highly r e s i s t a n t t o e i t h e r D I E or DBT demonstrated t h a t t h e mixture had broader repository a c t i o n against t h e drugr e s i s t a n t l i n e s than e i t h e r d r u g alone.21J22 Further evaluation of t h e DAD%cycloguanil p m o a t e mixture i n connection with t h e prevention and e r a d i c a t i o n of malaria i s i n progress.

-

3 . 4-Aminoquinolines . Several novel 4aminoquinolines have a l s o been t e s t e d f o r therapeutic and repository a c t i v i t y against trophozoite~ * most i n t e r e s t i n g of these, 4,4’-[1,4induced 2. b e r h e i i n m i ~ e . ~ ~ ,The piperazined&l-methylethyleneimino) 1b i s [ 7-chloroquinoline] (III)? prot e c t e d mice against intervening challenge with 2. berghei f o r 8 weeks followi n g a s i n g l e o r a l dose of 500 mg./kg. The compound has a marked a f f i n i t y f o r l i v e r and kidney, and a delay i n t h e r e l e a s e of t h e drug from l i v e r parenchym a l and Kupffer c e l l s i s apparently responsible f o r t h e s t r i k i n g repository e f f e c t s . No r e p o r t s have y e t been published t o i n d i c a t e whether or not t h e drug has s i g n i f i c a n t repository a c t i o n i n o t h e r laboratory animals or i n man. The synthesis of I11 and many r e l a t e d compounds has been reported.25

-

4. Silicone Rubber Implants. S i l a s t i c capsules have been used successfully i n experimental animals f o r t h e sustained r e l e a s e of antimalarial and antischistosomal agents .26

-

B. Suppressive Agents. From t h e dawn of chemotherapy progress has been shackled by t h e appearance of drug r e s i s t a n c e . Sporadic observations of l e s s ened s e n s i t i v i t y of malarial p a r a s i t e s t o quinine, quinacrine, pamaquine, chlorguanide, and pyrimethamine appeared p r i o r t o 1960, but t h e impact of t h i s phenomenon on malaria eradication was considered i n s i g n i f i c a n t .? Recent i n v e s t i g a t i o n s i n d i c a t e t h a t c e r t a i n s t r a i n s of 2. falciparum from South America t h e most widely and Southeast Asia are r e s i s t a n t t o t h e 4aminoquinolines used drugs i n malaria chemotherapy and t h a t some of t h e s e strains a r e a l s o l e s s susceptible t o many, o r i n a f e w instances widely used a n t i m a l a r i a l The possible i m p a c t of these findings on t h e prevention, treatment, and e r a d i c a t i o n of malaria i s obvious and underscores t h e need for new a n t i m a l a r i a l d r u g s . 3, 6-8,27,30

-

-

s,

-

1. Sulfones and Sulfonamides. It has been known for several decades t h a t sulfones and sulfonamides have a n t i m a l a r i a l action, but none has been used extensively i n t h e treatment of human malarias .3,8,21 Recent observations, however, have revived i n t e r e s t i n t h e possible u t i l i t y of t h e s e substances both a s repository (vide supra )21,22 and s u p p r e s ~ i v e ~ ~ -agents. ”5

Certain l i n e s of 2. berghei, p. cynomolgi, and p. allinaceum made r e s i s t a n t t o 4,4’-diaminodiphenylsulfone (diaphenylsulfone, ? DDS7 7 IIa - or t o DIIT or pyrimethamine were s t i l l susceptible t o t h e heterologous drug with only a l o w order of cross-resistance.31 Further, a 1:l mixture of DIEI) and DDS proved

Chap. 14a.

Antiparasitic s

El slager

139 -

highly e f f e c t i v e against t h e parent, DHT-resistant, and DDS-resistant l i n e s of 2. berghei, and t h e r a t e of emergence of r e s i s t a n c e i n t h e parent s t r a i n was s i g n i f i c a n t l y l e s s with t h e mixture than with e i t h e r drug alone.31 In man d a i l y o r a l doses of 25 t o 50 mg. of D B protected 22 of 26 volunteers from i n f e c t i o n by s t r a i n s of r e s i s t a n t 2. falciparum i s o l a t e d i n Southeast Asia.30 Pyrimethamine ( 5 0 mg. d a i l y f o r 3 days) o r sulfadiazine (2.0 g. d a i l y f o r 5 days) given alone did not cure i n f e c t i o n s with t h e m l a y a (Camp.) s t r a i n of

chloroquine-resistant 2. falciparum; these doses of pyrimethamine and s u l f a diazine administered concurrentlgb although not rapidly effe t i v e , cured i n f e c t i o n s i n 5 of 6 volunteers. Sulforthomidine (Fanasi& (IV) given weekl y i n 500 mg. doses alone o r i n combination with 25 mg. of pyrimethamine cleared symptomless asexual parasitemia i n East African schoolchildren i n f e c t e d with pyrimethamine-resistant 2. falciparum. 32 Although t h i s study indicated t h a t sulforthomidine acted t o o slowly f o r use a g a i n s t acute a t t a c k s , 25 semiimmune p a t i e n t s were subsequently t r e a t e d with s i n g l e 1 . 0 g. doses, and a l l but 3 were cured within 3 days.33 Sulforthomidine appears t o have no sporontocidal a c t i o n against 2. falciparum.34 The above findings, together with i n d i c a t i o n s t h a t t h e combined o r a l use of a sulfone or su1fonami.de with pyrimethamine o r chlorguanide may l e a d t o p o t e n t i a t i o n of a n t i m a l a r i a l effects,8,24.t31.t35 pinpoint an area worthy of extensive i n v e s t i g a t i v e e f f o r t s .

Iv

I11

-

2. 4-Aminoquinolines. In therapeutic s t u d i e s a g a i n s t a normal 2. berghei s t r a i n i n mice, I11 had an o r a l CD50 of 1 0 mg./kg. d a i l y x 4.23 A l though chloroquine-resistant s t r a i n s studied t o date have proved uniformly r e s i s t a n t t o other 4 - a m i n o q ~ i n o l i n e s , ~ a chloroquine-resistant s t r a i n of 2. berghei s u r p r i s i n g l y was reported t o be cross-resistant t o III.24

3 . New S t r u c t u r e s . - Several new a c t i v e chemical moieties have been reported. 3-Piperonylsydnone ( V ) was a c t i v e a g a i n s t 2. berahei a t a dose of 1 0 mg./kg." The glyoxal dithiosemicarbazones VIa and b showed high a c t i v i t y a g a i n s t P. pallinaceum i n t h e chick; t h e median e f f e c t i v e doses per 0s were 0.7 rug./&. x 7 and 0.5 mg./kg. x 7, respectively.37 It i s not y e t c l e a r whether these or r e l a t e d compounds will have p r a c t i c a l application.

-

C. Radical Curative Agents. The 8-aminoquinolines are t h e only substances having s u f f i c i e n t a c t i v i t y against secondaq t i s s u e schizonts t o be of p r a c t i c a l value i n achieving r a d i c a l cure of r e l a p s i n g malarias. However, primaquine and c e r t a i n o t h e r 8-aminoquino~inesproduce acute hemolytic anemia

140

Sect. I11

- Chemotherapeutics HC=N-NBCSNHX

a,X=H b, X = C&OCHs

I

HCd-NHCSNBX

V

F l y n n , Ed.

VI

i n persons whose red c e l l s are unusally susceptible t o drug-induced hemolysis. The rapid degradation and elimination of primaquine necessitates d a i l y @&inist r a t i o n , which c o n s t i t u t e s - a f u r t h e r drawback l i m i t i n g i t s use. The discovery that 4- (2-bromo-4,5-dimethoxyphenyl)-1,1,7,7-tetraethyldiethylenetriamine (RC-12) ( V I I ) has a pronounced effect on t h e exo-erythrocytic stages of 2. cathemerium i n canaries3 stimulated an appraisal of the a c t i v i t i e s of RC-12 against 2. cynomolRi i n t h e monkey.38 Although t h e drug per se had l i t t l e promise as a schizonticidal or suppressive drug, it showed s i g n i f i cant promise as a prophylactic or r a d i c a l curative agent and m i g h t f i n d use when: (1)t h e cambination of chloroquine-primaquine i s not e f f e c t i v e i n causal prophylaxis, as appears t o be t h e case where there i s chloroguine resistance; ( 2 ) there i s a need f o r a curative agent which can produce benefits i n l e s s than 14 days; and (3) there a r e fears of enhanced susce t i b i l i t y t o the hematot o x i c i t y of primaquine. Clinical studies are planned. Si:

m

S

VIII

VII

6- [ [4-(Diethylamino)-l-methylbutyl]amino]-5,8-dimethoxyquinaldine (B-505) (VIII)39 a l s o produced r a d i c a l cures of 12. cathemerium infections i n canaries

but the relative t o x i c i t y of t h i s compound precluded c l i n i c a l studies i n man. ?i

-

D. Test Methods. The demonstration of multiple drug resistance i n 2. falciparum (vide supra) spurred research t o develop better experimental models for studying t h i s phenomenon. Strains of 2. bernheihave been induced t o acaquire resistance t o representatives of a l l the major classes of suppressive drugs, and the r e s i s t a n t l i n e s have been employed i n cross-resistance tests t o determine the i n t e r r e l a t i o n s h i p among t h e various chemical types,20,31,40-44 Anopheles ste&ensi has been shown t o be a suitable experimental vector of 2. berghei t o serve as a source of sporozoites and f o r cyclic transmissions and the existence of primary t i s s u e stages of 2. berRhei has been c o n f i r ~ n e d . ~ ~ , ~ ~ The r e l a t i v e ease of infecting the splenectomized gibbon with blood-induced infections of 2.S c i m r u m indicates that t h i s species may be a suitable l a b oratory model f o r studies of fa3.cimm.m malaria. 47

Chap. 14a.

Elslag e r

Antipa r a s itic s

141 -

Leishmania i s responsible f o r millions of smoldering u l c e r s among inhabi t a n t s of endemic regions i n Africa, t h e Americas, and t h e Near and Far East. Antimonials a r e usually e f f e c t i v e i n leishmaniasis, but t h e necessity f o r r e peated (10-30)i n j e c t i o n s and t h e well-known contraindications of t h e s e drugs impose serious r e s t r i c t i o n s on t h e i r widespread use. Inasmuch a s some regression of e a r l y l e s i o n s of dermal leishmaniasis was observed i n malaria p a t i e n t s t r e a t e d with c h l o r g ~ a n i d e ,it ~ ~was of i n t e r e s t t o i n v e s t i g a t e t h e p o t e n t i a l usefulness of t h e repository drug cycloguanil pamoate ( I ) (vide supra). Early r e s u l t s from Costa Rica and Mexico a r e encouraging; a s i n d e intramuscular i n j e c t i o n of cycloguanil pamoake (280-350 mg. base) cured 26 of 30 p a t i e n t s with b r a ~ i l i e n s i s ~and ' 31 of 40 percutaneous and mucocutaneous i n f e c t i o n s of sons with cutaneous leishmaniasis produced by &. nexicana .49

L.

IV. CHAGAS' DISEASE A t l e a s t 7 m i l l i o n persons i n South and Centrql America a r e i n f e c t e d with Trypanosoma cruzi, t h e causative agent of Chagas' disease. In c o n t r a s t t o t h e success attending t h e development of e f f e c t i v e drugs f o r t h e African trypanosomiases, t h e treatment of cruzi i n f e c t i o n s has been generally unsatisfactory. There i s some evidence t h a t n i t r o f u r a n s may be e f f e c t i v e a g a i n s t acute i n f e c t i o n s , but no curative agent has y e t been found.50 The discovery t h a t strong chemotherapeutic i n t e r e s t . 51 The nature modifications of t h e N1-

N1- (3-dimethylaminopropyl )piperazines (IX) e x h i b i t a c t i v i t y against 2. c r u z i i n mice i s of p a r t i c u l a r of t h e R s u b s t i t u e n t i s r e l a t i v e l y non-specific, but (3-dimethylaminopropy1)piperazine moiety d r a s t i c a l l y

X reduce a c t i v i t y . In comparative s t u d i e s with 2. cruzi i n mice, piperamide maleate (X), given o r a l l y i n s i x t e e n 2.5 or 10 m g x doses over 7 days, prolonged t h e median survival t i m e of t r e a t e d mice s i g n i f i c a n t l y longer than mice t r e a t e d with these doses of furaltadone or primaquine.sl However, t h e curat i v e value and t h e s a f e t y of piperamide maleate and r e l a t e d substances52 have y e t t o be established.

v.

IS

The i d e a l antiamebic agent must be capable of e r a d i c a t i n g Ehtamoeba h i s t o l y t i c a from both t h e bowel and from e x t r a - i n t e s t i n a l s i t e s and should b y v i r t u a l l y non-toxic. Agents with sustained, prophylactic antiamebic p r o p e r t i e s a r e a l s o needed. Although many s a t i s f a c t o r y antiamebic agents a r e available, no s i n g l e preparation i s capable of exerting a l l t h e a c t i o n s required. Theref o r e , t h e search f o r new agents continues.=

Sect. I11

142 -

- Chemotherapeutics

F l y n n , Ed.

-

A. 2-Amino-5-nitrothiazoles. Over a decade ago it was reported t h a t 2-acetamido-5-nitrothiazole (aminitrozole ) ( X I ) possessed curative a c t i o n

against i n t e s t i n a l amebiasis i n r a t s and dogs and k i l l e d a d u l t Schistosoma mansoni i n mice a t well-tolerated 0 s e s . ~ 4 More r e c e n t l y a new 2-amino-5n i t r o t h i a z o l e d e r i v a t i v e ( f m b i l h a h , 32, 644-Ba ) ( X I I ) having strong therapeut i c e f f e c t s against amebiasis and schistosomiasis (vide i n f r a ) was discovered.55 !The minimum i n h i b i t o r y concentration of 32,644-& against g. h i s t o l y t i -

XI

XI1

ca i n v i t r o w a s 10 pg./ml.

G emetine

This i s approximately t h e same potency exhibited

or 4,7-phenanthroline-5,6-quinone under comparable conditions. 56

!Phe compound strongly suppresses amebic l i v e r abscess i n hamsters following d a i l y o r a l doses of 60-120 mg./kg. f o r 7 days (chloroquine was e f f e c t i v e a t 100-200 mg./kg. ). I n t e s t i n a l amebic i n f e c t i o n s i n rats and guinea-pigs were markedly reduced following d a i l y o r a l doses of 30-60 mg./kg. f o r 4 and 5 days, respectively .56 Results of preliminary human t r i a l s i n d i c a t e t h a t 32,644-&, administered i n d a i l y doses of 20-25 mg./kg. f o r 5 t o 10 days, i s e f f e c t i v e against both amebic dysentery and amebic l i v e r abscess, although s i d e e f f e c t s including frequent ECG changes and occasional neuropsychic episodes were noted.57 More extensive t r i a l s w i l l be required t o a s s e s s t h e r e l a t i v e s a f e t y and e f f i c a c y of t h e drug compared with o t h e r forms of treatment. Rel a t e d heterocyclic compounds a r e a l s o reported t o have antiamebic and a n t i schistosomal p r o p e r t i e s .58

-

B. 7-Quinolinols. Another new amebicide of p o t e n t i a l i n t e r e s t i s 5chloro-7- 3-diethylaminopropylaminomethyl ) -8-quinolinol (clamoxyquin PAA-3854 CI-433) ( X I I I ) s9 The r e l a t i v e amebicidal concentrations of clamoxyquin hydrochloride and emetine hydrochloride a r e 20-40 pg./ml. and 2.5 pg./ml. respect i v e l y . 6o The e f f e c t s of clamoxyquin hydrochloride and chloroquine against amebic h e p a t i t i s i n hamsters were roughly comparablej clamoxyquin hydrochloride,

.

,

,

OH

c1 XI11

XIV

pamoate, and s a l i c y l a t e w e r e a c t i v e against i n t e s t i n a l amebiasis i n r a t s i n gavage doses ranging from 75 t o 600 mg./kg. d a i l y f o r 4 days." Clamoxyquin hydrochloride and pamoate cured amebic dysentery in do s i n d a i l y doses of 3.13 t o 50 mg./kg. and 12.5 t o 25 mg./kg. respectively.6' Relative t o t h e 8-quinol i n o l s i n c l i n i c a l use t h e clamoxyquin s a l t s were deemed worthy of c l i n i c a l

Chap. 14a.

Antipar as itic s

Elslager

143 -

t r i a l . Clamoxyquin hydrochloride was t o l e r a t e d w e l l i n man following d a i l y 1 5 mg./kg. doses f o r 5-10 days and was highly e f f e c t i v e against various forms of Additional s t u d i e s w i l l be required before the r e l a i n t e s t i n a l amebiasis."' t i v e importance of the clamoxyquin salts i n amebiasis chemotherapy can be assessed

.

C. Anisomycin. - The determination of t h e s t r u c t u r e and stereochemistry of the potent antiprotozoal a n t i b i o t i c anisamycin (XIV)62 may serve t o stbiul a t e f u t u r e research on t h e synthesis and biological properties of r e l a t e d substances.

VI,

SCHISTOSOMIASIS

Nearly 200 m i l l i o n people a r e infected with schistosomiasis, and on a world b a s i s these i n f e c t i o n s a r e ~ p r e a d i n g . ~The degree of prevalence, t h e seriousness of t h e infection, and t h e paucity of adequate drugs make schistosomiasis one of t h e g r e a t e s t challenges i n p a r a s i t e chemotherapy today. Seve r a l promising new agents have been discovered, but t h e i r usefulness i n man has yet t o be adequately evaluated. Therefore, one s t i l l must r e l y on the a n t i monials with some assistance from lucanthone hydrochloride (ma) against Schistosoma haematobium and c e r t a i n s t r a i n s of S. mansoni. Research on schistosomiasis6Y'64 and other trematode infections63 has r e c e n t l y been reviewed.

-

A. 2-Amino-5-nitrothiazoles. To date 1-(5-nitro-2-thiazolyl)-2-imidazolidinone ( A m b i l h d 32 644-E!a) ( X I I ) has been evaluated more extensively i n ~ c h i ~ t o s a m i a s i than s ~ ~ i n ~amebiasis ~ ~ ~ ~(vide ~ ~ supra). " ~ ~ ~ When ~ adult S . mansoni were exposed t o 32,644-Ba i n v i t r o for prolonged periods (100 hr.), oviposition was i n h i b i t e d a t concentrations of 1 pg./ml. and worms were k i l l e d a t 100 p g . / m l . 5 5 ~ 6 5 The destruction of the v i t e l l o g e n i c gland i n the female coincides with a reduction i n body-length i n both male and female worms and i n t h e s i z e of t h e ovary i n t h e female.57 The drug.was highly a c t i v e against S . manSOn155,65 and 2. j a ~ o n i c u mi ~ n ~mice i n o r a l doses of 100 mg./kg. d a i l y f o r 10 days and had a strong prophylactic and chemotherapeutic e f f e c t on experimental 2. mansoni infections i n monkeys .57 Temporary, r e v e r s i b l e impairment of sljermatogenesis occurred i n mice- the drug apparently a c t s d i r e c t l y on t h e germinal epithelium of t h e t e s t e s . 66 Results of preliminary c l i n i c a l t r i a l s i n man i n d i c a t e t h a t 72 644-h has considerable e f f i c a c y against 2. h a e m a t ~ b i u m ~ ~and , " ~2. mansoni5 j Q 8 i n o r a l doses of 20-40 mg./kg. d a i l y f o r 5-15 days; S . japonicum i n f e c t i o n s appear t o be more refractory.57 Three types of side e f f e c t s were encountered which merit s p e c i a l a t t e n t i o n : (1)frequent ECG changes, involving mainly t h e T waves; ( 2 ) neuropsychic e f f e c t s , ranging from severe headaches t o convulsive a t t a c k s with infrequent hallucinations; (3) t r a n s i t o r y impairment of spermatogenesis .57 The ultimate place of 32,&4-& i n schistosomiasis chemotherapy w i l l depend l a r g e l y on t h e incidence and s e v e r i t y of these side e f f e c t s a t e f f e c t i v e dose l e v e l s when compared with t h e side e f f e c t s encountered i n t h e c l a s s i c a l treatments. The drug has a l s o been reported t o be e f f e c t i v e i n t h e treatment of Dracunculus medinensis (guinea worm) i n f e s t a t i o n i n humans .69

144

Sect. III

-

Chemotherapeutics

F l y n n , Ed.

-

B. Thiaxanthenones. Incubation of lucanthone (XVa) with sclerotiorum produces t h e a c i d - l a b i l e hydroxymethyl d e r i v a t i v e Surprisingly, t h e (Xvb) together with smaller amounts of t h e aldehyde five-day ED50 f o r lucanthone and hycanthone a g a i n s t S . mansoni i n hamsters were 8.0 mg./kg. and 0.78 mg./kg., respectively.70 The unusual potency of hycanthone l e d t o t h e hypothesis t h a t it might indeed be t h e e l u s i v e a c t i v e metabol i t e of lucanthone. The a c i d s e n s i t i v i t y of Xvb could explain why it had

escaped detection by previous i n v e s t i g a t o r s . Accordingly, monkeys were medicated with lucanthone, t h e w i n e s were c a r e f u l l y e x t r a c t e d avoiding a c i d treatment, and t h e e x t r a c t s were examined by thin-layer chromatography. In agreement with e a r l i e r work,71 t h e sulfoxide (XVd) was t h e major metabolite, and only t r a c e s of XVb w e r e detected. When t h e urine e x t r a c t s were incubated with glucuronidase p r i o r t o chromatography, however, hycanthone appeared as a major component.7o Idthough t h e sulfone XVe i s reported t o be t h e major urinary metabolite i n hycanthone was a l s o found i n mouse urine a f t e r glucuronidase incubation but t o a much l e s s e r extent t h a n i n t h e monkey.7o The results of t h e s e elegant s t u d i e s support t h e hypothesis t h a t hycanthone i s an important a c t i v e metabolite of lucanthone; it now becomes apparent why a 2-toluidine function i s an absolute requirement f o r a c t i v i t y among all lucanthone r e l a t i v e s . Other s t u d i e s u t i l i z i n g s u b t i l i s suggest t h a t t h e major i n vivo a c t i o n of lucanthone i s t o complex with DNA, thereby blocki n g DNA-dependent RNA synthesis .73 Various o t h e r schistosomicides r e l a t e d t o hycanthone and t h e aldehyde XVc have been prepared.74 It has a l s o been disclosed t h a t lucanthone N-oxide (XVI) i s considerably l e s s t o x i c than lucanthone .75

-

Tris (paminophenyl )carbonium Salts. Tris (paminopheny1)carbonium C. (TAC) pamoate (XVII)76 and c e r t a i n antimonials have broad antischistosome a c t i v i t y i n experimental animalsT7 and i n man.78 However, both groups have important l i m i t a t i o n s . Thus t h e slow-acting TAC pamoate must be administered for s e v e r a l weeks t o achieve a strong degree of e f f e c t while t h e f a s t - a c t i n g antimonials have a r e l a t i v e l y narrow therapeutic index.

In a recent study of t h e j o i n t a c t i o n of TAC s a l t s and t a r t a r emetic a marked degree of synergism was observed against S. mansoni i n v i t r o and i n mice, Moreover, n e i t h e r drug had p a r t i c u l a r l y a f t e r pretreatment with TAC pamoate?” an appreciable a f f e c t on t h e gross t o x i c i t y of t h e other f o r normal mice. An encouraging degree of j o i n t antischistosomal a c t i o n by TAC pamoate and t a r t a r

Chap. 14a.

Antiparasitics

-OOC

.OH

145

Elslager

H(

XVIII

XVII

emetic was also observed in monkeys. The therapeutic advantage of such a combination hinges largely upon whether o r not the co-administration of TAC pamoate produces higher o r more sustained antimony levels in the blood or tissues. A subsequent study utilizing 124Sb-labeled tartar emetic demonstrated that co-administration or pretreatment with TAC pamoate had no significant effect on the disposition of antimony in mice o r monkeys." Available evidence suggests that the mode of action of TAC pamoate and the trivalent antimonials is dissimilar. Briefly, the antimonials interfere with schistosome glycolysis by inhibiting phosphofructokinase, which catalyzes the reaction: fiuctose-6-phosphate

f

ATP

F'ructose-1,6-diphosphate

+ ADP

Recent studies with 14C-labeled glucose indicate that tartar emetic blocks both the Ehbden-Meyerhof and the hexose-monophosphate pathways of glycolysis Organic antimonials have also been reported to inhibit schistosome glutamicpyruvic transaminase

.

.

The TAC salts apparently act by an entirely different mechanism.e2 In mice subcurative doses of TAC chloride have no demonstrable effect on the carbohydrate and protein metabolism of the worms but produce a localized paralysis of the acetabulum, pharynx, and oral sucker which histochemically has been correlated with a pronounced decrease of cholinesterase activity in the central ganglia. This paralysis is completely reversed in vitro by the cholinergic blocking agent mecamylamine, suggesting an accumulation of acetylcholine in these structures. Simultaneously, glycogen depletion is observed in the papillae located below the cuticle of the worm. Either one or a combination of these changes could account for the antischistosomal effects observed.82

-

D. Cucurbitine. For several years reports emanating from mainland China have claimed that the dried, ripe seeds of the pmpkin varieties Cucurbita pep0 L. and C. moschata Duch. are effective in the treatment of schistosomiasis. The amino acid cucurbitine (XVIII) has recently been isolated from C. moschata Duch." Cucurbitine reportedly inhibits the growth of immature 2. japonicum, produces degenerative changes in the genital organs of the worms resulting in depressed egg-producing capacity, and is virtually nontoxic.s3 It would appear worthwhile to confirm the structure of cucurbitine and study its effects on 2. haematobium and 2. mansoni. VII.

CLONORCHIASIS

Clonorchiasis is caused by the human liver fluke Clonorchis sinensis (syn.

Sect. 111 - Chemotherapeutics

146 -

F l y n n , Ed.

Opisthorchis s i n e n s i s ) and i s widely d i s t r i b u t e d i n t h e Far East, especially i n Indochina, Central and South China, Formosa, Korea, and Japan. Approximatel y 19 million people are a f f l i c t e d with t h i s disease, y e t no r e l i a b l e treatment i s known. Therefore, preliminary r e p o r t s t h a t t h e f a s c i o l i c i d e a,a,a,a',@,a'hexachloro-2-xylene (XIX) i s highly e f f e c t i v e against C. s i n e n s i s i n experimen-

t a l animalss4"' and i n a t well-tolerated doses a r e most encouraging. Expanded human t r i a l s a r e indicated.

The world-wide incidence of i n t e s t i n a l helminthic i n f e c t i o n s i n man approaches t h e staggering f i g u r e of two b i l l i o n . * The prevalence of these i n f e c t i o n s and t h e i r complex and far-reaching e f f e c t s upon body function cause them t o rank among t h e most important disease problems of man. F i r s t reports" on tetramisole ( X X ) , a potent new anthelmintic with broad a c t i o n against nematodes, a r e highly encouraging. The drug i s reported t o be e f f e c t i v e against Ascaris lumbricoides and Enterobius vermicularis i n man" and against a v a r i e t y of adult and immature g a s t r o i n t e s t i n a l and pulmonary nematodes i n laboratory animals, poultry, and livestock. ,l' s2 Although devoid

xx

XXI

XXII

of antihistaminic, anticholinergic, adrenolytic o r other c l a s s i c a l pharmacol o g i c a l properties, tetramisole e x e r t s a rapid paralysing a c t i o n on a variety of nematodes i n v i t r o a t low concentrations.

''

The evolution of tetramisole points once again t o t h e importance of strong biochemical support i n drug development. Thus, t h e observation t h a t t h i a z o t h i en01 (XXI)'~ was a c t i v e against nematodes during a routine screening program i n chickens stimulated a search for a c t i v e metabolites of XXI and l e d t o t h e isol a t i o n and i d e n t i f i c a t i o n of XXII a s t h e major transformation product i n t h e feces. The c y c l i c metabolite XXII was a c t i v e a t s i g n i f i c a n t l y lower dose l e v e l s t h a t t h e parent compound (XXIX and these results prompted the s n t h e s i s ?' . and b i o l o g i c a l evaluation of an extensive s e r i e s of r e l a t e d compounds Tetramisole emerged a s t h e most promising substance f o r expanded s t u d i e s .

Antipa rasitics

Chap. 14a.

147 -

Els lage r

IX. BIBLIOGRAPHY

&I.

m., a.s.

(1) E. H. Hinman, 2. Tro Med. 2, 125 (1966). (2) Chronicle, 3 3 d a (3) L. J. Bruce-Chwatt, Trans. Trop. g . 2, 105 (1965). (4) E. F. Elslager and P. E. Thompson, Pharmacol.,& 193 (1962). ( 5 ) P. E. Thompson, B. J. Olszewski, E. F. Elslager, and D. F. Worth, &.' J& 481 (1963 -J. Tro Med. (eRexution, JJ, 361 (1964).

.

&. &.

m.,

m.,

w.,

1965,

S. Goodman and A. Gilman, Ed., t h e Mamillan Co., New York, N.Y.,

1087-1117.

ed., L. pp.

(10)E. F. Elslager and P. E. Thompson, Abstracts, 9th National Eledicinal Chemistry Symposium of t h e American Chemical Society, Minneapolis, Minn., June 1964, p. 6A. (11)E. F. Elslager and D. F. Worth, U. S. Patents 3,223,705 (1965) and 3,248 422 (1966). (12) E. F. Elslager and D. F. Worth, U. S. Patents 3,161,641 (1964); 3,236,849 (1966); 3,244,716 (1966)j and 3,248,422 (1966). (13) G. R. Coatney, P. G. Contacos, and J. S. Lunn, J. Trop. &d. &g.,

&I.

-

13 383 (1964)")(14) R. D. Powell, R. L. DeGowin, and R. B. Eppes, M.,&, 913 (1965). (15) P. G. Contacos, G. R. Coatney, J. S. Lunn, and J. W. Kilpatrick,

L;zI 386

u.,

(u@+)*

(16) 1. A. McGregor, K. Williams, G. H.'Walker, and A. K. Rahman, Brit. E .

2 . , 3 695 (1966).

- m.,

(17 P. E. Thompson, B. Olszewski, and J. A. Waitz, &. 2. m.Med. 343 (1%5)* (18)W. Chin, J. S. Lunn, J. Buxbaum, and P. G. Contacos, w., &I 922

&

(1965)

(20) A.

(19) P. G.

(1965

Contacos, G. R. Coatney, J. S. Lunn, and W. Chin,

a

w.,

925

Bishop, ParasitolopV, 53, 10 (1963); 407 (1965). (21) E. F. Elslager and D. F. S r t h , Nature, 206 630 (1965). (22) P. E. Thompson, B. Olszewski, and J. A. &z, &. 2.

r14 U1

343 (1965)-

&. Med. a.,

(23) J. Schneider, M. Bouvry, and J. Le Quellec, Abstracts, I n t e r n a t i o n a l Colloquium on , P a berghei, I n s t i t u t e of Tropical Medicine "Prince Leopold," Antwerp, Belgium, December 1964. (24) J. Hill i n "mperimental Chemotherapy," V o l N, R. J. Schnitzer and F. Hawking, Ed., Academic Press, New York, N.Y., 1966, p. 448. (25) Rhone-Poulenc S. A., B r i t i s h Patent 973,903 (1964); Eire Patents 461/62, 1097/62, 591/63, and 620/63 (1963). (26) K. G. Powers, J. Parasitol., 2, 53 (1965). (27) C. G. H u f f , 'Itrop. m e , & 339 (1965). (28) R. L. De Gowin and R. D. Powell, 519 (1965). (29) R. L. De Gowin and R. D. Powell, J. &&.B. 851 (30) 3. % (131, 38 ( i s ) . (31) P. E. Thompson, A. Bayles, B. Olszewski, and J. A. Waitz 198 (1965).

&.-z.

m.

m,

- a*,*,

@.

w.,

s., a,

, Am-

148

Sect. IU

- Chemotherapeutics

'Flynn, Ed.

(35) P. C. Basu, N. N. Singh, and N. Singh, B u l l . World Health Qrgan., & 699 -_ ( .1-964.). (36) W. H. Nyberg and C. C. Cheng, 2. B., 531 (1965). (37) P. A. Barrett, E. Beveridge, P. L. Bradley, C. G. D. Brown, S. R. M. Bushby, M. L. Clarke, R. A. Neal, R. Smith, and J. K. H. Wilde, Nature, 1340 (1965). (38) L. H. Schmidt, R. N. Rossan, R. Fradkin, J. Woods, W. Schulemann, and L. Kratz, presented a t t h e 14th Annual Meeting of t h e American Society of Tropical Medicine and Hygiene, New Orleans, L a . , Nov 1965. (39) F. Schonhofer and W. Schulemann, B r i t i s h Patent 970,949 (1964). (40) R. L. Jacobs, 2. Parasitol., 481 (1965). (41) W. Peters, 9. Parasitol., 80, 90, 97 (1%5)(42) W. Peters, Nature, (43) P. E. Thompson, B. %ze:ski, A. Bayles, and J. A. Waitz, 2. P a r a s i t o l . , /s 51 (21, 54 (1965). (44) P. E. Thompson, A. Bayles, B. Olszewski, and J. A. Waitz, Science, 1240 (1965). (45) M. Yoeli and H. Most, &. 2. Trop. €&., 700 (1965). (46) M. Yoeli, J. Vanderberg, R. Nawrot, and H. Most, & 927 (1965). (47) R. A. Ward, J. H. Morris, D. J. Gould, A. T. C. B u r k e , and F. C. Cadigan, Jr., Science, 1604 (1965 ) (48) A. Pel& Chavarria, E. Kotcher, and C. Lizano, 2. Assoc., 1142 (1965). (49) F. Beltrh, F. Biagi, and S. Gonza'lez, presented a t t h e 14th Annual hketing of t h e American Society of Tropical Medicine and Hygiene, New Orleans, La., NOV 1965. H&., 211 (1964). (50) F. Hawking, 2. Trop. (51) A. S. Tomcufcik, R. I. Hewitt, P. F. Fabio, A. M. Hoffman, and J. Entwistle, Nature, 2ez, 605 (1965). , (52) American Cyanamid Co., South African Patent 3833/63 (1964) and Belgian Fatent 654,017 (1965). (53) G. Woolfe i n "Drug Research," Vol VIII, E. Jucker, Ed., Birkhauser AG, Basel, Switzerland, 1965, pp. 13-52. (54) A. C. Cuckler, A. B. Kupferberg, and N. Millman, Antibiot. Chemother540 (1955). C. R. Lambert, M. Wilhelm, H. S t r i e b e l , F. Kradolfer, and P. Schmidt, Ek e r i e n t i a , 452 (1964). %adolfer and R. Jarumilinta, Trop. E.Parasitol., 210

@.

&

a

.

e.

s. & u., &. w.

a

2

&.

(19651* (57) "Th6rapeutique Nouvelle de l a Bilharzioae e t de 1'Amibiase, Symposium de Lisbonne, June, 1965, Acta WOE., SuppL. 9, 1-314 (1966). ( 5 8 ) Ciba Ltd., South African Patents 3781/64 (1965) and 4336/65 (1966); Neth. Patents 6,505,225 and 6,505,226 (1965). (59) J. H. Burckhalter, W. S. Brinigar, and P. E. Thompson, 2. & 4070 (1961). I'

a. e., Meisenhelder, J. e-

(60) P. E. Thompson, A. Bayles, P. McClay, and J. E. sitol., 817 (1965). (61)R. Hugonot, E. Granotier, and J. P. Farges, Therapie, & 329 (1965). (62) J. J. Beereboom, K. Butler, F. C. Pennington, and I. A. Solomons, 2. B., 2334 (1965).

a-

Chap. 14a.

Antiparas iti cs

Elslager

149 -

s., &,

(63) G. Uinrmler, 2. Tropenmed. Parasitol., 95, 164, 337 (1964). (64) World Health Organ. Tech. Rep. 317. (65) C. R. Lambert, Trop. Parasitol., 292 (1964). (66) C. R. Lambert, V. S. P. S i n a r i , and J. Tripod," Acta T r o ~ . , & , 155 (1965). (67) C. R. Lambert and F. S. I& C r u z F e r r e i r a , B u l l . World Health Organ.,& 73 (1965)(68) P. Jordan, Brit. w .z., A 276 (1966). (69) G. Raffier, Acta T r o ~ . ,.& 350 (1965).

a., s.

(70) D. Rosi, G. Peruzzotti, E. W. Dennis, D. A. Berberian, H. Freele, and

S. Archer, Nature,%

(71) R. Strufe,

1006 (1965).

w. Chem.

%.--.

Abhandl. Forschungsstaetten Farbenfabriken Bayer, & 337 (1963). (72).Gijnnert, B u l l . World Health O r g a n . , a 702 (1961). (73) I. B. Weinstein, R. Chernoff, I. Finkelstein, and E. Hirschberg, Pharmacol. 1 297 (1965). m e r h 'n4g Drug Inc., South African Patent 4081/64 (1965). (75) Farbenfab. Bayer A. G., Belgian Patent 663,790 (1965). (76) F. W. Short and E. F. Elslager, U. S. Patent 3,218,339 (1965). (77) E. F. Elslager, F. W. Short, D. F. Worth, J. E. Meisenhelder, H. Najarian, and P. E. Thompson, Nature, 628 (1961). (78) J. R. da Silva, A. Prata, C. A. Argento, H. American0 do B r a s i l , and L. F. Ferreira, Hospital (Rio de Janeiro), & 25 (1964). (79) P. E. Thompson, J. E. Meisenhelder, A. K. Moore, and J. A. Waitz, B u l l . World Health Organ., 2 517 (1965). (80) J. A. Waitz, R. E. Ober, J. E. Meisenhelder, and P. E. Thompson,

s.

z,

w.,

81) J. A. Waitz, Life Sci., 3 377 (1964). (82) E. L. S c h i l l e r and E. Buding, 2. P a r a s i t o l . Supplement, 51, 43 (1965). (83) J. H. Zhou, F. Y. Fu, and H. P. Lei i n "Proceedings of t h n e c o n d International Pharmacological Meeting, Prague, 20-23 August, 1963," Vol V I I , K. K. Chen, B. Mukerji, and L. Volicer, Ed., t h e Macmillan Co., New York, N. Y., 1965, p. 25. N. N. Plotnikov and L. I. Sinovich, Meed. Parazitol. & Parazitarn. Bole301 (1964); Diseases g., l m ) . 9 6 5 % . Yokogawa, H. Koyama, H. Yoshimura, and C. S. Tsai, J. Chiba Med. SOC., & 286 (1965). ( 8 6 ) H. L. Chung, Chinese Med. J., & 232 (1965). (87) J. Liu, (19657. ( 8 8 ) N. N. Plotnikov, No N. Ozeretskovskaya, V. K. Karnaukhov, N. S. Zalnova, G. M. Faibusovich, G. I. Kukhta, and M. I. Alekseeva, Med. Parazitol. i Egmzitarn. Bolezni, 676 (1964); IIl.op. Diseases B u l l . , & 32771965). Tv. N. mozdov, Diseases & 59 (1966). (90) M. Yokogawa, M. Tsuji, K. Araki, T. Nomoto, M. mtsumoto, M. Koyama, R. Yoshiba, S. Fukuchi, A. Hiratsuka, and M. Kitamura, Japan. 3. P a r a s i t o l . , & 526 (1965)(91) D. Thienpont, 0. F. J. Vanparijs, A. H. M. Raeymaekers, J. Vandenberk, P. A. Demoen, F. T. N. Allewijn, R. P. H. Marsboom, C. J. E. Niemegeers, K. H. L. Schellekens, and P. A. J. Janssen, Nature, 3 & 1084 (1966). (9) J. K. Walley, Vet. Record, J&, 4 0 6 m ) . (93) Janssen &arm., N. V., Belgian Patent 653,087 (1965). (94)Janssen Pharm., N. V., Neth. Patent 6,505,806 (1965).

(a)

-

m.

=.-

a

J.

us,

m.

u.,

150 -

Sect. 111

Chapter Ikb.

-

F l y n n , Ed.

Chemotherapeutics

Animal Antiparasitic Agents

Dale R. Hoff, Merck Sharp & Dohme Research Laboratories Rahway, New Jersey

I. Introduction Developments during 1965 in the field of animal health included reports of two new anthelmintics, tetramisole and bunamidine, which are representative of neu chemical classes. Other reports described modifications of the older antiparasitic leads as well as new therapeutic applications of established agents. A number of investigations into the biochemistry of metazoan and protozoan parasites appeared, and the results of such studies may be hoped ultimately to point out new approaches in the search for new antiparasitic agents. Lack of space rohibits discussion of these papers in this survey, but pertinent references are included for the use of interested readers.

E

Two books which became available during 1965 are important additions to the literature of antiparasitic chemotherapy. Gibson' s review2 of veterinary anthelmintics covers the literature throunh 1962 and into 1963: and Schktzer and Hawking's vol. IV3, conta&s an appendix which - volumes dealim with t-k&nosoPliasis, enterohepatitis, coccidiosis, babesiasis, theilekasis, anaplasmosis, filariasis, and myiasis.

11. Anthelmintics

A.

Anthelmintics for R.eatment of Roundworm Infections.

Tetramisole. Preliminary reports of an entirely new class of anthelmintics, hydrogenated derivatives of imidazop, l-k7thiazolesY appeared from the Janssen Laboratories4r4a. Discovery of the Mdazo~,l-b7thiazoles followed from the observation that 2-acetylimino-3-/?-hydro~-2-~2-thienyl)-ethyg-thiazoline (thiazothienol, I), under investization as an anthelmintic in poultry, was met bolized to the more potent 6-(2-thienyl)-~,6-dihydroimidazo_/2,l-b~thiazole (11)t. Further development of t h e lead led to 6-phenyl-2,3,~,6-~etrahydroimidazo fiYl-k7thiazole hydrochloride (111), for which the generic name tetramisole was proposed.

-

I

I1

I11

Tetramisole

Antiparasitics

Chap. 14b.

Hoff

151 -

Tetramisole was found by t h e Janssen groupb t o be a water-soluble (21% a t 2OoC. ), broad-spectrum anthelmintic which can be administered o r a l l y , o r by subcutaneous, intramuscular, o r i n t r a p e r i t o n e a l i n j e c t i o n . Doses of 40 mg/kg, o r a l l y o r by i n j e c t i o n , e f f i c i e n t l y removed immature and mature forms of- all t h r e e of t h e common chicken nematodes, Ascaridia g a l l i , H e t e r a k i s gallinarum, and obsignata. Tpxicity t o chickens was s a i d t o be low, doses up being t o l e r a t e d . Doses of 5-20 mg/kg. . o r a l l y o r by i n j e c t i o n , were reported e f f e c t i v e i n t h e removal of a wide’range of mature and immature g a s t r o i n t e s t i n a l nematodes i n sheep, as w e l l as t h e lungworm, D i c t ocaulus v i v i arus. Dose levels up t o 40 mg/kg, o r a l l y , o r 10 mg/kg., parent& by sheep, S i m i l a r r e s u l t s were r e p o r t e d with c a t t l e . The drug was a c t i v e a t 20 mg/kg, o r a l l y , a g a i n s t Toxascaris, Toxocara, and Uncinaria i n dogs, but was poorly a c t i v e a g a i n s t Trichuris. I n allauthors catalogued a c t i v i t y with t e t r a m i s o l e a g a i n s t a t l e a s t 56 s p e c i e s of nematodes i n t h i r t e e n hosts, i n c l u d i n g man. No a c t i v i t y a g a i n s t f l u k e s ( F a s c i o l a h e p a t i c a ) o r tapeworms (Moniezia ) was observed.

&

z.

Tetramisole was found t o have a r a p i d paralyzing a c t i o n in v i t r o a g a i n s t a v a r i e t y of nematodes. Walleys confirmed some of t h e observations described above concerning t h e e f f i c a c y of tetramisole. I n t e s t s with 1,076 sheep and 18 goats, tetramis o l e a t 15 mg/kg gave good r e s u l t s a g a i n s t all mature and immature nematodes, with t h e exception of Trichuris. Adult worms were removed by a 5 mg/kg dose. S l i g h t s i d e - e f f e c t s , c o n s i s t i n g of h y p e r i r r i t a b i l i t y and loss of a p p e t i t e , were observed with t h e 15-20 mg/kg o r a l doses. These e f f e c t s were more pronounced a t doses above 50 mg/kg, and one sheep died a f t e r a 80 mg/kg dose. Clonic convulsions were induced by t h e h i g h e s t l e v e l s . These r e p o r t s are p r e l i m i n a r y and l i m i t e d , hence f i n a l assessment of t h e e f f i c a c y and s a f e t y of t e t r a m i s o l e must await r e p o r t s o f wider i n v e s t i gations. Thiabendazole. A d e t a i l e d r e p o r t concerning t h e toxicology and pharmacology of thiabendazole (IV) w a s contained i n a paper by Robinson, Stoerk, and Graessle6. The o r a l a c u t e LD 0 of thiabendazole i n mice, rats, and r a b b i t s w a s 3600, 3100, and g r e a t e r t.2 an 3800 mg/kg, respectively. In sub-acute t o x i c i t y t e s t s , mice and rats grew normally when f e d thiabendazole i n t h e d i e t f o r 30 days. A t l e v e l s of 0.2%, 0.4$, o r 0.6% i n t h e r a t i o n , o r with s i n g l e o r a l doses of 100 o r LOO mg/kg f o r 30 days, all mice and r a t s survived; and, except f o r reductions i n food consumption and weight gain, no outward signs of t o x i c i t y were seen. The e f f e c t i v e oral dose of thiabendazole a g a i n s t nematode i n f e c t i o n s i n man and animals h a s been found t o be i n t h e range of 25-50 mg/kg.6,7 Thiabendazole was 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 e r a d i c a t i o n of Hyostrongylus rubidus i n pigs, using a single oral dose of 100 mg/kg.8

152

Sect. 111 - Chemotherapeutics

F l y n n , Ed.

Thiabendazole was active against the l a r v a l stages of Capillaria obsignata infectiohs i n chickens a t 1000 mg/kg, but was not e f f e c t i v e against mature parasites a t this high dose.9 Trials were conducted w i t h thiabendazole i n the treatment of nematode infections in horses.lO,ll A t h mg/kg and 88 mg/kg, orally, the drug was effective against immature and mature small strongyles and against Probstmayria vivipara and O x y u r i s equi. Campbell and Timinskil2 observed development o f itnmunity t o Ascaris i n r a t s cured of t h i s i n f e c t i o n by treatment w i t h thiabendazole.

H

IV Thiabendazole

Other Benzimidazoles. Deaths of sheep following the use of 2phenylbenzimidazole a t 250 mg/kg under conditions o f reduced water intake was investigated by Jones, Leaver, and Milne.l3 Toxicity was a t t r i b u t e d t o kidney damage caused by deposition of a c r y s t a l l i n e metabolite, most l i k e l y 5-0-glucuronyloxy-2-phenylbenzimidazole. The pH of t h e urine and r a t e of urine output were thought t o be c r i t i c a l f a c t o r s affecting deposition of t h e metabolite. A pH of 8.4 o r higher was considered important t o protect animals against the deposition phenomenon when water intake was low. Use of 2-phenylbenzimidazole formulations containing alkalizing i n redients, such as potassium c i t r a t e a , sodium carbonatea, or magnesium s a l t s 5 was proposed by t h e manufacturer (Imperial Chemical Industries) t o minimize the r i s k of t o x i c i t y with t h i s anthelmintic.

B

Preliminary reports of other new anthelmintic benzimidazoles have appeared, although d e t a i l s of biological t e s t i n g are not y e t availablel6-19. Quaternary Ammonium Anthelmintics. A group of related quaternized heterocyclic compounds have been under investigation f o r the treatment of Ascaris infections i n pigs and hookworms in dogs.

I n e a r l i e r reports20, 21, it was f aund that l-methyl-2-(pdimethylaminostyry1)-quinolinium methosulfate (V) protected pigs from l i v e r damage

caused by experimental infections with migrating ascarids. A t a l e v e l of O.Ol@ i n the d i e t , the drug lessened t h e severity of the infection. A l e v e l of 0.056% completely prevented l i v e r lesions when administration of t h e drug w a s s t a r t e d p r i o r t o establishment of the infection. This l e a d has now been extended t o the styrylp idinium analog22 ( V I ) , and t o c e r t a i n phenylbenzothiazolium salts ( V I I ) . 3 Diet l e v e l s of approximately 0.02%, o r lower, of V I o r V I I prevented l i v e r damage i n Ascaris-infected pigs, when medication was i n i t i a t e d two days before inoculation w i t h Ascaris larvae.

F

Chap. 14b.

Antiparasitics

Hoff

153 -

In a p a r a l l e l development, 2-(~-chlorostyryl)-pyridiniurn salts ( V I I I ) were reported t o be highly effective against mature and immature hookworms (Anc lostoma caninurn) in do s a t 5 mg/kg, and could be given i n t h e ration,* -ule24,25. The benzdithiylium salts IX and X, described by Wizinger and Soder26, and f o r which broad anthelmintic a c t i v i t y was claimed, would seem t o conform t o t h i s same general lead. Insufficient data have been published t o a l l o w f i n a l conclusions about the safety and u t i l i t y of these compounds.

VI

V

VII, R

=H

X@

Ix

o r CH3

X O X

Sect. I11

154 -

-

Chemotherapeutics

F l y n n , Ed.

Organophosphate h t h e l m i n t i c s . S veral additional studies of the use of t h e -organophosphates phthalophos27,28 (0,O-diethyl-0-naphthaloximide phosphate, X I ) and haloxon29,30 /6,0-di-( 2-chloroethyl)-0-( 3-chloro-4-methyl -7-coumarinyl)phosphate, XI27 appeared during the year. PhthaloDhos w a s found by Hebden and H a l l 2 7 t o be completely effective against Haemonchus contortus, Osterta l a Trichostro lus E i , and T. colubriformis i n sheep a t g d ( n a t u r a 1 - t this l e v e l , Fhthalophos had variable (38-94$ removal) efficacy against Nematodirus species, and poor a c t i v i t y against the l a r g e bowel worms Oesophagostomum columbianum and 0. venulosum. I n p a r a l l e l t e s t s , thiabendazole was completely effective in the removal of Oesophagostomum species a t 50 mg/kg.

a.,

Studies i n c a t t l e 2 8 showed phthalophos t o be highly effective against Haemonchus, Ostertagia, Trichostrongylus, and Cooperia species a t 5'0 mg/kg and 75 mg/kg, and t o reduce the l e v e l s of Bunostomum infections a t these doses. Hebden and H a l l 2 7 found no toxic e f f e c t s with phthalophos i n sheep a t up t o s i x times the effective doses a t up t o 300 mg/kg), and reported-only a transient diarrhea a t 400 mgfig.

(i.z.,

I n t r i a l s with haloxon, Baker29 confirmed e a r l i e r reports of the anthelmintic effectiveness and r e l a t i v e safety o f t h i s substance i n sheep a t Efficiency against O s t e r t a e circumcincta and Trichostrongylus 50 mg/kg. spathiger was not v i t r i n u s were 95% and 98% respectively, removed by this drug a t 50 mg/kg.

i i m '

(c1cH2cH,0),Po

XI1

XT Phthal ophos

Haloxon

Other Nematocides. A new piperazine derivative3l, 3 2 (l-methylp i p e r a z i n e m p h o n y l p i p e r i d i d e , X I I I ) a sulphonyl analog of diethylcarbamazine ( X I V ) , was found by Teusche1-3~t o eliminate lungworm (Dict ocaulus e form of two f i l a r i a ) infections i n sheep. The drug was administered i n consecutive d a i l y 20 mg/kg doses, subcutaneously. The t o x i c i t y of X I 1 1 r e l a t i v e t o d i ethylcarbamazine was not reported.

,

+

+

Of a s e r i e s of standard anthelmintics studied f o r the control of expefimental C a i l l a r i a obsignata infections i n chickens, methyridine ( X V ) proved highly e ec i v e against both mature and innnature parasites a t doses of 100-15'0 mg/kg given subcutaneously. Complete eradication of Capillaria

,

Chap. 14b.

Hoff

Antiparasitics

155 -

i n f e c t i o n s was achieved with 200 mg/kg of methyridine by t h e oral route33. Haloxon was found f u l l y e f f e c t i v e a g a i n s t a d u l t C a p i l l a r i a a t 50-60 mg/kg i n t h i s study, whereas e f f i c i e n c y a g a i n s t immature p a r a s i t e s was n o t s a t i s f a c t o r y .

XI11

XIV

XV

Die thylcarbamazine

B.

Me t h y r i d i n e

-

Anthelmintics f o r Treatment of Tapeworm I n f e c t i o n s .

Bunamidine. Bunamidine (N,N-dibutyl-4-hexylo phthamidine, XVI) i s r e p r e s e n t a t i v e of a new c l a s s of c e s t o c i d a l agents 3 4 T In i n i t i d experiments, t h e drug was r e p o r t e very e f f e c t i v e a g a i n s t Taenia p i s i f o r m i s , The same substance partially c l e a r e d o r a l l y , a t 25 mg/kg, i n t h e dog3 Dipylidium caninum i n dogs, and some v a r i a b l e e f f e c t a g a i n s t the a s c a r i d Toxascaris leonina was reported. I n c a t s , bunamidine a t a dose of 25 mg/kg Cats w i t h r e s u l t e d i n the complete e l i m i n a t i o n of Taenia taeniaformis. concurrent ascaxis i n f e c t i o n s (Toxocara ‘-experienced an average of 80% clearance of t h i s p a r a s i t e with t h i s dose of bunamidine.

2.

-

The preliminary announcement d i s c l o s e d a c t i v i t y a g a i n s t o t h e r s p e c i e s of tapeworms i n c a t s , dogs, and sheep, but without d e t a i l s of the t e s t s . Good a c t i v i t y a g a i n s t Echinococcus ulosus i n dogs was reported as an e s p e c i a l l y important q u a l i t y of bunamidhe S i d e r e a c t i o n s in dogs and c a t s c o n s i s t e d of occasional emesis and d i a r r h e a with 25 o r 50 mg/kg doses. Chronic treatment of pregnant dogs with 100 mg/kg /day of bunamidine s t a r t i n g f o u r weeks p r i o r t o d e l i v e r y was n o t harmful t o t h e mothers o r pups.35

c1 XVI Bunamidine

XVII Niclosamide

Sect. 111

156 -

- Chemotherapeutics

F l y n n , Ed.

e ~ ~ data on the e f f e c t of niclosamide Niclosamide. S t r ~ f reported (5,2 1 -dicaoro-4' -nitkosalicylanilide, XVII) on tlie metabolism of Hymenolepis diminuta. The compound was found t o i n t e r f e r e with glucose u t i l i z a t i o n i n m o m o g e n a t e s prepared from t h i s parasite.

In a c r i t i c a l trial with niclosamide i n do s infected with Taenia species, clearance was obtained w i t h 100-200 mg/kg.3

4

-

Male Fern Constituents., Blakemore, Bowden, Broadbent, and D r y ~ d a l e ~ ~ isolated some o n e phloroglucihol derivatives from Dryopteris rhizomes and tested them individually f o r cestocidal activity. Flavaspidic acid, aspidin, and desaspidin were active o r a l l y against H enole i s nana in mice, but the monocyclic phenol aspidinol was not a c t i v e % v T

In l a t e r p a p e r ~ 3 9 , 4 ~t,h e SKF' group synthesized simpler monocyclic phloroglucinol derivatives related t o Dryopteris constituents. Compounds of the type XVIII were a c t i v e against experimental g . , e infections i n mice o r a l l y a t 400 mg/kg. Tests with lower doses were not, reported. Many other members of the s e r i e s showed a l a s e r order of anthelmintic activity.

.

Cyclohexane4,&-bis-( 2-ethylisothiocyanate) (XIX) This compound was studied in 319 sheep infected with tapewormsw. A 25 mg/kg dose was considered adequate i n the treatment of simple Moniezia expansa i n f e c t i ~ n s . For multiple cestode infections, a l e v e l of 100 & v s e was recommended. Activity against other species of tapeworms was reported as well. The compound was said t o be well-tolerated, the minimum l e t h a l dose i n sheep being about s i x times the highest recommended dose of 100 mg/kg.

OH

0

XVIII

XIX

R'CO

CH,CH,NCS

R = H o r CH, Rl=

C.

i-C3H7;

n-C4H9; n-C5Hll;

Anthelmintics f o r "Ukm Against Liver Flukes.

Salicylanilides. The use of MapheneB, a mixture of three p a r t s -tribromosalicylanilide (XX) and one p a r t 5,4' -dibromosalicylanilide (XXI), f o r t h e treatment of l i v e r fluke disease i n c a t t l e was f i r s t proposed bjr Lienerd29 43.

3,5,4l

Chap. 14b.

Antiparas itic s

157 -

Hoff

H i l o m i d B (Astra Pharmaceuticals), a 1:l mixture of XX and XXI, was reported by Boray, Happich, and Andrewsh t o be effective (86-98g) against 10-12 week o l d Fasciola he a t i c a infections i n sheep tjhen administered i n o r a l doses of 30 m g / m oft60 mg/kg resulted in 90-100$ removal of larger immature flukes (six weeks or older). These authors observed t h a t mortality due t o acute l i v e r fluke disease usually occurs a f t e r seven o r eight weeks from the time of infections, and hence Hilomid 6 may prove effective i n controlling F. hepatica infections.

hoses

-

t5 ,

S a l i c y l a n i l i es related t o DiapheneQ

i n a patent by Stecker

and H i l o m i d B were described

A s e r i e s of chlorinated 21-hydroxysalicylanilides was described in I C I patents46,47. One member of t h i s series, 3,31,5,51,6-pentachloro-2,21dihydroxybenzanilide (oxyclozanide, XXII) w a s studied i n 1226 sheep and 530 c a t t l e by Walle$8. A 15 mg/kg o r a l dose was recommended for treatment of orally, f o r c a t t l e . Tfie author observed a four-fold sheep, and 10-15 mg/kg, margin of safety i n normal animals, but a narrower margin i n dehydrated animals, o r those suffering from l i v e r damage. The s t a t e d doses were reported effective f o r the eradication of mature flukes. Substantially'higher doses (50-60 mg/kg\, removed a high proportion of immature (over six weeks) flukes, but these doses are i n the toxic range. Toxic side-effects were observed in doses above 30 mg/kg: with deaths occurring a t 60 mg/kg. (sheep and c a t t l e ) .

Ry$

CONH O

XXI,

r

c1

Br XX,

-B

R=Br R = H

c1

XXII Oxyclozanide

Other Fasciolacides. Bayer 9015 (2,2~-dihydro~-3,31-dichl.ooo-~,~1d i n i t r o b i p w , XXIII)YJ,51 appears t o be the most potent member of t h e group of anthelmintic bisphenols which includes hexachlorophene, bithionol, and oxyclozanide. I n two recent reports, it was found effective against mature l i v e r and a t 6 mg/kg 51 i n sheep. I n other c r i t i c a l trialsSO, flukes a t 5 mg/kg%, 93-100$ clearance of mature parasites was achieved with 3 mg/kg, orally, i n 21 sheep. No gross t o x i c i t y due t o the drug was reported a t these dose levels, although some reduction i n r a t e of weight gain was seen i n some experiments a t 6 mg/kg. Lee, OINuallain, and Power51 indicated that the manufacturer had observed non-fatal toxic reactions with Bayer 9015 i n 20 sheep dosed with 20 mg/kg. Bayer 9015 a t 5 mg/kg exhibited 40-57% efficiency against immature flukes up t o 10 mm i n length, and 53-100$ a c t i v i t y against l a r g e r immature fonns51. The drug was ineffectiGe against the lancet fluke Dicrocoelium dendriticum i n one sheep which harbored t h i s parasite.

Sect. I11

158

-

Ghemotherapeutics

Flynn, Ed.

A series of novel organophosphates was reported i n Bayer p a t e n t s t o possess a c t i v i t y a g a i n s t l i v e r f l u k e s i n r a t s and sheep a t 5-15 mg/kg, o r a l l y and i n t r a m u ~ c u l a r l y 5 ~ , S 3 .

Kondos and McClymont54 produced evidence that t h e f a s c i o l a c i d a l a c t i v i t y of carbon t e t r a c h l o r i d e i s i n d i r e c t . Anthelmintic e f f e c t of t h e drug i n v i t r o w a s low but was enhanced g r e a t l y by a d d i t i o n of f r e s h l i v e r s l i c e s o r a r n l i v e r preparation. "he i n viva a c t i v i t y of ccl4 w a s much higher than could be explained by t h e o b s e r v e r i n v i t r o e f f e c t of t h e substance. The anthelmintic a c t i v i t y w a s a t t r i b u t e d t o a n unknown metabolic product o r products.

-

Although t h e range of a n t h e l m i n t i c s a c t i v e a g a i n s t t h e l i v e r f l u k e i s widening, t h e goal of a n agent a c t i v e a g a i n s t immature f l u k e s a t safe dose l e v e l s appears y e t t o be achieved. Some of t h e phenolic a n t h e l m i n t i c s described i n t h e papers c i t e d above r e p r e s e n t progress i n t h i s d i r e c t i o n , i n t h a t some e f f e c t a g a i n s t largerimmatures was seen a t elevated dose l e v e l s , b u t t h e reported t o x i c i t y of t h e products would l i m i t o r p r o h i b i t use of t h e s e higher doses. 111. Antiprotozoal Agents A.

Coccidiostats

Quinolines. Reference t o a n t i c o c c i d i a l q u i n o l i n e s appeared i n a number of Datents by Norwich. Comvounds of the tme XXIV were s a i d t o be e f f e c t i v e & t h e c o i t r o l of Eimeria tenella, E. a;&vulina, and E. n e c a t r i x Details of b i o l o g i c a l i n f e c t i o n s a t d i e t l e v e l s between 0 . 7 E d 0.1%W95b. t e s t i n g have n o t y e t been published. Buquinolate (XXIV, R=Rl=A-butyl, R21 e t h y l ) has been proposed f o r treatment o f c o c c i d i o s i s i n b r o i l e r chickens a t a concentration i n f e e d of 75 grams per t o n (0.00825$).57

XXIV

R and R 1 a r e e t h y l , propyl, o r butyl. R,

i s lower a l k y l .

Chap. 14b.

Antiparasitics

-+E.

Hoff

159 -

@minobenzoic Acid Anta o n i s t s . Exceptional a n t i c o c c i d i a l a t i v i t y was found with methyl 4-acetylarmno-2-e ombenzoate (ethopabate. X X V ) . ?8 This substance was reported a c t i v e a g a i n s t maxima chickens'at O;OOOl$ i n t h e d i e t . C o c c i d i o s t a t i c a c t i v i t y of e t h o p x w a s reversed by shultaneous a d m i n i s t r a t i o n of E-aminobenzoic acid. Ethopabate was a l s o found a c t i v e a g a i n s t E. b r u n e t t i and E. acervulina, but a l e v e l of 0.01% i n the f e e d was ineffectTveE. t e n e l l a .

-

NHCOCHH,

xxv Ethopabate Ryley59 examined t h e e f f i c a c y of a combination of 1,l.I -dimethyl-4, 4I-bipyridylium d i c h l o r i d e and sulfamethazine f o r t h e control of outbreaks of c o c c i d i o s i s i n chickens. The combination, known by t h e I C I trademark ParameeQ, was used i n a f i n a l concentration i n water amounting t o 0.02% sodium sulfamethasine and 0.0625% of t h e bipyridylium dichloride. P a r a m e z B w a s s a i d t o be e f f e c t i v e a g a i n s t Eimeria b r u n e t t i , E. maxima, and E. a c e r v u l i n a a t t h i s concentration without diminishing r a t e o f growth of the b i r d s . The bipyridylium compound alone was n o t e f f e c t i v e a t non-toxic l e v e l s . The author speculated t h a t t h e lower l e v e l of sulfa i n t h i s combination should reduce t h e r i s k of development of the haemorrhagic syndrome uhich has been observed wi.th t h e use of sulfonamide c o c c i d i o s t a t s .

Ball and Warren60 reviewed t h e h i s t o r y of sulfonamide combinations as c o c c i d i o s t a t s and s t u d i e d combinations of 4,4' -diaminodiphenylsulfone (DDS) with 2,~-diamino-5-(4-chlorophenyl)-6-ethylpyrimidine (pyrimethamine) and w i t h 2-amino-~-dimethylamino-~-(~-chlorophenyl)-6-ethylpyrimidinehydrochloride (May and Baker 4408), and a l s o combinations of pyrimethamine with sulfaquinoxal i n e . P o t e n t i a t i o n was seen w i t h all of t h e s e combinations, but the f i n a l u t i l i t y w a s not evaluated.

-

Other Coccidiostats. Preliminary r e p o r t s i n p a t e n t s claimed a n t i c o c c i d i a l a c t i v i t y f o r sulfonylurethanes61,62, lincomycin63,- y-pyridone&, . benzoquinones65, -&d n i t r o p y r r o l e s66. Nicotinamid e a n t a g o n i s t s ( 6-aminonico tinamide, pyridine -3 -sulf onamide, and 3-acetylpyridine) were r e p o r t e d by Ball, Warren, and Parnel167 t o possess c o c c i d i o s t a t i c a c t i v i t y . S i m i l a r observations have been made by McManus, Clark, Rogers, and Cuckler68, who noted t o x i c i t y of t h e s e compounds i n chickens. B,

Histomonostats

A study by Flowers, Hall, and Grumbles69 confirmed e a r l i e r r e p o r t s of the u t i l i t y of 1,2-dimethyl-5'-nitroimidazole (dimetridazole, XXVI) f o r t h e

160 -

Sect. 111

-

Chemotherapeutics

F l y n n , Ed.

prevention and treatment of enterohepatitis i n turkeys. The compound was judged equal t o o r b e t t e r than 2-acetylamino-5-nitrothiazole (Enheptin-A) f o r the prevention of enterohepatitis. 5-Nitro-2-furaldehyde acetylhydrazone ( X W I I ) w a s found by Hall, Flowers, and Grumbles70 t o be efficacious (comparable t o Enheptin-A and dimetridazole ) in the prevention of l o s s e s from enterohepatitis when administered prophylactically i n t h e feed a t 0.022%.

XXVI Dime tridazole

XXVI Enheptin-A

Lucas and Goose71 presanted a detailed description of a screening procedure f o r antiblackhead agents, using chickens infected with Histomonas meleagridis. Use of chickens instead of turkeys for screening was said t o be more convenient and economical.

IV. Organophosphate Insecticides A promising new organophosphate insecticide was announced by the Shell group72. This compound, an a-phenylvinyl phosphate (XXVIII) was said t o possess good a c t i v i t y against several species of insects, and i t s acute o r a l LDso was greater than 4000 mg/kg i n mice and r a t s . It i s close in structure t o chlorfenvinphos, (XXIX),which was evaluated i n f i e l d trials for the protection of sheep against the maggot f l y , Lucilia sericata73. Chlorfenvinphos was judge’d equivalent i n efficacy t o d i e l d r i n a t comparable concentration i n a d i p (0.05% compared with 0.04% dieldrin).

Cl XXVIII

XXIX

Details of the synthesis and t e s t i n g (mouse-mosquito assay and mouse t o x i c i t y t e s t s ) of a s e r i e s of phosphorylated benzenesul onamides i n the famophos s e r i e s were presented by Wagner, Baer, and Berkelhammer76

.

Trials with various older o r g ophosphates and other insecticides were reported by S h a m , Chhabra, and KapoorT ( c a t t l e grubs), Unwin76 ( c a t t l e grubs), Dnunmond and Medley77 ( c a t t l e t i c k s ) , and by Drummond and Graham78 ( c a t t l e grubs, Oestrus i n sheep, and Gastrophilus species).

Chap. 14b.

Antipa ras iti c s

161 -

Hoff

References

-

.'--,

Monteoliva, F. S. Rasero, T. L. Gorie and -F. --Mayor, ".-"..", Nature, L 205 'C, 1 111 (1965). -2 Rothstein, Comp. Biochem. Physiol.,- I = I 54.l (1965). TT.. Morales. Del CastillMorales, E e r i e n t i a , 20, lb (1964). S. F. A s h , and J. F. Tucker, Nature, 209, 306 .-9 Q. H. Gibson and M. H. Smith, Proc. Roy.3&. (London), . ondon), Ser. B, 163, 206 (1965). C. F. F e i s t , C. P. Reid, and F. M. Fisher, Jr.,- J. P a r a s i t o l . , - c 7 6 (1965). her. Jr. H. Oya, G. Kikuchi, T. Bando and H. Hayashi,,-qE 2, 229 (1965). 1 (1965). lh. L. G. Warren, n t n e r , J. Biol. Chem., 236, 915 (1961). li. I. Seidman and lj. S. Bloom and N. Entner, Biochim. Biophys. Acta., 99, 22 (1965). l k . E. Kmebec and E. Bueding, Comp. Biochem. Physiol., 15, 271 (1965). 11 W. P. Rogers, Comp. Biochem. Physiol., 311 (196n. l m . N. E. Downey, B r i t . Vet. J., 121, 362 (1965). m. P a r a s i k l . , L6, 353 (1965). laquero. and J. F. Duda. Duda, E Exp. Parasitol., In. J. R. Seed, M. A. Baquero, itment of t h e Domestic 2. T. E. Gibson, Recent Advances i n t h e A n t h e l x t i c Treatment Animals. Animals. Chapter i n Advances in Parasitology, Volume 111, Edited by Ben Dawes, Academic Press, London and New York, 1964. 3. R. J. S c h i t z e r , and F. Hawking, Experimental Chemotherapy, Vol. IV, Academic Press. New York and London.> 1966. 4. D. Thienpont, 0. F. J. Vanparijs, A. H. M. Raeymaekers, J. Vandenberk, P. J. A. Demoen, F. T. N. Allewijn, R. P. H. Marsboom, C. J. E. Niemegeers, K. H. L. Schellekens, and P. A. J. Janssen, Nature, 209, 1084 (1966). 6,505,806, (1965). 4a R. F. Arnold, C. G. Hartland, A. Siedsma, Ne-dnatent 5. J. K. Walley, Vet. Rec., 78, 406 (1966). 6. H. J. Robfison, H. C. Stoerk, and 0. E. Graessle, Toxicol. Appl. Phamacol., 7, 53 (1965). 7. H. D. Brown, A. R. Matzuk, I. R. I l v e s , L. H. Peterson, S. A. Harris, L. H. S a r e t t , J. R. Egerton J. J. Yakstis, W. C. Campbell, and A. C. Guckler, J. Am. Chem. SOC 83, 1761 (1961). 8. M. KarKvic, 0. Vrazic, and I. Drezancic, Vet. Arhiv., 34, 189 (1964); Vet. -Bull 0,35, 1398 (1965). 9. J. Hendriks, Tijdschr. Diergeneesk, 90, 1062 (1965'). 10. M. Meunier, Vet. Thesis, P a r i s ( 1 9 6 n 11. A. J. S n i j d e r s , S. G. Anema and J. P. Louw, J. S. A f r . Vet. Med. ASSOC., 36, 263 (1965). 12. W. C. Campbell, and S. F. Timinski, J. Parasitol., 51, 712 (1965). 13 L. H. P. Jones, D. D. Leaver and A. A. Milne, Res. Et. Sci., 6, 316 (1965). 14. Imperial- Chemical I n d u s t r i e s , A u s t r a l i a n Patent 25733/62. 15. Imperial Chemical I n d u s t r i e s , - E i r e P a t e n t 549/65. 16. T. J. Ursprung, U. S. P a t e n t 3,179,669 (1965). 1 7 T. J. Ursprung, U. S. Patent 3,179,670 (1965). 18. R. Aries, Address t o 84th Congress of t h e French Ass'n. f o r t h e Advancement of Sciences; Reported in Informations Chimie, October, 1965, p. 58. ) 1 9 Farbenfab. Bayer, Beglain-65 20. I. B. Wood, J. A. Pankavich and R. E. Bambury, U. S. P a t e n t 3,152,042 (1964). 21. I. B. Wood, H. Berger, J. A. Pankavich, and E. Waletzky, J. P a r a s i t o l . , 2 (suppl.), 60 (196h). 22. I. B. Wood, R. E. Bambury and H. Berger, U. S. Patent 3,179,559 (1965). 23 American Cyanamid, Netherlands Patent 6,501,092 (1965).

la. lb lc. Id. le If

..

M. M. J. A.

2,

I

I

-

vr I-

w., 9, .

u.

.

-

m,

2,

.

-.J

-

.

-&*A

\ A , " , / .

Sect. 111

162 -

.

- Chemotherapeutics

F l y n n , Ed.

25 26. 27 28.

American Cyanamid, Canadian Patent 698,704 (1964). I. B. Wood, J. A. Pankavich, and R. E. Bambury, U. S. Patent 3,177,116 (1965). R. Wizinger and L. Soder. U. S. Patent 3,2ll,748 (1965). S. P. Hebden and C. A. H i l l , Vet. Rec., ? 7, 207 (1965). D. D. Cox, A. D. Allen, Me T. Mullee, and E. M. Maurer, J. Parasitol.,

29 30. 31

N. F. Baker, Am. J. Vet. Res., 26, 651 (1965). C. J. Bosman, J. S. A f r . Vet. Med. ASSOC., 36, 251 (1965). E. Teuscher, Schweiz, Arch. Tierheilk., E F 7 4 (1964); Vet. Bull.,

32-

A. C. R. 0. V. C.

24 e

33

34.

35 36. 37. 38

39.

40

41

. 44.

42.

43

45

46

47:

48 49

50.

(196h).

52.

55

56

57* 58. 59 60.

61. 62.

63 64. 65

34,

4591

Marzer and A. Ilvespaa, U. S. Patent 3,211,608 (1965). C. Norton and L. P. Joyner, J. Comp. Path., 137 (1965). Baltzly, R. B. Burrows, M. Harfenist, K. A. Fuller, J. E. D. Keeling, D. Standen, C. J. Hatton, V. J. Nunns, D. A. Rawes, B. D. Blood, Moya, and J. L. Lelijveld, Nature, 206, 409 (1965). J. Hatton, Vet. Rec., 77, 4 m 6 $ ‘ J 7 R. Strufe, i n Proceedings of the I I I r d International Congress of Chemotherapy, S t u t t g a r t , Germany, July, 1963, Vol. 11, p. 1544, Hafner, New York, 1964, D. D. Cox and M. T. Mullee, J. Parasitol., 51 (suppl.) 30 (1965). R. C. Blakemore, K. Bowden, J. L. BroadbentFand K. C. Drysdale, J. Pham. Pharmacol., 6 l ., 464 (1964). K. Bowden and W. J. Ross, J. Pharm. Pharmacol., l7, 239 (1965). K. Bowden, J. L. Bro?dbent/ and W. J. Ross, B r i t . J. Pharmacol., 24, 7 l h (1965). M. Graber, Cahiers Med. Vet., 38, 87 (1965). E. Lienert, Arch. E Veterinzrmed, 17, 101 (1963). E. Lienert, T i e r a r z z . Umschau g, 8 n 1 9 6 3 ) . J. C. Boray, F. A. Happich, and J. C. Andrews, Vet. Rec., 77, 175 (1965). H. C. Stecker, Australian Patent 34,997/63 ( 1 9 6 r Imperial Chemical Industries, Netherlands Patent 6,409,241 (1965) Imperial Chemical Industries, Netherlands Patent 6,409,325;. J. K. Walley, V e t . Rec., 267 (1966). Nederl. Combinatie Voor Chem. Ind., Netherlands Patent 6,405,517 (1965). S. E. Knapp, P. A. Nyberg, V. T. Dutsen, and J. N. Shaw, Am. J. Vet. Res., 26, 1071 (1965). R. P. Lee, T. O’Nuallain and J. H. Power, Vet. Rec., 196 (1966). Bayer, German Patent 1,195,750 (1965). Bayer, Netherlands Patent 6,412,023 (196.5). A. C. Kondos, and G. L. McClymont, Nature, 206, 846 (1965). Norwich, Belgian Patent 640,906 (19E. J. Watson, Jr., Canadian Patent 697,792. Federal Register, 31, 4529 (1966). E. F. Rogers, R. L T C l a r k , H. J. Becker, A. A. Pessolano, W. J. Leanza, E. C. McManus, A. J. Andriuli, and A. C. Cuckler, Proc. SOC. Exp. Biol. E., 117, 488 (1964). J. F. Ryley, Vet. Rec., 77, 1498 (1965). S. J, Ball, and E. W. Warren, Vet. Rec., 77, 1028 (1965). Hoechst, Swiss Patent 380,714 (196b). Hoechst, Swiss Patent 392,487 (1965). R. E. Bloss, and R, E. Kohls, U. S. Patent 3,150,042 (1964). G. T. Stevenson, U. S. Patent 3,206,358 (1965). Hoechst, Netherlands Patent 6,415,174 (1965).

.53. 54. 51

2

(su~~L 33)(1965). ,

s,

.

.

2,

2,

-

C h a p . 14b.

66 67 68. 69

70. 71. 72.

73.

74.

Antiparasitics

Rhae-Poulenc, Netherlands Patent S. J. B a l l , E. W. Warren and E. W. E. C. McManus, R. L. Clark, E. F. Indust. Microbiol., 6, 4k (1964). A. I. Flowers, C. F. Hall, and L. (1965) C. F. Hall, A. I. Flowers, and L.

(1965’)

163 -

Hoff

6,405,568 (1965). P a r n e l l , Nature, 397 (1965). Rogers, and A. C. CucMer, Develop.

208,

C. Grumbles, Avian Diseases,

C. Grumbles, Avian Diseases,

2, 9,

395 400

121,

J. M. S. Lucas, and J. Goose, B r i t . Vet. J., 65 (1965). R. R. Whetstone, D. D. P h i l l i p s , Y. P. Sun and L. F. Ward, Jr., Abstr. Papers, Am. Chem. SOC., 12A (1965’). J. C. Wood, K. W. Page, P. R. M. Brown, M. S. Smith, and D. A. M. Fergu.son, Vet. Rec., 77, 896 (1965). F. A. Wagner, Jr., R. W. Baer, and G. Berkelhammer, J. Med. Chem., 377

2,

(1965)

2,

75 R. M. Sharma, R. C. Chhabra, and B. B. Kapoor, Vet. Rec., 77, 107 (1965 ) * 76. D. D. Unwin, Vet. Rec., 77, 593 (1965). 77. R. 0. Drummond and J. G. Medley, J. Econ. Entomol., 58, 1131 (1965). 78. R. 0. Drummond and 0. H. G r a h a m , Vet. Rec., 77, l b l 8 7 1 9 6 5 ) .

S e c t i o n IV - M e t a b o l i c Diseases and Endocrine Function E d i t o r : R. V. Heinzelman, The Upjohn Company, Kalamazoo, Michigan C h a p t e r 1 5 . A n t i d i a b e t i c Agents Rex P i n s o n , Chas. P f i z e r 6 Co., I n c . , Groton, C o n n e c t i c u t The hypoglycemic d r u g s t h a t are used t o d a y t o treat w e l l o v e r a m i l l i o n d i a b e t i c s are descended i n ,a d i r e c t l i n e from two e s s e n t i a l l y chance b o t h made more t h a n 20 y e a r s ago. The t r a n s i t i o n from t h e s e observations o b s e r v a t i o n s ( t h a t c e r t a i n a n t i b a c t e r i a l sulfonamides c a u s e d hypoglycemia' and t h a t a s i m p l e g u a n i d i n e d e r i v a t i v e reduced t h e blood s u g a r of r a b b i t s 2 ) t o s a f e , e f f e c t i v e t h e r a p y i s a real achievement of m e d i c i n a l c h e m i s t r y . N e v e r t h e l e s s , i t would be u n r e a l i s t i c n o t t o r e c o g n i z e t h a t i n s p i t e of t h e v a s t body of f a c t and c o n j e c t u r e now c o n t a i n e d i n t h e l i t e r a t u r e of d i a b e t e s , m e t a b o l i c b i o c h e m i s t r y and t h e c o n t r o l of metabolism; no a n t i d i a b e t i c drug h a s y e t come from r a t i o n a l a p p l i c a t i o n of knowledge a b o u t t h e s e p r o c e s s e s . Compounds u n r e l a t e d t o t h e s u l f o n y l u r e a s o r g u a n i d i n e d e r i v a t i v e s have, t o be s u r e , been s t u d i e d f o r t h e i r hypoglycemic p r o p e r t i e s some e x t e n s i v e l y -b u t a l l were a p p a r e n t l y f i i s t i d e n t i f i e d by a l a r g e l y e m p i r i c a l o b s e r v a t i o n of t h e i r e f f e c t on blood s u g a r i n e x p e r i m e n t a l a n i m a l s .

