Progress in Medicinal Chemistry, Volume 8

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Progress in Medicinal Chemistry 8

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Progress in Medicinal Chemistry 8 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 V l l Avenue, Cardiff

and

G, B. WEST,

B.PHARM., D.SC., PH.D., F.I.BIOL. Departrnent oJ Applied Biology. North E a t London Pol) technic, Longbridge Road, Dugenhum, EAsex

LONDON BUTTERWORTHS

THE BUTTERWORTH GROUP ENGLAND Butterworth & Co (Publishers) Ltd London: 88 Kingsway, WC2B 6AB AUSTRALIA Butterworth & Co (Australia) Ltd Sydney: 586 Pacific Highway Chatswood, NSW 2067 Melbourne: 343 Little Collins Street, 3000 Brisbane: 240 Queen Street, 4000 CANADA Butterworth & Co (Canada) Ltd Toronto: 14 Curity Avenue, 374 NEW ZEALAND Butterworth & Co (New Zealand) Ltd Wellington: 26-28 Waring Taylor Street, 1 Auckland: 35 High Street, 1 SOUTH AFRICA Butterworth & Co (South Africa) (Pty) Ltd Durban : 152-1 54 Gale Street First published 1971 Q Butterworth & Co. (Publishers) Ltd., 1971

ISBN 0 408 70314 8 Suggested U.D.C. number: 615.7:54 Filmset by Filmtype Services Limited, Scarborough, Yorkshire Printed in England by The Pitman Press, Bath

Preface

In this Volume, the policy of publishing reviews written by specialists concerned with the development and study of new drugs has been continued. We have included chapters covering the fields of pesticides, antibiotics, antivirals antifertility agents, and anti-malarial compounds. In addition, the biosynthesis, chemical synthesis and metabolism of the prostaglandins have been reviewed. The changes made in Volume 7 of the series have permitted reviews to appear in print sooner than has been achieved in the past. As in previous volumes, we are grateful to reviewers and others for their encouragement, criticisms, and suggestions. Our thanks are again due to the staff of Butterworths and to the authors, societies and publishers for permission to use illustrations and tables. G. P. Ellis G. B. West

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Contents Organophosphorus Pesticides :Pharmacology Ian L. Natoff, B.Pharm., MSc., Ph.D., M.P.S. Shell Research Limited, Tunstall Laboratory, Sittingbourne, Kent, England

1

The Mode of Action of Novobiocin A. Morris, B.Pharm., Ph.D., M.P.S. and A. D. Russell, B.Pharm., Ph.D., M.P.S. Welsh School of Pharmacy, University of Wales Institute of Science and Technology, Cardiff, Wales

39

Some Pyrimidines of Biological and Medicinal Interest-Part I11 C. C. Cheng, B.S., M.A.,Ph.D. Midwest Research Institute, Kansas City, Missouri 64110, U.S.A. Barbara Roth, B.S., M.S., Ph.D. Burroughs Wellcome and Co. (U.S.A.) Inc., 3030 Cornwallis Road, Research Triangle Park, North Carolina 27709, U.S.A.

61

Antiviral Agents D. L. Swallow, M.A., BSc., D.Phi1. Imperial Cheniical Industries Ltd., Pharmaceutical Division, Alderley Park, MucclesJeld, Cheshire, England

119

Antifertility Agents V. Petrow, Ph.D., DSc., F.R.I.C. Wm S . Merrell Company, Cincinnati, Ohio 45215, U.S.A.

171

Recent Advances in the Chemotherapy of Malaria R. M. Pinder, B.Sc., Ph.D. Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire, England The Prostaglandins M. P. L. Caton, B.Sc., Ph.D. The Research Laboratories, May and Baker Ltd., Dagenham, Essex RMIO 7x5,England Index

23 1

317

377

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Contents of Earlier Volumes

VOLUME 1 I 2 3 4

5 6

I

2 3 4 5

1

2 3 4 5

6 7

PHARMACOLOGICAL SCREENING TESTS-W. G. Smith HYPOTENSIVE AGENTS-R. Wien TRANQUILLISERS-M. W. Parkes DIURETIC DRUGS-H. Heller and M. Ginsburg ORAL HYPOGLYCAEMIC DRUGS-J. D. H. Slater ANTIFUNGAL AGENTS-E. P. Taylor and P. F. D'Arcy VOLUME 2 T H E PATENTING OF DRUGS-F. Murphy T H E TESTING A N D DEVELOPMENT OF ANALGESIC D R U G S - A . H. Beckett and A. F. Casy MECHANISMS OF NEUROMUSCULAR BLOCKADE-W. C. Bowman 2-HALOGENOALKYLAMINES-J. D. P. Graham ANAPHYLACTIC REACTIONS-G. E. Davies VOLUME 3 SOME CHEMICAL ASPECTS OF NEUROMUSCULAR BLOCK-J. B. Stenlake T H E CHEMOTHERAPY OF TRYPANOSOMIASIS-L. P. Walk ANTLTUSSIVE DRUGS-C. I. Chappel and C. Von Seeman T H E CHEMISTRY A N D PHARMACOLOGY OF T H E RAUWOLFIA ALKALOIDS -R. A. Lucas STATISTICS AS APPLIED T O PHARMACOLOGICAL A N D TOXICOLOGICAL SCREENING-G. A. Stewart and P. A. Young ANTICONVULSANT D R U G S - A . Spinks and W. S. Waring LOCAL ANAESTHETICS-S. Wiedling and C. Tegner

VOLUME 4 EXPERIMENTAL HYPERSENSITIVITY REACTIONS-P. 1 West

S. J. Spencer and G. B.

2 MECHANISMS OF TOXIC ACTION-J. M. Barnes and G. E. Paget 3 D R U G RECEPTOR INTERACTIONS-E. W. Gill 4 POLYPEPTIDES O F MEDICINAL INTEREST-H. D. Law 5 ANALGESICS A N D THEIR ANTAGONISTS: BIOCHEMICAL ASPECTS A N D STRUCTURE-ACTIVITY RELATIONSHIPS-A. H. Beckett and A. F. Casy VOLUME 5 I 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 A N D HEPARINOIDS-L. B. Jaques 4 T H E HISTIDINE DECARBOXYLASES-D. M. Shepherd and D. Mackay 5 PSYCHOTROPIC D R U G S A N D N E U R O H U M O R A L SUBSTANCES IN T H E CENTRAL NERVOUS SYSTEM-J. Crossland 6 T H E NITROFURANS-K. Miura and H. K. Reckendorf

1

2 3 4

5 6

VOLUME 6 T H E BRITISH PHARMACOPOEIA COMMISSION-G. R. Kitteringham PHARMACOLOGICAL ASPECTS OF T H E C O R O N A R Y CIRCULATION-J. R. Parratt SOME PYRIMIDINES O F BIOLOGICAL A N D MEDICINAL INTEREST-Part IC. C . Cheng T H E MECHANISM O F ACTION OF SOME ANTIBACTERIAL AGENTS-A. D. Russell T H E BIOSYNTHESIS A N D METABOLISM O F T H E CATECHOLAMINESM. Sandler and C . R. J . Ruthven T H E LITERATURE O F MEDICINAL CHEMISTRY--G. P. Ellis

VOLUME I SOME RECENTLY INTRODUCED DRUGS-A. P. Launchbury T H E BIOCHEMICAL BASIS FOR T H E D R U G ACTIONS O F PURINES-John H. Montgomery 3 T H E CHEMISTRY OF G U A N l D l N E S A N D THEIR ACTIONS AT ADRENERGIC NERVE ENDINGS-G. J . Durant, A. M. Roe and A. L. Grecn 4 MEDICINAL CHEMISTRY FOR T H E NEXT DECADE~-W. S. Peart 5 ANALGESICS A N D THEIR ANTAGONISTS: RECENT DEVELOPMENTS-A. F. Casy 6 SOME PYRIMIDINES OF BIOLOGICAL A N D MEDICINAL INTEREST-Part II C. C. Cheng and Barbara Roth 1 7

~

1 Organophosphorus Pesticides : Pharmacology * IAN L. NATOFF, B.Pharm., M.Sc., Ph.D., M.P.S. Shell Research Limited, Tunstall Laboratory, Sittinghourne, Kent, England

INTRODUCTION

2

INHIBITION OF CHOLIWESTERASES BY ORGANOPHOSPHORUS PESTICIDES Methods of measuring cholinesterase activity

5 6

METABOLIC ACTIVATION Sex differences Species differences Enzymatic influences TOXIC SYNDROME AND SYMPTOMS Effects in the central nervous system Enzyme inhibition in the central nervous system Effects in the autonomic nervous system Centrally mediated autonomic effects (i) Cardiovascular effects (ii) Metabolic effects Delayed neurotoxicity Teratogenic effects

9 10

10 11 12 12 13 13 14

DETOXICATION Route of administration Enzyme source Carboxylic ester hydrolases Dealkylation Nitro-reduction Stereospecificity Species differences Other means of inactivation

15 15 16 17 18 19 21 21 22

THERAPY OF INTOXICATION BY ORGANOPHOSPHORUS PESTICIDES Parasympathetic receptor blocking agents Oximes

23 23 27

CONCLUDING REMARKS

31

REFERENCES

32

*Submitted in final form in July 1970

1

2

ORGANOPHOSPHORUS PESTICIDES : PHARMACOLOGY

INTRODUCTION Pesticides and agricultural chemicals are used to maintain an adequate food supply for the ever-increasing world population. A large number belong to the organophosphorus family and are biologically active against economically important parasitic pests. It is not unprecedented therefore that this activity also should be evident in higher animals including domestic animals and man. The organophosphorus compounds currently in use have a margin of safety between their effective use concentrations and their minimum toxic concentrations to mammals. However, it is always possible that mammals may become over-exposed to these compounds or to toxic residues of these compounds following the application of excessive amounts to crops, either by hazard, by negligent handling or by intentional self-administration. A full understanding of the mechanism of their biological effects (i.e. the pharmacology) is therefore necessary so that these chemicals may be handled safely and effective remedies may be made available to counteract the effects of over-exposure. The principal biological effect of organophosphorus chemicals in the mammalian system is the inhibition of cholinesterases. This has been the subject Table 1.1

1. PHOSPHATES Approved name

Paraoxon TEPP TOCP

Synonym E 600 Mintacol Biadan Nifos T Vapotone -

2. VINYLPHOSPHATES Approved name Synonyni Monocrotophos Azodrin Nuvacron Dictrotophos Bidrin Carbicron Bomyl Chlorfenvinphos Birlane Supona Sapecron Somonil Ciodrin Crotoxyphos DDVP Dichlorvos Vapona Nogos Herkol Mafu

ORGANOPHOSPHORUS PESTICIDES

(R~O)~PO.OR~ R1 R2 Et 4-NO,.C,H4 Et

PO(0Et)z

o-tolyl

0-tolyl

(R' O)ZPO.OCRZ=CR3R4 R' R2 R3 Me Me H

R4 C0,NHMe

Me

Me

H

CO.NMe,

Me

CH,CO.OMe

H

CO.OMe

Et

2,4-CIz-C,H,R

H

c1

Me

Me

H

Me

H

c1

CO.OCHMe.Ph

CI

3

IAN L. NATOFF

Tetrachlorvinphos

Dedevap Tempal Gardona

Me

Mevinphos Phosphamidon

Phosdrin Dimecron

Me Me

3 . PHOSPHOROFLUORIDATE Approved name Synonym

DFP

Dyflos

4 . PHOSPHONOFLUORIDATES Approved name Synonym

Sarin Soman

-

Me

H 2,4,5-CI3C6HZ Me Me

CI

c1

H

CI

H CI

CO.OMe CO'NEt2

(R0)tPO.F R Pr

R'O-R~PO.F

R'

R2

Pr Me CHBu'Me Me

5. PHOSPHORODIAMIDATE Approved name Synonym Schradan OMPA Pestox 3 Sytam Tetrax

(R'2N)ZPO.R' R' R2

6 . PHOSPHODIAMIDIC FLUORIDES Approved name Synonym Dimefox Hanane Terra Sytam Mipafox Isopestox Pestox 15

(R'R2N)2P0.F R' R2 Me

Me

Pr'

H

(R' ,N)(R'O)PO.CN

7. PHOSPHOROAMIDOCYANIDATE Approved name Synonym Tabun -

R'

R'

Me

Et

8. PHOSPHOROTHIOLATES Approved name Synonym Malaoxon -

(R'0)2P0.SR2 R' R' Me CH-CO-OEt

I

Demeton-S

Systox

9. PHOSPHOROTHIONATES Approved name Synonyni Chlorthion Coumaphos Co-Ral Asuntol Muscatox Resitox Diazinon Basu d in Spectracide

Et

CH2.CO.OEt CH2,CHz'SEt

T*"'

( R'0)2PS.0R2

R Me '

RZ

Et , ' c1

\

Me

tc

4

ORGANOPHOSPHORUS PESTICIDES : PHARMACOLOGY

DMP

-

Et

-

Potasan E 838

Et

Parathion-methyl

Parathion

Fenitrothion

Demeton-0 Fenchlorphos

Dalf Folidol-M Metacide Nitrox 80 Partron Folidol Niran Fosferno Thiophos Murphos Folithio Sumithion Verthion Novathion Danathion Accothion Cytel Sonar Metation Systox Nankor Trolene Korlan Ronnel

9a. PHOSPHOROTHIONATE Approved nanie Synonym EPN PIN

3-Me-4-MeSC6H,

Me

4-NOZC6H4

Et

4-NOZC6HA

Me

3-Me-4-NOzC6H,

Et Me

CHZ'CHZ'SEt 3,4,6-Cl,C,HZ

(R'O)( R20).PS.0R3 R' R2 R3 Et Ph 4-NOZC6Hd

10. PHOSPHOROTHIOLOTHIONATES Approved name Synonym Dioxathion Delnav Navadel

Dimethoate

Disulfoton Ethion Azinphos-methyl

Rogor Cygon Perfekthion Roxion Phosphamide Fostion MM Disyston Dithio-Systox Dimaz Embathion Nialate Fenathion Guthion Cotnion

Me

c

Malathion Carbophenothion

Cythion Trithion Garrathion

IAN L. NATOFF Me

El

5 CH.CO.OEt CH2.C0.0Et 4-CICsH4.S.CHZ

of a great deal of study over the years, and much progress has been made towards an understanding of this effect. A standard reference work in this field was published in 1963 [l] and it is the purpose of this review to summarise some of the more recent developments. Table 1.1 lists the organophosphorus compounds which will be considered. The identification of acetylcholine as the chemical mediator in postganglionic parasympathetic transmission [2] and the subsequent demonstration of the natural occurrence of a cholinesterase led to the observation of the inhibition of this enzyme by physostigmine (eserine) [3]. Cholinesterases were identified later in horse serum and their actions studied using various substrates [4]. Studies of the distribution of acetylcholine and of cholinesterases have shown that they occur at four principal sites in the mammalian body [5-8]: (a) Post-synaptic membranes in the ganglia of the parasympathetic and sympathetic outflows of the autonomic nervous system [9]; (b) Post-ganglionic nerve endings in the parasympathetic nervous system ; (c) Neuromuscular junctions of the voluntary nervous system located on the post-synaptic membranes of the motor end-plate [lo, 113; and (d) Some synapses in the brain and in the spinal cord. Compounds which prevent the destruction of acetylcholine by cholinesterases at these sites therefore allow the accumulation of the endogenous neurohormone. At intoxicating concentrations, this effect is generally one of parasympathetic stimulation [ 121. INHIBITION OF CHOLINESTERASES BY ORGANOPHOSPHORUS PESTICIDES A review of the literature of the distribution, function and structure of acetylcholinesterase is too voluminous for the scope of this article, and the reader is referred elsewhere [l]. Cholinesterase enzyme is a protein, and a dietary deficiency of protein can result in lower cholinesterase activity in liver microsomes and serum of rats. Cholinesterase inhibition by parathion and by Banol (6-chloro-3.4-xylyl methylcarbarnate) (Upjohn) is more at lower dietary levels of casein than at higher levels, thus confirming that the toxicity of these enzyme inhibitors is greater at lower dietary protein levels [ 131. This observation indicates that a causal relationship exists between amino-acid intake and cholinesterase activity.

6

ORGANOPHOSPHORUS PESTICIDES : PHARMACOLOGY

The reaction of the organophosphate with the enzyme, EOH, may be regarded as taking place in two stages :

r

RO

I

RO-P-X

I/

RO

i

+ EOH

0

RO

I I

0

In the first instance, the inhibitor reacts with the serine hydroxyl moiety of the enzyme to produce the alkylphosphate ester [14]. The formation of this ester is reversible and the enzyme activity may be recovered by nucleophilic attack with compounds such as hydroxylamine [15] and oximes [16]. Alternatively, dealkylation of the enzyme-phosphate may then occur [17], a process known as ageing [18], and the covalent link between the enzyme and the phosphate is stabilised, so preventing any recovery of enzyme activity by oximes [19]. Thus, organophosphate inactivation of cholinesterase may be regarded as competitive, stoichiometric and irreversible [IS]. The rates at which phosphorylation and dealkylation occur differ markedly from one organophosphate to another, even within a homologous series, and Aldridge [20, 211 introduced the concept of measuring in vitro cholinesterase inhibitory activity as a function of the bimolecular rate constant, rather than as a function of concentration alone (see also [18, 22-25]). The duration of the inhibition of cholinesterase by organophosphates depends on the nature of the alkyl substituents [26]. The miotic action of dimethyl- and diethylphosphorofluoridates is shorter than that of the next higher a-methylated homologue, di-isopropylphosphorofluoridate (DFP) [27]. The rate at which ageing occurs appears to be related to the rate of dealkylation of the phosphate ester [19, 281. Generally, when a phosphorylated enzyme has aged, the inhibition is irreversible and the loss of enzyme activity is permanent. Recovery occurs only when new enzyme is formed in vivo [29-311. Organophosphorus pesticides inhibit not only cholinesterases, but also carboxylic ester hydrolases [32], trypsin and chymotrypsin [33], sometimes at concentrations lower than those required for cholinesterase inhibition.

METHODS OF MEASURING CHOLINESTERASE ACTIVITY

The incubation of a tissue homogenate or tissue slice with a choline ester reveals the presence of cholinesterase activity in the tissue [34]. This may be measured as a fall in the pH of the medium due to the production of acetic acid from acetylcholine [35], the manometric measurement of carbon dioxide released from bicarbonate by the liberated acetic acid, the titrimetric measurement of the amount of alkali necessary to maintain the incubation medium at

IAN L. NATOFF 7 a constant pH (pH stat method) [36, 371, or as a spectrophotometric colour change [38]. The rate of production of the acid may be used to derive the bimolecular rate constant [20, 211. Braid and Nix [24] recently suggested extending the measurement of the bimolecular rate constant by measuring the affinity constant, K,, and the phosphorylation constant, k Z p ,for an inhibitor against a given enzyme source. The bimolecular rate constant is equal to the ratio of k2JKa. Polarographic methods also have been reported [39]. The addition of an inhibitor of cholinesterase to this incubation medium results in the quantitative inhibition of these changes.

METABOLIC ACTIVATION Generally, the phosphorothionate pesticides have little or no cholinesterase inhibitory activity in vitro, yet in vivo they depress cholinesterase levels in different tissues of the body. Their 0x0-analogues however, are active both in vitro and in vivo. It has been shown that liver slices from mammals contain an oxidative enzyme system capable of converting the P=S linkage to P=O [40,41]. This enzyme activity has been demonstrated also to exist in the body wall of the locust [42, 431 and in the ganglia of the cockroach 1441. In mammalian liver homogenates, the enzyme system was found to be dependent on Mg2+, NAD* and nicotinamide [31], while O’Brien [45] reported N A D R to be a more efficient cofactor than NAD. The phosphorodiamidic fluorides also are activated in vivo. Dimefox is converted to an unstable active principle by microsomal enzyme activity, which may be prevented by p-diethylaminoethyl diphenylpropylacetate (SKF 525A), by 2,4-dinitrophenol, and by cyanide ions [46]. SEX DIFFERENCES

Sex differences have been demonstrated in the toxicity of phosphorothionate pesticides to rats in vivo and in the oxidative capacity of liver slices from male and female rats in vitro. In studying the influence of various factors on the activation of azinphos-methyl and O-ethyl-O-phenyl-O-p-nitrophenylphosphorothionate (EPN), the livers of male rats were found to be 2-3 times more active than those of females in converting these compounds to their oxoanalogues [47]. In young animals up to the age of 30 days, no difference is evident between males and females, but after 30 days the activity of the male liver rises while that of the female liver remains constant. Castration or administration of diethylstilboestrol prevents this rise in males, while admini*Nicotinamide adenine dinucleotide tNicotinamide adenine dinucleotide phosphate

8 ORGANOPHOSPHORUS PESTICIDES : PHARMACOLOGY stration of testosterone to females elevates their enzyme activity [47].Thus, sex hormones influence the synthesis of the phosphorothionate-oxidizing enzymes. Carcinogenic hydrocarbons have been reported to increase the activity of this oxidising enzyme system in immature males, adult females and partially hepatectomized adult males [47].This involves the synthesis of new enzyme protein which may be prevented by ethionine, a competitive antagonist to the incorporation of methionine into new protein [48].The microsomal enzyme inhibitor SKF 525A similarly prevents oxidation of the phosphorothionates, so reducing their toxicity [47].Similar observations on the influence of sex hormones, age and microsomal enzyme inducers on the toxicity of schradan [49,501,parathion [51, 521, fenitrothion [53], EPN, 0-(4-methyl7-hydroxycoumarin)-O,O-diethylphosphorothionate(E838) [51] and 0,Odiethyl-O-(3-methyl-4-methylthiophenyl)phosphorothionate (DMP) [54] in rats have been reported. Although the conversion of phosphorothionates to the active 0x0analogue is more rapid in male rats, it does not follow that the 0x0-analogues are more toxic to males than to females. Indeed, quite often the female has the lower LD50 value [47,511. This is true also for the directly acting organophosphates, tetra-ethylpyrophosphate (TEPP) and DFP [511. A possible explanation for this is that the microsomal enzyme system responsible for the oxidative desulphuration of the phosphorothionate is closely related to those enzymes concerned in the oxidative catabolism of the active 0x0metabolites. This explanation has been suggested for parathion [55]. The higher activity of the enzyme in males therefore leads not only to a more rapid production of the intoxicant, but also to a more rapid breakdown. The toxicity of parathion in male rats is increased by administration of stilboestrol while testosterone decreases its toxicity to females [52].

SPECIES DIFFERENCES

Differences in toxicity for a given compound are influenced by sex hormones only in rats. There have been no reports of this difference in animals of other species. However, species differences exist, and Hutson and Hathway [56] demonstrated the marked difference in the toxicity of chlorfenvinphos when administered orally to dogs, rats, guinea-pigs, mice and rabbits. The following oral LD,o values (mgjkg) were reported: rat. 10-15; mouse, 150-200; guinea-pig, 125-250; rabbit, 500-1000; dog, 3 5000. A biochemical study by these workers showed that the liver of the dog reduced the concentration of chlorfenvinphos in the circulation (assay of peripheral venous blood levels versus portal venous blood levels) and that at similar oral dose levels, the concentration of this vinyl phosphate in the blood of dogs is about one fifth of that found in rats. Concentrations achieved in the blood ofdogs far exceed

IAN L. NATOFF 9 those which are associated with death in rats, while chlorfenvinphos is taken up more readily by the brain tissue of rats than of dogs at similar blood levels. The cholinesterase activity of dog brain is far less sensitive to the inhibitory action of this compound in vitro than is that of rat brain. Thus, Hutson and Hathway showed that the species difference in the toxicity of chlorfenvinphos is determined by differences in distribution, metabolism [57] and sensitivity of the target enzyme. The differences in effects of cholinesterase-inhibiting organophosphorus compounds are found not only between sexes and species, but also between phyla. It is because of these differences that the pesticidal activity of these compounds may be exploited.

ENZYMATIC INFLUENCES

The toxicity of dimethoate to mice may be increased by pre-treatment with phenobarbitone [58] or pentobarbitone [59], whereas the toxicities of phosphamidon, dicrotophos and their N-dealkylated derivatives are decreased [58]. Dimethoate requires oxidative activation, yet the vinyl phosphates do not. In ltitro studies confirm the induction of mouse hepatic microsomal enzymes by phenobarbitone pre-treatment, which activate schradan, malathion and parathion [60]. Microsomal induction by pre-treatment with pentobarbitone stimulates not only the activation of certain organophosphates, but also their catabolism, thereby reducing their toxicity, for example mipafox [59]. TOXIC SYNDROME A N D SYMPTOMS The symptoms of intoxication of mammals by organophosphorus pesticides may be attributed, in general, to inhibition of cholinesterases. This leads to an accumulation of endogenous acetylcholine at post-synaptic sites, and a potentiation of its effects. The resulting hyperactivity of the cholinergic systems (nicotinic, muscarinic and central nervous) results in the symptoms of intoxication which include miosis, hypothermia, hypotension, vasodilatation, bradycardia, salivation, intestinal spasm, bronchoconstriction, motor paralysis and voluntary nerve-muscle junction blockade [61]. Death invariably results from respiratory arrest, which occurs most often by depression of the medullary centres in the central nervous system [62]. However, a species difference is evident, for neuromuscular blockade is predominant at the diaphragm of rabbits with lesser effects at the chest muscles whereas in the cat, a severe bronchoconstriction accompanying diaphragm blockade is seen. The monkey presents a central depression of respiration as a major symptom of the toxic syndrome [62]. The duration of respiratory depression in rats is a direct function of the dose of DFP, soman or sarin administered [63].

10

ORGANOPHOSPHORUS PESTICIDES

: PHARMACOLOGY

EFFECTS I N THE CENTRAL NERVOUS SYSTEM

Electroencephalographic changes due to the organophosphates TEPP and dichlorvos [64], and to physostigmine [65] have been reported, and these can be blocked by atropine sulphate. In addition, oxotremorine is a cholinergic stimulant at atropine-sensitive receptors both peripherally and centrally [66], and systemic or intracerebral injection of this compound or of carbachol in the rat produces hypothermia which may be blocked with atropine [67]. The intravenous injection of DFP, soman, tabun and paraoxon in rats also produces a hypothermia. Ionised organophosphorus cholinesterase inhibitors (with a quaternary nitrogen or an anion due to a hydrogen ion dissociation from the phosphorus group) are inactive intravenously, but produce a doserelated hypothermia on injection into the subarachnoid space [68]. Whereas atropine sulphate reduces this fall in body temperature, methylatropine is without effect as it penetrates the blood-brain-barrier only weakly [69, 701. These effects are species-specific for they are not evident in the mouse, guineapig or rabbit [68]. Barbiturate sleeping time in mice is increased by some organophosphates, and this may be due to an impairment of the breakdown of the barbiturate by oxidative degradation [7 11. However, the increase of hexobarbital sleeping time by TEPP in mice may be prevented by atropine sulphate, but not by methylatropine [72]. This again suggests the involvement of a central cholinergic component of action. E n q w r inhibition in tlzr centrd nervous system The direct injection of sarin into the pons and medulla of the rabbit produces tachypnoea, bradycardia, hypotension and respiratory arrest which are reversed by the intravenous administration of atropine [73]. Respiratory arrest also is produced when sarin is injected into the lateral reticular nuclei of the rabbit and bradycardia occurs following its introduction into the ventrolateral reticular formation, an effect which may be reversed by both atropine and vagotomy [73]. This suggests these to be the target sites in the brain of the rabbit at which cholinesterase inhibition results in respiratory arrest and bradycardia. Death due to cholinesterase inhibitors has been shown to be independent of the degree of inhibition of brain enzyme [51]. Maximum inhibition occurs within 24 hours of intoxication of rats with DFP, parathion, TEPP, schradan. E838 and EPN, but the rate of recovery differs for each compound [51]. Nevertheless, it has been claimed that death due to DF32Poccurs only when a critical level is reached in some organs, including the brain, of mice [74]. When brain cholinesterase is inhibited, the concentration of free acetylcholine rises. Symptoms of intoxication (for example fasciculations) are

IAN L. NATOFF 11 apparent when free acetylcholine in the brain reaches 60 per cent above normal-a situation which is seen at 75 per cent inhibition of brain cholinesterase activity 1751. The observation of adaptation of effectors to continuous cholinergic stimulation [7&78] suggests an interpretation to this phenomenon. Those organophosphates having a slow rate of onset of toxic action in vivo allow the gradual accumulation of acetylcholine, to which adaptation, or tolerance, may develop. Those organophosphates having a rapid onset of effect result in the sudden occurrence of relatively large quanta of the neurohormone at the receptor before adaptation may develop. Thus animals dying under the influence of a rapidly-acting organophosphate have lower levels of tissue enzyme inhibition than those dying as a result of slowly-acting compounds. It appears, therefore, that the toxicity of the compounds may be related not to the degree of cholinesterase inhibition, but to the rate of onset of cholinesterase inhibition. The injection of TEPP and a quaternary ammonium organophosphate, PPS (3-di-isopropoxyphosphinyloxy-N-methylpyridinium methylsulphate), into dogs results in 50 per cent inhibition of brain enzyme by the former and only 16 per cent by the latter [79]. Following injection of PPS directly into the lateral ventricle and subarachnoid space, cholinesterase inhibition is pronounced only in the immediate area of injection [79]. Studies on the acid oxalate and the quaternary methiodide of 2-diethoxyphosphinyl thioethyl dimethylamine [SO] in rabbits show that the intravenous LD50 dose of the methiodide produces no inhibition of brain cholinesterase, while the LD50 dose of the acid oxalate produces approximately 90 per cent inhibition of the brain enzyme activity. Intraventricular injection of the methiodide at 1/10 to ljl00 the intravenous LDSodose, however, produces approximately 90 per cent brain enzyme inhibition. Inhibitors of cholinesterase not only may cross the blood-brain barrier, but also may increase the permeability of the structure to certain compounds, for example anaesthetics [Sl] and sulphanilamide [82]. However, no causal relationship exists between the brain cholinesterase activity and the bloodbrain barrier permeability [82]. Whereas schradan does not inhibit brain cholinesterase on systemic injection [5 11, it facilitates the penetration of the blood-brain barrier by sulphanilamide [82].

EFFECTS IN THE AUTONOMIC NERVOUS SYSTEM

The effects of the cholinesterase inhibitors, DFP and physostigmine, on the autonomic nervous system were examined in the dog [83].Under conditions in which muscarinic effects are blocked by atropine, cholinergic nicotinic effects do not appear to be potentiated by cholinesterase inhibition. However, when autonomic ganglia are blocked with hexamethomium or mecamylamine, the responses to muscarinic stimulation are markedly potentiated.

12 ORGANOPHOSPHORUS PESTICIDES : PHARMACOLOGY This type of analysis demonstrates the predominant effect of cholinesterase inhibition by these compounds in the autonomic nervous system of the dog to be manifest at post-ganglionic parasympathetic nerve-endings. Centrally mediated autonomic efeects

(i) Cardiovascular effects Dirnhuber and Cullumbine [ 121 showed that the intravenous injection of physostigmine, sarin, DFP, TEPP and paraoxon in anaesthetised rats produces a protracted rise in blood pressure which is not affected by vagotomy or by adrenalectomy, but is depressed or prevented by spinalization, or treatment with atropine sulphate, hexamethonium, tolazoline [ 121, hemicholinium, mebutamate [84], bretylium, guanethidine [85], and reserpine [86]. The quaternary form of atropine, methylatropine, is without effect on the pressor response [87]. Varagic also demonstrated this phenomenon with physostigmine both in anaesthetised [88] and in conscious rats [89]. Neostigmine, a cholinesterase inhibitor containing a quaternary nitrogen, produces a fall in blood pressure on intravenous injection [88]. The intracisternal injection of neostigmine in the rat, however, gives rise to the pressor response [90]. It has long been considered that quaternary ammonium compounds are unable to penetrate the blood-brain barrier [91-931, although recent work with 3Hmethylatropine has demonstrated it to penetrate to the cerebrospinal fluid of the lateral ventricle of the dog at one-tenth the concentration measured in the subarachnoid space following subcutaneous injection [69]. Thus, the pressor response in the rat to the intravenous injection of some cholinesterase inhibitors relies on the structural integrity of the central nervous system and appears to be due to the central cholinergic stimulation of the autonomic sympathetic outflow following passage of the inhibitor across the bloodbrain barrier, resulting in the peripheral release of neurohormones from post-ganglionic sympathetic nerve endings. As the infusion of noradrenaline, L-dopa or serotonin only occasionally restores the response in the reserpinetreated rat [86], Varagic and Krstid [94] do not agree that the release of catecholamines from peripheral stores plays a significant role in the hypertensive effect. Those areas of the brain supplied by the vertebral arteries (for example pons and medulla oblongata) are of greater significance in this response than those supplied by the common carotid arteries [95]. The rat appears to be unique in its sensitivity to this pressor effect, for the dog responds to intravenous sarin with parasympathetic effects only [ 121, and perfusion of the IV ventricle of the cat with crotoxyphos Fails to exert a pressor action [871. The intravenous injection of neostigmine at high doses (100 pg/kg) after ganglionic blockade in the anaesthetised dog elicits a pressor response [96], and McEwen [97] reported intravenous injections of physostigmine at doses of 100 pg to 1 mg in atropinised rats (approximating 0.5 to 5 mg/kg) to elicit

IAN L . NATOFF 13 a pressor response of short duration. In this instance, the response is unaffected not only by adrenalectomy but also by adrenergic alpha-receptor blockade and ganglionic blockade. McEwen concluded that this was an effect of large doses of physostigmine directly on the smooth muscle of the arterial wall.

(ii) Metabolic effects Physostigmine has been shown to produce a dose-dependent rise in blood sugar with an accompanying dose-dependent fall in liver glycogen, and an increase in cardiac glycogen on intravenous injection in the rat [98, 991. Neostigmine has no glycogenolytic activity [98]. The fall in liver glycogen is blocked by propranalol, mebutamate, atropine sulphate and by pre-treatment with reserpine or guanethidine. The failure of hexamethonium, 50 mg/kg i.p., or adrenalectomy [98] to block this action of physostigmine agrees with the inability of hexamethonium at similar dose levels (1Cb-20mg/kg i.v.) and of adrenalectomy to abolish the pressor response produced by physostigmine in the rat [12, 881. Hexamethonium does not block the pressor effect of 1.5 mg/kg physostigmine [88], cf. McEwen 1971. The effect of physostigmine on carbohydrate balance suggests a central cholinergic stimulation (blocked by atropine sulphate and mebutamate) leading to the release of catecholamines from peripheral post-ganglionic sympathetic nerves. This stimulates the conversion of phosphorylase b to phosphorylase a [loo], (which may be mediated through the adrenergic 8-receptor), resulting in glycogenolysis [loll. Lipolysis due to injection of physostigmine also has been reported [102]. The decrease in the content of glycogen in the brain of rats due to physostigmine [ 1031is blocked by both atropine and propranalol. Electrical stimulation of the ventromedial hypothalamic nucleus of rabbits from which the sympathetic outflow arises causes a marked hyperglycaemia and decrease in liver glycogen [ 1041. DELAYED NEUROTOXlClTY

One of the symptoms of intoxication by organophosphates is motor paralysis, and it is related to the observed demyelination of voluntary nerve fibres. Work in this area has been reviewed most recently by Aldridge and Barnes [105]. Not all experimental animals show this phenomenon, and the species most closely resembling man in this regard is the fowl [106]. In both man and fowl, the symptoms of tri-o-cresylphosphate (TOCP), mipafox and DFP intoxication do not occur until 8 days after exposure [107]. The fowl presents a peculiar ‘goose-step’ gait, a flaccid paralysis of extensors or marked ataxia. In the cat, the symptoms are ataxia and extensor weakness, but not all organophosphates produce this neurotoxic effect. The essential neurotoxic lesion affects the axon, while secondary changes occur in the

14 ORGANOPHOSPHORUS PEST!CIDES : PHARMACOLOGY myelin sheath and Schwann cells. The hypothesis that neurotoxicity of organophosphates is associated with those having an affinity for pseudocholinesterase [1081 has since been abandoned, as certain organophosphates with no neurotoxic properties have an affinity for this particular enzyme [109]. Nevertheless, it seems likely that a biochemical lesion due to neurotoxic organophosphates or metabolic products thereof [ 1091 is responsible for this neurotoxic effect. The phenyl ester of phenylacetic acid (PPA) serves as a substrate for a small proportion of cholinesterase activity in homogenates of hen brain. This is not inhibited by TEPP or paraoxon, but can be inhibited by mipafox, a neurotoxic organophosphate [ 1101. The PPA-hydrolysing activity is associated with a protein which may be phosphorylated in vivo by neurotoxic organophosphates, but not by those without neurotoxic properties [ 1 1 11. .Inhibition of this ‘neurotoxic’ esterase with phenylbenzyl carbamate protects hens against the neurotoxic effect of DFP for up to 6 hours. This provides evidence that organophosphates having neurotoxic properties are capable of phosphorylating a specific ‘PPA-hydrolysing-esterase’ [ 1121. No common structural denominator is apparent between the organophosphates to which this biological effect may be attributed, although the inclusion of a 2-chloroethyl group has been considered to lead to neurotoxicity whereas diethylphosphate compounds are devoid of this effect [ 1071. Certain theories about the neurotoxic effect of TOCP have been put forward. Taylor [ 1131 observed it to increase the uptake of 32Pin the cervical spinal cord of cats possibly at the expense of the formation of proteins, and this may lead to an impairment of myelin formation. A reversible necrosis occurs at the end-plate region of striated muscle in rats poisoned with DFP, tabun and paraoxon, but not with soman [114]. This suggests that necrosis is a consequence of accumulation of excess acetylcholine, but it is unlikely that this phenomenon is related to demyelination, for paraqxon has been reported specifically not to produce demyelination [105]. TERATOGENIC EFFECTS

The increased awareness of the dangers of teratogenicity in drugs has led to an investigation of the potential of organophosphorus pesticides. Kimbrough and Gaines [115] claimed that the intraperitoneal injections of parathion, dichlorvos, and diazinon and the alkylating agents apholate (2,2,4,4,6,6hexahydro-2,2,4,4,6,6-hexakis( l-aziridinyl)-1,3,5,2,4,6-triazatriphosphorine) and tepa (tris( 1-aziridinyl)-phosphine oxide) at doses sufficient to produce toxic symptoms in pregnant rats at day 11 of gestation result in foetal malformations, resorptions or weight losses. Lower tolerated doses produce only an increase in the number of resorptions in the case of parathion, diazinon and tepa. Malathion has no effect on foetuses under similar con-

IAN L. NATOFF I5 &ions. Whether these effects are primarily due to the compounds themselves or their direct metabolites, or are secondary to their pharmacological actions was not determined. However, teratogenic effects were studied in hens’ eggs [I161 and no correlation was found between cholinesterase inhibition and teratogenesis. Moreover, where the incidence of malformations due to cholinesterase inhibitors such as dicrotophos or physostigmine are reduced by administration of nicotinamide and NADHZ, there is no parallel reduction in the degree of cholinesterase inhibition. An earlier survey has shown little or no evidence of teratogenic activity of pesticides to mammals [117].

DETOXICATION The majority of the organophosphorus inhibitors of cholinesterase are esters of phosphoric and phosphonic acids. Detoxication generally involves removal of an ester group by oxidative cleavage or hydrolysis. Once the biologically active organophosphorus ester has combined with the serine hydroxyl group of cholinesterase, it may then either revert to the original state by .spontaneous hydrolysis, or lose an alkyl group whilst attached to the enzyme, resulting in irreversible inhibition. The phosphorylated enzyme is then said to be aged. However, should the inhibitor lose an alkyl group by hydrolysis of the phosphate ester prior to contacting the enzyme, phosphorylation of the serine hydroxyl moiety does not occur. This initial ester hydrolysis is therefore a means of detoxication. ROUTE OF ADMINISTRATION

The route of administration of organophosphorus cholinesterase inhibitors determines the relative toxicity of the compound within a given species. Gaines [ 1181 first reported the carbamate cholinesterase inhibitor, Isolan (1-isopropyl-3-methyl-5-pyrazolyl dimethylcarbamate), to be more toxic by dermal application than by oral administration to rats. The injection of this compound and of dichlorvos via the hepatic portal venous system reduces their toxicities compared with systemic intravenous injection, suggesting a rapid action of the liver in their catabolism [119, 1201. Parathion, which is converted in the liver to its active analogue, paraoxon [41], is consequently more toxic by hepatic portal venous infusion. Furthermore, paraoxon is inactivated by hydrolysis in the liver [119, 1211. DF3’P is less toxic by intraperitoneal administration than by subcutaneous injection in mice [74], the uptake of the isotope by the liver being greater on intraperitoneal injection, with a corresponding decrease in isotope uptake by other organs in the body [122]. This is commensurate with the known ‘DFP-ase’ activity in the liver *NADH, is the reduced form of nicotinamide adenine dinucleotide

16 ORGANOPHOSPHORUS PESTICIDES PHARMACOLOGY [123] which has a low order of activity in rat and rabbit tissues [122]. Natoff found by classifying routes of administration of some organophosphates into ‘hepatic’ (intraperitoneal and oral) and ‘peripheral’ (intravenous and subcutaneous) routes that the differences in toxicity are compatible with the known rates of absorption, biochemical activation or breakdown of these compounds in mice [ 1241. ENZYME SOURCE

The rBle of enzymes associated with microsomes in the metabolic conversion (be it activation or inactivation) of foreign compounds is well recognised, and the effect of these foreign compounds in stimulating or reducing the activity of these enzymes is now accepted. Often a foreign compound stimulates the microsomal enzyme system to accelerate its own metabolic conversion, for example phenylbutazone [ 1251. Chlorthion, malathion and parathion inhibit the hydroxylation of testosterone by liver microsomes in vitro, and administration of chlorthion to rats in vivo was found to inhibit the hepatic metabolism of oestradiol-l7g, progesterone, deoxycorticosterone and testosterone [ 1261. Chlorinated hydrocarbon insecticides enhance liver growth and the synthesis of hepatic microsomal protein [ 125, 127-1291, and cause proliferation of the hepatic smooth surfaced endoplasmic reticuhm [130, 1311. This has been shown to result in a decreased toxicity to parathion, paraoxon, TEPP, DFP, TOCP and azinphos-methyl [132]. The protective effect of phenobarbitone and nikethamide [54] and the chlorinated hydrocarbon insecticide, aldrin, is blocked by ethionine [132, 1331. More recent work by Triolo and Coon [134] has shown that aldrin reduces the amount of free paraoxon in mouse plasma by increasing its binding to plasma protein. The toxicity similarly is reduced. The toxicities of schradan and neostigmine, which do not cross the blood-brain barrier [5 1,901, are not reduced by aldrin, neither is the inhibition of plasma cholinesterase by paraoxon, although depression of brain enzyme activity is prevented [ 1321. Therefore, aldrin probably exerts its protective effect against the inhibition of cholinesterase only in the central nervous system. Assuming the action of aldrin to be one of microsomal enzyme stimulation, these observations suggest a predominant increase in brain microsomal enzyme activity. This requires detailed study. Reduction of the toxicity of the phosphorothionate insecticide, EPN, to rats by nikethamide [135] is paralleled by a reduction in the cholinesterase inhibitory potency of this compound in vivo [136]. Nikethamide induces the synthesis of oxidative microsomal enzymes [ 1371 concerned in the detoxication of EPN in both male and female rats [138]. Induction of these enzymes by the daily administration of phenobarbitone, 50 mg/kg for 5 days, reduces the acute toxicities of parathion, demeton-0, EPN, disulfoton, dioxathion,

IAN L. NATOFF 17 &ion, carbophenothion and coumaphos in rats, and, to a lesser extent, in mice [139]. The toxicity of schradan to rats however, is increased by this treatment, indicating that the enzymes responsible for its activation are not involved with those responsible for its detoxication in this species. Although the liver is regarded as the principal site for detoxication of organophosphorus insecticides, hydrolytic enzyme activity has been demonstrated in mouse brain [140]. Enzymes which hydrolyse parathion and diazinon have been isolated using thin layer agar-gel electrophoretic techniques. The hydrolysis of malathion takes place at two parts of the molecule by enzyme activities present in two electrophoretic bands: the carboxylic ester site and the methoxyphosphate site. Dichlorvos and diazinon have similar degradative enzyme patterns, while a separate band is responsible for the degradation of D F P [140].

CARBOXYLIC ESTER HYDROLASES

Inhibition of the routes of detoxication of organophosphorus pesticides leads to an augmentation of their effects. Whereas ester hydrolysis of some organophosphorus insecticides by carboxylic ester hydrolases is a route of inactivation, the 0x0-analogues of many of these compounds may inhibit the activity of these hydrolases [ 141-1431. The mutual potentiation of the acute and subacute toxicities of both malathion and EPN [I441 in rats and dogs is due to inhibition by EPN of the hydrolytic detoxication of malathion, and of the malaoxon produced from it, by rat liver homogenates in vitro [145, 1461. The rate of detoxication of malathion is dependent on its tissue concentration [146]. In all species studied, the liver has the greatest carboxylic ester hydrolase activity, and whereas dog serum is devoid of this action, rat and mouse sera have some activity [146]. Sex differences in this enzyme activity are not seen in mice, but in rats the liver of adult males has four times the activity of that of adult females [146].A dose of EPN (1.5 mg/kg i.p.) which is inadequate to produce cholinesterase inhibition in the brain, serum and submaxillary gland of male rats, lowers the LD50value of malathion from 1100 mg/kg to 550 mg/kg [146]. Thus, this potentiation is not due to a summated effect of cholinesterase inhibition, but to an inhibition of the hydrolytic processes by means of which the detoxication of malathion proceeds. The diethyl succinate moiety present in both malathion and malaoxon makes these compounds suitable substrates for the carboxylic ester hydrolase enzyme [147,148]. However, not only is malaoxon a substrate for this enzyme but it also inhibits its activity [141, 1491 as does TOCP [143]. Indeed, pretreatment of rats with TOCP increases the oral toxicities of malathion, malaoxon and their aliphatic ester homologues [ 1411. In vitro studies in which malathion was incubated with liver homogenates from rats did not reveal the production of malaoxon. Pre-treatment of the

18 ORGANOPHOSPHORUS PESTICIDES : PHARMACOLOGY rats with EPN, however, yielded liver homogenates which produced malaoxon when malathion was used as substrate [146]. Thus, malathion is converted to malaoxon by oxidative desulphuration [141] and both the parent phosphorothiolothionate and the 0x0-analogue are detoxified by carboxylic ester hydrolases. Inhibition of the enzyme by EPN prevents the destruction of these organophosphates, but does not impair the activity of the oxidative desulphuration processes.

DEALKYLATION

The metabolism of dialkylaryl phosphorothionates by cow, rat and insect tissues may proceed by cleavage of both the alkyl phosphate and aryl phosphate bonds [150].

Alkyl -0

/-\0-

Aryl

The metabolism of fenchlorphos, and of its 0x0-analogue in rats and exemplifies these 0-dealkylations [I 5 11 (Figure 1.1).

COWS

Fenchlorphos

MeO\

HO’

‘ 0

\

CI

Figure 1 .I

MeO/

\OH

/ O

Me0

Me0

Metabolic pathway offerichlorphos [I511

Two enzyme systems have been identified for dealkylation. One is specific for O-demethylation, the other is active against alkyl groups in general. Studies on the metabolism of chlorfenvinphos have shown it to be O-deethylated by an hepatic microsomal enzyme system dependent on oxygen and NADPHS [152] (Figure 1.2).This enzyme system also attacks other alkyl groups. The enzvme reoonsible for the cleavage of methylparathion to demethylparathion *is assokiated with the soluble fraction of liver homogenates and *NADPH, is the reduced form of nicotinamide adenine dinucleotide phosphate

19 is inactivated by dialysis, and the activity is restored with reduced glutathione [153]. This enzyme system is specific to methyl groups only. Cis-mevinphos IAN L. NATOFF

-02 NAOPH2

- cy)

Microsomes

AH

Cl

cQ

CI

Figure 1.2

Cl

+MeCHO

Chlorfenvinphos

Metabolic pathway of chlorfenvinphos [152]

also is 0-demethylated by a reduced glutathione-dependent enzyme system, while the trans isomer is not [ 1541. Thus the two main dealkylating enzymes are : (1) 0-dealkylase enzyme, associated with the microsomal fraction of liver homogenates, dependent on oxygen and NADPH2 ; (2) 0-demethylase enzyme, present in the soluble fraction of liver homogenates, dependent on reduced glutathione.

Glutathione appears to be the methyl acceptor for this enzyme [155]. Oxidative N-demethylation is a pathway involved in the metabolism of some amide-substituted organophosphates. Dicrotophos is N-demethylated to monocrotophos via the N-methylol derivative [156, 1571. Monocrotophos, itself an active inhibitor of cholinesterase used in pest control, is further N-demethylated to its N-methylol and finally to the unsubstituted amide [157]. This latter step is of only minor significance [158]. The principal degradative metabolite of dicrotophos, dimethyl phosphate, produced by hydrolysis of the vinyl-phosphate linkage was found to exceed the production of 0-demethylmonocrotophos by a ratio of approximately 4 :1while only trace amounts of the N-demethylated metabolite were detected [ 1581. NITRO-REDUCTION

Parathion, paraoxon and EPN are subject to reduction of the aromatic nitro group to an amino group [I591 by mammalian, avian and piscine tissues [160]. Paraoxon is the most readily reduced of the three compounds, EPN the least. Paraoxon metabolism to aminoparaoxon is the route of inactivation by rat, chicken and guinea-pig livers in vitro [160]. However, enzymatic hydrolysis of the phosphorus-nitrophenyl linkage of the 0x0-analogues appears to be a major pathway of detoxication in mammals [142, 161, 1621 but bovine rumen fluid is capable of reducing parathion and EPN to their

20 ORGANOPHOSPHORUS PESTICIDES : PHARMACOLOGY respective amino derivatives in vitro [163]. The cow excretes 30 per cent of a dose of parathion as aminoparathion in the urine, whereas the rat excretes only about 0.5 per cent [159]. The reducing enzyme, present in rat liver, is similar in its properties to the nitroreductase enzyme system [ 1641. Essential co-factors appear to be NADP, FAD* and glucose-6-phosphate while nicotinamide contributes to the enzyme activity [160]. Reduction of the nitro group is one of the more important routes of inactivation of paraoxon [ 1601, and this enzyme system is not sex-dependent in rats or in other species [160]. When 35S-parathion is oxidised to paraoxon by rabbit liver microsomes in vitro, the 35S-metaboliteis bound to the microsomes. Concomitantly, the microsomes induce cleavage of the parathion molecule at the p-nitrophenyl substitutent. This is claimed to be specific to parathion, and does not occur with paraoxon [165]. Both activation of and cleavage of the p-nitrophenyl group from parathion are effected by oxidative liver microsomal enzymes dependent on NADPH2 and oxygen [165]. An extension of these studies [166] described the metabolic pathway for parathion shown in Figure 1.3. EtO

S

EtO

/ \ P\ YS -n n Po L:o\ \ //

*

Microsome7

EtO

OH

0 O- W

EtO

Dtethyl phosphorothtonic acid HO

0

\ // +so: P

/

‘o@o* -

2 EtO

Parathion

Parooxon EtO

\ No

\ D/

D

De-ethyl paraoxon

Diethyl phosphoric acid

/.

