PROGRESS IN MEDICINAL CHEMISTRY 7, Volume 7

Progress in Medicinal Chemistry 7

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Progress in MedicinaI Chemistry 7 Edited by

G. P. ELLIS, B.SC., PH.D., F.R.I.C. Department of Chemistry, University of Wales Institute of Science and Technology. King Edward VII Avenue, Cardiff and G. B. WEST, B.PHARM.,D.SC.,PH.D., F.I.BIOL Barking Regional College of Technology, North-east London Polytechnic, Longbridge Road, Dagenham , Essex

LONDON BUTTERWORTHS

T H E BUTTERWORT11 GROUP ENGLAND Butterworth & Co (Publishers) Ltd London: 88 Kingsway, WC2B 6 A B AUSTRALIA Butterworth & (’0(Australia) Ltd Sydney: 20 Loftus Street Melbourne: 343 Little Collins Street Brisbane: 240 Queen Street CANADA Butterworth & Co (Canada) Ltd Toronto: 1 4 Curity Avenue, 374 NIlW ZliALAND Butterworth & C o (New Zealand) Ltd Wellington: 49/5 1 Ballance Street Auckland: 35 High Street

SOIITtI AI:RI(’A Butterworth & <‘o (South Africa) (I’ty) Ltd Durban: 33/35 Bench Grove 1,’irst published 1970

@ Ih~tterwortli& (’0 (Publishers) Ltd.. I 9 7 0 Suggested CJ.D.(’. number: 6 15.7:54 ISBN 0 4 0 8 70080 7

Printed by plioto-litliograpliy and tiiadc in (;reat Britain at the Pitnian Press. Bath

Preface

A nuniber of changes have been made in Volume 7 of the series, and each volume will henceforth be published in two paper-backed parts at approximately sixmonthly intervals. On publication of the second part a complete volume will become available in a single case-bound edition. We hope that the other minor changes will also be appreciated by our readers. As in previous volumes, we are grateful to reviewers and others for their encouragement, criticisms and suggestions. We hope that the new arrangement of six-nionthly issues wlll permit reviews to appear in print at a shorter interval of time between submission and publication than has been achieved in the past. Our thanks are due t o the staff of Butterworths and t o the authors, societies and publishers for permission t o use illustrations and tables.

G. P. E l L l S G. B. W r m

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Contents 1 Some recently introduced drugs A. P. Launchbury, B.Sc., M.P.S.* Selly Oak Hospital, Birmingham, 29, England

2 The Biochemical Basis for the Drug Actions of Purines John A. Montgomery, Ph.D. Kettering-Meyer Labora tory, Sou thern Research La bora t o p , Birmingharn, Alabama 35205, U.S.A. 3 The Chemistry of Guanidines and their Actions a t Adrenergic Nerve Endings G. J. Durant, B.Sc., Ph.D., A.R.I.C. Smith Kline and French Research Institute, Welwyn Garden City, Hertfordshire A.M. Roe,M.A., D.Phil., F.R.I.C. Stnith Kline and Fretich Research Institute, Welwyn Garden City, Hertfordshire A. L. Green, B.Sc., P1i.D. Departrnent of Biocheni istry, University of Strathclyde, Glasgow

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69

124

4 Medicinal Chemistry for the Next Decade W. S. Peart, F.R.S., M.D., F.R.C.P. Medical Unit, St. Mary's Hospital, London, W.2.

71 s

S Analgesics and their Antagonists: Recent Developments

729

A. F.Casy, B.Sc., Ph.D., F.R.I.C., F.P.S. Faculty of Pharinacy and Pharniaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada 6 Some Pyrimidines of Biological and Medicinal Interest - Part I1 C.C. Cheng, B.S., M.A., Ph.D. Midwest Research Institute, Kansas City, Missouri 641 l U , U.S.A. and Barbara Roth, B.S., M.S., Ph.D. Burroughs Wellcome and Co. (U.S.A.) lnc., Tuckahoe, New York I0707, U.S.A.

Index

* Present address:

285

343 Pharrnitalia (U.K.) Ltd., Kingmaker House, Station House, Barnet, Hcrts

CONTENTS OF EARLIER VOLUMES VOLUME 1 1 PHARMACOLOGICAL SCREENING TESTS-W. G. Smith 2 HYPOTENSIVE AGENTS-R. Wien 3 TRANQUILLIZERS-M. W. Parkes 4 DIURETIC DRUGS-H. Heller and M. Ginsburg 5 ORAL HYPOGLYCAEMIC DRUGS-J. D. H. Slater 6 ANTIFUNGAL AGENTS-E. P. Taylor and P. F. D’Arcy

VOLUME 2 1 THE PATENTING OF DRUGS-F. Murphy

2 THE TESTING AND DEVELOPMENT O F ANALGESIC DRUGS-A. H. Beckett and A. F. Casy 3 MECHANISMS 01: NEUROMUSCULAR BLOCKADE-W. C. Bowman 4 2-HALOGENOALKYLAMINES-J. D. P. Graham 5 ANAPHYLACTIC REACTIONS-G. E. Davies VOLUME 3 1 SOME CHEMICAL ASPECTS O F NEUROMUSCULAR BLOCK-J. B. Stenlake

2 THE CHEMOTHERAPY OF TRYPANOSOMIASIS-L. P. Walls 3 ANTITUSSIVE DRUGS-C. I. Chappel and C. Von Seeman 4 THE CHEMISTRY AND PHARMACOLOGY OF THE RAUWOLFIA ALKALOIDSR. A. Lucas 5 STATISTICS’ AS APPLIED T O PHARMACOLOGICAL AND TOXICOLOGICAL SCREENING-G. A. Stewart and P. A. Young 6 ANTICONVULSANT DRUGS-A. Spinks and W. S. Waring 7 LOCAL ANAESTHETICS-S. Wiedling and C. Tegntr VOLUME 4 1 EXPERIMENTAL HYPERSENSITIVITY REACTIONS-P. S. J. Spencer and G. B. West

2 MECHANISMS O F TOXIC ACTION-J. M. Barnes and G. E. Paget 3 DRUG RECEPTOR INTERACTIONS-E. W. Gill 4 POLYPEPTIDES OF MEDICINAL INTEREST-H. D. Law 5 ANALGESICS AND THEIR ANTAGONISTS: BIOCHEMICAL ASPECTS AND STRUCTURE-ACTIVITY RELATIONSHIPS-A. H. Beckett and A. F. Casy VOLUME 5 1 POLYPEPTIDE ANTIBIOTICS OF MEDICINAL INTEREST-R. 0. Studer 2 NON-STEROIDAL ANTI-INFLAMMATORY DRUGS-S. S. Adams and R. Cobb 3 T H E PHARMACOLOGY OF HEPARIN AND HEPARINOIDS-L. B. Jaques 4 THE HISTIDINE DECARBOXYLASES-D. M. Shepherd and D. Mackay 5 PSYCHOTROPIC DRUGS AND NEUROHUMORAL SUBSTANCES IN THE CENTRAL . I NERVOUS SYSTEM-J. Crossland 6 THE NITROFURANS-K. Miura and H. K. Reckendorf VOLUME 6 1 THE BRITISH PHARMACOPOEIA COMMISSION-G. R. Kitteringham

2 PHARMACOLOGICAL ASPECTS O F THE CORONARY CIRCULATION-J. R. Parratt 3 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-Part 1C. C. Cheng 4 THE MECHANISM O F ACTION OF SOME ANTIBACTERIAL AGENTS-A. D. Russell 5 THE BIOSYNTHESIS AND METABOLISM OF THE CATECHOLAMINES-M. Sandler and C. R. J. Ruthven 6 THE LITERATURE O F MEDICINAL CHEMISTRY-G. P. Ellis

1 Some Recently Introduced Drugs A. P. LAUNCHBURY, B.Sc., M.P.S. Selly Oak Hospital, Birmingham, 29, England * INTRODUCTION Some preliminary considerations MODIFICATIONS O F PHY SICO-CHEMICAL IMPORTANCE Penamecillin Prednisolone stearoylglycollate Doxycycline DRUGS OF NOVEL STRUCTURE OR ACTIVITY A curariform muscle relaxant: pancuronium bromide A non-addictive analgesic: pentazocine An intravenous anaesthetic: propanidid A psychotropic drug: oxypertine A tricyclic antidepressive: ipnndole An anti-hypertensive sympathetic neurone blocker: debrisoquine An adrenergic a-receptor blocker: thymoxamine Three phenylethylarnines with complex sympatholytic or spasmolytic effects: (a) prenylamine (b) verapamil (c) mebeverine A diuretic: ethacrynic acid (with notes on frusernide) Two hypotensive diuretics: (a) chlorexolone (b) clopamide A mucoid-secretion modifying agent: bromhexine

A pulmonary anti-allergic for asthma therapy: cromoglycic acid A terpenoid ester for gastroduodenal ulcer: gefarnate A broad-spectrum antimicrobial nitrofuran: nifuratel The f m t anti-pseudomonal penicillin: carbenicillin A new antibiotic of unusual structure: rifamide A new antitubercular drug: rifampicin A selective bronchodilator: salbutamol Concluding remarks References *Present address: Pharmitalia (U.K.) Ltd, Kingmaker House, Barnet, Herts. 1

7

2 3 3

5 8

12 12 16 20 23 25

27 29 31 31 34 35 36 40 43 44 45 46

49 50 52 54 55 56 51

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SOME RECENTLY INTRODUCED DRUGS

INTRODUCTION In this brief review the variety of drugs t o b e discussed precludes a completely systematic approach. The compounds t o be studied have therefore been grouped under two main headings: ( 1 ) those which are modifications, designed primarily to improve physicochemical characteristics, of pre-existing drugs; and (2) drugs which are either completely new in structure or activity or are sufficiently different from previously available agents t o warrant separate notice.

Substances included in (2) form the major part of this work, because of their number and variety, but drugs in ( 1 ) are n o less important therapeutically.

Some preliminary considerations

Before a drug produces an effect, it must interact with, or pass through, one or more biological membranes. In a very few cases, the drug is required t o produce a pharmacological effect on the first membrane encountered during its absorption, distribution and excretion within an organism; generally it must penetrate external (plasma) cell membranes, many cytoplasmic membranes (for example the endoplasmic reticulum) and often organelle (for example lysosomal, microsoma1 or nuclear) membranes before reaching its required site o f action; in addition, some drugs are particularly intended t o interact with one or more of these barriers. With some estimates [ 11 placing membrane mass as high as 80 per cent of the total dry mass of the cell, it is not surprising that increasing attention is being given to the study of biological membrane structures and functions [2-4) . Interaction between membranes and chemical compounds often yields information of particular application to pharmacology [ S ] . While the early concept of cell membrane as a simple lipid film is a gross oversimplification, it has none the less enabled the behaviour of many drugs t o be better understood, provided allowance is made for the effects of the pH of the aqueous phases, t h e pK, of the drug, and the lipid-solubility of the non-ionized species [ 6 ] .By processes which have been described as ‘drug latentiation’ [7, 81 , the absorption-distiibution characteristics of drugs are being modified to take advantage of the fact that an un-ionized, lipid-soluble molecule penetrates biological membranes much more readily than a polar ionized one. Esterification (often double esterification) is being increasingly employed t o yield compounds which can be more readily absorbed (usually from the alimentary tract), subsequent esterase-hydrolysis releasing the parent drug in the tissues. Well-known examples of this approach include methyl nicotinate, glycol salicylate, betamethasone 17-valerate, fluocortolone 2 1hexanoate and ditophal (for epidermal absorption), and triacetyloleandomycin and other macrolide esters (for alimentary absorption). Triclofas deviates from

A. P. LAUNCHBUKY

3

this scheme only in that the ester is ionic-it is, none the less, a bland ‘transport form’ of the highly irritant trichlorethanol. In the present review, two further examples will be discussed; both are double esters b u t one (penamecillin) is o f a pharmacologically-active acid, the other (prednisolone stearoylglycollate) of an alcohol. In addition, an example o f reduction of polarity (or increase in lipid solubility) by another method-the elimination of a hydroxyl group-will be noted in the section o n doxycycline.

MODIFICATIONS OF PHYSICO-CHEMICAL IMPORTANCE Penamecillin

Benzylpenicillin is still the most active penicillin against the classically penicillinsensitive organisms [9-121 . It is, however, only poorly utilized after oral administration partly because of destruction by gastric acid and gut flora, but more particularly because, being strongly anionic and therefore only very slightly lipophilic, it is poorly absorbed. Almost all t h e drug which achieves a therapeutic effect (during oral administration of the sodium, potassium or calcium salts) is absorbed in the duodenal region; some 70-80 per cent of each dose is lost by degradation or faecal elimination [ 131. Simple methyl, lower alkyl and benzyl esters show improved absorption, but are of little clinical value since they neither undergo spontaneous aqueous hydrolysis [ 141 nor cleavage to active antibiotic by an enzyme present in man [ I S ] (though rodents can perform this reaction). Some alkanolamine esters which d o hydrolyse spontaneously gave early promise [ 141 though, of these, only penethamate hydriodide [ 161 received clinical application (asan injection, Estopen)and even this drug hasnow fxllen into disuse. The solution t o this problem lay, in effect, in conversion of the penicillin anion t o an alcohol (i.e. t o an ester of a gem diol) which was then further esterified with a suitable acid-though this wasnot the method o f synthesis. The penicillin molecule is extremely labile and synthesis of its double esters has been achieved by the use of a-acyloxyalkyl halides; these readily form the required compounds by direct reaction with a benzylpenicillin salt [ 171 , the activation energy for this process being low enough to avoid excessive penicillin degradation. Such double esterification, in the case of the acetoxymethyl derivative of penicillin G, results in rapid biological availability of benzylpenicillin since non-specific acetic esterases are ubiquitous in mammalian tissues and serum [25] . Once the acetate moiety is removed the remaining compound (which can be considered t o be the ester of penicillin, G with methanediol) undergoes rapid and spontaneous decomposition to active antibiotic and formaldehyde [16, 171 (the latter being promptly metabolized t o less vigorously reactive substances). The acetoxymethyl ester of benzylpenicillin is penamecillin (Havapen, 1 ). It is an almost white, free-flowing powder lacking the characteristic odour 2nd taste which make penicillins generally unpalatable when administered in tablet or

4

SOME RECENTLY INTRODUCED DRUGS

liquid formulation 161. This is an important factor in determining patient coopera t ion in therapy. The initial pharmacological report on penamecillin [ 181 , while containing errors of deduction regarding the site and rate of hydrolysis of the drug, showed it to be superior to the anion with regard to stability in gastric juice and the COOCH2 -0OC Me

(I1

persistence of serum antibacterial levels after single doses. When given orally to dogs, penamecillin itself was not isolated from circulating blood, even from the hepatic portal vein. The method of recovery (extraction into chloroform which, in contrast to acetone. removes penamecillin selectively in the presence of benzylpenicillin) was verified by in virro checks. Other in uitro experiments showed that extremely rapid hydrolysis to benzylpenicillin was effected by exposure t o tissue hornogenates and the serum of various animals. The possibility that the fatsoluble ester is taken up in lipidsvia the lymphatics was eliminated by examination of the contents of the thoracic duct two hours after oral dosage; n o penamecillin was detected. A re-examination [ 191 of the data presented in the earlier paper cleared some o f the apparent anomalies by the use of pharinacokinetic analysis. For example, it had been observed that both benzylpenicillin and penamecillin appeared in the circulation only as benzylpenicillin yet the levels resulting from a single dose of panamecillin persisted (at 0.1 pg/ml or above) for 10 hourscompared with 5-6 hours for an equivalent dose (on a molar basis) of benzylpenicillin, though the latter drug produced much higher levels, more rapidly, than did penamecillin. This phenomenon was shown to be due to prolonged absorption of penamecillin from the gut (over a period of 10 hours or so) which contrasted sharply with benzylpenicillin for which little absorption was demonstrated beyond the first h o u r after administration. This is a classic example of biological half-life estimations having been invalidated by failure t o correct for the behaviour of a poorly and slowly absorbed drug. Clinically, penamecillin has exhibited the expected activity against infections normally responding to benzylpenicillin [20-221, with the advantage that doses can be given at 8-hourly (instead of 4-6 hourly) intervals and still provide relatively smooth blood-level curves. Organisms producing penicillinases, or those normally tolerant to penicillin G, d o not, of course, respond to penamecillin. The main advantage of penamecillin lies in its lipid-solubility and resistance to degradation until absorbed into the alimentary mucosa, though the potential inherent i n these properties has not been widely appreciated by clinicians [24] . Its mrliti drawback at present is its very poor water solubility which limits the rate of dissolution of the drug particles and thereby limits the amount of drug

A . P. LAUNCHBURY

5

presented to the mucosa for absorption at any one time. While this low aqueous solubility (approximately 4.5 mg/lOO ml [ 191 ) in large measure maintains the intestinal reservoir of the drug, a more favourable rate of dissolution would enable some of the 90 per cent of each dose at present eliminated to be utilized therapeutically. There seems to be room for development here, and in the application of this principle of double esterification to the formulation of improved depot penicillin injections-where penamecillin’s low water solubility may be an asset [231 . Prednisolone stearoylglycollate *

For the first decade or so after the introduction into medicine of cortisone and hydrocortisone, most research endeavour attempted to emphasize therapeutically desirable effects (and reduce the proportion of unwanted properties) by modifying and substituting the steroid ring-system. This process has indeed resulted in drugs with greatly increased specificity over a wide range of endocrine functions and, in the anti-inflammatory field, produced compounds such as prednisolone, triamcinolone, dexamethasone, paramethasoneand betamethasone-to name only a few. These coiticosteroids have, generally, been administered only as the 2 1-acetate or the corresponding free alcohol when used orally or parenterally, though other esters and some 1 6 ~ 117cy-‘acetonides’ , are widely used topically. Of recent years, however, more attention has been paid t o the potential latent in 21 -esterification as. a means of modifying corticosteroid activity. While propionates, benzoates, decanoates and hexanoates have been common among parenteral preparations of other steroid groups (mainly androgens, oestrogens and progestogens), such esters are only now being examined as rational modifications in the oral steroid treatment of systemic disease. A series of 21-esters of prednisolone has now been described [ 2 6 ] , screened by the classic Ungar [27] test. Chemically, the series consisted of two parallel lines of compounds; (a) simple 21 -esters of prednisolone with short (propionic) t o long (stearic) chain aliphatic acids and with one aromatic acid (benzoic), and (b) prednisolone-2 1-glycollate esterified with the same acids at the glycollic hydroxyl. Methods of preparation and purification have been briefly given. Pharmacologically, the Ungar test showed that short-chain simple esters displayed higher activity than the corresponding ‘glycollic bridge’ compounds; the more complex trimethylacetates and enanthates provided the ‘break even’ point where activity in the two series was approximately equal while esters of longer chain or aromatic acids were more active in the glycollate form. The simple 21-stearate showed high and prolonged activity but the 21 -stearoylglycollate (11, Sintisone) was even more interesting in both these aspects and was chosen for detailed examination and subsequent clinical use. The basic concept behind studies such as this is that esterification may modify corticosteroid activity by (1) increasing lipid solubility of the drug and thereby *Now often termed sfeaglate.

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SOME RECENTLY INTRODUCED DRUGS

improving its absorption and distributlon after oral administration ( 2 ) ,modifying the rate of hydrolysis to active steroid alcohol by judicious selection of esterifying acid and ( 3 ) possibly introducing an element of selectivity for particular types of tissue (enzymes capable of regenerating the drug may not be equally active at all potential sites of action). As is indicated in the brief survey below, prednisolone-

(11)

21 -steaglate is rapidly and fairly completely absorbed from the gut, produces high, prolonged, blood levels (both in terms of drug present and of activity), and may possibly prove t o have some selectivity, though this awaits rigorous observation before warranting further comment. An early study [28] of the pharmacology of this prednisolone double ester examined the compound t o ascertain (a) its anti-inflammatory activity (intensity and duration), and (b) the degree t o which undesirable effects appeared a t dose levels ne-ded to achieve therapeutically interesting results. In both these respects, the drug was compared with prednisolone and the results, corrected for prednisolone content of the ester, are summarized in Table 1. I . The variations in Table 1.1.

Activity of prednisolone stearoylglycollate, corrected for prednisolone content, compared with prednisolone (activity = 1 ) [ 2 8 ]

Test Foreign-body granuloma Carageenin oedema Capillary protection Thymus involution Pituitary-adrenal inhibition Ulcerogenesis

I

Activity

0.75

relative potencies of the two drugs in the different tests are indicative of the complex effects of 21-double esterification on the activity (or susceptibility of tissues to that activity) of this type of drug. During the study it became apparent that absorption from the gut is little different from that of the free alcohol; the differences lie in the subsequent fate of the ester. There seems evidence of slower metabolic inactivation of the steroid, as might be expected, and hence a strong

A . P. LAUNCHBURY

I

probability that the greater activity of the double ester in many tests can be accounted for by more efficient utilization. In this respect the stearoylglycollate might be regarded as another example of ‘drug latentiation’-a circulating ‘depot’ from which prednisolone is steadily released. This interpretation is borne out by another study [29] which, as its prime objective, compared the blood-1evel:time relationships of prednisolone and its stearoylglycollate. The authors concluded that their results, showing consistently higher serum levels (the difference increasing progressively with time) after administration of the double ester, were only accounted for by reduction in the rate of inactivation of the ester compared with the parent steroid. The absence of any prolonged absorption from the gut was confirmed. Absorption was completed within two hours. Interference, by prednisolone and its stearoylglycollate, with the dynamics of calcium and nitrogen metabolism were compared [30] , using isotopic methods to measure calcium mobilization. As a by-product of this investigation, evidence was obtained that patients with impaired calcium absorption before treatment (‘hypoabsorbers’) show less disturbance of calcium balance during therapy than normal individuals. While both compounds accelerated calcium turnover in bone and increased its excretion, the ester produced significantly less effect than its parent. The loss of nitrogen characteristic of corticosteroid activity also appeared to be reduced. Both these advantages were apparent at dose levels adequate for therapy. Two further communications [3 1,321 reported that prednisolone stearoylglycollate, in substantial doses, was the least potent anti-inflammatory steroid (among the representative series studied) with respect t o pituitary-adrenal inhibition. The order of increasing suppressive potency in this test was: prednisolone stearoylglycollate, prednisolone, triamcinolone, dexamethasone, betamethasone. Techniques used in the comparative evaluations included the metyrapone test and gas-liquid chromatography. Clinically, the quantitative nature of spirometry led to its early use in evaluation of the effectiveness of the double ester in bronchial asthma [ 3 3 ] . By this method the newer drug was shown t o be twice as potent as prednisolone therapeutically. This is in terms of prednisolone equivalent (not a weight for weight compari5on with the ester which contains only 52.6 per cent steroid) and is an expression of the fact that the average daily suppressive dose, and the total drug consumed during the trial period, was only half that required by patients on the parent steroid. This, again, bespeaks better utilization of the administered steroid. Sideeffects, particularly fluid retention and steroid-induced glaucoma, were evaluated by other workers [34] in patients suffering from a wide range of disorders. No instance of steroid glaucoma and little fluid retention was detected during therapy with the double ester in clinically effective regimes. while prednisolone produced these complications in the familiar pattern. In this trial it was agreed that the approximate equivalence of 6.65 mg stearoylglycollate to 5 mg prednisolone alcohol was reasonable as a guide to therapy.

X

SOME RECENTLY INTRODUCED DRUGS

Dox ycycline

Hydrogenation of methacycline yields a t least two major products, the 6 a and 60 epimers of 6-deoxy-5-hydroxytetracyline. These were initially referred to, loosely, as ‘6-deoxyoxytetracylines’ but it soon became apparent that it could not be assumed that the configuration of the C6 methyl would necessarily be the same as in that of the parent 6-hydroxy compounds. Indirect evidence that the 6a epimer resulted from an improved hydrogenation reaction was obtained in early studies [35] in which it was also noted that one advantage accruing from 6-dehydroxylation was increased chemical stability; in the absence of the 6-hydroxyl, degradation t o anhydrotetracylines and isotetracylines is impossible. A further finding was that both the 6-methyl and the 6-hydroxyl groups may be removed without reduction in typical tetracyline activity-which was in fact increased t o the extent that 6-deoxy-6-demethyltetracyline was found t o be twice as active in v i m , weight for weight, as tetracyline. This compound, with only four remaining asymmetric centres, is the simplest molecule so far studied which retains typicd tetracyline-like activity and demonstrates that asymmetry a t c6 is not vital to such activity. None the less the actual configuration of the c6 substituents has quantitative consequences. This was clearly established by more detailed work [36] during the course of which both c6 epimers of doxycycline were synthesized. The 60 species was obtained by a stereospecific reaction involving Raney nickel desulphurization of the benzylmercaptan addition derivative of methacycline. Model studies confirmed the skeletal configuration. The 68 epimer was produced by noble metal catalytic hydrogenation of Ila-fluoro6-methylene-5-hydroxytetracycline.Bioassay of the two epimers against K. pneumoniue revealed that the 601 compound had almost three times the activity, weight for weight, of the 8 epimer; this has been confirmed against other organisms, and is in good accord with further studies of tetracycline [ 3 7 ] and derivatives possessing c6 asymmetry [38] ,

OH

CO-NH,

OH OH

0

OH

0

Doxycycline (6-deoxy-5-hydroxytetracycline,Vibramycin, 111) is, chemically, the most stable tetracycline in current use but its outstanding property is its high lipid solubility; these features combine to make it the most reliably and completely absorbed tetracycline derivative, with the longest half-life. The consequences of high lipid solubility were demonstrated in a comparison

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P. L A U N C H B U R Y

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[39] of the distribution in dogs of various tetracyclines after oral dosage. This study did not assess rate of absorption from the gut since the prime objective was to study the differences of distribution of the drugs (i.e. from serum t o interstitial fluids to tissue depots) and their variation with lipid solubility once the equilibrium state had been reached by repeated administration. After making allowances for serum protein binding, metal chelating powers and pK, values, it was shown that the distribution o f the antibiotics between tissue depots and interstitial fluid correlated well with the partition coefficients in chloroform/ water (used as an assessment of lipid solubility as previously described [40] ). The similarity between dog and man with respect to the consequences of physicochemical properties o f drugs was evident in this case as in many others. Since cloxycycline has high lipid solubility (K, CHC13/H20 at pH 7.4 x lo3 = 475 as against 95 for tetracycline, 7 2 for demethylchlortetracycline and methacycline, and only 7.2 for oxytetracycline) it would be expected t o attain high concentrations i n . tissues (its serum protein binding is similar t o that of demethylchlortetracycline and 1.5 times that of tetracycline); this was confirmed by recovery from various tissue homogenates. A more detailed investigation [41] of the absorption (including the effect on this of the presence of various foods), renal clearance and anti-infective properties of doxycycline showed it to be the first clinically-available tetracycline t o be rapidly and almost completely absorbed even in the presence of food. Hitherto, tetracycline absorption had always been unpredictable (421 (owing, for example, to their tendency to form insoluble complexes with metals) 11' taken during o r after meals, or even with milk. In some circumstances (for example ingestion of a glass of milk with the drug) the levels attained were slightly reduced. In n o case was doxycycline affected by normal dietary substances to anything like the degree encountered with older tetracyclines. Aluminium hydroxide was the only substance tested which reduced absorption of doxycycline to negligible levels (as happens with all tetracyclines). This is to be expected and it should be routine practice t o avoid any antacid with high adsorptive capacity durliig therapy with any tetracycline (or indeed, with many other drugs). Administration of I00 mg doxycycline, in the absence of foods, led t o almost complete absorption from the gut and a peak blood level of 1.8 pg/ml two hours after ingestion. Three times this dose was required.to produce a similar blood level in four hours in the case of 300 mg demethylchlortetracycline. The plasma half-life (after single dose) was 15 hours in the case of doxycycline and 12 hours for demethylchlortetracycline. This means that the half-life of doxycycline is seven hours longer than that of tetracycline. Plasma protein binding, measured by an ultracentrifugation method, was estimated at 93 per cent. This is somewhat higher than the figure quoted by other workers [49] who also obtained lower figures for the serum binding of other tetracyclines. It may well be that the binding forces are weak and the degree of I,i~~clingtherefore variable according to conditions during estimation; certainly

the attachment to serum proteins is loose enough to permit rapid migration of the drug, in quantity, into tissue fluids as discussed below. Renal clearance of doxycycline was strikingly different from that of other tetracyclines (1 2 per cent of creatinine clearance, compared with 26 per cent for demethychlortetracycline) and was considered to be the principal factor in determining both the serum concentration and its duration. This is a likely explanation since, as briefly indicated earlier, highly lipid-soluble drugs penetrate cell membranes readily. I t follows that lipid soluble materials present in the glomerular filtrate diffuse back through the tubular walls and return to the general circulation. This has been demonstrated for many bases (for example amphetamines [43-45] narcotic analgesics [46] );the tactorS involved have been comprehensively reviewed 1471 and summarized [48]. This phenomenon, apparently due to the loss of tile hydrophilic C6 hydroxyl, coupled with the dependable alimentary absorption would account for the increase in plasma half-life to 22 hours on repeated dosage in man. I n a pliarmacokinetic study [49], this and related aspects of doxycycline were stiiclied in man. Although the degree of serum protein binding as estimated by these workers was lower than in the report [41] discussed above, the differences seem inadequate t o call in question the validity of their conclusions; in addition. serum levels and protein binding data are not, of themselves, reliable indicators of therapeutic potential. Serum levels of doxycycline wei-e shown to be proportional to the dose (oral) f o r the series 50. 100, 150 and 250 mg. This is in contrast to the effect ot increasing the oral dose of tetracycline and most other derivatives of it, for hitherto the proportion of drug absorbed decreased as the dose increased. Nor could any reliable figure be assigned to this proportion [SO]. The report at present under review [49] also advances reasons for believing that the alimentary absorption of doxycycline is almost, if not quite, complete. This is important since many of the common side-effects of tetracycline therapy (such as gastrointestinal disorders and proctitis), are generally held to be due in large measure to residual drug remaining i n the gut [42] . While overgrowth with non-susceptible inicro-urganisrns ( t o r example Candida) may account for some of the effects, much o f the alimentary intolerance may well be due t o a direct irritant effect of the drug on the mucosa; a strong hint of this lies i n the fact that intramuscular injections of tetracyclines are generally formulated with a local anaesthetic t o reduce pain on injection. Doxycycline may, if only negligible amounts remain in the gut. be free of most of the traditional complications of tetracycline therapy; in t h i s connection i t is interesting that nausea is the only side-effect so far reported 141 1 and this only occurred when the drug was given after an overnight fast and contact with gastric mucosa was therefore maximal. As indicated Irom a previous study [41] , repeated dosage leads to an increase i n half-life from about I 5 to 22 hours. It was also established that this increase in apparent half-life was not due to decreased urinary excretion or to unusual accumulation of the drug; once equilibrium with storage depots (which release

A. P. LAUNCHBURY

11

lipid-soluble drugs only slowly) was achieved-generally within t w o days-no further accumulation was detected. In this study 25-30 per cent of the administered dose was recovered from the urine ( 2 4 hours collection) and this proportion appeared t o be independent of dose (in single dose studies) and of time (in chronic, multiple dose experiments). Since patients can rarely be relied upon t o take (or be given) medication after fasting,and since itis common experience that dosesare omitted more or less frequently the properties of doxycycline make it appear a promising successor t o the ‘first generation’ tetracyclines. This is even more likely since the antibacterial spectrum and activity is at least equal to that of tetracychne, and in the case of certain tetracycline-resistant bacteria doxycycline has (of all derivatives tested) shown the highest activity [ 3 5 , 4 1 ] . Clinical reports, while still not numerous, appear t o bear out the optimism aroused by the drug’s unusual physico-chemical properties. An outstanding example isan evaluation of doxycycline in sinusitis [ S 11, an often intractable and potentially dangerous infective disease. Antibiotics are widely used systemically in this condition primarily as prophylaxis against possible meningitis or osteitis; it is not generally believed that the sinusitis itself is greatly affected. The few reported attempts t o detect antibiotics in purulent sinus secretions, during systemic therapy, have failed. This is not surprising since the usual antibiotics used in such diseases (penicillins, streptomycin) are generally highly polar, often ionic, and are not expected to penetrate well under these circumstances (just as their low lipid solubility prevents their traversing the ‘blood-brain barrier’). Doxycycline’s ability t o penetrate lipoid membranes would be expected t o increase its chance, at least, of significant penetration into infected sinuses though the authors concerned claim t o have selected doxycycline, not for this fundamental reason, but for one of its consequences-the prolonged half-life-of which they had read a report [ 5 2 ] . During the trial, (blood levels as high as those reported by previous workers were attained, and all 1 2 patients produced sinus secretion concentrations of doxycycline of 05-7.5 pg/ml-approximately tenfold higher than the M.I.C.’s reported for the organisms isolated before the trial commenced. Sinus secretion ceased-in 4-9 days. This work indicates that many conditions, for which current tetracyclines are considered unsuitable, may prove treatable with more lipophilic agents. A different approach [ 541 t o increasing tissue permeability t o antibiotics is discussed under bromhexine. A further example of the ability of doxycycline to penetrate into secretions is provided by a report”[53] of a comparative trial of the drug and ampicillin in acute exacerbations in chronic bronchitis. It was found that 100 mg doxycycline daily and ampicillin 250 mg four times daily were clinically equally effective. Bacteriologically, however, it was found that H. irzfluerzzae re-appeared in sputum more often during ampicillin therapy than y i t h doxycycline. This was attributed, following earlier workers, to the failure o f ampicillin (in the dose used) to reach adequate levels in sputum. By inference, therefore, doxycycline (at one tenth of the daily dosage) achieves adequate levels more readily.

12

SOME RECENTLY INTRODUCED DRUGS

It is interesting to observe that in penamecillin, prednisolone stearoylglycollate and doxycycline, pharmacological effects predictable on the basis of physicochemical data are being observed in clinical practice. There can be little doubt that modification of physico-chemical properties will be increasingly explored in the future as a means of improving old drugs and designing new ones.

2 . DRUGS OF NOVEL STRUCTURE OR ACTIVITY A curariforrn muscle relaxant Pancuronium bromide

For many years (+)-tubocurarine has remained the standard non-depolarizing drug for all but short operations, in spite of its side-effects. Predominantly, these consist of (1) histamine release (often leading to bronchospasm) and (2) hypotension (resulting, in large measure, from autonomic ganglion blockade). Although many potential competitors have been examined, few have stood the test of time. Gallamine triethiodide has a shorter duration of action but raises the blood pressure and induces tachycardia. Like tubocurarine, it is a histamine-releaser. Alcuronium chloride is the other main contender in this field but it too liberates histamine though only to about the same degree as gallamine, i.e. rather less powerfully than tubocurarine. It is apparent that if any real advance is to be made in this field, resulting in the production of a neuromuscular blocking agent lacking histamine releasing properties, without cardiovascular effects but with a predictable period of action (and readily reversible with neostigmine and atropine), then some fundamental study of this type of activity is necessary. Two excellent reviews of work in this field have already appeared in this series, one concerned mainly with the structureactivity relationships [55] , the other more with the drug-receptor interaction [56] . Both authors express dissatisfaction with the terms used to characterize types of neuromuscular block. It is pointed out [55] that many so-called curariform agents have ‘a dual mode of action’, first an apparently acetylcholinelike depolarization of brief duration followed by a competitive-type block. Further, many compounds produce these effects in varying proportions in different species and tissues, and since many substances have been examined and classified on the basis of results obtained with a single preparation many need reevaluation now that techniques and terminology are more refined. Several proposed alternatives to the term ‘non-depolarizing’ to describe curariform neuromuscular block have been discussed and the alternative term ‘competitive antagonism’ [56] proposed asmore accurately descriptive of what is now believed to occur. It is, however, pointed out that however careful the laboratory study may be, it is still impossible to determine in vivo if curare-like drugs fulfil all the criteria of block by competition.

A. P. LAUNCHBURY

13

For the purposes of the present discussion the term ‘curariform neuromuscular block’ will be taken to refer to a post-neural membrane effect, resulting in paralysis of striated muscle, having the following characteristics: (i) blockade can be reversed by anticholinesterases, for example neostigmine and edrophonium, (ii) suxamethonium can antagonize the effect, (iii) partial-block by the drug in question can be augmented by small doses of tubocurarine or gallamine (these doses being insufficient to initiate full blockade unaided), (iv) partial neuromuscular blockade can be reversed with potassium chloride, (v) partial-block is reversible by adrenaline, (vi) after reduction of the tension generated by indirectly elicited tetanus, during partial block, post-tetanic twitches are, briefly, of increased amplitude, (vii) the degree of block is reduced by fall of temperature (in mammalian preparations). Most drugs with this type of activity have non-rigid molecules and for some time it was assumed that the fullyextended configuration of decamethonium represented an optimum interonium distance of approximately 14 A. Doubt was aroused by studies [57-611 indicating that full extension is unlikely in aqueous solution (factors determining configuration were reviewed [ 621 earlier in this series) and by observations of high activity of this type with molecules incapable of such interonium distances (for example toxiferine I [63] ,gallamine triethiodide [64] ). From such considerations it seemed likely that maximum activity would be associated with an interonium distance of 9-10 A, though the work discussed below has shown that this value is not critical. Against this background, and in view of the growing clinical importance of a proper understanding of drugs of this type, a contribution already referred to [55] concluded: ‘ . . . studies must establish not only absolute stereochemical configuration, but also the most probable conformation of the molecule in the biological system in which it produces its effect. Many of the most interesting and most potent neuromuscular blocking agents have nonrigid molecules, and studies of the biological cation of both completely rigid molecules, such us those bused on the steroid nucleus, and on molecules of known cbnformation should provide fruitful areas of work ultimately leading to new advances’ (present writer’s italics). The first of these advances reaches clinicians in the form of the steroid, pancuronium. The steroid nucleus provides an excellent rigid lattice for the attachment of pharmacologically active groups plus, in many cases, a hydrophiliclipophilic balance favourable to the ready penetration of derived drugs to their sites of action. In the study of curariform neuromuscular blocking agents the steroid fused-ring system offered the advantage of providing compounds of fixed interonium distances, thus enabling variations of activity with structure, or

14

SOME RECENTLY INTROI)UCl
charge density, to be separated. The first series of compounds based o n this structure were monoquaternary, having acetylcholine analogues ‘built in’ to the steroid A ring, in a variety of a and 0 configurations. The reasoning behind this approach (including many references) is given in the report [65]which appeared some time later. All ten compounds tested exhibited typical non-depolarizing neuromuscular activity. Though this was weak (the most potent drug showed only one sixteenth of the potency of tubocurarine, on a molar basis) the work did confirm that steroids with this type of activity had clinical potential, as had been anticipated from studies of aminosteroid alkaloids (e.g. malouetine 1661. and derivatives of funtumine and funtumidine 1651) and communications from other workers. Unfortunately, all thesc mono-quaternary Sa-androstanes proved to be both sympathetic and parasympathetic ganglion blockers, as had been suspected. Attention then turned t o bisquaternaory Sa-androstanes, substituted 3a, 17a t o give an interonium distance of 9-10 A. Pharmacological testing showed such compounds t o be potent non-depolarizing curariform agents (up t o one half the potency of tubocurarine, o n a molar basis) with weak anticholinesterase activity. The published report of these studies [67]also discussed the implications o f work in hand in other laboratories. The accumulated evidence indicated that a , 0-stereoisomerism had little effect on neuromuscular activity, steric impedance from angular methyl groups on C l o and C 1 3was absent and confirmed that this type of activity was not dependent on a ‘fixed’ interonium distance but varied within a range of quaternary separations. The obvious pointers offered by the foregoing compounds led t o the synthesis of further derivatives, this time incorporating the feature present in the earlier monoquaternary agents (acetylcholine-like structures ‘built into’ the steroid A and D rings). The series, based on 2~,16~-diamino-5a-androstane-3a,l7~-diol dimethohalide, was described [68] and one compound selected for detailed comparison with tubocurarine. The steroid selected was pancuronium bromide (IV) (20,160-dipiperidino-SaMe

I

CO

I

2 Br-

MeCO.0‘ H

atrdrostane-.b.. 1 7fl-diol cliacetate dimethohi-omide, NA07. Pavulon). Detailed pharmacology. iiicluding structure-activity relationships within this group, has been presented [601 . Pancuronium is one of the most potent curariform

A.

P. L A U N C H B U R Y

15

neuromuscular blocking agents k n o w n . y e t its histamine-releasit~gand ganglionblocking activities are iicgligiblc. According to the conditiuns of test ~ ~ I I I ~ I I I - U I ~ ~ U I I I

shows u p to ten times tlie potency (molar comparison) of tubocurarine, with equal duration of activity and an apparently similar mechanism of action. Toxicity is found to be low and related only to the neuromuscular blocking effects. Provided that artificial ventilation is available, 10,000 times the normal effective dose evinces no acute toxicity in anaesthetized cats for u p to two hours after injection;as usual. species vary in susceptibility to toxic effects. As might be expected o f a highly-charged cation. absorption from the gut is poor. No I~ormonal effects have been detected. Further studies [70-731 of the properties of pancuroniurn have appeared while the clinical advantages of the drug over existing agents have been reported in a separate publication [74] . In these clinical experiments on anaesthetixd human patients an electromyographic technique was used t o record action potentials from tlie hypothenar muscles following supramaximal stimulation of the ulnar nerve at the elbow. The height of tlie action potential was found to be, as previously reported. closely related to the degree of paralysis of tlie muscles under study. The drug was injected intravenously at controlled rates via tlie opposite hand. In no case was any evidence of histamine release found (no broncliospasm or skin wheals occurred). Tlie onset of action was considered more rapid than that of tubocurarine. and a complete absence of cardiovascular side effects (even in the presence of halothane) was particularly commented upon. Weight for weight, this study indicated pancuronium to be about five times as potent as tubocurarine. Tlie above and reports from other workers (awaiting publication at the time of writing), strongly suggest that this type of neuromuscular blocking agent constitutes a notable advance in anaesthesia. Tlie properties of pancuronium. which make it of special value in the poor-risk or asthmatic patient may be briefly summarized as: (i) myoneural blockade, similar in intensity t o that effected by 1 0 - 1 5 mg of tubocurarine, is rapidly produced by 2-4 nig of pancuronium and may, i f required. be maintained by incremental doses of 1-2 mg. (ii) Paralysis thus induced is easily reversed by an ticlio linesterases ( lOi examplr neostigmine), plus atropine, without subsequent recurarization. (iii) Circulatory effects are unusual in that even if hypotension is produced by. for example, halothane, the blood pressure often returns t o normal when pancuronium is given. In a similar manner an elevated or depressed pulse-rate frequently stabilizes at 80 to 90 per minute after administration of tlie relaxant (it is of interest in this context t o note that the tachycardia normally following reversal of block with neostigmine and atropine is generally prevented by pancuron ium). (iv) No liberation of histarnine can be detected. Hazardous bronchospasm is therefore unlikely to occur.

16

SOME RI-CENTLY INTRODUCED DRUGS

(v) I n man, tlie drug appears to be approximately five times as potent (on a milligramme basis) as tubocurarine. (vi) Even in long operations no evidence of tachyphylaxis or of cumulation was discernible. (vii) Premedicants such as promethazine and atropine d o not appear to interact with pancuronium. Anaesthetics vary in this respect; ether strongly potentiates the drug while tliiopentone only moderately reinforces it, halothane also ‘increases block b u t (as mentioned above) was prevented by pancuronium from producing its cliarai teristic liypoterisive complications. It is ccrtain that pancuronium is but tlie first of a series of steroids of this type There is Iorexaniple. need f o r a n agent with its favourable properties but with the brevity 01. action of suxametlionium. Achievement of this aim may not be too distant siiicc quaternary derivatives of the alkaloid conessine, possessing (in cats at least) the required cliaracteristics have recently been described [ 7 5 ], and dacitroriium bromide is now under intensive study by the team responsible for tlie develupmeiit ot’ pancuronium. A ’non-addictive‘ analgesic Pentazocine

Until recently (the last decade or so) studies of enormous numbers of potent analgesics seemed to indicate that effective analgesia and drug dependence of the morphine type were inseparable. In addition. ‘addiction’, ‘habituation’ and similar terms were widely used with very loosely defined connotations, so that considerable confusion resulted. The World Health Organisation has now published [76] a system of description o f such conditions, based on the term ‘drug dependence of the (name of drug specified) type’, together with a review of developments which led to its adoption. The last few years have produced a voluminous literature on structure-activity relationships among potent analgesics and their antagonists especially since research received ( i n 1054) ‘near-maximal stimulus’ with the discovery that morpliiiie-antagoiiists (nalorphine i n particular) were potent analgesics in man (see below for detail). Since nalorphine was known t o precipitate theabstinence syndrome when administered to laboratory animals (and man) with established drug dependence of the morphine type it seemed probable that, if the undesirable subjective (mainly hallucinogenic) effects of nalorphine could be reduced or eliminated, clinically valuable analgesics (comparable to morphine in effectiveness) lacking significant ‘addiction potential’ could be produded. The present position can now be summarized (at the risk of oversimplification) by grouping the resultant compounds under three headings:

(i) Analgesics with dependence liability, for example morphine (V), levorphanol (VI), phenazocine (VII) and methadone (VIII);

A. P. LAUNCHBURY

17

(ii) Antagonist-analgesics which will generally reverse the actions of compounds under (i) but will, if given alone or with a similar drug provide effective analgesia, at least in man, for example nalorphine (IX), pentazocine (X, Fortral, Sosigon, Talwin, Win 20,228), cyclazocine (XI); (iii) Antagonists which not only reverse most activities typical of compounds under (i) but also those of agents under (ii). This type of drug appears t o have Me

R,

Me Me

(

VIII )

little if any pharmacological activity of its own (and may be regarded as approaching in properties the much-sought ‘silent antagonists’, i.e. antidotes with activity only in the presence of the drug(s) for which they may be specific), for example naloxone (XII, Narcone, Narcan) and diprenorphine (XIII, MSOSO).

Drugs grouped under (ii) are the primary concern of this review as pentazocine in particular has now received wide clinical as well as experimental use, but a few further points require mention by way of background. Only when pentazocine is viewed against the complex background of drug-cell interaction, will the historic nature of its introduction become clear. Pentazocine may not be ideal but it is at present the only clinically available analgesic t o emerge from the protracted search for a non-dependence producing, effective analgesic with an acceptably low incidence of side effects.

18

SOMI, K1:CLNTLY INTRODUCED DRUGS

Knowledge o f the mechanisms of drug-dependent states, while still far from complete, has advanced far enough for it to be obvious that, even for the supposedly single case of morphine-type dependence, refined techniques are necessary to evaluate the liability of drugs to produce it. These can often, for screening at any rate, be developments of fairly standard techniques. T w o reviews of the chemistry and pharmacology of potent analgesics, and their analgesicallyactive antagonists have already appeared in earlier volumes of this series. The first [77] included a survey of test procedures, the second [78] discussed in more detail mechanisms of action. drug-receptor interactions and studies o f structureactivity relationships with special emphasis on the agonist-antagonist phenomenon. A third review appears i n the present volume (Part 2). Since then compounds included under (iii) (above) have appeared. As these substances antagonize both groups ( i ) and (ii) further techniques have had to bedevised to assess their properties. Examples of such drugs include naloxone [79] ,which has been shown to bevirtually inactive as an analgesic [ 8 0 ] (its low activity varies with dose in an anomalous manner), to lack respiratory depressant effects, and to be of negligible hallucinogenic potency [81,82]. More interesting still, naloxone antagonizes the analgesic effects of antagonist-analgesics such as nalorphine, pentazocine and cyclazocine (83,841 and finally demonstrates its unusual character by apparently lacking any significant pharmacological effects (for example analgesia) when administered alone [84] . A second example of such silent antagonists of effective analgesics is diprenorphine, very much more potent than naloxone in reversing effects produced by drugs grouped under both (i) and (ii) [SS] . Diprenorphine is one of the latest novelties to emerge from the extensive series [86] (‘Bentley’s compounds’) of very active substances based on a bridged C-ring tetrahydrothe; baine structure [87] . The modified procedures required to evaluate antagonist drugs of type ( i i i ) are illustrated in the reports [84,85] above and in a slightly earlier description 188) and discussion of techniques adapted particularly to demonstrate the presence or reversal of antinociceptive properties. The above discussion has included many drugs which are incapable, so far as is known, of inducing morphine-like drug dependence; for example, substances under (iii) are apparently free from this drawback [89] while those under (ii) (including pen tazocine) are unusual i n that although a degree of physical dependence can be induced by them (contrary to earlier findings [90]) the resulting condition is qualitatively different from that following use of‘type (i) drugs, “9 1-71 . This is not surprising since most type (ii) compounds will, like nalorphine itself, precipitate the abstinence syndrome [94, 981 when administered to drug-maintained subjects physically dependent on type (i) drugs. An important point is that with type (ii) analgesics drug-seeking behaviour fails to appear even i f withdrawal symptoms follow abrupt termination of use. This separation of properties involving the loss of craving [92, 95, 961 should reduce the risk of illicit consumption after termination of therapy. In fact. continuous treatment with drugs such as cyclazocine may well be a valuable additional treatment for narcotic-dependence (in patients with adequate motivation), since stabilization

A . P. L A U N C H B U R Y

19

on such a drug largely inhibits any pleasurable experience if, for example heroin, methadone or morphine are subsequently taken. Drugs of this type are currently on trial for this purpose and are being advocated [95, 99, 1001 as a promising development, since no ‘liking’ for cyclazocine appears to have arisen throughout the period of experience. The introduction of pentazocine into clinical practice appears even more o f a significant step if recent studies of dependence (on drugs of group (i), i.e. morptiinetype) at cellular level are considered. Tissue-culture work [ 1011 has shown that such drugs can become essential for the continued existence of cultured human cells and that administration of an antagonist (or withdrawal of morphine) can lead to metabolic derangement or death-depending on the conditions of the experiment. Such a system of it7 vitro testing may foreseeably supplement, or even supplant, existing in vivo methods of assessment of addictive-liability. A conveniently analytical, but general, review of present methods has recently appeared [ 1021. Against this background of extensive metabolic involvement, at the cellular level, of earlier morphine-like drugs the development of the antagonist-analgesics was almost inevitable. The surprising aspect is that early leads [ 103-105] were so slowly followed, though it has been pointed out [ I061 that the entrenched conviction that potent analgesic activity was inseparable from physical dependence liability [ 1071 prevented many hints from being seen. The discovery [ 108, 1091 that nalorphine was equipotent, weight for weight, with morphine as an analgesic stimulated research which is now, at an everincreasing pace, producing compounds similar to those briefly reviewed above. One of the major obstacles (apart from physical dependence liability) is the persistent tendency of nalorphine-like analgesics to produce subjective, even hallucinatory, mental changes. The history of the development of pentazocine (and similar benzomorphans) and corresponding derivatives of other analgesic parent structures (for exaniple cyclorphan) has been told many times and from various angles [ I 10-1 12, 1 151 ,some stressing synthetic methods for pentazocine and its congeners 1061 and others their preliminary screening [ 106, 1 131 in animals. The fact that nalophine, and other drugs grouped here under (ii) failed to show analgesic effects in many animal tests [ 1 141 and the effect of this (and other qualitative differences between drugs of groups (i) and (ii)) on the development of more informative tests has been noted above. As both pharmacological and clinical studies indicated at an early stage that pentazocine lacked morphinelike addiction liability (it is a weak morphine-antagonist), but was none the less of analgesic potency somewhere between that of morphine and pethidine, workers on b o t h sides o f t h e Atlantic coinnieiiced a thorough investigation of the drug and, to date, close o n two hundred reports have already appeared. Of these, only representatives can be quoted here though these have been chosen to cover, with their references, as much of the field as possible. In 1906 the World Health Organisation Expert Committee on Dependence Producing Drugscleared 11 161 pentazocine of need for restriction under narcotics

20

SOME RECENTLY INTRODUCED DRUGS

regulations,a view shared by the U.S. Committee on Drug Addiction and Narcotics. The drug is now widely used and a general concensus of opinion, based on comparisons with older analgesics in a wide range of conditions seems to be that 30-60 mg by intramuscular injection is equivalent to 10 mg morphine sulphate given by the same route. Much seems to depend on the nature of the pain-inducing disease (myocardial infarction is an example of the more refractory algesic stimuli, closely iollowed by some types of cancer and surgical, especially urological, procedures) and, of course, on concurrent medication. All authors agree [ 117-1291 that pentazocine is an effective analgesic, the majority finding it to possess the advantage (for most indications) of producing more sedation than equieffective doses of pethidine or morphine though some [ 1 18, 1301 disagree on this. point-these latter significantly, using rather lower doses than average. Comparatively reduced respiratory depression and induction of nausea (with only occasional vomiting), little effect on blood pressure or heart rate (though some tachycardia may occur) and apparent absence of troublesome constipating effect are all points marking out pentazocine as an advance on' older potent analgesics. Respiratory depression, when it does occur, it not reversed by nalorphine (as might be expected) but methyl phenidate (Ritalin) has been shown [ 1311 t o provide a measure of slow reversal of this complication; it would seem reasonable to suppose that drugs under heading (iii), such as diprenorphine [86] might provide better control if introduced into clinical practice. Respiratory depression of the newborn following use of analgesics during parturition is an important example of older analgesics having proved unsatisfactory. Pentazocine has been demonstrated [ 1321 to have minimal effects on foetal heart rate and respiration (and, incidentally, an apparent facilitating effect on progress of labour) and this may well be related to the finding [133] that pentazocine passes across the placenta more slowly than pethidine, thus reaching lower concentrations in the neonatal circulation. Although pain at the site of injection has been reported [134] a recent investigation [ 1351 seems to prove that pentazocine does not release histamine. This is in marked contrast to morphine and older drugs of its type, and it has, in consequence, been suggested [135] that pentazocine is suitable for use in asthmatics and patients suffering from other allergic diseases. While, thus far, pentazocine appears on all counts t o mark a new phase of analgesic development, long-continued study will be required t o delineate its precise properties and place in therapeutics. An intravenous anaesthetic

Propanidid

Eugenol, the major constituent of clove oil, is a local analgesic and has been used as such in dentistry (inter a h ) for very many years. By a series of modifications (involving more than one laboratory) a number of general anaesthetics suitable

A. P. LAUNCHBURY

21

for formulation into intravenous injection solutions, have been evolved. As will be seen from the structures (XIVa), propanidid (Epontol, FBA 1420) and Estil ( X I n , G 29505)-the only other drug of this type to receive clinical evaluationare related to eugenol only nominally. The development of this group, and of propanidid in particular, has been described [ 1 3 6 7 1 . R

q

i

C

H

2 CO N E t 2

(XLVa), R = C H 2 CO 0 Pr (XIVb) EstlL,R=CH2 CH CH2

For about 30 years, the thio- and 0x0-barbiturates have been the only widely accepted induction agents for intravenous use. They have several disadvantages; for example, although they have often been described as ‘short acting’ it would be more appropriate to describe them as ‘rapidly acting’ since clinical anaesthesia is followed by a long period of somnolence. This may last up to 24 hours and is due to redistribution of the drug into tissue depots, including fat [ 138-1411, though the relatively minor role of fat in this respect was only recently established [ 1401. Further, thiobarbiturates, for example, thiopentone, are generally metabolized by the liver to the corresponding oxobarbiturates [ 1421 which are still anaesthetically active. The inactivation of barbiturates by side-chain oxidation is slow, generally about 15-20 per cent per hour. N-Methylated barbiturates (for example, methohexitone) have the additional disadvantage of producing a more marked excitatory phase (muscle twitches, laryngospasm, etc.) before the onset of anaesthesia. Finally, the very alkaline solutions of sodium barbiturates (required to achieve adequate concentrations) are extremely painful and damaging to tissue if allowed to escape from the vein at the site of injection. There is, therefore, a place for a drug which can be administered intravenously for the induction of anaesthesia which is not only rapidly acting but also subject t o rapid metabolic destruction so that no cumulation or ‘hangover’ can occur. Propanidid is, in many respects, just such a drug. After administration, n-propanol is quickly released (within minutes) by ester hydrolysis while a few per cent of the injected material may also suffer loss of diethylamine to give a dicarboxylic acid. Neither the mono- nor the dicarboxylic metabolites are anaesthetic. The first (and most important) hydrolysis appears to occur predominantly in the liver [ 143-41 though some destruction has been attributed [ 1471 to non-specific serum esterases previously described [ 145-61. Demonstration of the second metabolite and evidence for the pharmacological inertness of this compound and of the solubilizing agent, (‘Cremophor EL‘) necessitated by the low. water-solubility of propanidid, has been obtained by radioactive tracer studies [ 1481. The position with regard to the role of serum cholinesterase in the inactivation of propanidid is not yet entirely clear though

22

SOME RECENTLY INTRODUCED DRUGS

recent evidence and a re-examination of previous work, seems to show that this enzyme is involved to some extent [ 1491. This may be relevant to the study of propanidid-induced potentiation of suxamethonium (see below). The use of propanidid in anaesthesia for outpatients has been discussed in a detailed survey (based on both clinical and pharmacological data) of the subject [ 1501 , while the more general applications (including a further survey of experimental and clinical experience) have also been comprehensively reviewed [151]. The place of the drug in dentistry [153-41 and in electro-convulsive therapy [ 1541 (with a description of a technique t o surmount difficulties arising from the concurrent use ot suxamethonium) has been evaluated: A refined radio telemetric technique has enabled a detailed study [ 1551 of the cardiovascular effects of propanidid to be made, resulting in strong evidence for a transient procainamide- or quinidine-like depression of myocardial conductive tissue. The above publications [ 150-51 quote a large number of relevant references. The effects of propanidid, injected intravenously, may be summarized as follows: (i) Loss of conciousness within one arm-brain circulation time. (ii) Coincident with (i) marked hyperventilation occurs, apparently due to the local anaesthetic-type effects of the drug on lung-stretch receptors (this phase can be used, if necessary, to hasten intake of volatile anaesthetic or assist blind nasal intubation). (iii) After some 30 seconds, hypoventilation (or even apnoea), occurs which does not appear to result from the over-breathing, but rather from a ‘biphasic’ effect of the drug. (iv) During the above no laryngospasm has been reported even when attempts have been made deliberately to provoke the reflex. (v) Blood pressure falls during induction and, as anaesthesia proceeds, quickly rises again to near-normal levels. As noted above [ 1551 this appears t o be mainly due to a temporary decrease in the contractile force of the heart through a quinidine-like effect. This can be of value in poor-risk patients since the antiarrhythmic effect protects against possible ventricular fibrillation until adequate anaesthesia renders this unlikely. (vi) Recovery, due to metabolic destruction of the drug, begins within 5 minutes of injection (unless anaesthesia is maintained by subsequent doses or other agents) and is usually complete within 10 minutes. There is, by virtue of the mchanism of recovery, no after-effect except, of course, for possible sequelae of the operation itself. Even large doses, resulting from repeated injection, produce no cumulation of the drug. (vii) The action of suxamethonium is intensified and prolonged by propanidid. As indicated above [ 1491 ,this seems related to the interaction by both drugs with serum cholinesterase; there is good reason to believe that the neuromuscular junction is not involved [ 1561 ;This interactionis only inconvenient, or potentially dangerous, if the anaesthetist is unprepared for it. Techniques can be modified accordingly [ 154, 1571.

A. P. LAUNCHBURY

23

(viii) Barbiturates are often antanalgesic. Some premedicants (for example, atropine and promethazine) also heighten pain-sensitivity, while others (for example, pethidine) do not. Propanidid is at least not antanalgesic even though its analgesic potency is open to question. This means that the patient can at least bespared intensification of pain and benefit from subsequent analgesic medication. (ix) Unlike barbiturates, solutions of propanidid do not cause excessive tissue damage if allowed to leak into perivascular tissue; while the local anaesthetic effect of the drug undoubtedly reduces any discomfort, no residual damage follows initial hyperaemia in these circumstances. The preparation has been injected intra-arterially in man (both by intention and misadventure) and no adverse effects have been detected histologically although short-lived, but profound, vasodilation of the perfused limb followed its administration. Propanidid seems, therefore, to possess many advantages over former intra-. venous anaesthetics. In particular, procedures of short duration (especially under out-patient conditions [ 1581 and induction in many poor-risk patients), seem particularly suitable for exploitation of its properties. A psychotropic drug

Ox ypertine

The subject of neurohumoral substances in the C.N.S. and their probable (and sometimes hypothetical) interactions with drugs was reviewed recently in this series [ 1591 ;included in that survey was a discussion of indole compounds (some) physiological, others psychotogenic) t o which attention is drawn (with special reference to the genesis of schizophrenia and similar psychoses). A publication devoted solely to the pharmacology of the central nervous system, with 21 specialist contributors, has also recently appeared [ 159al . The complexity of psychotropic drug action is well demonstrated by a close study of oxypertine, with its unusual combination of effects on brain noradrenaline, dopamine and serotonin, especially in the context of its apparent qualitative change of activity with increasing dosage and against the background provided by the reviews above. The drug was introduced in preliminary communications [ 160-21 discussing structure-activity relationships within the 1-(3-indolyl)alkyl-4-arylpiperazines. Many tryptamine derivatives have been studied (as notea above) but in this new series the terminal amine group was incorporated into a 4-substituted piperazine ring, in much the same way that open side-chain phenothiazines (for example, chlorpromazine) led to the piperazine side-chain group (for example, trifluoperazine). Authors vary greatly in the degree of importance attached to the indole and piperazine moieties. From a group of drugs based on the above parent structure oxypertine (XV, Forit, Integrin, Opertil, WIN 18,50 1-2)-a reserpine-

24

SOME RECENTLY INTRODUCED DRUGS

like moleculewas selected for further study and is now available for clinical use. Screening and evaluation included the usual animal behaviour tests [ 162-61 ; early deductions from the results of these have been confirmed by subsequent more detailed biochemical findings. These indicate that, for example, oxypertine (like chlorpromazine) is adrenolytic yet a potent releaser of noradrenaline from

Ph

presynaptic vesicles [ 160, 16 1, 167, 1681 . This amine-depletion seems fairly specific since dopamine and serotonin appear to be affected little, if at all [ 161, 167-91, and evidence has been presented that oxypertine acts at the level of the catecholamine storage granules, rather than at the cell membrane, in preventing uptake and storage of noradrenaline [168, 170, 1711. This release of noradrenaline would seem, on theoretical and experimental [167, 168, 172, 1731 grounds, to contra-indicate the concurrent use of M A 0 inhibiters and oxypertine. The absorption and distribution of oxypertine has been studied using tritiated drug and autoradiographic techniques [ 1741. The high lipid solubility of the drug is suggested as an explanation of its rapid accumulation in the brain (and other vital organs) and its rapid traversal of the placental barrier into foetal organs. Retention of the drug in the spleen was shown to be due to its uptake by blood platelets. Metabolites provided virtually all the radioactivity found in bile and urine and a proportion of that in brain (after approximately three hours). The unusual combination of release and antagonism of noradrenaline has been noted above, while another property separating oxypertine from other major tranquillizers is that it does not potentiate the analgesia produced by morphine or pethidine [ 1611. The compound is anti-emetic and weakly antihistaminic [ 16&2]. While many of the experiments referred to above suggested that oxypertine would be an effective major tranquillizer, with perhaps some alerting effects in withdrawn cases, only experiments in man demonstrated that, in fact, this might be the case. A number of studies [ 175-71 have indicated that low doses of the drug may show stimulating, anti-withdrawal effects on patients previously very difficult to reach and communicate with, thus rendering them more amenable to psychotherapy, while higher doses appear to have a more typically tranquillizing effect (often accompanied by drowsiness) on patients who would otherwise have received substantial doses of phenothiazines. Other clinicians do not, however, consider this feature to have been adequately demonstrated [ 1781 while another review [ 1791 setting out some of the problems inherent in clinical studies of drugs

A.

P. LAUNCHBURY

25

of this type, has attempted an appraisal of experience obtained up to 1965. It concludes that the balance of clinical evidence, taking into account its striking correlation with animal work, is in favour of the view that oxypertine may activate the depressed yet quieten the overactive, the degree of stimulation being inversely proportional to the dose. Toxicity is remarkably low for a compound of such activity. In mice, the LD50 value isabout three times that of chlorpromazine [ 1661 while none of the effects of the latter drug on the myocardium, liver, skin or eye have appeared in the studies of oxypertine. It is, however, still too early to appraise its chronic toxicity in man. As indicated earlier, dangerous interactions are likely to follow concurrent use of a MA0 inhibitor, though simultaneous use of anti-Parkinsonism drugs, for example, to control the relatively minor extra-pyramidal symptoms seems to present no unusual problems. Hypotension may occasionally occur with high doses. Oxypertine, with its novel structure and apparently promising therapeutic characteristics is a drug worthy of further study. Its specificity for catecholamine storage granules has already been exploited as a research tool [167, 170-3, 180 1811 and its continued use for this purpose can be expected to yield further fundamental information. A tricyclic anti-depressive lprindole

The complexity involved in the study of psychotropic agents has been noted above. This aspect is even more clear when consideration is given to the specific topic of mood. A recent review [ 1821 of some current opinion (on ‘amines, alerting and affect’), discussion [ 183-41 arising therefrom, and the continued development of new hypotheses (some alluded to below [ 186-91 ) all serve to bring home the fact that although much has been learnt, much achieved in therapeutics, the fundamentals of mood control still elude us. Against such a background of discovery, controversy, and ignorance the therapeutic value of newly introduced anti-depressive drugs can only be assessed on results in man. Animal experiments involvingmodel ‘depressive syndromes’ may be useful screening dev&es but failures of correlation between animal and human studies are unfortunately common. The series of compounds from which jprindole was selected has been described [ 1851 , further pharmacological details (particularly of iprindole) being conveyed [ 186-81 as contributory to the development of a new theory of brain function (in the context of stimulation and depression) involving novel methods of drug evaluation. Chemically, iprindole (XVI, Prondol, Wy-3263, originally called pramindole) like oxypertine, is an indole derivative. The classic and standard tricyclic anti-depressive, imipramine, has an

26

SOME RECENTLY INTRODUCED DRUGS

iminodibenzyl ring system; the radical change of steric shape involved in substituting this for the hexahvdrocyclo-octindolesystem does not seem to have been commented upon (most authors, on the contrary, claim close similarity for the two compounds) though it is likely to be important in terms of receptor-drug interaction. The 3-dimethylaminopropyl side-chain is the familiar one common

CH2

I

(

XVI 1

CH 2

I

YHZ

N Me2

to many psychotropics, antihistaminics, anti-cholinergics, local anaesthetics, etc. This is mildly surprising since, in the anti-depressants, the monomethylamino derivatives (for example, desipramine, nortriptyline) are claimed t o be the active metabolites [ 1891 . Unfortunately, no great detail on animal studies is available; the drug does, however, appear to possess the general properties of imipramine in tests involving motor activity, morphine-and reserpine-induced states [ 1861 amphetamine potentiation 11881 and lack of MAO-inhibiting activity [ 1901 . The only major difference emerging from these animal tests seemed to be that iprindole lacked most of the anti-cholinergic activity of imipramine. The clinical confirmation of this, while not yet conclusive, seems to indicate that anti-depressive and anticholinergic properties are not inseparable, as once appeared. Iprindole has received close clinical scrutiny. It has been compared with a placebo [ 190, 1911 with imipramine [192-1951 and with both [196]. All were double-blind controlled evaluations and established iprindole as at least as effective as imipramine, and one study [ 1951 included an examination of the doctor-patient interaction as a factor in such work (a similar discussion was felt necessary, as noted above [179] in the evaluation of oxpertine). In only two [ 192, 1941 of the above reports is it possible to estimate the frequency and severity of anticholinergic side effects, though in the one case [192J the care taken in the experimental design and the number of patients observed leaves little doubt that the dry mouth, constipation, etc. characteristic of imipramine therapy is either greatly reduced or even absent during iprindole treatment. This point is confirmed in an extension of this team’s work to include a 12 month toxicity study I1971 which, in addition, failed to produce evidence of haemopoitic, hepatic, cardiac, ocular or renal damage. Similar results followed other work. Another recent review [198] of published material pointed out that the animal work on atropine-like side effects had still not been published in full and, in any case, required independent corroboration. None the less, it was agreed that the clinical evidence (in spite of the difficulties of evaluation) did point to

A.

P. LAUNCHBURY

21

fewer problems of this type and recommended that the drug be used in patients for whom such effects might involve risk. It would appear, from the above, that the novel tricyclic system of iprindole has conferred properties which prbmise more patient comfort, and therefore more cooperation, in the treatment of depressive illness.

An anti-hypertensivesympathetic neurone blocker Debrisoquine

Since the publication of the review [ 1991 (of drugs used to treat hypertension) which appeared in the first volume of this series an enormous increase in understanding of sympathetic nerve function, and of drugs to modify it, has occurred. That review could only mention guanethidine briefly as the drug was thennew but at about the same time another drug, bretylium, became available. Although bretylium is no longer used in therapy it stimulated a process of re-appraisal of knowledge of sympathetic nerve physiology, from which radical new theories emerged [200] . This momentum has been maintained as still more postganglionic sympathetic neurone-blockers continue to appear. It now seems clear that stores of catecholamines in postganglionic sympathetic nerve pre-synaptic vesicles can, for the purposes of the present disassion, be divided into two types: (i) loosely bound transmitter readily releasable by tyramine and other indirectly-acting sympathomimetics [201-71, (ii) more firmly bound material only released (with depletion of stores) by reserpine, guanethidine, etc [208-2121 .

Drugs used in the treatment of hypertension vary greatly in their mode of interference with sympathetic nerve function. In the case of methyldopa the mechanism is complex and still largely unknown. Others, such as guanoxan, guanochlor or bethanidine, involve varying degrees of, for example catecholamine depletion, blockade of subsequent release of noradrenaline and, occasionally, a weak receptor-blockade. Bethanidine is perhaps the best-known of this series and

is now otten preferred to guanethidine. Debrisoquine (XVII, Declinax, Ro 5-3307/ 1) is a very close chemical relative of bethanidine (XVIII) as will be seen

28

SOME RECENTLY INTRODUCED DRUGS

from their structures. They are also almost identical pharmacologically although, as has been pointed out in a recent review [213] this fact is surprisingly rarely mentioned. The synthesis [214] of debrisoquine, its cardiovascular effects (in animal preparations), acute toxicity and biochemistry [20 11 and clinical pharmacology [215] have been reported (see also p. 167-168 of this volume). Like bethanidine, intravenous injection of a single dose of debrisoquine (in normal individuals) releases the ‘loosely-bound’ noradrenaline of the sympathetic neurones resulting in a brief pressor response [201, 21 51 . Clinically this could be dangerous in hypertensive crises or in patients suffering from phaeochromocytoma. Oral administration does not lead to this response since catecholamines are released more slowly and inactivated before their effects become evident. This pressor response t o injected drug is abolished by pre-treatment with reserpine (which depletes the transmitter stores), while the ability of tyramine to elicit pressor effects after pre-treatment with debrisoquine demonstrates that, unlike reserpine and guanethidine, debrisoquine does not greatly reduce the neuronal catecholamine stores. On the other hand, administration (intravenously) of noradrenaline t o debrisoquine-pretreated subjects leads t o an exaggerated pressor response indicating that debrisoquine effectively prevents noradrenaline release leading t o receptor ‘denervation supersensitivity’. The rationale and techniques of the above tests have been discussed [206]. It has been claimed [201] that this ability to block sympathetic function without major depletion of transmitter is a clinical advantage, especially in maintenance of adequate cardiac function. It is difficult t o see, however, how such a point can be valid since catecholamines ‘locked’ in storage can no more influence cardiac function than can the traces remaining after extensive depletion. Toxicity of debrisoquine is low. The ‘explosive’ diarrhoea and abdominal cramps commonly encountered during guanethidine therapy are rarely encountered. In this respect, again, bethanidine is similar. Unfortunately, there is little published information on detailed toxicology of debrisoquine (apart from acute toxicity, referred to above [201], which appears t o be very similar t o that of bethanidine and related drugs) though the manufacturers report negative findings in a variety of chronic toxicity studies in animals-with the exception of unexplained thyroiditis in a few dogs after one year’s high dosage. The same source reports debrisoquine to be free of endocrine, muscle relaxant, anticonvulsant or analgesic effects. Some evidence of C.N.S. stimulation and particularly of imipramine-like potentiation of amphetamine and cocaine is recorded, though this does not seem to have been noticed clinically. Haeniatological, renal, hepatic and thyroid functions have been reported normal [215-71 during debrisoquine therapy, nor could blood-sugar abnormalities be detected. Clinical reports [215-2221 agree that debrisoquine is an effective antihypertensive agent with a duration of action of 8-1 2 hours, permitting flexibility of dosage. Like other similar drugs it is potentiated by thiazide-type diuretics and other anthypertensives. In all these respects it appears to be interchangeable

A.

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29

with bethanidine so that patients intolerant t o the one may be transferred to the other.

An adrenergic a-receptor blocker

Thymoxamine

The theory, first proposed [ 2 2 3 ] in 1948, that responses to sympathetic stimulation are mediated through two types of receptor, a and 0 (permitting differing responses to a single transmitter) has, with various modifications, become accepted as a standard basis for discussion and study of sympathomimetic drugs and their antagonists. While fairly specific antagonists of 0-receptor effects have been developed (for example, pronethalol, propranolol), drugs available to block a-receptors (for example, dihydroergotaniine, phenoxybenzaniine, phentolamine, tolazoline) have lacked both potency and specificity. In addition some such drugs, especially the Iialoalkylamines (for example, phenoxybenzamine) exerted an irreversible effect by combining chemically with receptor material. Phentolamine has probably proved the most generally useful of the above; i t has, for example, been recommended a s routine antihypertensive treatment (given intravenously) in the acute pressor-crises of many M A 0 inhibitor-amine interactions and in the diagnosis of phaeochromocytoma. The drug is, however. of little value when given orally and is not specific for adrenergic a-receptors. Rather surprisingly, a much more specific, orally-active drug of this type has been available for at least 14 years. Largely unrecognized (for lack of detailed study) tliymoxamine (XIX, Arlytene, Carlytene, Moxisylyte. Opilon. Sympal) Me \

NMe2

has received sporadic use in Germany, over this period, as a ‘sympathicolytic’ for a variety of vascular disorders. Neither the original description [234] nor subsequent clinical recommendations (335A)j were sufficiently detailed t o indicate the mode ot‘ action of the drug, let alone its specificity. More recently the drug has been re-examined to elucidate its properties more thoroughly [23@1]. The first of these studies [230] demonstrated that thymoxamine satisfied the usual criteria [232-71 for competitive antagonism at a-receptor sites (against noradrenaline) but showed no 0-receptor blocking

30

SOME RECENTLY INTRODUCED DRUGS

activity (against adrenaline; guinea-pig auricle preparation) or anti-angiotensin or anti-serotonin activity. The potency, weight for weight, was judged to be intermediate between that of dihydroergotamine and piperoxan. Some an tihistaminic propertieswere detected but in this respect thymoxamine was of low potency when compared with mepyramine. The second study [ 2 3 1 ] , intended t o establish the quantitative aspects of thymoxamine a-blockade, compared it with phentolaniine. This paper is an example of the techniques required, and data evaluation problems encountered, during studies (in intact, concious animals) of drugs which produce a progressively lower baseline (in this instance, blood pressure). The first dose of either thymoxamine or phentolamine (into thejugular vein) resulted in a profound fall in blood pressure followed, in the case of thymoxamine, by a ‘rebound’ rise leading t o stabilization at below control level. After the complex dynamics of this initial adjustment subsequent admmistration of both drugs demonstrated a noradrenaline-antagonism with characteristics typical of competitive receptor block. A more general study [238] including postulated distributions of a,0 and myogenic receptors, also concluded that thymoxamine is a competitive antagonist of a-receptors of medium potency and with fewer other effects than any similar drug studied (tolazoline was found t o have the greatest variety of effects). The absence of 0-blockade and lack of effect on angiotensin or serotonin responses was confirmed. Clinical pharmacological work [ 2391 ,using plethysmography, showed that man responds t o thymoxamine as predicted from the animal work, and one such study [240] (again using plethysmography) challenged the widely held view that in occlusive arterial disease many non-atheromatous collateral vessels are always maximally dilated (before drug therapy) by demonstrating improvement in peripheral flow giving thymoxamine, bamethan, tolazoline and nicotinyl alcohol. Little effect on blood pressure has been observed after therapeutic doses of thymoxamine (orally) in man for treatment of Raynaud’s disease, labarinthine ischaemia (Meniere’s syndrome), various cyanotic states and excessively cold hands and feet. While its usefulness in these conditions is at least equal t o that of its predecessors, the role of thymoxamine as an adjunct t o orthopaedic and plastic surgery and in the diagnosis of phaeochromocytoma is still being investigated. Cutaneous blood flow and skin temperature have been shown t o be substantially increased (assessed against tolazoline) after application of a n ointment containing 10 per cent thymoxamine [240a]. Few toxic effects have emerged and such symptoms as diarrhoea, vertigo, headache and facial flushing are more likely t o be manifestations of profound a-blockade than of true toxicity. Since noradrenaline and thymoxamine compete for a-receptors overdosage with either drug can be countered by titrating the patient with the other. Thymoxamine is receiving increasingly wide attention as probably the most specific, non-toxic, a-receptor blocker currently available; many reports on experience with the drug are still awaiting publication.

A.

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31

Three phenylethylamines with complex sympatholytic or spasmolytic effects

[240bl

(a)

Prenylamine

The original communication [241] describing prenylaniine ( X X , Coiontin, Incoran, Irrorin, Reocorin, Synadrin 60, Segontin) directed attention primarily to the coronary vasodilating effects of the drug and its consequent cardiovascular activity and this application of the drug has tended t o dominate studies to date. From the chemical and pharmacological points of view however, the drug is of

Q / Me

CH2-CH

\

I

Ph

(XX)

considerable interest in possessing reserpine-like ability t o deplete catecholamines, dopamine and serotonin from neural stores, in addition t o some 0-receptor blocking activity. Prenylamine should be regarded as yet another compound of ‘sympathomimetic-based structure’ which, like bamethan, buphenine (nylidrin), verapamil and mebeverine, adds t o the sum of knowledge of structure-activity relationships (affecting both neuronal and receptor structures) within this group. Indeed, this theme can be taken further and attention drawn t o the common factor linking the potent 0-blocker propranolol and the a-blocker thymoxamine; both can be regarded as derivatives of 2-aryloxyethylamine. The ether linkage is, however, absent from the compounds at present under study. The ‘sympathomimetics’have been subjected t o a detailed study [ 2 4 2 ] determining their ability to prevent uptake (by rat heart) of noradrenaline, this being taken as a measure of the affinity of compounds under test for the transmitter transport system. Such basic information is often lacking, though important, since compounds with affinity for the transport systems, approximating t o or exceeding that of the transmitter itself, generally reduce the availability of the transmitter by permitting its diffusion from the neurone with subsequent exposure t o deactivating enzymes. Affinity for neuronal transmitter uptake systems does not, however, bear any obvious relationship t o affinity for either a- or 0-receptors; quite different stereochemical requirements seem t o apply in these situations. A further con-

32

SOME RECENTLY INTRODUCED DRUGS

sequence of inhibition, by many ‘sympathomimetics’,of noradrenaline recovery by the efferent neurone is that the small amount of transmitter released by tyramine for instance may be greatly potentiated because of its longer period of availability to receptors. In the above study prenylamine was found to have greater affinity for the transport system than for example (-)-adrenaline or phenylethylamine though less than tyramine or (-Fnoradrenaline. Early studies, using rats [243] and golden hamsters [244] indicated that prenylamine reduced the noradrenaline content of brain and heart (rats) and of adrenal catecholamines in the hamster. These studies inspired more detailed work and it was subsequently demonstrated [245j that the compound possessed weak reserpine-like activity in reducing concentrations of noradrenaline, dopamine and (to a lesser degree)serotonin concentrations in rat brain. These workers inclined to the view that the amine release from adrenal medulla (observed by them and others [243, 2441 was an artefact resulting from tissue damage since irreversible non-responsiveness to dimethylphenylpiperazinium stimulation occurred. Further study [246] of brain monoamine metabolism confirmed the close similarity between the effects of reserpine and those of prenylamine (except that the latter is generally less potent) and produced evidence that these two drugs compete for the same sites of action. Peripheral reserpinelike effects have been observed in man [247-81 and, as noted below, the possibility of central reserpinelike activity (culminating in suicidal depression) should always be borne in mind during use of drugs of this type. Preliminary studies [241, 249, 2501 of the cardiovascular and sympatholytic properties of prenylamine demonstrated that coronary blood flow and oxygenation could be increased under experimental conditions (in dogs) and that the drug interacted in complex fashion with sympathetically innervated organs, but the picture presented was somewhat confused because of the many uncontrolled variables and limitations of the actual techniques used. Anti-arrhythmic activity of potency comparable with that of quinidine, plus local anaesthetic properties, were also demonstrated [25 11 but the same worker was not able to reproduce these effects in intact live animals with any consistency. Large doses of the drug actually provoked cardiac fibrillation in some cases. A more thorough study [252] showed that prenylamine consistently potentiated injected noradrenaline and adrenaline but reversed the hypotensive effect of isoprenaline to a pressor one-due, according to further studies in phentolamine-prernedicated rabbits and guinea-pigs, to the unmasking of a-receptor response to isoprenaline. As is implicit in this result, prenylamine demonstrated(as in other tests also) &receptor blockade which may be of clinical consequence. The potentiation of adrenaline and noradrenaline noted above appears to be specific to these compounds and related to the amine-depleting effects of prenylamine. The pressor response to tyramine is reduced by prenylamine pre-treatment indicating that the readily releasable pool of catecholamines (discussed under debrisoquine) is concerned. The potentiation of administered adrenaline and noradrenaline is largely attributable t o inhibition

‘

A.

P. LAUNCHBURY

33

of their incorporation into sympathetic neurone-stores. Study of the effect of prenylamine on sympathetic-neurone function has shown [253] that, in vitro, response to nerve stimulation is reduced by the drug but that this impairment can be reversed by addition of noradrenaline or dopamine (though function gradually declines with time). This is what would be expected of a drug which impairs the ability of neurones to store transmitter (as does reserpine, for example). In vivo studies failed to demonstrate consistent sympathetic neurone blockade though ‘releasable’ amine depletion occurred. Intact animals do, of course, present complicating, compensatory, difficulties t o such studies; such factors also complicate clinical research. At the other end of the scale of complexity the catecholamine storage granules of adrenal medulla have been separated and used [254-6] to study the effects of several amines on ability of the granules to take up and retain transmitter. From these results it was concluded that not only was prenylamine an extremely potent catecholaminereleaser but that its ability to inhibit catecholamine uptake was second only to that of reserpine. Laboratory evaluation [241] of acute and chronic toxicity of prenylamine indicates that, in high doses, convulsions accompanied by respiratory paralysis (often with pulmonary oedema) led to death. Doses inadequate to produce this result led only to phenomena characteristic of reserpine-like drugs. Chronic administration failed to produce recognizable changes in any organs or tissues studied. No toxic effects, unattributable to amine depletion, have appeared during several years’ clinical use. Prenylamine has been employed clinically mainly for the treatment of angina pectoris. The problems of evaluating effectiveness of drug therapy of this disease (with its psychic and vascular components) are freely discussed in two reports 1257-81 ;the benefit likely t o be observed with any medication (active or otherwise) is considered with the general conclusion that prenylamine is at least as effective as other drugs used in angina in reducing incapacitation and psychic stress. No objective improvement in the disease could be demonstrated in these and other studies [259, 2601 though these also reported the drug as beneficial in management of the disease. An interesting discussion of the probable mechanism of prenylamine’s relief of anginal symptoms proposes [252] that the coronary vasodilating activity is of minor importance. It is pointed out that marked increases in blood catecholamine levels have been observed in anginal patients after exercise and it is postulated that uptake of these agents by the myocardium increases its oxygen demand. Hypoxia resulting from this could well account for the pain experienced and such a mechanism is in accord with the finding that 0-blockers (for example propranolol) can provide relief. The observed inhibition of catecholamine uptake by prenylamine coupled with its moderate 0-blocking activity provide some basis for an explanation of its apparent effectiveness. In addition, the amine depleting effects (which also involve the myocardium) and central sedative properties (agnin due to amine depletion) must be contributory. A final note of caution may be based on the observation [260] of reserpine-

SOME RECENTLY INTRODUCED DRUGS

34

like side effects in some patients. A drug with such pharmacological similarities to reserpine could presumably produce a reserpine-like depression which might involve suicidal intent. While this has not yet been reported the psychology of angina patients is so complex that it may not be readily detected until well advanced. (b)

Verapamil

Though the drug has now been used to treat a considerable number of patients suffering from angina pectoris, verapamil (XXI, iproveratril, Isoptin, Cordilox)

CH, - CH,

\N-Me

suffers from a comparative paucity of published detail to define its properties. Verapamil was initially described [261-71 as a ‘benign’ coronary vasodilator, a specific cardiac sympatholytic, smooth muscle relaxant and anti-arrhythmic agent. Other workers [268] also claimed evidence of myocardial symparnetic blockade, of quinidine-like anti-axrhythmic effects [269] and freedom from t k occasional complication of coronary vasoconstriction attendant upon the use of potent &blockers (for example, propranolol) [270] . Particular emphasis has been laid on the claim that verapamil is a sympathetic &receptor blocker, but with activity of a low order so that the risks inherent in the use of potent agents of thb type (for example, propranolol) do not arise. The coronary vasodilating activity has not, as is now general practice, received stress. The view that the drug has indeed p-blocking activity has been strongly challenged [27 1] and t h i s aspect‘ will undoubtedly require further study. Clinically, the drug has been studied (for the most part) in poorly controlled trials with few precautions against subjective bias [272-4]. Two careful doubleblind assessmentshave however been reported, one [275] measuring reduction of weekly consumption of glyceryl trinitrate. The other [276], by far the most informative published to date, demonstrates the drug’s ability to increase the amount of exercise necessary to provoke an@ attacks,as well as its effectiveness in reducing the need for nitrate consumption. Clinically, this study failed to show any difference between propranolol and verapamil (in appropriate doses) and, in

A. P. LAUNCHBURY

35

discussing their findings, the authors attribute the beneficial effects of the drug in angina to a direct suppression of myocardial contractility consequent upon its quinidine-like properties. No details of toxicity appear in the literature; in clinical trials the only recorded adverse effects have been minor (nausea, dizziness etc.). It is amazing that verapamil, with an apparently interesting spectrum of activity and a chemical structure indicative of potentially wide-ranging effects, should have as yet received so little detailed published study. (c)

Mebeverine

The well known properties of reserpine (already reviewed in this series [277]) can be radically modified by reducing the molecule to fragments and then substituting in a variety of ways. A preliminary report [278] of work on these lines indicated that derivatives of 0-indolylethylamine generally exhibited reserpine-like activity while the related 8-phenylethylamines could be grouped into two series: (i) papaverine-like and atropine-like compopnds generally producing bradycardia and/or spasmolysis, (ii) adrenolytic C.N.S.depressants. The literature relative to this work was then reviewed [279] and the syvtheses and physical data on the compounds of these series published [280]. A more detailed examination of 71 phenylethylamines, clarifying the structural features required to produce shifts ofemphasis towards different types ofactivityhas followed [281 J ..It is clear from this work that spasmolytics with structure-activity relationships unusual among aminoakyl esters occur in this group, which is also structurally and pharmacologically a far cry from the original reserpine which inspired the work. From these esters, mebeverine (XXII, Colofac, Duspatalin, Duphaspasmin) was selected for

CGz CH2 I

,cH2-o~co~~--~Me

'C",

detailed pharmacological study. This showed [282] mebeverine to be some three times as potent as papaverine (against barium chloride induced spasm in isolated

36

SOME RECENTLY INTRODUCED DRUGS

guinea-pig ileum) and up to forty times as potent in relaxing the same organ in vivo. Peristalsis generally remained unaffected even when spasm was abolished; only very high doses caused intestinal paralysis. The same study examined the effect of mebeverine on the sphincter of Oddi (measured by bile flow and by muscle tone) and found a similar high activity (20-40 times that of papaverine) in this situation. The absorption (following administration by oral, intramuscular and intravenous routes), distribution and elimination of the drug has been studied by radiotracer techniques in rats and rabbits [283, 2841. The substituted benzoic acid was labelled with I 4 C and the alkanolamine remnant with 3H.Almost all radioactivity was recovered from the urine even after oral administration. Clinical pharmacological examination 12851 of mebeverine has utilized micro-balloon (for distal colon) and radio-'pill' (for small intestine) techniques. Radical reduction in colonic hypermotility (with persistence of a relatively normal peristalsis) without significant effect on small intestine function followed parenteral mebeverine (50 mg). The study then proceeded to a double blind trial, assessed by sequential analysis of objective criteria (frequency of defaecation, cramps). Mebeverine very soon demonstrated activity compared with placebo. Other studies have compared the drug with various anticholinergics and attempted assessment of its potential in paediatrics. While these confirm the lack of side effects and indicate relief of colonic spasm, little further light is shed on mechanisms of action or on potency-ratios against standard drugs in the clinical situation. Acute toxicity studies in mice reported by the manufacturers indicate a very low toxicity though further details on the reason for the unusually large difference between oral and intravenous LDS values (some 40-fold) would be interesting. Subchronic toxicity tests with oral and parenteral use of the drug in rats again indicate a low potential toxicity; no abnormalities were detected. Teratogenic activity could not be demonstrated in rats or rabbits; only limited experience of the drug in pregnant women is available but no complications have yet arisen. The structure of mebeverine suggests that investigation of its activity at sub-cellular levels (especially in relation to possible interactions involving catecholamines or serotonin) might be rewarding. It would also be of value to have details of activity (or lack of it) in organs and tissues other than the gut. A diuretic Ethacrynic acid (with notes on frusemide)

Knowledge of renal physiology, and to some extent of its modification by diuretics, has advanced considerably since the subject was reviewed in a previous contribution to this series [286]. Other work quoted [287] at that time strongly suggested that thiazides and diuretics of similar effectiveness exerted their primary

A.