--

--

B r i e f s e c t i o n s on d i a b e t e s as a d i s e a s e e n t i t y and on some of t h e hormonal f a c t o r s and m e t a b o l i c p r o c e s s e s t h a t are deranged i n d i a b e t e s have been i n c l u d e d . L i m i t a t i o n s b o t h of s p a c e and of t h e t a l e n t s of t h e r e v i e w e r p r e c l u d e e i t h e r comprehensive o r profound t r e a t m e n t of t h e s e a s p e c t s and Rather, t h e a i m i s t o i n v i t e n o t h i n g approaching t h i s h a s been a t t e m p t e d . a t t e n t i o n a g a i n t o t h e r e l a t i o n s h i p of t h e s e f a c t o r s t o t h e d i s e a s e and t o p r o v i d e a few l e a d i n g r e f e r e n c e s t h a t may s t i m u l a t e thought on p o s s i b l e new approaches t o a n t i d i a b e t i c drugs. Diabetes Mellitus D i a b e t e s i s a h e r e d i t a r y d i s e a s e 3 y 4 b u t t h e n a t u r e of t h e g e n e t i c I t i n v o l v e s a widespread d i s d e f e c t and i t s primary l e s i o n are unknown. o r d e r of metabolism b u t e x h a u s t i v e s t u d y h a s n o t y e t determined whether t h i s i s a c a u s e o r consequence of t h e hormonal, v a s c u l a r and n e u r o l o g i c a l abnormali t i e s of t h e d i s e a s e . Reasonably u p - t o - d a t e t r e a t m e n t of a l l of t h e s e a s p e c t s a p p e a r i n t h e symposium volume from t h e F i f t h Congress of t h e I n t e r n a t i o n a l D i a b e t e s F e d e r a t i o n ( T o r o n t o , 1964).5 A c h a p t e r on t h e unanswered q u e s t i o n s about human d i a b e t e s by Williams and Wood6 and t h e d i s c u s s i o n t h a t followed i t s p r e s e n t a t i o n c o n t a i n a h o s t of s u g g e s t i o n s f o r r e a d i n g . Another review by W i l l i a m s Y 7 and B e r s o n ' s 1965 Banting Memorial L e c t u r e 8 are a l s o h e l p f u l g u i d e s t o c u r r e n t thought on d i a b e t e s , Insulin The dominant c h a r a c t e r i s t i c of d i a b e t e s i s an a b s o l u t e o r r e l a t i v e l a c k of e f f e c t i v e i n s u l i n a c t i o n . I n some m a n i f e s t a t i o n s o f t h e d i s e a s e , a d e f i c i e n c y of p a n c r e a t i c 8-cell f u n c t i o n i s c l e a r l y i n v o l v e d , but i n o t h e r s , t h e d e f e c t a p p e a r s most l i k e l y t o be a f a c t o r o r c o n d i t i o n t h a t p r e v e n t s insulin action.

Chap. 15

Antidiabetics

Pinson

165 -

C a h i l l h a s w r i t t e n an o u t s t a n d i n g l y r e a d a b l e account of i n s u l i n e f f e c t s and t h e i r d e f i c i e n c y i n d i a b e t e ~ . ~A review of t h e i n s u l i n l i t e r a t u r e t o J u l y 1965" c o v e r s many a s p e c t s of i n s u l i n s t r u c t u r e , s y n t h e s i s , s t o r a g e and release but does n o t c o v e r i n s u l i n a c t i o n . The review of s t r u c t u r e , p a r t i a l and t o t a l s y n t h e s i s , and t h e i n f l u e n c e o f s t r u c t u r e on a c t i v i t y i s q u i t e e x h a u s t i v e . E v e n t s t h a t o c c u r d u r i n g i n s u l i n release from t h e p a n c r e a s have been observed by e l e c t r o n m i c r o s c o p y , l l and much i s a l s o known about B-cell metabolism.12 The d e t a i l s of t h e p r o c e s s by which g l u c o s e a c t u a l l y t r i g g e r s i n s u l i n release, however, are n o t y e t understood i n any d e t a i l .

One of t h e most fundamental a c t i v i t i e s of i n s u l i n i s t h e removal of a b a r r i e r t o t h e e n t r of g l u c o s e i n t o f a t and muscle c e l l s . The f u n c t i o n s

of i n s u l i n i n glucose13914 and amino acid15 t r a n s p o r t and t h e r o l e of sodium and potassium i o n s i n t h e s e p r o c e s s e s 1 6 are t h e s u b j e c t of a r e c e n t symposium. Important though t h e t r a n s p o r t e f f e c t of i n s u l i n i s , i t i s clear t h a t i t i s n o t the primary a c t i o n from which a l l o t h e r e f f e c t s r e s u l t . Some a c t i o n s of i n s u l i n , as of most hormones, have been t r a c e d t o e f f e c t s on t h e s y n t h e s i s of enzyme p r o t e i n . Some of t h e s e e f f e c t s have been p o s t u l a t e d t o occur a t t h e l e v e l of DNA d i r e c t e d RNA s y n t h e s i s , whereas o t h e r s a f f e c t p r o c e s s e s n o t y e t c l e a r l y i d e n t i f i e d . I n s u l i n e f f e c t s on l i v e r glycogen s y n t h e s i s and breakdownl7 have been e x p l a i n e d as p a r t of a g e n e r a l t h e o r y of i n s u l i n i n d u c t i o n of g l y c o l y t i c enzymes and s u p p r e s s i o n of gluconeogenic enzymes. l8 Regulation of g l u c o k i n a s e and hexokinase a c t i v i t y depends on g l u c o s e c o n c e n t r a t i o n f o r immediate c o n t r o l , b u t on i n s u l i n induced d e novo enzyme s y n t h e s i s , a p p a r e n t l y through c o n t r o l of new RNA p r o d u c t i o n , o v e r t h e l o n g e r term.19 H similar e f f e c t a p p e a r s t o be r e s p o n s i b l e f o r i n s u l i n i n d u c t i o n of s a t u r a t e d and monou n s a t u r a t e d f a t t y a c i d s y n t h e s i z i n g enzymes. 20,21 Evidence h a s also been obt a i n e d t h a t i n s u l i n s t i m u l a t i o n of h e p a t i c p r o t e i n s y n t h e s i s i s due t o an as y e t undefined e f f e c t on ribosomes.22 Plasma f a c t o r s t h a t i n f l u e n c e i n s u l i n a c t i o n . P a t i e n t s t r e a t e d w i t h i n s u l i n develop i n s u l i n a n t i b o d i e s , and t h e r e h a s been much s p e c u l a t i o n , now l a r g e l y d i s c o u n t e d , 8 t h a t an autoimmune r e a c t i o n may be involved i n t h e e t i o l o g y of d i a b e t e s . Immune i n s u l i n r e s i s t a n c e as a t h e r a p e u t i c problem, Considerable research has o c c u r s i n o n l y a small m i n o r i t y o f been devoted t o s t r u c t u r a l m o d i f i c a t i o n s of i n s u l i n i n an e f f o r t t o reduce Even homologous i n s u l i n s , however, have proved t o be a n t i antigenicity." g e n i c , i f a d m i n i s t e r e d i n p a r t i c u l a r ways. 24

patient^.^^,^^

I t i s now beyond d i s p u t e t h a t t h e r e e x i s t i n plasma, s u b s t a n c e s o t h e r than p a n c r e a t i c i n s u l i n and i n s u l i n a n t i b o d i e s t h a t e i t h e r have i n s u l i n l i k e a c t i v i t y o r are c a p a b l e of modifying t h e a c t i o n of i n s u l i n . 2 5 The chemical i d e n t i t y and e s p e c i a l l y t h e p h y s i o l o g i c a l s i g n i f i c a n c e of t h e s e plasma c o n s t i t u e n t s , however, s t i l l remains o b s c u r e . The s u b s t a n t i a l amount of res e a r c h on t h e s e s u b s t a n c e s o v e r t h e l a s t f i v e o r s i x y e a r s stems from two o b s e r v a t i o n s ; 1 ) t h a t t h e amount of i n s u l i n - l i k e a c t i v i t y a s s a y a b l e i n plasma by u s i n g a d i p o s e t i s s u e as t h e a s s a y system i s much.'greater than when muscle i s employed and t h a t b o t h show more i n s u l i n - l i k e a c t i v i t y than c a n be d e t e c t ,32 ed by r a d i o i m m u n ~ a s s a y ~ ~ and 2 ) t h a t v a r i o u s albumin f r a c t i o n s i s o l a t e d from plasma are c a p a b l e of i n h i b i t i n g t h e e f f e c t of i n s u l i n on i s o l a t e d muscle.

166 -

Sect.

IV - Metabolic

& Endocrine

H e i n z e l m a n , Ed.

The first observation has been pursued by several groups most extensively by Antoniades,26,27 Samaan and Fraser,28,29 and Froesch.jo Antoniades and his co-workers have based their investigation on the fractionation of plasma, using an ion exchange resin, into "free" and "bound" insulin. The "free" fraction is apparently pancreatic insulin, since it is detectable by radioimmunoassay and stimulates glucose uptake by muscle. The "bound" fraction is not detectable by radioimmunoassay, stimulates glucose uptake by adipose tissue but not by muscle (unless an adipose tissue extract is added). Samaan and Fraser also distinguish two types of insulin-like activity. One, which they term "typical", is inactivated by insulin antibodies whereas the othef,-or "atypical" insulin is not. Froesch and his collaborators, working along lines very similar to Samaan and Fraser, classify the two kinds of plasma insulin-like activity as antibody "suppressible" and "non-suppressible." Davidson has studied a material with insulin-like activity on the rat diaphragm that is obtained from plasma by acid ethanol treatment.33 Power is working with a serum protein which has insulin-like activity and augments the action of insulin on the rat fat pad and which may prove to have relevance to the theories of A n t o n i a d e ~ . ~ ~ The chief difference between "atypical" or %on-suppressible" insulin, on the one hand, and Antoniades "bound" insulin on the other,is that the plasma concentrations of the former do not vary in response to changing plasma glucose levels, but the latter does. From his work, Antoniades concludes that the plasma insulin system he describes has a physiological role in the modulation of insulin activity. He visualizes a system in which insulin becomes bound or unbound in response to physiological stimuli and that diabetes may represent, in part at least, a disorder of this system. Although Antoniades observations have been confirmedY35his interpretations have been challenged, especially by Berson.8 Froesch appears to be making the most progress toward characterizing "bound" or %on-suppressible" insulin. He and his colleagues are working toward the isolation and characterization of this fraction. Their data thus far suggest that it is a small peptide, not identical with pancreatic insulin, with a molecular weight of about 6000, which is complexed with a large basic protein with a molecular weight over 100,000.3° From present evidence, it is difficult to ascribe a physiological function to these substances. It seems clear that they are r.ot pancreetic insulin in equilibrium with a carrier protein complex as are many hormones such as cortisol and thyroxine. Plasma concentrations (except for Antoniades "bound" insulin) do not vary and the activity is-not found in lymph, where immunoassayable insulin levels closely parallel those in p 1 a ~ m a . l ~Perhaps ~ the isolation work Froesch is now doing will clarify their sratus. Vallance-Owen first reported that dilute solutions of an albumin fraction were capable of inhibiting the effect of insulin on the rat diaphragm and further showed that albumin isolated from the plasma of diabetics was capable of inhibiting insulin action at a lower concentration than was the correspondin fraction from n ~ n - d i a b e t i c s . ~ These ~ observations have now Is hypothesis that the inhibitor, which been ~ o n f i r m e d , ~ but ~ ,Vallance-Owen ~~ he named synalbumin, is the albumin bound B-chain of insulin now seems highly

C h a p . 15

Antidiabetics

Pinson

167 -

q u e s t i o n a b l e . Mixtures of B-chain and albumin i n h i b i t g l u c o s e uptake i n diaphragm o n l y a t c o n c e n t r a t i o n s o f B-cha'in many f o l d h i g h e r than seem p o s s i b l y a t t a i n a b l e i n p l a ~ m a . ~Because ~ , ~ ~ t h e albumin i n s u l i n i n h i b i t o r i s found i n a p p a r e n t l y h i g h e r c o n c e n t r a t i o n s i n t h e plasma of d i a b e t i c s t h a n i n t h e plasma of n o n - d i a b e t i c s , Valiance-Owen h a s suggested t h a t t h i s s u b s t a n c e may be involved i n the e t i o l o g y of d i a b e t e s . The p h y s i o l o g i c a l s i g n i f i c a n c e of t h e Vallance-Owen f a c t o r seems h i g h l y dubious. Undiluted plasma h a s no i n h i b i t o r y e f f e c t on diaphrams g v i t r o and no e f f e c t h a s been d e t e c t e d i n v i v o . Moreover, i n t r a v e n o u s i n f u s i o n s of i n s u l i n B-chain have no e f f e c t on blood s u g a r . 8 Other Hormones and Humoral F a c t o r s S e v e r a l o t h e r hormones have been s t u d i e d i n t e n s i v e l y i n c o n n e c t i o n w i t h d i a b e t e s because of t h e i r e f f e c t s on metabolism and i n s u l i n a c t i o n . 6 Growth hormone and t h e g l u c o c o r t i c o i d s have o f t e n been c l a s s i f i e d as " d i a b e t o g e n i c " because t h e i r e f f e c t s g e n e r a l l y oppose t h o s e of i n s u l i n . Growth hormone d e p r e s s e s t h e p h o s p h o r y l a t i n g c a p a c i t y and i n s u l i n r e s p o n s i v e n e s s of s k e l e t a l muscle and s t i m u l a t e s g l u c o s e o x i d a t i o n i n a d i p o s e t i s s u e w i t h o u t a l t e r i n g i t s r e s p o n s i v e n e s s t o i n s u l i n , b u t t h e mechanism of t h e s e e f f e c t s h a s n o t been e s t a b l i s h e d . The e v i d e n c e o b t a i n e d t h u s f a r , however, i s cons i s t e n t w i t h t h e view t h a t t h e y are t h e r e s u l t of t h e l i p o l y s i s s t i m u l a t i n g e f f e c t of growth hormone.41 The g l u c o c o r t i c o i d s markedly i n c r e a s e t h e a c t i v i t y of key enzymes of g l u c o n e o g e n e s i s and some e v i d e n c e s u g g e s t s t h a t t h i s e f f e c t i s d u e , i n p a r t a t l e a s t , t o i n c r e a s e d enzyme system s n t h e s i s r e s u l t i n g from a s t i m u Other e v i d e n c e , however, does l a t i o n of t h e p r o d u c t i o n of messenger RNA.43 not e n t i r e l y support t h i s hypothesis.43 Glucagon and e p i n e p h r i n e both oppose t h e a c t i o n of i n s u l i n by s t i m u l a t i n g g l y c o e n o l y s i s and g l u c o n e o g e n e s i s t h r o u g h a c y c l i c 3 ' , 5 ' - M medi'ated p r o c e s s .84 I n a d d i t i o n , t h e hyperglycemic a c t i o n of e p i n e p h r i n e i s augmented by two o t h e r a c t i o n s ; a d i r e c t i n h i b i t i o n of i n s u l i n release from t h e pancreas45,46 and a s t i m u l a t i o n of l i p o l y s i s i n a d i p o s e t i s s u e , t h u s i n c r e a s i n t h e c i r c u l a t i n g l e v e l of f r e e f a t t y a c i d s , which oppose i n s u l i n a c t i o n .7' 3 48 G lucagon , a l t h o u g h i t h a s t h e same e f f e c t as e p i n e p h r i n e on g l y c o g e n o l y s i s , h a s now been shown t o have t h e o p p o s i t e e f f e c t on t h e 8 - c e l l , where i t s t i m u l a t e s t h e release of i n s u l i n d i r e c t l y . 1 0 A symposium on t h e m e t a b o l i c e f f e c t s of c a t e c h o l a m i n e s from t h e Second Catecholamine Symposium (Milan 1965) h a s appeared.49 Crude p r e p a r a t i o n s of an e x e r c i s e f a c t o r 5 0 t h a t can a p p a r e n t l y i n crease g l u c o s e u t i l i z a t i o n i n muscle have now bean o b t a i n e d by G o l d s t e i n . From Sephadex column b e h a v i o r , t h e material a p p e a r s t o have a molecular weight of l e s s t h a n lOOO.5l Intermediary hetabolism diabetes.

A wide r a n g i n g d i s o r d e r of e n e r g y metabolism i s c h a r a c t e r i s t i c of

Whether t h i s derangement i s caused by i n s u l i n d e f i c i e n c y o r wholly

Sect. IV - Metabolic & Endocrine

168 -

H e i n z e l m a n , Ed.

or in part by some other lesion remains to be learned, as does whether it is a cause or a consequence of the vascular complications of the disease. Nevertheless, it remains the facet of the disease to which therapy has been' directed and by which the therapy is guided, whether it be diet, insulin, sulfonylureas or biguanides. The hormonal and feedback control of metabolism has been extensively discussed by several authors, but particularly clearly by Randle.5* He points out that there is a simple, primitive feedback control of metabolism, exerted through the effect of metabolic intermediates and substrates on enzymatic processes and that this mechanism of control is important, not omly to simple organisms that depend on it entirely, but also to complex organisms which also have an additional component of hormonal control. That effective management of diabetes can be obtained by drug effects on either hormonal or feedback control of metabolism is attested by the clinical success of the sulfonylureas and biguanides. The role of all members of the trinity of energy metabolism; the liver, muscle and adipose tissue,has been extensively studied. Most of the current knowledge about adipose tissue metabolism is contained in a recent volume in the Handbook of Physiology series53 and has also been discussed by Renold in a recent Banting Memorial Le~ture.5~The elegant and very useful quantitative work from Ball's laboratory on the fate of glucose carbon in adipose tissue has been summarized .55 These researchers have recently shown that, in adipose tissue, the operation of citrate cleavage enzyme and malic enzyme together can provide both the extramitochondrial acetyl CoA and the NADPH2 re uired (in addition to that from the pentose shunt) for lipogene~is.5~The control of metabolism in muscle has been reviewed by Randle.52 Sulfonylureas and their Congeners In the United States, three sulfonylureas are in wide clinical use in the management of maturity onset diabetes; tolbutamide (Orinase), chlorpropamide (Diabinese) and acetohexamide (Dymelor). In other parts of the world, carbutamide (Nadisan) and glycodiazine (Redull,a sulfonamidopyrimidine isostere of the sulfonylureas are also used.

0

S02NHCONHC4Hg

CH3

CH3CO~

~ S O 2 N H C O N H C 3 H 7 Cl

Tolbutamide S

O

2

N

H

C

O

N

H

o

Ch1orpropamide

NH2m2NHCONHC4Hg

Acetohexamide

Carbutamide

Glycodiazine

Chap. 15

Antidiabetics

Pinson

169 -

G l y c o d i a ~ i n eis ~ ~ one member of a large group of closely related compounds, In its pharmacology and clinical application, it appears to resemble tolbutamide most close1 having approximately the same hypoglycemic potency and duration of a ~ t i o n . ~ ”The hypoglycemic effect of glycodiazine is probably due to the action of the compound itself, since the pyrimidine ring has proved to be metabolically stable. The principal metabolite is the carboxylic acid (1) that results from cleavage of the terminal side chain methoxyl group and oxidation of the resulting primary alcohol. A toxicology investigation has also been reported.57

I

Tolazarnide

New sulfonylureas and their congeners continue to be widely reported, but in many cases, apparently from work done some time ago. It is regretable that there is no current structure-activity review, but one is clearly beyond the scope of this discussion. A compilation of a number of compounds that have been studied in animals and humans appeared a few years ago and is still useful.59 Sulfonylsemicarbazides have also been found to have significant hypoglycemic activity and one, Tolazamide,60,61 has received FDA approval,62 but had not, at this writing, been marketed. An extensive series of hypoglycemic sulfamylureas (11, 111) has been d e ~ c r i b e d . ~In ~ general , maximum hypoglycemic activity was found in

R1>S02NHCONHR R2 I1

R2

wS02N I11

X = CH2, N, 0, S R = alkyl, cycloalkyl and haloalkyl R1 and R2 = alkyl and alkoxy this series among the same alkylurea substituents (R) that give maximum activity in the sulfonylureas. The corresponding sulfamyl semicarba~ides~~ also had significant hypoglycemic activity as did a few of the related sulfamylcarbamates.65 Compounds in which the alkylurea portion was derived from a secondary amine, usually heterocyclic, were, in general, less activeP5 A number of the compounds were as active (reduction of blood glucose in fasted rats) as chlorpropamide. Further details of the pharmacology of the sulfamylureas have not been reported, but it seems safe to assume that their mechanism of hypoglycemic action would be very similar to that of the sulfonylureas. Several of the compounds are stated to show hypoglycemic activity in man but their clinical use in the management of diabetes has not been reported.

170 -

Sect.

IV

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

This work made good use of the now firmly established correlation, among compounds of this class, of hypoglycemic action with the presence of the compounds in the blood. Synthesis was therefore guided not only by the relationship established between structure and acute hypoglycemic activity, but also by an evolving concept of the influence of physical and chemical properties on absorption, metabolism and excretion, derived from a concurrent investigation.66 The metabolism and excretion of 15 of the compounds were determined in dogs and seven were studied in human subjects. The results of these studies were correlated with physical and chemical properties, of which pKa and lipid-water partition ratio proved to be the most useful. 0 he variants on the sulfonylurea structure have recently included and 2-cyanop-alkenyl-,679 2-acyl- ,69 E-a1koxyalkyl - ,69 E-haloalkyl , as well as 6-s~lfamoylbenzotriazole,~~ benzenesulfonyl-69 6-sulfamoylben~imidazole~~ and 5-indanylsulfonyl70 derivatives. A variety of alkyland aralkylurea71 substituents have been combined with these as well as with the now conventional benzenesulfonyl moieties. A series of compounds in which the urea function was part of a benzimidazolinone ring were devoid of hypog 1 ycemic activity .72

h

-

'

A sulfonarnidothiadiazole very similar to the sulfonamide that started it all has been reported to reduce blood sugar in alloxan diabetic rats, but the experiment was not controlled with a known ~ulfonylurea.7~ Considering its structural similarity to the earlier compound, incomplete alloxanization appears a likely explanation of this result.

Pharmacolom, Mechanism of Action. The pharmacology of the sulfonylureas has been reviewed recently.74~10

It is now almost universally accepted that the primary action of the sulfonylureas is to release insulin from the pancreatic b-cells. Overwhelming evidence indicates that the drugs have no significant hypoglycemic effect in animals or human diabetics who are without functioning @-cells. The details of the process by which the sulfonylureas release insulin, however, remain as much a mystery as the process by which glucose produces the same effect. A vast amount of evidence has been marshalled against the view that the sulfonylureas affect hepatic glucose output or peripheral glucose utilization except through insulin release, but the uestion of a direct effect on the liver has not been entirely re~olved.~?There have been suggestions that sulfonylureas affect the plasma factors that influence insulin action (see above) but the significance of the effect to their hypoglycemic action remains to be assessed.76 It has long been recognized that the hypoglycemic effect of the sulfonylureas is related to their concentration in the blood.77 A s with many acidic drugs, plasma half life is a key determinant of overall effect79 and studies of their physiological disposition have proved to be important in guiding their clinical use.59,78 The metabolism of tolbutamide and chlorpropamide has been reviewed.74 The metabolism of glycodiazine ( lasma half life, 3.5-4 hr. )80,81 and tolazamide (plasma half-life, 7 hrs.) 85 has been studied in human subjects.

Chap. 15

Antidiabetics

Pinson

171 -

From the point of view of metabolism, acetohexamide is the most interesting of the sulfonylureas. Although its plasma half-life is short ( 1 . 3 hrs.), it is reduced in the body to a metabolite, L(-)-hydroxyhexamide, which is not only more active (2-2.4 times acetohexamide), but is also longer lasting (half-life, 4.6 hrs. ).82,83 The net result is that hypoglycemic activity in the blood, which is due to acetohexamide and the metabolite, persists for about the same length of time as it does with tolbutamide. The fact that the metabolite is at least twice as active as the racemic form of the compound would imply that the D(+) form is essentially inactive, which seems incredible, considering how widely significant activity is found among the sulfonylureas. Unfortunately, synthesis of the compound has not yet been reported. Other metabolites of acetohexamide, which are essentially inactive as hypoglycemic agents, result from hydroxylation in the cyclohexyl g roup .83 The importance of solubility, and of solution rate, as a function of surface area, to rate of intestinal absor tion and hence to the time course of blood levels has been i n ~ e s t i g a t e d . ~ ~ , ~ ~ , ' ~Proper ,~~ consideration of these factors in dosage form preparation can be important determinants of overall efficacy.84 Binurnides The biguanides are widely used in the control of diabetes; phenethylbiguanide (phenformin, DBI, Dibotin) in the United States, and, in addition, butylbiguanide (buformin, Silubin) and N,N-dimethylbiguanide (metformin, Glucophage) in Europe. Their principal clinical application is in combination with sulfonylureas for the treatment of patients whose disease is poorly controlled on either agent alone and for smoothing the hypoglycemic response of insulin-dependent brittle diabetic^.^',^^ Few new compounds of the class have been reported recently, but a series of aralkylamino, aryloxyalkylamino, and arylthioalkylamino-s-triazines (V) designed from consideration of the structure of the biguanides when written in the cyclic, hydrogen bonded form (IV) has been prepared. The compounds had only weak hypoglycemic activity in rabbits.89

IV

V

Pharmacolop;y,Mechanism of Action. The mechanism of biguanide induced hypoglycemia has still not been established. Different investigators cannot yet agree on which of the many effects that have been observed in experimental systems are responsible for their clinical action The roblem has been reviewed and discussed from several points of view.74,90,9P,92,93 It seems clear that glucose utilization by anaerobic glycolysis is increased, the further metabolism of the resulting pyruvate is inhibited, and lactate

172

Sect. EV

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

t h e r e f o r e accumulates. T h i s i m p l i e s a n a c c e l e r a t i o n of a n a e r o b i c g l y c o l y s i s r e s u l t i n g from a r e d u c t i o n of o x i d a t i v e metabolic a c t i v i t y ; an e f f e c t f i r s t d e s c r i b e d by P a s t e u r and s i n c e known as t h e P a s t e u r e f f e c t , which Randie52 From t h e work of Passonneau and now p r e f e r s t o c a l l " r e s p i r a t o r y c o n t r o l " . LowryYg4P a r k l 3 and h i s co-workers ar,d Randleg5 and h i s associates, t h e mechanism of t h i s e f f e c t i s now understood i n some d e t a i l . A thorough d i s c u s s i o n of t h e s e c o n c e p t s i s c o n t a i n e d i n R a n d l e ' s review.52 Phenformin h a s been shown t o i n h i b i t some m i t o c h o n d r i a l e l e c t r o n t r a n s f e r r e a c t i o n s and t h i s h a s been suggested as i t s primary s i t e of a c t i o n . 9 0 S e v e r a l o b j e c t t o n s t o t h i s h y p o t h e s i s have been r a i s e d , however. F i r s t , t h e concentrations required t o i n h i b i t mitochondrial oxidation & I n view of v i t r o are much h i g h e r than have e v e r been observed i n t h e f r e q u e n t d i s p a r i t y between i n v i t r o and i n v i v o r e s u l t s and problems of t i s s u e and s p e c i e s s e n s i t i v i t y , t h i s o b j e c t i o n i s n o t , of i t s e l f , p a r t i c u l a r t h a t c l o s e l y r e l a t e d hypoglycemic l y s e r i o u s . However, a second o b j e c t i o n b i g u a n i d e s show a similar hypoglycemic e f f e c t but do n o t i n h i b i t m i t o c h o n d r i a l o x i d a t i o n , whereas o t h e r s , which are devoid of hypoglycemic a c t i v i t y , are n e v e r t h e l e s s p o t e n t i n h i b i t o r s of m i t o c h o n d r i a l e l e c t r o n t r a n s f e r r e a c t i o n s g 7 -- d e s e r v e s c a r e f u l c o n s i d e r a t i o n . T h i r d , i t h a s been shown t h a t low conc e n t r a t i o n s of buformin ( 1 - 5 p g / m l ) , which are i n t h e range a t t a i n e d i n v i v o , a c t u a l l y i n c r e a s e t h e a c t i v i t y o f t h e p e n t o s e shunt i n a d i p o s e t i s s u e and t h i s i s c i t e d as e v i d e n c e t h a t t h e hypoglycemic a c t i v i t y of t h e b i g u a n i d e s does n o t i n v o l v e i n h i b i t i o n of o x i d a t i v e p r o ~ e s s e s . 9 ~The c o n c l u s i o n t h a t o x i d a t i v e metabolism i s n o t i n h i b i t e d seems u n t e n a b l e , c o n s i d e r i n g t h a t hypoglycemic d o s e s of phenformin i n v a r i a b l y c a u s e i n c r e a s e s i n blood and u r i n a r y l a c t i c a c i d and t h a t p y r u v a t e t o l e r a n c e i s decreased.98 The buformin e f f e c t on a d i p o s e t i s s u e i s v e r y similar t o t h e e f f e c t of epinephrine99 and, cons i d e r i n g t h e r e a s o n a b l e s t r u c t u r a l analogy between buformin and t h e a l i p h a t i c p r e s s o r amines, may r e s u l t by a similar mechanism.

--

One of t h e most p e r t i n e n t i n v e s t i g a t i o n s of t h e e f f e c t of phenformin on metabolism i s t h a t of Williamson, Walker and Renold.lOO Using t h e i s o l a t e d p e r f u s e d rat h e a r t , t h e y found s u b s t a n t i a l i n c r e a s e s i n g l u c o s e u p t a k e and l a c t a t e f o r m a t i o n and d e c r e a s e d i n t r a c e l l u l a r ATP a t phenformin c o n c e n t r a t i o n s of 3 5 - 5 0 Gg/ml. The a u t h o r s i n t e r p r e t t h e s e f i n d i n g s i n t h e l i g h t of c u r r e n t c o n c e p t s of n e u c l e o t i d e and phosphate feedback c o n t r o l of p h o s p h o f r u c t o k i n a s e , a s t h e rate c o n t r o l l i n g s t e p of g l y c o l y s i s , a n d f u r t h e r s u g g e s t p o s s i b l e i n h i b i t i o n of p y r u v a t e d e c a r b o x y l a t i o n . Like many i n v e s t i g a t o r s of t h e b i g u a n i d e s , Williamson, Walker and Renold d i s c o u n t t h e r e l e v a n c e of t h e i r r e s u l t s t o t h e c l i n i c a l s i t u a t i o n because of t h e r e l a t i v e l y h i g h drug c o n c e n t r a t i o n s used. Such d i f f e r e n c e s seem much less i m p o r t a n t i f one c o n s i d e r s p o s s i b l e t i s s u e and s p e c i e s s e n s i t i v i t y . The rat i s remarkably i n s e n s i t i v e t o phenformin, showing l i t t l e hypoglycemic e f f e c t at 80 m /kg 101 It w h i l e human d i a b e t i c s respond w e l l a t d o s e s of 50-150 mg ( 1 - 2 mg/kg).'8 h a s been p o i n t e d o u t t h a t an a d d i t i o n a l component of hypoglycemic a c t i o n probably r e s u l t s from impairment of t h e r e c o n v e r s i o n of lactate, formed i n The i n h i b i t i o n o f p y r u v a t e decarboxylmuscle, t o g l u c o s e i n t h e liver.90,102 a t i o n mentioned above would a c c o r d w i t h t h e d e c r e a s e d l i o g e n e s i s t h a t h a s been suggested by r e c e n t c l i n i c a l o b s e r v a t i o n s ,103,104,1g5 s i n c e reduced a v a i l a b i l i t y of a c e t y l CoA would be expected t o i m p a i r l i p i d s y n t h e s i s . T h i s a s p e c t of t h e a c t i o n of t h e b i g u a n i d e s does n o t a p p e a r t o have been s t u d i e d

Chap. 15

173 -

Pinson

Antidiabetic s

directly. The possibility of a direct action of the biguanides on adenyl cyclase to cause cyclic 3',5'-AMP activation of phosphofructokinase has not apparently been considered but it is an intriguing one. Although cyclic 3',5'-MP is known to activate mammalian phosphofructokinase, a hormonal effect through adenylcyclase stimulation has not yet been demonstrated.Io6 Y 125 The interesting enigma that the biguanides do not cause hypoglycemia in non-diabetic individuals has received some clarification recently.lo7 Glucose turnover studies in non-diabetic human subjects have shown that glucose utilization is markedly increased but that hypoglycemia does not occur, apparently because the increased utilization is offset by a corresponding increase in glucose output from the liver. It seems reasonable to suggest that an effect on hepatic glucose output is the key difference, in this respect, between diabetics and non-diabetics. Hepatic glucose out ut in diabetics comes mainly from gluconeogenesis, which phenformin inhibits;f02 whereas the hepatic glucose output of non-diabeti.:s, under the conditions of this experiment, would come primarily from glycogmolysis, which phenformin accelerates.90 The distribution, metabolism, and excretion of henformin, metformin and buformin have been investigated.lo8, log ,llo,11'1 The physiological disposition of the biguanides is typical of that of other strong organic bases. The compounds diffuse from the blood, where concentrations are invariably low and not very persistent, into acidic gastric juice, are found in higher concentrations in tissue than in the blood, and are eliminated efficiently in the urine. The identity of their metabolites has not been established with certainty. Other Hypoglycemic Compounds Pyrazoles and Isoxazoles. Since 1963, a series of reports have described two interesting hypoglycemic and lipolysis inhibitin corn ounds, 3,5-dimethylpyrazole (VI) ana 3,5-dimethylisoxazole (VII).112,~13,1p4,115

CH3

pcH3 CH3

CH3

H

VI

VII

HOOC

(flCH3 N/

H

VIII

HOGC (flCH3 O N IX

Both compounds are remarkably potent hypoglycemic agents (50 and 200 times as active as tolbutamide) in glucose primed rats but are much less active in fasted animals. Both are apparently inactive, themselves, but depend for activity on metabolism to 3 -methylpyrazole-5-carboxylic acid (VIlI) and 3methylis0xaz0l~~5-carboxylic acid (IX), respectively.116,11' Although the carboxylic acid is a relatively minor metabolite of dimethylpyrazole (10-15% in the rat), it apparently accounts for all of the activity. The major metabolite, 4-hydroxy-3 5-dimethylpyrazole (70-75% in the rat) shows no hypoglycemic activity.li7 The two carboxylic acids are potent inhibitors of the release of free fatty acids from adipose tissue and the authors make the reasonable suggestion that this is their primary action, from which the hypoglycemic effect r e s ~ l t s . ~ The ~ ~ mechanism . ~ ~ ~ by which inhibition of

174

Sect. IV

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

lipolysis might promote glucose utilization has been discussed extensively by Randle,48 and these compounds might well be used in further examination of some of his hypotheses. 5-Methylpyrazole-3-carboxylicacid was effective in reducing the plasma free fatty acid levels of fasted human subjects118 but the concomitant effect, if any, on their blood glucose was not reported. None of a series of pyrazole congeners of 3,s-dimethylpyrazole was apparently more active than the parent compound.120 L-Leucine. The subject of leucine induced hypoglycemia, which occurs in certain sensitive individuals, particularly some children with "idiopathic hypoglycemia'' and patients with functioning islet cell tumors, has been very ably reviewed recently by Fajans.l*I Addition of L-leucine (12 g/day) to the regimen of sulfonylurea secondary failures resulted in good diabetic control in 8 o f 10 patients.122

A series of hexahydroindenio[1,2-~]pyrrols and indanamines have been reported to reduce the blood glucose of orally treated rabbits but details that might permit an assessment of their potential as oral antidiabetic agents are lackirg.123 References 1. 2. 3. 4. 5.

6.

I. 8. 9. 10. 11. 12. 13. 14. 15 * 16.

11. 18. 19. 20. 21.

22. 23.

M. Janbon. J. Chaptal, A. Vedel, and J. Schaap, lontpelller Meed.. 21-22, 441 (1942). 253 (1918). C. K. Watanabe, J. Biol. Chem., P. White. led. Clin. I. An.. 3. 855 (1965). A. E. Renold and 0 . F. Cahill, Jr. in "The Metabolic Basis of Inherited Disease", 2nd Ed., J. B. Stanbury, J. B. Wynngaarden and D. S. Fredrickson. Eds., lcQrau-H111 Book Co., leu York, 1965. B. S. Leibel and 0. A. Wrenshall, Lds. "On the Uature and Treahent of Diabetes", Sxcerpta ledloa Foundation. Xeu York, 1965. R. H. Williams and F. C. Wood. Jr., Reference 5. Chapter 51. -R. II. Williams. Ann. Intern. led., 63, 512 (1965). S. A. Berson and R. A . Yalou. Diabetes, 14. 549 (1965). 0 . F. Cahill. Jr. led. Clin. x. An., 49. 881 (1965). 0. M. Orodsky and P. H. Forsham. Ann. Rev. Physiol.. g.341 (1966). P. P. Lacy. C l b a Found. Collos. Xndoorlnol., 15,15 (1964). S. E. Brolin. B. Hellran and 8 . Knutson. Eds. "The Structure and Metabolism of the Pancreatic Islets", Pergaron Press, !Jew York, 1964. H. P. Morgan. J. R. Xeelg. R. P. Wood, C. Llebecq. €I Liebermeister . and C. 8 . Park, Federation Proc.. 24, 1040 (1965). R. Levine. 1011. I. 0. Wool, 1060. D. X. Klpnl8 and J. E. Parrish, E .1051. J. S. Bishop, R. Steele, I. Altszuler, A. Dunn, C. Bjerknes and R. C. de &do. Physiol.. 208, 301 (1965). Q. Weber and 1. L. Singhal. Life Scl.. 2, 1993 (1965). A. Sols, Reference 5. Chapter 9. D. 1. Klpnis and R. Hallchoff, J. Clin. Invest., 44. 1064 (1965). A. Oellhorn and W. Benjamin. Science, 148,1166 (1964). D. R. Rampersad and I. 0 . Wool, Science, 149, 1102 (1965). E. Sprols. Reference 5, Chapter 16.

a,

s.

s.

uJ.

Chap. 15

Antidiabetic s

Pinson

175 -

24. D. H. Lockwood and T. X. Prout, Uetab. Clin. -2.. l4, 530 (1965). 25. B. H. Segal, Uetab. Clin. XQ 758 (1964). 26. S. H. Oershoff. A. 1. Huber and B. H. Antonlades, Uetab. Clin. Q.. g ,325 (1966). 27. H. H . Antonlades. A. U. Huber. B. R. Boshell. C. A. Saravis and S. Y. Oershoff,

.,s,

28.

29. 30. 31. 32. 33. 34.

35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49 * 50.

51. 52. 53. 54.

55. 56. 57. 58.

59. 60. 61. 62. 63. 64. 65.

a

Xndocrinoloa. 709 (1965). 1. Samaan, J. Brown, R. Fraser and I. Trayner, Brit. Hed. J.. 1965(1), 1153. T. Welborn, I?. Samaan, R. Fraser and J. Brown. Mabetologla. 1, 74 (1965). B. R . Froesch, H. Burg1 and U. S. Uuller, Helv. Xed. Acts.. 2, 341 (1965). Q . F. Cahlll. Jr., V. Laurls, J. S. Soeldner, D. Slone and J. Steinke. Btab. c u 769 (1964). J. Steinke, S . Uikio and 0 . F. Cahill, Jr., Yew pllg. J. Hed.. 273, 1464 (1965). J. K. Davidson, B. E. Haist and C. H. Best, Diabetes, 12, 448 (1963). L. Power, C. Lucas, and J. Is. Conn. Hetab. Clin. Exp.. l4. 945 (1965). W. 1. Shaw in "Adipose Tissue", A. E. Renold and 0. F. Cahlll. Jr., Pds., Handbook of Phusiologg, Section 5. American Physiological Society. Washington, 1965. J. Vallance-Owen, Reference 5, Chapter 23. H. Alp and L. Recant, 3. Clin. Invest., 44, 870 (1965). U. L. Ashton. J. fndocrinol., 33, 103 (1965). J. U. Xnsinck, R . J. Mahler and J. Vallance-Owen, Blocher J., 2.150 (1965). R. L. Fenichel. W. H. Bechrann, and H. 11. Alburn. Biochemistry, 5 , 461 (1966). I). 1. Kipnis, Reference 5 . Chapter 18. R. C. Haynes, J r . , Advan. X n m e Regulation. 2, 111 (1965). A. B. Xisenstein, 121. X. Y. Sutherland and 0. A. Robison, Pharmacol. Rev., Is,145 (1966). A. 0. Kris. R. L. Xlller, F. P. Wherry and J. Y. Wason, Pndocrinoloeg!. 3, 87 (1966). D. Porte, Jr., A . Oraber. T. Kuzuya and R. H . Uilllans. J. Clin. Invest., &%1087 . (1965) D. Steinberg, Pharmacol. Rev., 18,217 (1966). P. J. Randle. Reference 5. Chapter 25. Pharmacol. lev., B, 145-314 (1966). U. S. Ooldstein, An. J. Physiol., 200, 67 (1961). X. S. Ooldstein. Paderation Proc., 2,441 (1966). P. J. Randle in "HOmeOltaSil)and Feedback Uechanlsms", Society for mperirantal Biology Synposia XVIII. Academic Press Inc., Yew York, 1964. p. 129-156. A. X. Renold and 0 . F. Cahill. Jr., "Adipose Tissue", Handbook of Physiology, Section 5. American Physiological Society, Washington. D. C., 1965. A. P. Renold, 0. B. Cmfford, U. Stauffacher and B. Jearenaud, Mabetologla, 1,4 (1965). J. P. Flatt and P. 6. -11, Reference 53, Chapter 26. H. S. Kornacker and P. 0. Ball, $roc. Hat. Acad. ScI. 0. S., 54. 899 (1965). The preparation of glycodiazine and a number of analogs, with Its PhaFlaColOgJr, metabolism and clinical use deacrlbed in a. series of 10 papers; Arzneirittel-Forsch.. 14. 373 (1964). R. D. H. Stewart and D. X. Anderson, Brit. Hed. J., 1965(21. 682. F . Q. XcUahon. H. L. Upjohn, 0. S. Carpenter, J. B. Wright. H. L. Oster, and U. X. Dulln. Current ,.-T 2, 330 (1962). R. Bressler and R. Katz, Current Therap. Res., 1, 219 (1965). P. H. Orinnell, T. 0. Skillman, R. Barse. and C. L. Holler, Current them^. Res.. 2, 433 (1964). Federal Register, 2,2561 (1966). J. H. UcUanus, J. W. HcFarland, C. P. Oerber. W. U. UcLaPore, and 0 . D. Laubach. J. led. Cher.. 8 , 766 (1965). J. 1. 1cwSnus and C. F . Oerber. J. Ued. Chem., 2, 256 (1966). J. W. UcFarland. C. P. Qerber. and Y. 1. XcLarore, J. Had. Chu.. 2. 781 (1965).

s., 13.

u..

-

Sect. I V

176 66. 61. 68. 69. 10. 71. 72. 13. 14.

75. 76.

11. 78. 19. 80. 81. 82. 83. 84. 85. 86. 81. 88. 89. 90. 91. 92.

93. 94. 95. 96. 91. 98. 99. 100. 101. 102. 103. 104. 105. 106. 101. 108. 109.

- Metabolic

& Endocrine

H e i n z e l m a n , Ed.

E. H. Wiseman. J. 1. Perelm, K. F. Pingor, and R. Plmon. J. Ned. m.,4, 771 (1965). D. P. Hayman. 0 . B. Jackman, V. Petrow, 0 . Stephewon. and A. 1. Wild, J. P h a m . Phamacol., 677 (1964). D. P. H a m , V. Petrow, and 0 . Stephanron, J. P h a m . Pharucol., Is. 538 (1964). J. Lederer, J. Ned. Cham., 2, 370 (1064). L. J. L o n e r DA. Blanahi, an8 A. Borron. Hetab. Clln. Un., 578 (1965). K. 0 . Jo8hl and J. 9. h p t a , J. 1ndi.n Chm. Soc.. g,320 (1965). J. B. Wright, J. Bb8, Cham., g, 589 (1965). D. Bargeton, J . Roquet, A. Rouquer. A. CROrsaIn, and A. Bledar, Aroh. Intern. phamacodmamie, 185,319 (1965). L. J. P. Duncan and B. F. Clarke, Ana. Rev. Pharucol., S, 151 (1966). A. R. Colwell, Metab. Clln. -2.. 13, 1312 (1964). 8. 1. htOkIla8e8, J. A. Borrp.8. R. ~ r l ~ - D . r a l o rH., 1. Pyle, S. J. Naturkle, 0 . Low-Castaneda, and A. Narble, New -1. J. md., 269. 386 (1963). K. N. Wort and P. C. Johawon, Mabetor, 2. 464 (1960). J . Sheldon, J. h8er8on. and L. Stoner. Diabetes. 362 (1965). B. It. Bloar and 0 . D. loubach. Ann. Rev. PharrPcol., 2, 61 (1962). K. 8. Kolb. 1. KraMr. and P. X. Schulre. Arzneirittel-Forsch.. 385 (1964). L. Oerhardr, H. 01bI.n. and K . H. Kolb, Armeirittel-Fonch., 2, m4 (1964). D. 1. Smlth, T. J. Veochio, and A. A. F0ri.t. Netab. Clln. Up., 2, 229 (1965). R. 1. HcNahon. F. J. Narshall, and H. W. Culp, J. P w o o l . U p . Therap., 272 (1965). P. ielson, X. I. Knoeahel, W. X. Hailin, and J . 0 . Wagner, J. P U N . s o l . , 2,509 (1962). X. Nelson, S. Long, and J. 0 . Wagner, J. Pham. S o l . , 53, 1224 (19M). W. I. Higuchi, 1. A. Nlr, A. P. Parker, and W. P. Hailin, J. Pham. S o l . , 54, 8 (1965) J . I. bodman, Xetab. Clin. Un.. 14. 1153 (1965). B. P. Clarke and J. P. Duncan, Lancet, 1966(11, 1248. If. Suter and H. Zutter, Helv. Chim. Acta, 48, 1940 (1965). D. P. Stainer an8 R. H. W l l l i o r s , Mabetar, 8, 154 (1959). H. Dnweke and I. Bach. netab. Clin. UP., l2. 319 (1963). J. Sterna, Netab. Clln. UE..2,191 (1964). 1. Beckmann, Deut. mod. Wochschr.. 90. 1589 (1965). J. V. P a 8 8 O Z U l ~ Uand 0. H. L O W . 1,10 (1962). P. B. Oarland and P. J. llPndle. Blochea. J., 93. 678 (1964). A. B. Falcone, R. L. llao and X. Shrago. J . Blol. Cham.. 237, 904 (1962). Q. Ungar, 5 . PSYChoYO8, and H. A. H a l l , Netab. Clln. U p . . 2, 36 (1960). S. S. Fajans, J . A . Xoorehouse, H. Doorenbos. L. H. Louis, and J. W. COM, Diabetes. 9, 194 (1960). 0 . P. Cahill. Jr.. B. Leboeuf, an8 It. B. Fllnn. J. Biol. Chem., 235. 1246 (1960). J. R. Ullllorson. 1. 5 . Walker and A. E. Itenold, Metab. Clln. an..l2, 1141 (1963). 0 . Wwr. L. Freedman an8 9 . L. Shaplro. Proc. 900. U p . B l o l . ned., 95, 190 (1951). S. J. Patrick, Can. J. bloc her.,^, 27 (1966). D. P. Patel and J. 1. Stowerr, Lancet, l 9 6 4 ( 2 ~ ,282. J. Sch-rtg, s. N l r s k y . and L. X. Schaefer, Lancet, 19SS(ll, 959. J. Pedarren, Acta Xndoorlnol.. 49, 419 (1966). T. X. npmoar. P b u v c o l . Rev., 2,173 (1966). 0 . 1. Searle, 9. Schilling, D. Porte. J. Barbacoia, J . DoQmeia. and 1. ti. Cavalieri, Diabetes. l5. 113 (1966). A - 1-Wicklr.c. J. Stewart. and 0 . 5 . S e r i f , Mabetes. 2. 163 (1960). R. -0kMAn. 0 . Hwbner. Arznoinittel-Forsch., 5 . l, 165 (1965).

Is.

s,

z,

z.

xD

,.-

-

*.

Chap. 15 110. 111. 112. 113. 114. 116.

Ant idiabe tic s

Pinson

177 -

R. BeckroM, Arzneirittel-Forsoh.. 18, 761 (1965). Q. Debry and F . P. Cherrler. Theraple. 2.352 (1965). U. 1. Dulln and 0. C. Oerrltsen, Proc. Soo. PD. Blol. Ned.. 113,683 (1963). 0 . C. Oerrltsen and W. 1. Dulln. Diabetes. & 507 (1965). A . B1r;zI. A. Jorl, 1. Veneronl. and S. Oarattint, Life Solences, 3, 1371 (1964). W. 1. Dulln, 0. H. Lund, and 0. 0. Qerrltsen, Proo. Soe. ZXP. Biol. Ned., 118,

499 (1965). 116. D. L. Slrlth, A. A. lOrl5t. and W. 1. Dulln, J. Hod. Chsr., S, 350 (1965). Therap., 150, 316 117. D. L. Slith, A . A. Forist, 0 . C. Oerrltsen. J. Pharmaool. PD. (1966). 118. 0. C. Oerrltsen and W. 1. Dulln, J. Phosrpcol. P p . Therap., 160, 491 (1965). 119. Y. 1. Dulln and 0. C. Oerrltsen, Proo. Soo. P p . Blol. Ned., 121. 777 (1966). 120. J. B. Wright, Y. 1. Dulln, and J. H. I(arklllle, J. Ned. Chem.. 2 , 102 (1964). 12l. 5 . S. FaJaM, New Kml. J. n o d . , 272, 1224 (1965). 122. H. R l f k l n , 5 . Podolsky, H. Ross. X . 0 . Conason, and S. Nost, Mabetes, l5, 222 (1965). 123. S. C. LphlFi a d B. Pathak, J. b d . Ch-., 1, 131 (1966). 124. 1. A. -5i0, J. S. Soeldner and 0. F. Cphlll. Jr., Dlabetologia, 1,125 (1965). 125. R. M. Denton 8nd P. J. Ilurdle, Bloohem. J., E,6P. (1986).

Sect. I V - Metabolic & E n d o c r i n e

178 -

Chapter 16.

H e i n z e l m a n , Ed.

Atherosclerosis

Joseph J . Ursprung, The Upjohn Company, Kalamazoo, Mich. Introduction. - Atherosclerosis appears t o be a "disease of regulation" (I. H. Page) i n which a g e n e t i c a l l y conditioned cardiovascular system i s rea c t i n g t o an ever-changing environment. The a b i l i t y t o regulate t h i s i n t e r a c t i o n determines i n t h e long run whether a t h e r o s c l e r o s i s w i l l , or w i l l not, p r e v a i l . The disease is a l e s i o n of l a r g e and medium-sized a r t e r i e s charact e r i z e d by i n t i m a l thickening with f o c a l deposits i n t h e intima of yellowish plaques containing l i p i d s , carbohydrates, blood products, fibrous material and calcium. I n t h i s c o n s t r i c t e d a r t e r y thrombus formation occurs i n t h e ulcerated atheromatous plaque which re,sults ultimately i n occlusion. Serum c h o l e s t e r o l l e v e l s and blood pressure a r e useful p r e d i c t o r s of t h e disease and obesity, smoking, l e v e l of physical a c t i v i t y , psycho-social influences, increased t r i g l y c e r i d e and carbohydrate metabolism, enhanced blood c l o t t i n g , hormonal balance and genetics a r e f a c t o r s which play a r o l e but t h e i r r o l e i s as yet unclear. There has evolved t h e concept t h a t a t h e r o s c l e r o s i s i s a multifaceted disease and t h a t no single cause e x i s t s . Two main theories, both of which a r e susceptible t o experimental investigation, e x i s t today. The f i l t r a t i o n concept of Aschoffl focused a t t e n t i o n on disordered l i p i d metabolism and s t i l l c o n s t i t u t e s the broadest approach t o atherogenesis while t h e thrombogenic theory proposed by Rokitansky (1844) and revived by M i d 2 l e d t o increasing i n t e r e s t i n intramural c l o t t i n g mechanisms and t h e i r r e l a t i o n s h i p with plasma l i p i d s . While l i p i d s appear t o bear a r e l a t i o n s h i p t o human a t h e r o s c l e r o s i s and associated thrombosis, t h e i r involvement s t i l l i s based on assumption r a t h e r than f a c t . Whether l i p i d s deposited i n t h e vessel come from t h e blood or a r e synthesized i n t h e blood v e s s e l i s s t i l l debated. Increasing a t t e n t i o n i s being paid t h e blood vessel, i t s e l f . I n t h e atherosclerosissusceptible White Carneau pigeon enhanced a o r t i c f a t t y a c i d and cholesterol e s t e r synthesis i s highly c o r r e l a t e d with t h e s e v e r i t y of t h e disease3. I n t h e rat, hypertension increases c h o l e s t e r o l synthesis4 and c h o l e s t e r o l levels5 i n t h e a o r t a as w e l l as i n t h e l i v e r and o t h e r tissues.' Chapman7 provides evidence t h a t t h e f o r c e which d i s r u p t s an a t h e r o s c l e r o t i c plaque t o form a thrombus proceeds from within t h e v e s s e l w a l l toward t h e lumen. Regardless, by far t h e g r e a t e s t e f f o r t has been concentrated on reducing blood liDid l e v e l s , p a r t i c u l a r l y c h o l e s t e r o l , i n t h e hy-perlipemic p a t i e n t . Diet. - Epidemiological s t u d i e s provide a basis f o r t h e hope t h a t d i e t a r y adjustment may reduce t h e incidence o r a t l e a s t delay the development of atheros c l e r o s i s and coronary h e a r t disease. Preliminary r e s u l t s from t h e national d i e t - h e a r t study8 and t h e "anti-coronary club'ls a r e encouraging and t h e A.M.A. Council on Foods and N u t r i t i o n recommends d i e t a r y treatmental and prophylaxis against coronary heart disease. The r i s k of coronary h e a r t disease seems t o bear a t l e a s t some r e l a t i o n s h i p t o c h o l e s t e r o l i n t h e blood as shown by t h e Framingham s t u d y . l o Among d i e t a r y agents which influence cholesteremia a r e t o t a l fats ,11 polyunsaturated fats ,I2 t h e r a t i o of polyunsaturated t o s a t u r a t e d fat,13 c h o l e ~ t e r o l ,amino ~ ~ a c i d content15 and carbohydrate .I6 A recent review17 discusses t h e r e l a t i o n s h i p of d i e t a r y f a t and coronary heart disease.

-

Chap. 16

Atherosclerosis

Ursprung

179 -

A technique using t h e isotopic steady state24a suggests a limited cholest e r o l absorption i n man and t h i s may be h i s major protection against a high cholesterol diet24 since t h e r e appears t o be no feed-back mechanism i n man t o compensate f o r d i e t a r y c h o l e s t e r o l intake.24b On a high c h o l e s t e r o l d i e t 6080s of t h e serum c h o l e s t e r o l i s derived from endogenous sources.24a This contrasts with a recent report t h a t t h e serum l e v e l of c h o l e s t e r o l i n humans i s proportional t o t h e square root of t h e d i e t a r y intake of cholesterol.25 The r a t e of synthesis of c h o l e s t e r o l i n t h e s m a l l i n t e s t i n e a l s o i s independent of c h o l e s t e r o l feeding o r fasting.26 E a r l i e r work with primates showed that they do not respond t o d i e t a r y cholesterol; however, a t h e r o s c l e r o s i s can be induced27 when s a t u r a t e d f a t s a r e added t o a high c h o l e s t e r o l d i e t t o fac i l i t a t e absorption. Although c h o l e s t e r o l feeding w i l l produce myocardial inf a r c t s i n r a b b i t s , no c o r r e l a t i o n has been seen i n t h i s species between t h e occurrence of i n f a r c t s and t h e l e v e l of c h o l e s t e r o l i n t h e blood.23 The f a l l of serum c h o l e s t e r o l on feeding a d i e t high i n unsaturated f a t s occurs i n t h e absence of c h o l e s t e r o l excretion changes. This f a l l i n cholest e r o l l e v e l s i n t h e rabbit28 and i n man2’ seems t o be due t o plasma-tissue r e d i s t r i b u t i o n . An a l t e r a t i o n i n l i p o p r o t e i n s t r u c t u r e r e s u l t s such t h a t equilibrium between plasma and t i s s u e c h o l e s t e r o l pools favors t h e l a t t e r . 3 0 This c o r r e l a t e s with t h e finding that manipulating t h e d i e t a r y f a t intake i s not accompanied by a reciprocal change i n cholic acid turnover31 nor i s t h e r e a consistent r e l a t i o n s h i p between s t e r o l excretion and changes i n serum cholest e r o l concentrations. 32 The hypocholesteremic e f f e c t of d i e t a r y l i n o l e i c a c i d i n t h e r a b b i t appears t o be proportional t o t h e extent t o which l i n o l e i c a c i d i s incorporated i n t o plasma c h o l e s t e r o l e s t e r s . 34 Measurement of t h e l i p i d composition of a r t e r i a l t i s s u e and atheroma i n humans, however, has shown only an increased l i n o l e i c a c i d content i n atheroma i n those on a d i e t high i n uns a t u r a t e d fats as compared t o controls .33 A serum p r o t e i n deficiency i s reported i n humans with coronary a r t e r y An adequate intake of d i e t a r y p r o t e i n seems t o be necessary f o r disease.” e f f e c t i v e regression of coronary a t h e r o s c l e r o s i s i n chickend’In chickens on an atherogenic d i e t , cerebrosides and soy s t e r o l s reduce serum c h o l e s t e r o l and t h i s reduction c o r r e l a t e s with retarded development of a t h e r o s c l e r o s i s .20 It has a l s o been suggested t h a t a substance i n soy bean meal w i l l p r o t e c t rabb i t s on a high f a t d i e t against a t h e r o s c l e r o s i s .22 Serum Lipids. - Many attempts have been made t o reduce serum c h o l e s t e r o l by i n h i b i t i n g i t s biosynthesis. Triparanol w a s introduced some years ago f o r t h i s purpose but w a s withdrawn because of severe systemic reactionsa7 ( l e n t i c u l a r c a t a r a c t s , alopecia and i c h t h y o s i s ) . It a l s o appears t o increase a o r t i c a t h e r o s c l e r o s i s i n chickens38 and produces congenital malformations i n mice and rats.3s Compounds recently reported, which a r e s i m i l a r i n many respects t o t r i p a r a n o l , include some pyridyl analogs,40 c y a n ~ s t i l b e n e s ,triarylmeth~~ anols42 and other b a s i c ~ a r b i n o l s . ~ ~ Of some b a s i c e t h e r s of N-hetero-substituted a n i l i n e s which show cholest e r o l lowering a c t i v i t y , 4 3 compound 1 i s most a c t i v e . Three new c l a s s e s of

1.

2.

180

Sect. IV

H

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

6 3 0 4.

3.

inhibitors are shown in structures 2 [journal gives name as 4-(2-chlorophenyl)a-(p-methoxyphenoxymethyl) -1-piperazine] , 3 and 4.44 These compounds (2-4)were shown to accumulate ~-dehydrocholesterolin the plasma and tissues of rats and in this respect are similar to compounds related to AY-9944 reported by Ayerst.45 25-Azacholesterol blocks cholesterol synthesis at the desmosterol stage4" as has also been seen with 20,25-dia~a-~~ and 22,25-diazacholesterol.48 In man 20,25diazacholesterol lowers blood cholesterol but toxic effects are numerous 47b Myatonia and Keratoderma have been rep0rted4~and in each instance serum desmosterol was elevated and cholesterol was reduced at the time these toxic effects became evident. Some 4-azachole~tenes~~ have also been reported to inhibit cholesterol synthesis. Cyclization of squalene is inhibited by t0lbutamide5~while phenethylbiguanide inhibits conversion of farnesyl pyrophosphate to squalene 51 Phenyl- and biphenyl-substituted acids have been studied extensively f o r their cholesterol lowering activity.52 Activity is reported also with biphenyl ether-substituted acids.s2c The agent of most interest in this area remains pThe reader is chlorophenoxyisobutyric acid (CPIB, Clofibrate, atromid S) directed to the Atromid for background information. In bovine vascular tissue in vitr054, in rats55 and in man56 CPIB inhibits cholesterol biosynthesis, apparently between mevalonic acid and isopentenyl pyrophosphate 55 It was again confirmed that CPIB alone is as active as the combination of CPIB and androsterone (Atromid)57 and that it lowers triglycerides to a greater extent than cholester01.57b It also markedly decreases low density lipoprotein (a suggested mode of action f o r CPIB)S8 and reduces the glyceride content of high density lipoprotein. CPIB is reported to decrease the B/ol lipoprotein ratios9 and increase uric acid excretion.60 The norepinephrine-induced rise in free fatty acid levels is unaffected by CPIB in humans."l The effect of CPIB on the fibrinolytic system is variable62 but does not appear to be significant.63 Prospective studies of large groups has verified that the risk of myocardial infarctions is substantially greater in cigarette smokers than in non-smokers64 and this appears to be mediated via the nicotine-induced release of e~inephrine.~~ Nicotine, in dogs, has been shown to raise cholestero166 and blood sugar67 and this may be related to the elevation of free fatty acid levels?" Caffeine raises free fatty acid levels68 and it is suggested that coffee intake can be associated with heart disease.64b Agents which will regulate free fatty acid levels are of interest both in diabetes and atherosclerosis. 3,5-Mmethyli~oxazole,~~ 3,5-dirnethylpyra~ole~~ and its metabolite, 5-methylpyrazole-3-carboxylic acid7' dramatically lower fatty acids and blood sugar in animals. Infusion of norepinephrine produces a rapid rise in free fatty acid and in plasma triglycerides of l o w density lipoprotein.72 Nicotinic acid blocks this norepinephrine-induced rise in free fatty acid73r"l and lowers free fatty acid74 and triglyceride75 levels in the blood. It is also reported to reduce the cholesterol content of various tissues in cholesterol-fed

.

.

.

Chap. 16

Atherosclerosis

U r sp rung

181 -

~ l~u m i r a b b i t s .76 I n humans it produces abnormal carbohydrate m e t a b o l i ~ m . A num n i c o t i n a t e i s e f f e c t i v e as a hypocholesteremic agent and appears t o be b e t t e r t o l e r a t e d than n i c o t i n i c acid.78 A s e r i e s of methoxamine analogs7’ and a v a r i e t y of antidepressants80 a r e a l s o reported t o block t h e l i p o l y t i c e f f e c t s of epinephrine. Single i n j e c t i o n s of i n s u l i n cause acute f a l l s i n f r e e f a t t y acids i n man82 while i n another study a combination of potassium, glucose and i n s u l i n has been used t o t r e a t myocardial i n f a r c t i o n with some b e n e f i t .el Various agents reported t o lower c h o l e s t e r o l i n animals a r e chondroitin s u l f a t e A,83 vanadium,84 a mitochondria1 f r a c t i o n from starved rat liver8’ and i n man, 6-azauridine,eSa p-aminosalicylic acide6 and a r e l a t i v e of heparin, a s u l f a t e d polyanion SP54.e7 I n s u l i n i s reported t o decrease t h e c h o l e s t e r o l content of blood and a o r t a while increasing t h e l e v e l i n l i v e r and adrenalse7& i n cholesterol-fed r a b b i t s . I n p r i n c i p l e , feeding substances which remove b i l e acids from t h e i n t e s t i n e and which prevent t h e i r reabsorption should increase b i l e acid excretion and speed up c h o l e s t e r o l degradation. This i s , indeed, possible with agents such as f e r r i c chloride,88 aluminum hydroxide89 and with d i e t a r y saponins. so The b i l e acid-binding anion-exchange r e s i n MK-135 (Cholestyramine)s1 shows marked lowering of serum c h o l e s t e r o l i n t h e chicken, rabbit,s2 dogs3 and mans4 and has u t i l i t y i n r e l i e v i n g p r u r i t i s associated with b i l i a r y c i r r h o s i s .95 Steatorrhea i s a problem with t h i s drug97 but a r e p o r t i n d i c a t e s that when medium chain t r i g l y c e r i d e s a r e s u b s t i t u t e d f o r t h e long chain t r i g l y c e r i d e s i n t h e d i e t t h i s i s controlled.s8 Metamucil, an o r a l hydrophilic c o l l o i d , i s also reported t o lower serum cholesterol.S9 P-SitosterolLo0 plays a r o l e i n i n h i b i t i n g absorption of c h o l e s t e r o l . The a n t i b i o t i c s neomycin, N-methylated neomycin, but not streptomycin or Nacetylated neomycin, reduce c h o l e s t e r o l i n t h e chick,lol presumably by i n t e r f e r i n g with absorption processes. Paramomycin a l s o lowers serum c h o l e s t e r o l while reducing b a c t e r i a l f l o r a . l o 2 The r o l e of hyperglyceridemia i n a t h e r o s c l e r o s i s i s s t i l l cloudylo3 but it appears that t h e t r i g l y c e r i d e l e v e l i s no more u s e f u l than c h o l e s t e r o l l e v e l s i n i d e n t i f y i n g subjects with ischemic h e a r t disease .Io4 P a t i e n t s with elevated t r i g l y c e r i d e s a l s o have a high incidence of elevated f a s t i n g blood sugars and elevated f r e e f a t t y a c i d s l o s and thus have an increased r i s k of myocardial i n f a r c t i o n and l a t e n t diabetes. It i s perhaps s i g n i f i c a n t that i n t h e rat u t i l i z a t i o n of fat for energy a c c e l e r a t e s c h o l e s t e r o l b i o ~ y n t h e s i s . ~ 5 The development of some v a r i e t i e s of hy-pertriglyceridemia i s accompanied by a corresponding increase i n t h e turnover of palmitate but not l i n o l e a t e . This may represent increased synthesis of l i p i d from carbohydrate.106 I n s u l i n decreases t h e plasma l e v e l s of glyceride i n p a t i e n t s with hy-pertriglyceridemia?08 Protamine s u l f a t e , an i n h i b i t o r of l i p o p r o t e i n l i p a s e , i n h i b i t s t h i s a c t i o n . CPIB appears t o be t h e b e s t agent t o date f o r lowering elevated t r i g l y c e r i d e l e v e l s . 5713 Hormones. - I n t e r e s t i n estrogens stems from reports t h a t coronary a r t e r y disease i s a r a r i t y among premenopausal women1o9 and t h a t it i s much g r e a t e r i n c a s t r a t e d women than i n those having undergone hysterectomy but retain t h e i r ovaries,losb although t h i s finding i s not without i t s c r i t i c s . 1 1 0 On t h e o t h e r side, c a s t r a t e d men a r e less prone t o develop coronary disease than normal menyll Estrogens i n humans increase a-lipoprotein ( r i c h i n phospholipid P) l e v e l s but t h e i r e f f e c t on f5-lipoproteins ( r i c h i n c h o l e s t e r o l C ) i s v a r i a b l e . Thus,

182 -

Sect. IV

-

Metabolic 81 E n d o c r i n e

H e i n z e l m a n , Ed.

t h e C/P r a t i o w i l l decrease but t h e r e may o r may not be a drop i n c h o l e s t e r o l l e v e l s . l 1 2 Lecithin appears t o be t h e only major phospholipid f r a c t i o n which i s increased.113 Triglyceride l e v e l s a r e a l s o increased by estrogen administ r a t i o n . l l 4 Estrogens p r o t e c t t h e coronary a r t e r i e s but not t h e a o r t a of t h e chicken.l15 Experiments i n rats had l i k e e f f e c t s but r a b b i t s f a i l e d t o show b e n e f i t from estrogens.l16 Efficacy i n humans s t i l l remains a will-of-thewisp. S i g n i f i c a n t evidence of t h e i r prophylactic value against myocardial i n f a r c t i o n has not as y e t been demonstrated.117 Mental functioning i n p a t i e n t s with cardiovascular and cerebrovascular disease i s reported t o improve with estrogen treatment Since t h e s i d e e f f e c t s of estrogen i n t h e male a r e a problem, much e f f o r t has been devoted t o t h e search f o r compounds having t h e l i p i d p r o f i l e of an estrogen but devoid of i t s feminizing properties.l18 Among recent compounds studied a r e a s e r i e s of 1-methyl l3-alkyl estrogens120 and a s e r i e s of 3-alkyloxy-l7,17-(or 16,16)ethylenedioxy e s t r a t r i e n e s . 1 2 l Compounds 5"' and 6123 a r e reported t o show a possible s p l i t between t h e i r feminizing and l i p i d p r o p e r t i e s . On t h e p o s s i b i l i t y t h a t a metabolite i s responsible f o r

B

C-W2

d

H

3

0

C

H

z

,&b

CH30

CH30

5.

-cH3cH30 HO

6.

@-

OH

' 7.

t h e l i p i d e f f e c t s of estrogens, a s e r i e s of 2-hydroxy- and 2-methoxy-estradiols and t h e i r 16-oxygenated analogs w a s investigated. Com ound 7 i s reported t o have a l a r g e s p l i t between t h e properties i n question. p24 Conjugated equine estrogens increase blood p l a t e l e t levels125 during lipemia following i t s administration but o r a l contraceptives have l i t t l e e f f e c t , i f any, on blood coagulation o r f i b r i n o l y s i s l Z 6 (including p l a t e l e t adhesiveness) except perhaps t o increase f a c t o r V I I l e v e l s .Iz7 Estrogen therapy increases plasma hydrocortisone apparently by increasing t r a n s c o r t i n l e v e l s . 12' Gonadoptropins increase side-chain cleavage of c h o l e s t e r o l but not 20a -hydroxy cholesterol inThis i s i n d i c a t i n g t h a t 20a -hydroxylation i s stimulated by gonadotropins l i n e with t h e recent report that estrogen may decrease c h o l e s t e r o l by increasing t h e turnover r a t e , and therefore, t h e catabolism of c h 0 1 e s t e r o l . l ~ ~The f a c t that it i s not a c t i v e o r a l l y stimulated some e f f o r t i n t o finding an androsterone analog which i s a c t i v e o r a l l y . 3a-Methoxy-17-methyl-5a -androstan-l7@-ol i s reported t o lower c h o l e s t e r o l levels134 and enhance excretion of cholesterol i n t h e b i l e of rats.135 Methalone, an anabolic s t e r o i d , a l s o decreases t h e l e v e l of c h o l e s t e r o l i n man.'" Cortisone increases l e v e l s of triglyceri.de, phospholipid and c h o l e s t e r o l i n rats but does not a l t e r i n t e s t i n a l absorption o r t h e r a t e of incorporation of a c e t a t e i n t o cholesterol.137 I n c h o l e s t e r o l fed r a b b i t s adrenal hypertrophy precedes deposition of c h o l e s t e r o l and it i s suggested t h a t t h e adrenal gland

Chap. 16

Atherosclerosis

U r sp r ung

183 -

may a c t as a regulator intervening i n t h e process r e s p o i s i k l e f o r atheros ~ 1 e r o s i s . l I~n~man, hyperlipemia has been seen i n acute ad-renocortical insufficiency and successfully t r e a t e d with hydrocortisone .13' The use of adrenocorticotropic g e l has given encouraging r e s u l t s i n some cases of acute myocardial infarction.14' The hypothyroid s t a t e i s associated with an elevated plasma c h o l e s t e r o l l e v e l while t h e hyperthyroid s t a t e i s associated with low c h o l e s t e r o l l e v e l s i n plasma.141 It w a s again pointed out that i n a l a r g e number of p a t i e n t s hospitalized with acute myocardial i n f a r c t i o n t h e serum PBI l e v e l s a r e lower than The effectiveness of thyroactive substances i n lowering serum c h o l e s t e r o l and i n reducing atherogenesis has been amply demonstrated i n various animal species14' but t h e i r c l i n i c a l use remains r e s t r i c t e d because of t h e i r a c t i o n on b a s a l metabolic r a t e , and i n p a r t i c u l a r , on oxygen consumpt i o n of t h e myocardium and aggravation of angina. 14' Therefore , i n t e r e s t i s focussed on drugs whose e f f e c t on l i p i d metabolism i s accentuated r e l a t i v e t o t h e i r hypermetabolic e f f e c t . Many thyroxine analogs have been made, some of which a r e reported t o have a d e s i r a b l e " s p l i t " i n t h e two a c t i v i t i e s . 1 4 4 A comprehensive review of t h i s sub Sect has been published r e ~ e n t 1 y . l ~ ~ The thyroxine analogs of most i n t e r e s t a t t h e present time a r e D t h y roxine ( D T , ) and Dtriiodothyronine ( D T 3 ) . A controversy s t i l l e x i s t s as t o whether t h e r e i s any separation of l i p i d and calorigenic e f f e c t s of these two agents as compared t o t h ~ r 0 x i n e . l D ~T ~ 3 i s claimed t o lower serum l i p i d s i n p a t i e n t s with e s s e n t i a l hy-perlipemia but showed no e f f e c t i n nomolipemic controls.'47 As with D T 4 it a l s o appears t o show a worsening of angina o r arrhythmia i n humans. 14' Some more recent thyroxine-like compounds which have been studied extensively include Dtrichlorothyronine,149 4'-methoxy-3 ,3'-5triiodothyroacetic acid150 and a s e r i e s of t h y r ~ a l k a n o l s which l ~ ~ a l s o appeared t o show a " s p l i t " i n animals. !l?hyroidectomy i n rats has no e f f e c t on oxidation of e i t h e r labeled cholest e r o l o r sodium octanoate. Preliminary nuclear changes i n c h o l e s t e r o l take place a t about t h e same r a t e regardless of t h e thyroid s t a t e . This and o t h e r data a r e consistent with the e a r l i e r hypothesis t h a t t h e e f f e c t of t h e thyroid on cholesterol metabolism may be mediated a t t h e l e v e l of nuclear hydroxylation of t h e ~ t e r 0 i d . l ~ ' Increased thyroid l e v e l s decreases t h e production of e s t r i o l from e s t r a d i o l and increases production of 2-methoxyestrone. A recent shows t h a t 2-hydroxyestrone becomes t h e major metabolite i n subjects with high thyroid l e v e l s and t h a t i n myxedema 2-hydroxyestrone l e v e l s a r e diminished. The production of androsterone diminishes i n hypothyroidism and it has been postulated that t h i s produces increased l i p i d l e v e l s .132 These complicated endocrine i n t e r a c t i o n s make a study of thyroid a c t i v i t y d i f f i c u l t and i t s e f f e c t on l i p i d metabolism remains t o be c l a r i f i e d . Thrombosis and F i b r i n o l y s i s . - I n t e r e s t i n t h e thrombogenic theory of a t h e r o s c l e r o s i s proposed by von Rokitansky and revived by t h e s t u d i e s of D u g u i e has an experimental basis. A r t i f i c i a l production of f i b r i n deposits o r thrombi on vessels w i l l give rise t o intimal thickening and eventually a t h e r o s c l e r o s i s .156 Fibrin-like material has been demonstrated i n a t h e r o s c l e r o t i c l e s i o n s . lS7 locali z a t i o n of atheromatus changes may be r e l a t e d t o mechanical forces which lead t o t h e formation of thrombi a t s t r e s s points.15' The influence o f l i p i d s i n t h e pathogenesis of a t h e r o s c l e r o s i s might be f e l t not only i n t h e i r deposition i n t h e intimal l e s i o n but a l s o i n t h e i r clot-promoting effectsL5' (phospholipids

184

Sect. I V

-

Metabolic & E n d o c r i n e

H e i n z e l m a n , Ed.

and f r e e f a t t y acids can produce p l a t e l e t aggregation16') and t h e i r capacity t o i n h i b i t f i b r i n o l y s i s . 161,1ssb Chemical a t t a c k i n t h i s area has been a t i n h i b i t i n g t h e i n i t i a l event, p l a t e l e t aggregation, and a t d i s s o l u t i o n of t h e thrombus ( f i b r i n o l y s i s ) . For background t h e reader i s d i r e c t e d t o a comprehensive review on p l a t e l e t s and atherosclerosisl6* and t h e recent workshop on f i b r i n o l y s i s 163 Adenosine diphosphate (ADP) w i l l aggregate p l a t e l e t s i n v i t r o and t h i s aggregation can be prevented by enzymatic conversion of t h e ADP t o ATP.le4 This induced aggregation requires a complexable c a t i o n and fibrinogen. 16' The formation of thrombin and ADP a few seconds a f t e r blood i s shed has been demonstrated i n vivolB6 and thus both of these agents a r e present before t h e p l a t e l e t s a r e a v a i l a b l e . Epinephrine a l s o induces p l a t e l e t aggregation'" and thrombosis i n rats167 along with norepinephrine and serotonin. Pronethalol, a @-blocker, blocks t h e epinephrine-induced p l a t e l e t rise and @-Blocking agents have a l s o been found u s e f u l i n a l e v i a t i n g angina of effort1" and propranalol i s reported t o reduce t h e mortality from acute myocardial infar~ti0n.l~' I n i t i a l reports17' t h a t l i n o l e n i c acid caused a decrease i n p l a t e l e t adhesiveness and reduced t h e incidence of experimental thrombosis have been controverted by t h e same and by others.173 Among materials which are reported t o reduce p l a t e l e t adhesiveness a r e M p y r a m i d ~ l e ,a~s~e ~ r i e s of adenosine analogs,175 histamine17' and methyl mercuric nitrate.177 M.A.O. i n h i b i t o r s were shown not t o p r o t e c t against experimental thrombosis,178 i n disagreement with an e a r l i e r report. Dextran appears t o have some c l i n i c a l u t i l i t y i n thrombus inhibition17' and has been successfully used i n severe ischemia."' It appears t o i n h i b i t p l a t e l e t f a c t o r I11 by coating t h e platelet The r o l e of anticoagulants i n the treatment of myocardial i n f a r c t i o n i s unclear. Experiments with rabbits,lE3 dogs184 and rats185 have shown t h a t heparin w i l l prevent experimentally induced thrombosis. Conflicting reports suggest that anticoagulants may prolong t h e time f o r p l a t e l e t aggregation186 but heparin i n man showed no consistent changes i n p l a t e l e t economy or platel e t adhesiveness187 and only a feeble e f f e c t i n protecting against thrombotic occlusion a f t e r a r t e r i a l reconstruction.18' Increased s e n s i t i v i t y t o heparin w a s seen i n p a t i e n t s following acute myocardial infarction."' I n patients who suffered a myocardial i n f a r c t i o n while on anticoagulant therapy, t h e r e was a decrease i n anticoagulant l e v e l s as compared t o those with no myocardial infarction.'" I n p a t i e n t s with coronary heart disease t h e hematocrit and whole blood v i s c o s i t y is s i g n i f i c a n t l y higher than i n normals."' The r o l e played by t h e l i p i d - c l e a r i n g a c t i v i t y of heparin is s t i l l under i n v e s t i g a t i o d S 2 Long term anticoagulant therapy a f t e r myocardial infarction, using heparin"' and t h e c o u m a r i n ~appears ~ ~ ~ t o be b e n e f i c i a l and it has been recommendedlS5 t h a t t h i s form of treatment be used routinely. I n a cephalin f r a c t i o n which stimulated t h e c l o t t i n g mechanism, it w a s found that phosphatidyl-ethanolamine w a s t h e only component which showed cons i s t e n t high activity."' I n a study of p a t i e n t s with myocardial i n f a r c t i o n these authors1'' showed t h a t , although t h e t o t a l cephalin f r a c t i o n was similar t o t h a t i n t h e controls, t h e p a t i e n t s had cephalin with higher c l o t t i n g a c t i v i t y and markedly higher o l e i c a c i d content. I n s u l i n produces a c c e l e r a t i o n of blood c l o t t i n g and decreases f i b r i n o l y t i c a c t i v i t y l S 8 i n dogs, a t t r i b u t e d t o t h e sympathico-adrenal mechanisms for r e s t o r a t i o n of normal glycemia since it i s

.