/ ’ \

EtO

0

OH

HO

\

OH

Ethyl phosphoric acid Phosphoric ocid Figure I .3 Metabolic parhitny qf purrithion [ 1661

Paraoxon metabolism in rat liver was studied by Kojima and O’Brien [121]. Using the tritiated compound, they found four distinct enzyme systems to be involved, the principal metabolite being diethyl phosphate, produced by hydrolytic cleavage of the p-nitrophenyl group. This activity, which they *FAD is flavin adenine dinucleotide

IAN L. NATOFF 21 termed ‘paraoxonase’, was present in three fractions of liver homogenates : the mitochondria, the microsomes, and the crude soluble fraction. De-ethylparaoxon also was identified following enzymatic dealkylation in the crude soluble fraction, but de-ethylating enzyme activity, using chlorfenvinphos as substrate, was shown to be associated with the microsomal fraction of liver homogenates [ 1521.

STEREOSPECIFICITY

Stereospecificity in the cholinesterase inhibitory activity and in the metabolism of the geometrical isomers of mevinphos and Bomyl [167] has been demonstrated. Using cholinesterase enzymes prepared from fly head, mouse brain and bovine erythrocytes, cis-mevinphos is about 100 times as potent as the trans isomer in inhibiting the enzyme activity in vitro while cis and transBomyl are equiactive [167]. Fly head enzyme has the greatest sensitivity to these inhibitors. The degradation of cis and trans-Bomyl proceeds at equal rates in homogenates of mouse liver and whole fly, while trans-mevinphos is degraded faster than its cis isomer. Thus the degradation rates are compatible with the enzyme-inhibitory activity of the isomers. The difference in the rates of detoxication of the two isomers of mevinphos by mouse liver homogenates was subsequently shown to be due to the involvement of two different enzyme systems [ 1541. Cis-mevinphos is mono-demethylated at the phosphate ester site by a reduced glutathione-dependent enzyme (cf. [155]) while trans-mevinphos undergoes cleavage at the phosphate-vinyl link by an enzyme system independent of glutathione [ 1541. Extending these studies to the thiono-analogues of mevinphos [ 1681, stereospecificity again is evident between the geometrical isomers. Cis-thionomevinphos is activated to the 0x0-analogue by fly slices (but not by fly homogenates or microsomes) and by mouse liver slices, homogenates and microsomes. Trans-thionomevinphos is not activated by any of these systems. SPECIES DIFFERENCES

An example of a species difference in the detoxication of an organophosphorus compound is provided by studies on chlorfenvinphos [57]. Whereas no sex differences were observed in rats in the elimination pattern of chlorfenvinphos, a marked difference between dogs and rats was apparent in its elimination. Chlorfenvinphos is much less toxic to dogs than it is to rats, and the elimination of the ~ i n y l - ’ ~inC the urine and faeces of both species over 4 days following oral administration of the labelled compound is of the same order (approximately 95 per cent). However, within the first 24 hours, dogs excrete 86 per cent of I4C in the urine while rats excrete only 67.5 per cent.

22 ORGANOPHOSPHORUS PESTICIDES: PHARMACOLOGY This suggests a more rapid rate of clearance of chlorfenvinphos from the body of dogs compared with that of rats. OTHER MEANS OF INACTIVATION

Yet another biological method of inactivation of organophosphates was demonstrated for the vinyl phosphates by Boyer [1691 who showed monocrotophos, dicrotophos, chlorfenvinphos and tetrachlorvinphos to be adsorbed to mouse and human blood plasma proteins. In determining the potency of tetrachlorvinphos against human plasma cholinesterase activity in vitro, Boyer found that of the total concentrations studied, only 25 per cent was available for enzyme inhibition. In the case of chlorfenvinphos, the actual amount inhibiting the human plasma enzyme activity was only one-third of the total added to the in vitro system. Using mouse plasma as the enzyme source, the following submultiples of the total amount added were available for enzyme inhibition : tetrachlorvinphos, 1/63 ; chlorfenvinphos, 1/19. The differences estimated using fly head homogenates as the enzyme source showed negligible degrees of adsorption. Although this phenomenon is of importance in determining the in v i m potency of cholinesterase inhibitors against a specific enzyme source, Boyer does not regard it of relevance to the overall toxicity of the compounds in vivo, for in the mouse, in which plasma binding is a strong factor, no correlation was found between the oral LD50 value and the predisposition to protein binding (expressed as ‘sorption isotherm constants’). However, as toxicity is the resultant of many factors (rate of absorption, rate of detoxication, rate of penetration to the target site, rate of excretion and rate of cholinesterase dephosphorylation), adsorption to proteins may be regarded as only one of the factors determining the in vivo biological effect [169]. Triolo and Coon [134] reported aldrin to reduce the in vivo toxicity of paraoxon in mice by increasing the binding of the organophosphate to plasma protein. From the foregoing, the routes of metabolism of organophosphorus insecticides in mammals may be classified as: (a) Replacement of sulphur with oxygen to produce the active moiety (for example, phosphorothionates and phosphorothiolothionates) ; (b) Oxidative dealkylation (for example chlorfenvinphos) or reduced glutathione-dependent demethylation (for example methylparathion) of the phosphate ester; (c) Oxidative N-dealkylation (for example monocrotophos, dicrotophos, Dhosohamidon. schradan) : (d) Cleavage of the phosphate ester (for example parathion, paraoxon, EPN); (e) Carboxylic ester hydrolysis (for example malathion, malaoxon) ; ( f ) p-Nitro-reductase (for example parathion, paraoxon) ; 1

.

IAN L. NATOFF '23 (g) Protein adsorption (for example monocrotophos, dicrotophos. chlorveniphos, tetrachlorvinphos).

THERAPY OF INTOXICATION BY ORGANOPHOSPHORUS PESTICIDES PARASYMPATHETIC RECEPTOR BLOCKING AGENTS

Blockage of cholinergic receptors prevents the symptoms of intoxication by cholinesterase inhibitors, which result from the accumulation of endogenous acetylcholine. There are four major classes of cholinergic receptor : (a) nicotinic cholinergic receptors in the ganglia of the autonomic sympathetic and parasympathetic nervous systems ; (b) nicotinic cholinergic receptors at the voluntary nerve muscle junction ; (c) muscarinic cholinergic receptors at the post-ganglionic nerve endings of the parasympathetic nervous system; and (d) cholinergic nerve synapses in the central nervous system. Treatment of cholinergic symptoms by autonomic ganglionic blockade would be undesirable in that the effect of such treatment is to abolish sympathetic as well as parasympathetic tone. Removal of sympathetic tone adds to the existing bradycardia and hypotension which form part of the syndrome of intoxication by cholinesterase inhibitors. Drugs which block at the neuromuscular junction may be divided into depolarising and competitive blocking agents. An example of the former is suxamethonium, which, prior to inducing blockade, facilitates depolarisation at the neuromuscular junction, so augmenting the depolarisation that is already present due to accumulation of endogenous acetylcholine by an additive effect. Competitive blocking agents, such as gallamine triethiodide and ( +)-tubocurarine reduce the effect of acetylcholine depolarisation without any initial facilitation, but as one of the prime sites at which this effect occurs is the diaphragm, the dangers of an uncontrolled paralysis of respiration make these compounds unsuitable for the treatment of intoxication by cholinesterase inhibitors. Blockade of autonomic post-ganglionic parasympathetic receptors is by far the most suitable type of treatment for some of the major symptoms of cholinesterase inhibitor poisoning available at the present time, and the solanaceous alkaloids (for example atropine, hyoscine) have long been established as efficient therapeutic agents. Synthetic muscarinic blocking agents also are available, some of which are confined principally to the peripheral areas of the body by their quaternary ammonium groupings, so increasing the specificity of their loci of effect. However, as many organophosphates act in the central nervous system, the use of centrally acting

24 ORGANOPHOSPHORUS PESTICIDES: PHARMACOLOGY cholinergic blocking agents, which also act peripherally, is generally the more acceptable for therapy. The use of these compounds in combination with a reactivating oxime leads to an augmentation of the therapeutic effect of both drugs by removing the cause of intoxication at the enzyme level with the oxime and by obviating the more severe parasympathetic and central symptoms of intoxication by specific receptor blockade. Oxotremorine stimulates cholinergic receptors, both peripherally and centrally, which are sensitive to the blocking action of atropine sulphate [66]. This compound, therefore, has been used as an indicator of the-specificity of various blocking agents. There is a widely varying ratio between the peripheral and central potency of many cholinergic blocking drugs [170]. Brimblecombe and Green [ 1711 examined the peripheral and central actions of some cholinergic blocking agents, using oxotremorine-induced salivation in mice as an indication of peripheral cholinergic stimulation, and oxotremorine-induced tremors in mice to indicate central cholinergic stimulation. Atropine sulphate was found to be thirty-seven times more effective against the central component of oxotremorine activity than against the peripheral component, while hyoscine hydrobromide was twenty-two times more potent against the central effects than against the peripheral effects. Taking oxotremorine-induced analgesia and tremors as criteria of the central effects of oxotremorine, a good correlation was found between the EDsovalues of different cholinergic blocking agents in reducing both of these effects [ 1721. Quaternary cholinergic blocking drugs (methylatropine, propantheline (8-di-isopropylaminoethyl-9-xanthenecarboxylate methobromide)) which have principally a peripheral locus of activity, were without effect on analgesia and tremor. The synthetic cholinergic blocking agent, caramiphen (1-phenylcyclopentanecarboxylic acid 2-diethylaminoethyl ester) has been shown to protect experimental animals against intoxication by a number of organophosphate and organothiophosphate inhibitors of cholinesterase (for summary of data, see [173]),its effect being enhanced when used in combination with P2S* or TMB-4t. Caramiphen is approximately equiactive with atropine in protecting guinea-pigs against sarin intoxication [173, 1741 but Leslie [172] showed caramiphen to have one-tenth the activity of atropine in blocking oxotremorine-induced analgesia and one-fifth the activity against tremors produced by this agent. These data suggest that the organophosphates cause death by mechanisms additional to those resuiting in stimulation of oxotremorinesensitive receptors. In spite of the continuing search for an improved muscarinic cholinergic blocking agent, atropine and its salts are still most widely used in the treatment of intoxication by organophosphate and carbamate inhibitors of cholinesterase. Therefore, the characteristics of this compound in the *N-methylpyridinium-2-aldoxime methanesulphonate tN.N’-trimethylenebis(pyridinium-4-aldoxime) dichloride

a

mammalian body have received attention. Differences in the locus of predominant effect of atropine are divided not only between the central and peripheral areas of the body, but within these areas themselves. Thus, atropine sulphate is more potent in blocking the responses of the dog stomach to parasympathetic nerve stimulation than those of the duodenum and colon [175] and studies on the distribution of tritiated methylatropine in mice and dogs show a preferential uptake of the 3H-radioactivity in the salivary gland, ciliary body, heart and stomach after subcutaneous injection [ 1761. Methylatropine is twenty-five times less potent in producing an effect following oral administration compared with its subcutaneous injection in dogs [1761 which is further reflected in the excretion rate of this compound. Within 2 hours of its subcutaneous injection, 40 per cent unchanged methylatropine appears in the urine of mice, and 80 per cent in that of dogs. After oral administration, only 15-20 per cent appears in the urine at 6 hours, of which less than 8 per cent is unchanged [176]. Using 14C-atropine sulphate, it was shown that following the injection of 0.5 mg/kg subcutaneously to a dog, the plasma concentration was twice as high as the brain concentration at 30 minutes. At 2 hours, the plasma, brain and CSF concentrations were equal while at 6 hours, the brain contained twice as much 14C-atropine sulphate as the plasma [ 1771. This may be related to 50 per cent of the administered dose appearing in the urine at 6 hours which would lower the plasma concentration following establishment of an equilibrium distribution between the brain and the plasma. The influx and efflux of the drug from the brain appear to proceed at the same rate over 10 hours [177]. Plasma concentrations in excess of 0.1 pg/ml result in a decrease of the net tubular secretion of the drug, and a decreased urinary excretion may result in an increased biliary secretion. The distribution and excretion of the drug is strongly influenced by pH [177]. Intravenous infusions of atropine sulphate into cats at different rates result in the attainment of various levels of the compound in the plasma. These are accompanied by the appearance of atropine in the effluent collected from the perfused cerebral ventricles and the spinal subarachnoid space, demontrating the penetration of the compound from the plasma to the CSF. The concentration in the latter fluid is not related to the plasma concentration [ 1781. The plasma concentration of atropine sulphate and methylatropine which produce tachycardia in beagles is about 0.1 pg/ml. The injection of atropine sulphate into the lateral ventricle is more effective in producing tachycardia than it is on subcutaneous administration indicating the alkaloid salt to be absorbed in the cerebral ventricles [179]. Further evidence that atropine sulphate exerts an effect in the brain following systemic administration whereas methylatropine does not was obtained in rabbits 11301. The duration of the EEG after-discharge is increased four times following the intravenous injection of 3 mg/kg of atropine sulphate while methylatropine is without effect. The intraventricular injection of the latter compound, however, increases the duration of the after-

26 ORGANOPHOSPHORUS PESTICIDES: PHARMACOLOGY discharge. This demonstrates that the quaternary salt is unable to gain effective access to the brain, and that the effect, when it occurs, is attributable to the alkaloid base itself [ 1801. By blocking the receptors to acetylcholine, atropine has been shown to increase the release of free acetylcholine in the presence of a cholinesterase inhibitor. The incubation of rat cerebral slices with 6 x 1 0 - 9 ~atropine sulphate increases the rate of release of acetylcholine in the presence of soman and a high potassium ion content (25-50 mM). (-)-Hyoscyamine is somewhat more potent at 1.2 x 1 0 - 9 ~ .The potency for releasing acetylcholine is apparently related to the antimuscarinic properties of these compounds [181]. Polak [182] also showed atropine to inhibit the uptake of acetylcholine by rat cortex slices, and this uptake appears to be ATP-dependent as it proceeds against a concentration gradient and is inhibited by 2,4dinitrophenol and anaerobiosis. Prevention of re-uptake therefore may contribute to a net increased rate of release. Szerb [183] showed that (1 mg/kg i. v.) atropine sulphate in cats increases the output of acetylcholine into cups containing physostigmine, 1pg/ml in Locke’s solution, positioned on the cortical surface [184, 1851 from 5 to 17 ng per 10 minute intervals. Methylatropine has only one-half to one-quarter the activity of the tertiary salt under these conditions. This suggests a closer potency ratio of the two salts of atropine regarding central nervous effects than has been suggested elsewhere (for example [172, 1SO]) but Szerb [ 1831 suggests this may be due to a breakdown of the blood-brain barrier at the site of acetylcholine collection. Locally applied methylatropine increases acetylcholine output similarly to locally applied atropine sulphate [ 1831. Atropine is 50-70 times more effective in decreasing the level of acetylcholine in different areas of the brain after systox treatment than in controls. Benactyzine (j?-diethylaminoethyl benzilate) is equiactive with atropine under these conditions but is more potent in the absence of systox [186]. The decreased brain substance level of acetylcholine may be attributed to an inhibition of its uptake from extracellular media [181]. The assumption that methylatropine, which is a quaternary salt of the alkaloid, does not cross the blood-brain barrier following systemic administration, is based on its lack of biological effect following doses which would be strongly active for atropine sulphate [172, 180, 1871. The recent work of Albanus, Sundwall and Winbladh [69], using tritiated methylatropine, objectively demonstrated the presence of this compound in the CSF. Following subcutaneous injection of 0.5 mg/kg in dogs, a steady concentration was reached in the CSF of the ventricle within 2 hours, and was maintained over 6 hours. The concentration in the CSF of the subarachnoid space reached a steady concentration of 0.05 pg/ml within 60 minutes of injection, this concentration being ten times that in the ventricular CSF. However, although the quaternary salt may enter the ventricular CSF, it appears that the doses necessary to obtain supra-liminal concentrations in

IAN L. NATOFF 27 this region would result in supra-maximal concentrations peripherally: This must be considered in relation to the observation that some organophosphates may increase the permeability of the blood-brain barrier to some basic compounds (821. Albanus, Sundwall and Winbladh [69] made their observations in the absence of organophosphates. A relationship between the inhibition of uptake of acetylcholine in rat cortex slices and the antimuscarinic activity [181] and the affinity of the inhibitory substances for the acetylcholine binding sites [ 1821 has been suggested. Choline, hemicholinium-3, physostigmine, atropine and other similarly charged compounds inhibit the uptake ; DFP, soman and tabun do not. These organophosphates are thought to react with the esteratic site on the enzyme molecule, while choline, hemicholinium-3, physostigmine and atropine have a basically charged nitrogen capable of combining with the anionic site. Thus, inhibition of uptake does not appear to be a function of cholinesterase inhibition, but may be a function of the affinity of the inhibitors for the anionic site of the uptake mechanisms with which acetylcholine combines [ 1821. Atropine sulphate therefore has wider application than methylatropine in blocking the receptors both peripherally and centrally at which accumulation of excess acetylcholine may arise following cholinesterase inhibition. Indeed, atropine sulphate is more effective on a molar basis than methylatropine in protecting rats against intoxication by organophosphates [ 1881. The degree of protection afforded to mice by atropine against DFP is mostly independent of the dose of the blocking agent used [189, 1901. However, it has been shown that the duration of the effect of atropine against the toxicity of oxotremorine in mice is a function of the dose of atropine [ 1911. A direct relationship between anticholinergic potencies of a series of drugs in the central nervous system and their therapeutic efficiency in treating poisoning by sarin has been reported [ 1741. This relationship does not hold for the peripheral cholinoceptor blocking activity of these drugs.

OXIMES

The phosphorylation of the serine hydroxyl group of cholinesterase by organophosphorus compounds inhibits the activity of this enzyme [ 1921. This phosphorylation is reversible when the phosphoryl ester so formed is of the dialkoxy type, but becomes irreversible when deaikylation takes place. Wilson [ 151 found nycleophilic attack of the dialkyl phosphorylated enzyme with hydroxylamine to result in regeneration of the enzyme activity. It has been suggested that some oximes act initially by forming a complex with the phosphorylated enzyme [193-1951. Later work [196] showed the incorporation of a quaternary nitrogen into the nucleophilic molecule (for example N-methylpyridinium-2-aldoxime salts ; 2-PAM ; P-2-S ; prali-

28 ORGANOPHOSPHORUS PESTICIDES : PHARMACOLOGY doxime ;hereinafter referred to as PAM) (I) increases the nucleophilic attacking properties by attachment of the charged nitrogen to the anionic site of the enzyme. The rate of recovery of cholinesterase activity in an enzyme preparation inhibited with parathion is lo6 times faster with PAM than with hydroxylamine [ 1971. Intoxication of rats with soman may be prevented by the administration of PAM within 5 minutes of injecting the organophosphate. A delay of more than 10 minutes does not allow any protection to this intoxication [198] suggesting that ageing of the inhibited enzyme has taken place. However, the inhibition of cholinesterase by soman both in vivo and in vitro may be reversed by PAM and it has been demonstrated that the rate of ageing of this inhibited enzyme is dependent on temperature [ 1991. It also has been suggested that steric factors influence the rate of ageing of cholinesterase inhibited with soman [200]. Other oximes have been examined for their reactivating potency against phosphorylated cholinesterase [201-2031. Among these, the bis-quaternary pyridinium-dioximes have a high order of potency. It was suggested by Hobbiger, Pitman and Sadler [195] that the bis-quaternary nature of these compounds enhances their binding to the anionic site of the enzyme, so improving the nucleophilic attack by the oxime group. TMB-4 (N,N’trimethylenebis(pyridinium-4-a1doxime)dihalide) (11) has 20 times the reactivating potency of PAM against cholinesterase inhibited by isopropoxyand ethoxy-organophosphates, and the ethoxyphosphorylated enzyme is about 20 times easier to reactivate with TMB-4 than is the isopropoxyphosphorylated enzyme [204]. This is in keeping with the known relative rates of ageing of cholinesterase inhibited by organophosphates having different alkyl ester groupings [28]. Although the mono- and bis-pyridinium oximes are highly dissociated molecules, the percentage of the active anion present at normal physiological pH is greater in the bispyridinium compounds than in the monoximes [205]. Obidoxime (Lu-H6, Toxogonin, N,N’-oxydimethylenebis-(pyridinium-4a1doxime)dihalide) (111) appears to be more effective against inhibited cholinesterase of the red blood cell than against that of plasma following intoxication of dogs with parathion [206]. It is less effective following malathion intoxication. The use of non-charged nucleophilic compounds as reactivators of inhibited cholinesterase was envisaged to allow greater activity in the brain owing to their ability to penetrate the blood-brain barrier following systemic administration. Monoisonitrosoacetone (MINA)(IV) and diacetylmonoxime (DAM) (V) were shown to be effective against inhibited cholinesterase [207] and to reduce the mortality of mice following organophosphate intoxication [208], but these oximes are not without their toxic side-effects. For example, MINA has been shown to be metabolised to cyanide in vivo [209]. Using equimolar doses of MINA and DAM in sarin-poisoned rats,

IAN L. NATOFF -29 Rutland [210] showed MINA to be the more potent of the two oximes against phosphorylated esterase from blood, whereas DAM has the greater activity CH=NOH

I

CHI-CH2-cH*

rn

NOH

2x

NOH

II

cur

I

c=o

c=o

MC

Mt

(IVI

IVI

I

11111

I

1111

Ill r

CH=NOH

I

against sarin-inhibited brain esterase. MINA also was shown to be the more potent of the two oximes in reactivating cholinesterase inhibited by DFP in autonomic ganglia [211]. Although oximes reactivate dialkylphosphorylated cholinesterase in many cases, the quaternary compounds nevertheless do not have access to many sites at which cholinesterase inhibition occurs. Therefore, as an adjunct to oxime therapy, atropine sulphate is widely used. Although MINA was reported to be superior to PAM or atropine sulphate when used alone in protecting sarin-poisoned rats [2 121, the combined effect of atropine and PAM is superior to that of atropine and MINA. Mice intoxicated with diazinon are protected by atropine sulphate, and this protection is enhanced with oximes [2 131. The improved protection by the combination of atropine sulphate and oximes is a synergistic phenomenon, being greater than the summated effects of either antidote alone [lSS, 214-2181. Mice intoxicated with dichlorvos are better protected by the combination of atropine sulphate with obidoxime than with PAM [216], and this combination of atropine sulphate with obidoxime also was found to be superior to a combination of atropine sulphate with PAM in the therapy of rats intoxicated with dicrotophos, dichlorvos, chlorfenvinphos and mevinphos [188]. Because of the rapidity with which quaternised oximes are excreted from the mammalian body [219], it has been suggested that their combined use with atropine is more effective when given therapeutically rather than prophylactically in organophosphorus intoxication [2 151. An interesting mechanism for this synergism was suggested by Ramachandran [220] in that atropine may improve the turnover of oximes in vital areas of the body of intoxicated animals. Further protection of experimental animals from organophosphate intoxication was demonstrated by Brodeur and Alary [221] who found the application of atropine sulphate with PAM to increase the survival rate

30 ORGANOPHOSPHORUS PESTICIDES: PHARMACOLOGY of rats to a greater extent when these animals had been pre-treated with phenobarbitone, used as a microsomal stimulant. This suggests that the added effect is of an increased rate of catabolism of the organophosphates. As the N-alkylpyridine compounds are quaternary ammonium derivatives, it is likely that they exert their action mainly at peripheral nicotinic and muscarinic sites. Thus, organophosphate inhibition of the rat diaphragm may be reversed by these compounds in vitro [222], and Wolthuis and Meeter [223] showed neuromuscular blockade and inhibition of cholinesterase by D F P in vivo to be reversed by obidoxime and by PAM at the voluntary neuromuscular junction, but only very weakly in the brain. PAM and MINA also restore tetanus to the rat diaphragm in vitro after organophosphate exposure [224]. PAM is superior to both TMB-4 and obidoxime against soman poisoning whereas obidoxime is superior to PAM against tabun. Thus the effectiveness of the oxime varies with the cholinesterase inhibitor. Paraoxon reduces the cardiac output of cats. This is not tractable to treatment with methylatropine, but does respond to both PAM and obidoxime [225]. This effect is not therefore related to peripheral muscarinic hyperactivity at vagal nerve endings, but is more likely related to a potentiation of the nicotinic effects of acetylcholine at parasympathetic ganglia. The quaternary ammonium compound PAM has been shown to have nicotinic stimulant properties at autonomic ganglia in the anaesthetised dog [226] and in muscle preparations of the ascaris nematode [227]. PAM was thought to have a curare-like action at concentrations above 6 n m [228] which later was shown to be due to its reducing the presynaptic release of acetylcholine [229]. The non-quaternised oxime, MINA, also has been reported to have anti-nicotinic properties [230]. Much inferential evidence exists suggesting that the mono- and bispyridinium oximes (which are quaternary ammonium compounds) exert an effect in the central nervous system following systemic administration. The presence of small amounts of PAM, obidoxime and TMB-4 has been demonstrated in the brain following the injection of therapeutic amounts of these compounds [204, 23 I], and PAM has been shown to reduce the degree of inhibition of brain enzyme following the intoxication of rats [232] and rabbits [233] with paraoxon. The toxicity of TEPP to mice is reduced by the injection of PAM both intraperitoneally and intracerebrally. This protection is increased by atropine sulphate [234]. The amount of free acetylcholine in the brains of rats following paraoxon intoxication is reduced by PAM [235] and obidoxime [236]. In this latter study, obidoxime was found to be twice as potent as PAM on a weight basis. Further evidence that PAM penetrates the blood-brain barrier was provided by the observation that it affects the EEG of the cat [237] and raises the dose of sarin required to produce a ‘grand-mal’ EEG pattern in the rabbit [238]. Gosling and Lu [239] reported 3H-labelled quaternary methonium compounds to accumulate in the choroid plexus and the cells of the arachnoid membrane.

IAN L. NATOFF 31 Edery [240] suggested organophosphates and oximes to act on structures which line the cerebral ventricles, probably affecting 'functional' cholinesterase on the cell surface rather than the reserve enzyme in the tissue mass [232]. 14C-PAM was shown to result in an accumulation of radioactivity in the cerebral cortex of rats [241], and to enter the brains of rabbits following intravenous administration [242]. Falb and Erdmann [243] reported 14Cobidoxime to be present in the brains of mice and rats after intravenous injection. Other biological barriers also are permeable to these oximes. The presence of PAM has been demonstrated in the cerebral ventricles and the foetus of pregnant rabbits following intravenous administration [244], suggesting passage across the placental membrane, and both PAM and TMB-4 are absorbed across the intestinal wall [245]. Although PAM is absorbed more efficiently than TMB-4, the rate of elimination of these compounds through the kidneys [219] exceeds their rate of absorption [245], making their oral administration impractical for therapeutic purposes. The rate of absorption of PAM across the wall of jejeunal sacs is seven times slower than that of its tertiary analogue [246]. The intestinal absorption of obidoxime is poor [247]. While oximes are of great value in the therapy of intoxication by organophosphates, they are not without their unwanted side-effects. Thus, while it has been reported that PAM does not protect rats against intoxication by such carbamates as sevin (1-naphthyl-N-methyl carbamate) [248, 2491, it was suggested that PAM reduces the protective effect of atropine against this carbamate in one dog [248]. Evidence also has been presented showing that PAM has a direct inhibitory effect on electric eel cholinesterase in vitro [250]. Although chronic toxicity studies with PAM and with TMB-4 have revealed relatively few undesirable effects, such as gastro-intestinal disturbances [251] and a decrease in the coagulation time of blood [252], other oximes (2-fury1 methyl ketone 0-carboxyoxime methyl ester and 2-fury1 methyl ketone 0-acetyloxime) cause aggregation of rabbit and guinea-pig platelets, but not those of rats, possibly by the release of endogenous adenosine diphosphate (ADP) [253]. Hydroxylamine, however, inhibits ADPinduced aggregation of platelets.

CONCLUDING REMARKS The pharmacology of the organophosphorus cholinesterase inhibitors has many facets. Much has been discovered concerning their mode of action and the therapeutic measures to be adopted in the event of intoxication by these compounds. Accepting the specificity of the antidotes used in these therapies, laboratory studies with them may indicate the principal locus of the toxic effects of the organophosphates. Because of the multiplicity of the biological effects of the organophosphorus pesticides, whether these effects

32 ORGANOPHOSPHORUS PESTICIDES : PHARMACOLOGY are primary or secondary, no straightforward ruling may be made regarding their action. Although in some instances generalisations are possible, each organophosphate is unique and must be considered individually. Much of the quantitative data on enzyme inhibition in vivo and in vitro until recently has been expressed in terms of the degree of effect. Increasing importance is now being given to the rate of onset of effect. This review has summarised the more recent developments in the understanding of the mode of action of the organophosphorus compounds in mammals. The reader is referred elsewhere for the effect of these compounds in insects and in other pests.

REFERENCES

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2 The Mode of Action of Novobiocin A. MORRIS*, B.Pharm., Ph.D., M.P.S. and A. D. RUSSELL, B.Pharm., Ph.D., M.P.S. Welsh School of Pharmacy, University of Wales Institute of Science and Technology, Card& Wales

INTRODUCTION

40

DISCOVERY AND CHARACTERISTICS O F NOVOBIOCIN

40

MECHANISM O F ACTION O F NOVOBIOCIN Changes in bacterial morphology Effects on bacterial cell wall synthesis Effects on the synthesis and integrity of the bacterial cytoplasmic membrane Effects on bacterial protein synthesis Effects on bacterial nucleic acid syntheses: RNA synthesis DNA synthesis Induction of a magnesium deficiency Effects on enzyme systems and electron transport

41 41 43 46 41 47 47 48 49 54

STRUCTURE-ACTIVITY RELATIONSHIPS

54

ADSORPTION OF NOVOBIOCIN ON TO BACTERIA

55

RESISTANCE TO, AND CROSS-RESISTANCE WITH, NOVOBIOCIN

55

CONCLUSIONS

56

REFERENCES

56

*Present address: Bacterial Chemotherapy Unit, Glaxo Research Laboratories Ltd.. Greenford, Middlesex, England

39

40

THE MODE OF ACTION OF NOVOBIOCIN

INTRODUCTION A study of the mode of action of an antimicrobial drug attempts to elucidate its biochemical effects on susceptible organisms. In addition, such a study provides an insight into hitherto unknown structures and processes present in micro-organisms and may offer clues to the chemist who wishes to design a drug with a specific effect. Success in elucidating the precise mechanism of action of a drug depends on the complete understanding of the sensitive cell and, although much is known about microbial structure and metabolism, the existing gaps in knowledge are still sufficient to hamper experimental design and the interpretation of experimental results. The mode of action of many antibacterial agents is still unknown. An attempt has been made in this review to clarify the diverwand sometimes conflicting reports concerning the proposed mechanism of action of novobiocin.

DISCOVER+ AND CHARACTERISTICS OF NOVOBIOCIN Novobiocin (I) is the official name given to an antibiotic discovered by no fewer than five independent groups of workers [l-51. It is a dibasic acid, and before its chemical structure was completely elucidated it was defined as

R I

I

PJ H2

(1)

Novobiocin

7-[4-carbamoyloxytetrahydro-3-hydroxy-5-methoxy-6,6-dimet hylpyran-2yloxy]-4-hydroxy-3-[4-hydroxy-3-(3-methyl-2-butenyl)benzamido]-8-methylcoumarin. In the light of present knowledge [6], a better name would be

41 A. MORRIS AND A. D. RUSSELL ~-[3-0-carbamoyl-5,5-dimethyl-4-O-methyl-a-~-lyxosyl]-4-hydroxy-3-[4hydroxy-3-(3-methylbut-2-enyl)benzamido]-8-methylcoumarin. Novobiocin is marketed under the following trade names: Albamycin, Biotexin, Cathocin, Cathomycin, Inamycin, Spheromycin, Vulcamycin and Vulkamycin. The antibiotic is soluble in aqueous solution above pH 7.5 [7] and in polar organic solvents. It is stable in the dark, but is slightly lightsensitive [7]. In practice, it is used either as the mono- or di-sodium salt or as the calcium salt. Novobiocin possesses a fairly wide range of activity and is active mainly against Gram-positive bacteria, particularly Staphylococcus aureus, having a minimum inhibitory concentration of between 0-1 and 5 p g / d for most strains of this organism. Other organisms that are susceptible include Neisseria, Haemophilus and Brucella species and certain strains of Proteus, but it is rarely used against these organisms in clinical practice. It is normally reserved for use against penicillin-resistant staphylococcal infections. For a more detailed account of the range of activity and the clinical applications of novobiocin, two earlier reviews should be consulted [8, 91. Both of these reviews also describe the effects of inoculum size, pH, sodium chloride, metal ions, and serum on the activity of novobiocin against various bacteria.

MECHANISM OF ACTION OF NOVOBIOCIN CHANGES IN BACTERIAL MORPHOLOGY

Novobiocin induces filamentation in Gram-negative rods [ 1&13] with subsequent vacuolation [12] and loss of intracellular materials [14]. It is, however, debatable whether the induction of filamentous forms is characteristic of a particular biochemical effect. Some workers have advocated that filamentation indicates a specific inhibition of cell wall synthesis [15, 161, but a number of antibacterial agents that exert their effects elsewhere in the cell also induce filamentation, for example mitomycin C [17], acridines [18, 191, nalidixic acid [20], ultraviolet light [21] and m-cresol [22]. It is probable, therefore, that filamentation induced by novobiocin is not wholly related to a specific effect of the antibiotic. Novobiocin also causes chaining in Streptococcusfaecium [23], although this effect is not produced in all cocci [13]. The induction of spheroplasts in Escherichia coli by novobiocin has also been reported [24] and, in fact, the antibiotic has been recommended for preparing spheroplasts in Gramnegative bacteria [25]. In contrast, however, it has been shown by various workers [2628] that novobiocin does not induce spheroplasts in Serratia marcescens or in various strains of E. coli and [28] that it may even prevent spheroplast induction caused by benzylpenicillin in hypertonic medium (Figure 2.1).

42

THE MODE OF ACTION OF NOVOBIOCIN

100

80 In

a+

-a In 0

0 L

aJ f

-

Q

o

60

v)

v

?

L

0 C

0 L

al

$

LO

0,

m 0

c C

aJ

L U

0

a 20

, 0

c

2

w

CI

v

3 L Time ( h o u r s )

5

P 6

Figure 2.1. Effect of novobiocin on the conversion of E.coli cells into spheroplasts by benq.1penicillin. Benzvlpenicillin concentration throughout, 250 units/ml. Novobiocin concentrations (pgiml): 0, ( L O ; 50. x-x ; 100. 0-0;500, A-A. (From Morris and Russell [28], by courtesy of Microhios).

43

A. MORRIS AND A. D. RUSSELL EFFECTS ON BACTERIAL CELL WALL SYNTHESIS

Early workers [29] found that, like benzylpenicillin, vancomycin, ristocetin and bacitracin, novobiocin caused an excessive accumulation of cell wall precursor, uridine diphosphate-N-acetylmuramic acid-L-alanine-D-glutamic acid-L-lysine-D-alanine-D-alanine(UDP-MurNAc-L-ala-D-glu-L-lys-D-alaD-ala) in Staph. aureus and it was thus considered that novobiocin was a specific inhibitor of peptidoglycan synthesis with an effect similar to that of penicillin. However, subsequent studies led to the withdrawal of this hypothesis [26], since novobiocin caused the accumulation of other precursor-type compounds and also strongly inhibited both nucleic acid and protein synthesis in this organism. Thus, accumulation of particular precursors does not necessarily reflect the site of action of an antibacterial agent [30]. Other investigations have shown that novobiocin causes a non-specific inhibition of cell wall synthesis in Staph. aureus and Strept.faeciurn [23, 31, 321 and that in E. coli the novobiocin-induced inhibition of cell wall synthesis is secondary to the inhibition of nucleic acid syntheses [33]. With cellular extracts of Staph. aureus and Micrococcus lysodeikticus, novobiocin inhibits the formation of the alternating N-acetylmuramic acidpentapeptide and N-acetylglucosamineresidues

I --MurNALGlcNAcI

1

I

pentdpeptide

I I

1.

during cell wall peptidoglycan synthesis, but only at concentrations many times those needed to inhibit growth of the particular organism [34, 351; for example with Staph. aureus, 0.03 pg/ml is required for 50 per cent growth inhibition and as much as 10 pg/ml for 50 per cent inhibition of peptidoglycan synthetase [36]. Benzylpencillin also does not inhibit the peptidoglycan synthetase reaction, whereas ristocetin, vancomycin and bacitracin do at antibiotic concentrations closely related to those which are inhibitory to cell growth. Vancomycin and ristocetin allow the formation of the lipid intermediate, GlcNAc-MurNAc(pentapeptide)-P-P-phospholipid,but prevent its utilisation for peptidoglycan synthesis [36, 371. It may be concluded that novobiocin does not interfere with the formation or utilisation of the lipid intermediate involved in the above reaction [36-381. With other cell-free systems obtained from Bacillus licheniformis and Lactobacillus plantarum, novobiocin was found to inhibit both polyglycerophosphate and polyribitol phosphate syntheses [39, 401. However, the

44 THE MODE OF ACTION OF NOVOBIOCIN workers involved emphasised the need for caution in relating the primary mode of action to these inhibitions. Moreover, it has been observed [33] that B. megaterium is sensitive to novobiocin although this organism does not contain teichoic acid in its cell wall. In addition, another organism, M . lysodeikticus, which contains no teichoic acid in its cell wall, is also sensitive to novobiocin [31]. Novobiocin has been found to inhibit the synthesis of teichuronic acid by cellular extracts of B. licheniformis [41], but as this occurs only at concentrations of antibiotic much greater than the minimal concentration needed to inhibit growth, it is difficult to propose that this is its primary effect. An indication of whether an antibiotic exerts a specific effect on cell wall synthesis may be obtained by elucidating whether the antibiotic induces lysis of growing cells or the formation of spheroplasts, protoplasts or L-forms [42-44]. Warren and Gray [45, 461 found that Staph. aureus cells grown in subinhibitory concentrations of nafcillin underwent lysis on subsequent incubation with lysozyme and trypsin, whereas cells grown in subinhibitory concentrations of novobiocin were not rendered susceptible to the enzyme combination. Novobiocin has also been shown not to induce lysis of Staph. aureus [131 or of E. coli [27]. As already mentioned, although it was originally claimed that novobiocin induced the production of osmotically-sensitive forms, it is now clear that this is not so. Whereas it has been reported that the antibiotic induces the production of L-forms [47], there are a number of publications showing that this does not, in fact, occur [48-521. Roberts [52] for example, found that methicillin, ampicillin, cycloserine; cephalothin, ristocetin, bacitracin and vancomycin, but not novobiocin, induced L-forms in meningococci. The present evidence therefore indicates that novobiocin does not exert a specific effect on cell wall synthesis. Cell wall-deficient bacterial forms (protoplasts, spheroplasts and L-forms) are frequently used for determining whether a particular antibiotic exerts effects on the bacterial cell other than on the cell wall [53, 541, and this technique has been employed by many workers studying the effects of novobiocin. Thus, novobiocin is very active against penicillin-induced L-forms of E. coli [48], Proteus mirabilis [55] and Strept. faecalis [56, 571. Lysozymeinduced protoplasts of Strept. faecalis [58] and of B. megaterium [59], and L-forms of meningococci [51, 521 and of Staph. aureus [60] are as sensitive to novobiocin as are the corresponding parent forms. L-forms of Strept. pyogenes are lysed by the antibiotic [61], and similar observations have recently been made with penicillin-induced spheroplasts of E. coli and lysozyme-induced protoplasts of B. megaterium, both forms undergoing lysis in the presence of novobiocin although only when placed in a medium conducive to growth [27] (Figure 2.2). In addition, novobiocin has been found to interfere with the action of penicillins on Staph. aureus [62, 631. Thus, it seems highly unlikely that novobiocin exerts a specific effect on cellwall synthesis.

45

A. MORRIS A N D A. D. RUSSELL

0.6

0.5 0 0 In Y

0 5r

0.4

.-ul

. I -

C

al

- 0.3 73 0

.-V

I

8 0.2

I

0.1

1

I

0

I

1

I

I

2

1

1

3

(b)

E

'0-4

0 0

'? c

0

-0.3 .-ul . I -

C

a

73

0

0.1

-

0 -1

I

0

I

I

I

I

1 2 3 4 Time after addition (hours)

1

I

5

6

Figure 2.2. Effect of novobiocin on the growth (as represented by changes in optical density) o j (a) spheroplasts of E.coli, and (b)protoplasts of B.megaterium. Novobiocin concen0 (benzy!trations (pgiml), in ( a ) :0,O-O; 100, 0-0; 500, A-A; in ( b ) :0 - 4 ; penicillin 1.5 units/ml), A-A; 2.5, x-x ; 5, 0-0; 25, 0-0. (From Morris and Russell [27], by courtesy of Biocheniical Pharnzacology.)

46

THE MODE OF ACTION OF NOVOBIOCIN

EFFECTS ON THE SYNTHESIS AND INTEGRITY OF THE BACTERIAL CYTOPLASMIC MEMBRANE

In an early report [48], it was stated that novobiocin inhibits the synthesis of the bacterial cytoplasmic membrane, as it decreased the crypticity of E. coli strain ML 35 and caused a loss of ribonucleic acid (RNA) into the surrounding medium. These effects were not induced in resting cells and indicated that the antibiotic interfered with the synthesis of new membrane material. Another early investigation had shown that novobiocin induced the loss of 260 nm absorbing material from washed suspensions of Staph. aureus [MI. More recently, it has been observed that novobiocin-induced membrane damage occurs in ML strains of E. coli but not in non-ML strains [33]includTA- and W. Further investigations into this problem, ing KIz-3000,B,15 however, reveal that novobiocin also induces a loss of intracellular materials in non-ML strains [65], and as this was shown to occur in a hypertonic medium, it indicated a more specific effect of the antibiotic on the cytoplasmic membrane. In view of these findings and the earlier report by the same workers [27], it is probable that membrane damage induced by novobiocin in E. coli is not specific to any particular strain of this organism. A possible explanation for these conflicting observations has been suggested by Morris and Russell [66], who pointed out that, in their experiments [65], a minimal concentration (0.0025per cent w/v) of magnesium sulphate was present in the synthetic medium used because excess of MgZ ions markedly reduced the extent of leakage. Smith and Davis [33] did not investigate the effect of varying concentrations of magnesium sulphate on leakage. Several biochemical studies into the effects of novobiocin on membrane synthesis have been carried out. For example, the incorporation of radioactive glucose into the mannose fractions of the cytoplasmic membrane of M . lysodeikticus has been found to be inhibited by novobiocin [311, although similar inhibitions occur in other cellular fractions. With the use of protoplasts of B. meguterium, it has been shown [67] that the incorporation of radioactive amino acids and glycerol into both membrane and cytoplasmic fractions is inhibited by similar degrees. It was concluded that, although the membrane may be malfunctional, the cytoplasmic structure of novobiocintreated protoplasts can be similarly described. As already stated, lysis of L-forms of Strepr. pyogenes by novobiocin has been reported [61], indicating an effect of the antibiotic on the membrane. However, puromycin was also found in this investigation to induce lysis, and the results should therefore be interpreted with caution, since puromycin, which chemically closely resembles the end-group of aminoacyl-tRNA, interferes with protein synthesis by forming a peptide link with the terminal carboxyl group of the incomplete peptide chains [68]. Thus, novobiocin exerts a fairly specific effect at the cytoplasmic mem+

A. MORRIS AND A. D. RUSSELL 47 brane of E. coli, and such an effect occurs in more strains than those suggested by Smith and Davis [33]. Little is yet known about membrane biosynthesis [69], and it is not possible to propose that the effects of novobiocin so far noted constitute a primary lesion.

EFFECTS ON BACTERIAL PROTEIN SYNTHESIS

A number of workers have shown that novobiocin inhibits protein synthesis in bacteria. For example, it inhibits B-galactosidase synthesis in both Staph. aureus and E. cofi [48, 641. Although M-protein synthesis in a Group A streptococcus is not inhibited by novobiocin [70], the inhibition of protein synthesis in Strept. faecium has been attributed to the inhibition of tRNA synthesis [23]. Novobiocin inhibits protein synthesis in Staph. aureus [26], but as the inhibition of other macro-molecules also occurs to a similar extent, this effect may not be the primary action of the antibiotic. The antibiotic also inhibits protein synthesis in E. cofi [33, 711 but this inhibition appears much later than the observed inhibition of deoxyribonucleic acid (DNA) synthesis. Thus, the inhibition of protein synthesis is probably not the primary effect of novobiocin on bacteria but is normally secondary to the inhibition of other metabolic processes. EFFECTS ON BACTERIAL NUCLEIC ACID SYNTHESES

R N A Synthesis

Brock and Brock [48] found that novobiocin induced the loss of RNA from E. coli, but they did not propose that the antibiotic exerted a specific effect on RNA synthesis. Later, Brock [23] attributed the strong inhibition of RNA synthesis in Strept. faecium to a novobiocin-induced magnesium deficiency. However, Smith and Davis [33, 711 showed that the inhibition of RNA synthesis in E. coli is secondary to the inhibition of DNA synthesis. The degradation and leakage of RNA is induced by novobiocin with ML, but not with non-ML strains of the organism [33]. In contrast, however, Morris and Russell [65] observed that novobiocin induced the loss of RNA-like material from non-ML strains of E. coli (see also Figure 2 4 , and although RNA synthesis was strongly inhibited, the inhibition of DNA synthesis occurred to an even greater extent [72]. It was concluded that the loss of RNAlike material into the surrounding medium was a result of a loss of membrane integrity. A recent report [73] states that the novobiocin-producing organism Streptomyces niveus is itself inhibited by the antibiotic and that this inhibition

48

THE MODE OF ACTION OF NOVOBIOCIN

is due to an inhibition of nucleic acid, and particularly RNA, synthesis. However, the majority of findings indicate that novobiocin exerts only a secondary effect on RNA synthesis.

0

I

I

I

J

1

2

3

Time(hours)

Figure 2.3. Effect ofnovobiocin on leakage of260 nm-absorbing niaterialfrorii log phase cells of E.coli in synthetic rnediurn. Novobiocin concentrations (pg/ml): 0, M;5, 0-0 ; 10, V-V ; 50, x-x ; 100, 0-0; 500. A-A. (From Morris and Russell [66]. by courtesy of Microbios.)

D N A Synthesis

The evidence that novobiocin exerts a specific and primary effect on DNA synthesis in E. coli was the subject of a recent report [33]. In all strains of E. coli employed in this study, DNA synthesis was inhibited earlier and to a greater extent than were other macroaolecular syntheses. The antibiotic did not cause degradation of DNA; its action was reversible, for when the

A. MORRIS AND A. D. RUSSELL

49

novobiocin was removed from a culture of the organism, DNA synthesis was immediately resumed at a rate comparable to that of an untreated culture. A similar observation has been made by Morris and Russell [72]. Smith and Davis [33] also reported that the T,,, value of bacterial DNA was not affected by novobiocin, indicating that the antibiotic does not form a stable complex with bacterial DNA. By means of partially purified E. coli DNA polymerase and native E. coli DNA, it was found that novobiocin inhibited polymerisation [33]. It also inhibited the replication of RNA phage M5-2 and DNA phages T2 and T3. Earlier studies [48, 741 had shown that novobiocin induced a decrease in the DNA content of E. coli cells, but this was not regarded as being of primary importance. Wishnow, Strominger, Birge and Threnn [26] subsequently reported that novobiocin strongly inhibited the incorporation into Staph. aureus of radioactive lysine and of inorganic phosphate into nucleic acid, but the inhibition of inorganic phosphate also occurred in other macromolecular fractions of the cell, and the primary effect of the antibiotic was not defined. More recently [72, 751, novobiocin has been shown to exert an immediate effect on DNA synthesis in both E. coli and Staph. aureus, this inhibition occurring to a greater extent than that of RNA and protein syntheses (Figure 2.4). A low concentration of novobiocin (20 pg/ml) does not inhibit protein synthesis in E. coli in nutrient broth at 37°C [72, 751. However, Morris and Russell [72] considered that the inhibition of DNA synthesis was secondary to an effect on the cytoplasmic membrane; a similar proposal has been made regarding the mode of action of phenethyl alcohol [76]. In view of the rapid effects of novobiocin on both spheroplasts and protoplasts [27] and the close association of the bacterial membrane and chromosome during growth [77, 781, such a hypothesis is feasible, although further work along this line is desirable. However, mitomycin C, which forms cross-links with DNA and inhibits DNA synthesis [17], also induces lysis of growing spheroplasts of E. coli [27], and this finding suggests that the inhibition of DNA synthesis precedes membrane damage. INDUCTION OF A MAGNESIUM DEFICIENCY

Early studies [72, 791 showed that metal ions, in particular those of magnesium, antagonised the action of novobiocin against Gram-negative organisms but not its action against Gram-positive bacteria. These findings have since been confirmed [13]. Evidence was later provided that novobiocin forms a complex with magnesium ions [32] and it was proposed that an intracellular deficiency of these ions was thereby induced by the antibiotic. Many of the effects induced by novobiocin were then shown by Brock [23, 32, 801 to be similar to those induced by a magnesium deficiency. However, a deficiency

50

THE MODE OF ACTION OF NOVOBIOCIN

I

I

I

I

(

2

1 (C)

I

I

1

,

I

I

2

I

1

I

I

2

Time after addition (hours)

Figure 2.4. EJect of novobiocin on ( a )growth, as recorded by changes in opticul density, ( b )DNA synthesis, (c) RNA synthesis, and (d)protein synthesis in E.coli in nutrient broth at 37°C. Novobiocrn concentrations (pgiml): 0. C k O ; 20, m-m; 100, 0-0; 500, A- A.(From Morris and Russell [72], by courtesy of Microbios.)

A. MORRIS AND A. D. RUSSELL 51 of magnesium ions does not provide the complete explanation of the mode of action of novobiocin. Against Neurospora crassa, for example, novobiocin exerts less activity when the magnesium content of the medium is lowered [8 11 and the novobiocin-induced inhibition of the particulate cytochromelinked oxidase of Mycobacteriurn phlei is not alleviated by magnesium ions [82]. DNA polymerisation in vitro is independent of the magnesium concentration, although the reaction is inhibited by novobiocin [33]. Magnesium ions are essential for the formation of cell wall precursors and, as novobiocin causes the accumulation of these same precursors, it is difficult to envisage the induction of a magnesium deficiency to explain fully the effect of novobiocin [26]. More recently, it has been shown [83] that magnesium ions antagonise the bactericidal effect of novobiocin against E. coli (Table 2.1) in both complex

Table 2.1 ANTAGONISM BY MAGNESIUM SULPHATE OF THE BACTERICIDAL EFFECT OF NOVOHIOCIN (500 pg/ml) ON LOG-PHASE CELLS OF E.coli IN NUTRIENT BROTH (OXOID) AT 37"c. FIGURES ARE VIABLE NUMBERS OF CELLS x 107/ml.(From Morris and Russell [83], by courtesy of Microbios). Concentration ( % w/v) of magnesium sulphate added to broth'

Time (h) after addition of novobiocin

0 0.5 1 3

0.025 14.0 7.1 3.1 0.6

14.0 8.1 3.8 0.8

0.05

0.1

14.0 8.8 4.7 1.9

14.0 9.3 8.7 4.1

0.25 14.2 13.2 11.9 9.2

0.5

14.2 13.3 12.4 10.7

'The broth itself contained traces of Mg' +,although the concentrationwas not determined.

and synthetic media, but do not against Staph. aureus. However, the inhibition of cell division induced by novobiocin is not antagonised by magnesium ions, which indicates that these ions do not interfere with the uptake of the antibiotic, as had been previously suggested [79]. In addition, it has been found [66, 831 that magnesium ions reduce novobiocin-induced loss of intracellular materials (Figure 2.5), the metal ions in some way stabilising the membrane. Further evidence that novobiocin does not exert its effects on E. coli by inducing a magnesium ion deficiency was obtained when both DNA and RNA syntheses were found to be inhibited to a similar extent in media containing a trace and an excess of the metal ions [72]. In addition to the report that novobiocin forms a complex with magnesium ions [32], there are two other accounts which show that such a complex is not formed [84,85]. In their study, Morris, Russell and Thomas [85] obtained difference spectra with a solution of novobiocin sodium (5 x l O P 3 ~and ) ) an equal ionic strength solution either magnesium chloride (5 x 1 0 - 3 ~or of sodium chloride and observed a small peak with the same ,A at 346 nm in each case. Potentiometric titration curves confirmed the lack of complex

52 THE MODE OF ACTION OF NOVOBIOCIN formation between novobiocin and MgZ+.Furthermore, resting protoplasts of B. megaterium are not lysed by novobiocin [27], even though they are dependent on magnesium ions for optimal stability (Figure 2.6). Thus, antagonism between novobiocin and magnesium ions may occur at the cytoplasmic membrane, which requires magnesium ions for stability [86,

0

1

2

3

Time ihours)

Figure 2.5. Antagonistic effect of MgZCiomon leakage of 260 nm-absorbing niateriul induced by novobiocin (100 pg/ml) from E.coli growing in synthetic medium conruining the following concentrations (% w/v) of niagnesiwn sulphate: 0.0025, &-0; 0.01, x -x ; 0.05, 0-0;0.1, A-A. Control (niagnesiuni sulphate 0.0025% w/v, novobiocin absent), 0-0. (From Morris and Russell [66], by courtesy of Microbios.)