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37

effect on sodium reabsorption at the level of the proximal tubule. It is now clear that this is not so and a recent thorough review [288] of diuretics and renal function has concluded that drugs of the thiazide-type (and also chlorthalidone, chlorexolone, quinethazone and clopamide) act on the distal convoluted tubule of the nephron, probably at a point proximal to the exchange-site responsive to aldosterone. It has also become more evident that the loop of Henle is of major importance in the regulation of sodium and water balance, and in agreement with this, both frusemide (furosemide, fursemide, Lasilix, Lasix) and ethacrynic acid (XXIII

(XXIII)

Edecrin, Edecril, Hydromedin, Endecril, MK-595)-the most effective diuretics known (in terms of total sodium, chloride and water loss they can induce)-have been shown to exert most of their effects on this portion of the nephron. The main properties of the loop of Henle may be summarized by the statement that thisvery long structure, consisting of a descending limb and an ascending portion (linked by a ‘hairpin bend’) in close proximity to each other, exhibits the properties of a countercurrent multiplier system. Anatomically, the loops of various nephrons are of differing lengths and penetrate various distances into the renal medulla from the glomerular regions of the cortex but, in man, at least 250.000 (a quarter of the total per kidney) penetiate right down to the medullary papillae. This region of the renal medulla is strongly hypertonic relative to plasma (5-10 times the osmotic pressure of plasma, according to species and circumstances) as a direct result of the activity of the loops and the very limited rate of solute removal by blood vessels (which themselves function as countercurrent exchangers through following closely the loops of Henle). As a result, tubular fluid entering the descending portion of the loop is progressively concentrated (mainly by simple osmotic transfer of water from the lumen to the hypertonic interstitial fluid). As the fluid passes up the ascen$ng limb, sodium is actively reabsorbed(followed, of course, by chloride) but since this section of the nephron is impermeable to water the tubular fluid becomes progressively more dilute. This is an efficient arrangement since the one sodium transport region is thereby responsible for some of the diluting and the whole of the concentrating activity of the nephron. The development of the countercurrent concept of loop function [289-29 11 and its confirmation and discussion [292-41 have been summarized 129.5, 2961. One or two further points require mention before ethacrynic acid can be discussed in context. The ascending limb of Henle’s loop has the unusual property of reabsorbing a constant proportion (approximately two-thirds) of the sodium content of tubular fluid, regardless of the actual load involved [2971 . This

38

SOME RECENTLY INTRODUCED DRUGS

implies that the loop has an important role in sodium conservation in that if an unusually heavy sodium load escapes proximal tubular reabsorption, this load would be radically cut at the loop. Further, in contrast to distal tubule capability, it has not been found possible to saturate the sodium transport mechanisms of the ascending limb of the loop [298, 2991. Even with heavy sodium loading, fluid leaving the ascending limb remains at the hypotonicity characteristic of the nondiuretic state [297-3001. No single postulated mechanism satisfactorily accounts either for the activity of the loop or for its control. Since the moderately active diuretics (thiazide type) apparently exert all their effects upon the distal tubule, and .ethacrynic acid and frusemide may well have subsidiary effects at this level, brief reference to this structure is necessary. In the absence of ADH (antidiuretic hormone, vasopressin) the walls of the distal convoluted tubule and of the collecting tubules are relatively impermeable to water. At a point close to the junction with the ascending limb of Henle's loop there appears to be a length of distal tubule capable of considerable sodium reabsorption; this may be so rapid that not only is the tubular fluid further diluted but the steepest transtubular sodium gradients obtainable in the entire nephron may be set up [301]. However, the transport mechanism is quickly saturated by heavy sodium load [297] with the result that, as more and more sodium is presented to the proximal portion of the distal tubule, the proportion escaping reahsorption increases. After traversing this region the tubular fluid encounters the section of distal tubule which, in the presence of aldosterone, exchanges sodium for potassium (or H" ). This aldosterone-mediated secretion of potassium (and =absorption of sodium) accounts for much of the potassium loss, and its unpredictability, occurring during diuretic therapy. Since this is an exchange procedure the total potassium loss obviously depends upon an adequate tubular sodium load at this site-this is provided by diuretics. The effects of aldosterone can. be reversed by appropriate drugs (of at least two types, for example spironolactone and triamterene) but this is outside the scope of the uresent discussion. So too, beyond mere mention, is the action of ADH in rendering the distal and collecting tubular walls more permeable to water; as the tubular fluid traverses the increasingly hypertonic medullary regions, so final adjustment of urinary concentration occurs in proportion to the level, of circulating ADH. Drugs acting only on the distal tubule (however complete the resulting block of sodium resorption at that site) are limited, in the effect obtainable, by the comparatively small contribution to total sodium reclamation made by this portion of the nephron. For example, even at their activity peak, thiazide diuretics cause the elimination of only 8 per cent of the total filtered sodium [302,303] Very much higher proportions of filtered sodium would be expected to contribute to diuresis if the mechanisms of the loops of Henle could be blocked (this has, in fact, proved to be the case in that frusemide and ethacrynic acid are both capable of clearing up to 20 per cent of filtered sodium [302,304-6]).

A. P. LAUNCHBURY

39

It has long seemed reasonable to suppose that the mercurial diuretics achieved their intense action by reaction with protein-bound sulphydryl groups in renal tubular cells and this hypothesis has received experimental support [307]. In the search for diuretics more effective than thiazides, but less toxic than mercurials, it is therefore understandable that attention should be directed to organic compounds with high reactivity to sulphydryl groups. On this basis a series of a, 0-unsaturated ketone derivatives of aryloxyacetic acids was synthesized, screened for diuretic activity and reported [308] .From this group ethacrynic acid was selected, on the basis of structure-activity relationships initially only briefly outlined [308] but subsequently developed [309-3111. These reports indicated that ethacrynic acid had an effectiveness consistent with that predicted by the original concept of sulphydryl groups as essential for the major part of the nephron’s sodium reabsorbing function; the relationship between affinity for proteinaceous thiol groups and diuretic effectiveness then received further study. Several reports link these properties in the case of ethacryric acid [3 12-41 ,though other workers [ 3 151 believe inhibition of an adenosine triphosphatase (implicated in membrane ion-pump mechanisms) to be a more likely explanation of the drug’s activity-especially as mercurial diuretics have also been found to inhibit the enzyme [316]. It has, however, been stressed [288] that these isolated biochemical effects cannot be directly related to the diuretic activity of any given drug, and until more is known of the fundamentals of ion transport such mechanisms will remain hypothetical. Ethacrynic acid and frusemide are now widely used as the most effective diuretics available; both drugs are capable of producing a diuresis frequently described as ‘torrential’. Both may be given orally or parenterally according to need. Unlike previously available drugs these powerful agents are equally effective in acidosis or alkalosis [3 141 and hyponatraemia, hypochloraemia and changes in available potassium modify the diuresis but little [317-9] . This intense activity can, of course, be dangerous and caution to avoid potentially fatal hypovolaemia and electrolyte disturbance has been urged [320]. Further, while thiazides and mercurials become increasingly ineffective with falling glomerular filtration rate, this does not appear to be a factor with the potent agents at present under discussion [318, 3211. There seems little doubt that studies with ethacrynic acid have established that this astonishing potency is aspociated with abolition (or extreme reduction) of the urinary concentrating function of the loop of Henle [305] ;some [322] would go further and argue that the magnitude of the response indicates additional sites of action-probably distal to the loop. A recent communciation [323] suggests that ethacrynic acid may modify the course (for the better) of ischaemic acute renal failure; this is in line with other recent findings indicating that the drug increases renal cortical blood flow (while reducing medullary flow) [324] and that the sodium content of the renal medullary interstitial fluid influences afferent arterial flow to the kidney [325]. A point which does not seem to have attracted much attention is the reported increase in effect of ethacrynic acid if a thiazide has been given the previous day [326] -such

40

SOME RECENTLY INTRODUCED DRUGS

a ‘carry over’ could account for some of the occasionally reported unpredictability . Toxicity of the drug appears to be low in the sense that although the haemoconcentration, electrolyte disturbance, etc., possible during therapy with so potent a drug can of themselves be lethal, prolonged studies using animals given compensating fluids and electrolytes have revealed no evidence of hepatic, renal or haemopoietic toxicity. Clinicallv, most unwanted effects (for example, transient slight uraemia, hyperuricaemia, hypotension, haemoconcentration leading to occasional thromboembolism) are familiar results of intensive diuresis. A few cases of profuse watery diarrhoea have occurred [320] and the report suggests that in such instances the drug should be permanently withdrawn. No evidence of teratogenicity has appeared in animal tests covering three generations; clinical use in pregnancy has likewise failed to provide hint of such potential though it should, naturally, be borne in mind. Although the cellular basis for the action of ethacrynic acid may not yet be established its micro-anatomical effects on the nephron are well attested. The speed of onset and intensity of action make the drug (like frusemide) a valuable addition to existing compounds, especially where diuresis is urgently needed (for example, pulmonary oedema) or in patients resistant to milder drugs of the thiazide type.

Addendum on ethacrynic acid and frusemide

Since the major part of this section was written, evidence has been published [326a] demonstrating that the above diuretics are possibly even more diabetogenic than the thiazides (judged by inhibition of 14C-labelled glucose utilization in adipose tissue), with a mechanism of action probably similar to that of the earlier drugs. This work seems more enlightening on this controversial point than any hitherto presented.

Two hypotensivediuretics (a)

Chlorexolone

The thiazide diuretics possess antihypertensive properties, in part consequent upon electrolyte and plasma-volume changes but mainly resulting from a direct cardiovascular depressant effect. This is clearly illustrated by the non-diuretic thiazide, diazoxide, which is an effective hypotensive [326b, c ] . It is not therefore, surprising that both these properties should be found (in varying proportions) in other, structurally related, compounds. One particular line of research, aimed at modification of the thiazide heterocycle (the o-chlorobenzenesulphonamide moiety was untouched as it was believed essential to activity-the subsequent advent of ethacrynic acid questions this belief) examined first the corresponding

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41

thiatriazines [327] . Although some diuretic activity could be demonstrated, potency was low and some of the compounds chemically unstable. Further manipulation of the ring to give a range of basic structures (all involving replacement of the endocyclic sulphonamide group) indicated that, of the compounds studied, the isoindolines showed the greatest promise. From a further series chlorexolone (XXIV, Nephrolan) was selected as the most active (as a diuretic), subjected to preliminary pharmacological study, and the results briefly communicated [328] . Detailed synthetic methods and physical properties, with structure activity studies, were later reported in detail [329]. It is interesting, though not unexpected, that the corresponding 3-0x0 compound proved to be inactive. Pharmacological studies showed its carbonic anhydrase inhibiting activity to be weak and indicated that chlorexolone exerted its diuretic action upon the same sodium re-absorbing mechanism as did the thiazides (see above, under ethacrynic acid). Against this background it is understandable that the structural variants represented by the clinically useful compounds (Figure 1.I ) based on the original o-halobenzenesulphonamide, should show approximately the same limitation of peak effect. The outstanding exception is frusemide, which parallels ethacrynic acid in effectiveness, though bearing a close chemical resemblance to the earlier drugs. The elucidation of the mechanism of this unexpected extension of activity to include the loop of Henle should he fascinating. (So too, would be an explanation of the diabetogenic liability ot the above chemical types, especially of diazoxide (XXV, Figure 1.1) in view of their structural similarity t o the hypoglycaemic sulphonylureas, for example, chlorpropamide (XXVI); is an antihypertensive, diuretic, hypoglycaemic drug possible?). The hypotensive effect of chlorexolone was initially studied in the concious, normotensive dog (anaesthesia introduces complications into the experiment, asdiscussedby the authors) and compared with that of hydrochlorothiazide [330]. Both drugs produced a fall in blood pressure, with bradycardia (connection with the hypotension uncertain) and the authors suggest that their findings, compared with the lack of published success with anaesthetized animals [33 1-3331 , indicate that trained, conscious dogs may provide a sensitive means of detecting blood pressure changes induced by drugs-especially of this general type. The metabolism of chlorexolone to three monoh’ydroxylated derivatives (all apparently substituted in the cyclohexane ring) in both dog and man has been reported [334]. No storage in body tissues could be detected and elimination via the kidney appeared to follow oxidation to the polar metabolites (no unchanged drug could be recovered from urine). These findings contrast with those relating to thiazides which often appear unchanged in urine. Acareful study of the human and clinical pharmacology of chlorexolone [335] showed the drug to be capable of lowering the blood pressure of hypertensive patients at doses well below those required for diuresis. The duration of action appeared to be up to 48 hours, which some [336] consider a disadvantage

02

C hloro t h i az i de

Diazoxide

Hyd r oc hlorot hi az ide (substituted at position 3 t o give most thiazides i n current use)

(XXV)

&net

hazone

Chlorexolone

(XXIV)

Clopamide

Frusemide

(XXVII)

Chlort halidone

C hlor propam i de

(XXVI) Figure 1.1.

A. P. LAUNCHBURY

43

(especially because of sleep disturbance) though sub-diuretic, antihypertensive doses would seem free from this objection. Toxicity studies in animals have failed to show evidence of damage in any tissue examined after 3 months' administration to rats of daily doses up to 20 000 times that required to produce diuresis. Similar absence of effect on all vital organswas found in dogs and rhesus monkeys. No teratogenesis or impairment of fertility was detected. Very large doses (up to 10 000 mg/kg) failed to produce ill-effects in acute toxicity tests on rats, though intravenous LDS0 values were in the region of 230 mg/kg. Chlorexolone is thus another effective oral diuretic which may also form the basis of antihypertensive therapy. While many cases appear adequately controlled onchlorexolone alone [336] more severely ill patientsmay require its potentiation with specific antihypertensive drugs. (b)

Clopamide

This drug bears some formal chemical resemblance to the benzothiatriazines referred to above [327] in that a nitrogen atom occupies the position correwonding to C3 in the thiazide series. (XXVll, Figure I. 1 ) Clopamide (Aquex, Brinaldix) has obvious features in common with quinethazorie and chlorexolone for example and can be regarded either as a piperidinocarboxamide or a substituted carboxylic hydrazide of the familiar o-chlorobenzenesulphonamide. The series of compounds from which clopamide was selected has been described [337] and subjected to the usual evaluative tests to define properties and indicate the substances most clearly meriting development [338] . Detailed pharmacological study I3391 indicates that clopamide resembles the thiazides and their other analogues closely. For instance, the site of action appears to be identical with that of the thiazides (combination of maximum dose of clopamide with chlorothiazide failed to produce increase in diureses, though this was achieved by combination with spironolactone), there is no effect upon renal plasma flow, glomerular filtration rate and no carbonic anhydrase inhibition. An important qualitative difference is an apparently equal effectiveness in acidosis or alkalosis; the output of sodium and chloride can, in addition, be up to 30 per cent more than that achieved by thiazides-perhaps indicating' firmer binding to tubular sites. The effects of a single dose of clopamide can persist for up to 48 hours [340] and the hypertensive patients show up to 2 0 mmHg fall in diastolic pres sure [341]. The drug may be at least as diabetogenic as the thiazides [342-41 though further study is needed on this point. It is obviously not nearly as potent as frusemide and ethacrynic acid and is therefore unlikely to have any action on the loop of Henle. It is, however, a clinically useful diuretic [345-81 even though some feel that its long duration of action is a disadvantage and see no reason for preferring it to the thiazides [349]. Adverse or toxic effects are those associated with effective thiazide-type

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SOME RECENTLY INTRODUCED DRUGS

diuretics. Acute toxicity tests in animals show it t o be of low toxicity, and chronic toxicity tests in dogs failed to show any detectable change in vital organs or any teratogenicity in rats or rabbits [339]. A mucoid-secretion modifying agent Bromhexine

It was recently observed that a plant,Adhutodu vusicu,long used in Indian herbal medicine (the source of many therapeutic leads) for the relief of cough and respiratory tract congestion, did appear to reduce the quantity and viscosity of sputum. The plant was studied and its active alkaloid named vasicine. The tricyclic quinazoline structure (XXVIII) was then reduced t o fragments and a series of derivatives prepared [350]. From these bromhexine (XXIX, Na 274, Bisolvon)

was selected [351] as the most active. As will be observed, its structure is but a dim reflection of that of the alkaloid from which it arose. The pharmacology of bromhexine is unusual and as yet incompletely known. The property which has received most attention is its apparent ability to disorganize radically the structure of viscid sputum. It has been shown 1352) that the adhesive, viscous, mucoid bronchial secretion characteristic of chronic bronchitis for example, derives these properties from a fibrous structure hitherto largely unsuspected. After staining with Toluidine Blue and observation by means of polarized light such sputum is seen to consist of a mass of interlacing fibres embedded in a gel-like matrix. These fibrous structures consist largely of acidic mucopolysaccharides, except when infection is prominent in the disease state when DNA becomes the dominant ‘fibrogen’. T’he importance of pathologicallyinduced sputum production in providing infection ‘reservoirs’, in reducing ventilation (especially of smaller airways) and in other ways contributing to irreversible lung damage is now recognized [353] . The effect of administration of bromhexine to patients suffering from excessive production of viscous sputum (due to a variety of pulmonary disorders) has now been. studied and reported [354] . It is assumed by the authors that the observed effects were directly due t o bromhexine though the possibility of activity via a metabolite seems worthy of study. During the study the viscosity of sputum and its fibrous structure were noted. Within five days of commencement of oral administration of bromhexine the fibrous structure was seen to be

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markedly disrupted, with reduction in the viscosity of the material. After 10 days only very small, dispersed fragments of fibrous material could be found but on cessation of treatment sputum viscosity rose rapidly and the ordered fibrous structure reappeared. The volume of sputum produced appeared to increase for the first few days of treatniznt and t o fall thereafter though this increase may be more apparent than real-previously immobile sputum would be expectorated during this early phase. The authors discuss their findings in relation to earlier, less detailed studies and suggested modes of action of the drug. Other workers have demonstrated improved lung function (including arterial oxygen level increase [355]) and increased response to bronchodilators [356] after bromhexine. Some evidence of weak activity against tubercle bacillus has appeared [357] ;this could complicate culture control of anti-tubercular therapy. An interesting report [54] of oxytetracycline levels in bronchial secretions has provided evidence that the drug often fails to reach therapeutic concentrations in sputum but that concurrent administration of bromhexine greatly increases its levels in such exudate. This property has been briefly alluded t o above in connection with penetration of doxycycline into sinus secretions. The authors [54] attribute this higher antibiotic concentration in sputum t o increased capillary permeability, without quoting authority. Toxicity of bromhexine appears to be low; the drug is accordingly recommended for long-term treatment of chronic bronchitis, for instance. Very occasionally a slight rise in serum transaminase levels is found early in treatment but as this is transient, even in patients with hepatic disease, its significance is doubtful. The drug can irritate the gastro-intestinal tract (and is therefore likely to aggravate gastric ulcer). This is interesting since earlier expectorants were often believed t o act through reflex secretion of watery bronchial mucus following gastric irritation; this cannot, however, be accepted as the primary mode of action of bromhexine which seems to be much more fundamental. Bromhexine is unlikely to exert its effects only on bronchial mucus; information regarding its activity in other organs and secretions involving acid mucopolysaccharides is awaited with interest. If its ability to enhance the diffusion of antibiotics (and possibly other drugs) is substantiated, a very wide scope for the use of bromhexine would be opened up. A pulmonary anti-allergic for asthma therapy Cromoglycic acid

The aetiology of asthma remains obscure but at least one type, extrinsic asthma, appears closely linked to allergy and provoking agents capable of initiating Type I reactions (involving reagin antibodies) can often be identified. This type often becomes evident early in life and typical allergens include pollens, cat hair, etc. Another common form of asthma is the socalled intrinsic type, often of late

46

SOME RECENTLY INTRODIJCED DRUGS

onset, in which provoking allergens can rarely be identified. Recipitin antibodies may be associated with these episodes; some unfortunates possess antibodies of both reagin and precipitin types. Reagin-mediated, extrinsic asthma tic episodes are mediated by substances such as histamine, serotonin and SRS-A (slow-reacting substance of anaphylaxis), and therapy often aims at modifying the responses to these products of antigen-antibody interaction. Cromoglycic acid interferes with the reaction mechanism apparently by preventing the release of histamine, and SRS-A, even though antigen-antibody association occurs. Chromones have found uses in medicine for many years and it is possible to see in cromoglycic acid (XXX, Intal, FPL 670) the end-product of a long series

including among its forerunners khellin and methylchromone. The observation that reaction t o inhaled antigen could be suppressed by chromones lacking bronchodlator (or vasodilator) properties provided the lead t o the bischromones. The structure, general pharmacology and toxicity of disodium cromoglycate have been the subject of a briefcommunication [358] . This indicated that cromoglycic acid is not a bronchodilator, or an anti-inflammatory agent, nor will it modify tissue reaction to injected histamine, serotonin, bradykinin or SRS-A. It does, however, modify or abolish reaction t o allergens in previously sensitized animals provided the drug i s administered prior t o the antigenic challenge. This was shown to involve inhibition of histamine, etc., release from mast (and probably other) cells as indicated above; the drug does not appear t o modify in any way antibody production. Very few general pharmacological properties could be found; minor cardiovascular effects (varying in character with species) followed large intravenous doses. Poor absorption from the gut led t o the development of a specially designed inha1er;most of the dose so administered reaches only the larger airways and the small quantity reaching peripheral lung was rapidly absorbed and excreted, largely unchanged, in urine and bile. Toxicity tests reported in the same communication failed to provide evidence of any effects of note, except at very high concentrations (in v i m ) or doses (in vivo). In a field as complex as that involving the treatment of asthma it is not surprising that the initial published clinical work [359] should have come in for more than the usual criticism and controversy. This reception is perhaps even more understandable in view of the fact that the trial material contained isoprenaline (to abolish non-specific bronchospasm due to the dry powder itself), that the

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value of objective spirometry (in particular, measurement of FEV, changes) as an indicator of clinical improvement was challenged, and that the mode of action claimed for the drug was novel. The initial publication was sharply criticized by other workers [360] who had reached diametrically opposed conclusions from their own studies and these, in turn, were examined and points of difference clarified by the original authors [36 1 ] . This exchange reveals the clinical evaluation of a new aid to asthma therapy a too complex for easy resolution of differences of approach and view. Two cautious, but hopeful, reviews [362,362a] concluded, from evidence then to hand that the currently available preparation of disodium cromoglycate with isoprenaline could benefit patients with allergic asthma, but both expressed the hope that results from cromoglycic acid without isoprenaline would soon clarify the position. Then came publication of detailed work on the involvement of cromoglycic acid in both reagin-(Type I) and precipitin-(Type Ill) mediated reactions to inhaled allergens [363]. Concurrently, the clinical status of the drug was re-reviewed [364] in a thorough discussion of the pathological background; the conclusion was the cromoglycic acid ‘represents a new phase of treatment’. Other clinical reports followed in quick succession [365-71 , all (including additional correspondents [368] ) o f the opinion that the drug showed considerable promise. The sole dissenters (the original critics) remained, however, unimpressed [ 3691 though their criticisms seem to have been fairly adequately answered [370]. In one study [367] a marked inhibiting effect on exercise-induced asthma was actually recorded as objective improvement in FEV, after exertion. Two reports [371, 3721 of trials using disodium chromoglycate without isoprenaline confirmed that the drug possessed the beneficial properties claimed and that the dry powder could, in the absence of isoprenaline, give rise to transient pulmonary irritation. The present position appears to be that the drug has been shown to inhibit release of allergic reaction tissue-mediators, that this probably accounts for its clinical effects, and that the drug is therefore primarily a prophylactic one. Corticosteroid dosage can be reduced, with precautions, during cromoglycate therapy which also often reduces bronchodilator consumption. The drug is not of use during an acute asthmatic attack and is of,limited value in late-onset intrinsic asthma. It is proving a valuable tool in the elucidation of sensitization and reaction mechanisms. A terpenoid ester for gastroduodenal ulcer Gefarnate

In spite of the extensive and time-honoured use of anticholinergics and antacids, the only agent hitherto shown t o be of significant value in promoting healing of

48

SOME RECENTLY INTRODUCED DRUGS

gastric and duodenal ulcers has been the triterpenoid, carbenoxalone. The drug has, however, sideeffects (including sodium and water retention); its properties and place in therapeutics have recently been thoroughly reviewed in a symposium [373].

In the search for new compounds with healing potential in gastro-duodenal ulcerative conditions, attention has been given to sporadic reports of weak variable activity of this type in many vegetables, notably in white-headed cabbage. A useful account of these studies and of the steps leading t o gefarnate (XXXI, Gefarnil, Gefarnyl) has appeared in the form of a historical review [374].

No active material was isolated from any plant though crude extracts did give evidence of activity from time to time. This activity was traced to the liposoluble fraction of the extracts and its disappearance during further processing was taken to mean that many weakly-active substances together probably provided the necessary potency (in crude preparations) which could not be maintained by separated, individual substances. Various tenuous threads of evidence suggested that Vitamin K-like compounds might be involved (though the actual vitamins proved inactive), and the terpenic (farnesylic and phytylic) side-chains were examined. Although phytol proved inactive farnesol showed promise; like the crude vegetable extracts it was demonstrably active given either orally or parenterally (to guinea-pigs with histamine-induced ulcers). Hydrogenation abolished activity (sometimes producing irritant, ulcerogenic, compounds) and the number, as well as the existence, of olefinic links appeared critical. Increase in polarity achieved by oxidation of farnesol t o the acid maintained activity while addition of a further carbon atom gave farnesylacetic acid with greatly increased activity. Ethyl farnesate retained the properties of the acid while esters formed from terpinols (for example, geraniol) provided the most active series studied. The synthesis of these esters has been described [375] and their initial screening, leading to the selection of gefarnate, reported [376, 3771. The main screening test was that of ability to heal gastric ulcers induced in guinea-pigs by repeated histamine administration [378] though more detailed and more broadly based studies followed. Gefarnate, a mixture of stereoisomers, was shown t o lack both water-retaining and diuretic activity, to have no effect on blood pressure, respiration, blood-sugar,

A. P. LAUNCHBURY

49

and to have no local or central analgesic activity. Gastric motility and acid secretion was unaffected by the drug. One action which is noted, but not followed up, is inhibition of hyaluronidase-this might seem to be relevant in the context of tissue integrity. Experimentally, gefarnate has protected rats against prednisolone-and phenylbutazone-induced ulcers [379] and similar properties have been observed in humans [380]. More detailed studies of the drug, involving complex trial design, have shown [381] an apparent remarkable superiority of gefarnate over conventional antacid-anticholinergic treatments in the clinical management of gastric and duodenal ulcers. Toxicity appears to be negligible to the extent that both in animals and in humans large doses for extended periods, orally and parenterally, have failed to provide evidence of abnormal function of any type [376, 377, 3821. As might be expected, large volumes of the oily ester initiated local tissue irritation. Clinically, results rivalling those obtained with other treatments in conjunction with complete rest have been obtained using gefarnate in ambulant cases [374,380,381,383], and other reports are in press. The route of administration appears immaterial in that approximately 80 per cent of gastric and 60 per cent of duodenal ulcers are reported as healed whether the drug was given intramuscularly or orally. The mode of action is unknown but current theories on the structure of cell membranes permit of speculation concerning action at this level. Further clinical trials and more detailed biochemical studies are required to elucidate the mechanism of action and to evaluate properly this interesting compound. A broad-spectrum antimicrobial nitrofuran Nifuratel

The nitrofurans, their syntheses, general properties and trends in development, were reviewed recently in this series [384]. In particular, the limited development of resistance and generally low toxicity were stressed. The addendum to that review noted a number of nitrofurans with antifungal and antitrichomonal activity, and from this line of development a series of compounds possessing antibacterial, antitrichomonal and anticandidal activity has emerged. Of these, nifuratel (XXXII, Macmiror, Magmilor, Polmiror) is the most active and its general properties, synthesis and pharmacology have been described [ 3851 .

I 4Na

dbSMe

I C

HN J y O 0

(xxxn)

50

SOME RECENTLY INTRODUCED DRUGS

A thioether oxazolidinone nitrofuran, the drug has an unusually wide spectrum of activity. Many Gram-positive and Gram-negative bacteria are sensitive, antitrichomonal activity appears to be at least 3-4 times (weight for weight) that of metronidazole while Cundidu species show anomalous behaviour in that only moderate sensitivity is demonstrable in vitro yet clinical results indicate elimination of Cundidu and prevention of overgrowth by this organism during therapy for infections caused by other microbes. This does not appear to result from active metabolite formation. The structure of the drug is unusual in possessing the thioether linkage; antimicrobial activity depends on this feature as oxidation to the sulphone or the dioxide radically reduces activity. Increase in size of the alkyl radical also diminishes activity, trichomonacidal activity being demonstrable only in the methyl ether. Toxicity is low in that acute and chronic animal tests agree with clinical pharmacological findings [3861 that no biochemical or histological changes in any vital organ could be detected following exposure t o the drug. One or two patients are reported to have experienced mild symptoms (probably due to acetaldehyde) after taking alcohol with the drug, though other reports indicate no such interaction. Under the circumstances (other nitrofurans, for example, furaltadone have provoked vigorous reactions in the presence of ethanol) it is probably wise to avoid concurrent consumption of the two drugs. Nifuratel is absorbed from the gut in amounts adequate to give systemic antibacterial and antitrichomonal activity though the main metabolite is bactericidal only. This systemic activity permits treatment of male partners who may be carriers of trichomonas-a frequent cause of re-infection in married women. Clinical trials t386-91 demonstrate that combined therapy (oral tablets and pessaries) gives the most predictable and rapid elimination of pathogens in vulvovaginitis due to bacteria, trichomonads or Cundidu spp., though good results have followed oral therapy alone. A further report [390] indicates that nifuratel may greatly simplify the treatment, in general practice (where pathological reports are rarely to hand), of vulvo-vaginitis of all likely aetiologies (except syphilis and gonorrhoea). Concurrent cystitis (presumably bacterial) also responded. Patients rarely required more than 14 days treatment to secure eradication of the three main pathogens noted above; in difficult cases one month’s treatment (with oral - , therapy of the partner where appropriate) sufficed. The advent of nifuratel is a reminder that the nitrofurans are by no means a spent force; much more will doubtless be heard of t h s drug and of congeners now under development. The first anti-pseudomonalpenicillin Carbenicillin

The number of penicillins synthesized and screened for activity has now reached astronomical proportions following the isolation [39 11 of 6-aminopenicillanic

A.

P. LAUNCHBURY

51

. acid

(in 1959) from which the semi-synthetic derivatives are obtained. It was soon observed that penicillin activity could be extended into the Gram-negative range by the introduction,at the a-carbon of the sidechain, of hydrophilic groups. Ionized radicals gave the greatest promise and ampicillin (the a-amino derivative) has now an accepted place in therapeutics for the treatment of infections due to Gram-negative bacteria. Further work, based on this approach, involved the a-guanidino analogue [392, 3931 but real progress came only when the a-substituent was made anionic rather than basic. Carbenicillin (XXXIII, Pyopen) 0

I

COOH

COOH I

H

exhibits a property, most unusual for a penicillin, of activity against Pseudomonus spp. Its general antibacterial spectrum resembles that of ampicillin, except that its activity is of a lower order, with the notable exception of the organisms named above (and of related, similarly difficult genera). Carbenicillin shares the remarkably low toxicity [394] characteristic of the penicillins; since it produces no physiological abnormality in mammals, it can be given intravenously in large doses in the treatment of severe Pseudomonas infections (20-30 g daily is not uncommon [395] ). The only problem likely to be encountered is that of convulsions, which have been reported after high doses of penicillins, but the likelihood of this complication occurring during carbenicillin therapy is small since the highly ionic molecule does not pass the blood-brain barrier in appreciable amounts. Intrathecal use (in meningitis) is more likely to carry this small risk. The absorption and excretion of carbenicillin in man has been reported [396]. The antibiotic is not absorbed intact from the gut; intramuscular injection (which is painful) often provides adequate serum levels (approximately 20 pg/ml) but infections with Pseudomonus strains having minimum inhibitory concentrations up to, or higher than, 100pg/ml require intravenous thkrapy to achieve such levels. No evidence of active metabolite formation has been obtained. Marked reductions in the half-life (and serum levels) of carbenicillin follow extracorporeal dialysis or peritoneal dialysis, the former producing the most striking effect [397]. These results were, of course, obtained in patients with severe renal failure. Patients with normal renal function rapidly eliminate the drug but, as with all penicillins, renal tubular secretion can be retarded by concurrent administration of probenecid. The assay of carbenicillin in biological fluids presents problems in that it may be accompanied by traces of benzylpenicillin (probably arising by a-decarboxylation of carbenicillin) to which the usual test organism, Surcinu lutea, is extremely

52

SOME RECENTLY INTRODUCED DRUGS

sensitive. Pseudomonas aeruginosa NCTC 10490 is used instead since it is unusually sensitive t o carbenicillin but relatively resistant to benzylpenicillin. Concentrations down to 2.5 pg/ml can be assayed in this way [ 3 9 6 ) . Microbiologically, interest is mainly directed t o carbenicillin for its activity against Pseudomonas aeruginosa, Proteus morgani, P. rettgeri and P. vulgaris [395, 3981, all more or less resistant t o ampicillin. Klebsiella and Strep. fuecalis are often resistant. While many other pathogens are sensitive to carbenicillin other penicillins are more effective against them. Carbenicillin, then, dovetails with ampicillin in the Gram-negative field. It is complementary t o methicillin or cloxacillin in that these drugs are competitive inhibitors of penicillinase (amidases and 0-lactamases) which would otherwise destroy carbenicillin. Not all Gramnegative bacteria are resistant to penicillins because of such enzyme production so that the enzyme-inhibiting penicillins have only a limited usefulness in potentiating ampicillin and carbenicillin. Synergism with other anti-pseudomonal agents such as gentamycin, polymixins Band E, and even streptomycin, has been reported and is currently receiving study [398, 3991. It may well be that, in difficult cases, treatment with a combination of gentamycin, carbenicillin and cloxacillin (or with a polymyxin and the penicillins) will be necessary. So far, clinical evaluation [396-91 has given hope of a measure of control of Pseudomonas with, for the first time, a non-toxic drug. In view of previous calamities a recent reviewer 14001 commented: ‘to make the best use of carbenicillin both the minimim inhibitory concentration for each strain of organism and the level of the antibiotic in serum must be established.’ Unfortunately, the next sentence reads. ‘We fear that in most hospitals this will remain a council of perfection’. It is to be hoped that this drug will not be squandered like so many of its predecessors.

A new antibiotic of unusual structure Rifamide

The rifamycins, like so many other antibiotics, have been developed semisynthetically over a period of some ten years. The isolation of rifamycin B, the clarification ,of its biological conversion products and their subsequent modification to rifamide (XXXIV, Rifocin-M) have been described [401, 4021. The drug is unusual in that a long complex aliphatic chain spans a hydroxylated napthalene system. Its antibacterial spectrum has been studied [403, 4041. It shows intense activity against many Gram-positive organisms, especially staphyloccocci, pneumococci and streptococci (against which it is often bactericidal) and, at higher concentrations, rifamide inhibits many Gram-negative bacteria including Esch. coli, Pseudomonas, Proteus, Salmonella and Shigella. Of particular interest is its high activity against Mycobaterium tuberculosis though other

A. P. LAUNCHBURY

53

rifamycins (including rifampicin), currently under study, show even higher activity against this organism. The mode of action [403-51 of rifamide is interesting in that i t appears to bring about a widespread disruption of bacterial metabolism. Protein and carbohydrate utilization is impaired [406] and R.N.A. polymerase inhibited, at least in vitro. Me

Me

MeCO 0 Me

(XXXW)

The in vivo distribution of rifamide has been studied in dogs and rats [407]. Following intravenous injection by far the highest concentrations of the drug were found in bile and urine; a fairly uniform distribution in most other tissues was noted with the exception of brain where the concentration was negligible. In the exudate of rat granuloma pouch high antibiotic concentrations occurred. Oral administration has been shown to give unpredictable blood levels [408] . The same study revealed that almost 80 per cent of parenterally administered drug was recoverable in bile (giving biliary concentrations above 1 mglml), while other workers [409] have demonstrated strongly antibacterial concentrations in the gall-bladder wall. Placental passage is negligible 14101 as might be expected from the blood brain barrier findings referred to above. The toxicity of rifamide has been studied [404,411] with almost consistently negative results. At very high doses, some species showed minor haemodynamic disturbances, whilst others showed minor renal or hepatic changes. No toxic effects have been observed in animals or humans at therapeutic doses. Pain may be encountered at the site of injection. In clinical experience the drug has shown the properties expected from earlier experimental work. I t has been studied in biliary disease [412, 4131 in general surgical work [414] andin a variety of pulmonary infections [415-81 .The results have justified early optimism; in particular the very high concentrations achieved in bile make the drug eminently suitable for infections of the biliary tract. Rifamide (and its analogues) would appear t o be a much-needed addition to the antimicrobial arsenal; because of its low toxicity and lack of cross-resistance with other anti-infectives it is t o be expected that much more will be heard of the

POM E RECENTLY INTRODUCED DRUGS

54

drug. Again it is hoped [418a] that the emergence of resistance will be delayed by the application of the basic principles of antibacterial therapy. A new anti-tubercular drug Ri farnpicin

The observation [403,404] that rifamide and its congeners showed activity against Mycobacteria led t o the synthesis of further members of this series. Kifampicin not only possesses this property t o a marked degree but also exhibits good absorption from the gut and prolongs serum levels [419-421]. Differing in structure from rifamide (XXXIV) only in the substituents on one benzene ring. rifampicin (rifampin, Rifadin, Rimactane, XXXV) is of considerable interest in

fi!:=N.N3Me OH

(XXXV)

H o - H 2 c q N H B " t

(XXXVI)

that it inhibits bacterial RNA synthetase (422-4231, and, more surprisingly, the RNA polymerase induced in mammalian cells by many DNA viruses 143-4-43-61. This implies that the viral-induced enzyme resembles bacterial RNA polymerase more closely than the mammalian enzyme of this type. It has been pointed out [427], however, that clinical exploitation of this effect is hampered by the relatively high concentrations required. In addition to its satisfactory absorption from the gut, rifampicin also appears superior to other rifamycins with regard to penetration into bone; in view of the high levels reached i n this tissue and the sensitivity of S. uureus t o the drug, rifampicin has been suggested as worthy of trial in osteomyelitis [428]. Experience in urinary tract infections has not been encouraging [429]. The activity of rifampicin against mycobacteria has been intensively studied [430-4321 both in vivo and in vitro, as has its activity i n combination with other drugs such as isoniazid [433] and ethambutol [434]. Rifampicin appears t o be particularly promising in the treatment of tuberculosis, especially in combination with isoniazid. The prolonged high blood levels attainable with a single daily dose should simplify therapy. The prospects, including questions still unresolved, have recently been reviewed [435]. It is t o be hoped that the usefulness of this effective, almost non-toxic addition t o the antitubercular range of drugs will not be jeopardized by injudicious use of other rifamycins since cross-resistance exists between all known members of this group-at least so far as mycobacteria are concerned.

A.

P. LAUNCHBURY

5s

A selective bronchodilator Salbu tarnol

The treatment of acute bronchospasm with the classic drug isoprenaline has a number of disadvantages. Relief of spasm is short-lived since the catecholamine is metabolized by catechol-o-methyltransferase to the inactive 3-methoxy derivative; cardiovascular responses may actually worsen hypoxaemia since cardiac output increases and pulmonary vasculature dilates. Ventricular arrhythmias leading t o fibrillation are consequent hazards. Bronchodilation and the cardiac effects described are usually designated P-adrenergic responses but evidence exists [436, 4371 that this group of receptors comprise at least two sub-groups. Marked correlations exist between lipolytic and cardiac stimulatory potencies 0-1)and between bronchodilator and vasodepressor activity @ 2 ) , when series of 0-activating sympathomimetics are examined. This indicates that drugs prefererentially active at one or other of these receptor sites might be found. In one series [437] it was found that the 3-hydroxyl group of catecholamines could be replaced by a hydroxymethyl with retention of much typical p-stimulant activity. The phenolic 4-hydroxyl was, however, found t o be essential for activity which was greatest (as might be expected) in amines with branched-chain N-substituents. Of these ‘saligeninamines’, salbutamol (Ventolin, AH 3365, XXXVI) was selected for further study and finally for clinical use. It exhibited [438] considerably more activity on bronchial smooth muscle than on other muscles affected by 8stimulants. For instance, the stimulant effect of salbutamol on isolated guineapig atria was only a two-thousandth of that of isoprenaline and the vasodilating effect, in the perfused dog limb, only one-tenth. The bronchodilator effect was, however, profound and prolonged. Metabolic studies indicated that at least 10 per cent of the drug is excreted as a glucuronide. that no detectable enzyme induction occurred over a three month period and that salbutamol is not affected by catechol-o-rnethyltransferase. Clinically, it has been confirmed that the drug is an effective bronchodilator with very little cardiovascular activity; it does not appear to increase hypoxaemia [439]. A more detailed study [ 4 4 0 ] , comparing salbutamol with isoprenaline and orciprenaline, demonstrated that 200 p g of sabutamol provided effective bronchodilation for a t least three hours without detectable cardiac stimulation. Equivalent bronchodilator doses of isoprenaline and orciprenaline were 1000 pg and 1500 pg respectively. The bronchial effects of isoprenaline, though initially intense, waned within one hour and cardiac effects were noted. In this trial little objective difference could be detected between salbutamol and orciprenaline at the dose levels used, though most patients expressed preference for salbutamol. By virtue of its prolonged effect and high degree of selectivity salbutamol may prove t o be a significant development in bronchodilators, apart from its intrinsic interest as a research tool.

56

SOME RECENTLY INTRODUCED DRUGS

Concluding remarks

In this brief survey of some recently introduced drugs, it is inevitable that some will not have received mention. The following brief notes are intended merely t o indicate the scope o f the subject. In an earlier review [441] the concluding paragraph alluded t o the need for, and difficulties in attaining, a long-acting local anaesthetic. The introduction of bupivicaine (Marcaine, AH 2250) which, with adrenaline, can give anaesthesia for up t o 10 hours, may be a step towards this goal [442] . The substituted procainamide, metoclopramide (Maxolon) as an anti-emetic [443] is worthy of note, while the findings of many workers [444-4471 that cyproheptadine (Periactin) stimulates appetite and can thereby increase body weight may solve some problems in paediatrics and geriatrics. In a completely different context, clomiphene (Clomid) [448] appears less likely than the gonadotrophins to lead t o multiple pregnancies in the treatment of some form of infertility in women. Drostanolone (dromostanolone, Drolban, Emdisterone, Masted, Methalone), offered as a palliative in the treatment of mammary carcinoma, has stimulated considerable research into anti-oestrogenic mechanisms [449], while the increasing use of allopurinal (Zyloric) in gout has revealed new aspects of purine metabolism [450]. Cholestyramine (Cuemid, Dowex 1-X2-C 1, Questran) is a quaternary ammonium cationic resin used primarily t o bind, in the gut, bile salts which appear t o be the main cause of pruritis in obstructive hepatic disease. Again, many unexpected facets of steroid and lipid metabolism are becoming clear following studies of the drug’s effects [45 1.4521. Polypetides of medicinal interest were reviewed earlier in this series (4531 and developments have continued apace. Angiotensinamide, pentagastrin, tetracosactrin and felypressin are now either widely used or growing in importance and serve t o illustrate how rapidly this branch of medicinal chemistry is expanding. An interesting example of the rare true synergy is t o be found in the combination of trimethoprim and sulphamethoxazole (Septrin, Bactrim). Since these two drugs attack sequential enzymes [454] involved in folate utilization, a large, almost irreparable, ‘metabolic crater’ results from the simultaneous use of the two drugs. Resistance to the combination is rare and feeble (so far) and it would seem reasonable t o expect that this principle of attacking sequential, rather than widely separated, enzymes will be developed further with other drugs. Other antiinfectives now gaining acceptance include capreomycin, ethambutol (both antitubercular), gentamycin (combining the spectrum of kanamycin and the polymyxins) and candidicin (for vaginal candidosis). Antibiotics with antineoplastic activity continue to appear and rubidomycin (daunomycin, daunorubicin) [455] is currently receiving wide study. The above, like the chapter it concludes, is not intended t o be comprehensive, but rather to pin-point areas which may be of particular interest requiring further study.

A. P. LAUNCHBURY

57

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A. P. LAUNCHBURY 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129.

59

M. S. Sadove, R. C. Balagot, S. Hatano and E. A. Jobgen,J. Amer. Med. A s s , 1963, 183, 666 H. Blumberg, P. S. Wolf and H. B. Dayton,Proc. SOC.Exp. Biol. Med., 1965, 118, 763 H. Blumberg, H. B. Dayton and P. S. Wolf,Proc. SOC.Exp. Biol. Med., 1966, 123, 755 G. F. Blane and D. Dugdall, J. Pharm Pharmacol., 1968,20,547 K. W. Bentley and D. G. Hardy,Proc. Chem SOC.,1963,220 K. W. Bentley,J. Amer. Chern SOC.,1967,89, 3267 G. F. Blane, J. Pharm. Pharmacol., 1967, 19, 367 F. F. Foldes and T. A. G. Torda, Acta Anaesth. Scand., 1965, 9, 121 H. Isbell, Fed. Proc., 1956, 15,442 0. Schrappe, Arzneim.-Forsch., 1959, 9, 130 W. R. Martin and C. W. Gorodetzky, J. Pharmacol. Exp. Ther., 1965, 150,437 H. F. Fraserand D. E. Rosenberg, J. Pharmacol. Exp. Ther., 1964,143, 149 W. R. Martin, H. F. Fraser, C. W. Gorodetzky and D. E. Rosenberg, J. Pharmacol. Exp. Ther., 1965, 150, 426 L. Brill and J. H. Jaffe, Brit. J. Addict. 1967,62, 375 H. F. Fraser and L. S. Hams, Annu. Rev. Pharmacol., 1967,7, 277 L. Lasagna, Arch. Int. Med., 1954, 94, 532 H. F. Fraser, Med. Clin N. Amer., 1957, 47, 393 W. R. Martin, C. W. Gorodetzky and T. K. Clare, Clin Pharmacol. Ther., 1966, 7,455 V. P. Dole and M. E. Nyswander, J. Amer. Med. Ass., 1965, 193, 646 G. Gorssen and 1. A. Skora, J. Amer. Med. A s s , 1964, 187, 328 W. R. Buckett, Brit. J. Addict. 1967,62, 387 J. Pohl, Z. Exp. Pathol. Ther., 1914, 17, 370 J. Weijlard and A. E. Erickson, J. Amer. Chem. SOC.,1942,64,869 K. Unna, J. Pharmacol. Exp. Ther., 1943, 79.27 S. Archer, N. F. Albertson, L. S. Harris, A. K. Pierson and J. G. Bird, J. Med C h e m , 1964,7,123 0. Schaumann, Brit. Med. J., 1956,2, 1091 L. Lasagna and H. K. Beecher, J. Pharmacol. Exp. Ther., 1954, 112, 356 A. S. Keats and J. Telford, J. Pharmacol. Exp. Ther., 1957, 117, 190 N. B. Eddy, Brit J. Addict., 1966,61, 155 Lancet, 1967, 1, 1310 E. L. May, Brit. J. Addict., 1967,62, 197 L. S. Harris, Arch. Exp. Pathol. Pharmacol., 1964,248,426 L. S. Harris and A. K. Pierson, J. Pharrnacol. Exp. Ther., 1964, 143, 141 S. Archer and L. S. Harris, Yale Sci Mag., Feb. 1965 W. H. 0. Expert Committee on Dependence Producing Drugs: Fifteenth Report (1966), World Health Organ Tech. Rep., Ser. 343: 6 M. S. Sandove, R. C. Balagot and F. N. Pecora, J. Amer. Med. Ass., 1964, 189. 199 M. S. Sandove and R. C. Balagot, J. Amer. Med. Ass., 1965, 193,887 R . A. Gordon and J. H. Moran, Can. Anaesth. SOC.J., 1965,12,331 L. J. Cass, W. S. Frederik and J. V. Teodoro, J. Amer. Med. A s s , 1964, 188, 112 H. Erb, Gynaecologia, 1966, 162,275 J. L. Wilkey, L. J. Barson and F. H. Rowe, J. Urol. (Baltimore), 1967,97,550 R. Scott, R. J. Abernethy and H. S. Livingston, Clin. TrialsJ., 1966, 3, 395 N. McSwan, J. M. Reid, E. R. Matthews and E. M. Henderson, Clin. Tri3ls J., 1966, 3, 339 A. S. Keats and J . Telford, J. Pharmacol. Exp. Ther., 1964, 143, 157 V. K. Stoelting, Anaesth. Analg. Cum Res., 1965,44,769 J. P. Conaghan, Brit. J. Anaesthesia, 1966, 38, 345 M. Swerdlow and A. Dalal, Anaesthesist, 1966, 15,43 Drug Ther. Bull., 1967, 5 , 33

60 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 159a. 160. 161. 162. 163. 164. 165. 166. 167. 168.

SOME RECENTLY INTRODUCED DRUGS W. Norris and A. B. M. Telfer, Brit. J. Anaesthesia, 1968,40, 341 J. Telford and A. S. Keats, Clin. PharmacoL Ther., 1965,6, 12 W. W. Filler and N. W. Filler, Obstet. Gynaecol., 1966, 28, 224 A. H. Beckett and J. F. Taylor, J. Pharm Pharmacol., 1967, 19, Suppl. 50s W. T. Beaver, Clin. Pharmacol. Ther., 1966, 7 , 7 4 0 G . M. Halpern, Lancet, 1968,1,1205 M. J. Thuillier and R. Domenjoy, Anaesthesist, 1 9 5 7 , 6 , 163 R. Hiltmann, H. Wollweber, W. Wuth and F. Hoffmeister, Intravenose Kurznarkose mil dern neuen Phenoxyessigsaurderivat Propanidid (Epontol), (Eds. K. Horaz, R. Frey and M. Zindler) Springer-Verlag, Berlin, 1965, p.1 B. B. Brodie, L. C. Mark, E. M. Papper, P. A. Lief, E. Bernstein and E. A. Rovenstine, J. Pharmacol. Exp. Ther., 1 9 5 0 , 9 8 , 8 5 L. C. Mark, Clin Pharmacol. Ther., 1 9 6 3 , 4 , 5 0 4 H. L. Price, Anesthesiology, 1960, 21, 40 R. K. Richards and J. D. Taylor, Anesthesiology, 1956, 1 7 , 4 1 4 W. D. Winters, E. Spector, D. P. Wallach and F. E. Shideman, J. PharmacoL Exp. Ther., 1955, 114, 343 J. Putter, Die intravenose Kurznarkose mit dem neuen Phenoxyessigsaurederivat Propanidid (Epontol). (Eds. K. Horatz, R. Frey and M. Zindler) Springer-Verlag, Heidelberg, 1965, p.61 W. Wirth and F. Hoffmeister, Die intravenose KurznarkoFe mit d e m neuen Phenoxyessigsaurederivat Propanidid (Epontol). (Eds. K. Horatz, R. Frey and M. Zindler), Springer-Verlag, Heidelberg, 1965, p.17 W. N. Aldridge, Biochem J., 1953, 53, 110 K. B. Augustinsson,Acta Chem. Scand., 1959a, 13, 571 A. Doenicke, Acta Anaesth. Scand., 1965, Suppl. XVII, p. 21-5 B. Duhm, W. Maul, H. Medenwald, K. Patzschke and L. A. Wegner, Die intravenose Kurznarkose mit dern neuen Phenoxyessigsaurederivat Propanidid (Epontol), (Eds. Horaz, R. Frey and M. Zindler) Springer-Verlag, Heidelberg, 1965, p.78 A. Doenicke, 1. Krumey, J. Kugler and J. Klempa, Brit. J. Anaesthesia, 1968,40,415 M. Zindler, ed. Acta Anaesth. Scand., 1965, Suppl. XVII, R. S . J. Clarke and J. W. Dundee, Curr. Res. Anestha, 1966, 45, 250 D. R. Cadle, T. B. Boulton and M. S. Swaine, Anaesthesia, 1968, 23, 65 T. H. Howells, Brit. J. Anaesthesia, 1968,40, 182 P. W. Jackson and Z. M. Woodhead, Anaesthesia, 1967, 2 2 , 7 0 4 M. Johnstone and P. T. Barron, Anaesthesia, 1968, 23, 180 T. H. Howells, Acta Anaesth. Scand., 1965, Suppl. XVII, p.40 M. Zindler, Acta Anaesth. Scand., 1965, Suppl. XVII, p.41 Drug Ther. Bull., 1 9 6 6 , 4 , 9 7 J. Crossland, Progr. Med. Chem., 1967,5, 251 Various authors, Brit. Med. Bull., 1965, 21, 1 D. W. Wylie and S. Archer. J. Med. Pharm. Chenz., 1962.5, 932 S. Archer, D. W. Wylie, L. S. Hams, T. R. Lewis, J. W. Schulenberg, M. R. Bell, R. K. Kullnig and A. Arnold, J. Amer. Chem. Soc., 1962,84, 1306 D. W. Wylie and S. Archer, Fed. Proc., 1962, 21, 322 R. E. Edwards, L. E. Moon and J. Pearl, Pharmacologist, 1962,4, 161 J. 0. Cole and R. E. Edwards, Animal Behaviour and Drug Action, (Ed. H. Steinberg) J. and A. Churchill, London, 1964, p.286 J. M. A. Hameed and T. J. Haley, Proc. Western PhatWacol. SOC.,1965,8, I J. M. A. Hameed and T. J. Haley, Brit. J. Pharmacol., 1966,26,186 M. Matsuoka, Japan J. Pharmacol., 1964, 14, 181 K. Fuxe, H. Grobecker, T. Hokfelt, J. Johnsson and T. Malmfoss, NaunynSchmeidebergs Arch. Exp. Pathol. Pharmakol., 1961, 256, 450

A. P. LAUNCHBURY

61

169. J. P. McAuliff, F. J. Rosenberg, A. Arnold, L. S. Harris and S. Archer, Fed. Proc., 1963, 22,567 170. T. Itoh, J. Kajikawa, Y. Hashimoto, R. Yoshidaand Imaizumi, Japan J. Pharmacol., 1965, 15, 335 171. M. Matsuoka, S. Ishii, N. Shimiza and R. Imaizumi, Experientia, 1965,21, 121 172. R. Hassler, and I. J. Bak, Dei Nerv., 1966, 37,493 173. S . Spector, K. Melmon and A. Sjoerdsma, Proc. SOC.Exp. Biol. Med., 1962, 1 11,79 174. M. M. Airaksinsen and J. E. Idampaan-Heikkila, Psychopharrnacologica. 1967, 10,400 175. W. P. K. Calwell. M. Jacobsen and A. Skarbek, Brit. J. Psychiat., 1964, 110,520 176. J. Durell and W. Pollin, Brit. J. Psychiat., 1963, 109,687 177. L. E. Hollister, J. E. Overall, I. Kimbell, J. L. Bennett, F. Meyer and E. Caffey, J. New Drugs, 1963, 3 , 2 6 178. Drug Ther. Bull., 1967,5,51 179. A. Skarbek and M. Jacobson, Brit. J. Psychiat., 1965,111, 1173 180. A. Randrup and I. Munkvad, Acta Pharrnacol. Toxicol., 1964, 21,272 181. J. Kajikawa, T. Itoh, Y. Hashimoto, H. Yoshidaand R. Imaizumi, Japan. J. Pharmacol., 1965, 15,446 182. Anon., Lancet, 1968, 1, 1237 183. H. Weil-Maherbe, Lancer, 1968,2,219 184. W . G. Dewhurst, Lancet, 1968, 2, 514 185. L. M. Rice, J. Med. Chem., 1964,7,313 186. L. Stein, First Hahnemann Symposium on Psychosomatic Medicine, Lea & Febiger, Philadelphia, 1962, p. 297 187. L. Stein, Fed. Proc., 1964, 23, 836 188. L. Stein, Recent Advan. Biol. Psychiat., 1962, 4, 288 189. J. R. Gillette, J. V. Dingell, F. Sulser, R. Kuntzman and B. B. Brodie, Experimentia, 1961, 17,417 190. E. A. Daneman, Psychosomatics, 1967,8,216 191. J. T. Hicks, NlinoisMed. J., 1965, 622 192. N. W. Imlah, K. P. Murphy and C. S. Mellor, Clin. Trials J., 1968, 5 , 927 193. M. S. Sutherland, S. S. Sutherland and A. E. Philip, Clin. Trials J., 1967, 4, 857 194. J. Johnson and J. G. Maden, Clin Trials J., 1967,4,787 195. N. K. El-Deiry, A. D. Forrest and S. K. Littman, Brit. J. Psychint., 1967, 113,999 196. W. T. McClatchey, J. Moffatt and G. M. Irvine, J. Ther. Clin Res., 1967, 1, 13 197. N. W. Imlah, presented at 4th World Congress OfPsychiatry, Madrid, 1966 198. Drug Ther. Bull., 1968,6, 39 199. R. Wein,Progr. Med. C h e m , 1961, 1, 34 200. J. H. Burn and M. J. Rand, Adv. Pharmacol., 1962, 1, 1 201. R. A. Moe, H. M. Bates, Z. M. Palkoski and R. Banziger, Cum Ther. R e x , 1964,6, 299 202. N. Weiner, P. R. Draskoczy and W. R. Burak, J. Pharmacol. Exp. Ther., 1955, 137,47 203. J. R. Crout, A. J. Muskus and U. Trendelenburg,Brit. J. Pharmacol., 1962, 18,600 204. L. T. Potter, J. Axelrod and I. J. Kopin, Biochem. Phnrtncol., 1962, 11, 25 3 205. L. T. Potter and J. Axelrod, J. Pharmacol, Exp. Ther., 1963, 140, 199 206. W. M. Abrams,Amer. J. Cardiol., 1963, 12,711 207. M. L. Mashford, J. B. Philipson, D. A. Wolochow and W. A. Mahon, Proc. SOC.Exp. Biol. Med., 1962, 111, 308 208. J. H. Burn and M. J. Rand, J. Physiol., 1958, 144, 314 209. J. H. Bum and M. J. Rand, Brit. Med. J., 1958, 1,903 210. A. J. Plummer, Hypertension: Recent Advances (Ed. A. N. Brest and J. H. Moyer), Lea & Febiger, Philadelphia, 1961, 399 211. R. A. Maxwell, Hypertension: Recent Advances (Ed. A. N. Brest and J. H. Moyer), Lea and Febiger, Philadelphia, 1961, p.437 212. J. W. McCubbin, Y. Kaneko and I. H. Page, J. Pharmocol. Exp. Ther., 1961, 131, 346

62 213. 214. 215. 216. 217. 218. 219. 220. 221. 222. 223. 224. 225. 226. 227. 228. 229. 230. 231. 232. 233. 234. 235. 236. 237. 238. 239. 240. 240a. 240b. 241. 242. 243. 244. 245. 246. 247. 248. 249. 250. 251. 252. 253. 254. 255. 256. 257. 258. 259. 260. 261. 262.

SOME RECENTLY lNTRODUCED DRUGS

Drug Ther. Bull., 1 9 6 7 , 5 , 4 4 W. J . Wenner, J. Med. Chem., 1965,8, 125 F. J . Talbot, Med, A n n District Columbia, 1965, 3 4 , 5 8 0 J . M. Bryant, J. Amer. Med. Ass., 1965, 193, 1021 M. H. Luria and E. D. Freis, Cum. Ther. Res., 1965,7, 289 F. A. Finnerty,Med. Clin. N. Amer., 1964,48, 329 N. Kakaviatos,Amer. J. Cardiol., 1964, 13, 111 A. H. Kitchin and R. W. D. Turner, Brit. Med. J . , 1966, 2, 728 D. Athanassiadis, Brit. Med. J., 1966, 2, 732 A. E. Gent and A. P. C. Bacon, Practitioner, 1967, 198,673 R. P. Ahlquist, Amer. J. Physiol., 1948, 153, 586 K. Greefand H. J . Schumann,Arzneim.-Forsch., 1953, 3, 341 H. Koch, Deut. Med. Wochenschr., 1954, 19, 176 F. Brauch, Deut. Med. Wochenschr., 1954, 17, 676 W. Lindenburg, Arztl. W., 1950, 25,434 W. Lindenburg,Med. K l i n , 1953, 18,633 G. Wichmann, Muench. Med. Wochenschr., 1953,95, 373 A. T. Birmingham and J . Szolcsanyi, J. Pharm. Pharmacol., 1965, 17, 449 A. T. Birmingham and J . Szolcsanyi, J. Pharm. Pharmacol., 1967, 19, 137 J. H. Gaddum, J. Physiol., 1937,89, 71 J . H. Gaddum, Pharm. Rev., 1957, 9, 211 H. 0. Schild, Brit. J. Pharmacol., 1947, 2, 189 H. 0. Schild, Pharrn Rev., 1957.9, 242 R. t:. Furchgott, Pharm Rev., 1955, 7, 183 0. Arunlakshana and H. 0. Schild, Brit. 1. Pharmacol., 1959, 1 4 , 4 8 G. Brownlee, Angiology, 1966, 17, 186 K. Myers, J . T. Hobbs and W. T. Irvine, ExcerptaMed. (Amsterdam) Congress Series, No. 126, 1966,80 S. S. Rose, Vascular Dis., 1967, 4 , 6 7 J . Harrison and A. P. Turner, J. Pharm. Pharmacol., 1968, 20, 161 See also J. R. Parratt, Progr. Med. C h e m , 1968,6, 11 E. Lindner, Arzneim.-Forsch., 1960, 10, 589 A. S. V. Burgen and L. L. lversen, Brit. J. Pharmacol., 1965,25, 34 H. H. Schone and E. Lindner, Anneim.-Forsch., 1960, 10,583 F. Wohlrab, Arch. Exp. Pathol. Pharmakol., 1962, 243, 382 A. V. Juorio and M. Vogt, Brit. J. Pharmacol., 1965,24,566 B. Werdinius, Acta Pharmacol. Toxicol., 1967, 25, 1 H. J . Kuschke, F. Eckmann. H. ldriss and P. Bieck, Verhandl. Deut. Ges. Med., 1964, 70, 191 I{. J. Kuschke. H. ldriss and F. Eckmann. Klin. Wochenschr.. 1965,43,617 W. Brassch and D. Fleck, Arzneim.-Forsch., 1961, 11, 336 E. Lindner, Arch. Int. Pharmacodyn Ther., 1963, 146,475 1 . Lindner, Arch. Int. Pharmacodyn Ther., 1963, 146,485 H . Obianwu, Acta Pharmacol. Toxicol., 1967,25, 127 H. Obianwu, Acta Pharmacol. Toxicol., 1967, 25, 141 A. Carlssen and B. Waldeck, J. Pharm Pharmacol., 1965, 17, 243 A. Carlss.cn, N. A. Hillarp and B. Waldeck, Acta Physiol. Scand., 1963,59, 215 A. Carlssen and B. Waldeck, Acta Physiol. Scand., 1 9 6 6 , 6 7 , 4 7 1 M. Neumann and A. A. Luisada, Amer. J. Med. S c i , 1964, 247, 156 K. Donat and C. A. Schlosser,Med. K l i n , 1966, 61, 352 A. Kappert, Z. nier., 1965,2, 82 J . A. Sosa and M. McCregor, Can Med. Ass. J., 1963,89, 248 E. Gerlach and B. Deuticke, Arzneitn-Forsch., 1963, 13, 177 M. Schlepper and E:. Witzleb, Arzneix-Forsch., 1962, 12, 559

..

A. P. LAUNCHBURY

263. 264. 265. 266. 261. 268. 269. 270. 271. 272. 273. 274. 215. 216. 277. 278. 279. 280. 281. 282. 283. 284. 285. 286. 287. 288. 289. 290.

291. 292. 293. 294. 295. 296. 297. 298 299. 300. 301. 302. 303. 304. 305. 306.

63

H. Haas, Pharmaz. Ztg., 1964, 109, 1855 H. Haas, Arzneim.-Forsch., 1964, 14, 461 H.Haas, Deut. Med. Wochenschr.. 1964,89,2117 H. Haas and G. Hartfelder, Arzneim.-Forsch., 1962, 12, 549 A. Fleckenstein, Deut. Ges. Inn. Med., 70th Meeting, 1964 K. I. Melville and B. G. Eenfey, Can J. Physiol. Pharmacol., 1965,43,339 K. I. Melville, H. E. Shister and S. Huq, Can. Med. Ass. J., 1964,90, 76 E. D. Luebs, A. Cohen, E. J . Zaleski and R. J. Bing, Amer. J. Cardiol.. 1966, 17,535 R. H. E. Grant, D. G. McDevitt and R. G. Shanks, Lancet, 1968, I , 362 P. Hoffmann, Med. Klin. (Muench), 1964, 59, 1387 K. Hofbauer, Wien. Med. Wochenschr., 1966, 116,1155 K. Fischer,Med. Klin. (Muench), 1965,60,841 M. Neuman and A. A. Luisada, Amer. J. Med. S c i , 1966,251,87 G. Sandler, G. A. Clayton and S . G. Thornicroft, Brit. Med. J . , 1968, 3, 224 R. A. Lucas, Prog. Med. C h e m , 1963, 3, 146 T. Kralt ,H. D. Moed, V. Claassen, T. W. J. Hendriksen, A. Lindner, H. Selzer, F. Brucke, G. Hertting and G. Gogolak, Nature, 1960, 188, 1108 T. Kralt, W. J . Asma, H. H. Haeck and H. D. Moed, Red. Trav. Chim. Pays-Bas. 1961, 80, 313 T. Kralt, W. J. Asma and H. D. Moed, Reel. Trav. Chim. Pays-Bas, 1961, 80, 330 A. Lindner, V. Claassen, T. W. J. Hendriksen and T. Kralt, J. Med. Chem., 1963,6, 97 A. Lindner, H. Selzer, V. Claassen and P. Cans, Arch. Int. Pharmacodyn. Ther., 1963, 145,378 A. C. Houtman, Isotope Laboratory. Weesp. Holland, 1964 P. Crosti, Instituto di Semeiotica Medica della Universita di Milano, 1964 A. M. Connell, Brit Med. J., 1965, 2, 848 H. Heller and M. Ginsburg, Progr, Med. Chem., 1961, 1, 132 R. H. Kessler, K. Hierholzer, R. S. Curd and R. F. Pitts, Amer. J. Physiol., 1959, 196, 1346 M. Goldberg, (Ed., W. F. M. Fulton), Butterworths, London, 1967, p. 42 W. Kuhn and K. Ryffel, Z. Physiol. Chem., 1942,216, 145 B. Hargitay and W. Kuhn, Z. Electrochem., 195 1,55,539 H. Wirtz, B. Hargitay and W. Kuhn, Helv. Physiol. Pharmacol., 1951. 9, 196 C. W. Gottschalk and M. MyUe, Amer. J. Physiol., 1959, 196, 927 K. J. Ullrich, K. Kramer and J. W. Boylan, Renal Disease (Ed. D. A. K. Black), Blackwell, Oxford, 1962, p. 49 C. W. Gottschalk, Amer. J. Med., 1964,36,670 R. W. Berliner, N. G. Levinsky, D. G. Davidson and M. Eden, Amer. J. Med., 1958, 24,730 R. W. Winters and R. E. Davies, Ann. Intern Med., 1961,54, 810 G . Giebisch, R. M . Klose and E. E. Windhager, Amer: J.'Physiol., 1964, 206,687 M. Goldberg, D. K. McCurdy and M. A. Ramirez,J. Clin Invest., 1965.44, 182 V. M. Buckalew, M. A. Ramirez and M. Goldberg,Amer. J. Physiol., 1967, 212,381 M. A. Cortney, M. Mylle, W. E. Lassiter and C. W. Gottschalk, Amer. J. Physiol., 1965, 209,1199 G. Giebiech and E. E. Windhager, Amer. J. Med., 1964, 36,643 R. F. Pitts, The Physiological Basis of Diuretic Therapy., Thomas, Springfield, IU., 1959, p. 55 L. E. Earley, M. Kahn and J . Orloff, J. Clin Invest., 1961,40,857 W. Suki, F. C. Rector and D. W. Seldin, J. CIin Invest., 1 9 6 5 . 4 , 1458 M. Goldberg, D. K. McCurdy, E. L. Foltz and L. W.Bluemle, J. Cfin. Invest., 1964.43, 201 L. E. Earley and R. M. Friedler, J. Clin. Invest., 1964,43, 1495

64

SOME RECENTLY INTRODUCED DRUGS

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SOME RECENTLY INTRODUCED DRUGS

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The Biochemical Basis for the Drug Actions of Purines 2

JOHN A. MONTGOMERY, Ph.D. Kettering-Meyer Laboratoory, Southern Research Institute, Birmingham, Alabama 35205, U.S.A.

I NT RO DUCT1ON

i0

METABOLISM Anabolism The phosphoribosyltransferases The nucleoside kinases Nucleoside phosphokinases Nucleotide reductases Methylases and demethylases Other anabolic enzymes Catabolism F’hosphatases and nucleotidases Nucleoside phosphorylases and hydrolases Deaminases Oxidases Summary

14 14 14 80 80 83 83 84

MECHANISM OF ACTION Enzyme inhibition The de ~ O Y Osynthesis of inosinic acid The ‘salvage’ pathways Purine nucleotide interconversions Other enzymes Incorporation into nucleic acids Inhibition of protein synthesis Interference with co-factors Summary

91 93 93 96 91 99 99 100 101 102

69

85 85 86 81 88 91

I0

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

EFFECTS On the host Toxicity Embryonic tissue Immune response Platelet aggregation Gout On Invading organisms Micro-organisms (bacteria and protozoa) Viruses and cancer

102 102 102 104 104 105 105 105 105 106

THE PROBLEM OF RESISTANCE Mechanisms Circumventions

109 109 110

ACKNOWLEDGMENTS

111

REFERENCES

111

INTRODUCTION

An analogue of a naturally occurring purine, purine nucleoside, or a metabolic product of either, is potentially a substrate for and/or an inhibitor of any of the enzymes necessary for the reactions depicted in Figure 2.1. In addition certain analogue nucleoside mono-, di-, and triphosphates can, by negative pseudofeedback [l-31, inhibit the de n o w synthesis of purine nucleotides. Thus the problem of the biological activity of purines is a complex one based on the normal metabolism of the natural purines. Although a detailed discussion of the metabolism of the natural purine nucleotides is beyond the scope of this review, the general scheme is shown in Figure 2.1, and references t o reviews and recent original literature on the enzymes involved are given. The general subject of purine nucleotides and their metabolism has recently been the subject of an excellent review [ 4 ] , and numbers of earlier reviews dealing in part with this topic have appeared [S-131. With a few notable exceptions-such as puromycin and its relatives, psicofuranine and decoynine, and certain 9-alkylpurines-which will be discussed later, purines and their nucleosides must be anabolized t o nucleoside phosphates in order t o exert their biological effects. This type of metabolic event has been called a ‘lethal synthesis’, because it results in the death of cells that carry it out. Enzymes in general vary widely in their substrate specificity, and this wide variation is found in the purine-metabolizing enzymes, as will be seen from the

1, i

,Co-factors

6

in

12

11

t HvDoxanthine

t

1.7

lnosine

. r

21

I

Uric acid

Allantoin

*

\\*

Xanthin-e

=

1

F

27 Guanine

20

-

IMP:

RNP

t

-

2L b Xanthosine 1

1.7

Guanosine

22

19 dGTP

281'- --

* 29L

GMP

1 3 0

3

2' Deoxyguanosine

l L \ I 1 10 GDP

co:facJ

dGMP L

Figure 2.1. Cellular metabolism of purine nucleotides. The meaning of the numbers over the arrows is explained on the next page.

dGDP

12

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

ENZYMES A N D 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.

Nucleoside phosphorylase [ 14-2 1b] Pentosyltransferase [ 14, 19, 22-26] Nucleoside kinase [27] S’-Nucleotidase or nucleotide phosphatase [28-37a] Nucleotide kinase [38,39] Adenine phosphoribosyltransferase [40-45a] Nucleoside hydrolase (bacteria) [ 14,46,46aJ Adenosine kinase [27,47,48] Adenylate kinase (myokinase) [39, 49-52] Nucleoside diphosphate reductase [53, 541 Adenine deaminase (bacteria) 155, 561 Adenosine deaminase [57-64a] Adenylosuccinate lyase [65+7] Nucleoside diphosphokinase [68-7 1] Myosin adenosine triphosphatase [72] Adenylate deaminase [73-771 Adenylosuccinate synthetase [65,67,78] RNA polymerase [ 791 DNA polymerase [ 79-8 1 1 Inosine kinase [82,83] Xanthine oxidase [84] Hypoxanthine-guanine phosphoribosyl transferase [ 43,85-8 Sb] Inosinic dehydrogenase [6S, 85c] Xanthine phosphoribosyltransferase (bacteria) [8] GMP reductase [86] Uricase [87] Guanine deaminase [88] GMP synthetase [65, 89,901 Guanosine kinase (bacteria, fish) [82,83] Guanylate kinase [9 1]

discussions in the sections that follow. There are in many cases, however, clearly delineated structural requirements for substrates. The natural purine nucleotides can be placed into two groups based on structure, and it is logical t o discuss the metabolism of a purine analogue, according t o the group in which it best fits, based on the data we have at hand. The two structural groups are pictured in Figure 2.2. The dotted areas represent the parts of the purine nucleotide structure that determine its ability t o serve as a substrate for the various cellular enzymes. The structure of the 1-2-3+ positions o f the pyrimidine ring* is the primary determinant, and it is surely not by chance that this same portion of folic acid and the folic reductase inhibitors (for example, methotrexate

JOHN A. MONTGOMERY

73

and daraprin) (bigure 2.3) is critical t o their activity also. The hydroxyl or hydrogen a t C-2’ of the sugar moiety (ribose or 2‘-deoxyribose) is important to the metabolism of nucleosides and nucleotides. Thus analogues resemble either adenine compounds on the one hand or hypoxanthine-guanine compounds on the other (the amino or oxy group* at C-2, N-3 can also be determinant). In some cases, such as 2-fluoroadenosine, the classification of an analogue is obvious and predictable; but in others, such as 6-(methy1thio)purine ribonucleoside, the classification is deceptive and was correctly made in retrospect. Specific structural features also determine the deactivation or destruction by catabolic enzymes of purine analogues. Selective destruction of these toxic materials may, in fact, be the single most important factor relating to their activity . *For convenience the group at C-2 of xanthylic acid is represented here as an hydrosyl group, although it actually exists in the carbonyl or lactam form. This liberty is taken with other structures throughout this chapter.

I------

HO

HO

X

-

Adenylic acid: X OH 2’ - Deoxyadenylic acid X

-

H

-I

X

Inosinic a c i d : R -H.X - O H Xanthylic acid: R-X -OH Guanylic acid : R=NH,. X-OH 2’- Deoxyguhnylic acid: R -NH2, X-H

Figure 2.2. Purine nucleotfdes

In the sections that follow, the metabolism of the various purine analogues is presented first, followed by a discussion of the current status of our knowledge about the mechanism of action of these compounds. Next the distribution and effects of these drugs on the host and invading organisms is considered with some concluding remarks o n the ever present problem of resistance.

14

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

F o l i c acid

Methotrexate

Pyrimethamtne PMC (23)

Figure 2.3. Folic acid and inhibitors METABOLISM Anabolism The phosphoribosyltransferases

The reactions of purine bases with phosphoribosylpyrophosphate (PRPP)[92] t o form ribonucleotides are catalysed by purine phosphoribosyltransferases (purine nucleotide pyrophosphorylases)[93-96~], four of which have been identified in bacteria for the natural purines adenine, hypoxanthine, guanine, and xanthine by protein fractionation procedures [97] and by analysis of bacterial mutants [98-1001 . In mammalian cells, however, high levels of only two separate enzymes have been identified, adenine phosphoribosyltransferase [93, 96, 96a] and hypoxanthine-guanine phosphoribosyltransferase [96-96c, 101] . Although it has been suggested that the hypoxanthine and guanine phosphoribosyltransferase activities are separate enzymes [102, 1031, their separation has not been achieved and the many examples of mutant cells lacking both activities [ 8 ] , and no example of a mutant lacking only one, argue against this view. Recently, evidence has been presented supporting the view that not only is a single enzyme involved,

JOHN A. MONTGOMERY

15

but also that hypoxanthine and guanine bind to the same site on this enzyme [96b, 9 6 ~ 1When . the catabolism of xanthine was retarded by pre-treatment with 4-hydroxypyrazolo [3, 4-d] pyrimidine, a potent inhibitor ot xanthine oxidase (vide infru), its incorporation into nucleic acid purines in mice could be demonstrated [ 1041, and a very low level of xanthine phosphoribosyltransferase activity has been observed in man [ 1051 . Xanthine was later shown to be a poor substrate for hypoxanthine-guanine phosphoribosyltransferase [85a]. Adenine phosphoribosyltransferase (41-44, 103, 1061 and hypoxanthineguanine phosphoribosyltransferase have both been investigated in some detail [85a, 85b, 96b]. It has been suggested that in the case of adenine phosphoribosyltransferase, PRPP reacts with the enzyme t o form an enzyme-ribose-5phosphate complex, which then reacts with adenine to form AMP [41]. The mechanism of the hypoxanthine-guanine phosphoribosyltransferase appears more complex [85b, 9 6 b J . Initial velocity analyses suggest that a t relatively high magnesium concentrations the predominant reaction sequence for IMP synthesis is also one involving an enzyme-phosphoribosyl intermediate, whereas at low magnesium concentrations the predominant mechanism is one involving a ternary complex, although admittedly IMP synthesis by the latter sequence would be relatively slow [85b]. A number of purine nucleoside phosphates have been found to inhibit and to stimulate the enzymes 142-44, 85b, 96b, 103, 1061. It has been postulated that at low concentrations ATP is bound t o an activator site on adenine phosphoribosyltransferase which is separate from the substrate site and that at high concentrations it competes with PRPP for the active site [41] It has been further suggested that exogenous adenine is converted to AMP and assimilated into nucleic acid at a discrete locus separate from the pathway immediately accessible t o endogenous purines [ 1061, a point which could have important bearing on the biologic activity of adenine analogues. The enzymatic conversion of many analogues of the naturally occurring purines directly t o their biologically active form, the ribonucleotides, in vivo [ 5 , 8 , 10, 13,391 underlines the importance of these enzymes t o the drug action of this class of compounds. 2-Aminoadenine (2, 6-diaminopurine. 1) [ 1071, 2-fluoroadenine (11) [ 1081 , 4-aminopyrazolo [3, 4-d] pyrimidine (VIII) [ 109). and 2- and 8-aza-adenine (IX and X) [ I 10, 1 1 I ] have all been shown to be substrates for the adenine phosphoribosyltransferase [I 12, 1 1 31 . Extensive studies on the metabolism of 2-aminoadenine (I) in E. coli [ I 14, 1 151, L cells [ 1161, and mice [ 1 171 have also shown its conversion by this enzyme t o the ribonucleotide. Conversion of 4-aminopyrazolo [3,4-d] pyrimidine (VIII) to its ribonucleotide by mouse tumours and host tissues has been observed [ 1 18, 1191. Although no evidence of the anabolism of N-methyladenine (111) [ 1201 t o the ribonucleotide was obtained in mice with Ehrlich ascites carcinoma [ 121, 1221, it is anabolized by bacteria [ 123. 1241 : and the enzyme responsible was partially purified from Salmonella ryphimurium [ 1251 . Human epidermoid carcinoma No. 2 cells resistant t o 2-fluoroadenine (H.Ep.-2/FA) have lost adenine phosphoribosyl-

76

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS 01 PURINES

(1x1 X

= N; Y = C H ( X ) X = CH; Y = N

( I ) R1 =NH ; R zNH, (11) R, :F; =kHz ( I l l ) R1 = H; R2 = MeNH (IV) R1 = C I ; R , NH, ( V ) R1 = R z = H (VI) R1 = H; Rz =Me (V11) R1 Hi Rz C I

k,

y 2

ON> N

H

( X I I ) R = H. X S,Y = CH (XIII) R = NH,,X =S,Y =CH (XVI) R = H,X =O,Y = N (XVII) R NH,, X =O,Y:N

(XIV) R = H ( X V ) R =NH,

(XVII1)R =OH,X=O,Y=N

transferase, and cross resistance studies with these cells have provided indirect evidence that, in addition t o the purine analogues named above, 2-chloroadenine (IV) [ 126],4-aminoimidazo [4, 5-d] pyridazine (XI) [ 1271 , and 6-methylpurine (VI) are also converted to their nucleotides by this enzyme [13, 1281. Studies with ''C-labelled analogues showed that purine ( V ) was not anabolized to its ribonucleotide by mice 11291 , whereas 6-chloropurine (VII) and 6-methylpurine (VI) were (by S180 ascites cells in vivo [ 1301 and by rats [ 1 3 1 ] , respectively). 6-Chloropurine (VII) also reacted with PRPP in the presence of an extract from S180 cells [130]. Compounds more closely resembling hypoxanthine or guanine, such as 6-mercaptopurine (MP. XII) [ 1201, 4-hydroxypyrazolo [3, 4-d] pyrimidine (XIV) [ 1091, thioguanine (TG, X l l l ) [ 132],8-azaguanine (AzaG, XVII) [ 1 1 I ] , and 4-hydroxy-6-aminopyrazolo [ 3, 4-d] pyrimidine ( X V ) [ 1331 are substrates for hypoxanthine-guanine phosphoribosyltransferase [7, 8, 85a, I 12, 134, 1351, and the fact that 6-mercaptopurine-resistant neoplasms are cross resistant to 6-chloropurine [8] , whereas H.Ep.-2 cells lacking adenine phosphoribosyltransferase are not [ 1281, indicate that 6-chloropurine may also be anabolized by liypoxanthine-guanine phosphoribosyltransferase; although its binding constant is high [85a]. Low concentrations of ADP or ATP inhibit the formation of 8-azaguanylic acid by hog liver extract [ 1 121 , whereas pre-conditioning Ehrlich cells in vivo with a series of doses of non-radioactive 6-mercaptopurine stimulated

77

JOHN A. MONTGOMERY

the conversion of labelled 6-mercaptopurine to thioinosinic acid [ 136, 136a] Presumably 2- and 8-azahypoxanthine [ I 1 I . 1371 are also substrates for this enzyme, but 8-azaxanthine (XVIII) [ 1371 is not and is not anabolized in mammalian cells, which apparently convert xanthine to its nucleotide poorly be means of the hypoxanthine-guanine enzyme. On the other hand, bacteria have a xanthine phosphoribosyltransferase and can anabolize 8-azaxanthine [98] . In fact, bacterial cells lacking the hypoxantliine-guanine enzyme and resistant to MP and AzaC are sensitive t o 8-azaxanthine, which is converted to the nucleotide and thence t o 8-azaguanylic acid (981. Little quantitative work has appeared on the determination of the rate of conversion of the various purine analogues to their nucleotides with highly purified phosphoribosyltransferases from mammalian cells or any other source, but there would appear to be a rough correlation between the cytotoxicity of theseanaloguesand their ability t o serve as substrates ( t h e K; values for a number of purines and purine analogues have been determined [45a, M a ] but this value is not a measure of conversion t o nucleotide). Table 2.1 lists a number of purine analogues and an estimate of their ability to serve as substrates for the phosphoribosyl transferases. The ribonucleotides of 6-mercaptopurine. 6-thioguanine, 6-chloropurine, and purine have been prepared chemically for biochemical investigations [ 138-1 4 I ] . Table 2. I . Substrates for adenine phosphoribosyltransferase Estiriiated activitJ*

2-F:luoroadenine 2Chloroadenine 2-Aminoadenine (2, 6-DAP) 4-Aminopyrazolo [ 3, 4-d] pyrimidine 4-Aminoimidazo [ 4 , 5 4 1 pyridazine 2-Aza-adenine 8-Aza-adenine 6Chloropurine N-Aminoadenine N-Hydroxyadeninc N-ally ladenine

N-Me thyladcnine Purine 1-Methyladenine 2-Hydroxyadcnine

Good Fair Fair Fair Good Fair Poor Poor to none Poor to none Poor to none Fair to poor Poor to none None None

Substrates for hypoxanthine-guanine phosphori )syltransferase 6-Mercaptopurine 6-Thioguanine 8-Azaguanine 4-Hydroxypyrazolo [ 3, 4-d] pyrimidine 4-Hydroxy-6-aminopyrazolb [ 3, 4-d] pyrimidine 6Chloropurine

Good Good Fair Poor Poor ?