Chap. 16

Atherosclerosis

Ur sprung

185 -

blocked by dihydroergotamine. P r o g e s t a t i o n a l agents a r e a l s o reported t o produce hypercoagulability i n w ~ m e n . ~ ” Thrombolyt i c therapy has r e c e n t l y been reviewed. 2oo The enzymes s t r e p t o ~ ~ ~appear , t o have u t i l i t y i n kinase, 201 staphylokinase202 and u r o k i n a ~ e201c acute thromboembolic v a s c u l a r d i s e a s e s . CA-7 (a f i b r i n o l y t i c enzyme from a s p e r g i l l u s oryzae) has produced l y s i s of arterial thrombi i n dogs 9204 Phenformin205 (DBI) and Metformin206 have shown promise as f i b r i n o l y t i c agents which may be u s e f u l i n t h e prophylaxis of a r t e r i a l occlusion. The authors have suggested t h a t phenformin a c t s l i k e a c o r t i c o i d and have indeed shown i t t o be b e n e f i c i a l i n rheumatoid a r t h r i t i s . 2 c ” An e a r l i e r report207 t h a t atromid has f i b r i n o l y t i c a c t i v i t y w a s c o n t r a d i c t e d recently.208 A l a r g e number of vasoactive drugs, both hyper- and hypotensive a r e reported t o have f i b r i n o l y t i c a c t i v i t y which i s r e l a t e d t o t h e i r vasoactive changes .209 Complamin (xanthinol n i c o t i n a t e ) has been shown t o possess f i b r i n o l y t i c a c t i v i t y i n but t h e p a t i e n t s very quickly develop a t o l e r a n c e t o t h e drug. Psycho-Social Factors. - One group of investigators211 has undertaken t h e study of behavior p a t t e r n s and t h e i r r e l a t i o n s h i p t o coronary artery d i s e a s e . They f i n d t h a t “Behavior P a t t e r n A” ( r e s t l e s s , competitive a t t i t u d e ) men have higher c h o l e s t e r o l levels,212 faster c l o t t i n g times212 and have a much higher incidence of arcus s e n i l i s and coronary artery d i s e a s e O 2 l 3 The same r e l a t i o n s h i p w a s found i n women.214 I n d i v i d u a l s harboring p a t t e r n A e x c r e t e more epinephrine during t h e i r working day t h a n t h o s e i n p a t t e r n B ( r e l a t i v e l y nonambitious, non-aggressive) .215 Subjects of p a t t e r n A have a l s o shown a high incidence of blood sludging a fte r a f a t t y m s a l whether t h e fats were s a t u r a t e d o r unsaturated, while sludging w a s v i r t u a l l y absent i n non-coronary prone ind i v i d u a l s .218 S i m i l a r r e l a t i o n s h i p s occur when animals a r e stressed.216 It i s implied t h a t s o c i a l f a c t o r s may account f o r t h e low incidence of myocardial i n f a r c t i o n i n Roseto, Pa.217 The i n h a b i t a n t s a r e s t r i k i n g l y obese, eat cons i d e r a b l e q u a n t i t i e s of animal f a t and t h e i r serum l i p i d p a t t e r n s do not d i f f e r from those found i n t h e Framingham study,loa y e t t h e y have a very low incidence of myocardial i n f a r c t s as compared t o t h o s e i n t h e Framingham study. Summary. - There i s c l e a r l y no u n i f i e d concept of atherogenesis. Knowledge i n t h e f i e l d i s c h a o t i c and treatment, o r prevention, i s s t i l l a matter of opinion. The p r e f e r r e d method f o r i n h i b i t i n g atherogenesis i s s t i l l c o n t r o l of t h e d i e t . O f i n t e r e s t i s t h e evidence which shows t h a t s u b s t i t u t i n g unsatur a t e d f o r s a t u r a t e d fats i n t h e d i e t causes a plasma-tissue r e d i s t r i b u t i o n r a t h e r t h a n i n h i b i t i n g s y n t h e s i s o r i n c r e a s i n g e x c r e t i o n r a t e s of c h o l e s t e r o l . Dietary c h o l e s t e r o l seems t o play only a minor r o l e , i f any, i n t h e development of human a t h e r o s c l e r o s i s . Drugs which lower serum l i p i d s are s t i l l s c a r c e . Efficacy of CPIB on long term treatment of t h e disease remains t o be seen. Cholestyramine, D T 4 , estrogens and n i c o t i n i c a c i d have t h e i r champions and w i l l f i n d s p e c i a l usage. Increased a c t i v i t y has focusSed a t t e n t i o n on p l a t e l e t adhesiveness, although no promising agent has emerged as y e t . The f i b r i n o l y t i c enzymes s t i l l are t h e b e s t agents f o r f i b r i n o l y s i s therapy. A s t a r t has been made i n unraveling psycho-social f a c t o r s and r e l a t i n g behavior w i t h serum l i p i d p a t t e r n s and proneness t o coronary h e a r t d i s e a s e . Research i n a t h e r o s c l e r o s i s must s t i l l be considered i n i t s infancy. With i n c r e a s i n g i n t e r e s t by medical s c i e n t i s t s and by governmental agencies, progress a t an a c c e l e r a t e d rate can be expected.

Sect. IV

186

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

R e f e renc e s 1.

2. 3. 4.

56.

L. Aschoff, V o r t r l g e fiber P a t h o l o g i e , F i s c h e r , J e n a , Germany, 1925, pp. 62-84. J . B. Duguid, J . P a t h o l . B a c t e r i o l . , 507 (1946). H. B. L o f f l a n d and T. B. Clarkson, Arch. P a t h . , &, 2 9 1 (1965). M. M. Daly, Q. B. Deming, V. M. Raeff and L. M. Brun, J. C l i n . I n v e s t . , 1606 (1963). M. M. Daly and V. R a e f f , B u l l . N. Y. Acad. Med., G, 225 (19651. H. N. Adel, 9. B. Deming, M. M. Daly, V. M. Raeff and L. M. Brun, J . Lab. C l i n . Med.,

2,

2,

66.

571 (1965).

80,

I . Chapman, Arch. P a t h . , 256 (1965). N a t i o n a l Diet-Heart Study, J. Am. Med. Assoc., &, 105 (1963). Medical News, J . Am. Med. Assoc., 31 (1965). 10. ( a ) T. R . Dawber & &., J. Publ. H e a l t h , s u p p l . , 4 (1957). ( b ) W . B. Kannel I n t e r n . Med., 33 (1961). ( c ) A. Kagan 5 &., F e d e r a t i o n P r o c . , 21, s u p p l . , 52 (1962). 11. A. Keys, J . M t . S i n a i Hosp., 20, 118 (1953). 12. E. H. Ahrens, Am. J . Med., 3, 928 (1957). 13. A. Keys, A. T . Anderson, F. F i d a n z a , M. H . Keys and B. Swahn, C l i n . Chem., 1,34 (1955). 14. W. E. Connor, G e r i a t r i c s , 16, 407 (1961). 15. G. V. Mann, C i r c . Res., 9 , 3 3 8 (1961). 16. 0. W. Portman and F. V. S t a r e , P h y s i o l . Rev., 2, 407 (1959). 17. A. N . Howard and G. A . Gresham, Chem. I n d . (London) 831 (1965). 18. D. F. Davis and A. C l a r k , Lancet, I , 29 (1965). 19. R . P i c k , S. J a i n , L. M. Katz and P. Johnson, J . A t h e r o s c l e r o s i s Res., 5, 16 ( 1 9 6 5 ) . 20. R . P i c k , S . J a i n , C. Kakita and P. Johnson, Proc. SOC. E x p t l . B i o l . Med., 850 (1965). 21. S t a t e m e n t , J . Am. Med. Assoc., 2,1194 (1965). 22. A. H. Howard, G. A. Gresham, D. Jones and I . W. J e n n i n g s , J . A t h e r o s c l e r o s i s Res., 5 , 330 (1965). 23 * H. Buchwald, J. A t h e r o s c l e r o s i s R e s . , 5 , 407 ( 1 9 6 5 ) . 24. ( a ) J . D. Wilson and C . A. Lindsey, J r . , J . C l i n . I n v e s t . , %, 1805 (1965). ( b ) C . B. T a y l o r . B. Mikkelson, J . A. Anderson, D. T. Forman and S . S. Choi, E x p t l . Mol. P a t h o l . , -4 , 480 (1965). 52 (1965). 25- F. Grande, J. T. Anderson, S. Chlouverakis, M. P r o j a and A. Keys, J . N u t r .,.J'8 26. J . M. Dietschy and M. D. S i p e r s t e i n , J. Clin. I n v e s t . , 44, 1311 (1965). -27. G . A. Gresham, A. N. Howard, J . Mc'been and D. E. Bowyer, B r . J. E x p t l . P a t h . , 8 4 (1965) 28. F. A. Bieberdorf and J. D. Wilson, J . C l i n . I n v e s t . , 44, 1834 (1965). 29. N . S p r i t z , E. H . Ahrens, J r . and S. Grundy, J . C l i n . I n v e s t . , 2, 1482 1 9 6 5 ) . ( a ) N . S p r i t z , J . C l i n . I n v e s t . , 9, 339 (1965). ( b ) N . S p r i t z , B u l l N . Y. Acad. Med., 30. 41, 244 (1965). 31. S. L i n d s t e d t . J. Avigan, D. S. Goodman, J . S j b v a l l and D. S t e i n b e r g , J . Clin. I n v e s t . , 44, 1845 (1965). 32, J. Avigan and D. S t e i n b e r g , J . Clin. I n v e s t . , &, 1845 (1965). 3 3 . S. Dayton, S. Hashimoto and M. L. Pearce, C i r c u l a t i o n , 32, 911 (1965). 407 (1965). 34. J . H . Moore and D. L. Williams, B r i t . J . N u t r . , 9, 35. J . Dupont, J . Am. O i l Chemists S O C . , 3, 907 ( 1 9 6 5 ) . 37. W. Hollander and A. Chobanian, Boston Med. Q u a r t . , g,37 (1959). 38. M. R . Malinow. M. A. Marini, B. Lema, M. Nebbie a n d J . A. B e r a r d i . Acta P h y s i o l . L a t i n o h.. 71 (1964). 39. F. V i c h i , P. L. Masi, P. P i e r l e o n i and S. Orlando, S p e r i m e n t a l e , 168 (1965). 40. ( a ) J. H. B u r c k h a l t e r eta.,Am. Chem. SOC. Abstr. o f p a p e r s , 147 mtg.. P h i l a d e l p h i a , Pa., Apr. 6-10, 1964, p. 20M. ( b ) W. B. Wright, D. A. Dunnigan and V. Biermacher, J . Med. Chem., 1,113 (1964). 41. ( a ) S. K. F i g d o r , E. C. S c h r e i b e r . R . B. S t e b b i n s , P. F. Moore and R. Pinson, J r . , J . Med. Chem., 1,508 (1964). ( b ) G. M. K. Hughes, P. F. Moore and R . B. S t e b b i n s , J . Med. Chem., 1,511 (1964). 42. J . H . S h o r t g . , J. Med. Chem., 8, 223 (1965). 43. F. T. Bach, J. C . Barclay and E. Cohen, Am. Chem. SOC. Abstr. of p a p e r s , 150 mtg., A t l a n t i c C i t y , N. J . , S e p t . 13-16, 1965, p. 18P.

7. 8. 9.

s.,

z,

m,

5,

u,

5,

-

14,

x.

Chap. 16 44. 45. 46. 47. 48. 49. 50. 51. 52.

53. 54. 55. 56 57 *

-

58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69 * 70. 71. 72 * 73 * 74. 75. 7677. 78 i 79. 80. 81.

82.

83.

84.

Atherosclerosis

UrsD rung

187

G. Rodney, M. L. Black and 0. D. Bird, Biochem. Pharmacol., 14,445 (1965). (a) M. Kraml, L. G. Humber, J. Dubuc and R. Gaudy, 5 . Med. Chem., 1,5OG (1964). (b) P . Kr. Cushman and J . G. Hilton, Metabolism, 14. 924 (1965). (c) R. Fumagalli, R. Niemiro and R. Paoletti, J. Am. Oil Chemists SOC., 9, 1018 (1565). R. E. Counsell, P. D. Klimstra, L. N. Nysted and R. E. Ranney, J . Med. Chem., 8, 45 (1965). (a) R. E. Ranney and D. L. Cook, Arch. Intern. Pharmacodyn., 51 (1965). (b) B. A. Sachs and L. Wolfman, Arch. Internal Med., 116,366 (1965). D. Dvornik, M. Kraml and J. Dubuc, Proc. SOC. Exptl. Biol. Med.. 116,537 (1964). (a) P. C. Anderson and J. M. Martt, Arch. Derm., p1, 181 (19651. (b) N. Winer, J. M. Martt, J. E. Somers, L. Wolcott, H. E. Dale and T. W. Burns, J. Lab. Clin. Med.,

a,

66, 758 (1965).

J. Doorenbos and M. T. Wu, J. Pharm. Sci., 2, 1290 (1965). J. E. Dalidowicz and H. J. McDonald, Biochemistry, 5. 1138 (1965). (a) R. Hess, Advances in Lipid Research, Vol. 2 , R. Paoletti and D. Kritchevsky, eds., Academic Press, N. Y., 1964, p. 333. (b) P. Morand, J. F. Bagli, M. Kraml and J. Dubuc, J. Med. Chem., 1,504 (1964). (c) D. Nardi, L. Mauri and F. Barzaghi, I1 Farmaco, Ed. Sci., 20, 456 (1965). Atromid Symposium, J . Atherosclerosis Res., 3, 351-739 (1963). S. W. Teal and W. Gamble, Biochem. Pharmacol., 14, 896 (1965). D. L. Azarnoff, D. R. Tucker and G. A. Barr, Metabolism, 2,959 (1965). P. J. Nestcl, E. 2. Hirsch and E. A. Couzens, J. Clin. Invest., 2. 891 (1965). (a) D. R . Avoy, E. A. Swyryd and R. G. Gould, J. Lipid Res., 6, 369 (1965). (b) M. M. Best and C. H. Duncan, Amer. J. Cardiol., 3,230 (1965). (c)- D. Berkowitz, Metabolism,

14,

966 (1965).

E. H. Strisower, A. V. Nichols, F. T. Lindgren and L. Smith, J. Lab. Clin. Med.,

748 (19651.

&,

I. A. Slowe, A. C. Carter and E. B. Feldman, Arch. Intern. Med., 116,523 (1965). G. Trevaks and R. R. H. Lovell, Ann. Rheum. Dis., 24, 572 (1965). C. H. Duncan, M. M. Best and G. C. Robertson, Lancet I, l g l (1965). C. M. Ogston, D. Ogston and G. M. McAndrew, Curr. Ther. Res., 1,437 (1965). B. Sweet, B. M. Rifkind and G. P. McNicol, J. Atherosclerosis Res., 2, 347 (1965). (a) J. T. Doyle, T. R. Dauber, W. B. Kannel, S. H. Kinch and H. A. Kahn, J. Am. Med. Assoc., 886 (1964). (b) 0. Paul e t g . , Circulation, 28, 20 (1963). H. Engelberg, J. Am. Med. Assoc., ,&I 1033 (1965). A. Kershbaum, S . Bellet and R. Khorsandian, Am. Heart J., Q, 206 (1965). A. Tsujimoto, S . Tamino and Y. Kurogochi, Japan. J. Pharmacol.. 3, 415 (1965). S. Bellet, A. Kershbaum and J. Aspe. Arch. Intern. Med., 116,750 (1965). W. E. Dulin, G. H. Lund and G. C. Gerritsen, Proc: SOC. Exptl. Biol. Med., 118,499 (1965). (a) G. C. Gerritsen and W. E. Dulin, Diabetes, 14, 507 (1965). (b) D. L. Smith, A. A. Forist and G. C. Gerritsen, J. Pharmacol. Exptl. Ther., 316 (1965). D. L. Smith, A. A. Forist and W. E. Dulin, J. Med. Chem., g, 350 (1965). L. A. Carlson, S. 0. Liljedahl and C. Wiser, Acta Med. Scand., lJ8, 81 (1965). (a) K. Stock and E. Westermann, Life Sci., 4. 1115 (1965). (b) R. P. Eaton, D. Steinberg and R. H. Thompson, J. Clin. Invest., 9, 247 (1965). (a) R. Bombelli, T. Farkas and R. Vertua, Med. Pharm. Exptl J. Ostman g g . , Acta Med. Scand., 615, 623, 631, 639 N. M. Schulman and G. Galansino, Proc. SOC. Exptl. Biol. Med R . S . Jacobs, M. S. Grebner and D. L. Cook, Proc. Soc. Exptl P. Lipton and J. A. Michels, Geriatrics, 20. 379 (1965). R. Ishigami g g., Bitamin, 2, 232 (1965). (a) M. Verstraete, H. Maes and J. Vermylen, Med. Pharmacol. Exptl.. 3, 401 (1965). (b) J. P. Miale, Curr. Ther. Res., 1,392 (1965). J. J. Burns, K. I. Colville. L. A. Lindsay, S. P. April and R. A. Salvador, Pharmacologist,

a,

z.

x,

-6 ,

186 (1964).

a,

90 (1965). V. Vitek. B. Mosinger and V. Kujalova, Nature, B. Mittra. Lancet 11. 607 (1965). D. P. Jones and R. A. Arky. Metabolism, 14, 1287 (1965). L. M. Morrison K. Murata, J. J. Quilligan, Jr., 0. A. Sahjeide and L. Freeman, Gerontologist. 3, 16 (1965) abstrl Korkhov, Farma 01 Toksik, 28, 83 (1965).

d

.

Sect. IV

188

-

Metabolic 81 Endocrine

H e i n z e l m a n , Ed.

e,

660 (1965) abstrg. 85. B. B. Migicovsky, Federation Proc.. d5a. A. 2 . Buttoo, M. C. G. Israels and J. W. Wilkinson,$rit. Med. J., I, 552 (1965). 86. P. Samuel, H. C. Price and 0. B. Shalchi, Proc. Soc. Exptl. Biol. Med., 654 (1965). 87. H. Hammerl, 0. Pichler and M. Studlap. Med. Klin., 6 0 , 761 (1965). 87a. L. A. Myasnikov, Byul. Ekspes-im. Biol. Med., 2,40 (1965). 88. M. D. Siperstein, C. W. Nichols, Jr. and I. L. Chaikoff, Circulation, 2 , 57 (19651. 89* S . Rodbard. C. Bolene, R. Pick and L. N. Katz. Circulation, 2, 479 (19651. 90. P. Griminger and H. Fisher, Proc. Soc. Exptl. Biol. Med.. 2, 424 (1958). 91. D. M. Tennent. H. Siegel. M. E. Zanetti, G. W. Kuron, W. H. Ott and F. J. Wolf, J. Lipid Res., 1,469 (1960). 92 A. N. Howard, G. A. Gresham, D. Jones and I. W. Jennings. Life Sci., 5. 639 (1965). 93. G. R. Jansen and M. E. Zanetti, J. Pharm. Sci., 2,845 (1965). 289 (1965). 94. S. A. Hashim and T. B. VanItallie. J. Am. Med. Assoc., 95. F. Schaffner, F. M. Klion and A. J. Latuff, Gastroenterology, g,293 (1965). 97* R. W. Harkins, L. M. Hagerman and H. P. Sarett, J. Nutr., 3, 85 (1965). 98* R. B. Zurier, S. A. Hashim and T. B. VanItallie. Gastroenterology, 3, 490 (1265). 99. J. E. Garvan, D. T. Forman, W. R. Eisman and C. R. Phillips, Proc. Soc. Exptl. Biol. Med., 120. 744 (1965). 100. (a) L. Swell, E. C. Trout, Jr., H. Field, Jr. and C. R. Treadwell, J. Biol. Chem.. 2286 (1959). (b) T. Gerson, F. B. Shorland and G. G. Dunckley, Biochem. J., 399 (1965). 101. P. DeSomer. H. Vanderhaeghe and H. Eyssen, Nature, 204. 1306 (1964). 102. W. J. Messinger and C. M. Samet. Angiology, 6, 29 (1965). 103. M. J. Albrink, Annals of Internal Med.. 5p, 581 (196:). 104. D. F. Brown, S. H. Kinch and J. T. Doyle, New Eng. J. Med., 947 (1965). 105. H. Braunsteiner et&.. Klin. Wshr., 9, 715 (1965). 106. P. J. Nestel, Metabolism, 14,1 (1965). 108. 5. Schlief and L. W. Kinsell, Proc. SOC. Exptl. Biol. Med., 120, 272 (1965). 109. (a) L. 0. Underdahl and H. L. Smith, Proc. Staff Meet. Mayo Clin., 22, 479 (1947). (b) B. J. Clawson, Am. Heart J., 22, 607 (1941). (c) A. B. Ritterbrand. I. A. Jaffee, P. M. Densen, J. F. Magagna and E. Reed, Circulation, 3, 237 (1963). 110. F. J. Schilling, 0. K. Christakis. N. J. Bennett and J. F. Coyle. Am. J. Public Health, 2, 461 (1964). 111. R. H. Furman, R. P. Howard, M. R. Shetlar and R. Imagawa, Am. Med. J., 2, 965 (1957). 112. (a) R . H. Furman and R. P. Howard, Ann. Intern. Med., 9, 969 (1957). (b) R. H. Furman, R. P. Howard, L. N. Ncrcia and E. C. Keety, Am. J. Med., &. 80 (1958). (c) M. F. Oliver and G. S. Boyd, Lancet 11, 690 (1959). i13. M. Hagopian and R. W. Robinson, J. Clin. Endocr., 3, 283 (1965). 114. J. W. Gofman, 0. DeLalla. F. Glazier and N. K. Freeman, Plasma, 2, 413 (1954). 115. R. Pick, J. Stamler, S. Rodbard and L. N. Katz, Circulation, 6, T76 (1952). 116. R. Pick, J. Stamler and L. N. Katz, Hormones and Atherosclerosis. G. Pincus, ed., Academic Press, N. Y., 1959, pp. 229-240. 117. (a) M. F. Oliver and G. S. Boyd, Lancet 11. 499 (1961). (b) J. Stamler, Atherosclerosis and its Origin, M. Sandler and G. H. Bourne, Eds., Academic Press, N. Y.. 1963, p. 231. (c) J. Marmorsten, F. J. Moore. C. E. Hopkins. 0. T. Kuzma and J. Weiner. Proc. SOC. Exptl. Biol. Med., 110, 400 (1962). 118. (a) R. Hess, Advances in Lipid Research, Vol. 2, R. Paoletti and D. Kritchevsky, Academic Press, N. Y., 1964, pp. 351-363. (b) V. A. Drill, D. L. Cook and R. A. Edgren, Hormones and Atherosclerosis, G. Pincus, ed., Acadenic Press, N. Y., 1959, Chapt. 19. 119. A. Corbellini, G. Geraldi, G. Ferrara and G. Lugana, Farmaco [Scid , 3, 1044 (1964). 120. J. Fisher & J. Chem. Soc., 4472 (1964). 121. G. C. Buzby & s.. J. Med. Chem., 1, 755 (1964). 122. R. E. Schaub, J. H. van den Hende and M. J. Weiss, J. Org. Chem., 2234 (1965). 123. K. Nakamura, Y. Masuda, K. Nakatsuji, T. Hirooka and K. Fujimoto. J. Atherosclerosis Res., 2. 420 (1965). 124. S. Gordon, E. W. Cantrall, W. P. Cekleniak, H. J. Albers, S . Mauer, S. M. Stolar and S. Bernstein. Steroids, 4,267 (19641. 125. R. W. Robinson and R. V. Lebeau, J. Atherosclerosis Res., 2, 120 (1965). 126. (a) L. C. Powell, M. M. Guest and T. P. Bond, Am. J. Obstet. Gynecol.. 2, 167 (19651. (b) R. R. Margulis. J. L. Ambrus, I. B. Mink and J. C. Stryker, Am. J. Obstet. Gynecol., 2,161 (1965). (c) E . A. Caspary and M. Peberdy, Lancet I, 1142 (1965).

B,

-

z,

-

s, ~

a,

s..

x,

e,

Chap. 1 6

U r sp rung

Atherosclerosis

127. 128.

189 -

J. M. Thomson and L. Poller, Brit. Med. J. 11, 270 ( 1 9 6 5 ) . (a) S. D. Grant, F. Ch. Pavlatos and P. H. Forsham, J. Clin. Endocr., 3,1057 (1965). (b) 0. I. Dodek, Jr., E. J. Segre and E. L. Klaiber, Am. J. Obstet. Gynecol., 2, 173 (1965). 129. (a) K. Lifshitz and N. S. Kline, Am. J. Psychiat., 122. 329 (1965). (b) R. B. Evans and J. Marmorsten, Proc. SOC. Exptl. Biol. Med., 118,529 (1965). 130. E. Forchielli, R. I. Dorfman, S. Ichii and K. Menon, Cancer Res., 3, 1076 (19653. 131. P. J. Nestel, E. 2 . Hirsch and E. A. Cousenz, J. Clin. Invest., &. 8 9 1 (1965). 132. L. Hellman, H. L. Bradlow, B. Zumoff. D. K. Fukoshima and T. F. Gallagher, J. Clin. Endocr., B,637 (1959). 133. R. H. Furman, R. P. Howard and P. Alanpovic, J. Lab. Clin. Med., 60, 876 (1962). 134. R. E. Counsel1 and P. D. Klimstra, J. Med. Chem., 1,119 (1964). 265 (1965). 135. R. E. Ranney and S. H. Grayson, Arch. Intern. Pharmacodyn. Therapie, 136. S. Castillo, P. A. Serrano and M. Segura, Lancet 11, 1090 (1964). 137. M. Friedman, J. van den Bosch. S. 0. Byers and S. St. George, Am. J. Physiol., 208, 94 (1965). 138. W. Albrecht and W. Schuler. J. Atherosclerosis Res., 5, 353 (1965). 565 (1965). 139. P. Bjbrnstad. E. Gjone and H. N. Haugen, Acta. Med. Scand., 140. N. B. Baroody and W. G. Baroody, Am. J. Med. Sci., *,402 (1965). 141. D. Kritchevsky, Metabolism, 2, 984 (1960). 142. J. A. Azar, J. Am. Geriat. Soc., 3, 212 (1965). 143. G. S. Boyd, G. L. Montgomery and M. F. Oliver, Drugs Affecting Lipid Metabolism, S. Garattini and R. Paoletti, eds., Elsevier. Amsterdam, 1961, pp. 412-422. 144. G. S. Boyd. J. Atherosclerosis Res., 1. 26 (1961). 145. R. Hess, Advances in Lipid Research, Vol. 2, R. Paoletti and D. Kritchevsky, eds., Academic Press, N. Y.. 1964, pp. 370-381. 146. (a) G. S. Boyd and M. F. Oliver, J. Endocrinol., 21, 33 (19601. (b) E. H. Strisower, Federation Proc.. 21, 96 (1962) Fbstr.3. (c) C. Moses, J. R. Jablonski, J. H. Sunder and L. B. Katz. Am. J. Med. Sci., 244. 7 3 1 (1962). (d) J. M. Merrill and J. LemleyStone, Am. J. Physiol., 513 (1962). 147. 0. Schwarz and K. H. Kuhnstock, German Med. Monthly, lo, 53 (1965). 148. R. S. Watts, R. G. Schoenfeld, Rocky Mountain Med. J., 62, 3 4 (1965). 149. N. J. Oakley, K. Adadevesh and P. Wise, J. Endocr., 28, 225 (1964). 150. B. Blank, C. M. Greenberg and J. F. Kerwin, J. Med. Chem., 1, 53 (1964). 151. W. J. Wechter, W. A. Phillips and F. Kagan, J. Med. Chem., g, 474 (1965). 153. D. Kritchevsky and S. A. Tepper. J. Cellular Comp. Physiol.. 66, 9 1 (1965). 154. J. Fishman. L. Hellman and B. Zumoff, J. Clin. Endocr., 3,365 (1965). 155. 3. B. Duguid, Connective Tissue, Thrombosis and Atherosclerosis, I. H. Page, ed., Academic Press, N. Y., 1959, pp. 13-32. 156. T. Astrup, pp. 223-237. 157. N. Woolf and T. Crawford, J. Path. Bact., 80, 405 (1960). 158. J. F. Mustard, E. A. Murphy, H. C. Rowel1 and H. G. Downie, Am. J. Med., 2,6 2 1 (1962). (b) B. Kommerell. 159- ( a ) J. M. Waldron and G. C. Duncan, Am. J. Med.. s, 365 (1954). J. Atherosclerosis Res., 2, 233 (1962). 160. J. W. Kerr. I. MacAulay, R. Pirrie and B. Bronte-Stewart, Lancet, 1296 (1965). 161. H. W. Grieg and I. A. Runde. Lancet 11, 461 (1957)-; 162. J. F. Mustard, E. A. Murphy, H. C. Rowsell and H. G. Downie, J. Atherosclerosis Res., -4, 1 (1964). 163. Fibrinolysis, G. R. Fearnley, ed., J. Clin. Path., 305 (1964). 164. R. J. Haslam. Nature, 202, 765 (1964). 165. N. 0. Solum and H. Stormorken, Scand. J. Clin. Lab. Invest., suppl., lJ, 170 (1965). 166. J. Marr, J. J. Barboriak and S. A. Johnson, Nature, 3, 259 (1965). 167. H. Selye, B. Tuchweber and B. Rohan, Nature, 208, 900 (1965). 168. P. D. McClure, G. I. C. Ingram and R. V. Jones, Thromb. Diath. Haemorrhag., 3, 136 (1965). 169. P. J. R. B. Keelan. Brit. Med. J., I, 897 (1965). 170. P. J. D. Snow, Lancet 11, 551 (1965). 171. (a) P. A . Owen, A. Hellem and A. Odegaard, Lancet 11, 975 ( 1 9 6 4 ) . (b) A. Nordoy, Thromb. Diath. Haemorrhage, g, 543 (1965). (c) P. A. Owren, Ann. Intern. Med., &, 167 (1965).

u,

x,

a,

e.,

x,

190 172.

173*

174.

175.

Sect. I V

-

Metabolic & Endocrine

H e i n z e 1 m a n , Ed.

P . A. Owren, A. Hellem and A. Odsgaard, Lancet 11, 849 (1965). G. Bjbrck and B. Edgren, Acta Ned. Scand.. 67 (1965). P. R. Emnoris &. , Lancet 11, 603 (1965). ( a ) G. V. R . Born, R. J . Haslam, M. Goldman and R. D. Lowe, Nature, 678 (1965). ( b ) Y. I . M i s i r l i o g l u . Lancet 1 1 , 850 ( 1 9 6 5 ) . ( c ) J . W. C o n s t a n t i n e , N a t w e , 3,

m,

z,

1075 (1965). J . W. C o n s t a n t i n e , Nature, 9 1 (1965). C. W. Robinson, J r . , S. C. Kreas, R. H. Wagner and K. M. Brinkhous, Exptl. Mol. P a t h o l . , 2, 457 (1965). 178 S. V. Chandra and S. H. Z a i d i , J. A t h e r o s c l e r o s i s R e s . , 5 , 249 (1965). 5cZ (1965). ( b ) W. M. 179 * ( a ) R. B. Sawyer and J. A. Moncrief, Arch. Surg., Chadduck, W. G. C r u t c h f i e l d and M. Roberts, S w g . Gynec. O b s t e t . , 121, 491 (1965). 180. J. J . Bergan, 0. H. T r i p p e l and A. H. Kaupp, Arch. S u r g . , 338 (1965). 182. R. A. Ewald, S. W. E i c h e l b e r g e r , Jr.. A. A. Young, H. J. Weiss and W. H. Crosby, T r a n s f u s i o n , 2, 109 (1965). 183. V. Ourewich and D. P. Thomas, J . Lab. C l i n . Med., 604 (1965). 184. R. D. Williams and F. K a r a f f a , Surg. Gynec. O b s t e t . , 309 (1965). 185. I. L u s t r i n Surgery, 857 (1965). 186. G. P. McNicol and A . S. Douglas, Lancet I , 729 (1965). 187. J. F. Mustard, F. B. G i l b e r t and E. A. Murphy, Lancet I , 575 (1965). 188. E. W . Salzman, Surgery, 131 (1965). 189. C. Dufaillt, Canad. Med. Assoc. J., 2,13 (1965). 190. K. Molne and P. F. H j o r t , Acta Med. Scand., 121, 571 (1965). 191. G. A. Mayer, Capad. Med. Assoc. J . , 1151 (1965). 192. P. T. Kuo, D. R . B a s s e t t and A. M. DiGeorge, C i r c . Res., &I 2 2 1 (1965). , J. A t h e r o s c l e r o s i s Res., 5, 253 (19653. 193 * L. E. B t l t t i g e r , L. A. Carlson 194. V. A. Cooperative Study, R. V. E b e r t . c h r . , J . Am. Med. Assoc., 929 (1965). 195. D. Leak, G e r i a t r i c s , 20, 150 (1965). 196. J . D. B i l l i m o r i a , V. J. 1 - a d and N. F. Maclagan, J . A t h e r o s c l e r o s i s R e s . , 2, 9 0 , 102 (19651 198. M. I . Mamikonyan, Byul. Eksp. B i o l . Med., 1049 (1965). 199 R. R. Margulis, J. L. h b r u s , L. B. Mink and J. C . S t r y k e r , Am. J. O b s t e t . Gynecol., 2, 161 (1965). 200. D. Jewson, Minnesota Med., 4&, 1363 (1965). 201. 856 (1965). ( b ) ( a ) D. Cavanagh and E. A. Albores, Am. J . O b s t e t . Gynecol., M. C . Rozenberg, A u s t r a l i a n J. E x p t l . B i o l . Med. S c i . , 245 (1965). ( c ) C . D. Jacobsen and A. B. Chandler., Scand. J. Clin. Lab. I n v e s t . , s u p p l . . 8 , 2 2 5 (1965). ( d ) J . E. Nilehu and B. Robertson, Scand. J. Haematol., 2, 267 (1925). ( e ) L. B. Fleming and E. E. C l i p t o n , J . Surg. Res., 5, 153 (1965). 202. B. Sweet, A. P. McNicol and A. S. Douglas, Clin. S c i . , 3,375 (1965). 203. A. P. F l e t c h e r &- &., J. Lab. Clin. Med., &, 713 (1965). 731 (1965). 204. W. H. E. Roschlau and A. L. Tosoni, Can. J. Physiol. P h a r n a c o l . , 205. G. R. F e a r n l e y . R. C h a k r a b a r t i and E. D. Hocking, Lancet I, 9 (1965). 206. R. C h a k r a b a r t i , E. D. Hocking and G. R. F e a r n l e y , Lancet 11, 256 (1965). 207. S. C. S r i v a s t a v a . M. J. Smith and H. A. Dewar, J. A t h e r o s c l e r o s i s R e s . , 1. 640 t1963). 208. B. Sweet, B. M. R i f k i n d , G . P. McNicol and M. Gale, J. A t h e r o s c l e r o s i s Res.. 3, 347

a,

176. 177. I

E,

s,

&.,

66, 121,

s,

x,

s,

&.

x,

s,

5,

z,

5,

(1965 1 *

Holemans, Am. J. P h y s i o l . , 208, 511 (1965). P. McNicol and A. S. Douglas, B r i t . Med. J . . I , 1149 (1965). Friedman, G e r i a t r i c s , 2, 562 (1964). Friedman, R. H. Rosenman and V. C a r r o l , C i r c u l a t i o n , 852 (1958). Friedman and R. H. Rosenman, J. Am. Med. Assoc., 1286 (19591.' H. Rosenman and M. Friedman, C i r c u l a t i o n , 4, 1173 (1961). Friedman, S. S t . George, S. 0. Eyers and R. H. Rosenman, J . Clin. I n v e s t . ,

209. 210. 211. 212. 213. 214. 215.

R. G. M. M. M. R. M.

216.

( a ) H. S o b e l , C . E. Mondon and R . S t r a u s , C i r c . R e s . , 11,971 ( 1 9 6 2 ) . ( b ) H. L. Ratcliffe, Circulation, 481 (1963). C. S t o u t , J. Morrow, E. N. Brandt, Jr. and S. Wolf, J . Am. Med. Assoc., 845 (1964). M. Friedman. S. 0. Byers and R. H. Friedman, J . Am. Med. Assoc., B, 882 (1965).

217. 218.

a,

a,

758 (19601.

a,

188,

2,

Chapter 17. , Non-steroidal Hormones and Their Antagonists Eugene C. Jorgensen, School of Pharmacy, University California, San Francisco, Calif. INTRODUCTION The non-steroidal hormones considered in this section are composed primarily of amino acid derivatives. Structures of the simpler substances, such as the thyroid hormones and oxytocin and vasopressin have been known for some time. Recent advances center around structural variations of known hormones, structural elucidation of new hormones, and efforts of the initial syntheses of hormones whose structures have been proposed on the basis of degradation studies. The compounds selected for review are limited to those whose structures are known, or have been proposed, and in which significant recent progress has been made in the synthesis of the hormone or of its analogs. A hormone is generally considered to be a discrete chemical substance, produced in a gland, secreted into the body fluids, and producing a specific effect on the activities of other organs. The "glandular hormones" clearly belong to this class. Recently, a number of peptides with intense pharmacological activities have been found to be generated by specific enzyme catalyzed hydrolysis of plasma proteins; these have been called "tissue hormones.'' An additional miscellaneous group related by peptide nature and pharmacological properties have thus far been isolated only from non-mammalian sources.

HORMONES OF THE PANCREAS .Insulin.--Recombination experiments of synthetic and natural A and B chains of insulin have been reported from German, Chinese and American groups. The Aachen group described a synthesis of the A-chain of sheep insulin and its combination with the native B-chain to produce biological and chemical properties like those of insu1in.l Synthesis of the B-chain and condensation with the native sheep A-chain produced about 0.655; insulin activity.2 Combination of synthetic A and B chains of sheep insulin produced biological activity 0.2 to 1.0% that of native insulin.3 An improved recombination of bovine insulin was reported to produce 44% insulin-like activit using a 1:l mixture of the preoxidized A-chain and the reduced B-chain.

tr

The Shanghai-Peking groups reported combinations of synthetic B-chain with the natural A-chain of bovine insulin, and of the synthetic A-chain with the natural B-chain to give crystalline products with 2-4% of the activity of natural insulin: 5 , 6 slight biological activity was obtained initially by combining synthetic A and B chains. Improved synthesis of A and B chains,' using azide condensations at the final stages of synthesis to preclude racemization, and use of excess of the A-chain and air oxidation at pH 10.68 led to a synthetic bovine insulin with initial activity of 1.2-2.5% that of natural insulin based on the protein concentration present. Purification by solvent extraction and crystallization in a citrate buffer containing acetone and zinc acetate yielded a crystalline product identical in crystal shape and biological activity (mouse convulsion and rabbit hypoglycemia) with natural bovine insulin. Chromatographic and isotope dilution studies with C-14 labeled synthetic insulin

192 -

Sect. IV - Metabolic & Endocrine

H e i n z e l m a n , Ed.

confirmed identity with the natural protein. The Pittsburgh group has followed earlier work on the combination of s p thetic A-chain and natural B-chain of sheep insulin (0.5-1.2% activity) ,l o with synthesis of the B-chain and combination of this with the natural Achain to produce insulin-like activity equivalent to that produced by recombination of the natural A and B chains.ll Considerable activity was also found when synthetic B-chain was combined with a partially purified preparation of synthetic A-chain. The B-chain of human insulin has been prepared as the S-sulfonate and combined with the natural A-chain of bovine insulin to produce 4-8% the activity of crystalline bovine insulin.12 Synthesis of human A-chain, and combination with synthetic human B-chain and with natural bovine B-chain yielded 2% and 8% insulin activity by the mouse convulsion method.13 The combined results of the German, Chinese and American groups indicate that totally synthetic preparation of pure human insulin is possible with presently available techniques, and that the tools are available for important studies of insulin analogs. Glucagon.--The amino acid sequence of the glycogenolytic pancreatic hormone, glucagon, was determined by Bromer and co-workers in 1957. Partial syntheses of the linear peptide, which contains 29 amino acids, have been reported by Schr6der14, and by Wtinschl5 , but biological activities have not yet been reported for these glucagon-related peptides, and the complete synthesis of glucagon is not yet reported. HORMONES OF THE PITUITARY The Melanocyatimulating Hormones (M2-5) and Adrenocorticotropic Hormones JACTH).--Presence of the same amino acid sequence from 4 through 10 may account for certain common biological properties of MSH and ACTH. B-MSH from various species possess additional N-terminal amino acids, and vary in the nature of amino acids 1-3 in the a-MSH structure. Melanocyte-Stimulating Activity.--The ability to produce skin darkening in amphibia has been associated with the pentapeptide sequence 6-10, His- PheArg-Try-Gly, common to both ACTH and MSH. This requirement has now been shortened to the tetrapeptide, His-Phe-Arg-Try ( 6 - 9 ) , which has been shown to possess both melanocyte-stimulating and lipolytic activity at levels comparable to those of the pentapeptide.l6 Alteration in the (6-10) pentapeptide sequence has produced one of the few examples of inhibition by close analogs in the peptide field. The citrulline analog, His-Phe-=-Try-Gly elicits no MSH-activity, but inhibits the activity of a-MSH.I7 Substitution of D-amino acids in the pentapeptide produces compounds which range in activity from MSH-like to antagonists. The all-D isomer, D-His-D-Phe-D-Arg-D-Try-Gly showed no melanocyte-stimulating or lipolytic activity, but lightened the color of a specimen predarkened by either the all-L pentapeptide, or by synthetic a-MSH. Premixture of equal amounts of all-L and all-D isomers blocked the darkening effect on the isolated frog skin.1° The L-D-D-L and L-L-L-D (6-10) peptide sequences

Chap. 1 7

Non-ste roidal H o r m o n e s

193 -

Jorgensen

retained MSH-activity, while the D-D-D-L, L-D-D-D and D-L-L-L analogs were inactive, or weak antagonists.19 The influence of chain length on MSH-activity has been summarized by Schwyzer.20 Synthetic analogs of ACTH, lengthened from the N-terminal to the C-terminal, show initial melanocyte-stimulating activity at the decapeptide (1-101, although the nonepeptide (1-9) was not investigated. Activity increases markedly at the tridecapeptide amide stage, and activity remains constant with further elongation of the C-terminal end. Acetylation of the amino group of the N-terminal serine residue generally enhances activity 10 to 100-fold in the series.

a-MSH ACTH

Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Try-Gly-Lys-Pro-Val-~~ H-Ser-Tyr-Ser-Met-Clu-His-Phe-Arg-Tty-G1y-Lys-Pro-Val1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3

Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Try-Pro14 15 16 17 18 19 20 21 22 23 24 NH 1 2 [Asp-Gl y-Glu-Ala-Glu-Asp-Ser-Ala-Glu

1

Ala-Phe-Pro-Leu-Glu-Phe 34 35 36 37 38 39

F2

(Beef) (Sheep)

(Pig)

Adrenocorticotropic Activity.--The total synthesis of B-corticotropin, with the amino acid sequence characteristic of the porcine species, has now been reported in detail.21 The techniques developed have aided the synthesis of smaller fragments which have been used to relate chain length and ACTH activity.20 Corticotropin activity is associated with the presence of a free terminal amino group. From the amino end, threshold activity is found with the 1-13 tridecapeptide amide, corresponding to the free amino form of a-MSH. Four to five amino acid residues after MSH activity has reached its plateau, ACTH activity sets in strongly, reaching an almost constant value at 20-24 residues, then climbing to twice this value at the 39 residues of ACTH. The hexacosapeptide corresponding to the first 26 residues of bovine ACTH has been reported to possess adrenal- and melanocyte-stimulating activtty comparable to the native hormone on a molar basis." Ramachandran, Chung and LiZ3 have pointed out the possible significance of the concentration of all the basic amino acid residues of ACTH in the region between positions 5 and 22, with a solid core (Lys-Lys-Arg-Arg) in positions 15-18. The positive charge contributed by the basic amino acids is considered possibly to contribute to receptor binding with a negatively charged surface. The ten-fold increase in potency from the heptadecapeptide (1-17) to the nonadecapeptide (1-19) may be attributed to the additional arginine at 18, which may contribute a positive charge to receptor binding.

194

Sect. IV - Metabolic & Endocrine

H e i n z e l m a n , Ed.

Further enhancement of activity by conversion of the terminal carboxyl group to an amide in this series,24 could be due to the decrease in negative charge contributed by the carboxylate ion, which would interfere with associative interactions at a negatively charged receptor. The approximately equal activity for the octadecapaptide and nonadecapeptide amides was felt to support this concept, 23 since both contained the same core of basic amino acids. The acidic residues concentrated in the carboxyl terminal part of the molecule are suggested as playing a secondary role in enhancing receptor binding at a positively charged site. Since the amino terminal end is most sensitive to alteration, and many functional amino acid residues implicated in the active sites of proteolytic enzymes are present, this region is considered to be the active site of the hormones, oriented by the binding affinities of more distant basic and acidic centers. The importance of the basic amino acids has been further indicated by the essential loss in steroidogenic ACTH-like activit with the hexadecapeptide, cr1’14’ 17-18-Gly1-ACTH, and heptadecapeptide, cr1-T4’ 16-18-G1y1-ACTH-18-N~ in which lysine residues have been removed from the normal 1-18 sequence of ACTH analogs. 2 5 Synthesis of the active analog, 17,18-diornithyl-8-corticotropin(1-24)-tetracosapeptide adds further evidence for the important role of basic amino acids in the 15-18 region of the ACTH molecule.26 Oxytocin and Vasopressin.--BoissonnasZ7 and Lawz8 have published recent comprehensive reviews of structural analogs of these hormones of the posterior lobe of the pituitary.

The following data extends from the structure-activity correlations of Law.28 The question of the importance for hormonal activity of the 20-membered disulfide ring has been investigated further. Analogs of arginine- and lysine-vasopressin and their 1-deamino derivatives in which the ring was open and the S-atoms replaced by H-atoms (e.g , Ala1-Ala6-Arg*-vasopressin) were devoid of hormonal-like or antagonistic activity.29 Additional methylene groups in the ring eliminated activity, as shown by the inactivity of the 21-membered ring compound, 4-8-alanine-oxytocin, when compared with the moderately active 20membered ring analog 4-glycine-oxytocin, and of 1-y-mercaptobutyric acid oxytocin, compared with the highly potent deamino-oxytocin (1-6-mercaptopropionic acid oxytocin) 31 Although the intact 20-membered ring requirement has thus far been upheld, isosteric replacement of the ring atoms is possible with retention of activity. Cyclic analogs of oxytocin in which one of the S-atoms of the disulfide bridge was replaced by a methylene group,32 or by a selenium

.

.

Chap. 17

N o n - s te roidal Hormone s

Jorgensen

195 -

atom (6-seleno-oxytocin and deamino-6-seleno-oxytocin) showed significant hormonal activity. Synthesis of diseleno-oxytocin has been reported, but final purification and biological evaluation was not carried out. 34 The stereochemical relationship between the ring and the side-chain is important, as shown by the loss in oxytocic activity with both 1-hemi-D-cystine- and 6-hemi-D-cystine-oxytocin. All-D-oxytocin has been prepared and shown to lack either hormone-like or inhibiting effects.36 Replacement of L-Tyr2 by D-Tyr2 in oxytocin and its 1-deamino analog produced analogs which retained significant, although slight, oxytocin-like activities as well as an inhibiting effect on oxytocin.37 The analogs L-Dab8- and D-Dab8-vasopressin38, containing the shorter basic sidechain derived from a,y-diaminobutyric acid, showed significant pressor and antidiuretic activities. The D-analog showed selective antidiuretic activity relative to its pressor and uterotonic effects. The configuration of the basic amino acid at the 8-position seems more important for pressor, rather than antidiuretic action, as shown by the selective antidiuretic effects of D-Arg8- and D-Lys8-vasopressin. Alteration of side chains has aided the definition of structural requirements for activity, and has produced analogs with selective and prolonged effects. Both vasopressin and oxytocin show enhancement of activity in the 1-deamino derivatives.40 '41 Systematic replacement by hydrogen of other functional groups in amino acid side chains of oxytocin has shown that the phenolic hydroxyl group is not vital, but does contribute significantly to activity. The carboxamido groups are essential at 5 (asparagbe) and 9 (glycinamide), but not critical at 4 (glutamine), since glutamine may be replaced b a-aminobutyric acid with retention of significant biological a~tivity.~' Removal of the carbox terminal NH2- group alone (9-deamido oxytocin) leads to loss of activity.43 Acylation of the amino end of vasopressin (Gly-Cysl-vasopressin) resulted in a partial reduction in activity, but a three-fold prolongation of effects.43 Preparation of further extended-chain analogs of oxytocin, including Pro-Cysl-, Phe-Cysl-, D-Leu-Cysl-, Leu-Leu-Cysl-, and Gly-Gly-Cysl- oxytocins has been reported.44 The observation of oxytocin-antagonistic properties for analogs of oxytocin with 0-ethyltyrosine, p-methylphenylalanine, and p-ethylphenylalanine replacing t y r o ~ i n e ,has ~ ~ been extended by synthesis of Gly-CyslTyr (Me) 2-vasopressin. This chain-lengthened functionally modified analog showed a small but prolonged antidiuretic effect, and a relatively strong inhibition of the pressor activity of lysine-vasopressin at a molar ratio of analog to hormone of 25:l. Complete and long-lasting inhibition was produced at a ratio of 200:l. Complete inhibition of the uterotonic effect of oxytocin on the isolated rat uterus was obtained at a ratio of 300:1.43 N-Carbamyl-0carbamyl-oxytocin showed no effect alone on the isolated rat uterus, but at an analog to hormone ratio of 50:1, produced complete inhibition of oxytocin administered one minute later. The mono-acylated derivatives, N-carbamyl- and O-carbamyl-oxytocin showed slight oxytocic (0.05 and 0.1%), and no inhibitory effects on the rat uterus.46 The requirements of position 8 have been studied in detail. An aliphatic side chain is important for oxytocic activity, since Gly8-oxytocin was inactive,

196 -

Sect. IV

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

while an ethyl, or even methyl group $resent in But8-oxytocin and Ala8-oxyrocin conferred a high degree of a ~ t i v i t y . ~ The importance of the basicity of the amino acid residue at position 8 and the phenolic group at position 2 for vasopressin-like activity was shown with analogs combining variations at the 2 and 8 positions. Phe2-Om8-vasopressin and Phe2-Om8-oxytocin showed selective pressor activity 48whi1e desaminol-Arg8- and desaminol-Phe2-Arg8-vaso ressins showed an enhanced antidiuretic effect. 49 Syntheses of desaminol -0rn - and desaminol-Phe2-Om8-vasopressins and oxytocins has also been described. Thialysine8-vasopressin, an isostere containing sulfur in place of a methylene group of the lysine side chain, showed antidiuretic activity greater than that of Lys8-vasopressin, with less pressor activity. Therefore, factors other than base strength appear to be involved in the role of the side chain in the 8 position.51 Reaction of oxytocin with acetone under mild conditions to produce an inactive derivative has been noted.52 Reaction is presumed,to occur with the free amino group , since deaminol-oxytocin is not deactivated under these conditions. Specific tritiation of oxytocin by catalytic deiodination has been reported. 53 HORMONES OF THE STOMACH Gastrin.--Heptadecapeptide amides gastrin I and gastrin I1 isolated in 1964 from hog antral mucosa, are believed to be related to the hormone released during digestion, which stimulates gastric secretions. On a molar basis, gastrin I1 is some 500 times more effective than histamine in stimulating gastric secretion. Gastrin I1 differs from gastrin I only in being the sulfate ester derivative of the tyrosine residue.

Pyr-Gly-Pro-Tyr-Met-(Glu)5-Ala-Tyr-Gly-Try-Met-Asp-Phe-NH2 Gastrin I Only the C-terminal tetrapeptide amide sequence, Try-Met-Asp-Phe-NH2 was found to be required to produce the full range of ghysiological effects of the natural hormones. St Structure-activity studiess5’ in the active C-terminal tetrapeptide sequence led to the following observations: (a) all N-acyl derivatives of the tetrapeptide are active, (b) substitution of all, or part of the Lamino acids by the D-isomers leads to virtual loss of activity, (c) replacement of tryptophane by other amino acids results in loss of activity, although 4-, 5-, and 6-methyl tryptophanes are highly active, (d) methionine may be replaced by norleucine or ethionine with retention of activity, byt all changes in the aspartic acid position led to inactive compounds, (e) 0-methyl tyrosine may replace phenylalanine, (f) the terminal amide cannot be converted to the methyl ester, the free acid, or a dialkylamide without loss in activity. TISSUE HORMONES Bradykinin, Kallidin and MethionyJ-ksyl-bradykinin.--Bradykinin and its N-terminal analogs are formed by enzymatic degradation of inactive precursors present in the a2-globulin fraction of plasma. R-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 1 2 3 4 5 6 7 8 9 Bradykinin, R = H

Kallidin, R = Lys

Methionyl-lysyl-bradykinin, R = Met-Lys

Chap. 17

N o n - steroidal Hormones

197 -

Jorgensen

From a comprehensive survey of analogs in 1964,57 it was concluded that the importance of individual amino acids for biological activity was in the order, 7,8>1>9>5>4>2>3>6. The Pro7 and Phe8 residues appear to be most essential for biological activity. The terminal arginine residues (1,9) may be replaced with basic amino acids, but neutral amino acids produce a loss of activity. The aromatic residue in position 5 is essential. Exchange of Gly4 and Pro - residues causes medium loss of activity, while the Pro3 and Ser6residues are relatively insensitive to change. The importance of the free carboxyl end of bradykinin, and of other active peptides for full biological activity, has been emphasized.58 Variations in the nature and configuration of the peptide chain have led to less active or inactive analogs. Retrobrad kinin,59 containing the reverse sequence of amino acids, all-

D- bradykininx8'60, and all-D-tetrobradykinin6O showed neither bradykinin-

like nor antagonistic activity. Replacement of either or both of the L-arginine residues with D-arginine resulted in loss of activity, and no production of inhibitory effect.61 Introduction of ester in place of amide links between residues 3-4 and 5-6, in the depsipeptides glycolyl-4- and glycolyl-6-bradybinin produced analogs with 0.5 to 1% activity.62 The inactivity of the c clic decapeptide, Gly7-cyclokallidin, 3as compared with the active linear Gly -kallidin, further demonstrated the importance of conformation for biological activity.

s

A number of octa- and nonapeptide analogs containing threonine in place of serine were prepared as potential antagonist^.^^ Tests on rat uteri showed variable and unpredictable mimetic and antagonistic response; only bradykinin-like effects were seen with the isolated guinea pig ileum or rat duodenum. The solidphase synthesis of b r a d ~ k i n i nhas ~ ~ been extended to provide a number of additional bradykinin analogs. Among these, replacement of both phenylalanine residues with 0-methyl tyrosine yielded an antagonist at low concentrations, but bradykinin-like activity at higher concentrations.66 To date, no bradykinin antagonistic peptide has been reported which is free of bradykinin-like activity at high dose levels. Extension of the N-terminal end of bradykinin generally leads to little change in activity.67 Met-Lys-bradykinin is about 113 as active on isolated muscle preparations, but 2-3 times as active as bradykinin in lowering rabbit blood pressure. The methionine sulfoxide retains the activity of Met-Lys-bradykinin, while the sulfone is only about 1/10 as active. N-terminal-extended bradykinin analogs are generally highly active, with (Lys-Lys-Argl)and (Lys-Lys-Lys-Argl )-bradykinin being 8-10 times as potent as bradykinin in lowering rabbit blood pressure, while being less active in producing contractions

of isolated smooth muscle.

Dihydrochlorprothixene and a related analog have been reported to act as competitive inhibitors of bradykinin on isolated guinea pig preparations. CYPrQheptadine acted as a non-competitive inhibitor. An8iotensin.--Angiotension I, a relatively inactive decapeptide produced by the action of renin on an a2-globulin blood fraction, is converted to the active octapeptide, angiotensin-I1 by the enzymatic removal of a C-terminal dipeptide,

Sect. IV

198

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

His-Leu. Species differences provide two equiactive angiotensins, containing valine or isoleucine in the 5-position. The composition of human angiotensin is not yet established. Asp-Arg-Val-Tyr-Ile 1 2 3 4 5

(Val)-His-Pro-Phe 6 7 8

The kinetics of the renin-substrate reaction have been studied employing a synthetic tetradecapeptide renin substrate. 69 Earlier studies which provided structural relationships relative to smooth muscle-stimulating or pressor effects have been summarized by Law.28 For significant activity, the carboxyl and phenyl groups of Phe8 and phenolic hydroxyl group of Tyr4 appear to be essential. Loss of activity on photolysis and decreased activity of analogs containing 6-substituents supported the essential role of the imidazole ring of His 5 The low activity of the M a 7 analog indicated the importance of Pro7. Substitution of leucine for valine or isoleucine in the 5-position resulted in a four-fold decrease in activity, but leucine could replace Val with complete retention of activity, indicating a selective influence of side chain branching at position 5. A low degree of specificity was generally indicated for Asp1 and Arg2. Aromatic residues (4,6,8).--Using Asp (NH2)1-Val5-angiotensin-II as the parent compound, Schr6der70 has replaced the HisC residue with PhenYlalanine and lYsine+ obtaining activities of 1% and 0.1%, respectively. This provides further evidence for the importance of histidine, with aromatic character apparently being more important than base strength. A surprising loss in activity occurred when 0-methyl tyrosine replaced Tyr4 (0.2%), considering the relatively strong activity reported for the Phe4 analog (10%). Exchan e of hydroxyl groups between positions 4 and 8 with Asp (NH2) 1-Phe4-Va15-Tyr8-angiotensin-II led to inactivity. Apparently the phenolic character of Tyr4 and aromatic character of Phea are the most important features, as shown by the 10% activity of the di-tyrosyl analog, Asp (NH2) 1-Va15-Tyr8-angiotension-II. The closely related Ile5-Tyr8-angiotensinI1 has been reported as only slight1 less active than Ile5-angiotensin-II.71 Increased se aration between the Tyr' and Phe8 residues, by exchange of the Val3 and Tyr residues, led to loss of activity.70

t

r

tes+dues ( 3

~gdrbxyproline%$lace

.--Tyrosine in place of Val3 resulted in 12% activo€ Pro7 reduced activity to 7%.

-Acidic and basic residues (1,2l.--Variations of Asp1 have yielded analogs with

hei htened and prolonged activities. The isomeric a-D-? 6-L- and 6-D-Asp'Val@-angiotensins-II, by comparison with natural a-L-Aspl-Val.'-angiotensin-lI and its Asp1-B-amide, show an increase in activity of about 50%, and a two to three-fold prolongation of pressor activity in the rat. 72 This has been attributed to a decrease in the rate of degradation of the modified angiotensins by Desamino-Val5-angiotensin-I1 showed a 50% decrease, but prolonaminopeptidases ~ ~ showed that gation of activity. This series has been extended by S ~ h r t j d e rwho the D-Asp1-, Glul-, Glu (NH2)l- and Pyroglu1-Va15-angiotensin-II analogs all showed about 50% enhancement of pressor activity over that of Val5-angiotensin11.

.

Chap. 17

Non-steroidal Hormones

Jorgensen

199 -

The activities reported for the Lys2 and Val2 analogs of Asp (~~,)~-Val5angiotensin-I1 (10% and 5 % ) added to the conclusion that the arginine at position 2 is not essential for activity.74 In a study mainly involving 2-8 heptapeptide analogs, X-Val-Tyr-fle-His-Pro-Phe , Havinga’l’’7 6 has concluded that the features of the arginine moiety contributing to activity are the positive charge or the capacity to hydrogen bond of the guanidinium or ammonium groups forming part of X in active peptides. Heptapeptides with D-amino acids in the X-position were more active than the corresponding L-compounds, However, 1-8 octapeptides with D-amino acids in the 2-position were less active than their L-counterparts. All-D-Asp(N€l~~Va15-angiotensin-II has been synthesized as a potential antagonist, but showed neither angiotensin-like nor antagonistic effects in both smooth muscle-stimulation and pressor tests.7 7 97 8

The solid-phase method of peptide synthesis has been used to prepare Ile5-, Asp(NH2) ’-Ile5-, and B-Asp’-Ile5-angiotensins-I1 .79 The problem of the secondary structure of angiotensin in solution is unresolved. Thin-film dialysis studies indicate a coiled or compact form of low axial ratio.80 Biological experiments involving inhibition of smooth muscle responses to angiotensin by urea and amino acids, indicate that the simple compounds produce either randomization of an organized angiotensin structure, or produce inhibition by direct interaction with the receptor.81 MISCELLANEOUS PHYSIOLOGICALLY ACTIVE PEPTIDES

Eledoisin --- -_ .^__ and - Physa1aemg.--The powerful vasodilating hypotensive undecapeptide eledoisin was isolated in 1962 from the salivary glands of a mollusc. In 1964 the four-fold more potent related undecapeptide, physalaemin, was isolated from the skin of a South American amphibian. Eledoisin

H-Pyr-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-W2

Physalaemin H-Pyr-Ala-Asp-Pra-Asp-Lys-Phe-Tyr-Gly-Leu-Met-M2 1 2 3 4 & 6 7 8 9 1011 5 2

Studies of numerous analogs have shown that similar structure-activity relationships exist in both compounds. The C-terminal amino acids (6-11) are highly sensitive to alteration, while the N-terminal residues (1-5) are relatively insensitive.82 A minimum of five amino acid residues at the C-terminal end (7-11) is needed for appreciable activity, while activity increases sharply with the hexapepti.de (6-11) and higher analogs. The hexapeptide (6-11) related to eledoisin retains 30%;activity in lowering blood pressure in the rabbit, while the heptapeptide (5-11) is essentially as active as e l e d o i ~ i n . ~The ~ corresponding physalaemin hexapeptide (6-11) is 2-2 to 3 . 8 times as active as eledoisin, and at least half as active as physalaemin in its hypotensive effect in the d ~ g . ~ ~a Iseries n of A~p(NH~)~-eledoisinanalogs, maximal h otensive activity in the rabbit was reached in the octa to decapeptide range,T3E while maximal hypotensive activity in the dog was shown at the C-terminal nonapeptide (3-11)

200 -

Sect. IV

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

related to eledoisin.86 Increased activity has been shown in eledoisin analogs with higher thioalkyl groups replacing the thiomethyl group of methionine, while alkylcysteinamide residues, with one less methylene group,or other amino acids, drastically reduce activity.87 The all-D isomer of the highly active hexapeptide analog of eledoisin (6-11) was devoid of either eledoisin-like or antagonistic activity. Systematic replacement of individual L-amino acids by D-amino acids in the active heptapeptide (5-11) resulted in essential loss in activity, except for the D-Ala6 analog, which retained 30% of the activity of the parent heptapeptide, in lowering blood pressure in the rabbit. 88 With few exceptions, both physalaemin and its close analogs are relatively inactive in producing contractions of the rat uterus as compared with eledoisin, in doses equiactive in reducing blood pressure.Q4 THYROID HORMONES Thyroxine and Triiodothyronis.--Recent contributions to studies relating structure and activity may be discussed in terms of the structure of thyroxine.

Phenolic hydroxy1.--Thyroxine-like

activity found for 4'-deoxy-3,5-diiodothyronines, and inactivity for corresponding 4'-methyl analogs was attributed to a probable metabolic introduction of the hydroxyl group to produce the active molecule.89

Outer ring substituents.--The ability of alkyl or aryl groups to replace halogen atoms on the phenolic ring with retention, or even enhancement, of biological activity, has been confirmed. Relative to L-thyroxine (loo), substituted 3,5-diiodothyronines showed the following antigoitrogenic activities in the rat: L-3'-iodo (775), DL-3l-ethyl (3401, L-3'-isopropyl (12501, L-3'-isoproBarker has noted similar potent pyl-5'-iodo (440), and L-3'-isobutyl (60). thyroxine-like effects in oxygen consumption and heart rate tests on rats for thyroxine analogs with outer ring alkyl substituents, or bearing a fused phenyl ring (3,5-diiodonaphthyr0nine).~~ In these tests, as well as a test measuring inhibition of thyrotropic hormone release,91 the 3 ' -isopropyl analog was the most potent tested. 3'-Tertiary butyl-3,5-diiodo-L-thyronine showed equal activity to L-thyroxine in the tadpole metamorphosis assay,92 and about twice the activity of L-thyroxine in the rat antigoiter assay.93 Further examples of decreased activity for outer ring 3',5'-disubstituted analogs, as compared with the correspondine 3'-monosubstituted compound were presented.89 A long standing apparent inconsistency in this regard was resolved by the demonstration that the 3'-chloro analog was more active than 3 ' ,5 '-dichloro-3,5-diiodo-L-thyronine. 94 The importance for biological activity of a distal positioning in space relative to the inner ring for the outer ring substituent has been emphasized by further examples. 89 The role of ring substituents in establishing skewed conformation for the di henyl ether nucleus has been studied on thyroxine analogs using NMR techniques.t 5

Chap. 17

N o n - 8 teroidal Hormones

Jorgensen

201 -

Ether linkage.--Three biphenyl analogs of tetra- and triiodothyroacetic acid, and of thyroxine were found to be essentially inactive in the heart rate test,89 indicating a requirement for the ether linkage. However, the biphenyl analog, 3,5-diiodo-4-(3 ,5 -dimethyl-4'-hydroxylphenyl) phenylalanine was reported to be equiactive to the corresponding diphenyl ether, and about 1.5 times as active as thyroxine in inducing metamorphosis in the immersed tadpole. g6 Iodinated 2-methyl-3-carboxy-5-hydroxybenzofurans, compounds lacking the diphenyl ether nucleus, and previously reported active in the tadpole metamorphosis assay, were inactive in the heart rate test. 89 Inner ring substituents.--The 3,5-dibromo analog of DL-thyroxine approached the activity of thyroxine in the rat oxygen consumption test,89 indicating the lack of a requirement for iodination on the alanine-bearing ring. Thyronines mono-iodinated, or lacking iodine on the inner ring were inactive in oxygen consumption tests. -Side chains.--Thyroxine analogs with alcoholic side chains of varying lengths have been prepared by reduction of the corresponding acids. 97 Among these,

3,5-diiodo-3',5'-dimethyl thyroethanol was reported to show in the rat a greater dissociation between hypocholesterolemic and calorigenic effects than either L- or D-thyroxine. A number of reports of synthesis and biological activities of a-methyl thyronines have appeared. O 0 a-Methyl-DL-thyroxinegg showed weak thyroxine-like activity in antigoitrogenic, cholesterol-lowering, and heart weight assays, and inactivity as a thyroxine antagonist in the antigoiter test. a-Methyl-DL-triiodothyronine and its D- and L-isomers were reported to be more active in the hypocholesteremic test than DL-a-methyl.thyroxine.' O 0

REFERENCES 653 (1965). 1. H. Zahn, H. Bremer, and R. Zabel, Z. Naturforsch., 3, 2. J. Meienhofer and E. Schnabel, 2. Naturforsch., x h , 661 (1965). 3. H. Zahn, 0. Brinkhoff, J . Meienhofer, E. F. Pfeiffer, H. Ditschuneit, C. Gloxhuber, Z. Naturforsch. E , 666 (1965). 4. H. Zahn, B. Gutte, E. P. Pfeiffer and J . w o n , Ann. Chem. 691, 225 (1966). 5. C. I. Niu, Y. T. Kung, W. T. Huang, L. T. Key C. C. Chen, Y. C. Chen, Y. C. Du, R. Q. Jiang, C. L. TSOU, S. C. Hu, S. Q. Chu and K. Z. Wang, 15, 231 (1966). Sci. Sinica (Peking), 14,1386 (1965); 6. Y. Wang, C. Y. Hsing, et al., Sci. Sinica, l4, 1887 (1965). 7 . T. P. Loh, C. H. Li and C. Y. Hsing, Acta Chim. Sinica, 2,509 (1965). 8. Y. C. Du, R. Q. Jiang and C. L. TSOU, Sci. Sinica (Peking), 14,229 (1965) C. A . , 6 2 , 18410 (1965). 9. Anon. The Total Synthesis of Crystalline Insulin. Kexue Tongbao, 17,241 (1966) 10. P. G. Katsoyannis, A. Tometsko and K. Fukuda, J. Am. Chem. Soc., 85, 2863 (1963). 11. P. G. Katsoyannis, K. Fukuda, A. Tometsko, K. Suzuki, and M. Tilak, J. Am. Chem. SOC., 86,930 (1964). 12. P. G. Katsoyannis, A. M. Tometsko, J. Z. Ginos and M. A. Tilak, J . Am. Chem. SOC., 88, 164 (1966). 13. P. G. Katsoyannis, A. Tometsko, and C. Zalut. J. Am. Chem. SOC., 88, 166 (1966).

m.,

.

202

Sect. I V

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

14. E. Schrtjder, Ann. Chem., fif8if, 231, 241, 250 (1965). 15. (a) E. WUnsch, Acta Chim. Hung, 4-4, 173 (1965). (b) E. Wiinsch and A. &i&, Chem. Ber., 2,101, 105 (1966). (c) E. Wtinsch and F. Drees, ibid., 9!3, 110 (1966). 16. H. Hotsuka and K. Inouye, Bull. Chem. SOC. Japan, 37, 1465 (1964). 17. M. Bodanszky, M. A. Ondetti, B. Rubin, J. J. Piala, J. Fried, J. T. Sheehan and C. A. Birkhimer, Nature, 194,485 (1962). 18. H. Yajima and K. Kubo, J. Am. Chem. SOC., 87, 2039 (1965). 19. H. Yajima and K. Kubo, Chem. and Pharm. Bull., Japan, 2, 759, 1326 (1965). 20. R. Schwyzer, Ann. Rev. Biochem., 33, 259 (1964). 21. R. Schwyzer and P. Sieber, Helv. Chim. Acta, 9, 134 (1966). 22. J. Ramachandran and C. H. Li, J. Am. Chem. SOC., 87, 2696 (1965). 23. J. Ramachandran, D. Chung, and C. H. Li, J. Am. Chem. SOC., 87, 2691 (1965). 24. H. Otsuka, K. Inouye, F. Shinozaki, and M. Kanayama, J. Biochem. (Japan), 58, 512 (1965). 25. H. Otsuka, K. Inouye, M. Kanayama and F. Shinozaki, Bull. Chem. S O C . Japan, 28, 679, 1563 (1965). 26. G. I. Tesser and R. Schwyzer, Helv. Chim. Acta, 3, 1013 (1966). 27. R. A. Boissonnas, St. Guttmann, B. Berde, and H. Konzett, Experientia, 17, 377 (1961). 28. H. D. Law, Progr. Med. Chem., 4, 86 (1965). 29. R. L. Huguenin and St. Guttmann, Helv. Chim. Acta, 48, 1885 (1965). 30. M. Manning and V. duvigneaud, Biochemistry, 4, 1884 (1965). 31. D. Jarvis, B. M. Ferrier and V. duvigneaud, J. Biol. Chem., 2%. 3553 (1965). 32. J. Rudinger and K. Jost, Experientia, 20, 570 (1964). 33. R. Walter and V. duvigneaud, J. Am. Chem. SOC., 87, 4192 (1965). 34. W. Frank, 2. Physiol. Chem.. 339, 222 (1964). 35. M. Manning and V. duvigneaud, 87, 3978 (1965). 36. G. Flouret and V. duvigneaud, J. Am. Chem. SOC., 8 7 , 3775 (1965). 37. S. Drabarek and V. duvigneaud, J. Am. Chem. SOC., 8 7 , 3974 (1965). 38. M. Zaoral and F. Sorm, Collection Czech. Chem. Commun., 31, 90, 310 (1966). 39. M. Zaoral, J. Kolc and F. Sorm, 32, 382 (1966). 40. R. L. Huguenin, E. StUrmer, R. A. Boissonnas, and B. Berde, Experientia, 2 l , 68 (1965). 41. L. A. Branda and V. duvigneaud, J. Med. Chem., 9, 169 (1966). 42. B. M. Ferrier and V. duvigneaud, J. Med. Chem., 2, 55 (1966). 43. M. Zaoral and F. Sam, Collection Czech. Chem. Commun., 30, 2812 (1965). 44. E. Kasafirek, K. Jost, J. Rudinger, and F. Sonn, w.,30, 2600 (1965). 45. A. L. Zhuze, K. Jost, E. Kasafirek, and J. Rudinger, iJbs., 29, 2648 (1964). 46. D. G. Smyth, Acta Chim. Acad. Sci. Hung., 44, 197 (1965). 47. P. A. Jaquenoud, Helv. Chim. Acta, 48, 1899 (1965). 48. R. L. Huguenin, w.,47, 1934 (1964). 49. R. L. Huguenin and R. A. Boissonnas, 49, 695 (1966). 50. R. L. Huguenin, 49, 711 (1966). 51. P. Hermann and M. Zaoral, Collection Czech. Chem. Comun., 30, 2817 (1965). 52. D. Yamashiro, H. L. Aanning, and V. duvigneaud, Proc. Nat. Acad. Sci. U.S., 54, 166 (1965). 53. Y. Agishi and J. F. Dingman, Biochem. Biophys. Res. Cow., l8, 92 (1965).

u.,

m.,

s.,

Chap. 17

54. 55. 56. 57. 58. 59. 60.

61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92.

93.

Non-steroidal Hormones

Jorgensen

2 03 -

H. J. Tracy and R. A. Gregory, Nature, 204; 935 (1964). Morley, H. J. Tracy and R. A. G r e F y , Nature, 207, 1356 (1965). J. M. Davey, A. H. Laird and J. S. Morley, J . Chem. SOC. (C), 555 (1966). E. Schrbder and R. Hempel, Experientia, 20, 529 (1964). J. M. Stewart and D. W. Woolley, Nature, 206, 619, 1160 (1965). S . Lande, J. Org. Chem., 27, 4558 (1962). K. Vogler, P. Lanz, W. Lergier and W. Haefely, Helv. Chim. Acta, 49, 390 (1966). E. D. Nicoloides, D. A. McCarthy and D. E. Potter, Biochemistry, 5, 190 (1965). L. Shchukina, G. A. Ravdel, M. P. Filatova, and A. L. Zhuze, Acta Chim. Acad. Sci. Hung., 44, 205 (1965). J. M. Meienhofer, Ann. Chem., 691, 218 (1966). J. M. Stewart and D. M. Woolley, Biochemistry, 2, 700 (1964). R. B. Merrifield, Biochemistry, 2, 1385 (1964). J. M. Stewart and D. M. Woolley, Federation Proc., 24, 657 (1965). E. Schrbder, Experientia, 2l, 271 (1965). J. G. Leme and M. R. eSilva, Brit. J. Phannacol., 25, 50 (1965). D. Montague, B. Riniker, and F. Gross, Am. J . Physiol., *,599 (1966). E. Schrtjder and R. Hempel, Ann. Chem., 684,243 (1965). K. M. Sivanandaiah, R. R. Smeby and F. M. Bumpus, Biochemistry, 5, 1224 (1966). B. Riniker and R. Schwyzer, Helv. Chim. Acta, 47, 2358 (1964). E. Schrader, Ann. Chem., 232 (1966). E. Schrtjder, 680, 132 (1964). E. Havinga, C. Schattenkerk, G. H. Visser, and K.E.T. Kerling, Rec. Trav. Chim., 83, 672 (1964). C. Schattenkerk and E. Havinga, 84, 635 (1965). K. Vogler, R. 0. Studer, W. Lergier, and P. Lanz, Helv. Chim. Acta, 48, 1407 (1965). E. Schrtjder, Ann. Chem., 692, 241 (1966). G. R. Marshall and R. B. Merrifield, Biochemistry, 5, 2394 (1965). L. C. Craig, E. J . Harfenist and A. C. Paladini, Biochemistry, 2, 764 (1964). P. A. Khairallah, R. R. Smeby, I. H. Page, and F. M. Bumpus, Biochim. Biophys Acta, 104,85 (1965). E. Stunner. Ed. Sandrin, and R. A. Boissonnas, Experientia, 20, 303 (1964). E. Schrbder and K. Liibke, Experientia, 20, 19 (1964). L. Bernardi, G. Boisio, F. Chillemi, G. DeCaro, R. DeCastiglione, V. Erspamer, and 0 . Goffredo, Experientia, 22, 29 (1966). K. Liibke, R. Hempel and E. Schroder, Experientia, 21, 84 (1965). L. Bernardi, G. Bosisio, F. Chillemi, G. DeCaro, R. DeCastiglione, V. Erspamer, A. Glaesser, and 0. Goffredo, Experientia, 20, 306 (1964). L. Bernardi, G. Bosisio, F. Chillemi, G. DeCaro, R. DeCastiglione, V. Erspamer, A. Glaesser, and 0. Goffredo, Experientia, 21, 695 (1965). E. Schrbder, K. Liibke, and R. Hempel, Experientia, 21, 70 (1965). S. B. Barker, M. Shimada, and M. Makiuchi, Endocrinology, 76, 115 (1965). 78, 29 (1966). M. Wool, V. S. Fang, and H. A. Selenkow, T. Bauman, G. W. Pipes, and C. W. Turner, 76, 537 (1965). C. M. Buess, T. Giudici, N. Kharasch, W. King, D. P. Lawson, and N. N. Saha, J. Med. Chem., 8, 469 (1965). 533 (1965). E. C. Jorgensen and J. A. W. Reid, J. Med. Chem., J. S o

w.,

m,

u.,

w., w.,

2 04

Sect. IV

-

Metabolic & Endocrine

H e i n e e l m a n , Ed.

94. E. C. Jorgensen and J. A. W. Reid, Endocrinology, 76, 312 (1965). 95. P. A. Lehman and E. C. Jorgensen, Tetrahedron, 21, 363 (1965). 96. M. H. Kullk and J. F. Bester, J. Phann. Sci., 54, 494 (1965). 97. W. J. Wechter, W. A. Phillips, and F. Kagan, J. Med. Chem.,L 474 (1965). 98. DL-a-Methylthyroxlne. Chkmie Grtinenthal, Neth. Appl. 6, 503, 266, Oct. 11, 1965. C.A. 64, 9819d (1966). 99. B. Blank, E. G. Rice, F. R. Pfelffer, and C. M. Greenberg, J. Med.Chem., -9,lO (1966). 100. G. M. K. Hughes and P. F. Moore, Abstract No. 19, Div. Med. Chem., h e r . Chem. SOC. Meeting, Pittsburgh, Pa., March, 1966.

20 5 Chapter 18.