871 but there is no direct evidence to show that the induction of a deficiency of magnesium occurs in the sensitive cell. It is also difficult to explain why the effects of novobiocin on Gram-positive cells are not alleviated by magnesium ions. This phenomenon, however, may be attributable to a difference in the mode of action of the antibiotic against different organisms or to differences in the assimilation of exogenous metal ions by different organisms

WI.

ES Figure 2.6. Effect of Mgz+ions (added at J) on the stability, as recorded by changes in optical density. of protoplusts of B.megaterium. Concentrations ( % w/v) of mugnesiuni suiphate: 0, W;0+?02, x-x ;0905, A-A: 0.05, !I-U; 0.1, 0-0; 0.125, 7 - 7 . (From Morris and Russell 1751, by courtesy of Microbips.)

54

THE MODE OF ACTION OF NOVOBIOCIN

EFFECTS ON ENZYME SYSTEMS AND ELECTRON TRANSPORT

Both respiratory processes and oxidative phosphorylation are inhibited by novobiocin in rat liver homogenates [89] and in Mycobucterium phlei [82]. The antibiotic also inhibits the activity of nitrate reductase in E. coli, succinic dehydrogenase and ethanol dehydrogenase in M. lysodeikticus, and adenosine triphosphatase (ATP-ase) activity of sonic extracts of Strept. fuecium [23, 32, 481. It also reduces the activity of ATP-ase of E. coli [90] and the enzymes necessary for polyglycero-phosphate and polyribitol phosphate [39, 401, but it does not inhibit peptidoglycan synthetase activity [34]. Pyruvate oxidation by washed suspensions of Staph. uureus [64,91], oxygen uptake by M. lysodeikticus with either ethanol, succinate or glucose as substrate, and the production of lactic acid from glucose by Strept. fuecium [23] are all inhibited by novobiocin. These inhibitions do not, however, appear to be of primary importance and are probably secondary to novobiocin-induced membrane damage. STRUCTURE-ACTIVITY RELATIONSHIPS Novobiocin does not belong to any particular chemically defined group of antibiotics although there are a number of groupings in the molecule which may be related to specific effects. The antibiotic is a coumarin derivative (I) (see p. 40) and has been shown to inhibit oxidative phosphorylation in Mycobacterium phlei [82]. A vitamin K compound is necessary for electron transport in this organism, and both novobiocin-induced inhibition of electron transport and inhibition of growth are reversed by vitamin K. The antibacterial activity of a number of synthetic 3-acylamino-4hydroxycoumarin derivatives [92] and of several other coumarins has been described [93,94]. However, as the carbamoyl group is of great importance to novobiocin activity, it may be that the activity of coumarin is not directly related to the activity of the antibiotic. Novobiocin also contains a phenolic grouping in its structure. Phenols interfere with membrane integrity [95, 961 and it is therefore tempting to speculate that novobiocin-induced membrane damage is attributable to the phenolic group [23]. However, unlike phenols, novobiocin has no activity against resting cells, and whereas novobiocin induces lysis of spheroplasts [27], phenol itself does not [97]. Noviose, the sugar moiety of novobiocin, also appears to be important for the activity of the antibiotic, as has been suggested for the sugar moiety of streptomycin [98]. However, removal of the carbamoyl group on the 3-hydroxyl results in the formation of decarbamoylnovobiocin (descarbamylnovobiocin, 11, R as in I) and complete loss of activity. Similarly, when the carbamoyl group is on the 2-hydroxyl, the resulting compound. isonovo-

A. MORRIS A N D A. D. RUSSELL 55 biocin (111, R as in I), is inactive [9]; this indicates that both the presence and positioning of this grouping are of major importance. Novobiocin, but not

MaR

HO

M e!#"R

Me0 ( 1 1 ) Decar barnoylnovobiocin

050 N HZ

(111 1 lsonovobiocin

decarbamoylnovobiocin, has been stated to bind magnesium ions [23], but recent findings have cast doubt on the significance of this observation [84, 851. It is not possible, therefore, to attribute the major effects of novobiocin to any particular part of its unique chemical structure, the intact molecule being necessary for full activity. ADSORPTION OF NOVOBIOCIN ONTO BACTERIA It has been reported that novobiocin is strongly bound to bacterial cells [99], the binding being intermediate in strength between that of penicillin and polymyxin. This binding is pHdependent and occurs with both sensitive and resistant organisms [1001. With the use of tritium-labelleddihydronovobiocin [23], it has now been found that, at O'C, the antibiotic is bound rapidly to Strept. faecium and in amounts roughly proportional to the external concentrations. At 37'C, however, more antibiotic was adsorbed after short incubation periods than after long periods, and this anomaly may be the result of some metabolism of the antibiotic. Besides its ability to bind to bacterial cells, novobiocin also binds to proteins [101, 1021, cellulose filter paper [lo31 and membrane filters [104]. It is possible therefore that binding to washed suspensions of bacteria occurs non-specifically ; this appears reasonable considering the inactivity of novobiocin against resting cells [13, 48, 831. RESISTANCE TO, AND CROSS-RESISTANCE WITH, NOVOBIOCIN There are many reports available dealing with the acquired resistance of bacteria to novobiocin [105-1081 and, in view of the rapidity with which resistance develops, the antibiotic is often administered in combination with other antibiotics. However, it has also been reported that an organism resistant to novobiocin is usually sensitive to other commonly-used anti-

56 THE MODE OF ACTION OF NOVOBIOCIN biotics, and the organisms resistant to particular antibiotics are sensitive to novobiocin [2, 108-1 111. These studies have involved the use of bacitracin, chloramphenicol, penicillin, tetracycline, polymyxin and streptomycin, and the results suggest that novobiocin exerts effects which are different from the other antibiotics used. Novick [112] has recently described the enhancement of penicillinase plasmid-negative segregants by novobiocin, the rare negatives originally present in the population being able to mutate more readily to slightly higher levels of resistance to novobiocin than the penicillinasepositive population.

CONCLUSIONS It is difficult, at present, to propose a mode of action for novobiocin. The antibiotic exerts a variety of effects on bacteria, and although a number of hypotheses has been proposed, complete evidence for each is still lacking. The effects on novobiocin on cell wall, RNA and protein synthesis and on enzyme systems can now be considered as secondary effects of the antibiotic, and the theory that novobiocin induces an intracellular deficiency of magnesium is far from convincing. Recent findings show that the antibiotic has an immediate and pronounced effect on DNA synthesis and this may be of primary importance. It has also been shown that novobiocin interferes at an early stage with membrane integrity and, in view of the close association between membrane and chromosome in the bacterial cell, these findings have to be carefully considered. REFERENCES 1. B. M. Frost, M. E. Salliant, L. McClelland, M. Solotorovsky and A. C. Cuckler, Antibiot. Annu., 1955/56,918 2. F. K . Lin and L. L. Coriell, Antibiot. Annu., 1955/56, 634 3. H. Welch and W. W. Wright, Antibiot. Chemotherapy, 1955,5, 670 4. G. Rolland, P. Sensi, G. A. DeFerrari, G. Maffii, M. T. Timbal and L. G. Silvestri, Farniaco Ed. Sci., 1956, 11, 549 5. M. Kuroya, K. Katagiri, K. Sat0 and M. Mayani,,J. Anfibiot. (Japan) 1958, A l l , 187 6. B. T. Golding and R. W. Rickards, Chem. Ind. (London), 1963, 1081

7. H. Hoeksema, M. E. Bergy, W. G. Jackson, J. W. Shell, J. W. Hinman, A. E. Fonken, G. A. Boyack, E. L. Caron, J. H. Ford, W. H. Devries and G. F. Crum, Antibiot. Chemotherapy, 1956, 6, 143 8. M. Finland and R. L. Nichols, Antibiot. Cheniotherapia, 1957,4, 209 9. P. E. Macey and D. F. Spooner, Experimental Chemotherapy (editors: R. J. Schnitzer and F. Hawking), Vol. 3. Academic Press, London and New York, 1964, p. 291 10. C. G. Smith, A. Dietz, W. T. Sokolski and G. M. Savage, Anfibiot. Chenlofherapy, 1956. 6, 135 1 1 . T. D. Brock, J . Bacteriol., 1956, 72, 320 12. A. Coppo, Antibiot. Chemotherapy, 1957, 7 , 297 13. A. Morris and A. D. Russell, Expeyientia, 1968, 24, 195

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.

A. MORRIS AND A. D. RUSSELL 57 A. Morris, Ph.D. Thesis, University of Wales, 1969 J. T. Park, Symp. Soc. Gen. Microbiol., 1958,8, 49 G. W. Bazill, Nature, 1967,216, 346 W. Szybalski and V. N. Iyer, Antibiotics: Mechanism of Action (editors: D. Gottlieb and P. D. Shaw), Vol. 1, Springer-Verlag, Berlin, 1967 J. Ciak and F. E. Hahn, Science, 156,655 J. H. S. Foster and A. D. Russell, Inhibition and Destruction of the Microbial Cell (editor: W. B. Hugo), Academic Press, London and New York, 1971 (in press) W. A. Goss, W. H. Dietz and T. M. Cook, J. Bacteriol., 1964, 88, 1112 J. P. Payne, P. E. Hartman, S. Mudd and A. W. Phillips, J. Bacteriol., 1956, 72,461 A. C. R. Dean and P. L. Rogers, Biochim. Biophys. Acta, 1967,148,774 T. D. Brock, Antibiotics: Mechanism of Actions (editors: D. Gottlieb and P. D. Shaw), Vol. 1, Springer-Verlag, Berlin, 1967, p. 651 F. E. Hahn cited by K. McQuillen, The Bacteria (editors: I. C. Gunsalus and R. Y. Stanier), Vol. 1, Academic Press, London and New York, 1960, p. 275 A. C. Baird-Parker and R. C. S. Woodruffe, Progress in Microbiological Techniques (editor: C. H. Collins), Butterworths, London, 1967, p. 89 R. M. Wishnow, J. L. Strominger, C. H. Birge and R. H. Threnn, J. Bacteriol., 1965, 89, 117 A. Morris and A. D. Russell, Biochem. Pharmacol., 1968.17, 1923 A. Morris and A: D. Russell, Microbios, 1970 (in press) J. L. Strominger and R. H. Threnn, Biochim. Biophys. Acta, 1959,33,280 B. D. Davis and D. S. Feingold, The Bacteria (editors: I. C. Gunsalus and R. Y.Stanier), Vol. 4, Academic Press, London and New York, 1962, p. 343 T. D. Brock, Antimicrob. Agents Annu., 1960, p. 297 T. D. Brock, Science, 1962, 136, 316 D. H. Smith and B. D. Davis, J. Bacteriol., 1967,93, 71 J. S. Anderson, M. Matsuhashi, M. A. Haskin and J. L. Strominger, Proc. Nat. Acad. Sci. U S . . 1965.53, 881 J. S. Anderson, P. M. Meadow, M. A. Haskin and J. L. Strominger, Arch. Biochem. Biophys., x966, 116,487 J. L. Strominger, K. Izaki, M. Matsuhashi and D. J. Tipper, Fed. Proc., 1967,26, 9 J. L. Strominger, K. Izaki, M. Matsuhashi and D. J. Tipper, Topics in Pharmaceutical Sciences, 1968, 1, 53 J. S. Anderson, M. Matsuhashi, M. A. Haskin and J. L. Strominger, J. Biol. Chem., 1967, 242,3180 M. M. Burger and L. Glaser, J. Biol. Chem., 1964,239, 3168 L. Glaser, J . Biol. Chem., 1964,239, 3178 R. C. Hughes, Biochem. J., 1966,101,692 J. R. Ward, S. Madoff and L. Dienes, Proc. Soc. Exp. Biol. Med., 1958,97, 132 R. E. 0. Williams, J . Gen. Microbiol., 1963, 33, 325 C. W. Molander, B. M. Kagan, H. J. Weinberger, E. M. Heimlich and R. J. Busser, J . Bacteriol., 1964, 88, 591 G. H. Warren and J. Gray, Proc. Soc. Exp. Biol. Med., 1963, 114,439 G. H. Warren and J. Gray, Proc. Soc. Exp. Biol. Med., 1965, 120, 504 H. Gooder, Microbial Protoplasts, SpheroplastsandL-forms(editor: L. B. Guze), Williams & Wilkins Co., Baltimore, 1968, p. 341 T. D. Brock and M. L. Brock, Arch. Biochem. Biophys., 1959,85, 176 E. L. Krawitt and J. R. Ward, Proc. SOC.Exp. Biol. Med., 1963,114,629 J. Rotta, W. W. Karakawa and R. M. Krause, J . Bacferiof., 1965.89, 1581 R. B. Roberts, Proc. Soc. Exp. Biol. Med., 1967,124,611 R. B. Roberts, Microbiol Protoplasts, Spheroplasts and L-forms (editor: L. B. Guze), Williams & Wilkins Co., Baltimore, 1968, p. 230 T. W. Chang and L. Weinstein, Nature, 1966,211, 763

58

THE MODE OF ACTION OF NOVOBIOCIN A. D. Russell, Lab. Pracr., 1968, 17, 804 U. Taubeneck, 2. Allg. Mikrobiol., 1962, 2, 132 J. 2. Montgomerie, G. M.Kalmenson and L. B. Guze, J . Lab. Ciin. Med., 1966,68, 543 J. 2. Montgomerie, G. M. Kalmenson and L. B. Guze, Microbiol Protoplasts, Spheroplasts and L-forms (editor: L. B. Guze), Williams & Wilkins Co., Baltimore, 1968, p. 306 58. G. D. Shockman and J. 0. Lampen, J . Bacteriol., 1962,84,508 59. R. Hancock and P. C. Fitz-James, J . Bacteriol., 1964,87, 1044 60. B. M. Kagan, Microbial Protoplasts, Spheroplasts and t-forms (editor: L. B. Guze),

54. 55. 56. 57.

61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100.

Williams and Wilkins Co., Baltimore, 1968, p. 314 C. Panos, M. Cohen and G. Fagan, J . Gen. Microbiol., 1967,46,299 G. H. Warren and J. Gray, Proc. SOC.Exp. Biol. Med., 1967,126, 15 G. H. Warren and J. Gray, Proc. SOC.Exp. Biol. Med., 1968, 128, 776 K. E. Anderson and R. 0. Burns, Sci. Studies St. Bonaventure Unit, 1957, 19, 57 A. Morris and A. D. Russell, Microbios, 1970, 2, 253 A. Morris and A. D. Russell, Microbios, 1970, 2, 35 M. D. Yudkin, Biochem. J . , 1963,89,290 D. Nathan, Proc. Nut. Acad. Sci. U S . , 1964, 51, 585 M. R. J. Salton, Annu. Rev. Microbiol., 1967, 21, 417 T. D. Brock, J . Bacteriol., 1963,85, 527 D. H. Smith and B. D. Davis, Biochem. Biophys. Res. Commun., 1965,18,796 A. Morris and A. D. Russell, Microbios, 1970, In press M. A. Al-Nuri and N. S. Egorov, Microbioiogya, 1968, 37,413; Chem. Abstr., 1968,69, 50033h T. D. Brock, J . Bacteriol., 1956, 72, 320 A. Morris and A. D. Russell, Microbios, 1970, In press S. Silver and L. Wendt, J . Bacteriol., 1967, 93, 560 F. Jacob, A. Ryter and F. Cuzin, Proc. Roy. Soc., Ser. B, 1966, 164,267 A. Ryter, Bacteriol. Rev., 1968, 32, 39 E. D. Weinberg, Antibiot. Annu., 1956, 1056 T. D. Brock, J. Bacteriol., 1962, 84, 679 S. C. Kinsky, J . Bacteriol., 1961,82, 889 M. M. Weber and G. Rosso, Bacteriol. Proc., 1963,95 A. Morris and A. D. Russell, Microbios, 1969, 1, 335 P. J. Niebergall, D. A. Hussar, W. E. Cressman, E. T. Sugita and J. T. Doluisio, J . Pharm. Pharmacol., 1966, 18, 729 A. Morris, A. D. Russell and I. L. Thomas, Experientia, 1967, 23, 244 C. Weibull, J. Bacteriol., 1953, 66, 688 K. McQuillen, The Bacteria (editors: I. C. Gunsalus and R. Y.Stanier), Vol. 1, Academic Press, London and New York, 1960, p. 275 D. W. Tempest, J. W. Dicks and J. L. Meers, 2. Gen. Microbiol., 1967, 49, 139 J. Frei, N. Canal and E. Gori, Experientia, 1958, 14, 377 Kessler and Rickinberg,cited by T. D. Brock, Antibiotics: Mechanism of Action (editors: D. Gottlieb and P. D. Shaw), Vol. 1, Springer-Verlag, Berlin, 1967, p. 652 W. W. Umbreit, G. Microbiol., 1956, 2, 398 K. Okumura, Yakugaku, Zasshi, 1960,80, 525 R. Selleri, 0. Caldini and G. F. Ferretti, Boll. Chirn. Farm., 1965, 104, 248 K. Hodak, v. Jakesovaand V. Dadak, Cesk. Farm., 1967.16, 86 J. Judis, J. Pharm. Sci., 1962, 51, 261 A. D. Russell, Progr. Med. Chem., 1968,6, 135 A. Morris, Unpublishedobservations M. Kogut and J. W. Lightbown, Experimental Chemotherapy (editors: R. J. Schitzer and F. Hawking), Vol. 3, Academic Press, London and New York, p. 3 C . G. Smith, cited by T.D. Brock and M. L. Brock, Arch. Biochem. Biophys., 1959,85,176 C . G. Smith, cited by T.D. Brock, Antibiotics: Mechanism of Action (editors: D. Gottlieb

A. MORRIS AND A. D. RUSSELL

101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112.

59

and P. D. Shaw), Vol. 1, Springer-Verlag, Berlin, 1967, p. 651 W. Ritzenfeld, Arzneim-Forsch., 1957,7,464 D. M. Tennent, R. C. Mason, W. K. Gunther, M. E. Valiant and M. Solotorovsky, Proc. Soc. Exp. Biol. Med., 1957, 94, 814 W. T. Sokolski, N. J. Eilers and J. W. Shell, Antibiot. Annu., 1956-57, 1031 J. W. Lightbown, Proc. 7th International Congress of Microbiological Standardisation, Edinburgh, 1962 W. J. Martin, F. R. Heilman, D. R. Nichols, W. E. Wellman and J. E. Geraci, Proc. Staff Meetings Mayo Clinic. 1955, 30,540 M. Finland, Antibiot. Annu,, 1955-56, 929 E. Jawetz, W. Bertie and M. Sonne, Antibiot. Med. Clin. Ther., 1957, 4, 40 W. F. Jones, R. L. Nichols and M. Finland, J. Lab. Clin. Med., 1956, 47, 483 L. P. Garrod and P. M. Waterworth, Brit. Med. J., 1956, 2, 61 M. Barber, A. Csillag and A. J. Medway, Brit. Med. J., 1958, 2, 1377 J. J. Vavra, J. Bacferiol., 1967,93, 801 R.P. Novick, Bacferiol. Rev., 1969, 33, 210

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3 Some Pyrimidines of Biological and Mledicinal Interest-Part TI1 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 Triangle Park, North Carolina 27709, U.S.A.

INTRODUCTION

62

5-HYDROXYPY RIMIDINES

62

BARBITURIC ACID A N D ITS DERIVATIVES

66

AMINOHYDROXYPYRIMIDINES (AMINOPYRIMIDONES) Cytosine and its analogues Isocytosine and its derivatives Miscellaneous aminohydroxypyrimidines

82 83 88 93

PYRIMIDINE AMINO ACIDS

94

NITRO- A N D NITROSOPY RIMIDINES

98

PYRIMIDINES WITH BIOLOGICAL ALKYLATING FUNCTIONS

100

ACKNOWLEDGMENT

105

REFERENCES

105

61

62

SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART

111

INTRODUCTION Parts I and I1 of this survey of monocyclic pyrimidines were published in Volumes 6 and 7 respectively; the present chapter completes the coverage of this topic by discussing pyrimidines of the types listed immediately above. 5-HYDROXYPY RIMIDINES Hydroxyl groups substituted at positions 2- and 4-(6-) of the pyrimidine nucleus exist mainly in the pyrimidone form [1-4]. The only ‘true hydroxyl’ position on this ring system is position 5. This hydroxyl group is of phenolic character and all 5-hydroxypyrimidines give a positive (blue-violet coloration) test with ferric chloride. Because of the relative difficulty in synthesis of all but a few compounds, most 5-hydroxypyrimidines have not been extensively studied. Both 5-hydroxyuracil (isobarbituric acid, I) and 5-hydroxyuridine (11) can be prepared from the corresponding 5-bromopyrimidines by mild basic hydrolysis [5-81. 5-Hydroxyuracil inhibits enzymatic degradation of uracil [9]and is one of three pyrimidine analogues (the other two are 5-bromouracil and 6-azauracil) that inhibit the rate of incorporation of uracil into nucleic

acid pyrimidines of Trypanosoma cruzi [101. 5-Hydroxyuracil also exhibits some antimalarial activity against Plasmodium berghei [ 1I]. 5-Hydroxyuridine (11) inhibits the growth of E. cofi.The inhibitory effects of 5-hydroxyuridine and 5-hydroxy-2‘-deoxyuridineare mildly synergistic [12].The inhibition is suggested as being due to interference with protein synthesis through direct blockage of the synthesis of RNA [13], since 5-hydroxyuridine was reported to inhibit dihydro-orotase activity in Novikoff ascites hepatoma [I41 and orotic acid incorporation into RNA [15]. 5-Hydroxyuridine-5’-monophosphateis a potent inhibitor of orotidylic acid decarboxylase [151 (see section on pyrimidinecarboxylic acids, Part 11, Volume 7), and 5-hydroxyuridine triphosphate itself slowly incorporates into RNA, strongly inhibits synthesis of RNA and acts as a competitive

C. C. CHENG AND BARBARA ROTH 63 inhibitor of uridine triphosphate in the RNA polymerase reaction [16]. However, uridine kinase activity was reported to be unaffected by 5-hydroxyuridine [17]. The ease of incorporation of Shydroxyuridine into RNA can serve to alleviate the frequency of the ultraviolet-induced mutations of an auxotrophic strain (a tryptophan-requiring strain) of E. coli, because the mutagenic substances are intercepted by the fraudulent RNA [ 131. In a study of the establishment of lysogeny in E . coli strain C by phage P2, it was found that treatment of infected cells with 5-hydroxyuridine increases the frequency of lysogenisation [18]. The selective inhibition of protein synthesis in infected cells tends to favour the lysogenic response over the lytic WI. Administration of 5-hydroxy-6-methylisocytosine(111) to mice, either subcutaneously or orally, markedly reduced the development of pulmonary U

H

ke

Me

(111)

(IV)

adenoma following a single intraperitoneal injection of urethan [19]. A structurally similar pyrimidine, 5-hydroxy-6-methyluracil (IV), has only slight antiblastomogenic effect [19]. The isolation of vicine from vetch seeds [20-23] (Vicia setiva, and later in other species of Viciu [24] as well as from beet juice [25] and peas [26,27]) was reported as early as 1870, but its structure, 2,4-diamino-5-fl-~-glucopyranosidoxy-6-hydroxypyrimidine, was not successfully elucidated [22, 28-37] until more than 80 years later 138, 391. Divicine (2,4-diamino-5,6dihydroxypyrimidine, V) is the aglycone of vicine. This pyrimidine can be H

synthesised either by the condensation of guanidine with ethyl a-(tetrahydropyran-2-yl)oxycyanoacetate, followed by hydrolysis [40,411, or by nitrosation or diazonium coupling of 2-amino-4,5-dihydroxypyrimidine followed by reduction [42,43]. A related pyrimidine, 2,4,5-trihydroxy-6-aminopyrimidine(convicine, isouramil, VI), is also present in vetch [23]. Both divicine and convicine are

64 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 gem-oxohydroxypyrimidines.It is commonly known that pyrimidines having this type of structure possess strong reducing ability [39,44-471 a characteristic not shared by vicine [29, 30, 391. This activity is believed to be the cause of favism-an acute haemolytic crisis following ingestion of fava beans (Viciafaba) by persons whose red blood cells are deficient in the enzyme glucose-6-phosphate dehydrogenase-since divicine and convicine (the 'fava pyrimidines') may arise from the parent fava glycosides either in the beans or in the digestive tract through the hydrolytic action of p-glycosrdases [481. Alloxan was originally found in mucus excreted during dysentery. When this compound was first obtained in 1818 by the treatment of uric acid with nitric acid, chlorine or iodine [49, 501, the product was thought to contain the elements of allantoin and oxalic acid, hence the name. (Many picturesque names were given for compounds of the uric acid series during the early part of the nineteenth century. At that time it was not quite possible to envisage structures, and the analytical methods used for elemental analyses were so subject to error as to give misleading conclusions). The synthesis of alloxan has been reported by a number of investigators [51-591. Alloxan crystallises as a tetrahydrate from water, which can be changed to the monohydrate by heating to 100°C. The last molecule of water is more U

WIT) H

H

IH1

r---

HN

(VIII)

H

oyJ ' c T o

IHI

I01

0

H

NH

OH HO

0

0

(1x1

---

101

O fJ HN

H

0

(XI

intimately combined with alloxan, which led the early investigators to suggest that alloxan is a 5,5-dihydroxypyrimidine derivative (VII). Alloxan (VIII) is in a higher oxidation state than a 5-hydroxypyrimidine, and may actually exist as a 5-ketobarbituric acid which is hydrated in this manner. The reduction potential of this compound is 0.06volts at pH 7 (2Q-25°C) [60]. Partial reduction of alloxan gives alloxantin (IX). The latter, being a substituted 5-hydroxypyrimidine, can be readily oxidised back to alloxan. Alloxantin, in turn, has a similar oxidation-reduction relationship with dialuric acid (5-hydroxybarbituric acid, X).The interconversion of alloxan and related compounds has been extensively discussed [61, 621.

C. C. CHENG AND BARBARA ROTH 65 Alloxan is well known for its diabetogenic action in a number of animals. Injection of alloxan into rats, after brief transitory phases of hyper- and hypoglycaemia, causes permanent diabetes [63-66]. The diabetogenic action can also be observed in the fish, hamster, dog, cat, sheep, monkey and pigeon, but not in man, toad, owl, or guinea-pig (of high blood glutathione levels) [67-701. The cause of the experimentally induced diabetes is believed to be due to the removal of zinc from insulin in the form of alloxan chelates [71]. The zinc removal results in the degeneration and resorption of the /?-cells of the pancreatic islets (Langerhan islets) and reduction in the number of /?granules while the a-cells and aciner tissue are unaffected [72-801. The /?-cell membrane may be a primary site of the diabetogenic action [8l], and the degree of /?-cell destruction (necrosis) is dose-dependent [82]. The presence of glucose-6-phosphatase in B-cells is noted in alloxan-treated rats [83]. Administration of alloxan also causes a reduction of milk production in rats (believed due to decrease in insulin secretion) [84] and an increase of 17hydroxycorticosteroid secretion in dogs [85]. Certain compounds structurally related to alloxan, such as 1-methylalloxan, alloxantin (IX), dialuric acid (X), etc., also possess some diabetogenic action, but alloxan is the most active. An intact pyrimidine nucleus appears essential for diabetogenic activity [70]. Alloxan apparently also acts by interacting with SH groups and lowering the blood glutathione, since its action is prevented by SH-containing compounds such as glutathione, cysteine, BAL (2,3-dimercaptopropanol) or thioglycolic acid administered immediately before or within a few minutes after injection of alloxan [82, 86, 871. However, the actual mechanism by which alloxan acts is still rather poorly understood. The effect of alloxan diabetes on congenital malformations of the foetus has been examined in mice made diabetic prior to conception. Alloxan can induce agnathia, cranioschisis, chaniorrhachischisis, cleft palate, microglossia and fused ribs in a number of animals tested [88]. Development of chick embryos at the early stage is also inhibited by alloxan [89]. Addition of glutathione completely protects the embryos [89]. Growth of the submandibular glands, parotid glands, thyroid glands and adrenal glands as well as general body weight are retarded in diabetic rats. Subcutaneous injection of protamine zinc insulin alleviates these inhibitory effects, but growth hormone is ineffective [90]. Both alloxan and glucose inhibit systemic anaphylaxis (hypersensitivity) in rats. The protective effect of alloxan is reversed by insulin [91,92]. Alloxan also prevents histamine shock in mice [91, 921 and protects the exteriorated pancreatic ultrastructure against severe X-ray irradiation damage [93]. Urease toxicity can be lessened by either alloxan or sodium phenobarbitol [94] (videinfra). When given in small non-diabetogenic doses after splenectomy, alloxan prevents the appearance of the parasites Haemobartonella [95]. It is also

66 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 slightly active against the parasites Eperythrozon [96] and relieves symptoms of some carcinoma patients, but frequently produces hypoglycaemia, with fatty degeneration of the liver [70]. At least one contradictory activity is noted between alloxan and dialuric acid: alloxan increases the rate of growth and the rate of fat synthesis of Aspergillus fumigatus. Dialuric acid has just the opposite effect [97]. BARBITURIC ACID AND ITS DERIVATIVES The term ‘barbiturate’ is by long usage considered synonymous with ‘hypnotic agents’. However, the salts of barbituric acid, and of many of its derivatives, have no observable hypnotic properties in most systems. A consideration of the non-hypnotic barbiturates is one of the main objectives of this section. For comparison, a short review of the structural requirements H

(XI)

for hypnotic activity and the physical properties of such compounds is included. The latter have been reviewed so often, and in such detail [98, 991, that it seems redundant to include more than a brief recapitulation here. The parent compound, barbituric acid (XI), is a tautomer of 2,4,6-trihydroxypyrimidine. It is not a reduced pyrimidine, as might be inferred from the frequently used chemical name, 2,4,6-trioxohexahydropyrimidine(see Part 11).A less confusing name is 2,4,6-pyrimidinetrione.Although barbituric acid is not itself a chemotherapeutic agent, it is by no means devoid of physiological activity in various systems. It is instructive to explore the characteristics of this original member of the series. Barbituric acid has been known for over a century. In 1863 Adolf Baeyer published the first of a series of papers on ‘the uric acid group’ [loo]. The constitution of these ureido derivatives was then unknown. Baeyer proposed that they could most simply be grouped ‘around the substance N2C403H4, which I will call barbituric acid.’* In 1864,after a series of brilliant deductions concerning the relationship between uric acid, alloxan, and its derivatives, including barbituric acid, Baeyer established the structure of this key compound as ‘malonylurea’ [102]. The structural formula (XI) was correctly *His colleague Theodore Swartz allegedly stated that Baeyer’s naming of the compound originated in its preparation from uric acid on St. Barbara’s day. Others claim that a Munich barmaid was thus immortalised. Fieser and Fieser [I011 present a less frivolous suggestion, as a derivation from Schusselbart, the bit or ‘beard’ of a key, from L. barba, beard, plus uric acid.

C. C. CHENG AND BARBARA ROTH 67 drawn by Mulder [lo31 in 1873, and the synthesis from urea and malonic acid was achieved by Grimaux shortly thereafter [ 1041. Crystallographic analysis of barbituric acid shows it to be in the triketo form (XI) in the crystalline state, both as the dihydrate [I051 and as the anhydrous compound [106]. In aqueous solution it also exists almost completely in the triketo form. The relaxation method indicates an enol/keto ratio of 0.013 [107]. Barbituric acid is a stronger acid than acetic acid. The first thermodynamic dissociation constant (25°C) is 4.035 0005 [log], and the second one is 12.28 [109]. The neutral compound has an ultraviolet absorption maximum at 209 nm ( E 13 100) and a shoulder at 254 nm ( E 500); for the monoanion, A- at 257 ( E 20 900) and 540 ( E 60), and for the dianion, A,,, 216 ( E 4300), Amx 259 ( E 15400) and 520 nm ( E 60) [109]. A study of the partition of barbituric acid between water and ethyl acetate at pH 7.4, 3.0 and 1.2 showed a 1OO:O ratio at all three pH values [l lo]. A colour test, specific for barbituric and thiobarbituric acids in amounts greater than 0.5 pg, uses pyridyl pyridinium dichloride as the colour reagent [I 1I]. The quantitative determination of barbituric acid in the presence of several 5,5-disubstituted derivatives, by means of ultraviolet spectroscopic determinations and titrations in chloroform-methanol mixtures, has been described [ 1 121. The synthesis of barbituric acid can be best accomplished from diethyl malonate and urea, with an alkaline catalyst, such as sodium ethoxide in ethanol [113]. Barbituric acid-2-I4C has been prepared from urea-I4C and diethyl malonate [I 141. The 4-14C and 5-I4C compounds have been obtained by the pyrolysis of diethyl oxalacetate-3-14C, which produced an equimolar mixture of 4- and 5-I4C barbituric acid after a rather lengthy procedure [I 151. Barbituric acid is formed as a product of uracil metabolism in a number of systems. Cell free extracts from soil bacterium, strain U-1, catalyse the oxidation of uracil, thymine, and other 5-substituted uracils in the presence of methylene blue and air, with the uptake of one atom of oxygen per mole of base. Barbituric acid was isolated from this system [116]. A partially purified preparation from Corynebacterium and Mycobacterium metabolised uracil and thymine to barbituric and 5-methylbarbituric acids, respectively [1 171. A culture classified as Nocardia corallilza degraded uracil, thymine, and cytosine. Cells adapted to pyrimidines converted uracil, thymine, and 2-thiouracil to the corresponding barbituric acids. Barbituric acid was oxidised by uracil grown cells to carbon dioxide, ammonia and urea. Free malonic acid does not appear to be an intermediate in this degradation. The oxidation was inhibited by isobarbituric acid and by sodium azide [118]. The addition of uracil to minced rat liver at pH 7.2 was found to increase oxygen consumption and produce barbituric acid. Added barbituric acid was further degraded by rat liver [119]. Barbituric acid and other cyclic ureas have been found effective in sup-

68 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 pressing the activity of urease in animal intestines. The result is to increase growth rate and feeding efficiency in growing animals [120]. With chicks, the addition of either barbituric acid or chlortetracycline to soybean or casein diets enhanced the 4-week gains by 17 to 25 per cent. The killed chicks were found to have a lowered ammonia concentration in the GI tract [121]. When carbohydrate supplements were used, the degree of response to barbituric acid was dependent on the carbohydrate source [122]. Among various pyrimidines investigated as inhibitors of the transport of uracil across the intestinal wall in vitro, barbituric acid had a relatively weak effect. On the other hand, strong inhibition was seen with other 6-substituted, as well as 5-substituted, uracils [1231. Barbituric acid was found to be useful in the production of rifomycin B from Streptomyces mediterranei. Ordinarily, a mixture of antibiotics is produced, difficult to separate on a commercial scale, but with barbituric acid present, rifomycin B is the only antibiotic produced [124]. Barbituric acid is an inhibitor of the destruction of penicillin by penicillinase, although not as good as some other compounds [125]. In an investigation of inhibitors of the biochemical sequence carbamyl-L-aspartate-+ orotic acid using rat liver homogenate, barbituric acid was the most potent inhibitor of several compounds tested [1261. Barbituric acid and several related pyrimidines were found to be good inhibitors of the regeneration of the oral apparatus of Stentor cueruleus, after surgical repoval, but were mildly toxic [127]. There are conflicting reports as to whether barbituric acid supports the growth of E. coli. An early report states that growth was supported with several strains of E. coli and Aerobacter [128]. Later it was reported that barbituric acid and several analogues did not support the growth of two strains of E. coli [129]. In a more recent report, a number of uracils and barbituric acids were investigated as E. coli inhibitors, and it was found that 5,5-dibromobarbituric acid was the only derivative with reasonable inhibitory activity [1301. Barbituric acid only partially inhibited RNA or protein synthesis in tissue cultures of embryonic chick skin at 833 pg/ml, whereas puromycin was inhibitory at 1.7-333 pg/ml [1311. Several urea derivatives, including barbituric acid, completely inhibited influenza PR-8 virus in vitro, but were without effect in vivo in chick embryos [132]. Barbituric acid had no effect on riboflavin synthesis when used as a rat dietary supplement. On the other hand, uric acid markedly decreased, whereas urea increased, riboflavin excretion [ 1331. Potassium linolenate is reported to be readily oxidised in the presence of barbituric acid or thymine; uracil and uric acid, however, were found to be anti-oxidants. The pro-oxidant effects of barbituric acid were inhibited by chelating agents [1341. Barbituric acid does not have the diabetogenic action of alloxan (5-

C. C. CHENG AND BARBARA ROTH 69 oxobarbituric acid; vide supra). However, it has been reported to cause some hydrotropic degeneration of the cells of the pancreas [135]. The injection of 150 mg/kg (i.v.) of barbituric acid into rats immediately prior to 60 mg/kg of alloxan protected them against diabetes [136, 1371. There was no protection when given afterwards [1381. Sodium barbiturate, as well as a number of other compounds, suppressed the fighting stance of the Cantonese fighting fish, suggesting that this fish is not a suitable animal for the differentiation of tranquillisers [139]. Barbituric acid slightly diminished the effect of 2,4-dinitrophenol on rectal temperatures [140]. Physicat Properties-The thermodynamic dissociation constants of some representative derivatives of barbituric acid are listed in Table 3.1 in com-

Table 3.1

THERMODYNAMIC DISSOCIATION CONSTANTS OF BARBITURATES (25°C)

R2

Substituents

R'

R4

R3

R2

H Me Me H H

H H Me H H

H H H Pr' Et

H H H H Ph

H H

H H

Me CHZCH=CH2

Ph CH2CH=CH2

H

H

CHZCH=CHZ

Bu'

H

H

Et

CH~BU'

H

H

Et

Et

H

H

Et

Bu

H

H

CH2CH=CH2

Pr'

Me

H

Me

(i=CH(CHt)jYH>

~~

4.035 4.348 4679 4.94 7.45 7.41 7.73 7.77 7.79 1.79 7.68 1.96 7.94 7,9798 7.91 7.9 1 8-00? 7.98 7.92 7.99 7.9 1

::::

_ _ _ ~

~

'Assessment of relaability by ref. 144. v. rel. tpK2 = 12-8 [I431

PKI

=

f0.0005; rel.

Evaluation*

ReJ

rel. rel. rel. approx. uncert.

108 108 108 108 108 141 I08 108 141 108 141 108 141 142 I08 141 143 I08 141 108 141 108 141

-

uncert. uncert. -

uncert. uncert. v. rel. uncert. uncert. -

uncert. uncert. -

~

=

f0.005;approx.

=

k0.04;uncert. = > 50.04

70 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 parison with those of the parent compound. The derivatives included in the table are those found in the recent I.U.P.A.C. listing [ l a ] , which presents the most reliable data available, with an assessment of the reliability. A larger series of constants was published at an earlier date [141], which includes not only the thermodynamic constants at 25"C, but also data at different ionic strengths and temperatures. In general, the values are slightly lower than those presented in the I.U.P.A.C. listing. The comparative values are given in Table 3.1. A recent review [99], which contains the earlier listings only, cites an erroneous constant for the parent barbituric acid. Mono- and dimethylation of barbituric acid in the 1- and 3-positions decreases the acidity in increments of approximately 0.3 pH units per methyl group. This is a normal inductive effect. No constant is available for 5methylbarbituric acid ; however, a 5-isopropyl substituent decreases the acidity by 0-9 pH units, which is disproportionately high for a simple inductive effect. The introduction of two alkyl groups into the 5-position produces the tremendous acid weakening of approximately four pH units. Furthermore, the ultraviolet spectra of the anions of 5,5-dialkylbarbituric acid derivatives show maxima at much lower wavelengths and extinction values than those of barbituric acid, which implies a lesser degree of conjugation and resonance forms. The following data for the 5,5-diethyl derivative

(mono[143] are illustrative: I,, (neutral species), 210 nm ( E 11 100);,A anion), 239 (1 1 OOO); I,, (dianion), sh 220 (15 OOO), 254 (7800). Corresponding data for barbituric acid are found in the previous section. The spectra of the 1- and 1,3-alkylated derivatives of barbituric acid, on the other hand, are very similar to that of the parent compound [108]. These facts indicate that proton loss from barbituric acid to produce a monoanion occurs chiefly from the 5-carbon, giving (XII), whereas the 5,5disubstituted derivatives, on the other hand, must suffer proton loss from a ring nitrogen to yield (XIIIa) or (XIIIb), or both. It would appear reasonable that the ratios of (XIIIa) and (XIIIb) are influenced by the nature of the R groups in the 5position. Treatment of barbituric acid with deuterium oxide replaces all four H atoms with D [145]. This is not unexpected, since protons on nitrogen are normally readily exchangeable, and verifies the fact that protons are readily lost from C 5 . The above results demonstrate that barbituric acid and its 1- and 1,3-alky-

C. C. CHENG A N D BARBARA ROTH . 71 lated analogues differ markedly from the 5,5-disubstituted derivathes in their physical properties, in that the former are almost completely ionised at physiological pH, whereas the latter are less than half ionised. The consequence of this is, of course, a marked difference in solubility and transport properties, as discussed below. Most barbituric acid derivatives containing 5,5-hydrophobic substituents are quite soluble in chloroform, ether, benzene, and lipids, as the neutral species. Some of the derivatives are also fairly soluble in water. The water solubilities, surface tension, and distribution coefficients between olive oil and water for a number of barbiturates were determined at an early date [146]. These are listed in Table 3.2 along with some recently calculated (and

Table 3.2

DISTRIBUTIONCOEFFICIENTS, SOLUBILITY AND SURFACE TENSION OF BARBITURATES

Substiruenrs

R'

R2

H Me Et Et CH2CH=CH2 CH2CH=CH2 Et Et CH2CH=CH2 Et Et Et CH~CBFCH~ Et CH2CH=CH2 Et

H Me Et Pr' Pr' CHzCH=CHj Bu Bus Bus CH~BU CH2Bui CHMePr Bus Ph CHMeC6H13 CHMeC6HI3

Surfice tension (20"C), %of' H 2 0 alone L 1461

olive oillH2O octonol/H20 (20°C) [146] [147] -

0.066 0.2 14 0.73 1.12 0.85 2.58 1.36 2.48 2-92 2.895 44 4.3 1.34 0.306 0.408

0.034* -

4.5*, 3.4t 8.9t I4 t 11 t 45t 28 t 140t 89t 89t -

26* -

-

-

2.419 6.00 1.36 4.02 1.465 1.90 1.987 2.16 0.554 0.530 1.20 0,684 0.97 3.06 0.414

983 90.0 94.5 84.0 873 75.5 84.0 75.0

77.5 75.0 87.0 95.0 67.2 75.5

'Experimentally determined values tCalculated from II substituent constants

in some cases experimental) distribution coefficients between octanol and water [147]. Using the equation for a straight line, we find by the least squares method that the correlation coefficient r between the olive oil and

72 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 octanol figures is 0.829, where n (the number of data) is 10. This is a positive, but not close, correlation. The standard error (s) is 27. The sodium salts of the barbiturates (monoanions) are usually quite soluble in water, and this is normally the preferred form of the drugs for therapeutic use. The stability and hydrolysis products of barbiturate salts have been reviewed in detail elsewhere [99], and will not be discussed here. The anions of the barbiturates are not soluble in lipids, and the non-ionised form of the hypnotic barbiturates is considered to be the active form of the drugs. Hence, an accurate knowledge of their dissociation constants and the pH values of solutions under the conditions of a given experiment are of considerable importance. The relationship of physical properties to physiological activity is discussed in the following section on the hypnotic barbiturates. Pharmacologicalactions: Hypnotic Agents and Anticonvulsants-Since the discovery of the first hypnotic barbiturate, diethylbarbituric acid (barbitone, barbital, Medinal, Veronal), which was introduced into medicine in 1903 by Fischer and von Mering, more than 2500 derivatives have been prepared for pharmacological evaluation. All derivatives listed in Chemical Abstracts and Chemische Zentralblatt through 1956 have been tabulated in a 1959 review [99] along with pharmacological data, where available. Many new derivatives have been prepared since this date; however, only a dozen or so barbiturates are in wide usage today. Many are marketed in combination with other drugs, as sedatives or tranquillisers, hypnotics, analgesics, or anticonvulsants [148]. In brief, the major requirement for hypnotic activity among the barbiturates is 5,5-disubstitution by two hydrophobic groups, both of which must have at least two carbon atoms. The derivatives of major commercial importance have a total of 6-10 carbon atoms in the 5-substituents. The substituents may be alkyl, alkenyl, alkynyl, or cycloalkenyl, and a single halogen, usually bromine, sometimes increases the potency. A phenyl substituent, as in phenobarbitone (phenobarbital, 5-ethyl-5-phenylbarbituric acid), confers selective anticonvulsant activity on the molecule. Although phenobarbital is also a sedative, it can be used as an anti-epileptic in non-sedative doses. When used in sedative doses, it is classified as a long-acting barbiturate. Barbitone, 5,5-diallylbarbituric acid, and the 5-allyl-5-isopropyl derivative (aprobarbitone, aprobarbital, allypropymal, Allonal, Alurate, Numal) are also long-acting hypnotic agents, used for sedation and sleep. Branching of the chain in a 5-alkyl substituent usually increases hypnotic activity, and shortens the duration of action. However, slight changes in structure may produce convulsants, rather than hypnotic agents. In some cases, the drug may have both stimulant and depressant properties, depending on the dosage and other factors. Some examples of short- to intermediate-acting hypnotic barbiturates of commercial importance are as follows (XIV-XVIII).

C. C. CHENG AND BARBARA ROTH H

H

.

73

H

' HN Y F E CH,CH,CHMe, t

0

Me 0 Secobutobarbitone Butabarbital

Amy lo bar bi tone Amobarbttal

0 Me Pentobarbi tone fentobarbi tat

(XVI

(XIVI

(XVI \

H

H

O V N Y O

O q N k 0

Quinal barb i tone Secobarbital

Vinbarbitone Vinbarbital

(XVII)

(XVIII)

These structures are to be contrasted with (XIX-XXII), all of which are stimulants which cause convulsions in warm-blooded animals [149-1 541. Compound (XX) is the most potent convulsant of these, with an LD,, H ..

H

0

H

Me

0

Me

H

HN C=CHCHMe,

0

XXI

XXll

(i.p.) of 3.5 mg/kg in mice. Convulsant properties may also appear when the alkyl chains are too long. Beyond a total of 10 carbon atoms in the 5-position, toxicity generally increases to a greater extent than does the hypnotic activity. The hypnotic effect of compounds such as amylobarbitone is considered to be exerted by the unchanged drug alone rather than by metabolites [155]. Side-chain oxidation of barbituric acid hypnotics generally reduces or obliterates sedative and hypnotic activities [155, 1561.