Table 2.2. SUBSTRATE SPECIFICITY OF ADENOSINE KINASE* A. Variation of substituents on purine ring

Mp moles nucleotide formed min? mg-’ p-otein

Cornpou nd Adenosine AdenoFine-1-oxidc 1-Methyladenosine 2-Fluoroadenosine 2-H ydrazinoadenosine

2-Aminoadenosine 2Chloroadenosine 2-Bromoadenosine 2-Methyladenosine 2-Methoxyadenosine 2-Dime thy laminoadenosine N-Methyladenosine N . N-dimethy ladenosine N-Aminoadenosine N-Allyladenosine N-Hydroxyadenosine Purine ribonucleoside 6-(Xlethylthio)purine ribonucleoside 6-Methylpurine ribonucleoside 6
148 597 158 349 68 54 10 10 10 10 10 571 235 216

< < < < <

55 30 510 487 468 4 24 309

B. Variations in the purine ring Tubercidin (7-deaza-adenosine) 1P-D-Ribo furanosyl-4-aminopyrazolo [ 3, 4-d ] pyrimidine 8-Aza-adenosine Formycin [ 7-amino-3~D-ribofuranosyl)pyrazolo[4,3-d] pyrimidine] l-Dea7a-6-me thylthiopurine ribonucleoside 3-Deaza-6-methylthiopurine ribonucleoside

643 460 279 149

< 20 < 10

C. Variation in the sugar moiety

9-[fl-DL-2Lu,3c~-dihydroxy-4~(hydroxymethyl)cyclopentyl] adenine Cordycepin ( 3 ‘deoxyadenosine) 2 ‘-1)eoxyadenosine 9P-L,-Ararbinofuranosyladenine 2-l:luoro-3:-deoxyadenosine 6-(Methy1 thio)purine-2’-deoxyribonucleoside 6-(Methylthio)-9$-D-arabinofuranosylpurine 6-(Methyl thio)-9$-L)-xyIofuranosyIpurine

*Liata f r o m reference 47.

?Data from reference 2 5 3

< < < < < <

60t 20 10 10 10 10 10 20

OH

OH

HobJ "kiJ Hok! OH OH

OH

(XXII)

(M I 1 1)

NH Me

I

How Rl

Rz

(XXIV) R2

I

The nucleoside kinases

Although the importance of pyrimidine nucleoside kinases has been recognized for some time, the importance of the purine nucleoside kinases, which phosphorylate purine nucleosides, has only recently become apparent. Although inosine and guanosine kinases have not been isolated and characterized [ 1421, evidence for their existence has appeared [82, 83, 143-1451. Adenosine kinase, which has been known for some time, has been purified 20-fold from homogenates of rabbit liver and Ehrlich ascites cells [48] and 175-fold from human epidermoid carcinoma No. 2 cells in culture [ 4 7 ] . In the latter case the preparation was free of adenosine deaminase and AMP kinase activities. Table 2.2 shows the substrate specificity of the kinase from H.Ep.-2 cells for purine nucleosides and ring analogue ribonucleosides. Most of the nucleosides used in this study were evaluated earlier for cytotoxicity [ 128, 1471. All of the nucleosides that are highly cytotoxic are good substrates for adenosine kinase, a finding in accord with other evidence that nucleotide formation is requisite for toxicity [8, 1 3 , 4 7 1 . At the same time it should be pointed out that, although compounds such as 9-/3-~1-arabinofuranosyladenine(XIX) [ 148, 1491 and 9-/3-D-xylofuranosyladenine (XX) [ 150, 15 11 are not good substrates for adenosine kinase (47, 481 and arc not very cytotoxic, they are, in fact, phosphorylated (48, 152, 1531 and are biologically active [ 154-157] , I t is particularly interesting that 6-mercaptopurine ribonucleoside (XXI) [ 1581 is an inhibitor of, but not a substrate for, the kinase [47] ; but 6(methy1thio)purine ribonucleoside (XXII) [ 159, 1601, resulting from the S-methylation of XXI, is an excellent substrate [47] and is readily phosphorylated in vivo [ 161, 1621 . The distribution of adenosine kinase in various normal and neoplastic tissues of man and mouse has been determined by using 6-(methylthio) purine ribonucleoside -3sSas substrate [ 162a].

Nucleoside phosph ok inases

In the preceding sections the conversion of purines and purine nucleosides to purine nucleoside monophosphates has been discussed. The monophosphates of adenosine and guanosine must be converted t o their di- and triphosphates for polymerization to RNA, for reduction to 2’-deoxyribonucleoside diphosphates, and for the many other reactions in which they take part. Adenosine triphosphate is produced by oxidative phosphorylation and by transfer of phosphate from 1,3-diphosphoglycerate and phosphopyruvate t o adenosine diphosphate. A series of transphosphorylations distributes phosphate from adenosine triphosphate to all of the other nucleotides. Two classes of enzymes, termed nucleoside monophosphokinases and nucleoside diphosphokinases, catalyse the formation of the nucleoside di- and triphosphates b y the transfer of the terminal phosphoryl group from adenosine triphosphate. Muscle adenylate kinase (myokinase)

81

(XXXIV) R = H (XXXV) R=CN

(XXXVI)

(XXXVII) R = H (XXXVIII) R-Me

82

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

specific for adenine nucleotides was the first phosphokinase t o be discovered. A large number of nucleotide phosphokinases have been described since with varying specificities toward nucleotide acceptors and phosphoryl donors. Further phosphorylation of nucleoside 5'-diphosphates has been attributed t o the action of nucleoside diphosphokinases, a representative of which has recently been purified 1400-fold from human erythrocytes and characterized [69-711. For detailed discussions of the phosphokinases the reader is referred t o a number of excellent reviews [38, 39, 68, 79, 1631. Although little work has been reported on the conversion of adenylic acid analogues to the di- and triphosphates by purified enzymes, a number of studies have been carried out on the metabolism of adenine and adenosine analogues by whole cells. A variety of adenine nucleosides are converted t o the di- and triphosphates: cordycepin (3 '-deoxyadenosine, XXIII) [ 164-1 691 , 3 '-deoxy3'-aminoadenosine (XXIV) [ 170-1 731 ,9-fl-D-xylofuranosyladenine (XX) [ I531 and 9-fl-D-arabinofuranosyladenine (XIX) [ 1521. Methylation of the 6-amino group, however,prevents the action of the phosphokinases so that only the monophosphates of N-methyladenosine (XXV) [ 1741 , N-methyl-:! '-deoxyadenosine (XXVI) [ 1751 and N-methyl-3'-deoxyadenosine(XXVII) [ 1761 are obtained, and the compounds are not substrates for crystalline rabbit muscle myokinase [ 1771 . Even so, purine ribonucleoside (XXVIII) [ 178, 1791 and 6-methylpurine ribonucleoside (XXIX) [ 13 I ] are both converted to mono-, di-, and triphosphates by rodents, and purine ribonucleotide was a substrate for myokinase [ 1801, but no evidence has been presented that 6-chloropurine ribonucleotide is further phosphorylated [ 1811. Substitution of a fluorine or an amino group at C-2 of adenylic acid does not interfere with phosphorylation and the higher phosphates of 2-fluoroadenosine (XXXI) [ 108, 1821, 2-aminoadenosine (XXXII) [ I 13, 183-1 851, and 2-methylaminoadenosine (XXXIII) [ 1 14, 1861 are formed. Changes in the ring also appear unimportant since the high phosphates of tubercidin (XXXIV) [ I 87-1901 . toyocamycin (XXXV) [191-193], 4-aminopyrazolo [3, 4-d] pyrimidine (VIII) [ 1 191 , and 2-aza-adenine (IX) [ 1851 have all been isolated from mammalian cells. Thus a surprising variety of changes can be made in the structure of adenylic acid without destroying the ability of the analogues to serve as a substrate for cellular phosphokinases, although bulk tolerance at C-6 would appear to be critical on the basis of the information at hand. 8-Azaguanosine 5 '-mono-, di-, and triphosphates have been isolated from the soluble fractions of micro-organisms [98, 194-1961 and of mouse tissues and neoplasms [ 1011 and 8-azaguanylic acid was shown to be a substrate for the phosphokinases of hog kidney and beef liver [ 1401 . 8-Azaguanosine mono-, di-, and triphosphates have also been synthesized chemically [138, 1 4 0 b l . Chromatographic evidence has been obtained for the formation of 6-thioguanosine di- and triphosphates in Ehrlich ascites cells [ 1971 and their formation is supported by the demonstrated incorporation of thioguanine into nucleic acids as thioguanylic acid (1981. The incorporation of both the a- and fl-anomers of 2'-deoxythioguanosine (XXXVI) [ 1991 into DNA without

JOHN A. MONTGOMERY

83

separation of the sugar from the base indicates that these nucleosides are substrates for a nucleoside kinase and that the nucleotides are substrates for phosphokinases [ 1441 . Proof of the incorporation of 6-mercaptopurine into RNA as the nucleotide, and thus as an integral part of the macromolecule, has not been presented [200] ,although its incorporation into DNAas 2’-deoxythioguanylicacid has been demonstrated [101] .Despite these reports, other investigators have been unable to demonstrate the incorporation of 6-mercaptopurine or thioguanine into nucleic acid or their conversion to the di- and triphosphates in viuo [8]. Furthermore, it seems abundantly clear that a number of adenylic acid analogues and 8-azaguanylic acid are readily phosphorylated and incorporated into nucleic acids of many types of cells, whereas the monophosphates of 6-mercaptopurine and thioguanine accumulate with little or n o further phosphorylation, and this rather clear difference in metabolism could explain the contrasting effects observed with these different types of compounds.

Nucleotide reductases

During the past 15 years data from experiments with different types of animal tissues and micro-organisms, using intact cells, crude extracts or purified enzymes, have firmly established the general occurrence of nucleotide reductases and have stressed their importance for DNA synthesis in essentially all types of rapidly growing cells [ 541 . It has been proposed that ribonucleotide diphosphates lose a hydroxide ion from C-2‘ to form a carbonium ion which is then sterospecifically reduced by a ‘hydride’ ion derived from thioredoxin 1541. Adenosine diphosphate and guanosine diphosphate (as well as uridine and cytidine diphosphates) are reduced in this manner. There is n o direct evidence for the reduction of analogue ribonucleoside diphosphates to the 2’-deoxyribonucleoside diphosphates by cells, but the incorporation of analogues, such as 6-mercaptopurine [201] or thioguanine [ 1981, into DNA as 2‘-deoxythioguanylic acid would seem likely to proceed by such a reduction, although a nucleoside deoxyribosyltransferase could be involved instead. Recently the triphosphates of tubercidin, toyocamycin. and sangvamycin [202a] were found to be substrates for the ribonucleotide reductase from bacteria [202b, c] . Tubercidin is also incorporated into DNA, presumably in the same way as thioguanine [ 190, 2021 .

Methylases and demeth ylases

The presence of small quantities of methylated purine and pyrimidine bases in nucleic acid of micro-organisms and animals has been widely documented. The methyl group is transferred frbm S-adenosylmethionine to these bases, and methylation occurs at the polynucleotide level rather than by transfer to the acid-

84

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

soluble nucleotide precursors with subsequent incorporation into DNA and RNA. The methylation of 2-aminoadenine in E. coli, however, takes place at the ribonucleoside phosphate level (XXXVII) to give 2-methylaminoadenosine mono-, di-, and triphosphates (XXXVIII), and the reaction is mediated by S-adenosylmethionine: 2-aminopurine N-methyltransferase [ 1 14, 1 15, 2031, which also catalyses the N-methylation of 2-aminoadenine itself, 2-aminopurine, 2-amino-6-methylpurine, and 2-amino-8-aza-adenine [203] . An S-adenosylmethionine-sulphydryl transmethylase system, present in tissues of a variety of mammalian species, particularly in the kidney, catalyses the S-methylation of 2-thiopyrimidines and 6-thiopurines [ 2041 . Methylation of 6-mercaptopurine to 6-(methy1thio)purine (XXXIX) has been found t o occur in the rat [205], and -6-mercaptopurine and its ribonucleoside are converted t o 6-(methylthio) purine ribonucleotide (XL) in H.Ep.-2 cells [206]. This latter methylation presumably occurs at the nucleotide level; otherwise 6-mercaptopurine ribonucleoside would inhibit cells resistant to 6-mercaptopurine. Although demethylation, which occurs in the liver, is normally considered t o be a catabolic process, it may result in conversion of an inactive form of a drug to the active form. Thus 6-(methy1thio)purine (XXXIX) is demethylated by the rat to 6-mercaptopurine [205]. This demethylation occurs in the liver microsomes and is an oxidative process which converts the methyl group t o formaldehyde [204, 2071. The 1-methyl derivative of 4-aminopyrazolo[3,4-d] pyrimidine (XLI) is demethylated slowly, but 6-mercapto-9-methylpurine (XLII) not at all [208]. The N-demethylation of puromycin (XLIII) [209, 2101, its aminonucleoside (XLIV) [2 1 11 ,and a number of related compounds, including N-methyladenine and N,N-dimethyladenine, occurs in the liver microsomes of rodents [212]. In the guinea-pig the rate-limiting step in the metabolism of the aminonucleoside appears to be the demethylation of the monomethyl compound, which is the major urinary metabolite [213] . The relationship of lipid solubility t o microsomal metabolism [21.4], and the induction of these demethylases in rats by pre-treatment with varibus drugs have been studied [215]. Apparently N-demethylations can occur in cells other than liver microsomes, since the demethylation of N-methyl-3 -deoxyadenosine (XXVII) to 3'-deoxyadenosine (XXIII) in K B cells [216] and of the aminonucleoside of puromycin (XLIV) t o 3 '-amino-3'-deoxyadenosine (XXIV) in L cells have been reported [217].

Other anabolic enzymes

Evidence for the formation of analogues of nicotinamide adenine dinucleotides, in which the adenine moiety is replaced by 2-aminoadenine (XLV) and 7-deazaadenine (XLVI), from 2-aminoadenine [218] and tubercidin [202] has been presented, but the details of these biosyntheses have not been investigated. Inosinic acid dehydrogenase converts inosinic acid to xanthylic acid which in

JOHN A. MONTGOMERY

85

OH OH (XLV) R=NH, (XLVI) R=H;

OH

OH

(XLVII)

; X=N

X=CH

OH (XLVIII)

turn is aminated to guanylic acid. The formation of 6-thioxanthylic acid (XLVII) from 6-mercaptopurine in Ehrlich cells [219] would seem likely t o occur via the dehydrogenation of thioinosinic acid. Cordycepin- I-oxide (XLVIII) is activated in Ehrlich ascites cells by reduction t o cordycepin [220] and a similar N-oxide reduction probably underlies the action of 6-mercaptopurine-3-oxide(XLIX) [221] . Catabolism Phosphatases and nucleotidases

In addition to the enzymes that catalyse the formation of nucleotides and polynucleotides, a large number of catabolic systems exist which operate at all levels of the internucleotide pathways. The ribonucleases and deoxyribonucleases that degrade polynucleotides are probably not significantly involved in purine analogue metabolism, but the enzymes which dephosphorylate nucleoside 5 '-monophosphates are known t o attack analogue nucleotides and may be of some importance t o their in vivo activity. Phosphatases of low specificity are abundant in many tissues [38], particularly the intestine [29]. Purified mammalian 5 '-nucleotidases hydrolyse only the nucleoside 5 'monophosphates [ 281 and

86

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

could be important to the activity of purines, such as 6-mercaptopurine, that are anabolized primarily to the 5 ‘-monophosphate level, since dephosphorylation must occur prior to cleavage of the nucleoside to the free base by a phosphorylase. A number of analogue nucleotides have been shown to be substrates for calf intestine phosphatase or snake venom S’-nucleotidase [ 112, 161, 2181.

Nucleoside phosphorylases and hydrolases

Nucleoside phosphorylases that catalyse the reversible cleavage of purine nucleosides to the free bases and ribose-I-phosphate are found in most cells, although a phosphorylase that will cleave adenosine has so far been identified only in bacteria. Recent studies have shown that ribo- and 2’-deoxyribofuranosyltransferase activity is associated with phosphorylase activity [ 19, 23.. 2221 and that both activities reside in one enzyme, which can be converted from one form to the other by substrate o r product binding [ 2 0 ] . Upon crystallization of the enzyme from human erythrocytes a marked decrease in the ribosyl transfer reaction was observed [ 21 b] . Purified enzyme extracts have been used to biosynthesize the ribonucleosides of 6-mercaptopurine, 8-azaguanine. 8-aminoadenine, 4-hydroxypyrazolo [ 3 , 4 - d ] pyrimidine, 6-methylpurine [ 15, 20, 2 1, 2231, and the 2’-deoxyribonucleosides of 6-mercaptopurine, 6-methylpurine, 8-azaguanine, and 2-aminoadenosine [ 15, 2241 ; since in vivo and in whole cells the equilibrium is shifted toward purine formation [ 18, 2251, these conversions can be viewed as evidence that these purine analogue nucleosides are subject to cleavage in whole cells. Ribonucleoside derivatives of 6-mercaptopurine and other purine bases are rapidly cleaved by Ehrlich ascites cells in vitro [ 2 2 6 ] . Nucleoside phosphorylase troin human erythrocytes cleaves 6-mercaptopurine ribonucleoside readily, but 6-methylthiopurine ribonucleoside only slowly [21 a] . In another study the phosphorolytic cleavage of the ribo- and 2‘-deoxyribonucleosides of adenine, 6-mercaptopurine, and thioguanine was observed [ 2 2 7 ] . Changes in the sugar moiety to xylose, arabinose, 3’-deoxyribose, 5 -deoxyallose. or 6’-deoxyallose preventedcleavage [ 1 73,2271 . Furthermore, 9-0-D-arabinofuranosylhypoxanthine and 9Q-~-xylofuranosylhypoxanthine, resulting from the deamination of the corresponding adenine nucleosides, are excreted in the urine of treated mice indicating that these nucleosides are not substrates either [ 152, 1531. The phosphorolysis of cordycepin, 3’-amino-3‘-deoxyadenosine, 2-fluoroadenosine 2-aminoadenosine, and 9Q-D-arabinofuranosyladenine, but not tubercidin, by an extract from S. faecalis [228] and of cordycepin by B. subtilis [229] has been reported. Studies with resistant H.Ep.-2 cell lines lacking adenosine kinase and adenine phosphoribosyltransferase indicate that 2-fluoroadenosine [ 128. 1471, 2-fluoro-3 -deoxyadenosine [ 1471 , and 6-methylpurine ribonucleoside [ 2301 are also cleaved to some extent in these cells, whereas tubercidin, 4-aminopyrazolo [ 3 , 4 - d ] pyrimidine ribonucleoside [23 1, 2321 , N-allyladenosine [ 2 3 3 ] ,

JOHN A. MONTGOMERY

87

N-aminoadenosine [ 1741 , 2-chloroadenosine [ 176. 2341 , 6-chloropurine ribonucleoside [ 178,235,2361 ,purine ribonucleoside [ 1781, and the (alkylthi0)purine ribonucleosides [I601 d o not appear t o be cleaved t o any appreciable extent [ 128, 1471. Irreversible hydrolytic cleavage of nucleosides catalysed by hydrolases has been observed in bacteria but not in mammalian cells [ 4 6 ] .

Dearninases

Adenine aminohydrolase has been found in micro-organisms, but not in mammalian cells, and the substrate specificities of the enzymes from Azotobacter vinelandii and Candida utilis were found t o be similar [55, 561. Among other purines, 2-aminoadenine, N-aminoadenine, and 6-chloropurine were found to be substrates 1551 Adenosine aminohydrolase (adenosine deaminase) is found in all types of cells and is apparently an important catabolic enzyme for the regulation of cellular metabolism. It has been isolated from a number of sources and the substrate specificities of the various enzymes are similar, since a low degree of specificity R

y

d

HOCH2

2

0

OH OH

OH R

(L)

RzNHNH,,R’:H

( L V I ) R z H , Y:N

(Li)

R:NHOH,

( L V I I ) R =OH, Y: CH

(LII)

R = O M e , R’zH

R’:H

(LIfI) RzNH,, R’zOH ( L I V ) RzNH,,

R’ZCi

( L V ) R = NH,,

R’ZMeS

is generally observed. Not only is the 6-amino group of adenosine hydrolysed, but the methylamino [59, 63, 237, 237a], hydrazino [ 5 9 ] , hydroxylamino [61, 63, 237a], methoxy [59, 2371, and chloro groups [59, 61, 6 3 , 64, 237, 238-2411 are all hydrolytically removed from the corresponding 6-substituted purine ribonucleosides (XXV, XXX, L-LII). 2-Fluoroadenosine (XXXI) has been reported by some investigators [ 2 4 0 , 2 4 2 ] to be very slowly deaminated and by others not a t all [239, 241 1 . Crotonoside (LIII), 2-chloroadenosine (LIV), and 2-(methylthio)adenosine (LV) are neither substrates nor inhibitors of adenosine

**

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

deaminase [ 2431 , but 2-aminoadenosine (XXXII) is deaminated to give guanosine [57, 61, 64, 237a, 2411. Changes in the purine moiety have an unpredictable effect on substrate specificity: 8-aza-2 -deoxyadenosine (LVI), 8-aza-adenosine [237a] , and formycin (LVII [244-2461 are deaminated [247-2491 whereas tubercidin (XXXIV) is not 2411. Though changes in the sugar moiety effect binding t o adenosine deaminase, many adenosine analogues are fair substrates. Thus, 2'-deoxyadenosine [ 571 , 3'-deoxyadenosine (XXIII) 157, 64, 239, 240, 248, 2501 , 3'-amino-3'-deoxyadenosine (XXIV) [216, 239-2411, 94-D-xylofuranosyladenine (XX) [227, 239, 240, 2501 , 9$-D-arabinofuranosyladenine (XIX) [227, 239, 240, 2501, 4'-thioadenosine [240, 2511 and ~ - [ P - D L - ~ O I . 301-dihydroxy-4~-(hydroxymethylcyclopentyl] adenine [ 252, 2531 are all deaininated and at rates that appear to affect their activity (since the inosine analogues that are formed are generally inactive). N-Methyladenosine (XXV) 1227, 239, 2501 , N-methyl-2'-deoxyadenosine (XXVI) [ 571 , 3 '-amino-N-methyl-3'-deoxyadenosine [ 2541 , and 6-methoxypurine ribonucleoside (LII) [63, 234, 2411 are all good inhibitors of adenosine deaminase and N-methyladenosine (XXV) has been used in combination with 94-D-arabinofuranosyladenine (XIX) t o increase its activity [255] . A number of 2-substituted N-methyladenosines [6 11 and 9-substituted adenines (see reference 256 and earlier papers by Schaeffer) are also inhibitors of the enzyme. Although adenylic acid deaminase is a well-known enzyme that has been isolated in crystalline form, little work has been reported on its substrate specificity; evidence for the deamination of 4-aminopyrazolo [3,4-d] pyrimidine ribonucleotide has been presented [ I 181, but it is not known if it catabolizes any of the other intracellularly formed adenylic acid analogues. Guanine aminohydrolase (guanine deaminase) was discovered in rabbit liver and is present in the tissues of other animals, in certain bacteria and in yeast [88]. The conversion of 8-azaguanine to 8-azaxanthine in vitro [257] and in vivo [258] is catalysed by guanine deaminase. Thioguanine is also a substrate for the enzyme purified from rabbit liver [88] .4(5)-Aminoimidazole-5(4)-carboxamide inhibits guanine deaminase [259] ; and, when coadministered with 8-azaguanine, it increases the anabolism of the latter 12601, resulting in increased toxicity and antitumour effectiveness with no net benefit. A search for an inhibitor that will selectively inhibit guanine deaminase of cancer cells has been undertaken [ 26 1 1.

r'

Oxidases

The literature on xanthine oxidase [84] and its companion catabolic enzyme uricase [87] has been extensively reviewed. Many purine analogues, with the exception of most 9-substituted purines [262], serve as substrates for xanthine oxidase both in vitro and in vivo, and if the product is a substrate for uricase, in species that possess this enzyme, the ultimate product is allantoin (LVIII). Thus 2-aminoadenine [S] ,N-methyladenine [ 1221, and purine [ 1291 are all catabolized

R

R

A )*

(LXX) R (LXXI)R

H2CQ C

H =H

=NH,

R

l+oH OH OH

(LXXII)R = Et, Bu

(LXXI I1 1

90

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

by rodents to allantoin, although intermediate oxidation products are also found. 6-Chloropurine is oxidized in vitro [263] and in vivo [264] to ‘6-chlorouric acid’ (LIX), which inhibited uricase in slices and homogenates of rat liver [264], but the major catabolic pathway for this analogue in the rat involves the loss of the 6-chloro group [264] . Other cytotoxic 6-substituted purines are also substrates for xanthine oxidase [ 265,265al. 4-Aminopyrazolo [3,4-d] pyrimidine is oxidized to 4-amino-6-hydroxv~vrazolo[3, 4-d] pyrimidine (LXI) 1266, 2671 , and its deamination product, 4-hydroxypyrazolo [3,4-d] pyrimidine, is oxidized to 4, 6-dihydroxypyrazolo [3,4-d] pyrimidine (LXII) [267,267a] . The catabolism of the thiopurines is complicated by the fact that in addition to ring oxidation, methylation and oxidation of the mercapto group also occurs. Since this subject has been amply reviewed [5, 12,2681 ,it will be discussed briefly here; the reader is referred to the reviews for most of the references to the original literature. Both 6-mercaptopurine and thioguanine, after deamination to 6-thioxanthine (LXIII), are further oxidized to 6-thiouric acid (LXIV), and thence to sulphate, and unidentified products. In the rat 6-mercaptopurine is in part methylated to 6-(methylthio)purine (see above), which is converted to hypoxanthine and inorganic sulphate. In rodents sulphate arises from 6-mercaptopurine via thiouric acid (LXIV), but in man, lacking uricase, it is probably formed via 6-(methy1thio)purine. Since 6-(methy1thio)purine ribonucleoside is not cleaved by nucleoside phosphorylase, its catabolism (and that of the nucleotide) must involve demethylation, which is known to occur in the case of 6-(methylthio)purine, in the rat (see above). S-Methylation of some form of 6-mercaptopurine in man has been established by the identification of 6-(methylsulphinyl)-8-hydroxypurine (LXV), ‘6-(methy1thio)uric acid’ (LX), and 6-(methylthio)-8-hydroxy-N-glucuronide (LXVII). The oxidation of 6-(methy1thio)purine to 6-(methylthio)-8-hydroxypurine (LXVI) is mediated much more rapidly by rabbit liver aldehyde oxidase than by xantliine oxidase, and the oxidation is not inhibited by 4-hydroxypyrazolo[3,4-d] pyrimidine [269] , which is known t o be an effective inhibitor of xanthine oxidase, and consequently, of the oxidation of 6-mercaptopurine [ 12,2681 . S-Methylation of thioguanine t o 2-amino-6-(methylthio)purine (LXVIll), which is deaminated to 2-hydroxy-6-(methyIthio)purine (LXIX) and may give rise to inorganic sulphate, the major end product of thioguanine catabolism, occurs to a much greater extent in man than in the mouse, in which deamination and oxidation to thiouric (and thence to sulphate) is the main pathway [ 12, 2681 6-Mercaptopurine and thioguanine have been converted t o S-heterocyclic derivatives (LXX and LXXI) [270, 2711 that are protected from rapid methylation and oxidation in vivo, and are split nonenzymatically t o the purinethiones by sulphydryl groups. Certain S-alkyl derivatives [272] of the purinethiones are metabolized like 6-(methylthio)purine, but the 9-alkyl derivatives [273-2751 are metabolized only to some extent t o their S-glucuronides (LXXII) [276, 2771 and are not affected by xanthine oxidase.

JOHN A. MONTGOMERY

91

Summary

2-Fluoroadenine, 2-chloroadenine, 2-aminoadenine, 2- and 8-aza-adenines, 4-aminopyrazolo [3, 4-d] pyrimidine and 6-methylpurine are converted to their ribonucleosides by adenosine phosphoribosyltransferase, and their ribonucleosides are converted to the ribonucleotides by adenosine kinase; most of the ribonucleotides are then converted to the di- and triphosphates. N-Aminoadenine, N-hydroxyadenine, N-methyladenine, purine, 7-deaza-adenine, and 7-aminopyrazolo[4, 3-d] pyrimidine are either not substrates or are very poor substrates for the phosphoribosyltransferase, but their ribonucleosides are excellent substrates for the kinase. The ribonucleotides of purine, 7-deaza-adenine and 7-aminopyrazolo [4, 3-d] pyrimidine are further phosphorylated to the di- and triphosphates, but the N-substituted adenylic acids are not. The nucleosides of adenine-cordycepin, 3'-amino-3'-deoxyadenosine, 9-fl-~-arabinofuranOsy1adenine, and 9-fl-D-xy~ofuranosy~adenine-are phosphorylated with varying degrees of ease and the resulting nucleotides are readily converted t o the di- and triphosphates. With the exception of 2-fluoro- and 2-chloroadenosine, tubercidin, and, obviously, purine and 6-methylpurine ribonucleosides, the adenosine analogues, whether they are presented to the cell as such or result from dephosphorylation of the nucleotide, are catabolized by adenosine deaminase to the corresponding inosine analogues, which, with some exceptions, have a low level of biologic activity, although the rate of catabolism varies a great deal. Most of the hypoxanthine analogue ribonucleosides are cleaved by nucleoside phosphorylase to hypoxanthine, which can be further degraded by xanthine oxidase and uricase. Most of the other hypoxanthine nucleosides are not further catabolized. Analogues of hypoxanthine or guanine such as 6-mercaptopurine, thioguanine, and 8-azaguanine are converted to their nucleotides by hypoxanthine-guanine phosphoribosyltransferase, but their ribonucleosides appear to be cleaved by nucleoside phosphorylase to the free bases rather than phosphorylated t o the ribonucleotides. 8-Azaguanylic acid is largely converted to the di- and triphosphate, whereas thioinosinic acid and thioguanylic acid for the most part are not. 8-Azaguanine and thioguanine are catabolized by guanase, and both the purinethiones are degraded by xanthine oxidase. Methylation and demethylation is an important factor in the activity of the thiopurihes and certain adenine or adenosine analogues also. MECHANISM OF ACTION

Most biologically active purines or purine nucleosides must be anabolized to 5'-nucleotides t o exert their effects. The 5 hucleotides are in some cases further phosphorylated to the di- and triphosphates and the number of potential sites of action of these compounds is obviously greater than the number of potential sites of action of compounds metabolized only to the monophosphate level. But even

=

Glycine +ATP

OH OH

OH OH

OH OH

I

I

OH OH

1

,Aspartate

Figure 2.4. The de novo synthesis of inosinic acid

AOP +HPOi-

CoF

OH OH

I

OH AH

JOHN A. MONTGOMERY

93

the latter compounds have been shown to be capable of interfering at multiple sites. It is the concensus of opinion today that, in fact, there may be no one metabolic block that can explain all the biological effects of most of the purine analogues and their metabolites. In the sections that follow all the biological effects of the various analogues that have been observed are discussed with emphasis on those that, in the opinion of the author, appear to be the most important to the drug action of these compounds. Enzyme inhibition The de novo synthesis of inosinic acid

The elucidation of the steps by which cells synthesize inosinic acid d e novo for purine nucleotide interconversions is largely due to the brilliant work of Buchanan and his co-workers [ 6 5 ] . The biosynthetic scheme as it is currently envisioned is shown in Figure 2.4. The first step of this sequence, which is not unique to d e novo purine nucleotide biosynthesis, is the synthesis of 5-phosphoribosylpyrophosphate (PRPP) from ribose-5-phosphate and adenosine triphosphate. Phosphoribosylpyrophosphate synthetase, the enzyme that catalyses this reaction [ 2781 , is under feedback control by adenosine triphosphate [279] . Cordycepin interferes with thede novo pathway [229, 280, 2811, and cordycepin triphosphate inhibits the synthesis of PRPP in extracts from Ehrlich ascites tumour cells [282]. Formycin [ 2831 ,probably as the triphosphate, 9-~-D-xylofuranosyladenine[ 1571 triphosphate, and decoyinine (LXXIII) [284-2861 (p. 89) also inhibit the synthesis of P W P in tumour cells, and this is held to be the blockade most important t o their cytotoxic action. It has been suggested but not established that tubercidin (triphosphate) may also be an inhibitor of this reaction [ 1931. The first irreversible reaction peculiar t o the d e novo purine nucleotide pathway, the formation of 5-phosphoribosylamine from PRPP and glutamine,,is also subject t o end product or feedback inhibition by the natural purine nucleotides, inosinic, guanylic, and adenylic acids and their higher phosphates [287] ; The regulation of phosphoribosylpyrophosphate amidotransferase, the enzyme that catalyses this reaction [ 2 8 8 ] , is allosteric in nature [ I ] . There are at least two separate allosteric binding sites on the' enzyme, one for inosineguanosine phosphates and one for adenosine phosphates, and both types of compound can act on the enzyme simultaneously [287, 2891. The inhibition is competitive against PRPP and mixed competitive-noncompetitive against glutamine. The ribonucleotides of the unnatural purine analogues, 6-mercaptopurine, thioguanine, and 4-hydroxypyrazolo[3, 4-d] pyrimidine, are good inhibitors of this enzyme [287, 2901. This feedback inhibition may be primarily responsible for the in vitro [291] and in vivo [290, 2921 activity of the purinethiones (for a review of the effects of anticancer agents on biochemical control mechanisms see reference 2).

94

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

The effect of 6-mercaptopurine on the incorporation of a number of I4Clabelled compounds into soluble purine nucleotides and into RNA and DNA has been studied in leukemia L1210, Ehrlich ascites carcinoma, and solid sarcoma 180. At a level of 6-mercaptopurine that markedly inhibited the incorporation of formate and glycine, the utilization of adenine or 2-aminoadenine was not affected. There was n o inhibition of the incorporation of 5(or 4)-aminoimidazole4(5)-carboxamide (AIC) into adenine derivatives and n o marked or consistent inhibition of its incorporation into guanine derivatives. The conversion of AIC to purines in ascites cells was not inhibited at levels of 6-mercaptopurine 8-20 times those that produced 50 per cent or greater inhibition o f d e novo synthesis [292]. Furthermore, AIC reverses the inhibition of growth of S180cells (AH/5) in culture by 6-mercaptopurine [293]. These results suggest that in all these systems, in vitro and in vivo, the principal site a t which 6-mercaptopurine inhibits nucleic acid biosynthesis is prior to the formation of AIC, and that the interconversion of purine ribonucleotides (see below) is not the primary site of action [292]. Presumably, this early step is the conversion of PRPP to 5-phosphoribosylamine inhibited allosterically by 6-mercaptopurine ribonucleotide (feedback inhibition is not observed in cells that cannot convert 6-mercaptopurine to its ribonucleotide [244]. Yet another piece of evidence that supports the allosteric inhibitor theory for 6-mercaptopurine was obtained from a study of the inhibition of adenocarcinoma 755 and sarcoma 180 cells in culture [295]. Ad755 cells are more sensitive t o inhibition by 6-mercaptopurine than S180 cells, and this sensitivity appears to be unrelated t o the ability of these cells t o anabolize 6-mercaptopurine to its ribonucleotide, since both cells exhibit this enzymatic capacity. But 6mercaptopurine is ten times more effective in inhibiting de novo synthesis in Ad755 cells than in S180 cells, whereas the EDs0 for growth inhibition is four times more for SI 80 than Ad755 cells. Studies on the mechanism of action of 6-mercaptopurine are complicated by the fact that its anabolic product, thioinosinic acid, is further metabolized by oxidation to 6-thioxanthylic acid [219] and by methylation t o 6(methy1thio)purine ribonucleotide [206, 2961 . the effects of which could be even more important than those of thioinosinic acid itself, since the methylthio compound is about 20 times as potent as a feedback inhibitor [289]. I t has been suggested that thioguanine’s multistep inhibition, one step of which is the inhibition of phosphoribosylpyrophosphate amidotransferase, results in a profound lowering of the intracellular concentration of guanine nucleoside phosphates and that this depletion causes a marked depression in cellular metabolism that presumably would lead t o cell death [91]. A number of other purines, purine ring analogues, and their ribonucleosides have been evaluated as feedback inhibitors [13, 173, 294, 297, 297a] by a modification [294] of the method of LePage andco-workers [291,298,299,300]. This method utilizes azaserine t o isolate the first few steps of the de novo pathway in whole cells by the specific blockade of the conversion of formylglycinamide

JOHN A. MONTGOMERY

95

ribonucleotide (FGAR) to the corresponding amidine. Compounds that can, after conversion to their nucleotides, act as feedback inhibitors of purine biosynthesis decrease the amount of FGAR accumulation in azaserine-treated cells; the extent of this decrease is a measure of the ability of an agent to inhibit by feedback. Using this technique,the EDso values for FGAR-accumulation inhibition in H.Ep.-2 cells for a number of purine analogues have been obtained. Table 2.3. CORRELATION OF THE CYTOTOXICITY OF CERTAIN PURINE ANALOGUES AND THEIR RIBONUCLEOSIDES WITH THEIR INHIBITION O F FGAR ACCUMULATION Base Nome cy t o toxicity

2-Fluoroadenine 1.4 2Chloroadenine 11.0 2-Aminoadenine 20 6-H ydrazinopurine 20 N-Methyladenine 1-Methyladenine >670 Adenine-1-oxide N-Allyladenine 4-Aminopyrazolo[ 3,441 pyrimidine 1.2 4-Aminoimidazo[ 4,541 pyridazine 23 7-Deaza-adenine? 20 8-Aza-adenine 21 2-Axa-adenine 5.2 Purine 80 6Chloropurine 214 6-Methylpurine 1.6 6-Methoxypurine >670 8-Azaguanine 3.4 6-Mercaptopurine 1.2 6-Thioguanine 0.14 6-(Methy1thio)purine 4 00 6-(Benzylthio)purine

>

EDSO(p molell) * ___Ribori leoside -FGAR FGAR Accumu. c y toAccumutoxicity latiori lation 1.8 16.6 35 1.8 14 18 1.8 6.4 0.1 >56 0.05 0.8

2.3

> 38 70 > 37 34

222 15

> 74 7.4 42 322 >400

> 33 0.6

ca

0.08 15.4 0.38 65 1.2 1.2 1.3

2.9 11.8 < 37 7.3 7.5 14.7 < 7.4 36 3.8

< < <

>so

3.8 3.7

< <

3.8 <7.7 3.7 10.4 >32 3.0 0.6 <0.3 15

<

<

* See text. 7 The aglycone of tubercidin.

A comparison of EDs0 values for feedback inhibition and for growth inhibition in H.Ep.-2 cells in culture is shown in Table 2.3. It is readily apparent that t o r most of the purine analogues listed, the correlation between feedback inhibition and cytotoxicity is good. The few discrepancies may be due to the fact that these particular compounds are not ieedback inhibitors, but metabolism (or lack of it) of the analogue in question may be important. In comparing these ED5, values, the difference in experimental conditions for cytotoxicity determination (long

96

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

exposures to drug, 96-1 20 h) and feedback inhibition (2-5 h exposure to d r u d must be considered. If both inhibition of growth and FGAR accumulation require the formation of a fraudulent nucleotide and its maintenance at a minimum intracellular concentration, discrepancies may result. Thus, although 4-aminopyrazolo [3, 4-d] pyrimidine does not appear to be a feedback inhibitor, its ribonucleoside is, and yet they are both converted intracellularly t o the ribonucleotide, which appears to be the true inhibitor. It seems reasonable to assume that long exposure to the base allows a sufficient conversion to nucleotide to inhibit cell growth, but if 4-aminopyrazolo[3, 4dlpyrimidine is not a good substrate for adenine phosphoribosyltransferase, enough nucleotide may not be formed in the short exposure period t o show feedback inhibition. On the other hand, the ribonucleoside is known to be an excellent substrate for the kinase (see above);soit is more cytotoxic than the base and is a good feedback inhibitor. In contrast, 8-azaguanine and its ribonucleoside [301] are about equitoxic, but neither acts as a feedback inhibitor and their biologic activity is most likely due to a different phenomenon (i.e., nucleic acid incorporation, see below). In addition to the analogues listed in Table 2.3, cordycepin [302] .3'-amino3'-deoxyadenosine [ 1731, and formycin [303] can inhibit the de nova pathway by blocking the phosphoribosylpyrophosphate amidotransferase. Thus, a number ofpurine analogues-after anabolism to nucleoside phosphates-can act as feedback inhibitors, and this inhibition may be the primary cause of their cytotoxicity.

The Salvage' path ways

Although cells synthesize inosinic acid de now for purine internucleotide conversions, they also utilize exogenously supplied adenine, hypoxanthine, and guanine by converting them directly to their ribonucleotides (see section or1 phosphoribosyltransferases, p. 74). The utilization of adenosine appears to involve this reaction also since it is largely deaminated to inosine which is cleaved to hypoxanthine, even though it can be phosphorylated directly to adenylic acid [ 147,3041 . Since inosine-guanosine kinase activity is low or missing in many cells, the utilization of inosine and guanosine must also involve cleavage to the bases, which are converted by the phosphoribosyltransferpe to ribonucleotides. Thus, these salvage pathways are of importance t o the economy of cells and their inhibition could be of some consequence. In this connection, a specific loss of hypoxanthine-quanine phosphoribosyltransferase activity has been observed in cases of gout associated with the overproduction of uric acid [305] and in a neurological disorder, the Lesch-Nyhan syndrome [306]. Although a causa! relationship has not been established, these disorders could be indicative of the importance of these enzymes. 6-Mercaptopurine is a competitive inhibitor of phosphoribosyl transfer from PRPP to guanine and hypoxanthine, [85a] but does not affect the reaction with

JOHN A. MONTGOMERY

97

adenine, which is catalysed by a different enzyme [ 102, 3071. 6-Mercaptopurine also inhibits the cleavage of inosine and guanosine preventing their utilization by the phosphoribosyltransferases, although in bacterial cells this inhibition results in greater utilization of the ribonucleosides indicating their phosphorylation by a kinase [308] . Competitive inhibition of adenine phosphoribosyltransferase by certain adenine analogues has been observed, but only at relatively high levels of these analogues [45a, 308a] . Even so this enzyme is inhibited competitively, with respect to PRPP, by AMP, ADP, and ATP [42, 43, 1031, and thus it is another potential site for the action of adenine analogues that are converted to ribonucleoside phosphates, particularly ones that are not incorporated into nucleic acid or easily degraded. Purine nucleotide in terconversions

The conversion of inosinic acid t o adenylic acid is a two-step process [651 The first step, the conversion of inosinic acid to adenylosuccinic acid, is mediated by adenylosuccinate synthetase [65] , which is inhibited by 6mercaptopurine ribonucleotide 1309-31 11 in a non-competitive manner, although the exact nature of this inhibition is not known [67]. The second step of the sequence, the conversion of adenylosuccinic acid t o adenylic acid, is also inhibited by 6-mercaptopurine ribonucleotide [66, 31 11, and in this case the inhibition is competitive with respect to the substrate [67]. Although tumour growth inhibition by 6-mercaptopurine has been attributed to the inhibition of the conversion of inosinic acid to adenylic acid [3@, 310, 3121, probably at the first step, this conversion by cell-free extracts from the exquisitely sensitive tumour adenocarcinoma 755 was inhibited only at high levels of 6-mercaptopurine ribonucleotide (3 131 . Furthermore, hadacidin (N-formylhydroxyaminoaceticacid) is an excellent inhibitor of adenylosuccinate synthetase [314, 3151, and yet it has little antitumour activity and is not cytotoxic, showing that this inhibition may be relatively unimportant to cells. Although 6-chloropurine ribonucleotide has little inhibitory effect on the enzymatic conversion of inosinic acid to adenylic acid, it markedly inhibits the conversion of inosinic acid to xanthylic acid by inosinic acid dehydrogenase [3 1 6 , 3171 . Evidence has been presented that the nucleotide combines reversibly with the enzyme in place of the normal substrate and then reacts, at its 6-carbon, with a sulphydryl group of the enzyme active site to form a stable thioether thus irreversibly inactivating the enzyme [3 17,3 17aJ . 6-Mercaptopurine ribonucleotide inhibits the enzyme in a similar manner through the formation of a disulphide bond between the mercapto group of the enzyme and the mercapto group of the nucleotide. In this case the reaction is reversible in the sense that the addition of glutathione slowly reactivates the inhibited enzyme by cleavage of the disulphide bond. If the enzyme is fully activated by glutathione before the inhibitor is added, the inhibition is purely competitive and a sevenfold excess of thioinosinic acid over inosinic acid is required for 50 per cent reduction in initial rate.

98

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

6-Chloropurine ribonucleotide and thioinosinic acid also react covalently with GhlP reductase, the enzyme that converts guanylic acid t o inosinic acid and ammonia [3 181 . The significance of these in uitro enzyme inhibition studies is uncertain, in view of the evidence that has been presented concerning the sensitivity of cancer cells t o feedback inhibition by these nucleotides. O n the other hand, 6-chloropurine inhibits the de nouo biosynthesis of nucleic acid guanine but not of nucleic acid adenine in sarcoma 180 ascites cells [3 191 . The second step in the conversion of inosinic acid to guanylic acid is the aminolysis of xanthylic acid with glutamine by xanthosine-5 ’-phosphate aminase [ 6 5 ] . Thisaminase, isolated fromE. coli B, is inhibited allosterically b y adenosine and adenylic acid [320], and it is also inhibited by psicofuranine (9-/3-Dpsicofuranosyladenine) (LXXIV) [284, 32 1-3261 , which apparently is not y

2

(LXXV) R -A -0-arabinofuranosyl,R’=H (LXXVI) R - Bu, R’-NH, ILXXIV)

metabolically activated [327] ; and this inhibition, which takes place only in the presence of xanthylic acid and inorganic pyrophosphate, is irreversible [328] . Psicofuranine(LXX1V) inhibition of an altered aminase from a resistant E. coli B mutant is readily reversible and dependent only on pyrophosphate. These observations indicate that psicofuranine inhibition is a two-step process. The first step-a pyrophosphate dependent, reversible reaction between the inhibitor and the enzyme-is followed by an irreversible step requiring xanthylic acid. The altered aminase is presumed t o have lost the ability t o undergo the second step. Binding studies show that the primary interaction of the aminase with psicofuranine is non-competitive and s u a e s t that it is allosteric at a site normally occupied by a metabolic regulator [89, 3281 . The psicofuranine-inhibited aminase cannot catalyse the formation of the adenyl xanthylic acid intermediate from adenosine triphosphate and xanthylic acid, nor can it catalyse its aminolysis t o guanylic acid [90]. The related antibiotic decoyinine (angustmycin A , LXXIII) [285] also inhibits xanthosine-5 ‘-phosphate aminase [329] , presumably in the same manner as psicofuranine (LXXIV), but less effectively. There is evidence that psicofuranine (LXXIV) and decoyinine (LXXIII) may be interconvertible in cells 13291.

JOHN A. MONTGOMERY

99

Other enzymes

ATP-GMP phosphotransferase is a highly specific enzyme that catalyses the phosphorylation of guanylic acid and 2'-deoxyguanylic acid by adenosine triphosphate. Of' the purine analogue nucleotides tested, only 8-azaguanylic acid was a good substrate; thioinosinic acid was neither a substrate nor an inhibitor. Thioguanylic acid was a competitive inhibitor of the enzyme and was itself phosphorylated only to a small extent [91,329a]. Cytidylate reductase, the enzyme that reduces cytidine diphosphate to 2 '-deoxycytidine diphosphate is inhibited not only by 1~-D-arabinofuranosylcytosine, but also by 9-0-D-arabinofuranosyladenine[330] and 9-0-D-arabinofuranosylpurine-6( 1H)-thione(LXXV) [33 1-3331 .9-Butylthioguanine (LXXVI), which is catabolized to unidentified excretion products but which is not debutylated, blocks the incorporation of adenine into DNA, but the exact mechanism of this action is unknown [334]. 9$-D-Arabinofuranosyladenine, presumably as the triphosphate, inhibits the incorporation of precursors into DNA [ 1541 , and the triphosphate inhibits noncompetitively the incorporation of thymidine triphosphate into DNA in extracts of ascites cells, suggesting a direct interaction with DNA polymerase, possibly at an allosteric site [ 1551. Other studies have confirmed the inhibition of DNA synthesis catalyzed by DNA polymerase [335,335a]. Incorporation into nucleic acids

The subject of the incorporation of anticancer agents into macromolecules [ 131 and other compounds [336] has been reviewed. A number of purine analogues are incorporated into nucleic acid, but the incorporation of these compounds requires that they be anabolized to nucleoside mono-, di-, and triphosphates, and it is difficult to separate the metabolic effects of the nucleoside phosphates from the metabolic effects of the fraudulent polynucleotides. 8-Azaguanine was the first purine analogue shown to be incorporated into polynucleotides [337] and, since its primary metabolic effect is on protein synthesis, the incorporation into RNA is considered the basis for its biologic activity [338] . In microbial systems 8-aza-adenine, 8-azahypoxanthine, 8-azaxanthine, and S(4)amino- 1H-1, 2, 3-triazo~e~4(5)-carboxamideare all incorporated into RNA as 8-azaguanylic acid [336]. Another guanine analogue, thioguanine, is also incorporated into both DNA and RNA of mammalian cells [ 1981, and a correlation between its antitumour activityand the extent of its incorporation into DNA has been observed [339,340], although some investigators feel that the metabolic effects of thioguanylic acid may be more universally important [ 13, 91, 3411 . The incorporation of a-and P-2'-deoxythioguanosine into DNA has also been reported [ 144. 144al. 'The incorporation of 6-mercaptopurine into DNA as 2 '-deoxythioguanylic acid has been observed but in cells resistant to the action of the drug, which makes the meaning of this observation unclear [201].

100

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

Several analogues of adenine or adenosine are reported to be incorporated into nucleic acids: 2-fluoroadenosine 13421, tubercidin 190, 192, 342a], toyacamycin [ 193,342a1, sangivamycin [342a, b] , cordycepin [ 168,343,3441, 4-aminopyrazolo[3, 4-d] pyrimidine [ 1 191 , formycin [344a] , . and 9-0-Darabinofuranosyladenine [ 152, 1541. The evidence for the incorporation of 9-0-D-arabinofuranosyladenine has been questioned [345] .

Inhibition of protein synthesis

I n the preceding section. the incorporation of 8-azaguanine into RNA was discussed. How this incorporation inhibits protein synthesis is not certain, but synthetic polymer or triplet nucleotides containing 8-azaguanine are less active in stimulating polypeptide tormation in vifro than the corresponding guanine compounds, even though no miscoding was detected [346, 347, 3 4 7 a l . Thus inhibition of protein synthesis by the analogue may be a result of the formation of 8-azaguanine-containing messenger RNA with altered template activity [ 3381 . The effects of puromycin (XLIII) on the synthesis of macromolecules has been studied in intact bacteria, mammalian cells, and whole animals. Puromycin (XLIII) was shown to inhibit protein synthesis immediately while DNA and RNA syntheses continue for a while at near normal rates. This pattern of macromolecular synthesis is now recognized as characteristic of inhibition of protein synthesis at a step subsequent to the formation of aminoacyl-sRNA. The studies leading to our present understanding of puromycin (XLIII) inhibition have been adequately reviewed [348] and will not be discussed in detail here. Puromycin, because of its resemblance to aminoacyl-sRNA (LXXVII), attaches t o the acceptor sites of the ribosome-messenger RNA complex adjacent to the sRNA's to which growing peptide chains are attached (the donor sites). Each carboxylactivated peptide is then transferred to the free amino group of puromycin, which subsequently detaches from the ribosome. Thus, the growing polypeptide chains are prematurely released from the ribosomes and protein synthesis is inhibited. In addition to the inhibition of protein synthesis, there is evidence that puromycin affects nucleotide metabolism and RNA synthesis, but thesc effects are probably due, at least in part, to breakdown to the aminonucleofide and demethylation of the aminonucleoside to 3 '-amino-3 -deoxyadenosine (see p. 84). Homocitrullylaminoadenosine (LXXVIII) [349] also inhibits protein synthesis, presumably in the same way that puromycin does [350]. A similar mode of action has been suggested for lysylaminoadenosine (LXIX) [ 3 5 1 , 3 5 2 ] . Nucleocidin (LXXX) [353] , an adenine nucleoside antibiotic whose structure has recently been elucidated, is also a potent inhibitor of protein synthesis. Studies with this compouna have led t o the conclusion that nucleocidin forms a complex with ribosomes which is inactive in peptide bond formation. Binding

JOHN A. MONTGOMERY

101

of either soluble RNA or messenger RNA to this complex is apparently not inhibited, nor is progression of the ribosomes down the messenger RNA molecule. The specific aspect of peptide bond formation which is inhibited in the complex has not been determined [354].

. ,N +L N H O c H ;

M~o

0

I c=o

I

CH,CHNH,

N b N5 ) ) -03

%d

POCH2

I

c=o I

R - CHNH,

Interference with co-factors

Tubercidin 5 ‘-monophosphate is converted to the ‘corresponding analogue of NAD when incubated with cell free extracts of S.fueculis (see above). Furthermore, the inhibition of S. fueculis by tubercidin can be prevented by a mixture ot amino acids, nucleosides, ribose-5-phosphate, and pyruvate suggesting that the primary action of the antibiotic is due to interference with N AD-dependent reactions [202]. This protective effect is not seen in mammalian cell systems [ 1931. Formation of the 2-aminoadenine analogue of NAD by H.Ep.-2 cells in culture has been demonstrated, and it appears quite likely that dinucleotides containing 2-aminoadenine and nicotinic acid or nicotinamide can be formed in living

102

THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

animals. Whether this NAD analogue plays a significant role in the inhibition of biological systems by 2-aminoadenine is not known at present [218]. 6-Mercaptopurine ribonucleoside triphosphate is a potent inhibitor of adenylyl transfer to nicotinamide mononucleotide and is itself converted to the NAD analogue in vifro [355, 3561. Since it has not been established that 6-mercaptopurine is converted to its triphosphate in vivo, the significance of these observations is uncertain. It has also been suggested, but not established, that 6-mercaptopurirfe may interfere with co-factor A [357,358]. Summary

Certain purine analogues such as psicofuranine and puromycin are known to inhibit per se a specific metabolic event-the aminolysis of xanthylic acid and the formation of peptide chains, and these specific inhibitions are thought to be the basis of their biologic activity. In contrast, 6-mercaptopurine is known to interact with a number of enzymes and to be incorporated to a small extent into DNA. Some of these interactions result in the catabolism of the drug, and others in its anabolism to at least three biologically active forms-6-mercaptopurine ribonucleotide, 6-methylthiopurine ribonucleotide, and 6-thioxan thylic acid. These active forms are capable of interacting with still other enzymes. Many of the interactions of 6-mercaptopurine and its anabolities are enzyme inhibitions, any one of which could be responsible for cell death, although today it appears more likely that it is the simultaneous inhibition of two or more of these enzymes by the multiple active forms that results in the potent effects of this drug. Other compounds such as 8-azaguanine almost surely owe their effects to incorporation in macromolecules such as RNA, which in turn interferes with the normal function of this material in the direction of protein synthesis. Thus there are a number of metabolic events that are interfered with by the various purine analogues. The biological consequences of these interferences are discussed in the sections that follow. EFFECTS On the host Toxicity

Table 2.4 lists the LDlo values for the acute toxicity of a number of purine analogues for BDFl mice [359]. In general the values agree fairly well with the cytotoxicity values for the same compounds. Some of the discrepancies are easily explained. For example, 6-chloropurine ribonucleoside is nndoubtedly more readily converted to inosine in vivo accounting for its high LDlo value, whereas 6-(methy1thio)purine is demethylated t o 6-mercaptopurine (see above). The high LDlo value for 6-mercaptopurine ribonucleoside, however, is not easily understood, since it is readily cleaved t o 6-mercaptopurine. Studies on the toxicology of some of these purine analogues in rodents and dogs at sublethal doses showed that rapidly dividing tissues, especially the intestinal mucosa and bone marrow, are most sensitive to these compounds

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Table 2.4. A COMPARISON OF THE TOXICITY OF SOME PURINE ANALOGUES

Compound 2-Fluoroadenosine 4-Aminopyrazolo[ 3 , 4 d ] pyrimidine N-Aminoadenine 2-Aminoadenine 3 :Amino-3 Ideoxy-N, N-dimethyladenosine Purine 6-Methylpurine 6Chloropurine 6Chloropurine ribonucleoside 6-Mercaptopurine 6-Mercaptopurineribonucleoside 6-(Methylthio)purine 6-(Methylthio)purine ribonucleoside 6-(Benzylthio)purine ribonucleoside 9-Ethyl-6-mercaptopurine 9-Butyl-6-mercaptopurine Thioguanine Thioguanosine 8-Azaguahine 8-Azaguanosine

1

ca 15 ca 75 70 34 ca 400 1.7 230 550 40 250 94 20 150 52 250 1.6 1.7 90 17

>

In BDFl mice qd 1-1 1, observed 1-12.

[360] (as well as all other anticancer agents). However, the relative effects of these drugs on the two tissues vary. For example, 6-mercaptopurine, 2-aminoadenine, and 6-chloropurine produce similar lesions in the intestinal epithelium when given in doses that cause bone-marrow depression; the effects of thioguanine are largely limited to the bone marrow, in which most haematopoietic elements are susceptible. 6-Methylpurine appears to selectively depress erythrogenesis. The xanthine oxidase oxidation products of purine and 2-chloroadenine crystallize in the renal tubules causing kidney damage [360]. Hepatic damage occurs with many analogues, but is particularly prominent with Caminopyrazolo [3, 4-dlpyrimidine [361]. These various toxicities, and skin rashes [362, 3631 are also observed clinically along with anorexia, nause?, vomiting, and diarrhoea. The limiting clinical toxicity with 6-(methy1thio)purine ribonucleoside is gastrointestinal toxicity, particularly of the upper tract [364] , whereas bonemarrow toxicity is usually limiting with 6-mercaptopurine and thioguanine. Administration of 3 '-amino-3 '-deoxy-N,N-dimethyladenosine(the aminonucleoside of puromycin) to rats produces a nephrotic syndrome that is clinically indistinguishable from the nephrotic syndrome of unknown origin frequently observed in children [365] . Rats, monkeys, and humans are susceptible to this nephrotoxicity and susceptibility has been related to specie ability to demethylate the aminonucleoside [ 2 131. N6-Methyladenosine prevents development of this syndrome [365a].

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Embryonic tissue

Because of their effects o n rapidly dividing tissue, the purine analogues have a marked effect on the developing embryo. The analogues differ not only in the amounts required to produce toxicity in the embryo but in their teratogenicity. I t is possible t o produce teratogenic effects in the chick embryo with a sublethal amount of 8-azaguanine but not with 6-mercaptopurine [366]. Compounds such as 6-mercaptopurine and thioguanine affect both cellular multiplication and differentiation [367] . The abilityofpurine analogues to affect rodent embryos in ufero at doses that are non-toxic to the mother is well documented. When 6-mercaptopurine [368], thioguanine [369], some of their S-substituted derivatives [370, 3711, and 6-chloropurine [373] are given to the rat at the time o f implantation of fertilized ova, a high percentage of the foetuses are destroyed. To exhibit peak activity, 2-aminoadenine must be given before implantation [369] . 6-Mercaptopurine, 6-mercaptopurine ribonucleoside, h-mercaptopurine-3-oxide, and N-hydroxyadenine are teratogenic when administered on the eleventh day of gestation [373a]. The teratogenicity of 8-azaguanine in mice depends o n the timing and amount of the dose [ 3731 . Immune response

The finding that the administration of 6-mercaptopurine to rabbits following exposure to bovine serum albumin prevented antibody formation 13741 formed the basis for a new area of chemotherapy for purine analogues and other antimetabolites and was soon followed by the use of these drugs for the therapy of autoimmune disease and the suppression o f homograft rejection. This subject has been reviewed in depth [ 12, 375, 375al , has occasioned a symposium [376], and has received much recent publicity as a result of human heart transplants. Certain purines are capable of specifically inhibiting the immune response during the induction period of the response, and the inhibition is increased by increasing the antigenic stimulus. There is a close resemblance between druginducedand antigen-excess repression of the response, and although the mechanism by which these compounds suppress is not clear, the suggestion has been made that it is probably related to their cytotoxic nature [ 121. Fortunately for the potential of immunosuppressive agents in the treatment of homograft rejection, they have much less effect on a secondary than o n a primary immune response, although they are useful in the treatment of a number of autoimmune diseases such as psoriases, and undesirable effects have been reported [ 3 7 5 ] . A number of thiopurines (thioguanine, 6-mercaptopurine, 6xmethylthio) purine) [ 121 ,and purine, azathioprine (6(( l-methyl-4-nitro-5-imida~olyl)thio] other derivatives o t 6-rnercaptopurine [377] ) have all been used to successfully prolong homografts, and azathioprine (Imuran) appears t o be superior in its action [ 2 6 8 ] .

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Pla tele t aggrega tion

Circulating platelets adhere to the site of damage of blood vessels aggregating into clumps or white bodies, which stop the flow of blood causing clots. Adenosine diphosphate causes the aggregation of platelets in blood plasma both in vitro [378] andin vivo [379], and this effect is antagonized by adenylic acid and even more by adenosine. Of the thirty odd purine nucleosides that have been evaluated as platelet aggregation inhibitors [ 3 8 E 3 8 2 ] only 2-aza-adenosine and the 2-haloadenosines showed significant activity. 2-Chloroadenosine was more active than adenosine, but also caused respiratory arrest in rabbits [383]. A correlation has been noted between the ability of these compounds to inhibit platelet aggregation and their vasodilator activity [382] . Gout

The pyrazolo[3, 4-d] pyrimidines are substrates for and inhibitors of xanthine oxidase [ 266,267].4-Hydroxypyrazolo[3,4-d] pyrimidine was first investignted for its ability to protect 6-mercaptopurine and other analogues from oxidation by xanthine oxidase [384], but it also inhibits the oxidation of the natural purines, hypoxanthine, and xanthine. Its profound effect on uric acid metabolism made it an obvious choice for the treatment of gout and its utility in the control of this disease has been demonstrated [385,386]. On invading organisms

Micro-organisms (bacteria and protozoa)

Much information on the mechanism of action and cross-resistance of purine analogues has been obtained in bacteria, some of which are quite sensitive to certain of these compounds in vitro. There is a great deal of variation in response of the various bacteria to a particular agent and of a particular bacterium to the various cytotoxic purine analogues. Some, if not most, of these differences are probably due t o differences in the anabolism of the various compounds. Despite the fact that certain purine analogues have quite a, spectrum of antibacterial activity in vitro, none has been useful in the treatment of bacterial infections in vivo because their toxicity is not selective-the metabolic events whose blockade is responsible for their antibacterial activity are also blocked in mammalian cells and thus inhibition of bacterial growth can only be attained at the cost of prohibitive host toxicity. In contrast, the sulpha drugs and antibiotics such as penicillin act on metabolic events peculiar to bacteria. It is of historical interest that Tetrohyrnenagelii, whose metabolism has been described in detail [387], is inhibited by 8-azaguanine [388] and other purine analogues [389, 3901. Of more importance to chemotherapy is the fact that pathogenic protozoa such as the trypanosomes respond in vitro to a number of

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purines such as 6-mercaptopurine [39 11 , thioguanine 139 11, and 2-aminoadenine [392]. More active than these compounds, in vitro and in vivo, however, is the aminonucleoside of puromycin, 3 '-amino-3 '-deox y-N,N-dimethy ladenosine, which is more effective than puromycin itself [ 39 1,3931 . Primaquine appears to be more effeGtive than the aminonucleoside on the flagellate forms of T. cruzi; so the combination was tested in mice and found extremely effective [394]. Puromycin alone was not effective against T. cruzi in humans [395], but was effective against early infection of T. gambiense [396]. T. equiperdum and T. gumbiense in mice respond to both puromycin and the aminonucleoside [397-4001. Puromycin was also effective in suppressing T. equiperdum, T. equinum. T. Evansi, and T. rhodesiense in mice, but was ineffective against T. congolense [398]. T. equiperdum infections in mice 14011 and T. congolense, T. gambiense, and T. equinum in mice and rats are cured by treatment with nucleocidin [4021. Although inferior to pyrimethamine plus sulphonamides, both puromycin and the aminonucleoside are active against Toxoplasma gondii in vivo [403]. These drugs are also effective against Endamoeba histolytica in vifro [404] , and puromycin is active against the infection in man [405]. The mechanism of inhibition of these protozoal infections by the most active drugs, puromycin and the aminonucleoside, is not known. Puromycin and nucleocidin both intertere with protein synthesis, but the aminonucleoside does not. It is known to be demethylated to 3'-amino-3'-deoxyadenosine, which is phosphorylated and interferes with nucleic acid metabolism (see above). Whether puromycin must be converted to the aminonucleoside before it can inhibit protozoa has not been established. Some purine analogues known to interfere with nucleic acid metabolism, however, are less effective as antiprotozoal agents, even in vifro, perhaps because their effects are primarily on the de novo pathway which many, if not all, protozoa do not use [406]. Viruses and cancer

2-Aminoadenosine, the first purine found to possess antiviral activity, inhibits vaccinia [384], spring-summer encephalitis [407], psittacosis [408], and poliomyelitis [409] viruses in cell culture. 8-Azaguanine has been reported a$ both active [410] and inactive [384] against vaccinia virus, and active against psittacosis and encephalomyocarditis. 6-Mercaptopurine interfered with the replication of both RNA and DNA viruses in Lass cells [41 I ] . 9-P-D-Arabinofuranosyladenine has a remarkable inhibitory action on the multiplication of the DNA viruses [41 l a ] , herpes, vaccinia [412, 4131, and cytomegalovirus [414], which also responds to thioguanine. Puromycin is active against a number of viruses in cell culture. In chick embryo cells it delayed the replication of western equine encephalitis [4 151 and inhibits Venezulian equine encephalitis [4 161 . It interferes with the replication

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of poliomyelitis [417-4191 and western equine encephalitis [419] in HeLa cells. I t is reported to inhibit [420] and not inhibit [421] encephalomyocarditis and t o inhibit reovirus [422] and influenza (4231, but not herpes [424] in L cells. If added early in the eclipse stage it inhibits adenovirus in monkey kidney cells [425]. I t also inhibits polyoma virus in mouse embryo cells [426]. Activity of an agent against a virus in cell culture is only an initial lead. Many false positives are found because there is no measure of host toxicity at viricidal levels or of the many other complicating factors. Unfortunately the purine analogues have shown minimal activity against viral infections in the whole animal. 2-Aminoadenine reduced the mortality of mice infected with springsummer encephalitis [427], but puromycin had no effect on herpes keratitis in rabbits [428]. A number of purine analogues failed t o inhibit Semliki Forest virus infections in mice [ 3721 . 9-p-D-Arabinofuranosyladenine was the only purine of a large number evaluated for in viuo activity against influenza and vaccinia viruses that was inhibitory and its effectiveness was confined to vaccinia 14291. In vivo studies with virus-induced cancers mostly limited t o virus leukemias in mice and the Rous sarcoma in chicks, have been concerned primarily with the anticancer aspect of the problem, and have placed little emphasis on the viral aspects. 6-Mercaptopurine, its ribonucleoside, thioguanine, and azathioprine all prolong the life span of mice infected with the Friend virus leukemia [430,43 1 ] . In addition t o these compounds, 9cyclopentyl-6-mercaptopurine,9-butyl-6mercaptopurine, 6xbenzylthio)purine ribonucleoside, and thioguanosine are also active [432] . 6-Mercaptopurine [433] and thioguanine were active against both the Friend and Rauscher viruses in an in vitro assay system [434]. 6-Mercaptopurine showed only minimal effects against the Moloney virus leukemia [435, 4361, although other purine analogues such as thioguanine and 6chloropurine ribonucleoside are reported t o increase survival time of infected mice [ 3 6 6 , 4 3 7 ] . Although inactive against the Kous sarcoma in the standard post-infection test, 6-mercaptopurine, 2-aminoadenine, and 8-azaguanine inhibited the development of the tumour if given prior t o infection of the chicks [417,418]. Most of the adenine and adenosine analogues discussed in the precedine sections are converted to adenosine triphosphate analogues and are highlY cytotoxic. Unfortunately, their specificity for cancercells is low so that, although they show some activity in sensitive experimental animal systems such as Ehrlich ascites carcinoma, they are not useful agents; and those that have been evaluated clinically (i.e., 2-aminoadenine [363] and 4-aminopyrazolo[3, 4-d] pyrimidine [438] ) are not effective, but are toxic t o man. The ribonucleoside [439] of N-hydroxyadenine [440], an inactive adenine analogue, may be an exception to thisstatement, since it is quite active against L1210 leukemia [439], but haemolysis at low dosage occurred in preliminary clinical trials [439a]. Since other N-substituted adenosines are phosphorylated to the monophosphate stage only, the active form of this analogue is probably N-hydroxyadenylic acid, rather

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than the triphosphate, which may be part of the reason for its selective action on cancer cells. Trimethylpurin-6-ylammoniumchloride [44 11 and some of its derivatives [442, 4431 inhibit the growth of adenocarcinoma 755, but not sarcoma 180 or leukaemia L1210 in mice; little additional information is available on this series of compounds. 6-Chloropurine, as its ribonucleotide, is active against a number of animal neoplasms [444] and human leukaemias [363], but is less effective than the purinethiones, which it resembles in its action. 8-Azaguanine is inhibitory to several experimental animal tumour systems [444], but is not highly active. Clinically its toxicity has been more apparent than its anticancer effects [362,363]. Of the purine analogues investigated thus far, the purinethiones are by far the most effective anticancer agents. 6-Mercaptopurine remains the agent of choice clinically [363, 445-4471 , since other thiopurines, such as thioguanine, 6-(methylthio)purine, azathiopurine, 9-ethyl-6-mercaptopurine, and 6-mercaptopurine ribonucleoside, which are also active in man, appear to offer no real advantage over it. 6-Mercaptopurine is useful in the treatment of acute granulocytic, acute lymphocytic, and chronic granulocytic leukaemia and choriocarcinoma [ 12, 363, 445-4471 . The combination of 6-mercaptopurine and 6(methylthio)purine ribonucleoside, however, is more effective in the treatment of acute adult leukaemia than either drug alone [448]. 6-Mercaptopurine is considerably less effective in the treatment of solid tumours [363] . The reason for the selective toxicity of 6-mercaptopurine remains to be established, but two factors may be of primary importance. 6-Mercaptopurine is anabolized primarily, if not exclusively, to the monophosphate level, and it is readily catabolized by xanthine oxidase, an enzyme that is low in most cancer cells compared to normal cells. Another factor that must be considered is the metabolic state of the target cells. Actively proliferating leukaemia cells are more sensitive to 6-mercaptopurine, as they are to all antimetabolites, than cells in the so-called Go or stationary phase. Although this does not explain the difference between 6-mercaptopurine and other purine analogues, it may explain the ineffectiveness of 6-mercaptopurine against solid tumours, most of the cells of which are in the non-dividing state. Certain derivatives of 6-mercaptopurine, such as 6-(methylthio)purine, 6-mercaptopurine-3-oxide [448a] , and 6-mercaptopurine ribonucleoside and its acylated derivatives apparently owe their activity to their in vivo conversion to 6-mercaptopurine [ 11,131. It would appear, however, that the 9-alkyl derivatives of 6-mercaptopurine, and its arabinosyl and xylosyl derivatives, are not metabolized-except in the case of the 9-alkyl derivatives, t o a limited extent to their S-glucuronides-and that their mechanism of action is quite different from that of 6-mercaptopurine. Thioguanine is 5-30 times as toxic to rodents (depending on schedule) as 6-mercaptopurine and somewhat more effective against rodent neoplasms,

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although its therapeutic index is not greater. It would seem likely that 6-mercaptopurine and thioguanine inhibit cancerous growths in a similar manner. Changes in the structure of thioguanine also give rise to active structures. but no significant improvement in effectiveness [ 11,131 . THE PROBLEM OF RESISTANCE Mechanisms

Both natural and acquired resistance constitute a serious problem to therapy witH purine analogues, particularly in the case of cancer. Why one acute leukaemia responds well to methotrexate but not to 6-mercaptopurine, whereas a morphologically identical leukaemia responds to 6-mercaptopurine but not to methotrexate, and a third, seemingly identical leukaemia responds to neither is a vexing problem that has so far defied solution [449]. In the case of acquired resistance, a patient may respond well to a drug initially and appear to be completely cancer-free, only to succumb later to a cancer that now does not respond to therapy with the same drug. Such a situation may indicate that the cancer cell population has, before resumption of treatment, multiplied to a size where it can no longer be controlled by a drug dose th?t the patient can tolerate [450], or it may indicate that a drug-resistant mutant population has replaced the original heterogeneous population containing almost entirely drug-sensitive cells. The necessity for most purine analogues to be converted to their nucleotides to show their inhibutory effects has been discussed. Cells deficient in hypoxanthine-guanine phosphoribosyltransferase activity cannot convert 6mercaptopurine, thioguanine, or 8-azaguanine to their ribonucleotides and are resistant to these analogues [8,98, 101,260,451455], but are still sensitive to adenine analogues such as 2-fluoroadenine, 2-aminoadenine, and 4-aminopyrazolo [3, 4:d] pyrimidine [456]. Conversely, cells deficient in adenine phosphoribosyltransferase activity are resistant to the various adenine analogues, such as 2-aminoadenine [ 113, 3041 and 2-fluoroadenine [ 1281, but are still sensitive to cytotoxic hypoxanthine-guanine analogues [ 128, 1471 . Although mammalian cells are naturally resistant to xanthine analogues, because they are deficient in xanthine phosphoribosyltransferase activity, bacteria possess this enzyme and are sensitive to 8-azaxanthine. Bacteria that have become resistant to 8-azaxanthine were shown to have lost their xanthine phosphoribo'syltransferase activity [98]. Resistance to 2-aminoadenine and 8-azaguanine in Salmonella typhimurium is apparently due, in some cases, to genetically controlled alterations of the phosphoribosyltransferases to forms of the enzymes that can still convert the natural substrates to nucleotides but not the purine analogues [457,458]. Cells deficient in adenosine kinase fail to respond to adenosine analogues, unless they are cleaved to adenine analogues that can be converted to ribonucleotides by adenine phosphoribosyltransferase. Cells deficient in both these enzymes fail to respond to adenine and adenosine analogues, but are still sensitive to hypoxanthine-guanine analogues [ 1471 . Resistance to the various purine

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THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES

analogues due to these enzyme deletions has been observed in bacteria, mammalian cells in culture, and neoplasms in experimental animals indicating that this is a ubiquitous and an important cause of acquired resistance [8]. Since these enzymes catalyse the so-called 'salvage' pathways of purine utilization they are not essential to cellular metabolism, and their loss does not, in general, affect the ability of cells to proliferate at normal rates, which may explain why resistance by these deletions occurs so readily and frequently. However, acquired resistance is thought to result from chemical selection and overgrowth of specific drug-resistant mutant cells from a heterogeneous population, and hence it is not surprising that more than one type of resistance t o a particular agent has been found. Other mechanisms of resistance to purine analogues that have been advanced are inaccessibility of the nucleotide-forming enzyme system t o the analogue [459, 4601, increased rate of degradation of the analogue or its metabolities [461-463], and a decreased affinity of the activating enzyme for the analogue [ 1001 . Circumventions

One of the first demonstrations that acquired resistance could be circumvented was the inhibition of S. faecalis resistant to 8-amguanine by 8-azaxanthine. Thus, cells that possess xanthylic acid phosphoribosyltransferase activity could form 8-azaxanthylic acid, which was then converted to 8-azaguanylic acid and incorporated in nucleic acids as such 1981 . Early attempts to inhibit H.Ep.-2 cells resistant to 6-mercaptopurine [464J resulted in the finding that a number of 9-alkyl derivatives of 6-mercaptopurine were highly active in this test system. 9-Alkylhypoxanthines and adenines were less effective [465]. 6-Mercaptopurine ribonucleotide is not active against cells resistant to 6-mercaptopurine, presumably because nucleotides cannot penetrate cell membranes intact [466] (its activity in sensitive cells is no doubt due to its cleavage back to 6-mercaptopurine [467] ). This stumbling block led to the synthesis of esters of 6-mercaptopurine ribonucleotide [468, 4691 that might penetrate cell walls intact and then either inhibit per se or be cleaved back to the ribonucleotide. Two such derivatives, bis(thioinosine) 5 ', 5 "'-phosphate [470] and the monophenyl ester of. 6-mercaptopurine ribonucleotide [ 13, 4681 do inhibit H.Ep.-2 cells resistant to 6-mercaptopurine, but some cross-resistance was noted. Because of the relatively low therapeutic index of all known purine antagonists, this cross-resistance did not otter encouragement for in vivo activity against resistant neoplasms, and, indeed, the monophenyl ester did not inhibit leukaemia L1210 resistant to 6mercaptopurine. More successful in this regard was the use of 6-(methylthio) purine ribonucleoside against MP-resistant cells. Thus, this nucleoside, which is converted t o the nucleotide by adenosine kinase, is highly active against both resistant H.Ep.-2 cells in culture and resistant L1210 leukaemia in mice [471]. Furthermore, it is therapeutically synergistic with 6-mercaptopurine in the

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sensitive line of L1210 [471]. Similar observations were made in the Ehrlich ascites carcinoma system [472]. The clinical utility of this combination [448] has been discussed above. AC KNOWL EDG EMENTS

The author gratefully acknowledges the helpful criticism of Dr. Lee L. Bennett, Jr. Thanks are due to Mr. Vladimir Minic and Miss Linda Scott for checking references to the literature and to Mrs. Frances K. Hoffman for preparation of the manuscript. REFERENCES

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THE BIOCHEMICAL BASIS FOR THE DRUG ACTIONS OF PURINES K. Sugiura, Gann, 1 9 5 9 , 5 0 , 2 5 1 K. Sugiura, Cancer Res. Suppl., 1 9 6 2 , 2 2 , 9 3 R. W. Sidwell, G . J. Dixon, S. M. Sellers and F. M. Schabel, Cancer Chemother. Rep., 1966,50,299 E. A. Mirand, N. Back, T. C. Prentice, J. L. Ambrus and J. T. Grace, Proc. SOC.Exp. Biol. Med., 1961, 108, 360 M. A. Chirigos. Ann. N. Y.Acad. Sci., 1965. 130,56 M. A. Chirigos, J. B. Moloney, S. R. Humphreys, N. Mantel and A. Goldin, Cancer Res. 1961,21,803 J. P. Glynn, A. R. Bianco and A. Goldin, Cancer Rex, 1964,24, 1303 A. Goldin, J. P. Glynn, A. R. Bianco, J. M. Venditti and J. B. Moloney, Proc. 3rd Inter. Congr. Chemotherapy, Stuttgart, 1963, p. 812; A. Goldin, J. P. Glynn, J. B. Moloney, S. R. Humphreys and M. A. Chirigos, Acta Unio Inter. Contra Cancrum, 1964,20, 157 C. A. Nichol, Cancer Res., 1965,25, 1532 A. Giner-Sorolla, L. Medrek and A. Bendich, J. Med. Chem., 1966, 9, 143; J. H. Burchenal, M. Dollinger, J. Butterbaugh, D. Stoll and A. Giner-Sorolla, Biochem Pharmacol., 1 9 6 7 , 1 6 , 4 2 3 I. H. Krakoff and M. R. Dollinger,Proc. Amer. Assoc. Cancer Res., 1969, 1 0 , 4 7 A. Giner-Sorolla and A. Bendich, J. Amer. Chem. SOC.,1958,80, 3932 J. P. Howitz and V. K. Vaitkevicius, Experientia, 1961, 1 7 , 5 5 2 F. R. White. Cancer Chemothcr. Rept., 1963.30,57 J. A. Montgomery, C. Temple, T. P. Johnston F. M. Schabel, unpublished and observations J. A. Stock, Experimental Chemotherapy Eds. R. J. Schnitzer and F. Hawking), Vol. 4: Academic Press. New York. 1966, p. 80 G. Zubrod, Arch. Int. Med., 1960, 106.663 E. Frei and E. J. Freireich, Adv. Chemother., 1965,2, 269 J. K. Luce, G. P. Bodey and E. Frei, HospitalPractice, 1967,42 G. P. Bodey, H. S. Brodovsky, A. A. Isassi, L. Samuels and E. J. Freireich, Cancer Chemother. Rept., 1968.52, 315 K. Sugiura, Cancer Chemother. Rept., 1968, 1, 383 J. H. Burchenal, Cancer Res., 1963,23, 1186 H. E. Skipper, F. M. Schabel and W. S. Wilcox, Cancer Chemother. Rept., 1964, 35.1 R. W. Brockman, M. C. Sparks and M. S. Simpson, Biochim. Biophys. Acta, 1957; 26, 67 1 R. W. Brockman, M. C. Sparks, M. S. Simpson and H. E. Skipper, Biochem. Pharmacol., 1959,2,77 R. W. Brockman,G. G. Kelley, P. Stutts and V. Copeman, Nature, 1961,191,469 P. Stutts and R. W. Brockman,Biochem. Pharmacob, 1963, 1 2 , 9 7 R. W. Brockman, CancerRes., 1963,23, 1191 R. W. Brockman,Clin. Pharmacol. Ther., 1961, 2,237 G. P. Kalle and J. S. Gots, Science, 1963, 142,680 J. C. Adye and J. S. Gots,Biochim. Biophys. Acta, 1966, 118, 344 A. R. Paterson, Can. J. Biochem. Physiol., 1960,38.1117 A. R. Paterson,Can. J. Bwchem. Physiol., 1962.40, 195 A. C. Sartorelli, G. A. LePage and E. C. Moore, Cancer Res., 1958, 18, 1232; D. B. Ellis and G. A. LePage, Cancer Res., 1963.23.436 A. L. Bieber and A. C. Sartorelli, Cancer Res., 1964,24, 1210 L. L. Bennett, P. W. Allan, D. Smithers and M. H. Vai1,Biochem. Pharmacol., 1969, 18,725 G . G. Kelley, M. H. Vail, D. J. Adamson and E. A. Palmer, Amer. J. Hyg., 1961, 73, 23 1 G. G. Kelley, G. P. Wheeler and J. A. Montgomery,Cancer Res., 1962,22, 329

JOHN A. MONTGOMERY

123

P. M . Roll, H. Weinfeld, E . Carroll and G . B. Brown, J. Biol. Chem.. 1956,220,439 J . A . Montgomery, F. M . Schabel and H. E. Skipper, Cancer R e x , 1962, 22,504 J . A . Montgomery, H . J . Thomas and H . J . Schaeffer,J. Org. Chem., 1961, 26,1929 H . J . Thomas and J . A . Montgomery, J. Med. Plzarm. Cllem.,1962,5, 24 J . A . Montgomery, G . J . Dixon, E. A . Dulmadge, H . T . Thomas, R . W. Brockman and H. E. Skipper, Nature, 1963, 199, 769 471. F . M . Schabel. W . R . Laster and H . E. Skipper, Cancer Chemother. Rep., 1967,51, 1 1 1 472. M . C. Wang, A . 1. Simpson and A. R. P. Paterson, Cancer Chemofher.Rep.. 1967,51, 101 466. 467. 468. 469. 470.

3 The Chemistryof Guanidines and their Actions at Adrenergic Nerve Endings G. J. DURANT, B.Sc., Ph.D., A.R.I.C. Smith Kline and French Research Institute, Welwyn Garden City, Herts. A . M . ROE, M.A., D. Phil., F.R.I.C. Smith Kline and French Research Institute, Welwyn Garden City, Herts. A. L. GREEN, B.Sc., Ph.D. Department of Biochemistry, University of Strathclyde, Glasgow. INTRODUCTION THE STRUCTURE AND PHYSICAL PROPERTIES O F GUANIDINES SYNTHETIC METHODS Guanidines Aminoguanidines PHARMACOLOGICAL TEST PROCEDURES Effect o n blood pressure Effect o n the sympathetic nervous system STRUCTURE-ACTIVITY RELATIONSHIPS FOR ADRENERGIC NEURONE BLOCKADE Guanethidine and close analogues Aryloxyalkylguanidines and related structures Araky lguanidines Miscellaneous guanidines Structural requirements for adrenergic neurone blockade OTHER PHARMACOLOGICAL EFFECTS ON SYMPATHETIC NERVES BIOCHEMICAL EFFECTS Depletion of noradrenaline Antagonism of noradrenaline depletion Effect of guanidines o n enzymes involved in noradrenaline metabolism Mechanism of guanethidine-induced depletion Relationship between noradrenaline depletion and adrenergic neurone blockade ADDENDUM REFERENCES I24

125 126 130 130 133 135 135 136 139 139 15 1 160 169 171 174 177 177 185 188 193 196 200 203

G. J . DURANT, A . M . ROE, A. L . GREEN.

125

INTRODUCTION

Guanidines have been studied for many years as a possible source of medically useful compounds, but the recent vast increase in the literature on guanidine derivatives (from about 200 references in Chemical Abstracts in 1958 t o over 1000 in 1965) stems principally from the discovery [ I ] of the potent hypotensive properties of guanethidine (oktadin, Azetidin, Dopom, Eutensol, Guanexil, Guethine, Ipoctal, Ipoguanin, Iporal, Ismelin, Izobarin, Normorif, Octadinum, Octatensine, Pressidin, Sanotensin, Su-5864, Visutensil, I).

Around 40 years ago, the short-lived use of synthalin (11) as a hypoglycaemic drug [ 2 ] led t o numerous studies on guanidines as potential insulin substitutes [3] . Synthalin itself was withdrawn when it was reported that it can cause liver damage [4], and the widespread interest in guanidines eventually lapsed until the introduction of guanethidine nearly 30 years later. It is chastening t o note how often during these early investigations on guanidines, marked cardiovascular actions were reported but not pursued. One such report referred t o phenethylguanidine (111) in the following terms ‘the latter compound, in larger doses, exerts a powerful effect on the flow of blood as Ph(CH2 ) 2 .NH.C=NH

I

evidenced by the difficulty of bleeding the animals: [ 5 ] . Synthalin itself caused a fall in blood pressure, and also had curare-like effects [6] which were subsequently rediscovered in the related compound decamethonium. However, since the object of this early work was t o find drugs which lowered blood sugar rather than blood pressure, these observations were apparently not followed up. During the 1930s several aralkylguanidines were shown by Japanese workers to lower blood pressure [7, 81, but the mechanism of this hypotensive action was not understood a t the time, and again it was not followed up. Synthetic guanidine derivatives have been used successfully in the treatment of a variety of diseases, but the major success has undoubtedly been their exploitation as antihypertensive drugs. Guanethidine has still not been displaced,

126

GUANIDINES A N D ADRENERGIC NERVE ENDINGS

although it now has many competitors such as bethanidine (benzanidine, Esbatal,' Eusmanid, B.W. 467C60, IV), guanoxan (Envacar, V), guanoclor (Vatensol, VI) and debrisoquine (Declinax, VII). The comparative clinical value of some of these compounds has been reviewed recently [ 9 ] . All of these drugs appear to lower blood pressure by blockade of sympathetic

Cl

nerves, and the present review is confined to this particular aspect of the pharmacology of guanidines. The structure, physical properties and synthesis of guanidines are summarized first, and then, after an outline of the methods used for testing these drugs, the relationships between structure and adrenergic neurone blockade are discussed. The relevant biochemical effects connected with these pharmacological actions are surveyed, with particular reference to possible mechanisms of action. The review is written primarily for the medicinal chemist, and detailed pharmacology is generally included only where necessary for understanding the structure-activity relationships. An excellent review of the pharmacological actions of adrenergic neurone blocking agents has been given by Boura and Green [ l o ] . The biochemistry of guanethidine itself has been reviewed by Furst [ 1 I ] , with particular emphasis on tissuz distribution and metabolism; consequently these two topics are not discussed i n detail.