Reproduction

John C . Babcock, The Upjohn Company, Kalamazoo, Michigan Introduction There i s every reason t o b e l i e v e t h a t H i s t o r y will record t h e year 1955 a s one o f those r a r e t u r n i n g p o i n t s i n t h e L i f e o f Man f o r i t was t h e n t h a t t h e f i r s t s t e p s toward e f f e c t i v e family planning and s e l f - c o n t r o l o f population p r e s s u r e s were made; t h e elements o f o r a l contraception began t o take shape. True, it was o n l y a beginning. But it was a beginning b u i l t on a s o l i d foundation, and it o f f e r e d hope: Hope t o young couples not ready t o have children; hope t o o l d e r couples who could ill a f f o r d t o expand t h e i r f a m i l i e s ; hope t o c h i l d r e n where each new mouth t o be f e d would t h r e a t e n f u r t h e r t h e i r s l i m chance f o r s u r v i v a l . And it o f f e r e d hope t o n a t i o n s a l r e a d y s t r a i n i n g every resource i n a f u t i l e e f f o r t t o maintain an a l r e a d y inadequate l e v e l of e x i s t a n c e . A t a t i m e when the laws o f exponential growth were beginning t o t h r u s t populationsinto c r i t i c a l p e r i o d s o f s u r v i v a l , t h e developnent o f o r a l contraception was l o n g overdue. . .and almost t o o l a t e ! ~ r o g e s t e r o n (e 3 ) the hormone o f t h e corpus luteum and fundamental p r e c u r s o r o f most steroid hormones, had been known s i n c e 1 9 2 8 . ~ I t had been i s o l a t e d and synthesized by s e v e r a l research groups by 1934.3 By 1937 i t was known t o block t h e estrus 5 Eight more y e a r s cycle i n t h e r a t 4 and t o block o v u l a t i o n i n t h e r a b b i t . were t o p a s s before hormonal therapy was t o be considered s e r i o u s l y a s a means o f e f f e c t i n g b i r t h c o n t r o l “ and s t i l l t e n more y e a r s before t h e r i g h t s t e r o i d s , i n t h e hands o f some people with imagination.. ..and a plan, produced results o f e x t r a o r d i n a r y importance. Thus Rock, Pincus, and Garcia came t o r e p o r t t h a t o v u l a t i o n could be i n h i b i t e d by administering any o f 1 t h r e e 19-norsteroids o r a l l y between day 5 and day 25 o f t h e menstrual cycle. Like so many important e v e n t s i n h i s t o r y , t h e outcome may have been determined by a f o r t u n a t e coincidence. Both norethynodrel (8) and norethindrone (6b) (which were s e l e c t e d f o r f u r t h e r study) were contaminated with b i o l o g i c a l l y s i g n i f i c a n t amounts o f a n e s t r o g e n i c impurity7 while norethandrolone ( 6 a ) , a weak androgen, capable o f producing s e r i o u s l i v e r dysfunction,’ was not pursued. The corpus luteum and i t s s e c r e t o r y product progesterone, a r e generally held responsible f o r maintaining pregnancy and blocking o v u l a t i o n during t h e extended period o f g e s t a t i o n . A number o f s y n t h e t i c p r o g e s t i n s a r e a l s o recognized t o be e f f e c t i v e o v u l a t i o n i n h i b i t o r s i n animals. I t i s t h e r e f o r e not s u r p r i s i n g t h a t i n v e s t i g a t o r s i n t h e f i e l d considered t h e 19-norsteroids f i r s t s t u d i e d t o be simply “ p r o g e s t i n s . ” ? Unfortunately t h e term continues t o be a p p l i e d erroneously t o o r a l c o n t r a c e p t i v e s i n general, whether containing e s t r o g e n a s an impurity o r as t h e major i n g r e d i e n t i n terms o f b i o l o g i c a l e f f e c t . Actually most c o n t r a c e p t i v e s marketed or under study involve proestrogen, i n combination o r s e q u e n t i a l l y . gestin

-

206 -

Sect. I V

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

Available S t e r o i d Contraceptives Currently, a l l contraceptives marketed contain e i t h e r e t h i n y l e s t r a d i o l (1)o r i t s 3-methyl e t h e r , mestranol ( 2 ) . The former i s a highly potent o r a l estrogen f o r which a l a r g e body o f c l i n i c a l experience e x i s t s . The l a t t e r w a s a contaminant i n e a r l y samples of norethynodrel and norethindrone and i s generally added t o those products i n an amount required t o maint&!za f i x e d composition. I t i s believed t h a t e t h i n y l e s t r a d i o l may be s l i g h t l y more potent than mestranol since 0.05 mg. o f the former and 0.08 mg. of the l a t t e r a r e needed d a i l y t o i n h i b i t ovul a t i o n i n women.’

&- &.CECH

-CZCH

H

CH30‘ 1 Ethinylestradiol

‘ 2 Mestranol

To date, all known progestins a r e derived formally e i t h e r from t h e s t r u c t u r e o f the male hormone testosterone (4a) or from t h e female hormone progesterone (3). Among the former, e t h i s t e r o n e (4b), o r 17e t h i n y l t e s t o s t e r o n e , although t h e o n l y a v a i l a b l e o r a l progestin f o r many y e a r s , l ” has apparent1 not been used i n o r a l contraception. I t has weak androgenic properties.” The 19-nor analo norethindrone (6b), i s . a l s o and i n t h e weakly androgenic, both i n animal s t u d i e s ”jl’ I t s 17-acetate i s generally s i m i l a r i n a c t i v i t y . Norethandrolone ( 6 a ) , f i r s t marketed a s an anabolic agent t o promote weight gain and nitrogen r e t e n t i o n , a l s o e x h i b i t s androgenic p r o p e r t i e s and can produce serious hepatotoxicity over a prolonged period a t high dosage Norethynodwl (8) and norethisterone a c e t a t e a r e r e a d i l y converted t o norethindrone (6b) i n 7Jivo14, 18-21 but the former i s s a i d t o e x h i b i t more estrogenic than androHigh potency was a l s o encountered i n ethynodiol d i genic character. 16y22 a c e t a t e (10) wherein t h e 3-ketone was reduced t o an alcohol2345 and i n 26 l y n e s t r e n o l (12), an analog of 6b i n which t h e 3-ketone has been removed. Numerous r e l a t e d analogs have been prepared and studied, including haloethyriyl and t r i fluoropropynyl d e r i v a t i v e s of 19-norte s t o s t e rone ,27, 28 17chlo roe thynyl-17f3- hydroxye st r-4,9,11- t ri en- 3-one, 29 7a-me t hyl-17-e t h i nyl19-nortestosterone, &, 21-dimethyl-17-ethinyltestosterone (14, Dimethi sterone ) 31 and synthetic 13f3-ethyl-17a-ethinyl-17f3-hydroxygon-4-en-3-one.32

.’

Jd?

d

3 Progesterone

4a 4b

R = H, Testosterone R = CECH, Ethisterone

Chap. 18

Reproduction

Babcoc k

CH3

OH

CH3

6a R = C2H5 Norethandrolone 6b R = CECH Norethindrone

rogesterone 5 Medrox acetag

d

0 4&-xzcH

0’

7a

7b

8 Norethynodrel

X = C 1 Chlonadinone -OAc X = C H 3 Megestrol acetate

AcO’

9 Melengestrol a c e t a t e

&-c-czcH 0 10

Ethynodiol d i a c e t a t e

OH

{!

0

&-

- -0COC 6HL3

,C E C - C H 3

dP l3a R

13b R

05

= CH3 Hydroxyprogesterone =

H

caproat e

tH3

1 4 Dimethisterone

20 7 -

20 8

Sect. IV

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

A powerful stimulus t o t h e development o f o r a l l y e f f e c t i v e analogs o f progesterone was t h e r e c o g n i t i o n o f s u b s t a n t i a l o r a l a c t i v i t y While t h i s material was more p o t e n t t h a n i n 17~-aceto~yprogesteroone?~ e t h i s t e r o n e , it was not widely s t u d i e d because f a r more p o t e n t analogs were r a p i d l y synthesized. O f t h e s e , t h e 6a-methyl d e r i v a t i v e , (medroxyprogesterone a c e t a t e ( 5 ) ,prepared almost simultaneously i n s e v e r a l l a b o r a t o r i e s , 33--35 found wide c l i n i c a l use. Unlike t h e 19-nortestosterone derivat,iws, it has not been a s s o c i a t e d i n c l i n i c a l use with v i r i l i z a t i o n o f t h e f e t u s ( i n c o n t r a s t t o i t s e f f e c t s i n lower s p e c i e s ) nor with h e p a t i c dyso f t h e f e w p r o g e s t i n s which can be used e i t h e r f ~ n c t i o n . ~ I~t ’i s~ one ~ o r a l l y or by i n j e c t i o n . By t h e l a t t e r route, i t forms a m i c r o c r y s t a l l i n e depot from which drug i s a v a i l a b l e for prolonged a c t i v i t y 4 ’ o f up t o s i x months f r o m a s i n g l e i n j e c t i o n . 4 1 34 (n).

I n t r o d u c t i o n o f a A6-bond produces megestrol a c e t a t e i n c r e a s i n g potency s l i g h t l y a 2 and a l t e r i n g t h e metabolic pathway.43-4s i s a l s o a potent The corresponding 6-chloro analog, chlormadinone (7a) p r o g e s t i n used with mestranol i n a s e q u e n t i a l formulation. Other c l o s e l y r e l a t e d 17a-acetoxyprogesterone analogs of i n t e r e s t include t h e 16-methylene compound, melengestrol a c e t a t e (9)48 and t h e 3-desoxy compound DMAP (11).49

26’47

The 1 7 - a c e t a t e group can be replaced with a 17-alkyl roup without loss of a c t i v i t y , both i n 6,1~~-dimethyl-6-dehydroprogesteronegoandi n i t s corresponding 3-hydroxy analog .51 S i m i l a r l y , t h e acetophenonide of lh,17adihydroxyprogesterone i s an e f f e c t i v e s r o g e s t i n and has been s t u d i e d i n a parent e r a 1 contraceptive formulation. It has been estimated t h a t a t l e a s t seven m i l l i o n women i n t h e United S t a t e s a r e c u r r e n t l y using o r a l contraceptives. For most, t h e mechanism of a c t i o n appears t o be suppression of t h e mid-cycle r e l e a s e of LH, preventing ovulation. I n a s m a l l percentage of s u b j e c t s , i n d i r e c t evidence f o r ovulation has been obtained. The combined a c t i o n of estrogen and p r o g e s t i n i n t h e combination i s thought t o produce an endometrium unfavorable f o r implantation and a c e r v i c a l mucous b a r r i e r d i f f i c u l t f o r sperm t o p e n e t r a t e .53 I n t h e s e q u e n t i a l method, e f f i c a c y depends p r i n c i p a l l y on t h e i n h i b i t i o n of ovulation during the period when estrogen i s administered. Recently, l o w doses o f progesterone alane have been r e p o r t e d t o provide a measure of contraceptive efficacy.54 Further r e p o r t s w i l l be awaited with interest S i d e e f f e c t s of t h e s t e r o i d combinations v a r y w i t h t h e i n d i v i d u a l s u b j e c t and t h e p r e p a r a t i o n and may i n c l u d e , b r e a s t tenderness, weight gain, breakthrough bleeding, amenorrhea, f l a t u l e n c e , t i r e d n e s s , nausea......many of t h e symptoms of e a r l y pregnancy. Thromboembolism has not been c l e a r l y r e l a t e d t o use of o r a l contraceptives b u t occasional l i v e r dysfunction, e s p e c i a l l y i n some populations, may be r e l a t e d . 11 a d d i t i o n t o t h e s t e r o i d s used f o r contraception i n t h e female, a number o f s y n t h e t i c hormones resembling t h e s y n t h e t i c estrogens but e x h i b i t i n g a n t i e s t r o e n i c a c t i v i t y i n animals have been s t u d i e d . These i n c l u d (MRL-41), g5 which i s capable of s t i m u l a t i n g ovulation i n some women ;lymiphene U-ll100A,58 and a number of c l o s e l y related analogs.

Chap. 18

Reproduction

20 9 -

Babcock

I n the male as i n t h e female, i n h i b i t i o n of f e r t i l i t y can be produced a r t i ’ i c i a l l y a t many stages. Jackson has reviewed t h e e f f e c t s of agents i n t h e male ( e s p e c i a l l y as studied i n the male r a t ) , and has concluded t h a t while there a r e many opportunities f o r pharmacological control, interference with hormonal mechanisms has r e l a t i v e l y l i t t l e prospect f o r success Research i n t h i s f i e l d can be complicated by the r e l a t i v e l y long period of treatment and study generally required t o observe changes i n spermatogenesis. Some agents, however, l i k e methylene dimethanesulfonate (15) a f f e c t sperm i n t h e f i n a l stages of development and, when used i n experimental animals a t appropriate dosage, can produce prompt development of i n f e r t i l i t y and rapid post -treatment recovery. 6 o Related methanesulfonates i n t e r f e r e with multiple stages of spermatogenesis, suggesting t h a t s e l e c t i v e control at a most appropriate stage may be possible.61

.”

62

63

Certain b a c t e r i o s t a t i c n i t r o f i r a n s (16) and thiophenes (17) were also found t o i n t e r f e r e with spermatogenesis i n t h e male, blocking development a t t h e primary spermatocyte stage. S i m i l a r results,which were r e v e r s i b l e were a l s o obtained with b i s - (dichloroacety1)diamines (18) .64--g6 These materials, which were o r i g i n a l l y studied as amoebacidal agents, were r e l a t i v e l y well t o l e r a t e d i n man,67 e.xcept f o r an ‘Antabuse-like ’ e f f e c t . Another potent agent which blocks spermatogenesis a t t h e primary spermatocyte stage is t h e d i n i t r o p y r r o l e ORF-1616 (19)68 which i s e f f e c t i v e when given once a month.” Hormonal agents, both s t e r o i d a l and non-steroidal, have a l s o been found capable of blocking spermatogenesis. Many e x e r t t h e i r a n t i f e r t i l i t y e f f e c t s through gonadotropin i n h i b i t i o n . When used f o r t h i s purpose, e s t r o gens have the obvious drawback of producing feminization and l o s s of l i b i d o . Androgens have been l o n g k n m t e m p o r a r i l y t o suppress spermatogenesis and i n s e l e c t e d cases, t o produce a rebound i n sperm p r o d ~ c t i o n . ~ 7’ 2 A number of progestins have r e c e n t l y been evaluated i n the male, including norethandrolone (6a) and 17a-hydroxyprogesterone caproate (l3a).72 These agents i n h i b i t Leydig c e l l function and gonadal hormone production. Medroxyprogesterone a c e t a t e (5)33 a potent progestin e f f e c t i v e both o r a l l y and p a r e n t e r a l l y , was observed t o produce a marked decrease i n sperm count and m o t i l i t y l a s t i n up t o s i x months when given as a s i n g l e 1000 mg i n j e c t i o n t o male v o l ~ m t e e r s . ’ ~ The long duration of a c t i o n of t h i s progestin i s t h e r e s u l t of t h e formation of an intramuscular depot of slowly released drug when administered as a microcrystalline aqueous suspension by t h e intramuscular route.

Clomiphene (MRL-41), an agent which can induce ovulation under some conditions i n women, i n h i b i t s t e s t i c u l a r and accessory gland weights i n t h e when administered male r a t t o t h e range seen a f t e r h y p o p h y s e ~ t p m y . ’ ~ , ~However, ~ o r a l l y to7goung men i n doses of 50-200 mg/day, clomiphene stimulated Leydig c e l l A b i o l o g i c a l l y s i m i l a r dihydronaphthalene d e r i v a t i v e , U - l l l O O A (21) ,58 functior,. w a s found t o i n h i b i t spermatogenesis at a dose of 0.5 mg/kg i n t h e rabbit.75 The hydrazine d e r i v a t i v e I C I 33828 (22) a l s o i s e f f e c t i v e i n both male and female r a t s and i n other species and has been studied i n

Sect. IV

210 -

- Metabolic

& Endocrine

H e i n z e l m a n , Ed.

C12CHCONH- (CH2)8-NHCOCHCl, Win 18446

(18)

ICI

33828 (22)

CH3 O /

U-lllOOA (21)

Among n u t r i t i o n a l f a c t o r s a f f e c t i n g f e r t i l i t y , t,he r o l e of Vitamin E and A have r e c e n t l y been r e v i e ~ e d . ~ ~ J ~ ~ The r a p i d l y developing knowledge of immunological phenomena as app l i e d t o f e r t i l i t y and i n f e r t i l i t y has a l s o been reviewed79J80 and can conf i d e n t l y be expected t o provide important new approaches during t h e next decade

.

Chap. 18

Babc oc k

Reproduction

21 1

Eeferences J . Rock, 0. P i n c u s , and C. R . G a r c i a , S c i e n c e , 891 (1956). G. W. Corner i n "Progesterone" A . C. B a r n e s , e d . Brook Lodge P r e s s , Augusta, Mich. 1561, P. 3. 3. For a summary of t h e s e e v e n t s and survey of p r o g e s t a t i o n a l a g e n t s , s e e L. F i e s e r and M. F i e s e r " S t e r o i d s " Reinhold Publ. Corp., New York 1959, p. 556. h. H. S e l y e , J. S. L. Browne and J. B. C o l l i p , E .&. E. 472 (1936). 512 (1937). 5. A. W. Makepease, I;. L. Weinstein and M. H. Friedman, &I. J . P h y s i o l . , 6. F. A l b r i g h t i n " I n t e r n a l Medicine i t s Theory and P r a c t i c e . " ed. J. H. Musser, Lea and F e b i g e r , P h i l a . 1945. 323 (1957). 7. a)) J. Rock, C . R . G a r c i a , and G. P i n c u s , Recent Progr. Horm. g . , b ) G. P i n c u s , J. Rock, and C. R . G a r c i a , Ann. N Y Acad. 72, 677 (1958). &', 307 (1959). c ) G. Pincus, vita=. 8. I . M. A r r i a s , N Y Acad. &. , 124, 1014 (1963). 9. E. Mears i n "Agents A f f e c t i n g F e r t i l i t y " , ed. E. C . R a n s t i n and J . S. P e r r y , C h u r c h i l l , London, 1966, p. 211. 10. H. II. I n h o f f e n , W. Logemam, W. Hohlweg, and A . S e r i n i , Ber. 71, 1024 (1938). 11. L. Wilkins. J . &. @. lJ2, 1028 (1960). 12. V. A. D r i l l , J. Endoc-inol., .2?, x v i i (1962). 13. R. L. E l t o n , E. F. N u t t i n g , and W. E. Saunders. Endocrinol., 381 (1962). 14. R . W. K i s t n e r , C l i n . Pharm. Therap., 1,525 (1960). 15. R. W. K i s t n e r , Marquette K., 59, 2 (1963). 16. Lebher-z, s e e r e f . 14. L. 1 (1961). 17. R. W. K i s t n e r , 18. G. Betz and J. Warren. New Eng. J . , &8, 1171 (1963). l8, 1040 (1959). 19. V. A. D r i l l , F A . S., 20. D. A. McGinty and C. D i e r a s s i , Ann NY Acad. E., 500 (1958). 21. H. J. Ringold, A n n N Y . e . 4 . 515 (1958). 22. H. S. Kupperman, UP. 2_5,86 (1962). 23. F. B. Colton and P. D. K l i m t r a , I n t e r n a t i o n a l Congress on Hormonal S t e r o i d s , l s t , Milan, L. M a r t i n i and A. P e c i l e , e d s . (Academic P r e s s , New York, 1965) v o l . 2 , p. 23. 24. G. P i n c u s , C . R . G a r c i a , M. Panigua and J . Shepard, S c i e n c e , 2 8 , 439 (1962). 25. G. Pincus, Advances Chemistry 45, 177 (1964). 26. X. S. d e w i n t e r , C . M. Siegmann, aiih S. A . S z p i l f o g e 1 , e . (London), x 9 , 905. 157 (1964). 27. V. Petrow, Rep. P r o g . &. 28. J. C . Babcock, Advances & Chemistrx, l9O (1964). 29. J . H. F r i e d , T. S . Bry, A . E. O b e r s t e r , H. E . B e y l e r , T. B. Windholz, J . Hannah, L. H. S a r e t t , and S . L. Steelman, J . &. g . ,L3, 4663 (1961). a 6 , 800 (19641. 30* G. W. Duncan, S. C . L y s t s r and J. A. Campbell, E.g. Pharmacol., 929 (1557). 31. A . David, F. H a r t l e y , D. R . N i l l s o n , and V . Petrow, J . ??. 8 . A . Edgren, H. Smith, D. L. P e t e r s o n , and D. L. C a r t e r , S t e r o i d s , &, 319 (1963). f3. J. C . Babcock, E. 9. G u t s a l l , M . E. H e r r , J . A. Hogg, J . C. S t u c k i , L E . Barnes and W. E . D u l i n , J. 5.chk. G . ,L O t O . 2904 (1958). 34. H. J. Ftingold, 3 . Perez R w l a s , E. B a t r e s , C . D j e r a s s i , J . &I. 3712 (1959). 1959, 478. 35. P. P . E a r i o n , B. E l l i s , V. Petrow, 2. 56. .7. W. Goldzieher, Texas 2. 57, 962 (1961). .I J . Obst. Gynecol., k 6 , 882 ( 1 9 6 3 ) . 57. J. W. Goldzieher and K . Rodgers, & 38. W. J. Rawlings, Brit. J., 336. 873 (1963). 39. F. P . Rhoades, J . 40. M. L. Helmreich and R . A. Huseby, S t e r o i d s , Suppl. 11, 79 (1965). 41. Unpublished S t u d i e s . 42. H. ,i. Ringold. E. R a t r e s . A . Eowers, J . Edwards, J . Z d e r i c . J . &. g. , 81, 3485

1.

2.

=.

w., 3, 3,

-

&.

2,

m.,

e.,

-

e.,

e.

m. z.m., 5,

51,

=.

2,

m.,2,

-

u.

u, 3,

3,

e.

x.e. m., s. 2,

e. m.,

E., w. 1962,

m. e., Ll,

?_

m. m. e., 62,

43. 44. 45.

=.

(1959).

k,

J . M. Cooper, H. E. 9. J o n e s , and A. E. K e l l i e , S t e r o i d s , 255 (19651. B. A . Cooke and D. K. V a l l a n c e , Biochem. J . , 3 l p (1964). B. A. Cooke, T. J . McDonald and D. K. V a l l e n c e . Biochem. J. % , 2 5 p (1965).

z,

-

212

Sect. IV

- Metabolic & Endocrine

H e i n z e l m a n , Ed.

I?. J . Ringold, E. Batres. A. Bowers, J . Edwards, and J. Zderic.

47.

J.

48.

G. R. R.

D. J.

53.

a)

54.

b) J.

55.

D.

56.

S.

57.

G.

58. 59.

D. H.

60.

B.

62.

w.

61.

63. 64.

65. 66. 67. 68. 69.

H.

E. A. F.

w. c.

G.

D.

70.

C.

71:

c. c.

72.

E,

u,

=.

e.,

---

(2)

~

a. &.

2.

2,

s. a. e. 3, m.

e.,

79.

s.

80.

A.

e. *.

9. &. ,5 1 , 3485 (1959). Zderic i n Comprehensive Biochemistry, M. F l o r k i n and E. H S t o t z , ed. E l s e v i e r 166. Publ. Co., New York. 1963 p. W. Duncan. S. C . L y s t e r , J . W. Hendrix, J. J. Clark, and H. D. Webster, F e r t l l . and_ 419 (1966). Steril. P. Blye. V. R . B e r l i n e r and R. E. Horn, Fed. , &, 701 (1965). Deghenghi and R. Gaudry, 2. &. -. E.& ,8 4668 (1961). J . lularshall, P. F. Morand, C. Revesz. and R . Gaudry, 2. E. 7, 355 (1964). F r i e d , E. F. Sabo, P. Grabowich. L. J. Lerner, W. B. Kessler, D. M. Brennan, A. Borman. Chem. and Ind. (London), 465 (1961). 0. Pincus i 7 n Agents Affecting Fertility'!,C. R. Austin and J. S. Perry, ed. Churchill, London, 1965, p. 195. E. Diczfalusy, E .K . 2.. 1394 (1965). Martinez-Manautoil, V. Cortez, J. Oiner. R. Aznar, J. Casasola. and H. W. Rudel, Pert. and S t e r i l . , 1 ~ , 4 9 (1966). E. Holtkamp. J. G. Greslin. C. A. Root, and L. J. Lerner, @. Biol.Ided., 125. 197 (1960). Roy. R. B. G r e e n b l a t t , V. B. Mahesh, and E. C. Jungck, F e r t i l . and S t e r i l . , 575 (1963 1. I. M. Swyer i n "Agents Affecting F e r t i l i t y " . C. R. Austin and J. S. Perry, ed. Churchill, London 1965, p. 180. Lednicer, J. C. Babcock, S. C. Lyster and G. W. Duncan, M. E. (London) 1 2 3 , 408. Jackson i n "Agents Affecting F e r t i l i t y " . C. R. Austin and J. S. Perry, ed. Churchill, London 1965, p. 62. W. Fox and H. Jackson, c i t e d i n r e f . 59. Jackson, B. W. Fox. and A. W. Craig, 2. Reprod. F e r t i l . , 3, 447 (1961). 0. Nelson and E. S t e i n b e r g e r , Fed. Proc., 103 (1953). E.,9 ,312 (1956). S t e i n b e r g e r , W. 0. Nelson and A. Boccabella. R. Surrey and J. R. Mayer, 2. & Pharm. I . Chem., 3 4 0 9 , (1959). Coulston, A. L. Beyler, and H. P. Drobeck. Toxicol. Pharmacol., 2 , 715 (1960). 0. Nelson and D. J. Patanellf,, Fed. 418,1961. G. Heller. B. Y . F l a g e o l l e , and L. J. Matson, E. P a t h o l . , 2 , 107 (1963). Karmes. Abst. 144th Meeting American Chemical Society. p3lL (196fl. J. P a t a n e l l i and W. 0. Nelson, Recent Progress Hormone Research, 0. Pincus, ed. Academic P r e s s , New York. 1964, p. 491. G. H e l l e r , W. 0. Nelson, I. B. H i l l . E. Henderson, W. 0. Maddock. E. C. Jungck, C. A. Paulsen. and G. E. Mortimore, F e r t i l . and S t e r i l . , 1. 415 (1950). w W. Charney, F e r t i l . and S t e r i l . , 10, 557 (1959). G. E e l l e r i n "Estrogen Assays i n Z i n i c a l Medicine", C. A. Paulsen ed. Univ. Washington P r e s s , S e a t t l e (1965). p. 283. MacLeon i n "Agents Affecting F e r t i l i t y " , C. R . Austin and J. S. Perry, ed. Churchill, London 1965, p. 93. 0. Nelson and D. J . P a t a n e l l i i n "Agents Affecting F e r t i l i t y " . C. R . Austin and J. S. Perry, ed. C h u r c h i l l , London 1965, p. 78. J . Eriasson, Fed. & TOO (1965). L. Walpole i n "Agents Affecting F e r t i l i t y " . C. R. Austin and J. S. Perry, ed. Churchill, London 1965. p. 159. Moore i n "Agents Affecting F e r t i l i t y " , C. R. Austin and J. S. Perry, ed. Churchill, London 1965, p. 18. N. Thompson, J . M. C. Howell and 0. A . J . P i t t i n "Agents Affecting F e r t i l i t y " , C. R . Austin and J. S. Perry, ed. Churchill, London 1965, p. 34. J . Behrman i n "Agents Affecting F e r t i l i t y " , C. R. Austin and J. S. Perry, ed. Churchill, London 1965. p. 47. Tyler and D. W. Bishop i n "Immunological Phenomena", Pergamon Press, New York, 1961, Chapt. 8.

46.

-

Chap. 19

Chapter 19.

Steroid Hormones

Diassi and L e r n e r

213 -

S t e r o i d Hormones and t h e i r Antagonists

P a t r i c k A . D i a s s i and Leonard J. Lerner, The Squibb I n s t i t u t e f o r Medical Research, New Brunswick, N . J . A c a t a l o g of b i o l o g i c a l l y a c t i v e s t e r o i d s which have appeared i n t h e s c i e n t i f i c and p a t e n t l i t e r a t u r e through 1963 has been published.'Y2 The following is a review of t h e more a c t i v e s t e r o i d s and steroid-hormone a n t a g o n i s t s which have been published recently.

Corticoids C o r t i c o i d s have a s t h e i r primary c l i n i c a l u t i l i t y t h e c o n t r o l of inflammation. Recent reviews of this and o t h e r r e l a t e d a c t i v i t i e s of c o r t i c o i d s have been published. 3-7 S e v e r a l new 16a-halocorticoids have been synthesized, of which 16a-chloro601,9a-difluoroprednisolone 21-acetate (1) i s 1,100 times a s p o t e n t a s hydrocortisone i n i n h i b i t i n g granuloma formation i n r a t s when administered subcutaneously. S e v e r a l 17-desoxycorticosteroids, including 16a-methyl-1-dehydrocorticosterone ( 2 ) and t h e i r 6af luoro-10 and 9a-f l u o r o - d e r i v a t i v e s (2a and 2 b ) , 9 have been prepared and t h e i r c o r t i c o i d a c t i v i t y measured.

214

Sect. IV

-

Metabolic & Endocrine

Heinzelman, Ed.

Comparison of the thymolytic activity of some new 21chlorosteroids with the corresponding 21-hydroxy compounds has shown11 the danger of projecting the biological relationship of one series of steroid compounds into another. The mechanism of the anti-inflammatory action of steroid hormones remains obscure; however, the regulatory effect of corticoids on metabolism may be an important factor.12-14 Additional reports15-17 have been made on the thymolytic action of corticoids. The antiglucocorticoid activity of 1-dehydrotestololactone on cortisone acetate without inhibition of antigranuloma or thymolytic activity has been reported,6 Reviews on antialdosterone compounds have been published. 18,l9 There is as yet no known potent naturally-occurring,sodium-losing steroid.20 Progestational Compounds Progestational agents have been tested for many biological criteria, several of which are translatable into clinical utility.21-23 Among these criteria are the proliferation of the uterine endometrium, maintenance of pregnancy in the overectomized animal and inhibition of ovulation. In general, synthetic compounds are initially evaluated in the ClaubergZ4 or the modified Clauberg ( M a ~ P h a i l )assays. ~~ It is difficult to compare activities reported from many laboratories because of variation in method, route of administration and reference standards. A number of new progestational1 active compounds have been reported. The 21-fluoro-derivative 3aT6 of chlormadinone ( 3 ) is approximately 1.5 times as active orally as 3 in the Clauberg assay, and the 3p-acetoxy compound 427 is 100 times norethindione by oral administration. The 7-dehydro-derivative 5a of medroxyprogesterone (5) has been synthesized28 and has an improved ovulation inhibiting/progestational index. The totally synthetic 4,9,11-trienes 6 and 6a have been found29 to be orally as active as is progesterone administered subcutaneously. A number of retrosteroids have also been prepared,30 the most active being the 6-fluoro-6-dehydro compound 7. The l7a-vinyl compound 8 has been compared31 to norethynodrel (9) with regard to progestational activity. Both compounds demonstrate the same spectrum of activities, although 8 has slightly higher progestational activity and lower estrogenic activity than norethynodrel.

Chap. 19

Steroid Hormones

21 5 -

Diassi and L e r n e r

Salts of steroids have been found to have progestational activity on oral administration. These include the 3-ammonium sulfate 10 and the 3-pyridinium sulfate 11.32 The 38-(1pyrrolidy1)-derivative 1233 and the tricyclic compound 1334 also have progestational activity. CH2X

I c=o

3 3a

X= H

6a

C=O

C=O

5 5a

4

X=F

R=H R = CH3

~7

y 3

c=o

OH

8

7

9

R = CH=CH2 R = C=CH

CH3

CH3

I

I c=o

10

y 3

I

CH3 I C"0

6

CH3

CH3

1

c=o

11

c=o

12

21 6 -

Sect. IV

- Metabolic & Endocrine

H e i n z e l m a n , Ed.

13 Androqens Androgenic steroids, in addition to having an effect on the growth and secretion of sex organs and accessory sex organs, produce effects on other tissues. These include bone, muscle, 3 5 36 ~ blood, lymphatic system, hair and skin. Recent on this subject, as well as on the clinical utility of androgens, have been published. Several new structural types have been shown to have androgenic activity. These include the 19-nor-retrosteroid 14,37 the tricyclic acids 15 and 16,3* the 17-pyrrolidinium tosylate 1739 and 6-methylandrost-5-ene-3~-01-17-0ne (18).40

OH *=.-

COOH

....CH2CH3 0

14

15 A4 16 A1

17

18

Chap. 19

Steroid Hormones

217 -

Diassi and Lerner

A number of compounds have been synthesized in an attempt to separate the anabolic activity of androgens from their virilizing properties. These include testosterone 17f3-adamantate (19),41 the retrosteroids 2042 and 21,43 the 3,3-azosteroid 22,44 the 2,3furazan 23,45 la,7a,17a-trimethyltestosterone 2446 and substituted 17B-tetrahydropyran-2-yl ethers,47 all of which have good dissociation quotients.

0-c I

OH

OAc

X

20 21

19

x=c1, X=F

22

A126

OH

23

OH

24

The biological properties of antiandrogenic steroids as well as their possible clinical utility has been reviewed. 35948~49 As a rule, progestational compounds as well as a number of weak androgenic compounds have shown antiandrogenic properties. Synthetic efforts have been directed to separate hormonal properties from antiandrogenic activity. Among the recently synthesized compounds which have antiandrogenic activity are 17,17-dimethyl-18norandrosta-4,13-diene-3-one (25),50 the 17a-ethyl-19-nor- compound 26,51 3B-seleno~teroids~~ and the tricyclic compound 13.34 The

218

Sect. IV

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

A-nor steroids 2753 and 2854 continue to show antiandrogenic activity which has been characteristic f o r this series, and the B-nor steroid 29553 56 shows similar activity. The 16a-bromo17-ketal 3055 had significant antiandrogenic activity in the testosterone-stimulated mouse. An example of a potent progestational compound which has strong antiandrogenic properties is the l,2-methylene compound 31.48~57 CH3

OH

OAc

OH 25 26

R=R'= CH3 R = H , R'=C2Hg

28

27

7 3 OH

c=o

....Br

0

Cl 29

30

31

Estrogens Recent reviews on estrogens and antiestrogens have been published .48~ 58-60

The 2-chloroethoxy ether 32 and the 2-fluoroethoxy ether 32a have shown61 estrogenic potencies in the mouse uterine assay of 4 times estrone when administered orally but only 0.5 and 0.05 times estrone on subcutaneous administration. The pentacyclic

Steroid Hormones

Chap. 19

219 -

Diassi and L e r n e r

phenol 33 administered t o t h e c a s t r a t e female mouse exhibited62 e s t r o g e n i c a c t i v i t y of 1/4000 t h a t of e s t r o n e . A number of C-19 f u n c t i o n a l s t e r o i d s 6 3 a l s o show weak e s t r o g e n i c a c t i v i t y .

& I

CH30

32 32a

P OH

0- CH2-CH2X

HO

x=c1

33

X=F

The l i p o d i a c t i c - e s t r o g e n i c r a t i o has been determined f o r a number of 3-desox e s t r a t r i e n e s , a n d t h e 4-methyl compound 34 has a r a t i o of 150.g4 S e v e r a l o t h e r compounds have been s t u d i e d f o r their e f f e c t on blood l i p i d s . These i n c l u d e t h e 168,178pyrazoline 35,65 t h e 2-methoxymethyl compound 36,66 t h e 138-ethyl compound 37 ,G7 t h e androstane-2-propionic a c i d 3868 and s e v e r a l 1-methylequilenine compounds.69 A l l of t h e s e compounds lower blood c h o l e s t e r o l . NH2 I

c-s I

OH

0

34

cp3

36

35 OH

37

t

CH3

38

220 -

Sect. IV

- Metabolic &

Endocrine

H e i n e e l m a n , Ed.

Corticoids, sex hormones and nonhormonal compounds are known to inhibit the biological activity of estrogens. A compound which shows both antiandrogenic and antiestrogenic properties is 26.51

Steroid Hormones

Chap. 19

Diassi and L e r n e r

221 -

References

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

7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21.

N . Applezweig, " S t e r o i d DrUgs,l' McGraw-Hill, New York, 1962. N . Applezweig, " S t e r o i d Drugs 11," Holden-Day Press, San Francisco, 1964. L. H. S a r e t t , A . A. P a t c h e t t and S. L. Steelman, " F o r t s c h r i t t e d e r Arzneimittelforschung," Vol. 5, Birkhauser Verlag, Basel and S t u t t g a r t , 1963. I . E. Bush, Pharm. R e v . , $ 4 & J l, 317 (1962). I . R i n g l e r , "Methods i n Hormone Research," Vol. 111, R . I . Dorfman, Ed., Academic Press, N.Y., 1964, pp 227-336. L. Lerner, A . Bianchi, A. R. Turkheimer, F. M. S i n g e r and A . Borman, A n n . N.Y. Acad. S c i . , 1071 (1964). C. A . S c h l a g e l , J . Pharm. S c i . , 5 4 ( 3 ) , 335 (1965). F. Kagan, R . D . Birkenmeyer and B. J. Magerlein, J . Med. Chem., w 7 , 751 (1964). D. Brancini, G . Rousseau and R. Jequier, S t e r o i d s , 6 ,451 (1965). A . Domenico, H. Gibian, U . Kerb, K. K i e s l i c h , M. Kramer, F. Neumann and G. Raspe, A r z n e i m i t t e l f o r s c h . , 46 (1965). M. H e l l e r , R . H. Lenhard and S. B e r n s t e i n , S t e r o i d s , 615 (1965). L.Kostyo, Endocrinology, 76, 604 (1965). Q. T . Smith and D. J. A l l i s o n , Endocrinology, 785 (1965). A . Kekwick and G . L. S. Pawan, J. Endocrin., &, 265 (1965). P . H. Wiernick and R . M. MacLeod, Acta Endocrin., 138 (1965). R . I . Dorfman, P . G. Holton and F. A . Kincl, Acta Endocrin., 49, 262 (1965). .wI L. Angervall and P. M. Lundin, Acta Endocrin., 3, 104 (1965). C. M. Kagawa, "Methods i n Hormone Research,' Vol. 111, R . I . Dorfman, Ed., Academic Press, N.Y., 1964, pp 351-404. R. Gaunt, J. C h a r t and A. A . Renzi, " R e v i e w s of Physiology, Biochemistry and Experimental Pharmacology," Springer-Verlag, B e r l i n , Heidleberg, New York, 1965, pp 114-172. J. M. George, G . S a u c i e r and F. C . B a r t t e r , J. C l i n . Endocrin., 621 (1965). T. Miyake, "Methods i n Hormone Research," Vol. 11, R. I. 1962. Dorfman, Ed., Academic Press, N.Y.,

J.

u,

u,

u,

J.

u,

J.

a,

a,

222 -

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.

Sect. I V

- Metabolic

& Endocrine

H e i n z e l m a n , Ed.

J. C. Babcock, "Molecular Modification in Drug Design,"

Advances in Chemistry Series, No. 45, American Chemical Society, Washington, D.C., 1964, p. 190. M. S. Kufferman, "Human Endocrinology," Vol. 1-3, F. A. Davis Co., Philadelphia, Penna., 1963. C. Clauberg, Zentr. Gynaskol., !j4, 2757 (1930). 145 (1934). M. K. MacPhail, J. Physiol. (London), R. F. Church and M. J. Weiss, J. Med. Chem., 8, 386 (1965). Y. Lefebvre and R. Gaudry, Canad. J. Chem., 1990 (1965). G. Cooley, B. Ellis and V. Petrow, Tetrahedron, 1753 (1965). G. Nomine, R. BucOrt, J. Tissier, A. Pierdot, G. Costerousse and J. Mathieu, C.R. Acad. Sci. (Paris), 4545 (1965). J. Hartog and P. Westerhof, Rec. Trav. Chim., g,918 (1965). P. de Ruggieri, R. Matscher, C. Lupo and G. Spazzoli, Steroids, A, 73 (1965). R. Deghenghi and C . Revesz, J. Endocrin., 301 (1965). A. P. Shroff, J. Med. Chem. , 8, 881 (1965). A. Boris, Endocrinology, 3, 1062 (1965). "Methods in Hormone Research," Vol. I1 and 111, R. I. Dorfman, Ed., Academic Press, New York, N.Y. A. Segaloff, "Molecular Modification in Drug Design," Advances in Chemistry Series, No. 45, American Chemical Society, Washington, D.C., 1964, pp 204-220. J. M. H. Graves, G. A. Hughes, T. Y. Jen and H. Smith, J. Chem. S O C . , 5488 (1964). L. Mamlok, A. Horeau and J. Jacques, Bull. SOC. Chim. France, 8, 2359 (1965). 3203 (1965). R. M. Scribner, J. Org. Chem., E. Howard, Steroids, 255 (1965). R. T. Rapala, R. J. Kraay and K. Gerzon, J. Med. Chem., &, 580 (1965). P. Westerhof and J. Hartog, Rec. Trav. Chh., 918 (1965). P. Westerhof, J. Hartog and S . J. Halkers, Rec. Trav. Chim., 863 (1965). R. F. Church, A. S . Kinde and M. J. Weiss, J. Am. Chem. SOC., 2665 (1965). M. Shimizu, G. Ohter, K. Keno, T. Takigoshi, Y. Oshimia, A. Kasahara, T. Onodera, M. Mogi and H. Tachiyawa, Chem. Pharm. Bull. (Tokyo), g , 895 (1965). F. Neumann and R. Wiechert, Arzneimittelforschunq, 1168 (1965) A. D. Cross and I. T. Harrison, Steroids, Q , 397 (1965). L. J. Lerner, "Recent Progress in Hormone Research,'* Vol. 20, G. Pincus, Ed., Academic Press, New York, N.Y., 1964, p. 435. R. I. Dorfman, Acta Endocrin., G ,40 (1965).

a,

u,

a, m,

a,

a,

a,

a,

a,

a,

Chap. 19

50.

51. 52. 53.

A. R. A. L.

54.

L.

55. 56.

R. H.

57. 58.

F.

59.

R.

60.

G.

61.

A.

62. 63. 64.

J.

M. A.

65.

R.

66.

K.

67.

H.

68.

L.

69.

A.

C.

Steroid H o r m o n e s

D i a s s i and L e r n e r

5,

223 -

Segaloff and R . B. G a b b a r d , S t e r o i d s , 433 ( 1 9 6 4 ) . Y. K i r d a n i and R. I . D o r f m a n , J . Med. Chem., 8, 268 ( 1 9 6 5 ) . Segaloff and R . B. G a b b a r d , S t e r o i d s , 2, 2 1 9 ( 1 9 6 5 ) . J. Lerner, A. V. B i a n c h i , M. D z e l z k a l n s and E . D z e l z k a l n s , S t e r o i d s , 5, 2 2 3 ( 1 9 6 5 ) . J. Lerner, A . V. B i a n c h i , M. D z e l z k a l n s and E . D z e l z k a l n s , Steroids, 5, 215 ( 1 9 6 5 ) . I . D o r f m a n , J . Fajkos and J. Josha, S t e r o i d s , 1, 675 ( 1 9 6 4 ) . L . Saunders, K. H o l d e n and J . F. K e r w i n , S t e r o i d s , 2, 687 ( 1 9 6 4 ) . N e u m a n n and K. Junkmann, E n d o c r i n o l o q y , -, 7 3 33 ( 1 9 6 3 ) . w. Emrrsens and L . M a r t i n , "Methods i n H o r m o n e R e s e a r c h , " V o l . 111, R . I . D o r f m a n , E d . , A c a d e m i c Press, New Y o r k , 1964, pp 1-75. N.Y., I . D o r f m a n , "Metabolic I n h i b i t o r s , " V o l . 1, A c a d e m i c Press, New Y o r k , N.Y., 1963. P i n c u s , "The C o n t r o l of F e r t i l i t y , 'I A c a d e m i c Press, New Y o r k , N.Y., 1965. D. C r o s s , E . D e n o t , H. C a r p i o , R . A c e v a d o and P. C r a b b e , Steroids, 557 ( 1 9 6 5 ) . M. G r a v e s and H. J . R i n g o l d , S t e r o i d s ( S u p p l . I ) , (1965). 2553 ( 1 9 6 5 ) . E . W o l f f and T. Morioka, J. O r g . C h e m . , H. G o l d k a m p , W. M. H o e h n , R . A . Mikulec, E . F. N u t t i n g and D. L . C o o k , J. M e d . Chem., 409 ( 1 9 6 5 ) . E . Schaub, J. H. van den H e n d e and M. J. Weiss, J. O r g . 2234 (1965). Chem. , N a k a m u r a , Y. Matsuda, K. N a k a t s u j i , T. H i r o o k a and K. F u j i m o t o , J. A t h e r o s c l e r . R e s . , 5, 420 ( 1 9 6 5 ) . S m i t h , G. A . H u g h e s , G. H. D o u g l a s , G. R . Wendt, G. C. B u z b y , R . A. E d g r e n , J . F i s c h e r , T . F o e l l , B. G a d s b y , D. H a r t l e y , D. H e r b s t , A. B. Jansen and K. Ledig, J. Chem. SOC., 4472 ( 1 9 6 4 ) . C h e v i l l a r d , C . B o u r n i q u e , H. K a g a n and R . P o r t e t , Therapie., 371 ( 1 9 6 5 ) . C o r b e l l i n i , G. G e r a l i , G. F e r r a r a and G. Lugaro, F a r m a c o ( S c i . ) ,3 ! , l 1044 ( 1 9 6 4 ) .

3,

a,

g,

a,

a,

2 24

Sect. I V

-

Metabolic & E n d o c r i n e

H e i n z e l m a n , Ed.

Chapter 20. Non-steroidal Antiinflammatory Agents Robert A. Schemer, Parke, Davis and Company, Ann Arbor, Michigan

Introduction. Non-steroidal antiinflammatory ( A I ) agents a r e of i n t e r e s t f o r t h e management of inflammation and pain i n scores of rheumatic and other inflammatory conditions. Some may be found useful a s a n t i asthmatic agents.' The l a s t few years have seen a rapid developnent i n t h i s area i n new c l i n i c a l l y a c t i v e drugs, i n new and improved laboratory t e s t methods, i n proposals of mechanisms of a c t i o n of A 1 agents, and i n t h e number of laboratory publications and patents disclosing new agents. A recent revie$ covers background material. The b e s t introduction t o current thinking i s found i n t h e published Milan Symposium on Nons t e r o i d a l A 1 Drugs3 and a chapter by Whitehouse4 on biochemical and pharmacological properties of A 1 drugs. There i s a need f o r improved antirheumatic agents. The s t a t u s of currently a v a i l a b l e agents i s i n dicated i n t h e theme of a recent A.M.A. panel discussion on t h e subject5: "Risk vs. Benefits i n Rheumatology," and i n other r e v i e w ~ . ~ , ~ Test Methods. Lnflammation i s a complex b i o l o g i c a l process .4 Attempts t o duplicate t h e c l i n i c a l s t a t e i n t h e laboratory have been l a r g e l y em, ~ a complex p i r i c a l . The r e s u l t i s a v a r i e t y of t e s t r n e t h o d ~ , ~and assortment of compounds claimed t o have A 1 a c t i v i t y . Order i s gradually appearing. With new c l i n i c a l l y a c t i v e agents a v a i l a b l e i n a d d i t i o n t o phenylbutazone (indomethacin, flufenamic a c i d ) , Winter% has reexamined various r a t p a w edema t e s t s . He concludes t h a t t h e widely used t e s t s employing formalin, egg white and serotonin a s p h l o g i s t i c agents a r e unsuitable ( i n s e n s i t i v e ) . He a l s o found t h a t s t e r i l e kaolin does not produce edema. These and other i r r i t a n t s (e.g., yeast and mustard) a r e known t o give p o s i t i v e r e s u l t s with antihistamines, antiserotonins and diverse o t h e r agents.='' The most r e l i a b l e t e s t s appear t o be t h e W erythema t e s t (W, guinea p i g ) , t h e antibradykinin t e s t (B, guinea pig bronchoconstriction), and t h e cotton p e l l e t granuloma t e s t i n t h e r a t (GC). A r a t paw edema t e s t using ~ a r r a g e e n i n ~ ~ ?(Ec) ~ ' , has ~ ~ been widely adopted. It i s a considerable improvement over older E t e s t s , although f a l s e "positive" r e s u l t s a r e obtained with high doses of a One a l s o finds frequent reference t o t h e v a r i e t y of medicinal agents." granuloma pouch assay ( G ) and a new and i n t e r e s t i n g t e s t system, an The l a t t e r t e s t , or ones adjuvant-induced a r t h r i t i s i n r a t s (A)53j54. l i k e it, o f f e r hope for finding drugs which w i l l provide preventive o r curative r a t h e r than symptomatic treatment of inflammatory diseases. Steroids a r e i n a c t i v e i n t h e UV and B t e s t s , but a c t i v e i n t h e GI GC, E and A t e s t s .

Chap. 20

Antiinflammato r y

Scherr e r

In t h e discussion which follows, a code system w i l l be used t o i n d i c a t e approximate a c t i v i t i e s r e l a t i v e t o phenylbutazone (PB), and t h e test method used. Thus 2 Ec i n d i c a t e s a compound i s about twice a s a c t i v e a s PB by t h e r a t p a w edema t e s t using carrageenin. (Other abbreviat i o n s : y=yeast, d=dextran.) In a d d i t i o n t o t h e above tests most A I agents a r e a c t i v e i n antinociceptive tests involving inflamed t i s sues3h,13>14 and i n various writhing rodent t e s t s (presumptive of analg e t i c a c t i v i t y ) , o f t e n i n proportion t o t h e i r A1 a c t i v i t y . They a r e a l s o a n t i p y r e t i c . These agents a r e commonly r e f e r r e d t o a s antiinflammatory a n a l g e t i c s or a n t i p h l o g i s t i c agents t o d i s t i n g u i s h them from s t r i c t l y c e n t r a l a n a l g e t i c s . A number of non-steroidal antirheumatic agents appear t o a c t by a d i f f e r e n t mechanism from t h e a n t i p h l o g i s t i c antimalarial^,^ p r o t e o l y t i c agents. These include gold preparations enzyme^,"^ and agents s p e c i f i c f o r gout.' These will not be covered i n t h i s review. Receptor S i t e . In 1964 t h e Parke-Cavis group15 proposed a hypothetical presented a receptor t o accommodate a number of known A1 agents; s i m i l a r receptor based on an a n a l y s i s of t h e indomethacin s t r u c t u r e and s u b s t i t u t i o n e f f e c t s . 'Phe main f e a t u r e s , outlined i n f i g . 1, a r e a l a r g e f l a t area, a trough t o accommodate an out-of-plane group (such a s an a r y l r i n g ) , and a c a t i o n i c s i t e t o accommodate an a c i d anion (or unprotonated amine?). It will be seen t h a t a number of agents described below can be f i t t e d t o t h i s receptor. The c o r r e l a t i o n i s probably higher among known W-active compounds. These agents w i l l be discussed by broad chemical c l a s s i f i c a t i o n s .

@ cationic

site

c1 fig. 1

I

Arglacetic Acids. !Phis c l a s s probably represents t h e area of g r e a t e s t research e f f o r t i n t h e A 1 f i e l d today. One of t h e most s i g n i f i c a n t events i n medicinal chemistry i n 1965 was t h e NDA approval and marketing of indomethacin (I, & r c k j 2 Ec, 85 GC, 3 W, 4. B, 25 A * potency i n man i s several-fold W ) . The chemistry,3b and c l i n i c a l review^^'^ a r e available.

225 -

226 -

Sect. I V

-

Metabolic & Endocrine

Heinzelman,

C l i n i c a l r e p o r t s have appeared17 i n d i c a t i n g t h a t t h e diethylaminoethanol s a l t of a-ethylbiphenylylacetic a c i d (namoxyrate, Warner-Lambert ) is an e f f e c t i v e A 1 agent and a n a l g e t i c . Superiority of t h e s a l t over t h e f r e e acid (mouse writhing t e s t ) i s t h e b a s i s of a patent claim." Boots Pure Drug has received a patent on a s e r i e s o f bipheqlyl- and a-methylbipheqly l a c e t i c a c i d s ( s u b s t i t u t e d on Ar), but omitting a-methylbiphenylylacetic a c i d i t s e l f .19 The l a t t e r , along with numerous carboxyl derivatives i s now covered by a Merck patent.20 Patents on 4- and 5-phenyl-1-naphthaleneacetic acids, and &-substituted and a-alkylated derivatives have appeared.21 All t h e above a r e claimed t o be A1 agents.

,

4-Isobutylphenylacetic acid (ibufenac, Boots Pure Drug; 0.2-0.4 W ) has been found t o be c l i n i c a l l y a s e f f e c t i v e a s a s p i r i n , a t h a l f t h e dose, i n various rheumatic conditions, but hepatotoxicity may l i m i t i t s usefulness.22 An extensive Merck patent has now appeared covering hundreds of Ar-substituted 4-alkyl- and 4-cycloalkylphenylacetic a c i d s a s A 1 agents. 23 The e f f e c t of a - s u b s t i t u t i o n on t h e A 1 a c t i v i t y of various a r y l a c e t i c acids was t h e subject of two recent publication^.^^'^^ Substituted 1naphthaleneacetic acids were superior t o t h e corresponding phenylacetic acids. The best compounds i n t h e 1-naphthalene s e r i e s (e.g. a-furfuryl, 0.25 W ) were not much more a c t i v e than t h e unsubstituted acid (0.12 W) Buu-Hoi26 f i n d s A1 a c t i v i t y i n pn-butoxyphenylacethydroxamic a c i d (<1 E c ) but doesn't compare t h i s with t h e corresponding carboxylic acid.cf * l2

.

N-Arylanthranilic Acids. This s e r i e s includes t h e most potent agent r e ported i n t h e W and antibradykinin t e s t s : N-(2,6-dichloro-g-tolyl) a n t h r a n i l i c acid27 ( I I a , CI-583, Parke-Davis; 15 W, 14 GC, 32 B ) , now under c l i n i c a l t r i a l . Flufenamic acid (In;1.6 UV, 3.6 GC, 4 B, 4.5-9 Ec, >1 A ) a l s o under c l i n i c a l t r i a l , and mefenamic acid ( I I c ; 0.5 W, 0.6 GC, 4 B, 2.6 Ed, being marketed i n a number of a r e a s a s an a n t i i n flammatory a n a l g e t i c , a r e other members of t h e s e r i e s . While t h e peripheral s i t e of pain r e l i e f i n f e r a b l e from t h e i r W a c t i v i t i e ~ ' has ~ been confirmed i n r a t s f o r both mefenamic and flufenamic acids,14 the a d d i t i o n a l existence of a "presumably central'' action15 has been questioned.14 The a n a l g e t i c a c t i v i t y of mefenamic a c i d i n man continues t o be confirmed.e'g'56 An a n t h r a n i l i c acid s u b s t i t u t e d with t h e a n t i m a l a r i a l quinoline nucleus, 2, 3-dihydroxypropyl N- (7-chloro-4-quinolyl )anthranilate28 (glaphenine, 0.1 W), has been introduced i n France a s an a n a l g e t i ~ . ~ 'A s e r i e s of 2-anilinonicotinic a c i d derivatives' i s claimed t o have a n a l g e t i c and

Chap. 20

2 27 -

Scherr er

Antiinflammatory

Ar

X=H X=OH X=H

Ai I1

n

X

H

Iv

V

P;yrazolidinediones. Phenylbutazone ( I I I a ) was t h e f i r s t of t h e potent non-steroidal A 1 agents. Its A I a c t i v i t y was recognized i n man by chance i n 1952. Due t o i t s t o x i c l i a b i l i t y i t s use i n t h e U.S. i s genA metabolite, oxyphenbutazone o r e r a l l y l i m i t e d t o s h o r t periods. metabolite I ( I I I b ) , i s commercially a v a i l a b l e and r e p o r t e d l y less t o x i c t h a n PB. Much s y n t h e t i c e f f o r t has been expended i n t h i s a r e a without notable success t o date. A r e c e n t patent3' claims I I I c t o be a c t i v e i n man a s an A 1 and a n a l g e t i c . An i n t e r e s t i n g v a r i a t i o n f o r i t s p o t e n t i a l f i t t o t h e h y p o t h e t i c a l r e c e p t o r i s found i n IV31 ( l + E k a o m ) . 4-~-Butyl-2-phenyli~oxazolidine-3,5-dione~~, one of a s e r i e s , i s r e p o r t ed t o be about equal t o PB i n i n h i b i t i n g a gnawing response i n guinea pigs induced by i n t r a p e r i t o n e a l t a l c . S a l i c y l i c Acids. With t h i s c l a s s we come t o one of t h e o l d e s t a n t i rheumatic agents and t h e f i r s t drug of choice i n t h i s f i e l d - a s p i r i n . While pharmacologists and o t h e r s had f e l t i t t o be so for some t i m e , t h e f i r s t conclusive c l i n i c a l evidence t h a t a s p i r i n i s an antiinflammatory agent appeared i n 1965.33 Aspirin (0.1 W, 2 B, 0.5 Ec, 0.2 A ) i s more a c t i v e t h a n s a l i c y l i c a c i d i n most pharmacological t e s t s f o r a n t i i n f l a m matory and a n a l g e t i c a c t i v i t y , e * g o 3e and i s probably a c t i v e a s such in vivo. Salicylamide does not have A 1 a c t i v i t y . A c y c l i c d e r i v a t i v e of t h e l a t t e r , benz-l,j-oxazine-2,4-dione (carsalam), apparently a v a i l a b l e i n Europe3*, i s claimed t o be an a n a l g e t i c and A I agent. Another c l i n i c a l l y a c t i v e c y c l i c d e r i v a t i v e i s 2- (~-chloroethyl)-2,3-dihydr0-4-0~06-aminobenz-1,3-0xazine~~ '3J (V,A350; 1 Ec) In l a b o r a t o r y t e s t s t h e l a t t e r i s more a c t i v e t h a n t h e desamino compound known a s chlorthenoxazine.

.

2 28 -

Sect. IV

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

Alkylamines. There i s a r a p i d l y growing l i s t of w i n e s for which A1 act i v i t y i s claimed. Some have r a t h e r close counterparts i n a c i d i c s e r i e s discussed above and also would appear t o complement t h e receptor area outlined. It w i l l be of s p e c i a l i n t e r e s t from a pharmacological standpoint t o follow t h e progress of t h e s e agents through c l i n i c a l t r i a l s since many of these a r e i n a c t i v e i n t h e W t e s t . Benzydamine, 1-benzyl-3- ( y-dimethylaminopropoxy ) -1g-indazole hydrochlor i d e (VI, Angelinini Francesco; < 1 Ecr 1 G, 0 W ) , i s reported t o be a c l i n i c a l l y a c t i v e antiinflammatory analgetic3g. S i l ~ e s t r i n i36 ~~ ? has discussed h i s approach t o screening and t h e i n t e r p r e t a t i o n of t e s t r e sults. I n a publication on t h e pharmacologys6 t h e authors s t a t e : "As viewed from a q u a n t i t a t i v e angle [benzydamine] seems t o be-according t o our own experience-the most powerful analgesic-antiinflammatory dru@; so f a r described". ( C l i n i c a l l y ? This statement i s d i f f i c u l t t o understand from t h e data given; a parently t h e emphasis i s on analgesic. ) A chemical paper has appeared3'. O(CH2 ) s N ( C H S ) ~

VI

coNH2 wd7-'!!-(rn2

CH2 . -

C6H5

VII

)2N(CK3)2

(CH3)2N

CH3

VIII

A s e r i e s of publications on a-i sopropyl-a- (@-dimethylaminoethyl )naphthylacetamide (VII, naphthypramide; 1 Ec) and r e l a t e d derivatives has appeared out of t h e I n s t i t u t o De Angelis8. As with t h e a r y l a c e t i c acid s e r i e s of Durant e t a1.,24 t h e naphthyl d e r i v a t i v e s were superior t o t h e phenyl analogs. More r e c e n t l y bisobutylnaphthypramide (analogous t o ibufenac) has been reported t o be d i s t i n c t l y superior t o naphthypramide3'. N,N,l-Trimethyl-a,a--diphenyl-3-pyrrolidine acetamide (AHR-840; 0 W ) i s reported t o be comparable t o PB i n a n a l g e t i c t e s t s on t h e inflamed r a t paw and t o i n h i b i t alloxan-induced writhing and granuloma formation.40

1-[3-Ethoxy-3- (E-chlorophenyl)propyl]-4-phenylpiperazine (2-3 G and GC) and several r e l a t e d compounds a r e reported t o have antiinflammatory a c t i v i t y .41 This property could be separated from antihypertensive and adrenolytic a c t i v i t y found i n other members of t h e s e r i e s . Other Amines. The compounds i n t h i s group d i f f e r from those j u s t preceding i n t h a t an m i n e function i s attached d i r e c t l y t o a heterocyclic r i n g . The o l d e s t A I compound of t h i s type i s aminopyrine, ( V I I I ; 0.14 W ) considered too t o x i c by t h e U.S. FDA5= f o r general use. 5-Amino-1phenyltetrazole (fenamol, Armour) i s reported t o be a c t i v e c l i n i c a l l y i n rheumatic condition^.^^ No pharmacological paper has appeared. A s e r i e s of ~-amino-2,l-benzisothiazoles was reported t o have antiinflam-

Chap. 20

Antiinflammatory

Sc h e r r e r

matory a s well a8 g a s t r i c a n t i s e c r e t o r y a c t i v i t y 4 3 ( e .g. 3-ethylamino4 B; 6-chloro-3-dimethylamino- 1 W, 0.5 B). There was no c o r r e l a t i o n between A 1 and a n t i s e c r e t o r y a c t i v i t i e s . A patent on a s e r i e s of 3aminoalkylamino-Q-benzisothiazoles with A 1 a c t i v i t y has appeared44 (Ed, Ecroton o i l 1 Other ARents. Two compounds under c l i n i c a l t r i a l a r e of considerable i n t e r e s t since they d i f f e r from known a c t i v e non-steroidal agents i n not containing "functional" s u b s t i t u t i o n . These a r e 2,3-bis (E-methoxypheny l ) i n d ~ l e(IX, ~ ~ indoxole, Upjohn; 1-5 Ec, 1 A) and l-benzylcycloheptimidazol-2(z)-oneq6 (x, RCH-314, Sankyo; > 1 Ed). The l a t t e r compound is a feeble uncoupler of oxidative phosphorylation ( i n v i t r o ) , leading W h i t e h o ~ s et o ~ ~predict oor A 1 a c t i v i t y . Another "nonfunctional" ser i e s has been reported4'which included 2- (pmethylsulfonylpheny1)imidazo[l,2-a]pyridine ( X I ) with a c t i v i t y , a t comparable 1/4 LDSols, g r e a t e r than PB i n Ec, Ey and various antinociceptive t e s t s . Several 3-amino s u b s t i t u t e d derivatives were a c t i v e and a 3-acetic acid s e r i e s (analogous t o indomethacin, e t c . ) i s promised.

OCH3

Ix

x

XI

Dimethylsulfoxide has been s a i d t o be b e n e f i c i a l i n t h e r e l i e f of rheuIt has r e c e n t l y been withdrawn from most matic pain (reviewed4'). c l i n i c a l t r i a l s because of reported eye damage. I n t h e view of t h e U.S. Food and Drug Administration5' (March 19661, t h e r e i s a l a c k of subs t a n t i a l evidence of t h e effectiveness of D K O . I n j e c t i o n of Freund's adjuvant i n t o t h e hind f o o t of a r a t causes an i n flamed l e s i o n i n t h e i n j e c t e d f o o t , followed a f t e r about t e n days by inflamed secondary l e s i o n s ( a r t h r i t i s ) i n t h e other feet .53 Phenylbutazone i n h i b i t s t h e primary l e s i o n s and, t o a l e s s e r extent, t h e secondary ~ ~t h e f i n d i n g of a compound l e s i o n s . An i n t e r e s t i n g d e ~ e l o p m e n tis which prevents t h e appearance of secondary l e s i o n s , while not a f f e c t i n g t h e primary l e s i o n s . The cmpound i s 2-butoxycarbonylmethylene-~-oxot h i a z o l i d i n e ( X I I j s l i g h t Ec, 0 Ekaolin). Only c l i n i c a l t r i a l will determine i f these r e s u l t s imply a new kind-or any kind-of antirheumatic a c t i v i t y .

229

2 30 -

Sect. I V

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

Mechanism of Action. W h i t e h ~ u s je4~ ~ r a t h e r persuasively presents arguments t o support t h e proposal t h a t an important property of non-steroidal antiinflammatory agents i s t h e i r a b i l i t y t o uncouple oxidative phosphorylation. The c o r r e l a t i o n between these a c t i v i t i e s i s high, e s p e c i a l l y considering t h a t uncoupling a c t i v i t y i s determined i n v i t r o . Another i n v i t r o t e s t has been devised51 based on t h e high a f f i n i t y of A 1 agents f o r l y s y l e-amino groups of plasma albumin. Biochemical i m p l i c a t i o n s , including a possible connection w i t h t h e uncoupling of oxidative phosphorylation, have been discussed. 52 The r o l e plasma kinins may play i n inflammation has been reviewed by Lewis.3J A mechanism of a c t i o n of A 1 p r o t e o l y t i c enzymes i n terms of t h e destruction of bradykinin has been ~ o n s i d e r e d . ~ It~ has been found'' t h a t inflammation brought on by carrageenin i n t h e r a t paw edema t e s t d i f f e r s from t h a t caused by some other i r r i t a n t s and may be due t o t h e r e l e a s e of bradykinin. While t h e l a s t few years have seen considerable progress i n t h e nons t e r o i d a l antiinflammatory f i e l d , t h i s seems t o be only t h e beginning of a period of even g r e a t e r a c t i v i t y and progress a s developments i n t h e biochemical, pharmacological and medicinal chemical a r e a s a r e t r a n s l a t e d t o developments i n c l i n i c a l a r e a s . References.

(1)

P. A. Berry and H. 0. J. Collier, B r i t . J. Pharmacol., 2 3 , 201

(2)

J. S. Nicholson i n "Reports on t h e Progress of Applied Chemistry," Val. 49, Society of Chemical Industry, 1964, pp. lg2-,203.

(3)

(1964

*

I n t e r n a t i o n a l Symposium on Non-steroidal Antiinflammatory Drugs, Milan, Sept. 1964, S. G a r a t t i n i and M. N. G. Dukes, Eds., Excerpta Medica Foundation, Amsterdam, 1965. a ) C. A. Winter, p 190. b ) T. Y. Shen, p 13c ) S. G a r a t t i n i , A. J o r i , D. Bernardi, C. Carrara, S. Faglialunga and D. Segre, p 1-51. d ) G. J. Martin, M.,p 90. p 1-39. e ) H. 0. J. Collier,

u., u., w., w.,

Antiinflammato r y

Chap. 20

Sc her r e r

231

f ) E. Arrigoni-Martelli, F. Fontanini, A. Garzia and W. F e r r a r i , -

-

-

i b i d . , p 280. g ) A. Turchetti, p 396; M. DeGregorio, i b i d . , p 422. p 180. h ) B. S i l v e s t r i n i , i ) M. W. Whitehouse, p 52. j) G. P. Lewis, p 114. M. W. Whitehouse i n "Progress i n Drug Research," Vol. 8, E. Jucker, Ed., BirkhaCser Verlag, Basel, 1965, pp 321-429. 194, (12) Adv. 23 (1965). Panel discussion, J.A.M.A., The Medical Letter, 7, 109 (1965). The Yearbook o f Drug Therapy, 1965-1966 Series, H. Beckman, Ed., Yearbook Medical Publishers, Inc. , Chicago, 1965, pp 541-566. Proceedings o f Conference on Gout and Purine Metabolism, A r t h r i t i s Rheum., 8, 591-910 (1965). Laboratories U.P.S.A., Neth. Appl. 6,414,717 (1965). C. J. E. Niemegeers, F. J. Verbruggen and P. A. J. Janssen, J. Pharm. Pharmacol., 16, 810 (1964). C. G. Van Arman, A. J. Begany, L. H. Miller and H. H. Pless, J. Pharmacol. Exptl. Therap., 150, 328 (1965). Boots m e mug, Belg. Fat. 621,255 (1963). C. A. Winter, L. Flataker, J. Pharmacol. Exptl. TheraE., 150, 165

m., u., u.,

w.,

(19651*

C. A. Winter, L. Flataker,

M.,148, 373 (1965).

R . A. Scherrer, C. V. Winder and F. W. Short, Abstracts Ninth Medicinal Chemistry Symposium, Minneapolis, June 1964; pp 11 a - i . C . A. Winter and E. A. Risley, Arzneimittel-Forsch., 15, 427 (1.965). C. W. Denko, E. A. De Felice and J. W. Shaffer, C u r r . Ther. Res., 7, 749 (1965); A. Cohen and F. A. De Felice, J. 35(1965) * Warner-Lambert, Fr. Fat. 2401M (1964); Aust. Pat. 24,053/62. Boots Pure Drug, I r i s h Pat. 56/65. Merck, Belg. Pat. 664,187 (1965). Farke, Davis and Co., B r i t . Pat. 1,009,288 (1965). F. D. Hart and P. L. Boardman, Ann. Rheum. D i s . , 24, 61 (1965). Merck, Neth Appl. 6 507 505 (1965). G. J. W a n t , G. M. Smith, R. G. W. Spickett and E. Szarvasi, Med. Chem., 8, 598 (1965). E. Szarvasi, L. Fontaine and C. Letourneur, B u l l . Soc. Chim. fi.,

.

, ,

J.

3113 (1964).

N - P. Buu-Hoi, G. Lambelin, C. Lepoivre, C. G i l l e t , M. Gautier and J. Thiriaux, C. R. Acad. S c i . , 261, 2259 (1965). C . V. Winder, J. Wax and M. Welford, J. Pharmacol. Exptl. Ther.,

148, 422 (1965). Roussel-Uclaf, S. A., U. S. Pat. 3,232,944 (1966). Prod. Pharm. 20, 621 (1965 ) Wallace and Tiernan, U. S. Pat. 3,135,764 (1964). Siegfried A. G., Fr. Pat. 1,393,596 (1965). K. mchel, H. Gerlach-Gerber, Ch. Vogel and M. Matter, Helv. Chim. Acta, 48, 1973 (1965).

,

-

.

232

Sect. I V

- Metabolic & Endocrine

K. Rremont-Smith and T. B. Bayles, J.A.M.A., 192, Prod. Pharm., 20, 3 2 6 ~ ,Jlily 1965.

H e i n z e l m a n , Ed.

1133 (1965).

E. Arrigoni-Martelli and I. Conti, Medicina E k D t l . , 10, 164 (1964). B. S i l v e s t r i n i , A. Garau, C. B z z a t t i and V. Cioli, ArzneimittelForsch., 16, 59 (1966). G. Pelazzo, G o Corsi, L. Baiocchi and B. S i l v e s t r i n i , J. Med. 9, 38 (1966). S. Casadio, G. Pala, T. Bruzzese, E. Crescenzi, E. Marazzi-Uberti and G. Coppi, J. k d . Chem., 8, 594 (1965); 5. Casadio, G. Pala, E. Crescenzi T. Bruzzese, E. Marazzi-Uberti, and G. Coppi, 8, 589 (1965); G. Pala, So Cgsadio, T. Bruzzese, E. Crescenzi and E. Marazzi-Uberti, 8, 698 (1965). T. Bruzzese and C. Turba, J. Med. Chem., 9, 264 (1966). W. €I Funderburk, . M. Foxwell and J. W. Ward, The PharmacoloRist, - 0 ,

x.,

w.,

7, 181 (1965).

R. P. Mull, C - Tannenbaum, M. R. Dapero, M. Bernier, W. Yost and G. de Stevens, J. k d . Chem. 8, 332 (1965). Medical World News, 6 (51, 56, (12 F e b . l s 5 ) j Drug Trade News, 40 (241, 47, (22 Nov. 1965). R - F a Meyer, B- L. Cummings, P. Bass and H. 0. J. Collier, J . Med.

8, 515 (1965).

-9,

A. G. Knoll, Ger. Pat. 1,174,783 (1964). E. M. Glenn, B. Bowman, W. Kooyers, T. Koslowske, and M. mers, The Pharmacologist, 7, 181 (1965). H. Nakao, N. Soma, Y. Sat0 and G. Sunagawa, Chem. Pharm. B u l l .

(Japan), 13, 473 (1965)I. F. Skidmore and M. W. Whitehouse, Biochem. Pharmacol., 14, 547

-

(1965 L. Almirante, L. Polo, A. Mugnaini, E. Provinciali, P. Rugarli, A. Biancotti, A. Gamba and W. bbmann, J. b d . Chem., 8, 305

(1965 . - _ 1. . The Medical Letter, 7, 42 (1965). F. D- C - Reports, 28 (ll), 522 (1966). I. F. Skidmore and M.-W. Whitehouse, J. Pharm. Pharmacol., 17, 671

(1965)* M. W- Whitehouse and I. F. Skidmore, i b i d . , 17, 668 (1965). B. B. Newbould, B r i t . J. Pbarmacol., 24,632 (1965). E. M. Glenn, Am. J. V e t . Research, 27, 339 (1966). D r q Research Remrts, 7 (481, pp S22-S34 (1964) (25 Nov. 1964). S. W. Bissell, Practitioner, 194, 817 (1965).

Chapter 21.

Agents A f f e c t i n g Blood Enzymes

Murray Weiner, Geigy Chemical Corporation, Ardsley, N. Y. This review i s concerned with agents which act by a l t e r a t i o n of enzymes i n t h e c i r c u l a t i n g plasma, and w i l l n o t d e a l w i t h canpounds which i n f l u e n c e

blood enzymes c o i n c i d e n t a l t o o t h e r pharmacologic e f f e c t s . The 'drugs t o be reviewed a r e p r i m a r i l y t h o s e which a l t e r c l o t formation or d i s s o l u t i o n , o r hemorrhage as i t r e l a t e s t o c l o t t i n g f a c t o r s . I n t h e area of d i r e c t l y a c t i n g ( h e p a r i n - l i k e ) a n t i c o a g u l a n t s , t h e r e a r e new semisynthetic heparinoids3 ,31,32 polynucleotides with phosphate groups r a t h e r than s u l f a t e s a s i n heparin4~34,35 and n a t u r a l agents which are chemic a l l y q u i t e d i f f e r e n t , b u t e x h i b i t d i r e c t a n t i c o a g u l a n t a c t i o n not n e u t r a l i z e d by p r ~ t a m i n e . ~ ' These s t u d i e s confirm that h e p a r i n - l i k e e f f e c t i s not a s o l e f u n c t i o n of t h e n e g a t i v e l y charged groups. A review of p o s i t i v e l y charged compounds, p a r t i c u l a r l y metal complexes5 i n d i c a t e t h a t h e p a r i n n e u t r a l i z i n g potency i s not a simple r e s u l t of p o s i t i v e charge. There i s i n t e r e s t i n t h e antithrombotic a c t i v i t y of s u l f a t e - f r e e low molecular weight d e x t r a n s which do n o t s i g n i f i c a n t l y prolong t e s t t u b e c l o t t i n g time.7 The unexplained hemorrhagic complications of t h e use o f dextran a s a plasma expander may have a common o r i g i n w i t h t h i s a l l e g e d antithrombotic activity. So f a r t h e newer heparinoids show l i t t l e i f any promise of s i g n i f i c a n t advantage over heparin i t s e l f . None has y e t reached t h e U. S . market. Oral e f f i c a c y has not been achieved w i t h p r a c t i c a l dosage of heparin o r i t s analogs e i t h e r a s a n t i c o a g u l a n t s o r as l i p o p r o t e i n l i p a s e a c t i v a t o r s . A paradoxical use of heparin t o prevent o r c o n t r o l hemorrhage has been described6 i n p a t i e n t s with t h e s o - c a l l e d "depletion syndrome," a c o n d i t i o n i n which c l o t t i n g a c t i v a t o r s r e l e a s e d i n t o t h e blood as a r e s u l t of d i s e a s e , surgery, or p a r t u r i t i o n , cause excessive consumption of c l o t t i n g f a c t o r s , i n cluding fibrinogen, which i n t u r n l e a d s t o hemorrhage. Heparin, by preventing t h e hypercoagulability which i n i t i a t e s t h e s e events, may also prevent or cont r o l t h e r e s u l t i n g hemorrhage. Accurate assessment of t h e c l o t t i n g p a t t e r n i s c r i t i c a l t o t h i s newly proposed use f o r heparin. Among t h e hypoprothrombinemic agents ( i n d i r e c t a n t i c o a g u l a n t s ) , s e v e r a l new a c t i v e indandione and coumarin d e r i v a t i v e s have been d e s ~ r i b e d * , ' ~ ,i~n -~ cluding some from n a t u r a l sources, some w i t h polymeric s t r u c t u r e s s and some which a r e presumed t o be of primary i n t e r e s t as coronary d i l a t o r s . " Additional information concerning hypoprothrombinemic agents include: f d r t h e r observations of t h e r e l a t i o n s h i p of vitamin K and a n t i - v i t a m i n K act i v i t y t o structure;" t h e i n f l u e n c e of o t h e r drugs i n c l u d i n g s t e r o i d s 1 3 on t h e d i s t r i b u t i o n and metabolic f a t e of ~ o u m a r i n s ; ' , ' ~ indandione t o x i c i t y of t h e kind not seen w i t h coumarins.2 The concept t h a t t h e i n i t i a t i n g event of c l i n i c a l thrombosis i s t h e clumping and breakdown of p l a t e l e t s has r e s u l t e d i n t h e study of agents which a r e not a n t l c o a g u l a n t s i n t h e sense of prolonging c l o t t i n g time, but which decrease excessive s t i c k i n e s s o r adhesiveness of p l a t e l e t s .15 A d i v e r s e g r o u p of compounds which have l i t t l e i n common s t r u c t u r a l l y o r pharmacologically have been reported t o reduce p l a t e l e t s t i c k i n e s s , i . e . , t h e hypolipidemic agent Atromid," t h e coronary d i l a t o r Persantin,17 and t h e u r i c o s u r i c agent Anturane .la

234

Sect. I V

-

Metabolic & Endocrine

H e i n z e l m a n , Ed.