74 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 It was found that in mice infected with Plasmodium berghei, the malaria parasite caused an increase in hypnotic sensitivity of the mice to a number of barbiturates. This increase in activity appears to be due in part to a decrease in the detoxifying action of the liver and possibly to adrenal injury [157]. Ultra-short-acting hypnotic agents are obtained in some instances by alkylation of a ring nitrogen of the barbiturate. l-Methyl-5-allyl-5-(1methyl-2-pentyny1)barbituric acid (methohexitone, methohexital, Brietal, Brevital, Metharbital) is one such example, which is used as an anaesthetic of very short duration. Patients are usually awake in 5-10 min, and ambulatory in 15 min. Some N-methylated barbiturates, such as the 5-ethyl-5-phenyl

Me

CH Pr

(XXI I I )

( XXlV)

(methylphenobarbitone, mephobarbital, enphenemal, Prominal) and 5,5diethyl derivatives (metharbitone, metharbital, endiemal), are readily demethylated in the body, so that they remain long-acting hypnotics. 1-Methyl5-ethyl-5-(p-aminophenyl) barbituric acid (XXIII, PAM) is of unique interest because of the separation between its anticonvulsant and sedative properties [1581. Substitution of the 2-oxygen by sulphur converts barbiturates into highly lipophilic, potent hypnotics, with a rapid onset of action and short duration of activity. A notable example of this class is thiopentone (XXIV, thiomebumal, thiopental, Nesdonal, Penthiobarbital, Pentothal), which is widely employed as an intravenous anaesthetic. The introduction of hydrophilic groups, such as hydroxyl, amino, or carbonyl, into the barbiturate side-chains results in complete loss of hypnotic activity. Many such derivatives have been made in recent years, with the object of producing other types of pharmacological action. Examples of such compounds are provided in the sections which follow. It was recognised at an early date that the hypnotic activity of the barbiturates was related to their lipophilicity. In 1899 Meyer and Overton [159] had proposed that all chemically indifferent bodies which are soluble in lipids act as narcotics to living protoplasm. It was then assumed that relative activities of various narcotics depend on their distribution between body fats and fluids. In 1933, the Meyer-Overton hypothesis was examined relative to the hypnotic barbiturates by determining the distribution coefficients and other physical properties for a series of such derivatives (see Table 3.2, previous section) [1461. A parallelism between oil solubility and hypnotic

C. C. CHENG AND BARBARA ROTH 75 activity was observed, and it was concluded that lipid solubility is required for transport of the drugs into the cell. In a series of careful studies, the anaesthetic effects of 30 barbiturates on the fertilised eggs and larvae of Arbuciu puncfulufu were quantitatively determined as a function of extra- and intracellular pH [160]. The concentrations of undissociated barbituric acid derivative and its corresponding anion required to produce a 50 per cent reduction in rate of cell division of eggs and cessation of movement of larvae were calculated from the total concentrations of drug, its pKa under the conditions of the experiment, and the pH. The required concentration of undissociated species for a given compound was found to be very nearly constant over the pH range of the experiment, whereas the anion concentration varied considerably. This is exemplified in Table 3.3 for phenobarbitone (pK; = 7.26). The results indicate very clearly that the barbiturates penetrate the cells only in the

Table 3.3

CONCENTRATIONS OF P H ~ N O B A K B I T O N EREQUIRED FOR ANAESTHESIA OF A . punctulutu AS A FUNCTION OF pH [160]

PH

6.42 7.92 8.67

I

Concentrdion x lo4

Total

Anion

Undissociuted molecule

14 95 470

2 78 453

12 17 17

form of the undissociated molecule-in other words, in the lipid-soluble form. Other workers [161] found that the distribution coefficients of the barbiturates are affected not only by the mass of the substituents, but by their inductive effects on the hydrophilic portion of the molecule, as defined by Taft’s polar substituent constants. The modern mathematical test of multiple regression analysis has been applied [147] to various series of barbiturate data, in an attempt to define the relative importance of lipid solubility, dissociation constants, and steric parameters to biological activity [147]. It was concluded from the high correlation with lipid solubility that ‘ “lock and key theory” is of little use in explaining their mode of action’. The series tested did not include any of the convulsant barbiturates, such as (XIX-XXII), however. These structures, compared with (XIV-XVIII), demonstrate that molecular geometry cannot be ignored in this series. It was further concluded that pKa differences play only a minor role in defining barbiturate activity. Since most of the pK, values of the series cited vary only slightly, and since, furthermore, the pH

76 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART I11 values in the individual experiments were not identical, it is apparent that only a small variance would arise from such mathematical treatment. Although true, as far as the mathematics goes, and in as far as the authors intended it, the statement can be misleading. It is evident from the finding of previous investigators [ 1601 that the relationship of pK, to experimental pH is of the utmost importance, since barbiturate anions are inactive as hypnotics. On the other hand, high lipid solubility has long been known to be of importance, not only with regard to increased hypnotic potency, but to a decrease in onset of action and duration of action, and more rapid metabolic degradation. These factors have been frequently discussed elsewhere [98,99]. The mechanism and sites of action of the barbiturates have recently been reviewed [98] and many recent monographs and reviews covering the pharmacology of these pyrimidines are cited therein. The finding that barbiturates, such as phenobarbitone, have a very significant effect on liver microsomal enzyme activity should be stressed, since this can markedly change the activity of other drugs, by altering their metabolic pathways [ 1621. Analgesic agents-Certain barbiturates, such as 5,5-diallylbarbituric acid (allobarbitone, allobarbital, Dial), which is one of the hypnotic barbiturates, have been found to have a synergistic effect on the analgesic action of acetophenetidine, acetanilide, and related compounds . Other barbiturates, such as barbitone and carbromal (Adalin, Bromadyl), display a marked antagonism, on the other hand [ 1631. A combination of 5-allyl-5-isobutylbarbituric CH, CHzNEtz

I

acid (butalbital, allylbarbituric acid, alisobumal, Sandoptal) with aspirin and caffeine is available for chronic tension and muscle-contraction headaches [I 641. The related 5-allyl-5-cyclopentenyl derivative is used similarly [165]. A series of 5,5-disubstituted barbiturates containing cycloalkylmethylene groups, such as (XXV) has been patented for its analgesic properties [166]. During a study of the effect of 1- and 3-substitution on the physiological properties of the barbiturates, it was found that the 1-diethylaminoethyl-3benzyl derivative of allobarbital (XXVI) has pronounced analgesic and antipyretic activity, in addition to a sedative effect. The compound has hypotensive action, but is not a hypnotic [167]. A number of 5-monosubstituted barbituric acids have been reported

77 C . C. CHENG AND BARBARA ROTH recently, which in many cases are also substituted on one or more ring nitrogens [168-1741. These are claimed to have analgesic activity, and in some cases, antipyretic and anti-inflammatory properties as well (see also

section on anti-inflammatory agents). The 5-substituent may be alkenyl, alkyl, or alkyl containing a keto function, for example. The ring substituents are usually phenyl or cycloalkyl. Formulas (XXVII) [ 1671, (XXVIII) [ 1731 and (XXIX) [ 1721 illustrate these general types. Tranquillisers and muscle relaxants-As a consequence of studies which demonstrated the pronounced modifications in pharmacological effects that result from N-substitution of the barbiturates [167, 175-1771, a series of ,CH*OR’

OMe

OC HzCH CH2OC NH2 OH

II

0

(XXXI) 0

(XXX)

0

I1

Pr

I

NH~COCHZC CHzOCNHz he

!I

(XXXII)

1-substituted derivatives of phenobarbitone was investigated which contained a carbamate moiety in the side-chain (XXX) [178, 1791. Such compounds were designed for comparison with well-known muscle relaxants of the carbamate type such as methocarbamol (XXXI) or meprobamate (XXXII). The most interesting compound of this group is the butyl ether (XXX; R = H, R’ = Bu), called Go-560. Extensive animal experimentation showed Go-560 to be a tranquilliser without cortical and subcortical effects. It was found to have muscle sedative action with a duration several times

78 SOME PYRIMIDINESOF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 longer than that of (XXXII) or of carisoprodol. The toxicology is similar to that of (XXXII), with a wide safety margin. Anticonvulsive properties are similar to those of methyl phenobarbital. The substance has depressant

c F3 (XXXIII)

(XxxIV 1

(XXXV1

action on the extrapyramidal centres, permitting the quieting of tremors, but there is no atropinic action. A related series of compounds with the carbamate moiety in the 5-position (XXXIII, for example A = (CH,),, R = Bu) is also claimed to have sedative and tranquillising properties [I 801. Introduction of a m-trifluoromethyl group into the phenyl group of phenobarbitone (XXXIV) is said to modify its properties such that skeletal muscle relaxation is produced, at doses much lower than those that result in motor impairment 11811. A barbiturate with a hydroxy group in the side chain (XXXV) (iprona!) was reported to have tranquillising activity in a clinical study when given orally for a 2 to 3 week period [182]. Its short-term hypnotic effect was found to be very weak, and it was reported not to be an anticonvulsant. However, it markedly potentiates the hypnotic effect of phenobarbitone and barbitone. This effect is not produced with non-barbiturate hypnotics, such as chloral hydrate or ether. Anticholinergics for Parkinsonism-Several recent patents [ 183-1 871 claim barbiturates which are said to be useful for Parkinson’s disease, or for suppressing tremorine-induced spasms in small animals. All of these H H ?J(

:ypq---J

H

OYN

M & . + z c HOz ~

;“:....3 IXXXVI)

(XXXVI1)

(XXXVIII)

compounds contain nitrogen in one of the 5-substituents, either as an amino or substituted amino group, or as a nitrogen heterocycle. They may be considered to bear a relationship to benzhexol (trihexyphenidyl, XXXVI),

79 C. C. CHENG AND BARBARA ROTH which has been used clinically since 1949 for the symptomatic treatment of Parkinson’s disease. Examples are (XXXVII) [184] and (XXXVIII) [186]. Anti-inflammatory agents-A number of 1,3-diphenyl-5-substitutedbarbituric acid derivatives (XXXIX) have been prepared for evaluation as antiinflammatory agents [ 188-1901 since their structures are closely analogous H

Ar

Ph

Ar

H

(XXXIX)

Bu

(XL)

(XLI)

x = 0,s R = a l k y l .cycloalkyl.aryl, alkenyl. a r a l k y l , a c y l . carbamoyl

to that of phenylbutazone (XL). The most active compound of series (XXXIX) is 1,3-diphenyl-5-(3-methyl-2-butenyl)barbituric acid, as indicated by the Randall-Selitto test, pleural effusion method, and cotton granuloma tests in rats [189], with i.p. administration. In general, the compounds are less toxic and less active than phenylbutazone, and in addition, they are very poorly absorbed orally. They do not exhibit hypnotic activity in the rat, even at doses approaching toxicity. 1-Cyclohexyl-5-butylbarbituric acid (XLI, paramidine) has been reported to have anti-inflammatory activity comparable to that of phenylbutazone, as indicated by rat paw oedema, mustard derived peritonitis, and other tests [191, 1921. The toxicity of paramidine is reported to be less than that of phenylbutazone [192]. The acute LDS0 (p.0.) in rats is greater than 2g/kg [192, 1931. Respiratory depression is the usual cause of death, which may be due to decompensation of the adrenocortical function. According to the reports of several investigators [194, 1951, sub-chronic toxicity tests show slight degeneration and destruction of the convoluted tubular epithels in the kidney. Results with phenylbutazone are similar in lower doses, but there is also degeneration of the liver cells and cardiac muscle fibres. Chronic toxicity studies in rats with paramidine and phenylbutazone over a period of 48 weeks show that both drugs are more toxic to the females, but the former drug is the less toxic of the two. At high doses, proliferation and bleeding of the adrenals occurred, followed by atrophy. The binding of (XLI) to serum albumin was investigated [196]. It appears that it is bound at two sites on the protein, and furthermore, that (XLI) and phenylbutazone have common binding sites. The latter is bound four times more strongly. Hypotensive agents-The hypnotic and sedative barbiturates do not

80 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 produce significant cardiovascular effects at normal oral dosage levels. Thiopentone occasionally produces an increase in blood pressure when used for anaesthesia [98, 1971. However, 1dialkylaminoalkyl-5,5diallylbarbituric acids, such as (XLII), which do not show hypnotic activity, do

(XLII)

(XLLII)

produce a fall in blood pressure which is unrelieved by atropine, antergan, or vagotomy [167]. Similar claims are made for compounds of type (XLIII), where R' and R2 may be dimethylamino, diethylamino, or morpholino, n may be 2 or 3, R3is ethyl, and R4 may be ethyl or phenyl [198]. Diuretics-The m-sulphonamidophenyl analogue of phenobarbitone is claimed to have diuretic activity [199]. Virucides, Growth Inhibitors or Stimulants-A number of barbiturates and thiobarbiturates with long alkyl chains in the 5-positions are reported to have antiviral activity. One such example is 5-lauryl-barbituric acid [200]. l,S,S-Trisubstituted derivatives, including 1-methyl-5-lauryl-5-propylbarbituric acid, the 1-allyl-5-lauryl analogue, 1-benzylphenobarbitone, 1-benzylbarbitone and related compounds are claimed to be active in vitro against the

Nakamiya strain of Japanese encephalitis virus in mice [201]. Thiobarbiturates of type (XLIV, R = C l l H 2 3to ClsH3,) are claimed to have antiviral activity, especially against influenza [202]. In a related patent the ally1 substitution is extended to include CI-C6 derivatives, which may be alkyl, alkenyl, chloroalkenyl, cyclohexyl or cyclohexenyl [203]. 5-Butyl-5-ethylbarbituric acid and the I-methylbutyl analogue (pentobarbitone), as well as two thiouracil derivatives, were found to have a direct virucidal action in vitro on poliomyelitis viruses in monkey testicular and kidney cultures [204]. Among a series of 5-halo, 5-acyl, 5-benzylidene, and 5-phenylcarbamoyl derivatives of 1-H, 1-methyl, and 1-phenylbarbituric acids, the sodium salt

C. C. CHENG AND BARBARA ROTH 81 of 5-phenylcarbarmoylbarbituric acid (XLV) shows potent anti-tumour activity in vitro and in vivo [205]. A nitrogen mustard derivative of barbituric acid has been reported without antitumour data [206]. 5-Diazobarbituric anhydride (XLVI) has been found to be amoebicidal at 0401 mg/ml vs. Endamoeba histolytica [207]. The inhibition is reversed by nucleotides, but not by the bases or nucleosides. It had previously been found that (XLVI) is a cytosine antagonist in the yeasts, Torula utilis and Saccharomyces cerevisiae Hansen [208]. This compound is prepared by the action of nitrous and nitric acids on 5-aminouracil-6sulphonic acid [209], and as a cyclic derivative of a barbituric acid, it has quite different properties from the pyrimidinetriones. N-Hydroxy derivatives of the barbiturates inhibit the growth of E. coli. A series of such compounds, exemplified by hydroxy-ipral (hydroxy-probarbital) (XLVII), was prepared as analogues of aspergillic acid (XLVIII) [2 101. The latter has high antibacterial activity, but is quite toxic. It is metabolically derived from valine and isoleucine. The inhibitory activity of (XLVII) seems to be associated, at least in part, with interference with valine and isoleucine

yH

M eEt( ! H ~ N x O OyJEt OH I HN

Pr’

N

CH2CHMe,

0 (

xLVI I )

(XLVII I )

synthesis, since these counteract its toxicity toward E. coli. However, kinetic results show no direct relationship [21 I]. 5-Allyl-5-(2’-hydroxypropyl)barbituricacid has been found to be a plant growth stimulant. The 5,5-diallyl derivative stimulated the growth of the main root, but had a less pronounced stem effect. 5-Allyl-5-(2’-hydroxy-3’iodopropy1)barbituric acid had harmful effects on plants [212]. Barbituric acid and some analogues cause striking changes in the epidermis of developing giant silkworms (Samia cynthia and Callosamia promethea), with very little in the way of other effects. Ribonuclease in sublethal doses behaves similarly [213]. The hypnotic barbiturates are capable of causing a fully reversible inhibition of cellular functions in simple organisms. They can, for example, inhibit cell division of fertilised eggs of the sea urchins (Arbacia punctulata), and movement of the larvae of the marine worm Arenicola cristata [160]. The blocking effects occur at or before the eight cell stage, and produce no immediate lethal effects. Complete recovery occurs when the eggs are returned to normal sea water after 2 hours or less, of exposure to the barbiturates.

82

SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART

I11

AMINOHYDROXYPYRIMIDINES (AMINOPYRIMIDONES) The base cytosine (XLIX, 4-amino-2(1H)pyrimidone), which, paired with guanine, is present in both DNA and RNA, may be considered the most important parent structure in this class of compounds. Its isomer, isocytosine (L, 2-amino-4(3H)pyrimidone), while not found per se in the nucleic acids, exists as the pyrimidine moiety of guanine (LI), and of the vitamin folk acid (LII). Many analogues of (XLIX) and (L) have been prepared for biological study. Other analogues are found in nature. This section is divided into three parts, including compounds of type (XLIX), (L) and other miscellaneous aminohydroxypyrimidines. Uracils, barbiturates, and 5-hydroxypyrimidines which contain amino substituents are found in preceding sections.

bI H2

CH2COOH

(LIII a )

(LIIlb)

(LIIl c 1

(LIIId )

(LIIIe)

(LILI f

C. C. CHENG AND BARBARA ROTH

83

CYTOSINE AND ITS ANALOGUES

There are seven possible tautomeric forms for cytosine (XLIX, LIIIa-f). That the actual predominant tautomeric structure is (XLIX) has been adequately shown from ultraviolet spectral comparisons with N- and 0-methyl derivatives in aqueous solution [214-2161, from proton magnetic resonance studies in dimethyl sulphoxide and other solvents [214,217], from infrared spectra in the solid state [218], and from X-ray crystallography [219]. Cytosine has pK, values of 4-45 and 12-2 for the dissociation of the cation and base, respectively [215]; therefore, in neutral medium, the molecule is uncharged. The corresponding 1- and 3-methylcytosines have pK, values of 4.55 [215] and 7-49 [214] respectively for the dissociation of the cations, from which the equilibrium constant for the tautomeric forms (XLIX) and (LIIIa) of cytosine may be calculated as c. in favour of (XLIX) [214]; pro tonation of cytosine occurs at N3 [214]. Knowledge of the fine structure of cytosine and its nucleosides is of considerable importance with relation to the hydrogen-bonded structure of nucleic acids. (This statement naturally holds for the other bases as well.) The original Watson-Crick model proposed [220] for base pairing of cytosine and guanine has been shown to be correct [221]. There are three hydrogen bonds, as shown in (LIV), whereas only two hydrogen bonds are present in the A-T or A-U pair. In principle, it is possible for other hydrogen-bonded --0

--0

arrangements to occur between these two bases, or between each of these with other bases. A number of recent studies have been made on the interaction of glycosidic-N-substituted bases in non-aqueous medium [222-2251 and on the mode of co-crystallisation [22&228]. All of these studies produced the remarkable result that hydrogen-bonded pairs are formed exclusively between the G-C and A-T pairs. Theoretical calculations of the van der

84 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 Waals-London interaction energies confirm the fact that these pairs form the strongest associations [229, 2301. In some of the models, the H-bonded arrangement was different from that of (LIV), but this appears to be the single structure present in the natural polymer. It has been suggested [231] that spontaneous mutations might occur if an incorrect tautomeric form of a base were to be built into a nucleic acid chain upon replication. Thus, if 0.1 per cent of cytosine, for example, were in the imino form (LIIIc), it would simulate uracil, and produce a 'mistake' in some of the codons of the polymer. As stated above, studies of dissociation constants indicate that a small percentage of cytosine may occur with the proton on N3 instead of N1 [214], as (LIIIa), and this could be conceivably associated with mutations. There has been considerable speculation along this line. Parisie-Parr calculations on the electronic structure of cytosine indicate that mutations are probably not due to rearrangement to the lactim (LIIIb) [232]. The biosynthetic pathway which leads to the pyrimidine nucleotides is described in the section on pyrimidinecarboxylic acids, where orotic acid is shown to be an intermediate on the route to uridylic acid (UMP). This is then converted to the di- and triphosphates in the presence of ATP and 0 ,C-0 P

2

II

aspartate transcarbamylase 1k

-

/NH2

o=c\

CY P

"&I CH

NH-~6

C-OH

O4

-~ -~

magnesium ion. The only known pathway for the formation of cytidine nucleotides involves the amination of UTP in the presence of ATP and ammonia to yield cytidine triphosphate (CTP) [233, 2341. This product plays a unique role in pyrimidine biosynthesis in that it acts as a feedback inhibitor which controls one of the initial steps in the sequence, the formation of carbamyl aspartate (LV), mediated by the enzyme aspartate transcarbamylase [235]. This represents the first well understood case of feedback inhibition, (using E. coli as the organism). It is specific to the final products of the sequence, i.e. to cytidine derivatives, and the entire CTP molecule contributes in binding to the transcarbamylase enzyme at an allosteric site (i.e. a different site from that of the substrate). Cytosine itself is not an inhibitor for the E. coli enzyme. Knowledge of this mode of inhibition has provided an added stimulus to

C. C. CHENG AND BARBARA ROTH 85 research on the preparation of cytidine analogues as potential inhibitors of nucleic acid synthesis. In cancer chemotherapeutic studies, it was found that when a single dose of tritiated cytidine is inoculated intraperitoneally into a donor rat, a sufficient amount of the radioisotope is incorporated to result in the heavy positive labelling of more than 99 per cent of the ascite tumour cells. When these isotopically labelled tumour cells are subsequently injected into the blood of recipient animals, the cells can be readily identified in autoradiographs of the lung tissue of these animals [236]. The chemical synthesis of cytosine is readily accomplished from /?-ethoxyacrylonitrile and urea [237].The synthesis under prebiotic conditions has been accomplished starting from methane and nitrogen, which yield cyanoacetylene in the presence of an electrical discharge. This, with potassium cyanate, forms a small amount of cytosine on standing [238]. Biochemically, cytosine is formed on catabolism of its nucleotides. It is then further degraded to uracil, dihydrouracil, and finally, to /?-alanine. There is considerable evidence that cytosine can be utilised in anabolic processes. When the leaves of Solanum nigrum were treated with cytosine, and allowed to undergo photosynthesis in the presence of 14C02, it was found that DNA synthesis was strongly stimulated [239]. In the case of tobacco leaves diseased with TMV virus, brushing the leaves with cytosine or guanine increased the virus titre. With normal leaves, a high level of cytosine decreased nucleic acid synthesis [240]. Cytosine and uracil were found to stimulate the incorporation of methi~nine-~’S into rabbit blood serum proteins after acute bleeding [241]. Local injection of cytosine or metacil produced an acceleration of healing in the osseous tissue of a rabbit’s forepad by 14 to 20 per cent [242]. Treatment of caffeine-induced ulcers in rats with cytosine4,6-dihydroxypyrimidine combinations produced a 42-72 per cent reduction in the lesions. Thymine was ineffective, but 6-methyluracil-2methyl-4,6-dihydroxypyrimidine combinations were also useful [243]. Cytosine, thymine or uracil reversed the inhibition of root growth of Pisum sativum caused by N-sulphanilyl-3,4-xylamide[244]. Cytosine and thymine reversed the growth inhibition of Aerobacter aerogenes produced by streptomycin, but not dihydrostreptomycin [245]. Several studies have shown cytosine to be effective in the prevention or treatment of artificially induced oedematous conditions in rodents. However, it is ineffective after adrenalectomy [246-2501. The injection of cytosine (5CrlOO mg/kg) into the stomach of mice with lymphoid leukaemia prolonged life and reduced the leukocyte count. In some cases a haematological and clinical remission occurred. Uracil by itself worsened the leukaemia, although it sometimes had beneficial results in combination with cytosine [251]. Other studies have shown beneficial effects with solid tumours [252, 253-2551. Cytosine was reported to increase antibody formation in rabbits with an intact spleen [256].

86 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 An adenine-arginine-requiring strain of Candida albicans exhibited back mutation to independence of arginine when grown in the presence of inhibitory levels of cytosine. Since guanine prevented this mutation, the authors concluded that the cytosine favoured the erroneous incorporation of nucleotides replacing guanine in replicating DNA [257]. Pre-incubation of a cytosine-deficient mutant of E. coli with lactose and casein hydrolysate, followed by cytosine, resulted in the formation of /I-galactosidase 12581. The presence of 5-methylcytosine in DNA of plants and animals was reported in 1951 [259, 2601. It was identified in five out of eighteen preparations from human haemopoietic and other tissues [261]. 5-Methylcytosine was found to occur in both serine-specifict-RNAs from brewer’s yeast [262] and a yeast tyrosine t-RNA [263], but not in alanine t-RNA from yeast P641. In mammalian DNA, 5-methylcytosine was found to occur almost exclusively in fractions of solitary pyrimidine nucleotides [265], and in the terminal groups of polypyrimidine series carrying monoesterified phosphate at C3,position of deoxyribose [266]. The ratio of 5-methylcytosine to cytosine was almost 1 :1 in these positions [266]. 5-Methylcytosine was reported to occur in the DNA of B. subtilis [267] and in developing sea urchin embryos [268]. DNA-methylases had been found prior to that [269], but the only known methylated product had been 6-methylaminopurine [259, 270-2721. The methylation of cytosine, as well as adenine, occurs in the intact DNA polymer, and different methylases are required for the two bases. These methylases are species specific, as well [267, 273-2751. Even different strains of a given bacterium, such as E. coli, appear to differ as to the presence of this base [267, 2751. The source of the methyl donor used in these studies was methi~nine-methyl-’~C.Several t-RNA methylase enzymes from E. coli have been separated and purified, each specific for cytosine, uracil, guanine or adenine. No agent other than S-adenosyl-methionine acted as a donor with a nucleic acid acceptor, and only naturally occurring t-RNA acted as the methyl acceptor [276]. 5-(Hydroxymethy1)cytosine has been found in the DNA of the TZ,T4 and T6 phages of E. coli [277, 2781. A glycosyl-substituted 5-(hydroxymethy1)2‘-deoxycytidylic acid was isolated from a T z r +phage [279]. A great many ‘fraudulent’ bases related to cytosine and its nucleosides have been synthesised, either with the chemotherapeutic object of inhibiting one or more enzymatically controlled anabolic processes involved in nucleic acid syntheses, or of attempting incorporation into the DNA or RNA, with the object of producing mutations of the normal replication. Chemical reactions have also been carried out on the intact nucleic acids, for this purpose. Among the latter, one which is highly specific to cytosine is the reaction with hydroxylamine. This apparently adds first across the 5,6-double bond, followed by conversion of the 4-amino group to -NHOH, elimination of the first hydroxylamine, and hydrolysis to uracil [280, 2811. The G-C pair is

C. C. CHENG A N D BARBARA ROTH . 87 thus converted to G-U, which will then lead to A-U and A-T, thus producing a mutation. Semicarbazide also leads to a similar sequence of reactions [282]. Cytosine is photochemically converted to uracil at 2537A [283]. DNA with a high G 4 content is more resistant to ultraviolet irradiation than DNA with a low content of G-C [284]. Biological activities-The 5-halo derivatives and the mercapto analogues of cytosine have been discussed in Part I of this review. Cytosines with fraudulent sugars have received extensive study. Although a discussion of this type of modification is beyond the scope of this review, the fact that cytosine arabinoside [285, 2861 (LVI, 1-b-D-arabinofuranosylcytosine, Ara-C, CA, cytarbine) is a potent antiviral agent against herpes and vaccinia virus [287-2931 as well as a potent antileukaemia agent must not escape mention. In the latter case cytosine arabinoside is not only effective in transplantable

experimental neoplasms (such as sarcoma-] 80, Ehrlich carcinoma and leukaemia L-1210) in animals [294-3001 but is also clinically active against both acute myelocytic and acute lymphatic leukaemia in man [301-3211. Resistance to this synthetic pyrimidine develops readily but the resistant strains are usually not cross-resistant to other antileukaemic drugs [3223241. Cytosine arabinoside has also been reported to inhibit growth of E. coli and Streptococcus faecalis [325, 3261 and has radiosensitisation action to Micrococcus radiodurans [327]. Its biological action is presumably due to its ability to inhibit the conversion of cytidine 5’-diphosphate (CDP) to cytidine 2’-deoxy-5’-diphosphate(dCDP) ;thus it interferes with the synthesisof DNA but not RNA [328-3361. The interference is reversed by 2‘-deoxycytidine [337, 3381. Inhibition of DNA polymerase by cytosine arabinoside has also been reported [339]. Cytosine arabinoside is enzymatically deaminated in the liver. The resulting product, uracil arabinoside, no longer possesses antileukaemia or antiviral activity [291, 292, 340, 3411. A variety of cytosine derivatives have been studied as deaminase inhibitors. The most active of these was a 4hydroxylamino derivative, N4-hydroxy-5-rnethyl-2’-deoxycytidine [342]. Another cytosine derivative with antiviral activity is the 5-(4-aminophenyl) cytosine (LVII). This substance, according to results of clinical trials, possesses good prophylactic activity against influenza A2 [343]. The LDw

88 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 (i.v.) is 510 mg/kg; P.O., > 10 g/kg. It also showed some activity in clinical tests against tick-borne encephalitis [344], and against herpes [343]. It does

(LVLI)

( LVll I

not exhibit antibacterial properties [345]. The compound was also found to have some inhibitory action on Rous sarcoma [346]. Compound (LVII) is very rapidly excreted into the kidney. It is metabolised to 5-(3’-hydroxy-4’-aminophenyl)cytosineand its 0-glucuronide, as well as derivatives acetylated on both amino groups [343]. It is a competitive inhibitor of succinate dehydrogenase (K, = 9-2 x 1 0 - 3 ~ ;K i = 5.0 x 1 O P 3 ~ ) , and it has been considered that this is its mode of action [343]. One role of this enzyme involves aspartate synthesis, via fumarate. This pyrimidine enhances the incorporation of orotic acid into nucleic acid but is not itself incorporated. The synthesis of (LVII) is accomplished by formylation of p-(acetamidophenyl)acetonitrile, followed by conversion to the methoxymethylene derivative with diazomethane, condensation with thiourea to give 2-mercapto4-amino-5-(p-acetamidophenyl)pyrimidine, and oxidation by peroxide, followed by hydrolysis in base to produce (LVII) [347]. Alternatively, 5phenylcytosine can be nitrated and reduced [348]. A number of analogues with other substituents in the benzene ring, such asp-F, p-C1, 3,4-(OMe)2, and 2,4-C12, have been claimed to have antirachitic activity [349]. The 2-carboxymethylthio-4-amino-5-(p-chlorophenyl)pyrimidine analogue was found to be very active against vaccinia virus [350]; (see Part I, Volume 6, of this review for further information). Attention is called to the pyrimidine antibiotics section in Part I of this review. Several of these natural products, including blasticidin, gougerotin, amicetin, bamicetin and plicacetin, are 1-substituted cytosines. Cytovirin is also a plant antiviral substance from Streptomyces olivochromogenus which yields cytosine on degradation [351]. ISOCYTOSINE AND ITS DERIVATIVES

Isocytosine (L) is a weaker base than its cytosine isomer, with pK, values of 4.01 and 9.42 for the dissociation of the cation and free base, respectively

C. C. CHENG A N D BARBARA ROTH 89 [352]. Like cytosine, it exists in the aminopyrimidone form shown as (L), although the proton may exist in part on N1 rather than N3 [353]. Isocytosine per se does not have very interesting biological activity but some of its derivatives have chemotherapeutic activity, and others are of pharmacodynamic interest. 6-Methylisocytosine (LVIII, superacyl) has been reported to stimulate antibody production in rabbits to an extent greater than with a vaccine alone [354]. It also markedly reduced the development of pulmonary adenoma at 100 mg/kg per day for 10, 25 or 50 days S.C.or p.0. to mice, following a single i.p. injection of urethan. 5-Hydroxy-6-methylisocytosine had the same effect [19]. In tests on the effect of pyrimidines on reticuloendothelial functions in mice, the absorptive capacity of this system increased with 5-(hydroxymethyl)uracil or with 6-methylisocytosine. The effect was most pronounced when superimposed on cortisone inhibition [355]. When administered subcutaneously to mice, 6-methylisocytosine increased the inhibitory effect of thio-TEPA 011 the growth of Ehrlich tumours in mice. It is believed that this pyrimidine prevents the development of metastases [356]. Sodium 5-sulpho-6-methylisocytosine and 6-methylcytosine produced a considerable reduction of leukocytes in mice with myeloid and haemocytoblastic leukocytosis; the effect was greater on immature leukocytes. The life span of the animals was not prolonged, however. The authors drew the conclusion that uracils substituted at C, by nitro- or sulpho groups generally stimulated cell proliferation, whereas the corresponding 5-substituted isocytosines, cytosines, or diaminopyrimidines inhibited it 13571. In the case of S180, Ehrlich ascites tumours, and Crocker sarcoma in mice, sodium 5-sulpho-6-methylisocytosine stimulated tumour growth, on the other hand [358, 3591. This was also true of the 5-nitro analogue [358]. 5-Hexyl-6-methylisocytosine was found to have activity vs. type I adenovirus [360, 3611. Other 5-alkyl analogues (C, to CI2) were inactive. No activity was found against polio (Mahoney) virus or vaccinia (VD-6) virus. Isocytosine was reported to increase TMV virus production in daylight [362]. On the other hand, it delayed enterovirus multiplication in monkey

testicular culture [363]. N-Guanido derivatives of isocytosine of type (LIX, for example, R’ = NMe,, RZ = NH,) have been reported to have antiviral activity against influenza and the common cold [364]. A number of pyri-

90 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 midine-5-(a-haloacylamides) were reported to inhibit the multiplication of vaccinia virus in chick embryonic tissue. The isocytosine derivative (LX), as well as the corresponding uracil and 2-amino-4,6-dimethyl analogues, were the most active of the compounds tested (mic., 10 mg/l) [365]. In 1948 it was reported that a large number of 2,4-diaminopyrimidines and some 2-amino-4-hydroxypyrimidines are antagonists of folic acid (LII), as determined by their inhibition of the growth of L. casei, which can be reversed by pteroylglutamic acid [366]. Since that date a large number of 2,4-diaminopyrimidines have been synthesised for this purpose (see Part I, Volume 6, of this review), and in many cases, the 2-amino-4-hydroxy derivatives were prepared as intermediates in this synthesis. It was eventually shown that the pyrimidines are inhibitors of the enzyme dihydrofolate reductase [367, 3681, times more tightly to and that the diaminopyrimidines are bound 1 ~ 1 O O O the enzyme than the isocytosines, in general. This is true of folic acid itself, vs. its 4-amino analogue (Ki(M) = 1.1 x lop7and 6 x lo-”, respectively vs. Ehrlich ascites enzyme) [369]. In whole cells, the respective m. i. c. (pglml) for 2,4-diamino-5-(3’,4’-dimethoxybenzyl)pyrimidine,the 2-amino4 hydroxy, and 2-hydroxy-4-amino analogue vs. P. vulgaris were found to be 4, 500, and loo0 in the order given [370]. In rat liver enzyme, the relative binding constants (Ki(M)) for pyrimethamine (2,4-diamino-5-(p-chlorophenyl)-6-ethylpyrimidine) and its isocytosine analogue were 7-0 x lop7 and 7.5 x respectively [371]. Since the diaminopyrimidines which are half protonated at physiological pH are much more active inhibitors than those which are not [372], and since the isocytosines are essentially nonprotonated in the pH 7 region, it has been postulated that strong association with the enzyme involves electrostatic attraction to a negatively charged group on the latter [372,373]. It has also been suggested that the pyrimidine moiety may complex with the enzyme as a n-donor [374]. A number of isocytosine derivatives with bulky hydrophobic groups in the 5-position do bind quite successfully in a reversible manner to the reductase enzyme from pigeon liver, however. Furthermore, some of these compounds are bound as well to thymidylate synthetase as are their diamino counterparts, and some are bound better to synthetase than to the reductase enzyme. These results are illustrated in Tables 3.4 and 3.5. 2,4-Diamino-6-hydroxy-5-phenylbutylpyrimidine gave 50 per cent inhibition of dihydrofolate reductase at 8.4 PM concentration, compared to 0.027 p~ for the 2,4-diamino-6-methy! analogue. The corresponding value for the 6-methylisocytosine derivative was 30 p~ [382]. Based on these and related results, the first active-site-directed irreversible inhibitor of dihydrofolate reductase (LXI), was found [374, 3831, with Ki = 4 x ~ O - ’ M , and halftime for inactivation of the enzyme of 20 min. A related compound with the reactive group on the side chain of the 5-substituent (LXII) showed similar properties [384]. Since diamino derivatives are normally bound considerably better than the isocytosines, the 2,4-diamino analogue of (LXII) was pre-

C. C. CHENG AND BARBARA ROTH 91 pared. Although it showed reversible inhibition at K i = 5 x ~ O - * M ,it was not an irreversible inhibitor [385]. It was concluded that the pyrimidines can bind to the enzyme as different rotomers [374, 3851.

Table 3.4

INHIBITION OF DIHYDROFOLATE REDUCTASE A N D THYMIDY LATE SYNTHETASE BY

Me

R‘

R2

Dihy’drojolute Thymidylute ?Reductuse 7r S v n t h e t a s e 7 mMConc. % f / S mMConc. % f/S inhibi- inhibi- ratio inhibi- inhibi- ratio tor tion tor tion

OH -NHPh NH2 -NHPh OH -NHS02C6H4Me-p OH -NHC6H,COOH-p

Table 3.5

0.60 0.0022 0.60 0.077

43 50 20 50

130 0.37 400 13

0.62 0.80 0.28

50 50 50

-

-

50 63 II

Syn.1 Red.

0.39 70 0.027

_ _ _

Ref:

375 375 376 377

INHIBITION OF DIHYDROFOLATE REDUCTASE BY

R‘

R2

K i x lo6

--NHC,H,(CO-Glu)-/, -NHC~H~(CO-G~U)-I, -NHPh -NHPh -NHPh

Me Ph Me Ph CH2Ph

2 0.09 63 1.8 4

Ref:

378 379 380 38 1 381

92

SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART H

Q

111

H

(LXLI)

NHCOCH?RT

(LXI)

Another isocytosine analogue of pteroylglutamic acid which has been prepared is (LXIII), as well as its 7,lO-methenyl derivative (LXIV) [386].

(LXIV)

The former can be prepared by reductive alkylation of 2-acetamido-5-amino4(3H)pyrimidone with dimethyl N- {p-(N-(formylmethy1)formamido)benzoy1)-L-glutamate, followed by reformylation, partial deblocking, and acid hydrolysis to (LXIII). Both (LXIII) and (LXIV) were reported to have inhibitory activity against S. fueculis equal to that of aminopterin. They both inhibited dihydrofolate reductase (source not named) at 4 x ~O-’M, for 50 per cent inhibition. Compound (LXIII) is a better inhibitor of thymidylate ~ 50 per cent inhibition). synthetase than (LXIV) (3 x lo-’ vs. 7 x l O P 4for A number of isocytosines are of pharmacological interest. Some 5-alkyl6-aryl derivatives, such as (LXV), were found to be potent diuretics in rats [387,388].Compound (LXV) is orally active at 5 mg/kg. The sodium clearance increased even while the urine volume decreased. In the dog, however, the activity was low or inconsistent by the oral route, probably due to variable absorption. The mode of action seems to be like that of the xanthines. The

.93 C: C. CHENG AND BARBARA ROTH substance antagonised the pressor effect of deoxycortisone acetate (DCA) and transiently reversed metacorticoid (DCA induced) hypertension. It was H

H

Ph

Me

(LXV)

( LXVI

suggested that chronic administration may exert an antihypertensive action, at least partially as a result of the diuretic action. The compound has no ganglionic-blocking activity. In the rat, optimal activity in this series was found with 6-phenyl derivatives having methyl or ethyl in the 5-position. The 5-methyl-6-phenyl groups can be interchanged to give (LXVI) without loss of activity. The cytosine analogue of (LXV) and the 6-alkyl analogues of (LXV) were found to be inactive. The synthesis of (LXV) is accomplished from guanidine and ethyl a-benzoylpropionate [389]. Compound (LXVI) is similarly prepared from guanidine and ethyl a-acetyl-a-phenylacetate [390]. The related N-alkylated compounds (LXVII) are also claimed as diuretics [39Ij, and (LXVIII) as cardiovascular agents [392].

d N" , "

J

/ O R2

Ar

(LXVII) 1

2

( R and R may be alkyl, alkenyl. alkynyl, or substituted aikyl)

H

""YFR N / X

(LXVIII)

H2N R

(CH2)"

(LXIX)

Where R = alkyl,alkenyl X=furyl,thienyl,or pyridyl

Cyclic derivatives such as LXIX, for example R = CH,CH,OH, n = 3) have received mention as anti-ulcer, pepsin-inhibitory and antihypercholesterolemic agents [393]. MISCELLANEOUS AMINOHYDROXYPYRIMIDINES

Some 2-alkylamino analogues of the isocytosines have been reported to have activity as tranquillisers [394]. These are of the general formula (LXX) where R', RZ,and R3 may be methyl, for example.

94

SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART R’

(LXX)

I11

H

H

(

LXXI )

Some 2,4-diarylamino-6-hydroxypyrimidineshave been reported to cause marked growth inhibition of S. faecalis and L. arabinosis. This inhibition is not reversed by folic acid [395]. It is believed that the compounds interfere with the pyrimidine metabolism of the micro-organisms [396]. Such compounds were found to be more active than the 2-amino4arylamino analogues. 2-Dimethylamino6-hydroxy or 2-arylamino-4-amino-6-hydroxy 4-hydroxy-5-butyl-6-methylpyrimidine(LXXI), a compound in the same category, gives exceptionally good control of powdery mildew (Sphaerotheca fuliginia Schlecht) on cucurbits, particularly when applied to the soil as a liquid or in granules [397]. This pyrimidine has very low mammalian toxicity. Compounds of type (LXXII) have been patented as schistosomicidal agents and antibacterials [398]. Here R’ may be NH2 or SH, Rz may be OH, alkyl, SH, halogen, or substituted amino, or H when R’ = NH2, and A is an aliphatic chain of 2-12 carbon atoms.

Me

(LXXII)

Me ( LxxIlI)

The 5-amino derivative of cytosine is reported to inhibit riboflavin synthesis [399]. 4,5,6-Triaminopyrimidineinhibits both synthesis and growth inhibits synthesis, but by 60 per cent. 4,5,6-Triamino-2-hydroxypyrimidine not growth, of riboflavin [400]. Compounds such as (LXXIII) have been patented for their ‘microbiocidal’ properties [401]. PYRIMIDINE AMINO ACIDS Natural products containing a /?-substituted alanine moiety attached to a pyrimidine ring have attracted considerable attention in recent years. Lathyrine (tingitanine, LXXIV), a non-protein amino acid, was isolated from the seeds of Lathyrus tingitanus [402] (Tangier pea), L. cicera, L. aphaca, L. pratensis, L. niger and many other species of Lathyrus [402404]. The lathy-

C. C. CHENG A N D BARBARA ROTH 95 rine content in the seeds of L. tingitanus is rather high [405]-yields of 1 g of lathyrine from only 100 g of seeds have been reported [404]. This amino acid also occurs, in somewhat lesser amount, in the roots and rootstocks of plants of this species [406]. The results of analytical and physical measurements suggested its structure as @-(2-amin0-4-pyrimidinyl)alanine [407]. This

_. .

(LXXlVl

assignment was substantiated by syntheses utilising the condensation of oxalate with 2-substituted (methoxy, ethylthio, amino) 4-methylpyrimidines followed by oximination, reduction and hydrolysis [408-411], or by the condensation of 2-amino-4-chloromethylpyrimidineand ethyl acetamidocyanoacetate and subsequent hydrolysis [412]. Natural lathyrine is in the L-form [404]. It is proposed [413] that lathyrine is formed biogenetically from L-homoarginine [403, 407, 4141 (LXXVa, a-amino-E-guanidinocaproicacid) via L(y-hydroxy}homoarginine [415,416] (LXXVb). Both (LXXVa)and (LXXVb) were isolated from a number of Lathyus species [413,4141 and (LXXVb) has been shown by stereospecific synthesis to be the threo-isomer [417]. The principal site of lathyrine biosynthesis is believed to be the root of these plants,[416]. The high nitrogen content (308 per cent) and the low solubility of lathyrine at normal biological pH (L-homoarginine, with a similar nitrogen content, is more soluble) suggest that at least one of the functions of this amino acid is that of nitrogen storage [403, 4131. Lathyrine at 0.1-2 mM strongly promotes the growth of the pollen tubes of Lathyrus niger [418]. This amino acid also stimulates the growth of Rhizobium and Agrobacterium strains [419]. In dose levels of 1OWlOO mg/kg, it elicits no toxic symptoms in mice, chicks, or rats [404]. During the germination of seeds of Acacia willardiana, A . lemmoni, A . millefolia [420], Mimosa asperata [421] as well as from Pisum sativum L. var. Rondo (pea seeds) [422], a number of uracil-containing amino acids are formed in fairly high concentration. One of the amino acids isolated is willardiine (willardine, LXXVI). The structure of this compound, p-( 1uracilyl)alanine, was confirmed by a number of syntheses, including ( a ) the condensation of p-ethoxyacryloyl isocyanate with aminoacetaldehyde diethyl acetal, followed by a Strecker synthesis [423,424], ( b )alkali-catalysed condensation of the aforementioned acyl isocyanate with a 2-substituted 3-aminopropionic acid [425], (c) condensation of ethyl formylacetate with a-amino-B-ureidopropionicacid [426,427] and ( d )the reaction of uracil with

96 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 2-bromo-1,l -diethoxyethane [428]. The L-form of fl-( 1-uracilyl)alanine, obtained from the resolution of the synthetic DL-forms, is identical with the naturally occurring willardiine. Albizziin (L-a-amino-b-ureidopropionic acid, LXXVII), which accompanies willardiine in the seeds [420] bears a formal resemblance to willardiine. These two amino acids may well be biogenetically related.

(LXXVL)

(LXXVII)

From the young pea seedlings of Pisum sativum L. var. Rondo there was isolated another pyrimidine amino acid, the structure of which was initially proposed [429] as ~-fi-(5-uracilyl)alanine(LXXVIIIa). However, its physical constants by no means agree with those of the authentic fl-(5-uracilyl)alanine unequivocally synthesised earlier [430, 43 13. The structure has since been revised to L$-( 3-uracily1)alanine (LXXIX, isowillardiine) by examination of its ultraviolet absorption characteristics and chemical reactions [422] and substantiation by infrared, NMR, and mass spectrometry [432]. Its biological role has not yet been determined. 'NH3

I

CHzCHCO;

I

H

R

CH coy I +NH,

(LXXVIIIJ o R-H

(LXXIX)

b. R = M e

b-( 5-Cytosiny1)alanine [433] (LXXX) and P-(6-uracilyl)alanine (LXXXIa) [431, 434, 4351 have not yet been isolated in nature. The latter inhibits the growth of Lactobacillus citrovorum [436]. This inhibitory activity is not found in the corresponding 5-alanyl derivative [436] (LXXVIIIa), nor in fl-(5nitro-6-uracily1)alanine (LXXXIb) or j?-(6-methyl-5-uracilyl)alanine [437, 4381 (LXXVIIIb). No biological activity has been found as yet for fl-(4,6dihydroxy-2-pyrimidiny1)alanine[431J (LXXXII), a structural isomer of willardiine (LXXVI) and b-( 5-uracily1)alanine (LXXVIIIa).

-97

C. C. CHENG AND BARBARA ROTH

A compound having an aspartic acid moiety attached to the 5-position of a uracil ring has been isolated from the trichloroacetic acid extract of pea seedlings [439].The exact structure is not known. Some pyrimidine analogues of phenylalanine and tyrosine (LXXXIII, LXXXIV, LXXXVa and LXXXVb) have been prepared [44W4].Among these, fi-(4-hydroxy-2-pyrimidinyl)alanine(LXXXVb) was reported to have some activity resembling that of phenylalanine under certain experimental conditions [4424443.~-(4-Amino-6-carboxy-5-methyl-2-pyrimidiny~)alanine (LXXXVc) was initially designated as the structure of one of the components

(LXXXII I )

( LXXX IV )

( L x x x v ) a. R’: R2: R3=H b. R’= OH; R2=R:’H c. R’=NH,;R*=Me;

( LXXXVI)

R3=C02H

(LXXXVII) a R’= R2: Me; R3= H b. R’I R2: H .

R350H

of bleomycin A2 [445], an antitumour antibiotic reported to be inhibitory against rice sheath blight disease [446]as well as against human squamous cell carcinoma, Hodgkin’s disease, reticulosarcoma, brain tumour and other tumour systems with low toxicity [447450]. The structure of this amino acid was later revised to that of a fi-amino acid [451](LXXXVI). An antifungal antibiotic stendomycin, which inhibits a number of fungal

98 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART III plant pathogens, has been isolated from Streptomyces antimycoticus [452, 4531. Physical and chemical studies have revealed that it is not a homogenous compound, but rather a family of closely related lipid-containing polypeptide complexes, which differ from each other in their fatty acid constituents [454, 4551. The chief component consists of a chain of fourteen-unit polypeptides with a tetrahydropyrimidine a d n o acid stendomycidine (LXXXVIIa) linking unit 8 and unit 13 [454-456]. This pyrimidine can be regarded as derived from arginine. A closely related compound, tuberactidine [457a] (LXXXVIIb), was found to be a constituent of the antibiotic tuberactinomycin [457 b, c]. NITRO AND NITROSOPYRIMIDINES A wide variety of aromatic, heterocyclic, and aliphatic compounds with nitro substituents has achieved medicinal utility [458]. Certain 5-nitropyrimidines and their nitroso counterparts deserve mention as compounds of chemotherapeutic interest. Some of these have already been discussed in preceding sections, including 5-nitrouracil, 5-nitrocytosines and isocytosines and 5-nitrohydropyrimidines. 2-Amino-5-nitropyrimidine (LXXXVIII) has a high degree of suppressive activity in enterohepatitis (blackhead) of turkeys at non-toxic concentrations in the diet [459, 4601. When tested against turkeys inoculated with Histomonas meleagridis, a concentration of 0-1 per cent drug in the diet for 7-14 days almost completely prevented the mortality and development of caecal and liver lesions if given not later than 3 days after inoculation. There was 80 per cent mortality in the controls. Similar results were obtained against Heterakis gallinae ova inoculated orally. This is presumed to be the major mode of natural occurring infections. This activity was confirmed in the field. A related compound, 2-amino-5-nitrothiazole (LXXXIX), and its acyl derivatives, were also found to have similar activity. The synthesis of (LXXXVIII) can be accomplished by treatment of sodium nitromalonaldehyde with guanidine in the presence of aqueous piperidine 14611. 2-Amino-5-nitropyrimidine has only a moderate degree of anticoccidial activity at slightly toxic concentrations in the chicken (0.15 per cent). The

[ LXXXVIII 1 (LXXXLX) (XC) reverse is also true, in that highly active anticoccidials such as the sulpha drugs and nitrophenide (bis-(m-nitrophenyl) disulphide) were found inactive against the turkey enterohepatitis [459]. The nitroimidazole (XC) was reported to be an active antitrichomonal agent in clinical trials [462]. This led to further studies with (LXXXVIII),

C. C. CHENG AND BARBARA ROTH . 99 (LXXXIX), and related nitroheterocycles. Compound (LXXXVIII) was found to be highly active in mice infected with Trichomonas vaginalis, but it was not superior to the acetyl derivative of (LXXXIX) [463]. However, compound (LXXXVIII) shows high specificity in that it is much more active in vivo against T. vaginalis than against T.foetus. The curative doses (CD,,) against the former in mice were found to be 35.3 (p.0.) and 28.6 (i.p.) mg/kg; the therapeutic index is 7. The corresponding 2-hydroxy-hitropyrimidine is inactive. The concentration of (LXXXVIII) in plasma was found to increase to a maximum over an 8 h period. It was then slowly excreted. Compound (LXXXVIII) was selected as the best compound of many studied, because of the high blood levels obtained after a single oral dose in a monkey, and also because the drug presented was the biologically active form, in contrast with some acylated derivatives [463]. Efforts to determine the mode of action of the drug showed that the inhibitory action occurs during the prophase portion of the mitotic cycle [464]. Cells grown in the presence of (LXXXVIII) contain more glucose-6-phosphate and 6-phosphogluconate dehydrogenase, but less malic dehydrogenase, than the controls. The nucleic acids are not affected [465]. In contrast with 2,4-dinitrophenol, (LXXXVIII) does not appear to uncouple phosphorylation in resting cells. An accumulation of NAD occurs during growth, which is not responsible for the prophase blockage [464]. Cells which were made resistant to (LXXXVIII) also became resistant to 2-amino-5-nitropyridine and its 2hydroxy analogue, indicating that these inhibitors act at the' same locus [466]. The in vitro inhibitory effects of (LXXXVIII) could be reversed by 2-acetamido-5-nitropyridine [464]. Among a series of 2,4-diamino-5-nitroso-6-anilinopyrimidines (XCI) prepared as open chain analogues of riboflavin, the p-bromoanilino, p-iodoanilino-, and 3,4-dichloroanilino-derivatives were found to possess carcinostatic activity against adenocarcinoma 755 in mice [467] (see also Part I, Volume 6, of this review). A trace of activity was shown by analogues with a 5-cyano substituent, but not by those with a carboxamido group [468]. Electron-releasing substituents in the 5-position produced inactive products, but the activity of the nitroso derivatives was evidently not related to its electron-attracting properties. It was originally thought that 2,4-diamino-5nitro-6-@-bromoanilino)pyrimidine was inactive [468]. However, it was later found that this substance prepared initially was actually a 4-nitraminopyrimidine [469]. The actual 5-nitropyrimidine was active against the Walker 256 tumour in rats [469]. Since this test system is different from that used for the 5-nitroso analogue, there is no basis for comparing relative activities of the 5-nitro- and nitroso-derivatives. However, one or the other of these substituents is required for activity in this group of compounds. 4,6-Dihydroxy-5-nitropyrimidinehas been reported to inhibit the growth of SSK sarcoma in rats [470]. 2,4-Diamino-5-nitro-6-methylpyrimidine retards the growth of Crocker sarcoma in mice at 50 mg/kg [358].

100 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART

111

A number of 4,6-disubstituted 5-nitropyrimidines have been claimed to be useful as fungicides [471]. A 100 per cent control of typical fungi was attained with a 0.1 per cent concentration of 5-nitropyrimidines having the following substituents: 4,6-dichloro-, 4-chloro-6-amino-, 4,6-diamino, and 4,6-dihydroxy-. The 4,6-dichloro derivative (XCII) is the best. All compounds maintained some activity at 0401 per cent concentrations. U

(XCII)

(XCI)

Among the hexahydropyrimidines, some 5-nitro derivatives have been reported to have bactericidal activity which is useful in stabilising petroleum lubricants [472] (see discussion in the hydropyrimidines section). A related patent claims compounds such as (XCIII) for the control of bacteria in water flooding during oil recovery [473].