THE STRUCTURE A N D PHYSICAL PROPERTIES OF GUANIDINES

A prerequisite t o a full understanding of tlie nature of tlie interactions of guanidines with living tissue is an accurate expression of the molecular and physicochemical behaviour of both interactants. The molecular arrangement, and hence

127

G. J . DURANT, A. M. ROE, A. L. GREEN.

the physicochemical properties, of the biological surfaces with which this review is concerned are still unknown; but the position with respect to the guanidine partner in these interactions is reasonably well understood. Guanidine (VIII) and those substituted guanidines with which we shall be concerned are strong bases [12-191, and form stable salts with relatively weak acids [ 201 .Table 3.1 lists the published pK, values of some simple guanidines. The increased stability of symmetrical guanidinium ions is reflected in the high basic strength of guanidine and in the still higher basicity of N,N:N':trimethylguanidine. In strong acid, both the guanidinium ion and the aminoguanidinium ion can accept a second proton. The second pK, of guanidine has been estimated [ 151 Table 3.1. THE BASlCITY OF SOME SIMPLE GUANIDlNES*

R'

It2

R3

R4

RS

PK,

H H Me H Me H Me Me Me H

H H H Me Me Me Me Me Me H

H H H

H H H H H Me H Me Me H H H H H H H H

13.65 (131 13.6 (141 13.74 [18] 13.4 1141 13.4 1141 13.6 [ 1 4 ] 13.6 [ 141 13.9 [ 141 13.6 1141 13.9 [ 141 13.8 [ 141 10.8 (171 11.0 1381 10.6 [ 3 7 ] 10.5 (381 12.6 (371 10.85 [ 371 11.4 1381 11.5 i 3 8 j * 9.9 [ 4 1 7 ] 9.8 (4181 11.04 (4191 11.97 1211 11.3-11.4 [ 4 2 0 ]

H

H

Me H H H H H

H Me H H Ph H

H H H Me H Me H H H H H H H

H

Guanethidine ( I )

'The references should be consulted f o r the temperature and other conditions under which these measurements were made.

to be -10.9 on the Ho scale. The very large difference between the first and second pK, values of guanidine is thought to be associated with the loss of symmetry which arises when the guanidinium ion accepts another proton [ 151. The second pK, of (benzy1amino)guanidine is -3.2 on the Ho scale [21].

128

GUANIDINES AND ADRENERGIC NERVE ENDINGS

When the pK, of a guanidine is greater than 13, the ratio of guanidinium ion to guanidine is greater than 1 O6 : 1 under physiological conditions [22] ; aminoguanidine will exist as the protonated ion to the extent of at least lo4 :1, relative to the unprotonated species. In view of the foregoing, the ensuing discussion will concentrate on the structure and properties of the mono-protonated species, that is, on guanidinium ions. There is ample crystallographic evidence that the guanidinium ion is planar and symmetrical [23, 241. Both ions in the crystal of methylguanidinium nitrate exhibit almost perfect trigonal symmetry [25] , and the aminoguanidinium ion has also been shown to be planar [26]. The formal similarity between the guanidinium ion, the carbonium ion, and the carbonate anion has long been recognized [27], and many papers have been devoted to defining the precise electronic structure of guanidinium ions [28-361. Infra-red studies [31] and molecular orbital calculations [33,34] have led to the description of the guanidinium ion as a tri-amino carbonium ion with the nelectron charge distribution shown (IX) Most of the positive charge is located in the vicinity of the central carbon atom. The relevance of this description to the pharmacological properties of guanidinium ions will be discussed later. For typographical convenience, guanidines will be formulated in this review in the unprotonated form. Y

H2N.C-H

I

N H2

H\

, tO.086

rH \

NC+O .7L 2 /1.318

H

N

/

4

(VIII)

Some aspects of the structure and properties of amidines, including guanidines, in relation to their biological properties have been discussed by Fastier [36a] . The formation of relatively rigid amidinium carboxylate ion-, pairs, formulated as eight-membered rings stabilized by two hydrogen bonds, is thought to precede the formation of crystalline salts and is suggested [36b, 36c] as the basis for their biologcal activity. An alternative formulation of a guanidinium carboxylate ion-pair has been proposed [36d]. The affinity of amidinium groups for anionic sites, such as carboxylate and phosphate, has been stressed [36a]. The similarity of the guanidinium ion, in which the distance between the carbon and the hydrogen atoms is about 2.1A, to the hydrated sodium ion [Na(OH,),]+, in which the distance between the sodium and the hydrogen atoms is about 2.3A, has been pointed out [33, 36a], and the physiological

129

G. J . DURANT, A. M. ROE, A. L. GREEN.

properties of these ions have been compared [33, 36al. A comparison, which is more relevant to this review and is pursued later, may be made with the trimethylammonium group. Finally, it has been suggested [21] that the activities of certain aryloxyalkylguanidines depend on their ability to adopt an internally hydrogen-bonded conformation such as (LXXVIII) and (LXXIX) (p. 172 ). The strength of the hydrogen bond in these systems is related to the basicity of the oxygen atom, which in turn is markedly affected by the position and nature of the substituents in the aromatic ring. Although not strictly relevant to the present discussion, some other physico-chemical aspects of guanidines are summarized here, since no review of this subject has been published previously. The structures of aromatic guanidines and their conjugate acids, in which the T-electrons of the ring can interact with the delocalized electrons of the guanidine system, have been studied. From the ultra-violet absorption spectra and acid dissociation constants of pchlorophenylguanidine, structure (X) was preferred [ 371 over structure (XI); in contrast, the most probable structures for phenylguanidine and its conjugate acid were considered [ 171 to be (XI) and (XII). A more extensive study of some para-substituted phenylguanidines, which includes an estimate of the

Ar N H - G N H

1

ArN=C-NH2

I

ArNH-C=hH,

I

Hammett p constant (+ 2.30) for the formation of the conjugate acid and the Up constant of the guanidinium substituent (+0'317 to + 0.443), supported the conclusion [ 3 M ] that the base has the structure (X). A different view [ 391 was based on the fact that since p for substituted anilines is + 2-77, dissociationof the phenylguanidinium ion most likely takes place from the nitrogen atom which bears the aromatic ring. This argument, however, neglects the delocalization of charge in the ion. The directing effect of the guanidinium substituent in electrophilic aromatic substitution is a measure of the interaction of this substituent with an aromatic ring. The guanidinium substituent is not such a powerful metadirecting group as those substituents in which the positive Charge is localized on the atom which bears the aromatic ring [ 4 0 ] . The ultra-violet spectra of guanidines have often been determined in connection with the measurements of dissociation constants [ 17, 38, 411, and other studies have been reported [42-441. There have been many reports on the infra-red spectra [31, 35, 42, 45-48], the nuclear magnetic resonance spectra [ 38, 49-5 1 ) (see also p. 134). the Raman spectra [52-541, force constants [ 35, 361, and mass spectra [SS] of substituted guanidines. Guanidine forms salts with such relatively weak acids as nitromethane, phthalimide, phenol and carbonic acid [ 2 0 ] . lnteractions between carboxylate anions of proteins and added guanidinium ion are thought [19, 561 to be weaker than the interactions with ammonium ions; the role of guanidinium-carboxylate interactions in stabilizing natural protein conformations has been discussed [36c]. A few reports of metal complex formation by guanidines 157-601, and aminoguanidines [61] have appeared.

130

CUANIDINES AND ADRENERCIC NERVE ENDINGS

Much attention has been given recently to the chromatographic behaviour of guanidines [ 62-69), and various techniques for the detection and determination of guanidines are described there and elsewhere [ 70-761.

SYNTHETIC METHODS Guanidines

Guanidines have been prepared by a wide variety of methods, of which two are of much greater importance than the others. These two methods, (a) the addition of amines to cyanamides and (b) the displacement of an alkylmercaptan by an amine from an alkylisothiouronium salt, together with close variants, are discussed first, and this discussion is followed by a survey of less frequently used procedures. Since an excellent summary of some of the methods that have been used to obtain guanidines is available [77] , the following discussion concentrates on those aspects of synthetic chemistry that are likely to be of interest to medicinal chemists. Patents are only cited when the experimental methods described supplement those available elsewhere. It is worth stressing at this point that although many statements have been made concerning the relative merits of one method of synthesis compared with another, there is little consistency and much contradiction to be found. Except where a comparative study by the same workers has been carried out, it is unwise to rely on these generalizations as a guide for preparing novel guanidines. It is probable that much of the confusion arises from the use of inappropriate methods of isolation rather than from anomalous reactivities.

Method (a)

RzNH + RZN-CN + R Z N G N H

I

Guanidine itself was obtained by Erlenmeyer [78] in 1868 from the action of ammonia on cyanamide, a method that soon led to the synthesis of phenylguanidine from aniline hydrochloride and cyanamide [79,80]. This method has been used to prepare many arylguanidines, and the use of substituted cyanamides has given NJ”diary1- [81] , N-aryl-N’-alkyl- (or N-aryl-N:N:dialkyl-) [82], and N-aryl-N‘benzoyl-guanidines[83] . Arylguanidines are sometimes advantageously prepared by using the benzoylguanidine as an intermediate [83]. Reaction between an amine salt and cyanamide has been used successfully for the synthesis of many mono- and poly-alkylguanidines [84-95], and also of alkoxyguanidines [96, 971 and aryloxyguanidines [98]. The reaction is usually carried out in boiling water or ethanol for from 1 t o 24 hours. Higher temperatures have been employedusingsealed tubes [99, 1001 or butanolasasolvent [82,101].

G. J. DURANT, A. M. ROE, A. L. GREEN.

131

Very dilute acetic acid [lo21 and ethyl acetate [lo31 have also been used as solvents. The fusion of amine salts with cyanamide or a substituted cyanamide is often more satisfactory than using a solvent [ 107-109, 2281, particularly for sterically hindered guanidines such as t-butylguanidine [228] . The preparation of alkylguanidines by fusing amine salts with dicyandiamide at 180" for three hours has been advocated [104], however it has been shown that, depending on the conditions, a guanidine or a biguanide can result [105, 106). Odo studied the formation of methylguanidine from cyanamide and aqueous mixtures of methylaminc and methylamine hydrochloride in various proportions [ 1101 . He concluded that the reaction occurred by a reversible nucleophilic attack of the free amine on cyanamide, and that an acid was required t o shift the equilibrium in the direction of the guanidine. This method of formation of guanidines is considered especially suitable for arylguanidines [77] . The dihydrochloride of p-aminobenzylamine (XIII) reacted with cyanamide to give a mono-guanidine which was assumed [ 11 11 to be the arylguanidine (XlV) on the grounds that benzvlamine hydrochloride did not react under the same conditions, whereas aniline hydrochloride did. An isomer was obtained [ 1 1 1] when p-aminobenzylamine reacted with S-methylisothiouronium sulphate, and this isomer was formulated as (XV). However, these structural

assignments need verification. It is relevant to note that the free base (XIII) reacted with cyanamide t o give the bis-guanidine [ 11 I ] . Further, guanidines have been obtained by reaction of cyanamide with salts of benzylamine [82] and p-nitrobenzylamine [87, 1011. The product from the latter was reduced [87, 1011 to authentic p-aminobenzylguanidine (XV) dihydrochloride, which decomposed only 3-5" above the reported [ 1 1 1 ] decomposition point of the dihydrochloride of the isomer assigned structure (XiV).

Method (b)

RZNH + RzN.C=NR

I

X

-

R2 N G N R

I

+ HX

NR2

The most frequently used synthesis of guanidines involves the displacement by an amine of a suitable group, X, from the amidine-type compound shown. The preparation of phenylguanidine from ammonia and phenylthiourea was claimed [ 1121 in 1879 but the authenticity of the product has been questioned

132

GUANIDINES A N D ADRENERGIC NERVE ENDINGS

[79]. The generally recognized originator of this synthesis of guanidines is Rathke who showed that ammonia reacts with S-ethyl-N,N'-diphenylisothiourea to form N,N-diphenylguanidine [ 1131 . That this product can be formulated in two ways: PhNH.C(:NPh)NH, or PhNH.C(:NH)NHPh, was clearly recognized at that time. Similarly, guanidine itself was obtained from S-ethylisothiourea and ammonia [ 1141. Although 0-ethylisourea was reported not t o react with ammonia or with aniline [ 7 9 ] , the synthetic potential of this method became apparent when various polymethylguanidines were prepared from S-alkylisothiouronium hydriodides [ 14, 115, 1161 or, more conveniently, from S-methylisothiouronium sulphate [ 14, 1171. Aniline was reported not t o react with the latter salt [ 1171, but a later paper [ 1181 described its conversion into phenylguanidine. Many different alkvl- and awl-guanidines have been obtained by this procedure [SS, 86, 1 1 1, 119-1321, as have alkoxyguanidines [97, 1331, and benzoylguanidines [ 134, 1351 . It is usual t o carry out the reaction in water or ethanol, or in mixtures of the two, at temperatures ranging from 60" to the boiling point, for from 2 t o 1 2 hours. Butanol has also been used as a solvent [82, 1361 and, exceptionally xylene [I371 and dimethylformamide [138], or even n o solvent a t all [I361 The alkyl mercaptan which is evolved in this reaction can be absorbed in a solution of sodium hydroxide and hydrogen peroxide [135] or in a charcoa1:cupric chloride trap [ 1391 . The guanidines are often conveniently isolated via their relatively insoluble bicarbonates [ 118, 1391 . King and Tonkin [82] used this method t o prepare many guanidines and, more recently, other workers [109, 140, 1411 have studied its scope. I t has been stated [ 141 ] that primary amines are generally more reactive than secondary amines, but there are many exceptions [109, 140-1431. A series of comparative experiments has demonstrated [ 1441 that the structural requirements for a successful reaction are often fairly critical; for example, RCHzCMe2.NH2 gave the guanidine when R = OH, NHPr, or NHBu, but not when R = H or NMez. In spite of this work, and the successful preparation [139] of cr-methyl- and a-ethyl-benzylguanidine, as well as trans-2-phenylcyclopropylguanidine,it has recently been stated [ 1411 that this method is not successful when benzylamines are substituted by a methyl group on the cr-carbon atom. Substitution of two methyl groups on the p-carbon atom of aliphatic amines slows down the reaction with S-methylisothiouronium sulphate [228] ; two methyl groups on the a-carbon atom, as in t-butylamine, prevent reaction altogether [228] . When the amine and the S-alkylisothiouronium group are part of the same molecule, an intramolecular synthesis of a guanidine can occur with great ease [145, 1461. A special effect attributed to the presence of a cyclic system is the reaction between aziridines and S-methylisothiouronium salts t o give 2-methylthioethylguanidines [ 147, 21 51. There have, of course, been many applications of this reaction t o the preparation of guanidino-acids such as arginine [ 6 6 , 9 0 , 149-1561 .

133

G. J. DURANT, A. M. ROE, A. L. GREEN.

An important development of the synthesis described above is derived from the observations of Scott, O’Donovan and Reilly [ 1591, which were taken up by others [ 1091 . l-Guanyl-3,5-dimethylpyrazolenitrate reacts with alkyl- and arylamines in hot water, ethanol, or without solvent to give good yields of guanidines. Use of this pyrazole nitrate or other salt for obtaining guanidines now competes with older methods if yield and ease of isolation of the product are the main considerations [95, 138, 140, 143, 160-1641. The foregoing methods are those most generally applicable t o the synthesis of guanidines. There are many other ways in which guanidines have been obtained, some of which have occasionally been used to prepare compounds whose biological properties will be discussed later. Although there are many claims in patents to the preparation of alkylguanidines by reaction of guanidine with alkyl halides, this method, which was first studied by Schenck [ 1651 ,has only rarely been found satisfactory. Primary and secondary alcohols in 80-85 per cent sulphuric acid react with guanidines to give the monoand di-alkyl derivatives [ 1661 . A process for direct alkylation of guanidine by an alkyl tosylate has been developed [95, 138, 1671. The acylation of guanidines can be controlled more easily than alkylation; and esters, acid anhydrides, and acid chlorides have been used successfully [ 134, 168-1 731 . Guanidines have been prepared by the reaction between an amine, or an amine salt, and a host of other reagents, such as a thiourea in the presence of lead or mercuric oxide [83, 157, 1581, carbodi-imides [140, 174, 1751, calcium cyanamide [ 176, 1771 , isonitrile dichlorides [ 178-1 801 , chloroformamidines [ 18 11 , dialkyl imidocarbonates [ 1821 , orthocarbonate esters [ 1831 , trichloromethanesulphenyl chloride [184], and nitro- or nitroso-guanidines [ 185-1881. Substituted ureas can furnish guanidines, either by treatment with amines and phosphorus oxychloride [ 1891, or by reaction with phenylisocyanate [ 1901 or phosgene [ 191] . Aminoguanidines

Since the synthesis of aminoguanidines has been comprehensively discussed by Kurzer and Godfrey [61] ,only the more recent developments are included in this review. Most of the methods used for the synthesis,of guanidines are adaptable t o the synthesis of aminoguanidines, but the fact that a mono-substituted aminoguanidine can be one of three isomers (XVI), (XVII) or (XVIII) adds interest t o the preparative methods. RN H.NH.C=NH

I

NH2

(XVI)

R.N.C=NH

I 1 H2N NH2

(XVII)

RN HC=N H

I

NHNH2

(XVIII)

134

GUANIDINES AND ADRENERGIC NERVE ENDINGS

Until recently it was thought that mono-substituted hydrazines invariably reacted with S-methylisothiouronium salts t o give compounds of type (XVII) [129, 192, 1 9 3 ) ; however, examples have now come t o light in which the alternative isomer (XVI) has been isolated from this reaction [21, 131, 194, 1951. This formulation is based on (a) the failure of the products t o react with benzaldehyde, [21, 129, 131, 1941, (b) the unambiguous synthesis of the two possible alternative structures [21, 129, 1941, and ( c ) an examination of the nucleai magnetic resonance spectra of the mono- and di-protonated forms of some

RCH2 .NH.NH-

c

=NH

RCH2 .N-C=NH

I I

H2N NH2

NH2

+H']

I

- H'

+H']

(XIXa)

-H'I[

R C H l~

+H'

f - H'

I

+H+1 - H'

j.N H-C ~ +-N ~H~

I

NH2 (XIXb) pairs of isomers of type (XIX) and (XX): in trifluoroacetic acid and in 90 per cent sulphuric acid (XIX) and (XX) exist as the mono- (XIXa) and (XXa), and di-protonated forms, (XIXb) and (XXb) respectively. Under these conditions, the effect of the second positive charge on the chemical shift of the methylene protons is considerably greater when the charge is on an adjacent nitrogen (XIXb) than when on a more remote nitrogen as in (XXb) [21, 1941. This analysis

G. J. DURANT, A. M. ROE, A. L. GREEN.

135

enables a monosubstituted aminoguanidine of unknown structure to be assigned structure (XIX) or an isomeric structure. Compounds of type (XVI) have been obtained by reduction of guanylhydrazones [ 129,1921, by treatment of an N-substituted-N-benzylhydrazinewith 1-guanyl-3,5-dimethylpyrazolesulphate followed by reductive removal of the benzyl group [ 1941, or by reaction of a hydrazine with a substituted cyanamide [ 1961 . An N,N-dialkylhydrazine reacts with S-methylisothiouronium salts unambiguously to give an N,N-dialkylaminoguanidine[ 1971 . Structures of type (XVII) have been obtained by treatment of an N-alkyl-N-cyanohydrazinewith ammonia [ 1941. The use of I-guanyl-3,5-dimethylpyrazolesalts for preparing compounds of type (XVI) originated with Scott [ 159, 1981, and this reaction has also been used by others [ 163, 1941 . Compounds of type (XVIII) have been prepared by reduction of a nitroguanidine [ 1991,by reaction of an amine with S-methylisothiosemicarbazide [ 13 1, 194,199,2001 ,or by reaction of hydrazine with a substituted S-methylisothiouronium salt [ 131, 1931. PHARMACOLOGICAL TEST PROCEDURES Effect on blood pressure

Direct measurement of the effect of drugs on the blood pressure of animals has found little use as a screening procedure. This apparent deficiency in hypotensive drug testing stems mainly from three causes. Firstly, a fall in blood pressure is difficult t o interpret in the absence of considerable background information about the site and mode of action of the drug concerned; it might, for example, result from the liberation of histamine or from a myocardial depressi.on reducing cardiac output, neither of which is of therapeutic value. Secondly, many compounds of established utility as antihypertensive drugs in man, d o not lower blood pressure when administered in single doses t o anaesthetized animals. Thirdly, even when administered over a long period, many established antihypertensive drugs fail to lower the blood pressure of normotensive animals. This last problem can be partly overcome by use of animals which have been deliberately made hypertensive in one of the following three ways, (a) by surgical destruction of the buffer nerves in the regions of the carotid sinus and aortic arch'(neurogenic hypertension), (b) by reducing the blood supply t o the kidney, using clips on the renal arteries or encapsulation of the kidneys in cellophane or latex (renal hypertension), or (c) by giving animals a diet with a high sodium chloride content and administering adrenal corticoids, particularly deoxycorticosterone acetate (DOCA). The argument that such hypertensive animals do not represent a realistic model of human hypertension and that results obtained in them by the use of drugs may have no relevance to the human situation, has some validity; nevertheless, the effectiveness of known antihypertensive drugs can almost always be demonstrated in such animals. In man, the therapeutic value of antihypertensive drugs lies in

136

G U A N I D I N E S A N D A D R E N E R G I C N E R V E ENDINGS

their ability t o lower the blood pressure rather than in removing the underlying cause of the raised blood pressure. Even so, the technical problems entailed in the production of hypertensive animals and the regular determination of their blood pressure are considerable, and the use of such animals is generally confined t o providing confirmatory evidence of an antihypertensive action in drugs which, on other grounds,have been thought likely to possess such an action. A comprehensive and detailed review of methods for measuring arterial blood pressure, and of producing hypertensive animals, has been given by Boura and Green [201]. Effect on the sympathetic nervous system

The more usual methods which have been employed for testing guanidine derivatives as potential antihypertensive drugs depend on detecting some form of interference with the sympathetic nervous system. This.subject has also been reviewed recently in considerable detail [ 7 0 7 ] , and only those methods which have been, or could be, widely applied to elucidating structure-activity relationships are described here. By far the most popular test system is the cat nictitating membrane. In unanaesthetized cats, the nictitating membranes are normally kept retracted, and barely visible, by nerve impulses conveyed along the cervical sympathetic nerves. Abolition of these impulses by surgical denervation, or by sympathetic blocking drugs, causes the membrane t o relax until it covers about 70 per cent of the eye. The extent of relaxation can be measured by estimating the position of the membrane along the lower lid with a ruler or calipers, or by photographing the eyes and estimating the percentage covered by placing transparent paper with a square grid marked on it over the photographs. This latter procedure is often less satisfactory because of the closure of the palpebral fissure which generally accompanies relaxation of the membrane. Whichever method is used, it is better t o aim at a quick approximate result with minimal disturbance of the animals. When using this test, it is desirable for the experimenter to observe the cats at various times up to at least 38 hours after injection of the drug. since the onset of relaxation may be delayed u p to 74 hours or more. Furthermore. a range of drug doses should be used rather than a single arbitrary test dose since some adrenergic neurone blocking drugs also possess a contracting action on the membrane which. at higher doses. masks the sympathetic blockade and leads to anomalous dose-response curves [ 203. 7041 . In comparing results from different laboratories it is important t o note whether they are expressed as percentage relaxation. which generally means percentage cover o f the eyes (maximum about 70). or as a percentage of maximal relaxation (maximum 100). In anaesthetized cats. the tone of the nictitating membranes may be recorded with a writing lever o r strain gauge. Stimulation of the cervical sympathetic nerves at a frequency of 0.1 to 50 pulses/second causes the membranes to contract. The magnitude of the contractions is dependent on the frequency of

G. J . DURANT, A. M. ROE, A. L. GREEN.

137

stimulation, but it does not increase much at rates above lO/second. The potency of sympathetic blocking drugs can be assessed from the extent by which they reduce the magnitude of the contractions. Ideally, all drugs should be tested at a range of frequencies, as some drugs are more effective at low stimulation rates than at high ones, hence the relative potencies of two drugs may depend on the stimulation rate. This is rarely done, but when comparing a group of chemically closely related compounds the error due t o use of a single stimulation rate is probably less than that arising from individual variation among the small number of animals which are generally used. Relaxation of the nictitating membrane in conscious cats, and failure of the nictitating membrane t o respond t o sympathetic nerve stimulation in anaesthetized cats, may be brought about by a blocking action a t the sympathetic ganglia, at the post-ganglionic sympathetic nerve endings, or at the effector organ receptors. A decision as t o the primary site of action of any particular drug can usually be achieved without much difficulty. Ganglion blocking drugs cause mydriasis in conscious cats, by depressing the transmission of parasympathetic impulses through the ciliary ganglion, as well as relaxing the nictitating membrane. In anaesthetized cats, they block contractions of the nictitating membrane in response t o pre-ganglionic stimulation of the cervical sympathetic nerve without affecting responses t o post-ganglionic stimulation. Drugs which prevent the release of noradrenaline from the post-ganglionic sympathetic nerve endings cause relaxation of the nictitating membrane in conscious cats but n o mydriasis:and in anaesthetized cats, they block the contractions of the nictitating membranes produced by pre-ganglionic or post-ganglionic nerve stimulation without affecting contractions due t o injected noradrenaline. Drugs which block the a-receptors on the membrane itself cause relaxation without mydriasis in conscious cats; in anaesthetized cats, they tend t o inhibit the contractions of the nictitating membrane in response t o injected noradrenaline more readily than responses t o nerve stimulation, although these too are blocked a t higher doses. A further distinction can be made between adrenergic neurone blocking drugs such as xylocholine, bretylium and guanethidine, which act a t the post-ganglionic sympathetic nerve endings by inhibiting the release of noradrenaline in response t o nerve stimulation, and those, such as reserpine, which act by depleting the noradrenaline from the nerve endings. The effects of $rugs of the former type on the nictitating membrane can be readily reversed by injection of low doses of amphetamine. These differences are summarized in Table 3.2. Another method for use in unanaesthetized animals, which is akin to studying the relaxation of the nictitating membrane in cats, is the observation of ptosis in mice or rats. These smaller and less expensive animals can be used in much larger numbers than cats, and the method does not suffer from the anomalous doseresponse curves mentioned previously. Ganglion blocking drugs, adrenergic neurone blocking drugs and a-receptor blocking drugs can be simply distinguished by essentially the same means as those used in cats. However, the method has not so far been widely exploited. A full description, with illustrative examples

Table 3.2. DIFFERENTIATION OF SYMPATHETIC BLOCKING DRUGS

Nictitating membrane

Type of drug

Pupil

\

A

/-

Response t o preResponse t o postganglionic stimularion ganglionic srimulation

A

Response to noradrenaline

Reversal of block by amphetamine

Mydriasis

Normal*

Not

Yes

Ganglion blocking (e.g. hexamethonium, pempidine)

Blocked

Normal

Adrenergic neurone blocking(e.g. xylocholine, guanettfidine)

Blocked

Blocked

Normal*

Yes

No

Noradrenalinedepleting (e.g. reserpine)

Blocked

Blocked

Normal*

Not

No

&Receptor blocking (e.g. phentolamine, phenoxybenzamine)

Blocked

Blocked

Blocked

No

No

*The response of the nictitating memhrane may often be potentiated. depending b o t h o n the time the noradrenaline is given after the sympathetic blocking drug and o n the dose o f the latter. t A partial reversal of block may sometimes be encountered. but t h i s effect is short-lived a n d Mock subsequently re-appears.

G . J . DURANT, A. M. ROE, A. L. GREEN.

139

comparing the mouse ptosis and cat nictitating membrane tests, has been published elsewhere [203]. Numerous in vitro preparations, consisting of isolated smooth muscles with attached nerves, have been described for studying the actions of sympathetic blocking drugs, but only one of these, the isolated rabbit mesenteric nerveintestine preparation (or Finkleman preparation) [205] has been used at all extensively as a screening test. This preparation is a short segment (about 3 cm) of intestine, which is removed from a freshly killed rabbit, together with the mesentery containing the branches of the mesenteric artery which supply the segment. This piece of intestine is suspended in oxygenated Tyrode solution in a small jacketed water bath. The lower end of the segment is attached to the base of the bath and the upper to a writing lever. Electrodes are attached to the mesenteric artery enabling the mesenteric nerve, which runs periarterially, to be electrically stimulated. In the absence of stimulation, the segment undergoes spontaneous pendular contractions at a rate of about 10 per min. Stimulation of the mesenteric nerve, or addition of noradrenaline to the bath, inhibits these spontaneous contractions. Adrenergic neurone blocking drugs added to the bath abolish the inhibitory effect of nerve stimulation but not that of noradrenaline, whereas a-receptor blocking drugs abolish the inhibitory effect of noradrenaline as well as that of nerve stimulation.

STR UCTURE-ACTIVITY RELATIONSHIPS FOR ADRENERGIC NEURONE BLOCKADE

For the purpose of examining the effect of chemical structure on the pharmacological activity of guanidines at sympathetic nerve endings, compounds are discussed in terms of three main classes. These classes, namely, guanethidine and close analogues, aryloxyalkylguanidines, and aralkylguanidines (in addition to some miscellaneous guanidines) perhaps represent the attempts of chemists to manipulate a particular type of guanidine or related structure, more than a strict categorization on chemical grounds. Nevertheless,, there are indications of structural prerequisites for biological activity and apparent structure-activity relationships within these subdivisions. However, it is appreciated that compounds discussed in one subsection are often pertinent to others. As an example, the activities of guanethidine analogues containing a fused benzene ring are relevant to the discussion of aryloxyalkylguanidines and aralkylguanidines. Guanethidine and close analogues

Guanethidine (I) (p. 125) is a potent antihypertensive agent of great clinical importance [206]. A detailed discussion of the pharmacology of guanethidine is

140

GUANIDINES AND ADRENERGIC NERVE ENDINGS

beyond the scope of this review and, for further pharmacology, the reader is referred to other sources [ l o , 207-2091. Here, we merely indicate the major factors involved in the action of guanethidine at sympathetic nerve endings so that the activities of related compounds may be compared. The hypotensive action of guanethidine seems most likely t o be attributable t o blockade of transmission at adrenergic nerve terminals, and is associated with the inhibition of the release of the neurotransmitter, noradrenaline [207] .Guanethidine may therefore be classified as an adrenergic neurone blocking agent [ l o ] . As discussed later, the marked depletion of tissue stores of noradrenaline which is produced by guanethidine, appears unlikely to be responsible for the adrenergic neurone blockade. The adrenergic neurone blocking action of guanethidine is accompanied by an initial transient sympathomimetic activity [ 2071 caused by release of free catecholamines. Guanethidine also inhibits the release of noradrenaline induced by indirectly-acting sympathomimetic amines, such as tyramine and amphetamine [2071. ~. A great many compounds have been synthesized which are chemically related to guanethidine. Discussion of the relationship between chemical structure and biological activity withn this class of compound is complicated by the varying criteria of activity used by different workers. For instance, some [ 127, 2 l o ] have compared close analogues of guanethidine in terms of antihypertensive activity, whilst others [ 140,211, 2121 have used adrenergic neurone blockade as their criterion. In this review, most of the data are presented in tabular form which lists, where possible, both antihypertensive and adrenergic neurone blocking activities. Unless otherwise specified, relaxation of the nictitating membrane of the conscious cat is used as the criterion of the latter action. Other relevant features of the action of guanethidine analogues which appear in the literature are described in the text and indications of clinical activity are included. The Tables are compiled on a chemical basis which emphasises different structural aspects of the guanethidine molecule. Modification of the ring

Open chain analogues of guanethidine were originally reported to be devoid of antihypertensive activity [210]. However, it has subsequently been shown that numerous compounds in this category are active as adrenergic neurone blocking agents (Table 3.3). For example, it is reported that, compared with guanethidine, 2-(methylisobutylamino)ethylguanidine (XXI) has a more potent action of longer duration and more rapid onset [211] . 2-Cyclohexylaminoethylguanidine (XXII) was found to be similar to guanethidine when their actions on isolated sympathetically innervated organs were compared [213]. The adrenergic neurone blockade was persistent but could be reversed by amphetamine [213] . 2-Diethylaminoethylguanidine was found to beonlyslightly less active than guanethidine both in virro [213, 2141 and in vivo [140, 21 1,2121.

141

G. J. DURANT, A. M. ROE, A. L. GREEN.

Short andDarby [ 2 151 have recently reported that 2(2-methylthioethylamino) ethylguanidine (XXIII) relaxes the nictitating membranes of conscious cats, and Me2CH.CH2 .NCH2 .CH2-NH-C=NH

I

Me

I

C6H 11.NHCH2-NHC=NH

NH2

NH2

(XXII)

(=I)

has a hypotensive action, similar to that of guanethidine, in the anaesthetized cat. Comparison of closely related compounds indicated a considerable specificity in certain of the structural requirements for activity (Table 3.3). For example, Table 3.3. T H E ACTIVITY OF OPEN CHAIN ANALOGUES OF GUANETHIDINE R1R2NCH2CH2.NHC(:NH)NH2

R' Me Et H H Me Et H Me H Pr Pr. Pr f Pr' Me Me Et Me Pr Bu H H H Et H H

R2

Adrenergic neurone blocking activity

Inactive ( 1 4 0 , 2 1 3 ) Slight activity [211] Active [ 1 4 0 , 211-2141 Inactive [ 2151 Active [ 2 1 1 ] BU' Prf Active 12111 Pr' Active (2111 Ami Active [ 2 1 1 ] But Active [ 2 1 1 ] Active [ 2 1 1 , 2131 C6H11 Pr Active (2111 Slight activity [214] Pr f Active I2111 Pr' Slight activity 1 4 0 , 2 1 4 1 Bui Active 12111 C6Hll Active 1211, 2 41 C7H13 Active [ 1 3 5 , 2 41 C6Hll Active [ 2 1 1 ] CaH 15 Active [ 1351 C6Hll Active [ 2 1 1 ] C6Hll Inactive [ 2 1 1 ] MeS(CH92 Active (2151 EtS(CHd2 Inactive [ 2 1 5 ] MeO(CHd2 Active [ 2151 MeS(CHd2 Active [ 2 1 5 ] MeS(CHd3 Active [ 2 1 5 ] Et*N(CH& Inactive 12151 Me Et Et .

A n tihypertensive activity -

-

Inactive [ 2101 -

Active [ 2 1 5 ] -

Active [ 2151 Active [ 2151 Inactive [ 2 1 5 ] -

replacing sulphur by oxygen in compound (XXIII) maintains the adrenergic neurone blocking activity, although at a lower potency, but replacing the methyl group of (XXIII) by ethyl gives a compound which does not relax nictitating

142

GUANIDINES AND ADRENERGIC NERVE ENDINGS

membranes at 30 mg/kg [215]. The activity of these open chain structures is a clear demonstration that the heterocyclic ring is nof a prerequisite for adrenergic neurone blocking activity within guanethidine-like compounds. The association of peak antihypertensive activity with the eight-membered ring of guanethidine rather than other ring sizes has been discussed previously Table 3.4. THE ACTIVITY OF RING SIZE MODIFICATIONS OF GUANETHIDINE (CH2),N€H2€H2eNHC(:NH)NH2

n 4 5

6 7 8 9

Adrenergic neurone blocking activity

Antihypertensive activity

Active [ 2141 Active [ 2 1 4 ] High activity [ 2 1 4 ] Peak activity [ 2 1 4 ] -

Active [ 1271 Active [ 127, 2101 High activity [ 127,2101 Peak activity [ 1 2 7 , 2 1 0 ] High activity [ 127,2101 Active [ 2101

[127, 2101 (Table 3.4). Recently, Ozawa and Sato [214] have compared the pharmacological properties of several of these analogues in more detail. The activities of the compounds on the isolated rabbit ileum paralleled their reported MeS-CH2CH2.NHCH2-CH2.NH.C=NH R'R2N-CH2CHz.NH-C=NH

I

I

NH2

NH2 (XXIII)

(XXW

antihypertensive activities (XXIV, R'RZ = (CH,), 2 (CH2)6> (CH,),

> Etz

= (CH2)4).

Other compounds tested (for example (XXIV), R' R2 = Pr2; P r i ;C6Hi1, Me) were either very weakly active or inactive. In the anaesthetized cat, contractions of the nictitating membranes induced by noradrenaline or adrenaline were enhanced by the active compounds, but the contractions caused by tyramine were inhibited. The contractions elicited by stimulation of the pre-ganglionic superior cervical nerve were also blocked. Modifications of the moderately active piperidiw analogue of guanethidine (XXIV,RIRz = (CH2)5), in which the guanidinoethyl group is attached to different positions of the piperidine ring, show only very low adrenergic neurone blocking activity [ 1401. The effect of introducing methyl groups into the heterocyclic ring system of guanethidine and related compounds is recorded in Table 3.5. The high adrenergic neurone blocking activity of the methyl substituted piperidine analogues is of interest [211]. A potent unsaturated derivative of this type (XXV), guanacline (cyclazenine. Leron), has been introduced recently [216-2181. The 2,2,6,6-tetramethylpiperidine analogue (XXVI) of guanethidine also has hypotensive properties [129, 2191, but these are probably due to ganglionic

G . J. DURANT, A. M. ROE, A. L. GREEN.

143

blockade rather than toadrenergic neurone blockade. This finding is understandable, since the compound is essentially a derivative of the ganglion-blocking agent pempidine (XXVII) [ 1291 . Table 3.5. THE ACTIVITY OF RING-SUBSTITUTED GUANETHIDINE ANALOGUES RCH2CH2'NHC(:NH)NH2

R

u

e

a

Adrenergic neurone blocking activity

Active [ 2 1 1 ]

-

Active [ 21 11

-

High activity [ 2161 M

e

Antihypertensive activity

High activity [ 2161

0

Ganglion blocking activity [ 1291

Active [ 129,2191

ue Me

0:'

Slight activity 12101 Slight activity [421]

Diazacycloalkane analogues of guanethidine and related structures have been examined for both antihypertensive and adrenergic neurone blocking activity (Table 3.6). In contrast to what is found in the mono-aza series, the sixmembered piperazine ring system is associated with higher activity than the corresponding seven- and eight-membered structures. In the piperazines, the ring N-rnethyl compound (XXVIII) has the highest potency. Larger substituents lead t o loss of adrenergic neurone blocking activity (1401. In contrast to (XXVIII) the related morpholine and hexahydropyrimidinyl compounds are reported to be inactive. The high activity of bridged heterocyclic systems related to guanethidine was first reported by Schlittler, Druey and Marxer 12101 . Subsequent investigations

144

GUANIDINES AND ADRENERGIC NERVE ENDINGS

N.M e

Table 3.6. THE ACTIVITY OF DIAZACYCLOALKANE ANALOGUES OF GUANETHIDINE AND RELATED STRUCTURES R.CH2CH2.NH C ( :NH)NHz

R

R'

Adrenergic neurone blocking activity

Me Ph Me PhCHz

An tihypertensive activity Active [210,422] Slight activity [210,422

Active [ 140,4211 -

-

High activity [ 1401 Inactive [ 1401 Inactive [ 1401 Inactive [ 1401 -

Inactive [ 1401 -

Slight activity (2101 Inactive [210] Inactive [422] High activity [210] Active (2101 -

Active [ 2101 -

Slight activity [210] Inactive [210] Inactive [422] -

Slight activity 14221 Inactive [127, 2101 Inactive [ 127,2101

145

G . J . DURANT, A. M. ROE, A. L. GREEN

r'\

M eN

C H; C H; N H . C = N H

r\l. C H,. C '-I,. N H - C = N H

u

I

I

NHZ

NH2

(XXVIII)

i

sx1x:

Table 3.7. THE ACTIVITY OF BICYCLIC DERIVATIVES OF GUANETHIDINE RCHz €H*.NH €(: NH)NHz

R

Adrenergic netrrone blocking activity

A n tihypertensive activity

-

High activity [ 2 1 0 , 4 2 3 , 4 2 4 1

-

Active 1 2 1 0 , 4 2 3 , 4 2 4 1

Active [ 1 3 5 )

-

-

Slight activity [ 2101

-

Slight activity [ 2 1 0 ]

-

Active [ 4 2 4 ]

Active [ 2201

Active [ 2 2 0 , 4 2 4 1

Active [ 4 2 4 ]

Active [ 4 2 5 ]

I

XXX)

R=CH,.CH,.NH

C ( : N H ) NH,

Active (4251

146

GUANIDINES AND ADRENERGIC NERVE ENDINGS

have confirmed that bicyclic variants of guanethidine, bridged in several different ways, have antihypertensive activity, presumably acting by adrenergic neurone blockade (Table 3.7). The 2-aza-bicyclo [2,2,2] octane derivative (XXIX) has, however, shown undesirable side effects (severe diarrhoea in mice [220] , and the analogue with a three-carbon side chain caused cardiac arrest in dogs [220]. Bicyclic modification of guanethidine containing azaspiro-alkane ring systems (Table 3.8) have been studied in some detail. The most active compounds are comparable to guanethidine as sympathetic blocking agents, and show the following order of potency [221-2231: The total number of atoms in the azaspirocycle is optimally seven or eight, similar to the optimal requirements in the monocyclic guanethidine series. However, an additional factor with the azaspiro-alkanes is that the number of atoms in the heterocyclic ring should be as large as possible consistent with the previous condition. Compound (XXX) has a longer duration of action than guanethidine both in animals [221] and in man [224]. The antihypertensive activity of the seven-membered analogue of guanethidine in which a benzene ring is fused in the 4,5-position was reported in the review by Table 3.8. THE ACTIVITY OF AZASPIRO ANALOGUES OF GUANETHIDINE

k

m

n

1

2

2

Numbef of atoms in heterocycle

8

Adrenergic neurone blocking activity

High activitv 1221-223.4261 Active- [ 122,4271 Active [ 222,4261 Active [ 222,223,428) slight activity [222,223,426] Slight activity [ 222,223,4261 Inactive 1222, 223,4261 Inactive (222,223,4261 .

1 2 2 2

2 2 1 2

1 1 1 2

7 8 9

3

2

1

9

3 4

1 1

1 1

8 9

I

An tihypertensive activity

I

High activity (221-223,4261 Active [ 222,4271 Active [ 222,4261 Active [ 222,223,4281 Slight activity [222, 223,4261 Slight activity [ 222,223,4261 Inactive 1222. 223,4261 Inactive i222,223,426]

.

Schlittler, Druey and Marxer [210]. Subsequently, the analogous variant (XXXI) of guanethidine was shown to be an adrenergic neurone blocking agent [ 225,2261 Compound (XXXI) was found to be more potent than guanethidine on the guinea-pig isolated vas deferens preparation [227] . The sympathomimetic phase following the administration of compound (XXXI) to whole animals is reported to be less pronounced and more transient than that produced by guanethidine, and it has been suggested that the compound has a weaker amine-releasing

G . J. DURANT, A. M. ROE, A. L. GREEN.

147

capacity [226]. Central actions have also been observed with this compound [225].

NH,

Modifications of the side-chain

The ethylene side-chain present in guanethidine was formerly regarded as essential for optimum activity in compounds of this type [210]. However, the effect of introducing methyl groups has been reported only recently (Table 3.9). Rand and Wilson [213] studied the adrenergic neurone blocking potencies of a series of cyclohexylaminoalkylguanidines (XXXII), using isolated sympathetically innervated organs. Derivatives with methyl substituted side-chains (XXXII, R3 or R5 = Me)* were highly active in these preparations, being similar in potency to guanethidine, and they were possibly more active than the compound containing the unsubstituted ethylene linkage. The isobutyl analogue (XXXII, R3R4 = Me2) was also found to be a highly active antihypertensive agent in rats [213]. Short, Ours and Ranus [228] investigated methyl substitution in the side-chain of diethylaminoethylguanidine (XXXIII) and found that the isobutyl derivative (XXXIII, R3R4 = Me,) was considerably more active than the parent compound or other analogues (XXXIII, R3 = R4 = H, R5 = R6 = Me) in relaxing the

(XXXII) R'R2 = CIHll,H

R3 R5

I I

R' R2N-C-C-NHC

= NH

I I

I

R4R6

NH2

(XXXIII) R' R2 = Et

nictitating membranes of cats. Extending this to the hexamethyleneimines, these authors found that the isobutyl analogue (XXXIV, R3 = R4 = Me) was highly active, and in fact caused a remarkably long prolapse of the nictitating membrane (2 16 hours). However, this compound was reported to be disappointing below 10 mg/kg in hypertensive dogs [228]. Increasing the distance between the amino function and the guanidine group in compounds related t o guanethidine results in a decline in activity (Table 3.9) [ 127, 140, 2131. *Here and subsequently R = H unless otherwise specified.

148

GUANIDINES AND ADRENERGIC NERVE ENDINGS

Table 3.9. THE ACTIVITY O F SIDECHAIN MODIFICATIONS O F GUANETHIDINE AND ITS ANALOGUES R*A*NHC(:NH)NH2

R

A

0

High activity [ 127,207,2101 High activity [ 127,207, 2101 Slight activity [ 1271 Slight activity [ 1271 Inactive [210] Inactive [210] High activity [ 2281 Slight activity [ 2281 Active [ 2281 -

((392 (CH2)3

Active [ 1401 Active [ 1401 Inactive [ 1401 Active [ 2281 Active [ 2281 Active [ 228) Active [ 2281 Active [ 2281

-

(CH2)3 CMe2CH2

Active [ 140) Inactive [ 1401 Inactive [228]

-

(CH2)2 CH(Me)CH2 CH2CH(Me) CH(Me)CH(Me) CH(Et)CH2 (CH2)3

Active (2131 Active [213] Active [213] Active [213] Slight activity (2131 Slight activity [213]

(CH2)2

A MeN

N

NH

An tihypertensive activity

(CH2)2 (CH2)3 (CH2)4 CH(Ph)CH2 CH2CO CMe2CH2 CH2CMe2

CH(Me)CH2 CHzCH(Me) CMe2CH2 CH2CMe2 CH2CMe2CH2

Et2N

Adrenergic neurone blocking activity

-

-

-

Active [213] -

Modifications of the guanidine function

Modification of the guanidine function of guanethidine by alkyl group substitution does not seem to have been reported. In the related series of diethylaminoethylguanidines (Table 3.10) it was found that, whereas the analogue containing a methyl substituent on the terminal nitrogen atom had adrenergic neurone blocking properties, all other alkyl substituted guanidines tested were inactive [ 1401 . 2-Aminoimidazolines are cyclized N , N'-dialkyl guanidines: this variation of guanethidine was originally reported to be devoid of antihypertensive properties [210], although some activity has since been claimed for related compounds [ 1861 (Table 3.10). The guanidine function in compounds such as guanethidine is essentially completely protonated at physiological pH, and adrenergic neurone blockade is presumably caused by interactions of the cationic species at hypothetical

G . J. D U R A N T , A.M. R O E , A. L. G R E E N .

149

Table 3.10. T H E ACTIVITY OF G U A N E T H I D I N E A N A L O G U E S A L K Y L A T E D O N T H E G U A N I D I N E FUNCTION

Adreriergic rieuroiie blockirig activity Active [ 1401 Inactive 1401 Inactive 1401 lnac tive 140) Inactive 1401 Inactive 1401

Et2NCH2CH2.NHC(:NMe)NH2 EtZNCH2CH2’NHC(:NBu)NH2 Et2NCH2CH2.NMeC(:NH)NH2 Et2NCH2CH2.NHC(:NEt)NHEt Et2NCH2CH2.NHC(:NMe)NMe2

n

M e N ~ ,N.CH, U

A I I rilij,perretisive activitj,

CHz.NH.CI N B u I N H Z

Cyclic guariidines Active [ 861

Inactive [210]

G

Active 1,1861

N CH, CH, N H C’

.N-

Bu,N CH, CH, NH C

“ J

Active [ 1861

Active [ 186)

‘NH

EI,N

CH, CH, NH C

EL,N

C H 2 CHz NH

Active [ 1401

Inactive [ 1401

C4

receptor sites (see p. 199). It is natural therefore that strongly basic groups other than guanidine should have been incorporated into guanethidine and analogues for investigation of adrenergic neurone blocking activity (Table 3.1 1). Guanethidine was developed from a series of amidoximes (for example XXXV, Su 4029) which were potent antihypertensive drugs. However, compound (XXXV) had n o adrenergic neurone blocking properties when examined for relaxation of the cat nictitating membrane, although tissue stores of noradrenaline were extensively depleted and the pressor effects of amphetamine and other indirectly

C

N-CH,.

CH,. C=NOH

I NHZ

C

N-CH2*CH2*NH.C=NH

I

Me

(XXXV) (XXXVIJ acting sympathomimetic amines were suppressed [230-2321. In this series of amidoximes, peak antihypertensive properties are associated with the sevenmembered ring system. This is in contrast t o the guanethidine series, in which the eight-membered ring bestows maximum activity.

150

GUANIDINES AND ADRENERGIC NERVE ENDINGS

Table 3.1 1. THE ACTIVITY OF GUANIDINE MODIFICATIONS OF GUANETHIDINE AND ITS ANALOGUES RCHzCH2.X

Adrenergic neurone blocking activity

X

R

C(: N0H)NHz C(: NH)NHz / 7~~ NH .C( : NH)NH NH 2 _ ~ NH.NHC(:NH)NHz , S C ( N H )NH NHC(: NH)NHC(:NH)NHz

’

Active (4311 Active [ 2341

-

O.NH.C(: NH)NH2 C(:NOH)NHz C(:NH)NHz NH-C(:NH)Me

Inactive [230-2321 Active [ 2 1 0 , 4 3 0 ] Active [ 2331

N(NH~)C(:NH)NHZ NHC(: NH)NHNHz

Inactive [ 1401 Inactive [ 1401

CN EtzN

Inactive [ 230-2321

Antihypertensive activity Active [ 2 1 0 , 2 3 0 , 4 2 9 3 Active [ 4 3 0 ] Active [ 1 9 9 , 4 3 1 1 Active [ 2341 Inactive [ 2 lo] Inactive 12101 Inactive [ 4 3 2 ] Active [ 2 1 0 , 2 3 0 , 4 2 9 1 Active [ 2 1 0 , 4 3 0 1 Active [ 2331

The corresponding amidines also have antihypertensive properties, but whereas the amidine with the seven-membered ring exerts similar actions to guanethidine, the higher homologue is reported to act more like the amidoxime [210]. Some related N-substituted amidines are reported to be adrenergic neurone blocking agents and the amidine (XXXVI) is comparable to guanethidine in potency [233]. Adrenergic neurone blockade by the amidine (XXXVI) is accompanied by sympathomimetic effects and depletion of catecholamine stores [233]. Peak adrenergic neurone blocking activity within this group of N-substituted amidines is associated with the seven-membered ring system, and the presence of a two-carbon sidechain is essential [233] . Aminoguanidines related to guanethidine form another clas8 of compound with adrenergic neurone blocking properties. The fall in blood pressure due to compounds of type (XXXVII) is reported to be of shorter duration than that caused by guanethidine [ 1991. The isomer (XXXVIII) has antihypertensive actions very similar to those of

NH NH2

IXXXI’I I i

H2N

i XXXVIII 1

guanethidine in several animal species [234] ,the compound acts like guanethidine in causing adrenergic neurone blockade; and the sympathomimetic action of guanethidine is largely eliminated by this modification of the guanethidine molecule. Various other basic terminal groups have been investigated in the

G. J . DURANT,A. M. ROE,A. L. GREEN.

15 1

guanethidine series, but n o appreciable antihypertensive or adrenergic neurone blocking actions have been reported (Table 3.1 I ) . Aryloxyalkylguanidines and related structures

Several years before the discovery of guanethidine, Hey and Willey [23S] had found that choline 2,6-xylyl ether bromide (XXXIX, xylocholine, TbllO) blocked transmission at postganglionic sympathetic nerve terminals. I t was shown subsequently that this blockade resulted from inhibition of the release of the adrenergic transmitter at the nerve endings [236-2381. The clinical us< of xylocholine as an antihypertensive agent was, however, limited by marked niuscarinic effects. A large number of structures related t o xylocholine have been synthesized in attempts to obtain antihypertensive drugs of clinical utility. Although reduction or abolition of the gross muscarinic stimulation, with retention or enhancement of adrenergic neurone blocking activity, was achieved by suitable modification of the xylocholine molecule, n o drug of proven clinical

($-0.Cti2,CH2.&4e3

6r

I XXXIX)

value emerged from this study [ 2 3 9 ] . Erratic absorption is one common disadvantage of orally administered quaternary ammonium drugs and this may have been a problem here. It is interesting that xylocholine has a structure derived from phenoxyethylamine, since the classical work of Bovet and BovetNitti [ 2401 demonstrated that aryloxyethylamine derivatives (XL) have affinity for adrenergic receptor sites (as sympathomimetics or sympatholytics). A later example is the potent adrenolytic action of phenoxybenzamine (Dibenyline Dibenzyline), (XL, Ar = Ph, R' = PhCHz, R2 = CHzCH2CI), which is thought [ 24 I ] t o be due to the cationic species(XL1, Ar = Ph, R' = Ph CH,). As xylocholine acts at sites within the presynaptic nerve terminal and not at a-adrenergic receptors in the effector cells, the significance of this c o F m o n structural feature is uncertain. The discovery of guanethidine, a drug with a structure including the ArO .C H2 .CH .N R

' RZ

ArO-CHz.CH2-y\bH + / CH2 2

y a n i d i n i u m cation, and with a potent blocking action at peripheral sympathetic nerve endings prompted the investigation of compounds related to xylocholine

152

GUANIDINES AND ADRENERGIC NERVE ENDINGS

in which the quaternary ammonium group was -.replaced by the guanidine function. The adrenergic neurone blocking action of 2-(2,6-~ylyloxy)ethylguanidine (XLII) was reported independently by two groups [ 1 3 5 , 2 4 2 ] .

I NH2

(XLJI)

The blockade caused by (XLII) was equal in intensity t o that induced by similar doses of guanethidine and the duration of action was even longer [ 135, 2431. The potency of (XLII) on the Finkleman preparation was much greater than that of guanethidine [243]. The compound had initial sympathomimetic effects and produced a triphasic response on the blood pressure of anaesthetized cats and dogs. The antihypertensive action of (XLII) in man was comparable to that of guanethidine [243], and a mechanism of action similar to that of guanethidine was indicated. Muscarinic properties similar to those exhibited by xylocholine were originally attributed [242] to(XLI1) but Barron, Natoff and Vallance [243] were able to demonstrate that parasympathetic stimulation is not a feature of this guanidine analogue of xylocholine. Thus, replacement of the trimethylammonium head of xylocholine with the guanidine moiety removes the muscarinic properties whilst retaining or enhancing its adrenergic neurone blocking effects. Attempted analysis of structure-activity data in compounds related to (XLII) is particularly complicated since they often give anomalous dose-response curves for their effects on the nictitating membranes of cats [203]. Some results are listed in Table 3.12, and it is apparent that aryloxyalkylguanidines may appear only weakly active at high doses (50 mg/kg, s.c.) but show high activity at much lower dose levels. Also given in Table 3.12 are results from an assay for adrenergic neurone blockade employing ptosis production in mice [203]. It may be deduced from these results that whilst the 2,6-xylyl derivative is a highly potent adrenergic neurone blocking agent, this property is by no means unique to the 2,6-disubstituted structure, but is also possessed to a large degree by some monosubstituted compounds. Unsubstituted phenoxyethylguanidine does not appear to have a marked adrenergic neurone blocking action, and substituents undoubtedly greatly influence the magnitude of the blocking activity. There is evidence of steric factors with 2,6-disubstituted .compounds, since the high activity associated with (XLIII, A = 0 , Ar = 2,6-F2C6H3, 2,6-C12C,H3 or 2,6-Me,C6H,) declines when the size ot the substituent is increased, i.e. (XLIII, A = 0 , Ar = 2,6-Et2C6H3 or 2,6-Pri2C6H3)(Table 3.12). A somewhat similar reduction in adrenergic neurone blocking action associated with an increase in bulk of the groups flanking the ether linkage has been observed with analogues

G. J. DURANT, A. M. ROE, A. L. GREEN.

153

of xylocholine [239] . A recent study [244] of 2,6-disubstituted aryloxyethylguanidines and aryloxyethylammonium salts has shown that in the former series, the 2, 6-difluoro compound was the most active in conscious cats; the order of Table 3.12. THE ACTIVITY O F ARYLOXYALKYLGUANIDINES ArO*(CH2),*NHC(:NH)NHz

Ar

An tihypertensive activity

n Adrenergic neurone blocking activity* Ptosis score f

35% coverage of the eyes

50% coverage of the eyes

2 >SO[244] >SO (1351 0 2 2.5-5 [203] >SO [135] 5.2 2 2.5-5 [203] >SO [ 1351 4.8 2 1 0 [203] >50 [135] 2.0 10 [135] 5 -2 Active [ 135,2431 2 2.5 [203] I 0 [244] 50 (1351 2 2 >50 [135] 2, > 4 0 [ 1351 2 >SO [135] 2 2 0 [ 1351 2 < 5 [23<] 10 [244] 5 [21] 2 5 [244] 2 3-10 [239] 2 >SO [ 135) 2 > 3 8 [135] 2 50 [1351 Active [ 248.249) 3 Inactive (248, 2491 3 50 [135] Active [251] 3 Active [ 25 1] 4 >SO [1351 -

>

*Cat nictitating membrane; approximate dose (mg/kg, s.c.) causing the coverage shown.

Figures in italics represent interpolated data tPtosis assessed 2 h after injection into mice ( 2 0 mg/kg), maximal score usually

4-6 [ 2031

potency in the latter series increased when the 2,6-substituents were H < F < Me < C1 [244]. Related 2,6-xylyl thioethers (XLIII, A = S,Ar = 2,6-Me2C,H3) and amines (XLIII, A = NH or NMe, Ar = 2,6-Me2C,H3) are also highly active adrenergic neurone blocking agents, as evidenced by the nictitating membrane assay, whereas the unsubstitutedcompounds(XLII1,A = S or NMe, Ar = Ph) were apparently inactive at high doses [ 1351 (Table 3.13). However, 2-(methylpheny1amino)ethylguanidine (XLIII, A = NMe, Ar = Ph) is reported to be an adrenergic neurone blocking agent, with an action similar to that of guanethidine [245-2471. Groups flanking the ether linkage markedly affect the adrenergic

154

GUANIDINES AND ADRENERGIC NERVE ENDINGS

Table 3.13. THE ACTIVITY OF MISCELLANEOUS ARYLOXYALKYLGUANIDINES AND RELATED STRUCTURES ArA*NHC(:NH)NH2

Adrenergic neurone blocking activity.

Ar

A

2,6-Me2C6H3 2,6-h!e,C6H3 Ph 2,6-hIe2C6113 2,6-h'fe2C,H3 Z ,6-Me 2C6H 3 Ph

OCH(Me)CH2 S(CH2)2 S(CH2h NH(CH2)2 NMe(CH2)2 NEt(CH 2)2 NMe(CHd2

Antihypertensive activity

2 0 [ 135, 2421 10 [ 1 3 5 ] > 5 0 [ 1351 10 [ 1351 10 [ 1 3 5 ] >SO 11351 10, t [ 245,2461 >[135] 1C10117 O(CH212 >SO [ 1 3 5 ] 2C,Ol17 O(CH2)2 50 [ 1 3 5 ] Ph2CH O(CH212 > 4 0 [135] 2,6ClzC6H3.0.(CH2),.Nh!e.C(:NH)NH? 20 $ [ 211

~

-

~

_

_

_

_

-

Active [ 1351 -

Active [ 2 4 5 , 246) -

Active [ 135) -

_

*Cat nictitating membranes. Approximate dose (mg/kg, s.c.) causing SO% coverage of the eyes. Figures in italics represent interpolated data

administration $.Intravenous Transient activity

neurone blocking properties of homologous phenoxypropylguanidines (Table 3.12). 3-Phenoxypropylguanidine (XLIV, Ar = Ph, n = 3) does not cause ArACH2CH2.NHGNH I NH2 (XLII I)

ArO-(CH2),*NH-C=NH

I NH2

(XLIV)

adrenergic neurone blockade, but does produce hypotension, possibly by depletion of peripheral tissue stores of catecholamines [ 248-2501. The 2,6(XLIV, Ar = 2,6-Me2C6H3 dimethyl- and 2,6-dichloro-phenoxypropylguanidines and 9 , 6 - c I 2 C 6 H 3 , n = 3) however, d o show adrenergic neurone blocking activity [135,251]. I n the series of aminoguanidines (Table 3.14) the 2,6-dichloro- and 2,6dimethyl-phenoxyethyl compounds (XLV, Ar = 2,6-C12C6H3 or 2,6-Me2C6H3), are highly active adrenergic neurone blocking agents, whilst the unsubstituted compound is inactive [21, 2521. In this series, disubstituted compounds containing only one ortho substituent (for example XLV, Ar = 2,5-C12C6H3 or 2,3-Me2C6H3)also possess good adrenergic neurone blocking properties. Inhibition of the release of transmitter substance upon nerve stimulation, leading to adrenergic neurone blockade has been demonstrated with (XLV, Ar = ?,6-C12C6H3),i.e. guanoclor (VI). When examined in conscious hypertensive dogs,

155

C. J. DURANT, A.M. ROE, A. L. GREEN.

ArO.(CH,),.NH.NH.C=NH

I NH2

(XLV) guanoclor caused only slight depression of blood pressure on chronic administration; the analogues (XLV, Ar = 2,6-Me2C6H3and 2,5-CI2C6H3)were slightly more effective in lowering blood pressure. However, an antihypertensive action of guanoclor has been demonstrated in clinical trials (2531. Table 3.14. T H E ACTIVITY OF ARYLOXYALKYLAMINOGUANIDINES

ArO.(CHd;X

X

Ar

n

Ph Ph

2 NHNHC(:NH)NH2 3 NHNHC(:NH)NHz 2 NHNHC(:NH)NHz 3 NHNHC(:NH)NHz 2 NHNHC(:NH)NHz 2 NHNHC(:NH)NH2 2 NHNHC(:NH)NHz 2 NHNHC(:NH)NHz 2 NHNHC(:NH)NHz 2 NHNHC(:NH)NH? 3 NHNHC(:NH)NH2 2 NMeNHC(:NH)NHz 2 NHNHC(:NH)NHMe 2 NHNHC(:NMe)NHMe 2 N(NHdC(:NH)NHz 2 N(NH2)C(:NH)NH2 2 NHC(:NH)NHNH2 2 NHC(:NH)NHNHz

2-MeOC6H4 2-MeOC6H4 ~ C I C ~ H ~ 2-MeC6H4 2,5ClzC6H3 2,6ClzC6H3 2.6-MezC6H 3 3,5-Me&H3 2 ,6-MezC6H 3 2,6-MezC6H3 2,6-Me2C6H3 2 ,6-Me2C6H 3 2,5Cl&H 3 2,6Cl&H 3 2,6-Me2C6H3 2,6ClzC6H3

Adrenergic neurone blocking activity * >20 (21) >20 [21] 2 0 [21] >20 [2l] > 2 0 [21] > 2 0 [21] 5 [211 5 [211 5 1211 > 2 0 [21] > 2 0 [21) 5 [211 > 2 0 [21] >20 [21] > 2 0 1211 2 0 I211 2 0 [ 131) Inactive [ 1941

*Cat nictitating membranes. Approximate dose (mg/kg,

s.c.) to

Antihypertensive activity -

-

Active (211 Active (211 Active [ 2 1 ] -

-

-

-

cause 30-5046 coverage

Of

the eyes.

1 4-Benzodioxans

The affinity of phenoxyethylamines for adrenergic receptor sites is enhanced by cyclization to analogous aminomethyl- 1 ,Cbenzodioxans such as piperoxan (XLVI, R'R2= (CH,),) which block a-receptors [240] . The introduction ofguanidine 194,138, 252, 2541 and quaternary ammonium [94] groups into 2-aminomethyl- 1,4-benzodioxan leads to compounds with adrenergic neurone blocking properties similar t o those of guanethidine and the

156

GUANIDINES AND ADRENERGIC NERVE ENDINGS

openchain aryloxyalkylguanidines and quaternary ammonium derivatives. The pharmacology of the guanidine derivative, guanoxan (Envacar) (XLVII), has been studied in some detail [94, 254, 2551. It has many properties typical of

( XLVI

1

j

XLVII i

adrenergic neurone blocking agents, causing relaxation of the nictitating membrane of conscious cats and abolition of contractions of the nictitating membranes evoked by stimulating the post-ganglionic cervical sympathetic nerve of anaesthetized cats. Experiments o n the spleen indicated that during nerve stimulation, guanoxan prevented the release of noradrenaline from nerveendings, and that this effect was reversed by amphetamine. Long-lasting depletion of noradrenaline following administration of guanoxan has been reported 194, 2541. The antihypertensive action of guanoxan, which is comparable t o that of guanethidine, has been demonstrated in animals and in man [94, 256, 2571. Structure-activity relationships amongst compounds related t o guanoxan have some interesting features (Table 3.15). Introduction of a single methyl group into position 5 of the aromatic ring results in an almost complete loss of adrenergic neurone blocking activity. Moving the position of this substituent to positions 6, 7 or 8 gives rise t o compounds with increased activity. The last of these compounds (XLVIII, R = %Me, n = 1) at a dose of 30 mg/kg, caused the

most pronounced relaxation of the nictitating membranes observed in this series [ 1381 . This apparent enhancement of adrenerBc neurone blockade by orfho substitution is somewhat analogous t o the increase in blocking activity resulting from ortho substitution in phenoxyalkylguanidines. Introduction of more than one methyl group into the aromatic ring giving 5,7-, 6,7-, or 5&disubstituted compounds, resulted in loss of all activity 194, 1381. The effect of chlorine substitution follows a rather similar pattern [ 1381 . The adrenergic neurone blocking activity of guanoxan is maintained in the homologous compound in which the side chain is increased by one carbon

157

G. J. D U R A N T , A . M . ROE, A. L. G R E E N Table 3.15. T H E A C T I V I T Y OF 2GUANII)INOALKYL-1,4-BENZODIOXANS

Formula (XLVIII)

R

n

H 5-Me 6-Me 7-Me 8-Me 5,s-Mez

1 1 1 1

5CI 7CI 6,7432 H H

Adrenergic neurone blocking activity *

10 [94,138,254] >20 [138] 20 [ 138) 5 [ 138) 20 [ 138) > S O (94,1381 >20 [138] 20 [ 1381 20 [ 1381 20 [ 138) 20 [ 1381

1 1 1 1 1 2 3

Antihypertensive activity High activity [94,138,2541

-

Active 1138)

Formula ( X L K )

R'

RZ

H

H H Me

Met H H H

R3

Adrenergic neuronr blocking activity *

Antihypertensive activity

Me 10 (94)20 [138] H 20[95] H >20[95] -CHZ-$ -CHz-*

20 [95]

* >20 1951 Formula (L)

R

H H H H H H H H H H H 5,8-Mez

X NHC(:NH)NHMe NHC(:NMe)NHMe N M e C ( : NH)NH2 ,pCHZ NH.$ 1 NHCHz NHC(:NH)NHNHz N H .C ( :N H N H 2,N H N H 2 NH.NHC(:NH)NHz NHC(:NH)NHC(:NH)NH* $(:NH)NH* NMe3 hEt3 hMezEt

Adrenergic neurone blocking activity*

Antihypertensive activity

>20 [ 1381 >20 [138] > 2 0 [138]

>20[138] > 2 0 [ 1381

>20 [138] >20 [21] > 20 [ 1381 20 [ 1381 25 [ 941 > 25 [94] 10-20 1941

*Cat nictitating membranes. Approximate dose (mg/kg, s.c.) t o cause 30% [ 941 or 30-50% 1 2 1 , 9 5 , 1381 coverage of the e y e s . ?Trans. SSyn. * * Anti.

158

GUANIDINES AND ADRENERGIC NERVE ENDINGS

atom (XLVIII, R = H, n = 2 ) , although amme-depleting power in rat tissue is decreased 11381. The isomeric branched chain structure (XLIX, R' = R2 = H, R3 = Me) also has potent adrenergic neurone blocking properties but causes less depletion of the noradrenaline in mouse hearts than guanoxan I941 . The isomeric 3-methyl substituted compound (XLIX, R' = Me, R2 = R3 = H) (either the pure trans-compound 12571 or the cis-:trans-niixture [ 1381) had a shorter duration of action than guanoxan. Syn- and anti-isomers* of the cyclopropyl analogue (XLIX, R' = H, R2R3 = CH2) have also been investigated [ 9 5 ] . The former was much shorter acting than guanoxan [ 9 5 ] , whilst the latter compound was devoid of adrenergic neurone blocking activity.

N-Methylation of guanoxan leads t o complete loss of activity in the nictitating membrane assay [ 1 3 8 ] . This is similar t o the findings in the open chain phenoxyalkylguanidines [ 1 3 5 , 2 4 2 1 . Analogues containing cationic groups other than guanidine have also been examined (Table 3.15). Various amino- and diamino-guanidine derivatives are reported t o have a very low order of adrenergic neurone blocking activity [21, 1381. Some activity was found with related amidine and amidoxime derivatives [ 1381 . ATongst quaternary a m m o n i u T derivatives, the compounds (L, R = H, X = NMe, and R = 5,8-Me2, X = NMe,Et) caused relaxation of the nictitating membranes in cats, although they were less active than guanoxan [94]. 1.3-Benzodioxoles

1,3-Benzodioxole analogues (LI, R = H, 4-Me, 4,7-Me2, X = NHC(:NH)NH,) of the active 1,4-benzodioxans have been synthesized [94] , but their adrenergic

(LI) neurone blocking activity was less than that of the corresponding 1,4-bemodioxans [94] (Table 3.16). However, the quaternary ammonium compound (LI, *The terms syn- and onti- refer to the configuration of the guanidine group with respect to the methylene group of the dioxane ring.

G. J. DURANT, A. M. ROE, A. L. GREEN.

159

+

R = 4-Me, X = NMeEt,) had a more potent adrenergic neurone blocking action than any of the guanidines in this series [94] . Table 3.16. THE ACTIVITY OF 2GUANIDINOALKYL-1,3-BENZODIOXOLES(LI)

R

X

H 4-Me 4.7-M~ H 4-Me 4-Me 4-Me

N H C ( : NH)NH2 NHC(:NH)NH2 NHC(:NH)NH2 &Me3 kMe3 kMeEtz kEt3

Adrenergic neurone blockingactivity* (941

25-50 10-25 25

>

-t -t

10- 25 50

*Cat nictitating membranes. Approximate dose (mdkg, s.c.) to cause 30% coverage o f the eyes. ?Powerful nicotine-like drug, toxic to conscious cats.

2,3-Dih ydrobenzo furans The guanidine and quaternary trimethylammonium deriva$ves of the 2,3-dihydrobenzofuran (LII, R = 7-Me, X = NHC(:NH)NH2 or NMe3) which

bear a direct relationship to xylocholine (XXXIX) and the corresponding guanidine (XLII) (and also to bretylium and aralkylguanidines) have a prolonged adrenergic neurone blocking action [93]. The release of noradrenaline from the spleen during nerve stimulation is inhibited by both these compounds, but the noradrenaline content of rat hearts is not apprecia6ly lowered 24 hours after a single dose of either of these drugs. In cats the guanidine (LII, R = 7-Me, X = NHC(:NH)NH2) has a sympathomimetic action on the blood pressure and the nictitating membranes resembling that of guanethidine. In large doses the quaternary compound (LII, R = 7-Me, X = NMe3) has a sympathomimetic action that for some time antagonizes the relaxation of the nictitating membranes resulting from the adrenergic neurone blockade. This action, which is probably due to release of catecholamines, causes anomalous dose-response curves when relaxation of the nictitating membranes is used as a method of assay for adrenergic neurone blocking activity [93].

160

GUANIDINES AND ADRENERGIC NERVE ENDINGS

Substituted 2,3-dihydrobenzofuranyl guanidines (LII, X = NH.C(:NH)NH2) were studied for their relative activities in relaxing the nictitating membranes of cats, and the following order of potencies was obtained [93] : R = 5-CI > 7-Me = 5-Me > H > 6-Me (Table 3.17). Quaternary ammonium analogues of the S c h l o r o and 5- and 6-methyl substituted guanidines were inactive as adrenergic neurone blocking agents Table 3.17. THE ACTIVITY OF 3-GUANIDINO-2,3-DIHYDROBENZOFURANS (LII)

R H 7-Me 6-Me 5-Me 5C1 H 7-Me 6-Me 5C1 ?-Me 6-Me 5-Me 5C1 7-Me

X NH€(:NH)NHl NHC(: NH)NH? NHC(:NH)NH2 NH C(:N H ) NH 2 NHC(:NH)NH2 &Me3 kMe3 &Me3 kMe3 kMezEt kMe,Et $MezEt NMezEt kMeEtz

Adrenergic neurone blocking activity* [ 931

0.15 0.35 0.10 0.35 0.65 0.10 0.20 0 0 0.30 0 0 0 0.20

*Relative activities (xylocholine = 1.0) from weight for weight comparisons of the subcutaneous doses necessary t o relax the nictitating membranes of cats t o cover 30% of the eyes.

(Table 3.17) suggesting, perhaps, that in this type of compound substituents ortho t o the ether linkage are more important for activity in the quaternaries than in the guanidines. Aralkylguanidines

Following the discovery that xylocholine blocked transmission at sympathetic nerve terminals, intensive chemical and biological investigations of related compounds revealed that various benzyl quaternary ammonium salts were potent and selective inhibitors of adrenergic nerve function. A review of compounds of this type has been published by Copp [239] . N-2-BromobenzylN-ethyl-N. N-dimethylammonium tosylate (LIII) (bretylium) has been studied extensively [237, 2581, and suppression of adrenergic nerve function has been demonstrated in numerous test situations in various animal species. That adrenergic neurone blockade was caused by inhibition of the release of the neurotransmitter substances during nerve stimulation, was shown by

G. J. DURANT, A. M. R O E , A. L. G R E E N

CH2 ;Mule,

Et

161

Tos

Table 3.18. THE ACTIVITY OF SUBSTITUTED BENZYLGUANIDINES (LIV)

R

'

RZ

R3

H H H H H H H H ti H Ii H I1

I1 H H ti H H H ti H H H li H H I1 H H ti ti ti

H 11

H ti ti H H H H I1 I1 Me H (+)Me H ( )hie H Me H Me H Me H (+)Me H (

)Me

Me Me

H H H

R4

R5

A drenergic neurotie

A ntihyperrensive

blocking activity Cat nictiraring Ptosis in membranes. micet [ 2591

activin

H H H H H H H H H H H ti

> 2 5 $ [ 1391 0 < I S [ 141, 239) 1s [ 1401 > 3 0 11411 > 3 0 [ 1411 10 [ 1411 > 3 0 [ 1411 6 0 [ 141 I 3 - 5 0 11391 + <3U [ 2 3 9 ) . > 3 0 [ 141115[140],>50[139] + > 3 0 [1411 15 [ 1411 I S [ 141) 30 [ 1411 5-10 [ 1391 ++ 30 [ 239. 141 I -

H

> 3 0 [ 141)

H H H

H H I{ H H H

t1

I1

H

H H H H H H H

30[1411 2 5 [ 2441 <15[1411 so [ 2441 >so [ 1391 >50 11391 1.25-2.5 [ 1391 1.25-2.5 [ 1391 1.25-2.5 [ 1391 2.5-5.0 [ 1391 2 . 5 - 5.0 [ 1391 1.25-2.5 [ 139) 2.5-5.0 [ 1391 1.25-5.0 [ 1391

H

H H

H H H H H H H H 11 11

H H tl H H li It t1

H H H H 11

'H

H H H H

H

< <

-

<

Inactive [ 141) -

-

Active [ 1411 -

Inactive [I411 -

-

Active [ 141) -

<

-

+ '

0

++ +++ ++ +++ +++ +++

+++ +++

Hypertensive [ 1391 Active [ 1391

162

GUANIDINES AND ADRENERGIC NERVE ENDINGS Table 3.18. continued

R'

4CF3 2.4-Mez H 4C1 H 4C1 H 2CI 4CI H 2CI 3-Me 3CI 4C1 4CF3 H 2421 H

H

R2

R3

R4

R5

Me

Me Et Et H H H H H H

H H H H Me Me H H H H

H H H H H H Me Me Me Me

H H H H H H H H H Me

H H H H H H H H H

H H H H H H H H NH,

Me Me Me Me Me Me Me Me H

Me Me

Me Me Me Et Et Pr H

Adrenergic neurone blocking activity Cat nictitating Ptosis in membranes* mice? [ 2591

< 15 [ 1 4 1 ] 5-10 [ 1391 2.5-5.0 [ 1391 > 2 5 [139] > S O [ 1391 > 3 0 [ 1411 > 3 0 [239] 10[239] >30 [141] 1-3 [10;239,242,260] <30 [ 141) 3-10[10,239,242,260] <_S_o[239] < 3 0 [239] > 3 0 [141] > 3 0 [ 1411 10 [ 2391 w t (2391 30 [ 2391 > 2 0 1211

-

+++ + 0

-

-

+++** +++** -

Antihypertensive activiv

Active [ 1411 Active [ 2601 Active [ 2601 -

-

-

-

-

-

-

'Approximate dose (mg/kg, s.c.) t o cause-25-30% coverage o f the eyes. Figures in italics represent interpolated data. tEstimated 2 h after injection of 20 mg/kg (0 = negligible, + = slight, ++ = moderate, +++ = marked). $1.~.administration. "Ptosis production in rats [ 3 5 8 1 . ttTransient activity [ 2393.

measurements of noradrenaline release from the spleen of cats 12371. In shortterm experiments in numerous animal species only slight depletion of tissue catecholamines has been found with bretylium. The low capacity of bretylium for releasing catecholamines is probably its major distinction from guanethidine in its pharmacological action. Bretylium has been used for control of hypertension, but its value as a clinical agent is limited by the rapid development of tolerance [ 101 . Cuanidine analogues of benzyl quaternary ammonium salts have afforded a series of compounds with an interesting range of activities. Although benzylguanidine (LIV)* itself causes only barely significant adrenergic neurone *See footnote on P. 147

G. J. DURANT, A. M. ROE, A. L. GREEN.

163

blockade [ 139, 2591, the introduction of substituents into the aromatic ring, on the a-carbon atom, or on the terminal nitrogen atoms, can lead t o compounds exhibiting a high degree of activity. Available data for adrenergic neurone blocking activity and indications of antihypertensive action are summarized in Table 3.18. Where necessary to provide a standard (although very approximate) measure of potency, namely the minimum subcutaneous dose required to produce 25-30 per cent coverage of the eyes, the liberty has been taken of extrapolating or interpolating semiquantitative data on the effects of these drugs on the nictitating membranes in conscious cats. Where available, adrenergic neurone blocking potencies assessed from the production of ptosis in mice are included. The essentially semiquantitative nature of these assessments of adrenergic neurone blocking activity must be emphasized.

(LV. R=H) (LVI, R=CL)

The first published report [242] on the adrenergic neurone blocking activity of aralkylguanidines showed that N , N', N'Lbenzyldimethylguanidine (bethanidine, Esbatal, LV) and its ortho-chloro analogue (LVI) were about twice as active as guanethidine in relaxing the nictitating membranes of cats. Subsequently, the higher potency of bethanidine (LV) in this test was demonstrated [ 2601 . Both compounds resembled bretylium and guanethidine in potentiating the effects of adrenaline and noradrenaline on the blood pressure and the nictitating membranes. The compounds differed in the extent to which they inhibited the response of the nictitating membranes and the blood pressure of cats to tyramine given 24 hours later: bethanidine (LV) was much more active, and comparable to guanethidine in this respect, whereas the weak action of the ortho-chloro analogue (LVI) resembled that of bretylium (LIII) [260] . Again (LV), but not (LVI), caused appreciable depletion of the pressor m i n e content of the spleen of cats on chronic administration. Bethanidine has proved an effective and well-tolerated antihypertensive agent ihn man. A low incidence of side-effects, particularly the diarrhoea which is often troublesome during treatment with guanethidine, has been reported [ 10, 261, 2621. Adrenergic neurone blocking activity is increased when substituents are introduced into the orrho- and para-positions of benzylguanidine (Table 3.18); active compounds include the bromo (a particularly close analogue of bretylium), chloro and trifluoromethyl derivatives (LIV, R' = 2-Br, 2-C1, 2- and 4CF3) [140, 141, 239, 2421. The 4-trifluoromethyl derivative, which has a relaxing effect on the nictitating membrane of cats comparable in intensity and duration to bethanidine, also decreased blood pressure in hypertensive dogs [ 1411 . Certain

164

GUANIDINES AND ADRENERGIC NERVE ENDINGS

ring-disubstituted benzylguanidines, notably 2,4- and 2,6-dichlorobenzylguanidine, are also active adrenergic neurone blocking agents (LIV, R' = 2,4-C12, 2,6-C12) [ 141.2393. Derivatives of 1-phenylethylguanidine (LIV. R2 = Me) are potent adrenergic neurone blocking agents, as has been demonstrated by their effects on the nictitating membranes of anaesthetized and conscious cats, on the Finkleman preparation, and in mice [ 139, 2591. This type of compound is of particular interest as the existence of an asymmetric centre permits the separation and the study of optical isomers. Racemic 1-phenylethylguanidine and also the (+)-isomer are almost inactive in conscious cats, whereas the (-)-isomer is very active both in conscious and anaesthetized cats. The (+)-isomer causes a rise in the blood pressure of anaesthetized cats, in contradistinction to the (-)-isomer which causes a prolonged fall in blood pressure and only a transient increase in heart rate. Furthermore, the (+)-isomer antagonizes the adrenergic neurone blocking action of (-)-1 -phenylethylguanidine, xylocholine and guanethidine in cats and mice [263]. It is this antagonism that accounts for the inactivity of the racemic compound. In contrast, both optical isomers of 1-p-tolylethylguanidine (LIV, R' = 4-Me, R2 = Me) are potent adrenergic neurone blocking agents, as are the racemates of analogues having ortho ring substituents [ 139, 2591 (Table 3.18). Racemic 1-p-trifluoromethylphenylethylguanidine(LIV, R' = 4-CF3, R2 = Me) causes prolonged relaxation of the cat nictitating membrane [ 1411. This compound decreases the blood pressure of renal and neurogenic hypertensive dogs, and is reported to be notable for its lack of side effects [ 1411. Methylation of the terminal nitrogen atoms of benzylguanidine greatly enhances its activity, and retains or enhances the adrenergic neurone blocking activity of ortho- and meta-substituted compounds [239] . However, the activity of para-substituted compounds such as the trifluoromethyl derivative (LIV, R' = 4-CF3, R4 = R5 = Me) is apparently diminished by this substitution [ 1411. A study of structural modifications of the guanidine portion of bethanidine (LV) and its ortho-chloro analogue (LVI) indicates that adrenergic neurone blocking activity is optimized in this system by N, Nrdimethylguanidine substitution (LIV, R' = H or 2-C1, R4 = R5 = Me) [239]. Compared with the potent adrenergic neurone blocking activity displayed by numerous derivatives of benzylguanidine, the activity of derivatives of phenethyl; guanidines (LVII) and longer chain compounds is generally of a much lower order (Table 3.19). The parent compound (LVII) has a very weak relaxing activity on cat nictitatingmembranes and does not produce ptosis in mice [ 139,2591 .P-Hydroxyphenethylguanidine (LVII, Ar = Ph, R' = OH) also has only very weak adrenergic neurone blocking properties [264, 2651 . In contrast, the ortho-chloro derivative of the latter (LVII, Ar = 2.C1C6H4, R'.= OH) has a much stronger adrenergic neurone blocking action in cats and mice. These two guanylated arylethanolamines also differ in that depletion of tissue catecholamines is very marked with the former compound but is insignificant with the latter orthochloro compound [265]. It appears, therefore that the ortho-chlorine atom

165

G. J. DURANT, A. M. ROE, A. L. GREEN. Table 3.19. THE ACTIVITY O F PHENETHY LGUANIDINES AND RELATED COMPOUNDS (LVII) Ar

R'

Ph

H

R2 R3

H

H

R4 Rs

H

H

4C1C6H4 H H H H 3,4-(Me0)2 H C6H3 H H H H 2,6C1&6H3 H H H H 2,6-F2C6H3 H H H 2,6-Me2C6H3 H H H H Ph M e H H H 4-HOC6H4 H H H H 4-MeOC6H4 H Me H H Ph O H H H H Ph (-)OHH H H Ph (+)OH H H H 4ClC6H4 OH H H H 2CIC6H4 OH H H H 3,4Cl2C&3 OH H H H 4-MeOC6H4 OH H H H Ph OH H Me H Ph (-)OHH Me H Ph OH H H Me Ph H H NH2 H PhChIe2CHzNHC(:NH)NH2 4C1C6H4CH2CMe2NHC(:NH)NH2 rrans-PhCHCHNHC(:NH)NH2

H H H H H H H H H H H H H H H H H Me H

Adrenergicneurone blocking activity Cat nictitating Ptosis in membranes* mice?

An rihypertensive

activity

>25 $ [ 139,211 >SO (2441 0 ** [ 2 5 9 ] 3 0 j t [ 141) 3 0 [ 1401 > 2 0 (211 50 12441 25 [ 244) >SO 12441 0 [ 2591 50 [ 1391

>

-

> 2 5 11391 5-25$$ [265] > 2 0 [318] -

5 [2651 -

-

-

>20 30 >30 >25

>

-

0(259] + [ 2651 + [ 2651 + (2651 + [ 2651 + [ 2651 0 [ 2651 0 [ 2651 0 [ 265) tt [2651 0 [265]

-

Hypertensive 14331 -

0 (2591

[21] [ 2283 [228] [139]

\I

CH z Ph(CH2)$4HC(:NH)NHz. cis-PhCH=CH C H 2NHC(: NH)NH 2

> 2 5 (1391 Active [ 266 ]

0 [2591 -

Active [ 266,2671

*Approximate dose (mg/kg, s.c.) t o cause 25-300/0 coverage of the eyes, see text. Figures in italics represent interpolated data. ?Estimated 2 h after injection of 2 0 mg/kg 1 2 5 9 1 , or 100 mg/kg 1 2 6 5 1 (0 = negligible, + = slight, ++ = moderate, +++ = marked) $1.". administration. **Ptosis production in rats has been reported 13581 ransient response Erratic and only partial response

iY

causes a complete change in the mode of action at adrenergic nerve endings.

ArCHCH-N-C = NR4

I 1 II

R' R2 R%HR5 (LVII)

166

GUANIDINES AND ADRENERCIC NERVE ENDINGS

Cinnamyl and phenyl c yclopropyl guanidines

Cinnamylguanidines have antihypertensive properties 1266, 2671 (Table 3.19) and the cis-isomer (LVIII), which is reported t o be more active than the isomer with the trans configuration [266], has an action which is more rapid in onset and of shorter duration, than that of guanethidine [266, 2671. Blockade of adrenergic nerve fibres has been demonstrated and a similar mode of action to guanethidine appears likely [267] . The trans-phenylcyclopropyl derivative (LIX) is inactive as an adrenergic neurone blocking agent [ 139, 2591 ,but it is claimed to lower blood pressure [268]. The cis-phenylcyclopropyl isomer has not yet been described. As pointed out earlier, the cyclopropyl derivatives in the 1,4-benzodioxan series also demonstrate the importance of stereochemical configuration in the action of guanidines at adrenergic nerve endings. CH,.NH.C=NH

I

N H2

Ph

/H

'c-c H'

'1 \NH.C=NH

c H2

I NH2

(L[X)

(LVIII Heteroaromatic alkylguanidines

Various guanidines linked by an alkylene chain to heteroaromatic rings have shown activity at adrenergic nerve endings; the reported activities of compounds in this class are listed in Table 3.20. These include the thenylguanidine (LX) Table 3.20. THE ACTIVITY OF HETEROAROMATIC ALKY LGUANIDINES R'.(CH~~.NHC(:NR~NHR~ A drenergic neurone

R'

RZ

n

hlocking activity*

4-Pyridyl 2-Pyridyl 4-Imidazolyl 2-F~r~l 2-Thienyl 10-Phenothiazinyl 2CFrlO-Phenothiazinyl

H H H H Me H H

2 2 2 1

> 3 0 [ 1401

1

3 3

30 [ 1401

> 3 0 [ 1401 >30 [ 1401 1 t [2391 >40 [ 1351 >50 [135]

A n tihypertensive activity

Inactive [210] Active [ 2101 Inactive [ 1271 -

'Cat nictitating membranes. Approximate dose (mg/kg, s.c.) to cause 25-3096 coverage the eyes. Figures in italics represent interpolated data. +Short acting [ 2391

Of

which has a very potent, but transient, action in cats [239]. In contrast to the activity of (LX), the replacement of benzene by its isostere, thiophene, in benzyl quaternary ammonium derivatives, leads to inactive compounds [2391 .