I n t h e search f o r c l o t d i s s o l v i n g a g e n t s , t h e t r e n d has been away from f i b r i n o l y s i n i t s e l f and toward a c t i v a t o r s of n a t u r a l l y o c c u r r i n g precursors of f i b r i n o l y s i s . D i f f i c u l t i e s w i t h s t r e p t o k i n a s e a n t i g e n i c i t y has caused more a t t e n t i o n t o be paid t o non-antigenic urokinasel' i n s p i t e of considera b l e production problems r e l a t e d t o t h e need f o r l a r g e volumes of human u r i n e as t h e s t a r t i n g material. I t s f u l l t h e r a p e u t i c value i s y e t t o be proved e m p i r i c a l l y i n t h e c l i n i c . This need f o r e m p i r i c a l proof of e f f i c a c y i s a t l e a s t as g r e a t f o r a group of non-enzymatic compounds which a c t i v a t e f i b r i n o l y t i c proenzymes. Some of these12J2' appear t o be outgrowths of the observ a t i o n that p a r e n t e r a l n i c o t i n i c a c i d induces an i n t e n s e but s h o r t - l i v e d f i b r i n o l y t i c response i n man,21 while others22 probably can t r a c e t h e i r o r i g i n i n d i r e c t l y from t h e w e l l known i n v i t r o f i b r i n o l y t i c a c t i o n of conc e n t r a t e d s o l u t i o n s of urea. The i n h i b i t i o n of excessive f i b r i n o l y t i c a c t i v i t y as a means t o c o n t r o l hemorrhage has r e s u l t e d i n t h e marketing of E-aminocaproic acid2= and t h e development of a c t i v e analog^.^^'^' Large doses of EACA a r e r e q u i r e d i n s p i t e of i t s high potency and good absorption because of i t s very r a p i d urinary excretion. Another approach t o t h e c o n t r o l of b l e e d i n g i s based on t h e presumed a c t i v a t i o n of Hageman Factor (Factor XII) by e l l a g i c a ~ i d , ~ ~ even , ~ though ' p a t i e n t s with marked Hageman d e f i c i e n c y do n o t s u f f e r from bleeding problems. The mechanism of t h e a l l e g e d i n f l u e n c e of v a r i o u s s t e r o i d s on thrombotic2' and hemorrhagic3' tendencies remains unclear. This reviewer has previously expressed concern about t h e absorption, and, t h e r e f o r e , e f f i c a c y of o r a l l y administered enzyme p r e p a r a t i o n s whose a c t i o n i s presumed t o be a r e s u l t of t h e i r e n t r y i n t o t h e blood i n a c t i v e form.26 He i s not aware of any new d a t a t o a l t e r t h i s p o s i t i o n and has y e t t o be s a t i s f i e d t h a t any of t h e pharmacologic a c t i o n s of o r a l enzymes a r e due t o t h e presence of t h e i r s p e c i f i c enzymic p r o p e r t i e s i n blood or t i s s u e a f t e r oral administration. References 1. K. Pyorala, Ann. Med. Exp. et Biol., 9, Suppl. 3 (1965). T. Hargreaves and M. Howell, Brit. Heart J., 3 , 932 (1965). 311 (April 20, 1964). 3. A. M. A. Council on Drugs, J. Am. Med. Assoc., 4. s. Yachnin, Blood, 553 (1964). 290 (1965). 5. c, Singh and B. K. Bhattacharya, Arch. Int. Pharmacodyn., 6. c. Merskey, A. J. Johnson, 3. H. Pert and H. Wohl. Blood, &, 701 (1964). 7 . E. F. Cox, Surgery, 12. 225 (1965). Farmakol. Toksikol., 3,438 (1965). 8. V. I. Zavrazhnov, ,J'2 331 (No. 3 ) (1964 9. N. V. Petryaevskaya and A. V. Valdman, Farmakol. Toksikol., 10. J. Lowenthal and 3. A . MacFarlane, J. Pharmacol. Exp. Therap., 130 (1965). 11. c. N. Mathur, & &., Arch. Int. Pharmacodyn., 218 (1965). 12. H. Somer, Arzneimittel-Forsch.. 2, 1337 (1965). Fed. Proc., 2.No. 2 (March-April, 1965). 13. M. Weiner, & d,, 14. M. Weiner, Am. Therap. SOC. 66th Annual Mtg., New York, June 1965. Abstract pages 34-35. 15. c. Merskey and A. Drapkin, Blood. 3, 567 (1965). 16. c. Symons, A. DeTaszeghi and I. J . Cook, Lancet, 2, 233 (1964). Lancet, 2, 603 (1965). 17. P. R. Emmons, & 18. H. A. Smythe, & Can. MedT Assoc. J., X, 818 (April 10, 1965). 903 (Nov. 22, 1965). 19. A. M. A . Council on Drugs, J. Am. Med. Assoc., 20. F. Pericoli and R. Nuzzi, Policlinico (Prat.), E, 1589 (Nov. 11, 1963). 21. M. Weiner, Am. J. Med. Sci., 294 (Sept., 1963). 22. K. N. Von Kaulla, Experientia, 21, 439 (Aug. 15. 1965). 2.

188.

a,

u,

a.,

z,

a., a.,

e,

z,

x,

Chap. 21 23. 24. 25. 26. 27. 28. 29. 30.

31.

32. 33. 34. 35. 36.

Blood Enzyme Agents

Weiner

188,

235 -

A . M. A. Council on Drugs, J . Am. Med. Assoc., 56 ( A p r i l 6 , 1 9 6 4 ) . F. Markwardt, G a l . , Arch. I n t . Pharmacodyn., 223 (No. 1 - 2 ) (1964). S. Okamata, & &. , Keio J . Med., 2, 177 (Dec.. 1964). M. Weiner, Drugs and Enzymes, Proc. 2nd I n t e r n . Pharm. Mtg., Prague, August, 1963 R . E. B o t t i and 0. D. R a t n o f f , J. Lab. C l i n . Med., 385 ( S e p t . , 1964). E. E. C l i f f t o n , & g . , Proc. SOC. Exp. B i o l . Med., 120, 179 ( O c t . , 1965). N . C . Nevin, P. C . Elmes, and J . A. Weaver, B r i t . Med. J . . 1,1586 (June 19, 1965 J . Van d e r V i e s . Acta E n d o c r i n o l . , 48, 630 ( A p r i l , 1965). C . L. J a r r e t t and L. B. J a q u e s , Canad. J . P h y s i o l . Pharmacol., 93 ( J a n . , 1964 2. Yosizawa, Biochem. Biophys. Res. Commun.. 336 ( J u l y , 1964). M. D e z e l i c and M. Trkovnik, J. Med. Chem., 1,284 (May, 1 9 6 4 ) . A . C. S a r t o r e l l i and R . D. Magus, Pharmacologist, l, 171 ( A b s t r a c t 202) ( F a l l , 1965) S. Yachnin and G. M. Ruthenberg, J. Immunol., $, 12 ( J a n . , 1965). 3. M. Khanna, g g . . J. Pharm. S c i . , 2, 1016 ( J u l y , 1960).

z, fi,

16,

g,

Editor:

I. 1.A.

-

Section V T o p i c s i n Biology Tabachnick, Schering Corporation, Blocmfield, New J e r s e y

Chapter 22. Molecular Aspects of Drug-Receptor I n t e r a c t i o n s Barry M. Bloom, Chas. P f i z e r and Co., Inc., Groton, Conn. "...it h a s always been clear t h a t no d r u g a c t i o n can b e understood completely u n t i l i t i s pursued t o t h e level of molecular i n t e r a c t i o n s , and now such p u r s u i t s can i n c r e a s i n g l y become r e a l i s t i c g o a l s of r e s e a r c h . . .'I Avram G o l d s t e i n 1

-

I n t h e absence of any g u i d i n g o r c o n s t r a i n i n g p r e c e d e n t s , t h i s f i r s t of a n annual series of b r i e f reviews on molecular a s p e c t s of drugr e c e p t o r i n t e r a c t i o n s w i l l emphasize s e v e r a l newer t e c h n i q u e s and concepts of broad a p p l i c a b i l i t y t h a t appear d e s t i n e d t o p l a y a n i n c r e a s i n g r o l e i n uncovering knowledge a b o u t t h e molecular n a t u r e of d r u g - r e c e p t o r i n t e r a c t i o n s . The f i r s t t h r e e s e c t i o n s d i s c u s s such t o p i c s b r i e f l y and p r o v i d e l e a d i n g r e f e r e n c e s . The f o u r t h s e c t i o n reviews r e c e n t developments i n r e c e p t o r t h e o r y of a more s p e c i f i c n a t u r e . Space c o n s i d e r a t i o n s have l i m i t e d i t s scope t o p u b l i c a t i o n s d e a l i n g w i t h t h e autonomic and c e n t r a l nervous systems. S i n c e t h e p r e s e n t s u b j e c t i s n o t w e l l s u i t e d t o review on a c a l e n d a r b a s i s , coverage has been expanded t o i n c l u d e o t h e r i m p o r t a n t p u b l i c a t i o n s of r e c e n t vintage. The formidable problem of s e e k i n g t o understand t h e a c t i o n of a drug i n molecular terms can only be f a c e d a f t e r c e r t a i n more fundamental q u e s t i o n s are answered. Where does t h e drug act morphologically7 How does i t a c t f u n c t i o n a l l y ? Which of i t s a c t i o n s d i r e c t l y i n v o l v e t h e r e c e p t o r s i t e of i n t e r e s t ? F i n d i n g t h e answer t o t h e s e q u e s t i o n s can o f t e n prove t o be a major problem. N e v e r t h e l e s s , t h e p r e s e n t review l a r g e l y i g n o r e s t h e s e the interp r e l i m i n a r y c o n s i d e r a t i o n s t o emphasize t h e u l t i m a t e a n a l y s i s p r e t a t i o n i n molecular terms of t h e i n t e r a c t i o n between drug and r e c e p t o r . It h a s become i n c r e a s i n g l y f e a s i b l e t o c o n s i d e r drug a c t i o n a t t h i s l e v e l i n r e c e n t y e a r s because of t h e p r o g r e s s made toward understanding n u c l e i c a c i d b i o l o g y , p r o t e i n t e r n a r y and q u a t e r n a r y s t r u c t u r e , t h e n a t u r e of enzymes, and t h e mechanism by which t h e a c t i v i t y of t h e l a t t e r i s c o n t r o l l e d . The s e c t i o n t o f o l l o w d i s c u s s e s t h e importance of t h i s new knowledge as a s o u r c e of d i r e c t i n s i g h t r e g a r d i n g t h e n a t u r e of r e c e p t o r s .

--

Sources of I n s i g h t Regarding t h e Nature of Receptors. I n s e e k i n g t o understand t h e n a t u r e of d r u g - r e c e p t o r i n t e r a c t i o n s i n molecular terms, t h e g r e a t e s t o b s t a c l e s u s u a l l y relate t o t h e macromolecular p a r t n e r -- t h e r e c e p t o r . The problem i s most o f t e n approached by i n d i r e c t i o n , by assuming " t h a t small molecules whose b i o l o g i c a l a c t i o n i s s p e c i f i c and s t r u c t u r e dependent d i s p l a y a h i g h d e g r e e of molecular complementarity toward t h e s i t e a t which they act."2 Although a p p r e c i a b l e p r o g r e s s h a s been made i n t h i s way and a good d e a l more can be a n t i c i p a t e d , i t i s w e l l known t h a t t h e

Chap. 2 2

Receptor Theory

Bloom

237 -

s y n t h e s i s and meaningful b i o l o g i c a l e v a l u a t i o n of l a r g e numbers of analogous molecules can be an exceedingly l a b o r i o u s e x e r c i s e t h a t o f t e n proves r e l a t i v e l y unrewarding.

As w e come t o a p p r e c i a t e more f u l l y t h e s u b t l e t y and dynamism of enzyme-small molecule i n t e r a c t i o n s (assuming t h a t w e a c c e p t t h e i n e v i t a b i l i t y t h a t d i r e c t l y o r i n d i r e c t l y drugs act upon enzymes) i t becomes i n c r e a s i n g l y apparent why t h i s c l a s s i c a l approach i s so o f t e n l i m i t e d i n e f f e c t i v e n e s s . I n t h e eloquent words of a Lancet e d i t o r i a l 3 e n t i t l e d The Shape of Molecules t o Come: "...the s o - c a l l e d a c t i v e s i t e of an enzyme i s not a fixed circums c r i b e d area endowed w i t h c a t a l y t i c p r o p e r t i e s but a moving c o n s t e l l a t i o n of atoms, a g e s t u r e r a t h e r than a limb.. . . ' I The r e a l i z a t i o n t h a t s i g n i f i c a n t conformational changes can and probably do occur i n r e c e p t o r systems as t h e r e s u l t of t h e i r binding drugs a s s u b s t r a t e s , i n h i b i t o r s , a l l o s t e r i c e f f e c t o r s , o r otherwise, suggests one very good rGason why i t i s SO d i f f i c u l t t o conceive t h e i r nature merely by assuming molecular complementarity w i t h i n t e r a c t i n g drugs. More d i r e c t means of g a i n i n g i n s i g h t a r e -1early needed. Perhaps t h e b e s t such means p r e s e n t l y a v a i l a b l e i n v o l v e s t h e e x t r a o l a t i o n of newer knowledge and concepts regarding t h e s t r u c t u r e of p r o t e i n s f :' 5 , t h e regulat i o n of enzyme a c t i v i t y 6 , and t h e n a t u r e of feedback c o n t r o l mechanisms a t t h e molecular leve17; f o r example, t h e views of Koshland899 on enzyme f l e x i b i l i t y and induced f i t , and those of Monod and h i s colleagueslo-12 on a l l o s t e r i c p r o t e i n s and t r a n s i t i o n s , and on t h e molecular i n t e r a c t i o n s c o n t r o l l i n g c e l l u l a r metabolism. The kind of s t r u c t u r a l information c u r r e n t l y being garnered by v a r i o u s instrumental techniques may a l s o be expected t o p r w i d e a powerful stimulus t o thought about t h e nature of r e c e p t o r s . A case i n p o i n t are the Nobel Prize-winning, X-ray c r y s t a l l o g r a p h i c s t u d i e s on t h e s t r u c t u r e of hemoglobinl3-18. Not t h a t one e x p e c t s t o i s o l a t e , c r y s t a l l i z e and analyze the d i f f r a c t i o n p a t t e r n s of very many r e c e p t o r s i n t h e next l i t t l e while! But i n t h e sense of g a i n i n g i n s i g h t regarding fundamental p r i n c i p l e s , one must recognize the p o t e n t i a l v a l u e of a method t h a t can d e t e c t and d e f i n e remote conf ormational changes i n a macromolecular s t r u c t u r e t h a t a r i s e from i t s i n t e r a c t i o n with a small molecule. Separate X-ray ana y s e s of reduced hemoglobin and oxyhemoglobin have afforded evidence of a 7 increase i n d i s t a n c e between t h e heme groups of t h e two @-chains, which occurs upon oxygenation! These d r a m a t i c f i n d i n g s should serve t o temper any reservat i o n s about t h e l i m i t e d v a l u e of s t r u c t u r a l information o b t a i n a b l e from p r o t e i n s i n t h e c r y s t a l l i n e state.

B

The f a s c i n a t i n g hemoglobin s t o r y w a s w e l l reviewed during 1965 i n t h e above-mentioned Lancet e d i t o r i a l 3 , by P e r u t z l g , and by Muirhead20. Other X-ray s t r u c t u r e a n a l y s e s that r e v e a l t h e p o t e n t i a l i t i e s of t h e technique f o r studying complexes of p r o t e i n s with small molecules d e a l w i t h hen egg-white lysozyme-inhibitor complexes2lr22, t h e binding of a z i d e , c u p r i c and z i n c ions t o sperm whale myoglobins23,24, and a-chymotrypsin i n h i b i t e d with d i isopropyl fluorophosphate and with a number of s u l f o n y l f l u o r i d e i n h i b i t o r s 2 5 .

2 38

Sect. V

-

Topics in B i o l o g y

T a b a c h n i c k , Ed.

D e s p i t e t h e g r e a t t e c h n i c a l c o m p l e x i t i e s of X-ray c r y s t a l l o g r a p h i c a n a l y s i s , t h e s w i f t p r o g r e s s being made i n t h e automation26 of both instrument a t i o n and methods f o r h a n d l i n g t h e d a t a produced, serves t o engender hope t h a t s i g n i f i c a n t c o n t r i b u t i o n s t o o u r u n d e r s t a n d i n g of t h e n a t u r e of r e c e p t o r s w i l l e v e n t u a l l y be forthcoming from t h i s q u a r t e r , e a r l i e r p e s s i m i s t i c comments27 notwithstanding

.

methods tions. ments28 protein induced point.

Space l i m i t a t i o n s p r e c l u d e a review of o t h e r newer i n s t r u m e n t a l s i g n i f i c a n t t o t h e problem of c h a r a c t e r i z i n g d r u g - r e c e p t o r i n t e r a c B r i e f l y , o p t i c a l rotatory d i s p e r s i o n and c i r c u l a r d i c h r o i s m measured e s e r v e mention as a v e r y e f f e c t i v e means of d e t e c t i n g changes i n t e r n a r y s t r u c t u r e . A r e c e n t study29 of c o n f o r m a t i o n a l changes i n chymotrypsin by v a r i o u s s u b s t r a t e s and i n h i b i t o r s i s a case i n

The a p p l i c a t i o n of n u c l e a r magnetic resonance s p e c t r o s c o p y ( NMR) t o b i o l o g i c a l problems h a s been w e l l d e s c r i b e d by t h e p i o n e e r i n g Cohn30 and J a r d e t s k y 3 1 3 3 2 groups. The method may be of p a r t i c u l a r v a l u e i n s t u d y i n g t h e n a t u r e of d r u g - r e c e p t o r b i n d i n g ( e . g . , a r e c e n t a n a l y s i s 3 3 of thiaminei n d o l e complexes). A s o p h i s t i c a t e d example of t h e use of b o t h NMR and e l e c t r o n s p i n resonance s p e c t r o s c o p y t o d e f i n e t h e s t r u c t u r e of enzymes u b s t r a t e - a c t i v a t o r complexes i s provided by r e c e n t s t u d i e s 3 4 , 3 5 on p y r u v a t e k i n a s e , which t a k e advantage of t h e paramagnetic p r o p e r t i e s of manganous i o n t o e l u c i d a t e t h e r e a c t i o n pathway and t h e s t r u c t u r e of t h e t e r n a r y i n t e r mediates.

-

Although n o t s t r i c t l y a n i n s t r u m e n t a l t e c h n i q u e , t h e u s e of &t e r i u m i s o t o p e e f f e c t s t o i n v e s t i g a t e t h e n a t u r e of b i o r e c e p t o r - s u b s t r a t e complexes w a r r a n t s mention here because of i t s r a t h e r g e n e r a l a p p l i c a b i l i t y . A r e c e n t review36 by B e l l e a u , who h a s led i n t h e development of t h i s approach, summarizes i t s p r e s e n t s t a t u s . S t u d i e s are d e s c r i b e d t h a t relate t o r e c e p t o r and enzyme-bound a c e t y l c h o l i n e , t o t h e h i s t a m i n e - h i s t a m i n a s e system, t o t h e n a t u r e of monoamine o x i d a s e and dehydrogenases more g e n e r a l l y ( d e t e r m i n a t i o n of whether t h e y are f l a v i n - d e p e n d e n t or p y r i d i n e n u c l e o t i d e - d e p e n d e n t ) , and t o "morphine N-demethylase" and t h e morphine r e c e p t o r , Quantum Mechanical Approaches t o Determining L o c a l i z e d E l e c t r o n D e n s i t i e s - U n t i l such t i m e as a macromolecular r e c e p t o r system can be d i r e c t l y c h a r a c t e r i z e d w i t h f a c i l i t y , i t w i l l remain n e c e s s a r y t o understand as much as p o s s i b l e a b o u t t h e u n d e r l y i n g n a t u r e of t h e small molecules t h a t i n t e r a c t w i t h it most s t r o n g l y and s p e c i f i c a l l y . One c h a l l e n g i n g a s p e c t of t h a t a n a l y s i s i n v o l v e s t h e d e t e r m i n a t i o n of l o c a l i z e d e l e c t r o n i c d e n s i t i e s t h a t may p l a y a s i g n i f i c a n t r o l e i n t h e r e c e p t o r i n t e r a c t i o n . The u s e of m o l e c u l a r o r b i t a l c a l c u l a t i o n s toward t h a t end has been urged r e p e a t e d l y by , p r o v o c a t i v e a n a l y s e s have been p r e t h e Pullmans37 , and S ~ e n t - G y % r g y i 3 ~and s e n t e d f o r i m p o r t a n t series of e l e c t r o n - d o n o r molecules of p h a r m a c e u t i c a l i n t e r e s t such as the p u r i n e b a s e s , p h e n o t h i a z i n e s , i n d o l e s and t h e l i k e . The a d v e n t of high-speed computers h a s g r e a t l y f a c i l i t a t e d t h e a n a l y s i s of complex o r g a n i c m o l e c u l e s , and t h i s w i l l d o u b t l e s s stimulate f u r t h e r i n t e r e s t i n a p p l i c a t i o n s t o d r u g - r e l a t e d problems.

Chap. 2 2

Receptor Theory

Bloom

239 -

I n a s s e s s i n g t h e p o t e n t i a l i t i e s of t h e approach, i t i s n e c e s s a r y t o r e c o g n i z e t h a t t h e u s e of molecular o r b i t a l c a l c u l a t i o n s h a s on o c c a s i o n spawned some u n l i k e l y c o n c l u s i o n s r e g a r d i n g t h e n a t u r e of d r u g - r e c e p t o r i n t e r a c t i o n s . Although i t i s probably c o r r e c t t o a t t r i b u t e some of t h e s e t o t h e i n h e r e n t c o q l e x i t y and l i m i t a t i o n s of quantum mechanical c a l c u l a t i o n methods , a more common f a i l i n g a p p e a r s t o be l a c k of r e c o g n i t i o n t h a t a m u l t i p l i c i t y of f a c t o r s of t e n u n d e r l i e t h e observed b i o l o g i c a l a c t i v i t y . Recent s t u d i e s by t h e Hansch39~40and Purcel141 groups d e m o n s t r a t e a means of a v o i d i n g t h i s p i t f a l l . T h e i r f i n d i n g s from molecular o r b i t a l c a l c u l a t i o n s are p l a c e d i n t o p e r s p e c t i v e by weighing them a l o n g s i d e o t h e r , o f t e n u n r e l a t e d molecular c h a r a c t e r i s t i c s such as p a r t i t i o n c o e f f i c i e n t s , as a b a s i s f o r drawing conclusions. Quantum b i o c h e m i s t r y h a s been d i s c u s s e d i n s e v e r a l b o o k s 3 7 ~ 3 8 , 4 2 ~ 4 3 , A r e c e n t a r t i c l e on one of t h e most s t i m u l a t i n g b e i n by Szent-Gydrgyi38. quantum chemistry i n d r u g designg4 o f f e r s a convenient i n t r o d u c t i o n t o t h e s u b j e c t . T e x t s on c a l c u l a t i o n methods are now a v a i l a b l e i n p r o f u s i o n . Int r i g u i n g a n a l y s e s based on molecular o r b i t a l c a l c u l a t i o n s have been made of c h a r g e - t r a n s f e r complexes of i n d o l e s 4 5 , c h o l i n e s t e r a s e i n h i b i t o r s 4 6 , and h a 1l u c i nogenic i ndo les and m e thoxy l a t ed phenethylamines47

-

The r e l a t e d s u b j e c t of c h a r g e - t r a n s f e r complex f o r m a t i o n h a s been reviewed by K o s ~ w e r ~who ~ , d i s c u s s e s b i o l o g i c a l systems. Analyses of such complexes49, e s p e c i a l l y t h o s e i n v o l v i n g f l a v i n n u c l e o t i d e s 5 0 ~ 5 1 ,have been described. Mathematical Approaches t o t h e C o r r e l a t i o n and A n a l y s i s of S t r u c t u r e A c t i v i t y Data - Attempts t o s y s t e m a t i c a l l y c o r r e l a t e t h e physicochemical prope r t i e s of molecules w i t h t h e i r b i o l o g i c a l a c t i v i t i e s have been recorded s i n c e t h e t u r n of t h e c e n t u r y . Needless t o s a y , many e a r l i e r e f f o r t s involved o n l y t h e s i m p l e s t forms of d a t a m a n i p u l a t i o n and a n a l y s i s . I n e v i t a b l y , what l i t t l e knowledge about drug r e c e p t o r s t h e y produced w a s hard won. From about 1951 on, i n t e r e s t has focused i n c r e a s i n g l y on t h e p o s s i b i l i t y of u t i l i z i n g l i n e a r f r e e energy r e l a t i o n ~ h i p s 5as ~ expressed i n t h e Hammett e q u a t i o n t o i n t e r r e l a t e t h e physicochemical and b i o l o g i c a l c o n t r i b u t i o n s made by a s t r u c t u r a l moiety t o t h e p a r e n t molecule. A s t h e r e s u l t of e f f o r t s by Hansch and h i s colleagues39,40,53-64, t h i s mathematical approach h a s g a i n e d s t e a d i l y i n s o p h i s t i c a t i o n , and w i t h t h e advent of "simple- language'' computer programs , i n e f f e c t i v e n e s s as w e l l . Of p a r t i c u l a r p e r t i n e n c e h e r e , r e c e n t l y p u b l i s h e d r e g r e s s i o n a n a l y s e s have demonstrated t h a t t h i s approach c a n o f f e r i n s i g h t i n t o t h e n a t u r e of d r u g - r e c e p t o r i n t e r a c t i o n s . C o n t r i b u t i o n s t o knowledge from t h i s s o u r c e have been of two k i n d s , t h o s e a r i s i n g by t h e d i s e n t a n g l e t h e a b i l i t y of t h e ment from i n t r i n s i c a c t i v i t y of d r u g dynamic f a c t o r s b i o l o g i c a l l y - a c t i v e molecule t o r e a c h i t s s i t e of a c t i o n e f f e c t i v e l y , and t h o s e provided by i d e n t i f i c a t i o n of t h e s p e c i f i c hydrophobic bonding, e l e c t r o n i c , and t o a lesser d e g r e e s t e r i c f a c t o r s t h a t i n f l u e n c e i n t r i n s i c activity.

--

I t makes p a r t i c u l a r l y i n t e r e s t i n g r e a d i n g t o trace t h e advent of t h e Hansch e q u a t i o n i n c h r o n o l o g i c a l f a s h i o n , a g a i n s t a backdrop of e a r l i e r attempts65-69 t o t r e a t b i o l o g i c a l d a t a m a t h e m a t i c a l l y . I n 1962 Hansch55 s u g g e s t e d t h a t l a c k of s u c c e s s e x p e r i e n c e d i n a p p l y i n g Hammett l i n e a r

240 -

Sect. V. - Topics in Biology

T a b a c h n i c k , Ed.

free energy relationships to rationalize the effect of aromatic substituents on plant auxin activity was the result of ignoring differences in the rate of penetration of various congeners to their site of action. He thereupon introduced the comparative free energy-related substituent constant, n, to reflect that parameter, leading to the modified Hammett equztion wbLich now bears his name in vernacular usage: log

1 C

-

-K,2

+ K', + P O + K"

where C is the molar concentration of drug producing a specified biological response, K, K' and K" are constants for a given series of congeneric drugs, and p and u retain their famciliar significance. From its humble beginnings in the plant kingdom, the use of this mathematical expression came into full flower in 1965, with six important publications40,59-63 emanating during that period from the computer of its originator. The impressive results obtained in these studies should serve to dispel any lingering doubts as to the breadth of applicability and incisiveness of this method. The n term is c m o n l y defined as the difference between the logarithm of the partition coefficient (usually 1-octanol/water) of the parent molecule and that of a derivative bearing the substituent in question. 9n occasion it has proven possible to use parachors or chromatographicallydetermined substituent constants in place of partition coefficients as the basis for the n term. Other electronic parameters have been successfully employed in place of the Hammett a constant when more pertinent. These include pKa values and electron densities obtained by molecular orbital calculations. Xnterestingly enough, relative to the discussion in the previous section, it was lack of success in attempting to develop a quantitative relationship between the biological activating ability of functional groups and their relative electronegativity as determined by molecular orbital calculations that first led Hansch to appreciate the need for a term reflectins the different penetration characteristics of various congeners. The Hansch approach is not without shortcomings. By and large it cannot cope too successfully with steric factors, or with metabolic inactivation processes, except in certain favorable instances. Difficulties can be encountered in predicting n from partition coefficients, particularly where substituents that can ionize at physiological pH are involved. Despite these limitations, it seems safe to predict that as computers become commonplace, along with programs for treating data of this type with facility, use of the Hansch equation (or possibly extensions of it) will increase appreciably The power of the method is well-exemplified by a recent analysis64 of structure-activity relationships among phenylcarbamates, anilides and phenylureas inhibiting photosynthesis, which concludes that the primary effect on biological activity of aromatic ring substituents in such structures arises from the hydrophobic bonding power of the substituents. This conclusion was in disagreement with a previous paper70 that stated, on the basis of molecular orbital calculations, that a high electron density in the ortho position of the aromatic ring was the factor responsible for

Chap. 22

Receptor Theory

Bloom

24 1 -

b i o l o g i c a l a c t i v i t y . I n t e r e s t i n g l y enough, t h e l a t t e r p a p e r h a s s u b s e q u e n t l y been withdrawn'l, due t o a fundamental error i n t h e molecular o r b i t a l calcul a t i o n s employed, which i n v a l i d a t e d i t s c o n c l u s i o n s . The two p a p e r s taken t o g e t h e r c o n s t i t u t e a n i n t e r e s t i n g case s t u d y of c o n t r a s t i n g methods of analyzing structure-activity relationships. A d i f f e r e n t approach t o t h e mathematical t r e a t m e n t of s t r u c t u r e a c t i v i t y d a t a , based on t h e assumption t h a t t h e r e i s some a d d i t i v i t y of s u b s t i t u e n t c o n t r i b u t i o n s i n analogous s t r u c t u r e s has been d e s c r i b e d by F r e e and Wilson/Z. The method h a s been a p p l i e d r e c e n t l y t o a n a n a l y s i s of t h e c h o l i n e s t e r a s e i n h i b i t o r y p r o p e r t i e s of a l a r g e series of N-alkyl subI n i t s p r e s e n t s t a t e of s t i t u t e d 3-carbamoylpiperidines by Purce1173. development, i t d o e s n o t seem t o o f f e r t h e same p o t e n t i a l i t i e s f o r g a t h e r i n g i n s i g h t i n t o t h e m o l e c u l a r n a t u r e of d r u g - r e c e p t o r i n t e r a c t i o n s as does t h e Hans ch e q u a t i o n .

S t u d i e s on Autonomic and C e n t r a l Nervous System Receptors. A n a l g e s i c s - ??0rtoghese7~-76h a s r e i n t e r p r e t e d e x i s t i n g s t r u c t u r e a c t i v i t y d a t a i n terms of d i f f e r i n g modes of a n a l g e s i c - r e c e p t o r i n t e r a c t i o n s . H e h a s suggested t h a t t h e mode of b i n d i n g of two d i f f e r e n t series of compounds can be recognized as similar o r d i f f e r e n t , by d e t e r m i n i n g whether comparable a c t i v i t y changes o c c u r when s u b s t i t u e n t s on t h e b a s i c n i t r o g e n atom are v a r i e d i n i d e n t i c a l f a s h i o n . A 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 h i p c a n be demonstrated t o e x i s t when b i n d i n g modes are similar. T h i s method, which o b v i o u s l y h a s broad i u p l i c a t i o n s , h a s r e c e n t l y been a p p l i e d t o a n a n a l y s i s of t h e mode of b i n d i n g of c h o l i n e s t e r a s e i n h i b i t o r s 4 1 . E a r l i e r views on a n a l g e s i c r e c e p t o r s are summarized i n s e v e r a l r e c e n t reviews77-80.

-

General A n e s t h e t i c s g l During 1965, t h e compelling h y d r a t e microc r y s t a l t h e o r y of a n e s t h e s i a p u t f o r t h by PaulingBZ, and i t s " i c e b e r g e f f e c t " c o u n t e r p a r t o f f e r e d c o n c u r r e n t l y i n 1961 by Miller83, have been challenged i n a p a p e r by Paton and h i s c o l l e a g u e s 8 4 , which asserts t h a t t h e c r i t i c a l phase € o r g e n e r a l a n e s t h e t i c a c t i o n is a non-aqueous one. A t a conference on Forms of Water i n B i o l o g i c a l Systems, however, t h e h y d r a t e m i c r o c r y s t a l t h e o r y was f a v o r e d and new evidence c i t e d i n s u p p o r t of i t 8 5 .

-

Local A n e s t h e t i c s Recent s t ~ d i e s 8 6 - 9s u~g g e s t t h a t l o c a l a n e s t h e t i c s compete w i t h calcium i o n f o r b i n d i n g s i t e s on s p e c i f i c phospholipid molecules which are p r o b a b l y a s s o c i a t e d w i t h t h e plasma membrane c a t i o n t r a n s p o r t system of n e r v e cells. The n a t u r e of t h e p h o s p h o l i p i d b i n d i n g s i t e involved h a s been s u g g e s t e d t o be a p h o s p h o d i e s t e r on t h e b a s i s of Ln v i t r o i n t e r a c t i o n s t u d i e s w i t h model compoundsgo. S t r u c t u r e - a c t i v i t y c o r r e l s t i o n s among local a n e s t h e t i c f a m i l i e s have been made on t h e b a s i s of d e t e r m i n a t i o n of c a r b o n y l bond o r d e r by i n f r a r e d s p e c t r o s c o p y 9 1 , r e g r e s s i o n a n a l y s i s of s t r u c t u r e - a c t i v i t y d a t a ( t h e Hansch approach)39, and molecular o r b i t s 1 c a l c u l a t i o n s 9 2 . I n t h e l a t t e r case, t h e a n a l y s i s was i n t e r p r e t e d t o f a v o r c h a r g e - t r a n s f e r complexing w i t h t h i a m i n e as t h e b a s i s f o r l o c a l a n e s t h e t i c acti'on. Compelling c r i t i c i s m of t h i s v i e w p o i n t i s o f f e r e d i n a n e x c e l l e n t review93 of t h e e n t i r e s u b j e c t of t h e mode of a c t i o n of l o c a l anesthetics.

242

Sect. V

-

T o p i c s in Biology

T a b a c h n i c k , Ed.

Phenothiazine C e n t r a l Depressants - Chlorpromazine aptl i t s analogs have been favored s u b j e c t s f o r mechanism s p e c u l a t i o n s s i n c e t h e i r advent as t r a n q u i l i z e r s . A number of u s e f u l s t u d i e s have been published which a t t e m p t t o d e f i n e t h e i r mode of a c t i o n a t t h e l e v e l of c e l l and o r g a n e l l e membranes and i n d i v i d u a l enzyme systems. Molecular o r b i t a l c a l c u l a t i o n s 9 4 , 9 5 , c r y s t a l p h o t o e l e c t r i c t h r e s h o l d s t u d i e s 9 6 , o p t i c a l a b s o r p t i o n and NMR s p e c t r a measurem e n t s 9 7 ~ 9 8have a l l confirmed t h a t t h e p h e n o t h i a z i n e s have s i g n i f i c a n t e l e c t r o n - d o n o r p r o p e r t i e s , which Szent-GyBrgi38 h a s suggested t o be t h e fundamental b a s i s f o r t h e i r b i o l o g i c a l a c t i v i t y . Another important c l u e i s t h e observation99 based on s u r f a c e t e n s i o n measurements, t h a t chlorpromazine complexes w i t h CoupPBd with t h e f i n d i n g t h a t t h e c e n t r a l nervous system d e p r e s s a n t potency of analogous phenothiazine s t r u c t u r e s c o r r e l a t e s w i t h t h e i r a b i l i t y t o i n h i b i t r a t b r a i n N a + , K + - a c t i v a t e d , Mg++-requiring ATPaselOl, t h i s raises t h e p o s s i b i l i t y t h a t enzyme-bound ATP may c o n s t i t u t e a r e c e p t o r s i t e f o r such d r u g s . Strong evidence t o suggest- complexing t o o t h e r n u c l e o t i d e s i n v i v o a l s o e x i s t s 1 0 2 , 1 0 3 . An i n t e r a c t i o n between f l a v i n a d e n i n e n u c l e o t i d e - r e q u i r i n g enzymes and h e n o t h i a z i n e d e r i v a t i v e s , has been noted i n s e v e r a l s t u d i e s . A r e c e n t o n e l o e s t a b l i s h e s a c o r r e l a t i o n between e f f e c t i v e n e s s i n i n h i b i t i n g such an enzyme (D-amino a c i d o x i d a s e ) and c e n t r a l d e p r e s s a n t potency. The g e n e r a l h y p o t h e s i s l 0 5 ~106 t h a t t h e t r a n q u i l i z i n g a c t i o n s of p h e n o t h i a z i n e d e r i v a t i v e s r e l a t e t o i n h i b i t i o n of both energyproducing r e a c t i o n s ( s u c h as o x i d a t i v e phosphorylation a t a l e v e l where FAD i s involved) and e n e r g y - u t i l i z i n g r e a c t i o n s ( s u c h as ATP h y d r o l y s i s supporti n g membrane t r a n s p o r t p r o c e s s e s ) seems worthy of r e t e n t i o n .

e

Autonomic Receptors - A number of papers from a symposium on t h e I n t e r a c t i o n of Drugs w i t h Receptors h e l d i n A p r i l , 1965, a t t h e School of Pharmacy, Chelsea C o l l e g e of Science and Technology, London, have been publ i s h e d i n Advances i n Drug Researchl07. Of t h e s e , one t h a t c l e a r l y r e v e a l s t h e impact of modern m e c h a n i s t i c biochemical t h i n k i n g i s t h e g e n e r a l i z e d conformational p e r t u r b a t i o n t h e o r y of drug a c t i o n o f f e r e d by Belleau108,109. T h i s view, which h a s been i n f l u e n c e d p r i m a r i l y by knowledge about c h o l i n e r g i c r e c e p t o r phenomena, s u g g e s t s t h a t d r u g molecules r e g u l a t e t h e c a t a l y t i c a c t i v i t y of t h e i r p a r t i c u l a r t a r g e t enzyme system by inducing a s p e c i f i c o r d e r i n g (agonism) o r n o n - s p e c i f i c d i s o r d e r i n g (antagonism) conformational change i n i t s p r o t e i n - b i n d i n g s u r f a c e . Because it goes beyond the earlier occupancy and rate t h e o r i e s of drug a c t i o n i n o f f e r i n g a p l a u s i b l e physicochemical b a s i s f o r r e c e p t o r phenomena a t t h e molecular l e v e l , t h i s theory i s d e s e r v i n g of t h e most t h o u g h t f u l a t t e n t i o n . The accompanying c r i t i q u e of o t h e r s o - c a l l e d t h e o r i e s of drug a c t i o n emphasizes t h e need t o r e c o g n i z e t h e elementary molecular p r o c e s s e s i n v o l v e d , and i n t h i s r e g a r d o f f e r s u s e f u l p e r s p e c t i v e on t h e overall problem. S e v e r a l o t h e r p a p e r s l l o - 1 1 6 d e a l i n g w i t h c h o l i n e r g i c a g e n t s are worthy of n o t e , p a r t i c u l a r l y t h o s e regarding muscarinic receptors.

A t t h e Chelsea meeting, Bloom and Goldman o f f e r e d a d e t a i l e d molecular v i s u a l i z a t i o n of t h e n a t u r e of catecholamine-adenine mononucleot i d e i n t e r a c t i o n s i n a d r e n e r g i c mechanisms117. I n t h i s s o - c a l l e d dynamic r e c e p t o r h y p o t h e s i s , t h e catecholamines are p o s t u l a t e d t o f u n c t i o n by enhancing t h e rate a t which s p e c i f i c p h o s p h o r o l y t i c enzymes c o n v e r t ATP t o ADP ( a - r e s p o n s e ) o r c y c l i c 3' ,5'-AMP ( p - r e s p o n s e ) . The a d r e n e r g i c r e c e p t o r s

Chap. 22

Receptor Theory

Bloom

243 -

are v i s u a l i z e d as enzyme-substrate complexes ( i n c o n t r a s t t o t h e c l a s s i c a l

concept: r e c e p t o r = enzyme), w i t h t h e catecholamine hormones f u n c t i o n i n g i n t h e manner of a c t i v a t o r s u b s t a n c e s . The catecholamines are s e e n t o i n t e r act d i r e c t l y w i t h t h e bound n u c l e o t i d e s u b s t r a t e , t h e r e b y enhancing i t s r e a c t i v i t y r a t h e r t h a n t h a t of t h e enzyme t o which i t i s bound. T h i s d i f f e r s i n concept from t h e above-mentioned conformational p e r t u r b a t i o n t h e o r y of c h o l i n e r g i c agonism, a l t h o u g h t h e two views are n o t incompatible. E k l l e a u h a s r e c e n t l y d i s c u s s e d t h e dynamic r e c e p t o r h y p o t h e s i s and o f f e r e d c e r t a i n a l t e r n a t i v e views, e s p e c i a l l y r e g a r d i n g t h e role of t h e c a t e c h o l moiety a t $ - r e c e p t o r s , i n a paper118 t h a t i s one of more t h a n s e v e n t y - f i v e from t h e Second Catecholamine Symposium published as fin 800-page volume of Pharmac o l o g i c a l Reviews. P r a t e s i and G r a n a l l g have summarized t h e i r e x t e n s i v e studies of s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s among a d r e n e r g i c s u b s t a n c e s . T r i g g l e and h i s colleagues120- 123 have c o n t r i b u t e d new a d r e n e r g i c r e c e p t o r s t u d i e s i n v o l v i n g i r r e v e r s i b l e i n h i b i t o r s of t h e f5-haloalkylamine t y p e , as h a s Chap1nanl2~. I v e r s o n l 2 5 h a s made t h e i n t e r p r e t a t i o n of pharmacological experiments i n v o l v i n g a d r e n e r g i c r e c e p t o r s a b i t easier w i t h h i s s y s t e m a t i c s t u d i e s of s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s among s u b s t a n c e s i n h i b i t i n g catecholamine uptake mechanisms. Noteworthy attempts have been made t o c h a r a c t e r i z e a d r e n e r g i c a - r e c e p t o r s through t h e use of r a d i o l a b e l led blocking a g e n t s of t h e a l k y l a t i n g p-haloalkylamine type1269 127. Woolley and h i s c o l l e a g u e s have suggested t h a t a s p e c i f i c s u b s t a n c e w i t h t h e p r o p e r t i e s of a g a n g l i o s i d e i s t h e s e r o t o n i n r e c e p t o r i n smooth muscle and brain128- 132. T h e i r compelling arguments are based p r i m a r i l y on evidence t h a t s e r o t o n i n - s u s c e p t i b l e t i s s u e may be rendered s e l e c t i v e l y i n s e n s i t i v e by t r e a t m n t w i t h neuraminidase and EDTA, and t h a t t h i s p r o c e s s may be r e v e r s e d by t r e a t m e n t of t h e d e s e n s i t i z e d tissue w i t h a p u r i f i e d sample of a p p r o p r i a t e g s n g l i o s i d e s . I t would seem i n o r d e r t o s u g g e s t t h a t a t t h i s s t a g e of knowledge t h e g a n g l i o s i d e i n q u e s t i o n might b e t t e r b e c a l l e d a v i t a l component of t h e s e r o t o n i n r e c e p t o r system, r a t h e r t h a n the s e r o t o n i n receptor. S e v e r a l r e c e n t l y published a r t i c l e s 1 3 3 d e a l w i t h d r u g - r e c e p t o r i n t e r a c t i o n s more g e n e r a l l y . G i l l 1 3 4 emphasizes an a n a l y s i s of t h e p h y s i c a l f o r c e s which determine t h e size and shape of d r u g and r e c e p t o r molecules. Burger and P a r u l k a r l 3 5 p o i n t o u t v a r i o u s i m p l i c a t i o n s of b i o r e c e p t o r t h e o r y t o drug d e s i g n . The review by Gourleylf6 on b a s i c mechanisms of drug a c t i o n i n c l u d e s u s e f u l s e c t i o n s on c e l l s u r f a c e s and membranes. Watkinsl37 has o f f e r e d a n e x p l a n a t i o n of t h e a c t i o n of a c e t y l c h o l i n e , y-aminobutyric a c i d and g l u t a m i c a c i d on c e l l membrane p e r m e a b i l i t y i n terms of t h e s t r u c t u r a l and charge d i s t r i b u t i o n similarities between t h e s e a g e n t s and t h e "polar head groups" of l e c i t h i n , phosphatidylethanolamine, and p h o s p h a t i d y l s e r i n e . Burgen138 and Mackayl39 have each published g e n e r a l i z e d mathematical t r e a t m e n t s of t h e d r u g - r e c e p t o r i n t e r a c t i o n problem. C o l l i e r 1 4 0 has d e a l t w i t h r e c e p t o r i n d u c t i o n i n a g e n e r a l t h e o r y on t h e g e n e s i s of drug dependence.

244 -

Sect.

V

-

Topics in Biology

T a b a . c h n i c k , Ed.

References

,-P 2, 95 (1965). 1. A. Ooldsteln. The 2. 8. 1. Bloor and Q . D. Uubach. AM. Rev. Phorrscol., 2, 67 (1962). 3. Xdltorlal (anon.). Lancet, 895, (April 24, 1965). 4. P. Dunnill, S o l . Progc. (London). @. 609 (1965); T.W. Ooodwin. J.I. Harris, and B.S. Eartley, Lds., "Structure and Actlvlty of Enzymes." Acadrrric Presa, London, 1965. 5. H. Sund and I(. Weber, *rrsrew. Chem. Intern. Xd. ILnnl.. 9. 231 (1966). 6. X. A . Yewshol~e. Scl. Pro=. (London), E,237 (1965). 7. D. X. Atkinson. Science, 2, 851 (1965). 8. D. X. Koshland, Jr., J. Cellular 0-D. PhPs1ol., 245 (1959). 9. D. X. Koshland, Jr.. Fed. Proc., 23, 719 (1964). 10. J. Xowd, J.-P. Chongeu~. pnd F . Jacob, J. Wol. Blol.. 2, 306 (1963). 11. J. Xonod, J. Wyman, and J.-P. Change-, J. Wol. Blol.. 2. 88 (1965). 12. J . Wonod, EmloorlnologZ, 412 (1966). 19. a. F. Perutz, Scianae, H.863 (1963). 14. J. C. Kendrew, Sclonce. 139. 1259 (1963). 15. 8. Nulrhead and 1. F. Perutz, Xature, 189. 633 (1963). 16. 1. F. Perutr, W. Bolton, R. msrond, H. aulrhead, and H. C. Watson, nature. 303,687 (1964). 17. a. P . Peruts. J . woi. B I O ~ . , ~ .646 (196s). 18. a. F . Peruts. J. a. Kendrew, and H. C. Watson, J . 101. ~ i o l . ,l3, 669 (1965). 19. 1. F. Peruts, Chemlstru I n Britain. 2, 9 (1965). 20 H. Hulrhead, 901. Progr. (London), 53, i7 (1965). 21. C.C.P. Blake, D.F. Koenlg, O.A. Ualr, A.C.T. Yorth, D.C. Phillips, and V.B. S a m , Nature, 208, 757 (196s). 22. L. R. Johnson and D. C. P h i l l i p s , nature, 206. 761 (1965). 23. L. Stryer, J. C. Keadrew, and €I C. . Watson. 3. Xol. Blol., 3, 96 (1964). 24. L. J. Banaszak, H. C. Watson. and J. C. Kendrew, J. Xol. Blol.. 2,130 (1965). 2s. P. B. S l g l e r , B.A. Jeffery, B.W. Uatthewa. and D.M. Blow, J. 101. Blol..lS,l75 (1968). 26. Y. Hoppe, m e w . Chem. Intern. Xd. Zasl 2. 508 (1965); 5. 287 (1966). 27. 1. l4. Kosower, Wolecular Biochemlstry." W c O r a w - H i l l . Uew York, 1962, p. 286. 28. L. Vellue and 1. Legrand. Amew. Chem. Intern. Ld. -1.. , 838 (1965); L. Velluz. M. Legrand, and 1. OrosJean, "Optical Circular mchrol8m." Academic Press. Yew York, 1965. 29. H. Welner and D. L. Xoshland, Jr., J . 101. Blol., l2, 881 (1965). 30. A. Kowolsky and 1. Cohn. Ann. Rev. Blochem., 33, 481 (1964). 31. 0 . Jardetsky I n "Struoture and Properties of Blololecules and Biological Systems," J . Duchesne, ad., Interscience. London, 1964, Chap. 13. 32. 0 . Jardotsky and C. D. JPfdetsky, Methods Blocha. *nnlx., 2, 235 (1962). SCi. U. 5 . . 808 (1965). 33. H. 2. Sable and J. L. BlpgloW, P ~ c .Yatl. A-d. 34. A. S. Mldvan and 1. Cohn, , 2, 910 (1963). 35. A. S. Mildvan and 1. Oohn. J. Biol. Chem.. 249, 238 (1965); 241, 1178 (1966). 36. B. Belleau i n "Isotopes I n I x p e r l m n t a l Pharmwology." L. 5 . Roth, Xd. The University Of Chicago Press. Chlmgo. 1965, Chap. 36. ~~ New York. 1963. 37. B. Pullpran and A . Pullman, "Quantum B l ~ c h a r i s t r y ,Zntarscience, 38. A. Szent-Oy%rgyi, "Introduction t o a SubPolecular Biology," Academic Press,Uew York, 1960. 86. 1616 (1964). 39. C. Iiansch and T. Bujita. 40. C. Hansch. P. W. Deutsch. and R. W. Smith, J. Am. Chem. SOC., g.2738 (1965). 41. W. P . Purcell. J. Med. Chem., 2, 297 (1966). 42. P.-0. I%wdin and B. Pullman, E d s . , "Molecular Orbitals I n Chemistry, Physics and Bioioty," Academic Press, hew York, 1964. 43. B.Pullman and M.Weissbluth. Eds., "Molecular Biophysics," Academic Press, New York. 1965.

s,

s,

-

.,

s.

-.,

Chap. 2 2 44.

45. 46. 47 * 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 15. 76. 71. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90.

91.

Receptor Theory

245 -

Bloom

R . 8 . Schnaare and A . 13. Hartin, J. Pharm. S c i . , 54, 1707 (1965). J . p. Oreen and J. P . Mabrieu, Proc. Natl. Acad. Sol. U.S., 64, 659 (1965).

W. B. Neely, Hol. P h e m O O l . , 2, 137 (198S). 8 . H. Snyder and C. R. Merril, Proc. Natl. Acad. Sci. U.S., 54, 258 (1965).

X . M, Kosower, . -orP , 2, 81 (1965). L. D. Wright and 0 . 8. ItcCormick, ~ e r i e n t l a 20, , 601 (1964). D.P. Fleischman and 0 . Tollin, Proo. Natl. Acad. Sci. U.S., 53, 38, 237 (1965).

T. Sakurai and H. Hosoya, Biochlm. Bio~hyS.Acta, 112, 469 (1966). P. R. Wells, Chm. Rev., 171 (1963). R. IN. Muir and C. Hansch, Ann. Rev. P l a n t Physiol., 2, 157 (1966). 1. 11. Muir and C. Ransoh i n "Fourth I n t l l . Conf. on Plant Qrowth Regulation," Iowa S t a t e U. Press, Ames, Iowa, 1961, p. 249. C . Hansch, P. P. Naloney, and T. F u j i t a , Nature, 194. 178 (1962). C. Hansch, 1. M. h l r , T. P u j l t a , P.P. Naloney, F. Oelger. and M. Streich, J . Chem. SOC., 65, 2817 (1963). T. F u j i t a . J . Iwasa, and C. Hansch, J . Am. Chen. Soc., 3, 5175 (1964). C . Hansch and A. R. Steward, J. Mod. Chem., 1, 691 (1964). J. Iwasa, T. F u j i t a , and C. Hansch, J. Ned. Chem.. 2, 150 (1965). C . Hansch and Z. W. Dautsch. J. Ned. Chem., 8. 705 (1965). C. Hansch, A. R. Steward, and J. Iwasa, J. ned. Chem.. 8, 868 (1965). C. Hansch. A . R. Steward, and J . Iwasa, 1. 87 (1965). C. Hansch, A. R. Steward, J . Iwasa. and P. W. Deutsch, Mol. P h a m c o l . . 2, 205 (1965). C. Hansch and E. W. Deutsch. Bloohim. Biophys. Acta. 112, 381 (1966). R. Zahradn(ik and 1. Chvapil, Xxperientia, l6, 511 (1960). It. Zahradncik, Arch. Intern. Pharmacodrn, 135, 311 (1962). R. Zahraddk, Xxverientla, l8, 534 (1962). K. BoYoek, J. Kopeck:, n. IWVUCOVS, and D. viachov:, UDerientia, 20, 667 (1964). J . KopecG, K. BoYek. and D. Vlachov5, Ilature, 207, 981 (1965). M . Kaarin, W. F. Bertsch, and R. F. Wood, Proc. l a t l . Acad. Sol. U.S.,53, 1118 (1965). H. Kamrin, W. B. Bertsch, and 1. F. Wood, Proc. Natl. Acad. Scl. U.S. , 54, 1285 (1965). 9 . M. Free, Jr. and J. W. Wilson, J. Ned. Chen., 1, 395 (1964). W. P. Purcell, Biochlm. Biophys. Acta, 105,201 (1965). P. S. Portoghese, J. Phnm. S c i . , 54, 1017 (1965). P. S. Portoghese and T. 1. Riley, J. Pharm. S c i . , 54. 1831 (1965). P. 5 . Portoghese. J. ned. Chon., S , 609 (1965). L. B. N e l l e t t and L. A. Woods i n "Drug Research," Vol. 5 , P. Jucker, Xd., B i r k h h s e r Verlag, Basel. 1963, p. 253. A. H. Beckett and A. F. Casy, Progr. Med. Chem., f , 171 (1965). 9. Archer and L. S. Harris i n "Drug Research." Vol. 8. S. Jucker. Pd., B i r w u s e r Verlag, Basel, 1965, p. 261. 0 . deStevens, Xd.. llAnalgetics," Academlc Press. Stew York, 1965. R. M. Featherstone and C. A. Huehlbaecher.Pharmaao1. Rev., Is,91 (1963). L. Pauling. Soience, E, 15 (1961). 5 . L. Miller, Proc. E a t l . Acad. S c l . U . S . . 47, 1515 (1961). K . W. Miller, W.D.M. Paton, and P. B. Smith, Ilaturc, 206. 574 (1965). J . F. Catchpool, Ann. 1.11. Acad. Sol., 125. 595 (1965). M. B. Feinstcin, J. Oen. Physlol., g,357 (1964). D. X . Ooldman. Biophys. J., 3, 167 (1964). 1. P. Blaustcin and D. X . Ooldman. Fed. Proc., 24, 584 (1965). 1. P. Blaustcin and D. S. Ooldman, Fed. Proc.. 25, 511 (1966). 1. B. Felnstcin and M. Palmre, Biochi.. Biophys. Acta. 115, 33 (1966). A. M. Oalinsky, J. X. flearien, A. J. Perkins, and S. V. Susina. J. Wed. Chem.. 5. 320 (1963).

a,

~

.

,

246 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107.

108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 121. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140.

Sect. V

-

Topics in Biology

T a b a c h n i c k , Ed.

P. Yoneda and Y. Yltta, Chem. Phara. Bull. (Tokyol, 2, 574 (1965). J. M. Rltchle and P. Qreengard, AM. Rev. Pharmacol., 2. 405 (1966). 0. Karrulan, I. Isenberg. and A. Szent-Qi6rgy1, Science. $0, 1191 (1959). M. K. Orloff and D. D. Fltts, Blochim. B I o D ~ ~ sActa, . 47, 596 (1961). L. I. Lyons and J. C. Hackle, Mature. 197, 589 (1963). R. Foster and P. Hanson, Biochlm. Biophys. Acta, 112,482 (1966). R. ?oater and C. A. Fyfe, Biochlm. Blophys. Acta, 112, 490 (1966). I. Blei. Arch. Biochar. Blophys.. 321 (1965). 3. 647 (1963). P. Hele. Blochim. B ~ O D ~ Y SActa. . P. W. Davis and T. 1. Brody. The Pharmacologist, 2 , 141 (1965). D. Richter In "The Scientific Basis of Drug Therapy In P~ychiatry,~' J. Harks and C. 1. Pare, Lds., Pergaron Press. New York, 1965, p, 63. 9 . 5abay and S. R. Harris, Blochem. Pharmacol., l4, 17 (1965). S. Qabay and 9. R. Harris. Biochem. Pharmcol., l5, 317 (1966). L. Decsi, PagchophaMcologla, 2, 224 (1961). L. Decsl In "Drug Research." Vol. 8. I. Jucker.Pd. ,Blrkh.&user Verlag. Base1.1965,p.139. II. J. Harper and A.B. Si.nonds,Ms., "Advances In Drug Researoh." Academic Press, London, Vol. 2 (19651, Vol. 3 (1966). B. Belleau. Advan. Drug Res., 2, 89 (1965). B. Belleau, J. led. Chem., 1,776 (1964). A. Bebblngton and 1. U. Brirbleconbe. Advan. Drug Res.. 2 , 143 (1965). A.S.V. Burgen, Brit. J. Phamcol.. 2.4 (1965). W.D.M. Paton. Proc. Intern. PhalPPcol. Heetlng, 2nd.Prame. 1963, 2. I1 (1964). W.D.M. Paton and H . P . Rang, Proc. Roy. SOC. (London).Ser.B. 163,1 (1965). H. P. Rang, Proc. ROY. SOC. (London), Ser. B, 488 (1066). B.W.J. Pllenbroek. R.J.P. Nivard, J.N. van Rossum, and L.J. Ariens, J. Pharm. Pharmacol.. l7, 393 (1965). D. J. hlggle, "Cheaical Aspects of the Autonomic Yervous System," Academlc Press London, 1965, Chap. X11, p. 159. B. H. Bloom and I. 1. Goldman. Advan. Drug Res., 2, 121 (1966). B. Belleau, Phareacol. Rev., l8, 131 (1966). P. Prates1 and P. Qrana. Advan. Myg Res.. 2, 127 (1965). D. J. Trlggle, J. Theoret. Biol., 1, 241 (1964). H. Klmelberg and D. J. Trlggle, J . Theoret. Blol., 3, 323 (1965). D. J . Trlggle, Advan. Drun Res., 2, 173 (1965). D. J. Trlggle, l'Chemlcal Aspects of the Autonomic Nervous System," Academic Press, London, 1965, Chap. XXI, p. 296. W . B. Chapman, K. Clarke, and R.D. Strickland. Proc.Rou.Soc.(London) =,116 (1965). L. L. Iversen, Advan. Drug Res., 2. 1 (1965). S. Dlksteln and F. 0. Sulman, Biochem. Pharmacol., 2, 139, 881 (1965). J. P. Lewis and J. W. Miller, Fed. Proc., 24. 388 (1965). D. W. Woolley and B. W. Gonrmi, Nature, 202, 1014 (1964). D. W. Woolley and B. U. O m , Proc. Matl. Acad. Scl. US., @, 14 (1964). Proc. Natl. Acad. S c l . U.S.. 53, 959 (1965). D. W. Woolley and B. W. -1, D. W. Woolley and B. W. 0-1, Fed. Proc., 2.194 (1965). 5. Wild, D. W. Woolley. and B. W. Qomml, led. Proc.. 2,352 (1966). W. Haefely, Schwelz. Ned. Wochschr., 95, 460 (1965). I. U. 0111, Progr. Ned. Chen., 4, 39 (1965). A. Burger and A. P. Parulkar, Ann. Rev. Pharmacol., 5 , 19 (1966). D.R.H. Oourley In "Drug Resear-ker, Xd., Blrauser, 1964, p.11. 3. C. Watklns. J. Theoret. Biol., 3. 37 (1965). A. S. V. Burgen, J. Pharn. Phamacol.. 2,137 (1966). D. Hackay. Brit. J. Pharmacol., 26, 9 (lQ66); J. Pham. Pharmacol.. l8, 201 (1966). H. 0. J. Collier. Nature, 205, 181 (1965).

m.

*,

Chapter 23. D.A.

F a t e and D i s t r i b u t i o n of Drugs

Buyske and D. Dvornik, Ayerst L a b o r a t o r i e s , Montreal, Canada.

I n s p e c t i o n of l i t e r a t u r e c i t a t i o n s indexed i n 1964-65 i n t h e Chemical A b s t r a c t s and Derwent Pooled Pharmaceutical L i t e r a t u r e Documentation (Ringdoc) r e v e a l e d over 800 r e f e r e n c e s d e a l i n g with some a s p e c t of t h e metabolism of drugs o r chemicals, e x c l u s i v e of normal biochemical c o n s t i t u e n t s of t h e mammalian organism. The parsimonious page r e s t r i c t i o n s imposed by t h e e d i t o r s and t h e l i m i t e d ambition of t h e a u t h o r s d i d not a l l o w a complete coverage of t h i s g r e a t number of p u b l i c a t i o n s . We have, t h e r e f o r e , confined our examinat i o n t o p e r t i n e n t p a p e r s appearing i n t h e following 20 j o u r n a l s : Arch.Int. Pharmacodyn. ; Arch.Biochem.Biophys. ; Bi0chem.J. ; Biochem.Pharmaco1. ; Curr. Therap.Research; Endocrinology; Fed.Proc.; J.Biol.Chem; J . C l i n . I n v e s t . ; J.Med. Chem.; J.Pharmacol.Expt1.Therap.; J.Pharm.Pharmaco1.; J.Pharm.Sci.; Life Sci.; Metabolism; Nature; Proc.Soc.Exp.Bio1.Med.; S c i e n c e ; The Pharmacologist; and; Toxicol.Appl.Pharmaco1. I n a d d i t i o n , Ringdoc a b s t r a c t s ( s i n c e 1964) were examined and s e l e c t e d r e f e r e n c e s a r e a l s o included. It i s e s t i m a t e d t h a t t h e sel e c t e d j o u r n a l s t o g e t h e r w i t h t h e a d d i t i o n a l r e f e r e n c e s c i t e d i n t h e t e x t cont a i n over 90% of t h e p u b l i s h e d work on t h e b i o t r a n s f o r m a t i o n s of drugs. To t h o s e few a u t h o r s whose p a p e r s we may have missed, we o f f e r our unprejudiced apologies. E x c e l l e n t reviews on drug metabolism have appeared s i n c e 1961 i n every VOlume of t h e Annual Reviews of Pharmacology, except 1966. T h i s major source of i n f o r m a t i o n h a s been supplemented by t h e r e c e n t review by Shuster' and, by a s t i m u l a t i n g g e n e r a l d i s c u s s i o n on t h e f a t e of drugs and t h e r a p e u t i c implicat i o n s i n a r e c e n t c h a p t e r by Brodie2. The a p p r e c i a t i o n of how t h e understandi n g of t h e metabolic f a t e of a drug can be o f importance i n i t s proper usage and i n t h e i n t e r p r e t a t i o n of i t s t o x i c i t y h a s been w e l l s t a t e d by Burns3 and c o l l e a g u e s 4 ; an o u t s t a n d i n g example i s a l s o found i n t h e work by t h e M i l l e r s 5 on a s e l e c t group o f chemicals and t h e i r c a r c i n o g e n i c i t y . The proceedings from s p e c i a l pharmacologically o r i e n t e d symposia h e l d i n Prague6 i n 1963 and i n New York7 and Chicago8 i n 1964 have been p r i n t e d and c o n t a i n a wealth of i n f o r m a t i o n p e r t i n e n t t o drug metabolism. The s p e c i a l i z e d f i e l d of .the metabol i s m of n a t u r a l and s y n t h e t i c s t e r o i d s i s covered i n t h e r e v i s e d encyclopaedic compilation of Dorfman and Ungar9 and needs no f u r t h e r r e f e r e n c e . With t h i s c o n c e n t r a t i o n of knowledge s o r e a d i l y a v a i l a b l e , i t appeared t h a t t h e r e a d e r s i n t e r e s t would b e s t be served by b r i n g i n g t h e a v a i l a b l e informat i o n up t o date. Considering t h e chemical background of t h e l i k e l y r e a d e r of t h e Annual Reports i n Medicinal Chemistry, we have o r i e n t a t e d t h e review t o t h e a c t u a l b i o t r a n s f o r m a t i o n s t h a t have been r e p o r t e d i n 1964-65. Thus, w e have not reviewed t h e voluminous and i l l u m i n a t i n g r e p o r t s on t h e n a t u r e of t h e enzymes t h a t metabolize drugslo; t h e mechanism by which enzymes c a t a l ze t h e s e t r a n s f o r m a t i o n s l l ; on t h e dependence of enzymic a c t i v i t y on s p e c i e s g 2 , l 3 , strain14-16,age17,18, ~ e x l g - ~ lon , t h e i n d u c t i o n of i n c r e a s e d q u a n t i t i e s of t h e s e enzyme systems by chemicals and d r u g 2 2 , and on t h e r e l a t i o n s h i p of "drug metabolizing enzymes" and t h e metabolism of s t e r o i d a l h o r m o n e ~ ~ 3 - ~ 5 We have s e l e c t e d t h e above 1964-65 r e f e r e n c e s t o enable t h e r e a d e r t o make convenient acquaintance with t h e s e s p e c i a l i z e d a r e a s . The review is w r i t t e n f o r a r e a d e r a c q u a i n t e d with approaches used by t h e medicinal chemist. Data on compounds, whose metabolism i s reviewed, a r e

.

2 48

Sect. V

-

T o p i c s i n Biology

T a b a c h n i c k , Ed.

arranged i n t a b u l a r form and inc1ude:main pharmacodynamic property;chemical name, o r , i f a v a i l a b l e , g e n e r i c name as l i s t e d i n t h e Merck Index; s t r u c t u r a l formula(an a s t e r i s k above t h e name i n d i c a t e s study done with r a d i o a c t i v e drug) with arrows indicatine; s i t e s of metabolic transformation; t h e s e a r e a l s o desc r i b e d i n t h e accompanying t e x t . Abbreviations used a r e : UCC, unchanged ( p a r e n t compound; (MI, major ; (m) ,minor; ( t r ) , t r a c e ; ( A ) , b i o l o g i c a l l y a c t i v e ; conj., conjugation; gluc., glucuronide; s u l f . or SO4, s u l f a t e . Whenever deemed d e s i r a b l e , a comment was added. The compounds a r e arranged a r b i t r a r i l y according t o i n c r e a s i n g s t r u c t u r a l complexity. Inspection of t h e chemical f a t e of t h e 90 d i v e r s i f i e d s t r u c t u r e s i n d i c a t e s t h a t t h e compounds underwent r e l a t i v e l y few t y p e s of biotransformations. T h i s s e r v e s t o i l l u s t r a t e t h e concept of minimal s u b s t r a t e s p e c i f i c i t y . It i s g e n e r a l l y accepted t h a t f o r e i g n compounds, i n c l u d i n g drugs, are transformed by a s p e c i a l i z e d group of enzymes t o products t h a t a r e l e s s l i p o p h i l i c and t h u s more r e a d i l y excreted from t h e body. The concept s t i l l appears t o be t r u e t h a t l i p i d s o l u b i l i t y f a c i l i t a t e s t h e metabolism of a foreign organic compound. However, i n r e c e n t s t u d i e s , l i p i d s o l u b i l i t y had l i t t l e e f f e c t on t h e demethylation of a s e r i e s of similar compounds i v i t r o l l 7 , o r on t h e metabolism of s u l f o n a t e d arylazanaphtol dyes i n vivo118. Hence, o t h e r unknown f a c t o r s may o p e r a t e t o c o n t r o l t h e rate of metabolism.

-

Most of t h e chemical transformations shown i n t h e t a b l e s might have been a n t i c i a t e d , but t h e r e d u c t i o n of a s u l oxide37, t h e l o s s of a sulfonami.de group55 and t h e formation of an N-oxide , a r e examples of some new and unp r e d i c t a b l e transformations. While work on t h e metabolic f a t e of new compounds may r e v e a l some unique biochemically moderated r e a c t i o n s , i t i s l e s s l i k e l y t h a t any f u t u r e study of drug metabolism w i l l be of such fundamental importance t o biochemistry as h a s been t h a t of t h e a c e t y l a t i o n of sulfonamides: a study which, by prompting t h e discovery of coenzyme A, h a s eventuall y l e d t o t h e understanding of a s u b s t a n t i a l p a r t of t h e intermediary l i p i d metabolism. However, t h e growing pool of b a s i c knowledge of how drugs a r e metabolized w i l l indoubtedly i n s p i r e more chemists t o i n f l u e n c e t h e metab o l i c f a t e of a drug by chemically o r s t e r i c a l l y modifying t h e a n t i c i p a t e d s i t e s of metabolic r e a c t i o n s .

6,

A major impetus t o study t h e metabolic f a t e of a new drug comes from t h e growing number of drugs whose main pharmacological o r b i o l o g i c a l a c t i v i t y i s due t o i t s metabolite(s1. Some examples r e p o r t e d i n 1964-65 include: hypot e n s i v e e f f e c t of N,N-diallymelamine due t o i t s N-oxide85; hypoglycemic a c t i v i t y of 3,5-dimethylpyrazole due t o t h e corresponding 3 - c a r b o ~ y l a t e; ~ ~ s c h i s t o s o m i c i d a l property of "Miracil D" due t o i t s 4-hydroxymethyl metab o l i t e l 0 1 ; c a r c i n o g e n i c i t y of urethane%, DAB52 , and p o s s i b l y of dimethylbenzanthracene69 a r e due, i n p a r t , t o metabolites.

S u f f i c i e n t d a t a a r e i n t h e l i t e r a t u r e t o f o s t e r t h e hope t h a t e v e n t u a l l y , f o r a given compound, i t may be p o s s i b l e t o p r e d i c t t h e type as w e l l as t h e r a t e of i t s metabolic transformation. However, v a r i a b l e s imposed by n a t u r e such as s p e c i e s , sex, age, g e n e t i c background, e t c . , combined with those introduced by man, e.g. d i f f e r e n c e s i n experimental design, dosage schedules with t h e p o s s i b i l i t y of enzyme induction, e t c . , w i l l continue t o t e s t both t h e i n g e n u i t y of t h e compiler' and t h e c a p a c i t y of h i s computer.

Chap. 23

Fate of Drugs

Buyske and Dvornik

SOME NETABOLIC TRANSFORMATIONS REPORTED

249 -

IN 1964-65

MABOLITES AND COWWENT

coww)(D

Metabolite8 are S-methylcysteine; methylmercapturic acid; rethylthioacetic acid; N-(methylthioacetyl) glyc ine Study showed metabolites of iodomethane &d Smethyl-L-cysteine are the same, thus confirming S-alkyl-L-cysteine as intermediate metabolites in the metabolism of halogenoalkanes to alkylmercapturic acids (26).

4

cn,r Iodomthnr Iluodllator

p*m2

7 7 cH2m2

Hydrolysie of a single nitroester (M) (A). Dinitro metabolites are resistant to further metabolism. Nitro-glycerin can react non-enzymatically with reduced gluthathione to liberate nitrite. However, a hepatic enzyme catalyzed reaction (27).

Nitroglyoed~

Vuodllator

Hydrolysie of all four nitro esters to pentaerythritol as only urinary metabolite (28). Follow-up study of same group who showed PEZ" is degraded stepwise in blood to the tri-, diand mono-nitro ester and free pentaerythritol ( 2 9 ) .

4

-

~onteoryttwito~ trtranitrato' kneethotia flypnotlo

I

y 2

"7%

CH20H

flydroxybutpic acid

*

kdatiw U l d hypnotio

7%

cn2 fJHk€l&ONHCONl$

: u b r o u l * Br

?

$-Oxidation (rat) (30). Findings indicate breakdown via $-oxidation rather than via Krebs cycle (terminal hydroxyl oxidation) (31 isolation of la elled hippuric acid after d 4 C ] - and not [l-lpC]hydroxybutyrate and Na benzoate; trapping of radioactivity with 3,bdihydroxybu tyrate , a suggested intennediate in $-oxidation. Y-Hydroxybutyrate reported as a normal constituent of brain tissue (31). Only urea-C-atom containing metabolites were studied. 3-Hydroxylation, (M) mice; (m) rat, dog. Debromination. In vitro, with tissue slices, like in vivo, faster metabolism in mice than rats and dogs. Only debrominatioa detected in rat and dog tiseues. Median anesthetic dose (mg/kg) of carbromal, 3-hydroxycarbromal and 2-ethylbutyrylurea in mice (i.p.1 were 256, lo00 and 506 respectively (32).

2 50

Sect. V

Tranqulliwrn Fp-H2 CH3CH2CH-C-CH

1'1

Cl120CONHR2

% %

hbutamte(33) H

CH(CH3),

H

(CHz)3CHJ

H2NCOOR

R-cH3 C t 5

"-c35 n44llg Urethane M d related carbamates

0 H

H C-S-CH,

' t

Mmethylsulfoxide

Aatituberculoaln

'i""

t

d

CH20H

I

CHNFI(CHE)2nricR

I

I

CZH5

Topics in Biology

T a b a c h n i c k , Ed.

p-Hydroxylation(M) ; N-dealkylation(m) ; P-hydroxylation and N-dealkylation; conj.(gluc.)(tr). Hydroxylation of B-carbon of propyl side chain is typical site of prevalent metabolic transformation of analogs of meprobamate. In vitro, p-hydroxylation not affected by a-methyl substitution (33) In spite of metabolic conversion to meprobamate, tybamate may act per se (35).

cari.oprodol(*) "ybamate(35)

Carcinogen

tt

-

5'2'

N-Hydroxylation, N- and 0-acetylation(m), rat, rabbit, man. SCarbethoxylation and S-ethylation followed by N-acetylation and S-oxidation reflected in isolation of S-ethylmercapturic acid, its sulfoxide and of N-acetyl-S-carbethoxycysteine. Same metabolites after N-hydroxyurethane. Carcinogenicity of urethane may be due to its metabolism to hydroxyurethane which can cause biological carbethoxylation and ethylation (36)

Heduction to dimethyl sulfide (cat) and dimethyl sulfone (rabbit) (37). Reduction of an oxidized sulfur to a lower oxidation state in mammalian system is an unusual biotransformation.

Oxidation of both alcohols to aldehyde (m) and subsequently to dicarboxylic acid metabolites (HI; man, rat and dog metabolism similar (38)(39)0 L and D isomers compared in dog and found L isomer quantitative conversion to metabolites greater than D isomer (40).

Ethbutol*

Antileprosy

Dit0Ph.l

c

Ethylmercaptan (MI, methyl ethyl sulfoxide (M) and methyl ethyl sulfone m) formed. Used 3% and only examined radioactive metabolites (41).

Chap. 23

Fate of Drugs

Buyske and Dvornik

a1

\

t

Epoxide formed across double bond (MI i epoxide at 1,4 transformed to a 1,4-(OH)2 (m)* Although related epoxy tetrahydronaphtalenes react with glutathione and are metabolized to mercapturic acid conjugation, this did not occur with this compound (42).

,4-epoq-l, 4-dihydronaphthalone narcticidea Cl

-@$ c1

cx3

c1

Epoxide only metabolite formed in rabbit and rat microsomes.

leptaohlor

Cofactor requirements, 02 and cellular localization of enzymes similar to other oxidative reactions on drugs. Lack of subsequent oxidation of epoxides to hydroxyl metabolites taken as evidence that epoxidation and hydroxylation are similar, but separate reactions dependent on the structure of the substrate (43).

c1

Udrin

Cl Pentachlomcyclohemne

IDT

7:; f

251 -

Dechlorination; dehydrochlorination (m); dechlorination and dehydrochlorination (tr); bis-dechlorination (tr); oxidative cleavage. Established that, in rats, DDD, the dichloro analogue is, or can be, a metabolite of DDT. The suggested pattern of metabolic transformation of DDT is based on metabolic studies with metabolites of DDT (44).

252 -

Sect. V

Btirlral

4

-

Topics i n Biology

T a b a c h n i c k , Ed.

N-methylation only metabolite and occur6 only in dog. Not metabolized in man, mouse or monkey (51)

l-Adamantanamine

Reductive cleavage to amino- and diaminometabolites; p-hydroxylation of the aminometabolite; N-dealkylation. In rats excr. of diamino metabolites may depend on their binding to tissue; this may vary with substitution. Carcinogenicity may be related to tiesular binding of metabolite(s) , e.g. DAB (Rl=Rz=H) and J'-methyl-DAB (R~=CHJ, R2=H) are carcinogenic, 2-methyl-DAB (Rl=H, R ~ = c H ~is) not ( 5 2 ) .

Ring hydroxylation (M) and glucuronidation (M), no reduction of azo linkage (53).

R -2

R -1

H

SO Na

3

'

60

H

Na

2-Phonylaso-l-~phthol-4-

and 5-sulfonlc acids

0-dealkylation primary metabolite, but does not occur if carbon atom adjacent to the oxygen is tertiary (!%)a

Chap. 23

Fate of Drugs

253 -

Buyske and Dvornik

--

In rats (p.0.): UCC (6%); conj. (gluc. 4%; 504, 4%) ; hydrolysis and oxidation (1’7%); further oxidative cleavage (H); 0-dealkylation (5%). In man (p.0.): 85% excr. as gluc. with tr. of other metabolites. Prolonged adrn. or larger doses cause in rats metabolic tolerance. Carbamate group metabolically more stable in humans than rats (45).

luscle r e l u a n t

-

/OH

alorpheniain oarbarate*

tadiopaque agent8

C H

Glucuronidation in cats (only species -Studied). Found dopanoic acid and related compounds, tyropanic and bunamiodyl, form large amounts of glucuronides, with iodine atoms intact, and excrete in the bile (46).

l2

I kpanoic acid kntiparkinwn

N-Uesme thy1 orphenadrine (M) N-Bis-desmethyl orphenadrine (m) cleavage of the ether linkage of the amino alkyl chain (m> (47).

,

kphenadrine

inalgeeio

In man (p.0.): isolation of new metabolites formed by 0-dealkylation (M); conj. with cysteine (2%) and N-acetylation (tr). Also after p-acetamidophenol admin. Position of S-link not determined. Low acetylating capacity of interest, since usually only observed in guinea pigs (481,

.1

hoe tophenst i d i n e

kroinogen

~

-I\ u

2

/

I

~

w *

N-2-Fluors~la~etadd0

o

Hydroxylation in 5,7 and on N; sulfate conjugation on OH at 7 (M); glucuronide conjugation c on H OH at~ 5,7 and on N (a); deacetylation. Extension of earlier work on metabolism of this carcinogen, Contrary to popular belief, the cat is capable of forming glucuronides ae a minor metabolite (49). Hamster forms metabolites similar to other species (50).

2 54 Cnrbonia

Sect. V

wdr.w

Topics in Biology

T a b a c h n i c k , Ed.

Complete cleavage of sulfonamide group and replacement with mercaptan (m) mercapturic acid (M) and mercaptoglucuronide (MI. Reaction sequence initiated by the transitory metabolite glutathione conjugate (55).

,

inhibitor

knwthi.wl.-z-*ulfoaa8ioM.hd.

-

*

Antibaot.ri.1

==(

N‘-glucuronide (M). Previous work showed formation of N-glucuronide, without determining on which nitrogen. “his work established conclusively that conjugation occurs on sulfonamide nitrogen (56).

8ulfadi..thodIn htib.Ot.rirl

y@!!cycp,

-

N3cn

N-4 Acetylated metabolite (MI; small amount of glucuronide forraed. Metabolism studied only in the heifer (57).

8ulfaothoxypyrid.rinr hticonrulunt

5’

N-Dealkylation (M) (A) ; only the unsubstituted sulfonamide has activity. Dealkylation varies inversely with the bulk of alkyl substituent

(58)

CH3 t o (CH2)qCH3

R2- I or CI 3 N-allprl-4-braoboarao~ulf~des Blpoeba..ia

cq-Q-.”! Aooto&udQ

(m ~ p ’ o l o h r r g ~ )

0-18,536 (R= hexahydmnsepdnyl)

I

I

In acetohexamide: reduction of Ac to 1CH CHOH (M)(A); hydroxylation of cyclohexane (m7(*) (59) In man, both drugs are rapidly absorbed and rapidly transformed to p-a-hydroxyethyl metabolites, which remain hypoglycemic. Since the metabolites have a lopger biological half-life than the parent drugs, their effect correlates well with the total serum sulfonylurea level (60).

Chap. 23

Btimulnnt

Fate of Drugs

Buyske and Dvornik

255 -

Ring hydroxylation (m) rat, dog; gluc. conj. (M) rat, (m) dog; oxidative deamination; hippuric acid (MI monkey, dog;(m) rat. This is a study comparing the metabolism in rat, dog and monkey (61).

No metabolism occurs in rabbit liver homogenates capable of metabolizing tyramine, epinepherine, norepinepherine and amphetamine (62). M@ t u u i n o l

Hallw1nog.n

Aatihyport.Mi+.

In cats(63) , and man(&) : UCC (cat) ; oxidative deamination (MI, cat, man. Distrib. in cat brain(63) indicates binding to spec. intracell. components. Hax. conc. of mescaline corresponded to approx. the estimated max. intox. Mescaline may be deaminated in brain. In man, trimethoxyphenylacetic acid (the major metab,) produced no definite physiol. or psychol. changes (64). In rats (i.p.)* 1-Methyldopa and l-methyldopamine: UCC .(M); 0-methyl. (m); decarbox. (m); conj. (gluc.) (m). d-Methyldopa: only UCC and 0-methyl. 1-Methyldopamine: UCC, 0-methyl. and conj. (gluc.). 1-Methyldopa better abs. from gut than d-; 1-causes spec. aminoaciduria, possibly by competing for renal tub. reabs. of neutral amino acids; only 1- is decarbox. These diff. may be responsible for therap. inact. of d- form ( 6 5 ) . Hydroxylation and sulfate conjugation at carbon 1 plus the unusual addition of a formyl group on the amino nitrogen ( 6 6 ) .

2 56 -

Sect. V

Anticoagulant

-

Topics in Biology

T a b a c h n i c k , Ed.