H°CH2\

Et’

c 3 N 0 2 CHN Me

(XCIII 1

(XCIV)

(XCV)

Reduced pyrimidines containing nitro substituents on the ring nitrogen atoms have proven of interest for agricultural use. For example, 1,3-dinitro3,4,5,6-tetrahydro-2( 1H)pyrimidone (XCIV) is claimed to inhibit growth of suckers on tobacco plants, and derivatives are said to have herbicidal activity 1H)pyrimidone (XCV) is claimed to be a [474]. 1,3-Dinitro-5,6-dihydro-2( fungicide giving good control of Phytophthora infestans [475]. PYRIMIDINES CONTAINING BIOLOGICAL ALKYLATING FUNCTIONS A large number of derivatives of the original nitrogen mustard (‘HN,’,MeN(CH2CH2C1)2)and other biological akylating agents, such as ethylenimines, epoxides, etc., have been prepared as potential anticancer agents [476]. The thought behind this extensive chemical exercise is that the alkylating agents consist of a carrier and the alkylating group, and that differences in selectivity of action upon the tumour, or ability to reach the site of desired action, with minimum damage to the host, are dependent upon the carrier [477].

101 C. C. CHENG A N D BARBARA ROTH The biological importance of uracil, relative to nucleic acid and protein syntheses, led to its early selection as a candidate carrier [478]. Uracil mustard (5-(bis-(fi-chloroethyl)amino)uracil, XCVIa) can be synthesised by the treatment of 5-aminouracil with ethylene oxide in aqueous acetic acid, followed by the chlorination of the resulting 5-(bis-(fi-hydroxyethyl)amino) uracil with thionyl chloride in diglyme [479, 4801. H

R (XCVI)

a R=H b R=Me

Uracil mustard was found to be orally active in small animals against Walker 256 carcinoma, Jensen sarcoma, Murphy-Sturm lymphosarcoma, Guerin carcinoma, adenocarcinoma 755, Cloudman S91 melanoma, L 12 10 leukaemia [479, 481,4821, sarcoma 45, sarcoma 180 and Ehrlich carcinoma [482484], Dunning rat leukaemia, Dunning rat lymphosarcoma, lymphoma K4, lymphosarcoma L2, and mouse reticulum-cell sarcoma [485486]. The activity is equal to that of chlorambucil, and side effects are less than with HN2 [481]. The LDS0values in mice and rats were reported as 3.7 mg/kg (i.p.) and 7.5 mg/kg (p.0.) [479]. Chronic toxicity tests (3 months in rats) show the m.t.d. to be 1 mg/kg per day. Histological changes are most marked in the bone marrow and testis, with considerable leukopaenia and eosinopaenia. Metabolism of 17-ketosteroids is inhibited [487]. No automatic or cardiovascular activity was observed in anaesthetised dogs. Uracil mustard is not a uracil antagonist for E. coli Bu-, which requires this nutrilite [479]. However, in rats with Walker 256 carcinosarcoma, incorporation of ~racil-2 -’~ into C the RNA of intracellular tumour and spleen fractions is markedly inhibited [488]. Uracil mustard also inhibits the incorporation of nucleic acid precursors into RNA and markedly inhibits DNA synthesis in tissues of A/J mice [489]. Inhibition of arginine incorporation into nuclear proteins was likewise noted [490]. Uracil mustard has undergone quite extensive clinical trials, and has been found effective in the treatment of chronic lymphocytic and granulocytic leukaemias, Hodgkin’s disease and lymphosarcoma. Occasionally solid tumours, notably ovarian carcinoma, have responded. Dramatic symptomatic relief is obtained with Hodgkin’s disease and multiple myeloma, and the drug is well tolerated [491-496]. It is not effective in acute granulocytic leukaemia [491], in acute leukaemia of children (4971, or metastatic Wilm’s tumour [498]. It is of limited usefulness with rare neoplasms in children [499]. Gastrointestinal disturbances, leukopaenia, and thrombocytopaenia are often noted in patients under treatment with uracil mustard. It is less toxic than cyclophosphamide (cytoxan) [500].

102 SOME PYRIMIDINES OF BIOLOGICAL AND MhDICINAL INTEREST-PART 111 Uracil mustard had only very weak activity in suppressing antibody synthesis in rats, when given 24 to 48 hours after the antigen [500, 5011. Daily intraperitoneal injections of 10 mg of uracil mustard for 20-30 days in mice causes alternating elevation and depression on normal phage neutralising antibody activity [502]. It is not uncommon to note that many compounds possessing anticancer activity may also show carcinogenic properties under different biological and physiological conditions. Uracil mustard was found to be a potent inducer of lung tumours in mice. When injected intraperitoneally, this biological alkylating agent can increase the incidence and average number of pulmonary tumours in A/J mice [503]. Approximately ten times as many nodules are induced in the animals treated with uracil mustard [503, 5041 as with nitrogen mustard [504-5081. Possibly the marked carcinogenic activity of uracil mustard is due to the fact that it may be incorporated into RNA as a uracil analogue during biosynthesis, as well as alkylating specific receptor sites. This would consequently alter protein function [488, 503, 509-5 111. A number of analogues of uracil mustard have been prepared. The 6-methyl analogue (dopan, XCVIb) was reported prior to uracil mustard [512]. Clinical trials show it to have a decided therapeutic effect in Hodgkin’s disease, myelogenic leukaemia and reticulosarcoma [513-5241. A fluoro analogue (fluoropan, XCVIIa) is reported to have caused complete regression of osteogenic sarcoma of the hip [525], and is therapeutically effective against Hodgkin’s disease [526]. However, it failed to show inhibitory activity against Walker 256 carcinosarcoma. In general, replacement of chlorine by a fluorine atom often leads to an increase in toxicity without a corresponding improvement in antitumour activity [523, 527-5301. A mixture containing dopan, fluorodopan and hydroxylated derivatives was claimed to be less toxic than dopan and active against the growth of osteogenic sarcomas [531]. H

a. R=Me b. R = H

(XCVJI)

H

X = H,CI R’= low mol. wt. a l k y l or CH2CH,Cl 2 R = H,alkyl. cycloalkyl, aralkyl, akkenyl or a r y l (XCVIII)

‘One-armed’ analogues [532], as well as an ‘aliphatic’ analogue, with a methylene group separating the uracil and alkylating moieties, have also

103 C. C. CHENG AND BARBARA ROTH received mention [533-5361. 5-(Bis-(fi-chloroethyl)amino)methyluracil (ypenyl; XCVIII, X = H, R' = CH2CH2CI, RZ = H) [536] was found to cause chromosome damage of Viciufabu [537]. 6-(Bis-(~-chloroethyl)amino)uracil (XCIX), the position isomer of (XCVIa), was prepared by the treatment of 6-chlorouracil with bis(fihydroxyethy1)amine followed by chlorination with thionyl chloride [538, 5391. This compound fails to display tumour-inhibitory activity. '

N (CH2CHzCI 12 (XCIX)

Other mustard-substituted pyrimidines synthesised include benzylidine derivatives of barbituric acid [540] (C), cytosine and thiocytosine mustards [541] (CI) and DL-willardiine mustard [542] (CII). No significant anticancer activity was found among these compounds. Compound (CI) may actually have an ionic structure such as (CIII).

(CII)

(C I l l )

Hexahydropyrimidines have also been used as carriers for the nitrogen mustard function. Compounds of type (CIV), prepared by the condensation of substituted 1,3-diaminopropanes with substituted benzaldehydes containing a p-bis(fi-chloroethy1)amine moiety, exhibit tumour-inhibitory activity against Walker 256 carcinoma in rats [543,544]. The degree of activity of these compounds is believed to be dependent on the electron-donating ability of the substituents on the aryl moiety [543].

104 SOME PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART 111 Some pyrimidines containing two ethylenimine functions have also been found to possess anticancer activity. 2,4-Bis(aziridinyl)-6-chloropyrimidine [545-5481 (CV, ethymidine, etimidin) significantly inhibits growth of many transplantable mouse and rat tumours (Walker carcinosarcoma, sarcoma 180, sarcoma 45, etc.) [545-547, 549-5521. The LDloo of ethymidine on single intravenous administration to rats and mice is 15 mg/kg. In toxic doses ethymidine has a cholinolytic effect [553] and depresses haemopoiesis [512]. A decrease of sulphhydryl group content in blood serum of rats was noted .upon intraperitoneal administration of this pyrimidine derivative [554]. Clinically, ethymidine exerts a beneficial influence on lung cancer, cancer of the ovaries and the lymph nodes of the neck [512, 555, 5561. Its side effects include nausea, vomiting, impaired hearing and haemopoiesis [5 121. Change in blood serum protein fractions was noted during chemotherapy in patients with ovarian cancer [557]. Chlorination of aminopyrimidines, followed by treatment of the resulting phosphoramidic dichloride with ammonia, can lead either to the breakage of the N-P linkage [558] or to the formation of phosphoric triamides [559].

(CVJ

When 2-aminopyrirnidia- IS refluxed with pnosphorus oxychloride and then treated with ethylenimine, there is obtained P,P-bis( 1-aziridinyl)-N-2-pyrimidinylphosphinic amide [560] (CVIa, phosphazine). This product has high anticancer activity against transplanted carcinoma in mice, rats and rabbits [561-5631. The tcxicity of phosphazine [564] was claimed to be much less than that of thio-TEPA or dipin [565]. In mice, phosphazine at 10 M decreases the cellular concentration of glycine-14Cand its incorporation into proteins of the Brown-Pearse carcinoma by 26.5 and 20 pc. cent, respectively [566]. Phosphazine injected intraperitoneally into rats bearing Yoshida sarcoma sharply arrested the accumulation of lysine and its incorporation into cancer cell proteins [566]. A methyl homologue of (CVIa), methylphosphazine [5631 (CVIb), was found to be more active and less toxic than (CVIa), thio-’I’EPA or cytoxan in animal systems [563].

-’

C. C. CHENG AND BARBARA ROTH

105

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 Renke Laube for assistance in the literature search, and to Miss Soula Culver for typing this manuscript.

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BIOLOGICAL A N D MEDICINAL INTEREST-PART

I11

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111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135.

I07

A. Lazarow, Proc. Soc. Exp. Biol. Med., 1946,61,441

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468. 469. 470. 471. 472. 473. 474. 475. 476. 477. 478. 479. 480.

D. E. O’Brien, F. Baiocchi, R. K. Robins, and C. C . Cheng, J . Med. Chem., 1963,6,467 D. E. O’Brien, C . C. Cheng and W. Pfleiderer, J . Med. Chem., 1966.9, 573 I. F. Grekh, Sovretn Probl. Onkol., 1965, 74; Chem. Abstr., 1966,64, 14826e V. P. Lobov, V. V. StOpkdn, T. 1. Cherepenko and V. M, Cherkasov, Prikl. Biokhim. i Mikrobiol.. 1965, 1,248; Chem. Abstr., 1965,63, 13737~ E. B. Hodge, U.S. Patent 3 183 188 (1965); Chem. Absfr., 1965,63, 283Od E. P. Hodge, U.S. Patent 3087891 (1963); Chem. Abstr., 1963,59,4945e H. A. Hageman and J. A. Riddoll, U S . Patent 3 226221 (1965); Chem. Abstr., 1966,64, 10340b Dow Chemical Co., Netherlands Patent Appl. 300737 (1965); Chem. Abstr., 1966, 64, 13329e Cancer Chemother. Rep., 1963,26, lists 2255 nitrogen mustards, 132 sulfur mustards, 648 aziridines, and 384 epoxides and episulfides H. R. Ing, Trans. Faraday SOC.,1943,39, 372 For a discussion on the active transport of pyrimidines across the intestinal epithelium, see L. S. Schanker and J. J. Jeffrey, Nature, 1961, 190, 727 D. A. Lyttle and H. G. Petering, J . Amer. Chem. Soc., 1958, 80, 6459 D. A. Lyttle, C.S. Patent 2969364 (1961); Chem. Abstr., 1961, 55, 25999f

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PYRIMIDINES OF BIOLOGICAL AND MEDICINAL INTEREST-PART

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481. 482. 483. 484. 485. 486. 487. 488. 489. 490. 491. 492. 493. 494. 495. 496. 497. 498.

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500. 501. 502. 503. 504.

505. 506. 507. 508. 509. 510.

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123 511. S. J. Smith and H. Busch, Texas Rep. B i d . Med., 1964,22, 731 512. L . F. Larionov and G . N. Platonova, Vopr. Onkol., 1955, 1 [5],36 513. L. F. Larionov, Cancer Chemotherapy (trans]. A. Crozy), Pergamon Press, London, 1965 514. V. G. Nemets, L. F. Larionov, E. G . Airapetyants and G. N. Platonova, U S S R Patent 116912 (1959); Chenz. Abstr., 1959,53, 17438i 515. L. F. Larionov, Brit. J . Cancer, 1956, 10, 26 516. N. N. Blokhin, Malignant Neoplasnzs Proc. X. Session USSR Acad. Med. Sci., 1956 517. Y . M. Ivanova, Vopr. Onkol., 1957,3 [5], 556 518. H. G. Vishnyakova, Vopr. Onkol., 1958.4 [5], 569 519. R. A. Mokeyeva, Sovetskaya Med., 1958, [91,42 520. Y. M. Ivanova, Prob/ems of the Chemotherapy of Malignant Tunzours (Vopr. Khinrioterapii Zlokachestvennykh Opukholei), Moscow, 1960, 388 521. L. F. Larionov, Acta Unio Intern. Contra Cancrunz., 1957, 13, 393 522. J. Louis, J . Chronic Diseases, 1962, 15, 273 523. H. Dubicki, F. Zielinski, and F. W. Starks, J. Pharm. Sci., 1964,53, 1422 524. I. V. Sikorskaia, Sovet Zdravookhr Kingiz, 1965, 2, 34 525. L. F. Larionov, M. D. Chadakova, and E. I. Arkhavgel'skaia, Vopr. Onkol., 1969,7, 112 526. L. P. Bulanov and V. P. Zolotstev, 4th Sci. Cont. Inst. ESP. Clin. Oncol. Sunznz. Rept.

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527. 528. 529. 530. 531. 532. 533. 534. 535. 536. 537. 538. 539. 540. 541. 542. 543. 544. 545. 546. 547. 548. 549. 550.

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4

Antiviral Agents

D. L. SWALLOW, M.A., B.Sc., D.Phi1. Imperial Chemical Industries Ltd., Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, England

INTRODUCTION

120

ALICYCLIC COMPOUNDS Adamantane derivatives Other multicyclic compounds Monocyclic compounds

122 122 126 132

INTERFERONS AND INTERFERON INDUCERS Double stranded ‘RNA’ Statolon Pyran Other interferon-inducing agents

133

HETEROCYCLIC COMPOUNDS

138 138 139 141 142 143

3,4-Dihydroisoquinolines Benzimidazoles Thiosemicarbazones of heterocyclic ketones Triazinoindoles Other heterocyclic compounds

134 135 136 136

PURINES AND PYRIMIDINES

147

AMIDINES AND GUANIDINES

151

AROMATIC COMPOUNDS Phenylglyoxal Miscellaneous aromatic compounds

153 153 154

ALIPHATIC COMPOUNDS

155

PEPTIDES AND AMINO ACIDS

156

NATURAL PRODUCTS Gliotoxin and its analogues Rifampicin Daunomycin and other antibiotics

157 158 159 160

CONCLUSION

160

ADDENDUM

161

REFERENCES

162

119

120

ANTIVIRAL AGENTS

INTRODUCTION The present state of the art of antiviral chemotherapy is similar in many ways to that of antibacterial chemotherapy in the late 1930s with only one or two drugs in current use and a great deal of research being done to find more effective agents. Will the antiviral equivalent of penicillin be found in the next few years or has it already been discovered in interferon? The problems to be overcome are great, not least the primary one of attacking selectively an intracellular parasite which takes over the cell’s metabolic processes in order to reproduce itself. A virus is so much simpler in its constitution than a bacterium that many of the points of attack on the latter are just not possible with the former. Thus a virus lacks a muramic acid type cell wall and the enzymes associated with such a structure, and consists only of a single nucleic acid molecule either ribonucleic (RNA) or deoxyribonucleic (DNA), protected from external attack by a protein coat. Associated with the protein coat may be lipid material and in some cases enzymic material. Only the nucleic acid is infectious but the antigenic coat of protein must determine the site of attack of the virus within the host. Our knowledge of the physical and biochemical phenomena which take place once the attack has begun is by no means complete although considerable progress has been made in the last few years. Suffice it to say that the end-product may be several hundreds to several thousand new viruses per cell and the death of the cell. This in turn releases cellular contents into the environment; it is thought that allergic type reactions from these, and loss of cell function give rise to some of the symptoms of the disease. In order to cover all the possibilities of influencing a virus infection or its sequelae, the term ‘antiviral agent’ has been defined in very broad terms [l] as ‘a substance other than a virus, a virus-containing vaccine or specific antibody, which can produce either a protective or therapeutic effect to the clear, detectable advantage of the virus infected host. Any material that can significantly enhance antibody formation, improve antibody activity, improve non-specific resistance, speed convalescence or depress symptoms would also be considered an antiviral agent despite the fact that such an agent has no direct action on the invasion, synthesis or migration of the virus’. Most workers in the field would restrict themselves to the first sentence of this definition (as in this review) but they would not deny that drugs which would enhance any of the five factors in the second sentence may be very useful in the therapy of virus diseases. Having defined ‘antiviral agent’ one should emphasise the specificity aspect of its activity-its effect must be to the clear, detectable advantage of the virus-infected host, be it a tissue culture cell or a living animal. So many authors appear to ignore this essential fact in claiming activity for their compounds. If a compound is toxic to a cell culture, then the metabolic processes of the culture are no longer functioning properly and the virus

D. L. SWALLOW .121 cannot use them as efficiently as it normally would for replicating itself. Hence its growth appears to be inhibited, although the compound has no specific effect on the virus. Phrases like ‘X has high antiviral activity accompanied by high cytotoxicity’ have been much too common in the literature under review, and even descriptions like ‘Compound X was tested at the highest dose which did not cause cytotoxicity and was found to have good antiviral activity’ are almost meaningless. It should be established as a criterion for activity of a compound in an in vitro test system that there should be a ratio of at least four between the minimum toxic dose and the lowest active dose. This should give a sufficiently meaningful result until further rigorous assessment of toxicity can be made, for example, by growing the cells in the presence of the drug for several days and finding the maximum non-toxic concentration. The commonly used practice of looking for druginduced cytopathic effect can be misleading since a compound which stops cellular RNA and DNA synthesis simultaneously may leave cells looking undamaged. In animal test systems, the assessment of how local cellular or overall toxicity of a drug affects virus growth is even more difficult, but here again a ratio of about four between the lowest dose causing overt toxicity and the lowest active dose would be a useful starting point for further investigation. Having stated these criteria it is difficult to put them into practice in the present review as such a large amount of material would have to be omitted either because no toxicities are mentioned, for example, in patent claims or because compounds have been claimed active when tested at or near or even above a quoted toxic dose. Some idea will be given of the wide variety of chemical types which have been claimed as active with structure-activity relationships, where reported ; the validity of the results with respect to the question of toxicity will be indicated. Some of the more blatant claims, particularly in the patent literature where one sometimes sees five or six types of activity claimed for the same compound have been omitted. The survey covers a wide range of literature from about 1964 to July 1970. During this time several general reviews of antiviral chemotherapy have appeared 12-14]. More specialised reviews have appeared on controlling viral infections by means of vaccines [ 151 and interferons [ 16221, the mechanism of viral multiplication and mode of action of antiviral agents [23], potential antiviral agents having lipophilic characteristics [24], the pharmacology of viruses (sites of inhibition by antiviral agents) [25], the prevention and treatment of influenza [26] and the evaluation of antiviral drugs in volunteers [27]. Screening of compounds for antiviral activity is carried out by a large variety of methods. Some of these have been collated [28] and progress over the last few years can be assessed by comparison with a more recent collection of papers [29]. This review is divided on the basis of broad chemical types, with subdivisions for smaller groups or individual compounds within the main class.

122

ANTIVIRAL AGENTS

ALICYCLIC COMPOUNDS ADAMANTANE DERIVATIVES

There has been frenzied interest in these, until recently, rare compounds. The origins of adamantane (I), its synthesis and chemistry have been fully reviewed [30].

I -Aminoadamantane This compound had been known [31] for some time before its antiviral properties were reported. Its activity in tissue culture against various strains of influenza, activity against influenza in mice and its lack of toxicity [32, 331 led to quite early clinical studies in volunteers [34] in which it was shown that the incidence of influenza in susceptible persons was reduced by 60 per cent by treatment before, during and subsequent to virus challenge with 100 mg twice daily of 1-aminoadamantane hydrochloride (amantadine, 1-adamantanamine, Symmetrel). Many subsequent clinical trials were carried out both in natural epidemic infections of influenza A2 [for example 35-37] and with controlled virus challenge of attenuated strains [for example 38,391. These confirmed that the drug used prophylatically reduced considerably the possibility of infection, although it did not eliminate it completely. Studies were also carried out on the possible therapeutic effects of amantadine, treatment with 100 mg twice daily being started about 20 hours after the volunteers felt the first signs of illness [40]. There was a significant increase in the number of drug-treated patients over placebo in those whose infection resolved rapidly, whereas placebo-treated patients predominated in the ‘slow resolver’ group. Toxicological studies [41] confirmed the very bland nature of amantadine. Side effects of dosage at 200 mg/day, particularly if taken in two 100 mg doses were very slight, the most common complaint being wakefulness. In a study involving thousands of both drug and placebo-treated patients [42] including children, adults and aged persons, the average incidence of all complaints was 11 per cent in drug-treated and 8 per cent in placebo-treated. Complaints specifically attributable to central nervous system effects averaged 1.5 per cent in drug treated and 0.7 per cent in placebo. One very interesting side-effect discovered after the compound was marketed in January 1967 was its beneficial effect in Parkinson’s disease [43, 441.

D. L . SWALLOW 123 Mode of action studies on 1-aminoadamantane indicated that it prevented an early stage in the virus attack on the cell [45]. Detailed work showed that absorption of virus on to cell surfaces was apparently unaffected, but that penetration of the virus into the cell was blocked [46]. More recent studies [47] showed that it is the stage of ‘uncoating’ of the virus particle that is prevented. One unusual result obtained with this drug was that it appeared to aggravate an influenza infection in ferrets [48]. However, as the PR8 strain of influenza used is not particularly susceptible to adamantanamine and as the drug was dosed at very nearly a toxic level, the result is somewhat suspect. 1 -Aminoadamantane is thus an antiviral agent of very considerable merit and will no doubt continue to be vigorously investigated until it is either accepted, replaced by a better compound or until a new strain of influenza completely insensitive to the drug emerges.

a-Methyl-I-adamantane methylamine hydrochloride (Rimantadine) Me 1-Ad-dH-NG3el This compound from the same ‘stable’ as amantadine is reported to be more active in vitro against influenza A2 than its predecessor and also inhibits rubella, rubeola, respiratory syncytial and parainfluenza viruses in vitro [49]. It was active in mice and also in ferrets against influenza strains and as it showed a lack of toxicity similar to that of adamantanamine it was soon tested in man. In a double-blind trial in 55 volunteers, it was shown to reduce the occurrence and severity of artificially induced influenza A2 when dosed before, during and after infection [50].In a naturally occurring influenza A2 outbreak, it had some therapeutic effect when dosed when symptoms were first observed [36]. Rimantadine provided significant protection to mice from lethal infections of influenza A2 when treatment was begun at the time of appearance of the first symptoms which were from 2 4 days after infection [51]. Thus it appears to have a real curative effect in this species, but whether this is solely an antiviral effect or an antiviral effect coupled with an effect on the allergic-type lung tissue reaction and consolidation which is the main cause of death in influenza-infected mice, is not yet known. The compound also has in vitro activity against Rous and Esh sarcoma viruses at 7.4 and 5.8 pg/ml respectively, doses which are well below the 4&50 pg/ml toxic to the chick embryo fibroblast cells used in the test. It does not cause inactivation of the viruses extracellularly at 50 pg/ml[52], and so has a specific effect on these tumour-forming RNA viruses. More will be heard about this wide spectrum antiviral agent in the future.

124

ANTIVIRAL AGENTS

Other Adamantane Derivatives

Almost every conceivable variation on nitrogen-containing adamantane molecules has been claimed in the patent literature to have antiviral activity against influenza strains and related RNA viruses but occasionally DNA viruses are included in the spectrum of activity. It is difficult to assess the validity of these claims unless a scientific paper is published and even then, a compound claimed active at 50 or 100 pg/ml is not really significant compared with AdNH2 active at 1-2 p g / d [33]. In a small series of 1-substituted adamantanes, it was found that good activity against influenza A2 was present when the compound could ionise, for example AdNH2, AdCH2NHz, AdCOOH, but that activity was lost when ionisation could not occur, for example AdNHCOCH3, AdCONHz [53]. Neither of these findings may be true for more complex adamantanes. The antiviral spectrum of a series of 1-alkylaminoadamantanes has been reported [54],and the antiviral activity against influenza A in vitro and A2 in vivo of some mono- and di-substituted adamantanes has been examined [55]. In this paper, it was shown that 1-adamantylurea, 1-adamantylguanidine and 1-adamantylbiguanide at pg/ml all produced about the same reduction of virus growth in vitro as 1-aminoadamantane at the same concentration. In mice, all three compounds were active but less so than l-aminoadamantane. The guanidine and biguanide had undesirable side effects. Further substitution on the urea and guanidine nitrogens led to elimination of activity [56]. The di-substituted compounds were of structure (11) where R = -NH2 -COOH, -OH, -Br, and -Me. R = H was used as a positive marker and all drugs were tested in v i m at 1 pg/ml. R

When R = NH2 or COOH the compounds were completely inactive and very nearly so when R = OH, but R = Br and Me were equiactive with R = H. The same pattern was evident in the in vivo tests in mice. It was suggested that the adamantane moiety of (11, R = H) is absorbed by the phospholipid layer of the cell to be protected leaving the polar grouping protruding. The presence of another, presumably hydrated, polar grouping in the molecule prevents this absorption and produces the unfavourable effect on antiviral activity. A brief paper on 1-adamantylthioureas substituted on the terminal

D. L. SWALLOW 125 nitrogen [57l claimed (without details) increased survival time of mice infected with influenza A2 when the substituent was Me and Ph but not with H, Et, Pr, Pr' and Bu. Other 1-substituted adamantanes which have been claimed as antiviral agents in the patent literature are the following:

R

Reference

NH.NH2

1581

CO-CHO CO*FH* NHPh OR' R\H,ALkyl O.CH2.COzH

I591 1601

I611

2-Substituted adamantanes have recently been claimed as antiviral agents. These have become more accessible through the elegant oxidation/isomerisa: tion of adamantane or 1-hydroxyadamantane with 96 per cent sulphuric acid to give 2-adamantanone (111). 2-Aminoadamantane (IV, R' = N H 2 ; R2 = H) has been claimed active against all strains of influenza [63] and its methylamino analogue (IV, R' = NHMe; R2 = H) is also said to be active [64].

Further substitution has led to (IV, R' = CH2NHR3 where R3 = H, Me, Et; R2 = OH or NH2) [65] and thence to the spiro-compound (V) in which R = H, Me, Et, Pr or -CH2C=CH. These compounds were claimed to have very powerful antiviral activity against influenza viruses [66]. More recently the N-alkyl series have been reported to be active in vitro and in vivo against influenza A2/Japan, A2/Hong Kong and Sendai virus and in vitro against Coxsackie A21 and two strains of rhinovirus. The N-methyl analogue was said to be significantly more active than 1-aminoadamantane against influenza viruses [67]. Some more complex adamantanes appear to have different antiviral spectra from the simpler ones. Compound (VI) is claimed to be active in mammals against viruses causing encephalitis or encephalomyelitis [68] while (VII) is active against herpes virus in mice and tobacco mosaic virus in plants [69].

126

ANTIVIRAL AGENTS

YMe

b2

NCONH2 Me QMe

OTHER MULTICYCLIC COMPOUNDS

The amino- or alkylamino-derivatives of almost every tri-, tetra- and pentacyclic octane, nonane, decane and undecane have been claimed in the patent literature to have activity against influenza virus. Little or no details are given about the activity or toxicity of these compounds, but their synthesis often represents a great deal of chemical skill and ingenuity. For those interested in the latter topic, a list of the structural types is appended. Reference

Structural Type

Pentacyclo-octane (cubane) Pentacyclo-nonane (homocubane) Pentacyclo-decane (bishomo-cubane) Pentacyclo[6.2.1.0237.04,10.0s*9]undecane Pentacycl0[6.2.0.0~*~ .04,'.Os,']decane Tetracycl0[5.3.0.O~*~.O~~~]decane Tetracyclo[5.2.0.02~6.04~9]nonane

1721

Tetracyclo [4.3.0.02*4.03 s7]nonane Tricyclo[S.2.2.02v4]nonane

1 1

Tricyclo[4.3.1.03v8]decane Tricycl0[4.3.0.0~~~]nonane Tricyclo[3.3.1.03-7]nonane Tricyclo[3.3.0.03.7]octane Tricyclo[4.3.1.13,8]undecane Bicyclic compounds

As in the previous section, the majority of amino and alkylamino bicycloheptanes, -octanes, -nonanes and -decanes have been claimed to have activity

D. L. SWALLOW I27 against influenza but in most cases without any detail being supplied. 'Two exceptions are the bicyclo[2.2.2]octanes and bicyclo[2.2.l]heptanes. 49 Amines of types (VIII), (IX) and (X) were tested for activity in mice against influenza A/swine/S. 15. [78]

I

NR' R~

The test, based on the daily mortality of the mice, was designed to give a quantitative comparison of the antiviral activity of a series of compounds tested at different times. The results were expressed as an Antiviral Index 5O(AVI,,), i.e. that dose, in milligrammes per kilogramme body weight which allowed only a half-log,, (3-2-fold) increase in the infectivity of an intranasal20 LD,, dose of virus. The compounds were dosed intraperitoneally 30 minutes before infection. On this basis 1-aminoadamantane, which was tested earlier in this way, had an AVISOof 4.6 mg/kg. Only alkyl groups were investigated as substituents in these molecules. All three types had very similar structure activity relationships in that R = Me conferred highest activity and changing it to H or a larger alkyl decreased activity. The corresponding CF3 analogues were inactive (AVISo>32 mg/kg). Taking the simplest cases in the three types where R = Me and other substituents were hydrogen the following AVISOwere recorded : Compound type (VIII) n Compound type

=

0

n = l

2.8

7.9

Compound type (IX)

2.2

Compound type (X)

3.1

Substitution on the amino groups of types (IX) ant- (X) wit.. one methyl group retains the activity of the unsubstituted molecule but two methyl groups reduce activity markedly. Higher alkyls than methyl abolish activity. Substitution on the methylene of type (X) with 2 = Me, Y = H or 2 = Y = Me enhances activity considerably and one of these compounds (XI) is the most active recorded in the paper with an AVIS00.5mg/kg.

128

ANTIVIRAL AGENTS

Me

There was no mention of the toxicity or the possible mode of action of these compounds in the paper, but one might expect them to be non-toxic a t the low AV150doses quoted. The examples of bicyclo[2.2.llheptanes are 32 racemic endo-2-aminobornanes (XII) substituted on the nitrogen with alkyl, benzyl and substituted benzyl [79].

These and 10 N-acyl compounds were tested against influenza A2 in mice. Compounds were dosed either orally, subcutaneously or intraperitoneally 1 hour before and 1, 5, 24, 30, 48 and 72 hours after intranasal infection with 100 LD50 of virus. Each dose was about 1/10 LDlo of drug. All control mice died in &5 days and results were expressed as percentage number of survivors on days 6, 10 and 21 after infection. Although the authors claim that no compound was more active than the simple racemic amine hydrochloride (XII, R' = RZ = H) or its (+)-enantiomer comparison is made difficult by the fact that different compounds are dosed at either 5, 10,25, 50 or 125 mg/kg no doubt depending on the LDlo values for that compound, and a few by routes other than orally. For example, the survival rate with three compounds over the 6,lO and 21 days periods is practically identical (XII, R' = R2 = H), (XII, R' = Me, RZ = H), and (XII, R' = RZ = Me) but the first is dosed orally a t 50 mg/kg and the other two orally at 10 mg/kg. Taking compounds at equal dose levels of 50 mg/kg the order of activity is shown in Table 4.1.

129

D. L. SWALLOW

Table 4.1

ANTI-INFLUENZA VIRUS ACTIVITY IN MICE OF SALTS OF ENW-2-AMINOBORNANE .AND ITS DERIVATIVES DOSED ORALLY AT 50 mg/kg [79]

Activily as percentage survivors at day:

R'

RZ

X

6

10

21

H racemate

H

c1

84

44

22

H (+)-isomer

H

AcO

82

48

34

racemate

H

c1

78

46

33

racemate ~ F

H

CI

57

31

20

- c H , o c t racemate

H

c1

50

30

14

- c t + - c F , racemate

H

Br

42

34

29

H

Br

42

22

10

H

CI

36

29

16

H

c1

24

9

3

H

c1

13

8

8

H

c1

10

4

1

- w,-@

-C

I=\

H

~

+

-CH,

U1

-CY

\ / -0-

\

CWMeracemate No,

- c y e a r . - c b o i

racemate

racemate racemate

The effect of interposing a second methylene between the aromatic ring and the nitrogen was to reduce activity considerably. Addition of a third methylene in the p-fluorophenyl series (dosed at 10 mg/kg) gave an inactive compound. Activity decreased sharply as the size of the halogen atom increased in thep-halogenobenzyl derivatives (p-Br at 125 mg/kg gave slightly lower figures that p-CI at 50 mg/kg). A second halogen atom ortho to the methylene gave an almost inactive compound. All the N-acyl derivatives were of low or zero activity. A small group of guanidinoalkyl-aminobornanes of type (XIII) where R = H, Me and n = 2, 3 have been reported to have weak activity against influenza virus [80]. High activity has been claimed (without details) for endo-(~-2-bornane)methylamines of type (XIV) [8 11.

130 ANTIVIRAL AGENTS Methano- and ethano-hexahydroindanes (XV) and (XVI) respectively with an amino or aminoalkyl group at various positions in the molecule are said to be active in mice against influenza A and A2 [82, 831. In a specific example the 1-amino derivative of (XV) gave a 0.5 log reduction of infectivity of influenza A2 at 12 mg/kg in mice. Amino derivatives of other bicyclic systems claimed to have antiviral activity are :

System

Reference

Bicyclo[3.2. lloctane

~ 4 1

Bicyclo[3.2.2]nonane

~ 5 1

Bicyclot3.3.llnonane

1861

Bicyclo[3.3.2]decane

~ 7 1

Bicyclo[6.1.O]nonane

[881

Decalin derivatives

A variety of structures based on the trans-decalin nucleus have recently been reported to have activity against influenza A (strain DSP) in tissue culture [89]. The cellular toxicity, based on the principle that healthy cells take up the dye neutral red, whereas unhealthy or dead cells take up less or none, was generally very low, whereas some activities determined by quantitative haemadsorption [90] were extremely high. Three types of active compound (XVII-XIX) were described. R'

Y

(XVII]

(XVIII)

(XIXI

D. L. SWALLOW 131 Vinyl ethers of type (XVII) gave a 50 per cent end-point of the assay at about 2 pg/ml when R' = H and R2 = lower alkyl, but toxicity increased as the size of the alkyl group increased from 400 pg/ml when R2 = H to 50 pg/ml when R2 = Bu. Specificity for the configuration of R' and RZwas shown by the amide derivative. When R' = H and R2 = CONHz activity was 0.06 pg/ml and toxicity 400 pg/ml, but when R' = CONH2 and R2 = H the compound was almost inactive. Reduction of the active amide to the aminomethyl compound also caused loss of activity. The vinyl ethers (XVII) were readily converted to type (XVIII) by a variety of reagents. Weak acids gave (XVIII, X = 0, Y = B-OH), while strong acids ( H + A-) gave X = 0, Y = u-A. Alcohols (R'OH) in the presence of HCl gave X = 0, Y = u-OR'. None of these compounds were of great interest although all were active, but if (XVII) was treated with hydroxylamine or 0-alkylhydroxylamines a highly active series was obtained (XVIII, X = NOR', R' = H or alkyl; Y = & O H ) . In the simple oximes both activity (0-2 pg/ml) and toxicity (400 pg/ml) remained almost constant when R was varied, but if either R or R' = Me and the other alkyl was varied then both activity and toxicity increased with the size of the alkyl group, maintaining a ratio of about 600 between toxic and active doses. The trans-4a-decalyl amides (XIX) were the most thoroughly investigated group since the highest activities were found in this type. Unlike the alicyclic amines so far reported trans-4a-decalylamine was completely inactive, but as the size of the amide group increased so did the activity. Some indication of the relation between structure and activity in this series can be gained from Table 4.2, which contains a selection of the 110 compounds examined.

Table 4.2 STRUCTIJRE AND ACTIVITYAGAINST INFLUENZAA OF ~ ~ ~ ~ S - ~ A - U E C AAMIDES LYL CULTURE

I N TISSUE

(89)

IXlXl

R

H COMe COCH,CI COEt COPh COCH,Ph COCH,-@I COCH,

-

a

Activity

Toxicity

Pg/ml

Pg/ml

200 12.5 1.9 0.6 2 0.05

>200 200 25 200 250 200

0.0 1

200

100

>I00

132

ANTIVIRAL AGENTS

R

Activity m/ml

0.03 0,003

>200

0,004

>200

0.07 0.29

>250

0.13

50

COCH2-N

0.08

200

COCH’NPh

0.008

COCH,OPh COCHiSPh COCH’S

0

Cl

COCH2CH2Ph COCH2CH2CHZPh

cocH28 w T

250

>loo

< 12.5 12.5 50 200 200

0.04 0.5 0.02 5 -0.8

COCHPh COCHPh2 COCF’Ph CONHPh CH2CHzPh

< 16

Highest activity was associated with an aromatic ring separated from the CO of the amide function by one or two groups. The first group must be a methylene or substituted methylene and the second can be CH2 or preferably 0, S , N-Me. When the first CH2 was absent, activity was very low, and when the aromatic ring was fully reduced the product was inactive. All the cisdecalin analogues were of very low or zero activity. Mode of action studies indicated that the compounds prevented the virus from entering the cell, but not absorption of the virus on to the cell surface. Tested in vivo in mice, the compounds had very low activity, probably due to very rapid metabolism or inactivation which in turn led to extremely low blood levels only a short time after dosing. MONOCYCLIC COMPOUNDS

Cyclo-octylamine hydrochloride (XX) and its N,N-dimethyl derivative have been claimed to have activity against both RNA and DNA viruses [91, 921. ~

~

N

Y

H

C

,

D. L. SWALLOW 133 The maximum concentration permitting optimal growth of chick embryo cells for 5 serial passages was 50 pg/ml. The compound was used at this concentration in haemagglutination inhibition tests against myxoviruses (influenza and parainfluenza) but was tested at 100 pg/ml in plaque inhibition tests against polio and vaccinia viruses. It was active at these concentrations against 21 viral strains. Tested against influenza A2 in mice using an oral !+dose regimen (3 and 0.25 hours before infection and 3,24,30,48, 54,72,78 hours after) the compound increased survival rate by 3-6 days at 25 mg/kg per dose. Doses of 50 and 100 mg/kg gave greater protection and were well tolerated; 200 mg/kg dosed in the above schedule produced 20 per cent mortality. The above results in mice were confirmed by similar tests against influenza in ferrets which were dosed at 50 mg/kg subcutaneously [93]. This seems to be an interesting compound but is less active at equivalent doses than 1-aminoadamantane. Two other monocyclic amine types (XXI, XXII) have been claimed to have anti-influenza activity but no details are available [94,95].

NHz

I

Me-C-R

R=H,Me

INTERFERONS AND INTERFERON INDUCERS To cover adequately the advances made in the study of that apparently ideal, broad-spectrum antiviral agent interferon would require a whole review in itself. The topic has very recently been fully reviewed [96] and the reader is referred to this and to the earlier references to interferon [ 16-22]. Since the interferons have characteristic specificities, human interferon would have to be used for treating virus infections of humans. Crude human interferon has been prepared from human leucocytes infected with Sendai virus, but attempts are being made to establish human cell lines which can be grown in very large amounts and at the same time yield a high titre of interferon when infected [97]. The leucocyte interferon is particularly difficult to purify but sufficient progress has been made to enable some of its properties to be described [98].Aspects of the safety testing of this product have been described [99] and a small trial of the interferon has recently been carried out in man against a rhinovirus infection [loo]. This was inconclusive and further work awaits a new batch of interferon.

134 ANTIVIRAL AGENTS It is thought by some workers that the answer to the problems of human interferon production lies in its induction when required in the human subject, and in addition to viruses a number of more or less ‘chemical’agents have been found which will do this. On the whole they do not produce high titres of interferon in humans, but they will do so in cell cultures and in animals. The following are the main groups of current interest. DOUBLE-STRANDED

‘RNA’

It is thought that interferon production in a virus infected cell is triggered off by the cell’s recognition of the replicative form of the viral nucleic acid, which at some stage must exist in a double-stranded conformation. This conclusion arose from the testing of a number of ‘foreign’ double-stranded RNAs for inducing ability [loll. The most widely investigated is the poIyinosinic acid poly-cytidylic acid complex (poly I : C) probably on account of its commercial availability and its powerful antiviral properties in cell systems and animals. The physical properties of a standardised preparation of poly I:C have been reported [loll and its molecular and crystal structure investigated [ 1021. The antiviral activity of this and several other doublestranded synthetic polyribonucleotides is said to depend largely on their thermal stability [103], given a sufficiently large (>3000) molecular weight. The interferon-inducing ability of known chain length oligonucleotides of cytidylic and inosinic acids, complexed with either poly-I or poly-C or with each other, falls off rapidly as the chain length of the oligomer is lowered below 35-40 units [104]. The interferon-inducing ability of poly I :C was first demonstrated in cell systems in 1968 [lo51 and this was shortly followed by details of its curative effects on a herpes virus infection of a rabbit’s eye [106], and on various virus infections of animals [107]. Since then there have been a large number of papers on its antiviral activity in vitro and in vivo. Human cell cultures appear to be protected against the common respiratory viruses at doses of poly I :C ( 0 1-1.0 pg/ml) which are much lower than those required to induce detectable interferon (50 pg/ml) [log]. This and similar observations pose difficult questions as to the mode of action of poly 1:C. It has been suggested that small doses of the compound result in the synthesis of a ‘pre-interferon’ which can produce an antiviral state without detectable free interferon. This pre-interferon can be converted to detectable interferon by antimetabolites, endotoxin or by larger doses of poly I :C itself [ 1091. The high toxicity of poly I:C in animals was soon apparent. In mice the LDS0value was 500 pg/mouse and they were ill at 100 pg. Both in these values and in the potentiation of toxicity by the presence of a small amount of lead acetate, poly I :C was similar to the bacterial endotoxin of E. coli [110]. In rabbits as little as 0.5 pg/kg intravenously produced a significant pyrogenic

D. L. SWALLOW 135 reaction. Administration intranasally produced fever as did also endotoxin. A dose of 5 pg/kg produced a high level of circulating interferon with the same time course as the degree of fever. These effects were not seen with poly I or poly C alone and appear to be due to the double-strandedness of the complex [l 113. At 2.0 mg/kg in rabbits it was 100 per cent embryotoxic dosed on days 8 and 9 of pregnancy and 80 per cent embryotoxic at 1.0 mg/kg [1121. A variety of double-stranded molecules (poly I : C, double-stranded RNA from two sources and Reovirus RNA) showed similar toxic effects in mice and rats, which included atrophy of the thymus gland, transformation of lymphocytes and runting of baby mice dosed at birth. Two primate species treated with poly I :C did not show these effects but neither was circulating interferon detected [113]. In spite of these adverse effects in animals, cautious trials have been carried out in humans and protection against rhinovirus infections demonstrated at 0.1 mg/kg per day intranasally for 6 days [114]. When dosed intravenously at 0.3, 1.0 and 2.5 mg/m2 per day on day 1 and then on days 8-35, the lowest dose produced no circulating interferon and no side effects, but both were present at the higher doses. Fever seemed to be the commonest side effect, but the authors concluded that a dose of 1.0 mg/m2 per day is relatively safe [115]. In another report, single doses of from 2 pg/kg to 300 pg/kg intravenously had been given to human patients with no adverse effects other than reproducible fever in one patient. Circulating interferon was detectable in 2 patients but not in 2 others [ 1 161. Thus poly I : C is a toxic but efficient inducer of an antiviral state. It is effective in animal models against such serious human infections as rabies [I 171 and Japanese B encephalitis [118] and could possibly be used to treat such life-threatening infections in humans, but for general use in minor infections it appears at the present time to be far too toxic. Other double-stranded molecules have been examined and found to show inducing and toxic effects [ 1 191. The polyadenylic-polyuridylicacid complex (poly A:U) is a much weaker inducer, and less toxic, than poly I:C but it is much less thermally stable and more readily degraded by enzymes. Substitution of the phosphate groups by thiophosphate gave a more stable complex and increased its ability to stimulate interferon production [ 1201. Some single-stranded RNAs, for example poly I, poly C, yeast RNA when in the presence of relatively large amounts of polybasic substances such as diethylaminoethyl dextran, poly-~-lysine,protamine, histone or methylated albumin, could induce interferon production in cells, but much higher concentrations of RNA were required than of the double-stranded variety [121, 1221. There is some recent evidence that a single-stranded virus RNA will induce interferon even without potentiation [ 1231.

136

ANTIVIRAL AGENTS

STATOLON

The active entity of this interferon-inducing extract from Penicillium stoloniferum has been finally identified as a double-stranded RNA, probably from a virus infection of the fungus [124, 1251, so it is no longer a unique antiviral agent. The same applies to helenine, the antiviral extract from Penicillium funiculosum [1261. PYRAN CO-POLYMER

(Maleic acid-divinylether co-polymer)

r

1

(XXIII)

This purely synthetic polymer was an antitumour agent which was found to stimulate high levels of interferon in animals [127] and in man [128]. Structure (XXIII) for the repeating unit is probably correct being derived from several obviously incorrect descriptions in the literature [129, 1301. Interferon induction could not be demonstrated in vitro with pyran copolymer, but only in intact animals. Similar effects were shown to occur with a large variety of polyanionic polymers based on maleic acid or acrylic acid [ 1291. Variation of inducing ability with molecular weight of these polymers showed that optimum size was 17 00&34 OOO [ 1311. Although Pyran co-polymer suppressed both ordinary and oncogenic virus infections in animals it produced a number of serious side effects. In man, at intravenous doses of 15-16 mg/kg per day for 8 days it produced fever concurrently with circulating interferon on days 1-4 but not thereafter, but at all doses above 8 mg/kg (which did not produce detectable interferon) it produced thrombocytopenia and was deposited as particulate material chiefly in the spleen but also in blood cells, liver and bone marrow [128]. However, it has recently been claimed ‘from experience in mice and in over 60 human patients to be less toxic than poly I: C’ [132]. OTHER INTERFERON-INDUCING AGENTS

Phagicin, from A-phage infected E. coli K-12 [133] and an extract from Huemophilus influenza type B [ 1341 are possibly double-stranded RNAs, and may therefore act by interferon induction. The bacterium Brucella abortus which causes abortion in cattle and relapsing fever in humans, is a potent inducer of interferon in mice. Both live and heat-killed bacteria were equally effective indicating that replication of the

D. L. SWALLOW 137 organism was not a prerequisite for activity. A single intravenous injection of organisms protected mice for more than a week against vaccinia virus. The active principle has not been identified but it is probably neither an RNA nor an endotoxin. It is destroyed by organic solvent extraction of the organisms and is regenerated to varying degrees on combining the aqueous residue and organic extract. Thus it could be a lipopolysaccharide. It is not antigenic and a second dose in the same animal still produces circulating interferon [135]. Antivirin, an unidentified material isolated from both established and primary cell lines grown in the absence of interferon-inducing factors is said to inhibit both RNA and DNA viruses [136]. The material is stable to trypsin, periodate oxidation and to heating for 1 hour at 100°C. It is a little odd that this material comes from cells which presumably will support viral growth, but the original paper is in Japanese and the abstract is uninformative. Polyacrylic acid and polymethacrylic acid, which belong to the same class as pyran copolymer, will protect newborn mice from a lethal infection with vesicular stomatitis virus [ 1371. Their mode of action in vitro [1381 and in vivo [ 1391 has been described. Polyvinyl sulphate will also induce interferon in mice but not in in vitro systems [140]. Some properties of these two compounds have been compared with pyran copolymer [ 1291. Polyacetal carboxylic acids are a new group of antiviral poly-anions which have interferon-inducing properties [ 1411. They are produced from suitable long chain polymeric sugars such as amylose and starch which contain 2,3-cis-diol configurations in each sugar residue. This is first oxidised with periodate to split the 2,3 bond and give a dialdehyde, which in the second stage is oxidised with sodium chlorite to a dicarboxylic acid. The 1,6-linked sugar backbone remains intact and a poly-carboxylate polymer is obtained. This will induce interferon in animals but somewhat higher doses than pyran copolymer are required. It is much less toxic than pyran and this is probably due to its biodegradability. The antiviral activity in mice of the most active compound, derived from amylose, is typical of an interferon inducer [142]. The most recent and in some ways the most remarkable interferon inducer is not a polymeric substance but a simple organic molecule 2,7-bis(2diethylaminoethoxy)fluoren-9-onedihydrochloride (XXIV). 0

EtzNC H z C H z O m O C H z C H ?NE t 2

I

I

2HCL

(XXIV )

It is claimed to induce interferon in mice on oral administration (all other inducers have to be given parenterally) [143] and to produce broad spectrum

138 ANTIVIRAL AGENTS antiviral activity comparable to statolon [144]. No further details are a t present available.

HETEROCYCLIC COMPOUNDS

Two types of compound (XXV) and (XXVI) have been of interest in this series.

CH2 CON HZ

(XXV)

( XXVI )

Compounds of type (XXV) were first investigated as inhibitors of viral and bacterial neuraminidase and activity was found to depend more on steric factors than electronic. Compound (XXV, R = p-C1) inhibited influenza A2 neuraminidase by 38 per cent at 500 pg/ml and was thought to be antiviral because of this [145]. Twenty-two compounds related to (XXV) were compared for their anti-neuraminidase activity and severe steric limitations for binding on the hypothetical receptor site on the enzyme were found [146]. The preferred conformation was where the phenyl and isoquinoline rings were approximately co-planar and the oxygen and nitrogen atoms were cisoid. The corresponding tetrahydro- and fully aromatic isoquinolines were inactive as were N-substituted compounds. Changes in the l-phenoxymethyl substituent which removed activity were substitution on the methylene, replacement of 0 by CH2 and bis-ortho substitution on the phenyl ring. A single ortho-methyl substituent allowed retention of activity as did 2,5-dimethyl, but the 2,3-dimethyl analogue was of low activity. Surprisingly, replacement of the-CH,O-by trans-C=C allowed retention of activity. These results have been submitted to Hansch analysis and a highly significant relationship between inhibition and the hydrophobicity constants for the molecules has been found [ 1471. Reports of antiviral activity have been mainly confined to (XXV), R = p -C1 and R = p-OMe). The compounds were active in vitro only when pre-incubated with virus and have no effect on the replicative cycle of influenza, parainfluenza, measles, herpes and Newcastle disease viruses. There was however, some inhibition of growth of ECHO, rhinovirus, rubella and respiratory syncytial virus when the compounds were added

D. L. SWALLOW 139 together with virus to the cells [145]. The effect of contact time and temperature of incubation for viral inactivation have been investigated [ 1481 as has the nature of the interaction between drug and virus [149]. The compounds are relatively free from toxicity and have been tested in human volunteers at doses of 1-5 g/day for 7 days without side effects. Compound (XXV, R = p-OMe) was partially successful in preventing infections in man with influenza A2 and influenza B, but (XXV, R = p-C1) was inactive against influenza B [150]. A positive result was obtained with the latter compound and virus in another trial although this result was based on a reduction of symptoms and there was no difference in serological response [ 1511. Neither of the two compounds showed significant activity against a rhinovirus infection in human volunteers although (XXV, R = p-C1) was active in v i m against several rhinovirus strains [ 152, 1531. The other compound in this group, l-carbamoylmethyi-3,4-dihydroisoquinolinium chloride (1-dihydroisoquinolineacetamide hydrochloride, DIQA, XXVI) is claimed to be active in vivo but not in vitro against two strains of influenza, ECHO 9, Columbia S.K. and herpes viruses. In mice only the dose (100 mg/kg intraperitoneally) given simultaneously with virus was effective in decreasing mortality. The LDS0 value was 375 mg/kg by the same route and the compound also had a hypnotic effect on the animals with a HD50 value of 100 mg/kg [154]. However, there was no hypnotic effect in monkeys at 300 mg/kg and none in humans with a single dose of 4 g. It has been suggested that the antiviral effect is due to delayed penetration of virus into the cells [155]. Recently some substituted l-(o-aminophenyl)-3,4-dihydroisoquinolines were examined for inhibition of viral neuraminidase, together with some 2-(o-aminophenyl)benzimidazoles.The most potent compounds were tested in mice against influenza PR 8 but were all inactive [ 1561.