G. J. DURANT, A. M. ROE, A. L. GREEN.

167

2-(2-Pyridyl)ethylguanidine (LXI) is said to be more active than the Qisomer (LXII) as an antihypertensive agent [210].However, the reverse order of activities

Q-w,.

NH.c=NMe

I

CHI CH,. N H * C= NH

NHMe

I

NH2

(LX)

(L XI)

(LXIII)

(LXII)

is reported for adrenergic neurone blocking activity in cats [ 1401. The related 3-pyridylmethylguanidine(LXIII) has a weak ephedrine-like action [2691 . C H i C=NOH

I

CH,.C=NOH

I

NH2

(LXIV)

(LXV)

The bicyclic amidoximes (LXIV) and (LXV)cause a reduction in blood pressure in various animal species, but apparently they are not adrenergic neurone blocking agents [270].The activity of (LxIV)is thought to be mainly due to catecholamine release and subsequent depletion, whilst the action of (LXV)involves blockade at a-receptors [270].

-.

Tetrahydroisoquinoliner

R

\

l

a

N

- C=N R 2

I

‘Et

(LXVI)

NHR3

(LXVII)

1,2,3,4-Tetrahydroisoquinolineis an example of a cyclized ardkylamine Moderate adrenergic neurone blocking activity has been demonstrated by the

GUANIDINES AND ADRENERGIC NERVE ENDINGS

168

quaternary ammonium derivative (LXVI) [239] (Table 3.21), whereas the corresponding guanidine (LXVII), debrisoquine, has a potent action resembling that of bethanidine (LV). The drug is an effective antihypertensive agent, both pharmacologically and clinically [271-2761 . Appreciable depletion of tissue catecholamines does not occur [271]. Like bethanidine, debrisoquine has a shorter duration of action than has guanethidine, and does not cause diarrhoea, a common drawback with guanethidine. Table 3.21. THE ACTIVITY OF TETRAHY DROISOQUINOLINE DERIVATIVES (LXVII)

R'

RZ

R3

H 6,7-(MeO)z 7-Br H H

H H H Me -CH2CH2-

H H H Me

Adrenergic neurone blocking activity

Antihypertensive activiv

Active [271] Active [ 2771 Active [ 278) Inactive [ 2791

Active Active Active Active Active

[271-2761 [ 1431 [277] 12781 [279]

The ring-substituted derivatives (LXVII, R' = 6,7-(Me0)2 and R' = 7-Br) are also potent anti-hypertensive agents [ 143, 2771 . The pharmacological profile of the latter (guanisoquin) includes adrenergic neurone blocking activity on cat nictitatingmembranes, where it has about one-fourth the potency of guanethidine, pronounced catecholamine depletion and local anaesthetic activity. Although the hypotensive effect is considered to be primarily due to catecholamine depletion and adrenergic neurone blockade, centrally mediated interference with vasomotor tone and peripheral vasodilatation are also considered to be partly responsible 12771. - The dimethylguanidine (LXVII, RZ = R3 = Me) causes vasodilatation but lowers blood pressure as a consequence of adrenergic neurone blockade [278]. Apparently, the related imidazoline (LXVIII) is not an adrenergic neurone blocking agent but a hypotensive, which acts by vasodilatation and blockade of a-receptors [279] .

iL XVlIIl

(LXIXI

The tetrahydrobenzazepine (LXIX) related to debrisoquine (LXVII) is reported to be a potent adrenergic neurone blocking agent (three-fifths as potent as guanethidine in anaesthetized cats) [280] . Marked sympathomimetic activity is not associated with (LXIX) and in this respect it is similar to the hexahydrobenzazo-

G. I. DURANT, A. M. ROE, A. L. GREEN.

cine analogue (XXXI) of sympathomimetic activity.

169

guanethidine, which also lacks appreciable

Miscellaneousguanidines Alkylguanidines

Hypotensive effects caused by simple alkylguanidines such as methyl- and ethylguanidine (LXX, R' = Me, Et), and even by guanidine itself, were recorded by Ales [281] in 1926. More recently, these and other simple guanidines have been shown to affect neuromuscular and ganglionic transmission [282,283]. A review of the general pharmacology of alkylguanidines and related amidines has been published [36a]. Methyl- and N, N -dimethyl-guanidine (LXX, R' = Me, RZ=H or Me) show antagonism against ganglion blocking agents, whereas N,N-diisopropylguanidine (LXXI) is itself a powerful ganglion blocking agent. This last activity is understandable in view of the structural similarity of (LXXI) to pempidine (XXVII). Me

R' NH .C=NH

I

I

CH -M e

\

NHR~

IL XX)

CH -Me

I

Me

(Lxxr)

Short, Ours and Ranus [228] found that,whereas straight chain alkylguanidines (LXX, R' = Bu or CIH1,) were devoid of adrenergic neurone blocking activity, some branched-chain compounds, for example (LXX,'R' = MeJ or Me3CCHz), were active (Table 3.22). The presence of a tertiary carbon atom separated from the guanidine group by not more than one carbon atom appears to be essential for appreciable adrenergic neurone blocking activity in simple alkylguanidines [228] . t-Octylguanidine (LXXII, guanoctine) is an antihypertensive drug with a guanethidine-like action both as an adrenergic neurone blocking agent and as a depletor of endogenous noradrenaline [ 2841 . Unlike guanethidine, t-octylguanidine shows little sympathomimetic activity. A direct vasodilatation has been demonstrated, but it is doubtful whether this plays a very important role in the action of this drug. The possibility of a central

GUANIDINES AND ADRENERGIC NERVE ENDINGS

170

component in the hypotensive action of this guanidine has also been suggested [284]. The compound has been investigated clinically and is reported to be an effective antihypertensive drug with few side effects [285]. Table 3.22. THE ACTIVITY OF ALKYLCUANIDINES R'NHC(:NH)NHR~

R'

R2

Adrenergic neurone blocking activity *

Bu nC8H 17 But But Me 3CCH 2CMe Me 3CCH &Me2 Me3CCH 2 Me3CCH2CH2 E t 3CCH 2 Me 3CCH 2CH(Me)CH 2CH EtC(Me)2

H H H PhCH2 H Me H H H H CN

>30 (2281 >30 12281 <15 (2283 >3b[2283 < 2 (228,2841 30 [228] 15 [ 2281 >30 [228] < 1 5 [228] 30 [ 2283 Inactive [ 2 2 9 . 2 8 6 ]

Antihypertensive activity

-

Active (284,2853

< >

-

Active [ 229, 286, 2871

*Cat nictitating membranes, approximate dose (mg/kg, s.c.) t o cause Z S Q coverage of the eyes.

N-Cyano-N'-t-amylguanidine(LXXIII, guancydine) is reported to be a potent and moderately long-acting hypotensive agent [ 229, 2871 . However, adrenergic neurone blockade is not part of the mechanism of action of this drug, but antagonism of angiotensin, an action not associated with adrenergic neurone blockade, has been found with this cyanoguanidine [229, 2861 .

Ye

Et .C.NH.C=NH

I

Me

(LXXII)

I

NHCN

(LXXIII)

A rylguanidines

Certain aromatic guanidines (and biguanides) are known to cause a reflex fall of blood pressure and heart rate by an action on receptors in the heart [288] . However, there have been no reports of arylguanidines exhibiting activity at sympathetic nerve endings. o-Bromophenylguanidine (LXXIV) does not affect cat nictitating membranes [239] .

G. J. DURANT, A.M. ROE, A. L. GREEN.

171

2-Arylaminoimidazolines (LXXV, R = ArNH) are related chemically to guanidines as well as to 2-imidazolines. Substituted 2-imidazolines exhibit a range of activities varying from vasodilatation, often accompanied by a-adrenergic blockade (for example tolazoline, Priscol, Priscoline, Vasodil, Benzidazol, LXXV, R = PhCH2), to vasoconstriction (for example naphazoline, Privine, Rhinoperd, Niazol, LXXV, R = I -CI0H7CH2). Some 2-arylaminoimidazolines have been investigated by Hutcheon and co-workers [279], who found that the naphthalene derivative (LXXV, R = I-CloH7NH) causes vasoconstriction and a rise in blood pressure, together with some a-adrenergic blockade. Derivatives of 2-anilinoimidazoline(LXXV, R=PhNH) have recently proved to be a very interesting class of hypotensive drugs. The most widely investigated compound (LXXVI, clonidine, Catapres, Catapresan, ST 155) causes a prolonged decrease of blood pressure, accompanied by bradycardia, inhibition of pressor reflexes, and decrease of cardiac output [289,290] .No structure-activity data have appeared, although the structural features required for activity are said to be fairly specific [290]. Compound (LXXVI) has proved effective clinically in the treatment of hypertension [ 29 1-2931 . An initial vasoconstrictor action has been identified with direct stimulation ofa-receptors [294] . The effects of stimulating adrenergic nerves are prevented only at high doses, and no depletion of heart catecholamines takes place following administration of compound (LXXVI) [289]. It appears most unlikely that the compound causes a reduction in blood pressure by blockade of the peripheral sympathetic nervous system, and a central action appears more

I LXXIVJ

(LXXVJ

(

LXXVI 1

likely [295, 2961. Recently, it has been found that the hypotension in dogs and cats is very pronounced following intracisternal administration, and the drug has been classified as an antihypertensive agent with a specific blocking effect on cardiovascular sympathetic centres [ 297,2981 . Thusderivatives of arylguanidines often possess marked antihypertensive activity, although this may not result from an action at sympathetic nerve endings, and the compounds cannot be classed as adrenergic neurone blocking agents. Structural requirements for adrenergic neurone blockade

From the discussion on close analogues of guanethidine, it may be concluded that adrenergic neurone blocking activity requires the structural unit (LXXVIIa) comprising a nitrogen atom linked to the guanidine via an alkylene chain, in which R' - R8 are selected alkyl (or aralkyl) radicals or hydrogen. Depending on

172

GUANIDINES AND ADRENERGIC NERVE ENDINGS

the nature of the groups R' - R8, a further CHz or NH group may be inserted between the guanidine and the carbon atom bearing Rf and R6. In the branched chain structures (LXXVIIa, various R3 - R6 = Me), the side-chain nitrogen atom R3 RS R'RzN.~.~.NH4i=NR7 Ar C-NHC=N R Ar0.C-C .NH-C=NR

II

R4 R6

I I

.-

NHR*

I1

I

1 1

NHR

I

NHR

(LXXVlla) (LXXVIIb) (LXXVIIC) is no longer an essential requirement for activity and may be replaced by hydrogen or an alkyl group. The activity of aralkyl- (LXXVIIb) and aryloxyalkylguanidines (LXXVIIc) demonstrates that adrenergic neurone blockade does not demand the presence of an additional basic centre. The activity of benzylguanidine and phenoxyethylguanidine is greatly enhanced by appropriate substitution or cyclization. However, the complexity of structure-activity relationships is such that no satisfactory general structure-activity theory or receptor model has yet been proposed. A lipophilic group linked to a guanidinium (or similar) ion appears to be the onlycommon feature amongst the active compounds. An attempt has been made by Augstein, Green, Monro, Wrigley, Katritzky and Tiddy [21] to relate the adrenergic neurone blocking activity of aryloxyalkyl-guanidines and

( LXXVIII

1

(LXXIX)

(Reproduced by courtesy of the American Chemical Society)

-aminoguanidines to an intramolecularly hydrogen bonded conformation. In an extension of Belleau's hypothesis [299] concerning the conformational requirements for the blocking activity of 0-haloalkylamines on a-adrenergic receptors, they suggested that, if the conformations (LXXVIII) and (LXXIX) are adopted, the guanidine group is brought to the optimum distance from the aromatic centre required for effective adrenergic neurone blockade. This idea affords one explanatior for the loss of activity caused by methylation o f N ' in the guanidine (LXXVIII, Y- = CI, n = 2) and the retention of activity in compound (LXXIX, Y = R = Me). Hydrogen bonding should be prevented in the former, but

G. J. DURANT, A. M. ROE, A. L. GREEN.

173

should not be affected in the latter. This concept also affords an explanation for the inactivity of 3(2,6-dichlorophenyI)propylguanidine [ 2 I] , since it lacks a suitable group adjacent t o the aromatic ring with which the guanidine group can form a hydrogen bond. In ortho-disubstituted anisoles, steric restrictions lead t o the &-carbon being displaced from the plane of the aromatic ring and the resulting inhibition of resonance will increase the basicity of the ether oxygen. Ortho-disubstitution of phenoxyalkylguanidines should therefore enhance the hydrogen bond donator properties of the oxygen atom and facilitate the interactions shown in (LXXVIII) and (LXXIX), providing a neat explanation for the increased activity of (LXXVIII) and (LXXIX) when Y # H. It should be noted, however, that the relative activities of some bicyclic aryloxyethylguanidines do not support this idea that a hydrogen-bonded conformation can bring the aromatic ring and the guanidinium function into the optimum distance. Thus guanoxan (XLVII) is active, as is the syn-cyclopropyl analogue (XLIX, R' = H, RZR3 = CH2), but the anti-isomer, in which the guanidinium function can hydrogen bond to the 1-oxygen atom, is inactive. (LII) cannot Further, although the active 3-guanidino-2,3-dihydrobenzofurans form a hydrogen bond between the guanidinium function and the oxygen atom, in these compounds the distance between the aromatic ring and the guanidinium function is similar to that found in the active benzylguanidines. The above speculation [21]may be extended to include the related quaternary ammonium compounds such as xylocholine (XXXIX). It is probable that the volumes of the guanidinium ion and the trimethylammonium group are similar. The ionic radius of the guanidinium ion (IX) is about 3A; the ionic radius of the tetramethylammonium ion has been estimated [300] to be 34A, although rather smaller values have also been proposed [301-3031 . Crystallographic analyses of muscarine iodide [304], choline chloride [305] and acetylcholine bromide [306] have revealed that the carbon to nitrogen distance is about 1 .SA,and that a hydrogen bond (C-H--Odistance 2.87-3.07A) exists in the crystals of these compounds. In xylocholine the two ortho subslituents inhibit the delocalization of t h q oxygen 7r-electrons and it is possible that the oxygen atom is sufficiently basic tot cause the hydrogen-bonded conformation (LXXXltp be adopted.* Some correlations have been pointed out between the nicotine-like stimulant activities of substituted aryloxyethylammonium salts and the angle of twist between the side-chain and the aromatic ring (and hence the basicity of the oxygen atom) [307,308). The possibility that 0---H-C bonding might stabilize a conformation that has adrenergic neurone blocking activity has not been suggested previously. This concept could be extended to include alkylamino-

* The conformation (LXXX) has been confirmed [ 306al by a single-crystal X-ray analysis o f xylocholine bromide. The methyl-carbon t o oxygen separation is 2.949 k 0.009A

174

GUANIDINES AND ADRENERGIC NERVE ENDINGS

ethylguanidines, such as guanethidine, where a N---H-M bond can be envisaged.

(LXXX) OTHER PHARMACOLOGICAL EFFECTS ON SYMPATHETIC NERVES

Though the main action of guanidine derivatives is to interfere with the liberation of noradrenaline at adrenergic nerve endings, guanidines may affect the results of sympathetic nerve stimulation in other ways as well. Thus, guanethidine has a transient blocking action on sympathetic ganglia [207, 3091 and reversibly blocks a-receptors [3101 ; guanoxan causes transient blockade of a-receptors in dogs (2541 and rats [311], but not in cats [254] ; phenethylguanidine is also a weak antagonist of adrenaline on isolated rabbit intestine and uterus [3121 .Such actions, which are not confined to these three guanidines [94], are usually elicited only by high concentrations. This has discouraged any extensive study of the structure-activity relationships for these minor actions and makes it unlikely that any of them contribute significantly to the hypotensive action of the drugs. It was first noticed clinically that guanethidine-induced hypotension could be abolished by small doses of methamphetamine [313]. Amphetamine and related compounds have since been shown to reverse the adrenergic neurone blockade produced in animals by guanethidine [314-3161, guanoxan [254], bethanidine [260], (-)-1 -phenylethylguanidine [3151, and many other adrenergic neurone blocking drugs [226,245,3 151 . This reversing effect appears not to be associated with the sympathomimetic effect of amphetamine, since noradrenaline and tyramine are both inactive [3 14, 3 171 . Only very small doses of amphetamine are required (often less than one tenth of that of the blocking drug), and other types of sympathetic blockade are largely unaffected [315]. The specificity of this reversing effect is such that it can be used as a diagnostic test for distinguishing adrenergic neurone blockade from other types of sympathetic blockade (see p. 197). Antagonism of adrenergic neurone blockade by amphetamine has been attributed t o competition by amphetamine for the site on the nerve ending at which the guanidine derivative exerts its blocking action [314, 3161. It might consequently be expected that other guanidine derivatives, which themselves had no blocking action, would exert an antagonistic effect similar to that shown by amphetamine, and numerous examples of antagonism of adrenergic neurone blockade by guanidines have been reported [259, 315, 3181. The relative antagonistic potency amongst the compounds studied is largely independent of the nature of the drug causing adrenergic neurone blockade, although the absolute

G . J. DURANT, A . M . ROE, A. L. GREEN.

175

potency varies with both the nature and the dose of the blocking drug, which is what would be expected with drugs which competed for a common site. The available information on the relationship between structure arid potency in preventing guanethidine-induced ptosis in mice is summarized semiquantitatively in Table 3.23. The most active compounds are aralkylguanidines Table 3.23. ANTAGONISM BY GUANIDINE DERIVATIVES OF GUANETHIDINE-INDUCED PTOSIS IN MICE RNHC(:NH).NH2 R

Poteiicv*

PhCHz Ph (C H 3 2 Ph(CH33 PhCH(Me)CHZ PhCH(Et)CH 2 (+)-PhCH(OH)CH2 (-)-PhCH(OH)CH2 (+)-PhCH(Me) (+)-PhCH2CH(Me)

Reference

t

+ 0

+++ 0

+

0

+

0

+++ 0

+ 0

+ 0

I

Me

Me

PhCHz.NMeC(=NH)NH2 PhCHzCH(Me)NH2(Arnphetarnine)

+++ ++

*Based on dose required t o reduce intensity of ptosis due to by 50%. 0 10 mg/kg; + about 3 mg/kg; ++ about 1 mg/kg;

>

13151 13151

176

GUANIDINES AND ADRENERGIC NERVE ENDINGS

like mans-phenylcyclopropylguanidine(LXXXI) and N-benzyl-N-methylguanidine (LXXXII) which, when given alone, have no detectable effect on adrenergic neurones; however, two of the guanidines in the benzodioxole and benzodioxan series which did not possess adrenergic neurone blocking activity (Tables 3.15 and 3.16) were also inactive as antagonists. It is of interest that although amphetamine is a potent guanethidine antagonist, neither the guanidine (LXXXIII, R = H) directly derived from it, nor i t s p-methoxy analogue (LXXXIII, R = OMe) display any significant effect. Many of the antagonists in this test, such as benzylguanidine and phenethylguanidine, display weak blocking activity on the cat nictitating membrane [ 1391 . (+)-I-Phenylethylguanidine (LXXXIV) is an antagonist in mice and also antagonizes the relaxation of the nictitating membranes produced by adrenergic neurone blocking drugs in

(LXXXII)

conscious cats, but in anaesthetized cats it has a weak blocking action, whilst on the isolated rabbit ileum it is a potent blocking drug [ 1391 . If adrenergic neurone blockade is regarded as the result of an agonist action on an appropriate receptor, compounds like benzylguanidine might be regarded as partial agonists with moderately high affinity and low efficacy, and compounds like N-benzylN-methylguanidine (LXXXII) as pure antagonists with very high affinity and zero efficacy. Ph0.(CH2 ) 3 .NH.C=NH

p-RC,j H4 .CH2 .CH.NH.C=NH

I

Me

I

I

NH2

NH2

(LXXXV)

(LXXXIII)

(LXXXIV) Although this susceptibility to antagonism by amphetamine-like drugs is

G. J. DURANT, A. M . ROE. A. L. GREEN.

177

characteristic of adrenergic neurone blockade. some guanidines can also slightly delay the onset of the sympatholytic signs which follow the administration of reserpine [263.315]. Thiseffect most likely results from the monoamine oxidase inhibition produced by many guanidine derivatives. an action which is discussed later. 3-Phenoxypropylguaiiidine (LXXXV). but not guanethidine. antagonized ptosis due to either reserpine or tetrabenazine when given before. or together with. these drugs, but was ineffective when given 21 hours after reserpine [ 2191. Prevention of reserpine-induced sympathetic blockade is commonly used as a test for antidepressant activity and. on the basis of these observations. phenoxypropylguanidine was given a clinical trial in depressed patients. but without success [319]. It is by n o means certain that the ptosis caused by reserpine is primarily ot central origin [320], and consequentl). the ability to antagonize reserpine-induced ptosis should not be automaticall!. regarded as an indication of a central antidepressant action.

BIOCHEMICAL EFFECTS Depletion of noradrenaline Guanethidine

The characteristic pharmacological action of adrenergic neurone blocking agents istoinhibit responses to stimulation otadrenergic nerves. an action unaccompanied by any similar blockade of responses to injected noradrenaline. The demonstration [321] that the somewhat similar s!-mpathol~.ticaction of reserpine was associated with almost complete elimination of noradrenaline from the tissues suggested that guanethidine might act in a like manner. and only a short time after the antihypertensive activity of yanetliidine was first reported. the drug was indeed shown t o cause a marked fall in tissue noradrenaline [ 3 U . 4371. These original observations have been amply confirmed. Table 3 . 3 . containing a selection from the voluminous literature on the subject. shows that. in a variety of species. guanethidine causes a fa11 in the noradrenaline content of every peripheral adrenergically-innervated tissue examined. In all species, depletion does not appear to reach its greatest extent until at leist four hours after injection. irrespective of the dose of drug. The rate of recover!' is variable. but after large doses of guanethidine the noradrenaline level does not return to normal for several days. Although some depletion of brain noradrenaline has been reported, in this tissue both depletion and recovery are rapid and not dose-dependent [ 3 7 _ 2 ] .Guanethidine, being largely protonated at neutral pH. does not readily cross the blood-brain barrier, and only trace amounts have ever been detected in the brain after injection [323-3251. This is true also of bethanidine (IV) [326] and guanisoquin(LXVI1, R' = 7-Br, R 2 = R3 = H ) [ 3 2 7 ]. In the very young chick, in which the blood-brain barrier is poorly developed. guanethidine does cause

Table 3.24. EFFECT O F SINGLE INJECTIONS O F GUANETHIDINE ON TISSUE NORADRENALINE

Species Mouse Mouse Mouse Mouse Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Guinea-pig Guinea-pig Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit

Tissue Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Heart Brain Adrenals Brain Hypothalamus Brain Brain Spleen Intestine Adrenals Heart (atria) Heart Heart Heart Spleen Brain Adrenals Superior cervical ganglion

Dose (mg/kg) and rou re

Time (h) after injection

1.25,25,20 i.p. 0.3,1,3,10 S.C. 10 S.C. 20 S.C. 15 i.p. 15 S.C. 20 i.p. 10 S.C. 35 i.v. 25 i.v. 8 i.v. 10 S.C. 10 S.C. 5 S.C. 5,15 i.p. 5,10,20 S.C. 10 S.C. 15 S.C. 15 S.C. 150,200,400 i.p. 3,10,30 i.p. 30 i.p. 1 2 3 i.v. 1 2 3 i.v. 12.5 i.v. 12.5 i.v. 1 2 5 i.v.

16 4 1 24 2,24 1,2,4,6,18,48 5,24,72 1 4,24,48,72 0.5,1,2 1,5,10,20 1 1 1,3,6

15 i.v.

2 4 1,2,4,6,18,48 2,4,6,18,48 12 24 24 1,2,4,18 48,72,120 18 18 18

Percentage depletion of noradrenaline

Reference

25,40,80 35,60,80,86 51 40 50,o 25,35,65,80,75,30 90,70,30 50 90,95,75,50 10,20,50 10,70,85,80 0 0 48,60,28 0 10,60,15 15 30,40,75,80,95,70 40,80,80,75,15 0,10,50 0,74,83 78 35,61,78,85 5 3,3 7,25 60 0 0

351 259 434 94 383 334 435 340 323 323 349 340 340 436 341 322 322 334 334 323 404 340 437 437 437 437 437

50-60

329

Table 3.24. continued

Species

Tissue

Dose (mg/kg) and route

Time (h) after injection

Percentage depletion of noradrenaline

Reference

~~

Rabbit Cat Cat Cat Cat Cat Cat Cat Cat Dog Dog Monkey Chick Chick (1-5 day) Chick (21 day)

Heart Heart Brain Adrenals Hypothalamus Superior cervical ganglion Heart Spleen Nictitating membrane Heart (atria & yen tricles) Heart (atria) Heart Heart Brain Brain

1.25,5,20 i.v. 15 S.C. 15 S.C. 15 S.C. 15 i.p.

17 24 24 24

35,59,70 75 0 0 0

337 437 437 431 34 1

15 i.v. 10J5 S.C. 10,15 S.C.

18 18

50-60 61,83 73,94

3 29 264 264

10,15 S.C.

18

85,>95

264

10,30 i.v. 15 i.v. 15 10 S.C. 10 S.C. 10 S.C.

2.5 4,24 18 24 24 24

50.60 25,95 95 50 40 0

435 403 284 328 328 328

180

GUANIDINES AND ADRENERGIC NERVE ENDINGS

extensive depletion 13281, but the short-lasting effect on brain noradrenaline in other species is due perhaps t o a prolonged reflex stimulation of the sympathetic system rather than to a more direct action o n the brain [329] . The catecholamines in the adrenal glands are also fairly resistant t o depletion by guanethidine. Direct observation with the electron microscope of rat adrenal medullary granules showed that these were not depleted of their noradrenaline in animals treated with either guanethidine or reserpine, whereas noradrenaline storage granules in the pineal gland and vas deferens were depleted readily [ 3 3 0 ] . This resistance is not due t o any failure o n the part of guanethidine t o enter the adrenals 13231, but it may depend on differences in the catecholamine storage mechanisms in adrenal and nervous tissue. The catecholamine storage granules from chromaffin cells are larger and heavier than the storage granules isolated from sympathetic nerves, [ 3 3 1 , 3 3 2 ] and there are also other differences. Literature o n the effect of guanethidine o n other natural tissue amines is scanty. In rats, u p t o about SO per cent depletion of 5-hydroxytryptamine has been reported in the brain [21 I ] , thyroid [333] , duodenum [21 I ] and small intestine 1334, 33.51, but n o depletion has been observed in the spleen or ileum [21 1, 3341. Guanethidine does not affect rat-heart histamine [ 3 3 6 ] . The noradrenaline normally contained in the storage granules can be partly or completely replaced by structurally related sympathomimetic amines, either by injection of the amine itself, or of suitable precursors such as a-methyl-DOPA or a-methyl-rn-tyrosine. These amines can be depleted from the heart by guanethidine in the same way as the noradrenaline which they had replaced. a-Methylnoradrenaline [337] and metaraminol [338] are depleted less readily than noradrenaline from rabbit or rat hearts, whereas dopamine, octopamine and rn-octopamine are depleted more readily than noradrenaline [339] . The more rapid depletion of these last three compounds was attributed t o weaker binding in the storage granules [ 3 3 9 ] , but could equally well be due t o their greater susceptibility t o destruction by monoamine oxidase, since both a-methylnoradrenaline and metaraminol are resistant t o attack by monoamine oxidase. The effect on tissue noradrenaline levels of repeated administration of guanethidine over a period of several days or weeks differs little from that produced by a single large dose. Thus, repeated dosing failed t o lower brain ,noradrenaline in rats [322, 340, 3 4 : ] , dogs [254] or rabbits [ 2 5 4 ] , although there was some effect in cats [341] ; nor was there much effect on adrenal catecholamines in rats [322, 3401 or rabbits [254]. Adrenal catecholamines in dogs were lowered by about 50 per cent o n daily oral administration of 5 mg/kg of guanethidine for 2 8 days [254].

Other guanidines

Many guanidines, besides guanethidine, will lower tissue noradrenaline, although only two have been found to be more active, namely the spiro analogue

181

G . J . DURANT, A. M. ROE, A. L. GREEN.

(LXXXVI) [342] of guanethidine and P-hydroxyphenethylguanidine (LXXXVII) [264]. Guanoxan (V) has about equal activity, on acute administration, to that of guanethidine [94, 2541 . Structure-activity relationships are summarized in

c

OH

N.(CH2)2.NH.C=NH

I

I

PhCHC H2. NH C=N H

I

N H2

N"2

(LXXXv I )

(LXXXVI I)

Tables 3.25-3.27. As there is considerable diversity in the choice of species, route of administration, and time after injection in these studies, depleting Table 3.25. NORADRENALINE-DEPLETING ACTIVITY OF GUANETHIDINE ANALOGUES AND MISCELLANEOUS GUANIDINES RNHC(: NH)NH2 Depleting potency *

R

c

N (CH7)* (Quanethidine)

tt++

Reference

see Table 3.24

tt

234

t+t+t

342

0

438

+t

438

+t tt

*

2 84 344

*Relative to guanethidine (++++) o n a scale increasing from 0 t o +++++

potency has been expressed only semi-quantitatively, that of guanethidine being used as a standard. Few useful generalizations can be drawn from these data. Although almost all the more active compounds contain the moieties ring-C-C-guanidine or ring-

182

GUANIDINES AND ADRENERGIC NLRVE ENDINGS

0-C-C-guanidine,

the presence of either of these systems does not necessarily

Table 3.26. NORADRENALINE-DEPLETING ACTIVITY 01.'ARALKY LGUANIDINES ArA.NHC(: NH)NH2 Ar

Deplrtirig poter1c.v *

A

+

CH2 CH2 CH 2 CH2 CH2 (CH2)2 (CH2)2 w2)3 CH(Me)CH2 CH(F t)CH2

0

+

0

0

+++ + ++ ++ +

7r2

trans-HC-CH CH(Me) CH(Me) CH(Me) CH(Me) CH(Me) CH(Me) CH(Me) CH(1-t) CH(I!t) CH2CH(Me) (-)CH(OH)CH2 (+)CH(OH)CH? CH(OH)CH2 CH(OH)CH2 CH(OH)CH2 CH(0H )CH 2

++ + + 0 0

++ 0 0

+

+ 0

+++++ +++ 0

+++ +++ 0

Reference 25 9,3 5 8,4 34. 358.434 259 259 259 2 5 9,3 5 8,4 3 4 434 259,358 259 391 259 259 259 259 259 259 259 259 259 259 259 265 265 265 265 265 265

ArA.NC(: NR2)NHR2

I

R' Ar

R'

R2

PI1 CH2 Ph CH2 2CI-

H Me

Me H

C6H4 C H 2

H H H Me

Me Me Me H

Ph Ph Ph

A

(CH32 CH(OH)CH2 CH(0H)CHz

Depleting poteticji*

+ 0 0

+ +

+++

Rrferrrlce 358,434 315 358,434 358 265 265

G. J . D U R A N T , A. M . ROE, A . L. GREEN.

183

bestow depleting activity; furthermore, 3-plienoxypropylguanidine (LXXXV) and 1 -(p-chloropheny1)ethylguanidine (I~XXXVIII),which d o not conform to the above structures, are nevertheless active depleters. A ring substituent may enhance activity, as in the carboxamidinotetrahydroisoquinolines (Table 3.26)

Farinitla (LXVII, R ~ = K ” )

H 7-Br H

H H Me

0

364

i

277 278

+++

‘ S e e footnote to Table 3.25

or abolish it, as in 0-hydroxy-o-chlorophenethylguanidine, and in the benzodioxans and benzodioxoles. Substitution on the carbon atom adjacent t o the guanidine group usually lowers activity, although (LXXXVIII) is an obvious exception t o this rule. None o f the compounds in which more than one o f the guanidine nitrogens bear a substituent shows significant depleting activity. As found with guanethidine, doses of other guanidines sufficient to cause marked depletion of noradrenaline from tissues such as heart or spleen, fail to lower the catecholamine content of the brain or adrenals. This has been shown for 0-hydroxyphenethylguanidine (LXXXVII) [ 2651 , guanisoquin (LXVII, R’ = 7-Br, R2 = R3 = H) 12771, 3-phenoxypropylguanidine(LXXXV) [ 2 4 9 ] , mercaptoetliylguanidine [344] and guanacline (LXXTIX) 13451. Unlike what is found with guanethidine, repeated administration of some other guanidines can lead t o a marked fall in brain and adrenal catecholamines. This is so for guanoxan ( V ) which, when given orally t o dogs at 10 mg/kg per day for a month, caused an 80 per cent fall in the noradrenaline content of the hypothalamus. When given a t this dose for 12 months, there was a fall of over 80 per cent in heart, spleen, adrenal and brain amines (2541. A marked depletion of brain and adrenal amines has also been found on prolonged dosing with this drug in rabbits [ 2 5 4 ] . Even when given for only 3 days at SO mg/kg orally, guanacline (LXXXIX) lowered cat brain noradrenaline by 70 per cent [345] : but it is uncertain whether a comparable reduction would be found in other

GUANIDINES A N D A D R E N E R G I C N E R V E ENDINGS

184

species, since, under some conditions, guanethidine may also lower brain noradrenaline in cats [341].

Table 3.27. NORADRENALINE-DEPLETING ACTIVITY OF O X Y G E N C O N T A I N I N G G U A N I D I N E DERIVATIVES Fornzula (XLVIII) (p. 156)

R

n

H 5-Me 5,8-M~2 H

1 1 1 2

Depleting potency*

++++ + 0

++

Reference

94, 138,254 138 94 138

Formula (XLIX) (p. 158)

R'

RZ

R3

H

H

Me

+

94

Formula (LI, X = N H C ( : N H ) N H d (p. 158)

R

++ 0 0

94 94 94

Formula (LII) (p. 159)

R

X

7-Me N H C ( : N H ) N H z

+

ArO-A.NHC(: NH)NH?

93

G. J . DURANT, A. M. ROE, A. L. GREEN.

185

Antagonism of noradrenaline depletion

Two related aspects of guanidine biochemistry are considered under this heading. Firstly, the depleting action of guanidine derivatives may be prevented by many drugs, including other guanidines, and secondly, guanidine derivatives frequently antagonize the noradrenaline depletion produced by non-guanidines, such as reserpine. The many compounds which reduce the extent of the noradrenaline depletion produced by guanethidine can be broadly sub-divided into the following groups: (a) cocaine, (b) antidepressants, such as imipramine, (c) amphetamine and related compounds (including the guanidines like N-benzyl-N-methylguanidine(LXXXII), (d) monoamine oxidase inhibitors, and (e) non-depleting adrenergic neurone blocking agents. Compounds in the first three of these groups decrease the uptake of guanethidine into the heart as well as reduce the extent of noradrenaline depletion and adrenergic neurone blockade. A reduction in the uptake of guanethidine has been produced by cocaine [346, 3471, imipramine (3461, desipramine [348] , amphetamine [347, 3491 and methylamphetamine [350]. However, the block of guanethidine uptake cannot be the sole factor responsible for the reduction in noradrenaline depletion, or for the prevention of adrenergic neurone blockade, since tyramine, which neither reverses such blockade [3 14, 3 181 , nor prevents guanethidine-induced noradrenaline depletion [35 1 1, nevertheless decreases the uptake of guanethidine [346, 3471. The primary pharmacological effect of cocaine and antidepressants at sympathetic nerve endings is potentiation of the action of noradrenaline at the receptors. Since adrenergic neurone blockade results from a reduction i n the amount of noradrenaline released on sympathetic nerve stimulation, sensitization of the receptors to this decreased noradrenaline output might tend to offset the impairment of sympathetic nerve. function. However, antidepressants and, t o a lesser extent, cocaine are more effective as antagonists when given before the adrenergic neurone blocking drug than when given after it [314, 3521, which is more in keeping with prevention of the uptake of guanethidine than with sensitization of the receptors t o noradrenaline. Another drug which prevents, but does not reverse, guanethidine-induced adrenergic neurone blockade is phenoxybenzamine [353] which also inhibits guanethidine uptake [348]. The reduction in guanethidine-induced noradrenaline depletion produccd by large doses of cocaine [354] or antidepressants [352] can scarcely be attributed t o a sensitization of receptors to noradrenaline, and is more likely to be due t o the prevention of entry of guanethidine into the nerve endings. Support for this interpretation of the protective effect exerted against guanethidine-induced noradrenaline depletion comes from the observation that desipramine and other antidepressants prevent the depleting action of guanethidine, but not that of reserpine [352, 3551. Guanethidine is not very lipid-soluble at physiological pH and is taken up into adrenergic nerve endings by an active transport mechanism

186

GUANIDINES A N D ADRENERGIC N E R V E ENDINGS

in the nerve cell membrane (3251, similar to that which takes up noradrenaline, and which is known to be readily inhibited by antidepressant drugs [ 3 S h ] . In contrast, the much more lipid-soluble reserpine can enter the nerve endings by straightforward passive diffusion. Since cocaine also blocks the active uptake of guanethidine, it is rather unexpected t o find that, unlike the antidepressants, it appears to provide protection against reserpine-induced depletion comparable t o that exerted against depletion by guanethidine [354]. Amphetamine differs from cocaine and the antidepressants in being almost as effective in reversing adrenergic neurone blockade as it is i n preventing it. When given several hours after guanethidine, amphetamine not only abolishes the adrenergic neurone blockade, but also displaces the guanethidine bound in the nerve endings (3491. It probably competes directly with guanethidine for a common binding site [316]. Amphetamine is not active as an antagonist of reserpineinduced sympathetic blockade or noradrenaline depletion (264,3571 . N-BenzylN-methylguanidine (LXXXII) appears to act in the same way as amphetamine [263, 2641, but a direct effect of this drug on tissue guanethidine levels remains to be demonstrated. I t is of interest that phenethylguanidine (111) will prevent the adrenergic neurone blockade caused by guanethidine [315], even though i t is itselfalmost asactive a depleting agent as guanethidine [259. 358). The extent of the noradrenaline depletion produced in mouse hearts by a combination of guanethidine with sufficient phenethylguanidine to abolish the adrenergic neurone blockade, was the same as that produced by this dose of phenethylguanidine when given alone [ 3I 51 . Drugs of the above three types counteract the adrenergic neurone blocking action of guanethidine at lower doses than are required t o prevent the noradrenaline depletion. Monoamine oxidase inhibitors behave differently, in that they greatly lower the extent of noradrenaline depletion [35')-362] at doses which have no marked effect on adrenergic neurone blockade [ 3 14, 3591. Gessa, Cuenca and Costa 13621 reported that the monoamine oxidase inhibitor iproniazid protected rat-heart noradrenaline from depletion by guanethidine at doses which were too low to inhibit monoamine oxidase, but Fielden and Green [359] later showed a correlation between the extent of monoamine oxidase inhibition produced by iproniazid in rat hearts and the protection afforded by it against guanethidine-induced noradrenaline depletion. As mentioned earlier, if, the noradrenaline in mouse hearts is replaced by octopamine, guanethidine depletes this octopamine in the same way as it depletes noradrenaline. Pretreatment of mice with a monoamine oxidase inhibitor prevents the depleting action o f guanethidine on the octopamine. However, if the heart noradrenaline is replaced instead by a-methyloctopamine, which is resistant to attack by monoamine oxidase, the depleting action of guanethidine is not reduced by prior inhibition o f monoamine oxidase [363]. This difference in the effect of monoamine oxidase inhibition on guanethidine-induced depletion of octopamine and a-mettiyloctopamine. and the similar difference noted in depletion of noradrenaline and a-methylnoradrenaline (3371 , confirms that the protection

G. J. D U R A N T , A . M . R O E , A . L. G R l l N .

I87

afforded by monoamine oxidase inhibitors against depletion by guanethidine is a direct consequence of monoarnine oxidase inhibition. Most adrenergic neurone blocking drugs differ from guanethidine in that they d o not cause an appreciable fall in tissue noradrenaline content. If given before or together with guanethidine, many of these non-depleting adrenergic neurone blocking drugs counteract the noradrenaline depletion caused by the guanethidine. This protective action was first shown with bretylium [ 3 3 4 ] , but has since been demonstrated for a variety of guanidines including bethanidine (1v) [ 3 5 8 ] ,

o-CIC, ti,.CH,.NHT=NMe

I NHMe

o-chlorobethanidine (XC) [ 3 2 3 ] , debrisoquin (V11) [3(,4] and 1-(2,4-xylyl)ethylguanidine ( X U ) [ 2 6 4 ] . The ability t o counteract guanethidine-induced depletion is not very closely correlated with potency in causing adrenergic neurone blockade-thus o-chlorobethanidine (XC) is more effective than bethanidine in preventing depletion[358], although less active as an adrenergic neurone blocking agent 11,601, and other factors are doubtless involved in the prevention of depletion. One of these could be competition with guanethidine for the active transport system responsible for the specific uptake of guanethidine into adrenergic neurones 1325, 3491, but again this cannot be the only factor, since bretylium will prevent guanethidineinduced noradrenaline depletion at doses too low to prevent the specific uptake of guanethidine [348] . Monoamine oxidase inhibition may also play an important role in this protective effect. As discussed below, some adrenergic neurone blocking agents are capable of inhibiting monoamine oxidase in vivo. In particular,o-chlorobethanidine,one of the most effective guanidines in preventing noradrenaline depletion, is also one of the most potent monoamine oxidase inhibitors. The prevention by drugs of noradrenaline depletion Droduced by guanidines other than guanethidine has not been extensively studied. However, a comparison has been made between the ettect o t drugs on depletion by guanethidine, and their effect on depletion by (-)&hydroxyphenethylguanidine (LXXXVII), a compound which is more potent t h d i guanethidine as a noradrenaline depleter, but which causes only weak impairment of sympathetic transmission (2641. Depletion by both drugs was reduced by amphetamine, the guanidines (LXXXII), (LXXXIII, R = OMe) and (XCI), and by iproniazid. Besides preventing depletion by guanethidine, many non-depleting adrenergic

188

GUANIDINES AND ADRENERGIC NERVE ENDINGS

neurone blocking drugs, such as bretylium, o-chlorobethanidine or 1 -(2,4-~ylyl)ethylguanidine, also markedly reduce the extent of depletion by reserpine [264, 354, 365, 3661. Competition for uptake is unlikely to be involved here, but monoamine oxidase inhibition could well be a major factor. Guanethidine itself exerts a weak protective effect against reserpine-induced depletion [367-3691 ,but this is almost certainly not due to monoamine oxidase inhibition, since guanethidine has virtually no effect on this enzyme (see below). The protective effect of guanethidine or bretylium on reserpine-induced depletion is abolished if amphetamine is given at the same time [370]. (+)-I-Phenylethylguanidine (LXXXIV) and N-benzyl-N-methylguanidine(LXXXII), which are selective antagonists of guanethidine-induced adrenergic neurone blockade and noradrenaline depletion, have little effect on either the sympatholytic or depleting actions of reserpine [263, 2 6 4 , 3 151. No detectable protection is afforded by bretylium [371] , guanethidine [372] or o-chlorobethanidine [365] against depletion of rat heart noradrenaline by tyramine, nor does o-chlorobethanidine affect the sympathomimetic effects of tyramine. It is significant in this connection that, whereas the noradrenaline released from the tissues by reserpine or guanethidine is excreted mainly in the form of deaminated metabolites [373] , the noradrenaline released by tvramine is excreted mainly as free base or 0-methylated metabolites 13741, thus monoamine oxidase is not involved in the depletion of noradrenaline by tyramine. The absence of Protection by adrenergic neurone blocking agents against tyramineinduced depletion thus implies that the protection they exert against depletion by guanethidine or reserpine is mediated to a considerable extent by inhibition of monoamine oxidase. Effect of guanidines on enzymes involved in noradrenaline metabolism

The major routes for the synthesis and metabolism of noradrenaline in adrenergic nerves [375], together with the names of the enzymes concerned, are shown in Figure 3.1. Under normal conditions the rate controlling step in noradrenaline synthesis is the first, and the tissue noradrenaline content can be markedly lowered by inhibition of tyrosine hydroxylase [376] . Tissue noradrenaline levels can also be lowered, but to a lesser extent, by inhibition of dopamine+-oxidase [ 377,3781 . However, the noradrenaline depletion produced by guanethidine is unlikely t o result from inhibition of synthesis, since intracisternal injection of guanethidine does not prevent the accumulation of noradrenaline which follows brain monoamine oxidase inhibition, even though it does cause depletion of brain noradrenaline [323]. Although numerous compounds have been tested as inhibitors o f tyrosine hydroxylase 13791, no guanidines appear yet among them. Guanethidine itself has no significant effect on DOPA decarboxylase [323, 3801 nor on dopamine(3-oxidase [259,38 1,3821. Neither guanoxan, which is also a potent noradrenaline

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depleter, nor a variety of aralkylguanidines affect dopamine$-oxidase [ 253, 2591 , but this enzyme is inhibited by some aryloxyalkylaminoguanidines.Guanoclor (XCII, R = CI) and its 2,6-dimethyl analogue (XCII, R =Me) cause 50 per cent inhibition [252] at a concentration of about 0.1 mM. Of the other isomeric CO, H

I

H o o C H 2 C H N H 2

HO

hydrorylase

Tyrosine

&

Ho =T

CO2H

I

DOPA decarboxytase (aromatic amino acip,

CH ,CHNH,

decarboxyhsel

DOPA

Dopamine

/Noradrenaline Catcchol- 0 - m e t h y l

3 - 0-Meth yl norad rena line

3 , 4 -Dihydroxymandelic aldehyde

Figure 3.1. Synthesis and metabolism of noradrenaline

2,6-dicNorophenoxyethylaminoguanidines(XCIII) and (XCIV), the former has about the same activity as guanoclor, whereas the latter is inactive [194].

2,6-R2Cs H3.O.CH2.CH2 .NH.NH.C=NH

I

v

NH,

(XCII) Dopamine$-oxidase contains copper and is inhibited [343] by copper-chelating agents such as 2,9-dimethyl-o-phenanthroline (XCV). The aminoguanidine structure R N H . N H G N H is formally akin t o the -N=CH-CH=Nmoiety in o-phenanthroline and thus guanoclor may inhibit dopamineQ-oxidase by chelation with the copper. If this is so though, the total lack of activity of the

190

CUANIDINES A N D ADRENERGIC NERVE ENDINGS

isomer (XCIV) is surprising. Guanoclor does not cause noradrenaline depletion o n acute administration [382] ,'but daily administration over a protracted period

(XCIII)

(XCIV) reduced catecholamine levels in the heart, spleen, hvpothalamus and adrenals of dogs [382], and in the adrenals [382, 3851 and hearts [385],but not the brains 13851 of rats. The rather greater effect on amines in the adrenals compared with those in the heart [38S] is in complete contrast to what is found with guanethidine, and supportsthe belief that this depletion results from an inhibition

Me

(XCV1 of synthesis. There is n o correlation between the adrenergic neurone blocking potency of aryloxyalkylaminoguanidinesand their effect on dopamine-8-oxidase [211. I t should be mentioned that inhibition of dopamine$-oxidase has been suggested [236] as a possible mechanism for the adrenergic neurone blocking

action of xylocholine (XXXIX). This hypothesis was based on the observations that dopamine, but not noradrenaline or any of its other precursors, prevents or reverses the blocking action of xylocholine o n the isolated rabbit ileum, and that xylocholine inhibited the formation of 14C-noradrenaline from ''C-dopamine by human chromaffin tumour tissue [ 148,2361. However, later experiments with this tissue I3861 showed that appreciable inhibition of this conversion only occurred at very high concentrations of xylocholine (> 20 mhf) which are unlikely t o be reached by sympathetic-blocking doses in vivo. Dopamine may antagonize adrenergic neurone blockade in the same way as amphetamine, or may act like cocaine and the anti-depressants, since it has a potent blocking action o n the re-uptake of liberated noradrenaline (4391 . At a concentration of 6.3 mM, guanethidine had n o effect o n catechol-0methyltransferase 1440). No other guanidines appear t o have been tested against this enzyme. In contrast, the inhibition by guanidines of monoamine oxidase, the other important enzyme involved in noradrenaline destruction, has been

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191

extensively investigated. Guanethidine itself has little effect on monoamine oxidase 13x4, 387-3801, but many other guanidines can cause profound inhibition. Structure-activity relationships are summarized in Tables 3.28 and 3.20. As precise potencies depend considerably on the assay conditions, the results are expressed semi-quantitatively only; however, for the aralkylguanidines (Table 3.2X). on which most work has been done, the internal consistency among the results from the three sources is good. In general, the phenethylguanidines are more active than analogous benzylguanidines . and, in both series, inhibitory potency is increased by ring substitution (except hydruxyl). Disubstitution in the o,o- or o,p- positions gives the greatest activity. Trans-2-plienylcyclopropylguanidine (LXXXI) is surprisingly only weakly active, in contrast to its parent amine (tranylcypromine), which is a potent irreversible inhibitor of monoamine oxidase. Interpretation o f results for the N'-methylated aralkylguanidines (Table 3.29) is less clear. as there appear to be marked species differences. Monoamine oxidase in rat tissues 13871 is more sensitive to inhibition by compounds of this type than that in cat o r guinea-pig tissues 13891, and the increase in potency found on o-chloro substitution is less marked in other species than it is in rats. For any single aryl group N'-methylation or N'N"-dimethylation may either slightly increase or decrease activity. Inhibition by these compounds is competitive with substrate, and is reversible [2.59, 3871. These properties have been used to show that o-chlorobethanidine (XC)combines with heart, liver and kidney monoamine oxidase in vivo [387, 3901 since prior administration of this drug decreases or prevents the long-lasting inhibition produced by subsequent injection of an irreversible inhibitor such as iproniazid, plieniprazine, pargyline o r tranylcypromine. Brain monoamine oxidase is not protected under these conditions, presumably because of the inability of the guanidine to cross the blood-brain barrier [390] . Other indirect evidence that guanidines can inhibit monoamine oxidase in vivo is the protection afforded by p-methoxy-a-methylphenethylguanidine(LXXXIII, R = OMe) against depletion by both guanetliidine and reserpine 12641 . This compound is a fairly active monoaniine oxidase inhibitor 12591 , but has no significant adrenergic neurone blocking activity [ 139, 2.591 nor does it prevent guanethidine-induced adrenergic neurone blockade [ 39 I ] . Low concentrations of bethanidine, o-chlorobethanidine, debrisoquin and bretylium potentiate the uptake of m-octopamine into rabbit heart slices without potentiating the uptake of metaraininol (3921. Monoamine oxidase inhibitors, like iproniazid and pheniprazine, act similarly [393]. Since m-octopamine is a substrate for monoamine oxidase, whereas metaraminol is not, these observations can be most readily explained if the adrenergic neurone blocking drugs were inhibiting monoamine oxidase within the adrenergic neurones. There is n o correlation between the monoamine oxidase inhibitory activity of guanidine derivatives and their ability to cause adrenergic neurone blockade or depletion of noradrenaline 12.59) . The contribution made by monoamine oxidase inhibition to the antihypertensive properties of guanidines remains to be

CUANIDINES AND ADRENERGIC NERVE ENDINGS

192

Table 3.28. INHIBITION OF MONOAMINE OXIDASE BY ARALKYL- AND

ARYLOXY ALKY LCUANIDINES ArA.NHC(:NH)NHZ Ar

A

Potency*

+ ++ ++

259,387,389 389 389 259,389 389 389 389 259,389 387,389 389 389 3 89 387 259 3 89 389 389 389 259 259 259 259 259 259 259 259 259 259,387 387 3 87 387 259

++

++

++ ++ ++ ++ ++ ++ ++

+ +++ +++ +++ ++ +++ + ++ ++ + ++ ++ ++

0

+

++ +++ + 0

+ Ph 4-M e oc6H 4 Ph Ph 2-Pr ‘csH4 2 ,6-Me2C6H3

Reference

+ +++ ++

259 259 259 389 389 389

++ ++

+++

*Approximate concentration producing 50% inhibition at a substrate concentration of 1 mM or less:- 0 (i1 mM), + (SO0 @), ++ (100 +++ ( 2 0 ++++ ( 2 I-(M). Within any one category there will obviously be a considerable spread of relative potencies.

w),

w),

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193

clarified. The potent irreversible monoamine oxidase inhibitors. originally studied clinically for their antidepressant properties, have almost invariably been found t o cause a fall in blood pressure [394] which does not seem t o result from peripheral sympathetic blockade [395] . Table 3.29. EFFECT OF N-METHYLATION OF BENZYLGUANIDINES ON INHIBITION OF MONOAMINE OXIDASE AKH *NHC(:N R ')NH R~

Ar

R

'

R2

Ph

H Me Me Me

H H Me Me

H

H H Me Me

0CIC6144

Me

Me Me I-Naphthyl

H

Me Me

Potency*

Reference

i +i

i ti

+t t+i

++

+++t

H H Me

+ti +i

++

389 389 389 387 389 389 389 387 389 389 389

* S e e Footnote to Table 3.28.

Mechanism of guanethidine-induced depletion

Guanethidine may reduce tissue noradrenaline levels in one or more Jf three ways, (a) by inhibiting the synthesis of noradrenaline, (b) by accelerating the enzymic destruction of noradrenalme, or (c) by interference with the storage and release mechanisms for this neurotransmitter. As discussed in the previous section, however, there is no evidence that guanethidine affects any of the enzymes involved in noradrenaline synthesis or metabolism, so,the site of action must consequently be one or more of the processes concerned with noradrenaline storage or release. This has been shown to be so for reserpine, whose action within the nerve endings has been localized fairly precisely to the membrane surrounding the noradrenaline storage vesicles, where it inhibits the active transport mechanism responsible for transferring noradrenaline from the cellular cytoplasm into the vesicles [396,397] . However, as first pointed out by Kuntzman, Costa, Gessa and Brodie [323], there are a number of differences between the depleting actions of guanethidine and reserpine, w h c h imply that although both drugs bring about depletion by disrupting noradrenaline storage, they nevertheless do so by different mechanisms.

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GUANIDINES AND ADRENERGIC NERVE ENDINGS

Perhaps the most crucial difference between these two depleting agents lies in the relationship between the extent of depletion and the tissue concentration of the drug. Reserpine is a lipid-soluble weak base, which appears to enter the tissues by passive diffusion. The drug has largely disappeared from the tissues by the time depletion is maximal, and only trace amounts remain during the subsequent protracted period of intense depletion [398]. Guanethidine is a strong base, poorly lipid-soluble at physiological pH, which enters sympathetically-innervated tissues by an active transport process [325, 3491. The noradrenaline depletion, and the adrenergic neurone blockade, are associated with the uptake of up to three molecules of guanethidine for each molecule of noradrenaline lost [349] , and spontaneous recovery in noradrenaline content follows the disappearance of bound guanethidine [323] .The guanethidine presumably enters the noradrenaline storage vesicles, since it can be released into the circulation when the sympathetic nerves are stimulated [399] . The reversal of guanethidine-induced adrenergic neurone blockade by amphetamine is also associated with the rapid displacement of the guanethidine from the heart [349]. The specific uptake of guanethidine into rat hearts can be inhibited by prior treatment of the rats with reserpine, and reserpine given subsequent to guanethidine will cause the release of guanethidine which is already bound 1347,3491. Depletion by guanidines, unlike depletion by reserpine, is not dependent on sympathetic innervation, and is unaffected by denervation of peripheral tissues [400], or by ganglionic blockade [264,401]. The ability of some drugs to antagonize the depleting action of guanidines without appreciably reducing that of reserpine has been described earlier (p. 185). The form in which noradrenaline is liberated by guanethidine differs from that liberated by reserpine. When the hearts from rats, which had been pre-treated with tritiated noradrenaline, were perfused with solutions containing either guanethidine or reserpine, most of the labelled material appearing in the former perfusate was in the form of free noradrenaline or its 3-O-methyl derivative, whereas in the latter perfusate it consisted mainly of acidic products arising from the oxidative deamination of noradrenaline [402] . However, this difference does not persist throughout the whole course of depletion. In dogs given guanethidine intravenously, the sympathomimetric effect of guanethidine and the release of free noradrenaline into the coronary sinus had subsided within four hours although the atrial noradrenaline content was still 75 per cent of normal. After another 20 hours the atrial noradrenaline content had fallen to below 10 per cent of normal, thus the bulk of the atrial noradrenaline must have been released in a pharmacologically inert form, presumably as deaminated metabolites [403] . This conclusion is supported by the finding [373] that the noradrenaline metabolites excreted into the urine after administration of either guanethidine or reserpine to rats, which had been pre-treated with tritiated noradrenaline, were predominantly those arising from deamination. Furthermore, as a dose of iproniazicl sufficient to inhibit rat-heart monoamine oxidase by 90 per cent reduced the extent of guanethidine-induced depletion of noradrenaline from 80 per cent to only 20 per cent

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[359] ,most of the noradrenaline released by guanethidine is presumably disposed of by oxidative deamination. The apparent necessity for guanethidine to be bound within the sympathetic nerve endings before it can cause depletion, suggests that one possible mechanism for depletion by guanethidine is simply a direct physicochemical displacement of the noradrenaline from its granular binding sites within the storage vesicles. Most of the differences between depletion by guanethidine and reserpine can be plausibly explained on this basis. One difference which is not readily accounted for is the greater sympathomimetic effect displayed by guanethidine, which is associated with the release of noradrenaline initially in the form of the free base [402]. Many authors have attempted to explain such results by assuming that there is more than one pool of noradrenaline within the nerve ending, and that these pools show varying degrees of sensitivity to different depleting agents [365, 4041. The precise morphological location of such pools has always been obscure, and recent work on the kinetics of noradrenaline depletion by tyramine has cast doubt on the whole concept of readily releasable and resistant pools, although it does not exclude the possibility that noradrenaline is distributed in a state of rapid equilibrium between several compartments [405]. Crout [406] suggested that the more readily released noradrenaline was simply that in the storage vesicles closest to the outer membrane ofthe nerve cell. Since guanethidine is taken up through the nerve cell membrane by an active transport process, the noradrenaline may be displaced initially from those storage vesicles sufficiently close to the membrane to permit the noradrenaline to pass out of the nerve terminals into the circulation without first coming into contact with the intracellular mitochondria1 monoamine oxidase. If reserpine, which is more readily diffusable, acted equally on vesicles throughout the nerve terminal, most of the noradrenaline would be destroyed by monoamine oxidase in the interior of the cell, and very little would escape from the cell in an un-metabolized form. Another possible factor contributing to the sympathomimetic action of guanethidine is its blocking action on the neuronal uptake of noradrenaline [356, 4071. Whereas reserpine acts solely on the noradrenaline transport mechanism in the membrane surrounding the storage vesicles, guanethidine interferes with the uptake of noradrenaline through both the vesicular membrane and the nerve cell outer membrane [408]. although it is not clear whether this interference is a true inhibition, or whether it merely results from guanethidine being a Eompeting substrate for the same uptake mechanism [348]. Although the noradrenaline transport process in the vesicular membrane is more sensitive t o guanethidine than that in the cellular membrane [409], in vivo the latter is affected first [410]. Thusafter guanethidine, although not after reserpine, any noradrenaline which is released from the nerve terminal cannot be taken back again into the cell. More free noradrenaline is therefore liberated and the sympathomimetic effect is consequently enhanced. Lundborg and Stitzel [4 101 have suggested that guanethidine-induced noradrenaline depletion can be adequately accounted for solely by the inhibition of noradrenaline uptake at both the cellular and vesicular membranes; but if this

196

GUANIDINES AND ADRENERGIC NERVE ENDINGS

were so, depletion by guanethidine, like depletion by reserpine, should be slowed down by procedures, such as cutting the pre-ganglionic sympathetic nerve, (decentralization), or ganglion blockade, which prevent access of nerve stimuli to the noradrenaline storage vesicles, and, as described above, this does not happen. Another suggested explanation for guanethidine-induced depletion is that guanethidine liberates noradrenaline from its stores by persistent activation of the normal process of physiological release [323]. This hypothesis has important consequences in connection with the mechanism by which guanethidine produces adrenergic neurone blockade, and will be discussed below. Relationship between noradrenaline depletion and adrenergic neurone blockade

The sympathetic blocking action of reserpine is closely related t o the depletion of noradrenaline in peripheral tissues [321] . Sympathetic blockade after administration of reserpine is slow in onset and does not become significant until 85 per cent or more of the tissue noradrenaline has disappeared [411]. This is not the situation with guanethidine. In rats and mice, ptosis and other signs of sympathetic blockade are apparent within 15-30 minutes of injection, whereas noradrenaline depletion does not become maximal for several hours [264, 334, 349, 4121. One of the most striking illustrations that guanethidine can block sympathetic transmission without appreciable depletion of noradrenaline comes from the work of Gaffney, Chidsey and Braunwald f4111 who showed that the increase in the heart rate of dogs induced by stimulation of the cardioaccelerator nerve was blocked by intravenous guanethidine within 30 minutes of injection, at which time the atrial noradrenaline in the same animals was not significantly below normal. It is clear from observations such as these that the adrenergic neurone blocking action of guanethidine cannot be attributed to a gross deficiency in the tissue concentration of the sympathetic neurotransmitter. Since numerous guanidine derivatives are known to cause adrenergic neurone blockade without lowering tissue noradrenaline, the question obviously arises whether the adrenergic neurone blocking action of guanethidine is causally related at all to the depletion of noradrenaline. Costa, Kuntzman, Gessa and Brodie [358] suggested that it is, and, on the basis of some limited structure-activity studies, attempted to differentiate between adrenergic neurone blocking agents which caused noradrenaline depletion, and those which not only did not cause depletion but which also blocked the depletion due t o guanethidine. These two groups appeared to be distinguishable on the basis of their chemical structures, in that all the members of the first group were derived from ethylguanidine whereas those in the second group were derivatives of methylguanidine. Subsequent studies have blurred this simple structural distinction between depleting and non-depleting guanidines, and there are numerous exceptions to this generalization. Even so, twoclasses of adrenergic neurcne blocking agent might still exist, although lacking such a precise structural differentiation. The main arguments

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supporting the existence of two different mechanisms of adrenergic neurone blockade have been drawn from a comparison between the properties of guanethidine and bretylium [348,349] .With hindsight, it might have been better if a nondepleting guanidine had been used for making this comparison rather than bretyhum, since some of the observed differences may arise solely from the fact that bretylium is a quaternary ammonium salt whereas guanethidine,although largely protonated at physiological pH, will nevertheless exist t o a very small extent as the free base. This structural difference might affect such properties as tissue distribution and transport across cellular membranes. Other than the major difference between the two drugs, namely that guanethidine causes noradrenaline depletion whereas bretylium prevents depletion, the experimental biochemical differences between the actions of bretylium and guanethidine are largely concerned with the way in which these two drugs are taken up into sympatheticallyinnervated tissues, particularly the heart. Guanethidine enters the tissue by an active transport process which is capable of being saturated and which can be blocked by amphetamine, noradrenaline, and reserpine [325,347,349]. There is also a passive uptake process for guanethidine which is not affected by these three drugs, but this process does not appear to be pharmacologically important. The uptake of bretylium is not readily saturated at high concentration and is not blocked by noradrenaline or reserpine, although it is lowered by amphetamine [348] From measurements on the specific uptake of guanethidine into rat hearts [3491 ,there appears to be acorrelation between the intensity of adrenergic neurone blockade and the concentration of guanethidine in the intracellular sites concerned with noradrenaline storage. The explanation proposed for these differences [348, 349,3581 is that bretylium causes adrenergic neurone blockade by preventing the depolarization which is normally produced when nerve impulses reach the terminals of the postganglionic sympathetic nerve fibres, whereas guanethidine causes adrenergic neurone blockade by persistently depolarizing the nerve terminals such that no further depolarization, and hence no response, can be induced by nerve stimulation. This depolarization is assumed to occur by an action at the storage granule level, whereas bretylium most likely inhibits depolarization by an action on the outer membrane of the nerve terminal. By assuming the existence of these two distinct mechanisms of adrenergic neurone blockade it is possible to explain (a) how guanethidine produces depletion in a differeit way to reserpine, (b) why guanethidine-induced depletion is associated with a marked sympathomimetic effect (this effect is known to be indirect since guanethidine has no sympathomimetic action in animals whose noradrenaline has already been depleted by pretreatment with reserpine [413] ), (c) why sympathetic blockade precedes the depletion produced by guanethidine, (d) how bretylium causes adrenergic neurone blockade without also producing depletion, (e) how bretylium blocks depletion due to guanethidine at doses which do not prevent uptake of guanethidine, (f) why guanethidine does not block its own depleting action, which it might be expected to do if it caused adrenergic neurone blockade in the same way as

198

GUANIDINES AND ADRENERGIC NERVE ENDINGS

bretylium, and (g) why amphetamine and amphetamine-like compounds, antagonize both guanethidine-induced depletion and adrenergic neurone blockade. However, there are many objections, six of which are discussed below, to the hypothesis that there are two distinct mechanisms of adrenergic neurone blockade for guanidine derivatives, and to the particular mechanism proposed for the adrenergic neurone blocking action s f guanethidine. Firstly, a close parallelism exists between the ability of amphetamine and amphetamine-like guanidines t o prevent adrenergic neurone blockade by guanerkidine and by 1(2,4-xylyl)ethylguanidine (XCI) a compound which does not deplete tissue noradrenaline [315]. Such a parallelism would scarcely be expected if the two adrenergic neurone blocking drugs acted at totally different sites. Secondly, there is no sharp division between depleting and non-depleting adrenergic neurone blocking agents, there is rather a broad spread of depleting activity, and many structure-activity relationships are not easily reconcilable with the existence of two distinct mechanisms for adrenergic neurone blockade. For example, guanoxan (XCVI, R = H) has about the

R

NH2

same activity as guanethidine in causing adrenergic neurone blockade and in causing noradrenaline depletion, and thus might be presumed to block sympathetic transmission by the same mechanism. The very closely related compound (XCVI, R = Me) is slightly more active than guanoxan as an adrenergic neurone blocking agent, but is a much weaker depleter of noradrenaline [94],and this leads to the unlikely postulate that it causes adrenergic neurone blockade by the same mechanism as bretylium, and in a quite different way to guanoxan. Thirdly, e)$-hydroxyphenethylguanidine (LXXXVII) which is more active than guanethidine as a depleting agent and which, on the basis of antagonism experiments, appears to deplete by the same mechanism, nevertheless causes only weak impairment of sympathetic transmission. Fourthly, it is questionable how far the adren, ergic neurone blocking action of bretylium is responsible for its ability t o prevent, guanethidine-induced depletion. Thus, bretylium and non-depleting adrenergic neurone blocking agents containing a guanidine group also block depletion by reserpine and by (-)$-hydroxyphenethylguanidine [ 264, 3661 , and in neither the benzylguanidine nor the phenethylguanidine series [3 1.51 is there a correlation between adrenergic neurone blocking activity and ability t o prevent guanethidineinduced depletion. If bretylium blocks depletion by some other mechanism (for example, by inhibition of monoamine oxidase), there would be no need for an explanation for the failure of guanethidine to blocking its own depleting action.

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Fifthly, in the cat spleen, guanethidine and bretylium both block the increase in noradrenaline output into the circulation produced by stimulation of the splenic nerve, and there is no marked increase in the spontaneous output of ndradrenaline following the injection of guanethidine [4 141 . ASdiscussea earlier, alternative explanations are available which will adequatelv account for the greater sympathomimetic action of guanethidine compared with that of reserpine. Sixthly, experiments using electrophysiological techniques support the view that both guanethidine and bretylium block sympathetic transmission by holding the nerve terminal membrane polarized at, or even above, the resting level [4 151 . Almost all the above evidence can be most satisfactorily interpreted on the assumption that two sites of action are involved, and these have similar but not identical specificity. One of these would most likely be situated on, or close to, the ewer membrane of the nerve terminals. Occupancy of this site can lead to adrenergic neurone blockade, as with bretylium, or antagonism of adrenergic neurone blockade, as with amphetamine or N-benzyl-N-methylguanidine.The second site, or group of sites, would be located on, or in, the noradrenaline storage vesicles, and occupancy of these sites would lead to noradrenaline depletion. (-)-0-Hydroxyphenethylguanidine would be an example of a drug combining strongly with this second site, but not with the first, whereas guanethidine and guanoxan may be presumed to act at both sites. Perhaps the most puzzling problem in terms of this two-site hypothesis is the failure so far to find any drug which prevents or reverses the adrenergic neurone blocking action of guanethidine without also lowering the extent of the noradrenaline depletion which it produces This might be interpreted as evidence that the two phenomena are causally related, but a possible explanation for this difficulty is that one route of access of guanethidine to the noradrenaline storage vesicles is via the sites concerned with adrenergic neurone blockade [318], but there is as yet no direct evidence to support this suggestion. One further factor which may confuse the interpretation of the action of antagonists of depletion and of adrenergic neurone blOCKaae IS the possibility that they may block the transport mechanisms required for the specific uptake of guanethidine. Since uptake must precede any other effect, antagonists of uptake would prevent the adrenergic neurone blockade and the depletion caused by guanethidine, but would not necessarily reverse these effects once they had appeared. Even if this two-site hypothesis is essentially correct, many unsolved problems remain. Where and what are the sites whose occupation leads to adrenergic neurone blockade? How does their occupation by a drug stabilize the nerve terminal against depolarizaiion by nerve impulses? Is there, perhaps, a small biochemically specific or morphologically localized pool of noradrenaline from which the neurotransmitter is released when the sympathetic nerves are stimulated? If so, could all adrenergic neurone blocking drugs act by depleting this special pool'! Since both labelled guanethidine and bretylium have been shown to be released from sympathetic nerve endings when the nerves are stimulated

200

GUANIDINES AND ADRENERGIC NERVE ENDINGS

[399,416], is the sympathetic blocking action of either of these drugs connected with their ability to function as ‘false’ transmitters? Despite the large amount of work already carried out in this field, there is still plenty of scope for further fundamental investigation. ADDENDUM

The most significant papers published since the above account was written are summarized below. Numerous reports [441-444] have established the clinical utility of the newer adrenergic neurone blocking guanidines: bethanidine (IV), guanoxan (V), guanoclor (VI), and debrisoquine(VI1). There are clinical papers recommending the use of guanacline (XXV), (see P. 142) particularly in corn bination witha-methyl DOPA (the mixture is known as Tadip), for the treatment of hypertension [445-448]. However, severe side effects have been reported [449,450]. Chemistry

Two groups [45 1,4521 have discussed the restricted rotation revealed by nuclear magnetic resonance in some penta-substituted guanidine bases, but which is not apparent 14521 in the hexa-substituted guanidinium salts. Further work on the chromatography [453, 4541 and mass spectra [455] of guanidines has appeared (see p. 129). 1-Adamantylguanidine could not be obtained directly from 1 -adaman-tylamine [456] ;it was obtained in good yield, however, when 1-adamantylcyanamide was fused with ammonium chloride [456]. This is the method of choice for obtaining sterically hindered guanidines 12281 ,(see p. 13 1). Some substituted guanidines have been obtained [457] by reaction of amines with the disulphide H,N(HN:)CSSC(:NH)NH,. Papers on the structure and pK,’s [458], and the synthesis [458, 4591 of acylguanidines have been published. Reaction of guanidine with alkyl-, alkenyl-, and benzyl-halides, followed by distillation under basic conditions, is reported to give useful yields of amines [460] . A novel electrophilic substitution of benzene to give N-?ethyl-Nphenyl-guanidine amongst other products has been published [461] . Structure-Ac tivity

Some substituted azetidine analogues of guanethidine are devoid of adrenergic neurone blocking activity [462], results which accord with previous findings on ringmodified guanethidine analogues (see p. 142). In addition to its adrenergic neurone blocking activity (see p. 156) guanoxan has been shown [463] to have a protracted a-adrenolytic action in cats and dogs. Its resolution has been reported [464] and the two isomers were equipotent adren-

G . J. DURANT, A. M. ROE, A. L. GREEN.

201

ergic neurone blocking agents. The (+)-isomer caused more catecholamine release in rabbit ear arteries and in rats, and it also had greater a-adrenoreceptor blocking activity. Some heterocyclic analogues of guanoxan have been described [465], and the only compounds with pronounced adrenergic neurone blocking activity were those with six- or seven-membered heterocyclic rings and with the grouping Ar-0-C-C-guanidine. The results could be accommodated [465] by the same author's, earlier hypothesis [21] on structure-activity requirements for aryloxyal kylguanidines. N-Cvano-N't-amylguanidine (LXXIII, guancydine) (see p. 170), is a hypotensive with an obscure mode of action [229] A recent report 1.4661 suggests that the hypotensive activity of cyanoguanidlnes is not directly related to that of the analogous guanidine, for example, the cyanoguanidine derived from guanethidine is inactive. A preliminary clinical report [467] suggests that guancydine may be a useful antihypertensive agent with minimal side-effects. related compounds has been reviewed [468]. An attempt to include all adrenergic neurone blocking guanidines into a general receptor scheme has been made by Rand and Wilson [469] ,who have postulated that all adrenergic neurone blocking agents act at a hypothetical cholinergic receptor, which is assumed to be involved in adrenergic transmission. However, the complexities of structure-activity data for adrenergic neurone blockade are too difficult to accommodate within this receptor scheme. Biochernistry

Two metabolites of guanethidine (I) have now been isolated from liver homogenates and identified [470] as guanethidineN-oxide (XCVII) and 7x2-guanidinoethy1amino)heptanoic acid (2(6-~arboxyhexylamino)ethylguanidine (XCVIII). The latter compound undergoes ring closure in hot alkaline solution to 7x2iminoimidazolidin-I-y1)heptanoicacid (1-(6-~arboxyhe~yl)-2-iminoirnidazolidine (XCIX). All three compounds had less than one tenth of the antihypertensive activity of guanethidine in renal hypertensive rats. The mechanism by which amphetamine antagonizes guanethidine-induced adrenergic neurone blockade (see p. 185) has been studied further. From work on the uptake of tritiated guanethidine and amphetamine into subcellular fractions of mouse hearts, it was concluded that guanethidine has a much higher affinity than amphetamine for the neuronal noradrenaline storage sites, and consequently, that the antagonism of adrenergic neurone blockade is not due to displacement of guanethidine by amphetamine from these stores [471]. Competition for the process responsible for the uptake of guanethidine into the nerve endings has been proposed as a more likely mechanism for antagonism by amphetamine and cocaine [472]. Amphetamine may also enhance the release of noradrenaline by nerve stimulation, or sensitize the adrenergic receptors to noradrenaline [472, 4731, but if these actions were predominant amphetamine should also

202

GUANlDlNES AND ADRENERGlC NERVE ENDINGS

exert a marked antagonism to blockade of sympathetic ganglia or of a-receptors, which is not the case [ 3 151 . An excess of calcium ions in the bath medium can reverse [474] the adrenergic

NHz (XCVIII)

(XCVII)

NH (XCIX) neurone blocking action of guanethidine on the Finkleman preparation (see p. 139) but the nature of this effect is not clear. Calcium ions enhance the release of guanethidine from adrenergic nerve endings, but this release appears t o be from non-specific binding sites and not from the noradrenaline storage granules [475]. The release of both acetylcholine and noradrenaline as neurotransmitters is known t o be dependent on the extracellular calcium ion concentration [ 4 7 6 ] . Boullin and OBrien [477] have investigated the interaction of guanethidine with the uptake and storage of 5-hydroxytryptamine in blood platelets, a system which in many respects resembles the adrenergic nerve ending [478]. Guanethidine isactively taken up and bound by blood platelets. The uptake is strongly inhibited by 5-hydroxytryptamine in the medium, but the binding sites within the platelet appear t o be different from those for 5-hydroxytryptamine since the accumulation of guanethidine is unaffected by the 5-hydroxytryptamine concentration within the platelet, and guanethidine uptake and release are unaffected by reserpine. The significance of monoamine oxidase inhibition as a contributotp factor, to the antihypertensive action of some adrenergic neurone blocking drugs (see p. 186), has received further support. The tetrahydroisoquinoline derivatives debrisoquine (VII) and guanisoquin (LXVII, R' = 7-Br, R2 = R3 = H) are particularly potent amine oxidase inhibitors in vitro [479] . In rats, the adrenergic neurone blocking action of debrisoquin is short-lived compared to its antihypertensive action in hypertensive rats or man, which suggests that the antihypertensive action of this drug may lie, at least in part, in inhibition of neuronal monoamine oxidase [479].

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The possibility that adrenergic neurone blocking agents act by depleting some essential, but relatively small, localized store of noradrenaline at adrenergic nerve endings (see p. 185) is supported b y the observation [480] that in cats treated with bretylium, noradrenaline depletion is found in some subcellular fractions of the spleen, even though it is insignificant in the whole unfractionated homogenate. This depletion and the adrenergic neurone blockade are b o t h prevented by previous administration of amphetamine. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26, 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 36a. 36b.

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G. H a t t i n g , L. T. Potter and J. Axelrod, J. Pharmacol. Exp. Ther., 1962, 136, 289 C. W. Nash, E. Costa and B. B. Brodie, Life Sci., 1964, 3,441 D. C. Harrison, C. A. Chidsey, R. Goldman and E. Braunwald, Circ. R e x , 1963, 12, 256 A. J. Muskus, Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol., 1964,248,498 N. H. Neff, T. N. Tozer, W. Hammer and B. B. Brodie, Life Sci., 1965,4, 1869 J. R. Crout, Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol., 1964,248,85 H. J. Dengler, H. E. Spiegel and E. 0. Titus, Nature, 1961, 191,816 R. Lindmar and E. Muscholl, Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol., 1964,247,469 P. A. Shore and A. Giachetti, Biochem. Pharmocol., 1966, 15, 899 P. Lundborg and R. E. Stitze1,ActaPhysiol. Scand., 1968.72, 100 T. E. Gaffney,C. A. Chidsey and E. Braunwald, Circ. R e x , 1963, 12,264 T. L. B. Spriggs, Brit. J. Pharmacol., 1966,26,271 C . N. Gillis and C. W. Nash, J. Pharmacol. Exp. Ther., 1961, 134, 1 G. F. Abercrombie and B. N. Davies, Brit. J. Pharrnacol., 1963,20, 171 R. Cabrera, R. W. Torrance and H. Viveros, Brit. J. Pharmacol., 1966,27,51 J . E. Fischer, V. K. Weise and I. J . Kopin, Nature, 1966,209, 778 P. Walden and H. Ulich, 2. Elektrochem., 1928, 34,25 L. Metz, 2. Elektrochem., 1928,34,292 G. B. L. Smith and E. Anzelmi, J. Amer. Chem. Soc., 1935,57,2730 W. J . C. Dyke, quoted in ref. 213 H. Ozawa, Y. Gomi and I. Otsuki, Yakugaku Zasshi, 1965,85,112 R . P. Mull, R. H. Mizzoni,M. R . Dapero and M. E. Egbert, J. Med. Pharm. Chem., 1962, 5,944 H. Krieger, Suomen Kemistilehti B , 1962, 35,218 H. Najer, R . Ciudicelli and J . Sette. Bull. Soc. Chim. Fr., 1962, 1593 J . Klepping, G. Nouvel, A. Escousse, B. Franc and J. T. Didier, Therupie, 1965, 20, 205 H. Najer, R. Giudicellj and J. Sette, Bull. Soc. Chim. Fr., 1964,2572 H. Najer, R. Giudicelli and J . Sctte, Bull. SOC.Chim. Fr., 1965, 2 1 18 H. Najer, R. Giudicelli, J . Sette and J. Menin, Bull. Soc. Chim. Fr., 1965, 204 R. P. Mull, P. Schmidt, M. R. Dapero, J . Higgins and M. J . Weisbach, J. Amer. Chem. Soc., 1958,80,3769 R. P. Mull, R . H. Mizzoni, M . R. Dapero and M. E. Egbert,J. Med. Pharm. Chem., 1962, 5.651 D. Jack, R. C . W. Spickett and G. J. Durant, British Patent 952,194, Chem. Abstr.. 1964,61,14647 L. A. Paquette, J. Org. Chem., 1964, 29, 3545 R. Truchot, J . Klepping, K. Michel, A. Escousse and H. Tron-Loisel. C. R . Soc. Biol., 1964,158,1249 J . W. Daly, C. R. Creveling and B. Witkop, J. Med. Chem., 1966,9,280 0. Krayer, M. H. Alper and M. K. Paasonen, J. Pharmacol. Exp. Ther., 1962,135,164 A. K. Pfcifer, E. S. Vizi and E. Satory, Biochem. Pharmacol., 1962, 1 I , 397 R. Cass, R. Kuntzman and B. B. Brodie, Proc. Soc. Exp. Biol. Med., 1960, 103, 87 1 T . L. Pai, W. C. Chen,C. M. Wang, K. S. Ting and J . Y. Chi, Sci. Sinica (Peking), 1964, 8,1521 L. L. Iversen, J. Pharm. Pharmacol., 1964, 16,435 E. T. Abbs, Life Sci., 1962, 1.99 Anon., Brit. Med. J., 1969, 2, 365 J . Bath, D. Pickering and R. Turner. Brit. Med. J.. 1967.4, 519 Anon, Practitioner, 1969,202, 294 J. Bariety,PresseMed., 1968, 76, 205 L. Beeker, Med. Welt., 1968,68, 1684 K. D. Bock and V. Heimsoth,Deutsch. Med. Wochenschr., 1969,94,265 U . Kumpmann,Med. Welt., 1968.46.2553

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448. E. Stampfer. Wien. Med. Wochenschr., 1968, 118,551 449. W. Teichmann. H. V. Frankberg and A. Rosenberg, Med. Klin., 1968,63. 260 450. M. L. M. Parker and M. Bullen,Med. J. Aust., 1969, 1, 159 451. H. Kessler. Tetrahedron Letters. 1968, 2041 452. V. J. Bauer, W. Fulmor, G. 0. Morton and S. R. Safir, J. Amer. Chem. SOC.,1968,90, 6846 453. Y. Robin and N. van Thoai, Bull. Soc. Chim. Fr., 1967,3965 454. 1 . di Jesso,J. Chromatogr., 1968,32, 269 455. A. G . Loudon, A. Maccoll and K . S. Webb, Advan. Mass. Spec., 1968.4, 223 456. H. W. Geluk, J . Schut and J . L. M. A. Schlatmann,J. Med. Chem., 1969. 12.712 457. Danish Patent 107,550, Chem. Abstr., 1968,68, 114241 458. K. Matsumoto and H. Rapop0rt.J. Org. Chem., 1968,33,552 459. R. Krug and K. Nowak, Rocz. Chem., 1967,41, 1087 460. M. Olomucki and P. Hdbrard, Tetrahedron Letters, 1969, 13 461. A. Heesing and R. Peppmoeller, 2. Naturforsch., 1968, 23, 1325 462. E. Bellasio and G. Cristiani J. Med. Chem., 1969, 12, 196 463. K. Vidal-Beretervide, L. E. Pritsch and H. Trinidad, Arzneim-Forsch., 1969, 19, 947 464. J . B. Stenlake, D. D. Breimer, G. A. Smail, A. Stafford and W. C. Bowman, J. Pharm. Pharmacol.. 1968,20. 82 S 465. J. Augstein, A. M. Monro, G. W. H. Potter and P. Scholfield,J. Med. Chem., 1968, 11, 844 466. S. M . Gadekar, S. Nibi and E. Cohen, J. Med. Chem., 1 9 6 8 , l l . 81 1 467. J. Hammer and E. Freis, Clin. Res., 1969, 17, 60 468. R. P. Mull and R . A. Maxwell, Antihypertensive Agents (Ed. E. Schlittler), Academic Press, New York and London, 1967, p. 115 469. M.J. Rand and J . Wilson, Circ. Res., 1967.21, Suppl. 3 , 89 470. F. B. Abramson, C. 1. Furst, C. McMartin and R . Wade, Biochem. J., 1969, 113, 143 471. H. 0. Obianwu, R. E. Stitzel and P. Lundborg,J. Pharm. Pharmacol., 1968,20,585 472. J . F. Cerbens, M. W. McCulloch and J. Wilson, Brit. J. Pharmacol., 1969,35,563 473. H. 0. Obianwu, Acta Physiol. Scand., 1969,75. 102 474. J. H. Burn and F. Welsh, Brit. J. Phormacol., 1967, 31,74 475. D. J . Boullin,Brit. J. Pharmacol., 1968.32, 145 476. W. W. Douglas, Brit. J. Pharmacol., 1968, 34, 45 1 477. D. J . Boullin and R. A. O’Brien, Brit. J. Pharmacol., 1969, 35.90 478. A. Pletscher, Brit. J. Pharmacol., 1968,32, 1 479. M. A. Medina, A. Giachetti and P. A. Shore, Biochem. Pharmacol., 1969, 18,891 480. E. T. Abbs and M.I. Robertson,Brit, J. Pharmacol., 1969, 36, 191P

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4 Medicinal Chemistry for the Next Decade W.S. PEART, F.R.S.,M.D., F.R.C.P. Medical Uriit, St. Mary's Hospital, Lotidon, W.2. INTRODUCTION

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DRUGS F O R T R E A T I N G RAYNAUD'S PHENOMENON AND CHILBLAINS

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DRUGS F O R TREATING URINARY T R A C T INFECTIONS

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INTRODUCTION In writing a chapter in which an attempt is made to suggest diseases for which drugs are required, a particular point of view has t o be assumed and a decision made as to whether one is looking at the whole world situation or at the diseases of the Caucasian races. Looking at the world situation, it is clear that what is needed is food, since malnutrition and its attendant diseases probably claims more victims than other maladies, but this raises the question of increase of world population and methods of containing it, since at present there seems no easy solution to the food problem and it certainly is not yet proven that Malthus was completely wrong. Until, for example, the cow does not'compete successfully with the Indian peasant, no great advances are to be expected. Since these problems can only immediately be met by improvements in agriculture as well as in contraception, it is difficult to imagine any pharmacological therapeutic approach which would be helpful, except perhaps a contraceptive food. On the one hand, the brave effort of Pirie [ 1 , 2 ] to provide protein from grass and leaves seems to founder on questions of palatability, whereas questions of contraception on the other founder for quite other psychological reasons. We therefore have the paradoxical situation that where contraception is needed most, the population growth outstrips the food supply, and in our highly civilized communities, 21 5

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with major disorders of gluttony and its attendant fatness, contraception is a matter more of social convenience than necessity [ 3 ] .