Detailed reexamination in several species

of coumarin hydroxylation at C-7. Metabolite formed in rabbit and coypu in large amounts; cat, pigeon and guinea pig only 10 to 25% as much; no 7-hydroxylation in rat, mouse and locu8ts (67).

Co\aarin Natural eatroganic isoflavones

In sheep, i n this study, p-ethylphenol and p-cresol are major metabolites. Only fonnononetin undergoes reduction of ring double bond and ketone (68).

OH H

H

2

CH

3

hniatein Fomoamnetin

Major metabolite is isomeric monohydroxymethyl compound. Minor metabolites include hydroxylation at 3 or 4 and dihydroxylation at 3*4 and 8,9. All metabolites appear to be carcinogenic (69).

In rats, dogs: UCC (tr); metab. septd. by electrophor., not identified. Indicated: in vitro, primary and/or sec. amine formation; . in vivo. ring hydroxylation and conj. (gluc.) Radioact. mainly excr. in feces, notably in dogs. Metabolites similar in rats and dogs differ in rel. amounts (70).

Analnesic

Bandol *

K.N/ I

R

R

CH

T z H

C1 H Cl 3 a e t h y l b e n ~ l C1 CH;

Cyolieine Chlorcyclisine Nor-chlorcyclidne kolisina

N-Dealkylation (M). Norchlorocyclizine penetrates placenta (71) and may be cause of congen. malform. induced by meclizine and chlorocyclizine (72). Demethylation faster in male rats; slow in dogs, accel. by chron. admin. Chlorocyclizine is metab. slower than cyclizine* possibly due to plasma protein binding. N-demethylation causes loss of antihistaminic act. (73).

Fate of Drugs

Chap. 23

257 -

Buyske and Dvornik

UCC (approx. 2%), dog; ring hydroxylation presumd ably at both p-positions, followed by conj. (gluc. )(MI, dog, cat, rat, man, In dogs, good intestinal abs. Unchanged drug is major plasma component up to 6 hr after dose. 0-Demethylation not likely (74)

.

bphenoxalon~

J

atiinflA5atory

H \3oQ3:-I

I

0

*coo

Cl

UCC (tr)(man); conj.(gluc.) of parent drug (MI, man, monkey, rabbit, guinea pig, rabbit, rat, except dog; N-des-chlorobenzoylation(m); N-des-chlorobenzoylation and conj.(gluc.)(M); 0-demethylation and conj. (gluc. ). Total gluc. excr. consistent and characteristic for species. Poor urinary gluc. excr. in rats and dogs. Renal excr. of indomethacin and metab. as gluc. is predominant clearance route (75)

tndomothncin *

Latimalaria

Studied only in rats; hydroxyethyl group removed (M) in preference to ethyl (m); trace amounts of free alkyl amino metabolite. No 4-amino quinoline detected (76)

c1

3ydroxychlomquine

1

CH2CH2NHCH3

II ,-H.tboxy-N-wt~ltlypt~ine

.

.

In rats; oxid. deamination (approx. 90%); postulated : 6-hydroxylation followed by conj (gluc) Isolated from the grass Phalaris tuberosa, causing "staggers" in sheep. In rats, the drug readily distrib.,rapidly and quant. metab. and excr. as 5-methoxyindoleacetic acid. N-Monomethyltryptamine remains substrate for MA0 (78). In rabbits, man: UCC (tr); 6-O-demethylation(M); N-demethylation(m); dehydrogenation(tr1; conj. (glUc. 95%; s u l f . 5 ) . Major metabolite, 0,N-bis-desmethylversidyne (33-55% of dose). Fluorirnetric det. of versidyne and metab. based on dehydrogenation with Hg-acetate (79)-

Versidpe

c1

2 58 -

Sect. V

H Hietamine

H Iaidacolylace t i c acid

Topics in Biology

T a b a c h n i c k , Ed.

UCC (tr) mouse; oxidative deamination (m), followed by conj. (riboside)(M); rat, mouse. Nmethylation ( l a ) , mou~e. In mice, circulating histamine is rapidly metabolized and its N-methyl metabolite detected. Both parent drug and its metabolite are stored in tissue depots. Imidazolylacetic acid and its riboside are major metabolites and also retained in tissues. The riboside predominated in the rat (80).

+

(--Jcn2cn*NH2

-

*

No metabolites detected in rats in vivo (i.p.) or in vitro (liver slices and homogenates). As reported (80), imidazolylacetic acid is metabolically inert, hence stable terminal metabolite of histidine and histamine (81).

kfypoglycedo

Oxidation of ring methyl to COOH (M)(A) with probable glycine conjugation. Hydroxylation at C-4 and conjugation to produce major metabolite (7% of dose) (82).

H 1

5,5-Mmethylpyramle

In rats: UCC (15%); ring hydroxylation and conj. (gluc.) (40-50%); N-acetylation (25-3m); hydrazone of pyruvic acid ((5%). LDw (slg/kg) compared: apresoline, 35; hydrazone of pyruvic acid, 40; N-acetylapresoline, 100. No hydrazine liberated (83).

3ypotenaive

f)Q ipresoline

e(I

I

L,

OH?

L,b-Mhydroxyphthalazlne

UCC, rat

(3&), rabbit (45%); conj. (gluc.) rat (3.599, rabbit (none

(50%) ; N-methylation,

detected); oxidative cleavage to phthalic acid, rat (%). 0-Acetyl metabolites may have been formed but are readily hydrolyzed in the urine ( 8 4 ) .

Chap. 23

Fate of Drugs

Buvske and Dvornik

259

In dog, monkey, man: UCC; conj. (gluc.) of parent drug (m); ?-methyl hydroxyl. (M)(A) with part. conj. (gluc.) and oxid. (tr.) On chronic adm. of high doses (dog, monkey) no accum. of parent drug and/or metab(s). On bicarb. supplement., conj. of parent drug and its 7-CH2OH metab.(I) is decreased. In man, I is excr. like parent drug, with less tend. to conj. In vitro, I is as antibact. as parent drug (94).

hntibacterial

'ZH5 Nalidixic acid Tranquilizers

Chlorpromazine (I)( 9 5 ) : chon. adm. in man: conj. (glut.): N-demeth. (trj. Purple pigment. obs. in some psych. patients after high dose (96,971 may be lightinduced alter. of ?-OH metab(s) enhancing tyrosinase act. and incr. melanin prodn. In dogs, excr. is %ide-chain" dependant. NMethylpiperazinyl enhances biliary and reduces urin. excr. of parent cpds and/or their metabolites (98). Fluphenazine (11)enanthate(99): hydrol. to 11; S-oxid. ; hydrol. and S-oxid. ; C02 (tr). Excr. of radioact., urine 5374, feces 22%; bulk of tiss. radioact. not extracted. Prolonged act. due to slow release of ester from tiss. depots. Act. of ester due to 11. Adm. as soln. in oil essent. for prolong. act., aqu. soln. short dur. of act. Major metab. of promazine and I have N(mono-, bis-) desmethyl. chain on hydroxylated ring and S-oxidn. (100). UCC: ?-OH: S-oxid.:

-2 R -N(C$+

-N

/--\

Prornazine

N-CH

3

u€'rochlorperazine*

Fluuhenazine enanthate * Schistosornicide

1-Methylaminoe thylnmino4-me thylthiaxauthone hydrochloride

4-Methyl hydrox. (M)(A), in A.sclerotiorum; conj. (gluc. ) (M) , monkey; (m) , mouse; S-oxid. (m), monkey; oxid. of 4-CH20H metab.(I) to aldehyde (m) in A.sclerotiorum. In hamsters, I (hycanthone) has ED% (P.o.), parent drug 8.0 mg/kg. Act. of parentxdrug apparently due to I, hence 4-methyl required for biol. act. In mice, the 6-chloro analogue also converted to 4-CH20H metabolite (101).

260

Sect. V

-

Topics in Biology

T a b a c h n i c k , Ed.

Mono- and bis- N-dealkylation (M); dihydroxylation on chain double bond (M) B N-Oxide in ring (unique biotransformation) is active hypotensive, formed in small quantity in rat and dog, but not in man (85,86).

Antitwin

UCC, rat (1-5s); deamination to diethylamine (rat, 10%; mice (tr); dogs, 5-12%) and to a neutral 3-phenyloxadiazole deriv. (rat, 1-s; dog, tr). Oxidative ring cleavage to benzoic acid, conj, with glycine (rat, 5-15; mice, dog, man) and gluc. (rat, mice, dog, man). Exploratory study (87).

In iamb~(88): UCC (2%) ; 5-hydroxylation (M), only 10% unconj.; conj. (gluc. 70%. sulf. 14%). Similar findings in goats, calves and swine. Rapidly abs., metab. and distrib. Only tr. detected after several days. Small amounts of metab. in milk of lact. animals disappear after several days (89). In dogs(%), man(%,91): UCC, man only; Ndemeth.; N,N-bis-demeth.; N-acetyl. of N,Nbis-desmethyl metab.; deamin. of N,N-bisdesmethyl metab. to 4-OH deriv. Part. ring Ndemeth. and intramol. cond. to rubazonic (I) and N-methylrubazonic (11) acid. I and I1 inhib. ox. phosphor. more than indomethacin. But, in vivo less act. than parent drug ( 9 2 ) . kdatirr

hthyl-)-n-b.wlkrbiturio acid

Oxidation of C-5 of hexyl chain to OH,CO and GOOH, as major metab.; a l s o oxid. at carbons 4 (M), 3 and 2. Penultimate carbon predominant, but not exclusive site of oxidation (93).

'

Chap. 23

Fate of Drugs

Suyske and Dvornik

26 1 -

10-Hydroxylation; ring hydroxylation; 10,llbis-hydroxylation; 5,lO-bis-hydroxylation; conj. (mostly gluc. 1 5,lO-Bis-hydroxylation detected in rat and children; l0,ll-hydroxylation predominant. in rabbit and dog (102).

btlconvulonnt

1 /

Rapid disappearance of both imipramine (I) and DMI (mice, rabbits), In rat and man I is metab. to DMI faster than DMI is metab. further. In vitro (liver microsomes): from rat, I demeth. mainly to DMI and 2-OH metab.; DMI metab. slowly. From rabbit, DMI metab. faeter than I; N-demeth. minor pathway. From human, slow metab. of I and still slower of DMI. Species variation as obs. in the pharmacol. response of I and DMI may reflect metab. fate which differs from species to species (103)

In dogs: UCC (10%); ester hydrolysis (60%); N-demethylation (3%). N-Demethylation greatly accelerates ester hydrolysis. Radioact. excr. mainly in feces in rats (5%), but urine in dogs (73%). Unlike with ethoheptazine (104),no hydroxylated metab. detected: implied interference by 3-methyl suggestive of 2- or 3-hydroxylation of ethoheptazine (105).

blpeio

Pranqlilimr

Libriu

r

Hydrol. cleav. to 2-0x0 metab. ("lactam") (MI dog, man; further cleav. to "opened lactam" (m), dog, man; metab, of "opened lactam" to alkali-sens. undef. "conj." (m) , man. Rat: unident. basic metabolite (M) (106) Librium is extens. metab. in rat, dog, man. Major metab. pathways similar in dog, man, diff. in rat. Major site of metab. transform. is Nl-C-2 link. N-oxide or benzophenone moiety not changed. %actamticentr. as act. as librium in mice (antifight. test, anticonvuls. and muscle relax, effects), but inact. in cond. avoid. behaviour (rat) (107). (A),

26 2

Sect. V .

-

Topics i n Biology

T a b a c h n i c k , Ed.

Diaze m ( I ) : N-demeth. (tr)(A) ; N-demeth. and 3 - h d r ) ( A ) ; N-demeth., 3-OH and conj. (gluc. (M). Blood metab. (dog, man) show rapid form. of N-desmethyl-I (half-life longer than I), In dogs, oxaeepam (11) gluc. is major metab. of I and 11. In man, gluc. of 3-OH-I and I1 det. in urine(l08) In man, stored I and metab. are released

:H i

I

t

A'

2

3

H

OH

Diaaepm Oxazepm

oY2cH4

*

OH Oxezepam* succinate

slowly (109). N-Demeth., p-OH and conj. (gluc.) (rabbit) (110). N-Demeth. increases antiagress. eff. (monkey); 3-OH enhances anticonvuls. and muscle relax. eff. (mice). Pharmacol. act. of parent cpd not due to rnetabs. (107). Dur. labour I was in maternal and fetal circul., tr. in amniotic fl. (111). Oxazepam and succinate metab. similar in dog, pig, man: m a i n l y m l u c . 95%). Complex in rat (112).

Antifungal

c1

CH3

6-Demethylation followed by conjugation to produce major metabolite, A second metabolite in some conjugated form not identified (113, 114),

?aychotherapsutic agent

N-Desethyl tion (M) , 0-demethyl tion ( m ) , deacetylation (m), a total of 11 metabolites identified in urine of dog and man (115,116).

Benzquinnmide

*

Chap. 23

Fate of Drugs

Buyske and Dvornik

263

REFERENCES

1. L. Schuster, Ann. Review Biochem., 2,571 (1964). 2. B.B. Brodie in I'Absorption and Diatribution of Drugs1*,T.B. Binns, Ed., E. and S. Livingstone Ltd., London, 1964, pp. 191-251. 327 (1964). 3. J.J. Burns, Am. J. Wed., 4. J.J. Burns, S.A. Cucinell, R. Koster and A.H. Conney, Ann. N.Y. Acad. Sci., 273 (1965). 125 (1965). 5. J.A. Miller and E.C. Hiller, AM. N.Y. Acad. Sci., 9, 6. Proceedings, 2nd International Pharmacol. Meeting, Vol. 4 and 8, The MacMillan Co. , N.Y. , 1965. 7. llEvaluationand Mechanism of Drug Toxicity1*,H.E. Whipple, Ed., Ann. N.Y. Acad. Sci., Vol. 123, Art. 1, 1965, pp. 1-366. 8. "Isotopes in Experimental Pharmacology*1,L. J. Roth, Ed., University of Chicago Press, 1965. 9. R.I. Dorfman and' F. Ungar, llMetabolismof Steroid HormonesN, Academic Press, N.Y., 1965. 10. H. Remmer and H.J. Merker, Ann. N.Y. Acad. Sci., 3,79 (1965). 11. A. Rubin, T.R. Tephly and G.L. Mannering, Biochem. Pharmacol., 13, 1007 (1964)12. J.K Dingell, F. Suleer and J.R. Gillette, J. Pharmacol. Exptl. Therap., 3, 14 (1964). 156 (1965). 13 R.P. O'Brien, Ann. N.Y. Acad. Sci., 14. R.L. Cram, H.R. Juchaw and J.R. Fonts, Proc. SOC. Exptl. Biol. Ned., 118, 872 (1965). 15. Do=. Evans, Ann. N.Y. Acad. Sci., 3,178 (1965). 16. J.H. Peters, G.R. Gordon and P. Brown, Life Sci., 5, 99 (1965). 245 (1965). 17. J.R. Fouts and L.G. Hart, Ann. N.Y. Acad. Sci., 18. J.R. Fouts in l*BiologicalCouncil Symposium on Embryopathic Activity of Drugs", J.M. Robson, F. Sullivan and R.L. Smith, Eds., J. and A. Chqrchill Ltd., London, 1965, p. 43. 19. R. Kuntzman, W. Jacobson, K. Schneidman and A.H. Conney, J. Pharmacol. Exptl. Therap., 146, 280 (1964). 20. R. Kato and J.R. Gillette, J. Pharmacol. Exptl. Therap., 150,

u,

x,

a,

=,

285 (1965).

21. R.A.

Neal and K.P.

185 (1965).

DuBois, J. Pharmacol. Fxptl. Therap.,

148,

L.G. Hart and J.R. Fouts, Biochem. Pharmacol., &, 263 (1965). A.H. Conney, K. Schneidman, M. Jacobson and R. Kuntzman, Ann. N.Y. Acad. Sci., 98 (1965). 24. E.E. Werk, J. MacGee and L.J. Sholton, J. Clin. Invest.,

22.

23

9

25. 26. 2728.

s,

9,

1824 (1964).

D. Kupfer, T. Balazs and D.A. Buyske, Life Sci., 3 , 959 E.A. Barnsley and L. Young, Biochem. J,, 2, 77 (i965).

(1964).

P. Needeman and J.C. Krantz, Biochem. Phannacol., 2,1225 (1965). F.J. Dicarlo, J.M. Hartigan, C.B. Coutinho and G.E. Phillips, Proc. SOC. E x p t l . Biol. Med., 311 (1965). 29. F.J. Dicarlo, J.M. Hartigan and G.E. Phillips, Proc. SOC. Ekptl. Biol. Med. 118,514 (1965). 3 0 0 S.S. Walkenstein, R. Wiser, C. Gudmundsen and H. Kimmel, Biochim. Biophys. Acta, 5 , 640 (1964).

118,

264 -

Sect. V

-

Topic8 i n Biology

T a b a c h n i c k , Ed.

Bessmann and W.N. Fishbein, Nature, 200, 1207 (1963). Butler, J. Pharplacol. Exptl. Therap., 23 (1964). 33. J. Edelson and J.F. Douglas, The Pharmacologist, 2, 160 (1965). 34 J. Edelson, A. Schlosser and J.F. Douglas, Biochem. P h a m c o l . , 901 (1965). 35 J.F. Douglas, B.J. Ludwig, A. Schlosaer and J. Edelson, unpublished data. 198 (1965). 36- E. Boyland and R. Nery, Biochem. J., 37. K.I. W i l l i a m s , K.S. Whittemore, T.N. Mellin and D.S. Layne, Science,

31

S.P.

.

32.

a,

T.C.

2,

2,

38.

39.

149, 203 (1965).

E.A. Peets, W.M. Sweeney, V.A. Place and D.A. Buyske, Amer. Rev. of Respirat. Diseases, 91, 51 (1965). D.A. Buyske, W. S t e r l i n g and E. Peets, Ann. N.Y. Acad. Sci.,

m,

42. 43.

A r t . 2, 711 (1966). E.A. Peets and D.A. Buyske, Biochem. Pharmacol., lJ, 1403 (1964). G.A. E l l a r d , J.M.B. Garrod, S. Scales and G.A. Snow, Biochem. Pharmacol. l4, 129 (1965). P. Sims, Biochem. J., 92, 608 (1965). T. Nakatsugawa, M. Ishida and P.M. Dahm, Biochem, Pharmacol., &,

44

J.E.

40. 41.

.

,

1853 (1965).

Peterson and Wm. H. Robison, Toxicol. Appl. Pharmacol.,

321 (1964).

6,

45. D.R. Buhler, J. Pharmacol. Exptl. Therap., 14 , 232 (1964). 46. E.W. Mcchesney, Biochem. Pharmacol., z, 13$(1964). 47. W. Hespe, A.M. DeRoos and W.Th. Nauta, Arch. Int. Pharmacodyn., 156, 180 (1965). 48. O.R, Jagenburg and K. Toczko, Biochem. J., 2,639 (1964). 49. J.H. Weieburger, P.H. Grantham and E.K. Weisburger, Biochem. Pharmacol., 469 (1964). 50.

51 53

0

54.

a,

J.H. Weisburger, P.H. Grantham and E.K. Weisburger, Toxicol. Appl. Pharmacol., 427 (1964). W.E. Bleidner, J.B. Harmon, W.E. Hewes, T.E. Lynes and E.C. Hermann, J. Pharmacol. Fxptl. Therap., QO, 484 (1965). P.J. Robinson, A.J. Ryan and S.E. Wright, J. Pharm. Pharmacol., l6, suppl. , 8OT (1964). J.F. B a r r e t t , P.A. P i t t , A.J. Ryan and S.E. Wright, Biochem. Phamacol., 873 (1965). A.H. Coney, M. Sansur and U. Harfenist, The Pharmacologist, 2,

6,

14,

160 (1965). 55. D.F. Colucci and D.A. Buyske, Biochem. Pharmacol., 2, 457 (1965). 829 (1965). 56 J.W. Bridges, M.R. Kibby and R.T. W i l l i a m s , Biochem. J., 57. W.H. Linkenheimer, S.J. Stolzenberg and L.A. Woznik, J. Pharmacol. Exptl. Therap., &, 280 (1965). 58. D.L. Smith, H.H. Keasling and A.A. F o r i s t , J. Med. Chem., 8, 520 (1965). 59. R.E. McMahon, F.J. Marshall and H.W. Culp, S . Pharmacol. Exptl. Therap., 149, 272 (1965). 229 (1965). 60. D.L. Smith, T.J. Vecchio and A.A. F o r i s t , Metabolism, 61 T. E l l i s o n , L. Gutzait and E.3. VanLoon, J. Pharmacol. Exptl.

s,

e,

Therap.,

i n press.

62. T.E. G r a m and H.N. Wright, Biochem. Pharmacol., 2,1911 (1965). 63 N. Neff, G.V. Rossi, G.D. Chase and J.L. Rabinovitz, J. Pharmacol. Exptl. Therap., 144, 1 (1964).

Chap. 23

Fate of D r u g s

Buyske a n d Dvornik

*,

265 -

Charalampous, A. Orengo, K.E. Walker and J, Kinross-Wright, J. Pharmacol. Exptl. Therap., 242 (1964). J.A. Young and K.D.G. Edwards, J, Pharmacol. Exptl. Therap., %,

64. K.D.

102 (1964). E. Boyland and D. Manson, Biochem. J. 2,7P (1965). P.J. Creaven, D.V. Park and R.T. W i l l i a m s , Bioc'tiem. J., 96, 390 (1965). T.J. Batterham, N.K. Hart and J.A. Lamberton, Nature, 509 (1965). E. Boyland and P. Sims, Biochem. J., 2,780 (1965). G. Braun, J. Krapcho and S.M. H e s s , Proc. SOC. Exptl. Biol. Wed.,

zc

118, 933 (1965). Narrod, A.L.

71

S.A.

72

C.TKing,

147, 380 (1965).

76

Weaver and S.A.

147, 391 (1965).

King, J. Pharmacol. Exptl. "herap., Narrod, J. Pharmacol. Exptl. "herap.,

A, Klutch, L. T s a i and J.J. Burns, J. Pharmacol. Ekptl. Therap., 29 (1965). J.A. Morrison, Arch. I n t . Pharmacodyn., 385 (1965). R.E. Harman, M.A.P. Meisinger, G.E. Davis and F.A. Kuehl, J. Pharmacol. Exptl. Therap., &, 215 (1964). E.W. McChesney, W.F. Banks and D.J. Sullivan, Toxicol. Appl. Pharmacol.,

73. R.%ntzman, 74. 75.

S.A.

Wilk and C.T.

149,

m,

7, 627 (1965). 77. S.-Wilkinson, J. Chem. SOC., 1958, 2079. 78. R.G. Raborsky and W.M. McIsaac, Biochem. Pharmacol., 2,531 (1964). 79. D.E. Schwartz, H. Bruderer, J. Rieder and A. Broesi, Biochem. Pharmacol. , 2,777 (1964). 80. S.H.

Snyder, J. Axelrod and H. Bauer, J, Pharmacol. Exptl. Therap.,

144, 373 (1964).

K.cHosebach,

82.

D.L. Smith, A.A. F o r i s t and G.C. Gerritsen, J. Pharmacol. Exptl. Therap., 150, 316 (1965). W.M. HcIsaac and M. Kanda, J. Pharmacol. Exptl. Therap., 7 (1964). W.M. McIsaac, Biochem. Pharmacol., Q, 1113 (1964). G.R. Zins, 3. Pharmacol. Exptl. Therap., 150, 109 (1965). G.R. Zins, J.J. Urpsprung and J.R. Weeks i n "Isotopes in Experimental Pharmacology", L.J. Roth, Ed., University of Chicago Press, 1965, pp. 361-382. B. S i l v e r s t r i n i , B . Catanese, G. Corsi and P. Ridolfi, J. Pharm. Pharmacol., 38 (1964). D.J. TOCCO, R.P. Buhs, H.D. Brown, A.R. Matzuk, H.E. Mertel, R.E. Herman and N.R. Ttenner, J. Med. Chem., 2, 339 (1964). D.J. TOCCO, J.R. Eggerton, W. Bowers, V.W. Christensen and C. Rosenblum, J. Pharmacol. Exptl. Therap,, 149, 263 (1965). F. Petchold, Arzneimittelforsch., 972 F.R. Preuss and K.M. Voigt, Naturwiss,, 537 (1964). G.W. Smith, M.E. Parsons and M.W. Whitehouse, J. Pharm. Pharmacol.,

87.

88. 89. 90. 91 92

93

94.

(1964).

W.W.

Huep and G. Rieck, Z. Physiol. Chem.,

m,150

81.

a,

16,

2,

(1964).

s,

16, 830 (1964). E.K Maynert, J. Pharmacol. Exptl. "herap., 150, 476 (1965). E.W. McChesney, E.J. Froelich, G.Y. Lesher, A.V.R. Crain and D. Rosi, Toxicol. Appl. Pharmacol., 6, 292 (1964).

266

Sect. V

-

Topics in Biology

T a b a c h n i c k , Ed.

146,

95.

T.L.

96. 97.

A.C. Griner and K. Berry, J. Can. Med. ASSOC., 663 (1964). A.S. Zelickson and H.C. Z e l l e r , J. Am. Med. ASSOC., 8 .l8 , 394 (1964). E.J. VanLoon, T.L. Flanagan, W.J. Novick and A.R. Maas, J. Pharm. Sci., 1211 (1964). A.G. Ebert and S.M. Hess, J. Pharmacol. Exptl. Therap., 412 (1965). . _. V. Fishman and H. Goldenberg, J. Pharmacol. Exptl. Therap., 150, 122 (1965). D. Rosi,-G; Peruzzoti, E.W. Dennis, D.A. Berberian, H. Free and S. Archer, Nature, 1005 (1965). M. Kraml, K. Sestanj and D. Dvornik, presented i n p a r t a t the M t h Ann. Meeting of the Canad. Fed. Biol. SOC., Vancouver, B.Co,

98.

99. 100.

.

101

102.

103. 104.

105

106. 1070 108.

109. 110.

111. 112

0

116. 117. 118.

Perry, C.F.A.

Culling, K. Berry and S. Hansen, Science,

81 (1964).

90,

2,

3,

208,

June, 1966.

J.V. Dingel, F. Sulser and J.R. G i l l e t t e , J, Pharmacol. Exptl. 14 (1964). Therap., S.S, Walkenstein, J.A. MacMullen, C.M. Knebel and J. S e i f t e r , J. Am. Pharm. Assoc. Sci. Ed., 20 (1958). S.S. Walkenstein, R. Corradino, R. Wiser and C.H. Gudmundsen, 121 (1965). Biochem. Pharmacol., BOA. Koechlin, M.A. Schwarta, Go Krol and W. Oberhansli, J. Pharmacol. Exptl. Therap., 399 (1965). L.O. Randall, C.L. Scheckel and RoF. Banzinger, Curr. Therap. Res., 79 5% (1965). H.Ze Ruelius, J.M. Lee and H.E. Alburn, Arch. Biochem. Biophys., 111, 376 (1965). M.CSchwartz, B.A. Koechlin, E. Postma, S. Palmer and G. Krol, J. Pharmacol. Exptl. Therap. 423 (1965). G. Jommi, P. Manitto and M.A. Silands, Arch. Biochem. Biophys., 562 (1964). J.A.F. DeSilva, L. D'Arconte and J. Kaplan, Curr. Therap. Res.,

3,

2,

g, 148,

, &,

108,

69 115 (1964).

Walkenstein, R. Wiser, C.H. Gudmundsen, H.B. Kimmel and R.A. Corradino, J. Pharm. Sci., 1181 (1964). K.K. Wong, Fed. Proc., 3, 42 (1965). S. Symchowicz, Fed. Proc., 426 (1965). E.H. Wiseman, E.C. Schreiber and R. Pinson, Jr., Biochem. Pharmacol., 3, 1421 (1964). B.K. Koe and B. Pinson, Jr., J. Med. Chem., 2, 635 (19641, 92 (1965). P. Maze1 and J.F. Henderson, Biochem. Pharmacol., J.F. B a r r e t t , P.A. P i t t , A.D. Ryan and S.E. Wright, Biochem. Pharmacol., 873 (1965).

S.;.

, z? 2,

2,

g,

e wish t o express our thanks t o the many colleagues Acknowledgment. W who hare promptly responded t o our c a l l f o r documentation of t h e i r recent work on drug metabolism.

Chap. 24

Cell Metabolism Chapter 24.

Smith

2 67 -

Regulation of Cell Metabolism

Charles G. Smith, The Upjohn Company, Kalamazoo, Michigan This review w i l l consider recent h i g h l i g h t s i n two areas of metabolic regulat i o n : 1) I n h i b i t o r s of nucleic acid r e p l i c a t i o n , t r a n s c r i p t i o n and t r a n s l a t i o n processes, thought t o a c t d i r e c t l y at t h e enzyme level, and 2) hormonal regul a t i o n of c e l l u l a r metabolism at nucleic acid and p r o t e i n synthesis sites from t h e p o i n t of view of t h e medicinal chemist who is seeking t o apply t h i s knowledge i n t h e development of new and u s e f u l drugs.

During t h e p a s t two years, many publications on t h e mechanism of r e p l i c a t i o n of t h e genetic m a t e r i a l deoxyribonucleic acid (DNA), i t s t r a n s c r i p t i o n t o y i e l d s p e c i f i c ribonucleic acids (RNA) and t h e t r a n s l a t i o n of RNA t o give s p e c i f i c p r o t e i n s have appeared. A wide v a r i e t y of agents have been reported t o a f f e c t various s t e p s i n these sequences. Thus, u l t r a v i o l e t irradiation‘, 2, a c r i d i n e dyes’, 4, t h e a n t i b i o t i c s phleomycin4, novobiocin’, actinomycin D39 4~ 8e, ‘Oa, nogalamycin4r chloramphenicol* - 1 3 9 ’07 , ~ t r e p t o m y c i n ’ ~ - ’ ~ ,erythromycinla, lS, puromycin4, 20-’2, gougerotin=, 24, pluramycin26, daunomycin49 26, miracil 28, chromomycin4, cycloheximide4r 34-37, rnika~nycins’~, a c t inospectacin=, cycloheximide analoguesa, r i f amycin3@ mithramycinm, mitomycin and p o r f i r ~ m y c i n ~ ’ - ~streptomycin-like ~, antibiotic^'^ , edeineS1, peptide a n t i b i o t i c s 6 cinerubin4, oliomycin4, n a l a d i x i c acidlo’, ’04, corticoster~ids~ 56J ~56J ’ ~’04, ~ ~ e c d y ~ o n e ~ ~142, -p* nucleosidin’12, androgens4’, 47, 5 3 ~6 4 ~’ 0 4 J t h y r o t r o p i c hormones7r histoness’-68, FlIiA7O9 7 1 , ACTP’J 6 o J 7 2 ~lo4, vitamin D7% 74, para-thyroid hormone7s, growth hormone7’, 7 7 , insulin47, ‘04, Vinca alkaloids7e, aldoK m J a f l a t ~ x i n”, ~ ~and ~ ~a l k y l a t i n g agents8% have s t e r ~ n e ~vitamin ~, all been reported t o a c t e i t h e r d i r e c t l y or i n d i r e c t l y at some s i t e i n the control of DNA, RNA or p r o t e i n metabolism. Since DNA is t h e genetic m a t e r i a l which determines t h e nature of t h e c e l l (genotype) and R N A m a y be looked at as the photocopy thereof which determines phenotypic expression, we a r e indeed dealing i n these processes with t h e most b a s i c controls of c e l l u l a r function. Several i n t e r e s t i n g review a r t i c l e s or symposia have been published i n t h e recent p a s t which summarize b a s i c concepts and developments i n t h i s e x c i t i n g field4, 47, e4-ee, s o ,

’,

“,

,

”,

”,

Direct I n h i b i t o r s of Nucleoprotein Metabolism a t t h e Macromolecular Level A simplified schematic diagram of t h e r e p l i c a t i o n of DNA and t h e t r a n s c r i p t i o n thereof t o RNA which i n t u r n d i c t a t e s c e l l u l a r p r o t e i n i s shown i n Fig. 1. The proposed s i t e s of i n h i b i t i o n by s e l e c t e d agents a r e a l s o shown i n t h i s f i g u r e and w i l l be described i n some d e t a i l below.

-

I n h i b i t i o n of DNA r e p l i c a t i o n can be e f f e c t e d I n h i b i t i o n of DNA r e p l i c a t i o n . by agents which i n h i b i t t h e DNA polymerase enzyme p e r se or agents which e f f e c t t h e primary or secondary s t r u c t u r e of DNA such t h a t it can no longer i n t e r a c t properly with t h e enzyme. One example of t h e former type of i n h i b i t o r m a y be the a n t i b i o t i c novobiocin’, while i n h i b i t o r s of t h e l a t t e r type include u l t r a v i o l e t and x-irradiation‘, and t h e a n t i b i o t i c 6 actinomycin D3, 4, nogalamycin7, phleomycin4J , mitomycin C and p o r f i r ~ m y c i n ~ ’ , 43. One common f e a t u r e of t h e l a t t e r i n h i b i t o r s involves r e a c t i o n with DNA such t h a t the separation of t h e strands of t h i s double h e l i c a l molecule, which i s

268 -

Sect. V

-

Topics in Biology

T a b a c h n i c k , Ed.

required for its replication by DNA polymerasee1 is inhibited. Thus, ultraviolet light causes dimerization of t ine residues which may crosslink the molecule and prevent strand separatior The sites of action of actinomycin D and nogalamycin are discussed below. Phleomycin binds to the DNAprimer at the A-T moieties and prevents its interaction with DNA polymerase', The structures of these antibiotics are not presented here but can be seen in the reviews by Goldberg' and Umezawae2. Clearly these agents are not relaked chemically to one another and in fact insufficient chemical similarity exists to draw any structure-activity relationships. Other antibiotics which combine with DNA or inhibit DNA polymerase include chromomycina, streptonigrin', and edeine4, which are discussed in more detail in the review by Goldberg',

'.

Inhibitors of DNA transcription. - Three noteworthy inhibitors of this site of metabolic activity are actinomycin D3, ', nogalsmycin7 and daunomycina. The inhibition of DNA replication by actinomycin and nogalsmycin is a secondary site of action observed at high concentration. Time-course inhibition and doseresponse studies have shown that the primary site of action for both of these agents is the inhibition of RNA synthesis, which is followed by DNA and protein inhibition in mammalian cells', 7. Actinomycin D is known to inhibit RNA synthesis by binding to the guanine residues of DNA where it fits Into the minor groove, thus inhibiting the f'unction of RNA Nogalaq,rcin polymerase which apparently also occupies this position', 03, also shows base specificity but appears to bind preferentially to the adenine or thymine portions of DNA, or possibly both, thereby inhibiting the RNA polymerase It is interesting to note that actinomycin binds specifically to a reaction'. base in DNA but does not intercalate, whereas nogalemycin shows evidence of intercalation in addition to base specificity. Thus, the latter antibiotic, although it contains only one nitrogen atom*, increases the viscosity of DNA markedly and its binding to DNA is reversed by salts7, properties cormnon to On the other hand, it shows strict base specificity the acridines', under the experimental conditions used, both with respect to physical chemical changes of the DNA and its reaction in the RNA polymerase system7. Although direct structure activity correlates cannot be made at this time with respect to predicting base specificity or intercalating ability of these molecules, certainly considerable rational effort can now be directed at the question of modifying the interaction of such agents with the DNA primer in order to increase or alter base specificity, salt displacement reactions, and so forth. Likewise, altering organ specific absorption of these agents may indeed also lead to unique chemotherapeutic effects, impossible to obtain with the present compounds.

-

Inhibitors of RNA translation. Several antibiotics have been shown to inhibit protein synthesis by interfering with some step in the DNA-MA-protein sequence subsequent to RNA synthesis. Chloramphenicol is rcportedm'13 to inhibit the attachment of messenger-FiNA to ribosomes. This is a fascinating concept since it can also explain the specificity of chloramphcnicol in inhibiting bacteria. The life span of messenger-RNA in bacteria is measured in minutes.', while that in mammalian cells is measured in hoursg7. Thus, in the relatively short period in which significant concentrations of chloramphenicol are present in - - - - - -*R. B. Kelly and P. F. Wiley, private communication

Chap. 24

Cell Metabolism

Smith

269 -

an animal after d i s c r e e t dosing, s u f f i c i e n t a n t i b i o t i c may be expected t o be absorbed by the b a c t e r i a t o seriously impair t h e i r a b i l i t y t o synthesize prot e i n because of the rapid turnover of the messenger-RNA and i t s required regeneration. A mammalian c e l l on the other hand w i t h a much slower turnover would be expected t o be l e s s inhibited. When s u f f i c i e n t a n t i b i o t i c i s given over an adequate period of time, t o x i c i t y and cytotoxicity a r e indeed observed w i t h chloramphenicol even i n animal cellsee. Streptomycin causes misreading.of the m-RNA a f t e r the l a t t e r i s bound t o ribosomes t o form a polysome, and other a n t i b i o t i c s of t h i s chemical type appear t o a c t i n the same way -i n vitro14-17. Cycloheximide i n h i b i t s p r o t e i n synthetase, which catalyzes the formation of t h e peptide bond, while puromycin s u b s t i t u t e s f o r a molecule of transfer-RNA and accepts the growing peptide following which it uncouples the system and releases nascent peptide i n t o solution4, 34-37. Again, no common chemical c h a r a c t e r i s t i c s are evident which would suggest

-a p r i o r i t o a knowledgeable investigator t h a t a p a r t i c u l a r a c t i v i t y of the type observed should be possessed by t h a t molecule.

Knowing t h e s t r u c t u r e s of these substances4 and having available enzymatic systems which a r e thought t o represent t h e i r primary s i t e s of action, however, gives the medicinal chemist a very powerful t o o l w i t h which t o investigate analogues and derivatives f o r increased s p e c i f i c i t y or s e l e c t i v i t y of action, increased o r decreased binding capacity, and the l i k e . Comparative studies of compounds which prevent the attachment of m-RNA t o ribosomes i n systems w i t h short-lived vs. those w i t h long-lived messengers hopefully w i l l lead t o compounds w i t h the p o t e n t i a l of chloramphenicol f o r a n t i b a c t e r i a l a c t i v i t y . A common f e a t u r e of all of the i n h i b i t o r s discussed thus far is the d i r e c t action which they can be shown t o possess e i t h e r on the s p e c i f i c primer or enzyme involved i n the reaction. This represents a boon t o t h e medicinal chemist who is i n t e r e s t e d i n structurea c t i v i t y relationships, since he can t e s t d i r e c t l y f o r the e f f e c t s of analogues and derivatives of given compounds on such parameters as DNA melting, intercalation, e f f e c t s on RNA and DNA polymerases, peptide synthetase, combining of messenger t o ribosomes, etc. Thus far, the new moleculaz species showing the selective inhibitions reviewed above have originated from the empirical approach of a n t i b i o t i c screening, which needs no j u s t i f i c a t i o n f o r continued exploitation. It is stimulating t o speculate on the a b i l i t y t o design even more potent o r more s e l e c t i v e agents i n the future, making use of the s t r u c t u r e - a c t i v i t y relationships already known. It is i n t e r e s t i n g t h a t c e r t a i n of these inhibitors, p a r t i c u l a r l y thoae binding t o DNA (some of which cause damage which requires a c e r t a i n degree of r e p a i r by the organism), are highly t o x i c t o animals and other forms of l i f e as well (irradiation, actinongwin D, nogalamycin and mitomycin). Certain of the other agents, however, are capable of exerting profound e f f e c t s against microorganisms w i t h low orders of t o x i c i t y t o a n i m a l c e l l (chloramphenicol, streptomycin, erythromycin and novobiocin). Continued studies on the mode of action of various agents i n h i b i t i n g d i f f e r e n t s i t e s i n the pathway from DNA t o protein m a y allow t h e medicinal chemist i n the not-too-distant future t o determine whether the i n h i b i t i o n of a c e r t a i n s t e p i n t h i s process i s l i k e l y t o lead t o s e l e c t i v e t o x i c i t y t o one form of organism r a t h e r than others. Such conclusions, however, must await the discovery or synthesis of additional i n h i b i t o r s known t o a c t at s p e c i f i c s i t e s i n the above pathways.

270

Sect. V

-

Topics in Biology

T a b a c h n i c k , Ed.

Hormonal Controls Steroids. - A series of papers appeared in 1963 on the mechanism of action of corticosteroidq, androgens and e s t r o g e n ~ ~ ~ - ~3-6.8, O ' s e . Various studies published in recent years have demonstrated the induction of a variety of enzymes in liver ( glucose-6-phosphatase, fructose-l,6-diphosphatase, phosphohexose isomerase, PEP-carboxykinase, tryptophane pyrroyase, transaminases, arginase, glutamic dehydrogenase, arginino-succinase and arginine synthetase) after administration of corticosteroids to anirnalsloo, ee-p- '9 'O1. synthesis of these enzymes is preceded by synthesis of RNA, also elicited by corticoid administratione4-P* la. Early studieslo0 demonstrated the induction of tryptophane pyrrolase and tyrosine-a-ketoglutaxatetransaminase by cortisone or substrates, administered independently to adrenalectomized animals. Since puromycin inhibited the induction of these enzymes by steroids or their substrates, protein synthesis has presumed necessary. Actinomycin on the other hand was shown to abolish the cortisone mediated induction, but not the tryptophane induced enzyme activity, thus differentiating between these processes and suggesting that the steroid induction of enzyme, but not that of substrate, was mediated by the synthesis of m-RNA. Detailed studies by Weber and co-workersse'P' on the induction of hepatic gluconeogenic enzymes by corticosteroids led these authors a l s o to conclude that the process involves de novo protein synthesis and that it is sensitive to puromycin, actinowcin and ethionine. In view of the fact that the gluconeogenic enzyme level is a l s o elevated in the liver of chronic alloxan diabetic rats, and the observation that actinomycin D and puromycin did not inhibit this increase in enzyme, these investigators postulated that the insulin molecule may act as a physiological repressor of the synthesis of gluconeogenic enzymes and that the corticoids act by reversing this repression. Investigations by Kenney and collaborators have demonstrated an increased synthesis of nuclear RNA under stimulation in vivo by hydrocortisoneeg'Po 126. The stimulation of RNA synthesis by hydrocortisone was also inhibited by actinomycin D. The conclusion from these investigations is that the steroid hormone induces the synthesis of RNA which in turn results in an increase in the levels of specific enzymes. Whether this is indeed the primary, rather than a secondary, effect of the steroids cannot be answered at the present time, although some Regardinvestigations suggest that the latter m a y be the caseLo1, sg-p* less of whether the effect is primary or secondary, however, studies on the structure-activity relationships of the various hormones with respect to nucleic acid and enzyme induction in specific tissues and the prevention thereof by inhibitors with known sites of action might provide a new basis for the synthesis of analogues or the separation of biological activitied that could result in more favorable therapeutic applications of the steroid molecule.

--

'*.

Similar types of investigations with androgenic hormones demonstrate an increased level of RNA polymerase in prostate following administration of testosterone to castrated animals as well as an increased capacity for aminoacyl-t-RNA synthesis and amino acid Incorporating capacity of ribosomes These early changes in nucleic acid polymerase activity in vitroe4-po are accompanied in vivo"'P22 by increases in soluble proteins, activities of proteases, and a-amylases and actual weight of prostatic and seminal vesicle tissue. All of these changes are blocked by the administration of actinomycin D and can only be observed after administration of the steroid to the animal. No effect on RNA polymerase activity has been observed upon addition of

--

".

Chap. 24

Cell Metabolism

Smith

27 1

testosterone or other sex hormones to the enzyme system in vitro. In the case of estrogen action on the uterus of castrated animals, the data are among the most convincing for a direct and probably primary effect of the hormone on the induction of protein and RNA synthesis. Detailed studies by Jensene6'P' 317 J Gorskie4-p. and their co-workers have demonstrated the binding of estrogen to the uterus of the castrated rat with little or no detectable metabolism following this event. Mueller and co-workers4' -P. 22g demonstrated increased rates of protein and RNA synthesis as early as six hours after estrogen injection with RNA accumulation beginning at approximately 12 hours and protein accumulation at 24 hours. No increase in DNA synthesis was seen even at 24 hours after administration of the hormone. The early changes in RNA and protein synthesis were shown to be blocked by puromycin, actinomycin, and cycloheximide@'P22e~ indicating that the initial effects of estrogen are also demonstrated dependent upon RNA and protein synthesis. Gorskie4'P* an increase in uterine RNApolymerase after treatment of the animal with 17p-estradiol. Based on investigations of early effects of inhibitors, Mueller and co-worker~~~-P* 22(1conclude that the initial step in the action of estrogen is the activation of a protein which in turn stimulates synthesis of RNA polymerase, RNA, and subsequently the various proteins which comprise total aterine proliferation.

--

As noted by Gorski et aLe4-p. these facts are consonant with the molecular requirements for action of any hormone: 1) An interaction of the hormone with a receptor molecule of the target cell, 2) initiation of the primary response of the tissue to the hormone, and 3 ) amplification of this response by secondary effects to give rise to the ultimate expression of hormone action. it m t Zie remembered that most of the As was emphasized by Gorskie4-P* evidence to date, bearing on the question of 'primary or secondary response of mammalian cells to steroid hormones at the molecular level, does indeed rest on studies with inhibitors rather than direct measurements. However, this does not detract from the potential significance of the effects observed on the enzymes of nucleic acid metabolism, e.g. , the increased synthesis of nucleic acid per se and ultimately of organ specific proteins, or the potential application thereof in testing for new drug effects in simplified in vitro systems. Such screens might indeed result in the discovery of agents with unique biological properties which would go undete&ed in the standard whole animal model systems.

Protein hormones.- Studies of the type mentioned above performed by Wool and collaborator^^^ -pa e8 have demonstrated the ability of insulin to induce early RNA and protein synthesis in muscle. In a c a r e m series of experiments, these investigators show that the induction of nucleic acid and protein synthesis could not be reproduced by simple increased penetration of glucose or amino acids into the cells under the influence of this hormone. They further showed that the inductions by insulin were quantitatively markedly reduced when actinomycin D was administered in vivo although the percentage stimulation over the control by insulin w a s still noted in the presence of the inhibitor. Several recent studiesgg-m*1 8 1 ~1 7 7 ~ lDgclearly demonstrate the induction of glucokinase in rat liver by insulin. Although other factors such as glucose, thyroid and pituitary hormones m a y -' s o play a role, insulin is indeed

272 -

Sect. V

-

Topics in Biology

T a b a c h n i c k , Ed.

a major factor in this induction. Weber and co-workers showed that insulin also functions as an inducer of the other two key hepatic glycolytic enzymes: phosphofructokinase and pyruvate kinase113, '14. The insulin dose-dependent induction of the three hepatic key glycolybic enzymes1lS and recently a l s o that of glucose 6-phosphate dehydrogenase was describedlls. Inhibitor studies again demonstrate that de novo protein synthesis is involved as well as RNA synthesis in the induetion of these enzymes. Similar stuhies by Benjamin et al.lo2 on the induction by insulin of enzymes required for fatty acid synthesis in liver again suggest that de novo synthesis of protein is involved in this process, preceded by RNA induction. Ilyineg-po has further demonstrated a direct antagonistic effect by certain hormones on specific enzymes (e.g., hexokinase antagonism by corticosteroids and reversal by insulin). Clearly the effect of insulin on glucose penetration in muscle is not mediated by de novo protein synthesislog. Weber and co-workersgs'p' suggested that insulin may act as a physiological suppressor of hepatic gluconeogenic enzymes. Thus, the above studies on insulin strongly suggest a multiplicity of effects, some of which are instantaneous and do not involve de novo protein or RNA synthesis (e.g., glucose penetration, activation of hexokinase in the presence of steroid), while others occur more slowly and appear to be mediated by de novo synthesis of RNA and protein (e.g., induction of enzymes for glucose utilization and fatty acids synthesis), As in the case of the steroids, it is impossible at this point to state with certainty whether these represent primary or secondary results of hormone treatment, but it is clear that increased penetration of amino acids or glucose cannot 'account for the results observed. W O O ~ ~ ~ - P .raises the possibility that insulin increases the rate of transport of m-RNA from the nucleus to the cytoplasm as a primaxy event, followed by a secondary stimulation of nucle-ar RNA synthesis. Again, whether primary or secondary, the increased synthesis of m-IWA appears to be necessary to maintain a sustained effect of insulin on the induction of enzymes in the glucose and fatty acid pathways.

--

--

--

In addition to studies with insulin, similar investigations, again relying heavily on the use of inhibitors, have suggested that a critical site of action of growth hormonee4'P* lS3 and certain other peptide hormone^^^'^^ is the induction of de novo RNA and protein synthesis which ultimately results in the expression of their hormonal activity at the organ level. Studies by Tata and co-~orkers~~-P. 1739 los on the mode of action of thyroid hormone demonstrate an interesting sequence in which the induction of RNA polymerase precedes the production of RNAper se, which in turn precedes protein synthesis and tissue growth. Such data are impressive from the standpoint of the required sequence for ultimate expression of hormone effect. General Summary Incontrovertible evidence is now available in the biochemical literature demonstrating the significance of 1) DNA polymerase and double-stranded DNA in the replication of the genetic material and 2) RNA polymerase in the transcription of DNA to various classes of RNA which in turn are translated via gn elaborate mechanism to synthesize specific proteins. A variety of small molecules, notably antibiotic substances, have been shown to influence specific sites in these sequences, both in vitro and in whole organisms. The concentrations which are effective in vitro can be expected in many cases to be realized & vivo and likely do account for the primary biological effect observed with these agents. Studies with the corticosteroids, androgens and estrogens, as well as histones, protein hormones, thyroxin and vitamin D have implicated these agents 'as inducers of de novo protein synthesis in a variety of test situations either by direct stimulation of DNA transcription or RNA translation. Obviously, all

-

--

Chap. 24

Cell Metabolism

Smith

273 -

of the data collected are not completely consonant but a general summary of these investigations does favor the concept that some of these hormones induce the synthesis of RNApolymerase which in turn results in an increased rate of synthesis, and in some cases accumulation of,the various classes of RNA and ultimately of tissue-specific protein. Whether the effects on RNA synthesis are the primary ones of the hormones or secondary to some other more basic molecular action such as effect on permeability or translocation of RNA cannot be concluded from the data at hand. However, it is evident that 1) the effects are real, 2) they can be observed in vitro and in vivo and 3) the expression of the hormone-specific action in the target tissue is blocked by specific inhibitors of RNA and protein synthesis. It is of further interest that, in most cases, the hormonal effects on nucleaprotein metabolism cannot be demonstrated directly at the enzymatic level & vitro as can the inhibitory effect of the antibiotics. It is also evident from thevailable studies that certain of the hormone effects which appear to be instantaneous are not mediated by RNA and de novo protein synthesis (e.g., glucose penetration and tnsulin, fat mobilization and protein hormones, etc.) whereas the induction of enzymes and certainly the growth of tissue as the result of hormone administration are mediated by de novo macromolecule synthesis. Whether in the last analysis the effects observed to date are found to be primary, secondary or even further removed from the initial molecular effect of the hormone, the data in hand clearly point to the DNA transcription and RNA translation series of reactions as having profound importance in metabolic regulation mediated through hormones and antibiotics. In this regard, with the availability of purified enzymes, nucleic acid primers and means to measure products of these reactions, the medicinal chemist should look with renewed enthusiasm at the structureactivity studies in the hope of 1) divorcing multiple effects of hormones, 2) discovering new activities in previously unsuspected molecules, or 3) splitting activities in known types of chemical agents. The possible use of selective inhibitors to separate overall metabolic expression of drug or hormone effects in vivo should not be excluded. Likewise, concentration of agents in specific organs or selective binding at various sites within the cell, and competition for binding by related or unrelated molecules must be considered in the overall picture Of selective drug effects in the intact animal. and approaches open to the medicinal chemist seeking new chemotherapeutic agents.

1

Figure 1.

DNA

Act inomycin -Nogalamycin

P=-m-Rn

AA

t-RNA

y*1

-

, Porfiromycin IW

I

Mitomycin C

/1 ---

i-r-

AA-t-RNA

DNA

IlNovobiocin PhleomybhA

JkPr0tei.n Ribollomes --3iI5E&phenicol Poly sames Streptomycin Tetracycline Cyclohexlmide Puromycin Erythromycin Gougerotin

- - - ---

Protein + ribosome + t-FNA

Sect. V

274

-

T a b a c h n i c k , Ed.

Topics in Biology REFERENCES

68.

F. J . Bollum and R. B. Setlow. Biochim. Biophys. Acta 599 (1963) J. K. Setlow and R. B. Setlow. Nature 56c (1963) C. S c h o l t i s s e k and R. R o t t , Nature 204, 39 (1964) I. H. Goldberg, Am. 6. Med., 2,722 (1965) ( 5 ) A. Falaschi and A. Kornberg, Fed. Proc. 11, 940 (1964) 161 D. H. Smith and B. D. Davis, Biochem. Biophys. Re$. Com. 796 (1965) B. K. Bhuyan and C. G. Smith, Proc. Natl. Acad. Sci. 2, 566-572 (1965) D. 5. F i s c h e r , Biochem. Pharmacol. 2. 971 (1965) A. D. Wolfe and F. E. Hahn. Biochim. Biophys. Acta 146 (1965) 2 . Kucan and F. Lipmann. J. Biol. Chem. 516 (1964: S. M. Wolfe and A. S. Weisberger, Proc. Natl. Acad. S c i . 991 (1965) A. S. Weisberger, S. Wolfe, and S. Armentrout, J. Exptl. Med. 120. 161 (1964) A. S. Weisberger and S. Wolfe, Fed. Proc. 3, 976 (1964) S. M. Friedman and I. B. Weinstein, Fed. Proc. 4, 409 (1965) T. D. Brock. Fed. Proc. 3.965 (1964) S. Pestka, J. Biol. Chem. 367 (1966) J. Davies, L. Gorini, and B. D. Davis, Mol. Pharmacol. 1, 93 (1965) A. D. Wolfe and F. E. Hahn. Science 1445 (1964) K. Tanaka and H. Teraoka, Biochim. Biophys. Acta 204 (1966) D. Nathans, Fed. Proc. 9, 984 (1964) D. Nathans, Proc. Natl. Acad. S c i , 2, 585 (1964) M. A. Darken, Pharmacol. Rev. l6, 223 (1964) J. M. Clark, Jr. and A. Yen Chang, J. Biol. Chem. 24q. 4734 (1965) S. R. CaSJenS, A. J. Morris, and A. P. Stevens, Biochim. Biophys. Acta 677 (1965) N. Tanaka, K. Nagai, H. Yamaguchi, and H. Umezawa, Biochem, Biophys. Res. Corn. 21. 328 (1965) E. Calendi, A. DiMarco, M. Reggiani, B. Scarpinato, and L. V a l e n t i n i . Biochim. Biophys. Acta 25 (1965) M. C. Liau, L. S. Hnilica, and R. B. H u r l b e r t , Proc. Am. Assn. Cancer Res. 6, 40 (1965) (281 I. B. Weinstein. R. Chernoff, I. F i n k e l s t e i n , and E. Hirschberg, Mol. Pharmacol. 1. 297 (1966) Y. Kaziro and M. Kamiyama, Biochem. Biophys. Res. Com. 2,433 (1965) R. H. Connamacher and H. G. Mandel. Biochem. Biophys. Res. Com. 20. 98 (1965) M. Hierowski, Proc. N a t l . Acad. S c i . 594 (1965) T. J. Franklin, Biochem. J. 90, 624 (1964) 0. Suarez and D. Nathans, Biochem. Biophys. Res. Com. l8, 743 11965) H. L. EMis and M. Lubin, Science &, 1474 (1964) A. C. T r a k a t e l l i s , M. Montjar, and A. E. Axelrod, Biochemistry 5. 2065 (1965) B. Colombo, L. F e l i c e t t i , and C. Baglioni, Biochem. Biophys. Res. Corn.

m,

la,

a,

a,

a,

m,

z,

114.

108,

x.

z.

2,

389 (1965)

z,

M. B. Sporn. C. W. D i n g m a n . H. Phelps. and G. N. Wogan, Science 1539 (1966) F. 0. Wettstein. H. Noll, and S. Penman, Biochim. Biophys. Acta 8 J 525 (1964) L. L. Bennett, Jr., V. L. Ward, and R. W. Brockman. Biochim. Biophys. Acta 478 (1965) C . Calvori, L. F r o n t a l i , L. Leoni. and G. Tecce, Nature 417 (1965) J. W. Yarbro, B. J. Kennedy, and C. P. Barnum, Cancer Res. &. 36 (1966) A. Weissbach and A. L i s i o , Biochemistry 2. 196 (1965) M. I. Lerman and M. S. Benyumovich. Nature 1231 (1965) W. E. P r i c e r . Jr.. and A. Weissbach, Biochemistry 2. 200 (1965) S. Segal, 0. Davidson. and-K. Wada, Proc. Natl. Acad, Sci. 3, 782 (1965) G. Talwar, S. Segal. A. Evans, and 0. Davidson, Proc. Natl. Acad. Sci. 1059 (1964) A. Korner; R. Mayne and J. Barry; 0. Barnabei, C. Ottolenghi and U. Tomasi, Biochem. J. 2.21P. 24P, 25P resp. (1965) P. Karlson, Conference on Mechanismof Hormone Action. Proceedings, G. Thieme Verlag. S t u t t g a r t , Germany,(1965) N. Lang and C. E. Sekeris. L i f e Sci. 2, 161, 169 (1964) 0. Barnabei, B. Romano, and G. DiBitonto, Arch. Biochem. Biophys. 266 (1965)

x,

a.

z,

=,

x,

Chap. 24

Cell M e t a b o l i s m

275 -

Smith

L. Garren, R. Howell, and G. Tomkins, J. Mol. B i o l .

2,

100 (1964)

M. Hierowski and Z. Kurylo-Borowska, Biochim, Biophys. Acta H. L. Ennis. J. Bact. j & 1102, 1109 (1965)

s, 578,

590 (1965)

w.

W. D. Wicks and F. T. Kenney, Science 1346 (1964) S. Liao, J. Biol. Chem. 240. 1236 (1965) N. Lang and C. E. Sekerkis, L i f e S c i . 3. 391 (19648 T. P. Lin. Science 333 (1966) D. J. Begg and H. N. Munro. Biochem. J . 33P (1965) D. J. Begg and H. N. Munro, Nature 483 (1965) J. Vernikos-Danellis and M. H a l l , Nature 766 (1965) E. D. Bransome, Jr. and E. Chargaff, Biochim. Biophys. Acta. jlI 180 (1964) M. E. Dahmus and J. Bonner, Proc. Natl. Acad. S c i . 2. 1370 (1965) M. Sluysar, P. J. Th-ang, and P. Emmelot, Biochim. Biophys. Acta 249 (1965) J. Brachet, Nature 204, 1218 (1964) L. Gurley, J. I r v i n . and D. Holbrook, Biochem. Biophys. Res. Com. 2.527 (1964) Ru-Chih Huang and J. Bonner. Proc. Natl. Acad. S c i . 2, 960 (1965) S. Schwimmer and J. Bonner, Biochim. Biophys. Acta 67 (1965) V. A l l f r e y , R. Faulkner, and A. Mirsky, Proc. Natl. Acad. Sci. 2,786 (1964) I. Weinstein, R. Chernoff. I. F i n k e l s t e i n , and E. Hirschberg, Proc. Am. Assn. Cancer Res. 6, 68 (1965) M. Liau, L. i n i l i c a , and R. Hurlbert, Proc. Natl. Acad. S c i . 626 (1965)) A. L. Koch, Nature 3, 800 (1965) R. L. S o f f e r , J. Mol. Biol. lo, 359 (1964) L. Sokoloff, C. F r a n c i s , and P. Campbell, Proc. Natl. Acad. Sci. 728 (1964) J. Z u l l , E. Czarnowska-Misztal. and H. DeLuca, Science 182 (1965) J. Zull. E. Czarnowska-Misztal, and H. DeLuca. Proc. Natl. Acad. Sci. 2,177

z,

s, x, x,

108,

108.

z,

z.

m,

(1966 1

x,

E i s e n s t e i n and M. Passavoy. Proc. SOC. Exp. Biol. Med. 77 (1964) E. Pegg and A. Korner. Nature 904 (1965) R. Tustanoff and W. B a r t l e y , Biochem. J. jl,595 (1964) Creasey and M. Markiw. Biochim. Biophys. Acta &, 601 (1964) T. Castles and H. Williamson, Proc. SOC. Exp. Biol. Med. 2.308 (1965) R. E. Olson, Science 926 (1964) J. I. C l i f f o r d and K. R. Rees. Nature 312 (1966) G. P. Wheeler, Cancer Res. 22, 651 (1962) P. D. Lawley and P. Brookes, Biochem. J. &3, 127 (1963) Symposium Hormonal Control P r o t e i n Biosyn., J. Cell. Comp, Physiol. Suppl. 1. 1-183 (1965) Cold Spring Harbor S m . Want. Biol. 28 (1963) Natl. Cancer I n s t . Moriograph 12 (1963) H. Munro and J. B. Allison, Mammalian P r o t e i n Metab., V O l . I. (1964) F. H. Crick, B r i t . Med. Bull. 2l, 183 (1965) W. Kersten, H. Kersten, W . Szybalski. and M. F i a n d t , Biochemistry 5, 236 (1966) J. Watson, Molecular Biology of t h e Gene, W. A. Benjamin. I n c . . N.Y. Amsterdam, (1965) (91) F. Bollum, J. Cell. Comp. Physiol. 62, Suppl. 1, 61 (ig63) (92) H. Umezawa, Recent Advan. Chem. Biochem. A n t i b i o t . , Tokyo, (1964 44 (1963 (93) T. H. Goldberg. E. Reich, and M. Rabinowitz, Nature 684 (1964) (94) E. Reich, Science (95) L. S. Lerman, J. Mol. Biol. 2, 18 (1961) \ 1631 (1962 (96) C . Levinthal, A. Keynan, and A. Higa, Proc. Natl. A c a . Sci. (97) H. P i t o t . C. Peraino, C. Lamar, and A. Kennan. Proc. Natl. Acad. s c i . 845 (1965) (98) C. Smith, W. Lummis, and J. Grady, Cancer Res. 9, 847 (1959) (99) G. Weber, R. Singhal, N. Stamm, E. F i s h e r , and M. Mentendiek, Advan. Enzyme Regula%ion 2, 1 (1964) (100) 0. Greengard, M. Smith, and 0. Acs, J. Biol. Chem. 2J8, 1548 (1963) (101) P. Ray, D. F o s t e r , and H. Lardy, J. Biol. Chem. 3396 (1964) 110.2) W. Benjamin and A. Gellhorn. J. Lipid Res. 1, 285 (1966) l l c (1964) (103) J. Castles and I. Wool, Biochem. J. (104) C. Kidson and K. Kirby. Nature 599 (1964) 588 (1966) (105) A. Skalka. A. Fowler, and J. Hurintz, J. Biol. Chem. R. A. E. W.

a,

a,

z,

3

a.

9,

s,

a,

s,

a,

a,

s,

27 6

Sect. V

-

Topics in Biology

T a b a c h n i c k , Ed.

W. Dietz, T. Cook, and W. Goss, J. Bact. 2,768, 774, 780 (1966) D. Vazquez. Biochim. Biophys. Acta 277 (1966) M. G e l l e r t . C. Smith, D. N e v i l l e , and (t. F e l s e n f e l d , J. Mol. Biol. l l . w5 (1965) J. Tata and C. Widnell. Biochem. J. 98, 604, 621 (1966) H. Yamaguchi and N. Tanka, Nature 201, 499 (1964) J. Davies, P. Anderson, and B. Davis, Science M , 1096 (1965) J. R. F l o r i n i , H. H. Bird, and P. H. Bell, J. Biol. Chem. 241, 1091 (1966) 0. Weber, N. B. Stamm, and E. A. F i s h e r , Science 65 G. Weber and R. L. Singhal, L i f e Sciences 4, 1993 (1965) 0 . Weber, M. A. Lea, E. A. Fisher, and N. B. Stamm, Enzymol. Biol. Clin. 6 . i n p r e s s (1966) G. Weber 3nd H. J. Hird Convery, L i f e Sciences 4, i n p r e s s (1966)

g,

a,

(1965)

27 7 Chapter 25.

Agents Which Affect Enzyme Activity

A. Horita, University of Washington School of Medicine S e a t t l e , Washington

The discovery of numerous drugs acting on s p e c i f i c enzyme systems has res u l t e d i n t h e rapid development of biochemical pharmacology. This is evident from t h e newer c l a s s i f i c a t i o n s of drugs such as t h e i n h i b i t o r s of carbonic anhydrase, choline esterase, and mnoamine oxidase. Probably an even greater number of agents act on enzyme systems, o r a t least at t h e biochemical level. However, our lack of knowledge of d e t a i l e d biochemical mechanisms prevents us fran describing t h e i r actions on s p e c i f i c enzyme systems. Such phenamena as drug effects on transport mechanisms, on uptake and release of transmitters, and on c o n t r a c t i l e mechanisms most l i k e l y occur by drugs acting on enzymes. The biochemical mechanisms involved i n these events are not w e l l understood, but newly acquired evidence points t o t h e involvement of enzymatic processes. Eventually it may be possible t o explain many, although by no means a l l , heret o f o r e unknown mechanisms a t t h e biochemical level. Several excellent reviews on t h e effect of drugs on enzyme o r biochemical systems have been written earlier.1 92 This report a l s o discusses drug effects on enzyme systems, but a comprehensive coverage of t h e known enzymes is not feasible. Rather, t h e pertinent literature froln late 1964 through 1965 w i l l be reviewed i n t h e selected area of drug effects on enzyme systems which are concerned with t h e metabolism of several of t h e biogenic amines. This area was selected because it represents sane of t h e best examples of current thought on the biochemical approach t o pharmacology. INHIBITORS OF CATECHOLAMINE METABOLISM The pathway of epinephrine and norepinephrine metabolism is now w e l l established. Both amines are derived fran t h e amino acid tyrosine in t h e f o l lowing sequence: Tyrosine + DOPA + Dopamine +Norepinephrine +Epinephrine

I

I

1

1

I

M o n o a m i n e O x i d a s e Catechol 0-Methyl Transferase I n a c t i v e

1

P r o d u c t s

Tyrosine Hydroxylase Inhibition I n t h e above metabolic scheme of catecholamine formation t h e conversion of tyrosine t o DOPA by tyrosine hydroxylase represents t h e rate limiting step.3 The properties of t h i s enzyme were described i n d e t a i l by Nagatsu, Levitt and Ude~x€riend~ who , ~ found it d i s t r i b u t e d i n various organs and associated with p a r t i c l e s sedimenting a t 15,000 x g. I n beef brain tyrosine hydroxylase is concentrated i n t h e caudate nucleus; t h i s is of i n t e r e s t s i n c e t h e caudate is thought t o contain t h e adrenergic synaptosomes.6 Because of its r e l a t i v e l y low a c t i v i t y i n vivo, tyrosine hydroxylase appears t o be an i d e a l s i t e of a c t i o n

278

Sect.

V

-

Topics in Biology

T a b a c h n i c k , Ed.

for drugs to inhibit synthesis of norepinephrine. Already several agents have been discovered that act as inhibitors of tyrosine hydroxylase. Compounds described as inhibitors of purified beef adrenal tyrosine hydroxylase include amethyl tyrosine, 3-halogenated tyrosine derivatives, mono- and di-iodotyrosines, and several catechol compounds, especially 3,4-dihydroxyphenylpropylacetamide (H 22/54). The mechanism of inhibition of H 22/54 appears to be a competitive action with the cofactors of tyrosine hydroxylase, since it was reversed by dhethyltetrahydropteridine. The anti-enzyme effect of 3-iodo-L-tyrosine vivo was also irvestigated,8'9 and a decrease in norepinephrine of certain tissues as a result of tyrosine hydroxylase inhibition in the intact animal has been demonstrated. The latter investigators also found that in rats, the pressor response to the indirect acting sympathomimetic agent, tyramine, was markedly attenuated after the administration of 3-iodotyrosine. This tyramine blockade may be presumed to be the result of the diminished stores of tissue norepinephrine.

-

Equally interesting as a tyrosine hydroxylase inhibitor is the compound a-methyl-p-tyrosine (NPT). Unlike the a-methyl-m-tyrosine (MMT), MPT does not cause a release of endogenous catecholamines, nor is it decarboxylated to form a-methyl-p-tyramine. The compound causes a lowering of tissue catecholamines by inhibiting tyrosine hydroxylase and not by displacement of endogenous amines as in the case of the meta-hydroxylated congener. As expected, MPT also prevented the rise in tissue catecholamines after administration of the MA0 inhibitors. The preliminary pharmacological properties of MPT are also of interest in that certain reserpine-like effects were demonstrated. This agent may be a valuable aid in clarifying the much debated mechanism of reserpine. Weisman and Koell found that NPT was also able to block the stimulant properties of amphetamine, but potentiated the avoidance-disrupting properties of chlorpromazine. They interpreted these results to mean that, at least in part, amphetamine exerted its central effect indirectly via levels of free norepinephrine. In man NPT decreased catecholamine synthesis in subjects with pheochromocytoma or essential hypertension as measured by changes in urinary metabolites. A fall in blood pressure was noted in the pheochrcxnocytoma patients, but not in those with essential hypertension. However, in all cases sedation was present. Drugs such as MPT have potential not only as powerful tools for investigating the biochemical steps in catecholamine synthesis and regulation, but also as possible therapeutic agents in disorders of catecholamine biochemistry. However, one needs to temper this enthusiasm until further investigations confirm the specificity of this inhibition. It is possible that other metabolic pathways of tyrosine may also be affected and could lead to other complications.

DOPA Decarboxylase Inhibition The conversion of DOPA to dopamine is catalyzed by DOPA decarboxylase, a pyridoxal dependent enzyme possessing a rather broad substrate specificity. This last property has resulted in the proposal of the alternate nomenclature of "arcnnatic amino acid decarboxylase," a term which is appropriate in view of its ability to decarboxylate 0- and m-tyrosine, DOPA, and 5-hydro~ytryptophan,~ and possibly several other aromatic amino acids.

Chap. 25

Enzyme Affectors

Horita

27 9 -

Over t h e past decade a considerable number of DOPA decarboxylase inhibit o r s have been described, but i n no instance has it been possibl'e t o produce adequate inhibition in vivo t o cause depletion of t i s s u e catecholamines. The earlier view of t h e mechanism of action of a-methyl DOPA and a-MMT acting v i a decarboxylase inhibition is no longer tenable. These a-methylated amino acids are i n h i b i t o r s of amino acid decarboxylase, but they themselves are a l s o decarboxylated t o varying degrees, r e s u l t i n g i n t h e formation of a-methyl dopamine and a-methyl-m-tyramine, respectively. These amines then become hydroxylated a t t h e $-carbon, and because of t h e i r chemical s i m i l a r i t y t o norepinephrine, displace t h e sympathetic mediator in t h e a h n e r g i c nerve granules. I n t h i s location they act as norepinephrine s u b s t i t u t e s and may be released by nerve stimulation and may or may not be released by c e r t a i n pharmacological agents.14-18 Thus, t h e anti-decarboxylase a c t i v i t y of these a-methylated amino acids plays an i n s i g n i f i c a n t role i n t h e pharmacological and biochemical actions t h a t they produce. Another d i f f i c u l t y i n evaluating t h e decarboxylase i n h i b i t o r a c t i v i t y of these agents is stressed by Moran and S o u r k e ~who ~ ~found t h a t they inhibit e d several enzymes involved i n amino acid m e t a b o l i s m . Since t h e finding of t h e anti-decarboxylase a c t i v i t y of t h e a-methyl amino acids, more potent and s p e c i f i c agents have been described. Notable among these are N-(3-hydroxybenzyl)-N-methylhydraoine (NSD 1034) and N-(DL-seryl)-N'-(2, 3,4-trihydroxybenoyl) hydraoine (RO 4-4602). Both of these compounds are able t o prevent t h e conversion of administered DOPA t o dogamine, but affect only poorly t i s s u e l e v e l s of dopamine o r norepinephrine. ' Dopamine 8-Hydroxylase Inhibition The f i n a l s t e p i n t h e biosynthesis of norepinephrine is accomplished by t h e hydroxylation of t h e $-carbon of dopamine by dopamine 8-hydroxylase. An excell e n t review of t h e biochemistry of t h i s system has been compiled recently,22 and t h e reader is referred t o it f o r d e t a i l s on t h i s enzyme system. The authors have a l s o canmented on possible i n h i b i t o r s , but conclude t h a t t o date no competit i v e i n h i b i t o r of dopamine 8-hydroxylase which is e f f e c t i v e i n vivo is known. It should be emphasized t h a t t h e substituted benzyloxyamines and benzylhydrazines as described earlier23 were e f f e c t i v e i n v i t r o , but poorly or not effect i v e i n lowering endogenous norepinephrine l e v e l s . A t present t h e only $-hydroxylase i n h i b i t o r s which are e f f e c t i v e i n vivo a r e t h e copper chelating agents, and disulfiram has received the greatest use f o r t h i s purpose. Relatively l a r g e doses of disulfiram w i l l lower t i s s u e levels of norepine hrine but w i l l raise d ~ p a m i n e . ~ Similar ~ - ~ ~ observations w e r e made by CollinsE7 who used t h e com ound diethyldithiocarbamate, a metabolite a l s o demonstrated t h a t disulfiram retarded of disulfiram. Bhagat and G i l l i a m ' 8 t h e norepinephrine r e p l e t i o n i n r a t heart after depletion by qrramine p r e t r e a t ment, suggesting t h a t norepinephrine synthesis had been blocked. With t h e lowering of t i s s u e norepinephrine due t o i n h i b i t i o n of dopamine $-hydroxylase by disulfiram, t h e physiological response t o nerve stimulation of organs innervat e d by adrenergic fibers is reduced c ~ n c o m i t a n t l y . ~ ~

280

Sect. V

-

Topics in Biology

Tabachnick,Ed.

Monoamine Oxidase Inhibition In many organs innervated by adrenergic f i b e r s t h e regulation of dopamine and norepinephrine l e v e l s is t o a l a r g e extent t h e function of monoamine oxidase (MAO). This is apparent from t h e increase i n mine levels r e s u l t i n g after blockade of MAO. The enzyme works through an oxidative deamination process and, i n t h e presence of aldehyde dehydrogenase, forms the pharmacologically inactive carboxylic acid metabolite of t h e mine.

I n s p i t e of t h e declining therapeutic use of MA0 inhibitors, interest i n these compounds s t i l l prevails as evidenced by t h e numerous recent scientific publications. The picture of MA0 is s t i l l f a r from complete, and t h e inhibit o r s are useful t o o l s i n i t s biochemical and physiological investigations a s well a s in determining t h e mechanism of other mine-related compounds, such as reserpine, a-NMT, and t h e i n d i r e c t acting sympathomimetic amines. t h e past two Several important c l i n i c a l contributions i n t h i s area d u r i years w e r e (1) t h e reviews on t h e t o x i c i t y of the MA0 i n h i b i t o r?Or31 s i n which an attempt was made t o explain t h e d i r e c t adverse reactions as w e l l as t h e interactions t h a t occurred between t h e MA0 i n h i b i t o r s and various substances, and (2) a comprehensive review on t h e c l i n i c a l pharmacology of tranylcypromine.32 From these and previous articles it is q u i t e apparent that a l l of t h e long acting MA0 i n h i b i t o r s have many actions, and most of these are r e l a t e d t o t h e inhibition of MA0 and t h e subsequent a l t e r a t i o n of amhe levels. This is probably t r u e f o r t h e hypotensive action which is inherent with most of t h e long acti MA0 inhibitors, including t h e newest addition, modaline s u l f a t e (W3207B1.' The mechanism of t h e blood pressure lowering effect of t h e MA0 inh i b i t o r s has been a t t r i b u t e d t o various causes, but t h e most attractive hypothesis based on MA0 inhibition is t h a t of Kopin and associates.34y35 These authors postulate that after MA0 i n h i b i t i o n an accumulation of octopamine (B-hydroxytyramine) occurs a t adrenergic nerve terminals because of t h e l o s s of t h e normal deamination pathway. The octopemine i s thought t o replace p a r t of t h e normal s t o r e s of sympathetic mediator, and upon stimulation, it is released as a "false transmitter." Octopamine, however, is a t best a poor s u b s t i t u t e f o r norepinephrine, and t h i s r e s u l t s i n a diminished sympathetic response and hypotension. Other B-hydroxylated sympathomimetics have subsequently been found t o behave i n a s i m i l a r manner t o octopamine i n replacing t h e normal sympathetic transmitter. 36 The "false transmitter" concept has received support from Farmer3' who demons t r a t e d t h e impairment of sympathetic nerve responses after administration of DOPA and dopamine i n t h e presence of MA0 inhibitor. The literature abounds with descriptions of observations of t h e interactions of MA0 i n h i b i t o r s with d i f f e r e n t substances. Same of t h e interactions are d i f f i c u l t t o i n t e r p r e t ; t h i s is especially t h e case with inhibitors having t h e phenethylamine or phenethylhydrazine structures. Such agents have the a b i l i t y t o (1) i n h i b i t MAO; (2) cause increase i n t i s s u e l e v e l s of 5-HT, dopamine, norepinephrine, and probably amines which are normally absent, such as octopamine, t r y m i n e , and tryptamine; (3) cause release of norepinephrine and dopamine because of its i n d i r e c t sympathomimetic effect; and (4) produce other d i r e c t effects unrelated t o MA0 inhibition, such as an amphetamine-like c e n t r a l stimulation. It is no wonder then t h a t phenelzine or tranylcypromine could enhance t h e t o x i c i t y of amphetamine, probably due t o summation of effects of

Chap. 25

Enzyme Affectors

Horita

28 1 -

t h e two drugs.38 This also may be t h e case with t h e potentiation of t h e hyperthexmic response of amphetamine, ephedrine and deoxyephedrine after MA0 inhibition.39 Inhibition of t h e enzyme cannot be the s o l e mechanism, f o r these sympathomimetic substances are not substrates of MAO. However, t h e i n t e r a c t i o n occurring between t h e MA0 i n h i b i t o r s and t h e antidepressants of t h e imipraminelike agents appears far more complex."0 The protection against depletion of catecholamines and/or 5-hydroxytrypta-

mine (5-HT) by reserpine, tetrabenazine and guanethidine by t h e MA0 i n h i b i t o r s is well known, although t h e mechanisms of these i n t e r a c t i o n s are still not clear. One explanation is t h a t t h e e x c i t a t i o n observed i n mice given reserpine following a MA0 i n h i b i t o r consists of two phases--the first a t t r i b u t a b l e t o t h e l i b e r a t i o n and accumulation of catecholamines, and second, of S H T , i n t h e a c t i v e form i n t h e c e n t r a l nervous system as a consequence of MA0 inhibition.41 Fielden and Green42 a l s o a t t r i b u t e d t h e protection by iproniazid of t h e depletion of heart

norepinephrine induced by guanethidine t o t h e i n h i b i t i o n of MA0 and not t o a "bretylium-likefl action of t h e i n h i b i t o r . I n s p i t e of these evidences it is s t i l l d i f f i c u l t t o resolve whether t h e "protection" of mine depletion by reserpine o r guanethidine is i n fact protection from release, o r protection from degradation of t h e released amine. The l a t t e r p o s s i b i l i t y would appear more reasonable, but work by Clementi43 as w e l l as earlier investigations which demonstrate protection by iproniazid of t h e morphological i n t e g r i t y of adrenal medull a r y granules from reserpine requires f u r t h e r c l a r i f i c a t i o n . Other recent studies also point out t h e v a r i a t i o n of complexities of t h e actions of MA0 i n h i b i t o r s , and these generally conclude t h a t the anti-MA0 a c t i v i t y itself is not always t h e primary mechanism f o r t h e effects t h a t they

observe .44-4*

I n discussing drug i n t e r a c t i o n s with MA0 i n h i b i t o r s it should be pointed

out t h a t under certain conditions t h e action of t h e MA0 i n h i b i t o r may be modi-

f i e d . Thus, t h e red blood cell and its constituents, heme and hemoglobin, are capable of decreasing t h e anti-MA0 a c t i v i t y of several of t h e hydrazine-type It was concluded t h a t t h e i n h i b i t o r s , but not t h e noa-hydrazine hydrazines reacted with t h e heme portion of t h e red c e l l t o become chemically inactivated. A similar observation was made by Vitek and Rysanek'O who found t h a t D-cycloserine blocked the actions of isoniazid but not pheniprazine and tranylcyprcunine. The authors concluded t h a t isoniazid probably formed an inactive hydrazone with cycloserine. Antagonism of t h e i r r e v e r s i b l e i n h i b i t i o n of MA0 may a l s o be produced by p r i o r exposure of t h e enzymes t o reversible inh i b i t o r s . HoritaS1 took advantage of t h i s fact t o produce a s e l e c t i v e block of brain MA0 by pretreating rats with BW 392C60 ( a highly charged r e v e r s i b l e inhib i t o r of MA01 and followed by pheniprazine. I n so doing t h e peripheral MA0 w a s protected from t h e long acting effects of pheniprazine, while brain MA0 was inhibited without hindrance by t h e reversible i n h i b i t o r . Considerable work on t h e nature of i n h i b i t o r s of MA0 a c t i v i t y s t i l l continues. Hustzi and BorsyS2 investigated t h e anti-MA0 a c t i v i t y of diethyltryptamine and its analogues and found t h a t differences i n a c t i v i t y occurred. As with earlier authors they noted t h a t t h e s u b s t r a t e used i n t h e preparation also affected g r e a t l y t h e inhibitory a c t i v i t y . The variance of i n h i b i t o r a c t i v i t y with d i f f e r e n t substrates w a s a l s o noted by Burger and NaraS3 who

28 2 -

Sect. V

-

Topics in Bkology

T a b a c h n i c k , Ed.

employed various enzyme preparations, including pure beef plasma MAO. Their data are especially i n t e r e s t i n g because of t h e demonstration that t h e anti-MA0 a c t i v i t y of several i n h i b i t o r s varied so greatly depending upon t h e source and purity of t h e enzyme preparation. Fuller and WaltersS4 described t h e inhibition of MA0 by s u b s t r a t e amines and t h e i r a-methyl derivatives. No consistent relat i o n s h i between degree of i n h i b i t i o n and efficacy as substrate could be found. H ~ r i t a , ~on ’ t h e other hand, claims ‘that c e r t a i n of t h e arylalkylhydrazines, especially 6-phenethylhydrazine (phenelzine) and y-phenylpropylhydrazine, i n addition t o being i r r e v e r s i b l e i n h i b i t o r s of MAO, may serve as substrates of t h i s enzyme p r i o r t o exerting t h e i r i n h i b i t o r action. The evidence f o r t h i s hypothesis is based around t h e l o s s of anti-MA0 a c t i v i t y of phenelzine when exposed t o preparations of MAO, and t h e prevention of t h i s l o s s by p r i o r treatment with other MA0 inhibitors. Similar results w e r e seen i n t h e i n t a c t r a t . Catechol 0-Methyl Transferase (COMT) Inhibition such less work has appeared on t h e inhibition of COMT. Perhaps t h i s is t h e case because of t h e a v a i l a b i l i t y of fewer s p e c i f i c i n h i b i t o r s of t h e enzyme. The importance of COMT i n t h e metabolism of catecholamines is equally as great as for MAO, although t h e s p e c i f i c metabolic roles are q u i t e d i f f e r e n t . COMT, which o-methylates t h e r i n g OH- groups of catecholamines, generally plays a greater role i n t h e metabolism of endogenously administered o r released catecholamines, while MA0 is s a i d t o be m o r e important f o r t h e i n t r a c e l l u l a r regul a t i o n of t h e amines.