BENZIMIDAZOLES

The early studies on the antiviral effects of benzimidazoles by Eggers and Tamm are now regarded as ‘classical’. Since then interest has concentrated on benzimidazole derivatives which have a more selective toxicity towards the virus rather than the overall toxicity shown particularly by 5,6-dichlorol-(f?-D-ribofuranosyl)-benzimidazole(DRB) and its trichloro analogue (TRB) [ 1571. a-Hydroxybenzylbenzimidazole (HBB, XXVII) and its derivatives have been most thoroughly studied since HBB was found to inhibit selectively the replication of several picornaviruses (for example, polio and ECHO) by interfering with viral RNA synthesis [ 1581. In a more up-to-date survey of structural requirements of HBB analogues for selective inhibition of ECHO 6 virus very careful attention was paid to determination of the minimum toxic concentration of drug after both 3 and

140

ANTIVIRAL AGENTS

--Q H

OH

6 days incubation of the cells, as well as determining the concentration which gave 75 per cent protection of the cells from the virus [159]. Of the 42 compounds tested only 14 had a selectivity ratio (toxicity/activity) greater than four at day 3, while only two passed the extremely stringent test at day 6. These were HBB itself and 2-(a-methyl-a-hydroxybenzyl)benzimidazolewith ratios of 5.1 and 8.6 respectively at 6 days. Larger a-substituents than methyl lowered activity and this and other results indicated that decreased acidity of the carbinol function was important for activity. Structure-activity relationships were fully discussed. The effect of N-substitution in HBB has been particularly studied by an English group over several years. These derivatives were highly active against polio virus in tissue culture [160] as were N-substituted a-methoxybenzylbenzimidazoles (MBB) [ 1611. The N-propyl derivative was most active in both series. Seven N-substituted and three other HBB derivatives were tested against a range of other viruses in vitro and high activity was claimed against three Coxsackie strains, three ECHO strains and one rhinovirus strain, although all compounds were tested at half their minimum toxic concentration [ 1621. The rhinovirus strain was remarkably sensitive to these compounds, N-propyl HBB giving complete inhibition of viral CPE at 0.16 pg/ml. Against eleven other strains however, there was only slight activity. One of the few in vivo tests with benzimidazole derivatives is reported in this paper. Mice infected with Coxsackie A9 virus were treated with N-propyl HBB at doses bordering on the toxic level, but only slight activity was demonstrated. A large series of 2-ureidobenzimidazoles of type (XXVIII) together with similar benzthiazoles, benzoxazoles and naphthiazoles have been tested for immunosuppression and activity against Coxsackie A21 virus in mice [163]. A single i.p. dose of drug at 16, 32, 64 or 128 mg/kg was given 3 hours before an i.p. infection with 1 LDloo value of virus.

D. L. SWALLOW 141 Best activity was obtained when RZ = naphthyl, p-fluorophenyl or rn-nitrophenyl. A small substituent at Y, for example Me enhanced, and a large substituent, for example Ph decreased activity from Y = H. Z = H or Me were very similar in activity. Eight out of 122 benzimidazole derivativeswere claimed to have an EDs0 value of about 16 mg/kg but no estimate was given of the toxicity of these compounds.

THIOSEMICARBAZONES OF HETEROCYCLIC KETONES

The pioneering work on isatin-j?-thiosemicarbazoneswhich led to methisazone, an effective drug against smallpox, has occupied the attention of most reviewers [7-10, 1641 and will not be repeated here. Similarly, the analogous 3-methyl-4-bromo-5-formylisothiazole thiosemicarbazone was reviewed fully in the same references. More recent work on methisazone (N-methylisatin-j?-thiosemicarbazone) has extended its antiviral spectrum from purely DNA viruses to certain RNA viruses in tissue culture. These include foot-and-mouth disease, polio, certain rhinoviruses, some arboviruses and influenza A and B. The extent of inhibition is dose dependent and is said not to be due to any toxic effect on the cells in which the viruses are grown [165]. The possibility of using any of this class of compound for treatment of these diseases in man was thought improbable. N-Methylisatin-j?-4,4-dibutylthiosemicarbazone (Busatin) inhibited the growth of all three strains of poliovirus in vitro by 1 0 per cent at 3 p ~ . Maximum inhibition of >99 per cent was produced at 10 p ~ However, . at 20 p~ the compound inhibited cellular growth and DNA synthesiscompletely after 1 hour contact, but RNA synthesis was only slightly affected. Busatin was thought to block a stage in the viral RNA synthesis. All the above effects were reversible on removal of the compound from the medium [166]. in which the acyl function was Several N-acylisatin-8-thiosemicarbazones -CO--(CH2),Me(n = 5, 6, 8, 10, 12, 14 and 16) were tested in vitro against influenza, herpes, vaccinia and rhinovirus [167]. No details of active or toxic doses were given although n = 8 was said to be active against vaccinia and herpes at non-toxic concentrations, n = 10 had slight activity against rhinovirus and n = 12 slight activity against influenza A. N-( Dialkylaminomethy1)isatin-j?-thiosemicarbazoneswere apparently not active against polio and influenza at non-toxic doses [168], and indane-1,3dione bis-alkylthiosemicarbazones claimed to be active against polio were the subject of another very poorly detailed paper [169]. 2-Oximino-1,3-indandione bisthiosemicarbazone had activity at 8 pg/ml in vitro against vaccinia virus but no toxic level was determined [170].

142

ANTIVIRAL AGENTS

TRIAZINOINDOLES

Cyclisation of the isatin-j?-thiosemicarbazones leads to as-triazino[5,6-b] indole-3-thiols (XXIX) in which the thiol is readily displaced by various substituted amines. The use of amino alcohols in this reaction has led to an interesting series of compounds which had activity in tissue culture against several strains of rhinovirus. The most active and least toxic of these (XXX, R = Me) had a selectivity ratio (min. toxic dose/min. active dose) of 5-12 for four different rhinovirus strains [ 1711.

R

(XXXI)

The tetracyclic compound (XXXI, R = Me) prepared by the action of hydrazine on (XXIX, R = Me) followed by acetic anhydride was also active and had selectivity ratios of 4 and 10 against two rhinovirus strains, but was inactive against the other two. In this paper, the maximum concentration of compound which permitted normal cell maintenance was determined for each compound and called the maximum well-tolerated dose. For (XXX, R = Me) this was 252 pg/ml and for (XXI, R = Me) was 42 pg/ml. In another report [ 1721 these three compounds and three other analogues were tested for inhibition of 32 rhinovirus strains by agar overlay and gradient plate techniques. They were tested at 80 and 100 pg/ml as the top dose regardless of the toxicity of individual compounds. It was noted that with all compounds virus suppression occurred only in the presence of a drug and as soon as it was removed from the cultures virus growth began again. Thus the compounds are not virucidal and probably prevent an early step in the infection process. Further work with (XXX, R = Me) established that it can inhibit 19 strains of rhinovirus at concentrations of 2&50 pg/ml and the maximum well-tolerated dose was 250 pg/ml. It was active orally in mice against vac-

D. L. SWALLOW 143 cinia and was tested orally at 16 mg/kg, three times per day for 8.5 days in chimpanzees against rhinovirus, with only a suggestion of activity [173]. Two out of six chimpanzees died under barbiturate anaesthesia, used during manipulation, but there were apparently no drug-related effects. The compound is said to be 'well tolerated' in humans at 3 x 1 g per day orally [ 1741, and clinical trials are in progress.

OTHER HETEROCYCLIC COMPOUNDS (excluding

purines and pyrimidines)

There have been few compounds of real interest in this group and many showing only borderline activity at or near a toxic dose. Some of the better documented compounds will be reviewed. Derivatives of 1-piperazine carboxylic acid have been thoroughly examined for activity against influenza A (strain PR 8) h mice [175]. Of 100 compounds tested, 19 were found active after being given to mice orally in a single maximum well-tolerated dose. Virus was administered intranasally and activity was based on percentage survivors at 14 days after infection. It was found that the structural features of either (XXXII) or (XXXIII) were necessary for activity. R-N

n

N-COZ

u

(CH2)" N Etz

R

Me,Et,Pr

n n

= 2,3;2:0 = 2, Z:S

(XXXI1 1

n

Me N/-X-N

wNMe

W x=

c0,soz

(XXXIII)

The lead compound in this series (XXXII, R = Me, Z = 0, n = 2) remained the most active although it required high doses to achieve its effect -800 mg/kg gave 74per cent survivors at day 14 with no deaths from the drug itself. Treatment of the animals simultaneously with drug and virus gave the maximum effect and no benefit was obtained from multiple dosing [176]. Rhodanine (XXXIV) has been found to inhibit the multiplication of ECHO 12 virus, one of the enteric RNA viruses. Other strains of ECHO and various other RNA and DNA viruses were insensitive. It produced 95 per cent inhibition of virus growth in monkey kidney cells at 17 pg/ml. Cellular RNA synthesis and morphological appearance were unaffected by 150 pg/ml. This work indicated that rhodanine prevented synthesis of the viral protein coat. All the substituted rhodanines examined were inactive or only slightly active and generally more toxic than the unsubstituted compound [1771.

144

ANTIVIRAL AGENTS

ZJ"

S

(XXXIV) The mode of action study on rhodanine is one of an interesting series of investigations into the mode of action of various antiviral agents. A slightly earlier paper reported that N'-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (XXXV) specifically blocks release of vaccinia virus from chick embryo cells. This effect is shown at 3.1 pg/ml at which there is complete inhibition of viral cytopathic effect. The compound has no effect on cellular growth and metabolism up to 50 pg/ml, does not inhibit any other viruses and shows its antiviral effect only in certain types of cell cultures. Intracellular multiplication of the virus is not inhibited and it is only the release of newly formed virus from the cell that is prevented. The compound was inactive in vaccinia infected mice and rabbits [178]. It can thus be classed as a very interesting and specific virological tool. A large series of phenoxathiins (XXXVI) were investigated for activity against poliovirus in vitro. Three drug concentrations were tested 10, 2 and 0.4 pg/ml but no toxicities were reported except for two compounds toxic at 10 pg/ml. Many of the compounds gave 100 per cent reduction of plaque forming units at 2 pg/ml in the agar overlay test used. Structure activity relationships were discussed [1791. C S NH2

(XXXVI )

Me

(XXXVII)

[ XXXVIIl)

Quinoline-4,7-bisthiocarboxamide (XXXVII) is said to inhibit the infectivity of Newcastle disease virus completely after 1 hour of extracellular incubation at 1 mg/ml [180]. By analogy with the dihydroisoquinolines this compound may be active in vivo. A series of 2,6-dialkoxypyrans and 2,6alkoxy-d 3-dihydropyrans were also examined for extracellular virus inhibitory activity. The most active compound of the series against influenza virus was (XXXVIII) but no details of the test were given [181]. Further compounds in this series have recently been reported [182]. Two barbiturate derivatives have been reported to have activity against myxoviruses. The sodium salt of 5-(3,4-dichlorophenyl)-5-ethylbarbituric acid (XXXIX) has been extensively investigated on account of its activity

145

D. L. SWALLOW

in vitro and in vivo against Coxsackie A21 virus, causative agent of an upper

respiratory infection in man. It gave a good dose-response effect on a plaque reduction test, 50 per cent reduction being produced by 40 pg/ml and 82 per cent at 65 pg/ml at which dose there was some effect on cellular metabolism. In mice, complete protection was afforded by a single dose of 64 mg/kg either orally or intraperitoneally, while 95 per cent of control animals died from the infection.'This dose could be given up to 4 hours before and to 24 hours after infection and a high degree of protection obtained. No acute singledose toxicity was quoted but at about 144 mg/kg per day orally some animals died after 5-6 days. There was evidence of a slight sedative effect at 72 mg/kg per day but none at lower levels. Since Coxsackie A21 virus can be used experimentally in man the compound is being evaluated in volunteer trials [183].

NH

(XXXIX 1

IXL)

All analogues of (XXXIX) had very much lower activity. Changing the ethyl group to methyl or n-propyl reduced activity by at least a half, as did altering the chlorine substitution from 3,4 to 2,4 or the barbiturate to a thiobarbiturate. The thiobarbiturate (XL) also had high structural specificity. The ally1 group could only be replaced by n-propyl and the R group had to be a straight chain alkyl of 14 or 15 carbon atoms for maximum activity. Alkyl chains of 11-16 carbon atoms were investigated. These compounds were active in mice against influenza A2, a single oral dose of 45 mg/kg giving 50 per cent survival at day 6 after infection. To obtain the same effect at day 10 an initial dose of 250 mg/kg was required. The compound was poorly absorbed from oral administration and rats dosed daily for 7 weeks at 1500 mg/kg were unaffected. Similarly, rabbits survived 750 mg/kg. However, in tissue culture the highest concentration tolerated by KB cells was 5 pg/ml[184]. Several thenoyl amides of type (XLI) have been tested in ovo against influenza A2 and produce a 20-fold reduction in virus growth over controls. No dose levels were quoted, but in a test against pseudorabies virus in mice complete protection from 100 LD,, values of virus was achieved with a dose said to be 4 mM ! [ 1851. The imidazo[2,1-b]thiazole derivative (XLII) is one of a small series of compounds claimed to have high activity against rhinoviruses [186]. The

146

ANTIVIRAL AGENTS CO,H

UP

Ph-C=NN

H,

IXLII)

2-Ph group can be replaced by H, the 3-Me by H and the 6-Ph byp-hydroxyphenyl or H. The preferred compound (XLII) has a minimum inhibitory concentration in human KB cells against rhinovirus HGP and strain 2060 of 2 pg/ml and the maximum non-toxic concentration is 60 pg/ml. Against HGP in HeLa cells the figures were 10 pg/ml and 20 pg/ml respectively. The activity of several thiazolidine acetic acid derivatives of type (XLIII) against herpes virus in tissue culture has been reported [187] and structure activity relationships discussed [188]. The most active of these compounds Me

I

(XLIII, R' = PhC=, R2 = p-tolyl) had low toxicity to cells and has been tested against dermal herpes infections in man [189].

S

(XLIII)

The compound was administered as an ointment (no concentration of drug reported) to the site of the lesions. If the first application took place between 3 and 11 hours after first symptoms were noted, there was no further development of new papules and the existing ones healed in 2-3 days. The average duration of an untreated attack is 7.7 f 1-24days and in 31 patients treated with early application of drug the attack was shortened by 7.2 f 1.54 days. When the drug was first applied between 12 and 24 hours after symptoms appeared, then the attack was shortened by 3.4 & 1.42 days (46 patients). Preliminary trials have started against herpes keratitis of the eye. This seems to be a potentially useful drug against a common infection.

D. L. SWALLOW

147

PURINES AND PYRIMIDINES The distinction between what is an active and what a toxic compound in a group whose principal biochemical effect is to act as an antimetabolite, is difficult to draw. Very often the distinction can only be made between pronounced effects on rapidly proliferating species such as viruses and some cancer cells, and less pronounced effects on normal, slowly multiplying cells. In spite of these theoretical difficulties a large number of purine and pyrimidine analogues have been tested as antiviral agents, principally against DNA viruses, both in cell cultures and in animals and humans. The most well known of these is 5-iodo-2’-deoxyuridine (IDUR, XLIV, R = I). The origins and development of this compound have been very fully reported by many authors [4,7, 9, 131 and its mode of action together with that of its analogue (XLIV, R = F) and cytosine arabinoside has recently been reviewed [25].

HoH*@OH H

(XLIV)

Its principal use is against superficial herpes virus infections of humans in which it produces a moderate cure rate without harming the tissue to which the drug is applied. Since dermal herpes simplex is usually self-limiting and of short duration, the assessment of clinical effect is not easy when double-blind trials are carried out. Some authors claim that a statistically positive result is obtained and an almost equal number conclude that the difference between drug and placebo treated patients is not significant. Some patients, however, consider the treatment to be beneficial [190]. Results in ocular herpes keratitis are usually more positive and there is no doubt of the value of the drug in this form of infection [191, 1921. Some patients have been found to be resistant to treatment but in only 2 out of 12 such patients in one trial was the isolated virus found to be actually more drug-resistant than the normal wild-type virus. In this paper [192] the value of IDUR in herpes keratitis of man was critically assessed from the information available in mid-1969. Herpes virus encephalitis is a grave but fortunately rare disease of man which has in some cases been successfully treated with either intravenous or

148 ANTIVIRAL AGENTS intrathecal administration of IDUR. Again a marked spontaneous remission rate makes assessment of therapeutic effect difficult in man [193]. Results in guinea-pigs were also very erratic and although all control animals died it was not possible to determine whether the cause of death in the treated animals was due to virus or drug. IDUR given intravenously at 80 mg/kg per day in an adult produced abnormal liver function, a reduction in blood leucocytes and platelets and bloody diarrhoea [1931but these were reversible on cessation of treatment and no long-term effects have been noted. Other workers were unable to find any toxic effects of the drug when it was administered intravenously to children and infants suffering from cytomegalovirus infections. In these cases the dose was 100 mg/kg per day, but again it was doubtful if the drug had any effect on clinical progress [194]. In a trial of IDUR against herpes zoster, the compound as a 40 per cent solution in dimethyl sulphoxide was administered continuously to lint covering the site of infection. This gave only marginally positive results in younger age group patients, but those aged >60 benefited considerably and the duration of pain from the infection was reduced from 30 to an average of 23 days. When the treatment was continued for >4 days, no further lesions appeared [ 1951. An interesting paper on the molecular conformation of IDUR has appeared in which two types of molecule were found in the asymmetric unit cell in the solid state. It was suggested that different types of action of DNA may be related to the pucker of the sugar rings which can either be exo or endo with respect to the 5'-CHzOH and can occur at either the CIZ or C', positions [196]. The chemical changes have been rung on IDUR and many analogues have been tested against DNA viruses. The 5-methylamino analogue (XLIV, R = NHMe) is said to be of comparable activity but much less toxic with 10 mg/egg having no effect on the embryo. Activity in the 5-amino or substituted amino series was specific to this compound. Even the ethylamino analogue was 20 times less active [197]. The 5-trifluoromethyl analogue (XLIV, R = CF,) has been known for several years and is more potent than IDUR against herpes keratitis in rabbits. It is also active against IDUR resistant herpes [ 1981. The clinical pharmacology of this compound has recently been reviewed [199] and clinical trials in 18 children and 24 adults with advanced neoplastic disease reported. Blood disorders were the main toxic effects at doses from 5-30 mg/kg per day. There were 5 therapeutic responses in children at the highest dose level, but none in adults, who could only tolerate 5 mg/kg [200]. Studies on the incorporation of the compound into the DNA of vaccinia virus have been reported [201]. 5-Ethyl-2'-deoxyuridine (XLIV, R = Et) is almost as active as IDUR against vaccinia virus in vitro [202], but more effective and giving fewer relapses against dendritic herpes infections in humans than IDUR [203]. Several nucleosides altered in the sugar moiety have been reported.

D. L. SWALLOW 149 3‘-C-metliylcytidine is active against neurovaccinia in mice at 1.O mg/mouse but its 2’-C-methyl analogue was inactive at 2.0 mg. The corresponding analogues of adenosine are somewhat more active at 0-5 mg and 1.0 mg/ mouse respectively. No toxicities were given [204]. 5‘-Methanesulphonylamino-5‘-deoxyadenosine gave 72 per cent inhibition of herpes simplex in monkey kidney cells when tested at the highest non-toxic concentration, but the corresponding 5’-amino-5’-deoxy analogue was inactive. However, removal of the 2’-oxygen in this latter compound restored activity [205]. The two most widely investigated nucleosides with unusual sugar residues are 9-/3-~-arabinofuranosyladenine (XLV, Ara-A) and 1-b-D-arabinofuranosy1 cytosine (XLVI, Ara-C)

OH

’

OH

Ara-A is active against a variety of DNA viruses but not RNA viruses in vitro. It has significant therapeutic action against herpes keratitis in the eyes of hamsters and in herpes simplex and vaccinia infections of mice, when given either parenterally or orally. It is very rapidly de-aminated in vivo to 9-b-~-arabinofuranosyl hypoxanthine (Ara-Hx) but this fortunately retains almost all the activity of its precursor. Ara-A appears to have superior therapeutic activity to either Ara-C or IDUR and is apparently less toxic. The acute LDSo value by intraperitoneal dosing in mice was 4677 mg/kg while orally no deaths occurred at 7950 mg/kg. The weight loss in mice and suppression of weight gain produced by prolonged dosing at levels greater than 465 mg/kg per day was not reversed on cessation of treatment. The properties and activity of Ara-A have been reviewed [206] and also form a series of reports [207-2 121. Cytosine arabinoside (XLVI) (Ara-C) has a similar spectrum of activity to Ara-A but has also been found active in man against herpes keratitis [213] and against severe generalised varicella (chickenpox) [214]. More recently it has been tested against generalised herpes infections in man at doses of 0.2-3.0 mg/kg per day intravenously for 5 days. Virus disappeared from the patients in 1-7 days, but it was recommended that the blood picture be carefully watched during and after dosing [215]. Ara-C is also active against several oncogenic viruses in cell culture and in mouse embryos,

150 ANTIVIRAL AGENTS including those of murine leukaemia and sarcoma [216]. It has produced up to 30 per cent remissions in some types of human leukaemia when dosed intravenously [217]. In this same reference, a new synthesis of Ara-C was reported, which, starting from D-arabinose, constructed the pyrimidine ring in situ in four high yield stages. 5-Fluoro-Ara-C [218] and 5-iodo-Ara-C [2191 also have antitumour and anti-herpes activity and have been compared with Ara-A and IDUR. The iodo-compound completely suppressed herpes growth in rabbit kidney cells at 5 pg/ml but this dose produced 60 per cent reduction in cellular protein synthesis. Both 2-amino-4-mercaptopurine (XLVII) and its 9-b-~-ribofuranoside have high activity in vitro against cytomegalovirus. This virus is repeatedly found in patients on immunosuppressive treatment, is highly infective and can produce severe congenital disease in newborn infants. SH

( X LV I I )

The activity was determined by reduction of viral cytopathic effect and toxicity by examination of cells visually and their ability to grow at normal rate immediately after adding drug-free medium. The ratio of minimum toxic dose and active dose was 100 for both compounds. Five related compounds had a ratio of 32, and 19 other assorted purines and pyrimidines were inactive [220]. The N’-allyl-N3-ethylpyrimidine(XLVIII) is the best of a small series in which the substituents at the 3, 5 and 6 positions were varied. Activity was found against herpes virus in vitro and in rabbits eyes [221]. The compound has been evaluated in man and a 1 per cent solution was said to be effective against all recurring herpetic skin diseases [222].

( XLVIII)

(XLIX

151

D. L. SWALLOW

A sulphur containing pyrimidine (XLIX) is reported to be active against poliovirus (RNA virus) in vitro, reducing virus cytopathic effect by 70 per cent at 10 pg/ml and virus yield by 3-4 log,, units at 50 pg/ml. The maximum non-toxic dose was 100 pg/ml [223]. Another group of pyrimidine derivatives active against an RNA virus (Coxsackie A21) are 2-pyrimidyl ureas of type (L) [224].

The compounds were tested in mice at what was probably a maximum tolerated single dose although no toxicities were given. Under these conditions the most active compounds were : R

Activity mg/kg

3,4-diC1 2,5-diCI 3-c1 * 3-NO2 2-Et0

20 22 26 31 34

AMIDINES AND GUANIDINES The activity of this group of compounds is usually confined to RNA viruses, and guanidine itself has long been known as a specific inhibitor of the synthesis of viral RNA polymerase [225]. Once the polymerase has been formed then guanidine has no effect on its activity [226]. A large series of substituted acetamidines and propionamidines of type (LI) have been investigated for activity against RNA viruses. All were inactive against polio virus but some were active in mice against influenza. No details of dosage or toxicity are yet available but one compound was chosen as the best from each group. These were: NH

II

R*NH(CH z),,.C-NH

(W

2

HCI

152

ANTIVIRAL AGENTS

(LI, R

=

(LI, R

=

(LI, R

=

r * ~- O c o ~t-50~-

n

=

2) from 24 analogues [227]

n

=

2) from 31 analogues [228]

n

=

2) from 10 analogues [229]

The second of these compounds was claimed to be comparable in activity to 1 -aminoadamantane hydrochloride. Three amidines (LII-LIV) considered to be equivalent or superior to 1-aminoadamantane were found as a result of screening 280 amidine containing compounds against influenza in mice.

No dosage or toxicity levels were given. Dosing of the animals was started 3 days after infection and antiviral effect was estimated by prolongation of survival, decrease in lung consolidation and by titration of virus from the lungs [230]. Among guanidine derivatives canavanine (LV) has been reported as an inhibitor of Semliki Forest virus (RNA) at 100 pg/ml in tissue culture [231]. HOOC

\CH.CH2.CH2.0NH.C \ / H2N NH2

It probably acts as an arginine analogue as its activity can be reversed by addition of arginine to the system. Canavanine produced a much lower rate of RNA synthesis in infected cells than in untreated, infected cells, although the rate of protein synthesis was unaffected. Several aromatic derivatives of guanidine (LVI-LVIII) as their sulphates were active against influenza PR8 and Sendai virus in vitro. The minimum inhibitory concentrations ranged from 35-125 pg/ml but again no toxicities were given [232].

153

D. L. SWALLOW OH

Little has been reported recently on the antiviral activities of biguanides and the controversy over the claims of N'N'-anhydro-bis(p-hydroxyethyl) biguanide hydrochloride (ABOB) to be active in man against influenza, seems to have died down. N-Furfuryl biguanide hydrochloride (LIX) is

slightly active against Newcastle disease virus, influenza A and polio type 1 virus in chick embryo cells. The minimum toxic dose was 62 pg/ml, and 31 pg/ml gave complete suppression of plaque formation. There was almost no difference in number of plaques at 16 pg/ml to untreated cells, although the plaques were smaller in size [233]. A series of 32 p-alkoxyphenylsulphonylbiguanides were found inactive against influenza A, respiratory syncytial virus and rhinovirus by a plaque inhibition test [234] but eight out of 38 compounds in an earlier series had slight activity against rhinovirus. However, in a tube dilution assay, the compounds were inactive [235].

AROMATIC COMPOUNDS This group consists of compounds containing an aromatic ring which do not fit conveniently into any of the other main groups.

PHENYLGLYOXAL DERIVATIVES

These have been most thoroughly investigated by various Italian groups, since the antiviral activity of xenylamine (LX, R' = Ph, R2 = C 0 2 H ) which is a glyoxal diacetal was reported [236]. Acompound of current interest is LX (R' - NOz, R2 = COCHC12). This was moderately active in mice

154

ANTIVIRAL AGENTS

(LX)

against both influenza A (PR8) and influenza A2, a dose of 2.47 mg/mouse given orally hour after infection and daily thereafter for 6 days gave 33 per cent survivors on day 6 compared with 6 per cent in untreated controls. The same degree of protection was afforded by 0.75 mg/mouse of l-aminoadamantane (AdNH2) dosed subcutaneously 4 hour before infection and daily thereafter. Tested against 50 LD50values of influenza A2 and starting dosing 24 hours before infection, the compound still gave 30 per cent survivors but AdNH, gave 70 per cent. With 100 LD,,, values of virus, both compounds gave 30 per cent survivors. There was little or no synergism between the two compounds [237]. Of 41 arylglyoxal N,N-disubstituted hydrazones tested against influenza PR8 in embryonated eggs at the maximum tolerated dose only two compounds (LXI), were active [238].

+

1

’3

R=Ph ; R = --N

R

1

eCOC H=NR’

2

d=Ph; R =-N

-

NMe

W ILXI 1

A similar series of 29 compounds, phenylglyoxal hemiacetals and Schiff’s bases from either 7-amino or 5-amino-8-hydroxyquinolinewere nearly all found active against influenza PR8 in eggs but were inactive against vaccinia. Again they were tested at the maximum tolerated dose which varied from 0.62 to 10 pmol/egg [239]. Some of the phenylglyoxal derivatives allow rapid development of viral resistance. In experiments to define the mode of action of these compounds, influenza PR8 was found to develop total resistance to 4,4’-bis-biphenylglyoxal dihydrate (xenaldial) after only three serial passages in eggs [240]. MISCELLANEOUS AROMATIC COMPOUNDS

Activity against herpes virus has been found in 4’-[2-nitro-l -@-tolylthio) ethyllacetanilide (LXII) both in cell cultures and in the skin of rabbits. Extensive tests in baby rabbits showed a very definite advantage of the compound over placebo treatment. The safety of the compound for topical application to man as a 5 per cent suspension in ointment was established,

D. L. SWALLOW 155 but double-blind clinical trials in 80 patients showed no advantage over placebo treatment [241] NO2

I

M e C O NH O ! ? - S O M e

(LXII)

Ammonium aurintricarboxylate (LXIII) along with several other triphenylmethane dyes has been found active in cell-free systems at 10 4~in preventing the binding of a bacteriophage RNA to the ribosomes of E. coli. In viral OH

OH

(LXIII)

(LXIV)

infections a messenger RNA copied from the viral RNA or DNA attaches to the polyribosome complex of the host cell and directs the synthesis of viral proteins. This is one of the points at which viral replication could be inhibited without affecting the host cell [242]. A search for a specific inhibitor of the action of RNA replicase (RNA dependent RNA polymerase) another point at which viral replication could be inhibited independently of host cell metabolism, led to the screening of lo00 compounds against bacteriophage QB RNA replicase with E. coli as host cell. Thirty active compounds were obtained of which (LXIV) was the most active. This inhibited phage multiplication in E. coli by 99 per cent compared with control at 10 pg/ml whereas host cell growth was only inhibited by 50 per cent [243]. These authors hopefully conclude 'thus, further studies may produce a universal anti-RNA virus drug without side effects'. ALIPHATIC COMPOUNDS Two compounds are of considerable interest in this group, 3-ethoxy-2oxobutanal hydrate (Kethoxal, LXV) and calcium elenolate (LXVI).

156

ANTIVIRAL AGENTS

OCHO CH2C06

MeCH CO CHO H20 I OEt

MeOOC

'12

Ca2'

Me

(LXV)

(LXVI)

Kethoxal has been known since 1957 as a virucidal agent for a wide variety of RNA and DNA viruses when incubated at 1 mg/ml with the virus in saline for 30 minutes at 37°C. It had, however, no effect under these conditions on picornaviruses, for example polio and Coxsackie A21, and was inactive against systemic viral infections of animals. It has now been found active in tissue culture against Coxsackie A21, 50 pg/ml of compound preventing any new virus being formed, and has also shown activity in hamsters infected with parainfluenza 3 virus intranasally and treated with drug intranasally. The data indicated that the effect was not entirely due to thg virucidal effect of Kethoxal [244]. Calcium elenolate (LXVI) whose structure has just been revised from an open chain aliphatic dialdehyde is also a wide spectrum extracellular virus inactivator, as might possibly be expected from comparison of its structure with that of Kethoxal. The acid is obtained from extracts of olive plants and its calcium salt has a minimal effective concentration of 0.75 per cent when tested intranasally in hamsters infected with parainfluenza 3. This concentration applied six times in 28 hours had no toxic effects and aborted the virus infection. A concentration of 0.6 per cent tested intranasally in rabbits over an extended period produced mild to moderate changes in the nasal epithelium, but a 1.0 per cent nasal spray four times daily for 14 days was well tolerated in humans [245-2471. The possibility that ascorbic acid has an antiviral effect still excites some authors and a motley collection of simple aliphatic molecules including acetone, dimethyl sulphoxide and peracetic acid have been reported to have antiviral effects. PEPTIDES AND AMINO ACIDS The synthesis and testing of 175 di- and tri-peptides for activity against a range of viruses in vitro led to the discovery that 21 compounds were active against measles virus and 28 against herpes virus. The chief characteristic of all the active compounds was a benzyloxycarbonyl or carbobenzoxy (PhCH20CO-) protecting group on the N-terminal amino acid. Other protecting groups on the peptides did not confer activity and the de-protected peptides were either inactive or very weakly active [248]. Seven compounds were investigated in detail and the most active of these, carbo-

D. L. SWALLOW 157 benzoxy-L-phenylalanlyl-L-nitroarginine,had a high selectivity ratio against measles virus. At a virus input of 10 IDs0 almost complete inhibition of multiplication was produced by 20 pg/ml while the minimum toxic dose to the cells was 625 pg/ml. With a higher virus challenge of 100 IDs0 the minimum dose required was 62.5 pg/ml. The compounds were also effective ifadministered to the cells up to 24 hours after infection. Single oral doses of 25C2000 mg/kg in rats, dogs and monkeys gave antiviral activity in the serum, and were not toxic to the animals. In spite of these very interesting results, evaluation is not being continued because of the recent availability of a new and effective vaccine against measles [249]. The antimetabolic activity of various unnatural amino acids has been investigated as a possible source of antiviral activity. Slight activity has been found in D-penicillamine [250], D,L-thienylalanine [2511 and selenocystine [252].The last compound is claimed to be a specific inhibitor of virus induced RNA polymerase.

NATURAL PRODUCTS A very large number of products isolated from fermentation liquors or mycelia of fungi or directly from plants, bulbs and shellfish, have been reported to have antiviral activity. One frequently finds that while a particular compound may appear to have high activity and low toxicity when determined by say an agar diffusion assay, another method of assay will indicate that the compound is highly toxic to cells at low doses. Four such compounds which have been investigated in some detail are ascochlorin (LXVII),

CHO

MeO

Me

I Me

(LXVIII)

(LXVII)

M e O e C H = C -C=CH

-

I I

NC NC

Me

Me

(LXIX)

OCOCH=CHMe

(LXX)

O

O

H

158 ANTIVIRAL AGENTS mycophenolic acid (LXVIII), trichothecin (LXIX) and xanthocillin X monomethyl ether (LXX). Ascochlorin, whose structure was determined by X-ray crystallography [253] was reported to be active against herpes virus and Newcastle disease virus in an agar diffusion-plaque inhibition assay at 6 pg/ml and cytotoxic between 50 and 200 pg/ml. However, in a tube dilution assay using HeLa cells it was inactive against these viruses and the cellular LDS0value was 0 3 pg/ml [254]. In a similar way mycophenolic acid (LXVIII) was claimed [255, 2561 to have a wide spectrum of activity against DNA viruses by an agar diffusion-plaque inhibition assay, but more recently it has been shown that both BHK cells and chick embryo fibroblasts are prevented from multiplying by 0.3-1.0 pg/ml. The latter cells do not change morphologically when growth is prevented, but can recover from a dose as high as 100 pg/ml after it has been washed away from the cells. No antiviral activity could be observed at less than toxic doses [257]. Trichothecin (LXIX) stopped protein synthesis in chick embryo cells at 16-3 mg/ml and no inhibition of virus could be observed at lower concentrations [258]. Similarly, xanthocillin X monomethyl ether (LXX) was said not to be toxic to chick embryo cells at 16.5 mg/ml on an agar diffusion assay but in tube cultures toxicity was evident at 3-6 pg/ml. The LDS0 value to HeLa cells was 0.3 pg/ml[259]. It has more recently been shown to stop cellular protein synthesis and virus replication completely at 15 pg/ml [260]. Other compounds in the same class are antimycin A3, piericidin A, brefeldin A, verrucarin A, funiculosin, borrelidin, and cylindrochlorin. GLIOTOXIN AND ITS ANALOGUES

Gliotoxin (LXXI) is the original member of a unique series of natural products all of which contain the epidithia-diketopiperazine ring system

(LXXI)

OH

(LXXII)

(LXXII). Gliotoxin was originally found to be active in vitro against polio, herpes and influenza viruses in a variety of cell types with high ratios between the toxic and active doses. It was fairly toxic to mice, the maximum tolerated doses being 7.5 mg/kg i.v. and 10 mg/kg per day orally [261]. Later work showed that this and all other compounds containing the nucleus (LXXII) were highly active, specific, irreversible inhibitors of viral RNA synthesis.

D. L. SWALLOW 159 Concentrations of compound from 250 to 15 000 times greater were required to affect cellular RNA synthesis [262, 2631. The most active compound was the nucleus itself with a methyl group on each nitrogen and the other substituents hydrogen, which at only 0.0003 pg/ml gave 50 per cent inhibition of Coxsackie A21 viral RNA synthesis; 5 p g / d was required to give 50 per cent inhibition of cellular RNA synthesis. It was also shown that if the S-S link were broken leaving two SH-groups the compound was still just as active, but if the sulphur atoms were blocked by a chemically stable group or removed altogether, then activity was completely lost. The compound was active at 0.06 pg/ml in W.1.-38 cells against four strains of rhinovirus, with a selectivity ratio of 4. The other compounds so far identified in this interesting series are aranotin [264], acetylaranotin [264], apoaranotin [264], sporidesmin [265], chetomin [266] and chetocin [267]. Oryzachlorin [268] may possibly contain the nucleus (LXXII) but the structure has not yet been fully elucidated. In chemical studies on acetylaranotin [269] it has been shown that the -S-Sbond is very reactive due to considerable angular strain, and it is probably some reaction at this point which mediates its very speclfic and irreversible antiviral activity.

RIFAMPICIN

The group of rifamycin antibiotics suffered from the disadvantage of being inactive by oral administration. Chemical modification of rifamycin-SV led to rifampicin (LXXIII) which had the required oral activity [270]. Biochemical studies proved that its antibacterial action was due to inhibition of bacterial DNA-dependent RNA polymerase. It had no effect on mammalian RNA polymerase. Some DNA viruses also produce a DNA-dependent RNA polymerase and rifampicin was shown to have slight activity at 50 pg/ml and marked activity at 100 pg/ml against vaccinia virus. It began to show toxic effects in cells at 500 pg/ml [271]. Me

Me

;1:9:$

Me

Me0

X= H

in Rifarnycin SV

n

X: CHZN-N-NMe

in R i t a m p i c i n

0 Me

0

OH

(LXXIII)

160 ANTIVIRAL AGENTS Other workers reported similar observations almost simultaneously, extending activity to cowpox and adenovirus. Replication of these viruses was inhibited completely at 100 pg/ml. Seven different cell lines began to show signs of drug toxicity after 3 4 days incubation with 100-150 pg/ml of rifampicin, but they all recovered when drug was removed [272]. The organisms of trachoma, psittacosis and lymphogranuloma venereum, once thought to be viruses but now included in the bacteria, were also shown to be very sensitive to rifampicin [273]. Later studies on the mode of action indicate that the antibiotic is a specific inhibitor of vaccinia virus assembly inside the cell, although the necessary components have been formed [274]. The only biochemical function so far found to be inhibited is the induction of a late particulate polymerase [275]. This is an interesting development of an antibacterial agent, but so far there are no reports of antiviral tests in animals. DAUNOMYCIN AND OTHER ANTIBIOTICS

This antibiotic of the anthracycline group was claimed to be as active as methisazone against vaccinia virus, but it was in fact toxic to the host cells over the range claimed for activity [276]. Formycin A (LXXIV, R = NH2) and B, (LXXIV, R = OH), and pyrazomycin (LXXV) are natural products having weak antiviral activity. The first two are active against influenza in vitro [277] and the last has activity against vaccinia in mice [278].They are unique in being nucleoside analogues with the rare C-riboside link. U

(LXXIV I

(LXXV 1

CONCLUSION As our knowledge increases of the biochemical events which occur from the time at which a virus comes in contact with a host cell to when new viruses

D. L. SWALLOW 161 emerge from the same cell, we begin to find more ways of interrupting the process. Several of the compounds reviewed are specific inhibitors of virus directed processes and have considerable potential as antiviral agents. As always, the transfer of these properties from an in vitro system to a test animal and then to a human patient is a greater task than the finding of the compound for the in vitro test. A less complex approach is either to inactivate the virus before it enters the cell (and this was very nearly successful with the dihydroisoquinolines), or to prevent it entering as with the adamantane derivatives and decalins. Great progress has been made in the past few years in improving both in vilro and in vivo tests, and workers are becoming more aware of the necessity for selective action of their compounds on viruses. These facts together with the tremendous rate of growth of the subject lead one to hope that within perhaps a decade effective drugs will be found to overcome the virus diseases of man which cannot be controlled by vaccines.

ADDENDUM

The orally active interferon-inducing agent, 2, 7-bis(2-diethylaminoethoxy) fluoren-9-one dihydrochloride (p. 137), has been given the name tilorone hydrochloride. Two articles [279, 2801 and three papers have been published since this review was written. The antiviral tests covered at least nine RNA and DNA viruses which were inoculated into mice dosed with 250 mg/kg of drug 24 hours before infection [28 I]. Significant protective effects were observed against Semliki Forest, vesicular stomatitis, EMC, mengo, herpes simplex and vaccinia viruses, but the compound was least effective against viruses of the influenza group. Treatment of the animals with drug at times greater or less than 24 hours before infection gave less than optimum results. This correlated with the time-course of antiviral activity appearing in mouse serum after a single dose of tilorone, which reached a fairly sharp maximum at 24 hours [282]. The mode of action of tilorone was described in this second paper and the conclusion was reached that interferon was the active agent. The third paper describes some preliminary toxicology on tilorone [283]. In mice the LD,, value for a single oral dose was 959 mg/kg and in rats 852 mg/kg. For intraperitoneal dosing, the values were 145 and 244 mg/kg respectively. However, single oral doses from 3 mg/kg upwards produced vacuolation and granulation of peripheral leucocytes and alterations in the reticulo-endothelial system. These effects were seen also in dogs and monkeys. In the latter species, vacuoles appeared 10 hours after dosing and persisted for up to two weeks, while the granulation appeared 14 hours after dosing and persisted for up to 3 months. Similar changes have been observed after intraperitoneal injection of Poly I:C in these species. Mice dosed at 250 mg/kg daily failed to gain weight. In spite of these apparently adverse

162 ANTIVIRAL AGENTS effects, the drug has been given to human ‘volunteers’ at doses of 150 rng to 5 g. No circulating interferon was detected in the serum, even after the highest dose. A patent has appeared covering chemical variations on tilorone [284] and these were recently reported covering structure-activity relationships [285].

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200.

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~~

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Correction to Volume 7 p.63 item 288 : After : editor’s name Insert : Modern Trends in Pharmacology and Therapeutics p. 124 Structure - activity relationships for adrenergic neurone blockade From : ‘Blockade’ To : ‘References’ Resite all page numbers to next lower entry

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5 Antifertility Agents V. PETROW,Ph.D., D.Sc.,F.R.I.C. Wm. S. Merrell Company, Cinncinnati, Ohio 45215, U.S.A.

PREFACE

172

INTRODUCTION

172

STEROID BIOGENESIS Progesterone/Testosterone biosynthetic pathway Oestrogen biosynthetic pathway 19-Nortestosterone biosynthetic pathway

172 175 175 176

THE DISCOVERY OF THE OESTROGENS AND PROGESTAGENS USED IN CONTRACEPTION

178

THE BIOLOGICAL ASSAY OF OESTROGENS AND PROGESTAGENS Oestrogens Progestagens Inhibition of ovulation Inhibition of gonadotrophin

I82 182 183 186 187

CONTRACEPTIVE REGIMENS The combination pill Sequential products Serial regimens The minipill Depot preparations The precoital pill The weekend pill Postcoital use of oestrogens

190 190 191 191 192 I95 196 196 196

CERVICAL FACTORS IN FERTILITY CONTROL Postmenstrual and follicular phases Luteal phase

I97 198 199

THE EFFECT O F ORAL CONTRACEPTIVES UPON LACTATION

200

SELECTION O F AN ORAL CONTRACEPTIVE

20 1

ADVERSE EFFECTS Carbohydrate metabolism Lipoproteins Throm boem bolism Liver function Transcortin Hypertension Ophthalmological effects

202 202 203 208 214 214 217 217

171

172

ANTIFERTILITY AGENTS Neoplastic disease Amenorrhoea or anovulation following use of oral contraceptives Cutaneous effects of oral contraceptives Respiration Miscellaneous

218 220 220 220 22 1

ACKNOWLEDGEMENTS

221

REFERENCES

221

PREFACE The present review concerns the use of steroids in the control of fertility in the woman. Animal data is included only insofar as it appertains to the main theme or to bioassay of relevant types. This limitation of subject matter seems desirable owing to limitations of space and, in addition, by the lack of correlation that often exists between the animal and the human model. So great are species variations in the area of reproductive physiology, that the reviewer is tempted to quote the dictum enunciated by Alexander Pope in another connection, ‘The proper study of mankind is man.’ INTRODUCTION No other development in steroid chemistry has had so great an impact upon the human imagination as the introduction of the contraceptive ‘pill’, which was first approved, as such, by the American Food and Drug Administration. in 1960. In the succeeding years, this combination of steroids and variants thereof has been hailed by some as a major breakthrough in the control of the population explosion and by others as bad medicine that should be banned pending further evaluation [l]. Time alone will tell. The dominant role of the steroid chemist in contraception research, however, is now being slowly eroded by new developments involving entirely different types of chemical structures (cf. p. 221). It is therefore an opportune moment to review the achievements of the past and point the way to possible successes of the future. STEROID BIOGENESIS The role of steroid hormones in the control of fertility has its origins in foetal biochemistry. Although the sex of the embryo is determined at the

V. PETROW 173 time of fertilisation, it is impossible to distinguish between the male and female foetal gonads up to the 7th week of development. At this point the 'indifferent gonad' begins the typical morphological changes that lead to its differentiation into the ovary or testis. The factors controlling this initial differentiation are unknown and there is no experimental evidence to show that steroids are involved [2]. As the indifferent gonad undergoes sexual specialisation, so it begins to function as a steroid-secreting unit. It seems likely that it is these steroids secreted by the foetal gonads that participate in some processes of sexual differentiation, particularly at the level of the genital tract (see Figure 5.1) [2, 31. The role of the foetal gonads does not end at this point. As suggested by Young [4] and others, it seems likely that the gonadal hormones play a

Figure 5 . I

174 ANTIFERTILITY AGENTS dominant role in foetal/neonatal life in organising the sexually bipotential hypothalamus into cyclic or female, and continuous or male, patterns of gonadotrophin secretion. It is not surprising in these circumstances that the ovary and testis carry within themselves almost identical mechanisms of steroid hormone production but differ in the extent to which these diverse mechanisms are operated. The old ideas that made the pituitary the determining factor in reproduction have given way to the concept that control of the pituitary/gonadal axis resides in the hypothalamus which, in its turn, is influenced by the cerebral cortex. This control stems from follicle stimulating hormone- and luteinising hormone-releasing factors (FSH-RF and LH-RF), respectively, acting upon the pituitary which, in its turn, releases follicle stimulating hormone (FSH) and luteinising hormone (LH), respectively (see Figure 5.2). Ceribral

Cojlex

Hypothalamus FSH ond LH

J.

Releosing f a c t o r s

Pituitary FSH and

LH

Gonad

Ovary Follicle

-

-

Testis

fstrad’‘~~1’3

Corpus Luteurn

LHIFSH1

Testosteron

- Nortestosterone’

=.

,*

,*Oestradiol

‘Testosterone

+

FSH

LH

lLH’

iFSHl

Sertoli cells

Leydig cells

17ar - Hydroxyprogester6ne I

/

Progesterone Strorna

f

’

\\

\

I Progesterone

F S ~ , ~

Androgens

Androgens

Figure 5.2.

These gonadotrophins, in their turn, speed hormone production by the gonads. In both men and women there is a continuous secretion of the hypothalamic releasing factors so that there is a continuous production of gonadal hormones. In addition, in the female alone, there is superimposed an explosive cyclical release of FSH-RF and LH-RF which are essential for the ovulatory process [5]. In its simplest form it is possible to equate the follicular phase of the menstrual cycle with the preovulatory FSH surge and subsequent rupture of the follicle with mid-cycle LH release. Steroid biogenesis in the testis, in contrast, follows a continuous pattern with no dramatic changes in hormone output. Both ovary and testis utilise cholesterol as raw material for

V. PETROW 175 steroid hormone biosynthesis. The cholesterol is first converted into 20~,22Rdihydroxycholesterol which is then degraded into pregnenolone (I) which is the basic-but probably not exclusive-progenitor of the gonadal hormones [6, 71. This product then undergoes biogenesis, inter uliu via its oxidation product progesterone, as outlined in simplified form in Figure 5.2, which demonstrates in striking manner the qualitative biosynthetic equivalence of the male and female gonadal structures. The analogy ends at this point. Testosterone production by the ovary is minute. Oestradiol[8,9] production by the testis is hardly significant.

PROGESTERONE/TESTOSTERONE BIOSYNTHETIC PATHWAY

As shown in Figure 5.3, pregnenolone (I) is oxidised to progesterone (11) and thence converted via 17~-hydroxyprogesterone(111) into testosterone (V). Testosterone is the most active of the naturally occurring androgens [9, 101. Androstenedione (VI) and dehydroepiandrosterone (DHA) (IV) form less potent but quantitatively more significant secretion products. COMe

( I 1 Pregnenolone

(1V)Dehydroepiandrosterone

I

(11) Progesterone

( V ) Testosterone

(111) 1 2 - Hydroxy progesterone

(VI) Androstenedione

Figure 5.3. Progesteronelandrogen biosynthetic p a t h w q

OESTROGEN BIOSYNTHETIC PATHWAY

Although androstenedione (VI) represents the main precursor of oestrone (E,)/oestradiol (E2)(see Figure 5 . 4 , it has recently been shown [I 13 that there

176 ANTIFERTILITY AGENTS is an absolute and relative increase in oestriol (E3) [XI production during the luteal phase of the menstrual cycle. It seems that as much as 48 per cent of luteal phase E3 may be derived from DHA and not from El-E2. Steroid pathways of biogenesis in the ovary are in a constant state of flux throughout the menstrual cycle.