PSYCHOTROPIC DRUGS This naturally leads t o consideration of these drugs which have long been in the para-scientific literature, from witches’ pills and potions conferring either magical powers on the taker or correcting some defect insusceptible to ordinary medicine. In a sense, every drug given t o a patient is rendered psychotropic by the doctor, his mood and the appearance of the medicament. As has been remarked on many occasions, placeboes cure more ills than ordinary drugs create. Modern literature still contains burgeoning references to such drugs, and the ‘brain washing’ techniques used on prisoners in modern wars give evidence as t o the effectiveness of certain drugs in this regard. The ultimate is always a drug which in some way gives control of the mind t o another, and in a sense the modern pharmacology of psychotropic drugs is attempting t o d o just this. When knowledge on this score is further refined so that drugs are found which have their action on certain specified parts of the brain, then their use as potential weapons will have long since been c,onsidered. How much would governments have welcomed the chance t o use this sor,t of drug on a recalcitrant population of students rather than the more obvious external irritant courses which were available. Further, can governments resist the temptation when such drugs become available? The dose of LSD (lysergic acid diethylamide) required is so small, its ease of production so great, that it offers itself already in this form as a means for influencing whole populations. So these dangers of producing drugs for the treatment of the major mental disorders of civilized societies carry their own large risk; scientists working in this field have to be well aware of them and their political implications. They therefore have to be prepared t o be vocal politically [4]. Anxiety and depression are two common conditions that doctors meet almost daily. They include an enormous variety of symptoms and are often not recognized for what they are, leading t o enormous waste of effort in investigating patients whose trouble is psychological from start to finish. It is interesting t o look at the range of activity in this field from the psycho-analyst seeing his handful of patients annually. to the cavalier approach of the determined chemical therapist, believing that the modern armamentarium contains most of what is needed [ 5-71 . These drugs are used on an ever widening scale in general practice and I seriously doubt if any advance will emerge in our understanding of psychological disease, which is a paradox which runs through the whole of therapeutics. Consider how small a contribution knowledge of the action of digoxin has made to an understanding of cardiac failure; the use of insulin to that of diabetes; even of aspirin to headache, and certainly of hypotensive drugs t o the mechanism of hypertension. 1 would like t o put forward a general principle relating t o new drugs and their mode of action: When a drug is introduced in the treatment of a

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disease because of a known actiori. this actiori will subsequently be showri to have nothing to do with its true mode o f actiori. iior with the cause of the disease. The whole group o f psychotropic drugs needs a niuch more critical evaluation than it is receiving at present. I t is often doubtful whether i n anxiety they are better than phenobarbitone. and only perhaps in the case of depression is wide use contributing to the belief that they have a worth-while action in many patients [6] . We desperately need t o know in the use of these drugs, by a close study of a few patients, what they may be doing. and there is no field in which such study is more needed. I would niuch prefer to see this study being supported by large grants than for screening the blood and urine of patients with psychiatric disorders. hopefully waiting for something to turn up. If one of these drugs were taken and seriously pursued. the rewards might be very great indeed. My belief then here is very simple. I t is not that we need more drugs for these common diseases of man. but more intensive study of those we have. This field in particular throws into sharp relief the difficult relationship between the production of drugs for diseases and the doctors producing diseases with the drugs. In no other sphere is the proper study of man required and the pharmaceutical industry is clearly more handicapped here than anywhere else for nowhere else does animal experiment fall so far short of human behaviour. It would pay the industry handsomely to insist on the development of research units in this field. It would be a direction in which medicine requires to be led and from which the dividends in research could be rich. Nowhere else is seen quite so clearly the need for a positive action by the pharmaceutical industry since it has just as great a need of the medical profession as the latter has of it. I t is pointless for doctors to decry the activities of drug companies if. while willing to make use of the drugs produced by research on the one hand, they fail to offer the right sort of research facilities o n the other. Fortunately there are signs that medicine in general is coming to appreciate the need for close collaboration and the major question raised is what may be the best form for such collaboration. The first essential is for university hospitals each t o have thriving departments of clinical pharmacology or therapeutics, and I see no future myself in medical schools for departments of pure pharmacology except jointly with therapeutics. Tlie development of drugs and the initial assessment of their action will always, and increasingly so. be better, done in modern pharmaceutical laboratories, but the investigation of their actions in nian will always be better done in departments of medicine. A change of attitude in a sense is required from within medicine so that this is not regarded as drug testing, but rather the use of drugs to give information about the nature of disease processes and the way in which beneficial or even the reverse effects are pro duced. In relation to the therapeutic endeavour which is a part of every doctor’s daily life, the amount of serious effort going into this field in British universities is much too small, but I believe that the initiative will have to come very markedly as an act of forward-looking policy from the pharmaceutical industry

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itself. Study of the so-called side-effects alone could not but yield tremendous information. For example the Parkinsonism of certain patients receiving phenothiazine derivatives 181 must surely throw light, not only on the mechanism of action of the drug, but also on Parkinsonism itself.

DRUGS FOR TREATING MALIGNANT OBESITY It might seem a large leap to turn to fatness, with which may be tied smoking. It is strange that the most effective drug treatment for fatness that I know, namely smoking, should be a lethal habit [9-111 which has so much in keeping with that other malignant condition, obesity. I use the term malignant obesity advisedly, since all figures for morbidity and mortality prove the point [ 12, 131 , whether in the induction of diabetes mellitus, osteoarthritis or, more importantly, cardiovascular disease. The question is whether a drug is needed to cure the addictions to either smoking or food. The answer is probably not, since in choosing a new drug, the difficulty of a fresh addiction is clear, and unless it has addictive properties which are less than cigarettes or calories, then it would be of little value. It is quite likely that gluttony allied to smoking kills more people than most other diseases in Western civilizations, since it seems likely that vascular disease is closely related to both. It is difficult to be more precise than that at present but the ,evidence becomes stronger with the passage of time, and has led to campaigns to try to stop the smoking habit as well as to modify diet [14]. Do we then need a drug which abolishes those sensations of hunger which cause people to eat more than they need, or do we need to understand more about the reasons for people eating food regularly in excess of requirement [ 15- 171 ? Drugs directed at the mental aspect of over-eating have not been conspicuous by their success, with the one exception of smoking. Why is it that when people stop smoking their weight rises? Does eating become a mere substitution, something to do when the urge to light a cigarette becomes irresistible, or does some pharmacological effect of smoking reduce the appetite? It would be a worthwhile field for study since many smokers believe that the latter is the truth. A great deal depends upon knowledge of whether the sensation of hunger and .satiety is a central one or whether it arises from the stomach itself [15-171. That the sensations which we call hunger are felt largely in the epigastrium, does' not prove that the afferent signals are of greatest importance, but suggests that it would be worth trying to cut them off completely, since the only people who lose weight consistently over 5 years in an obesity clinic are the doctors running it. Since the same might be said of the control of smoking despite all evidence on mortality from cancer of the bronchus, then it surely would be better to try and tackle the gluttony problem via the afferent pathway. There seems to be nothing of synthetic nature and of no calorific value that is really bulky enough to compare readily with half a pound of mashed potatoes. I would therefore like to direct the attention of physiologists and pharmacologists to this afferent signal, for

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solution of this problem would make a greater contribution to morbidity in Western civilisations than any other single factor. It is striking, for example, that the intestinal by-pass operation designed to take food quickly from the stomach to the terminal ileum, so reducing the absorption in the jejunum, while theoretically a good idea [ 181 , failed largely because of psychiatric breakdown in many patients, which points to the underlying disability in many gluttons, but also I am sure because the gastric afferent pathway was not blocked. ANTI-INFLAMMATORY DRUGS There are a large group of diseases ranging from polyartheritis nodosa to asthma which have the common characteristic of an inflammatory response in the tissues and which are susceptible to the action of cortisone-like steroids. Some of these diseases have altered immunological states. Perhaps the best example of this is the inflammatory response induced in the transplanted kidney where prednisone is usually essential to reverse the attempts of the host to reject the foreign kidney [ 191. 'At the dose levels used in most of these conditions, the benefits of the drug are purchased at a considerable price in terms of the effects which lead to illness or death: decalcification of the bones, muscle wasting, peptic ulceration with bleeding or perforation, cataract, and, most serious of all, unsuspected infection until it is too late to reverse the situation [ 2 0 ] .So far n o adequate substitute has been found in most of these conditions and as alternatives, we are really left only with salicylate, phenylbutazone or indomethacin. No antiinflammatory steroid has yet been produced which separates these other undesirable effects from the anti-inflammatory ones, despite much effort, and it seems that the lengthy side chain at C,, is necessary in some way for most functions. Any drug which is found as effective but without the side-effects would be a boon. It is, of course, likely that prednisone is not working in quite the same way in all the conditions where it is effective, and the use of the term antiinflammatory is perhaps too loose. More needs to be known about the way in which the inflammatory response is induced and this might be more profitable since neither phenylbutazone [2 1 ) nor indomethacin [ 2 2 ] are equally effective in all conditions. A clue as to one mode of action may be provided by the aseptic necrosis of bone seen in patients receiving moderately large doses of prednisone over some months [23]. The hips and kn'ees are particularly liable to damage and the whole head of the femur may be resorbed completely, suggesting that the blood supply has been cut down markedly. If t h s can occur in normal bone, then the effect on the circulation in general might be worth studying, particularly in relation to the inflammatory response. DRUGS FOR TREATING RAYNAUD'S PHENOMENON AND CHILBLAINS The two commonest vascular conditions for which there is no really adequate treatment represent a continual challenge. While the average patient with

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Raynaud’s phenomenon does not usually suffer serious disability unless it is a manifestation of some underlying disease like scleroderma or lupus erythematosus where ischaemia of the fingers will ultimately occur in severe degree, nevertheless this condition is a source of great discomfort and at times pain, in large numbers of people when the fingers turn blue, white and numb in cold conditions. The same may be said of the painful swelling of chilblains, and particularly in some women when they ulcerate around the ,ankle, these lesions, so like frostbite, are a source of great trouble. The question of mechanism of disease is extremely important here since all therapeutic efforts have been very poor indeed in both conditions. To take Raynaud’s phenomenon first, the description by Sir Thomas Lewis of the vascular responses in the fingers of sufferers, suggested that the digital arteries were abnormally sensitive to cold [24, 251. The meaning of this in terms of either smooth muscle response to cold or the liberation of noradrenaline in the vessel wall, or an increased sensitivity of the vessel wall to noradrenaline itself, has not so far been elucidated, despite the claims that an excess of noradrenaline appears in the venous blood draining the hands of patients during an attack [ 2 6 , 271. An approach by cervical sympathectomy, while it produces transient improvement, over the course of a year or two is usually attended by a return of symptoms [28]. While this may bear on the possibility of performing adequate sympathectomy in the upper limbs as compared with that in the lower [29], the situation therapeutically still remains very difficult [30]. It is almost possible to guarantee patients warm feet from lumbar sympathectomy, but surgeons fight shy of performing sympathectomy on the upper limb since the failures are great. There is no really good evidence that much improvement has followed the use of either ganglion-blocking, sympathetic-blocking or noradrenaline-depleting drugs. Whether the drugs as administered in therapeutic doses to people with normal blood pressure really d o succeed in increasing hand blood flow has not been clearly demonstrated, with perhaps the exception of reserpine [30]. The situation therefore remains that the best advice is to keep the body and hands warm. Any therapeutic agent would have to have a very selective effect since in the average case it is the fingers which are involved, followed by the foot. There might be a case for a vasodilator which could be introduced through the skin, and of course one of the real difficulties is to get drugs to move through the skin into the subcutaneous tissue and then to remain there in high enough concentration to be effective. One of the ways of doing this has been to apply a cream of nitroglycerine in lanolin to the skin and some effect has been claimed [3 1 ] . This would hardly be practicable although in severe cases in the winter some patients would be very grateful if all they had to do was to wear gloves overnight impregnated with some cream containing a substance which slowly diffused into the subcutaneous tissues. The easiest way of introducing suitably charged substances into the subcutaneous tissue is by electrophoresis [32], and it would not be too fanciful t o use a bath with the appropriate drug in the right concentration and a low applied voltage which would take the substance into the fingers alone. The problem then would

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be the appropriate substance which would stay in the tissues long enough to have an-effect. It might be possible to d o this with such drugs as reserpine so that only a local effect is obtained. To turn to chilblains, this is perhaps, if anything, commoner as a condition and equally troublesome, for the affected areas, so often the back of the ankle with its swollen, painful, red, and subsequently itching lesions, last for a long time, sometimes ulcerate and become infected. There is an even greater lack of knowledge about the basic cause of chilblains although cold has a great deal to do with it and probably most people are capable of getting a chilblain in contrast to Raynaud’s phenomenon, which is much more difficult to induce in normal subjects. An out-pouring of fluid into the subcutaneous tissues as part of the inflammatory response is clear, which is why the analogy to frostbite is clear. The nature of the injury and its mediation is unknown, and while vitamin I> and nicotinic acid, not to mention calcium salts, have been prescribed freely, the evidence that they are effective is anecdotal. Prevention here is perhaps easier than in the case of Raynaud’s phenomenon, but again the degree of susceptibility varies and a similar principle to that enunciated for Raynaud’s phenomenon might be useful. DRUGS FOR TREATING PROGRESSIVE GLOMERULONEPHRITIS Following an attack of glomerulonephritis with inflammation and swelling in the glomeruli, the patient may recover completely with no further trouble, or may enter a progressive course in wliich the glomeruli become more and more damaged with an end-result as complete renal failure. In this disease the CI-3 fraction of complement may be deposited in-the glomeruli, associated with antigen-antibody complex [33], and certainly in various forms of nephritis the complement level may remain low and the length of time for which this occurs may be related to the prognosis [34]. The concept that antigen-antibody complexes when they are deposited in tissues in some way leads to an inflammatory response, is a most important modern concept in certain diseases, particularly gloinerulonephritis. The relation to infection initially by haeinolytic streptococcus of a particular type is clear, yet theprogression often seems to occur without further infection. If this progression could be halted in some way, then many patients, now otherwise dying with renal failure, could be saved at the outset of their attack of glomerulonephritis. If complement could be shown to be an essential part of the inflammatory reaction, then it might be possible to consider ways in wliich the action of complement could ue interfered with. Certain parts of complement are metal-dependent with calcium playing a very important role [35], and it would be possible to consider whether interference with the metal could be influential. Since parts of complement have esterase activity [35] , it may be that the antigen-antibody complex causes its inflamniatory change by bringing this esterase activity into close proximity with susceptible

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tissues. Whether there are any ways of interfering with the esterase activity of complement, I do not know, but it might be worth investigating. There,are certain natural inhibitors of this esterase activity and this is emphasized by the occurrence of those families deficient in t h s factor who readily develop angioneurotic oedema and in whom a local inflammatory reaction in which complement is essential is suspected [36]. It might be too speculative to suggest that those subjects who develop progressive glomerulonephritis are similarly deficient, but two approaches might be possible. Firstly, to test out the complement levels and the inhibitor levels in subjects with glomerulonephritis, progressive and non-progressive; and secondly, to wonder whether the natural inhibitor of complement might be a therapeutic agent in diseases where complement seems to be involved.

DRUGS FOR TREATING URINARY TRACT INFECTIONS Whether urinary tract infection in females is really responsible for a large number of foetal malformations as Kass believes [37], or whether it is subsequently responsible for a large number of patients with hypertension in later life, is not sure, but the problem of dealing with recurrent urinary infection is a real one. Certainly the progressive march of upper urinary tract infection is hard to halt, and the emergence of antibiotic resistant organisms is almost always the rule, so t h s group of patients is almost impossible to treat effectively. While some almost live in symbiosis with the organisms, this is by no means always the case. This problem is made all the greater because the reservoir of the infection which exists is not exactly known, by which is meant the exact distribution of organisms in the urinary tract and kidney [38]. In some cases re-infection with different organisms is clear, but this is by no means always the case and it is a real pity that mandelamine is effective only in a urine of such low pH (5) that it is difficult to achieve without the ingestion of large amounts of ammonium chloride or similar acidifying drugs [39]. Mandelamine can be extremely effective and since it relies upon the production of formaldehyde in the urine, which is a general toxic agent for bacteria, it has great merits as a theoretical approach. What is needed, therefore, is a drug which can lower the pH of the urine in a more convenient fashion than by increasing the load of hydrogen ions, as is done at present. This requires a manipulation of the function of the tubules in a way which would be of physiological interest if it could be done specifically. The converse attack would be the liberation of a substance like formaldehyde with general toxicity to bacteria, to which resistance was not acquired and which is liberated at an alkaline pH, since it is much easier to induce the kidney to excrete a urine of high pH than low, and further, many common urinary organisms produce ammonia from urea leading to a high pH in infections. Whether there are such substances which might be liberated at pH 8.0-8.5, I do not know, but again the effort might be worth while.

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ANTILYMPHOCYTE DRUGS The field o f transplantation immunology is dominated by the concept of recognition of the foreign tissue by a macrophage or lymphocyte, and the subsequent transfer of this information by this cell t o a proliferating cell in lymph nodes, capable of either attacking the foreign tissue itself or producing antibodies t o it [40]. While in some animals at least the information may have to be conveyed to the thymus first [41, 431, this does not seem to be the case in all, and particularly not in adults. The drugs used to make transplantation of foreign tissue successful initially were broadly anti-mitotic drugs, of which the only one that has seriously been useful in man is azothioprine [43]. The way in which this really works is unknown and whether it is by an effect on cell division in response to the foreign antigenic stimulus or by some other method, is speculative. This drug, allied to prednisone, which may be acting by its anti-inflammatory effects or by some other effect on the lymphocyte population, since, for example, i t is known to depress the number of circulating lymphocytes, has provided a means of maintaining renal homografts for years [43]. The complementary action of both drugs is necessary for this purpose in most recipients of such grafts. Latterly the use of antilymphocyte serum (ALS), which seems t o have its major action on circulating lymphocytes, has altered the picture completely, so that even heterografts, where tissue from another species may be transplanted, for example human skin on t o the mouse, are possible [44]. A state of tolerance t o the heterografts may be induced by giving the antilymphocyte serum before and during the early stages of the hetero-transplant so that the antilymphocyte serum can be stopped and the graft survives, perhaps indefinitely. The action seems t o be directed mainly at the circulating pool of lymphocytes and naturally the question that is raised is whether there are any other drugs more convenient to produce and to handle which may have a special action purely on lymphocytes. The use of ALS, which naturally means the introduction of large amounts of foreign protein (since it is mainly derived from the horse or rabbit), into man, has been attended by surprisingly few untoward reactions [45], but a chemical substance which is directed at the circulating lymphocyte specifically, would be a great boon. Clearly if the metabolic processes of the lymphocyte were as nicely arranged as the lymphoblastic leukaemic cell, whjch seems t o be dependent on asparagine t o maintain its metabolic activities, then it would be possible to use an approach similar t o that in which asparaginase was used t o remove most of the available asparagine from the blood stream leading to initial success in the treatment of leukaemic children [46, 471. No one would have forecast that asparagine was of such importance and it may be wondered whether a study of the proliferation requirements of lymphocytes in the Same way that nutritional requirements of bacteria have been studied, might be worthwhile in the hope that a heavy dependence on one amino acid might emerge. Certainly any approach which could specifically control the lymphocyte would be of real value.

224

MEDICINAL CHEMISTRY FOR THE N E X T DECADE

DRUGS FOR TREATING HIGH ARTERIAL PRESSURE With the large number of drugs available for the treatment of raised arterial pressure, it would be thought that there is hardly any need to call for further development in this field. The very proliferation of drugs, however, suggests the contrary, and there are various reasons for this which are worth discussing. The first is that here, perhaps more than in many fields, the proper study of the action of these drugs is in man himself, since after the usual animal toxicity tests have been carried out, this is where the answer to the question as to whether or not the drug lowers the blood pressure in man must be gained as fast as possible. Unfortunately, the animal models for hypertension are rather unsatisfactory and usually consist of either rats rendered hypertensive by renal encapsulation or clipping, or dogs made hypertensive by the same manoeuvres or, alternatively, by severing the depressor nerves from the carotid sinus. It is soon apparent that it is possible to lower the blood pressure of these preparations by drugs which have little effect on man or, while producing no obvious side-effects in the animals, produce considerable ones in man. The psychological effects which are so common to most of the drugs used in hypertension, usually escape notice in animal experiments, and certainly any suggestion of a mental effect in the animals will usually be seen to a far greater extent when the drug is used in man. I do not believe that there is any drug used in man in which we are clear as to how it is lowering the blood pressure. I have only to recall the classic controversy as to whether the hexamethonium group of drugs and its congeners introduced subsequently lowered the blood pressure by an effect on peripheral resistance or by an effect on venous tone with a reduction of cardiac output, to illustrate the thesis [48-521. The drugs which have received most investigation for their actions in man are perhaps this group and the diuretic group, with most emphasis on the thiazides [ 5 3 ] . In the latter case, the mode of action is still uncertain and if we turn to the drugs which in some way or another interfere with sympathetic efferent pathways, we still have to say that it is uncertain how the drugs lower the blood pressure in a particular patient. It is clear that postganghonic sympathetic block occurs [54, 551 but the relation of this to cardiac output and central vasomotor centre change is uncertain. Drugs such as guanethidine and aldomet, quite apart from the emphasis given to their peripheral nerve activity, have considerable central actions which most patients are only too unhappy to point out to the doctor [ 5 6 ] .Even if they do not complain of them, should they chance to stop taking the tablets for a brief period, they commonly will tell the doctor how much better they feel. Whether in fact a major action is on the central centres controlling blood pressure is not known, partly because this is a difficult area to study. Some drugs which were designed to have a markedly central action and may in some patients affect mood considerably, like pargyline, a mono-amine oxidase inhibitor, may lower the blood pressure of some patients very markedly [ 5 7 ] . This may still, however, depend also on peripheral effects of this drug. One of the striking things is how little

W. S. PEART

225

light has been shed on mechanisms of hypertension by the use of these various drugs. When hexamethonium was introduced [58], it was thought that analysis of its effect would throw new light on the participation of the nervous system, either as directly causing hypertension or participating in some way. This has, of course, not been true, nor has it been true of any other drug introduced so far into this field. Yet in practice the usual rule in treating patients with hypertension of all sorts is that a certain percentage respond to one of the commonly used drugs, some of whom may not respond to others, yet patients in n o way different, as far as can be seen, may respond to yet another drug. Too little use has been made of these observations. It may be that the drugs are in fact picking out different categories of patients. They may be different in respect of their metabolism of the drug, which would indicate that a metabolic product was more important that the effect on blood pressure of the main drug being given, or alternatively, the drug might be acting on a nervous centre in one group of patients which was more active than in other patients with high blood pressure and therefore might be regarded as being causally important. It would therefore still be well worthwhile trying in a restricted number of patients t o work out which mechanisms were important, for any way of subdividing this heterogeneous yet major group of patients with hypertension of unknown origin would be of help in trying to work out mechanisms. It might then be more sensible, perhaps rather than developing new drugs, to concentrate one’s attention on the old and t o ascertain why it is that one patient responds very well as opposed to another who responds poorly. That there are differences between patients in respect of effects on the nervous system of these drugs is shown perhaps best in the male in respect of impotence. The sympathetic blockers, again with their definite central effects, have a very variable effect in this respect. In many men, an effect on ejaculation is produced, while in many complete impotence results [ 5 6 ] .This may be secondary to the first, which in theory can be explained on the basis of sympathetic blockade, but in many men it is a simultaneous happening. These observations may tell us more about nervous factors controlling potency but they may also indicate other differences of the action of the drug which might be helpful t o examine further. Techniques for investigating the circulation in man as a whole simultaneously have not been easy, and this has been a big handicap in the investigation of hypotensive drugs. It is, for example, important t o be able to d o repeated measurement3 of cardiac output and blood flow through skin, muscle, splanchnic area and kidneys contemporaneously. With the available techniques this is not simple, and the biggest advance will certainly be represented by advances in precision of these measurements [59]. This may be why a proper analysis of any one drug in a particular patient has not frequently been undertaken. The territories of the circulation which still seem to be very much open for study in the hope that control of blood pressure would be more readily achieved uniformly, are perhaps the afferent baro-receptor pathways, and this is brought out by the example of patients with diabetes and tabes dorsalis where postural hypotension occurs and the defect may be on the

226

MEDICINAL CHEMISTRY FOR THE NEXT DECADE

afferent side rather than the efferent sympathetic pathway as shown by the work of Sharpey-Schafer [60, 611. Some of the recent effects of stimulating the carotid sinus nerves electrically in patients with severe hypertension, despite the relative crudity of this approach, show that it is possible to lower the blood pressure of such patients who have previously been extremely resistant to the action of a multitude of drugs [62]. Advance along these lines probably does await more knowledge of the afferent pathways involved in such patients but it is 'to be hoped that study of this sort of patient will continue. Allied to this, the study of those patients with postural hypotension associated with central nervous system defects of varying type [63,64] may throw light on other areas of the brain controlling blood pressure whose pharmacology may be worth closer study. The other large territory which has always seemed to me to offer a big challenge is blood flow in muscles. Since, at rest, blood flow is extremely low and only on exercise does it rise considerably, methods of increasing blood flow through muscles might be profitably studied. They seem to be under separate nervous control, as has been shown by the effects of stimulating various parts of the central nervous system [65], and the fact that very few of the currently used drugs seem to have very much effect on muscle blood flow, suggests that the pharmacology of these parts of the brain again could be worth study. COMMENT

I hope in this survey of various problems in the field of therapeutics, I have shown that there are many fields in which consideration of the basic mechanisms, both of the disease and of the known therapeutic agents, raises questions which it would be fruitful to answer. I suppose that the main message I would wish to convey is that perhaps we do not always need new drugs, useful though they may be, but rather closer study of those we already have. This carries important implications in terms of the organisation of such research as between industry and medicine. REFERENCES

N. W . Pirie, Science, 1966, 152, 1701 N. W . Pirie, Sci. American, 1967, 216, 27 R. G. Potter, R. Freedman and L. P. Chow, Science, 1968, 160, 848 J. Walsh,Science, 1968, 160, 1318 W . Sargant and P. Dally, Brit. Med. J., 1962, 1,.6 M. E. Jarvik, The Pharmacological Basis of Therapeutics, 3rd edn. (Ed. L. S. Goodman, and A. Gilman), Macmillan Co., New York, 1965, p. 159 7. M. N. McGuiness and C. M. B. Pare, Hosp. Med., 1968, 2, 1148 8. F. J. Ayd, J. Neuropsychiat., 1962, 3 , 177 9. R. Doll and A. B. Hill, Brit. Med. J., 1964, 1, 1399 and 1460 10. E. C. Hammond, Nat. Cancer Inst. Monogr., 1966, 19, 127

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

W. S. PEART 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54.

227

J. L. Haybittle, Brit. J. Prev. SOC.Med., 1966, 20, 101 Obesity and Disease, Office of Health Economics, London, 1969 C. C. Seltzer, New Engl. J. Med., 1966, 274, 254 The National Diet-Heart Study, American Heart Association Monograph 18, Circulation, 1968, 37, Suppl. 1. J. Mayer, New Engl. J. Med., 1966, 274, 610 J. Mayer, New Engl. J. Med., 1966, 274, 662 J. Mayer, New Engl. J. Med., 1966, 274, 722 L. A. Lewis, R. P. Turnbull and I. H. Page, Arch. Intern. Med., 1966, 11 7,4 R. Y. Calne, Renal Transplantation, Williams & Wilkins Company, Baltimore, 1963 R. H. Travis and G. Sayers, The Pharmacological Basis of Therapeutics, 3rd edn. (Ed. L. S. Goodman, and A. Gilman), Macmillan Co., New York, 1965, p. 1608 W. Graham, Can. Med. Ass. J., 1958, 79, 634 A. M. Katz, C. M. Pearson and J. M. Kennedy, Clin. Pharmacol. Ther., 1965, 6 , 25 J. P. Jones, E. P. Engleman, H. L. Steinbach, W. R. Murray and 0. N. Rambo, Arthrit. Rheum., 1965, 8 , 449 T. Lewis, The Blood Vessels of the Human Skin and Their Responses, Shaw & Son, London, 1927 T. Lewis, Vascular Disorders of the Limbs, 2nd edn., Macmillan, London, 1946 J. H. Peacock, Circulation Res., 1959, 7, 821 J. H. Peacock, C. Shaldon, C.'Tyler and F. E. Badrick, Lancet, 1962, ii, 1077 R. W. Gifford, E. A. Hines and W. McK. Craig, Circulation, 1958, 17, 5 C. W. Robertson and R. H. Smithwick, New Engl. J. Med., 1951, 245, 317 J. H. Peacock, Lancet, 1960, ii, 65 M. S. Kleckner, E. V. Allen and K. Wakim, Circulation, 1951, 3, 681 F. R. B&ny and E. H. Cooper, Clin. Sci., 1957, 16, 275 E. R. Unanue and F. J. Dixon, Advan. Immunol., 1967, 6, 1 C. S. Ogg, J. S. Cameron and R. H. R. White, Lancet, 1968, ii, 78 H. J. Miiller-Eberhard,Advan. Immunol., 1968, 8, 1 R. J. Pickering, H. Gewurz and J. R. Kelly, Clin. Exp. Immunol.. 1968, 3,423 E. H. Kass, Biology of Pyelonephritis, Henry Ford Hospital Int. Symposium (Ed. by E. L. Quinn and E. H. Kass), J. & A. Churchill Ltd., London, 1960 A. Percival, W. Brumfitt and J. d e Louvois, Lancet, 1964, ii, 1027 J. V . Scudi and J. F. Reinhard, J. Lab. Clin. Med., 1948, 33, 1304 Advances in Transplantation, Proc. 1st Intern. Congr. Transplantation Society, Paris, 27-30 June 1967 (Ed. by J. Dausset, J. Hamburger, and G. Mathd), Munksgaard, Copenhagen, 1968 J. F. A. P. Miller, Lancet, 1961, ii, 748 I. F. A. P. Miller, Proc. R o y . Soc. B., 1962, 156, 415 R. E. Gleason and J. E. Murray, Transplantation, 1967, 5, 360 E. M. Lance and P. Medawar, Lancet, 1968, i, 1174 T. E. Starzl, C. G. Groth, P. 1. Terasaki, C. W. Putnam, L. Brettschneider and T. L. Marchioro, Surg. Gynecol. Obstet., 1968, 126,4023 J. D. Broome,Nature, 1961, 191, 1114. W. C. Dolow, D. Henson, J. Cornet and H. Sellin, Cancer, N . Y . , 1966, 19, 1813 H. R. Gilmore, H. Kopelman, J. McMichael and 1. G. Milne, Lancet. 1952, ii, 898 L. Rokita and S. M. Sancetta, Circulat. Res., 1953, 1, 499 M. Hamilton, K. S. Henley and B. Morrison, Clin. Sci., 1954, 13, 225 P. A. Restall and F. H. Smirk, Brit. Heart J . , 1952, 14, 1 J. C. Rose and E. D. Freis,Amer. J. Physiol., 1957, 191, 283 G. W. Pickering, W. I. Cranston and M. A. Pears, The Treatment of Hypertension, Charles C. Thomas, Springfield, 196 1 R. A. Maxwell, A. J. Plummer, F. Schneider, H. Povalski and A. I. Daniel, J. Pharmacol. Exp. Ther., 1960, 128, 22

228 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65.

MEDICINAL CHEMISTRY FOR THE NEXT DECADF, R. A. Maxwell, A. J. Plummer, H. Povalski and F. Schneider, J. Pharmacol. Exp. Ther., 1960, 129, 24 C. T. Dollery, D. Emslie-Smith and J. McMichael, Lancet, 1960, i, 296 J. A. Oates, A. W. Seligmann, M. A. Clark, P. Rousseau and R. E. Lee, N e w Engl. J. Med., 1965,213,129 W. D. M. Paton and E. Zaimis, Brit. J. Pharmacol.. 1949,4, 381 J. Brod, Z. Hejl, A. Hornych, J. Jirka, V. Slechta and B. Burianova, Clin. Sci., 1969, 36, 161 E. P. Sharpey-Schafer, J. Physiol. (Lond.). 1956, 134, 1 E. P. Sharpey-Schafer and P. J. Taylor, Lancet, 1960, i, 559 T. Reich, J. Tuckman, A. F. Lyon, M. Mendlowitz and A. Jacobson, Surg. Forum, 1967, 18, 174 R. H. Johnson, G. J. Lee, D. R. Oppenheimer and J. M. K. Spalding, Quart. J. Med., 1966, 35, 276 R. Bannister, L. Ardill and P. Fentem, Brain, 1967, 90, 725 J . T. Shepherd, Physiology of the Circulation in Human Limbs in Health and Disease, W. B. Saunders Company, Philadelpha, 1963, Chapter 2

5 Analgesics and their Antagonists: Recent Developments A. F. CASY, B.Sc.,Ph.D., F.R.I.C., F.P.S. Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada. * MORPHINE DERIVATIVES

2 30

6,14-ENDOETHENOTETRAHY DROTHEBAINES

233

MORPHINANS AND BENZOMORPHANS

236

PHENYLPIPERIDINES, PY RROLIDINES AND RELATED COMPOUNDS

241

ACYCLIC AND MISCELLANEOUS CLASSES

24 I

ANTAGONISTS OF ANALGESICS

255

STEREOCHEMICAL ASPECTS AND RECEPTOR THEORIES

265

This review is intended as an extension of previous chapters on andgesics published in Volumes 2 and 4 of this series [ 1, 21 and chiefly covers literature of the period 1962-1969. During this time one comprehensive volume on analgesics [ 3 ] and two monographs dealing with morphinan and benzomorphan derivatives respectively (part of the Synthetic Analgesics series) [4, 51 have appeared. Reviews on analgesia and addiction [6] and on narcotic antagonists [7] are now available (the former includes a useful collection of hot-plate activity data in mice obtained under standardized conditions), while the annual reports in medicinal chemistry sponsored by the American Chemical Society have made the task of keeping upto-date in this ever proliferating area far easier [ 8 ] . Books on concepts of pain and its clinical measurement have also appeared [9] and the quantitative aspects of pain and analgesia are discussed in a recent Royal Society of Medicine supplement [ 101. The so-called ‘chemical anatomy’ of morphine-like analgesics is described in a new series devoted to medicinal research [ 1 11 . In this chapter, new material is classified and evaluated in terms of the more important structural groups of analgesics and analgesic antagonists. Although many biochemical aspects of analgesic action have been studied, results obtained continue to be complex and ill-defined [ 121 and no special section is devoted to this topic in the present review.

* Present address: School of Chemical Sciences, University of East Anglia, NORWICH NOR 88C. 229

230

ANALGESICS AND THEIR ANTAGONISTS

MORPHINE DERIVATIVES Relatively little work on novel morphine structures has been reported since the previous reviews in this series, although an improved route to morphine itself has been described [ 131 ; thebaine-adducts are considered separately below. B/C Trans-morphine (1) has been prepared [ 141 and shown to be about one fifteenth as active as morphine in the hot-plate test. T h s result is surprising in view of the enhanced activities of similar isomers of morphinan and benzomorphan analgesics. Nordihydrodeoxymorphine (EDs0 value of 2.2 mg/kg) is significantly active in mice by the hot-plate test after subcutaneous injection (cf. morphine, EDs0 value of 1.17 mg/kg) [ 151, a result which adds to the relatively sparse number of examples of analgesically-active secondary amines. Replacement of N-methyl by an amino group abolishes activity in the hot-plate test [ 161 . There is still interest in codeine, the best known variant of morphine, and an extensive review on its analgesic and antitussive effects has been made under the auspices of the World Health Organization [ 171 .

More information on 14hydroxy derivatives is now available. Both 14hydroxymorphine (EDso value of 2.9 mg/kg) and the same analogue of dihydromorphine (ED5,, value of 1.1 mg/kg) are moderately potent in mice [ 181 (cf. morphine EDs0 value of 2.1 mg/kg) while the 6-ethylene ketal (11) (the most active member of a series [ 191 ) is more potent than morphine in mice and rats after oral administration [ 201 . Demonstration of intramolecular hydrogen bonding in 14-hydroxycodeinone (111) and its dihydroanalogue [ 211 shows that the 14-OH group has. an axial configuration, and the close proximity of this substituent to the nitrogen atom in (Ill) may provide a clue to its potencyraising influence. Derived esters are active, some with very high potencies [22]. Thus in the esters (IV), Table 5.1, potency rises with increasing size of R, reaches a maximum when R is n-hexyl, and then diminishes. Benzoyl esters are inactive but phenacyl, cinnamoyl and related derivatives are highly effective. Transport factors are probably involved in these structure-activity relationships (the lipid solubility of the drug will rise as R in (IV) becomes larger) but conformational changes resulting from interactions between the 14-acyl group and the solvated basic centre may also be important.

23 1

A . F. CASY

Table 5.1. ANALGESIC ACTIVITIES OF SOME ESTERS OF 14-HYDROXYCODEINONE IN MICE (TAIL CLIP METHOD) [ 2 2 ]

IlVl

R

COMe COEt COPr COBu CO.CsH 11 CO.C6Hi3 CO.C,H,S CO.C9H 19 co.C1 1 H23 CO.CH2Ph CO.CH2CH2Ph CO.CH = CHPh CO.CH = CHMe

Relative activity (morphine = 1) 4

'

18.8 28.7 38.8 47.2 60.1 5.1 1.1 0.03 52 115 177 31

The methoxymethyl group effectively masks the morphine phenolic function (see (V)), a result which indicates that the chemical lability of such ethers is not reflected in uivo [23]. The same blocking group has been usefully employed in an improved synthesis [24] of 6-methylenedihydrodesoxymorphine (VI). The pharmacology of morphine methochloride originally reported [25] in 1868 has been further studied in rats [26]. The effects of morphine and its quaternary salt are quite different when the drugs are given systematically, the methochloride having curare-like actions (it causes neuromuscular paralysis) rather

232

ANALGESICS AND THEIR ANTAGONISTS

than analgesic actions. However, after intracerebral injection (i.c.), both drugs produce analgesia and have similar potencies, a result which shows that the absence of morphine-like properties in the methochloride is due to the failure of the completely ionized quaternary salt to penetrate the C.N.S. The low potency of morphine-N-oxide after subcutaneous and intraperitoneal administration [ 271 probably arises from the same cause. It is to be hoped that more studies of analgesics after i.c. administration will be made, since this route, which largely obviates distribution differences, allows more meaningful structure-activity relationships to be formulated. Me ED50 values in mice

R -

$& f

R

RO

H. 1 1 7 m g l k g MeOCH2, 28 8 m g / k g

(V) Me

EDSOvalues in r a t s 0 O L 5 mg/kg (morphine 1 0 2 m g l k g HO

CH2

(VIJ

The proposal that the effects of heroin are mediated chiefly by its deacetylated metabolites, morphine and 6-monoacetylmorphine (MAM) [ 281 , and that heroin (and MAM) function primarily as carriers to facilitate morphine availability at C.N.S. receptor sites is supported by studies in new-born rats [29]. Whde rats show a pronounced increase in their resistance to morphine 16 days after birth , (probably associated with development of a blood-brain barrier), there is little change in toxicity to heroin with increasing age; hence ready access of heroin to the brain is concluded, even after the barrier has developed. Codeine and morphine are found to have reciprocal synergistic actions in the rat [ 3 0 ] .When a low (non-analgesic) dose of codeine is given with an analgesic dose of morphine, the mean hot-plate reaction times are found to be significantly longer than after morphine alone. A similar but more persistent effect is noted after codeine plus a low dose of morphine. Novel methods for the detection of nanogramme quantities of morphine have been reported. In one procedure morphine, after extraction from biological

A. F. CASY

233

fluids, is oxidized to pseudomorphine by ferricyanide and the fluorescence of the oxidized product determined in a filter fluorometer [ 3 1 ] . In another, recovered morphine is converted t o its trimethylsilylether which is then determined by gas-liquid chromatography [32] . Mass spectrometry is another procedure of promise to metabolic and distribution studies, and mass spectral data upon morphine, codeine and some related alkaloids are now available [33] .

6,14-ENDOETHENOTETRAHYDROTHEBAINES More details are now available upon analgesics obtained by Diels-Alder reactions between thebaine (the diene component) and dienophiles such as vinyl methyl ketone [34, 351. The extremely high potencies in animals [36] of many 6,14-endoethenotetrahydrothebainederivatives (V11) formed from the ketonic adducts and organo-metallic reagents has been confirmed [ 3 5 , 3 7 , 3 8 ] . Etorphine (VIIa), for example, is 8 5 0 (mice), 1 700 (rats) and 8 600 (guinea-pigs) times as active as morphine in the animals specified after subcutaneous administration [38]. Its analgesic action is morphine-like since it is competitively antagonized by nalorphine. The ability of this drug to cause catatonia at very low dose levels has led to its use for immobilizing large animals for game conservation and veterinary purposes [39, 401. In the dog etorphine ( 3 pg/kg) synergises with methotrinieprazine (2 mg/kg) t o cause a state of neuroleptanalgesia [ 4 1 ] . Other effects of etorphine are typically morphine-like; small doses cause Straub tails and mydriasis in mice while in rats and guinea-pigs the drug causes greater degrees of respiratory depression than morphine at equi-analgesic dose levels. Neonatal respiratory depression was not seen in the young rat delivered from mothers receiving analgesic doses of the drug by the sublingual route [ 4 2 ] . Mice rapidly acquired an acute tolerance t o the analgesic effects of etorphine [43]. The unusually high potencies of etorphine and related compounds may be due, in part at least, t o their rapid penetration of the C.N.S. and high ability t o concentrate at receptor sites (radically different CC14/H20partition coefficients for (Vllb) (> 2 000) and morphine (<0.001) have been reported [37]). Thus the peak effect of etorphine is seen one minute after intravenous administration in mice and rats, 5- 1 5 minutes being the corresponding figure for morphine. Further, while EDSo values for morphine and compounds of comparable potencies are in the milligramme range when conventional administration routes are employed, doses far closer to the thebaine adducts S.C. and i.v. EDso values are reGorted after intracerebra] and intraventricular routes, for example, morphine 25 pg (rats), (-)-methadone 7-9 p g and levorphanol 12 pg (in mice) [26, 441. Rat brain tissue (maternal or foetal) rapidly takes up tritium-labelled etorphine, the brain concentration being above that in circulating blood in as short a period as 10 minutes after intramuscular administration. Under the same conditions, the blood concentration of labelled dihydromorphine (of lower CC l 4 /Hq 0 partition coefficient) never exceeds brain levels [45] . Structure-activity relationships in these derivatives may be summarized as follows (figures in parenthesis are molar potencies in rats relative to morphine = 1) [35].

234

ANALGESICS AND THEIR ANTAGONISTS

(1) The free phenols (VII) and (VIII) (R = H) are far more potent than 0-methylated analogues, as in the morphine, morphinan and benzomorphan series, for example, (VIIa) (3 200); (VIIa) R = Me (96). (2) Potency is greatly influenced by the size of C-7 t-alcohol substituent. As the R' chain length in the series (VII) (R = RZ = Me) is increased, activity rises to reach a maximum at Pr" (96) and then declines; benzyl(l50) and phenethyl (500) congeners are particularly potent. In the phenolic series (VII) (R = H, R2 = Me) highest activities are conferred by Bun ( 5 200) and iso-C5H1 (9 200) alkyl substituents, phenethyl (2 200) and cyclohexyl (3 400) derivatives also being amongst the more potent. The geometry of the t-alcohol substituent is also important as seen by the differing activities (89 versus 0.7) of the diastereoisomeric alcohols (VIIa) (R = Me) [36]. The probability of the OH group binding to the receptor, indicated by these results, is supported by the fact that activity losses occur when the t-alcohols (VIII) are dehydrated. (3) Reduction of the 6,14-endo-etheno bridge further enhances potency, for example (VIIa) ( 3 200) and (VIIIa) (1 1 000), the latter alcohol being the most potent analgesic so far reported. (4) In several frep phenols WII) (R' = Me to C s H l l ) , activity decreases steadily as the N-substituent is increased in size from Me to n-hexyl (in corresponding morphinan and benzomorphans activity is lowest for Pr" but rises when the size of the N-substituent is further increased) [4, 51. The influence of N-phenethyl (and related arylalkyl groups) is not yet available but should be of interest because replacement of N-Me by this function in other fused-ring analgesics uniformly yields more potent derivatives. N-Substituents which confer antagonistic properties on morphine, only do so in the thebaines (VII) when the chain length of the t-alcohol substituent R' is short. The base (VIIc) is a highly active competitive antagonist of morphine, having a pAz value of 8.2 (cf. nalorphine 5.6) [38]. The congener (VIIb), with a longer chain R' group, is a potent analgesic in rats [ 35, 361 although showing certain nalorphine-like side-effects; its analgesic effects, when fully developed are not antagonized by nalorphine. Replacement of N-methyl in etorphine (VIla) by N-allyl, giving (VIIe), results in a potency fall but the product still has pronounced analgesic properties (potencies over 100 times that of morphine are found in animals) [46] . The claim that the depression of respiration seen after administration of ( W e ) is significantly less

A. F. CASY

R

R'

RZ

235

Desimation

H

Pr

Me

Etorphine M99

H H

C5Hll Me

CHzC3H5* C H Z C ~ *H ~

M320 Cyprenorphine, M285

OCOMe

Pr

Me

Acetorphine, M183

H

pr

C3H5t

H

C 6 H l l $ Me

R & S 218-M M125

* cyclopropylmethyl;

t ally1

$ cyclohexyl

than that after etorphine or morphine at equi-analgesic doses, has been challenged by Bousfield and Rees [47] from experiments in which analgesia and respiratory depression were measured concurrently in the same animal. By conventional tests, secondary amine analogues of 4-phenylpiperidine analgesics lack activity (normorphine is equi-potent with morphine by the intracisternal route [48] ); many secondary bases of the thebaines,(VII), however, are morphine-like analgesics. These findings lend support to the view that the low potencies reported for many nor-analogues of analgesics are due to distribution factors and not to the failure of such derivatives to associate at the receptor. Bentley hss summarized the structure-activity relationships of 6,14-endoethenotetrahydrothebaines in papers presented at the 1967 and 1968 meetings of the Committee on Problems of Drug Dependence of the National Academy of Sciences (Washington, D.C.) and speculated upon factors governing the uptake of these analgesics at the receptor. It is to be hoped that these summaries will be published in a more generally available source. A crystallographic study of (VIIa) (R = Me) has confirmed the overall structural resemblance of the endo-ethenothebaines and morphine [49] ; the former may be regarded as morphine derivatives in which the 6 and 14 carbon atoms of ring C are bridged by a bimethylene chain. T h s bicyclic feature lies ?-OH A ' M e

*

Figure 5. I

largely behind the plane containing the nitrogen atom and the aromatic ring A (Figure 5.1).If the C-7 t-alcohol substituent binds to the receptor surface (which

236

ANALGESICS A N D THEIR ANTAGONISTS

must then be assumed to be more extensive than originally proposed) [2] the high activities of the thebaines (VII) and (VIII) may be accounted for, not only by distribution factors, but also by their being more acceptable to the receptor than morphine and like agonists [35]. The stereochemistry of some thebainedienophile adducts and of some diastereoisomeric alcohols of type (VII) has been elucidated by an elegant application of PMR spectroscopy [SO] . Little clinical data is available on these compounds. Compound (VIId) (60 pg) is as effective as morphine (10 mg) in patients with recent thoracotomy [51] and has a quick onset and short duration of action in male volunteers [52]. It is, however, a more potent respiratory depressant than morphine in equianalgesic doses and produces a high incidence of nausea and vomiting. The morphineantagonist (VlIc) ( 1 mg/70 kg) produced a greater degree of analgesia than a placebo in post-operative patients but induced psychotomimetic effects akin to those associated with nalorphine [53]. In monkeys, the physical dependence capacity of the thebaine derivatives is high [54]. The N-demethylation pathway is common to the metabolism of M125 (VIIf') and morphine, both drugs being converted to secondary amines by liver microsomes under the same condition [ S S ] . Reaction rates were depressed when microsomes from rats repeatedly treated with M125 or morphine were used. In the same work, rats wereeshown to become tolerant to the pharmacological effects of M125 (shock avoidance and tail clip tests) and cross tolerance to morphine was also found.

MORPHINANS AND BENZOMORPHANS Little new work on analgesically active niorphinan derivatives has appeared recently. Gates and Klein [ 5 6 ] have shown that a shift in the ethanamine bridge terminus of the morphinans from C-13 to (2-14, giving metamorphinans (IX),

R=Me

or C%-c-C3H5

abolishes the characteristic analgesic (IX, R = Me) and antagonistic (IX, R = CH2c-C3H5) activity of corresponding morphinans.

A . F. CASY

231

May's group are still actively working in the benzomorphan field. Their recent endeavours have centred on the isolation of both a- and pdiastereoisomers of the benzomorphans (X) and upon variations of the 5 and 9 alkyl substituents.

a- cis R' /R2

0-trans R'/R2

HO IX )

with respect to ring B

There is good evidence that the a-forms (X, R' = R2 = Me) have a cis and the 0 a trans 5,9-dimethyl configuration with respect to the hydroaromatic ring B (with the piperidine ring as reference, (a- is the pans and 0-the cis isomer). The rates of quaternization of a- are faster than those of the 0-isomers, reaction in the latter being hindered by the 9-methyl group (cf. (XI) and (XI); in addition,

Me

H

8-form

a-form

(XI)

(XI11

the a-9-methyl group has a higher PMR chemical shift than the 0-group because it lies within the diamagnetic screening zone of the aromatic group [57]. A recent X-ray analysis of the a-N-ally1 derivative (X) (R = CH2-CH = CH2, R' = R2 = Me) supports these assignments [58]. Differences in the PMR characteristics of hydrohalide and methiodide salts of a- and 0-isomeric benzomorphans have been interpreted in terms of differing conformations of the piperidine moiety (ring C) [59]. Without exception, 0-diastereoisomers are substantially more potent than the a-forms in spite of their stereochemistry differing from that of morphine

238

ANALGESICS AND THEIR ANTAGONISTS

(Table 5.2 cf. pairs 1-2, 3-4, etc.). Isomorphinan derivatives (trans series) are likewise more potent than corresponding cis morphinans [60] . Potency differences among diastereoisomeric benzomorphans with antagonist properties are less pronounced (see later) [61]. The influence of the R' and RZ chain length in (X) (R = Me) upon activity differs somewhat in the two series. In a-isomers, little change occurs when R' (C-5) is increased from Me to Pr (Table 5.2 Nos. 5, 7 and 15) or RZ (C-9) from Me to Et (Table 5.2 Nos. 5 and 9); when R2 is Pr, however (Table 5.2, No. 13), most activity is lost. All the 0-isomers (R' and R2 = Me, Et or Pr) are potent compounds (Table 5.2, Nos. 6 , 8, 10, 12, 14 and 16). Table 5.2. ANALGESIC ACTIVITIES OF SOME (f)-5,9-DIALKY L-2-METHYL BENZOMORPHANS IN MICE (HOT-PLATE METHOD)

p'Me B--R2 ,....R>

R

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

H H H H HO HO HO HO HO HO HO HO HO HO HO HO H H

a

Me Me Et Et Me Me Et Et Me Me Et Et Pr

Me Me Et Et Me Me Me Me Et Et Et Et Pr

Pr

Pr

fl

Pr Pr Pr Pr

Me Me Me Me

U

0 Q 0 a 0 a 0 0.

fl U

0 a

0

0 U

27.3 8.9 5.0 4.2 3.0 0.44 4.9 0.07 1.5 0.47 4.2 0.28 71.2 0.87 2.9 0.12* 4.8* 11.5*

-

37.1 36.7 38.6 23.9 8.2 31.7 1.1 14.8 17.2 -

6.5

72.1 4.5*

5 68 62 62 68 5,68 57,68 57,68 57,68 57,68 5 5 68 68 68 63 64 64

* determined with CDGP mice (morphine EDSo 1.2 mglkg s.c.) [ 7 1 ] ; all other results refer t o GP mice (morphine EDs0 2.1 m d k g s.c., 3.7 mg/kg oral).

0-Derivatives with a C-5 ethyl or propyl group (8 and 12) are particularly potent and these substituents can partly compensate for the lack of a phenolic hydroxyl function, normally a prerequisite for high activity (Table 5.2. cf. Nos. 3 , 4 and 17). When RZ in metazocine (a- (X), R = R' = RZ = Me) is replaced by hydrogen,

A . F. CASY

239

a threefold activity decrease results [65, 661 but the same analogues with R' = Et (EDs0 value of 1.5 mg/kg) [ 6 7 ] , Pr (ED50 2.1) [68], Bu (EDs0 2.0) and CSH I 1 (ED50 3.4) have morphine-like potencies while the hexyl analogue (EDSO 10.8) is significantly weaker [67-691. The compound (X) (R = Me, R' = R2 = H), a derivative of the unsubstituted parent nucleus of the series, has a potency (EDs0 value of 4.5 mg/kg) similar t o that of the corresponding 5-methyl homologue (EDs0 3.3 mg/kg). Removal of the 9-methyl group is more detrimental t o activity (as mentioned above) and these results, coupled with the influence of 9-methyl geometry on activity in a-0 pairs, suggest that 9-methyl is the more important pharmacodynamic feature of the molecule. The unsubstituted parent nucleus itself (X, R = R' = R2 = H, 2-OH replaced by H) is about one-ninth as active as morphine [70] . A 2'-hydroxyl group significantly enhances activity in a- and p-5, 9-dimethyl and diethylbenzomorphans (X) (R = Me), but 2'-amino, chloro, fluoro and nitro substituents reduce potency [71]. In metazocine (a-X, R = R' = R2 = Me), activity is lost when R is replaced by Et, Pr and Bu (the Pr derivative is a nalorphine-like antagonist [ 7 2 ] ) but is restored byN-CSHl (EDs0 2.1) andN-hexyl (EDs0 1.5) [69]. The advantageous effect of replacing N-Me in metazocine by 2-phenethyl (giving phenazocine, narphen), is well-known and other N-aralkyl groups also raise potency [73] (see XlII), paralleling results of the same changes in morphinan analgesics [ 4 ] . The R

EDs0 values (mg/kg)

Me 3.0 CHzCH2Ph 0.25 C H ~ C H ~ C ~ H ~ P - N H ~ 0.1 1 CH, CH2-2-thienyl 0.055

f g T -

'Me

HO

(XI11 1

compounds (XIII) (R = ally1 or CH2c-C3H 5 ) are potent nalorphine-like antagonists and these, together with the N-2, 2-dimethylally1 derivative (pentazocine), are discussed under antagonists [72]. Triggle's group [74] prepared some N-(2-bromoethyl) analogues of a-metazocine with a view to determining whether agents capable of forming covalent bonds with anionic binding sites (e.g. a carboxylate or phosphate anion) might prove effective analgesics, an idea which derives from studies of the mode of action of adrenergc blocking agents such as dibenamine. Although bromoethyl derivatives were more effective than the related (non-alkylating) hydroxyethyl compounds in two cases (hot.plate test in mice), the significance of this result could not be assessed because the halogen formshad a prolonged depressant action not seen in the hydroxyethyl derivatives or in a-metazocine. A 9-hydroxy substituent reduces the activity of 5, 9dimethylbenzomorphan derivatives, the influence of a 0-group (axial 9-Me as in XI) being the more detrimental. The di-0-acetyl derivative ofXI1 (axial 9.OH) however, is twice as active as morphine while the same ester of the 2, 5-dimethyl congener (9-Me absent) is, surprisingly, even more effective [75].

240

ANALGESICS AND THEIR ANTAGONISTS

Many benzomorphan derivatives with morphine-like potencies or better have a low physical dependence capacity (PDC) in monkeys; especially potent members, however, such as phenazocine and the 0-isomers (Table 5.2, Nos. 8 and 16) have high to intermediate PDC properties [S, 63, 681. Unusual results have been reported-for the enantiomorphs of a-(X) (R = Me, R' = RZ = Et); the (+)-isomer, although only of codeine-like potency has an intermediate PDC, whereas high doses of the potent laevo isomer completely fail to suppress abstinence in withdrawn monkeys [76]. The same isomer behaves as an analgesicantagonist (in spite of its N-methyl function) since it precipitates mild abstinence symptoms in non-withdrawn animals. Similar results are obtained for enantiomorphs of the corresponding 5-propyl-9-methyl racemate. The 5, 9-diethyl laevo isomer has the same absolute configuration as morphine since it has been synthesized from the dicarboxylic acid (XIV) derived from natural thebaine [77]. T h s result, together with ORD [78] and stereoselective absorbent data [79] , establish the configurational identity of laevo isomers of morphine, morphinan and 5,9-dimethylbenzomorphan derivatives at their common centres of asymmetry. ORD results further establish the Cs configurational identity of the laevo isomers of a- and 0-metazocine [79]. The Japanese workers also prepared (-)-3-hydroxy-6, N-dimethyl-C-normorphinan (XV) and the 6-0x0isomorphinan (XVI) from thebaine [80, 811 ; the former is nineteen and the latter twelve times as potent as morphine in rats by the tail-flick test.

f$$Y

C02 H C02 H

Me0

OPh

fgEie HO

(XIV)

0

HO

( X VI

(XVII

There has been recent interest in 5-phenylbenzomorphans since such derivatives may be thought to combine the diphenylquaternary carbon feature of methadone with the 4-phenylpiperidine unit. The derivatives XVIIa displayed

(XVII)

only weak activity in the tail-flick test [82], but the racemic 9-methyl-5-phenyl compoupd (XVIII) (there is evidence that the 9-Me is axial) [8b] is about twice as active as morphine in mice (EDs0 value of 0.51 mgJkg) [54], as is the laevo

A . F. CASY

24 1

isomer in man [53]. The related N-carboxamide (XVIlb) is claimed to be an orally effective analgesic (40 mg = 6 0 mg codeine in post-operative patients) [83] but its morphine-like classification is doubtful as it lacks a basic centre.

Hd (XVIII)

Since 1963, hot-plate assays at the National Institutes of Health, Bethesda, have been performed upon Caesarian Derived General Purpose (CDGP) mice; these animals are healthier and faster growing than the previously used General Purpose (GP) mice, and are about twice as sensitive to analgesic drugs, for example morphine EDs0 values (mg/kg): 2.1 (GP) and 1.2 (CDGP) mice; pethidine: 9.9 (GP) and 4.7 (CDGP) mice [71].

PHENYLPIPERIDINES. PYRROLlDlNES AND RELATED COMPOUNDS New derivatives of pethidine (XIX) (R = Me) and its reversed ester in which the Nmethyl group is replaced by other functions continue to be reported (Table 5.3). N-substituents such as PhCO(CH2)2 (Mannich ketones), PHCHOH(CH2)2 and PhNH(CH2)2 which are known to yield potent pethidine analogues [ 11 also give highly active reversed esters (Table 5.3, Nos. 6 , 9 and 12). The potency raising influence of a 3-methyl ring substituent, as in N-methyl and N-phenethyl reversed esters [ 1, 901 is not seen in the Mannich base analogues (Table 5.3, Nos. 6 and 7). The p-anilinoethyl derivative (Table 5.3, No. 12) related to piminodine (XIX, R = PhNH(CH2)3) has a much reduced activity when the anilino atom is tertiary (Table 5.3, Nos. 13 and 14). Compound 9 (Table 5.3), the most potent member of this series gives a diester (XX, R = PhCH(OCOEt)(CH,),) of similar potency [9 11 . Its pethidine counterpart, phenoperidine, has been used clinically, for example in thoracic surgery [92] and neuro1eptanalge:ia [93], and its optical resolution reported. The R-(+)-enantiomer (XIX, R = PhCH(OH)(CH2)2), although four times less active than the laevo isomer is, nevertheless, seven times more potent than morphine in mice [94]. Some labile amide derivatives of norpethidine have been tested in the expectation that they might hydrolyse readily to norpethidine in the CNS; the carbonate (XIX, R = C0,Et) and pethidine were equipotent orally in rats (hot wire-tail test) while the monosuccinimide (XIX, R = CO(CH2)2C02H)and pyruvamide (XIX, R = COCOMe) were inactive in mice by the hot plate test [95]. Linkage of norpethidine and its reversed ester t o the dibenzocycloheptene nucleus (the latter structure is the basis of psychotropic agents such as amitriptyline) has led to several compounds which show

242

ANALGESICS AND 'I'HEIK AN'I'AtiONISI'S Table 5.3. ANALGESIC ACTIVITIES OF SOME N-SUBSTITUTED NORPETHIDINES AND CORRESPONDING REVERSED ESTERS

No. 1

(XW

9 10 11 12 13 14

PhNHCCH2CH2

II

(XX)

0 PhCHOHCH2 C4H70t-(CH2)2 C4HvOt-(CH2)3 C5HllN2*-(CH2)2 PhCOCH2 C H 2 PhCO.CH2.CH2 (3-Me analogue) PhCCH2CH2

(XX) (XX) (XX) (XX) (XX) (XX)

NOH PhCHOHCH2CH2 PhSCH2CH2 PhSO.CH 2 .CH2 PhNH.CH2CH 2 PHNMeCHZCHZ PhNEtCH2CH2

(XIX) (XW (XIX) (XIX) (XX) (XX) 8

R

Structure

II

Activity (pethidine = 1)*

Ref:

8 x codeine in mice

84

I in mice 28 in rats 26 in rats 0.6 in mice 1346 in rats 893 in rats

85 86 86 87 88 88

314 in rats

88

3219 in rats 2.1 43 1301 15 4

88 88 88 88 89 89

* unless otherwise stated.

t C 4 H 7 0 = 2-tetrahydrofuryl C5 H

N2

=

4-methylpiperazino

morphine-like potencies in animal tests for analgesia. One of the more potent derivatives is the reversed ester (XXa) (five times morphine in the hot plate test), the 4-piperidinol analogue being devoid of activity [96]. Incorporation of the adamantyl moiety into the ester function of pethidine, giving (XXb), is claimed to be advantageous both in terms of potency and duration of action [971. Normally t-alcohols corresponding to reversed ester analgesics are inactive [2] . The free alcohol (XXIa) however, isolated during attempts to prepare the 0-propionyl ester, is highly potent in rats and its activity is, in fact, reduced on

A. F. CASY

24 3

Me (XXb)

(XXa)

I

(CH2)2*yPh COEt

Activity (pethidine:l) (a) R = O H , 5 0 ( b ) R=OCOEt,3 (c)R=H.ll

(XXI

esterification (cf. XXJa and b) [98]. N-Acetyl (6 X pethidine), N-butanoyl (32 X pethidine) and 3-methyl (12 X pethidine) analogues of (XXIa) and the 4-phenylpiperidine (XXIc) (4-OH removed) are all significantly active. Even higher potency levels are reached in the branched chain congeners (XXIIa-b) the activity of the former being particularly noteworthy [99]. Analogues of (XXIIa) and (XXIIb) with OH replaced by hydrogen are less active, but still

I R

Activity in mice (morphine=l) (a) CHzCHMe,N(COEt)Ph 150 (b) CHMeCH z.N(COEt)Ph 20 (c) CHzCHMe.N(COzEt)Ph 35 (d) CHzCHMe.N(CO-2-furyI)Ph 70

(XXII)

superior in potency t o morphine. Replacement of N-phenyl by benzyl in (XXIIb) reduces activity to half that of morphine. All the active derivatives are antagonized by nalorphine and mice develop tolerance towards their action. The physical dependence capacity (PDC) of (XXIIa) (OH replaced by H, 15 X morphine) in addicted monkeys is high. Structure-activity relationships in these N-substituted propionandides resemble those of acyclic basic anilides such as diampromide (XXIII), rather than reversed esters 6f pethidine, and the derivaMe

I

Ph I

Ph(CH,),.NCHMeCH,N.COEt

(XXIII)

tives (XXI) and (XXII) seem to be more appropriately classified with the former. On this basis the entire 4-phenylpiperidin-4-01 moiety (and modifications) serves as the basic unit with N-phenyl rather than N-piperidylphenyl as the prime aromatic feature of the molecule.

244

ANALGESICS AND THEIR ANTAGONISTS

The morphine-like potency [ 1001 of the 4-(2-furyl) ether (XXIV) raises the question of the general acceptability or otherwise of a 4-alkoxy group as the C-4 oxygen function in 4-phenylpiperidine analgesics. Comparison of the relative

Ar

%Me

NI (CH,):,

Ph

OEt

0

I (CH2)2.COPh

Ar Activity in mice

(a) Ph

4 x pethidine ( b ) 2-fury1 1.6 x pethidine

(XXV)

(XXIV)

activities of several 4-phenyl and 4(2-furyl) pairs such as (XXVa) and (XXVb) shows that the heteroaryl group is not an essential feature of active 4-alkoxy-4(2-furyl) piperidines [ 1011 . Hence, 4-alkoxy groups fulfil structural requirements for activity in Qphenylpiperidines although not so effectively as 4-acyloxy functions (cf. XXVIa, b and c) [ 1011.

8

I (CH2)2Ph

R Activity in mice (pethidine=l)

(a) OEt (b)OCOMe (c) OCOEt

0.6 5.7 17

(XXVI1

A route to 3-methyl analogues of (XXVIa) is now available [ 1021 and should provide some potent ethers because the activity of (XXIV) depends greatly on the 3-substituent [ 1001. Some 3-phenylpiperidine derivatives with significant analgesic activities have been reported [103]. The most active members, for example, (XXVIIa and b),

OH I

CH2.COPh

Hot-plate EDSOvalues (mg/kg) in mice (a) R = H 12.1 (pethidine 12.5) (b) R=Me 7.15 (morphine 4.12)

(XXVII)

have N-phenacyl or phenethyl substituents and their action is antagonized by N-ally1 congeners; the latter also antagonize morphine, a result in contrast with the properties of N-ally1 analogues of pethidine and its reversed ester [lo41 discussed later. The compound (XXVIIb) had no PDC in monkeys over the dose range 2- I6 mg/kg. The pyrrolidine derivative (XXVIII) (profadol), the optimum

A. F. CASY

245

structure of anew series related to prodilidine (XXIX) closely resembles (XXVIIa) [105]. Its analgesic potency in rats, assessed by an antinociceptive test, is over

I

1

Me

Me

(profadol)

(prodi Lid ine)

(XXVII 11

twice that of codeine (the laevo isomer is somewhat more active than the racemate and dextro enantiomorph by the intraperitoneal route) and its action in this test is antagonized by nalorphine [ 1061 . It has no PDC in monkeys and, indeed, unmasks physical dependence in these test animals, an unexpected result in view of its apparent analgesic properties [107, 1081. In patients with cancer, profadol proved to be about one fourth as potent as morphine and produced similar side effects [ 1071 . Azetidine analogues of profadol have been studied,

goHQ

MeN

QNCOEt

R ED50 in mice (mg/kg) (CHI)* COPh (CH2)zPh

0.5 2.0

Morphine

1.7

R

(XXVLII a)

(XXIXa)

the derivative (XXVIIIa) having a potency estimated as 2.4 times that of codeine in rats [ 1091. Higher potencies were seen in certain 3-pyrrolidinylanilides (see XXIXa) which combine features of both profadol and diampromid (XXIII) [110]. A study of ring contraction and expansion upon activity in reversed esters of pethidine together with other data show that analgesic properties are retained (although in reduced degrees) in 7-membered ring analogues of active piperidine derivatives but are absent or weak in 5-member congeners [ 1 1 11. Ph

I

(CH,lz Ph

(ml The azacycloheptane (XXX), the most active non-piperidine derivative of the reversed ester series (7 X pethidine), was a typical morphine-like analgesic; it

246

ANALGESICS A N D THEIR ANTAGONISTS

produced Straub tails and was antagonized by nalorphine, and tolerance developed towards its effects in mice. Reduction of Schiff base (XXXI) formed between 1-phenethyl-4-piperidone and aniline, and acylation of the resultant dibase gives the highly potent analgesic [ 1 121 fentanyl (Sublimaze,XXXIIa). In mice (tail-clip method), fentanyl Ph

(q

Gh COEt

1 LAH 2 (EtC0120

(a1 R

(b) R

(0R

N

(CH2)2Ph Me

-H =

R

(XXXI I I is almost 200 times as active as morphine by the subcutaneous route and has a faster onset and shorter duration of action than the standard drug. It shows the usual morphine-like effects namely, Straub tails, mydriasis and constipation in mice; in dogs and cats it causes respiratory depression but is devoid of emetic action [ l 131. A clinical study in post-surgical patients and healthy volunteers showed 0.2 mg fentanyl to be equianalgesic with 10 mg morphine (intramuscular route) but the two drugs had similar side-effects [ 1141 . The respiratory depressant action of fentanyl in healthy males is judged to be somewhat greater than that of pethidine at equianalgesic dose levels [ I I S ] . Its use in neuroleptanalgesia, an anaesthetic technique in which a mixture of a potent narcotic analgesic and a tranquilizer are given intravenously usually as an adjunct to nitrous oxide, has been advocated [93, 1161. One combination studied, Innovan, is a mixture of fentanyl and the butyrophenone tranquilizer droperidol (Droleptan, XXXIII). The latter has no analgesic effect in mice but potentiates the action of fentanyl [ 1 171 . The N-methyl analogue of fentanyl H

COEt H

I

N'CH2Ph

(XXXIV)

(XXXIII)

(XXXIIb) has a radically lower potency than the parent (hot plate test in mice: N-Me derivative, inactive at 100 mg/kg; fentanyl, EDs0 0.01 mg/kg [ 1181). In this respect fentanyl is allied t o acyclic basic anilides such as diampromid but it

A . F . CASY

24 7

is safer t o regard it as representing a distinct class of analgesic [ 1 181 . The drastic activity fall consequent upon insertion of a methylene group between the amido nitrogen and phenyl in (XXIIb) is mirrored in fentanyl and its N-benzyl analogue (XXXIV), the latter being only 2.3 times as active as pethidine [ 1 191 . Fentanyl is metabolized in the rat by oxidative dealkylation, the secondary amine (XXXIIc) being the major product isolated from urine and faeces apart from the unchanged drug [ 1201. The quinolizidine analogue of pethidine is about half as potent as the parent compound in mice by the tail-clip test [ 120a] ; the same analogues of the reversed ester of pethidine are discussed later ( p . 2 7 5 ) .

ACYCLIC AND MISCELLANEOUS CLASSES A number o f novel structures containing a quaternary diphenyl carbon centre, as in methadone, have been described. Certain basic amides (XXXV) of U-ethylbenzilic acid are active by mouth in mice and rats. The most active members are somewhat more potent than pethidine and carry 0-arylethylamino N-substituents such as phenethyl and 2- (or 4-) pyridylethyl. Detailed pharmacoOEt Me I 1 Ph2 C C . N C H 2 .CH2 .NMeR

I/

0

(XXXV)

logy of the derivative (XXXV) (R = CH2CH2Ph), etomide, is available; orally, it is one-third more potent than pethidine in mice (tail-flick test) and equipotent with codeine and propoxyphene [ 1221 . Structurally, the amides (XXXV) bear some resemblance to diampromid and other basic anilides but are best considered as mild analgesics since their effective oral dose in man is high ( 1 50 mg) [ 12 I ] . The respiratory effects of 125 mg etomide given orally are judged equivalent to those of 100 mg codeine in healthy males [ 1231. The related benzilic ether (XXXVI) has its basic feature in an ester function and is claimed to be five times as potent as pethidine [ 1241 . The derivative (XX)tVII) in which part of the

methadone side-chain has been incorporated into a piperidine ring, is also reported as a potent analgesic [ 1251 .

ANALGESICSA N D T H E I R ANTAGONISTS

248

Janssen has linked the cyanide precursor of normethadone to norpethidine to produce diphenoxylate (XXXVIII); this complex is not an analgesic but has the

IXXXVIII)

constipating action of morphine derivatives and is used as an antidiarrhoeal agent [ 1261 . The related 4-aminocarboxamide (XLI) piritramide (pirinitramide), obtained from N-benzyl-4-piperidone (XXXIX) via the cyano-amine (XL) is an

cs

KCN p

w

I

I

CH2 Ph

CH2Ph

(XXXIXl

(XLl

analgesic however, and is twice as active as morphine in mice (hot-plate test); it causes Straub tails, excitement and mydriasis in these animals and is antagonized by nalorphine [ 1281 . In an extensive clinical evaluation lasting for nearly 5 years, piritramide (marketed in Sweden as Piridolan) effectively relieved pain in postoperative patients at dose levels of about 15 mg [ 1291 . It had a long duration of action and its side-effects were comparable with those of morphine; physical dependence and tolerance were not experienced. Several spiranes such as (XLIIa and b), obtained by condensing 4-anilino analogues of (XLI) with formamide [ 1301, have low hot-plate EDs0 values in U

-

-

(CH,),

N

X

J

R I

Ph

(xL"l

(a) R - Me (bJ R - COEt

Hot-plate

E D50(mg/ k g ) 0 29 0-6 '

mice; the examples given show no physical dependence capacity in monkeys and behave as powerful chlorpromazine-like sedatives in these animals [54] .

A . I:. CASY

249

Attempts to modify the ester substituent of the aminocyclobutane (XLIII) which has aspirin-like activity, resulted in ring-opening and led to the 6aminoketones (XLIV) [ 1311. Some of these ketones, notably (XLIV) ( R = Ph or

M

-

t

-

I

Me

cH .N hie /

Me K

C6H1 had tail-flick activities in the codeine-dextropropoxyphene potency range (oral and intraperitoneal routes in rats) and nalorphine antagonized the actions of both codeine and (XLIV) (R = C6 H I ) in this test [ 1321, A few nuclear-substituted variants of (+)-propoxyphene (XLV, Ar = Ph) have been reported. Para substituents (Cl, F. Me and OMe) in the 1-phenyl group reduce the activity of the acetoxy ester analogue of propoxyphene while o-F and Ph

I

ArCH,. C*CHMe.CH,- NMe,

I 0.COEt (XLV)

o-CI (but not @Me) groups enhance potency [133, 1341 (see XLVI). The

dW2-

f!CHMe- CH2.NMe2 0-COMe

(XLVI)

R

H CI r:

ED,, values (ing/kg) F.C. in mice 12 15 12

Me inactive (75) morphine [ 1351 12 p r o p o s y p h e n e [ 1351 66

stereochemistry of propoxyphene has been established [ 136, 1371 , the more active 2S:3R (+)-isomer having the same C-3 configuration as(-)-isornethadone and dextromoramide. In an excretion study in man, 25 per cent of a 65 mg dose of propoxyphene was recovered in the urine within 48 hours as a mixture of unchanged drug and a metabolite [138] ; the latter corresponds with the previously identified biotransformed product, de-N-methylpropoxphene [ 1391 . Propoxyphene plasma levels may be determined by a gas chromatography procedure in which the N-pyrrolidino analogue of the drug itself is used as a mass internal standard [ 1401 . Two types of cyclized propoxyphenes have been

250

ANALGESICS AND THEIR ANTAGONISTS

prepared; activity is lost in the piperidine analogue (XLVII) [ 1411 but is retained in tetrahydrodecalins (XLVIII). The racemate (XLVIII) (Ar = Ph, R = Et) is as Ph

O.COEt

Qph

a c H 2 . N M e 2 ArCH,

Me

O.COR

(XLVIII)

(XLVII)

potent as dextropropoxyphene while the corresponding acetoxy ester has a morphine-like potency [142] in rats (the enhanced potency of acetoxy over propionyloxy esters is generally true in the propoxyphene group). deStevens and others [ 1431 have separated the (f)-diastereoisomers of (XLVIII) (Ar = Ph, R = Et) and report one isomer to be a weak and the other a reasonably active (0.1 times morphine) analgesic; a cis H/OCOEt configuration is provisionally assigned to the more active form. The 2-pyridyl acyclic (XLIX; 2 X dextropropoxyphene) and cyclic (XLVIII, Ar = 2-pyridy1, R = Et; 5 X morphine) derivatives are both more active than corresponding phenyl analogues [ 1431 , while the N-ethoxyethylamino congener of (XLIX) is inactive [144]. The 1-

indanol (L) and its tetrahydrodecalin congener, of similar structure to the cyclic propoxyphenes (XLVIII), are both more active than codeine in mice by mouth [ 1451. Analgesically active 3-substituted-8-propionyl-3,8-diazabicyclo [3,2,1] octanes (LI) have been reported [ 146-1481. These are piperazines with the 1,s positions

(LII

linked by a bimethylene bridge and their molecular framework resembles that of tropane. The first significantly active example was the 3-methyl derivative (LI) (R = Me) which had a low order of potency, compared with morphine, in rats by Randall and Selitto’s assay [ 1461 . N-Substituents of proven efficacy in other

A . F. CASY

25 1

analgesic classes yield more active analogues (Table 5.4), the 3-cinnamyl derivative being the most effective (10 X morphine) [147]. Compounds of similar orders of potency resulted when this compound was substituted in the aromatic ring (for example by C1, NO2 and Me) and the aliphatic side-chain (y- more active than S-Me or Br isomer) [148]. Reversal of 8-propionyl and 3-arylalkyl substituents severely depressed activity [ 1481 . While these bridged piperazines Table 5.4. ANALGESIC ACTIVITIES OF SOME 3-SUBSTITUTED-8-PROPIONYL3,8-DIAZABICYCLO[3,2,1]OCTANES IN RATS [ 1471

R in (LI)

Morphine HC 1

Intraperitoneal dose (mg/kg)

0.1 0.3 0.5 1 1.5 3 5

Per cent increase in pain threshold

32 41.5 28 99 142 46.5 75.8 143 11 76 81 330 31.5 91 >170

may well have morphine-like properties, they were assayed by a test specifically designed to detect anti-inflammatory drugs [ 1491. The effect of nalorphine upon their actions has not been reported.

If they are viewed as /3-prodine analogues (see LII and LIII), an aromatic function at position 8 would be expected to enhance activity; an 8-cinnamoyl group is detrimental in (LIII, R' = Me) [146] but data on smaller aryl groups such as benzoyl has not been reported.

252

ANALGESICS AND THEIR ANTAGONISTS

Activities of the order of one-third to one-fifth that of morphine are claimed for the Mannich bases (LIV) (n = 1 or 2) obtained from phenylpiperazine and cycloalkanones [ 1501 . There is revived interest in the metabolism and excretion of methadone (Me2NCHMeCH2Ph2COEt) in man, one reason being the use of this drug in treating addiction [ 1511. Only two basic excretion products were detected in the urine of males given methadone orally; these were unchanged drug and 2-ethyl-I ,5-dimethyl-3,3-diphenyl-1-pyrroline (LIVa) [ 1521 . The hydriodide of (LIVa) was synthesized and its endocyclic structure established from IR and PMR data, so confirming earlier proposals based on the results of animal experiments [153]. The pyrroline (LIVa) most probably arises as a result of spontaneous cyclization of N-demethylmethadone. The metabolism of methadol (Me2 NCHMeCH2CPhz CHOHEt) and 0-acetylmethadol have not been reported but there is chemical evidence that their N-demethylated products should be stable under physiological conditions. Thus, although catalytic reduction of the N-benzyl ketone (LIVb, X = COEt) yields a cyclic product, that of the secondary alcohol (LIVb, X = CHOHEt) gives the methylamino analogue of methadol

(LIV 1

(LIVa)

(LIVb)

[154]. Metabolites of this nature may well play a role in the mediation of the analgesic effects of the parent dimethylamino compounds in view of their significant analgesic properties [ 155, 1561 . Metabolites of this nature may well play a role in the mediation of the analgesic effects of the parent dimethylamino compounds in view of their significant analgesic properties [ 155, 1561.

PHENOTHIAZINE DERIVATIVES

Tranquillizing drugs based on the phenothiazine nucleus potentiate the action of narcotic analgesics [ 1571 although there are some reports to the contrary [ 1581 and certain derivatives, notably methotrimeprazine (levomepromazine, Levoprome, Veractil) (-)-(LV), are claimed to have intrinsic analgesic activity. In mice, (->(LV) is more potent than morphine in a variety of tests [ 159, 1601 for example hot-plate EDs0 value of 1.02 mg/kg, that of morphine 2.1 mg/kg,

25 3

A . F. CASY

while the two drugs are equiactive in rabbits by the tooth pulp stimulation test [ 1611. Blane found(-)-(LV) and chloropromazine to be inactive in the rat

(LV)

tail-pressure test although both drugs effectively abolished phenylquinone-induced writhing in mice [ 1621 . No analgesic studies with the (*)- or (+)-forms of (LV), or nalorphine antagonism of the (-)-isomer have been reported. Clinically, many reports are available that establish the analgesic utility of parenteral (-)-(LV) (10-20 mg), for example, in the management of pain due to post-operative conditions [ 1631, cancer [ 1641, labour [165] , and for pre-anaesthetic medication [166]. The lack of analgesia in cancer patients after chlorpromazine (25 mg, intramuscularly) [ 1671 reported by a group who later found methotrimeprazine (20 mg) to be effective under the same circumstances [164], emphasizes the unique property of the latter phenothiazine derivative. The side-effect profie of (-)-(LV) is different from that of morphine; the incidence of nausea and vomiting is less and there is no significant respiratory depression [ 164, 1681 . Furthermore, (-)-(LV) has no PDC in monkeys or former opium addicts and only partially suppresses abstinence in the latter subjects [169]. I t is evident, therefore, that methotrimeprazine represents a significant advance in analgesic therapy, although there is doubt as to its classification as a narcotic analgesic. BENZIMIDAZOLES

Little recent clinical data on the potent 2-benzylbenzimidazoles developed by Ciba [ 1701 have been reported, probably because of their pronounced sideeffects and addiction liabilities, but a number of chemical and pharmacological studies have been made. Activity is retained when the methylene group linking the two ring systems of the most potent 2-benzylbenzimidazole derivative (LVI) ( 1 000 X morphine in mice) is altered to NH [I711 or CHzO [172], but R

~

l

N N

I

~

y

~

(oL V I )EY =CH,. t R = NO, ( L V I I ) Y=CH,O. R=Me

CH,

I

CH 2- NEt

potency levels are reduced to two and five times that of morphine respectively. Structure-activity relationships of the 2-phenoxymethyl derivatives mirror those

254

ANALGESICS A N D THEIR ANTAGONISTS

of the original molecules. Thus 5-N02 and 4'-OEt substituents are specific for high activity while a 2-diethylaminoethyl basic side-chain at C-1 is far more effective than 2-dimethylaminoethyl [ 1721. A 5-methyl group can partly substitute for 5-NO2, the derivative (LVII) being morphine-like in potency. A series of 5- (and 6-) C1, MeO, and CF3 substituted [173], and branched-chain analogues [ 1741 of (LVI) have been reported but pharmacological results are not yet available. Seki, Sasazima and Watanabe [175] have made a series of l-(2-t-aminoethyl)-2-(arylthio)benzimidazoles (LVIII), several of which are superior in potency to morphine in mice. A detailed study of (LVIII) (R = OEt) R

0 ) ) S yI - J

7

H2

C Hz. N E t z

OPr OEt NHEt NHPr morphine

ED50mdkg (mice i.p.) 44.3 .5 3.1 7.6 7.3

(LVIII)

revealed it to be a typical narcotic-analgesic; it caused addiction and Straub tails in rats and its analgesic and respiratory depressant actions were antagonized by levallorphan [176]. The NEt, group of (LVIII, R = OEt) was replaced by pyrrolidino to advantage but other t-amino groups, such as piperidino, EtN(CH,),Ph and N-diallyl, were potency lowering [177]. The ally1 derivative was not an analgesic antagonist. Some 2-benzylindenes based upon the benzimidazole analgesics have been reported, several of which, for example, (LVIIIa, R = H) have potencies in the codeine-pethidine range [ 1781 . Their relationship to the benzimidazoles as analgesicsis doubtful, however, because insertion of a para-ethoxy group (R = OEt in LVIIIa) depresses potency, in marked contrast to its influence in the heterocyclic series. CH,F

CH,

I C H2.N Et2 EtO@+OMe (LVIII Hz-NMeZ b)

( LVIlIo)

The 6-dimethylaminomethylpregna-3,5-diene (LVIIIb) is reported to be more active than pethidine in the mouse writhing test (EDSO vnlues are 8.4 and 24 mg/kg respectively) and to have significant analgesic properties in the hot

A . F. CASY

25s

plate (mice) and tail-flick (rat) tests [ 1791. Its action in the last test is not, however, antagonized by nalorphine (although the respiratory depression it causes in rabbits is reversed by this antagonist), hence its classification as a narcotic analgesic is doubtful.

ANTAGONISTS OF ANALGESICS Much attention continues to be directed towards compounds of this class as a result of their now well-established analgesic properties in man, and the subject has been well reviewed [7, 180, 1811. The discovery that nalorphine was equipotent with morphine in man, accidently revealed during studies of morphinenalorphine mixtures [1S8, 182, 1831, led to the clinical evaluation of other narcotic antagonists (both proven and potential) and has culminated in the development of the valuable drug pentazocine. Specific compounds of importance are considered below.

Nalorphine (N-allylnormorphine, Lethidronej This drug cannot be used clinically because of its undesirable psychotomimetic side-effects (for example hallucination and feelings of unreality) which are characteristic of many narcotic antagonists that act as analgesics. Nalorphine was initially felt to lack addiction liability [ 1841 but there are now two reports demonstrating abstinence symptoms after chronic administration [ 185, 1861. In the Lexington study [186] former addicts became tolerant to the subjective effects of nalorphine and exhibited an abstinence syndrome when abruptly withdrawn from the drug. However, this syndrome was qualitatively different from that of morphine; it caused only mild discomfort and did not seem to give rise to compulsive drug-seeking behaviour, and the authors concluded the abuse potentiality of nalorphine to be very low. The long-assumed competitive nature of nalorphine-analgesic drugs receptor interactions has been placed on a firmer footing by quantitative studies utilizing Schild's pA2 value (the negative logarithm of tbe dose of antagonist which reduces the effect of a double dose of analgesic to that of a single dose) [ 1871, and the Gaddum drug-ratio (molar ratio of agonist arid antagonist producing a SO per cent analgesic effect) [ 1881 . Both parameters were constant for a variety of nalorphine-analgesic combinations as is required for the competitive condition. This view of antagonist action is complicated, however, by the report that simultaneous administration of nalorphine or levallorphan (2 mg/kg) with ''C-labelled levorphanol ( 2 mg/kg) in the dog resulted in a marked diminution of brain levels of levorphanol as compared with controls. Plasma levels were little affected [189]. Arguments for and against the concept of narcotic antagonists acting competitively with agonists have recently been summarized by Martin [ 1801 . The

256

ANALGESICS AND THEIR ANTAGONISTS

inhibition of N-demethylation of morphine by nalorphine appears to involve both competitive and non-competitive mechanisms [ 1901 . During studies of the use of nalorphine as an anaesthetic supplement (it is less effective than morphine), it was found that whde the respiratory effects of small intramuscular doses (10 mg) were similar to those of morphne (same dose level), large cumulative doses (70-80 mg) given intravenously did not induce severe respiratory depression [ 1911. These results indicate that the ceiling depression of nalorphine is attained at a low dose (approx. 10 mg) and provide an explanation for the greater degree of antagonism observed when nalorphine antagonizes large doses of morphine (substitution of a smaller nalorphine for a greater morphine depression) compared with small doses. In a similar study [ 1921, 13.5 mg nalorphine were found to produce the same degree of respiratory depression as 10 mg morphine in healthy subjects.

Pentuzocine ((+)-2’-hydroxy-5,9-dimethyl-2 (3,3-dimethylallyl)6,7-benzomorphan, Win 20,228, Talwin, LIXa). R

H 0‘

(LIX)

(a) R = C H 2 C H = C M e 2

This compound is the most promising clinical analgesic yet developed from analgesic antagonists. It gives no positive response in the tail-flick and hot plate tests when given in non-toxic doses to mice and rats and is only a feeble antagonist of the effects of morphine on the tail-flick reaction [ 1931. In man, however, it is an effective analgesic in a wide variety of pain situations as is evident from the numerous clinical reports now available; results of some of the more recent ones are shown in Table 5.5. On average, a 30-40 mg intramuscular dose is equivalent to 10 mg of morphine although higher doses have been found necessary in cancer patients [200]. In an oral study, 50 mg of pentazocine was as effective as 60 mg codeine or 600 mg aspirin [ 2 0 2 ] .Sideeffects similar in both nature and degree to those of morphine are observed but these do not appear to detract seriously from the clinical utility of the drug. Respiration is depressed but not in pronounced degrees (21 mg pentazocine and 10 mg morphine are estimated to have similar respiratory depressant actions in healthy volunteers [203] ); in both healthy subjects and surgical patients, depression was countered by the analeptic methyl phenidate [204,205].