Recent investigations on t h e i n h i b i t o r a c t i v i t y of various compounds as possible COMT i n h i b i t o r s include those of Ross and Halja~maa..~~ Using t h e extract from mouse brain and t r i t i a t e d norepinephrine as substrate they found the esters of g a l l i c acid, D-catechin, 4-methylesculetin, 3,4-dihydroxymandelamide, 8-hydroxyquinoline and Y-methyltropolone w e r e as a c t i v e o r greater than pyrogallol as i n h i b i t o r s of COMT. The same authors also determined t h e i n vivo efficacy of these compounds, but unlike the i n v i t r o r e s u l t s , they observed t h a t t h e compounds varied in potencies between brain and peripheral structures.” Furthemore, many of t h e compounds t e s t e d interfered with other adrenergic mechanisms, such as stimulation o r blockade of adrenergic +receptors. Pyrogallol appeared t o be t h e most s p e c i f i c i n h i b i t o r f o r brain COHT i n vivo, while the tmpolones w e r e most e f f e c t i v e against COMT i n peripheral structures. I n intact animals, Burba and MurnaghanS0 found t h a t demethylpapaverine potentiated t h e epinepbine-induced pressor responses and isoproterenol-induced depressor responses and correlated t h i s t o t h e inhibition of COMT which they had determined i n vitro. The COMT system should not be confused with the microsomal enzyme t h a t omethylates dihydroxy compounds. 59 s b 0 A number of differences exists between t h e two systems, including d i s t r i b u t i o n , species, pH optima, and substrate specificity. INHIBITORS OF 5-HYDROXYTRYPTAMINE (SEROTONIN, 5-HT) METABOLISM Although t h e height of popularity enjoyed by 5-HT i n the 1950’s as an area of pharmacological, biochemical and pathological research has subsided, much i n t e r e s t and investigative a c t i v i t y continue i n an attempt t o elucidate its

Enzyme Affectors

Chap. 25

Horita

28 3 -

properties and possible biological functions. Several outstanding reference sources on the various aspects of 5-HT and other indoles are available.61,62 The most recent comprehensive coverage on this subject is the monograph by Garattini and Valzelli6 which includes references through 1964. The metabolic pathway of 5-HT, like the catecholamines, involves the steps of hydroxylation and decarboxylation in its synthesis, while deamination plays the major role of degradation. Several other systems are also known, but these probably play a lesser part in the regulation of 5-HT metabolism. The following scheme represents the major steps in the formation and breakdown of 5-HT. Tryptophan

+

5-Hydroxytryptophan + 5-Hydroxytryptamine (Serotonin)

5-Hydroxyindole Acetic Acid

+

Minor Inactivation Pathways

Inactive Products

Currently, the enzyme involved in the first step of 5-HT biosynthesis, namely tryptophan hydroxylase, is undergoing experimental scrutiny. Its ability to transform tryptophan to 5-hydroxytryptophan (5-HTP) in intact animals was seen earlier, but until recently this enzyme could not be demonstrated in vitro. Tryptophan hydroxylase activity has now been reported to occur in dog brain,64 rat liver,65 and in neoplastic mast cells of the mouse.66-68 It should be stressed that in the liver and neoplastic mast cell the enzyme may not represent the true tryptophan hydroxylase of brain tissue since the former also act as a phenylalanine hydroxylase. In vivo conversion of tryptophan by brain to eventually form 5-HT has been demonstrated. 69-72

--

Inhibitors of tryptophan hydroxylase have been sought, but thus far highly active agents are not known. a-Methyl-DOPA, a, 8, B-trimethyl-DOPA, and to some extent, a-propyl-3,4-dihydroxyphenylacetamide have been found to inhibit rat liver tryptophan hydroxylase.7 *74 Fuller75 also surveyed a number of compounds as potential hydroxylase inhibitors, and many of the tyrosine and phenylalanine derivatives showed variable activities in vitro. 6,7-Dihydroxycoumarin demonstrated considerable in vitro activity, exerting some 60% inhibition at a concentration of 10'6M. 5-Hydroxytryptophan decarboxylase, the enzyme responsible for the conversion of 5-HTP to 5-HT, is presumably identical with the aromatic amino acid decarboxylase discussed under the catecholamines. Inhibitors which were described under the aromatic amino acid (DOPA) decarboxylase would also apply here. The degradation of 5-HT occurs primarily via monoamine oxidase, and this enzyme system and its inhibitors have been described earlier. 5-HT serves as a better substrate than norepinephrine; consequently, the inhibition of MA0 in intact animals generally causes a more rapid and greater increase in tissue levels of 5-HT. This increase appears to occur within specific serotonergic neurons. 76

28 4

Sect. V

-

Topics in Biology

T a b a c h n i c k , Ed.

INHIBITORS OF HISTAMINE METABOLISM Like the other two biogenic amines discussed above, histamine biosynthesis involves the decarboxylation of its amino acid precursor, histidine. The enzyme, histadine decarboxylase, is not identical with the aromatic amino acid decarboxylase which converts both DOPA and 5-HTP to their corresponding amines. The breakdown of histamine is catalyzed by the enzymes d i d n e oxidase and imidazole N-methyl transferase. The former system oxidatitrely deaminates histamine, much like MA0 on monoamines, while the latter enzyme inactivates histamine by methylating on the nitrogen at the 1-position. Upon formation of this intermediate it becomes susceptible to deamination via MA0 to undergo further transformation. With the resurgence of Interest in the possible biological role cf histamine a8 a regulator of the microcirculation and as a neurotransmitter, studies on the manipulation of histamine synthesis by inhibiting histadine decarboxylase are increasing. Actually there are at least two histidine decarboxylase enzymes present in maumalian tissues. Only the specific histidine decarboxylase is of concern here, for it appears to be the primary source of endogenous tissue histamine. Inhibition of this enzyme has been produced both in vitro and in vivo by D-2-hydrazino-3-C4-imidazolyll propionic acid (hydrazinoxistidine1 and by 4brom0-3-hydroxy benzyloxyamine (NSD-1055). 77 After administration of these compounds tissue levels of histamine decrease to varying degrees. L e ~ i n e ~ ~ also demonstrated the effectiveness of these compounds to reduce the basal secretion of acid in the rat stomach, presumably through a decreased formation of histamine. Thus, compounds which are effective inhibitors of specific histidine decarboxylase may aid in determining the biological functions of histamine. In addition to inhibition, histidine decarboxylase also undergoes the unusual property of increased activity under certain conditions, including the influence of various pharmacological agents. Adrenergic agents such as epinephrine, pronethalol, DOPA and dopamine have been shown to increase histamine formation in mice, presumably through an increased activity of histidine decarboxylase. 79 D M n e oxidase (DAO) has long been known to be a major degradation pathway for 'histamine and other diamines. Being a pyridoxal dependent enzyme earlier investigators employed carbonyl trapping agents, such as semicarbazide, isoniazid and aminoguanidine as inhibitors. The last named is still one of the most potent diamine oxidase inhibitors known. Other recent additions to the list of inhibitors (most of which are poorer than aminoguanidine) of diamine oxidase include mescaline and papaverine. REFERENCES 1.

E.A.

Zeller and J.R. Fouts, Ann. Rev. Pharmacol.,

2.

L. Decsi, Prom. Drmg Res., g, 53 (1965).

3.

Ther., 148, 1 (1965).

3,

9 (1963).

M. Levitt, S. Spector, A. Sjoerdsma and S. Udenfriend, J. Pharmacol. Exp.

Enzyme Affectors

Chap. 25 4.

28 5 -

Horita

T. Nagatsu, M. L e v i t t and S. Udenfriend, Biochem. Biophys. Res. Commun., 14. 543 (1964).

--

-

5.

T. Nagatsu, M. L e v i t t and S. Udenfriend, J. Biol. Chem., 239, 2910 (1964).

6.

S.P. Bagchi and P.L. McGeer, L i f e S c i . ,

7.

S. Udenfriend, P. Zaltman-Nirenberg and T. Nagatsu, Biochem. Pharmacol., 14, 837 (1965).

5,

1195 (1964).

-

8.

M. Goldstein and Z. Weiss, L i f e Sci., 4, 261 (1965).

9.

S. Spector, R.O. Mata, A, Sjoerdsma and S. Udenfriend, Life Sci.,

10.

(1965).

k,

1307

147,

S. Spector, A. Sjoerdsma and S. Udenfriend, J. Pharmacol. Exp. Ther., 86 (1965).

11. A. Weismann and B.K.

Koe, L i f e Sci.,

z, 1937 (1965).

12.

A. Sjoerdsma, K. Engelman, S. Spector and S. Udenfriend, Lancet, 7422, 1092 (1965).

13.

J. Awapara, T.L. (1964).

19.

M.O.

15.

H.J. Schuemann and H. Grobecker, Arch. Exp. Path. Pharmakol., (1965).

16.

A. Carlson, A. Dahlstrom, K. F’uxe and N.A. 270 (1965).

17.

R. Lindmar and E. Muscholl, Arch. Exp. Path. Pharmakol.,

18.

N.E. Anden, Acta Physiol. Scand., 64, 136 (1965).

19.

J.F.

20.

B.B. Brodie, R. Kuntzman, C.W. (1962).

21.

A. P l e t s c h e r , M. Da Prada and F.A. (1964).

22.

S. Kaufman and S. Friedman, Pharmacol. Rev.,

23.

B. Nikodijevic, C.R. 140 -’ 224 (1963).

24.

J. Musacchio, J.J. Kopin and S. Snyder, Life Sci.,

Perry, C. Hanly and E. Peck, Clin. Chim. Acta, 1 0 , 286

Day and M.J.

Rand, B r i t . J. Pharmacol.,

22,

72 (1964).

251,

48

H i l l a r p , Acta Pharmacol.,

249,

22,

529 (1965).

-

Moran and R.L.

Sourkes, J. Pharmacol. Exp. Ther.,

3,252

Hirsch and E. Costa, Life Sci.,

(1965).

5,

81

S t e i n e r , I n t . J. Neuropharmacol.,

17, 71

5,

559

(1965).

Crevgling and S. Udenfriend, J. Phannacol. Exp. Ther.,

5,

709 (1964).

286 25.

Sect. V M. Goldstein,

(1964).

-

Topics in Biology

T a b a c h n i c k , Ed.

B. Anagnosle, E. Lauber and M. Mc Kereghan, Life Sci., 3, 763,

15, 445

26.

Y. Hashimto, Y. Ohi and R. ImaizUmi, Jap. J. Pharmacol.,

27.

G.G.S.

28.

B. Bhagat and J. Gilliam, J. Pharmacol. Exp. Ther., 150, 41 (1965).

29.

H. Thoenen, W. Haefely, K.F. Gey and A. Kurlimann, Life Sci., 4, 2033

-

(1965).

Collins, J. Pharm. Pharmacol., 17, 526 (1965).

-

(1965).

190, 456

30.

L.I. Goldberg, J.A.M.A.,

31.

F. Sjoqvist, Proc. Roy. SOC. Med.,

32.

R.M.

33.

H. Freeman, I. Karacan, W.E. Walerman, A.J. Khan, F.L. Zimmer, A. Feldstein, C.G. Tedeschi, E.A. De Felice, Int. J. Neuropsychiat., 1, 513 (1965).

34.

I.J. Kopin, J.E. Fischer, J. Musacchio and W.D. Sci. USA, 52, 716 (1964).

35.

(1964).

5, 967

(1965).

Atkinson and K.S. Ditman, Clin. Pharmacol. Ther., 6, 631 (1965).

-

Horst, Proc. Nat. Acad.

I.J. Kopin, J.E. Fischer, J.M. Musacchio, W.D. Horst and V.K. Weise, J. Pharmacol. Exp. Ther., 147, 186 (1965).

36.

J.E. Fischer, W.D. Horst and I.J. Kopin, Brit. J. Pharmacol., (19651.

37.

J.B. Farmer, J. Pharm. Pharmacol., 17, 640 (1965).

38.

R.T. Brittain, D. Jack and P.S.J. (1964).

24,

Spencer, J. Pharm. Pharmacol.,

477

16, 565

39.

S. Ferri and A. Ornesi, Atti Accad. Med. Lombard., 19, 455 (1964).

40.

A.H.

41.

F.G. Graeff, J.G. 17 (1965).

42.

R. Fielden and A.L. Green, J. Pharm. Pharmacol.,

43.

F. Clementi, Experientia, 21, 171 (1965).

44.

A. Tothill, Brit. J. Pharmacol.,

45.

A. Jori, C. Carrara, S. Paglialunga and S. Garattini, J. Pharm. Pharmacol.,

46.

T.H. Tsai and W.W.

17, -

Loveless and D.R. Maxwell, Brit. J. Pharmacol., 25, 158 (1965). Leme and M. Rocha e Silva, I n t . J. Neuropharmacol.,

25,

17, 463

4,

(1965).

217 (1965).

703 (1965).

Fleming, Biochem. Pharmacol.,

2, 369

(1965).

Chap. 25

Enzyme Affectors

T s a i and W.W.

47.

T.H.

48.

U. Zor, S. Dikstein and F.G.

49.

A.

50.

V. Vitek and K. Rysanek, Biochem. Phararacol.,

51.

A. Eorita, I n t . J. Neumpharmacol., 4, 337 (1965).

52.

2. Huszti and J. Borsy, Biochern. Pharmacol.,

53.

A. Burger and S. Nara, J. Med. Chem.,

54.

R.W.

55.

A. Horita, B r i t . J. Pharmacol.,

56.

S.B. Ross and 0. Haljasmaa, Acta Pharmacol.,

57.

S.B. Ross and 0. Haljasmaa,

58.

J.V.

59.

J. Axelrod, J.K. (1965).

60.

J. Daly, J.K.

E,40

Fleming, J. Pharmacol. Exp. Ther.,

32,

Sulman, J. Endocr.,

(1965).

35 (1965).

1,97 (1965).

ToXie. kppl. Ph-Wl.,

-9

28 7 -

Horita

5,

13, 1151 (1964).

859 (1965).

-

Fuller and C.P. Walters, Biochem. Pharmacol., 14, 159 (1965).

Burba and M.F.

24,

245 (1965).

E., 21,

21,

205 (1964).

215 (1964).

-

Murnaghan, Biochem. Pharmacol., 1 4 , 823 (1965).

-

Inscoe and J. Daly, J. Pharmacol. Exp. Ther., 149, 16

Inscoe and J. Axelrod, J. Med. Chem., 8, 153 (1965).

61, V. Erspamer, Progr. Drug R e s . , 3-,

151 (1961).

62.

G.P. L e w i s , "The Hormones," Vol. 4, G. Pincus, K.V. Thimann and E.B. Astwood, Ed., Acad. Press Inc., N e w York, New York, 1964, p. 387.

63.

S. Garattini and L. Valzelli, "Serotonin," Elsevier Publ. Co., New York, New York, 1965.

64.

D.G.

Grahame-Smith, Biochem. Biophys. Res. Commun.,

65.

R.A.

Freedland, Biochim. Biophys. Acta,

66.

R.J.

67.

S. Hosoda and D. G l i c k , Biochim. Biophys. Acta,

68.

W. Lovenberg, R.J.

69. 70.

73, 71

16, 586

(1964).

(1963).

Levine, W. Lovenberg and A. Sjobrdsma, Biochem. Pharmacol., (1964).

(1965).

13, 1283

111, 67 (1965).

Levine and A. Sjoerdsma, Biochem. Pharmacol.,

14, 887

-

S. Consolo, S. Garattini, R. Ghielmetti, P. Morselli and L. Valzelli, L i f e 4 625 (1965). -'

Sci -'* L.J.

Weber and A. Horita, Biochem. Pharmacol.,

E, 1141

(1965).

288

Sect. V

-

Topics in Biology

T a b a c h n i c k , Ed.

71.

E.M. Gal, M. Morgan, S.K.

72.

E.H. G a l , M. Morgan and F.D. Marshall, Life S c i . , 4 , 1765 (1965).

73.

W.P.

74.

W.P. Burkard, K.F. (1965).

75.

R.W.

76.

V. Bartonicek, Med. Pharmacol. Exp.,

77.

R.J. Levine, T.L' (1965).

78.

R.J.

79.

P. Graham, G. Kahlson and E. Rosengren, J. P h y s i o l . ,

80.

E.A.

Pharmacol.,

13, 1639

Burkard, K.F.

C h a t t e r j e e and F.D. (1964).

Marshall, Biochem.

Gey and A. P l e t s c h e r , L i f e S c i . ,

3,

27 (1964).

Gey and A. P l e t s c h e r , Hed. Pharmacol. Exp.,

Fuller, L i f e Sci.,

2, 388

5, 1 (1965). 13, 184

(1965).

-

S a t 0 and A. Sjoerdsma, Biochem. Pharmacol., 1 4 , 139

Levine, Fed. Proc.,

24,

1331 (1965).

C a r l i n i , M. Santos and M.R.P.

172, 174

Sampaio, E x p e r i e n t i a ,

21,

(1964).

72 (1965).

28 9 -

Section VI

Topics i n Chemistry

Editor: E. E. Smissman, University of Kansas, Lawrence, Kansas Chap. 26 Synthetic Peptides By George W. Anderson, Lederle Laboratories, American Cyanamid Co., Pearl River, New York

Introduction. Bnphasis i n t h i s short review will be placed on develapnents of methods of synthesis and t h e i r applications t o the synthesis of biologically important peptides. Procedures are now good enough f o r the rapid synthesis of peptides containing up t o a dozen amino acids, and with more difficulty, ccmrplex peptides such as insulin (51 amino acids). The develgment of "solid phase" synthesis and i t s autcpnation by Merrifield (1)prcinises much. Meanwhile, the synthesis of dozens of analogs of such important peptides as oxytocin and bradykinin has already been acccrarplished, and structure-activity relationships are becaning clearer. Reviews. O f particular importance i s the book by Schroeder and Lubke (2), The Peptides, V o l . I. Methods of Peptide Synthesis which was published i n 1965. This i s a c r i t i c a l , w e l l written, ccmrprehensive survey, which covers the literature t o nearly the end of 1964. The tables l i s t i n g amino acid derivatives which are useful i n synthesis a r e especially valuable. A short review by the present author intended as a quick survey of t h e present s t a t e of the f i e l d of synthetic peptides also appeared i n 1965(3). Conference Report. The proceedings of the Seventh European Peptide Symposium, held in Budapest on September 3-8 1964, have been published as a special The 38 research reports axe indicative volume of Acta Chimica Hungari a of the problems receiving attention i n 1964, and t o a large extent i n 1965. Seven papers were concerned with new protecting groups or removal of protecting groups, six with racemization, two with aminoacyl incorporation into peptide chains, five with methods of forming the peptide bond, f i v e with special problems and thirteen with the synthesis of naturally occurring peptides and analogs. Since more manpower was a l s o involved i n the l a t t e r , it i s obvious that the actual synthesis of peptides i s receiving the most effort. Along with t h i s synthesis, of course, problems of methodology are met and solved. Sane of these papers w i l l be referred t o i n specific discussions t o follow. Methods of Forming the Peptide Bond. Among the more interesting active e s t e r s are the 1-hydroxypiperidine e s t e r s discovered by Young and associates (5). These are generally unreactive i n peptide formation i n neutral or basic conditions, but react readily i n the presence of acetic acid. Activation by protonation i s proposed:

(2).

2 90 -

Sect. VI

-

Topics in Chemistry

S m i s s m a n , Ed.

A s racemization, generally speaking, i s a base catalyzed reaction, it was

expected and experimentally verified t h a t N-hydroxyyiperidine esters of acylamino acids were not racemized by triethylamine. In contrast, other active esters such as p-nitraphenyl and N-hydroxysuccinimide e s t e r s are racemized by trietbybmine, although they a r e stable under neutral or acidic conditions ( 6 ) Since the l a t t e r e s t e r s can be used f o r peptide formation without racemization, the principal virtue of the N-hydroxy-piperidine esters would appear t o be s t a b i l i t y during t h e i r synthesis and storage. Unfortunately, Young and associates found that the direct synthesis of such esters of benzoyl-L-leucine, a t e s t case analogous t o w h a t would be encountered i n the synthesis of active esters of peptides, was acccPlrpanied by racemization when custamasy procedures were used. This i s also true i n t h e synthesis of other active esters. Active e s t e r s of peptides can be made by s t a r t i n g with an amino acid active ester and building on the amino end, and then these can be used without racemization. However, u n t i l a simple method f o r the d i r e c t synthesis of active e s t e r s of acylpeptides without racemization i s found, t h e i r practical use i s r e s t r i c t e d t o derivatives of acylamino acids where the acyl group i s benzyloxycarbonyl or the like. The good r e s u l t s with such esters makes t h e i r use quite papular today. Further experience with N-hydroxysuccinimide (7), (30) and 2,4,5-trichloraphenyl(8) esters has been favorable, indicating that these derivatives w i l l compete with pnitrophenyl e s t e r s i n popularity. A new class of reagents for making all three types of e s t e r s was reported by S W i b a r a and Inukai (9); t h i s consists of the trifluoroacetyl e s t e r s of the respective phenols or N - h y d r o x y s u c c i d e . These reagents m e so reactive that attempted isolation brings about deccqosition, but excellent r e s u l t s were obtained by the use of crude reagents. Furthemore, it was not necessary t o i s o l a t e the active esters before reaction with amino acid e s t e r s t o form dipeptides, which were obtained i n excellent yields. Continuation of the investigation of esters of p-phenylazophenol by Barth (10) has shown t h a t they may be considered t o be equivalent t o p-nitrophenyl e s t e r s with the additional advantage that they are detectable by t h e i r color or UV fluorescence. Among new active esters proposed i n 1965 are those derived f r a (13), 8-hydroxyquinoline (11) a-selenonaphthol (12), 3-bydro-idine 3-nitroacetaphenone (14) and 1-phenyl-3-methylpmazolone-5 (14) The use of acylguanidines as intermediates in peptide synthesis (15) i s reminiscent of e s t e r s of N-hydroxypiperidine i n that they are activated by protonation. The scope of this method i s yet t o be determined. Two investigations of 1 - d i a l k y ~ o - 1 - a l k y n e s (ynamines) (16), (17) have shown t h a t raqemization occurs i f these reagents a r e used t o activate acylpeptides, hence their use should be r e s t r i c t e d t o t h e activation of suitably protected amino acids. Rapid Synthetic Procedures. The outstandhg d e v e l q e n t t o date i s the "solid phase" method of Merrifield (1). The success of t h i s procedure depends upon making the peptide insoluble by attachment a t the carboxy end t o a polymer, and adding acylamino acid units t o the graying chain quantitatively. t.Butyloxycarbonylamino acids ( B E amino acids) in large excess along with

,

.

Chap. 26

Peptides

Anderson

29 1 -

dicyclohexylcarbodiimide reagent are used, with subsequent removal of the BOC grpup by acid at each step. All by-products and excess reagents are removed by washing at each step. The procedure has been autamated, with the addition of an amino acid unit to the growing peptide chain every four hours; bradykinin was synthesized in this fashion. By non-automatic procedures, a large number of bradykinin analogs were made (18), and scme angiotensins (19). A preliminary communication in 1963 by Letsinger and Kornet (20) reported a "solid phase" method of building from the carboxy end of a peptide chain using isobutyl chloroformate as the reagent. This has not been followed up, probably because conditions to avoid racemization were not known. Recent work in the writer's laboratory (21) has disclosed conditions which should be safe, hence this method can now be used. An interesting variation of the Merrifield approach using a soluble polymer support and N-hydroxysuccinimide esters of BOC amino acids was reported in 1965 by Shemyakin and associates (22). A new approach to rapid synthesis in aqueous solution using a water soluble carbodiimide as reagent and lengthening the peptide chain from the carboxylic end was reported by Knorre and Shubina at Budapest (23). Although ingenious, this method faces difficulties, the greatest being the possibility of racemization. Another procedure using a water soluble carbodiimide builds the peptide chain from the amino end (24). This method in its present form suffers from variable yields and the number of manipulative procedures. One can see a definite advantage of an automatic machine for making s m a l l amounts of peptides, but there may be no advantage if large quantities are desired. The development of a safe general procedure (other than the azide method, which has disadvantages) of combining peptides without racemization would diminish the importance of the one-at-a-timeprocess for moderate-sized or large peptides. Further improvements of rapid conventional procedures which give good yields might also nullify some of the advantages of the machine approach. In the author's experience, the mixed carboxylic-carbonic anhydride procedure (21) offers excellent possibilities for such develqments. Amine-Protecting Groups. The ease of removal of the benzyloxycarbonyl group by hydrogen bromide in acetic acid and the increasing recognition of the dangers of handling peptides under alkaline conditions have focussed attention in recent years on protecting groups which can be removed by mild acidic conditions. Of a number of mine-protecting groups of this type, the most widely used is the t-butyloxycarbonyl group (25). Favorable results with t-amyLoxycarbonyl (26), benzhydryloxycarbonyl (27), p-methoxybenzyloxycarbonyl (28), and 0-nitrophenylsulfenyl ( 2 9 ) groups reported during the year shows that these may have canrpasable use. Cleavage of the o-nitrophenyl group by Raney nickel is of interest (30). A preliminary report on use of the acetoacetyl group for amine protection ( 3 )is noted. Electrolytic reductive splitting of benzoyl and tosyl groups from amino acids and peptides has been accauplished in the presence of tetramethylammonium chloride (32) Carboxy Protecting Groups. Benzhydryl esters of several acylamino acids have been synthesized by the azeotropic distillation method or by the reaction of the acid with diphenyldiazomethane (2'0, (33). They are comparable to t-butyl esters in acid lability.

.

292

Sect. VI

-

S mi s s m a n , Ed.

Topics in Chemistry

Selective reactivity of an activated acylamino acid at the amino group of glycylhydrazide or glycylglycylhydrazide was demonstrated, hence a hydrazide might be useful as a protecting group (34). Special Protecting Groum. In contrast to S-diphenyhethylcysteine, S-4,4'dimethoxydiphenyhethylcysteine gives a color on thin-layer chranat LMS upon treatment with hydrogen chloride, which should be a useful pruperty 35). A useful, comprehensive survey of the protection of the guanidino group of arginine was presented at Budapest by Guttmann and Pless (36). New groups proposed for this function are p-nitrobenzyloxycarbonyl, which is resistant to acidolysis and easily cleaved by catalytic hy&ogenation, and 3,4,5,6tetrachloro-2-isopropylphthalyl(TIP), which is resistant to acidolysis and hydrogenation but readily removed by aqueous acid.Bajusz (37) reported good results with the t-butyloxycarbonyl (BOC) group. When the nitro group is used, it subsequently be = . w e d by electrolybic reduction (38). Conversion of Seriie-Co&aining Peptides to Cysteine-Contaw Peptides The conversion of 0-tosyl-L-serine peptides to S-acetyl or S-benzoyl-L-cysteine peptides by thioacetate or thiobenzoate ions has been studied in detail (39). This simple procedure p r d s e s to be particularly useful in enzyme studies. Racemization Studies. The cambination of acylpeptides with amino acid or peptide derivatives without racemization of the carbon alpha to the carboxylic group (starred in equation below) continues to present problems.

T

.

*

Acy1"HFCOOH

R

f

_j

f

Acy1.NI-I HCONH~HCOOR" R'

In this case, the acyl stands for an acylamino acid or acylpeptide. If R is H or part of the prolyl residue, there is no danger, and hence many synthetic schemes involve coupling of peptides when glycine or proline are C-terminal. Otherwise, the safest procedure is activation through an azide, and this often gives unsatisfactory results. Other activation procedures can often be used with proper control of conditions, but the uncertainties make these unpopular. Hence the building of peptide chains by the addition of acylamino acids to the amino end of a peptide chain has become the most popular procedure today. This procedure has been successful, perhaps as best exemplified by Merrifield's "solid phase" procedure. However, considering the flexibility and yield advantages of safe procedures for the cambination o f peptides, it is surprising that very little basic work to solve the problem was reported during 1965. It is the author's opinion that some of our ccaumon activation methods will give satisfactory results when properly used, and more basic research with them is needed to determine the safe conditions. P r a i s i n g results for the mixed carbonic anhydride method have recently been reported (21). G. T. Young and associates have continued their research on racemization mechanisms, with proof that racemization of benzyloxycarbonylglycyl-Lphenylalanine p-nitruphenyl ester by triethylamine proceeds through an oxazolone mechanism (40). Frcm previous work fra the laboratories of Young and of others, it seems likely that this is the camon mechanism of racemization of activated acylpeptides during peptide synthesis.

Chap. 26

Peptide s

Anderson

293 -

B. Liberek has also continued his studies on racemization of amino acid derivatives where direct abstraction of the a hydrogen is the favored mechanism (41). Taschner and associates have shown that cyananethyl esters made with branoacetonitrile axe less likely to be'racemized than those made with chloroacetonitrile (42). Basic informstion on racemizatian in several methods of peptide synthesis was reported by Anderson, C a U a b a and Zimmerman (43). Further studies by Beyennan, Weygand and associates with 1,2,4-triazole as a catalyst for the reaction of several active esters in peptide synthesis have given no indication of racemization, whereas imidazole, as expected, can cause racemization (44). Schaabel (45) has shown that azlactones fran formyl- and trifluoroacetylamino acids and fran benzyloxycarbonyldipeptides are readily made by treatment with dicyclohexylcarbodiimi.de. Most of these are at least partially optically active, and reaction with ethyl glycinate results in more or less strong racemization which is diminished in the presence of acetic acid. Indirect evidence was obtained that symnetrical anhydrides of benzyloxycarbonylamino acids are formed by the reagent, and these are racemized partially during synthesis and aminolysis. The conclusions drawn about racemization of the symmetrical anhydride from benzyloxycarbonylglycyl-L-phenylanine are also based on indirect evidence, and they need to be verified by experiments with isolated and characterized anhydride. In agreement with observations that benzyloxycarbonylamino acids are not normally racemized by dicyclohexylcarbodiimi.de, Vajda and associates (46) conclude that no azlactones are formed. Acylureas and diketopiperazines were detected, and these might - have been derived f r o m smetrical anh.vdrides. Synthesis of Biologically Important Peptides. This is the major-goal of peptide synthesis, hence it is not surprising that the most effort has been _ _ spent in this fieid. It is not possible in this short review to refer to a l l the papers in this field which were published in 1965. Analogs of oxytocin, vasopressin, angiotensin eledoisin, bradykhin and kallidin continued to receive much attention. In a discussion of the possible mechanisms of action of peptide hormones, Boissonnas (47) estimates that the total Ilumber of anslogs which have been synthesized up to the end of 1965 of vasopressin plus &ocin at about 130, of aMSH plus ACTH at 25, angiotensin 50, bradykinin 60 and eledoisin 120. In the antibiotic field, the syntheses of valine- and isoleucinegramicidin A(48), p o r n i n B1(49), and Colistin-A(S0) should be noted. In the enzyme field, the investigations of Hofmmn and associates (51) on the synthesis of peptides related to the N-terminus of bovine pancreatic ribomclease deserve special ccpmnent. Formation of the peptide bond under aqueous conditions (organic solvent plus water) was frequently acccmplished with good results, using active ester or azide intermediates and fYee peptides. With peptides of increasing size and water solubility, one can anticipate that the method w i l l becane correspondingly important. The last paper in the series, which discusses structure-activity relationships, well illustrates the importance of synthetic peptides far the developnent of understanding of biological mechanisms.

294

Sect. VI

-

Topics in Chemistry

S m i s s m a n , Ed.

Before 1965, the synthesis of insulin peptides with biological a c t i v i t y was reported fran three laboratories; Further progress Frau a U three has

recently been reported. I n brief camnnunications (52), KatsoyagaiS and associates have reported the canbination of synthetic A and B chains of human insulin with a 2$ yield of insulin activity, and hybrids of synthetic human A and natural bovine B (4 t o 8%)and synthetic human B with natural bovine B (@). K u n g and associates (53) reported the isolation of c r y s t a l l b e synthetic bovine insulin with a c t i v i t y greater than 20 I.U./mg. (natural insulin Z a h n and associates (54) found t h a t pre-oxidation approximates 24 I.U./mg.). of the A chain imprwed the yield of insulin upon oxidative recambination of the A and B chains. Work fran the three laboratories indicates that p a p e r conditions f o r recanbination of the A and B chains are the most h p o r t a n t factor f o r high a c t i v i t y yields. Since the formation of three disulfide bonds (one intra-chain, two inter-chain) i s involved, t h i s i s understandable. A number of detailed synthetic papers frm the three groups w i l l not be referred t o here. A paper concerning preparative and analytical problems incurred by Zahn's group (55) i s reccmnended as an indication of the scope of the problem of insulin synthesis. Insulin, with 51 amino acids, i s the largest peptide (or s m a l l protein) synthetized t o date. Indicative of new approaches constantly being developed i s the synthesis of an insulin fragment by Zervas e t al. (56). Here, by selective use of S- and N- protecting groups, a peptide containing the 6-12 amino acid fragplent of the A chain of sheep insulin, with the i n t r a disulfide link, was synthesized. A c q l e t e A chain with t h i s l i n k wuuld make ccaubination with the B chain simpler, and muld l i k e l y acccPlrplish i n a more elegant way the result of the A chain pre-oxidation step of Z a h n . D-Amino Acid Analogs. Although some peptide antibiotics contain D amino acids, peptide hormones do not. Before 1965, it had been shown with several hormones that the replacement of one L-amino acid with the D-isomer usually lowered a c t i v i t y but did not destroy it. It remained t o be seen what the effect of ccunplete replacement would be. During the year, results were reported f o r (57), D-oxytocin (58), and D-Val% several hormones. With D-bradyAngiotensin II-As$-f3-Amide (59) no hormone a c t i v i t y or inhibition of the a c t i v i t i e s of the L-analogs were found. P a r t i a l replacement of Ir-BminO acid by D-amino acid residues i n eledoisin lowered a c t i v i t i e s t o a variable degree (60). The melanocybe-stimulating a c t i v i t y of a pentapeptide fragment of a-melamocyte-stimulating hormone was, in contrast, inhibited by the a l l - D analog (61). With the C-terminal. tetrapeptide derivative of the hormone gastrin, BOC*try-met-asp-phe.I9$, a c t i v i t y was l o s t by partial. o r cauplete replacement of the L - a o acids by the corresponding D-amino acids, and inhibitory a c t i v i t y was not found (62). Cyclic Peptides. The principal i n t e r e s t here also is the synthesis of naturally occurring peptides or analogs, and several such syntheses were reported. The p-nitrophenyl e s t e r method was favored f o r t h e cyclization stbp, although other Coupling procedures were also used. Syntheses of polymyxin B1(49), colistin-A (50), evolidine(63) and an active analog of gramicidin S(64) were reported.

Chap. 26

Peptides

Anderson

295 -

An interesting technique involving ester attachment of an amino acid t o an insoluble poly(nitrophenol), building a peptide chain t o this by stepwise addition at the -0 end, and O i n a J l Y intra-molecular cyclization was

disclosed (65).

Sect. VI

-

Topics in Chemistry

S m i s s m a n , Ed.

REFWENCES R. B. Merrifield and J. M. Stewart, Nature, 207, 522 (1965). E. Schrijder and K. Lubke, The Peptides, Vol. I, Academic Press, New York,

1965*

G. W. Anderson, Ccmprehensive Biochemistry, Vol. 6, 258, Elsevier Publishing Co., Amsterdam, 1965. Acta Chim. Acad. Sci. Hung. 44, No. 1-2 (1965). S. M. Beaumont, B. 0. Handford and G. T. Young, ref. 4, p. 37; B. 0. Handford, J. H. Jones, G. T. Young and T. F. N. Johnson, J. Chm. SOC. 1965, 6814. G. W. Anderson, F. M. Callahan and J. E. Zimmerman, Ref. 4, p. 51. M. Lijw and L. Kisfaludy, ref. 4, p. 61. P.-A. Jaquenoud, Helv. Chim. Acta., 48, 1899 (1965). S. Sakakibara and N. Inukai, Bull. Chem. SOC. Japan, 1979 (1965). A. Barth, Ann., 683, 216 (1965). H.-D. Jakubke, Z. Naturforsch, 20b, 273 (1965). H.-D. Jakubke, Ann., 682, 244 (1965). E. Taschner and B. Rzeszotarska, Ann., $0, 177 (1965). G. Losse, K.-H. HOPand G. Hetzer, Ann., 684, 236 (1965). E. Hoffmann and D. Diller, Can. J. Chem., 3, 3103 (1965). F. Weygand, W. KZnig, R. Buyle and H. G. Viehe, Chem. Ber., 9, 3632

s,

(1965)

A. J. G. R. G.

S. Van Mourik, E. Ha;rryvan and J. F. hens, Recueil, 84, 1344 (1965). Stewart and D. W. Wooley, Federation Proc., 24, 657 (19%).

M. R. L. W.

Marshall and R. B. Merrifield, Biochemistry,T 2394 (1965). Letsinger and M. J. Kornet, J. Am. Chem. Soc.7'5, 3045 (1963). Anderson, J. E. Zimmerman and F. M. Callahan, J. Am. Chem. SOC.,

88, 1338 (1966).

MTM. Shemyakin, Y. A. Ovchinnikw, A. A. Kinyushkin and I. V. Kozhevnikwa, Tetrahedron Letters, No. 27, 2323 (1965). D. G. Knorre and T. N. Shubina, ref. 4, p. 77. J. C. Sheehan, J. Preston and P. A. Cruickshank, J. Am. Chem. SOC., 3, 2492 (1965). L. A. Carpino, J. Am. Chem. SOC., Q, 98 (1957); F. C. McKay and N. F. Albertson, ibid, B, 4648 (1957); G. W. Anderson and A. C. McGregor, ibid, B, 6180 (1957). S. Sakakibara, M. Shin, M. Fujino, Y. Shimonishi, S. Inone and N. Inukai, Bull. Chem. SOC. Japan, 38, 1522 (1965). R. G. Hiskey and J. B. Adams, Jr., JFAm. Chem. SOC. 87, 3969 (1965). F. Weygand and E. Nintz, Z. Naturforsch., 2OJ, 429 (1g5). L. Zervas and C. Hamalidis, J. Am. Chem. SOC., 3, 99 (1965). J. Meienhofer, Nature, 205, 73 (1965). F. D'Angeli, F. Filira, and E. Scoffone, Tetrahedron Letters, No. 10, 605 (1965). L. Horner and H. Neumann, Chem. Ber., g,3462 (1965). A. A. Aboderin, G. R. Delpierre and J. S. Fruton, J. Am. Chem. SOC. -9 87 5469 (1965).

Chap. 26

P ept ide s

Anderson

&,

297 -

H. T. C h e w and E. R. Blout, J. Org. Chem., 0, 315 (1965). R. W. Hanson and H. D. Law, J. Chem. SOC., 7285. Ref. (4), p. 23. Ref. (4), p. 3. P. M. Scapes, K. B. Walshaw, M. Welford and G. T. Young, J. Chem. SOC., 782. I. Photalri and V. Basdakos, J. Am. Chem. SOC., 3489 (1965); C. Zioudrou, M. Wilchek and A. F'atchornik, Biochem., 18u (1965). 43; I. b t o n o v i c s and G. T. young, Chem. ccmunun., NO. 17, Ref. 398 1965). B. Liberek and A. Michalik, r e f . (4), p. 71. E. Taschner, B. Rzeszotarska and A. Kuziel, r e f . (k), p. 67. Ref. (4), p. 51. H. C. Beyerman and W. Maassen van den B r i n k , r e f . (4), p. 99; H. C. Beyerman, W. Maassen van den B r i n k , F. Weygand A. B o x , W. Konig, L. Schmidhammer and E. Nintz, Recueil, 84, 213 (19651. E. Schnabel, Ann., 688, 238 (1965). T. V a j d a , A. Kueiel, F. R u f f , B. Rzeszotarska and E. Taschner, r e f . (41, P. 45R. Boissonnas, Ann. Endocrinol., Paris, 26, 632 (1965). R. Sarges and B. Witkap, J. Am. Chem. Soc., 3, 2020 (1965). K. Vogler, R. 0. Studer, P. Lanz, W. Lergier, and E. BGhni, Helv. Chim. Acta, 48, 1l61 (1965); K. Vogler, Ref. (4), p. 143. R. 0. SGder, W. Lergier, P. Lanz, E. Bohni and K. Vogler, Helv. Chim. Act% 48, 1371 (1965). K. Hof?mann, R . Schiechen, R. D. Wells, Y. Wolman and N. Yanaihara, J. Am. Chem. SOC., 3, 611 (1965); K. Hofmann, W. H a a s , M. J. Smithers, R. D. Wells, Y. Wolman, N. Yanaihara and G. Zanetti, ibid, 620; K. Hofmann, W. H a a s , M. J. Smithers, and G. Zanetti, ibid, 631; K. Hofmann, R. Sckrmiechen, M. J. Smithers, R . D. Wells, Y. Wolman, and G. Zanetti, ibid, 640; F. M. Finn and K. Hofmann, ibid, 645. P. G. Katsoyannis, A. M. Tmetsko, J. Z. Ginos, and M. A. T i l e , J. Am. Chem. Soc., 88, 164 (1966); P. G. Katsoyannis, A . TcPnetsko and C. Zalut, ibid, 166TK u n g Yueh-ting, Du Yu-cang, Huang Wei-teh, Chen Chan-chin, Ke Lin-tsung, Hu Shih-chuan, Jiang Rong-qing, Chu Shang-quan, Niu Ching-i, Hsu Je-Zen, Chang Wei-chun, Cheng Ling-ling, L i Hong-shueh, Wang Yu, Loh Teh-pei, Chi Ai-hsech, L i Chung-hsi, S h i Pu-tao, Yieh Yuen-hwa, Tang Kar-lo and H s i n g Chi-yi, Scientia Sinica, &, 1710 (1965). H. Zahn, B. Gutte, E. F. P f e i f f e r and J. Ammon, Ann., 225 (1966). H. Zahn, J. Meienhofer and E. Schnabel, r e f . (4), p. 109. L. Zervas, I. Photaki, A. Cosmatos and D. Borovas, J. Am. Chem. SOC.3, 4922 (1965). J. M. Stewart and D. W. Wooley, Nature, 206, 619 (1965). G. Flouret and V. du Vigneand, J. Am. Chem. SOC., 3, 3775 (1965). K. Vogler, R. 0. Studer, W. Lergier and P. Lanz, Helv. Chim. Acta, 48,

a,

t4),

3,

5,

a,

14.07 (1965).

298 -

(60) (61) (62) (63) (64) (65)

Sect.

VI

-

Topics in Chemistry

S m i s s m a n , Ed.

E. SchrBder, K. Liibke and R . Hempel, Experi’entia, 21, 70 (1965). H. Yajima and K. Kubo, J. Am. Chem. SOC., 5, 2039 (1965). J. S. Morley, J. H. Tracy and R. A. Gregory, Nature, 1356 (1965). R. 0. Studer and W. Lergier, Helv. C h h . Acta, 48, 460 (1965). H. Aoyagi, T. Kato, M. Ohno, M. Kondo, M. Wahi, S. Makisumi and N. I d y a , Bull. Chem. SOC. Japan, 2139 (1965). M. Fridlsin, A. Patchornik, and E. Katchalski, J. Am. Chem. SOC., 5,

m,

2,

4646 (1965).

Chap. 27 NUCLEOSIDES AND NUCLEOTIDES

Howard J. Schaeffer Department of Medicinal Chemistry School of Pharmacy State University of New York at Buffalo Over the past few years, several excellent reviews (1,2) and books

(3,4) have appeared on nucleosides and nucleotides. During 1965 consid-

erable interest in the nucleosides and nucleotides has been maintained as evidenced by the significant research activity in this area. Because brevity has been requested ia this report, some of the papers published during 1965 could not be included.

The preparation of nucleosidles by the fusion (5,6,7) of purines with fully 0-acylated sugars in the presence of an acid catalyst is being used extensively because of several advantages: (a) it avoids the necessity of preparing a heavy metal salt of the purine and thereby insures that the nucleoside will not be contaminated with a metal which could interfere with studies on biological activity, (b) it obviates the necessity of preparing a chloro sugar, and (c) the yield of the nucleoside is frequently higher than in the conventional synthesis. It has been reported (8) that (-&b E-nitropheny1)hydrogen phosphate is an effective catalyst for the synthesis of purine nucleoside by the fusion method. For example, the fusion of theophyline with penta-Q-acetyl-B-D-glucopyranose in the presence of &-( 2-nitropheny1)hydrogen phosphate gave a 47% yield of the 7-substituted nucleoside. A synthesis of ~-oxo-3-ribofuranosyldihydropyrimidine by the fusion of 4-hydroxypyrimidine and a tetraacylribose d rivative was described (9). Condensation of purines, benzimidazole, or N%-benzoy lcytosine and acylglycosyl halides in nitromethane in the presence of mercuric cyanide gave the corresponding nucleosides in higher yields than in the conventional method (10). A variety of nucleosides were prepared by the condensation of the b&-( trimethylsily1)ether of 5substituted (HaCH3,F,C1,Br,I and -CH2OC&C6H5)uracils with several glycosy1 halides( 11). The utility of the directive influence of a remwable blocking group at N-3 on an adenine nucleus has been demonstrated by the synthesis of 7a-D-ribofurano~yladenine~ which was shown to be identical to the nucleoside from pseudwitamin Bu. The synthesis of this nucleoside was accomplished by the condensation of the mercuri salt of N6-benzoyl-3benzyladenine with 2,3,5-tri-O-acetyl-D-ribofuranosyl chloride followed by deacylation and remwal of the 3-benzyl group by catalytic hydrogenolysis (12). Other investigators who studied the product distribution in the ribosylation of mercuri salts of certain purines and deazapurines found that the ratio of the 9-substituted to 7-substituted isomer was usua€ly greater than one for those purines which contained a nitrogen at position 3 (13). In the benzimidazoles, the ratio of isomeric products was closer to unity (13). However, certain exceptions

300

Sect. VI

-

Topics in Chemistry

S m i s s m a n , Ed.

were noted, and it is apparent that steric effects, as well as electronic effects are important in determining the position of reaction in purines and deazapurines. Ikehara and Tada have prepared an 8,2'-cyclonucleoside by allawing 2-chloro-8-mercapto-9 (2,5 -diacetyl-3 tosyl-@-g-xylofuranosy1)adenine to react with sodium methoxide. Raney nickel desulfurization of the 8,2'-cyclonucleoside, followed by hydrogenolysis of the 2-chlorine atom with a palladium catalyst led to 2'-deoxyadenosine (14). The synthesis of 9-p-D-ribofuranosyluric acid was accomplished by converting 8bromoguanosine into 8-benzyloxyguanosine,which on treatment with nitrous acid followed by catalytic hydrogenolysis of the benzyl group gave the desired product (15). In addition, 8-aminoadenosinewas synthesized from 8-bromoadenosine ( 15).

-

-

Robins and Robins (16) have employed the fusion method for the synthesis of 2'-deoxy-g-a-andf3-ribofuranosylpurine. Thus, fusion of 6 -chloropurine and 1,3,5 -tri-0-acetyl-2-deoxy-D-ribofuranosein the presence of chloroacetic acid gave, after deblocking, 6-chloro-9-a-and 9-p-D-ribofuranosylpurines. Similar stu ies were carried out with 2,6dichloropurine, 2,6,8 trichloropurine, -acetyladenine, benzimidazole, 5,6-dimethyl-,5,6-dimethoxy-, and 5,6-dichlorobenzimidazoles (16,17). An NMR study of the 2'-deoxyribofuranosylpurines and benzimidazoles revealed that the f3-anomerexhibits a pseudo-triplet with JHI' 7 C.P.S. and a peak width = 14 C.P.S. for the anmeric proton whereas the aanomer exhibits a mltiplet of four with JH' = 3 and 7 C.P.S. and a peak width = 10 C.P.S. for the anomeric proton (16,17). The method of anomeric assignment by NMR for the 2' -deoxy-D-ribofuranosylnucleoside appears to be general and is not dependent on the heterocyclic base (16)

&

-

The synthesis of 9-( 3-deoxy-f3-D-galactof uranosy1)adenine was accomplished by condensation of 1,2-di-0-acetyl-~,6-di-O-benzoyl-3deoxy-D-galactofuranose with chloromercuri-6 -benzamidopurine in the presence of titanium tetrachloride (18). In addition, 1,2-isopropylidene-3-deoxy-D-galactofuranosewas reacted with periodic acid and then with sodium borohydride to give a 3-deoxy-L-arabinosederivative. Condensation of the appropriately blocked arabinose derivative with chloromercuri-6-benzamidopurinein the presence of titanium tetrachloride gave 9-(3-deoxy-a-L-arabinofuranosy1)adenine ( 18). In a related sequence of reactions, 1,2-di-0-acetyl-~,6-di-0-benzoyl-3-deoxy-D-glucofuranose on condensation with chloromercuri-6 -benzamidopurineusing titanium tetrachloride gave 9 ( 3-deoxy -p-D-glucofuranosy1)adenine ( 19) 1,2-Isopropylidene-3-deoxy-D-glucofuranose was converted into 1,2-di0-acety1-5-0-benzoy1-3-deoxy-D-ribofuranose which on condensation with chloromercuri-6-benzamidopurineand titanium tetrachloride gave 9-(3deoxy-f3 -D-ribofuranosy1)adenine ( 19). 1,2-1sopropylidene-3-deoxy-Dglucofuranose was converted into 1,2-di-O-acetyl-~benzoy1-3-deoxy-Dribofuranose which on condensation with chloromercuri-6-benzamidopurine and titanium tetrachloride gave 9-(3-deoxy-$-D-ribofuranosyl)adenine (19). A different synthesis of some 3'-deoxyribofuranosylnucleoside was reported and involved the condensation of 3-deoxy-2,5-di-O-benzoylribofuranosyl bromide with chloromercuri-6-benzamidopurine(20). In

-

.

Chap. 27

Nucleotides

Scha eff er

301

additionathe bromo sugar was also condensed with chloromercuri-2,6dibenzamidopurine (20)with chloromercuri-6-chloropurine(20) and with chloromercuri-I@-acetylguanine (21). From the 6-chloropurine nucleor side, a variety of 6-substituted derivatives were prepared (2O), and it was found that 9-(3-deoxy-$-D-ribofuranosyl)-6-methylaminopurinewas as potent an inhibitor of uridine incorporation into RNA in KB cells as was 9-(3-deoxy-p-D-ribofuranosyl)adenine (22). Two groups (23,24) have described the synthesis of 9-(5-deoxy-pD-xylofuranosy1)adenine. With the view of converting the xylonucleoside into the arabinonucleoside, a study of the monotosylation was undertaken (23). It was found that the reaction of 9-(5-deoxy-p-Dxylofuranosy1)adenine with E-toluenesulfonyl chloride in pyridine gave the 3'-O-tosylate. The formation of a 3'-O-tosylate rather than the expected 2'-0-tosylatewas rationalized on the be3is of hydrogen bonding between the 3I-hydroxyl and the N-3 nitrogen of the adenine nucleus which could make the 3I-hydroxyl more reactive. Consequently, it was reasoned (23) that monotosylation of the dianion of 9-(5-deoxy-B-Dxylofuranosy1)adenine should produce the 2'-O-tosy?ate,and under these conditions should give 9-(2,3-anhydro-5-deoxy-~-D-lyxofuranosyl)adenine. These results were obtained and on opening the epoxide with sodium benzoate in DMF, the desired 9-(5-deoxy-(3-D-arabinofuranosyl)adenine was obtained along with smaller amounts of the corresponding xylonucleoside (23). Two groups of investigators (25,26) have synthesized 5-trifluoromethyl-6-aza-2'-deoxyuridine. Montgomery and Hewson have devised an improved procedure for the preparation of 5-allyl-2'-deoxyuridinewhich involves the fusion of the %
In an elegant series of papers, Doerr, Codington and Fox describe their work on the 2' 5'4 and 3 ' ,5 ' -epoxides of pentanofuranosyluracils (31,32). Thus, when 1-(5'-O-rnesyl-2'-3'-epoxy-B-D-l~osyl)uracil is allowed to react with sodium benzylate 1-( 2Iy5'epoxy-3'-0-benayl-p-Darabinosy1)uracil is formed. Catalytic hydrogenolysis of the benzyl group produced 1-(2' ,5 ' -epoxy-B-D-arabinofuranosyl)uracil which was also prepared by the treatment of 1-(5'-O-mesyl-~-D-arabinofuranosyl)-

302

Sect. VI

-

Topics in Chemistry

S m i s s m a n , Ed.

uracil with aqueous alkali. The synthesis of 1-(3',5'-epoxy-@-D-xylofuranosy1)uracil was accomplished by allowing 1-(5 ' -0-mesyl-p-D-xylofuranosy1)uracil to react with aqueous alkali (31). The synthesis of the 1-(2 I, 5 ' -and 3 ' ,5 ' -epoxy-@-D-1yxofuranosyl)uracils was also described (32). Base treatment of 1-(5 -O-mesyl-f3-D-lyxofuranosyI) uracil gave 1-(2 ',5' -epoxy-p-D-1yxofuranosyl)uracil. The proof of structure of this compound(2' ,5 ' -epoxy derivative) was established by synthesis of the other isomer. Thus, l-(3',5 '-epoxy-p-D-xylofuranosy1)uracil was converted into its 2'-0-mesylatewhich on treatment with sodium t-butoxide gave 2,2'-anhydro-1-(3',5 '-p-D-1yxosyl)uracil. Aqueous base cleaved the 2,2'-anhydro bond resulting in the formation of 1(3I , 5 ' -epoxy-p-D-1yxofuranosyl)uracil which on hydrolysis with aqueous acid gave 1-p-D-lyxofuranosyluracil (32). Recently, a facile synthesis of 2,2'-anhydroarabinopyrimidine nucleoside has been described by the reaction of 5 -0-trityluridinewith thiocarbonyldiimidazole (33).

-

Because of an interest in the synthesis of the antibiotic amicetin, it was necessary to explore the general area of disaccharide nucleoside synthesis (34). The 1-halo sugar derivatives of heptaacetyl lactose and cellobiose were converted to their corresponding cytosine nucleoside derivatives by two established procedures. The f3-D-configuration of the nucleoside bond was established by degradation to a known compound. In addition, an amino sugar disaccharide nucleoside by the condensation of ~-(2,~,~-tr~-0-acetyl-~-D-glucopyranosyl)-~-ethoxy2( 1H) -pyrimidone and 2-deoxy-2-benzamido-3,4,6-tri-O-acetyl -&D-glucopyranosyl bromide (34). Several reports have appeared in the literature on the synthesis of pyrimidine nucleosides derived from 2-deoxyhexoses (3536). The synthesis of 9-(p-D-glucuronosy1amide)adenine has also been described (37). The utilization of the 2,h-dinitrophenyl group as a blocking. group for the synthesis of 2'-amino-2'-deoxynucleoside has been described, and it appears that this blockinggroup offers utility when it is desired to prepare the anomeric nucleosides since it shows no tendency to participate at C-1 (38). For example, the condensation of 3,4,6-tri-Oacetyl-2-deoxy-2-(2,~-dinitroanilino-a-D-glucopyranosylbrF-idewith chloromercuri-6-acetamidopurinegave, after deblocking, the a- and f3anomers of 9-(2-amino-2-deoxy-D-glucopyranosyl)adenines (38). The use of the benzylsulfonyl group as a nitrogen blocking group in the preparation of amino sugar nucleoside synthesis was demonstrated by the preparation 9 -(2-acetamido-2-deoxy-~-D-glucopyranosyl)adeninefrom tri-0acety1-2-benzylsulfonylamino-2-deoxy-a-D-glucopyranosy1 chloride and chloromercur1-6-benz-amidopurine( 39)

.

Several studies have appeared on the conversion of ribonucleosides into 3-amino-3-deoxyhexosylnucleosides (40,41,42,43). Periodate oxidation of uridine followed by condensation with nitromethane gave a mixture of isomers from which the isomer with the gluco-configuration was obtained in approximately 60$ yield. Hydrogenation of the nitro group with Raney nickel gave l-(3-amino-3-deoxy-p-D-glucopyranosyl)uracil (40) and when the hydrogenation was carried out with a platinum catalyst, the corresponding 5,6-dihydro derivative was obtained (40,41) In related studies (42,43), adenosine was oxidized with periodate and

Chap. 27

Nucleotide s

Sc ha effer

303 -

the dialdehyde on condensation with nitromethane gave a mixture of isomers. Hydrogenation of the nitro group of these isomers allowed the separation of 9-( 3-amino-3-deoxyhexopyranosy1)adenines of the gluco-, galacto- and manno- configurations (42). Finally, Brown and Read have shown that the product obtained by treating periodate-oxidized adenosine-5'phosphate with methylamine and then with sodium borohydride is 2- (9-adeny1) -4-methy1-6-phosphoryloxymethylmorpho1ine (44). Broom and Robins have reported that the reaction of diazomethane and adenosine in a homogeneous solution of water and l,2-dimethoxyethane produced 2'-O-methyladenosine (45). This selective reaction of the 2'hydroxyl group of adenosine may find extensive application to the synthesis of polynucleotides if a functional group can be introduced, such as benzyl, which can be easily removed at a later stage. In order to study the position of attachment of the aminoacyl group to the terminal residue in aminoacyl-S-RNA, Reese and Trentham have studied acyl migrations in ribonucleosides (46,47). The preparation of 3',5'-di-O-acetyluridines and, for the first time, 2',5'-di-Oacyluridine was reported, and a method (46) was developed to determine the position (2' or 3') of an acyl group which takes advantage of the fact that anhydronucleoside formation occurs more readily with a 2'-0mesyl-3' -0-acyl derivative than a 3 ' -0-mesyl-2' -0-acyl derivative of uridine. Thus far, the mesylation analysis procedure is useful for 2'or 3l-derivatives of uridine. A study of thequilibration of 2',5'-di0-acyluridines and 3 ',5 ' -di-0-acyluridinewas undertaken, and it was found that the 3',5'-diesters were only marginally more stable than the 2',5'-isomers. Thus, it was found by an acetylation study as well as an equilibration experiment that the ratio of the 3',5'-diester to 2',5'diester was approximately 1.6 to 1 (47). The synthesis of some 2'(3')0-aminoacyl derivatives of ribonucleosides and ribonucleoside-5'phosphate by the use of ortho ester derivatives of amino acids has been described(48). A variety of new 2',3'-ketal derivatives of ribonucleoside were synthesized with a view to extending the types of blocking groups for those reactions which convert ribonucleosides to 5I-substituted derivatives (49). A study of the rates of hydrolysis of the 2',3'-ketals of uridine at EH 2 and at 26O revealed that the half-life of the reaction varied from 0.5 hour for the crotonylidene derivative to more than 40 hours for the diphenylmethylidene derivatives. The other ketals studied with half-life shown in parentheses were: cyclooctylidene(2.5), cycloheptylidene ( 3 ) , cyclopentylidene (4), methyl-t-butylmethylidene (20), isopropylidene (20) and diethylmethylidene (40). The methoxymethylidene derivative was also studied (49) and was found to have a half-life of 0.08 hour for the formation of a mixture of 2'- and 3'-0formyluridine. Hampton has suggested that a 5 '-0-alkoxyalkyl derivative of a nucleoside may have utility as a 5'-hydroxy blocking group because of its lability under acid or even neutral conditions (50). In the case of uridine the 5'-O-alkyoxyalkyl derivative is selectively prepared by an exchange reaction between the nucleoside and 2,2-dimethoxypropane (50). In an interesting paper on the periodate oxidation of pseudouridine-

304

Sect. VI

-

S m i s s m a n , Ed.

Topics i n C h e m i s t r y

3'-phosphate (I) a t EH 8.9, it w a s found t h a t t h e products of oxidation were 5 -formyluracil,, 5 -carboxyuracil, inorganic phosphate, formaldehyde, i s Ilowered toward 7, t h e oxidative and unidentified products. As t h e @ r e a c t i o n is slower, and 5-carboxyuracil appears t o be t h e only heteroc y c l i c product formed (51). Based on t h e observation t h t pseudouridine and pseudouridine-2'(3 ' )-phosphate i s isomerized i n e i t h e r acid o r a l k a l i t o give a mixture of two anomeric furanose derivatives and two anomeric pyranose derivatives, t h e following mechanism of periodate oxidation of I w a s proposed (51):

0

0

>

+--

OH

OP

I1

I

CHO

0

1

CH-CHO

+

Isomers of I

(ko

€IN

Hoe --->

0

HCHO

+

bP

CHO IV

OP

OA

I11

a p r i o r i , One might expect t h a t t h e 5-carboxyuracil formed from I m i g h t a r i s e by "weroxidation"of I V . However, when IV w a s exposed t o potassium periodate, t h e r a t e of oxidation of IV t o t h e 5-carboxy d e r i v a t i v e was t o o low t o account f o r t h e amount of 5-carboxyuracil from I. Therefore, a second pathway w a s proposed t o account f o r t h e formation of V I I I and involves t h e d i r e c t oxidative hydroxylation a t C - 1 ' t o give V which by one of t h e pathways shown can give p c a r b o x y u r a c i l . Similar experiments were performed on pseudouridine-3 I ,3-' -phosphate (51).

Chap. 27

Hyoyo Nucleotides

30 5 -

Schaeffer

Ioh-

__j

104-

1-

OP

v

5.

OH

0

OP

VI

Q 0

HN

VIII

VII Dekker has described a unique method for the separation of nucleoside mixtures on Dawex-1 (OH) using methanol and water mixtures. This separation procedure is based primarily on ion-exchange and consequently, should find wide application both for micro- as well as largescale syntheses (52). In an interesting report by Leonard and Laursen, the preparation of 3-B-D-ribofuranosyladenine (3-isoadenosine) and the corresponding 5'phosphate w a s described (53). Because 3-isoadenosine supported the growth of an adenine-requiring Escherichia -mutant and inhibited the growth of certain mammalian cell cultures, coenzyme analogs of 3-isoadenosine were prepared (54). 3-Isoadenosine-5-triphosphate replaced ATP in a hexokinase enzyme system and in the production of light in the luciferin-luciferasesystem. 3-Isoadenosine-5' -phosphate was'converted in into the 3-isoadenosineanalog of nicotinamide-adenine dinucleotide (NMN-3-iso-AMP)and this isomer was studied in a variety of dehydrogenase enzymes. Surprisingly, alcohol and glutamic dehydrogenase "-3iso-AMP was reduced more rapidly than NAD (54). The observation that the 3-isoadenosine derivatives are active in enzyme systems will probably lead to a better understanding of the reaction mechanism and binding sites of certain enzymes.

306

Sect. VI

-

Topics in Chemistry

Smi s s m a n , Ed.

aorona and his coworkers have continued their extensive investigation on polynucleotides (55,56,57,58,59). For the synthesis of ribodinucleotides bearing a 3'-phosphomonoester group, a method was developed which involves the condensation of a fully protected purine or pyrimidine 3'-phosphate with uridine-2',3'-cyclic phosphate. This general approach should be applicable to all bases (55). Some (3l-5') linked diribonucleoside phosphates containing 3- and 5-methyluracil have, in general, been synthesized by the condensation of a 3I-nucleotide blocked at the 2 I - and 5 -positions by trityl groups with a 2 I , 3 -isopropylidene derivative of a second nucleoside (60). In addition, a general procedure for the isolation of "minor" nucleoside from the hydrolysates of ribonucleic acid has been described (61). The synthesis of dinucleoside phosphates by the "Anhydronucleoside MetAod" (62,63,64,65) is finding extensive utilization. For example, the synthesis of cytidylyl-(3' -5' ) adenosine was readily accomplished by the condensation of lt(2,3'-anhydro2 ' ,5 ' -di-O-trityl-@-D-xylorufanosy1)cytosine with adenylyl-5' benzoic anhydride (66). The dismutation of adenosine 5'- triphosphate as i t s tributylamine salt in anhydrous pyridine has been found to producetwwseries of compounds: a homologous series of adenosine 5'-polyphosphates containing up to seven phosphate groups and a second minor series of a,@-di(adenosine5 ')polyphosphates. The presence of an excess of ortho or-pyrophosphate leads to the formation of AMP and ADP (67). A study of-the reaction mechanism of he dismutation of ATP to adenosine 5'-polyphosphates utilizing a-3'P-labeled-ATP and$ -32P-ldabeled-ATP in pyridine led to the conclusion that pyridine attacks the'Il-phosphorus of ATP to give ADP and a covalently bonded N-phosphorylpyridinium ion which is the reactive phosphorylating agent (68). a-Adenosine-5' -phosphate has been synthesized by the condensation of 5-diphenylphosphoryl-D-ribofuranosylbromide 2 ' ,3 -cyclic carbonate with chloromercuri-6-benzamidopurine and it was shown that a-adenosine5I-phosphate is not a substrate for snake venom 5'-nucleotidase (69). For'a program on the synthesis of phosphonic acid analogs of nucleoside phosphates, the preparation of 5'-adenylyl methylphosphonate and 5'adenylyl chloromethylphosphonatewas accomplished by the condensation of adenosine 5'-phosphate and the alkylphosphonic acid in the presence of dicyclohexylcarbodiimide (70). The synthesis of adenosine-5'-methylenediphosphonate was accomplished by the condensation of 2 I , 3 ' -isopropylideneadenosine with methylenediphosphonic acid (71). Baker, Tanna and Jackson have attempted to simulate 5'-phosphoribosyl binding to thymidylate synthetase and succinoadenylate kinosynthetase by the utilization of 5I-O-carbamates of nucleosides but the 5I-O-carbamoyl derivatives of 6-mercaptopurine ribonucleoside, thymidine, and 2 deoxy-5-fluorouridinewere noninhibitory against these two enzymes (72).

Chap. 27

Nucleotides

Schaef f er

307

REFERENCES

1.

J. A. Montgomery and H. J. Thomas, Advai.Carbohydrate Chem., &l 301 (1962).

2.

T. L. V. Ulbricht, Angew Chem. Intern. Ed. Engl.,

3.

A. M. Michelson, 'The Chemistry of Nucleosides and Nucleotides," Academic Press, Inc., New York, N. Y., 1963.

4.

H. G. Khorana,"Some Recent Developments i n the Chemistry of Phosphate Esters of Biological I n t e r e s t , "John Wiley and Sons, Inc., New York, N. Y., 1961.

5.

T. Simadate, Y. Ishido, and T. Sato, Nippon Kagaku Zasshi,

6.

Y. Ishido and T. Sato, Bull. Chem. S O C . Japan,

7.

M. J. Robins, W. A. Bowles, and R. K. Rows, J. Am. Chem. SOC., 86, 1257 (1964)-

8.

T. Hashizume and H. Iwamura, Tetrahedron Letters, 3095 (1965).

9.

W. P f l e i d e r e r and R. K. Robins, Chem. Ber.,

10.

N. Yamaoka, K. Aso and K. Matsuda, J. Org.Chem.,

(1961) *

11. E. Wittenburg, Angew. Chem., 12.

476 (1962).

a

1063

82, 938

1347 (1961).

98, 1511 (1965). 149 (1965).

(1965).

J. A . Montgomery and H. J. Thomas, J. Am. Chem. SOC., 8 L 5442 ( 19651

-

13. Y. Mizuno, N. Ikekawa, I. Itoh and K, Saito, J. Org. Chem., 3,

4066 (1965).

14. M. 3Ckehara and H. Tada, J.

15

Am. Chem. S O C . ,

& 8

606 (1965).

R. E. Hohes and R. K. Robins, J. Am. Chem. SOC., 8 J

16. M. J. Robins and R. K. Robins, J.

Am. Chem. S O C . ,

17

C. P. Whittle and R. K. Robins, J. Am. Chem. SOC.,

18.

J. Prokop and D. H. Murray, J. Pharm. Sci.,

19. D.

H. Murray and J. Prokop, J. Pharm. Sci.,

1772

(1965).

8 L 4934 (1965).

82, 4940

(1965).

54. 359 (1965). 54, 1468 (1965).

20.

E. Walton, F. W. Holly, G. E. Boxer, R. F. Nutt and S. R. Jenkins, J. Med.Chem., & 659 (1965).

21.

S . R. Jenkins, F. W.

( 1965)

Holly and E. Walton, J. Org. Chem.,

2851

308

Sect. VI

-

S m i s s m a n , Ed.

Topics i n C h e m i s t r y

22.

C . 0. Gitterman, R. W. Burg, G. E. Boxer, D. Meltz and J. H i t t , J. Med. Chem., 8_, 664 (1965).

23

E. J. Reist, V. J. Bartuska, D. F. Colkins and L. Goodman, J. Org. Chem., 3401 (1965).

9

24.

R. H. Shah, H. J. Schaeffer and D. H. Murray, J. Pharm. Sci., 15 (1965).

25.

M. P. Mertes, S. E. Saheb and D. Miller, J. Heterocyclic Chem.,

26.

T. Y. Shen, W. V. Ruyle and R. L. Bugianesi, J. 'Heterocyclic Chem.,

493 (1965).

&

2,, 495 (1965).

27. J. A. Montgomery (1965)

and K. Hewson, J. Heterocyclic Chem.,

& 313

&

28.

E. Benz, N. F. Elmore and L. Goldman, J. Org. Chem.,

29

T. Y. Shen, H. M. Lewis and W . V. Ruyle, J. Org. Chem.,

( 19651*

3067

(1965).

& 835

C. P. J. Glaudemans and H. G. Fletcher, Jr., J. Org. Chem., 28, 3004 ( 1963)

I. L. Doerr, J. F. Codington and J. J. Fox., J. Org. Chem.,

467 (1965) *

a 476

32.

J. F. Codington, I. L. Doerr and J. J. Fox, J. Org. Chem.,

33 *

J. J. Fox and 1-Wempen, Tetrahedron Letters,

34

C. L. Stevens and P. Blumbergs, J. Org. Chem.,

35.

G. Etzold and P. Langen, Chem. Ber.,

36 *

W . W.

37

M. L. Wolfrom and P. McWain, J. Org. Chem., &log9

38

M. L. Wolfrom, H. G. Garg, and D. Horton, J. Org. Chem.,

39.

M. L. Wolfrom and R. Wurmb, J. Org. Chew.,

40.

K. A . Watanabe, J. Beranek, H. A. Friedman, and J. J. Fox, J. Org.

41.

( 19651

643 (1965).

2, 2723 (1965).

98, 1988 (1965).

Zorbach, H. R. Munson, and K. V. Bhat, J. Org. Chem.,

3955 ( 1965)

( 19651

&em.,

a 3058

(1965). & 1556

(1965).

3% 2735 (1965).

F. W. Lichtenthaler, H. Palbrecht and G. Olfermann, Angew. Chem.,

1zI

131 (1965).

Chap. 27

Nucleotides

30 9 -

Schaeffer

42.

J. Berhek, H. A. Friedman, K. A. Watanabe and J. J. Fox, J. Heterocyclic &em. 5 188 (1965).

43

F . W. Lichtenthaler, H. P. Albrecht, G. Olfermann and J. Yoshimura, Angew. Chem., 77, 731 (1965).

44.

D. M. Brawn and A. P. Read, J. &em.

45

A. D. Broom and R. K. Robins, J. Am. Chem. SOC.,

46.

C. B. Reese and D. R. Trentham, Tetrahedron Letters, 2459

47

C. B. Reese and D.

48.

J. Zemlicka and S. Chladek, Tetrahedron Letters, 3057 (1965).

49.

A. Hampton, J. C. Fratantoni, P. M. C a r r o l l and Su-chu Wang, J. Am. Chem. Soc., 8z( 5481 (1965).

SOC., 5072

(1965). 8 J

1145 (1965).

(1965)

R. Trentham, Tetrahedron Letters, 2467 (1965).

A. Hampton, J. Am Chem. SOC.,

4654 (1965).

M. T ~ m a s z , Y. Sanno and R. W. Chambers, Biochemistry,

( 19651

&

1710

52.

C. A. Dekker, J. Am. Chem. SOC. 8 J

53.

N. J. Leonard and R. A. Laursen, Biochemistry

& 354 (1965).

54.

N. J. Leonard and R. A. Laursen, Biochemistry

& 365 (1965).

55.

D. Sd11 and H. G. Khorona, J. Am. Chem. SOC.

8L 350 (1965).

56.

D. ,5811 and H. G. Khorona, J. Am. Chem. SOC.

8L 360 (1965).

402'7 (1965).

57- T. M. Jacob and H. G. Khorana, J. Am. Chem.

SOC.

8 J

368 (1965).

58. E. Ohtsuka, M. W. Moon and H. G. Khorana, J. Am. Chem. SOC. 8L 2956 (1965). 87, 2971 (1965).

59.

T. M. Jacob and H. G. Khorana, J. Am. Chem. SOC.

60.

R. Thedford, M. H. Fleysher and R. H. H a l l , J. Med. Chem.

61.

R. H. Hall, Biochemistry

62.

Y. Mizuno, T. Sasaki, T. Kanai and H. Igarashi, J. Org. Chem. & 1533 (1965).

( 19651

5

486

& 661 (1965).

J. Zemlicka and J. Smrt, Tetrahedron Letter, 208 (1964). J. NBgyvary and J. S. Roth,

w , 6 1 7 (1965).

310

Sect. VI

-

T o p i c s in Chemistry

=,

S m i s s m a n , Ed.

2451 (1965).

65.

K. L. Agarwald and M. M. Dhar,

66.

Y Mizuno and T. Sasaki, Tetrahedron Letters, 4579 (1965).

67.

D. L. M. Verheyden, W . E. Wehrli and J. G. Moffatt, J. Am. Chem. Soc. 8 3 2257 (1965).

68.

W. F. Wehrli and J. G. Moffatt, J. Am. Chem. Soc.,

69.

M. Ikehara, E. Ohtsuka, E. Honda and A. Nomura, J. Org. Chem.,

70.

T. C. Myers and L. N. Simon, J. Org. Chem.

71.

T. C. Myers, K, Nakmra and A. B. Danielzadeh, J. 1517 (1965).

72.

B. R. Baker, P. F. Tanna and J. D. F. Jackson, J. Pharm. Sci.

82 3760 (1965).

1077 (1965).

987 (1965).

443 (1965). Org. Chem.

a

31 1 -

Chap.28 Alkaloids

William I. Taylor CIBA Pharmaceutical Company Work on s y n t h e t i c r e s e r p i n e d e r i v a t i v e s c o n t i n u e s , new 18O-phenoxyacetic a c i d and phenylmercaptoacetic a c i d esters of methyl r e s e r p a t e have been prepared’ and w e r e found t o be much more weakly a c t i v e than r e s e r p i n e . Racemic 17-desmethoxydes e r p i d i n e s y n t h e s i z e d by t h e Woodward r o u t e , i n c o n t r a s t t o an e a r l i e r c l a i m Y 2 i s s a i d t o show no s e d a t i v e a c t i v i t y . = Mediodespidine ( I ) , a t e t r a h y d r o i s o q u i n o l i n e analogue of r e s e r p i n e , e x h i b i t s a r e s e r p i n e - l i k e blood p r e s s u r e d r o p i n Maccacus rhesus monkey given an intravenous dose of 0.5 mg. K. However, no s e d a t i v e e f f e c t was observed u n t i l a dose l e v e l of 10 mg. K. had been reached.‘ T h i s r e s u l t i s t o be c o n t r a s t e d An w i t h t h e statement t h a t I (OCH20 = 2 OCH3) i s i n a c t i v e . ’ e x c e l l e n t summary of t h e voluminous work on r e s p e r i n e congeners has appeared.