H

O

\-

W

I [ V l ) Androstenedione

n

I

l

l

J

Me i

2

HO

HO

(1x1DHEA

H

,

(XiOestriol Figure 5.4. Oestrogen biosynthetic pathway

19-NORTESTOSTERONE BIOSYNTHETIC

PATHWAY

In addition to the above two main biosynthetic pathways, there is presumptive evidence for a third subsidiary pathway leading to 19-nortestosterone (XIII) (see Figure 5.5). Biogenetic conversion of androstenedione (VI) into El and E2 is believed to proceed through 19-hydroxy intermediates (XI) which lose formaldehyde (or formic acid) during aromatisation. Similar transformations have been carried out in the laboratory. Human benign hypertrophic prostate slices have been shown to convert testosterone into 19-nortestosterone (XIII) in v i m [12]. In addition, 19-norandrostenedione (XIV) has been identified in the spermatic vein blood of the stallion [13] and in the follicular fluid of the mare [14]. 19-Nortestosterone (19-norandrostenedione) may consequently be regarded as the structural and biogenetic link between the androgens and the oestrogens. As indicated in Figure 5.2 the production of oestradiol (VIII), 19nortestosterone (XIII), testosterone (V), 17u-hydroxyprogesterone (111) and

V. PETROW 177 progesterone (11) is dependent, either directly or indirectly, upon FSH and L H release by the pituitary gland. By applying the concept of negative feedback action to the above biological systems, it can be concluded that the above hormones, in their turn, may inhibit FSH and/or L H production either directly by action upon the ovary or pituitary, or through the hypothalamus. As-inhibition of FSH inhibits development of the ovarian follicle, and inhibition of L H prevents its rupture, it follows that the above five steroids represent natural starting points for the hormonal control of fertility and in particular for the inhibition of ovulation. Their application to fertility control, however, though predicted in general terms as early as 1921 [15], was not possible owing to their almost complete lack of oral activity. New products had to be discovered in the pharmaceutical laboratories of the

(VI)Androstenedione

(XIII) 19-Nortestosterone

( X I ) 19-Hydroxy-

I

-H,CHO

(XIV)19Norandrostenedione

( X I I ) 19-0x0-

I

(VII) Oestrone

Figure 5.5. 19-Nortestosterone biosynthetic puthwuv

world that would be active by mouth and, in addition, would meet the strictest requirements of safety ever levelled against any type of therapeutic product by the health regulatory agencies of the world. When, at long last, such contraceptive steroids had become available, they were found to be derivatives of oestradiol, ( 17cr-hydroxy)progesterone, 19-nortestosterone and testosterone. Their discovery, it is true, had been governed by chance, by an element of serendipity, and by a courageous projection of animal data to the human model, yet it seems difficult to avoid the conclusion that their success as contraceptive steroids stems from their chemical derivation from those very hormones that control the reproductive process in man.

178

ANTIFERTILITY AGENTS

THE DISCOVERY OF THE OESTROGENS AND PROGESTAGENS USED I N CONTRACEPTION The discovery of oestrone (VII) in 1929 by Doisy and independently by Butenandt was followed in 1934 by the isolation of progesterone (11) from corpus luteum tissue and of dehydroepiandrosterone (IV) from urine (see review by Petrow [ 161). Although their chemical structures were not fully elucidated, a relationship between them and cholesterol was assumed. Progesterone, in contrast to oestrone, was only slightly active by mouth so that the need for a more active product was readily apparent. Dehydroepiandrosterone (IV) had become readily available in 1935 from oxidative degradation of cholesterol (acetate dibromide). As it was a C19 keto-alcohol and as progesterone was a Czl diketone, Kathol, Logemann and Serini [17] and Ruzicka and Hofmann [I81 independently conceived the idea in 1937 of adding two carbon atoms onto (IV) by reaction of the keto group with potassium acetylide to give 17a-ethynylandrost-5-ene-3/?, 17P-diol (XV). The following year, Inhoffen, Logemann, Hohlweg and Serini [19] converted the last compound into ethisterone (XVI), which was the first of the orallyactive progestagens. The same year, Inhoffen and Hohlweg [20] condensed

OH

(XVll) R=H, Ethynyloestradiol ( E E ) (XVIIa) R=Me. Mestranol ( XVIIb)

R=Cyclopentyl;

quinestrol

oestrone (VII) with potassium acetylide and obtained ethynyloestradiol (EE) (XVII), which was the first-and still remains the most important-f the orally active contraceptive oestrogens. A decade later its 3-methyl ether, mestranol (XVIIa), which represents the second most important contraceptive oestrogen. was found as a minor impurity in early commercial samples

V. PETROW 179 of norethynodrol (XIX) and norethindrone (XVIII) [21], being formed from the oestrone 3-methyl ether used as starting material for their production (vide infra). Following its identification and the recognition of its contribution to the contraceptive efficacy of these two 19-nor steroids, it was added to them to provide variants of the combination pill. One more contraceptive oestrogen needs mention. Quinestrol (XVIIb), the 3-cyclopentyl ether of ethynyloestradiol, was prepared by Ercoli, Pellegrini and Falconi [22, 231 as part of a broad programme dealing with the cyclopentyl ethers and enol ethers of steroidal hormones. 3-Etherification of (XVII) with cyclopentyl alcohol enhanced and prolonged the oral and decreased the subcutaneous activity of the oestrogen. Meli, Wolff and Hourath [24] subsequently showed that in the rat storage in, and release from, body fat is the mechanism responsible for the increased oral activity of the product. Similar findings were reported in man [25]. The depot action of this oestrogen was exploited by Greenblatt [26] in his pill-a-month regimen. The development of the progestagens used in contraception likewise had its origins in 1938 when ethisterone (XVI) was prepared (videsupra) and, for the next decade, no further progress was apparent. In 1949, Birch and Mukherji [27] reduced oestrone glyceryl ether with sodium in liquid ammonia and obtained 17P-hydroxyoestra-5( lO)-en-3-one, which may be regarded as the parent structure corresponding to norethynodrel (XIX). In the following year, Birch [28] completed the sequence by converting the 5( lO)-en-3-one into 19-nortestosterone (XIII). The resulting compound was initially a chemical curiosity-a steroid hormone lacking the C1,-angular methyl group. Its relevance to ovarian steroidogenesis was entirely unsuspected at the time, nor was it possible to predict that its derivatives would usher in the contraceptive revolution. In 1951, Djerassi, Miramontes and Rosenkranz [29] converted 19nortestosterone into 19-norandrostenedione (XIV) and thence into norethindrone (19-norethisterone) (XVIII). Two years later, Colton [30] prepared norethynodrel (XIX). These two 19-nor steroids achieved immediate success as orally active progestagens. In retrospect, the enhanced biological potency of (XVIII) over ethisterone (XVI) is not surprising as 19-nor steroids form the biogenetic link between the androgens and the quantitatively more potent oestrogens. At the time, however, the results were baffling.

Progestagens derived from 19-Nortestosterone

&=CH

0

’

@SH

&=CH

I

(XVIII) R=H, Norethindrone (XVIIIA) R=Ac, 19-Noresthisterone acetate

(XIXJ Norethynodrel

(XX) Lynestrenol

180

( X X I ) Norgestrel

ANTIFERTILITY AGENTS

(XXII)Ethynodiol diacetate

(XXIII) Quingestanol acetate

Other 1%nor steroidal progestagens were prepared in rapid succession. In 1954, Colton prepared ethynodiol(l7a-ethynyloestr-4-ene-3P, 17P-diol)by reduction of norethindrone with sodium borohydride [311. Eleven years later its diacetate ‘ethynodiol diacetate’ (XXII) was introduced as a contraceptive in combination with mestranol. Engelfried, Kaspar, Popper and Schenck [32], following up earlier work on the acetylation of ethisterone, prepared 19-norethisterone acetate (XVIIIA). The 3-deoxy analogue (XX) of norethindrone was prepared by Szpilfogel [33] and given the generic name of lynestrenol. Ercoli extended his studies on the 3-cyclopentyl derivatives of steroid hormones (vide supra) to 19-norethisterone acetate, obtaining the highly potent quingestanol acetate (XXIII) [34]. Finally, Herchel Smith-a pupil of Birch-in collaboration with Hughes prepared the totally synthetical ‘norgestrel’ (XXI) [35], which represents the last member of the first generation 19-nor progestagens. Parallel with these discoveries, another group of progestagens based upon the naturally occurring 17a-hydroxyprogesterone (111) was being developed. The last compound had been isolated from adrenal glands by Pfiffner and North [36] and had been found to be inactive in the Clauberg assay for progestational activity by the intramuscular route (see p. 183). Its 17-esters were examined by Junkmann [37] in his search for a depot progestagen, for which purpose he had finally selected the 17-caproate. Surprisingly, he failed to examine them for oral activity. It fell to the Upjohn group [38,39] to make this important advance and establish the progestational activity of 17aacetoxyprogesterone when administered by the oral route. A year previously, Burstein, Dorfman and Nadel [40] had found that metabolic deactivation of hydrocortisone in man occurred through 68hydroxylation. It occurred to the reviewer that such metabolic deactivation might be prevented by 6-methylation. To this end 6cc-methylethisterone was prepared by Adams, Ellis, Petrow and Stuart-Webb [41] and found [42] to be c. 6-5 times more potent than the parent ethisterone (XVI) in the Clauberg assay (p. 183). Extending the series, Barton, Burn, Cooley, Ellis, Petrow and Stuart-Webb [43] prepared dimethisterone (XXIV), which is the only derivative of testosterone presently in use. In addition, (XXIV) is surprisingly effective in endometrial carcinoma [44].

181

V . PETROW Progestagens derived f r o m testosterone

&

=C.Me

0

/ I

Me

( X X I V ) DI methisterone

Progestagens derived f r o m 17a - Hydroxyprogesterone COMe

I

Me

(XXV) Meclroxyprogesterone acetate

COMe

COMe Me I.-OAc

I

Me (XXVI)

(XXVII)

Megesterol acetate

Chlormad i non acetate

CL

(XXVIII) Algest one acet ophen ide

Other 6-methylated progestagens followed in rapid succession. Spero [45, 461 prepared medroxyprogesterone acetate (XXV), which was the first

17a-hydroxyprogesterone derivative to find use in oral contraception. The related megesterol acetate (XXVI) [47] was prepared at about the same time

182 ANTIFERTILITY AGENTS and found to be considerably more potent than (XXV) in the rabbit assay. The analogue (XXVI) in which the 6-methyl group is replaced by 6-chloro was obtained independently by two groups [48-501. The product, chlormadinone acetate (XXVII), resembled megesterol acetate in biological and contraceptive potency. One other progestagen needs mention. In 1955, Cooley, Ellis, Hartley and Petrow [Sl] successfully converted 16-dehydroprogesterone into the 16a,17aglycol and characterised the product as an acetonide. The potential of this derivative of 17a-hydroxyprogesterone as a progestational agent was recognised by Fried, who introduced [52] the acetophenide (XXVII1)-to which the name algestone acetophenide was given-into contraceptive practice as a parenteral ‘depot’ progestagen (cf. p. 195). THE BIOLOGICAL ASSAY O F OESTROGENS AND PROGESTAGENS Although specialist volumes are available covering this topic [53], some reference to it seems desirable in order to permit a better understanding of the field. A measure of detail has been included in those assays that appertain directly to contraceptive potency and utility. OESTROGENS

Allen- Doisy assay

This assay depends upon the induction of vaginal cornification in the ovariectomised rodent some 24-60 hours after treatment with the oestrogen. Rats and mice may be employed and the hormone may be administered by mouth, subcutaneously, intramuscularly, intraperitoneally, intravenously or intravaginally. Attention to detail, such for example as the maintenance of uniform conditions and avoidance of time to time variations are necessary in order to obtain reproducible results. Vaginal smears are best taken some 24 hours after injection, transferred to a glass slide, stained with methylene blue solution and scored when dry under a low power microscope. A smear containing nucleated or cornified epithelial cells and no leucocytes is regarded as a full positive response. The rat unit is defined as that dose of oestrogen that produces an oestrus smear in 50 per cent of the treated animals during a period of 24 hours. It should be noted that progestagens used in oral contraceptive preparations induce a variety of changes in vaginal cytology and in the growth of the uterus. Changes in vaginal histology may be determined using spayed rats

V. PETROW 183 (or mice). In the ‘acute’ version of the assay, the animals are dosed with the test substance on two successive days. Alternatively, they may be treated by gavage for periods of two weeks. After autopsy, the vaginae are examined histologically. Metrotopic effects (uterine growth stimulation) are generally evaluated in immature intact mice/ovariectomised rats.

Anti-oestrogenic activity

The method of Lerner, Holthaus and Thompson [54] and variants thereof is not fully quantitative, but does permit compounds to be graded in approximate order of activity. Spayed adult female rats are injected subcutaneously (s.c.) with oestradiol in olive oil at zero time. The inhibitor is injected S.C. at different sites at zero and eight hours. Vaginal smears are taken at 56, 64 and 72 hours. Positive smears contain nucleated or cornified epithelial cells with only a few leucocytes. Antioestrogenic compounds at effective dose levels fully inhibit vaginal cornification with zero positive response. Weak oestrogens, androgens, progestagens and corticoids all interfere to some extent with the action of oestrogen at the peripheral level. Neither the adrenals nor the pituitary are necessary for this antagonism. Comparison of ethynyl oestradiol (EE) with mestranol in a variety of animal assays shows that EE is c. twice as active as mestranol [55]. Similar results have been obtained in the human [56]. PROGESTAGENS (see

Table 5.1)

Corner-Allen assay

Corner and Allen [57l devised the first assay for progestational activity. Young female rabbits are castrated, treated for five days S.C.with the test compound and sacrificed on the sixth day. Uterine segments are then compared with controls. The assay was modified by Clauberg [58] who primed immature rabbits for eight days with oestrogen, treated them with the test substance S.C. for five days and sacrificed them on the fourteenth day. Refinements, particularly appertaining to standardisation of endometrial development were introduced by McPhail [59], thereby permitting quantitative, albeit crude, comparisons between progestagens. The characteristics of the endometrial development induced by different progestagens have been carefully documented by Elton [60]. Simple, low columnar luminal epithelium resembling that produced by oil-treated controls is produced by progesterone. High columnar epithelium is characteristic of testosterone and the androgenic progestagens. Tall columnar cells

184 ANTIFERTILITY AGENTS with extensive vacuolisation develop under the influence of oestrogenic progestagens.

McGinty assa-v Another useful but less frequently employed assay was developed by McGinty, Anderson and McCullough [61] in which immature female rabbits (or spayed adult females) are primed with oestrogen and the test compound instilled directly into the lumen of the uterus. The contralateral uterine horn is treated with the vehicle alone and serves as a control. In the related Hooker-Forbes assay [62, 631 the test substance is instilled into the ligated segment of the uterine horn of the castrated female mouse. Carbonic anhydrase assay

An interesting assay for progestational activity was developed by LutwakMann [64, 651, who observed that the carbonic anhydrase concentration of Table 5.1

PROGESTATIONAL ACTIVITY A N D OTHER PROPERTIES OF COMPOUNDS (ACTIVITIES RELATIVE TO THOSE OF PROGESTERONE OR ETHISTERONE)

Progesterone Ethisterone Dimethisterone Norethindrone Norethisterone acetate Norethynodrel Lynestrenol dl-Norgestrel Ethynodiol diacetate

-

1 11-12 [XI]

3~ 4 1 1 ~341 (s.c. prog) -

40 [79] (parenteral prog.) 2 x NEA [86]

Quingestanol acetate Medroxyprogesterone acetate 10&300 [46] Megestrol acetate Chlormadinone acetate 50 x Noreth ~4x1 Algestone acetophenide 1-2 Noreth) [741

1 ~

-

~

+ ve [83]

10 [83]

? +ve +ve [73]

~

0.12 [84] -

91 [73] 1-2 [79] -

McPhail 5&60 [46] -

100 [791

McPhail 1-2 [74]

+ ve-[73] + ve [73]

V. PETROW

1.85

the rabbit endometrium varies directly with progestational activity. The .role of this enzyme in the reproductive process is unknown [66] but may involve some facet of pH control. Oral administration of carbonic anhydrase inhibitors such as acetazolamide to the female rat fails to interfere with the reproductive process [66a]. Deciduoma ,formation

The endometrium of the oestrogen-primed, progesterone treated rodent is extremely sensitive to local stimuli, such as scratching, histamine, etc., when it produces a maternal placental tumour termed a deciduoma. As consistent deciduoma formation may be produced in pseudopregnant animals by ovariectomy, traumatisation of one uterine horn with a needle and progesterone administration, this experimental model and variants thereof provides a good assay for progestational activity. The degree of swelling is scored by comparison with deciduoma produced in intact and untreated females by similar traumatisation. Deciduoma formation induced by progesterone is inhibited by oestrogens and in the intact untreated animal by testosterone. The following results have been reported for the contraceptive progestagens : Medroxyprogesterone acetate : S.C.2-4X progesterone in inducing deciduoma formation in spayed mice. Inactive by the oral route [67]. Chlormadinone acetate : S.C. c. = medroxyprogesterone acetate; orally inactive in spayed mice 1671. Dimethisterone : weakly active in mice [68]. Lynestrenol : inactive in mice [69]. Norethisterone: active [68]. Norethynodrel : inactive p.0. and S.C. but active when administered locally [70]. Ethynodiol diacetate : active [71J. Maintenance of pregnancy

Maintenance of pregnancy represents a classical biological property of progesterone. In performing this assay, Saunders and Elton 2721 spay female rats on day eight of pregnancy (day one represents the day sperm are found in the vaginal smear) and immediately begin supportive treatment with the progestagen until day 18, when the animals are sacrificed and the uteri examined for implantation sites. The following steroids maintain pregnancy : progesterone [73] norgestrel [73] medroxyprogesterone acetate [73]

186

ANTIFERTILITY AGENTS

megesterol acetate [73] chlormadinone acetate [73] algestone acetophenide [74] The following are inactive: testosterone [73] ethynyloestradiol/mestranol norethisterone [73] and its acetate [75] norethynodrel [73] lynestrenol [69] ethynodiol diacetate [73] dimethisterone [76] Drill [77] has pointed out that the ability to support pregnancy correlates with a positive result in the McGinty assay (p. 184) involving intrauterine administration of the steroid. Likewise. inactive steroids are inactive in this assay. INHIBITION OF OVULATION

All steroids* in current contraceptive use and at sufficiently high doses inhibit gonadotrophin(s) and ovulation. Makepeace, Weinstein and Friedman [87] originally noted that progesterone inhibits copulation-induced ovulation in the rabbit. This test was used by Pincus and Merrill [88] in their classical work on steroidal ovulation inhibitors. The compound is administered to the mature female rabbit 24 hours before mating. Activity is assessed by the absence of ovulation points 24-48 hours after mating. Ovulation may also be induced in the rabbit by copper salts [89, 901. The test.suffers from the defect that the rabbit, unlike the woman, is an induced ovulator. Its ovarian follicles consequently tend to develop and die in continuous succession without definite periodicity, thus permitting ovulation to occur at random times following coitus with its resultant gonadotrophin stimulation. Continuous follicular development leads to continuous follicular oestrogen production so that the female rabbit is normally in a state of pro-oestrus. The human female, in contrast, is a spontaneous ovulator with cyclical development and rupture of the ovarian follicle. Her follicular development and ovulation consequently respond to added oestrogen and in this respect differ sharply from that of the female rabbit which is relatively insensitive to this hormone. In spite of this difference in ovulation mechanisms, the rabbit represents an extremely convenient animal model for determining ovulation time, which can be calculated with accuracy following induced ovulation. The use of rodents offers certain advantages in that they, like the human *

Dydrogesterone in currently used doses as a progestational agent does not inhibit ovulation. I t is presently under study in a ‘pill-a-month’ regimen (p. 195).

V. PETROW 187 female, respond to exogenous oestrogens, but again the analogy is not perfect. The assay involves the inhibition of induced ovulation in the immature rat treated with pregnant mare’s serum (PMS) [91] followed by flushing of the oviducts for freshly ovulated ova as evidence of ovulation. The PMS induces the development of the follicles and subsequent release of LH by the pituitary. The progestagen inhibits such LH release. Careful attention to detail is required for meaningful results [92]. Greenwald [93] recommends use of the hamster for this assay. The animals are injected on day one of metaoestrus, which is characterised by a copious vaginal discharge, and killed on day two of the next oestrus cycle. The ovaries are then removed, sectioned, stained and examined. In addition, ova can be flushed readily from the oviducts and counted. Results are obtained four days after injection. Oestradiol and testosterone prevent ovulation by producing atresia of the developing follicles. The progestagens, in contrast, act on the final stages of follicular maturation. An oestrogenic progestagen such as ethynodiol diacetate in low doses blocks ovulation of mature follicles, but with increasing doses induces atresia by virtue of its inherent oestrogenicity. In general terms, results obtained by the hamster assay agree with clinical observations of potency.

Ovulation-inhibiting activities of progestagens in the rabbit (oral) Potency Norethisterone : 1 [941 Chlormadinone acetate : 35 [94] Megestrol acetate 5 [941 Medroxyprogesterone acetate 2.6 [94] Algestone acetophenide: in mouse 5 x progesterone [95] Ethynodiol diacetate: +ve (rabbit) [71] Lynestrenol : Oral, rabbit, 3 x norethisterone [96] Norethisterone acetate : + ve [97] Norethynodrel: 25 per cent less effective than norethindrone [96] INHIBITION OF GONADOTROPHIN

(see Table 5.2)

Parab iotic technique

This technique is widely used for testing the antigonadotrophic activity of steroids. Two rats, generally c. 30 days of age, are surgically joined through body wall incisions. One of the partners is castrated at the time of surgical union. When two female rats are used, hypersecretion of gonadotrophin occurs in the castrated animal resulting in ovarian weight increase in the intact partner. By administration of a gonadotrophin-inhibiting steroid such

Table 5.2

Dimethisterone Norethindrone Norethindrone acetate Norethynodrel Lynestrenol dl-Norgestrel Ethynodiol diacetate Quingestanol acetate Medroxyprogesterone acetate Megestrol acetate Chlormadinone acetate Algestone acetophenide

OTHER PROPERTIES OF ANTI-FERTILITY COMPOUNDS

Androgenic

Anabolic

-ve [IOO] 2 % T.P [85] 2 3 % T.P [85] - ve [85]

-ve [IOO] 1 T.P [85] 4 % T.P [85]

I % T.P [85] -

- ve [ I I I ] -ve [ I 121 - ve [74]

-

Rat foetus masculinisa t ion

Antiandrogenic

Glucocorticoid

-

+ve [I041 +ve (chickcomb) [78]

f v e [lo21

-

+ve [98] +ve [I051 +ve [85] -

-

-

~

~

__ -ve[112] -

-

~

-

? +ve [ 8 5 ]

+ve [I061

~

+ve [98] -ve [1 I'll -ve[113] -ve [74]

~

- ve

[7 I] -

+ve (chickcomb) [78] +ve [lo41 +ve[114] -ve [I151 +ve (chickcomb) [78]

Adrenal A rropliy

-

-ve [I011 -ve [I021

-

-

-

+ve [I071

-

-ve [71] +ve [I091 - ve [ I I I] - ve [ I 121 -ve [I 151

--ve [I021 -

+ve [I021 +ve [ 1 101 ~

V. PETROW 189 as oestradiol, such ovarian hypertrophy may be prevented. Use of this technique shows that it is the oestrogenic component of the combination pill that is primarily responsible for the inhibition of ovulation observed with preparations of this type.

Androgenic and anabolic activity Male rats 23-25 days of age are castrated. Three weeks later the test compounds dissolved in corn oil are administered intramuscularly daily for seven days. One day after the test injection the animals are sacrificed and the seminal vesicles, ventral prostates and levator ani muscles removed and weighed. Comparison with data obtained from untreated controls permits evaluation of androgenic effect (seminal vesicles and ventral prostates) and anabolic effect (levator ani). The levator ani muscle responds to some extent to androgens per se. Nitrogen retention studies are therefore preferred as a more valid index for anabolic activity. Anti-androgenic activity Castrated albino rats are treated one day after surgery with testosterone propionate and the test compound, separately dissolved in sesame oil and injected daily for seven days at separate sites. Twenty-four hours after the last injection the rats are sacrificed and the body, seminal vesicle and levator ani weights determined. Masculinisation of the rat female foetus [98] Many cases. of human female pseudohermaphrodism have been reported following administration of progestagens during gestation. Such masculinisation has generally been attributed to inherent androgenicity of the progestagens as androgens administered during pregnancy are known to produce pseudohermaphrodism in the human [99]. Such androgenic activity may be determined by administering the progestagen daily subcutaneously in sesame oil from days 15 through 20 to the pregnant rat. Pups are removed by Caesarean section on the 22nd day of pregnancy and the anogenital distance determined. They are placed with foster mothers and sacrificed on day 20 for dissection and examination of the reproductive system. An androgenic progestagen such as norethynodrel administered as above in a dose of 0.5 mg daily increases the anogenital distance in the female pups from 1.5+ O G 4 mm to 1.810.03 mm. Progesterone is inactive in this assay.

190

ANTIFERTILITY AGENTS

CONTRACEPTIVE REGIMENS The most important regimens currently in use or under study include : (a) the combination pill in which ovulation is inhibited by a progestagen plus an oestrogen. (b) the sequentiallserial method in which ovulation is inhibited by an oestrogen, with progestagen added towards the end of each cycle of administration in order to induce withdrawal bleeding. (c) the minipill in which infertile menstrual cycles are enforced by daily administration of progestagen. ( d ) long acting oral, parenteral and vaginal preparations based upon progestagen or upon an oestrogen plus progestagen combination. ( e ) a precoital pill based upon a progestagen. ( f ) a weekend pill based upon an antiprogestational steroid and ( g ) the postcoital use of oestrogens. THE COMBINATION PILL

Counting the first day of menstruation as day one, administration of the combination pill is normally started on day five and continued for 21 days. Withdrawal of hormone administration on day 25 is generally followed by withdrawal bleeding about four days later on day 29, which is day one of the new cycle. In practice, small differences in withdrawal bleeding time are of no significance and a ‘three weeks on medication and one week off medication’ type of regimen is the general rule. The efficacy of the combination pill approaches 100 per cent. Its primary mode of action is undoubtedly inhibition of ovulation generally by suppression of the mid-cycle burst of LH. Varying degrees of suppression of FSH also occur depending upon the preparation used [116, 1171. In view of the pill’s efficacy, it is somewhat surprising that only 95 per cent of cycles appear to be anovulatory [118, 1191. It follows that in addition to ovulation inhibition, other contraceptive mechanisms are invoked. Thickening of the cervical mucin [120, 1211 is observed in ‘progestagenic combinations’ (vide infra). Some degree of endometrial atrophy with glandular regression occurs with consequent production of a uterine milieu unfavourable to implantation [122]. Other mechanisms are reviewed by Chang [123]. Recently Jakobovits, Gecse, Piukovich, Szontagh and Karady [124]have shown that 19-norsteroids diminish the intensity and frequency of peristalsis of the Fallopian tubes, whilst combinations of oestrogens and progestagens significantly reduce muscular tone. The authors suggest that these effects may be significant components in the contraceptive actions of these hormones. Ethynyloestradiol and mestranol are the oestrogens used in combination products. The following progestagens are used (cf. Maas [125] for tabulated

V. PETROW 191 list of o.c.’s currently available in the U.S.):norethindrone; 19-norethisterone acetate ; quingestanol acetate ; f-norgestrel ; lynestrenol; norethynodrel ; ethynodiol diacetate; medroxyprogesterone acetate; megestrol acetate; and chlormadinone acetate. The use of -norgestrel combined with ethynyloestradiol has recently been reported by McBride [ 1261.

+

SEQUENTIAL PRODUCTS

The sequential method of oral contraception involves the use of an oestrogen to inhibit ovulation, mainly by blocking FSH release. Progestagen is additionally administered towards the end of the cycle in order to promote maturation of the endometrium and to facilitate withdrawal bleeding. A typical regimen involves administration of 16 tablets of oestrogen starting on day five of the cycle, followed by five tablets of oestrogen progestagen. Withdrawal bleeding generally begins two-four days after completing the regimen and represents day one of the new cycle and so on. Other sequential regimens include, for example, the use of seven oestrogen tablets followed by 15 oestrogen progestagen tablets [127]. The sequential method gives a more normal endometrium than the combination pill [128, 1291, but the pregnancy rate is somewhat higher. In contrast to combination products, there is a significant risk of pregnancy following omission of a pill. There is also some presumptive evidence that ovulation may occur in ‘predisposed women’ following a major emotional episode. Ovulation is probably inhibited in c. 95 per cent of the cycles. The absence of progestagen at mid cycle as in combination products may account for the higher failure rate [130]. Mestranol and ethynyloestradiol are used as oestrogens. Dimethisterone, chlormadinone acetate, megestrol acetate, norethindrone and its acetate, lynestrenol and norgestrol are used as progestagenic compounds.

+

+

SERIAL REGIMENS

The serial regimen [131] was based upon the observation that sequential products generally gave such good cycle control that it was possible to include seven placebo tablets at the end of each course of medication (16 x oestrogen, five x oestrogen + progestagen, seven x placebo). In this way, one tablet a day could be taken continuously, thus obviating the need to remember to start medication on day five. In practice, it was not entirely successful as a significant number of women were found to behave atypically by starting withdrawal bleeding on days 27 or 28 instead of day 29. By taking seven placebo tablets between courses of medication, such women were effectively beginning medication on days six or seven of the new cycle, thereby permitting follicular development to get under way with consequent risk of pregnancy.

192 ANTIFERTILITY AGENTS In an attempt to overcome the increased risk of pregnancy on the serial regimen, McBride [132] introduced a 16-7-5 regimen with medication beginning on day three. An improved pregnancy rate of 0.18/100 woman years was reported [133]. A modified 15-8-5 regimen has been described by Andelman [ 1341. Sequential/serial methods are based upon doses of oestrogen greater than 50 pg and are thus proscribed by the Committee on Safety of Drugs in the U.K. Mestranol and ethynyloestradiol represent the oestrogenic components in the above preparations. Various progestagens have been employed (see sequential regimen).

THE MINIPILL

Much attention is presently being directed to the ‘minipill’, which comprises a low dose of progestagen administered daily and continuously whilst contraception is desired (see Petrow [135] for historical review). Ovulation is probably inhibited in perhaps one third of the women on this formof medication. Some confusion exists on this point and some workers, for example Van Leusden [136] using 500pg megestrol acetate, claim that in a small series (20 women) ovulation inhibition was not observed. Cervical mucin becomes scanty, spinnbarkeit disappears and cell-count increases [ 1371 with consequent prevention of sperm migration into the uterus [137]. This effect has been ascribed to the antioestrogenic activity of the progestagen [138]. The role of cervical factors in this method of contraception is sufficiently important to warrant a separate section (p. 197). Other contraceptive mechanisms also brought into play include effects upon sperm capacitation [ 1391 and upon the transport of ova [140]. Chlormadinone acetate* (500 pg) [141], megestrol acetate (500 pg) [142], norethisterone acetate, norgestrel (50 pg) [140] quingestanol acetate are all currently under study. As oestrogens are presently suspect for their alleged role in producing thromboembolism (see p. 208), considerable efforts are being devoted to the development of the ‘minipill’ concept. Side effects including frigidity, increase in weight, migraine, nausea and depression are minimal with this method. Blood changes predisposing to thrombosis, as evidenced by classical blood parameters, do not appear to be significant (see, for example, Jeppsson and Kullander [143], who used 500 pg chlormadinone acetate daily in their study). The main disadvantage lies in irregular bleeding or spotting, which may occur in c. 20 per cent of the women during the first cycle, dropping to 12 per cent by the fifth cycle. Such irregular bleeding seems to be the main * Clinical studies temporarily suspended in January

1970

193 reason why women discontinue medication. Pregnancy rates are probably of the order of 4 per 100 woman years, i.e. similar to the I.U.D. and thus acceptable. They increase markedly, however, following omission of medication [1441. There is, consequently, a body of expert opinion that supports the view that the current minipill is unlikely to offer a serious challenge to existing methods of contraception [145]. Improved cycle control with continuous chlormadinone acetate administration has been effected by concomitantly administering ethynyloestradiol on days 19 or 21 through 25 of each cycle [146]. Such methods, however, though attractive in theory, involve the use of memory packs, thereby eliminating the advantages inherent in daily administration of a single chemical entity. Considerable effort is being devoted at the present time to new drug delivery systems for continuous progestagen administration. These include administration of the progestagen in (a) silastic capsules implanted subcutaneously [147], (b)a silastic cylindrical ring placed in the woman’s vagina [148], and ( c ) a silastic I.U.D. placed in the uterus. All these methods show promise at the present time and may well provide second generation contraceptives based upon the minipill concept. Thus, for example, Croxatto, Diaz, Vera, Etchart and Atria [149] (see also [150]) have reported studies with silastic capsules containing megestrol acetate and introduced subcutaneously into the ventral aspect of the forearm through an 1 I gauge trocar. Two sizes of capsules were used delivering, in toto, 78 (three capsules) and 104 (four capsules) pg megestrol acetate per 24 hours, respectively, as determined by in vitro studies. Ninety-seven women were used in the study with 160 womanmonths of experience at the lower dose level and 595 at the higher dose level. The results shown in Table 5.3 were obtained: V. PETROW

Table 5.3

THE EFFECT OF MEGESTROL ACETATE (MA) ON HUMAN CONTRACEPTION

Conrrols

Total ovulatory cycles Cycles with B.T.B. Cycles with spotting Spinnbarkeit Endometrium-normal secretory irregular secretory irregular or inactive Pregnancies

Lower dose M A

Higher dose M A

92 8-3% 21.2% 5.8 f2.2

95 % 8.2 % 16.8% 4.4f 1.6 30 % 61 % 9% 4

~

5

The theoretical functional life of each implant was calculated to be approximately lo00 days, but this point was not established as the study was continued for only 12 months. The contraceptive effectiveness of the silastic

194 ANTIFERTILITY AGENTS implant was nevertheless established. In studies with the silastic vaginal ring impregnated with medroxyprogesterone acetate (vide supra) and inserted into the vagina on day five for 21 days, presumptive evidence of ovulation was obtained in some subjects, so that the method offers possibilities for development. Some degree of erosion and ulceration of the vaginal mucosa and submucosal tissue in the posterolateral fornices was noted. The intrauterine administration of progesterone, contained in a silastic capsule attached directly to a Lippes loop (see Figure 5.6) and introduced into the uterus in the usual way, has recently been reported by Scommegna,

\-

Figure 5.6. IUD with Silustic capsule containing progesterone

Pandya, Christ, Lee and Cohen [I511 (see Petrow [135] for early references). The 30 mm capsules released c. 300 pg progesterone/day and were effective for about three months. In a small group of volunteers, the expulsion rate was 13 per cent, which was not significantly different from that using the standard I.U.D. Endometrial suppression indicative of progesterone effect became apparent 18 hours after insertion of the capsule and was characterised by decidual or pseudodecidual reaction with atrophy of glands and prominence of arterioles and venous sinuses. Menstrual cycles and ovulation patterns were virtually unchanged, thereby furnishing presumptive evidence for a local rather than a systemic action of the progesterone. Further development of the concept should present no difficulty providing such dual action I.U.D.3 offer advantages over the conventional types. More potent progestagens would need to be employed in order to provide devices with longer effective lives. In addition, the concept could be extended to other compounds-not necessarily steroidal-with highly specific antifertility effects.

195

V. PETROW

DEPOT PREPARATIONS

Injectible progestagen preparations for intramuscular administration have proved both effective and acceptable. Initial studies used a depot form of medroxyprogesterone acetate [ 152, 1531. Dosage at the rate of 150 mg/three months was found to be satisfactory [154]. Inhibition of LH release occurred under these circumstances [ 1551, with increase in the viscosity of the cervical mucin and thinning of the endometrium. Lactation generally was not decreased. Irregular bleeding and a significant incidence of post treatment amenorrhoea lasting up to 18 months did not appear to detract from the acceptability of the method [156]. In some studies 10 oestrogen tablets/month were administered to regularise bleeding. The pregnancy rate was less than 0.5/ 100 woman years [ 1571. Other progestagens used in the same way include chlormadinone acetate and norethisterone enanthate [158, 1591. Preliminary experiments on rats, using medroxyprogesterone acetate in microcrystalline suspension, seem to indicate that administration of depot o.c.’s by jet injection through the skin may lead to more regular cycles without increased local pain during administration 11601. Monthly injections of algestone acetophenide (1 50 mg) and oestradiol enanthate (10 mg) in oil administered intramuscularly at the beginning of each cycle have also proved effective as a parenteral contraceptive [161, 1621. Application of the depot concept to an orally administered formulation was first achieved by Greenblatt [26], who used the long acting oestrogen, quinestrol, in combination with a short-acting progestagen such as chlormadinone, dydrogesterone, 6,17a-dimethylpregna-4,6-diene-3,20-dione or norgestrel. The combination .was administered monthly on day 25. Side effects included hypermenorrhea, nausea and breast soreness. Another preparation of the same type is based upon the administration of quinestrol on day one of the cycle, followed by quinestrol quingestanol acetate on day 22 of the cycle, the latter combination then being administered every four weeks without regard to the timing of withdrawal bleeding [163, 1641. The product proved to be an effective contraceptive with an incidence of side effects similar in type and frequency to those encountered with the conventional combination pill. A small scale study using quinestrol + dydrogesterone (XXIX) has been reported [165] with similar results. The method has been well received [ 1661. The use of a silastic vaginal ring impregnated with medroxyprogesterone acetate and inserted on day one of the cycle for 28 days represents yet another variation of the depot concept [ 1671. LH release and ovulation were inhibited as expected. Removal of the ring was followed by withdrawal bleeding in about 48 hours. In contrast to the depot injection, this technique provides rapid termination of drug action.

+

196

ANTIFERTILITY AGENTS

(XXIX)Dydrogesterone

(XXX)R = H (XXXn)R = M e

THE PRECOITAL PILL

Cox (168) has described the precoital use of ‘minipill’ progestagens. Using megestrol acetate (500 pg), he found that the effect of a single minipill upon cervical mucin was fully apparent four hours after administration and lasted c. 18 hours (cf. p. 192). By restricting coital activity from four hours through 14 hours after administration of the pill, excellent fertility control was obtained in a limited study. Further work will be awaited with interest. THE WEEKEND PILL

The Roussel-UCLAF group [169] had previously synthesised 17a-ethynyl17P-hydroxyoestra-4,9,11-trien-3-one (XXX), which, in admixture with 50 pg ethynyloestradiol, had been introduced into France as a conventional ‘combination pill’. Subsequent work by the group had led to the total synthesis of its 18-methyl analogue (XXXa) (R2323), which was found by Sakiz and Azadian-Boulanger [ 1701 to show pronounced antiprogestational activity with some androgenic activity and low or very low oestrogenic, anti-oestrogenic, progestational and pituitary inhibitory activities. Further study showed that the compound inhibited implantation in the assay of Banik and Pincus [ 17I]. On the basis of its marked anti-progestational and anti-implantation properties, the product was submitted for clinical trial as a ‘weekend pill’ (Pincus). Studies in progress employing a single weekly dose of the order of 2.5 mg appear encouraging, with BTB as the main adverse reaction. Ovulation is not inhibited, nor is there evidence of changes in blood biochemistry characteristic of the combination pill. Critical analysis of the data must await its publication. POSTCOITAL USE OF OESTROGENS

The postcoital use of oestrogens stems from the early studies of Parkes, Dodds and Noble [ 1721on the anti-implantation and abortifacient properties

V. PETROW 197 of oestrogens in the experimental animal. Small wonder that this class of compounds has been used sporadically as a postcoital/abortifacient pill for many years. It undoubtedly shows considerable efficacy when taken ‘the morning after’, but the usual regimen of one mg EE, taken twice daily for five days, so often induces nausea and vomitting [173] as to make the method unattractive. Administration of oestrogens during the implantation phase of pregnancy is ineffective. Thus Bacic, de Casparis and Diczfalusy [174] have administered up to 5 mg daily of EE for seven days starting on days 36 to 46 without evidence of abortifacient effect. Nausea generally occurred on day one only. It follows that ethynyloestradiol is not a reliable abortifacient in the woman. This result underlines the lack of correlation that often exists between the animal model and the human subject.

CERVICAL FACTORS IN FERTILITY CONTROL The introduction of the minipill and the recognition that its contraceptive efficacy stems to large extent from its effect upon cervical factors has focused attention upon the vital role of the endocervical canal in human reproduction [175]. Like Janus, the old Roman god of doorways, the cervical structure mounts guard over the entrance to the interior of the female, controlling all that would enter and all that would leave. This is achieved: (a) by variation in the diameter of the canal and the size of the os, (b) by variation in the quantity of the secretion of the endocervical glands that line the canal (cf. Table 5 . 4 , (c) by variation in the composition and physico-chemical properties of the secretion.

Type E mucus

Type

Figure 5 . 7 .

G mucus

198 Table 5.4

ANTIFERTILITY AGENTS CHARACTERISTICSOF CERVICAL MUCIN (OVULATION ASSUMED TO OCCUR ON DAY ~~

Day of cycle

8

Spinnbarkeit Fern test Viscosity Quantity Cells Sperm penetration Mucin

+ 0

10

12

++

+++

++

+++

+ +++

14)

~

+

++

++

16

14

++++ ++++ + +++ 0

18

20

22

++

+

0

0

0

+++

+ ++

+

+++

+

max

POSTMENSTRUAL A N D FOLLICULAR PHASES

Immediately postmenstrually, the cervical 0s is small and contains a plug of thick, turbid, tenacious much which effectively blocks entrance of foreign matter to the regenerating but still somewhat raw endometrium. With the start of the follicular phase of the menstrual cycle and under the influence of increasing levels of oestrogen, the cervix becomes more patulous with slow increase in calibre. Concomitantly, and under the action of the female hormone, the endocervical glands begin to produce a mucin which becomes increasingly more favourable in physico-chemical characteristics to the migration of sperm. This process continues up to the time of ovulation when a synchronous situation obtains that is optimal for fertilisation to occur. Thus : ( a ) the released ovum will be travelling down a Fallopian tube and will be ready for fertilisation, (b) the cervical 0s will have maximal calibre and the mucin-filled endocervical canal will have become an exquisitely designed conduit for migration of sperm. (c) the production of cervical mucin will be approximately maximal. (d)the physical structure and composition of the mucin will be optimal for sperm migration. This point needs further elaboration.

Characteristics of Mid-Cycle Mucin Mid-cycle mucin forms a thin, clear, watery liquid that can readily be drawn out into threads 1&15 cm long (‘spinnbarkeit’). It is rich in sodium chloride, which is a decisive factor in determining consistency and sperm penetration, so that, when dried on a slide, crystals of salt form in tree-like or fern-like patterns (arborisation). Viscosity is minimal and cellular debris virtually absent. The acellular part of cervical mucin is composed of cervical plasma and

V. PETROW 199 cervical mucoid. Cervical plasma is not unlike blood plasma minus fibrinogen. Cervical mucoid, which is responsible for the viscoelastic properties of the mucin, appears to consist mainly of a protein(s) and of mucopolysaccharides. Characteristically, it behaves as though its molecules were long, flexible and thread-like and arranged in random coil configuration [176]. As much is formed in the endocervical glands, so rods of high viscosity much are extruded into the endocervical canal (see Table 5.4) separated by lower viscosity mucin. Together these minute mosaic parts form a three dimensional mosaic pattern. As flow down the cervical canal occurs, so stretching and alignment of the mucoid molecules along the lines of strain takes place with the result that the intracanicular mosaic patterns become long and thin. The micelles in such mucin-termed E mucin-are believed to be present as long chains arranged in approximately parallel order [177] (see Figure 5.7) and of such calibre as to permit rapid migration of normal sperm but to present an effective barrier for immature or abnormal forms such as ‘pinheads’ and large. Penetration of such mucin by normal spermatozoa is rapid, occurring usually in 1&3 minutes. Their mobility, coupled with their shape which conforms to the theoretical aerodynamic ideal required for minimum deformation of lines of strain in the medium, carries them rapidly at the rate of 1.5-2.0 mm/min through the cervical canal and into the uterus where uterine contractions, possibly aided by oxytocin or prostaglandin, aid further spermatozoa1transport. Phagocytes from higher up the genital tract are immobilised by cervical mucus, but predation by phagocytes in the uterus and Fallopian tubes does occur [178]. In addition to permitting sperm to ascend into the uterus, type E cervical mucin forms only an imperfect barrier to bacteria. Cervical mucin from all phases of the menstrual cycle exerts a bactericidal effect but this is least pronounced during the ovulatory phase. In addition, as recently shown by Enhorning, Huldt and Melin [179], Proteus mirabilis can migrate along a nylon tube filled with such mucin but not with mucin collected at other phases of the menstrual cycle. The migration of bacteria is very slow, however, and it seems unlikely that, in vivo, many could move against the stream of descending mucus. LUTEAL PHASE

Following ovulation and development of the luteal phase, oestrogen levels fall and increasing quantities of progesterone pass into the blood stream. The effect of this change in hormonal domination upon the cervical structure is dramatic. Within 24-48 hours of ovulation, the calibre of the cervical os begins to diminish. The canal narrows and, as progesterone domination reaches its peak, and particularly during pregnancy, so the isthmus becomes a physiological sphincter [180].

200 ANTIFERTILITY AGENTS Equally significant changes occur in the cervical mucin (see Table 5.4). Sialic acid content increases as does the degree of coiling of the mucoid molecules. Spinnbarkeit falls and arborisation on drying disappears. The micelles of the resulting mucin, termed G-mucin [177] now form a dense network (see Figure 5.7) containing much cellular debris thereby providing conditions inimical to sperm migration and sperm penetration [181]. The mucus barrier is equally effective against bacteria [179] so that the uterus is now sealed off from the outside environment, thereby affording maximal protection to the descending and implanting zygote. Maximal consistency is reached during pregnancy. These changes are summarised in Table 5.4 [182]. As may be expected, ovariectomy leads to cessation of cervical mucin production. Endocervical mechanisms of fertility control are characteristic of the Weal phase-‘the safe p e r i o d ’ 4 f the menstrual cycle and are also of paramount importance in the ‘combination pill’ regimens [ 1201and in the various ‘minipill’ regimens [137, 183, 1841 and in the precoital use of progestagens [168]. It must be remembered, however, that the changes induced in endocervical mechanisms by progestagens are not yet fully understood and there is no doubt that gross examination of mucin is not a reliable guide to contraceptive action 11451. THE EFFECT O F ORAL CONTRACEPTIVES UPON LACTATION Oestrogens are known to inhibit lactation in the woman on a dose-related basis [185]. It may consequently be expected that oral contraceptives containing oestrogen will, in general, be inimical to lactation and, by and large, this is found to be the case (see for example [186-1881). Unfortunately, the picture is neither clearcut nor consistent. Results vary with (a) the ethnic group involved, (b) the socioeconomic background, (c) the day postpartem when medication was commenced, (d) the length of treatment, (e)the contraceptive regimen and v) the nature and quantities of the hormones employed. It is this complex combination of factors that is probably responsible for such diverse results as those recorded by Kaern [190], who found that administration of norethindrone (1 mg)+mestranol (0.05 mg) to 451 lactating women from day one through day eight led to a decrease in milk production, whilst Semm [191], employing lynestrenol (2.5 mg) + mestranol (0.075 mg), found (a) that 100 lactating women treated from day one through day ten postpartum had no change in milk production and (6)that 70 lactating women similarly treated from day 10 through day 31 postpartum incieased their milk yield. A similar divergence of data has been recorded for the sequential regimen. Thus, Katesi, Goss and Townes [192] started medication on the fifth day postpartum and observed no significant impairment of the ability to breast feed whilst Andres, Ibarra, Faundes and Guiloff [193] in contrast,

V. PETROW 201 observed dramatic inhibition of lactation. The minipill, per se, seems 'to exert little effect [194]. In these circumstances it seems difficult to avoid the conclusion that combination and sequential oral contraceptives are best avoided in the underdeveloped areas of the world where milk substitutes are not freely available. Postpartum breast engorgement can be effectively suppressed by both the combination pill and the sequential regimen [195-1961. There is decreased lochial flow and a predictable onset of menses, with the additional social benefit of fertility control.

SELECTION OF AN ORAL CONTRACEPTIVE The progestagens used in oral contraceptives differ from each other and from progesterone in their biological profiles. They are conveniently classified, from the standpoint of the present review, as follows: Progestagens which are potent anti-androgens and are devoid of oestrogenic and androgenic properties: chlormadinone and megestrol acetates, dimethisterone [104]; Progestagens devoid of androgenic, oestrogenic and anti-androgenic properties: algestone acetophenide ; Progestagens with slight androgenicity : medroxyprogesterone acetate ; Progestagens with some androgenicity and with oestrogenic metabolic products: norethindrone, norethisterone acetate, lynestrenol, quingestanol acetate, norgestrel ; Progestagens with some oestrogenic and androgenic properties: ethynodiol diacetate ; Oestrogens with some progestational and no androgenic activity : norethynodrel. No two contraceptive steroids are equal. In addition, their biological differences are carried over into the contraceptive preparations per se, be these combined, sequential or mini-regimens. It is therefore possible to classify oral contraceptives as progestational/androgenic-as in the norgestrel minipill-through to frankly oestrogenic-as in the norethynodrel/ mestranol combination 1125, 197, 1981 and to attempt a correlation between their overall hormonal characteristics and the incidence of those more common side effects that are believed to stem from hormonal inbalance [199], thus: Progestagen excess : decrease of libido, tiredness, depression. Increased appetite with resulting weight gain. Oestrogen excess : mastalgia, hypertension, fluid retention, gastrointestinal upsets, mucous discharge, uterine cramps, chloasma. Oestrogen deficiency : dyspareunia, depression of oestrogen sensitive tissues.

202 ANTIFERTILITY AGENTS Change of preparation often leads to acceptability. It must be remembered, however, that the incidence of the foregoing side effects varies with the ethnic type, with parity and age and with the psychosomatic type, so that many factors need to be considered in the selection of a preparation. ADVERSE EFFECTS Early clinical studies on oral contraceptives revealed certain troublesome side-effects such as nausea and breakthrough bleeding (BTB), but on the whole the preparations were remarkably free from adverse reactions that presented a serious hazard to health. As the use of oral contraceptives became world wide, so reports of adverse reactions became more frequent. In 1961 [200], the first case of pulmonary embolism following the use of oral contraceptives was reported and attributed to the vomiting caused by the medication. The following year Ockner and Davidson in a review [201] described a case of jaundice. Since then a whole literature has accumulated [202] dealing with the metabolic effects of contraceptive steroids. Many of the findings are non-controversial and many of the changes are apparently reversible on withdrawal of medication even after a few years treatment. Information is sadly lacking, however, on the results of long term continuous administration which, by definition, cannot be available at this time. There is less agreement on some of the very infrequently observed adverse side effects such as thromboembolism which may well be idiosyncratic and the result of an unusual combination of physiological events which may never be repeated, and thus outside the scope of normal statistical analysis. Also, as stressed above, no two oral contraceptive formulations are 'equal' in their total metabolic effects so that extrapolation of data from one preparation to another is not strictly valid. CARBOHYDRATE METABOLISM

Impaired glucose tolerance not infrequently occurs in pregnancy [203]. In addition, progressive biological resistance to endogenous insulin is a normal concomitant of advancing pregnancy. This is accompanied by increased plasma insulin levels with but little change in carbohydrate tolerance. Prediabetic and overtly diabetic women, in contrast, generally show a marked deterioration in carbohydrate tolerance during late pregnancy in spite of higher levels of circulating insulin [204]. The foregoing effects of pregnancy in normal women have recently been reproduced in both normal men and in hysterectomised women by i.m. administration of progesterone [205]. The results suggest that progesterone, like pregnancy, can evoke an enhanced plasma insulin response to insulinogenic stimuli. Oestrogens, too, affect

V. PETROW 203 carbohydrate metabolism. Thus, Talaat, Habib, Higazy, Abdel Noby, Malek and Ibrahim [206] have reported that administration of oestradiol dipropionate to nondiabetic women increases the arterio-venous gluco ,e difference and results in increased sensitivity to exogenous insulin. In these circumstances it is not surprising that similar changes are often observed during oral contraception medication. The picture is complicated, however, by significant differences in the effects produced by the various oestrogens and progestagens per se, as well as by such factors as age, parity, family history of diabetes, and the existence of subclinical and frank diabetes mellitus. Gershberg, Javier and Hulse [207l were the first to show impaired glucose tolerance in women treated with an oral contraceptive (a mestranol-norethynodrel combination). Patients with an obstetrical or family history of diabetes showed a greater tendency for the glucose tolerance to deteriorate. Since then a considerable literature has accumulated on the subject. In general terms, the present picture is as follows: 1. Mestranol' and mestranol-containing oral contraceptive preparations seem to impair glucose tolerance possibly more than ethynyloestradiol and oral contraceptive preparations derived therefrom [208], particularly where there is a history of gestational (subclinical) diabetes. This situation may be reversed in the presence of progestagen (vide infra). 2. Goldman, Ovadia, and Eckerling [209] have reported that the chlormadinone minipill ( 0 5 mg/day) does not appear to show consistent changes in glucose tolerance in groups of both normal and subclinical diabetics. The mean integrated plasma insulin response, however, is elevated in the normal but not in the diabetic subjects. Somewhat different results have been obtained by Larsson-Cohn, Tengstrom and Wide [210] who, using mainly young healthy university students for their study, failed to observe statistically significant differences in the glucose tolerance tests (i.v.) and in plasma insulin levels before and after administration of the norethindrone (0.5 mg daily) and chlormadinone (0.5 mg daily) minipills. It seems likely, however, that the contraceptive progestagens may produce significant deterioration in glucose tolerance in susceptible subjects. 3. Sequential type products, as may be expected, produce a monthly biphasic response. In general terms, glucose tolerance tends to decrease during the oestrogen phase of the cycle and to improve during oestrogen/progestagen administration [21 I]. Spellacy [212] reports that in a group of women on a sequential preparation based upon mestranol and chlormadinone, glucose and insulin levels which were elevated after 6 months of treatment, returned to normal after 12 months. Yen and Vela [213] studied sequential preparations based upon EE (100 pg) and dimethisterone (25 mg) and upon mestranol (100 pg) and ethynodiol diacetate (1 mg). After three months administration, glucose tolerance tests were unchanged, but were associated with raised serum insulin and serum hormone ( x 3 over normal fasting ambulatory levels).