A . F. CASY

251

Table 5.5. SOME CLINICAL RESULTS WITH PENTAZOCINE

Type o f patient or pain

Effective dose (intramuscular)

Post-opera t ive

20-40 mg

Moderate t o severe chronic pain

40 mg approximated b u t did not equal 20 mg morphine

Various medical and surgical disordee, e.g. angina pectoris pelvic disease, kidney colic

Infrequent and non-hazardous, some sedation; mild respiratory depression; euphoria absent; psychic effects absent Similar t o those of morphine (20 mg) but more severe; more frequent drowsiness Minimal and not severe

Reference 194

195

196

30-60 mg

Post-operative

30 mg equivalent to 10 mg morphine

Post-operative

38 mg equivalent to 10 mg phenazocine 30-60 mg

Post-operative

Side reactions

No severe side-effects and incidence similar for both drugs None serious; some sedation; n o nausea o r vomiting Low incidence of nausea; n o symptoms of respiratory depression; n o psychic effects; mild sedation

197

198 199

Cancer

1/6th as potent as morphine

Side effects quantitatively similar t o those of morphine; some withdrawal phenomena

Labour

45 nig

20 1 None serious; labour not retarded; increased uterine activity; no significant change in foetal heart rate

200

Pentazocine has been successfully used to relieve labour pain [201] and its obstetric use in place of pethidine is favoured b y j t s apparent inferior ability to pass the placental barrier [206]. A clinical trial of (+)- and (-)-pentazocine adds to the rare number of examples in which optical enantiomorphs have been evaluated [207]. In post-operative patients, response to 6 0 mg of the dextro isomer was less than that to 5 mgof morphine, while 25-29 mg of (-)-pentazocine was as effective as 10 mg of morphine. Hence most of the activity of the racemate resides in the laevo isomer, as anticipated from results in animals [208]. Several studies of the distribution, excretion and metabolism of pentazocine have been made. Peak levels of the tritium-labelled drug (and its cis-3-chloroallyl analogue) were present in the C.N.S. of a cat within 40 minutes of intramuscular administration [209], the comparable figure for morphine being 2 hours [210].

25 8

ANALGESICS AND THEIR ANTAGONISTS

Following intramuscular and oral administration to post-operative patients, plasma levels (detected by a sensitive spectrofluorometric method) coincided closely with onset, duration and intensity of analgesia [211]. The same analytical procedure applied to the urine of healthy men given pentazocine, showed the drug to be extensively metabolized; less than 13 per cent of a dose appeared in the urine as unchanged drug and 12-30 per cent was excreted as a conjugate [212]. The more extensive metabolism of pentazocine as compared with that of pethidine in man has also been reported [206]. Metabolism of pentazocine in the monkey involves attack upon a terminal methyl of the 2,2-dimethylallyl substituent as shown by the isolation of cis and trans (LIX; R=CH2 CH=CMeCH, OH) and cis H/C02 H (LIX; R=CH,CH=CMeCO, H) from urine [213]. Product identification was aided by PMR spectroscopic data and this study is a fine illustration of the potential value of n.m.r. spectroscopy to metabolism studies.

Cyclazocine [(+)-LIXb] and cycforphan [(-)-LX] Both these compounds have weak or negligible activities in the rat (tail-flick test), mouse [hot-plate test, EDs0 values (mg/kg) 19 (LIXb); 78 (LX); 5.5 (morphine)] and monkey (electric shock method, (LIXb) only tested) [2 141 and their effects, where significant, are probably due to muscle relaxing rather than analgesic properties [ 193, 2151. Both are potent morphine antagonists (5 and 3 times nalorphine for (LIXb) and (LX) respectively); N-cyclopropylmethylnormorphine is about twice as active as nalorphine and the relative antagonist

H 0'

(LX)

activities of these cyclopropylmethyl derivatives parallel the analgesic properties of the parent N-methyl compounds [215]. Cyclazocine also antagonizes the respiratory, cardiovascular and behavioural depression produced by morphine and pethidine in dogs [216]. Compounds (LIXb) and (LX) are both potent analgesics in man, doses of the former as low as 0.25 mg given by mouth or injection providing effective pain relief in post-operative patients [ 158, 2 171. Equi-analgesic doses of (LIXb) and morphine produced similar degrees of respiratory depression but use of the benzomorphan derivative also led to undesirable mental effects (confusion, depersonalization and dysphoria) [2 171 . Cyclazocine precipitates abstinence in subjects physically dependent on morphine but its chronic administration leads to evidence of tolerance, and an abstinence syndrome in subjects so intoxicated is observed when the drug is withdrawn [218]. This

A. F. CASY

259

syndrome resembles that due to nalorphine rather than morphine and its use has been recommended for the treatment of abstinent ambulatory addicts 12191 . Its respiratory depressant and subjective effects are antagonized by naloxone [220]. L~

Naloxone (N-allylnoroxymorphone, LXI) This derivative is one of the most potent morphine-antagonists yet examined (19 X nalorphine in rats [221]) and it gives no analgesic response in the mouse hot-plate and rat tail-flick tests [222]. It is inactive, furthermore, in the mouse

phenylquinone writhing test, a procedure which detects analgesic properties in other morphine-antagonists (see below) [221] ; the actions of these compounds in this test are, indeed, antagonized by (LXI) [223]. The analgesic properties of naloxone in man are equivocal - Lasagna found a 2 mg dose to approach morphine (10 mg), higher doses, surprisingly, being less effective ( 8 mg gave the response expected from a placebo) [ 1581 , while Foldes [224] reported the drug to be inactive. It does not induce respiratory depression and is a more effective antagonist of narcotic induced respiratory depression than nalorphine or levallorphan [225]. Neither psychotomimetic effects [ 1581, nor the mild abstinence syndrome that follows chronic administration of nalorphine or cyclazocine are seen after the drug [226]. Harris [8] regards naloxone as the most nearly pure antagonist so far tested. In a quantitative study of the antagonism of morphine analgesia (writhing test in mice), the PA? value of naloxone (7.01) was greater than that of nalorphine (6.7) (the latters action was complicated by its agonist properties in this test) [227] but the difference is less than that anticipated from results in rats [221]. The pA2 values for the antagonism of intestinal motility were lower for both drugs and these results are taken to indicate that the receptors for analgesia and inhibition of intestinal movement are different. Naloxone has been used for the treatment of dependence on heroin and has the advantage of potency, rapid action, absence of side-effects, and acceptability; on the debit side are its brief action and high cost [228]. The thebaine derived derivative diprenorphine (MSOSO, VIII, R=H, R’=Me, 2 R =CH2cC3H5)(see p. 236) has a similar pharmacological profile to naloxone

260

ANALGESICS AND THEIR ANTAGONISTS

[229]. It lacks agonist properties in the mouse writhing and in the rat-tail pressure tests [ 1621, and is a powerful antagonist of both conventional analgesics such as morphine and narcotic antagonist-agonists such as nalorphine. Both diprenorphine and naloxone antagonized the action of nalorphine in abolishing the response of rats to intra-arterial bradykinin, the former being more than 250 times the more potent. Animal tests The non-response of narcotic analgesic-antagonists in the usual animal tests for analgesia has stimulated a search for tests that can detect analgesics of this class. Several reports [208, 22 1,2301 advocate the use of the phenylquinone writhing test of Siegmund, Cadmus and Lu [231]. Blumberg, Wolf and Dayton [221] found a reasonable correlation between writhing prevention EDS, values and analgesic properties in man for a series of morphine antagonists (Table 5.6). Table 5.6. WRITHING TEST ANALGESIA AND CLINICAL POTENCY [221]

Compound Naloxone Levallorphan Pentazocine Nalorphine Cyclazocine Cyclorphan Morphine

* In

EDs0 values (mg/kg) Rat Mouse

>82 26 3.8 0.48 0.028 0.019 0.59

>41 1.7 0.95 0.20 0.0 12 0.0 18 0.20

Analgesic potency in man 0 O* 0.2 0.9 30 30 1.0

another clinical report, levallorphan (8 mg/70 kg) approached but was not equivalent

to a 10 m d 7 0 kg dose of morphine 1531

there being no correlation, however, between analgesic and narcotic antagonist potencies as is well illustrated by naloxone (potent antagonist, doubtful analgesic). Pearl and Harris [208] obtained similar results. In a modification of' the test involving use of acetylcholine rather than a quinone, a significant correlation between rank order of analgesic potency in man and ability to lessen the incidence of abdominal constrictions in the mouse was obtained for 27 analgesic drugs which included both narcotic and non-narcotic types [232]. The writhing test has been criticized on the grounds of its lack of specificity (drugs as diverse as ephedrine, pilocarpine and meprobamate also block the response) and the large number of factors that affect its sensitivity (for example, the agent that induces writhing, temperature and strain of animal) [233]. Tolerance to drug-induced writhing in mice has been reported [234].

26 1

A. F. CASY

Some anomalies have been noted, the most interesting being the case of dexoxadrol (Relane, (+)- LXII); this isomer (but not the laevo form) increases

Q-n O X 0

Ph

Ph

writhing after phenylquinone [235] yet is capable of relieving clinical pain (20 mg more effective than 600 mg aspirin), its side-effects including the induction of psychotomimetic states [236]. Bradykinin, a natural peptide which causes increased capillary permeability, lowers pain thresholds and stimulates nerve endings, has been advanced as the chemical mediator of writhing [237] and the possible relationship between bradykinin-induced responses in animals and pain in man has been discussed [238]. This peptide induces a characteristic syndrome in the rat (dextrorotation of the head and flexion of the right fore-limb) which is blocked by narcotic analgesics (for example, methadone) and anti-inflammatory agents [ 2391. Blane [ 1621 has evaluated narcotic antagonists in this test but obtained non-uniform results; thus levallorphan, a potent analgesic in man [53], was inactive whereas nalorphine and cyprenorphine (VIIc), also active in man, were effective antibradykinin agents. Narcotic-antagonist analgesics also depress the flexor reflex in the spinal dog [240] and the coaxially stimulated guinea-pig ileum [8], naloxone being ineffective in each case. Relative potencies of morphine, codeine, nalorphine and cyclazocine from the former test correlated with those obtained in the mouse writhing test. An assay procedure based on the Straub tail reaction in mice has been shown to be capable of ranking the activities of both agonists and antagonists, results which are consistent with previous estimates [241]. The guinea-pig ileum test has been developed [242, 2431 to provide quantitative data upon the agonist and antagonist properties of morphine-like drugs. The agonist activity is measured by the concentration 'of drug which causes 50 per cent depression of the twitch induced by coaxial stimulation (IDs0 value) and the antagonist activity by the equilibrium constant ( K e ) The latter is obtained a from the expression K , = DR-l - where a is the molar concentration of the

antagonist and DR is the ratio of the concentration of agonist (morphine) required to depress the twitch to the same extent in the presence or absence of a given concentration of antagonist. The smaller K , is, the more potent is the anID50 tagonist. Thirteen derivatives were tested and it was found that the ratio-

Ke

26 2

ANALGESICS A N D THEIR ANTAGONISTS

was less than two for all morphine ‘agonists’ (for example, morphine, levorphanol, pentazocine) but above two for potent ‘antagonists’ such as cyclazocine and nalorphine which produce analgesia in man. Naloxone, an effective antagonist in this test, was the only member of the series which had little or no agonist activity (even diprenorphine, VIll, R=H, R’=Me, RZ=CH2c-C3H5, was a potent agonist), a result which supports the opinion that the 14-hydroxy morphinone derivative is purely an antagonist. The guinea-pig ileum preparation may appear an unlikely model for the analgesic receptor (cf. [227]), but its use in this respect is supported by the ability of the procedure to (1) rank both agonists and antagonists in the order anticipated from results of analgesic evaluations in whole animals and in man, and (2) to differentiate between stereoisomers in the same sense as do tests for analgesia. Thus both the analgesic (writhing test in rats) and guinea-pig ileum agonist properties of (+)-cyclazocine reside in the laevo isomer, while in the case of profadol (in which the isomeric potency difference is small) the IDso values of the isomers differ only by a factor of about two with the laevo form the more active [244]. In addition, the 0-isomers of both prodine and 3-methylpethidine were identified as the more potent diastereoisomas in this test, again in agreement with the relative analgesic potencies of a/P pairs. On the basis of these findings, the guinea-pig ileum test of Kosterlitz may offer a reliable assay procedure for analgesia; an isolated-tissue test of this nature involves far fewer variables than do whole animal experiments (these are subject in particular to factors of drug transport) and might allow more meaningful speculations about drug interactions at the analgesic receptor to be made. The mode of action of morphine-like drugs on autonomic neuro-effectors in the guinea-pig ileum has been discussed [245] .

Structural requirements Thanks to the efforts of the Sterling Winthrop group [61, 721, some account may be given of structure-activity relationships in analgesic antagonists based on the 6,7-benzomorphan nucleus; potencies of a racemic a-series (cis-5,9-diMe) respecting abilities to antagonise the effects of pethidine in the rat tail-flick test are given in Table 5.7. Antagonist properties are conferred by a straight threecarbon chain N-substituent, potency decreasing with increasing unsaturation (Table 5.7, Nos. 1-3). Variable effects follow substitution of this chain - two terminal methyl (No. 4, pentazocine) or chloro (No. 5) groups depress potency 80-100-fold, while the derivative with a single 3-chloro group (No. 6) is one of the most potent members of the (+)-series. The reasonable activity of the 2methyl variant (No. 7) contrasts with the feeble performance of the 2-chloro derivative (No. 8). The cyclopropyl unit has double bond character and it is not surprising that it may stand in lieu of vinyl in No. 2 to give cyclazocine (No. 9),

A. 1.: CASY

263

Table 5.7. RACEMIC cis 5.9-DIMETHYLBENZOMORPHAN ANALGESICANTAGONISTS [ 721

No.

An tagorlist activity AD,, values (mg/kg)

R in (LXIX) ~~

1

2 3 4 5 6 7 8 9 10 11 12 13

--

~~

0.0 19 0.047 0.78 3.9 5.1 0.018 0.094 4.2 0.019 0.092 0.37 0.28 14.5

of equal potency with the chloro derivative (No. 6); activity is depressed fivefold in the cyclopropylethyl derivative (No. 10) and analogues with larger alicyclic rings are much less effective (Nos. 11-13). Replacement of 5-Me by 5-Et in compounds 2, 4, 9 and 11 does not alter the activity ranking. Comparisons among(+)-cis and -rruns pairs reveal a lack of marked stereo-specificity (Table 5.8. Nos. 1-4, 7-10, 11-14, 17-18, and 19-20), in contrast with the uniform superiority of rruns over cis isomers in benzomorphans with hot-plate activities in mice (Table 5.2). Regular variations in the antagonist properties of enantiomorphs are seen however, differences being much more pronounced in the cis and trans cyclopropylmethyl derivatives (Table 5.8. Nos. 12-13 and 15, 16) than less active antagonists such as pentazocine and its trans analogue (Nos. 2, 3 and 5 , 6). In all cases, activities of the racemates reside largely in the (-)-enantiomorphs of related configuration. The (+)-forms appear to reduce the effects of their antipodes since racemate activities are considerably less'than half those of the laevo isomers. The more active enantiomorphs of both the a- and /3-series (Table 5.8. Nos. 2, 5, 12 and 15) all have a 5-R configuration as do, also the analgesic derivatives u-(-)-and 0-(-)-metazocine [78] . The optimum 5,9-diMe geometry for writhing prevention is difficult to ascertain; in some cases, a trans isomer is more effective (for example, Nos. 4 and 10) while in others, the cis (for example, Nos. 1 1 and 17). Some of these results are complicated, however, by the fact that compounds Nos. 10 and 17 show hotplate activities in mice. Again, activity differences between antipodes are consistent, (-)pentazocine and (-)-cyclazocine being more active than corresponding (+)-forms (Nos. 2, 3 and 12, 13 respectively).

264

ANALGESICS AND THEIR ANTAGONISTS Table 5.8. STEREOISOMERIC BENZOMORPHAN ANALGESIC-ANTAGONISTS [61, 2081

H0’

R

R’

Form

CH2CH=CMe2

Me

(*) cis*

No.

Antaxonist activity ADSo-value (mg/kg)