’

OClH, (1)

H (11)

The c u r a r e a l k a l o i d , Macusine-B (11) has been shown t o b l o c k a - a d r e n e r g i c receptors and t o s t i m u l a t e @ - r e c e p t o r s y 7 b u t i s not of p r a c t i c a l use. The a c t i v i t y of Epena snuff powder of t h e Waica t r i b e (South American I n d i a n s ) i s due t o simple i n d o l e d e r i v a t i v e s . ’ The complete s t r u c t u r e , s t e r e o c h e m i s t r y and a b s o l u t e conf i g u r a t i o n of t h e antileukemic vinca a l k a l o i d s , l e u r o c r i s t i n e (111); R1 = CHO- R2 = COCH3) and v i n c a l e u k o b l a s t i n e (111; R1 = CH3; R2 = COCH3j has been unequivocally e s t a b l i s h e d by x - r a y a n a l y s i s of l e u r o c r i s t i n e methiodide.’ C l i n i c a l success has been achieved w i t h a modified v i n c a l e u k o b l a s t i n e , v i z . , I11 (R1 = CH3; R2 = C O * C H 2 * N ( C H 3 ) 2 one of a series of compounds prepared from d e s a c e t y l v i n c a l e u k o b l a s t i n e (111; R1 = CH3; R2 = H ) . l o There was a s t r i k i n g c u r a t i v e e f f e c t of t h i s p a r t i a l l y s y n t h e t i c compound on P-1534, t h e r a p y being e q u a l l y e f f e c t i v e by t h e o r a l or p a r e n t e r a l r o u t e . Remissions have been produced i n p a t i e n t s w i t h Hodgkins d i s e a s e and t h e r e was a l a c k of c r o s s r e s i s t a n c e between t h i s drug and o t h e r vinca a l k a l o i d s and alkylating agents.

312

Sect. VI

-

Topics in Chemistry

S m i s s m a n , Ed.

Mimosine ( I V ) about 1%i n t h e d i e t of r a t s , caused a complete i n h i b i t i o n of t h e e s t r o u s c y c l e and complete i n f e r t A normal c y c l e r e t u r n e d upon c e s s a t i o n of t h e ility. treatment. I t was suggested t h a t an antagonism t o vitamin Be could account for t h e above e f f e c t s . I n j e c t i o n of l a s i o c a r p i n e (V) hydrochloride i n t o r a t s r e s u l t s i n a high mast c e l l count i n t h e p a r a - a o r t i c lymph nodeg a p r o p e r t y n o t shared by t h e o t h e r p y r r o l i z i d i n e a l k a l o i d s tested.'= The N-oxide reduces m i t o s i s of t h e parenchymal c e l l s i n l i v e r s of r a t s . l 3

0

N

lasiocarpine

v

Chap. 28 1. 2.

3. 4.

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

11. 12.

13.

313 -

Taylor

Alkaloids

I. E r n e s t and M. Protiva, C o l l e c t . Czech. Commun., 3355 (1965). F. L. Weisenborn, 2. Chem. 2, 4818 (1957). I. E r n e s t , J. 0. J i l e k , J x r k o v s k y and M. P r o t i v a , C o l l -

Jirkovsky,

2,

&.

s.,

z,

e c t i o n Czech. Chem. Commun., 2395 (1965). V. Trcka, A. D m c and M. Vanecek, L i f e Sciences,

2257 (1965).

-

2,

B. B e l l e a u and J. Puranen, Canad. 2. Chem., E. S c h l i t t l e r i n Alkaloids, R. H. F. Academic Press, New York, 1965. B. E. Leonard, J. Pharm. Pharmacol., 566 ( 1965)* B. Holmstedt, Aich. I n t . Pharmocodyn. %er * , a 6 , 285

( 1965) * J. w. Moncrief

( 1965) *

and W.

N. Lipsconib,

2.

&.

Chem.

SOC.,

3,

J. G . Armstrong, R. W. Dyke, P. J. Pouts, J. J. Hawthorne and c. Jansen, Proc. Am. Ass. Cancer 6, 3 (1965); I. S. Johnson, W. W. H a r z o v e and M. F. Wright: i b i d . ,

6, 33

-

J.

(1965).

Hylin, I. J. Lichton, Biochem. Pharmacol.,

T.

W.

E.

D i e n e r , Nature,

1167 (1965).

J.

E.

e. ,

Peterson,

2.

,

1305 ( 1965) g h o l . Bacteriol.,

2,1167

9,153 (1965).

314 Chap. 29

Sect. VI

- Topics in Chemistry

S m i s s m a n , Ed.

Reactions of I n t e r e s t i n Medicinal Chemistry

by Edward E. Smissman Department of Medicinal Chemistry, U n i v e r s i t y of Kansas The medicinal chemist i s i n t e r e s t e d i n t h e whole spectrum of o r g a n i c r e a c t i o n s and i s u s u a l l y w e l l versed i n t h e l i t e r a t u r e of s y n t h e t i c o r g a n i c chemistry. T h i s p r e s e n t a t i o n w i l l a t t e m p t t o t r e a t s o m e new and/or novel r e a c t i o n s which w e r e r e p o r t e d d u r i n g 1965. A v e r y i n t e r e s t i n g book by House' appeared during t h e y e a r and o f f e r s a condensed and c r i t i c a l e v a l u a t i o n of modern b a s i c s y n t h e t i c methods. Since t h i s r e p o r t cannot be a l l i n c l u s i v e t h e r e a c t i o n s d e s c r i b e d w i l l be grouped i n a c l a s s i c a l manner a s f a r a s possible. Methods of r e d u c t i o n a r e c o n s t a n t l y being explored and hydrogenation w i t h homogeneous c a t a l y s t s have t h e advantage of being u n a f f e c t e d by t h e u s u a l c a t a l y s t poisons. The r e d u c t i o n can be performed under r e l a t i v e l y m i l d c o n d i t i o n s a s i l l u s t r a t e d by t h e hydrogenation of methyl l i n o l e a t e i n t h e presence of i r o n pentacarbonyl c a t a l y s t , F e ( C O ) 5 t o produce t h e monoene i n 65.5% y i e l d a t looo and 400 p s i . 2 The homogeneous c a t a l y t i c hydrogenation of a c e t y l e n e s gave q u a n t i t a t i v e y i e l d 8 of the corresponding a l k a n e s . 3 Hydrogenation occurred a t less than 1 atm. a t 20' using 0.005 m o l e (Ph~P)3FthC1, 2 moles of t h e a c e t y l e n e a l l d i s s o l v e d i n a mixture of ethanol-benzene. Diimide (H2N2) r e d u c t i o n s have been reported many t i m e s p r i o r t o t h i s year and continuous work on t h e i r s e l e c t i v i t y i s underway. Diimide was used i n t h e r e d u c t i o n of aldehydes and ketones t o t h e corresponding a l c o h o l s . A l i p h a t i c carbonyls w e r e reduced slowly and t h e y i e l d was l o w (max whereas aromatic aldehydes and ketones gave y i e l d s a s high a s [email protected] A new s y n t h e s i s of N-monosubstituted hydroxylamines i n volves t h e r e d u c t i o n of a l i p h a t i c ketoximes and aldoximes w i t h diborane. An i n t e r m e d i a t e i s formed which on h y d r o l y s i s under a c i d i c or b a s i c c o n d i t i o n s g i v e s t h e hydroxylamines i n y i e l d s of 50-90$.5 Nickel borane can be used i n s e l e c t i v e d e s u l f u r i z a t i o n . The y i e l d s obtained a r e comparable t o t h o s e obtained w i t h Raney-N; i n c e r t a i n i n s t a n c e s b u t i n o t h e r s , s u c h a s w i t h p - t o l y l s u l f o n a t e s , no d e s u l f u r i z a t i o n occurs." Reduction of ketones w i t h L i A l H 4 - A l C 1 3 w i l l g i v e s t e r e o s e l e c t i v i t y dependent on c o n d i t i o n s . I n t h e mixed hydride r e d u c t i o n under k i n e t i c c o n t r o l , nor-camphor gave 95% of t h e l e s s s t a b l e endo-norborneol and camphor gave 66-71$ of t h e less s t a b l e isoborneol, hydride approaching from t h e less hindered s i d e i n b o t h bases. E q u i l i b r a t i o n of norborneols w i t h excess The ketone l e a d s t o 89-9M of t h e m o r e s t a b l e exo-isomers. alkoxyaluminum d i o h l o r i d e s formed from t h e x t o n e ( o r a l c o h o l ) and mixed anhydride, i n t u r n f u n c t i o n a s reducing agents. Isobornyloxyaluminum d i c h l o r i d e i s a f a i r l y s t e r e o s e l e c t i v e reducing agent i n t h a t it produces l a r g e l y a x i a l alcohols.'

lw),

-

Chap. 29

Chemical Reactions

Smi s s m a n

315 -

Lithium trimethoxy aluminum hydride i s a s a l e c t i v e reducing agent which w i l l reduce n i t r i l e s and amides t o amines b u t r e a c t s slowly w i t h epoxides.’ A good method f o r t h e r e d u c t i o n of p y r i d i n e N-oxide and i t s a l k y l d e r i v a t i v e s involves t h e use of t h i o n y l chloride a t 50’. The m i x t u r e i s allowed t o s t a n d 30 min. and on i s o l a t i o n A new method f o r g i v e s t h e desired amine i n 60-85% y i e l d . ’ t h e r e d u c t i o n of aromatic n i t r o compounds w i t h s u l f u r and sodium hydroxide i n an acetone-methanol s o l u t i o n g i v e s high y i e l d s of t h e corresponding amine.” T e r t i a r y amides w e r e reduced t o t e r t i a r y amines w i t h sodium borohydride i n p y r i d i n e i n good y i e l d s . l1 Oxidation of primary and secondary amines w i t h a suspens i o n of a r g e n t i c p i c o l i n a t e i n a p o l a r o r g a n i c s o l v e n t or water y i e l d s aldehydes or ketones from most amines. Y i e l d s t o g i v e aldehydes and ketones w e r e b e t t e r w i t h secondary amines being i n t h e 30-90$ range.12 This r e a g e n t was found t o be s u p e r i o r t o mercuric a c e t a t e or s i l v e r oxide. Ozonization of t e r t i a r y amines and amides was found t o induce a Polonovski r e a c t i o n . When N,N-dimethylanthranilic a c i d was ozonized i n e t h y l a c e t a t e , methylene c h l o r i d e , or methanol s o l u t i o n a t Oo t h e d e m e t h y l a t e d groduct and t h e corresponding hydrobenzoxazone w e r e obtained. Lead t e t r a a c e t a t e was found t o r e a c t w i t h primary amides I t was suggested t h a t t h e mechanism i n t o form acylamines.” volves t h e formation and rearrangement of an a c y l n i t r e n e . The NH2

+

Pb(OAc)4

+ RN

r e a c t i o n can be used t o form a l k y l carbamates i f it i s perCyclohexanecarboxamide formed i n t h e presence of an alcohol.’’ gave a 9€$ y i e l d of t h e methyl N-cyclohexylcarbamate.

(3:

C-NH2

+

Pb(OAC)4

+

CHaOH

+O ; - ! ! - O - C H s

The scope of t h e o x i d a t i o n of many d i f f e r e n t types of hydroxyl f u n c t i o n w i t h dimethyl sulfoxide-carbodiimide h a s been The p a r t i c u l a r u t i l i t y of the m e t h o d f o r t h e oxidastudied.” t i o n of primary a l c o h o l s t o aldehydes and of s e n s i t i v e molecules such a s homoallylic a l c o h o l s i s emphasized. The method has been s u c c e s s f u l l y a p p l i e d t o a l k a l o i d s and t o an unusual dehydration of a hemiacetal t o g i v e a v i n y l ether. Oxidation of benzylalcohol, p s u b s t i t u t e d benzyl a l c o h o l s , and t r a n s - c i n namylalcohol t o t h e corresponding aldehydes was performed s u c c e s s f u l l y w i t h t - b u t y l chromate. l 7 Ketones can be regenerated f r o m their p-toluenesulfonylThe r e a c t i o n hydrazones by r e a c t i o n s w i t h l e a d t e t r a a c e t a t e . ” i n a c e t i c a c i d a t 20° evolves n i t r o g e n and was s u c c e s s f u l w i t h acetophenone, acetone, cyclohexanone, (+)camphor, ( - ) p i p e r i t o n e , and benzophenone. Lead t e t r a a c e t a t e was a l s o found t o

Sect. VI

316

-

S m i s s m a n , Ed.

Topics in Chemistry

c o n v e r t a l d e h des i n t h e presence of ammonia t o t h e corresponding n i t r i l e . ” B e s t y i e l d s w e r e obtained w i t h aromatic RCHO

+ NH3

Pb( OAC)

4

>

RC=N

aldehydes. Another convenient one s t e p conversion of aldehydes t o n i t r i l e s involves t h e r e f l w i n g of a s o l u t i o n of t h e aldehyde i n formic a c i d w i t h hydroxylamine hydrochloride and sodium methoxide.20 A number of aldehydes w e r e converted t o n i t r i l e s by t h i s method and it was found t o be p a r t i c u l a r l y s u c c e s s f u l i n t h e aromatic series. A new m e t h o d for halodecarboxylatioglof a c i d s u t i l i z e s lead t e t r a c e t a t e and i o n i c h a l i d e s a l t s . It i s a convenient method. A photochemical transformation a f f o r d s an e x c e l l e n t method f o r t h e decarboxylation of a c i d s . 2 2 This method involves RCO2H

+

Pb(oAC)4

+ Mx +

Rx

+ Cop +

Pb(OAc)2

+ HOAC + MOAC

t h e photochemical decarboxylation of acylhypoiodites. Primary, secondary, and t e r t i a r y a c i d s , a s w e l l a s t h e h i t h e r t o i n t r a c t able g l u t a r i c and a d i p i c a c i d s , can be decarboxylated without

difficulty. The continuous i n v e s t i g a t i o n of v a r i o u s a d d i t i o n r e a c t i o n g i v e s unique s y n t h e t i c methods f o r t h e p r e p a r a t i o n of new types of compounds. Phosphorous t r i b r o m i d e can be added t o o l e f i n s under a v a r i e t y of c o n d i t i o n s t o g i v e B-bromoalkylphosphorous dibr~mides.N ~ i~t r o s y l c h l o r i d e w i l l react w i t h a v a r i e t y of u n s a t u r a t e d compounds. Methyl methacrylate was found t o g i v e I CH3 I ONCH2 C02CH3

CH3 I CH2 CO2CH3

Fc1

I F

Q NO

I

I1

when heated w i t h n i t r o s y l c h l o r i d e i n a sealed tube b u t I1 was formed when t h e r e a c t i o n was performed i n t h e presence of aluminum c h l o r i d e . 2 4 With c y c l i c o l e f i n s t h e most h i g h l y subs t i t u t e d chloride i s obtained. The r e s u l t i n g compound can be used f o r the p r e p a r a t i o n a-alkylated-a,B-unsaturated ketcnes.=”

a

1. Acetone/ NOCl

Pyridine

2. H2SO4

CI xp. 29

Chemical Reactions

Smi s sman

317 -

The formation of a new c l a s s of a l i p h a t i c d i a z o compounds by t h e a d d i t i o n of s u l f o n y l a z i d e s ( A r S 0 2 N 3 ) t o ethoxyacetylene was r e p o r t e d . 2 e The product (111) can be reduced t o t h e c o r responding hydrazine, caused t o r e a c t w i t h a c e t y l e n e s , RSO2N :C ( OCzH5) CHN2

I11

triphenylphosphine, and mineral a c i d s . " Chloroacetaldehyde w i l l r e a c t w i t h o l e f i n s i n t h e presence of aluminum c h l o r i d e . Cyclopentene w i l l undergo t h i s r e a c t i o n t o g i v e an i n t e r m e d i a t e f o r t h e p r e p a r a t i o n of unique heteroc y c l i c compounds. 27

The a d d i t i o n of p o s i t i v e halogen compounds can be i l l u s t r a t e d by s e v e r a l r e a c t i o n s . Mono and dibromomalononitrile, v i a a f r e e r a d i c a l i n t e r m e d i a t e , w i l l add t o a l i p h a t i c 2 8 and a r y l subs t i t u t e d olefins2' i n good y i e l d s . A novel method f o r t h e cu CH3( CH2) s C H d H 2 + BrCH( CN) 2 4 CH3( CH2) 5 HCH&H( CN) 2

FB r

@C%CH2

+

BrCH(CN)2

~K> *HBCH(CN)~ Br

p r e p a r a t i o n of a , a - d i c h l o r o ketones involves the r e a c t i o n of a c e t y l e n e s w i t h N-chlorosuccinimide i n methanol t o . g i v e t h e dihalodimethyl k e t a l s which a r e r e a d i l y hydrolyzed w i t h d i l u t e a c i d s t o t h e ketones.30 The r e a c t i o n i s of g e n e r a l use and a f f o r d s t h e ketones i n y i e l d s of 60-8N. Enol a c e t a t e s derived from aldehydes having two a-hydrogens r e a c t w i t h b i s ( 1,2-dimethylpropyl)borane. The i n i t i a l addit i o n involves a s l o w "anti-Markovnikov" hydroboration b u t a r a p i d e l i m i n a t i o n and rehydroboration occurs.31 Enol a c e t a t e s derived ketones do n o t g e n e r a l l y r e a c t .

Sect. VI

318 -

-

Topics in Chemistry

S m i s s m a n , Ed.

Hydroxylamine-0-sulfonic acid will aminate olefinic, acetylenic, and aromatic compounds.32

@CH'=CH2

+

H2NOSOaH

pp;

>?FeC1 CH3OH FeS04

>

@ -CH2 oay, NH2

New uses of eneamines have been reported. A new heterocyclic synthesis involving an enamine cycloaddition with Ncarbethoxyaziridine and 1-pyrrolidinocyclohexene produced a 42% yield of IV.33 Dichlorocarbene forms an adduct with the piperdine

0

IVCooCaH5

eneamine of cyclopentanone which undergoes ring expansion to 2-chloro-2-cyclohexene-l-one.34 Ring expansion from the corresponding cyclohexanone adducts was not obtained. Benzene diazoniumchloride was found to react with l-piperidinocyclohexene. 35

n

0

a-Formyl ketones can be prepared from enamines by Vilsmeir formylation. Phosgene, dimethyl formamide, and l-morpholinocycloalkenes yielded the a-formylketones on hydr~lysis.~' Phosphorous acid tris(dimethylamide), [ ( M ~ Z N ) ~ P ] is , suitable in many cases in lieu of triphenylphosphine for the Wittig rea~tion."~ The increased rate of formation of the phosphonium salts and the water solubility of the resulting ( M ~ z N ) ~ P O formed along with the olefins simplified the preparation of the olefins. "he Wittig reaction is used in a new synthesis of a-branched-B-keto esters.38 The product of the Wittig is C1

+

Ph3P

p

COzEt)R' * PPhsIC1 C-COzEt o-200 3 [I&( I c 6H6 R'

Chap. 29

Chemical Reactions

319 -

Smi s s m a n

electrolyzed to give good yields of the corresponding ester. Other yields have found wide synthetic use. Dimethyloxosulfonium methylide and dimethylsulfonium methylide are both nucleophiles and both function to transfer methylene to certain electrophilic unsatur ed linkages including C=O, C=N, C=S, and in certain cases C=CO3$ A new synthetic route to ketones and "overall methylene insertion" are examples of the use of this system. Carbamyl-stabilized sulfur yields have been prepared via treatment of the corresponding sulfonium salts with sodium hydride. These yields react with Schiff bases to produce 3aryl amino cinnamate der atives to demonstrate the synthetic potential of the series.

48

A new synthesis in good yields of amino alcohols with tertiary alcohol groups involves a selective Grignard reaction.41

The reaction of vinyl Grignard reagents with a-unsaturated esters gives a preparative method for a,y'-unsaturated ketones.42 0

RCH=CHMgCl +

It

RYHCHCCH = YCH3 Ch2=CH R

CH CH C = CHC02Et 3 31

R

Triethylaluminum reacts with benzonitrile. 43 The reaction is believed to proceed through a 6-ring complex utilizing one of the 0

ethyl groups from triethylaluminum for ring formation. a,a'-Dibrominated dicarboxylic acids can be prepared by bromination in formic acid solution in the pr sence of red phosphorous and irradiation with a 200 watt kmp.t4 The yields are excellent. A convenient synthesis of cycloalk-2-eneones and a,P,a',P'-cycloalkadienones utilizes the direct bromination of 45 several cycloalkanones in ether followed by dehydrobromination.

'

Other interesting substitution reactions have been reported durin.g the year. N-formyloxyme 1-N-methylformamide proves to be a useful electrophilic reagent. The acid catalyzed reactions of this compound with carboxylic acids, alcohols, a mercaptan, 9-naphthol, anisol, phenol, and thiourea have been studied. All the reaction involve the carbonium ion,@ CH NMeCHO = as an intermediate, The phenolic hydroxyl doup will nucleophilic replacement by the mercapto group in acid solution. The following mechanism was proposed.

3 20

Sect. VI

-

Topics in Chemistry

S m i s s m a n , Ed.

7 RS

A simple s y n t h e s i s of p-hydroxy-a-amino m o d i f i c a t i o n of t h e S t r e c k e r r e a c t i o n . 4 8 CH2=CH-OAc

-&+

CH2CH( O A c )

I

OAc

1.NH40H KCN/ 2. HC1

>

a c i d s involves a 1:2-E2C00H

S e r i n e can be converted i n t o a p y r a z o l i n e b y a novel s e r i e s of I f t h e p y r a z o l i n e i s heated, cyclopropane reactions.*' COOH

2,4,6

(Me02C)3CeH2F = DL-Serine

NaHC03

> f"2-c\I

CH2Np

NH

CO 2 M e

&Me

d e r i v a t i v e s a r e obtained. Threonine a f f o r d e d s i m i l a r r e s u l t s . A new method was developed f o r t h e s y n t h e s i s of cyclopropane d e r i v a t i v e s by t r e a t i n g phosphonenolpyruvic a c i d t r i e t h y l e s t e r [ ( E t O ) 2P( 0) CH( :CH2) C 0 2 E t 3 (V) w i t h compounds c o n t a i n i n g a c t i v e methylene groups.50

VI, was reR-H, R'=NH2, n=O gave an active analgetic.

A new sgyies of 1-aminomethylbenzocyclobutenes,

ported,

Chap. 29

Chemical Reactions

Smi s s m a n

321 -

Tryptophan can be s e l e c t i v e l y a l k y l a t e d a t t h e indole-N p o s i t i o n w i t h sodium i n l i q u i d ammonia and v a r i o u s a l k y l h a l i d e s . 5 2 E t h y l a c e t o a c e t a t e can be benzoylated by using E t h y l b e n z o a c e t a t e i s obtained i n 45s aluminum c h l o r i d e . y i e l d . A method is described f o r t h e cleavage of benzyl ethers i n molecules c o n t a i n i n g m u l t i p l e bonds u t i l i z i n g sodium and butanol. 8-Phenoxyacrolein w i l l condense w i t h sodio d e r i v a t i v e s of a c t i v e methylene compounds.55 The l a t t e r compound w i l l undergo

’’

’‘

OCH=CHCHO

+

NaCH ( C 0 2 E t ) 2

---+

( E t 0 2 C ) 2CHCH=CHCHO

v a r i o u s r e a c t i o n s such a s a l k y l a t i o n and eneamine formation.

322 -

1. 2.

3. 4.

5. 6. 7. 8. 9. 10. 11. 12.

130 14.

15. 16.

17.

18. 19.

20.

21.

22.

23.

24.

25. 26.

27 28.

Sect. VI 0. House,

H.

-

S m i s s m a n , Ed.

Topics in Chemistry

"Modern S y n t h e t i c Reactions" W. A. Benjamin,

I n c . , N. Y., 1965. E. F r a n k e l , E.

and R. B u t t e r F r a n k e l , E. Emkeq

f i e l d , 2. Org. and V. Davison, F. H. J a r d i n e , Chem. Ind. 560 ( 1965). E. E. Van Tamelen, M. Davis, and M.

and J. F.Young,

--

71 (1965).

Vincent, Jr., and R.

H. Feuer, F. B.

Chem., 2,2877 (1965). W. E. Truce and F. M. Perry, E. H.

L. C.

3.

Deem, Chem. Commun.

F. S.

Bartlett, J. Org.

Org.

E l i e l and D. N a s i p u r i , 2. Org. Brown and P. M. Weissman, 2.

5614 (1965).

W. Hoefling, D. E i l h a u e r , and G. Reckling, G e r .

:!2iega%, Y. Yamada,

S.

9

Chem. Ind. 1496 ( 1965). K. Kagawa, and S. Ikegami, Chem. Pharm. B u l l .

394 (1965).

G.

R.

( 1965) *

36,

(East)

, 18047.

Bacon and W.

J. W.

Hanna,

2.

Chem. S o c . ,

4962

P. Kolsoker and 0. Meth-Cohn, Chem. Commun., 423 (1965). B. Acott and A. L. J. B e c k w i t h m e m . Commun., 161 (1965). B. Acott. A. L. J. Beckwith. A. H a s s a n a l i and J. W. Rednrxld. Tetrahedkon Letters 4039 (1965). K. E. P f i t z n e r and J. G. Moffatt, 2. &I. Chem. SOC. 5670 (1965). T. Suga, K. Kihara, and T. Matsura, B u l l . Chem. SOC. Japan

-

,

8, 1 1 4 1 (1965). & B h a t i , Chem. Commun., K. N. Parameswaran and

476 (1965).

a,

g.

Chem. 988 (1965). Van E s , J. Chem. SOC., 1564 (1965). J. K. Kochi; g x C h e m . Soc., 2500 (1965). D. H. Barton, H. P. Faro, E. P. erebryakov, and N. F. Woolsey, J. Chem. SOC., 2438 (1965). B. Fontal-and H. Goldwhite, Chem. Commun., 111 (1965). K. A. Oglobolin and V. P. Semenov, G. Organ. Khim., 27 (1965); c~:62, 16009. K. A. o g l o b x n and A. A. Potekhin, G. Organ. Khim., &, 0. M. Friedman,

T.

8,

2,

865 (1965); g : 6

, 6873.

$

98

P. Gruenanger , F i n z i , and C. S c o t t i , Chem. B e r . , 623 (1965). Y. I. Smushkevich, V. N. Belov, B. V. Kleev, and A. Y. Akimova, G. Organ. Khim., 1, 288 (1965); CA:62, 16073. K. Torssell and E. Ruusa, A % i v V9"f1965); 2:

B, 3,

T o r s s e l l and R.

, 6839. F.

Reed, Jr.,

2.

Ryhake,

Arkiv

Org. Chem.

e, 2, 537

, 2,2195

(1965); CA

(1965).

Chap. 29

31. 32.

33.

34. 35.

36. 37. 38. 39 40.

41.

42.

43.

323 -

Smi s s m a n

Chemical Reactions

B. D. B i g l e y and D. W. Payling, 2. Chem. Soc., 3974 (1965). F. M i n i s c i and R. G a l l i , Tetrahedron L e t t e r s , 1679 (1965).

J.

E.

D o l f i n i and J. D.

4381 (1965).

Simpson,

2. &I.

Chem. SOC.,

3,

- g.,

720 (1965). J. Wolinski, D. Chan, and R. Novak, Chem. V. I. Shevedov, L. B. Altukhova, and A. N. Grinev, g.Orga

K h i m . , 1, 879 (1965); =:2, 6893. W. Ziegznbein, Angew. Chem., , 380 ( 1965). H. Oediger and K. E i t e r , Ann.qhem , 682, 58 H. J. Bestmann, G. 620 (1965). E. J.. Corey and M.

(1965);

J.

Graf, H.

e: 9 , 1353 (1965).

A. Speziale, Chem. s. S a m e and!&: C. Lunibroso and ( 1965).

- %.,

( 1965) *

Hartung, E e w . Chem.,

Chaykovsky, J .

&I.

Chem. SOC.,

3,1,345 -

C. C. Tung K. W. R a t t s , and A. Yao, J. Am. 3460 (1965j. Rigny! Compt. Rend. 260, 1678 (1965). P. Maitte, B u l l . S o c T h i m . France 31-5

Nauk

44.

45. 46. 47.

48. 49. 50.

51. 52.

Org. ( 1965). . _ . H. Geipel, J. Gloede

, 1381

‘1

g, 1677 (1965j.

E.,

K.

P. H i l g e t a g , and H.

Gross, Chem.

F. M i c a e l , 0. Eickenscheidt, and I. Z e i d l e r , Chem. -*, Ber 3520

(1965).

, 216

(196).

son, J .

( 1965) *

e.-,

8,

255

54

S. Yamada, T. S h i o i r i , T. Haya, T. Hara, and R. Matsueda, Chem. Pharm. B u l l . , 88 (1965). K. Matsui and T. N o j i r i , Kagaku Azsshi, 86, 531 (1965); 8242. F ’ g ’ M a r k i e w i t z and C. R. Dawson, 2. Org. Chem., 30, 1610

55.

R. G e l i n and D.

53.

2:

-

( 1965)

-

Makula, COmPt.

Rend.

& I , 489

(1965).

3 24 -

Sect. VI a a P * 30

-

Topics in Chemistry

S m i s s m a n , Ed.

ANTIRADIATION AGENTS William 0. Foye

Department of Chemistry Massachusetts College of Pharmacy Boston, Massachusetts The medicinal chemistry of antiradiation agents is concerned here primarily with the development of new radioprotective agents and their activity. Such a discussion necessarily omits much of the work in radiation biochemistry and contiguous areas, although interesting new developments where chemical interactions have been shown to take place are included. The subJect of ohemical radioprotection and radiosensitization of tumor-bearing systems has been touched only briefly, covering events subsequent to the reviews cited, and is really worthy of a separate review. Methods of biological radioprotection and clinical aspects have not been covered in this report. Preparations of potentially active antiradiation agents without report of protective activities regretfully have been left for future reviewers when radioprotective results may have become known.

-

Reviews. New books concerning chemical radioprotectionl and t m n c i p l e s of radiation protection2 have appeared. Current concepts of chemical protection against IonizrLng radiation have been re~iewed,~" and the role of copper and peroxides in radiobiology has been discussedOs The 92 papers presented at the First International Symposium on Radiosensitizers and Radioprotective Dmgs in 1964 have appeared in a well-organized volume.6 The use and pharmacology of radioprotective aminothiols, disulfides, nitriles, and other compounds has been r e v i e ~ e d . ~ Radioprotective Compounds 2 Mammals.- Chemical modification of the MEA and MBQ structures has continued actively, although no real improvement over these two basic structures has-been re: ported in the past two years. A new direction f o r syntheses of potential radiation protectors appears to be among heterocyclic systems, where protective activity is being found apart from any sulfur content of the molecules. Several non-basic thiols have also been reported active, and sulfur-containing analogs of the amino acids have shown some radioprotective effects. Protection by metal chelating agents, metal chelates, and metal ions themselves has pointed to the importance of the role of metal Ions in cellular radiation effects. Heterocycles. Among a group of 22 thiazolines tested in mice vs. 850r (V-radiation), "aminoethylthiazoline" provided up to 30rprotection, presumably because of its ability to hydrolyze to protective fragments in v i v ~ . Of ~ 15 imldazoles and related N-heterocycles, benzimidazole gave rats 90% protection 2. a lethal dose of x-raysOg In a subsequent series of imldazoles and benzimidazoles, ~~naphthylmethyl-2-imidazoleprovided 100% protection to mice.

Chap. 30

Antiradiation

Foye

325 -

3,5-Dlmethyl-l-( dlmethylcarbamoy1)-pyrazole was found to give 50s protection to mice E. x-rays (LDloo).lf Several C-alwlated thiazolines protected mice against a lethal dose of x-rays, and the activity was comparable to that from the mercaptoamlnes obtained on hydrolysis of the thiazolinea.12 A copolymer consisting of S-vinyl(2,2-dimethylthiazolidyl)-N-monothiolcarbamate and N-vivlpyrrolidone was also found effective E. x-rays in mice.13

Two 5,7-dihydroxyisoflavones, representing a class of compounds for which radioprotective ability has been controversial, were shown to afford 100% protection to mice 7OOr when administered percutaneously but were not protective by the intraperitoneal route l4

E.

.

Thiols. -Acetamldinlum thiosulfates, RNHC( =NH2+) CH2SS03-, were radioprotect1ve1%o mice 2. a lethal dose of x-rays, and a substituted 2-amlnothiosulfiric acid, prepared froma-amlnobutyric acid, also gave good protection.16 The thiosulfonate of NH~CH~CH~SOZSCHPCH~NH~, afforded good protection to mice, and it also protected 2. marcescens at pH 7, where it is decomposed to cystamlne and hypotaurine, but not at pH 4, where it is stable.18 The aminothio acids derived fromdC-and/J-alanine and gigcine showed Good prono protective activity in rodents but did in bacteria. tection in mice was provided by the@-aminoethylamlde,of thioglycollic acid, HSCHaCONHCH2CHzNHa, as well afsby N,N -bis(mercaptoN-Acetylthioacetyl)hydrazine, however, s.800r (x-rays) glycollic hydrazide, HSCH2CONHNHCOCH3, and its disulfide increased the LDSO of x-radiation in mice.20

T#,

.

The mixed disulfide of MEA and o-mercaptobenzoic acid was protective in mice z. a lethal dose of x-rays; this compound showed remarkable structural specificity, neither the meta or para isomers nor a variety of other close relatives were protectivem21 Thiols lacking a basic function also appeared with radioprotective activity. 2,3-Dithiosuccinic acid (both meso and dl 1-Phenyl-1forms) protected 90% of mice 7OOr (x-rays).22 acetthio-2-nitroethane, and its next higher homolog, showed some activity in mice VS. an LDloo dose of x-rays, whereas the corresponding amlnothix, 2-mercapto-2-phenethylamlne, was

E.

Numerous other interesting amlnothiol derivatives were synthesized without report of activities. ~lOC-Diallcyl-~-amlnothiols, and addition of long chain however, were found inactive in mice, N-alkyl groups to AET and the trithiocarbonate of MJ3G, RMIC(=NH2+) CH2CH2SC(=S) S-, also resulted in loss of a~tivity.~” Other Compounds. Hexacoordinated chlorophyllin-metal chelatea= with Co,M&,Mn,V) have been claimed to be radioprotective in mice; and a series of 1,5-diphenylthiocarbohydrazides capable o f metal ion chelation provided 40-65s protection to rats 750rm27 An a w n analog, ~-2,4,5-trichlorophenoxyethanol,

E.

326

Sect. VI

-

Topics in Chemistry

S m i s s m a n , Ed.

E.

which reduces normal oxygen consumption, protected mice 800r (V-radiation).28 The proestrogen, chlorotrianisene (tri-p-anisylchloroethylene), has shown the surprising property of protecting 80% of mice(vs. 590-690r) when administered 5-30 days prior to x-irradiati o E 2*

E.

Pyromellitic and benzenepentacarboxylic acids, but not 102% when administered mellitic acid, were protective in mice in high doses.30 It was considered that the activity resided In the osmotic effect of these polyionic substances which could cause hypoxia, rather than in the chelation of calcium ions known to occur. 750r, were believed to Gallate esters, which protected rats inhibit chain oxidation

E.

E.

Radioprotection of Other Systems.- Glycine exerted a protective Soor, but showed little effect effect on the catalases in mice on ATPase, pyrophosphatase, or gl~taminase.~~ Selenomethionine and selenocystine showed a greater protective effect for amino acids, yeast alcohol dehydrogenase, and RNase than the analogous sulfur cornpo~nds.~~ The r@ioprotective effect of both glycerolg4 and dimethyl sulfoxide for catalase was attributed to complex formation with the of catalase. Protection of catalase by a++, pe*, and ~n ions, however, was explained by radical scavenging.36

irs

Iron and copper ions also inoreased radioresistance of ceruloplasmin and hemoglobin, and a similar effect was shown by Nipicolinic acid and Ni-glycine chelates.s7 Other chelates did not protect these proteins. A correlation between radioprotection of proteins b inorganic ions and hydration energy of the ions was discerned, Incorporation of iron in erythrocytes was not inhibited by radiation (75r in mice) in presence of MEA, histamine, or serotonin,g9 Ehrlich ascites cells in mice VS. 400r (V-rays) were protected by eitheroC- or 0-alanine followed by arginine.40 AET protected both cancerous and healthy cells in mice from x-rays, but did increase the survival rate of cancerous mice.41 The gastrointestinal tract of rats was protected by perfusion with MEG gOCr, but Dunning leukemia cells were not eradicatede4= The hematopoietic 5OOr system of mice was protected by cysteine thiosulfonate (x-rays), but Crocker sarcoma cells also received protection. 43 The subject of chemical radioprotection and tumors has been recently reviewed. 44

E. E.

Radiosensitizers.- A review has appeared on studies of radiosensitizers in radlothera y of tumors.45 Radiosensitization of Ehrlich carcinoma cells by -methyluracil and 5-hydroxymethyl-4methyluracil has been O b S e r ~ e d ,as ~ ~well as by propyl gallate and other antioxidant^.^^ Mice proved to be radiosensitive to bdeoxypyridoxine, INH, Dbtryptophan, and Dbkynurenine E. 590r; taurine afforded some protecti~n.~~ Dogs were found radiosensitive to quinoxalinedi-N-oxide, whereas mice had been protected by this compound.4s Colcemide and urethane increased radiosensitivity in

t

Chap. 30

Anti r a dia tion

Foy e

327 -

mice when administered twelve hours prigr, but protected when given 48 hours prior to x-irradiation. Erythrocytes were sensitized by iodoacetic acid and related alelating agents ncluding ethyl methanesulfonate, iodine, and N-ethylmaleimide.5f E, coli cells have shown radiosensitivity to $,8 -dichlorodietGl =one, whereas the monosulfoxide of cystamine was only slightly sensitizing, and ions gave some protection.s2 Folic acid analogs53 and spar~omycin~~ sensitized -E. -coli to ionizing radiation, and Cu* ions sensitized 2. flexneri under anaerobic but not aerobic condition~.~~

-

Modes of Radioprotection.- The various mechanisms proposed for chemicarradioprotection have been recently evaluated by Bacq: A convincing argument in favor of anoxia was made for the radioprotective action of histamine, acetylcholine, and the catecholmines, but it was believed that the thiols and disulfides protect by interactions with free radicals and oxygen. Arguments in favor of radical interaction, and transfer of energy to sulfur, in combination with mixed disulfide formation, have been advanced recently in regard to protection by thiols.56 None of the complexities of this problem of explaining radioprotection have been removed by recent results, but it appears pertinent nevertheless to cite evidence supporting some of the current concepts.

No correlation between hypothermia and radioprotection by

MEA, cysteine, cyanide, 5-hydroxytryptamlne, or diethyldithio-

carbamate has been reported from two l a b o r a t o r i e ~ ;both ~~~~~ groups conclude that the interaction of MEA, at least, with free radicals constitutes its major protective role. A direct relation between radical inhibitory action and radiation protection was observed for antioxidant phenols, pyridines, and gallic acid esters.5s A correlation between vasoconstrictive effect and radioprotection was reported for the indole aminesO6*

It has been postulated that the introduction of thiols or disulfides into cells upsets the thiol-disulfide equilibria present, resulting in the increased formation of free thiol groups which can react with radicalti Instantaneous repair of damage by H transfer then takes place. Some evidence for this idea has since been reported. Increased amounts of an endogenous compound with reactive thiol groups has been observed after MEA treatment,62 as well as an increase in the thiol level of spleen,63 and the The latter compound could release of intracellular gl~tathione.~~ eliminate H a 0 2 via the glutathione peroxidase pathwayO6' The complexation of enzymes by protective agents has been proposed, and evidence for the existence of a protective glyceroliron-catalase complex has been advanced.6s Spectrophotometric evidence for the existence of complexation between catalase and MEA, MEG, and diethyldithiocarbamate has been observed, and the subject of metalloenzyme complexation discussed.66 Radioprotection of lactatedehydrogenase by complex formation with D-lactate has been and decreased catalase inactivation in the presence of owgen has been attributed to formation of catalase-peroxide complexes. 88

3 28 -

Sect. VI

-

Topics in Chemistry

S m i s s m a n , Ed.

The problem of energy t r a n s f e r , from radiation-induced r a d i c a l s t o e i t h e r v i t a l o r p r o t e c t i v e molecules, l i e s a t t h e h e a r t of t h e molecular events t a k i n g p l a c e i n t h e course of r a d i a t i o n p r o t e c t i o n . Any of t h e c u r r e n t hypotheses of r a d i o p r o t e c t i o n must u l t i m a t e l y consider t h i s e f f e c t . However, some understanding of t h e process is emerging5s*e5*eB-71 mainly by use of e.s.r. spectroscopy. References

.

(1) Bacq, Z.M., "Chemical P r o t e c t i o n Against I o n i z i n g Radiation," Charles C Zhomas, S p r i n g f i e l d , Ill., 1965. (2) Eaves, G . , P r i n c i p l e s of Radiation P r o t e c t i o n , " I l i f f e Books, London, 1964. (3) Baraboy, V.A., "Current Concepts of Mechanisms $f t h e P r o t e c t i v e Effect of Chemical Compounds Against Radiation, Office Tech. 1964. Serv., J.P.R.S. Report 26,842, Washington, D.C. 4 Langendorff, HiI, Arzniemittel Forsch., 463 (1965). Copper and Peroxides i n Radiobiology and 151 Schubert , Medicine, Charles C. Thomas, S p r i n g f i e l d , Ill., 1964. (6) P a o l e t t i , R. and Vertua, R . , "Progress in Biochemical Pharmacology," Vol. 1, S. U r g e r A.G., B a s e l , and Butterworths, Washington, D.C., 1965. Shapiro, B . , Med. Clln. N. Am., 48, 547 Shashkov, V.S., & Radiobiologiya , R i n a l d i , R. and Bernard, Y., R i n a l d i , R., Bernard, Y., and

u,

u.,

(1965)

(11) b o x , J . M . , Freeman R.G., and T r o l l , D., A c t a Radiol., Therapy, Phys. B i o l . , 2, 66 11965) (12) Handrick, G.R., Atkinson, E.R., Granchelli, F.E., and Bruni, R.J., J . Med. Chem., 8, 762 (1965). (13) Overberger, C.G., Ringsdorf, H., and Avchen, B., U ., 8, 862

.

(1965) (14) Gazave, J . M . ,

Modigliani, D.C., and Ligny, G., Progr. Biochem. Pharmacol. Vol. 1, S. Karger A.G., Basel, 1965, P. 554. , 766 (1964). Bauer, L. and Sandberg, K . R . , J. Med. Chem., Gershon, H., and Rodin, R., U d . , 8, 864 (19 5 ) . F i e l d , L. F e r r e t t i , A., Crenshaw, R.R., and Owen, T.C., ibid.,

5

1,39 (1964). (18) Owen, T.C., Parker, M.S., and S t e r n , G.M., J . Pharm. Pharmacol., 108 (1965). (19) Atkinson, E.R., Handrick, G.R., Bruni, R.J., and G r a n c h e l l i , F.E., J. Med. Chem. 8, 29 (1965). (20) Rose, F.L. and Walpole, A.L., Pro r. Biochem. Pharmacol., Val. 1, S. Karger A.G., Basel, 19 5, P. 432. Crenshaw, R.R., and F i e l d , L., J . Org. Chem., 30, 175 (1965). Cugurra, F., and B a l e s t r i , E., Progr. Biochem. Pharmacol., v o i . 1, S. Karger A.G., Basel, 1965, P. 507.

z,

%

Chap. 30

Antiradiation

329 -

Foye

B h a t , K.V., and Mecarthy, W.C., J . Pharm. S c i . , 51, 1545 (1964). Stacy, G.W. Barnett, B.B., and Strong, P.L., J. Org. Chem., 311, 592 (1965) (25) Foye, W.O., Laiala, E.F. Georgiadis, M., and Meyer, W.L., J. Pharm. S c i . , 557 11965) 26 Kasugai, N., J. Pharm. SOC. J a b n , 84, 1152 (1964). (271 G o r d e t s k i i , A.A., e t a l . , Pato enez, Eksperim. P r o f i l a k t i k e i Porazhenii ~MOSCOW) , 1964, 179; Chem. Abstrs.,

s,

I

S t e e r s , C.W.,

and Norman, D.,

K. and Langendorff, M.,

Nature,

Strahlentherapie,

206,

207

128,109

Nature, 205, 816 (1965). A.A., Baraboi, V.A,, and C h e r n e t s k i i , V.P. , Vopr. Biofiz. i Mekhanizma D e i s t v i y a I o n i z i r . R a d i a t s i i , 1964, 159; Chem. Abstrs., 18617 (1965). (32) Capalna, S., Ghizari, E., S t e f a n , M., and Petec, G. , S t u d i i Cercetari F i z i o l . , 2, 369 (1964) ; Chem. Abstrs. , 62, 13488 (1965) Shimazu, F. and Tappel, A.L., Radiation Reg. , 310 (1964) Lohmann, W., MOSS., A.J., Perkins, W.H., and Fowler, C*F., Biophysik, 2, 16 (1964). (35) Lohmann W,, Moss, A.J., a n d ' p e r k i n s , W.H., J . Nucl. Med., 6 519 (1965) (36) Lo-m, W. Moss, A . J . , and Perkins, W.H., Radiation R e s . , 24, 9 (19655. (37) Krsmanovic-Simic, D. and Duchesne, J. , Compt Rend. , 260

a,

u,

6455 (1965). Ueno, Y., C o l l e c t i o n Czech. Chem. Commun. , 30, 2839 (1965) Koch, R. and S e i t e r , I., S t r a h l e n t h e r a p i e , 124, 99 (1964) Bregadze, I .F. , Radiobiologi-, 5, 97 (1965). Maisin, J.R.

Leonare, A . , and Hugon, J., J. Belge Radiol.,

a, 511 (1964).

(42) &&ms, J .L., Ambrus, C.M. , Pickren, J e w . t Felt23 E-, and Back, N., Cancer R e s . , E , 609 (1965) (43) Zebro, T., Stachura, J., Prochnicka, B. , and Syczepkowski, T. , Acta Med. Polona, 6, 155, 171 (1965). (44) Greco, S . , Gasso, G., and B i l l i t t e r i , A., Progr. Biochem. pharmacol., Voi. 1, S. Karger, A.G., Basel, 1965, P. 277. M i t c h e l l , J .So i b i d . , 335. Polsklna, R.I., Bychenkova, M., and Zalesskaya, L. , Acts unio 1219 (1964); Chem. Abstrs. 62, I n t e r n . Contra Cancrum, 9429 (1965) (47) Afanas'ev, G.G., Lipchina, L.P., and Pelevina, I.I., ibid., 1213 (1964) ; Chem. A b s t r s . , 62, 9428 (1965) (48) Melching, H.J. Abe, M., and S t r e f f e r , C., S t r a h l e n t h e r a p i e , 125,352 (19643 (49) m l e y , T.J., Trwnbull, W.E., and Cannon, J.A. , Progr. Biochem. pharmacoi., Voi. 1, S. Karger A.G., Basel, 1965, P o 359.

.

a,

a,

0

Sect. VI

3 30 -

-

Topics in Chemistry

Rothe, W.E., Grenan, M.M., and Wilson, S.M., i b i d . , 372. Bianchi, M.R., Boccacci, M., Misiti-Donello, P., and Q u i n t i l i a n i , M., I n t e r n . J . Radiation Biol., 8, 329 (1964) 8, 519 (1964). S t u y v a e r t , J . and Bacq, Z.M., iu., P i t i l l o , R.F., Lucas, M., Blackwell, R.T., and Wooley, C., J. Bacteriol., 1548 (1965) 773 (1965) P i t i i i o , R.F., e,t a l . , Nature Cramp, W.A., i b i d . 7 2 0 6 , 636 P i h l , A . and E e r , T , , Progr. Biochem. Pharmacol., Vol. 1, S. Karger A.G., Basel, 1965, P. 85. Bacq, Z.M.,Beaumariage, M.L., and Liebecq-Hutter, s o , I n t e r n . J. Radiation B i o l . , 2, 175 (1965). Magdon, E., Nature, 204, 484 (1965) Burlakova, E.B., et Dokl. Akad. Nauk S.S.S.R. ,

.

a,

(57)

S m i s s m a n , Ed.

.

z,

934 (1965).

(60) Zherebchenko, P.G.,

.

164,

e t a l . , Patogenez, Eksperim. P r o f i l a k t i k e i Porazhenii (Moscow), 1964, 193; Chem. Abstrs.,

Nature, 203, 162 (1964) M.M., Sokolova, O.M. , and Tarasenko, A.G., Dokl. Akad. Nauk S .S.S.R., 441 (1965) Jamieson, D., Nature, 207, 541 (1965). Revesz, L. and Modig, H., U d . , 207, 430 (1965). Lohmanrl,W., Progr. Biochem. Pharmacol., Vol. 1, S Karger A.G. Basel, 1965, p . 118. Foye, W.O., and Mickles, J . , i b i d . , 152. Dose, K., i b i d . , 161. Magdon, E., S t r a h l e n t h e r a p i e , 258 (1965). Alexander, P., L e t t , J.T., and Dean, C . J . , Progr. Biochem. Pharmacol., Voi. 1, S. Karger A.G., Basel, 1965, p . 22. Nicolau, C., Qrigorescu, S., and Nedelcu, C., i b i d . , 472. C a s t e l e i j n , G., Depireux, J., and Muller, A . , Intern. J . Radiation B i o l , 8, 157 (1964)

P.,

164,

126,

.

.

,

Chap. 31 PHARMACEUTICS AND BIOPRARMACEUTICS

Tgkeru Higuchi and Kenneth F. Finger School of Pharmacy, University of Wisconsin, Madison, Wisconsin W i l l i a m I. Higuchi School of Pharmacy, University of Michigan, Ann Arbor, Michigan The science of dosage forms and their influence on drug absorption and u t i l i z a t i o n is a r a p i d l y growing medicinal f i e l d . I n t h i s s h o r t review w e have attempted t o present a l i m i t e d b u t r e p r e s e n t a t i v e development i n t h i s area occurring within the p a s t two years. The materials treated, admittedly, are i n t h e areas of p a r t i c u l a r interest t o t h e reviewers. THERMODYNMIC AC’PIVITY,

SOLUBILITY AND COMPLEXING

Although thermodynamic p r o p e r t i e s of medicinal agentsin s o l u t i o n has received r e l a t i v e l y l i t t l e a t t e n t i o n i n the p a s t , there are a number of reasons why they should be of a major concern t o medicinal chemists. One can r e a d i l y show, f o r example, a direct r e l a t i o n s h i p between t h e physiological a c t i v i t i e s of drugs and their thermodynamic a c t i v i t i e s a t the s i t e of action. One should a l s o note t h a t t h e rates of drug t r a n s p o r t and absorption across membrane relate. more t o thermodynamic a c t i v i t y than t o concentration. Drug s o l u b i l i t i e s which are i n v e r s e l y proportional t o thermodynamic a c t i v i t y c o e f f i c i e n t control, of course, rates of d i s s o l u t i o n and a v a i l a b i l i t i e s of many drugs. And f o r p r a c t i c a l purposes it is more convenient and o f t e n necessary i n many instances t o administer parentera1 drugs i n s o l u t i o n thus r e q u i r i n g rather s o p h i s t i c a t e d understanding of s o l u t i o n chemistry t o permit d i s s o l u t i o n of less s o l u b l e pharmaceuticals. For these reasons t h e nature of t h e s t r u c t u r e s of s o l u t i o n s of drugs i n aqueous and nonaqueous s o l v e n t s and the mechanisms of a c t i v i t y of cosolvents i n these systems p a r t i c u l a r l y have been r e c e n t l y subjecQs of increased scrutiny. c o r r e l a t i o n of s o l u b i l i t y p r o p e r t i e s of nonaqueous systems t o a s i n g l e parameter such as dielectric constant has been attempted by s e v e r a l groups i n t h e l a s t two years w i t h l i m i t e d A t r y by Restaino and Martin t o f i t s o l u b i l i t y of benzoic acid i n various n-alkanols3 t o Hildebrande’s theory of s o l u t i o n proved t o be u n f r u i t f u l . I n these instances t h e s o l v e n t phase has been e s s e n t i a l l y assumed t o be a continuum. A more e f f e c t i v e recent a p p r o a ~ h ” ~ has been t o look a t solute-solvent-cosolventinteractions on a molecular basis, the t o t a l s o l u b i l i t y of c r y s t a l l i n e drug A i n solvent B and cosolvent C being represented by a summation

332 -

Sect.

VI

-

S m i s s m a q , Ed.

Topics in Chemistry

P'" Total solubility =

knnp

A"B~CP

L

n = 1 m - 0

P * O At saturation the thermodynamic activity of each species present is equal to that of the solid solute. The total solubility behavior furthermore must take into account the interactions between B and C species. The mechanism responsible for increases in solubility induced in aqueous solutions by addition of cosolvent continues to be largely associated with formation of solute containing species in these systems. The species may take the form of simple one to one or one to two molecular complexes between the solubilizing agents and the solubilized solutes as reported recently for a series of or less stoichiometrically defined hydroxyaromatic acid giant aggregates such as micelles or adducts between dispersed polyether surfactants and low molecular weight phenols. ""lo' l2 A rather interesting instance of solubilization and complex which apparently formation was reported by Lach and coworkers'" involves trapping of organic solutes into cage-like structures of cyclodextrins or Schardinger dextrins in aqueous solutions. The observed stability constants are apparently quite high and the chemical behaviors of the bound species are significantly altered in several instances. There may be some relatipgship between these and starch adducts reported by Goudah and Guth.

''

'"

DRUG STABILITY

Increasing attention has been directed towards elucidation of mechanism responsible for deterioration of medicinal preparations. Since drug formulations often involve relatively complex mixtures of polyfunctional organic and inorganic species, chemistries of their breakdown on storage under varying conditions for periods of months and years can indeed be quite involved. Mechanisms of degradation of cycloheximidef6 of porfiro~~ycin:~ of hydroxocobalmin," of Mannich coopounds,lB of diaminotriazine derivatives,=' of apomorphine,'' of iodoxuridine,22 and of methicillin*' are representative of some of these systems recently studied. Attention should be called to series of papers on an anaerobic loss of ascorbic acid. 24

Chap. 31

Biopharmaceutic s

Higuchi, Finger and Higuchi

333 -

Steroids continue to receive attention. Although prednisolone in queous solution undergoes oxidative breakdown catalyzed by metals, s J p * Jensen and Lamb have shown that autoxidation of fluprednisolone acetate is preceded by a hydrolytic step. 27 Participation of other ingredients in formulations affecting drug stability has become increasingly evident. Citrate and tartrate buffers, for example, have been shown to be in slow equilibria with their corresponding acid anhydrides which can react rapidly with any nucleophile which may be present.20’2s’30 Waake and G ~ t t m a n ,on ~ ~the other hand, have found that formation of a borate complex tend to stabilize riboflavine. Lach in his studies on cyclodextrins have shown that these form complexes with drugs often conferring great atability to the bound guest. 32 PHARMACEUTICS OF HETEROGENEOUS SYSTEMS Powders, Su sions and Emulsions: Rippie and his collaborators some noteworthy stur3ies on the segregation have report8 kinetics of particulate solids. These investigators have taken steel and glass balls of various sizes loadzd into cylindrical containers and subjected them to vertical sine wave motion. Samples were taken at various times and standard deviations from the mean composition were determined. Segregation of binary m i x tures of spheres followed an apparent first order approach to equilibrium. Employing this technique the authors have studied the influence of particle size, size distribution, particle densitysize interactions, and the dependence of the agitation upon the segregation rate. Other variables such as particle shape could be studied also. While the idealized experiments have not included (and may not be able to include) some of the factors important in real systems, e.g., particle-particle adhesion, particle wall adbeeion, and electrostatic effetrts, they have quantitatively clarified many aspects of the particulate mixing phenomena and demonstrated the importance of this kind of approach. Shlanta and M i l o s o ~ i c hhave ~ ~ described an apparatus for studying stress relaxation of p e e r beds under constant strain. The technique appears to be useful for studying elastic compressifactors that are bility, relaxation and flow under pressure basic to the understanding of the tabletting process. The Coulter Counter played an important role in research on emulsions and suspnsions of pharmaceutical significance. Lemberger and Mourad 7338 studied the influence of a number of variables on the deaggregation behavior of oil-and-water emulsions. R ~ , s ealso employing the Coulter Counter, studied the effect of emulsifier type and concentration on the particle size distribution of oil-in-water emulsions. Edmundson and Lees4’ determined the dissolution rate of a fine suspension of hydrocortisone acetate in water using the Coulter Counter. These and other work41J42J4swith

-

3 34

Sect.

VI - Topics in Chemistry

S m i s s m a n , Ed.

this instrument involving emulsions and suspensions point out the value of this tool in future work with pharmaceutical dispersed systems Dissolution Rate Behavior: A number of studies on the rate of dissolution have been recently reported. Wurster and his a s s ~ c i a t e ~ ~ ~ ~studied ~ ~ ~ *the ~ heffect a ~ e of complex formation on the dissolution rate of drugs and the dissolution behavior of the three different crystalline forms of prednisolone. In the latter study these investigators observed the unusual effect of agitation on the relative dissolution rates of polymorphs first reported by Hamlin et al.47 Theaissolution rate behavior of polyphase systems has recently received the attention of several workers since the report by Sekiguchi and Obir8 that the eutectic mixture of sulfathiazole and urea gives a much higher drug release rate than the pure drug alone. The authors had attributed the greater rate to the c smaller crystals in the eutectic mixture. In their recent work with the chloramphenicol-urea system, Sekiguchi et al.4e found that the drug-urea mixture of 1:4 (W/W) dissolved significantly faster than the eutectic composition. The authors proposed that urea solubilization of the drug was probably the more important factor. Goldberg et. G.’’ has suggested the possibility of solid solution formation in these systems as an alternative explanation. Higuchi et. al. s’ have presented a mathematical analysis of dissolution rates involving polyphase mixtures which should be helpful in resolving the various factors.

.

-i-

BIOPHARMACEUTICS Current thinking in regard to the absor tion of drugs is based largely on the early studies of OvertonSg and Collander and Barlund” who have advanced the concept of the lipoidal nature of the biological membrane and more recently upon the work of Schanker, Brodie, Hogben and c 0 1 1 a b o r a t o r s ~ who ~ have ~ ~ ~defined ~ ~ ~ ~ the ~ ~ ~ ~ role of lipid/water partition coefficients, ionization and pH in the passive transport of drugs and other foreign compounds across biological membranes. !Chese studies have shown that most drugs are absorbed by passive transport and that the rate of absorption is a function of the concentration gradient of the diffusing moiety across the menibrane which in turn is dependent upon the lipid/water partition ratio of the diffusing moiety. The diffusing moiety for weak electrolytes is the non-ionized portion of the dissolved drug. Passive absorption of lipid-insoluble molecules occurs through small aqueous channels or pores in the membrane if the molecules possess sufficiently small molecular volumes to permit passage through the pores. The transport of highly icpnized, lipidinsoluble compounds, such as the quaternary amines, is not satisfactoily e lained by the above mentioned lipid-partition theory. LevinsB has postulated that the transport of quaternary amines

Chap. 31

Biopharmaceutic s

Higuchi, Finger and Higuchi

may Be dependent upon the interaction of these highly ionized molecules with a phosphatido-peptide constituent of the membrane and it is this complex that is the diffusing moiety in the transport of quaternary &nes across biological membranes. Such an interaction satisfactorily explains the rapid, but short lived oral absorption characteristics of the quaternary amines observed in man and other animals. The mechanism of permeation of biol ical membranes has * been the subject of several recent reviews. eo31 From the biopharmaceutical standpoint, the absorption of a drug into the body encompasses other parameters in addition to the permeation of the biological membrane. Included among these are dissolution properties (rate of dissolution and absolute solubility), drug interactions affecting drug availability and diffusion of the drug from its site of dissolution to the absorptive surface. These parameters will constitute the subject material to be discussed in the following paragraphs. Rate of Dissolution and Drug-Absorption: Both absolute solubility and the rate of solubility are important parameters in the overall absorption processes, It-has long been known that a compound must possess sufficient aqueous solubility to be effectively absorbed from the gastro-intestinal tract as well as other obsorptive sites, More recently, Nelson" has clearly elucidated the role of the rate of solubility in the absorption process and pointed out the theoretical relationships that exist between the rate of dissolutfon of a particle and its surface area, diffusion layer pH and the concentration gradient of drug across the diffusion layer. The rate of dissolution principles have provided the theoretical bases for many recent biopharmaceutical studies. The effect of particle size of sulfisoxazole on its oral absorption in dogs has been studied by Fincher, Adams and BeaLeS These authors concluded that the rate of absorption of sulfisoxazole was a &unction of the particle size of the crystals administered, faster and higher blood levels of the drug being obtained with the smaller crystal sizes. Their results indicate that while the rate of absorption was affected by particle size, there was no alteration in the total percentage of administered dose absorbed thus giving rise to the possibility that the blood levels of sulfisoxazole could be controlled by regulating the particle size of the administered drug. The rate determining step in the absorption of salicylate from pharmaceutical dosage forms appears to be the dissolution p r o c e ~ s ? ~More ' ~ recently, ~ ~ ~ ~ Truitt and Morganes have studied the absorption of buffered and non-buffered dosage forms of acetylsalicylic acid in humans and have related their in vivo findings to differences in dissolution rates of the administered aspirin. Their results indicate that (a) statistically significant differences in the rate of aspirin absorption from buffered and non-buffered tablets do exist and (b) the enhancing effects of bufferbmg are primarily upon the rate of dissolution of the acetylsalicyclic acid.

''

--

.

335 -

3 36 -

Sect. VI

-

Topics in Chemistry

S m i s s m a n , Ed.

'Fhue, when the buffered a s p i r i n formulation w a s administered as a s l u r r y o r as a solution, buffering had no enhancing e f f e c t u p n the overall rate of absorption of a c e t y l s a l i c y l i c acid. Only when the tableted form of a s p i r i n w a s administered d i d the enhancing effect of buffering appear, suggesting that the r o l e of the buffer is t o a l t e r diffusion layer pH and thus increase the dissolution rate of the aspirin. The importance of dissolution rate on the rate of absorption of s a l i c y l a t e was a l s o shown bY Lieberman and Wood7' who found that higher blood s a l i c y l a t e levels and more rapid absorption of s a l i c y l a t e occurred when the analgesic w a s administered i n the form of a solution than when administered as either a buffered o r non-buffered tablet. Their data indicate t h a t buffering enhanced the overall absorption rate of a c e t y l s a l i c y l i c acid when administered i n tablet form. Levy and I i ~ l l i s t e r ~ were ~'~* a l s o able t o show a relationship between the rate of dissolution and the rate of absorption of a c e t y l s a l i c y l i c acid i n humans. These authors employed both

conventional compressed tablets and an experimental sustained release preparation employed produced a s i g n i f i c a n t l y reduced dissolution rate as compared t o the dissolution rate obtained f o r the compressed tablets. Similarly, the f i r s t order rate constants f o r absorption following the administration of the sustained release from were s i g n i f i c a n t l y lower than those obtained w i t h the compressed tablets. I n addition, the sustained release dosage form caused a delay i n the onset of absorption i n most patients. Despite the delay i n onset of absorption and the much slower rate of absorption, the t o t a l amount of s a l i c y l a t e absorbed f r o m the sustained release dosage from compared favorably w i t h that obtained from t h e conventional tablet. These r e s u l t s i n d i c a t e again the r o l e of the rate of dissolution i n the over-all absorption rate of acetyls a l i c y l i c acid. The importance of rate of dispersion and dissolution of a drug w a s made evident by the work of Calesnick, Katchen and Black7' who administered various dosage forms of diazoxide and obtained blood levels and biological response data i n humans. Comparing tablet and capsule forms w i t h an aqueous solution of the drug, these authors found the highest blood levels were obtained when the aqueous solution of diazoxide w a s administered, lowest blood levels being obtained w i t h the tableted form and the administration of the encapsulated drug producing blood levels intermediate between those obtained w i t h the solution and tablet forms. It w a s evident from their data t h a t the dispersion of the formulation contained w i t h i n the capsule w a s the rate determining s t e p i n the dissolution of the drug when u t i l i z e d i n this form. Very rapid dissolution rates were observed when the capsule contents were emptied and evenly dispersed throughout the dissolving media as compared t o the very slow dissolution rate observed when the i n t a c t capsule w a s placed i n the media. Similar findings i n tegard t o blood

Chap. 31

Biopha r m a c eu ti c s

Higuchi, Finger and Higuchi

337 -

levels obtained with encapsulated aspirin VS. tableted aspirin In this study, a significant delay in the appearance of salicylate in the blood was observed in those persons receiving aspirin in the form of capsules as compared with those receiving the analgesic in the form of tablets. In addition, the blood levels of total salicylate were lower in the group receiving the capsules than was found in those receiving the aspirin in the form of a tablet. Hollister and KanterTs compared enteric coated dosage forms of aspirin with compressed tablet preparations. They observed significant delays in onset of serum salicylate levels when the enteric coated preparations were employed. The delay in absorption found in their study was of such magnitude (three to six hours) that they concluded that enteric forms of salicylate should not be employed where an immediate analgesic or antipyretic effect was desired. Percutaneous Absorption: The influence of solubility and lipid/ water partition ratios in the percutaneous absor tion of the corticosteroids were studied by Katz and Shaikh. Fa The results of their study are in agreement with the theoretical postulates of Higuchi and their results show that the efficiency of percutaneous absorption absorption of the corticosteroids may be a function of the partition coefficient and the square root of the aqueous solubility. In their continuing studies pertaining to the factors influencing percutaneous absorption, Wurster and M u n i e ~ ~ have ~ , shown ~ ~ that the degree of hydration of the stratum corneum played an important role in the transport of methyl ethyl ketone. These workers have shown that dehydration delays percutaneous absorption while hydration of the skin above normal values enhances absorption. Thus, percutaneous as well as oral absorption of chemical substances appears to be a function of the lipid/water partition coefficient and aqueous solubility parameters of the diffusing moiety and intimately associated with the integrity and physico-chemical properties of the biological melldbranes. Drug Interactions: The interaction of dissolved drugs with either endogenous or exogenous materials present in the vicinity of the absorptive site may have a pronounced effect on the absorbability of the drug. Interaction of the drug with these materials may either facilitate or inhibit drug absorption. Sorby'' has shown that promazine adsorbed to the surface of both attapulgite and activated charcoal and this interaction of drug and adsorbent resulted in altered absorption patterns of promazine. When admixed with attapulgite, the absorption of promazine was significantly delayed, however, only minor decreases in the proportion of the administered dose absorbed was observed. On the other hand, when admixed with charcoal, the drug-adsorbent interaction resulted in both a decrease in the rate and the extent of absorption of promazine. Althou h absorption characteristics were not studied, BlaUg and crosse8 investigated the adsorption of antichlolinergic drugs by

were obtained by Wood.74

3 38 -

Sect. VI

-

Topics i n Chemistry

S m i s s m a n , Ed.

various antacids and found appreciable binding of atropine, methantheline, propantheline and oxyphenonium to the antacids, particularly magnesium trisilicate. In ldea of the frequent coadministration of anticholinergic drugs and antacids, definitive in vivo absorption studies are necessary to define the possible effects of the antacids on the therapeutic efficacy of the anticholinergic drugs. The intestinal absorption of water soluble dyes such as bromthymol blue, methyl orange and eosine-B and certain lipid soluble coTlexes of the dyes have been investigated by Levy and Matsuzawa.8 Their data suggests that the lipid/water partition ratio of the dye complexes did not reflect the intestinal absorption characteristics observed. The absorption characteristics of Certain metal-acid complexes of tetracycline and demethylchlortetracycline have also been investigated and it has been shown that some of the metal-acid-tetracycline complexes enhance the absorption of the tetracyclines.82 Diffusion: Diffusion of the drug from the site of dissolution to the absorbing surface may have a pronounced effect on the maintenance of the concentration gradient across the absorbing membrane and thus an effect on the rate of absorption. This parameter of the over-all absorption process was the subject of a report by Levy and J u ~ k o . ~ ~ These workers studied the oral absorption characteristics of both ethanol and salicylic acid in rats as a function of the viscosity of dissolving media. Their results indicated that diffusion of the drug molecules to the absorbing membrane was significantly decreased and the rate of gastrointestinal transit of the solutions was also decreased. Urinary Excretion: In lieu of the frequent use of urinary excretion data as an index of drug absorption and elimination rates in the evaluation of pharmaceutical dosage forms, it would appear to be of value to call the reader's attention to the recent works of Beckett et al.84resr8s pertaining to the excretion of drugs in man. These workers have investigated the influence of urinary PH and urine output on the renal excretion of several dru 8 . In regard to their studies on the excretion of amphetamine," the authors state that the alterations in excretion rates observed as a function of urinary pH and urine volume can be explained on the basis that the unionized portion of the amphetamine is reabsorbed by the Kidney and the passive reabsorption process is p H and volume dependent. The more alkaline the urine, the higher the percentage of unionized amphetamine present and hence a greater reabsorption of the drug and consequently a lawer excretion rate. Similar finding8 were reported by Beckett and collaborators for chlorpheniramine and methylamphetamine. 86 While these studies are largely applications and extensions of the basic studies reported much earlier,87 J 88 J 8s J 0 the kinetic approach to data evaluation and implications deduced from their findings throughout their reports are indicative of the value of these reports to the biopharmaceutical literature.

Chap. 31

Biopharmaceutic s

Higuchi, F i n g e r and Higuchi

339 -

REFERENCES

1.

2.

3.

4.

5. 6. 7. 0. 9.

10. . .11. 12

.

13 14. 15

16.

17 18. 19.

2 0.

.

21. 22

23.

24. 25. 26. 27. 28.

D.

52,

L.

Sorby, R.

1149 (1963).

G.

B i t t e r , J. G.

B i t t e r , J. of Pharm. Sci.

G. Sciarrone, and N. G. Lordi, I b i d , 53, 463, Bid, 1325, 1334 (1965). E. A. Restaino, and A. N. Martin, I b i d , 636 (1964). T. Higuchi and K. A. Connors, Advances i n A n a l y t i c a l Chemistry and Instrumentation. Vol. 117-210, Wiley, New York 1965 J. Meier, and T. Higuchi, J. of,Pharm. sci. 4 1183, 119651. T. Higuchi and F. D. Pisano, B i d , 4 T. Higuchi and A. Drubulis, -J1 5, 3z4(1961). E. G. Rippie, D. J. Lamb and P. W. Romig, I b i d , 1346 (1964 M. B a r r and L. F. T h e , I b i d , 46, 445 ( 1 9 5 r C. K. Bahal and H. B. Kostenbaaer, I b i d , 2, 1027 (1964). N. K. Pate1 and N. E. FOSS, I b i d , 2(1964). W. B. Breuninger and R. W. Goettsch, I b i d 4, 1487 (1965). J. L. Lach, and T. Chin, I b i d , 69--&4% w. A. P a u l i and J. L. Lach, I b i d , 54 1745 M. W. Goudah and E. P. G u t h , I b i d , 298 E. R. Garrett and R. E. Notari, I b i d , 4, 209, 56; (1965). E. R. G a r r e t t and W. Schroeder, I b i d 917 (1964). A. D. Marcus and J. L. Stanley, -isra: 91 (1964). K. T. Koshy and H. Mitchner, I b i r s , 1381 (1964). D. H. Szulczewski, C. M. Shearer and A. J. Aguiar, I b i d , f52, A.

N.

Paruta,B.

1349 (1964).

2

z,

If,

g, (lb).

z,

z,

- &,

5 $:

1156 (1964).

Me BUrkMnj I b i d j 2, 325 (1965). L. J. Ravin, C. A. Sim son, A. F. Zappala and J. J. Gulesich Bid, 976 1064 (1994). M. A. Schwartz, E. Bara, I. Rubycz and A. P. Granotek, I b i d ,

A.

- z, 9, 149 (1965)-

P. F i n h o l t , R. B. Paulssen, I. A l s o s and T. Higuchi, B i d , 52, g48 (1963)) 54, 124J 181 (1965). T. 0. O s t e r l i n g and D. E. Guttman, 1189 (1964). M. L. Lewbart and V. R. Mattox, 820 (1959). E. H. Jensen and D. J. Lamb, J. 53, 402 (1964) T. H i uchi, A. Shah, T. Miki and A. Herd, J. Am. Chem. SOC.,

,

&, 3855

(1963)J

3899 (1961).

Higuchi, S. 0. Eriksson, H. Uno and J. J. Windheuser, of Pharm. Sci., 53, 280 (1964). Higuchi, H. Uno and I. Shimada, B i d , 302 ( 1965). A. wadke, and D. E. Guttman, I b i r 1073 (1964). L. Lach and T. Chin, I b i d , Z x - 4 8 4 6 4 ) . G. Rip ie, J. L. O l s e n and M. D. Faiman, J. Pharm. Sci.

33

34 35. 36. 37.

J. L. Olsen, and E. G. Rippie, J. Pharm. M. D. Faiman and E. G. Rippie, J. Pharm. S. S h l a n t a and G. Milosovich, J. Pharm. A. P. Lemberger and N. Mourad, J. Pharm.

30. 31. 32

.

,

T. J. T. D. J. E.

29.

,

53,

9,

1360 71964).

p: ,A,

,

Sci. Sci., Sci., Sci.

5

,

147 1964). 719 I196 562 (19647. 229 (1965).

3 40

S e c t . VI

38. 39. 40.

A.

41.

W.

42. 43. 44.

45. 46. 47.

48. 49. 50. 51. 52. 53.

54.

55.

56. 57. 58. 59.

60, 61. 62.

63.

P.

E. L. I. C.

-

Topics in Chemistry

S m i s s m a n , Ed.

,

Lemberger and N. Mourad J. Pharm. S C i . 54, 233 ROWe, J. Pharm. Sci., 260 (1965). Edmundson, and K. A. L e e s , J. Pharm. Pharmacol.

54,

193 (1965).

, z, ,

9 7"

(1965).

, 17

I. Higuchi, J. Pharm. Sci. 405 (1964). H. Y. Saad, J. Pharm. Sci., (1965). I. Higuchi, 1 3 (1 65 D. 0, Kildsig, -1491 ( 19k51. P. W. Taylor, 16m;.$670 (1965). P. w. Taylor and D. E. Wurster, 5, 1654, (1965). W, E. H a m l i n , E. Nelson, B. B a l l x a n d J. G. Wagner, Ibid, 2, 432 (1962). K. Sekiguchi and N. Obi, Chem. Pharm. B u l l . , 2, 866 (1961). K. Sekiguchi and N. O b i , and Y. Uyeda, Ibid, 12, 134 (1964). A. H. Goldberg M. Gibaldi and J. L. K a x JTPharm. Sci.,

W. I. Higuchi and H. Y. Saad and W. D. E. Wurster and D. E. Wurster and

54, 1145

m,

(1965).

s,

W. I. H i uchi, N. M i r , and S. J. Desai, J. Pharm. Sci., 1405 (19 5). E. Overton, Arch. ges P h y s r l . , Pfluger' 8 92, 115 ( 1902). R. Collander and ? I Barlun . A c t a Botan. Fenn. 11, 1 (1933). P. A. Shore, B, B. Brodie and C. A. M. Hogben, J. Pharmacol. Em. Ther. + ll 361 (1957). L. S. Schan er, P. A. Shore, B. B. Brodie and C. A. M. Hogben, a i d , 120, 528 (1957). C. A. M. H o g b e n L. S. Schander, D. J. Tocco and B. B. Brodie, Ibid, 120, 540 11957). L. S. Schander D. J. TOCCO, B. B. Brodie and C. A. M. Hogben, Ibid ' 123, 81 11958). B. B. Brodie and C. A. M. Hogben, J. Pharm. Pharmacol., 345

2

,

-

--

-

2,

( 1957)

R. Levine, Preliminary Studies on t h e R o l e of a Carrier i n t h e Transport of Quaternary Ammonium compounds. I n Proceedings of t h e F i r s t I n t e r n a t i o n a l Pharmacol i c a l Meeting, V o l . 4, Ed. Hogben. Oxford: Pergamon Press ( 19 2). L. So Schander, Annual Rev. of Pharmacol., 1, 29 (1961). D. H. Sqayth, Abs. and D i s t . of Drugs, T. B. Binns, Ed., E. and S. Livingston LTD, Pub. Edinburgh and London, Pg. 1, R.

3

( 19641.

B.

B.

Brodie,

D i d , Pg.

16, (1964).

5,

R. R. Levine a m . W. P e l i k a n , Annual Rev. of Pharmacol. E. Nelson, J. Pharm. Sci., 46, 60 19571 H, Fincher, J. G. Adams, and H? Beal, Ibid 4, 704 ( 1.965)

L.

64, 65. 66. L. J. Edwards, Trans. Faraday SOC., Q, l l g l T 5 1 % 67. E. Nelson and I. Schaldemose, J. Pharm. Sci., 48, 489 (1959) 68. G. Levy and B. A. Hayes, N.E. 1053 (1960). 69. E. B. T r u i t t and A. M. Morgan, J. Pharm. 70. S. V. Lieberman and J. H. 71. G. Levy and L. E. Hollister, 72. G. Levy and L. E. 730 B. Calesnick, B. Katchen

J.

.

Chap. 31

B i o p h a r m a c eutic s

cia,9

54,

74. 75.

J. H. Wood, I b i d , 1207 (1965). L. E. H o l l i s t e r and S. L. Kanter, C l i n .

76. 77. 78. 79

Katz and 2. I. Shaikh, J. Pharm. id, 5g1 Wurster and R. Munies, Ib 4 J 554 1965) .D. E. Wurster and R. M U n i e S , Ibid, , 1281 (1965

80.

81. 82.

a3.

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

-6, M. D.

5 (1965).

E.

54,

Pharm.

and Therap.

L.

S. M.

,

(lm).

Sorby, I b i d , 677 BlaUg and M. Gross, G. Levy and T. Matsuzawa, E. G. Remers, W. C. Barringer, G. M. S i e g e r and A. Bid, 49 (1965). G. Levy and W. J. Jusko, I b i d , 54 219 (1965). A. H. Beckett and M. Rowland, J, Pharm. Pharmacol. D.

341

Higuchi, F i n g e r a n d Higuchi

53,

J

( 1965 A. H. Beckett and G.

17,

628

R.

H. Beckett and M. J. Orloff and R. W. B e r l i n e r , J. C l i n . Invest. M. H. Jacobs, Cold Spring Harbor Symposium, M. D. Milne, B. H. Scribbner and M. A. Crawford, Am. J. Med. 24, 709 (1958). W. J. Waddell and T. C. B u t l e r , J. C l i n . Invest. 36, 1217 A.

-

( 1957

g,

,

,

,

SUBJECT INDEX

Agents Which Affect Enzyme Activity, 277 Analgetics, Strong a n d Weak, 40 Anesthetics, G e n e r a l , 30 Angina P e c t o r i s , 78 Alkaloids, 31 1 Anorexigens, 51 Antiallergy Agents, 92 Antianginal Agents, 78 Anti-anxiety Agents, 1 Antiarrhythmic s, 8 5 Antibacterial Agents, Synthetic, 118 Antibiotic s, 109 Anticonvulsants, 30 Antidepr e s s a n t s, 12 Antidiabetics, 164 Antihypertensive Agents, 59 Anti-inflammatory Agents, non- s t e r o i d a l , 224 Antipara sitic Agents, Animal, 150 Antiparasitic Agents, Human, 136 Antipsychotic s, 1 Antiradiation Agents, 324 Antiviral Agents, 129 A t h e r o s c l e r o s i s , 178 Biopharmaceutic s, 33 1 Blood Enzymes, Agents affecting, 233

Cell Metabolism Regulation of, 267 Distribution of Drugs, F a t e and, 247 D i u r e t i c s , 67 Drug Receptor Interactions, Molecu l a r A s p e c t s of, 236 Enzyme Activity, Agents Which Affect, 277 F a t e and Distribution of Drugs, 247 G a s t r o i n t e s t i n a l Functions, Agents Affecting, 99 Hallucinogens, 12 Hormones, Non-steroidal a n d Antago n i s t s , 191 Hormones, Steroid a n d Antagonists, 21 3 Hypnotics, 30 Molecular A s p e c t s of Drug-Receptor Interactions, 236 Muscle Relaxants, 30 Nucleosides, 299 Nucleotides, 299 P e p t i d e s , Synthetic, 289 P h a r m a c e u t i c s , 331 P u l m o n a r y Agents, 92 Reactions of I n t e r e s t i n Medicinal C h e m i s t r y , 314 Regulation of Cell Metabolism, 267 Reproduction, 20 5 Sedative, 30 Stimulants, CNS, 12 Synthetic P e p t i d e s , 289

3 42