Writhing test activitv EDSOvahe(mg/kg)

~~~

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

(3 (+)

(*)trans (-) (+)

CH2CH=CMe2

Et

(k)cis (-) (+)

(*) trans

CH2 c - C ~ H S

Me

(*)cis

(3 (+)

(k) trans

(3

(+I CH2 c - C ~ H ~

Me

(*)cis (2)trans

CH2CHgCHCl

Me

(*)

* Configuration

cis

(2) trans

3.9 0.9 14.0 3.3 0.55 13 10.9 3.1 19.5 equivocal 0.019 0.006 2.5 0.014 0.005 19 0.37 0.06

0.018 0.047 0.1 3

3.1 1.4 Inactive (32) 1.9 -

4.1

0.32 0.1 0.005 Inactive (1) >2 -

0.08 0.61 peak activity at 3

0.54

with respect to ring B.

Some 1,4-benzodiazepine derivatives, loosely regarded as cyclic versions of basic anilide analgesics, antagonize pethidine in the rat tail-flick test [246]. Activity orders are about 200 times below that of nalorphine for some 4rallyl COEt

(LXIII)

and one 4-phenethyl derivative, and the member (LXIII) also reverses the respiratory and cardiovascular depression produced by pethidine in the dog.

A . F. CASY

265

STEREOCHEMICAL ASPECTS AND RECEPTOR THEORIES Acyclic analgesics Interesting developments in views upon the association o f analgesics with the analgesic receptor have originated from Portoghese and Larson's configurational studies of diampromid and other basic anilides [247]. When these analgesics were first developed, it was attractive t o regard them as members of the methadone group o n account of chemical similarities and mutual antagonism by nalorphine. The analgesic receptor is stereo-selective towards methadone and diampromid and both molecules have asymmetric centres of the same kind (LXIV) which have been related t o optically active alanine. However, while the more active (laevo) enantiomer of methadone is related to R-(-)-alanine (LXV) [248] the configuration of the more potent optical antipode of diampromid is identical with that of the S-(+)-amino acid (LXVI) [247].

An inverse configurational relationship between more active enantiomorphs of other anilides (LXVII) and (-)-methadone is also found (Table 5.9). This reversal o f optical specificity is not without precedent in analgesics related t o methadone (see below) but in all other classes studied, groups of analgesics with related asymmetric centres have identical configurations [2] . Members of a configurationally related group of agonists may reasonably be assumed to associate with the receptor in a similar manner although it may not be taken for granted that association modes for different groups of analgesic agents are alike even though studies with analgesic antagonists indicate that a common receptor is involved. Configurational data o n basic anilides indicate, therefore, that the assumed analogy with the methadone class'is not justified and that the two classes differ in their binding modes at the receptor [252]. Degrees of receptor stereoselectivity are generally less in the anilides (Table 5.9) than in methadone (R/RS potency ratio is two [6] ). Further, the arylalkyl nature of the basic function in these derivatives is of a type unusual t o acyclic analgesics. This fact, however, does not help to explain the configurational results because the more active antipode of the analogue with the 'normal' dimethylamino function also has an S-configuration (Table 5.9, R = Me). Probable conformations for methadone (LXVIII) and the N-benzylmethylaminoanilide (LXIX), based upon spectroscopic and X-ray crystallographic studies have been

266

ANALGESICS AND THEIR ANTAGONISTS

Table 5.9. ANALGESIC ACTIVITIES IN RATS OF ENANTIOMORPHS OF DIAMPROMID AND RELATED COMPOUNDS MeNCHMeCH2. N C O E t I R Ph (LXVII)

R

Ford *

SIRS potency ratio

Refi

8 4.3 inactive (50)

1.86

249$

3.7 3.6 11.7

1.o

249

12.5 8.9 11.9

1.4

250

1.6 1.4 inactive (50)

1.14

250

1.7

25 1

AD50 values (mg/kg)t

50'' 35 40

*

[a] D Sign of base given

t Tail-flick method

$ Hot-plate ED50 values in mice are (*) 15, (+) 12 and

(-1 2 4 0 1251 ]

5 Diampromid "

Hot-plate ED50 values in mice

proposed [253-2551. If it is assumed that these conformations are likely to resemble those adopted by the analgesic at the receptor site, and further, that the phenyl-s-methyl-basic centre orientation of the methadone conformation (LXVIII) is particularly inducive to drug-receptor association, a possiele reason may be advanced for the differing stereochemical and basic group features of methadone and basic anilide analgesics. Although the spatial arrangement of the three groups specified in (LXVIII) is not favoured in the basic anilide (LXIX), this compound is nevertheless an active analgesic and it is therefore probable that its mode of binding to the receptor differs markedly from that of methadone. Hence, ( 1) the stereospecificity of the receptor towards enantiomorphic forms of the anilide is not necessarily the same as that which it exhibits towards methadone isomers, and (2) binding sites, additional to those operating in the case of the methadone-receptor association, may be required for the effective uptake of basic anilide molecules upon the receptor surface - such sites could possibly be

267

A. F. CASY

provided by the arylalkyl N-substituent of basic anilides (absent in methadone and related compounds). It is significant, in this respect, that the dimethylaminoanilide (Table 5.9, R = Me) has a low order of analgesic potency while methadone

Me (

LXVIII)

(LXIX)

Representation of a probable conformation o f N - [ (2-benzylmethyl~mino)-propyl] propronanilide hydrochloride. Note: 1. End-on view of aromatic ring as shown. 2. Amido-carbonyl carbon eclipses amido-nitrogen. 3 . H and Me on C-2 may be interchanged. 4. For clarity, N.Me and CH2.Ph substituents have been omitted. (From Casy and HassanZs4, by courtesy of The Pharmaceutical Society of Great Britain.) Representation of a probable conformation o f methadone hydrochloride (N.Me groups are omitted). (From Casy and H a ~ s a n ~ by ’ ~ ,courtesy of The Pharmaceutical Society of Great Britain.)

and isomethadone analogues with benzylmethyl- and phenethylmethyl-amino functions are inactive [251, 2561. The greater analgesic potency of a-(-)-methado1 has long been an anomaly in the study of configurational relationships amongst diphenylpropylamine analgesics due to the fact that the more active a-methadol optical isomer is derived from the weak analgesic (+)-methadone rather than the potent laevo form. Evidence of the configuration of the C-3 alcoholic centre of the methadols is now available through PMR analyses of derived 2-ethyl-3,3-diphenyl-5-methyltetrahydrofurans [257,258] and the application of Prelog’s rule [259,260], and it appears from the data of Table 5.10 that the C-3 rather than C-6 configuration is of prime importance respecting the activities of these alcohols. Thus the two more active methadols [a-and pa(-)-]both have the 3s-configuration while the C-3 centre of the most active isomethadol [@-(+)I [263] belongs to the same steric series [264]. It follows from this view, that the S-members of methadol enantiomers lacking asymmetry at C-6 (normethadols) should be the more potent analgesic and this has been confirmed (Table 5.10). The,relative activities of (+)and (-)- a-methadol are reversed when the alcohols are acetylated (Table 5.10), the a-(+)-isomer derived from (-)-methadone being the more potent ester [261]. This remarkable inversion of stereoselectivity may be interpreted in terms of the C-6 centre reasserting its dominating role. Alternatively, however, it may be considered due to esters requiring an R-configurated C-3 centre for optimal activity.

268

ANALGESICS AND THEIR ANTAGONISTS Table 5.10. HOT-PLATE ACTIVITIES IN MICE OF SOME METHADOLS AND NOR-METHADOLS BY SUBCUTANEOUS ROUTE [ 261,2621 Mez N.CHRCH,.CPhzCHEt I 0 R’ (LXW (a) R = Me; (b) R = H

Precursor R-(-)-methadone (0.8)* S-(+)-methadone (25.7)*

EDSOvalues (mg/kg) Me5hadols Ace,tylmethadols (R = H ) (R =COCH3)

Form

Configuration

a-(+)-70a p-(-)-70a a-(-)-70a p-(+)-70a

6R:3R 6R:3S 6S:3S 6S:3R

24.7 7.6 3.5 63.7

RS RS R RS R

9.88 10.3 17.7

(?)-normethado1 HC1 (LXXb) (+)-normethadol-(?)-tartrate (+)-normethadol-(+)-tartrate (?)-acetylnormethadol HC 1 (+)-acetylnormethadol HC 1

0.3 0.4

1.8 4.1

-

4.44 2.7

* ED^,, value

The latter seems the more probable since the same steric reversal is seen in the case of the normethadols and the acetate esters, the less active R-alcohol yielding the more active antipodal form of the acetate (Table 5.10). Although the methadols and methadyl acetates are closely related in structure, their conformations at the receptor may well differ as a result, for example, of a hydrogen bonding donor group (OH) in one molecule being replaced by an acceptor group (MeCO) in the other [252]. This interpretation of differing stereospecificities of structurally related analgesics in terms of differing receptor binding modes is akin to that proposed in the case of the more active enantiomorphic cholinergic agents (+)-muscarine and (-)-muscarone which have opposite configurations a t the C-5 centre [265]. It is interesting that a-(-)-acetylmethadol (6S, 3.5) is far less potent than the dextro isomer after intraventricular administration and it has been suggested that the analgesic effects of the (-)-isomer are due to a metabolite rather than the intact drug itself [44] . Strong intramolecular hydrogen bonding has been demonstrated in diastereoisomeric bases (in carbon tetrachloride) and hydrochlorides (in chloroform), and preferred conformations proposed for methadols on this evidence [260, 2661. The ethyl ester analogue of methadone is a further stereochemical anomaly in the methadone group. The S-(+) ester (LXXI) is weaker than (+)-antipode [267] . Conformational differences between methadone and the ester (LXXI)

A. F. CASY

CPhz .COz Et

269

XCH2 .CHZCPhz C 0 2 E t

I

Me2N-C-H Me

(a) X = NMez (b) X = rnorpholino

(LXXII)

(LXXI)

could not be detected by spectroscopic means [268] and the explanation of configurational reversal, in this case, may lie in a differing influence of the s-methyl group upon activity in the two compounds. A 0-methyl group raises potency in normethadone and related ketones but depresses activity in the esters (LXXIIa and b) [6] and steric requirements for optimal activity in the two situations are not necessarily associated.

Conformational factors in 4-phenylpiperidine analgesics It is well known that a 3-methyl group enhances the activity of the reversed ester of pethidine (LXXIII, R = H) and that of the two resultant (*)-isomers, a-and 0-prodine (LXXIII, R = Me), the 0-form is the more potent. The configuration of

VkO"?Ye Me

I

(CH,),Ph

(LXXIII)

( LXXIV)

these diastereoisomeric esters has been the subject of several papers [ 2691 and is now firmly established as frans ( 3 Me/4 Ph) for a- and cis for Pprodine by X-ray crystallography [270]. The superiority of cis over trans geometry for activity in reversed esters has been confirmed in other 3-methyl analogues [271] and it is significant that the potent 4-ethoxy-4-(2-furyl) piperidine (LXXIV) analgesic has

"7,.-

ED, a

mg/ kg*

- (trans) 2.5

p - ( c r s ) leL

Me

OM^

Ph Co2Et

ED,

mg 1kg

a - ( t r a n s ) 3.6

p - (cis)

0.12

Me I

(LXXVI)

N

I

Me

(LXXV)

* Hot-plate

test in mice,

S.C.

route; ED,

morphine = 1.1 mg/kg [71]

ANALGESICS AND THEIR ANTAGONISTS

210

the same configuration. Still more recent examples are the isomeric 4 - o - t o l ~ l reversed esters (LXXV) [272] and the 3-methyl analogues (LXXVI) of pethidine H

Me

I

N ‘

H

Me

EDS0value( m g / k g )

E D50vaI u e ( mg / kg ) a - (Irons)3 6 B - ( C I S ) 0 42

a-(fruns)2 5 P-(c/s) 1 4

(L XXVl I I )

(LXXVII)

itself [273]. The previous patent report on these compounds [274] lacked stereochemical and pharmacological details. The report of a-3-allyl-1-methyl-4phenyl-4-propionoxypiperidine(LXXIII, R = C H 2 - C H = C H 2 ) being more active than its 0-isomer provides a possible exception but the configurations of this pair have not been unequivocally established [275] . Preferred conformations of a- and 0-esters (LXXIII, R = Me) as solutes (bases and conjugate acids) in deuterochloroform (CDC13) and water have been proposed on the basis of PMR studies [276]. Those of the a-esters do not differ significantly from the solid

Me

(LXXIX)

,+N I H

w

@;+

h

H

Me a-(12)

B-CS)

I LXXX) (Activity in parenthesis morphine.1)

7-13]

A . 1:. CASY

27 1

state conformation of a-prodine (LXXVII) [27O] and have equatorial 3-Me and 4-Ph groups linked t o a chair piperidine ring. The conformations of 0-ester salts in CDC13 differ only in having axial 3-Me groups and a different aromatic piperidine ring orientation (LXXVIII); but their conformations in DzO ( G water) are less easy to establish from PMR data. The deshielding influence of the charged nitrogen centre upon 3-methyl, normally greater in the 0-than the a-isomer, is much reduced when solvent CDCl is replaced by DzO [271].rhis result could be due t o an increase in the population of conformers in which 3-methyl is further removed from protonated nitrogen (a skew-boat of type (LXXIX) being most likely) or t o a dispersal of charge about nitrogen due to solvation with the chair (LXXVIII) remaining favoured. Further work on this problem is in hand [59] . Potency differences between trimeperidine (y-LXXX, promedol) and its a- and 0-isomers have also been reported [277]and it may be significant that the

most active form (a-) has steric structure in which significant populations of nonchair conformers are probable (the extreme chair forms entail either two axial methyl groups or one axial phenyl). A conformational study of the isomers (LXXX) is currently in hand [ 2781 . Ethyl 3-a-phenyltropane-2-carboxylate (LXXXI), an ester somewhat more potent than pethidine [279] would also be expected to have a significantly large skew-boat population because the chair conformer is destabilized by a-Ph/bimethylene bridge interactions; spectroscopic evidence supports this contention for related fl-ethyl- and phenyl-ketones. Further evidence upon the role of the 3-methyl substituent in reversed ester of pethidine is available from a recent investigation of enantiomorphs of a- and fl-prodine [280] (Table 5.1 1). It is to be noted that activity is governed by C-4 rather than C-3 geometry since the two more active optical antipodes (dextro a- and P-prodine) have identical C-4 but different C-3 configurations. This result supports the view that the importance of the 3-methyl substituent lies in its influence upon the conformation of the molecule. Its effect upon activity in (+)-a-prodine where it is equational and favours a chair conformation is minor as seen by the similar potencies of (+)+prodine and 3-desmethylprodine. Both

272

ANALGESICS AND THEIR ANTAGONISTS

Table 5.1 1 . ANALGESIC ACTIVITIES OF PRODINE ENANTIOMORPHS IN MICE (HOT PLATE TEST) [ 2801 Ph

OCOEt

OR

Compound

R

a-Prodine

Me Me Me Me Me Me H

0-Prodine 3-Demethyl prodine Pethidine

Isomer

Configuration C? c4

(+) (+)

RS R

(3

S

(k) (+) (-)

RS S R

-

-

RS S

R RS S R -

ED50 values (mg/kg) 1.7 0.9 22.0 0.35 0.25 2.6 1.3 12.0

axial and equatorial 3-methyl groups appear to impair drug-receptor association in the laevo enantiomorphs although the 0-isomer is still four to five times more potent than pethidine. Interpretations of activity differences between a pair of agonists in terms of events at the receptor usually rest upon the assumption that drug transport factors play only a secondary role. Although this might seem reasonable in the case of a- and 0-prodine in view of their isomeric nature, preliminary results indicate that their potency differences may be related primarily to differences in their ease of penetration of the C.N.S. (in rats brain levels of 0-prodine exceed those of the a-isomer) [280]. If these findings be substantiated, conformational differences may then be related chiefly to processes governing the transport and distribution of the diastereoisomeric pair rather than to drug-receptor associations. "his example emphasizes the need for data upon the intraventricular potency of narcotic analgesics whereby transport factors would largely be eliminated and it is to be hoped that procedures for the direct administration of drugs to the brain (technically difficult and often unreliable in their present state [281] ) will be improved in the near future. Alternatively, the development of a meaningful isolated tissue assay for analgesia would do much to improve our interpretation of structure-activity relationships in this field (see page 262). In an analysis of stereochemical factors in narcotic analgesics, Portoghese considers that the conformational requirements for most of the 4phenylpiperidine analgesics appear to be minimal [282] . His argument was based, in part upon (1) the fact that endo and ex0 isomeric azabicyclo[2,2,1] heptane analogues (LXXXII) of pethidine have similar orders of potency in mice (benzoquinone

A. I:. CASY

27 3

writhing test) after allowance is made for the greater ease with which the ex0 isomer penetrates the brain [282], and (2) the equi-, albeit weak (half pethidine)

(LXXXII) Endo (R = C 0 2 E t , R ' = P h ) 2 x pcthidinc Exo (R = Ph, R ' = C 0 2 E t ) 1 2 x pethidine

activities of the decahydroquinoline isomers (LXXXIII) and (LXXXIV) made with the aim of evaluating an e-Ph/a-Ph pair of reversed esters [283].

RO

(LXXXIII)

(LXXXIV)

The potencies of isomeric 2-acyloxy-2-phenylquinolizidines(which likewise differ in the orientation of phenyl with respect to the rest of the molecule) were also found to be alike in an electric stimulation test [284] , but significant activity differences were seen in mice when a tail-flick procedure was used (see LXXXV) [285] R

C Ph

O

W

P

h OCR II

(LXXXV)

0

w

*EDSO R = C O M e 64 R = C O E t , 18 Morphlne 6.2

* mg/kg S.C.

111

EDSO R = C O M e 38.5 R = COEt 7.9

mouse tail-flick test [ 2851

In the absence of distribution data (and knowledge of preferred conformation at least in the axial phenyl examples), the significance of potency differences amongst the isomeric esters (LXXXIII-LXXXV) cannot, however, be judged. Of the more rigid reduced acridine congeners (LXXXVI), only the e-phenyl isomer has been obtained and this lacks hot-plate activity in mice [286]. The 4-phenylpiperidine unit is common to simple piperidine derivatives such as pethidine and a-prodine, and also to morphine, morphinan, and benzomorphan

274

ANALGESICS AND THEIR ANTAGONISTS

analgesics. This fact allows of a superficial correlation between rigid and nonrigid cyclic analgesics but should not be interpreted too narrowly in terms of MI=

(LXXXVI)

molecular geometry. In rigid analgesics the 4-phenylpiperidine moiety is constrained to an axial-phenyl chair conformation with the aromatic plane parallel with one passing through a line joining C-2 and C-4 of the heterocyclic ring (Figure 5.2). In simple 4-phenylpiperidines, however, the likely conformations I

Figure 5.2

outlined in (LXXVII-LXXIX) possess piperidine-aromatic ring orientations which differ markedly from that shown in Figure 5.2. Thus, if flexible piperidine analgesics are to present the latter orientation to the receptor they must adopt highly unfavoured conformations and it seems more reasonable to postulate a receptor capable of adapting itself to a variety of agonist conformations of varying binding efficiences. Evidence for the differing association modes amongst the two classes is also provided from some comparative structure-activity relationships. Although it is generally true that N-substituents have similar influences upon analgesic activity in the two types (for example, the potency raising effect of the N-phenethyl group) there are some discrepancies. Thus, while N-cinnamyl and 3-phenylpropyl groups give potent pethidine analogues [ 2871 corresponding morphinan derivatives are inactive [288] . Conversely, although N-phenacylnormorphinan is 6.5 times as potent as laevorphanol, the same norpethidine derivative is only one-tenth as active as the parent N-methyl compound [91]. Of particular interest is a comparison of the influence of the N-ally1 and N-dimethylallyl functions upon activity. Replacement of N-methyl by the above substituents in morphine and related molecules leads to compounds which are analgesic antagonists (potent for N-allyl, weak for N-dimethylallyl) and which are devoid of

A . F. CASY

275

activity in the usual tests for analgesia in animals (as discussed above). The same analogues of pethidine and its reversed esters, however, are active analgesics in animals (some as potent as morphine) but lack antagonist properties [ 1041. (In contrast, the N-ally1 analogue of the 3-phenylpiperidine derivative (XXVIIa) is an antagonist [ 1031 and such derivatives may therefore have receptor association modes more allied to that of morphine.) The two classes also show different structure-activity relationships with regard to oxygen functions. In analgesics based on morphine, morphinan and benzomorphan, a free phenolic group i s an essential feature for activity (its removal or maslung results in sharp falls in potency) but the same function is not a prerequisite for high potency in pethidine and its congeners although it may be advantageous (for example, as in ketobemidone); on the other hand, all potent analgesics of the latter class possess non-aromatic oxygenated functions such as COZEt, OCOEt, OEt [2]. Series 1

Series 2

Ph OCOEt

Q' R

Figure 5.3. A plot of the log EDs, of analgesics in Series I 11s.the log EDSOof identically substituted compounds in Series 2

216

ANALGESICS AND THEIR ANTAGONISTS

Portoghese [252] was the first to emphasize the value of studying the influence of an identical N-substituent upon activity in different analgesic groups as a means of comparing modes of interaction with the analgesic receptor. He considers that if identically substituted compounds in two different series interact with receptors in similar manners, then the quantitative contribution of various substituents to analgesic potency should produce (other factors such as distribution being equal) proportionate variations of activity in both series. A plot of log activity in one series against log activity in the others should then be a straight line, demonstrating a linear free-energy relationship; dissimilar binding modes would lead to a scatter of points. For example, the plot relating to derivatives of pethidine and its reversed ester (Figure 5.3) based on well-defined data [91] provides much evidence that the two series have similar interaction modes at the receptor.

CONCLUDING REMARKS UPON STRUCTURAL ASPECTS

1. In spite of the continual appearance of novel structures characterized as having morphine-like actions which are reversed by antagonists such as nalorphine, no significant analgesic has been yet identified which lacks either a basic centre (of pKa permitting extensive protonation at physiological pH values) or aromatic features. Doubt has arisen as to the fundamental nature of these structural requirements for analgesics after a non-basic steroid was reported to be a more potent analgesic than morphine [289] ; this claim, however, was later withdrawn by workers from the original laboratory [290]. 2. The view that morphine-like analgesia is mediated at a single type of receptor has been generally upheld by further studies with analgesic antagonists, establishment of the competitive nature of analgesic-antagonist interactions being specially significant in this respect. 3 . In an earlier receptor postulate [291], attempts were made to interpret the uptake of a number of cyclic and acyclic analgesics in terms of a receptor based upon the rigid morphine skeleton, with the implication of similar association modes for all molecules. Binding interactions of so restricted a character are now incompatible with present stereochemical and structure-activity data aqd it is evident that a variety of receptor-agonist uptake modes of varying binding efficiencies obtain. Regardless of any particular binding mode, it is reasonable to assume that all analgesics and their competitive antagonists are involved in ionic bonding with an identical anionic centre and this site has been proposed as a pivot around which the various modes of binding may occur [252]. 4. During the last 10 years, several classes of molecule with analgesic potencies far in excess of that of morphine have been discovered, notably 2-benzylbenzimidazole derivatives, 6,14-endoethenotetrahydrothebainesand 4-phenylpiperidines with N-arylalkyl substituents. The high lipid solubilities of several of these analgesics have been established [37, 2921, but this property, although it

A. F. CASY

277

facilitates the ready access of such molecules to the C.N.S., cannot alone account for the observed potency levels. The 4'-ethoxy derivative (LVI) (5-N02 absent), for example, is 70 times more active in mice than the corresponding 4'-methoxy analogue [293] and it is unlikely that the two derivatives differ significantly in their solubility properties. It may be concluded, therefore, that these potent molecules provide a greater number of binding sites than do smaller, less active analgesics. Hence the overall dimensions of the analgesic receptor appear to be much larger than those originally proposed.

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187. B. M. Cox and M. Weinstock, Brit. J. Pharmacol., 1964, 22, 289 188. L. Grumbach and H. I . Chernov, J. Pharmacol. Exp. Ther., 1965, 149, 385 189. K. D. Wuepper, S. Y . Yeh and L. A. Woods, Proc. Soc. Exp. Biol. Med., 1967, 124, 1146 190. L. Leadbeater and D. R. Davis, Biochem. Pharmacol., 1964, 13, 1569 191. A. S. Keats and J. Telford, J. Pharmacol. Exp. Ther., 1966, 151, 126 192. J . W. Bellville and W. H. Fleischli, Clin.Pharmacol. Ther., 1968, 9, 152 193. L. S. Harris and A. K. Pierson, J. Pharmacol. Exp. Ther.. 1964, 143, 141 194. M. Sadove, R. C. Balagot and F. N. Pecora, J. Amer. Med. Ass., 1964, 189, 199 195. L. J. Cass, W. S. Frederik and J. V. Teodoro, J. Amer. Med. Ass., 1964, 188, 1 12 196. M. Ende, J. Amer. Geriat. SOC., 1965, 13, 775 197. V. K . Stoelting, Anesth. Analg. Curr. Res., 1965, 44, 769 198. J. P. Conaghan, M. Jacobson, L. Rae and J. N. Ward-McQuaid, Brit. J. Anaesth., 1966, 38, 345 199. S. C. Finestone and J. Katz, Anesth. Analg. Curr. Res., 1966, 45, 312 200. W. T. Beaver, S. L. Wallenstein, R. W. Houde and A. Rogers, Clin. Pharmacol. Ther., 1966, 7, 740 201. W. W. Filler and N. W. Filler, Obstet, Gynecol., 1966, 28, 224 202. T. G. Kantor, A. Sunshine, E. Laska, M. Meisner and M. Hopper, Clin. Pharmacol. Ther., 1966, 7, 447 203. J. W. Bellville and J. Green, Clin. Pharmacol. Ther., 1965, 6, 152 204. J. Telford and A. S. Keats, Clin.Phmmacol. Ther., 1965, 6, 1 2 205. A. S. Brown, Proc. Roy. SOC.Med., 1969, 62, 1 9 206. A. H. Beckett and J. F. Taylor, J. Pharm. Pharmacol., 1967, 19, 5 0 s 201. W. H. Forrest, E. G. Beer, J. W. Bellville, B. J. Ciliberti, E. V. Millar and R. Paddock, Clin. Pharmacol. Therap., 1969, 10, 468 208. J. Pearl and L. S. Harris, J. Pharmacol. Exp. Ther., 1966, 154, 319; J. Pearl, M. D. Aceto and L. S. Harris, J. Pharmacol. Exp. Ther.. 1968, 160, 217; J. Pearl, H. Stander and D. B. McKean, J. Pharmacol. Exp. Ther., 1969, 167, 9 209. R. A. Ferrari, Toxicol. Appl. Pharmacol., 1968, 12, 404 210. H. 1. Chernov and L. A. Woods, J. Pharmacol. Exp. Ther., 1965, 149, 146 21 1. B. A. Berkowitz, J. H. Asling, S. M. Shnider and E. L. Way, Clin.Pharmacol. Ther., 1969, 10, 320 212. B. Berkowitz and E. L. Way, Clin. Pharmacol. Ther., 1969, LO, 681 213. K. A. Pittman, D. Rosi, A. J. Merola and W. D. Conway, Biochem. Pharmacol., 1969, 18, 1673 214. B. Weiss and V. G. Laties, J. Pharmacol. Exp. Ther., 1964, 143, 169 215. L. S. Harris, A. K. Picrson, J. R. Dembinski and W. L. Dewey, Arch. Int. Pharmacodyn. Ther., 1967, 165, 112 216. L. S. Harris, Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol., 1964, 248,426 217. L. Lasagna, T. J. DeKornfeld and J. W. Pearson, J. Pharmacol. Exp. Ther., 1954, 144, 12 218. W. R. Martin, H. F. Fraser, C. W. Gorodetzky and D. E. Rosenberg, J. Pharmacol. Exp. Ther., 1965, 150,426 219. W. R. Martin, C. W. Gorodetzky, and T. K. McClane, Clin. Pharmacol. Ther., 1966, 7,455 220. D. R. Jasinski, W. R. Martin and J. D. Sapira, Clin.Pharmacol. Ther., 1968, 9, 215 221. H. Blumberg, P. S. Wolf and H. B. Dayton, Proc. SOC.Exp. Biol. Med., 1965, 118, 763 222. H. Blumberg, H. B. Dayton and D. N. Rapoport, Fed. Proc., 1961, 20, 31 1 223. H. Blumberg, H. B. Dayton and P. S. Wolf, Proc. Soc. Exp. Biol. Med., 1966, 123, 755 224. F. F. Foldes, Med. Clin. N. Amer., 1964, 48, 421 225. F. F. Foldes, M. Schapira, T. A. G. Torda, D. Duncalf, and H. P. Shiffman, Anesthesiology, 1965, 26, 3 20

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270. G . Kartha, F. R. Ahmed and W. H. Barnes, Acta Cryst., 1960, 13, 525; F. R. Ahmed, W. H. Barnes and L. D. M. Masironi, Acta Cryst., 1963, 16, 237 271. A. F. Casy, J. Med. Chem., 1968, 11, 188 272. M. A. Iorio and A. F. Casy, J. Chem. SOC.,(C), 1970, 135 273. A. F. Casy, L. G . Chatten and K. K. Khullar, J. Chem. Soc., (C), 1969, 2491 274. P. A. J. Janssen, U.S. Patent 3 004 977,1961; Br. Patent 941 748 1963 275. A. Ziering, A. Motchane and J. Lee,J. Org. Chem., 1957, 22, 1521 276. A. F. Casy, Tetrahedron, 1966, 22, 2711 277. I. N. Nazarov, N. S. Prostakov and N. I. Shvetsov, J. Gen. Chem. U.S.S.R., 1956, 26, 2798; N. S. Prostakov, B. E. Zaitsev, N. M. Mikhailova and N. N. Mikheeva, J. Gen. Chem. U.S.S.R.,1964, 34, 463 and refs there cited 278. A. F. Casy and K. M. McErlane, unpublished results 279. M. R. Bell and S. Archer, J. Amer. Chem. Soc.. 1960, 82, 151, 4638 280. P. S. Portoghese and D. L. Larson, J. Pharm. Sci., 1968, 57, 71 1 281. Dobbing, Physiol. Rev., 1961, 41, 130 282. P. S. Portoghese, J. Pharm. Sci., 1966, 5 5 , 865; P. S. Portoghese, A. A. Mikhail, and H. J. Kupferberg, J. Med. Chem., 1968, 11, 219 283. E. E. Smissman and M. Steinman, J. Med. Chem., 1966, 9, 455 284. I. Sam, J . D. England and D. Temple, J. Med. Chem., 1969, 12, 144 285. L. S. Harris, private communication 286. E. E. Smissman and M. Steinman, J. Med. Chem., 1967, 10, 1054 287. B. Elpern, L. N. Gardner and L. Grumbach, J. Amer. Chem. SOC., 1957, 79, 1951; B. Elpern, P. Carabateas, A. E. Soria and L. Gmmbach, J. Amer. Chem. SOC., 1959,81, 3784 288. N. B. Eddy, H. Besendorf and B. Pellmont, Bull. Narcot., 1958, 10, 23 289. L. R. Axelrod, P. N. Rao and D. H. Baeder, J. Amer. Chem. Soc., 1966, 88, 857; L. R. Axelrod and D. H. Baeder,Proc. SOC.Exp. Biol. Med., 1966, 121, 1184 290. D. R. Vanderipe, G. B. Hoey, W. R. Teeters and T. W. Tusing, J. Amer. Chem. SOC., 1966, 88, 5366 291. A. H. Beckett and A. F. Casy, J. Pharm. Pharmacol., 1954, 6, 986 292. A. F. Casy and J. Wright, J. Pharm. Pharmacol, 1966, 18, 677 293. A. Hunger, J. Kebrle, A. Rossi and K. Hoffman, Helv. Chim. Acta, 1960, 43, 800

Some Pyrimidines of Biological and Medicinal Interest-Part I1 C. C. CHENG, B.S.,M.A.,Ph.D. Midwest Research Institute, Kansas City, Missouri 641 10, U.S.A. BARBARA ROTH, B.S., M.S., Ph.D. Burroughs Wellcome and Co. (U.S.A.) Inc., 3030 Cornwallis Road, Research %angle Park, North Carolina. 27709, U.S.A. INTRODUCTION

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286

THE URACILS Uracils without amino substituents Uracils with amino substituents

295 296 306

HYDROPY RIMIDINES Dihydropyrimidines Tetrahydropyrimidines Hexahydropyrimidines

31 1 31 2 3 20 323

ACKNOWLEDGEMENT

324

REFERENCES

325

INTRODUCTION Part I of this review, in Volume 6 of this series, introduced the very broad topic of pyrimidines of biological and medicinal interest with a discussion of 2,4diaminopyrimidines, halogenated and sulphur-containing derivatives, 4-amino-5hydroxymethylpyrimidines, sulphonamides, and pyrimidink antibiotics. The current chapter (Part 11) is concerned largely with oxygen-containing pyrimidines. Subjects covered include pyrimidinecarboxylic acids, the uracils and hydropyrimidines. This review is not intended to be all-inclusive. It would require encyclopaedic length to do more than exemplify the structure-activity relationships in certain areas. The bibliography, likewise, presents a representative, rather than comprehensive listing, especially in cases where there are many patents. As in Part I, the discussion is restricted to monocyclic pyrimidines in which the pyrimidine moiety is an important element of the biologically active molecule. Certain classes of pyrimidines which have not been covered include those with quaternary functions, such as the trypanocidal pyrimidinium salts, and vitamin 285

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B1 analogues. Also omitted are the phosphorothioic acid derivatives, such as the insecticide diazinon, and related compounds. Unnatural nucleoside derivatives have been given only brief mention, since the base, rather than the sugar, is our target in this review. Since a knowledge of the correct tautomeric form of the pyrimidines is a requisite for understanding the mode of binding to active sites, as well as nucleic acid structure and modification, the formulae of the conventionally-named 2- and 4-hydroxypyrimidines are presented in the correct lactam, or pyrimidone, form in this chapter. Other physical properties of the pyrimidines, such as dissociation constants, protonation sites, and distribution coefficients, are presented in cases where there is a known relation to drug activity. Biogenesis and enzyme control mechanisms are discussed where they relate to an understanding of inhibitor action. For the convenience of readers, the activities of compounds in several longer sections are subdivided according to their chemotherapeutic, pharmacological and other uses. Since overlapping is inevitable in a classification based on functional groups, material is cross-referenced. This survey will be completed in Volume 8.

PYRIMIDINECARBOXYLIC ACIDS The most interesting and important member of this series is orotic acid or uracil 6-carboxylic acid, (I). This compound was synthesized in 1897 by the condensa-

tion of urea with the ethyl ester of oxalacetic acid [ 1, 21. However, it received little attention until it was isolated as an unknown substance from the whey of cow’s milk in 1905 [3] (Greek: oros = whey). Later it was also found in the milk of goats and sheep and, in smaller amounts, in the milk of humans, pigs and horses [4-71 (orotic acid was originally reported to be absent from human milk [8] ). Orotic acid was also isolated from the ‘distillers dried solubles’ [9, 101 and, because of its growth-promoting property, was initially claimed t o be either related to a ‘vitamin B I 3 ’ [ l 1, 121 or a part of the ‘vitamin B I 3 ’ molecule [ l o , 13, 141. Free orotic acid usually contains one mole of water of crystallization, which can be removed only by drying at high temperature. This led to the erroneous

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postulation that the product from milk was a 7-membered cyclic ureido derivative [15]. The synthetic product from urea also was complicated in that an intermediate substituted hydantoin was formed initially [ 1, 2, 161. Consequently, the early investigators did not recognize the relationship of their synthetic product [16-181 to the natural material. This delayed the true structural assignment for orotic acid until 1930 [19-221. A number of different or improved synthetic procedures for this important compound have appeared in the literature [21-381 since this date. In early biological studies of several uracils, orotic acid was found to be the only pyrimidine derivative to have the same effect as liver extract on Lactobacillus casei E (i.e., to replace the folic acid requirement) [39]. Orotic acid and uracil have about the same growth-promoting effect on certain streptococci [40] and pyrimidine-deficient mutants of Neurosporu [41] . Although orotic acid was found to be a precursor of some pyrimidine nucleosides and nucleotides in certain yeast [42], bacteria [6, 431, higher plants [44-471, many mammalian systems [48-521, as well as some tumour cells [53] , it was initially considered as either just a by-product [54-561 or as an unimportant intermediate [57] in the biosynthesis of nucleic acids. Continued studies indicated that orotic acid is indeed an intermediate in the de novo synthesis of pyrimidines [58-601. This synthesis starts from aspartic acid (11) which, by the action of aspartic transcarbamylase, reacts with carbamoyl phosphate (formed from ammonia and carbon dioxide in the presence of ATP [61-631 ) t o form carbamoyl-L-aspartate [64-7 11 (111, or ureidosuccinate). Citrulline (6-ureidonorvaline) has also been found to contribute to the formation of (Ill) [66]. Ring closure of the latter by dihydroorotase yields dihydro-orotic acid [72-74] (IV). This, in turn, is oxidized to orotic acid [72-841 (I) by the DPN- or TPN-dependent dihydro-orotate dehydrogenases [72, 75-80]. The orotic acid then reacts with PRPP [85, 861 in the presence of orotidylic pyrophosphorylase and Mg2+ to form the corresponding ribonucleotide orotidine-5’-phosphate [85-901 (V, OMP), which is then irreversibly decarboxylated by the enzyme orotidylic decarboxylase to yield uridine-5’phosphate [87,88] (VI, UMP). UMP is subsequently converted to the nucleotides of cytosine, uracil, thymine and other nucleic acid pyrimidines [91-1061. An interesting difference between the biosynthesis of pyrimidines and purines is that, with the latter, ribosidation occurs prior to the purine ring closure; with the former, a pyrimidine (orotic acid) is formed prior to ribosidafion and phosphorylation (a riboside of orotic acid was isolated from a pyrimidineless Neurospora [ 1071 and identified [ 1081). The formation of pyrimidines from small molecule precursors has been shown by tracer studies [53, 109- 1 121 . Confirmation of the role of orotic acid in the pyrimidine biosynthetic sequence has also been provided by incorporation experiments [41-50, 53, 113-1281. The significance of inhibition of this metabolic route is reflected in antiviral, antineoplastic, and antibiotic activities [ 1291 . The C6 atom of (IV) is asymmetric. The D and L-forms, as well as the racemate of (IV), have been prepared by heating L-, D- or DL-Na-carbethoxyasparagine, respectively, with ethanolic sodium ethoxide [74]. It was found that

PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST

288

CO2H H2N,

-

- _ L . .

I

corbamoyl

/CH2

phosphate*

FH

CO2H

I

HN,

,kHz

FH

^^

-

,

.

LU2H

COzH

H

H

_._..

/

R’

YR

5/ P’

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5‘ (V)

Biosynthesis of pyrimidines

L- and D L-dihydro-orotic acid supported growth of Lactobacillus bulgaricus 09. The D-isomer alone is not only without activity, but is reported to reversibly inhibit the growth-promoting property exerted by ureidosuccinic or orotic acid [74, 911. The ‘dihydro-orotic acid‘ prepared by fusion of maleic acid and urea [130] isinactiveinbothL. bulgaricus09 [128] and an enzyme system [72, 1311. A comparison of the urea fusion product and that prepared by catalytic hydrogenation of orotic acid revealed that the former is actually fumarylurea [ 1321 . Under normal conditions, many of the enzymes involved in the pathway for pyrimidine biosynthesis de novo may not operate at maximum efficiency but rather exist in an ‘inhibited’ state. This ‘inhibition’ is released during the course of regeneration (for example after partial hepatectomy [ 1331 or castration [ 1341 ). This increase in enzyme activity can be prevented by the administration of actinomycin D [ 1331 or an androgen such as testosterone propionate [ I % ] . Pretreatment of partially hepatectomized rats with methotrexate markedly decreases the incorporation of orotic acid into DNA of the regenerating livers [135]. Orotic acid incorporation into nuclear and cytoplasmic RNA is decreased in polycythemic mice [ 1361 . Aflatoxin B 1 , a known hepatocarcinogen which induced polysomal disaggregation in the livers of rats, also inhibits the in vivo incorporation of intraperitoneally injected orotic acid into liver RNA [ 1371. On the other hand, the incorporation of orotic acid into free nucleotides and the RNA of the monkey kidney is rapidly increased twofold after castration and is restored to normal within seven days by the administration of testosterone [ 1341 . It is of interest to note that, in experiments with rats, administration of orotic

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acid to the diet increased the activity of both hepatic aspartic transcarbamylase and dihydro-orotase [138]. Orotic acid is claimed to be a dietary essential for mice [139] . Carbamoyl phosphate synthetase formation in liver taken from tadpoles treated with thyroxine is enhanced by the addition of orotic acid, uracil or uridine (cytosine and adenosine had no effect). The synthesis of this enzyme is not affected by these pyrimidines in untreated animals. This indicates that there is a relative pyrimidine deficiency during thyroxine-induced metamorphosis [ 1401. Orotic acid in the diet (usually at a concentration of 1 per cent) can induce a deficiency of adenine and pyridine nucleotides in rat liver (but not in mouse or chick liver). The consequence is to inhibit secretion of lipoprotein into the blood, followed by the depression of plasma lipids, then in the accumulation of triglycerides and cholesterol in the liver (fatty liver) [ 141-1611. This effect is not prevented by folic acid, vitamin B 1 2 , choline, methionine or inositol [ 141, 1441, but can be prevented or rapidly reversed by the addition of a small amount of adenine to the diets [ 146, 147, 149, 152, 1621. The action of orotic acid can also be inhibited by calcium lactate in combination with lactose [ 1631. It was originally believed that the adenine deficiency produced by orotic acid was caused by an inhibition of the reaction of PRPP with glutamine in the de novo purine synthesis, since large amounts of PRPP are utilized for the conversion of orotic acid to uridine-5’-phosphate. However, incorporation studies of glycinel-14C in livers of orotic acid-fed rats revealed that the inhibition is caused rather by a depletion of the PRPP available for reaction with glutamine than by an effect on the condensation itself [ 1601 . Apparently the acceleration of de novo purine biosynthesis by orotic acid results from a release of feedback inhibition imposed by hepatic purine nucleotides. In a related study, it was found that orotic acid feeding can prevent hyperlipaemia, which normally follows the administration of Triton WR- 1339, a surface active agent [152]. The influence of orotic acid on lipid metabolism can be readily shown by the fact that depression of serum lipoproteins and milk production were observed in lactating goats when an aqueous suspension of orotic acid was administered orally [ 1641. Orotic acid added to rat diet also provokes an excessive biosynthesis of porphyrins in liver, erythrocytes and bone marrow. Administration of adenine monophosphate (AMP) counteracted this effect of orotic acid intoxication [ 1651 . Haemorrhagic renal necrosis in rats, caused by choline deficiency, can be relieved by orotic acid [166]. Simultaneous supplementation of the diet with adenine does not influence the protective effect of orotic acid. It has been suggested that orotic acid may lower the body requirement for choline through a metabolic interaction-orotic acid may stimulate the cytidine phosphate choline pathway of lecthin biosynthesis [ 1661 . One of the most interesting observations in the nutritional and metabolic study of orotic acid is its close relationship with vitamin B 1 2 and methionine.

290

PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST

These three compounds exert many similar effects in nucleotide metabolism of chicks and rats [167]. They cause an increase of the liver RNA content and of the nucleotide content of the acid-soluble fraction in chicks [ 1681 , as well as an increase in rate of turnover of these polynucleotide structures [ 169,1701 . Further experiments in chicks indicate that orotic acid, vitamin B, and methionine exert a certain action on the activity of liver deoxyribonuclease, but have no effect on ribonuclease. Their effect is believed to be on the biosynthetic process rather than on catabolism [ 1711 . Both orotic acid and vitamin B12 increase the levels of dihydrofolate reductase (EC 1.5.1.4), formyltetrahydrofolate synthetase and serine hydroxymethyl transferase in the chicken liver when added in diet. It is believed that orotic acid may act directly on the enzymes involved in the synthesis and interconversion of one-carbon folic acid derivatives [ 1721 . The protein incorporation of serine, but not of leucine or methionine, is increased in the presence of either orotic acid or vitamin B I 2 [ 1731 . In addition, these two compounds also exert a similar effect on the increased formate incorporation into the RNA of liver cell fractions in chicks [ 174-1761. It is therefore postulated that there may be a common role of orotic acid and vitamin B12 at the level of the transcription process in rn-RNA biosynthesis [ 174- 1761 .

BIOLOGICAL ACTIONS

Urethane possesses specific carcinogenic action in a number of animals. In an investigation of the effect of known precursors of nucleic acid pyrimidines [ 1771 , orotic acid was found to reduce the number of adenomas produced. The action of urethane may take place through its conjugation with another substance, possibly oxalacetic acid or a related compound. The conjugate interferes competitively with pyrimidine biosynthesis at the level of orotic acid [ 1781 . On the other hand, it was found in the course of studying the effect of nucleic acid precursors on viral carcinogenesis, that an increased number of metastases developed in the lungs of orotic acid-treated mice with spontaneous mammary tumours [ 1791. The nature of its action is not yet understood, but it is known that inhibition of decarboxylation of orotic acid derivatives ordinarily doespot parallel the effect on tumour growth [ 1801 . Although both orotic acid and uracil are utilized by Ehrlich ascites tumoui cells in mice [52], the presence of orotic acid completely inhibits the incorporation of bicarbonate into C 2 of the acid-soluble nucleotides in the same tumour system [181]. Orotic acid or 6-methyluracil (vide infra), when administered to rabbits with myocardial infarction induced by ligation of the anterior descending branch of the left coronary artery, can decrease the incidence of necrosis and increase the rate of regeneration for healthy cellular and fibrous connective tissue in the infarct region [ 1821. Rats with induced aortal stenosis which are treated with

C. C. CHENG, BARBARA ROTH

291

orotic acid or withpurinor (orotic acid:adenine:xanthine:hypoxanthine,3: 1 :1: 1) can cause an increase in cardiac mass and work capacity. These effects are attributed to the increased myocardial nucleic acid conlent and protein synthesis [183]. The development of cataract in rats by the addition of galactose to the diet can be counteracted during the early stages (first 2 weeks) by the administration of orotic acid [ 184al. After 3 to 4 weeks, however, no difference can be seen in the lens opacities between the orotic acid-treated animals and the control [ 184b-el . Orotic acid was also reported to be beneficial in human galactosemia [184b,fl. Oral administration of orotic acid to guinea-pigs can stimulate the phagocytic activity of leukocytes against injected chicken erythrocytes [ 1851. The sodium salt of orotic acid can also increase phagocytosis and the digestive ability of the leukocytes in the rats [ 1861 . Orotic acid and aspartic acid, when given orally to full-term, healthy newborn infants for the first 4 days of life, can significantly lower their serum bilirubin level [ 1871 . The effect of aspartic acid is immediate, whereas that of orotic acid is delayed [ 1871 . In the presence of adenine and kinetin, orotic acid or thymine can enhance the production of flower buds in vitro in stem segments of Plumbago indica [ 1871 . The production of buds is often inhibited by auxins and gibberelins [ 1881 . In radiation protection studies, it was found that orotic acid does not offer a protective effect in mice against the whole-body X-irradiation of 700 y or 550 y [189]. On the other hand, orotic acid, alone or with folic acid, definitely increased the survival rate of irradiated rats and guinea-pigs by 20-30 per cent, while hemopoiesis was not affected [ 1901 . Daily orotic acid injection into rats subjected to carbon tetrachloride (or dichloroethane, DDT, etc.) poisoning can partially prevent the toxic effects of the latter on the liver [191-1951. This protective action of orotic acid may be related to increased synthesis of nucleic acids depleted by the toxic substances [ 1941 . The diabetogenic effect of alloxan can be nullified by orotic acid, as well as by barbituric acid [196]. Orotic acid administered orally to rats daily can raise the content of y-globulins, decrease the diphenylamine reaction in the serum, and increase the activities of aldolase, glutamic-aspart,ic transaminase and glutamic-alanine transaminase in the blood [ 1951 . Certain salts of orotic acid, such as alkanolamine, aliphatic diamine and heterocyclic amine salts, are useful in the treatment of hepatitis and cirrhosis [ 1971 . Sodium orotate has a strong antipyretic effect against inflammations in rats induced by dextran, formalin or serotonin [ 1861. However, in adrenalectomized rats, sodium orotate has no antipyretic effect [ 1861. In human volunteer experiments, it was reported that sodium orotate has anti-inflammatory action similar to colchicine [ 1981 or sodium urate [ 1991 against induced acute gouty arthritis. Orotic acid hydrazide and its acid salts are effective in the treatment of plant infections and the removal of fungus growth from textiles and food [ 2001 . These

292

PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST

compounds also counteract the overgrowth of Cundidu ulbicuns during tetracycline therapy [200] . The hydrochloride salt ofN-(diethylaminoethy1)orotamide (VII) was claimed to be useful in the treatment of leukaemia and tumours [201] . The fact that many agents which interrupt the synthesis of pyrimidine nucleotides from orotic acid in animals can also inhibit the growth of experimental neoplasms suggests a search for additional antimetabolites whose locus of action is in this metabolic sequence. Two in vitro biological screening systems were developed for this purpose [202-2071. From a study of systems with oxidative energy sources, 5-bromo-[208-2091 (VIIIa), 5-chloro- [2 101 (VIIIb) and 5-diazo-orotic acid [211] (IX) were found to inhibit the conversion of orotic acid to the uridine nucleotides by 40-100 per cent [202].

CONH(CH,),

h

(WII) a . X = Br

(IX1

b . X =C1 c. X=F d . X =NO,

Tests in a system with a non-oxidative energy source revealed that 5-bromo(VIIa), 5-chloro- (VIIIb) and 5-fluoro-orotic acid (VIIIc) produce single or multiple blocking actions [204]. 5-Nitro-orotic acid [I91 (VIIId) is not an inhibitor, but it interferes with the balance between UDP and UTP and their conjugate compounds [204]. The fact that 5-diazo-orotic acid (IX) showed an inhibitory action against the oxidative system [202] but failed to show a corresponding action against the non-oxidative system is believed to be due to its effect on the enzymes of oxidative phosphorylation [204]. 5-Fluoro-orotic acid was found to be an effective insect chemosterilant. When fed at a 1 per cent concentration (w/w in milk powder) for 24 h , it induced complete and permanent sterility in female adult houseflies (Muscu domesticu) [205,206] . Other biological activities of .S-fluoro-orotic acid have been discussed in Part I of this review. Some sulphur-containing derivatives, such as 2-hydroxy-4-mercapto [36a] (X), 4-hydroxy-2-methylthio-[36a](XI), 2-hydroxy-4-methylthio-[36a] (XII), and 2,4-bis(methylthio)-6-pyrimidinecarboxylic acid [ 36a] (XIII), can cause more than 50 per cent inhibition of orotic acid metabolism by cell-free extracts from mouse liver o r L5178Y leukaemia cells at concentrations less than 2 x lC3 M[207]. 5-Diazo-orotic acid (IX) has recently been found to possess good inhibitory activity against leukaemia L-1210 in mice [212] . Among 5-alkyl- or 5-aryl-substituted 4-pyrimidinecarboxylic acids screened for antiviral, antimalarial and antimicrobial activities [2 131 , 2-mercapto-5methyl-6-amino-4-pyrimidinecarboxylicacid (XIVa) is active against influenza

7

293

C. C. CHENG, BARBARA R O T H H

HN

H

N

/

C02H

M>eS ,M re

/

N\

C02H

(XI

H

(XI)

CO2H

COpH

(XI11

(XIIII

virus and slightly active against Plasmodium berghei; 2-hydroxy-S-ethyl-6-amino(XV) and 2-mercapto-5-phenyl-6-amino-4-pyrimidinecarboxylic acids (XIVb) are effective against herpes simplex virus. Compound (XIVb) also inhibits the growth of S. aureus and Candida albicans in virro. 2-Mercapto-5-ethyl-6-amino-4pyrimidinecarboxylic acid (XIVc) also shows activity in virro against S. aureus, but neither (XIVb) nor (XIVc) is active in infected mice [213].

oy7NHz ’

HsT$NHzCO,H R

Et

COZH

(XIV)a. R = M e b. R = P h c. R = Et

A number of 4-pyrimidinecarboxylic acids of the general formula (XVI) and their esters, amides and nitriles are claimed to possess cardiovascular, hypotensive, and spasmolytic properties [ 2141 . Other biological activities and medicinal uses of 5-halo-substituted orotic acids, as well as those of uracil-6-sulphonic acid and related compounds, have been discussed in Part 1 of this review [ 2 151 . R’r.”;.rR2 k

R’, R2 = NH2, O H , SH, NHR’, NR’R’’, NHNHR’ and NH.C(=NH).NHz v

R

3

C02H

R3

=

H, halogen, CN, NO*. NHz, O H , SH

at least one R being the N atom of a heterocycle.

(XVI)

Many N 3 substituted (N,,according to the uracil numbering system) orotic acids (XVII), prepared by the treatment of the appropriate hydantoins with sodium hydroxide, were reported to be useful as antiviral drugs [216]. H

H

oy>o RN

/ I

C02 H

(xvir)

R - Et ,(CH2),Me,(CH2),Me,(CH~)~Me, 0 - M e CsH4, p - M e C & I ~ , P - C L C s H ~ e t c

H2 NHC//C H

PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST

294

Maleuric acid (N-carbamoylmaleamic acid, XVIII), when injected into mice bearing Ehrlich ascites tumours, can produce cytoplasmic abnormalities in all phases of mitosis. This acid also inhibits the incorporation of tritiated thymidine into DNA, and prevents the progression of premitotic cells into mitosis [217]. This substance, which is an open-chain analogue of orotic acid, may possibly be an antimetabolic of this pyrimidine or related compounds. Among the 5-pyrimidinecarboxylic acids and esters, some 2,4-disubstituted derivatives provide interesting biological activities. Uracil-5-carboxylic acid [ 171 (XIX) was found to inhibit the formation of orotic acid from carbamoyl-Laspartate [ 801 . Ethyl 2-mercapto-4-hydroxy-5-pyrimidinecarboxylate (XXa) possesses antimalarial activity against Plasmodium berghei in mice [218] . The corresponding acid [2 191 (XXb) has antitubercular activity in chick-embryo but not in mice [220] . A number of 2-(substituted amino)4-hydroxy-S-pyrimidinecarboxylic acids, esters and amides (XXI; R3 = OH, OR, N H 2 ) have diuretic acid activity [ 22 1] . Among these, 2-amino-4-hydroxy-5-pyrimidinecarboxylic (XXI; R ' , R2 = H ; R 3 = OH) exhibits tuberculostatic activity [222]. This compound is a diaza analogue of p-aminosalicylic acid. H

H

H

OYX HN

R'

R2

NY2;oR3 N /

C02H

(XIXI

(XX)a R-OEt

(XXI)

b R-OH

Rxy3NH2 N\

(XXII) a X - C L b X-OH

(XXUI)a

C02Et

X

S.

(XXIV)

R - Et b X-S. R - CH,Ph c x-0. R - Et

Ethyl 2-ethyl thio-4-chloro-5-pyrimidinecarboxylate (XXIIa), as well as the corresponding 4-hydroxy-(XXIIb) and 4-amino-(XXIIIa) derivatives, possessanticytogenic activity on Neurospora crassa [223, 2241. Compounds (XXIIIa, b and c) were found to inhibit the conversion of orotic acid to the uridine nucleotides [202] . Ethyl 2-methylthio-4-(halo-substitutedaniline)-5-pyrimidinecarboxylates (XXIV), particularly the o-bromo- and the o-chloro- derivatives, substantially inhibit the growth of five experimental mouse tumours (Krebs-2 ascites carcinoma, Ehrlich carcinoma clone 2, leukaemia L-1210, carcinoma 755 and lymphocytic neoplasm P-288) [225]. Compounds of this type are usually prepared by the base catalysed condensation of ethoxymethylenemalonic esters or related derivatives with urea, thiourea, guanidine, or substituted amidine-type analogues [2 12, 225-2371.

<‘. C. CHENG. B A R B A R A ROTH

295

Several N-phenylsubstituted anthranilic acids and esters are potent antiinflammatory, analgetic and antipyretic agents. Replacement of the benzene ring by the pyrimidine ring caused retention of the anti-inflammatory and analgetic properties. For example, 4-(a,a, a-trifluoro-m-to1uidino)-5-pyrimidinecarboxylic acid (XXV, R’ = m-CF,, R2 = H) is orally active as an anti-inflammatory agent, and ethyl4-(2,3-xylidino>5-pyrimidinecarboxylate (XXV, R‘ = 2.3-Me2, R2 = Et) exhibits analgetic activity on oral administration [238-2401 . The most effective compounds in both the benzene and pyrimidine series have identical substitution patterns. A number of ethyl esters of I-phenyl-2-(substituted phenyl)-4-thiono6-methyl-5-pyrirnidinecarboxylic acids (XXVI, R = H, m-NOz, p-Br, etc.) are useful as sedatives, tranquilizers and bactericides [ 2411 .

(XXV)

(XXVI)

( XXVll)

Uracil-6-acetic acid [242] (XXVII), wherein the carboxylic acid group is attached to the pyrimidine ring through a -CH2- linkage, was found t o inhibit the germination of conidia of Peroriospora tabacina in vitro [ 2431 . THE URACILS The uracils represent an important class of physiologically active pyrimidines. The bases uracil (XXVIII) and thymine (XXIX) are ubiquitous in nature as constituents of the nucleic acids, where they are linked at the N1 position to chains of ribose-5-phosphate (in RNA) and deoxyribose-5-phosphate (in DNA), respectively. H

oT3° HN

/

H

oT2° ’ HN

(XXVIIJ)

Me

(XXIX)

Numerous publications on uracil and thymine and their role in controlling the metabolism, reproduction, and growth of living systems - in particular in the transcription of genetic information and biosynthesis of proteins - have already appeared in the literature. Therefore, these two important pyrimidines will not be discussed again in the present review. Some pertinent literature references are provided [244-2641.

296

P Y R I M I D I N I S O t BIOLOGICAL A N D hII.L)ICAL INTERLST

During the past decade, some monosubstituted derivatives of uracil have emerged as chemotherapeutic agents. As already discussed in Part 1 of this review, 5-fluorouracil (5-FU) and its deoxyriboside (5-FUDR) are anticancer agents of remarkable activity [264a] , 5-iododeoxyuridine is a chemical antiviral agent of high selectivity, and 5-niercaptouracil possesses some interesting an tineoplastic activity. As discussed in the preceding section, the 6-carboxy derivative, orotic acid, is an important metabolic intermediate. Many substituted uracils have recently been reported t o have pharmacological activity, and among the Nsubstituted uracils, an important class of herbicides was uncovered. Several 6-aminouracils have been used as diuretics for a number of years. The uracils in this section are divided into two classes, depending on the presence or absence of the biologically important amino substituents. Compounds which are primarily of chemotherapeutic or agricultural interest are also separated from those chiefly of pharmacological importance. Although it is tempting to classify the N-substituted uracils separately from the N-unsubstituted derivatives, it is not advisable t o d o so on a structure-activity basis, since frequently the N-substituent serves merely to transport the pyrimidine to the active site, where it is enzymatically removed, and the product then is the active drug. This has been noted particularly in the barbiturate series (vide irifra). Also, there is often only a change in degree, rather than type, of activity with N-substitution.

A. URACILS WITHOUT AMINO SUBSTITUENTS

1. Derivatives of chemotherapeutic or agricultural interest

6-Methyluracil-6-Methyluracil(XXX, pseudothymine, methacyl) stimulates the incorporation of adenine and uracil into the insoluble RNA factor but itself is not incorporated [265]. Thiseffect on the synthesis of nucleic acid has been reported t o be analogous to the hormone-like (cytokinin-like) activity of kinetin (6furfurylaminopurine) [265]. This activity can be illustrated by its action on leaf growth, chlorophyll preservation and intensity of protein synthesis. This pyrimidine inhibits the chlorophyll decomposition in barley and bean leaf segments and in pinto bean primary leaf halves attached to the plant. Like kinetin, it also stimulates the surface growth of pinto bean primary leaf halves and the incorporation of l-'4C-labelled glycine into the insoluble protein fraction of floating disks from pinto bean primary leaves [266]. Aqueous suspensions of 6-methyluracil, when administered orally t o rats, accelerate and intensify the assimilation of alimentary carbohydrates and increase hepatic glycogen synthesis [267] . 6-Methyluracil was found to decrease the toxic effects of large doses of potassium benzylpenicillin or Bicillin 5 and to eliminate the undesirable effect of dichlorotetracycline on tissue cells [268, 2691. The combination of one of these antibiotics with 6-methyluracil decreases the number of degenerating cells

and the level of their alteration, while markedly increasing and even normalizing culture mitotic activity, apparently by increasing the resistance of the cells to the toxic action of high antibiotic doses. However, the toxic action of the antibiotics oxacillin and levomycetin is potentiated in the presence of 6-methyluracil [ 3681 . H

Me

(XXX) When administered into the newly developed chicken embryo, 6-methyluracil was found to possess strong teratogenic activity. The compound produces abnormalities in 85 per cent of the embryos at 4 nig/kg [ 3 7 0 ] . In pregnant rats this pyrimidine causes damage to 10-day-old embryos. Microanatomical examination reveals urogenital system abnormalities (e.g. hydronephrosis, uretal oedema) [271]. Daily oral administration of 6-methyluracil to rats during increased motor activity (experimental training) induces adaption, i.e.. this pyrimidine increases the myosin and glycogen content and promotes the activities of hexokinase. UDP-glucose-glycogensynthetase and phosphorylase, hence it renders more economical expenditure of glycogen during muscular work and more rapid resynthesis during rest [272] . Gastric ulcers induced in rats by repeated administration of caffeine can be reduced by the administration of 6-methyluracil. Treatment is more effective when this compound is combined with either 2-methyl-4.6-dihydroxypyrimidine or cytosine [ 2 7 3 ] . In a study of the influence of 6-methyluracil and uracil on experimental blastomogenesis in mice, it was found that 6-methyluracil reduces the frequency of urethan-induced lung adenoma whereas uracil increases the instances [274] . 6-Methyluracil, like orotic acid, can restore experimentally induced infarction in rabbits [ 179- 1821 . 5-Ethyluracil - The growth of thymine-requiring E. coli mutants is inhibited by 5-ethyluracil [275, 2761 (XXXI). In E. coli 15 T,u p to 15 per cent of the

“3 H

HN

H

’ Et

(XXXI)

Me (XXXII)

thymine in the DNA is replaced by this pyrimidine [276] . No effect was observed in E. coli B. With S. faecalis 5-ethyluracil is less inhibitory than with the E. coli mutants. The riboside [275] and the deoxyriboside [277] o f 5-ethyluracil

298

PYRIMIDINES OF BIOLOGICAL A N D MEDICAL INTEREST

are considerably more potent inhibitors than the pyrimidine itself. The DNA from phage T3, with host cells E. coli CR34, had 66 per cent of the thymine replaced by 5-ethyluracil when the deoxyriboside was used [277]. Although the ethyl DNA was found to be less stable than the natural polymer, there appeared to be ,no appreciable steric hindrance to formation of the helical structure. In other studies it was found that 5-alkyluracils inhibited the degradation of uracil and thymine by rat tissue supernatants. It is believed that these compounds are competitors for dihydrouracil dehydrogenase, which catalyses the reduction of uracil and thymine [278]. 5-Hydroxymethyl-6-methyluracil(pentoxyl) - Pentoxyl (XXXII), which is readily obtained from 6-methyluracil and formaldehyde [279a] or chloromethylether [279b], has received considerable attention for treatment of various anaemic conditions and infectious diseases. A combination of folic acid and pentoxyl rapidly obviates the symptoms of anaemia in lead poisoning [280, 281 1 . This compound appears to be beneficial in experimental myocarditis [282]. In the case of severe blood loss, pentoxyl was found effective in stimulating protein metabolism and restoring serum protein [283]. This pyrimidine is a leucopoietic stimulant. Oral administration stimulates production of agglutinins in rabbits [284]. Pentoxyl stimulates the incorporation of m e t h i ~ n i n e - ~ ’into S a- and yglobulins [285]. When orally administered daily to dogs, pentoxyl causes the appearance of embryo-specific a-globulins I2861 . It has been postulated that pentoxyl may stimulate the mitotic activity of the liver cells which synthesize a-globulins. Protein synthesis is effected through the formation of specific RNA, thus directing it toward the formation of embryo-specific globulins [286]. In experimental nephritis, it arrests the elimination of the products of protein metabolism from blood. This pyrimidine is considered to be an anticatabolizer as well as an anabolizer [287]. Pentoxyl also is reported to have a beneficial effect on wound healing [288], to eliminate leukopenia in rabbits which was induced by tetrathione, and to raise the myelokarocyte count [289]. Tyrosine metabolism, after disruption by infectious hepatitis, can be restored by corticosteroids and pentoxyl [290]. It has some prophylactic action on lethal X-ray doses and decreases metastases in rat experimental SSK sarcoma [29 11 . Pentoxyl and 6-methyluracil strongly inhibit rat lymphosarcomas and powerfully stimulate leukopoiesis in rabbits with leucopenia [292]. When pentoxyl or uracil is administered into the stomach of mice, the antitumour action of simultaneodsly administered thio-TEPA on solid Ehrlich tumours is increased [293]. In experimental typhoid disease in mice treated with colimycin, the addition of pentoxyl prevents immunological disorders and stimulates an immunological reaction [294]. This compound potentiates the action of sulphonamides in mice infected with type I1 pneumococcus [295] . In combination with streptomycin, pentoxyl is beneficial in the treatment of experimental tuberculosis in guineapigs [296]. However, a single pharmacological report, relative to effects on the central nervous system, was unfavourable [297] . No mechanism for these activities has been suggested. It would seem likely, however, that this pyrimidine acts as a one-carbon donor. Beneficial results in

C. C. CHENG, BARBARA ROTH

299

combination with folic acid, for example, suggest this, as does an expe‘riment showing methionine incorporation [285] .

Thymine derivatives - 5-[N-(2-Amino-4-hydroxy-6-methyl-5-pyrimidinylpropy1)-p-carboxyanilinomethyl]uracil (XXXIII) was synthesized for study as a possible intermediate in the enzymatic synthesis of thymidylate. It is active as an enzyme inhibitor against thymidylate synthetase isolated from E. coli [298] . Certain thymine derivatives containing a 2-thioimidazole moiety ( X X X V , R = alkyl) inhibit growth of Ehrlich ascites carcinoma (fluid form) in mice [299].

I

H

(XXXIII)

OYNYO

COOH

H

N

d

C

~

N

(XXXV 5-Cyanouracif This pyrimidine (XXXV) can be synthesized either by the condensation of pseudoalkylthiourea with ethyl ethoxymethylenecyanoacetate followed by acid hydrolysis [300], or by the treatment of the corresponding cyanoethoxyacrylamide with aqueous ammonia [301,302]. In a study of the catabolic pathway of pyrimidines, it was found that the reduction of uracil was blocked almost completely by 5-cyanouracil (XXXV) in an in vitro test with the rat enzyme dihydropyrimidine dehydrogenase [303]. 5-Halogenated uracils and thymine are weakly active in this regard, and 5acetyluracil and 5-trifluoromethyluracil are completely inert. 5-Acetyluracil - Ethyl 2-hydroxy-4-methyl-5-pyrimidinecarboxylate (XXXVI), which is prepared by the cyclization of the ureidomethylene derivative of acetoacetic ester, can be caused to rearrange into 5-acetyluracil (XXXVII) in dilute alkali [304]. Compound (XXXVII) can also be prepared from dikdtene and ethyl carbamate, followed by treatment with ethyl orthoformate and cyclization with ammonia [305]. ~

H

H

O y ’ ’ oT3Me “ Z M e

HN

IXXXVII

C02Et

HN

(XXXVIII O

y

,-’ NO2

HN

(XXXVIIII

5-Acetyluracil can cause the production of certain pyrimidine-specific antibodies [306] . This interesting activity is responsible for the antibody precipitation of some mouse myeloma proteins [307].

300

PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST

5-Nitrouracil - This pyrimidine (XXXVIII) can be prepared either by direct nitration of uracil in boiling fuming nitric acid [308, 3091 or by nitration of 2-thiouracil or a 2-alkylthio-4-hydroxypyrimidine[3 101 . It inhibits the growth of L. casei, and the inhibition is partially reversed by thymine [31 I ] . 5-Nitrouracil has been regarded as a uracil antagonist [312] as well as a folic acid antagonist [311]. Its growth inhibition against L. leichmannii was reversed non-competitively by thymine, thymidine and 5-methylcytosine, but not by uracil or cytosine. Interference with an enzyme of the folate system was inferred [3 131 . E. coli, S. faecalis R and Enterococcus Stei take up 5-nitro~raci1-2-'~C into the cell and this action is inhibited by thymine or 5-bromouracil. The DNA of cells grown in tagged 5-nitrouracil was found to contain less than 10 per cent of the radioactivity; the substance isolated was no longer 5-nitrouracil. Its constitution was not determined, but the same product was also formed in small amounts under identical experimental conditions with 5-aminouracil [3 141 . 5-Nitrouracil has been found to display a stimulating effect on the growth of both the epigeous and subterranean parts of plants, suggesting its application in agricultural practice [315] . 5-Nitro-6-methyluracil, like 6-methyluraci1, was reported to produce abnormalities in 75 per cent of the embryos by interrupting basic development of the organs [270]. IDiazouracil- Diazotization of 5-aminouracil yields 5-diazouracil [3 16-3 181 (XXXIX). This pyrimidine inhibits cell division in bacteria and yeast [319] , as well as cell growth in tissue culture [320]. For example, 5-diazouracil possesses signifi-

H i d N ; .

(xxxrx) cant in vitro activity against a number of gram-positive and gram-negative bacteria such as Staph. aureus, S. faecalis, Proteus mirabilis, Salmonella typhimurium, Lactobacillus casei, E. coli, Pseudo. aeruginosa, and group D streptococcus species, micro-organisms often responsible for infections in acute leukaemia patients [321, 3221. This pyrimidine also possesses significant in vivo activity against two gram-negative pathogens - E. coli and Pseudo. aeruginosa [321]. The cell division-inhibitory effect of 5-diazouracil is not reversed by D Lpentoyllactone or by L-tyrosine [322]. In a study of the effect of 5-diazouracil on E. coli it was shown that synthesis of cell constituents continued when cell division was arrested, and that cell mass was not reduced, since atypical filament cells were produced [323]. 5-Diazouracil also inhibits virus production in plants [324, 3251, and is effective as a prophylactic agent against poliomyelitis in mice (but not in monkeys) [326, 3271 . It interferes with the conversion of orotic acid into uridine nucleotides, but is apparently not a direct orotic acid inhibitor [202].

C. C . C H E N G . B A R B A R A R O T H

301

5-Diazouracil is a rapidly acting carcinostatic agent, with a wide range of sensitivities to different types of animal tumour cells. Lymphomas and leukaemias are most sensitive to this compound. In an oxidative system 5-diazouracil may act on the enzymes of oxidative phosphorylation [204]. It is a powerful inhibitor of glycolysis in mouse tumour cells and it does not appear to act as a thymine or uracil analogue [328]. In several aspects, the activity of this chemically active pyrimidine [329] resembles that of the biological alkylating agents such as nitrogen mustard [330, 3311 and, like nitrogen mustard, 5diazouracil probably acts primarily via inhibition of hexokinase or phosphohexokinase, or both [328]. There appears to be little correlation between inhibition and cell viability [331]. This compound also inhibits the oxidation of a ketoglutarate by rat liver homogenates, whereas uracil does not inhibit the same reaction [323]. Quantitative evaluation of 5-diazouracil against Walker 256 carcinosarcoma, Ehrfch ascites carcinoma, C3H-FX lymphoma and other tumour systems has been studied and detailed results in comparison with other standard 'active' agents have been reported [332]. Clinically, this pyrimidine was found to produce prohibitive gastrointestinal and vascular toxic reactions at intravenous doses from 0.3 to 10 mg/kg and total doses of 1.25 to 126 mg/kg for periods up to 6 0 days. It is not considered to be an agent with significant antitumour effect [333]. 5-(Substituted azojuracils - The coupling of 5-diazouracil (XXXIX) with certain aminonaphthalene derivatives yielded a number of 5-(4-substituted amino1-naphthy1azo)uracils (XL) which exhibit high schistosomicidal activity against Schistosoma mansoni 1334-3361. Compounds of this type were selected from among 500 azo-substituted-heterocyclic compounds after extensive studies in experimental animals [334]. H

yz:=NBRz

0

HN

b. R ' a. c.

=

H,R Z2 = NH(CH2)2NI:t2 NH2

K ' = NH(CH2),NEt2. R' = H

(XL) The two most active compounds in this series are 5- [4-(2-diethylaminoethylamino)- 1 -naphthylazo] uracil (XLa) and 5-(4-amino- 1 -naphthylazo)uracil (XLb), (ANU). In mice infected with the Puerto Rican strain of S. mansoni, compound (XLa) causes an 82-100 per cent reduction in live worms when administered in the diet at 167-489 m d k g per day for 14 days [334], whereas ANU (XLb) causes a 73-100 per cent reduction at 221-804 mg/kg per day for the same period [335]. These compounds are reported to be more effective than either lucanthone hydrochloride [334, 3361 or the tris-(p-aminopheny1)carbonium salts [334-3371. Substitution of the amino groups at other positions in the

302

PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST

naphthyl ring, such as (XLc), resulted in loss of activity [338]. It is suggested that 1,4-naphthdenediamine may be the active metabolite [335] . The uracil moiety in (XL), nevertheless, plays an important role as the obligatory carrier. Clinical trials on 132 people infected with S. mansoni and S. haematobium indicated that these drugs are moderately effective in suppressing egg production, but rarely curative. There was a high incidence of gastrointestinal side effects [334]. 6-Benzyluracils and related compounds - The enzyme thymidine phosphorylase catalyses phosphorolysis of pyrimidine-2'-deoxynucleosides, such as thymidine, with the formation of 2-deoxy-a-D-ribofuranose-1-phosphate and the pyrimidine. The chief chemotherapeutic interest in finding an inhibitor of this enzyme lies in the search for an adjunct for tumour therapy with 5-FUDR (the 2'-deoxy-~-D-ribofuranoside of 5-fluorouracil) which will inhibit the phosphorolysis of 5-FUDR in the tumour cell lines [339]. 6-Benzyluracil (XLIa) was found to be a good reversible inhibitor of thymidine phosphorylase. The inhibition is caused by hydrophobic (lipophilic) interaction between the benzyl group and the enzyme [340]. This inhibition is enhanced with the substitution of a bromo group at the 5 position of the pyrimidine ring (XLIb) [341]. When the para position of the benzyl moiety of (XLIa) is substituted with a bromoacetamido group, the resulting compound (XLIc) is a slow acting active-site-directed irreversible inhibitor [342, 3431. The meta-bromoacetamido analogue (XLId) is not an inhibitor. A study of compounds related to 6-benzyluracil (XLIa), wherein the methylene bridge is replaced by NH, 0, S or CO, reveals that the phenylthio (XLIe) H

H

4

HN

Q Y

( X L I ) Q.X=

Y =H. A=CH, b.X=Br, Y=H, A=CH, c.X=H. Y =p-NHCOCH,Br,A=CH, d.X=H, Y =m-NHCOCH,Br, A=CH, e . X = Y=H,A=S f .X=Y=H,A=NH g.X=H, Y =2, 3-c1z9 A=NH

(XLII)

and the benzylamino (XLIf) derivatives are the best inhibitors of this group (4.2 pM and 6.2 pM concentrations required for 50 per cent inhibition, respectively). The benzoyl derivative has low activity [344] . It was postulated that for

C. C . CHENG, I3AKHAR.A ROTII

303

hydrophobic interaction it was necessaiy f o r the benzene ring to be out of the plane with respect to the pyrimidine ring. Increasing the bridge length between the two rings also resulted in decreased inhibition. 6-Aminouracils (vidc infru) also inhibit this enzyme. The most potent thymidine phosphorylase inhibitors obtained to date are 6-anilinouracils containing substituents in the benzene ring [345] . The marked increase in activity produced by introduction of orrho substituents is in line with the postulate that the two rings must not be coplanar. The 2,3-dichloro derivative (XLlg) and 6-(2-anthrylamino)uracil (XLII) were found to be complexed 1 100 times better than the substrate, 5-FUDR. From these and related data, a proposed map of the lipophilic bonding region of E. coli B thymidine phosphorylase was drawn [345] as a working basis for future compounds. N-Alkylurucils - Several 3-alkyl- and 3,6-dialkyluracils were found in 1962 to have high phytotoxicity against a wide variety of plants and t o possess low mammalian toxicity [346] . One member of this class, 3-s-butyl-5-bromo-6methyluracil (XLIIla, bromacil) has gained wide acceptance as a potent herbicide. The corresponding 3-isopropyl analogue (XLIIIb, isocil) has also had wide testing. At low concentrations, these compounds cause a complete kill of a variety of annual broadleaf and grass weeds; under similar experimental conditions 5bromouracil and 6-methyluracil are inactive. A large number of related compounds with similar properties have been described in the patent literature [347-3601. Such compounds can be prepared by heating alkyl isocyanates with methyl 3-aminocrotonate to form methyl 3-(3’-alkyl-substituted ureido)crotonates; direct cyclization t o uracils can be effected by treatment with dilute alkali without isolation of the intermediate. Halogenation at Cs is then readily accomplished with bromine or chlorine.

(XLIII)

a

R

-

CH(Me)CHzMe ,X

b R = CHMe2, X

c

R =(CH&Me,X

-

-

Br

Br

=H

Bromacil and isocil have been found to be potent and specific inhibitors of photosynthesis at the chloroplast level [361]. The uptake of carbon dioxide is blocked in Chlorella pyrenoidosa and growth inhibition parallels the inhibition of carbon dioxide uptake. In Euglena grucilis and in spinach chloroplasts, the blockage of oxygen production was noted [361]. In growth studies with E. coli, it was found that bromacil (XLIIIa) was not a metabolic analogue of 5-bromouracil, and was not incorporated into the DNA

304

P Y K I M I D I N E S OF BIOLOGICAL A N D MEDICAL INTEREST

of this organism, nor into the DNA of mouse spleen and liver [362, 3631. Further studies on a bacteriophage [364] showed that these compounds, in contrast t o 5-bromouracil, did not increase the back-mutation rate of several AP72 mutants. The alkyl-substituted 5-bromouracils showed no evidence of influencing the effects of 5-bromouracil. Mutagenicity does not appear to be a characteristic of these compounds, and they seem t o be biologically inert relative to DNA synthesis. Both bromacil and isocil have recently been found t o be inhibitors of nitrate reductase formation in leaves [365]. The uracils with herbicidal activity d o not necessarily contain 5-halo substituents. 3-Cyclohexyl- 5-methyluracil [ 3 541 (XLIV), 1,3-di-isopropyI-6-methyluracil [352] (XLV) and 3-s-butyl-5-thiocyanato-6-methyluracil 13531 (XLVI), for example, are cited as having this type of activity. 3-Butyl-6-methyluracil (XLIIIc) possesses interesting selective activities. For instance, this pyrimidine kills many annual weed species without damage t o peas and peanuts, even when applied at twice the concentration needed to kill the weeds [346]. On the other hand, the related 5-bromo derivatives, such as (XLIIIb), are useful as industrial herbicides where it is desirable t o kill all plants [346] . Me

E< ,Me

Me

‘C ri

CH

HN+CN Me

(XLIV)

(XLV)

(XLVI)

l-Allyl-3,5-diethyl-6-c~orouracil(XLVII) and other related 1-allyl-5-alkyluracils [ 366, 3671 possess chemotherapeutic value against herpes and vaccinia viruses in tissue cultures, as well as against experimental Herpes keratitis in rabbits. In clinical evaluation, this compound is efficacious in the treatment of all types of recurring herpetic skin and mucous diseases, including Herpes genitalis and Stomatitis aphthosa [366] .

( X LVI I I

(XLVI l l )

2. Derivatives of pharmacological interest Although uracil possesses a structure very similar to that of barbituric acid, it is a much weaker acid (pKa: 9.38) [368]. Also unlike barbituric acid, it is not

<'. <'. CHENG. B A R B A R A ROTH

30s

appreciably ionized at physiological pH. Early studies to determine possible pharmacological activities of uracil and thymine were without success [ 3 6 9 , 3 7 0 ] . Later investigations showed that although uracil and thymine are devoid of hypnotic activity, they do potentiate the sleeping of mice in admixture with hexobarbitone [371] . Orally, 2 mM/kg of uracil, when administered with hexobarbitone. caused a 78 per cent increase in sleeping time. 6-Methyluracil produced similar effects. It was postulated that uracil and barbiturates might compete with liver enzymes involved in the biotransformation of hexobarbitone [37 1 1 . The pharmacological activity of a series of 3,5,6-trialkyluracils was studied, considering them as analogues of barbit one. and i t was found that they did possess sedative action. The 3,5-dibutyl-6-methyI derivative (XLVIII) was reported to be a comparatively potent sedative agent [372] . Partition coefficient and metabolic studies would be of interest in comparing such compounds with the barbiturates. Uracil and thymine are both reported t o have electroshock anticonvulsant activity [ 3 7 1 ] . A series of 5- and 6-alkyl derivatives was prepared [373] and tested for electroshock as well as for metrazole protection [ 3 7 4 ] . It was found that most compounds of this type were active in the electroshock test. There is a trend toward increased activity with increased size of the alkyl groups, and introduction of 1,3-dimethyl substituents is also of benefit. Against metrazoleinduced shock, however, there are n o obvious structure-activity relationships. It is claimed that certain N' substituted 6-chlorouracils, such as (XLIX), increase tolerance to electric shock [375]. 3-Phenyluracils with alkyl groups substituted in the 5- and 6-positions. such as (L), are reported to have analgesic and antipyretic activity [376, 3771. The I-position may also be alkylated. Such compounds are prepared by the treatment of a-alkyl-P-aminocrotonates with phenyl isocyanate. When the 5-substituent is an isopropyl group. the resulting compound is a sodiuretic without increase in potassium excretion.

(XLIX)

Hw# Me

(CH,

l3

Me

(L) The cycloalkylamine salts of 5-cyano-1 -uracilacetic acid and analogues, exemplified by (LI), are claimed t o produce marked inhibition of gastric secretion with virtually n o anticholinergic activity [ 3 7 8 ] . This activity is not inherent in the free acetic acid nor in the amine. There is n o distinction between optical isomers of the longer chain acids. The pyrimidine portion can be prepared by the treatment of a-cyano-0-ethoxy-N-(ethoxycarbony1)acrylamide with alanine or related derivatives [301, 3021.

306

PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST

The product of oxidation of uridine with periodic acid, 6 - ~ -1(-uracilyl)-5hydroxy-p-dioxane-2-D-carboxaldehyde (LII) is reported to give an adduct with H

H

HOOCCH,

CN

potassium bisulphite which has anti-inflammatory action when administered parenterally [379]. The cytosine analogue is reported to have similar activity. Additional information relative to dihydrouracils with anti-inflammatory activity is found in the section on reduced pyrimidines (hydropyrimidines).

B. URACILS WITH AMINO SUBSTITUENTS

1. Derivatives of chemotherapeutic interest

5-Arninouracils-5-Aminouracil (LIII), which is prepared by reduction of 5-nitro- or 5-nitrosouracil [380] or by the treatment of 5-bromouracil with

(LIII)

ammonia [381], is a growth inhibitor of L. casei [311], S. faecalis [382] and E. coli [383]. In L. casei, the inhibitory action was competitively reversed by thymine [31 I] , and in S. faecalis, by uracil and thymine [382]. 5-Aminouracil is assumed to be a thymine antagonist that blocks DNA synthesis [384-386]. It has been found to possess mutagenic activity in E. coli, and the action was not annulled by thymine or thymidine [387]. This pyrimidine is incorporated to a small extent into the DNA of Enterococcus Stei [314], but is apparently unincorporated for the most part as cytosine and thymine, plus an unknown radioactive derivative. In the presence of 5-aminouracil, a thymine-requiring mutant of E. coli was found to incorporate greater amounts of 6-methylaminopurine [388, 3891. 5-Aminouracil also induces a defective phage and increases DNA methylase activity in E. coli 15T [390]. Addition of 5-aminouracil to a medium containing Mycobactenurn tuberculosis inhibits the formation of adaptive enzymes for the oxidation of benzoic acid by the organism [391]. Other

C. C. CHENG. B A R B A R A ROTH

307

pyrimidines and purines, such as 2-thiouracil, 2-thiothymine,.6-mercaptopurine and 2,6-diaminopurine, have a similar effect, but 2-thio-orotic acid is inactive. On the other hand, 5-aminouracil and 2-thio-orotic acid inhibit the formation of the adaptive enzymes for the oxidation of myoinositol, but 2-thiouracil has no effect [391].

Y3:HMe

R2

o+ A :r

k1

( L I V ) a. R=2'-deoxyribosyl (LV)

b. R = H

5-Aminouracil produces a block in the mitotic cycle of various plants. For example, cessation of mitosis occurred in Vicia fuba roots incubated 24 h with this compound [392-3941 . Depending on different experimental conditions, thymidine or thymidylic acid may or may not alleviate these effects [392-3941. I t was concluded that 5-aminouracil depressed the rate of DNA synthesis, which led to an accumulation of cells in the S phase. After removal of the agent, DNA synthesis resumed. Similar results have been observed with Allium cepa and Huplopappus grucilis [395, 3961 . Inhibition of guanosine incorporation into RNA of meristematic cells in Viciufaba by 5-aminouracil was also reported [397] . Among various pyrimidines tested in uitro as inhibitors of the active transport of uracil across the rat intestinal wall, strong inhibition was seen with 5-aminouracil, 5-fluorouracil, 5-bromouracil and thymine [ 3981 . The deoxyriboside derivative of 5-methylaminouracil (LIVa) is a specific antiherpes agent [399]. The aglycone itself (LIVb) as well as 5-aminouracil, however, has no antiviral activity [399]. Some anilinouracils of the general formula (LV, R ' , RZ = H, alkyl) were claimed to suppress the immune response in animals [400].

Yi

~ : J , ,NH NH2

(LVI) a . X = H

NH2

trvrr)

OY! HNJO

NH i=w*

(LVII I )

b.X:Me c.X=Br d.X=I e.X= F f. X I NH2 g.X =NH-ri b i tyl

6-Aminouracils - 6-Aminouracil (LVIa) significantly inhibits Crocker sarcoma in mice [40 11 . The corresponding 5-methyl derivative, 6-aminothymine (LVlb),

308

PYRIMIDINES OF BIOLOGICAL A N D MEDICAL INTEREST

has no effect on the same tumour [401]. Compound (LVIb) and 6-aminosubstituted derivatives of 5-bromo-, 5-iodo, and 5-fluorouracil (LVIc-e) are powerful inhibitors of thymidine phosphorylase in vitro [402] . Studies t o elucidate the mode of formation of riboflavin in the riboflavinsynthesizing organism Eremothecium ashbyii [403] have indicated that 5,6diaminouracil (LVIf) is a probable precursor in flavinogenesis. An extension [404, 4051 of this observation states that the ribityl derivative of 5,6-diaminouracil is converted by E. ashbyii and Aerobacter aerogenes into 6,7-dimethyl-8ribityllumazine, the latter being the immediate precursor of riboflavin [406] . This postulate was later confirmed by the isolation of 5-amino-6-(ribitylamino)uracil (LVIf) from a riboflavinless mutant of Aspergillus nidularzs [407] . A structurally related 2-thio analogue, 4-amino-5-methyl-2-thiouracil(LVII) possesses growth inhibition against L. leichmannii. This inhibition could be reversed in a non-competitive manner by vitamin B I Z [408]. The condensation product of 6-uracilylhydrazine and 2-pyridinecarboxaldehyde (LVIII), shows a pronounced in vitro antitumour activity in the Miyamura test [409]. A number of 1-aryl-6-aminouracils (LIX; Ar = fluoro- or trifluoromethylsubstituted phenyl, X = H, NO or N H z ) were found to possess antiviral activity against rhinovirus 1059. Among these, 1 -(m-trifluoromethylphenyl)-5,6-diaminouracil (LIX; Ar = m-CF3, X = NH2), possesses antibacterial activity against Bacillus subtilis, Staph. aureus and Mycobacterium smegmatis [410] .

H

Me

OTy

A;’

X

OTJO HN

’

NHAc

NH2

I-Alkyl (or aryl)-6-aminouracils, obtained from N-alkyl or N-arylureas and ethyl cyanoacetate, are reported to be useful for prevention and therapy of diseases caused by viral pathogens [411]. 3-Methyl-5-acetamido-6-aminouracil (LX) has been isolated as a metabolite in human urine I4121 . Preliminary observations indicate that the excretion may be elevated by oral intake of caffeine, but this pyrimidine has also been detected clinically in a controlled, caffeine-free, low purine diet. Treatment of certain uracils, such as 1 -methyl-6-amino-,3-methyl-6-aminoor 1,3-dimethyl-5,6-diarninouracil with formaldehyde gave compounds containing between 10-30 per cent by weight of reversibly bonded formaldehyde. These new compounds were claimed to be useful as disinfectants or antiseptics [413].

309

C. C. CHENG. B A R B A R A R O T H

2. Derivatives of pharmacological interest A number of 1,3,6-trialkyluracils containing 5-amin0, 5-alkylamino or 5substituted amido groups, such as compounds (LXI) and (LXII), have been synthesized [414-4201 as analogues of antipyrine (LXIII). I

Me

(LXI 1

I

I

Me

Me Me

Ph

Me

(CH&Me

NMe,

Me

(LXII)

(LXIII)

The trialkyl-5-amino derivatives possess analgesic activity. Their quaternary salts with methyl bromide are very toxic. Most 5-dialkylamino derivatives, as well as the amides, showed analgesic, hypothermic, and antipyretic activity, with less toxicity than aminopyrine. They also appear to have a tranquillizing action. In attempts to prepare methylated xanthine analogues with greater therapeutic effectiveness as diuretic and cardiac drugs than theophylline (LXIV) and theobromine (LXV), it was found that some of the intermediate 6-aminouracils (LXVI) showed considerable activity as oral diuretics in experimental animals

(LXIV)

(LXV)

(LXVI)

[43,1]. A comparison of various derivatives, where R' and R2 are either H or alkyl, revealed that N-monosubstituted 6-aminouracils have little or no activity. Among the 13-dialkyl analogues, several C, t o C4 derivatives were highly active. Where R' and R2 are methyl and propyl, the isomeric propyl methyl derivative shows comparable effectiveness. These activities are of the same order as those of aminophylline, but the toxicities are less than half as great. From these studies the drug amisometradine (Rolicton (LXVI), R' = CH2 .C(Me)=CH2, R2 = Me) eventually developed. The mode of action of compounds of this type in man appears to involve specific inhibition of tubular reabsorption of sodium and chloride. This is not

310

PYRIMIDINES OF BIOLOGICAL A N D MEDICAL I N T E R E S T

accompanied by an increase in glomular filtration rate, nor by a change in the factors regulating acid-base balance [422-4241 . Clinical studies in non-edematous subjects showed an increase in urine volume and loss in body weight [425] . The maximum effect is achieved within 6-12 h [426]. A minimum of side effects have been noted with amisometradine in contrast with the analogue aminometradine (Catapyrin, Katapyrin, Mictine, Mincard, (LXVI), R' = CH2 .CH=CH2, R2 = Et), which produced anorexia, gastric irritation, and nausea in most patients. Amisometradine may be classified as a useful, but only moderately potent, oral diuretic. Its use is indicated for the treatment of patients with mild heart failure, or for prolonging the period between injections of organomercurials in patients with moderately severe to severe congestive heart failure, but not for initial diuresis in severe cases [426, 4271. The electrolyte excretion is potentiated by carbonic anhydrase inhibitors [428] . Preparation of compounds of type (LXVI) may be accomplished by the condensation of substituted ureas R'NHCONHR' with cyanoacetic acid in acetic anhydride. The intermediate R' NHCONR' COCH2CN can then be cyclized in base [429, 4301 . With monosubstituted ureas, the initial condensation proceeds almost exclusively on the unsubstituted nitrogen of the urea. When both nitrogen atoms are substituted, condensation occurs primarily, although not exclusively, on the nitrogen to which the smaller substituent is attached [ 4 2 1 ] . With N' -ethyl-N'-allylurea, for example, the product is predominantly (80%) 1-allyl3-ethyl-6-aminouracil (aminometradine). Alternative preparative synthetic procedures have also been reported [421]. Pharmacological actions - 5-Alkyl derivatives of (LXVI) are also cited as having diuretic activity [43 11 . Certain 1,3-dimethyl-6-alkylaminouracils are reported to have diuretic activity similar to that of theophylline [432]. 1,3Dimethyl-5-ethyl-6-(methylamino)uracil(LXVII) was found to be among the most active of the compounds tested. Diuretic and hypotensive activity are also claimed with anils of 5-amino derivatives, such as (LXVIII) [433] .

5-Carboxymethylthio derivatives of 1,3-dialkyl-6-aminouracils (LXIX) and their amides possess anti-inflammatory activity, as evidenced by their ability to inhibit local edema formation [ 4 3 4 , 4 3 5 ] . Bronchodilator and CNS stimulant activity are also claimed. The related 1-aryl derivatives (LXIX; R' = Ar) are reported t o have anti-inflammatory, hypocholesterolemic, pepsin-inhibiting, and anti-ulcer properties [436] .

C. C. CHENG, BARBARA ROTH

311

5-Cyanoethyl derivatives of 1-alkyl and 1,3-dialky1-6-amino uracils (LXX) are reported to have activity as anti-ulcer agents. Inhibition of appetite and hypocholesterolemic activity are also claimed [437]. RZ

R2

I

I

NH2

( L X XI

(LXIX 1

5-Ureido derivatives of I-alkenyl-3-alkyl-6-aminouracils, such as (LXXI), are reported to have particular activity as appetite inhibitors. They also have antisecretory, anti-iritic and mild diuretic activities 14381 . The related 5-sulphonamides, such as (LXXII), are said to inhibit the appetite without producing other pharmacological responses such as pressor-depressor, or diuretic effects [439].

(LXXI)

(LXXII)

The 5-chloro derivative of (LXVI), where R’ is propyl and RZ is ethyl, is reported to have activity as a smooth muscle relaxant [440],as determined in both experimental animals and isolated muscle.

HYDROPY RIMIDINES Difficulties are often encountered in conducting a careful search of the literature relating to hydropyrimidines or reduced pyrimidines. This is due largely to the nomenclature used to designate these compounds. The names of many compounds arbitrarily given as dihydro-,tetrahydro-, and hexahydropyrimidines, depending on the number of ‘tautomerizable’ groups present in the pyrimidine derivatives, seem unnecessarily confusing. True hydropyrimidines (see following illustration) are those that contain less than three exocyclic and endocyclic

PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST

312

double bonds [441], and only compounds of this type are discussed in this section. H ..

ON Y y M:

1-Methyluracil l-Methyl-2,4-dioxo-l,2,3,4-tetrahydro-l~,3~-pyrimidine, Not a hydropyrimidine (three exo- and endocyclic double bonds). A less confusing nomenclature is l-methyl-2,4(1H,3H)-pyrimidinedione, which is used by Chemical Abstracts as an alternate listing.

oy3NHz HN

5.6-Dihydrocytosine 2-0x0-4-amino-l , 2,s ,6-tetrahydro- 1H-pyrimidine. A dihydropyrimidine, not a tetrahydropyrimidine (two exo- and endocyclic double bonds) 4-Amino-5,6-dihydro-2( Iff)-pyrimidone (Chem. Absfr.) assigns the correct oxidation state, tautomeric form and location of the proton; the danger is that the latter is not always known, however.

Relatively little attention has been focused on hydropyrimidines. The real significance of hydropyrimidines, especially dihydropynmidines, in biochemical reactions is perhaps on the threshold of being uncovered. Biological intermediates are, in general, very rarely generated from a single precursor or through one single reaction route. The well known conversion of ureidosuccinic acid to orotic acid through dihydro-orotic acid (IV) in pyrimidine biosynthesis has been discussed in the pyrimidine carboxylic acid section [73, 76, 811. Dihydrouracil, which is believed to be unrelated to the orotate system, is also incorporated in the anabolism of pyrimidines by certain biological systems [442-4481. It has long been recognized that dihydropyrimidines are important intermediates in the catabolism of pyrimidines [72,449-4621 . Since most reactions in biological degradative systems are reversible [463, 4641, it is reasonable to assume that many hydropyrimidines and their derivatives may play important roles in nucleic acid biosynthesis. The report that dihydrocytidylic acid was isolated from liver [465] has led to the postulation that some dihydrocytosine derivatives may be precursors of cytidine and related compounds [466-4701. Recent discovery of the repeated appearance of the 5,6-dihydrouracil unit in rrunsfer-RNA (solubleRNA) [471-4791 further suggests the importance of this hydropyrimidine in protein synthesis, although the exact function of the 5,6-dihydroura'cil unit in RNA is not yet known. In this connection it should be noted that the sugar moiety can be removed from dhydrouridine under hydrolytic conditions with much greater facility than from uridine itself [480] , which has possible biological implications. The corresponding 5,6-dihydrocytosine unit has not been found in RNA. Perhaps this is a result of the ready hydrolysis of the 4-amino group of 5,6-dihydrocytosine derivatives [468, 48 1-4821 , to yield the corresponding 5,6-dihydrouracils. Preparative methods for dihydro-, tetrahydro-, and hexahydropyrimidines have been comprehensively illustrated and discussed [441,483-4851.

C. C. CHENC,. B A R B A R A ROT14

313

The pyrimidone ring system is not fully aromatic, since such compounds exist as amides, rather than hydroxypyrimidines, and the n-system does not extend fully around the ring. In the case of the uracils, the 5,6-double bond possesses allylic character, and various relatively stable adducts can be obtained, for example, with bromine or chlorine water [ 7 10, 486-4881 . Catalytic reduction across the 5,6-double bond is also readily accomplished [441, 489-4911, The discovery nearly a hundred years ago that ultraviolet light had a lethal action on bacteria [ 4 9 2 ] , led to the eventual finding that the most sensitive point of attack lay in the pyrimidine bases of the nucleic acids of the cells [493-4971 . In model photochemical studies with the free bases and nucleosides, it was found that irradiation of uridylic acid, uridine. or uracil solutions at 230-280 nin results in the disappearance of the characteristic pyrimidine ultraviolet absorption around 260-270 nni [ 4 9 8 ] . This absorption maximum can be largely restored in the dark by acidification o r heat treatment of the irradiated solutions [498-5031 , and the reconstituted substance behaves biologically like the original [SO 11 . Subsequent study using 1,3-diinethyluracil revealed that the disappearance of its absorption maximum, upon irradiation, is due to the addition of a molecule of water across the 5,6-double bond [SO?] to yield 1,3-dimethyl6-hydroxy-5,6-dihydrouracil[ 502-506] (LXXIII). The possibility of formation (LXXIV) has been of the isomeric 1,3-dimethyl-5-hydroxy-5,6-d~hydrouracil ruled out by unequivocal experiments [SO?, 5031 and indeed would be unlikely since there is a greater electronic deficiency at C6 than a t C 5 . The mechanism was postulated as a 1,4-addition [ 503-5051 , analogous to the acid-catalysed reaction with a carbonyl conjugated system [504]. The fact that hydration in the absence of light has not been observed under any conditions suggests that the photohydration intermediates are high-energy species, either in the electronically excited singlet or upper vibrationally excited ground state [507]. Under similar experimental conditions, uracil, uridine and related derivatives yield analogous dihydropyrimidines [499, 506-5 121 . Reversal o f the irradiation effects by acid, or by heat treatment in the dark, is believed due t o removal of the water molecule with reformation of the 5,6-double bond. Cytosine, its Me

( LXXII I )

Me

ILXXl v 1

nucleosides and nucleotides, and 1-methylcytosine have been reported t o undergo similar reversible photolysis reactions [5 13-5 181 . (Some uracil is formed from irradiated cytosine due t o deamination [519] ). Thymine (5-methyluracil) and related 5-substituted uracils d o not yield isolable dihydropyrimidines under similar irradiation conditions [507,515,5201 . Uptake of a water molecule was at first discounted [521]. Subsequent study

314

PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST

revealed that the photo-addition of water does take place, but the reversal dehydration reaction is much faster under ordinary experimental conditions [522] ,which hampers the isolation of the dihydrothymine derivatives. In attempts to isolate the aforementioned irradiated products of thymine derivatives at lower temperature, the photochemical reactions were carried out in frozen aqueous solutions containing either thymine or 1,3-&methylthymine. The resulting products were not hydrates, but had elementary analyses corresponding to the starting material. Molecular weight determination indicated that the products were dimers, and infrared and ultraviolet spectral data suggested cyclo addition across the 5,6-double bond to form a cyclobutane system

' I

0

cis-syn

0

0 0

trans -syn

trans-onti

LXXVd

LXXV c

[523-5341. There are four possible structures for such a product (LXXVa-d). The thymine ice-dimer was proven to have the cis-syn configuration (LXXVa) by transformation into an isomer shown to be a derivative of 3,5,9-triazatric y c l 0 [ 5 , 3 , 0 , 0 ~ decane ~ ~ ] of cis-cis-cisconfiguration (LXXVI) [535-5361.

"8: CI ..

Me H

HNKNCoNH2 0 (LXXVI )

Irradiation of I-methylthymine in frozen aqueous solution produces both the cis-syn and trans-syn isomers, whereas 3-methylthymine yields only a small

C. C. CHENG, BARBARA ROTH

315

amount of one dimer, of the cis-syn configuration [537]. 1,3-Dimethylthymine in frozen solution produces two dimers, and one of these is different from the above two types, indicating that it has one of the anti configurations [530,537] . Uracil behaves similarly in frozen solution to undergo cyclo addition [527, 538, 5391. The synthesis of the trans-syn and trans-anti dimeric uracils has recently been accomplished [ 5401 . The ultraviolet irradiation of DNA produces two photoproducts from thymine. The major product is identical with thymine ice-dimer (cis-syn, LXXVa), and accounts for 91 per cent of the DNA-derived photoproducts [541, 5421. A uracil-thymine dimer is also obtained in smaller quantity, which arises from deamination of a cytosine-thymine dimer [543, 5441. The cis-syn thymine dimer from DNA is believed to be formed from intrastrancl dimerbation of adjacent thymine residues. Photoaddition of two unsaturated molecules in the solid state can arise only if they are initially located in proximity in the crystal lattice [545, 5461. The formation of interstrand dimers would require gross distortion of the helical structure of DNA in order for the bases to approach the limiting distance (c. 4 A.) Hence, such dimers would be formed in only very small amount. However, the composition of the photoproducts may differ under varying experimental conditions [547-5501. It has been found that photochemical reactions of monomers in aqueous solutions, in frozen aqueous solutions, and in solutions containing organic solvents or photosensitizers differ from each other [527, 551-5581. The formation of cyclobutane dimers has been widely observed with a$-unsaturated ketones (5591. Diradicals are favoured as the reaction intermediates. The probability of dimer formation depends on the distance between the two reacting molecules, and therefore photodimerization occurs mainly in the solid state. It has been established that frozen aqueous solutions of 1,3-dimethylthymine are heterogeneous, with optical differences occurring in different layers [ 5341 . On the other hand, corresponding solutions in glycerol form a rigid glass on freezing, and the dimethylthymine molecules are separated by about 100 A. There is no reaction on irradiation, apparently since the molecules are much too far apart. Other experiments established that this is not a temperature effect in this case. In DNA, on the other hand, the temperature of irradiation can make a big difference [548]. Thin ‘dry’ films of thymine have been‘found to form dimers in variable amounts. This was found to be a function of moisture content. The structure of thymine crystals is dependent on water of crystallization. In its absence, the crystal framework collapses, and no dimerization occurs [528] . Biological, chemical, X-ray diffraction, infrared absorption, e.s.r., n.m.r., luminescence, and quantum studies show that dimer formation is universally observed in irradiated frozen solutions of thymine, thymidine, uridine, thymidylic acid and related compounds, and in DNA [560-5761. The purines of DNA, on the other hand, are little affected [577, 5781. Thymine dimers obtained in frozen solution can be converted to the original monomers by ultraviolet

316

PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST

irradiation after thawing [ 525-5281 (i.e., in homogeneous solution), and can be broken in frozen aqueous glasses at 80"K, followed by melting t o form the monomer [ 5 7 9 J . Earlier, it was found that ultraviolet irradiation damage in micro-organisms could be partially reversed by subsequent irradiation with light of lower wavelength [580, 5811 . Orotic acid readily forms dimers even when irradiated in liquid medium [582, 5831, 5-Bromouracil (5-BrU) in DNA is dehalogenated, rather than forming cyclobutane-type dimers. Such DNA derivatives are more sensitive to ultraviolet irradiation than normal DNAs [ 584-5941 . Irradiation of 5-bromcuracil and derivatives in aqueous medium produces 5,5'-diuracil [590, 5911 . However, derivatives such as 3-sbutyl-5-bromo-6-metliyluracil have been reported to yield cyclobutane dimers either by irradiation of frozen aqueous solutions, or by catalysis with free radical initiators, such as aluminium chloride, ferric chloride, peroxides or azonitriles [595] . 5-Hydroxymethyluracil is reported to dimerize very slowly in frozen water at 2537 A [596] . The fundamental research in the photochemistry of the nucleic acids, the monomeric bases, and their analogues has stimulated new experiments in certain micro-organisms and approaches in such diverse fields as template coding and genetic recombination [ 597-6 161 . Although a large volume of compelling evidence has indicated the formation of thymine dimer as the principal ultraviolet damage in DNA [617], some experiments clash with this interpretation. Thymine dimer and its nucleoside and nucleotide derivatives failed to give competitive inhibition of DNA repair in the presence of yeast photoreactivating enzyme, even at high concentrations [618] . Furthermore, some cells can remove thymine dimers very efficiently from the nucleic acids, so such ultraviolet damage can have very little to do with inactivation of the cells. Alteration in deoxycytidine C may be important in non-photoreactive damage to DNA [604]. The ultimate meaning of these results remains to be ascertained. Pharmacologicalactions - Certain dihydrouracils and their 4,6-dioxo analogues and related compounds have interesting pharmacological uses. The anticonvulsant [618a] and antispasmodic activity of a group of compounds of type (LXXVII; R' = alkyl, R2 = aryl) have been extensively studied [619-6361. The most important member in this series is primidone, (hexamidine, primaclone, Mysoline, Mizodin, Mylepsin, Mylepsinum, Lepsiral, LXXVII, R' = Et, R2 = Ph). H

HN

0

(LXXVII)

This compound is a closely related analogue of the anticonvulsant barbiturate phenobarbitone (phenobarbital; Part 111, Volume 8), in which the 2-carbonyl

<‘. C. CHENG, BARBARA ROTH

317

group has been replaced by a methylene linkage. A comparison of the activities of these two drugs gives indication that the 2 - 0 ~ 0function is not an important element in binding to receptor sites. Primidone depresses the respiratory quotient of brain and liver. It is effective against epilepsy with only slight side effects. In studies of convulsions caused by hyperoxia in white mice, a dose of 400 mg/kg can cause a delay of the onset of convulsions by a t least 10 min, and at 500 mg/kg it causes delay and attenuation of epileptic symptoms 16271. The effectiveness of this compound as an anticonvulsant agent against electroshock and metrazol (leptazol) seizures is shown by a report that a single dose of 5 nig/kg of primidone abolishes the tonic extensor component of electrically induced seizure in 60 per cent of rats, and 20 mg/kg prevented maximal metrazol seizures in over 50 per cent of rats [620]. At doses from 0.1 to 0.33 g/kg given to rabbits a t 30 min before electric shock, the electroencephalograms show effects ranging from complete antispasmodic action to alleviation of the normal convulsion [628] . Antispasmodic effects appeared first in the cortex, then in optical papilla, and lastly in the caudate nucleus 16281 . Administration of primidone t o dogs with experimentally induced epilepsy (reflex epilepsy is introduced using strychnine, and faradic epilepsy by electric stimulation of the cortical motor zone) reveals that 3 mg/kg of primidone intravenously, or 5 mg/kg orally prevents the localized strychnine clonus and inhibits reflex epilepsy in all the animals; 5 mg/kg intravenously inhibits faradic epilepsy in 6 0 per cent and 7 mg/kg in all the animals [621] (for comparison, 12 nig/kg of phenobarbitone is required to inhibit reflex epilepsy, and doses up to 40 mg/kg d o not prevent localized strychnine clonus). Primidone suppresses the bioelectrical activity of the cortex somewhat sooner than that of the subcortex [630]. Clinically, it was found that for epilepsy patients, the optimal dose for adults is 1.6-2 g/day; for children it is 1-1.5 g/day. Under this dosage complete disappearance of epileptic seizures is noted in over 20 per cent of cases [619, 6251. Primidone, which decreases the amino acid change in the pyramidal layer of the hippocampal sections (the Ammon’s horn) 16241 , has low acute and chronic toxicity in terms of either the LDS0 or doses producing neurological symptoms or morphological change [620] . Toxicity (agitation followed by coma) produced by extra large doses of primidone can be rapidly terminatea by the intravenous injection of bemegride (Megimide) [63 I ] . Primidone is rapidly metabolized by liver tissue [6?2]. Accumulation of phenobarbitone is noted upon repeated administration of primidone [635, 6361. The close relationship between the drugs primidone and phenobarbitone can be demonstrated by the fact that when the two drugs are given together, their effects are simply additive [616]. Anticonvulsant activity is still retained when the N I and N 3 positions of compounds (LXXVII) are formylated [637]. It is not unlikely that the formyl groups are lost in viva Dhydrouracils substituted with two hydrocarbon units in the 5-position,

PYRIMIDINES OF BIOLOGICAL A N D MEDICAL INTEREST

318

such as (LXXVIII), are strong and effective soporifics [638]. These compounds are analogues of the barbiturates in which the 6-carbonyl group has been replaced by a methylene unit. The compounds having N-substitution were reported to have certain physical and pharmacological advantages over those without N-substitution [638] .

0

R3'

1 3 ;

R ' , R2 = aliphatic, alicyclic or aromatic hydrocarbon radicals = H or hydrocarbon radicals

R3,R4

(

LXXVlIl

1-Alkyl and l-aralkyl-5,6-dlhydrouracils (LXXIX), prepared by condensation of N-(2-~arbamoylalkyl)aralkylamineswith urea or by treating a suitable primary amine with an ester of acrylic acid followed by cyanic acid, are CNS depressants and anticonvulsants [639, 6401, as well as anti-inflammatory agents [641]. Such compounds are to be compared with the corresponding barbituric acid derivatives in which not more than one hydrogen in the 5-position is substituted, and also with barbiturates in which the 5,5-substituents are similar to the R' and R2 groups of the 5,6-positions here. H ~1

(LXXIX)

R' = H, alkyl or Br RZ = H or alkyl R 3 = alkyl, aryl or aralkyl of the type of Ar-Y where Y = alkylene radical which can be substituted with either one o r two aryl groups

Among the dihydro analogues of thiouracil, 2-mercapto-6-methyl-5,6-dihydrouracil (LXXX) retains some of the antithyroid activity exhibited by 2-thiouracils [642] (see Part I of this review, Volume 6). This compound can be obtained either by the condensation of thiourea with ethyl crotonate or by the h y d r o genation of 2-mercapto-6-methyluracil.

sTF H

J $ N z H

HN

Me

(LXXX)

H

H

Ph-CH,

(LXXXI)

Ar

(LXXXII

l-Benzyl-2-mercapto-5,6-dihydrouracil (LXXXI), when given orally at a dose of 50 mg/kg per day to rats for 2 days, stimulates both cortisol and hexabarbitone

C. C. C H E N G , BARBARA R O T H

319

metabolism by rat liver homogenates [643]. These effects were similar to those of 2-(o-chlorophenyl)-2-~-chlorophenyl)1,1-dichloroethane. (0, p’-DDD). This pyrimidine also increases the lipid peroxidase activity. The stimulation of hepatic microsomal activity by this compound has been suggested to be due to an increase in the de novo synthesis of microsomal enzymes [643] . 6-Aryl-5,6-dihydroisocytosines(LXXXII; Ar = c6 H 5 , p-MeOC6H4, 3,4C 1 2 C6 H3, etc.) are useful diuretics. These compounds may be administered orally [644]. Herbicidal and chemotherapeutic activity have also been noted in other dihydropyrimidines. Certain 5,5-dihalo-6-methoxydihydropyrimidines, especially the 3-substituted derivatives such as (LXXXIII), are reported to have herbicidal activity against many grasses and broad-leaf weeds [645] (herbicidal activity of some corresponding N-substituted uracils has been discussed in the preceding section). The compounds can generally be prepared by halogenation of uracil derivatives in alcohol [646, 6471 . Me ,€t ‘C H I

( LXXXIII )

(LXXXIV)

When the halogenation is carried out with 5,6-dihydrouracils in aqueous suspension at pH 1-3, the resulting products are 1,3-dihalouracils (LXXXIV, R = alkyl or aryl groups of C l - C l o , X = halogen [648-6501). Compounds of this type, which can readily be purified by treatment with cold, concentrated, sulphuric acid followed by dilution [651] , are claimed to be useful as fungicides, nematocides, bactericides (e.g., completely inhibiting the growth of Erwinia amylovora, Xanthomonas phasioli, Micrococcus pyogenes var. aureus, E. coli, Altemaria oleracea and Monilinia fmcticola at 1 p.p.m. whle causing neither plant nor seed injury) as well as bleaching and sanitizing agents [648-6501.

2-Hydroxy-4-methyl-6-aryl-5,6-dihydro-5-pyrimidinecarboxylates (LXXXV) and other related compounds (5-C0OH, 5-COOEt, 5-COMe, etc.) possess activity against viruses of the psittacosis-lymphogranulomatrachomagroup when Ar = the 5-nitrofuryl group [652-6551 . These compounds show chemotherapeutic activity against Lymphogranuloma inguinale [652] and allied diseases, but fail to influence murine infections with Salmonella dublin or Streptococcus pyogenes [655]. When Ar = substituted phenyl groups (e.g., p-Me2N.C6H4-), the compounds have an inhibitory effect on the growth of sarcoma 180 and Ehrlich tumour, and lengthen the life span of mice with transplanted leukemia LIO-1 [656,657].

320

PYRIMIDINES OF BIOLOGICAL A N D MEDICAL I N T E R E S T

At a dose of 200 mg/kg, 5-bromo-5,6-dihydrothymine (LXXXVI) retards the tumour growth of Crocker sarcoma [658] . 5-Fluorodihydrouracils (LXXXVII; X = F, R = H, alkyl) are useful as germicides [659] . S-Thiocyanato-5,6-dihydrouracil (LXXXVII; X = -SCN, R = H) shows a high level of antibacterial activity [660]. H

(LXXXV)

(LXXXVI)

H

(Lxxxvrr)

The activity of tetrahydropyrimidines is presented in the order of substitution at the 2-position: 2-unsubstituted, 2-alkyl (and 2-alkenyl), 2-aralkyl, 2-aryl, 2-hydroxy, 2-niercapto, 2-amino, etc. 5-Hydroxymethyl-l,2,3,4-tetrahydropyrimidines (LXXXVIII; R' , RZ = H, Me), prepared by metal hydride reduction of corresponding 2-0x0-5-ethoxycarbonyl- 1,2,3,4-tetrahydropyrimidines [661] , are useful as antihistaminic and antibacterial agents [66 I ] . 4,4,6-Trimethyl-3,4,5,6-tetrahydropyrimidines substituted with long chain alkyl groups at or near a nitrogen atom often possess high fungitoxic and other therapeutic activities [662-6641 , provided that the total number of carbon atoms per molecule is at least nine [663]. 2-Alkyl-4,4,6trimethyl-3,4,5,6-tetrahydropyrimidines (LXXIX), prepared by reaction of 2,4diamino-2-methylpentane with the appropriate acid or ester, are active against fungus spores of Alternaria circinans (Bert. and Curt.) Bolle and Monilinia fructicola (Wint.) Honey. Maximum activity is reached at an alkyl chain length of 17 carbon atoms (i.e., the 2-heptadecyl derivative), beyond which fungitoxicity decreases [662, 6641. The three methyl groups on the ring enhanced the effectiveness of these compounds, since 2-heptadecyl-4,4,6-trimethyl-3,4,5,6tetrahydropyrimidine is markedly more fungitoxic than 2-heptadecyltetrahydropyrimidine.

(LXXXVIII )

(LXXXIX)

( XC)

1-Dodecyl-2-methyl-1,4,5,6-tetrahydropyrirnidine (XC, n = 1 l), at a concentration of 3-100 y/ml, controls and prevents fireblight (Erwinia amylovora) on Bartlett pear and other related plants [665, 6661. The effect is said to be

321

C. C. CHENG, B A R B A R A ROTH

comparable to that of streptomycin sulphate (Agrimycin 17) [665] . Many compounds of type (XC), where substitutions at the 1-position are always of long, straight aliphatic chains, are fungicides at 10-80 p.p.m. but not phytotoxic below 1 000 p.p.m. (tests are made on Alfernaria oleracea, ibfoniliniafructicola and Macrosporium sarcinueforme) [667] . 2-Alkenyl- 1,4,5,6-tetrahydropyrimidines (XCI, R' = unsaturated aliphatic radical of 2-22C, RZ = H or Me but at least one R2 = H) inhibits the growth of tobacco mosaic virus and other plant viruses [668] . 1,2-Diaralkylsubstituted tetrahydropyrimidines (XCII, R = H or Me) are active as antihistamines and bronchodilators [669]. Adrenolytic and sympatholytic activities are noted with some compounds of a similar structure [670] (XCIII). R'

(XCII)

(XCIII)

R'. R2, R3= H , alkyl RL= H, Me,OH

2-(2-Thienyl)alkyl- and 2-(2-thienyl)alkenyl- 1,4,5,6-tetrahydropyrimidines possess anthelmintic activity against intestinal parasites [67 1-6741 . These compounds are active against helminths of the families Ancyclostomafidue, Strongylidae and Trichostrongylidue in sheep, cattle, goats, dogs, cats and horses by the oral or perenteral routes. At oral concentrations of 12.5 and 25 mg/kg, respectively, the tartrate salt of trans-1-methyl-2-[ 2-(2-thienyl)vinyl] 1,4,5,6-tetrahydropyrimidine(XCIV, pyrantel tartrate) is effective against preexisting infections- of Nematospiroides -dubius and Nippostrongylus brasiliensis

U

C

H ICH

Me'

yJ N

702 H H-C-OH I

HO-F-H

CO,H

(XCIV)

in mice. The compound is also useful in treating Nippostrongylus muris and Syphaczu obvelata in rats. Although compounds with alkyl side chains are also claimed to have similar activity [671] ,the vinyl linkage is believed to be important to its anthelmintic activity [672]. The pamoate salt of pyrantel has been used successfully in the clinic (79 patients) for treatment of pinworms, at doses of 5 and 6 mg/lb. A slightly higher dose gave 9 3 per cent cures of ascarides (49 patients) and 8 2 per cent cures of hookworms (40 patients) [675] . The drug is reported to inhibit cholinesterase [676] .

322

PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST

A variety of chemotherapeutic and pharmacological activities are displayed by certain 2-oxymethyltetrahydropyrimidines.2-Aryloxyaralkyl- 1,4,5,6-tetrahydropyrimidines of the general formula (XCV) are said to have diuretic, spasmolytic, eurhythmic, antibiotic and antiemetic activity [677] . Oxyphencyclimine (XCVI) possesses anticholinergic, antispasmodic, and sedative properties [678] . At 0.5-1 .O mg/kg, this tetrahydropyrimidine prevents perforation of epidemoid ulcers and reduced extension and number of glandular ulcers. Doses above 2 mg/kg prevented formation of both ulcers in rats [679] .

(XCVI)

IXCV) 6 R’-R = ti, Me.Et X,Y = alkyl.alkoxy, halo

Catalytic reduction of cytidine in water over rhodium on alumina yields the tetrahydro derivative [ 1-(/3-D-ribofuranosyl)-4-aminote trahydropyrimidin-2( 1H)one] and l-(/3-D-ribofuranosyl)tetrahydropyrimidin-2(lH)-one as the major products [680] . The former hydrolyses readily to give tetrahydrouridine, which is a potent inhibitor of human liver deaminase. The latter compound is also formed by sodium borohydride reduction of 5,6-dihydrouridine. 1,4,5,6-tetrahydropyriHalogen-substituted 1’,2’,3’,4’-tetrahydro-l-naphthylmidines, such as (XCVII), and their non-toxic water-soluble salts, are useful as hypotensive agents [681] 2-(p-Aminophenyl)-l,4,5,6-tetrahydropyrimidines (XCVIII), prepared from p-aminobenzoic acid ester and a,o-diaminoalkanes, are active as local anaesthetics over a wide pH range [682].

(XCVII) R= H,Me

IXCVIII

It is of interest to note that a simple tetrahydropyrimidine, such as the 2-hydroxy derivative (XCIX), which actually is a cyclic urea, with the two protons probably on the nitrogen atoms, rather than the tautomer shown here,

C. C. CHENG, BARBARA ROTH

323

has been claimed to be active as a fungicide, an insecticide, a bactericide, as well as having plant growth controlling properties [683].Many anthelmintic 2alkylthio 1,4,5,6-tetrahydropyrimidinesare useful in controlling phytopathogenic agents which attack not only plants, but also humans and animals [684,6851. Compound (C) can be used to control wilts on tomato, cotton, watermelon or flax pea, and rusts and mildews caused by obligate parasites, without harming the host [684].Compounds of this type are so low in phytotoxicity that they do not even inhibit germination of cucumber seed [684].In general, the 2-SH analogue is not active unless it is alkylated [686]. 2-Aralkylthio-l,4,5,6-tetrahydropyrimidines (CI, N 6)are useful in inhibiting the local oedema associated with inflammatory states [687].These compounds are also claimed to have antispasmodic, diuretic, hormonal and anthormonal activities [688].

<

Spirocyclic tetrahydropyrimidines such as (CII) have been investigated as hypotensive drugs, which probably exert their effects by preventing the release of the adrenergic transmitter substance at sympathetic nerve endings [689]. 3-Dodecyl-2-iminohexahydropyrimidine-1-acetic acid (CIII, which contains an exo double bond at position 2 and is therefore a tetrahydropyrimidine) is useful as a germicide and a fungicide [690].

The physical, chemical and biological properties of hexahydropyrimidines are analogous to those of alkyl diamines. Most biologically active compounds in this series are N1 ,N3-disubstituted derivatives. 1,3-Bis-[3’-(substituted amino) propyl] hexahydropyrimidines (CIV), prepared by condensing substituted alkylamines with trimethylene dhalides, possess fungicidal, bactericidal and antiviral properties [691].These compounds are also corrosion inhibitors [692].The 1,3-bis(diethylaminopropyl) derivative, for example, inhibits 98-1 00 per cent of germination of spores of Macrosporium sarcineforme and Sclerotina fmcticola in fungitoxicity tests [692].When a carboxyl linkage is inserted into each side

3 24

PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST

chain, the resulting 1,3-bis-(3-diethylaminopropyloxycarbonyl) derivative (CV) can be used as a curarizing agent in shock therapy and surgical procedures to produce muscle relaxation [693].

(CIV)

ICV)

Addition of 100-1 000 p.p.m. of 1,3-disubstituted S-alkyl-s-nitrohexahydropyrimidines (CVI) to petroleum lubricants such as cutting oils, penetrating oils, grinding lubricants, hydraulic fluids and the like, inhibit bacterial growth in these media. In particular, 1,3-bis(hydroxy-t-butyl)-S-ethyl-S-nitrohexahydropyrimidine, can render protection of petroleum lubricants for more than 4 2 days at a concentration of 1 000 p.p.m. [694]. The corresponding S-aminohexahydropyrimidines [695] (CVII), believed to be thiamine antagonists [696], possess R'

R'

(CVI1

(CVII)

even stronger bactericidal activity. In addition, these compounds were found to have insecticidal and antifungal activity and have been used as insect repellents, nematocides, dermatoses, ophthalmic ointments and stabilizers in glues and adhesives (which contain natural products such as casein, starch, etc.) [696-7061. The most active ones have the eight-carbon radicals (as the 1methylheptyl or 2-ethylhexyl) substituted at both the 1 and 3-positions. 1,3-Bis(2-ethylhexyl>5-amino-S-methylhexahydropyrimidine (hexetidine, sterisil; CVII, R' = CH2.CHEt.(CH2)3Me, RZ = Me) is not only bacteriostatic and fungistatic, but germicidal and fungicidal [701]. Hexetidine also inhibits oxygen consumption of methylene blue-stimulated erythrocytes when inosine or glucose-6-phosphate is used as substrate [702]. ACKNOWLEDGMENT

The authors wish to express their appreciation to Dr. William B. House and Dr. George H. Hitchings for their encouragement, to Dr. Eugene G. Podrebarac for many helpful discussions, and Miss Rene'e Laube for assistance in the literature search, and to Miss Soula Culver for typing this manuscript.

C. C. CHENG, BARBARA ROTH

325

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PYRIMIDINES OF BIOLOGICAL AND MEDICAL INTEREST R. Hull and E. W. Hurst, Chem. Ind., 196 1, 1898 E. W. Hurst and R. Hull, J. Med. Pharm. Chem., 1961, 3, 215 N. I. Volfson, h o e . USSR Acad. Med. Sci., 1952, No. 4, p. 235 L. F. Larionov, Cancer Chemotherapy (translated by A. Crozy), Pergamon Press, London, 1965 I. A. Aksamitnaya, Vopr. Onkol.. 1963, 9, 29 F. Hoffman-LaRoche & Co. A-G, Netherlands Patent 6 404 756 (1964); Chem Abstr., 196S,62,14693d J. Gut, J. Morivek, C. Pirkinyi, M. Prysta;, J. i k o d a and F. :om, Collect. Czech. Chem. Commun., 1959, 24,3154 A. Takamizawa and K. Hirai, Japan Patent 1184 (1967); Chem. Abstr.. 1967, 66, 55505m W. E. Rader, C. M. Monroe and R. R. Whetstone, Science, 1952, 115, 124 N. V. de Bataafsche Petroleum Maatschappij, Dutch Patent 78 483 (1955); Chem. Abstr., 1956, 50, 6740a P. A. Harvey, P. A. Eastburg and V. L. Reidy, Antibiot. Chemotherap. 1955, 5, 135 F. R. Fronek and E. J. Klos, Plant Dis. Rep., 1963,47, 348 W. W. Abramitis and R. A. Reck, US. Patent 3 135 656 (1964); Chem. Abstr., l964,61,6311f Armour & Co., Brifish Patent 793 749 (1958); Chem. Abstr., 1958, 52, 18999f W. A. Darlington, US. Patent 2 965 540 (1960); Chem. Abstr., 1961, 55, 87451 Ayerst, McKenna and Harrison Ltd., British Patent 952 802 (1964); Chem Abstr., 1964, 6 1 , 4 1 6 2 ~ J. A. Faust, L. S. Yee and M. Sahyun, J. Org. Chem, 1961, 26, 4044 Pfuer Corp., Belgium Patent 658 987; Chem. Abstr., 1966, 64, 8192c W. C. Austin, W. Courtney, J. C. Danilewicz, D. H. Morgan, L. H. Conover, H. L. Howes, Jr., J. E. Lynch, J. W. McFarland, R. L. Cornwell and V. J. Theodorides, Nature, 1966, 212,1273 Chas. Pfuer & Co. Inc., J. Amer. Med. Assoc.. 1966, 196, 728 H. L. Howes, Jr., and J. E. Lynch, J. Parasitol., 1967, 53, 1085 H. L. Howes, J. E. Lynch and G. F. Smith, Amer. SOC. Trop. Med. Hyg. 17th Meeting, Atlanta, Georgia, Nov. 1968 P. Eyre, Vet. Rec., 1968, 83, 605 C. A. Dornfeld, US. Patent 2 893 993 (1959); Chem. Abstr., 1959, 53, 20102e Modern Drug Encyclopaedia, 1965, 204 P. Naranjo, G. Hidalgo and E. Banda-Naranjo, Arzneim.-Forsch., 1961, 11, 662 A. R. Hanze,J. Amer. Chem. Soc., 1967,89,6720 M. Sahyun and J. A. Faust, US. Patent 2 948 724 (1960); Chem. Abstr., 1961,55, 2701f Chas. Pfuer & Co. Inc., Brifish Patent 770 592 (1957); Chem. Abstr.. 1957, 51, 14825b J. Bindler and J. A. Rumpf, Swiss Patent 360 843 (1962); Chem. Abstr., 1963, 58, 9576a P. N. Gordon, US.Parent 2 988 478 (1961); Chem. Abstr., 1962,57, 6363 P. N. Gordon, U S . Patent 3 219 522 (1965); Chem. Abstr., 1966, 64,6671f L. G. Nickell, P. N. Gordon and A. Goenaga, Plant Dis. Rep., 1961,45,756 R. C. Tweit, US. Patent 2 969 362 (1961); Chem. Abstr., 1961, 55,15520g R. C. Tweit, U.S. Patent, 3 025 295 (1962); Chem. Abstr., 1962, 57, 85891 R. L. Salvador and M. Saucier, Can. J. Chem., 1968,46, 751 J. Bindler and E. Model, German Patent 1 126 393 (1962); US. Patent 3 108 903 (1963); Chem. Abstr., 1964, 61, 667e W. E. Craig and J. 0. Van Hook, US. Patent 2 675 381 (1954); Chem. Abstr.. 1956, 50,411d

C. C. CHENG, BARBARA ROTH

692. 693. 694. 695. 696. 697. 698. 699. 700. 701. 702. 703. 704. 705. 706.

34 1

J. 0. Van Hook and W. E. Craig, US. Patent 2 675 387 (1954); Chem. Abstr., 1955, 49,4729b Laboratories Bruneau & Cie. S.a.r.l., French Patent M2750 (1964); Chem Abstr.. l965,62,574h E. B. Hodge, U.S.Patent 3 183 188 (1965); Chem Abstr., l965,63,2830d M. Senkus, U.S.Patent 2 387 043 (1945); Chem. Abstr.. 1946,40,613 Warner-Lambert Pharmaceutical Co., British Patent 793 379 (1958); Chem Abstr., 1958,52, 20923g M. Senkus, US.Patent 2 415 047 (1947); Chem. Abstr., 1947,41,3252 F. A. Barkley, F. J. Turner, R. S. Pianotti, P. L. Carthage and B. S. Schwartz, Antimicrobial Agents Annual, 1950, 507 L. S. Fosdick, R. R. Read, R. G. Sanders and R. B. Edwards, US. Patent 2 837 463 (1958); Chem. Abstr., 1958, 52, 16703b W. G. Fredell, R. C. White and R. G. Sanders, J. Amer. Pharm. Assoc. Proct. Pharm Ed., 1958, 19,428 A. L. Welsh and M. Ede, J. Invest. Dermatol., 1958, 30,171 F. J. Lionetti, W. L. McLellan and F. Cornunale, Biochem. Pharmacol., 1959, 2, 226 E. B. Hodge, German Patent 1 134 244 (1962); Chem. Abstr., 1962,57, 12962a R. G. Sanders and B. D. Church, US.Patent 3 072 529 (1963); Chem. Abstr., 1963, 58,66538 E. B. Hodge and G. J. Lafferty, French Patent 1 364 172 (1964); Chem Abstr., l965,62,7988c G. P. Larrick, Fedeml Register, 1964, 29, 15228

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lndex Italicised page numbers indicate that the subject is referred t o on succeeding pages

Abstinence syndrome, 18, 258, 259 Acetorphine M183, 235 5-Acetyluracil, 299 Adenine, 73, 74, 96 allyl-, 95 amino-, 75, 86, 88, 9 4 , 9 5 , 101, 109 chloro-, 95 fluoro-, 95, 1 0 9 methyl-, 75, 9 5 nucleosides, 82 1-oxide, 95 phosphoribosyl transferase, 72, 75, 96 Adenocarcinoma, 755, 94, 95 Adenosine, allyl-. 8 6 amino-, 82, 86, 87 3'-amino-3'deoxy-, 96, 106 3'-amino-3'-deoxy-NN-dimethyl-, 103, 106 3'~amino-N-methyl-3'-deoxy-, 8 8 chloro-, 87, 91, 105 deaminase, 8 8 fluoro-, 86, 100, 1 0 3 kinase, 8 0 lysyhmino-, 1 0 0 N-methyl-, 82, 88, 103 N-methyl-deoxy-, 8 2 methylamino-, 8 2 phosphates, 80, 83, 93, 105 thio-, 8 8 S-Adenosy Imethionine, 83 Adenylic acid, 73, 93, 97 analogues, 82 deaminase, 8 8 from inosinic acid, 97 Adenylosuccinate synthetase, 97 Adenylosuccinic acid, 97 Addiction liability, 255 Adrenergic neurones, blockade, 138, 171, 174, 202, 203 antagonism of blockade, 174 Adrenergic a-receptor blocker, 29 Adrenolytic action, 24 Aflatoxin B , , 288 Agonists, 262 AH 2 2 5 0 , 5 6 Alcuronium chloride, 12 Aldomet, 200 N-Alkyluracils, 303 Allergy, drugs for, 45 Allopurinal, 56 Amidines, 128 4-Amino-6-hydroxypyrazolo [ 3,4-d] pyrimidine, 9 0 4(5)-Aminoimidazole-5(4)-car boxaniide, 88, 94 343

4-Aniinoimidazo [4,5-d] pyridazine, 76, 95 Amino-nietradine, 310 6-Aminopenicillanic acid derivatives, 50 4-Aniinopyrazole(3,4-d)pyrin~id1ne, 75, 82. 8 8 , 9 0 , 9 1 , Y5, 1 0 7 , 1 0 9 5(4)-Amino-1H-1 ,2,3-triazolc-4(5)carboxamide, 99 5-Aminouracils, 306 6-Aminouracils, 307 Amisometradine, 309 Ampicillin, 1 1 , 5 1 Anabolism of purines, 74 Anaesthetics, intravenous, 20 Analgesics, 16, 229 antagonists, 17. 18 conformation factors, 269 stereochemical aspects, 265 Angustmycin A, 98 Animal tests for analgesia, 260 Antagonist-agonists, 260 Antagonists, 262 Antagonists of Analgesics, 255 Anti-allergy drugs, 45 Antibiotic drugs, 3, 8, 50, 5 3 Anti-cancer effect of purines, 107 Anti-depressive drugs, 25 Antidiuretic hornione, 38 Antihypertensive drugs, 27, 125, 151 pharmacological tests for, 135 Anti-inflammatory drugs, 219 Antilyniphocyte drugs, 223 Antimicrobial drugs, 3 , 8, 49, 52, 5 4 Antinociceptive test, 245 Antitrichomonal drugs, 49 Antiviral effect of purines, 106 Aquex, 43 9-p-D-Arabinofuranosyladenine, 80, 99,100 antiviral effect, 106, 107 9-p-D-Arabinofuranosylhypoxanthine,86 9-p-D-Arabinofuranosylpureine-6( 1 H)thione, 99 1-p-D-Arabinofuranosylcytosine, 99 Arlytene, 29 Arterial pressure, drugs for, 224 Asthma, drugs for, 45 Aza-adenine, 75, 82, 95, 99 2-amino-, 85 8-Ata-adenosine, 88, 105 8-Aza-2-deoxyadenosine, 8 8 8-Azaguanine, 86, 88, 91, Y5, 105 8-Azaguanosine, 103 phosphates, 82 8-Azdguanylic acid, 76, 82, 91, 99

344 8-AzaguanyEc acid (contd.)phosphates, 8 2 9-Azahypoxanthine, 9 9 Azathioprine, 104 8-Azaxanthine. 77, 88, 99, 1 0 9 , 1 1 0 Azetidin, 1 2 4 Azothioprine, 223 Bacteria a n d purines, 105 Bactrim, 56 Bamethan, 31 Barbiturates, 2 1-23 Benzanidine, 126 Benzidazol, 1 7 1 Benzimidazole a n d analgesics, 253, 2 7 6 1,4-Benzodiazepine derivatives, 264 Benzodioxan derivatives, 155 Benzodioxoles, 1 5 8 Benzomorphan, 234, 273 Benzomorphans, 238 Benzylpenicillin, 3, 51 6-Benzyluracils, 302 Betamethasone 17-valerate, 2 Bethanidine, 27, 1 2 6 , 1 6 3 , 1 7 4 , 177, 187, 200 0-chloro-. 187 Blood-brain’b&cr, 191, 232 Bradykinin, 261 Bretylium, 1 5 9 , 188, I Y 7 , 203 Brinaldix. 4 3 Bromacil, 303 Bromhexine, 1 1 , 4 4 5-Broniouracil, 3 16 Bronchitis, antibiotic treatment, 1 1 Bronchodilator activity, 4 6 , 55 Buphenine, 31 Bupivicaine, 5 6 B.W.467C60, 1 2 6 Candida species. drugs for, 5 0 N-carbamoylmaleamic acid, 294 Carbanioyl phosphate synthetase, 289 Carbenicillin. SO 6Carboxylic acid, 286 Carlytene, 29 Catabolism, 8 5 , 290 thiopurines, 90 Catapres, 171 CatapresAn, 171 Catapyrin, 310 Catecholaniines and prcnylaniine, 32 release, 27 Chilblains drugs for, 219 Chlorexolone, 4 0 4 3 6-Chloropurine, 76, 9 5 r i bonu cleo t i de, 82 Chlorothiazide, 4 2 Chlorpromazine, 253 Chlorpropaniide, 4 2

INDEX Chlorthalidone, 4 2 Cholestyramine, 56 Chromones as drugs, 46 Clomide, 5 6 Clomiphene, 5 6 Clonidine, 1 7 1 Clopamide, 4 2 , 4 3 Cloxacillin. 5 2 Codeine,-230,232, 247, 249, 261 Co-factors, 101 Colofac, 3 s Cordilox, 34 Cordycepin, 85, 86, 9 3 , 96, 100 1 -oxide, 85 phosphatc, 9 3 Corontin, 31 Corticosteroids, 5 Cromoglycic acid, 4 5 Crotonoside, 8 7 Cuenid, 5 6 5-Cyanouracil, 299 Cyclazenine, 142 Cyclazocine, 17, 18. 258, 261, 263 Cyclorphan, 258 Cyprenorphine, 261 Cyprenorphine, M285, 235 Cyproheptadine, 5 6 Cytidine, 322 Cytidine diphosphate, 9 9 2’deoxy-, 9 9 Cytidylate reductase, 99 Cytotoxicity of purines, 9 5 Daunoniycin, 5 6 Daunorubicin, 5 6 Deaminases, 72, 8 7 7-Deazadenine, 84, 9 5 Debrisoquine, 27, 32, 126, 1 8 7 , 200 Decamethonium, 1 3 Declinax, 27 Decoyinine, 9 3 , 98 De nie t h y lases, 8 3 Demethylation, 83 N-Demethylation, 256 Demethylclilortetracycline, 9 2’-Deoxyadenylic acid, 7 3 2’-Deoxyguanylic acid, 73 Deoxyribofuranosyltransferase, 86 Lkoxyribonuclease, 290 Deoxyribonucleases, 85 2‘-Dcoxythioguanylic acid, 83, 9 9 3-Desmethylprodine, 2 71 Dexoxadrol, 261 Dextromoraniide, 249 Diampromide, 243 Diastercoisonieric, 238 5-Diazouracil, 3 0 0 Diazoxide, 4 2 Didhydro-orotic acid, 288 Dibenylenc, 15 I

INDEX Dibenzylene, 151 Dihydroorotase, 287 Dihydrobenzofurans, 1 5 9 Dihydroergotamine, 29, 30 Dihydrofolate reductase, 290 Dihydrouracils, 316 Dihydro-orotic acid, 287 Dihydropyrimidines, 31 2 Dihydropyrimidine dehydrogenase, 299 4.6-Dihydroxypyrazolo [ 3,4-d J pyrimidine, 90 1.3-diniethylthymine, 314 Diphenoxylate, 248 2p,16(3-Dipiperidino-5a-androstane-3a,l7pdiol diacetate dimethobromide, 1 4 Diprenorphine, 17, 18, 259, 262 Disodium cromoglycate, 47 Disulphide bonds, 97 Ditophal, 2 Diuretic drugs, 36 hypotensive action, 40 thiazide, 38.40, 4 3 DNA. 288, 295, 306. 307. 315 DNA synthesis, 83, 99, 100 deoxythioguanosine in, 99 polymerase, 99 virus, 106 Dopamine and psychotropic drugs, 2 3 and svmpathetic neurone action. 3 3 Dopamine-p-oxidase, 188 Dopom, 125 DOPA decarboxylase, 188 a-methyl-, 200 Dowex 1 -X2-C1, 56 Doxycycline, 8, 4 5 Drolban, 56 Droleptan, 246 Drornostanolone, 56 Droperidol, 246 Drostanolone. 56 Drug latentiation, 2 Duphaspasmin, 35 Duspatalin, 35 Edecrin, 37 Edecril, 37 Electric shock test, 258 Emdisterone, 56 Edecril, 37 6,14, Endoethenotetrahydrothebaines, 233, 276 Enzymes, 72 adenylosuccinate synthetase, 97 anabolic, 74 catabolic, 73, 85 deaminases, 87 dehydrogenasc, 7 3 demethylases, 8 3 deoxyribofuranosyltransferase, R6 deoxyribonuclcases. 85 guanase, 91

345

Enzymes, (contd.1guanidines and, 1 8 8 hydrolases, 72, 8 6 inhibition, 93 kinases, 72, 80 methylases, 8 3 nucleotidases, 85 oxidases, 72, 8 8 phosphinases, 80 phosphoribosyltransferase, 72, 91, 1 0 9 phosphoribosylpyrophosphate synthetase, 93 phosphorylases, 8 6 . 9 1 purine metabolism, 7 0 reductases, 72, 8 3 ribofuranosyltransferase, 72, 86 uricase, 72, 9 0 xanthine oxidase, 72, 105, 1 0 8 Envacar, 126, 156 Epontol, 21 Esbatal, 126, 163 Estil, 21 Estopen, 3 Ethacrynic acid, 36, 4 0 5-Ethyluracil, 297 Etomide, 247 Etorphine, 233 Etorphine M99, 235 Eusmanid, 126 Eutensol, 125 FBA 1420, 21 Ientanyl, 246 Fluocortolone 21-hexanoate, 2 2-Fluoroadenine, 75, 95 2-Fluoroadenosine, 8 2 Folic acid, 74 Forit, 23 Formycin, 88, 9 3 . 9 6 , 100 Formylglycinamide ribonucleotide, 94 N-Formylhydroxyaminoacetic acid, 9 7 Formyltetrahydrofolate synthetase, 290 Fortral, 1 7 FPL 6 7 0 , 4 6 Frusemide, 3 6 , 4 0 4 2 Funtumidine, !4 Funtumine, 14 Furosemide, 37 Fursemide, 37 G 29505, 21 Gallamine triethiodide, 12, 1 3 Ganglion blockers, 138 Gastro-duodenal ulcer, drugs for, 47 Gefarnate. 47 Gefarnil, 4 8 Gefarnyl, 4 8 Gentamycin, 52 Glomerulonephritis. drugs for, 221 Glutathione, enzyme activation. 97

346

INDEX

Glycol salicylate, 2 Gout and purines, 105 Guanacline, 142, 200 Guanethidine and aldomet, 224 Guancydine, 201 Guanethidine, 27, 125, 200 analogues, 139 biochemical effects, 177 blood-brain barrier, 177 depletion of noradrenaline, 193, 201 metabolism, 126, 201 N-oxide, 201 Guanexil, 125 Guanidine, properties, 127 Guanidines, alkyl, 1 6 9 amino-, 133 antagonism of ptosis, 175 aralkyl-, 160 aryl-, 170 aryloxyalkyl-, 15 1 benzyl-, 161 blood-brain barrier, 191 effect o n enzymes, 1 8 8 heteroaromatic alkyl-, 166 phenethyl-, 125 physical properties, 126, 200 structure-activity relationships, 139, 200 synthesis, 130, 200 Guanidinium ion, 127 Guanine, 73, 88, 96 9-butylthio-, 9 9 deaminase, 88 kinase, 9 6 Guanisoquin, 168, 177, 183 Guanoclor, 27, 126, 154, 189, 190, 200 Guanoxan, 2 7 , 1 2 6 , 1 5 6 , 1 7 4 , 1 8 1 , 200,201 Guanylic acid, 73, 85, 93, 9 9 t o inosinic acid, 9 8 Guethine, 125 Guinea pig ileum test, 261, 262 Hadacidin, 97 Havapen, 3 Hexahydropyrimidines, 323 Hexamethonium, 225 Hexamidine, 31 6 Hexetidine, 324 Hexokinase, 297, 301 Homocitrullylaminoadenosine, 100 Hot plate test, 241, 246, 248, 254, 256, 258, 259 Hydrochlorothiazide, 4 1 , 4 2 Hydromedin, 37 4-Hydroxypyrazolo[ 3,4-d] pyrimidine, 75, 8 6 , 9 0 , 93, 105 Hydropyrimidines, 31 1 14-Hydroxycodeinone, 230 5-Hydroxymethyl-6-methyluracil Pentoxyl, 298 Hydroxymorphine, 230

(-)-3-Hydroxy-6, N-dimethyl-C-normorphinan, 240 Hypoglycaemic action, 125 Hypotensive drugs, 40 Hypoxanthine, 73, 36 Imipramine, 25, 26 Imuran, 104 Incoran, 31 Innovan, 246 Inosine, 96 kinase, 72, 80, 96 Inosinic acid, 73, 93 conversion t o adenylic acid, 97 conversion t o xanthylic acid, 97 dehydrogenase, 84, 97 from guanylic acid, 9 8 synthesis, 92 Intal, 46 Integrin, 23 Ipoctal, 125 Ipoguanin, 125 Iporal, 125 Iprindole, 25 Iproveratril, 34 Irrorin, 31 Ismelin, 125 Ismorphinan derivatives, 238 Isocil, 303 Isomethadol, 267 Isomethadone, 249 Isoprenaline, 47, 5 5 Isoptin, 34 Izobarin. 125 Katapyrin, 310 Khellin, 46 Lasilix, 37 Lasix, 37 Lepsiral, 3 16 Leron, 142 Lesch-Nyhan syndrome, 9 6 Leukemia, nucleosides, 107, 109 Levomepromazine, 25 2 Levoprome, 252 Levorphan, 16 Levorphanol, 233, 255

M I 2 5 , 2 3 5 , 236 M320, 235 MS050, 259 Macmiror, 49 Magmilor, 49 Maleuric acid, 294 Malignant obesity, drugs for, 21 8 M A 0 inhibitors, 24, 26, 29 Mercaine, 5 6 Masteril, 5 6 Maxolon, 56

IN I1 I;X Mebcverine, 3 I , 35 Metaniorphinanr. 236 Metazocinc, 238, 239 Mcthacycline. 9 Methadol, 267 Mcthadol metabolism of, 252 Methadone, 16. 1 9 , 233, 247. 252. 2 6 5 , 2 6 8 Metliacyl, 296 Methalone, 56 Methotrexatc. 74 in Icukaeiiiia, 109 Me thotrinieprazinc. 233, 25 2 Methylases. 8 3 6-Methylenedihydrodcsoxymorpl1inc,23 1 Methyl nicotinatc, 2 6- [ ( 1 -Met h y l-4-n i t ro-5-I 111Ida zo I y I )t hio 1 purine, 104 Methyl phcnidatc. 256 3-Methylpcthidine, 262 6-Methylpurine, 7 6 6-(MetIlylthio)purine ribonuclcoside. 8 0 1 -Methylthymine, 3 / 4 3-Mcthylthyminc, 3 / 4 5-Metliyluracil, 3 / 3 6-Metliyluracil, 296 Mctoclopramidc. 56 Mictine. 310 Mincard. 3 10 Mizodin, 31 6 MK-595. 37 6-MonoacctyIinorpIiinc. 232 Mo n 0-a mi n e o \ida \c in I1 i b i t ion, I S 7. 20 2 Mono-a 111i tic ox i dasc inhibit or. 2 24 Morpliinan. 234, 273 Morphine, 16, 19, 232. 239. 256, 273 antagonist, 236 derivatives, 230 detection of, 232 methochloride. pharmacology of, 231 -N-oxide. 232 Moiisylyte, 29 Musacrine, 268 Muscarone, 268 Mylcpsin. 316 Mylepsinum, 3 I6 Myroline. 31 6 NA 97, 14 Nalorphinc I6 19, 245. 249, 260, 261 Na lorp hinc (N-a I I y In or niorpliinc, Let hidrone), 25 5 Naloxonc. 17. 18. 259. 262 Na loxone N-a I I y In or o \r y 1110rpli onr , 2 5 9 Narphen, 239 Napliazolinc, I71 Narcan, 17 Narcone, 17 Ncphrolan. 41 Ncuroniu\cular block. 1 3 -1 5 Ncuronc blocker. 27 Niazol. 17 I

Nicotinainidc adenine nuclcotidcs. 84 Nifuratcl, 4 9 Nitrate rcductase. 304 Nitrofurans. 4 9 5-Nitrouracil. 300 Noradrenaline, I38 antagoniwi of depletion, 185 dcplction. 177, IY3. 201 metaboli\m. 189 \yntliesi\, 189 Nordiliydrodeoxymorpliinc,230 Normcthadonc. 269 Norinorif. I 2 5 Norinorpliinc. 235 Norpethidine. 24 1 Nucleic acid. purines i n , 100 Nucleocidin. 100 Nucleosidc. antibioticr, 84. 100 kinases. 72. 8 0 phorpliokinarcs. 8 0 phosphor y la se s . 86 reduetaw, 72 Nucleotide\. 91, I 0 9 rcdircta.;cs. 83 synthis. 93 Nylidrin. 3 1 Octndinuni. 125 Octatensin. 125 rrz-Octopaminc, 186. 191 Oktadin. 125 Opertil. 23 Opilon, 29 Orotlc acid. 286. 316 Orot id y lie decar bo xy lase, 2 8 7 Orotidylic pyrophosphorylasc, 287 Oiidases. 72. 88 Oxpliencycliminc, 3 2 2 O\ypcrfine. 23. 26 I’ancuronium hroniidc. 1 2 I’arkinronisin, 21 8 Pavulon. 14 Prnamecillin, 3 Pcnctliamate liydriodidc. 3 I’entaiocine, 16. 239. 256. 2 6 2 , 263 Penicillin G , 3 Periactin. 5 6 Pcthidine, 241, 247. 268, 273 Phcnazocinc. 16. 239. 240 I’hcnopcridinc. 24 I I’hcnotlii~izincderivatives. 2 1 8. 2 5 2 I’llcno\ybcnzamlnc. 29. 15 I . 1 85 5-PlienyIbenzoiiiorphans, 240 I’licn t ola mine. 29 Plicn y let hy la n i incs. 3 1 I’ll0S~’llatases.72. 85 Phorpliolicxokinase. 301 5-~lior~~lioribosyla1i111ie~. 94 I’liosplioribosylpyrophos~~hatc, 74-93

347

348

INDEX

Phosplioribosylpyrophosphate (confd.)aniidotransfcrase, 9 3 synthetase, 9 3 Phosphoribosyltransferase, 72, 91, 9 6 , 109 Phosphorylase, 297 Piminodine, 241 Piperazines as analgesics, 250 Piperoxan. 30 Piridolan, 248 Pirtraniide (pirintramide), 248 Platelet aggregation, 1 0 5 Polmiror. 4 9 Polymisin. 5 2 Pramindole. 25 , Prednisolone 2 1 -esters. 5 Prcdnisolone stearoylglycollate, 3. 5 Prenylarninc, 31 Pressedin, 125 I’rimaclonc, 3 16 Primidone, 31 6 Primaquine. 106 Priscol. I 7 I Priscoline, 17 I Privine, 17 1 Prodilidine. 245 Prodine, 262, 268, 272, 273 Profadol. 244. 245. 262 Proniedol. 271 Prondol, 25 Pronethalol. 29 Propanidid, 20 Propranolol, 29. 33. 34 Proposypliene, 249 Protozoa and purines. 105 Pseudomorphine, 23 3 Pseudothymine, 296 Psicofuranine, 98, 102 9-p-l~-Psicofuranosyladenine,9 8 Psychotropic drugs. 23, 2 5 , 216 Purine, 77, 88. 9 5 . 103 2-amino-. 84 6-bcnzylthio-. 95. 103. 107 9-butyl-6-mercapto-, 103, 1 0 7 6-chloro-, X7, 90. 95, 9 7 , 98. 102 9i.tliyl-6-mercapto-. 103 6-hydrazino-, 9 5 6-1nercapto-, 7 7 , 83, 84, 86. 90. Y4, 107 nucleoside, 80. 102. 1 0 3 nucleotide, 83, 97 6-~1iethyl-.76, 95, 1 0 3 6-iiictliyltliio-, 84, 9 5 . 102 nucleo\ideb. 70, 7 5 nucleotidcs. 70, 9 3 oxidation. 105 3-oxide, 85, 108 rihonuclcoside. 8 2 substituted. 77 thiopurine\, 104 to\icity. 102 Purine analogues, 72 cytoto\icity, 9 5

Purine analogues (confd.)metabolism, 73, 7 4 Purines. 69, 315 anti-cancer effect, 106 antiviral effect. 106 as enzyme substrates, 7 0 effect o n bacteria, 105 lethal synthesis, 7 0 substituted, 8 8 Purinor, 290 Puroniycin, 84, 100, 102 antiprotozoal effect. 106 antiviral effect, 106 Pyopen, 5 1 Pyrantel tartrate, 321 Pyrimethaniine, 7 4 , 1 0 6 Pyriinidinecarboxylic acids, 286, 293, 294 Pyrimidines, S-containing, 292 Pyrimidines. herbicidal. 303, 3 1 9 Pyrimidone ring, 31 3 Quaternary animonium steroids, 1 4 Questran. 56 Quinethazone, 4 2 Quinolizidines, 273 R & S 21 8-M, 235 Raynaud’s phenomenon drugs for, 21 9 a-Receptor, blocking of. 138 Receptor site, 266 Receptor theories, 265 Receptors for analgesia. 259 Relane. 261 Reocorin, 31 Reserpine, 21, 34 a n d noradrenaline. 1 9 3 Resistance t o purines. 1 0 9 Respiratory depression, 233, 254, 256. 258. 259 Rhinoperd. 171 Ribofuranosyltransferase, 8 6 Ribonuclease, 290 Ribonucleases, 85 Ribonucleotides, 74, 75 reductase. 8 3 Rifadin. 5 4 Rifamide. 52 Rifdmpicin, 53. 5 4 Rifanipin, 5 4 Rifamycin, 52. 5 3 Rifocin-M, 5 2 Rimactane. 5 4 RNA. 288, 290. 295, 296. 307. 31 2 RNA. messenger, 100, 101 messenger, 100, 101 virus, 106 RO 5-3307/1. 27 Rolicton. 309 Rous sarconia. 1 0 7 Rubidoniycin. 56

IND1:X Salbu t a nio I, 5 5 Sangivamycin. 100 phospliatec. 8 3 Sanotensin. 125 Scgontin. 31 Septrin. 5 6 Serinc hydroxymethyl transferasc. 290 Sintisone. 5 Sinusitis. antibiotic t r c a t n i s n t . I 1 Sosigon,'l7 Spironohctone. 4 3 ST 155, 171 Stereo-specificity in analgesics, 263 Sterisil. 324 Steroids. anti-inflammatory. 5 basic. 1 4 ganglion blocking, 14 Straub tail effect, 233. 246. 248 Straub tail reaction, 261 Streptomycin. 5 2 SU-5864, I 2 5 Subliniaze, 246 Suxariiethoniuni, 16. 22 Syrnpal, 29 Sympathetic nervous system. 136 blocking of. 1 3 8 ganglia, 1 7 4 Sympathetic neurone blocker. 27 Sympatliicolytic drugs, 29, 151 Sympatholytic drugs. 31 Synipatliomimetic drugs. 3 I . 151 Synadrin, 31 Syntlialin. 1 2 5

Tail clip test. 236, 247 Tail-clip method, 246 Tail-flick test, 240, 247. 256. 258. 259. 264 Tail-flick (rat) test. 255 Tail-pressure test, 253 Talwin, 17. 256 Terpenes as drugs, 47 Tetracycline, 9 6-deoxy-5-hydroxy-, 8 derivatives, 8 Tetrahydroisoquinolines as neurone blockers. 167. 202 ' Tetrahydropyrimidines, 320 Tetrahydrothebaines. 18 Thebaine. 233. 240 Thebaines. 235 Thiobarbiturates, 21 Tliioguanine, 77. 90. 91. 95. 103 toxicity, 108 Thioguanylic acid, 9 I , 99 6-Thioguanosine, 103, 107 phosphates, 8 2 Thioinosinic acid, 7 7 , 91, 9 3 , 9 7 2-Thio-orotic acid, 307 Thiopentone, 1 6 , 21 Thiopurines, 90, 91, 95, 107, I08 6-Thiouric acid, 9 0

349

Thio\antliinc. 90 6-Thio\antliylic acid. 9 4 . 102 Thy inid ine phosphor y la b e , 3 0 2. 30 8 Thymidine. tripliospliate. 9 9 Thymine. 295. 305. 313 Thymine derivatives. 299 TM 10. 151 Tolazoline. 29. 30. 171 ToGferine I , I 3 Toyocamycin. 100 p11osp11ate. 82. 8 3 Transaniinase. 291 Trans-morphine. 230 Transplantation immunology, 2 2 3 Tranquillizinp drugs. 25 2 Triacetyloleandoniycin, -1 Triclofos. 2 Trimcperidine. 27 1 Trypanosomes a n d purines. I 0 5 Tubercidin. 84. 8 8 i n iiuclcic acid. 1 0 0 p11osp11ate. 82, 83. 93. 101 Tubocurarine. 12-16

L1 D P-glu cose-gly cogens y n t he t ase. 2 9 7 Ultraviolet irradiation of D N A . 315 Uracil. 286. 295 Uracils. 295 Uric acid. 9 6 metabolism. I 0 5 Uricace, 9 0 Uridine-5'-pliosphate, 287 Urinary tract infections. 222 Vasodil. 171 Vasodilator action of iiucleosidcs. 1 0 5 Vasopressin. 3 8 Vatensol. 126 Ventolin. 5 5 Veractil. 252 Verapainil. 31. 3 4 Vibramycin. 8 Viruses and purines. 1 0 6 Visutensil. 125 Win 18,501-2, 2 3 Win, 20,228. 17, 256 Writhing test. 253, 254, 259. 260, 262. 273 Wy-3263. 25 Xanthine, 74 oxidase. 72, 75. 88, 90. 91. 105. 1 0 8 Xanthosine-S'-phosphate a m i n a x , 98 Xanthylic acid, 73, 84, 102 from inosinic acid, 9 7 Xylocholine, 151, 159, 1 7 3 9-p-D-Xylofuranosyladenine, 80 9-p-D-Xylofiiranosylhypozanthine, 86 Zyloric. 5 6

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