204 ANTIFERTILITY AGENTS 4. In general, combination type oral contraceptive products produce effects upon glucose and insulin levels. Initially a rise in both glucose and insulin levels is observed. As insulin levels rise, so glucose levels tend to fall to pretreatment levels. With prolonged administration, glucose levels may rise in certain women [212]. Both intravenous and oral glucose tolerance becomes relatively impaired [214, 21 51. Exceptions to the foregoing generalisation have been reported. Thus, Starup, Date, and Deckert [216] found that in a group of normal women after 12 months of treatment with megestrol acetate (5 mg)/mestranol (0.1 mg) no changes in fasting blood glucose, glucose disappearance rates, serum insulin concentrations, and plasma insulin responses to intravenous glucose, could be detected. 5. Prolonged treatment of women for 3 4 years with combination products, particularly those containing mestranol, may lead to an acquired form of subclinical diabetes [207, 2081 but there is no evidence at present that in the normal woman such diabetes does not regress on discontinuation of treatment.* Equally, there is no reason to assume that in the preclinical diabetic the administration of oral contraceptives will slow down the natural progression of the disease. This fact must be borne in mind in considering the effect of oral contraceptive administration in such cases. 6. Blood glucose levels rise significantly in overt diabetics on such preparations as the mestranol/ethvnodiol diacetate combination product. .~ Mestranol appears to be the component primarily responsible for this effect 12181. Wynn and Doar [215] point out that there are several metabolic patterns shared by nonobese women on oral contraceptive therapy, nonobese subjects on glucocorticoid therapy and obese nondiabetic subjects. In particular, the fasting plasma glucose levels are generally normal in these three groups, but oral glucose tolerance is impaired and the fasting blood pyruvate level and/or the increase above the fasting level after glucose administration may be raised abnormally. It seems likely that, as in pregnancy, the foregoing metabolic shifts to the prediabetic state stem largely from oestrogen [219]. The latter hormone is known to produce diabetes when administered together with cortisone to the normal rat [220]. In the diabetic human, subdiabetogenic doses of corticoids produce marked glycosuria when administered concomitantly with oestrogen [221]. Large doses of oestrogens potentiate the antiinflammatory effects of cortisone in the normal woman which fact may explain, inter a h , the remissions in rheumatoid arthritis that often occur during pregnancy. In summary, therefore, it seems reasonable to put the hazard resulting from changes in glucose tolerance and insulin levels * Wynn

[217] reported verbally at the 3rd International Congress on Hormonal Steroids, Hamburg, 7-12 Sept. 1970 that permanent diabetes has resulted as sequelae to the combination pill.

V. PETROW 205 following oral contraceptive administration into the same category as the generally accepted risks associated with obesity and pregnancy.

LIPOPROTEINS

Women prior to the menopause have long been known to have a lower incidence of atherosclerosis than men although their relative immunity declines after the menopause. As gonadal hormones affect plasma lipids (see Table 5.5) and as the latter are widely believed to be a key factor in this disease process, the effect of oral contraceptives upon plasma lipids is a matter of some importance. By means of precipitation methods of ultracentrifugation, plasma lipoproteins have been divided into 3 main groups, as shown in Table 5.6. Most of the plasma cholesterol is found in the p- or low density lipoprotein (LDL), with a carry over to the CI- or high density fraction (HDL). Triglycerides, which are regarded by some authorities as a more significant parameter in atherosclerosis, are found primarily in the very low density pre-P fraction (VLDL). Some general observations on plasma lipids are of relevance to the present discussion. In normolipemic subjects, administration of oestrogens increases HDL (i.e. phospholipids) and VLDL (i.e. triglycerides) with little effect upon cholesterol [222]. The plasma of young women is normally richer in HDL and poorer in LDL than in young men of comparable age, but this difference in the p/a ratio can be largely eliminated by treatment of the men with oestrogen [223]. It is not surprising in these circumstances that androgens have the reverse effect [224] upon serum triglycerides and phospholipids with variable effects upon serum cholesterol [225]. Progesterone in physiological doses has little effect upon serum lipids [223, 2261. Serum lipid concentrations vary during the menstrual cycle with serum cholesterol and phospholipid minimal at approximately the time of ovulation [227]. Serum lipids, triglycerides and cholesterol increase during pregnancy concomitantly with increased placental oestrogen synthesis [228, 2291. The effect of oral contraceptives upon plasma lipids, cholesterol and triglycerides follows generally the predominant hormonal characteristics of the product. It must be noted, in this connection, however, that synthetic androgens can override the effects of oestrogens upon serum lipids with resulting markedly elevated LDL and cholesterol levels [230]. As the progestagenic components of oral contraceptives vary from the significantly androgenic norethisterone acetate to the antiandrogenic megestrol and chlormadinone acetates, the effects of different preparations upon the serum lipid and cholesterol picture are definitely not equal. Oral contraceptive preparations based upon EE and a progestagen derived from progesterone produce the anticipated effects upon lipids (Table

Table 5.5

VLDL Pre-B

EFFECT OF GONADAL HORMONES UPON PLASMA LIPIDS

LDL

HDL

B

a

Triglycerides

t

Oestrogen

t

t

Androgens

1

1

Progesterone

+-------------------

Ovulation Pregnancy 13- 18th week

t

t

t

Cholesterol

slight

phi?!:1

Ratio

t -Tc-G. t' - cP 1 -c- . 1 - c TG' P

207

V. PETROW

Table 5.6

Electrophoretic mobility

B pre-8 a

SOME PROPERTIES OF PLASMA LIPIDS AND LIPOPROTEINS

Density

Triglycerides

Cholesterol

Phospholipid

low very low high

++ +++

+++ + ++

++ + +++

+(+I

5.7) with raised triglycerides and phospholipids and minimal effects upon plasma cholesterol levels [23 11. Variable results have been obtained with

EE/ 19-norethisterone acetate preparations. Aurell, Cramer and Rybo [232] have reported raised cholesterol and triglyceride levels in a group of women after 12 months treatment. Brody, Kerstell, Nilsson and Svanborg [233], in contrast, found no significant changes in these parameters under similar circumstances. Mestranol/ethynodiol diacetate follows an oestrogen-like pattern [233], as does mestranol/norethynodrel [234]. Wynn and Doar [235] have made the interesting observation that serum triglycerides tend to fall from pre-treatment levels after discontinuation of oral contraceptives, but it is not yet known how long such an improvement is maintained. It is not yet known whether the elevated triglyceride levels (above) stem from an increased rate of production or from impaired removal from the plasma. The finding that plasma postheparin lipolytic activity is decreased during oral contraceptive administration [236] suggests an impairment of TG removal [237] as in pregnancy [238]. In trying to assess the pathological significance of the foregoing effects of oral contraceptives upon plasma lipids, it should be borne in mind that TG levels are markedly affected by such factors as dietary intake, age, obesity, preclinical diabetes and race. No information is available at the present time on whether the foregoing changes in carbohydrate and lipid metabolism may accelerate the atherogenic Table 5.7

Oestrogen

EE EE EE Mestranol Mestranol

EFFECTS OF ORAL CONTRACEPTIVES UPON PLASMA LIPIDS

Progestagen

Triglycerides

Cholesterol

Phospholipidr

Megestrol acetate Medroxyprogesterone acetate Norethisterone acetate Ethynodiol diacetate Norethynodrel

t t

0 0 0, t 0 0

t t

0, t

t t

1

t

208 ANTIFERTILITY AGENTS process and, if so, whether there is regression of such atherosclerotic lesions on discontinuation of treatment [239]. THROMBOEMBOLISM

There is presumptive evidence that disorders of carbohydrate and lipid metabolism may lead to thromboembolism. As contraceptive medication affects both these parameters, the question whether it also predisposes to thromboembolism-which in gross form may be fatal-is of the utmost importance. Unfortunately, all evidence of a possible connection is epidemiological in nature and no conclusive laboratory evidence to link oral contraceptives with thromboembolism has been obtained. Some of the difficulty stems from the very low incidence of this adverse reaction which may be the result of an unusual combination of biochemical events in a susceptible individual. Thus, for example, Jick, Shone, Westerholm, Inman, Vessey, Shapiro, Lewis and Worcester [240] have observed a deficit of patients with blood type 0 among women who developed thromboembolism whilst taking oral contraceptives (see also [241]). In addition, it seems likely that, under normal conditions, microthrombi occur not infrequently and resolve spontaneously. The circumstances that precipitate progression of such dynamic lesions to a macroclot are not known and are probably of such temporary character as to be no longer in evidence when causal laboratory investigations can be undertaken. For this reason, it seems logical to present the epidemiological data first and then comment upon relevant biochemistry. Following the first reported case of pulmonary embolism on an oral contraceptive ( a mestranol (75 pg)/norethynodrel (5.0 mg) combination [200]), regulatory authorities have followed the picture with concern. In the U.S. a committee appointed by the Food and Drug Administration published a report in 1963 [242], on 350 cases of thromboembolic disease in women taking a mestranol/norethynodrel combination. Reported mortality among white women was 12.l/million, with a comparable mortality in the general population of an estimated 8=4/million.In the U.K. in 1967, a ‘Preliminary Communication to the Medical Research Council by a Subcommittee’ appeared [243] covering, inter a h , 261 fatal incidents. In this retrospective study, two control groups of women aged 1 5 4 4 were broadly matched for age and parity. The following conclusions were reached: (a) The risk of venous thrombosis or pulmonary embolism was increased approximately x 6 in pregnancy or in the puerperium and approximately x 3 in women talung an oral contraceptive; (6) A higher proportion of women (14/29) hospitalised for thromboembolism had been using oral contraceptives relative to the controls (3/36) (c) No relation was apparent between oral contraceptive use and death from coronary thrombosis.

V. PETROW 209 Final reports on this study appeared in 1968 [244, 2451, which, together with a further report [246] (see [265] for review) expanded earlier conclusions as follows : ( a ) risk of death attributed to oral contraceptives in healthy women 1.5/100OOO users/annum aged 2 6 3 4 years 3-9/100OOO ,, 3 5 4 ,, (b) the relative risk userslnon users was as follows : deep vein thrombosis or pulmonary embolism 6.3: 1 (death 8.3: 1) cerebral thrombosis 6.1:1 (death 5-7:1) coronary thrombosis 0.9: 1 (death 1.7: 1) Drill and Calhoun [247] followed up the U.K. study by an analysis of data obtained in the U.S. This failed to reveal a direct connection between thromboembolic disease and oral contraception. Their calculations have been criticised by Frederiksen and Ravenholt [248], who additionally concluded [249] that ( a ) the upper limits of excess mortality from thromboembolism in the years of U.S. attributable to oral contraceptives in women 2age is 3 4 deaths/100000 users (b) the complications of pregnancy, childbirth and the puerperium lead to 3.1 deaths/100000 women ( c ) users of oral contraceptives who are heavy smokers have an incidence of thromboembolism 23 times that of women who do not smoke or employ oral contraceptives. Heavy smokers on the pill likewise have a higher incidence of thromboembolism than non smokers. Frederiksen and Ravenholt estimated that in 1966 in the U.S. less than 171 deaths could be attributed to oral contraceptives, whilst more than 300 OOO could reasonably be attributed to cigarette smoking. They drew the logical conclusion that it would be safer to stop smoking cigarettes and use oral contraceptives than vice-versa (see also [250]). Kay, Smith and Richards [251] report that preliminary analysis of data based upon 32000 women recruited for a prospective oral contraceptive study, showed that oral contraceptive users are more likely to be heavy smokers than non-users of the pill. Vessey and Doll [245, 2521, in contrast, have been unable to find evidence that smoking potentiates the liability to venous thromboembolic disease. It seems unlikely that both opposing views can be correct and, until the differences are resolved and reconciled, other associated conclusions by these groups of workers must be accepted with reservation. Regulatory action followed the studies of Vessey, Doll and their collaborators. In December 1969, Scowen [253], in his capacity as chairman of the Committee on Safety of Drugs in the U.K., advised the medical profession to no longer prescribe oral contraceptives containing more than 50 pg of 9

3,

210

ANTIFERTILITY AGENTS

oestrogen as the Committee had evidence that the incidence of thromboembolism is higher among women taking larger doses of oestrogen ( >75 pg) than among women taking preparations containing the smaller dose (50 pg). The report on which this recommendation was based appeared in April 1970 [254]. In it, reports of thromboembolism following use of named oral contraceptive preparations that had been reported to regulatory authorities in the U.K., Sweden and Denmark were analysed. The number of incidents reported for each product in each area was compared with total usage in that area as estimated from market research estimates of sales. Their findings were as follows (see also Table 5.8): (a) a positive correlation was found between the dose of oestrogen and thromboembolism. (b) no significant difference was noted between EE and mestranol and between combined and sequential preparations. (c) there was a significant excess of reports associated with the combination of 50 pg EE 4 mg megestrol acetate and a significant deficit of reports associated with the 100 pg mestranol+2.5 mg norethynodrel combination. The Committee found the latter result to be particularly difficult to explain as norethynodrel, in animals at least, is known to have an oestrogenic effect. It is, perhaps, relevant to stress the latter point. Norethynodrel is generally regarded as the most oestrogenic progestagen in current use. Thus, Drill [85] has shown that in the uterine weight assay it has 4 per cent of the uterotrophic activity of oestrone. Norethindrone, in the same assay, has a

+

Table 5.8

RATIO OF OBSERVED/EXPECTED NUMBERS OF REPORTS OF THROMBOSES WITH COMBINED TYPE ORAL CONTRACEPTION PRODUCTS

Oestrogen and progestagen content of preparation

Ratio of observed to expected reports of total thromboses

Progestagen

Lynestrenol ( 5 mg) Megestrol acetate (2 mg) Norethisterone (2 mg) Ethynodiol diacetate (1 mg) Norethisterone ( 1 mg) Megestrol acetate (4 mg) Lynestrenol (2.5 mg) Norethisterone acetate (1 mg) Norethisterone acetate (3 mg) Norethisterone acetate (4 mg) Norethynodrel (2.5 mg) Norethisterone acetate (2.5 mg)

*M = Mestranol

1.99 1.74 1.55 1.31 1.08 1.00 0.82 0 80 078 0.63 0.59 0.55

V. PETROW 21 1 potency of 001 x oestrone. Megestrol acetate ii inactive. Furthermore, its progestational activity in the Clauberg assay is low [255]. Its combination with mestranol has therefore been generally regarded as the most oestrogenic of currently used oral contraceptive preparations. It is true that this viewpoint is derived from animal data and not from direct clinical comparison. It is, nevertheless, difficult to reconcile it with the finding of a positive correlation between the dose of oestrogen and the risk of thromboembolism with de facto exclusion from consideration of the progestagenic component. Returning to the main theme, in April 1970, Oliver [256] also presented data supporting the view that oral contraceptives do not, on their own, increase the risk of myocardial infarction but may do so in women prone to ischaemic heart disease. The Food and Drug Administration in the U.S.A., whilst generally accepting the U.K. data, took the stand that good therapy demanded that the lowest effective dose of oestrogen that is otherwise acceptable should be prescribed [257]. In a statement issued on May 5,1970, the Australian Minister of Health reported that the Australian Drug Evaluation Committee was of the opinion that although the U.K. survey provided evidence of a relationship between thromboembolism and oestrogen dosage, it was clear that other factors were also operative and important. In their view withdrawal of oral contraceptive preparations containing 100 pg or more of oestrogen was not warranted at the present time, but use of lower dosage formulations was advisable f2581. Nanni [259] has recently drawn attention to certain discrepancies and particularly the lack of patient control data in the U.K. study. He pointed out that although the preparation containing 150 pg of oestrogen represented only one of 15 brands used in the study, it had a strong effect upon the outcome. When this preparation was excluded from the statistical calculations, no significant trend was apparent among the remaining formulations. Exclusion of data is often informative in such circumstances as hormonal products not infrequently show biphasic effects with reversal of a characteristic biological activity when administered in doses above certain threshold levels.

Mechanism of Blood Coagulation

Injury to blood vessel endothelium with resultant exposure of blood platelets to collagen or some other non-endothelial surface represents the most usual stimulus to microclot formation. As platelets have a surface 6( -) charge and exposed collagen fibres a surface 6( +) charge, adhesion occurs. This causes release of platelet ADP and other factors causing localised platelet aggregation and formation of a white platelet thrombus. Such aggregation concerns platelet function and the vascular wall only and is independent of blood coagulation factors with the exception of thrombin.

212 ANTIFERTILITY AGENTS The latter factors exist as proenzymes in the blood and exert no direct effect upon coagulation until they are activated. Such activation generally begins with an injured vessel wall which serves as a foreign surface to activate factor XI1 (to give factor XIIa-i.e. activated factor XII) which starts the blood coagulation process that results in fibrin formation (see Figure 5.8). The ‘cascade’ shown in this diagram represents a powerful biochemical amplifier mechanism. The active factor XIIa, in conjunction with factor XI, activates lxII

6 + Charge

~

Platelets

I

on

surloce

+ WlaI

IXII

(1x1

Aggregation

I 1x111) Plasminogen Prothrombin

Fi br i nogen

Inhibitors +Activators J-lXIIIa)

~

(Fabric for fibrobbast migration and laying down of collagen to repair injury

St$h&d

FIbrinolysts

4

lnhi bitors

-

Pla&nin

fragments

Figure 5.8. Mechanism of coagulation and jibrinolysis

factor IX, which, in its turn, in conjunction with factor VIII, activates factor X. The active factor Xa forms an essential component of prothrombin activator. The latter converts prothrombin into thrombin which converts fibrinogen into fibrin. The deposited fibrin traps red blood corpuscles with formation of a red thrombus. Fibrin stabilising factor (Factor XIII) then causes cross-linking of fibrin fibrils thereby substantially increasing their resistance to lysis. The fibrin deposit forms a haemostatic barrier preventing extravasation of blood, limiting the area of injury and forming the fabric on which fibroblasts migrate and lay down collagen. In such repair processes, fibrinolysis limits the amount of fibrin remaining at the site of injury and plays a vital role in the resolution of clots. Biochemistry of blood coagulation and oyjibrinolysis

The more important points in the biochemistry of blood coagulation and of fibrinolysis are summarised below : ( a ) Assay methods for the quantification of blood factors give relatively poor reproducibility. This deficiency in methodology must be borne in mind in attempting interpretation of assay data.

V. PETROW 213 (b) Individual levels vary significantly, e.g. factors VIII and IX may vary by f50 per cent from ‘normal’. ( c ) The role of the blood factors in coagulation is not fully understood. Thromboses may occur in patients with low levels of fibrinogen, factors V, VII, VIII (antihaemophilic factor) and XI1 (Hageman factor) [260, 2611. (d) Many patients with thrombophlebitis do not show detectable changes in their coagulation factors. ( e ) Lipids exert important effects upon in vivo coagulation [262, 2631. It seems likely that hypercoagulability of blood is related to the levels of activated coagulation factors and not to the total quantities of unactivated factors present. As such activated factors are rapidly cleared from the blood, and as the process of thrombus formation is in dynamic equilibrium with thrombolysis, it has not yet been possible to define rigidly that concatenation of circumstances that leads to macroclot formation, or to be certain that the changes in coagulation factors induced by the pill are, per se, directly responsible for thrombembolic episodes attributed to this medication. As mentioned elsewhere [264], their role may be both causative and permissive.

Efect of pregnancy upon blood coagulation factors

The association of thromboembolic phenomena and pregnancy has long been recognised. In practice, the risk of death from thromboembolic complications in pregnancy is small (see Table 5.9) (taken from Doll and Vessey [265])(see also [266])and somewhat less than the risk believed to be associated with oral contraceptive usage. It is pertinent to point out that the latter risk compares favourably with that involved in such a socially-accepted activity as driving a motor car and is considerably less than the chance of dying from cancer or from the complications of pregnancy. No further comment seems necessary. Table 5.9

ESTIMATED RISK OF DEATH

Estimated death ratei100 000 per annum

Thromboembolism Nonpregnant, non-0.c. During puerperium O.C.users Motor accidents Cancer All risks of pregnancy (including delivery and puerperium)

2&34

3544

0.2 1.3 1.5 4.9 13.7 22.8

0.5 2.3 3.9 3.9 701 57%

214 ANTIFERTILITY AGENTS EfSect of oral contraceptives upon clotting mechanisms A selection of data is shown in Table 5.10. There is wide agreement that the changes induced by these agents are modest in character and not immediately apparent as predisposing to thromboembolism. It is significant that oestrogens per se can induce increased fibrinolytic activity. Also oestrogens have long been regarded as representing one of the factors that protect young women from coronary heart disease. Oestrogens in higher than Physiological doses,’ however, as when used in suppression of lactation in women [276-2781, or in the treatment of carcinoma of the prostate in men [279], appear to increase the risk of thromboembolism. Extending this concept further, it is proposed that thromboembolism resulting directly from oral contraceptive usage represents an idiosyncratic hypersensitivity of the vascular system towards oestrogens. As progestagens undoubtedly modify the effects of oestrogens upon blood coagulation (vide supra), they, too, must play a role in the induction of thrombosis in the ‘oestrogen hypersensitive’ woman. The influence of gonadal hormones upon uterine fibrinolytic activity has recently been reviewed by Astrup [280]. Publications have appeared on the effects of oral contraceptives and of their constituents upon blood platelet adhesiveness (see inter alia [281, 2821). Both oral contraceptives and oestrogens may increase platelet sensitivity to aggregating agents, but the pathological significance of data obtained under such artificial test conditions is still presumptive and equivocal.

LIVER FUNCTION

Cholestatic jaundice is a well-known though uncommon complication of pregnancy [283]. In addition, 17a-alkyl derivatives of testosterone are known to be capable of causing cholestasis [284]. It is therefore not surprisin.g that jaundice occasionally occurs during oral contraceptive use. Most of the reports have come from Scandinavia [285]. As in pregnancy, there is also decreased hepatic excretion of BSP. In general, patients with a previous history of reduced hepatic excretory function, jaundice, or pre-existing hyperbilirubinemia are mainly at risk [286]. Withdrawal of oral contraceptives normally results in recovery in a matter of days.

TRANSCORTIN

Raised oestrogen levels during pregnancy profoundly affect cortisol metaboliism. Increased levels of unbound cortisol are also found in the plasma of oestrogen-treated subjects [287] accompanied by raised transcortin levels

Table 5.10

EFFECT OF HORMONES AND ORAL CONTRACEPTIVESUPON PLASMA CLOTTING FACTORS (n = normal)

Clot ring

Pregnancy [267] Enovid 5 [268] Enovid 2.5 [268] Orthonovin 2 [269] Deladroxate [270] Depo-Provera [270] Testosterone [271] Oestrogen [272] C-Quens [273] Norinyl 1 [274] Menstruation [275] Norethindrone [270]

Lysir

I1

VII

X

XI11

Fibrinogen

ttt t

tt

1

1

t t n

t

t

n n n

Urokinase

tt n 1 n n n

Euglobulin Plasminogen Fibrinolytic activity n n

t 1

1

t t t n

1

t ?t

?t t

t

?t t n

n

n

[t 1 1

216 ANTIFERTILITY AGENTS [288]. (See Table5.11) [289]. Similar changes are induced by oral contraceptive preparations. As oestrogen levels during oral contraceptives administration do not reach levels found in pregnancy, remissions of rheumatoid arthritis that often occur in pregnancy are not normally observed. Table 5.11

PLASMA CORTISOL AND TRANSCORTIN LEVELS I N PREGNANCY AND AFTER OESTROGEN

Plasma cortisol

(Pg%) Normal Pregnancy (3rd trimester) Oestrogen

12-14

35 40

Plasma transcortin (mg cortisol/100 ml plasma) 17-25 45-55 40-60

In practice, migraine, headache and depression represent the more serious of the adverse effects encountered by oral contraceptive users. Migraine and headache The apparent incidence of headache in oral contraceptive users varies somewhat with differing question techniques at various centres [290]. At the same time, it seems well established that severe migrainous headache may be brought on by oral contraceptive preparations [2913. The pioneering work of Mears and Grant [292] reveals a direct correlation between well-developed arterioles in endometrial biopsy specimens and incidence of migrainous headaches. Interestingly, Grant [293] has pointed out that individual response depends not only upon the individual and the ratio between the progestational and oestrogenic components of the preparation, but also upon the particular steroids. It is relevant to add that occasional reports on the use of progestagens in the treatment of migraine have not been too encouraging [294]. Depression There is little doubt that combination products often relieve some of the symptoms associated with premenstrual tension [295, 1191. At the same time, there is little doubt that depression is the most distressing side effect encountered on oral contraceptive medication [296] and may affect up to 20 per cent of women [297-2991.

V. PETROW 217 Grant and Pryse-Davies [300], starting from the concept that depression may be treated with monamine oxidase inhibitors, developed evidence to support the thesis that depression is linked with the use of strongly progestagenic compounds which raise endometrial monoamine oxidase levels in the endometrium. Oestrogens in adequate dosage apparently protect against this depressant effect. The clinical situation is complicated, however, by the action of the regulatory authority in withdrawing usage of oral contraceptives containing more than 50 pg oestrogen/tablet as the latter dose seems inadequate to prevent the ‘pill syndrome’ in a significant number of women [301]. Pill-induced depression is frequently associated with frigidity [302]. Evidence concerning the effect of oral contraceptives upon libido is contradictory [303], but the medication does appear to erase the depression in libido that frequently occurs during the luteal phase of the menstrual cycle 13041. Rose [305] reported the excretion of grossly increased amounts of xanthurenic acid in the urine of women taking combination products. A similar increase in tryptophan metabolites occurs in pregnancy and has been interpreted as indicating pyridoxine deficiency [306]. Dewhurst [307] subsequently postulated a causal connection between dysfunction of trytophan metabolism and certain types of depression. Winston [308] developed the concept further by suggesting that depression from oral contraceptive medication be treated with pyridoxine. Price and Toseland [309] have proposed routine inclusion of pyridoxine in oral contraceptive preparations. Developments will be awaited with interest. HYPERTENSION

Laragh, Newton and Sealey [3 101 have drawn attention to the occasional induction of hypertension by oral contraceptive medication in susceptible individuals. Increases in plasma renin and aldosterone were observed, together with a striking increase in plasma angiotensinogen. There was a marked enhancement of angiotensin formation upon addition of a fixed amount of endogenous renin to the plasma. OPTHALMOLOGICAL EFFECTS

In 1965 Walsh, Clark, Thompson and Nicholson [311] (see also [312]) suggested a possible link between oral contraceptives and neuro-opthalmologic damage. In the same year Kuvin, Harner and Smith [313] reported the dilatation of retinal veins in monkeys given various oral contraceptives. In general the degree of dilatation varied with the dose. Meyer, Leibowitz,

218 ANTIFERTILITY AGENTS Christman and Niffennegger [314] reported a significant fall in intraocular pressure in patients treated with norethynodrel for primary open-angle glaucoma. They suggested the possible use of progestagens for therapy of this condition. Studies by Faust and Tyler [315], and by Connell and Kelman [316] have failed to reveal a significantly different incidence of eye abnormalities in pill users. Both groups of workers pointed out the limitations inherent in studies involving relatively small groups of patients. Two cases of retinal oedema secondary to the use of oral contraceptives have been reported by Goren [317]. Four cases studied by Salmon, Winkelman and Gay [318] led these authors to the conclusion that neuro-opthalmic sequelae may be secondary to vascular disease. A possible connection between oral contraceptives and ocular complications in users of contact lenses has been mentioned by several workers (see for example [319]). NEOPLASTIC DISEASE

The tumorigenic action of oestrogen in the experimental animal has been known for many years [320]. In addition, more than 70 years ago, Beatson [321] pointed out that in women certain forms of cancer of the breast, which are now known to be oestrogen dependent, regressed after removal of the ovaries. It is therefore not surprising that steroidal contraceptives containing oestrogens have been regarded by some authorities as potential cancer producing agents. The possibility-no matter how remote-is clearly a matter of great concern and worthy of the most careful study. Unfortunately, no mechanism exists for resolving this problem to the satisfaction of the present generation of pill users as induction of cancer by hormones is believed to require up to 25 years of continuous administration to become apparent. Ten years of oral contraceptive experience is not regarded by many as sufficient time to permit a definitive conclusion [322]. It must also be remembered that it is impossible to prove a negative. No pains have been spared by the pharmaceutical companies, however, to establish freedom of their products from carcinogenicity by means of animal experiments. Certain built-in features of these studies however, make interpretation of the data so obtained a matter of extreme difficulty and controversy. Cancer of the breast is the most common malignant neoplasm in the human female. It also occurs with high frequency in many strains of mice and is not uncommon in rats. It is particularly common in the bitch and is frequently observed in females more than five years of age. The monkeys and great apes, in contrast, as well as such domestic animals as the cow, are virtually immune to this disorder [323]. Toxicological requirements for oral contraceptives (see the recent article by Djerassi [324]) involve daily administration of the medication to dogs at

V. PETROW 219 0, 10 and 25 times the clinical dose for 7 years and to monkeys at 0,lO and 50 times the clinical dose for 10years. The species selected, it will be noted, have respectively a high and a low incidence of naturally occurring mammary tumours. The design of the experiment, however, requiring, as it does, continuous daily administration of many times the clinical dose of steroid, appears to be so far removed from human practice as to make relevance to the human situation almost a matter of belief. It is doubtful if many materials in continuous culinary use could pass such exaggerated toxicological requirements. It must also be noted that the biological activity of a steroid may be reversed at unphysiological dose levels. Thus, for example, the incidence of mammary tumours in ovariectomised women on oestrogen replacement therapy is certainly no greater-and may be less-than in a control population [325]. In contrast, it is reasonably certain that high doses of oestrogen can sometimes cause mammary tumours in both men and women. In addition, presumptive evidence now exists that low oestrogen levels in women may be associated with an increased incidence of cancer of the breast [326]. There is, consequently, a cogent argument to support the thesis that more meaningful data on carcinogenic risk associated with oral contraceptive products may result from studies that reproduce the pattern of human usage as far as possible, both with respect to dose-which should be based upon activity in the species per se and not extrapolated from clinical data-and mode of administration (which is cyclical for all oral contraceptive preparations with the exception of the minipill). In 1970, three oral contraceptive preparations (two based upon chlormadinone acetate and one upon medroxyprogesterone acetate) were withdrawn following the appearance of breast nodules in beagle bitches. These were seemingly related to the progestagenic components. No data on dose levels are available, nor have any reports appeared to date on the formation of metastases, so comment must be limited. In attempting to assess the significance of such data vis-a-vis human usage, it should be remembered that the canine mammary tumour is extremely rare in the ovariectomised bitch and in the dog and is generally regarded [327] as related to ovarian function. It follows that administration of ovarian hormones or their partially synthetical congeners to this experimental model would be expected to increase the incidence of mammary growths. It needs still to be established, however, that the appearance of such tumours is an expression of carcinogenic potential per se and not a simple expression of a greater but normal hormonal action. Although direct comparison is lacking, it may be anticipated that both chlormadinone acetate and medroxyprogesterone acetate, by virtue of their C6-substituent, would have a longer half-life in vivo than the progestagens derived from 19-nortestosterone. Atypical endocervical hyperplasia has been infrequently observed in women on oral contraceptives [328] and has hitherto been regarded as a benign lesion. In addition, histopathologic alterations similar to those of

220 ANTIFERTILITY AGENTS normal pregnancy have been reported [329]. Various endometrial changes have been characterised [330]. It also appears that oral contraceptives may produce hyperplastic (pregnancy) gingivitis [33 11. AMENORRHEA OR ANOVULATION FOLLOWING USE OF ORAL CONTRACEPTIVES

The incidence of spontaneous secondary amenorrhea in a random female population is not known. In women exposed to stress-which may include not only such stressful situations as leaving home for college or for travel but even marriage-the incidence has been variously estimated to be between two and seven per cent [332]. In oral contraceptive users, Rice-Wray, Corren, Gorodovsky, Esquivel and Goldzieher [333] have reported that 87-98 per cent of 98 women on oral contraceptives ovulated within the first three posttreatment cycles, although the length of the first post-treatment cycle was generally prolonged, with combination regimens exerting a greater effect than sequential treatment. Similar results have been obtained by other workers [334]. There is consequently some doubt that contraceptive regimens per se can cause amenorrhea in normal subjects. A different situation obtains in the case of women with low endogenous oestrogen production and low gonadotrophin secretion [335] when oral contraceptive induced amenorrhea may occur infrequently. It must be remembered, however, that there is a strong tendency for secondary amenorrhea to undergo spontaneous remission without treatment after 12-18 months and this generally occurs in oral contraceptive users. Where treatment is required, resumption of menses may often be induced by clomiphene administration. CUTANEOUS EFFECTS OF ORAL CONTRACEPTIVES

Melasma can occur both in pregnancy and following oral contraceptive administration in a small number of women. After cessation of therapy, the melasma induced by the pill fades more slowly than that accompanying pregnancy [336] and may be permanent. Acne may occasionally improve on oral contraceptive medication [337]. As with pregnancy, oral contraceptive users are more prone to vaginal moniliasis [338]. A similar situation occurs with telangiectasia. Other minor adverse effects are discussed in an excellent review by Jelinek [339]. RESPIRATION

Increase of minute ventilation and reduction of alveolar carbon dioxide tension has been observed during pregnancy [340] and during the luteal phase of the menstrual cycle [341]. These effects have been attributed to progesterone

V. PETROW 22 1 [342]. Progestational agents of the 17a-ethynyl type do not appear to show this effect [342, 3431. The clinical significance of these observations has not been established.

MISCELLANEOUS

Oestrogens seem to have an inhibiting effect upon bone resorption but do not stimulate new bone formation. There is consequently presumptive evidence that sequential type preparations may be of value in slowing down the osteoporotic process in the perimenopausal woman on oral contraceptive medication [344]. Other metabolic effects ascribed to oral contraceptives include increases in serum iron, total iron binding capacity and serum copper, which are apparently due to the progestagenic components [345]. In addition, quantitative immunodiffusion studies show that both pregnancy and oral contraceptive combination products change the serum levels of certain proteins and in particular those synthesised by the liver [346]. There is now wide acceptance of the need for massive measures to control population growth. In consequence, many different approaches to fertility control are presently under study in research laboratories all over the world. Important new developments include the experimental intravenous [347, 3481, and intracervical [349] administration of prostaglandins El (and F2a) as abortifacients and their intravaginal use on a monthly basis [350]. With the liberalisation of attitudes towards abortion, acceptability of compounds that act upon the implantation/immediate post-implantation stages of pregnancy seems inevitable. As steroidal compounds make pregnancy possible and have not hitherto shown teratogenicity, [351] their use as building blocks for such contraceptives of the future seems assured. Acknowledgements: The author thanks Mary Malcolm Newton for the artwork and Charlotte Bagenstose for help with the references. He is indebted to Dr R. D. MacKenzie for comments on the section on thromboembolism and to Dr Dorsey E. Holtkamp for valuable advice.

REFERENCES 1. P . M . A . Newsletter, 1970, 12, (a), 1 2. A . Jost, Proc. Int. Congr. Horm. Steroids, h d , Milan, My 23-28, 1966 (pub. 1967) p. 74 3. P. Bouin and P. Ancel, C. R . Soc. Biol., 1903,55, 655 4. W. C. Young, Sex and Internal Secretions Vol. II. (Ed. W. C. Young) Williams & Wilkins, Baltimore, 1961, p. 1173 5. C. A. Barraclough, Recent Progr. Horm. Res., 1966,22, 503

222

ANTIFERTILITY AGENTS

6. K. Shimizu, M. Gut and R. I. Dorfman, J . Biol. Chem., 1962, 237, 699 7. R. 1. Dorfman, E. Forchielli, S. Ichii and J. Kowal, Proc. Int. Congr. Endocrinol., Znd, London, Ag. 17-22, 1964 (pub. 1965) p. 1087 8. L. M. Fishman, G. A. Sarfaty, H. Wilson and M. B. Lipsett, Ciba Found. Colloq. Endocrinol., 1967, 16, 156 9. K. B. Eik-Nes, The Androgens ofthe Testis (Ed. K. B. Eik-Nes), Marcel Dekker, New York, 1970, p. 1 10. K. B. Eik-Nes and P. F. Hall, Viiam. Horm. (New York), 1965,23, 153 11. J. J. Barlow and C. M. Logan, Steroids, 1966, 7 , 309 12. W. E. Farnsworth, Steroids, 1965,6, 519 13. H. R. Lindner, J . Endocrinol. 1961, 23, 171 14. R. V. Short, Nuture (London), 1960, 188,232 15. L. Haberlandt, Muenchen. Med. Wochenschr. 1921,68, 1577 16. V. Petrow, Chem. Rev., 1970,70, 713 17. J. Kathol, W. Logemann and A. Serini, Naturwissenschaften, 1937, 25, 682 18. L. Ruzicka and K. Hofmann, Helv. Chim. Acra, 1937, 20, 1280 19. H. H. Inhoffen, W. Logemann, W. Hohlweg, and A. Serini, Ber. Deut. Chem. Ges. B., 1938.71, 1024 20. H. H. lnhoffen and W. Hohlweg, Naiurwissenschaften, 1938, 26, 96 21. D. A. McGinty and C . Djerassi, Ann. N . Y . Acad. Sci., 1958, 71, 500 22. A. Ercoli, R. Pellegrini and G . Falconi, Proc. World Congr. Ini. Fed. Gynecol. Obstet., Vienna, 1961, Communication No. 173 23. A. Ercoli and R. Gardi, Chem. Ind. (London), 1961, 1037 24. A. Meli, A.Wolff and W. L. Honrath, Steroids, 1963, 2, 417 25. J. A. Epstcin, A. J. Sobrero, A. Rodriguez-Perez and J. Krocin-Karasek, Clin. Rex., 1965, 13, 241 26. R. B. Greenblatt, Fert. Sterd., 1967, 18, 207 27. A. J. Birch and S. M. Mukherji, J . Chem. Soc., 1949,2531 28. A. J. Birch, J . Chem. Soc., 1950, 367 29. C. Djcrassi, L. Miramontes and G. Rosenkranz, U.S. Patent 2 744 122; Chem. Abstr., 1957, 51, 510b 30. F. B. Colton, U.S. Patent 2 691 028; Chem. Absir., 1955,49, 11729h 31. F . B. Colton, British Patent 776 427; Chem. Abstr., 1958, 52, 4621 32. 0. Engelfricd, E. Kaspar, A. Popper and M. Schcnck, German Patent 1 017 166; Chem. Abstr., 1959,53, 22096e 33. S. A. Szpilfogel, Dutch Patent 1 017 166; Chem. Abstr., 1959, 53, 22096e 34. A. Ercoli, British Patent 922 51 1 equivalent to U.S. Patent 3 019 241 ; Chem. Abstr., 1962,57,4730c 35. Herchel Smith and G . A. Hughes, British Patent 1041 280; Chem. Abstr., 1966, 65, 170281, 36. J. J . Pfiffner and H. B. North, J . Biol. Chem., 1941, 139,855 37. K . Junkmann, Arch. Exp. Paihol. Pharmukol., 1954, 223,244 38. The Upjohn Co., British Patent 848 881; Chem. Abstr., 1962.57.23488 39. M. E. Davis and G. L. Wied, J . Clin. Endocrinol. Metub., 1957, 17, 1237 40. S. Burstein, R. I. Dorfman and E. M. Nadel, Fed. Proc., 1954. 13, 188 41. W. J. Adams, B. Ellis, V. Petrow and I. A. Stuart-Webb, British Patent 802 005; Chem. Absir., 1959, 53, 9293b 42. A. David, F. Hartley, D. R. Millson and V. Petrow, J . Pharm. Pharmacol., 1957, 9. 929 43. S. P. Barton, D. Burn, G. Cooky. B. Ellis, V. Petrow and I. A. Stuart-Webb, British Patent 841 887; Chem. Absir. 1961,55,4589g 44. W. B. Wentz, Obstet. Gjnecol., 1964, 24, 370 45. G. B. Spero, U.S. Patent 3 377 364, equivalent to British Patent 866 381 ; Chem. Absrr., 1961.55, 22383d

V. PETROW

46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 66a. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88.

223

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224

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748

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R. L. Elton, Contraception: The Chemical Control ofFcrti/ity (Ed. D. Lednicer), Marcel Dekker, New York, 1969, p. 242 G. S. Greenwald, J . Endocrinol., 1965, 33, 25 F. A. Kincl and R. I . Dorfman, Acta Endocrinol. (Copenhagen), 1963, Suppl., 73, 17 L. J. Lerner, E. Yiacas and A. Borman, Inr. J . Fert., 1964, 9, 547 F. A. Kincl and R. I. Dorfman, Steroids, 1963,2, 521 G. K. Suchowsky, J . Endocrinol., 1962, 24, XX C. Revesz, C. I. Chappel and R. Gaudry, Endocrinology, 1960.66, 140 G. Nellhaus, New Engl. J . Med., 1958, 258, 935 F. Hartley, J. Endocrinol., 1962, 24, XVI S. Higashiyama, Advan. Obsret. Gynecol. (Osaka), 1965, 17,219. Excerpta Med. Sect. 3: Endocrinol., 1966, 20, 213 G. Tokuda, Y. Aoki and H. Higashiyama, Hormon To Rinsho, 1962,10,843 G. K. Suchowsky and G. Baldratti, J . Endocrinol., 1964, 30, 159 G. R. McKinney and J. P. Braselton, Steroids, 1970, 15,405 G. K. Suchowsky and K. Junkmann, Endocrinology, 1961,68,341 J. A. Thomas and E. T. Knych, Jr, Acra Endocrinol. (Copenhagen), 1966,51,224 J. A. Blaquier, Acta Physiol. Lat. Amer., 1964, 14, 255 P. A. Desaulles and C. Krahenbiihl, Acta Endocrinol. (Copenhagen), 1964,47,444 D. M. Brennan and R. J. Kraay, Acta Endocrinol. (Copenhagen), 1963,44, 367 B. G. Steinetz, V. L. Beach, G. DiPasquale and J. V. Battista, Jr, Steroids, 1965, 5, 93 A. David, K. Edwards, K. P. Fellowes, J. M. Plummer, J. Reprod. Fert., 1963,5, 331 T. Miyake, F. Kobayashi, K. Horibe, E. Itoga, H. Kakushi, Y. Nomura, M. Kadowaki, K. Odaguchi, K. Hara, F. Furukama and M. Ide, Folia Endocrinol. Jap., 1965,41, 1079; Excerpta Med. Sect. 3: Endocrinol., 1966, 20, 421 F. A. Kincl and R. I. Dorfman, Acta Endocrinol. (Copenhagen). 1962,41,274 G. Hecht-Luchari, Geburtsh. Frauenheilk., 1966,26620 L. J. Lerner, D. M. Brennan and A. Borman, Proc. SOC.Exp. Biol.Med., 1961, 106,231 E. Diczfalusy, Amer. J. Obstet. Gynecol., 1968, 100, 136 R. S. Swerdloff, D. R. Mishell and W. D. Odell, Clin. Res., 1969,17, 110 E. Rice-Wray, 0. Gonzalez, S. Ferrer, A. Aranda-Rosell, M . Maqueo and H. Munguia, Amer. J . Obstet. G.vnecol., 1965, 93, 115 J. W. Goldzieher, L. E. Moses and L. T. Ellis, J. Amer. Med. Ass., 1962, 180, 359 J. A. Bowman, Jr, Amer. J. Obstet. Gynecol., 1968, 102, 1039 E. Carbia, A. Alvarado-Duran and M. Lopez-Llera, Amer. J . Obstet. Gynecol., 1968,102,

122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132.

J. Song, M. S. Mark and M. P. Lawler, Jr, Amer. J . Obsret. Gynecol., 1970,107, 717 M. C. Chang, Res. Reprod., 1970, 2(3), 1 A. Jakobovits, A. Gecse, I. Pinkovich, F. Szontaghand I. Karady. Inr. J . Fert., 1970,15,36 J. M. Maas, Clin. Med., 1970, 77(3), 14 W. G. McBride, Curr. Ther. Res. Clin. Exp., 1970. 12, 177 D. Tenhaeff, Muenchen Med Wochenschr., 1970,112, (18). 851 Medical News, J . Amer. Med. Ass.. 1969, 209, 363 E. R. Friedrich, Obstet. Gynecol., 1967, 30,201 D. C. Hines and J. W. Goldzieher, Fert. Steril., 1968, 19, 841 G . C. Liggins (lecture at Hanmer Springs, N.Z., 1964, unpublished) W. G. McBride, World Congr. Int. Fed. Gynecol. Obster.. 5th. Sidney, S . , 1967, p. 354

92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112.

113. 114. 1 15. 116. 117. 118.

1023

V. PETROW

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. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174.

225

W. G . McBride, Med. J . Aust.. 1970, 1, 212 M. 8. Andelman, Fert. Steril., 1970, 21, 314 V. Petrow, Chem. Brit., 1970, 6, 167 H. A. Van Leusden, J . Reprod. Fert., 1969, 19, 537 M. Roland, Fert. Steril., 1970, 21, 211 J. Martinez-Manautou, J . Giner-Velasquez and H. Rude], Fert. Steril., 1967, 18, 57 R. B. L. Gwatkin and D. T. Williams, Nature (London), 1970, 227, 182 G. L. Foss, E. K. Svendsen, K. Fotherby and D. J . Richards, Brit.Med. J., 1968,4,489 J . Martinez-Manautou. I.P.P.F. Med. Bull., 1968, 2(5), 2 P. E. Lebech, P. A, Svendsen, E. Ostergaard and F. Koch, Acta Obstet. Gvnecol. Scund., 1969,48, Suppl. 3, 22 S. Jeppsson and S . Kullander, Fert. Steril., 1970, 21, 307 J. Martinez-Manautou, I.P.P.F. Bull., 1967, 1(4), 1 E. Mears, M. P. Vessey, L. AndolSek and A. Owen, Brit. Med. J . , 1969,2, 730 J. Zafiartu, Brit. Med. J., 1967, 4, 771 H. J. Tatum, E. M. Coutinho, J . A. Filho and A. R. S. Sant’Anna, Amer. J . Obsret. Gynecol., 1969, 105, 1139 D. R. Mishell, Jr. and M. E. Lumkin, Fert. Steril., 1970, 21, 99 H. Croxatto, S. Diaz, R. Vera, M. Etchart and P. Atria, Amer. J . Obsret. Gynecol., 1969, 105, 1135 S. Tejuja, Amer. J . Obstet. Gynecol., 1970, 107. 954 A. Scommegna, G . N. Pandya, M. Christ, A. W. Lee, and M. R. Cohen, Fert. Steril., 1970.21. 201 J . Zaiiartu and C. Navarro. Proc. Pun-Amer. Congr. Endocrinol., 6th, 1965 (pub. 1966) p. 150 I.P.P.F. Medicuf Bulietin, 1968, 2(3), 1 D. R. Mishell, Jr., M. El-Habashy. R. G . Good and D. L. Moyer, Amer. J . Ohstet. Gynecol., 1968, 101, 1046 D. R. Mishell. Jr.. Amer. J . Obstet. G.vnecol., 1967, 99. 86 I.P.P.F. Med. Bull. 1967, 1(5), 3 I.P.P.F. Med. Bull., 1969, 3(4), 2 J. Zaiiartu and C. Navarro, Obstet. Gynecol., 1968, 31, 627 J. Zafiartu, Int. J . Fert., 1968, 13, 415 T. K. Welty, J . B. Josimovich, J . H. Gerende and R. A. Hingson, Fert. Steril., 1970, 21, 673 J. Wiese and T. R. Bollerup, Acta Obstet. Gynecol. Scund., 1966, 45, Suppl. 9, 60 A. Esquivel and L. E. Laufe, Obstet. Gynecol., 1968,31, 364 M. Maqueo-Topete, E. Berman. J. Soberon and J. J. Calderon, Fert. Steril., 1969, 20, 884 E. Guiloff, E. Berman, A. Montiglio, R. Orsorio, and C. W. Lloyd, Fert. Steril., 1970, 21, 110 A. D. Claman. Amer. J . Obstet. Gynecol., 1970, 107, 461 I.P.P.F. Med. Bull., 1968, 2(3), 2 D. R. Mishell, Jr., M. Taias, A. F. Parlow and D. L. Moyer. Amer. J . Obstet. Gynecol., 1970, 107, 100 H. J. E. COX,J . Reprod. Fert.? 1968, Suppl. 5, 167 Roussel-UCLAF S.A., Neth. Patent 6 401 555; Chem. Abstr., 1965, 62, 10498f E. Sakiz and G . Azadian-Boulanger, Proc. Int. Congr. Horm. Steroids, 3rd. 1970, p. 46 U. K. Banik and G. Pincus, Proc. Soc. Exp. B i d . Med., 1962, 111, 595 A. S. Parkes, E. C. Dodds and R. L. Noble., Brit. Med. . I 1938, . 2, 557 I.P.P.F. Med. Bull., 1969,3(3), 6 M. BaCiC, A. W. De Casparis and E. Diczfalusy, Amer. J . Obstet. G,vnecol., 1970. 107. 53 1

226 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 186. 187. 188. 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. 21 1. 212. 213. 214. 215. 216. 217. 218.

ANTIFERTILITY AGENTS

The Cervix Factor, An Invitational Symposium, J . Reprod. Med., 1969, 3, 121 R. A. Gibbons and F. A. Glover, Biochem. J., 1959,73,217 E. Odeblad, Acta Eur. Fertil., 1969, 1, 99 I. G. Pitkiyanen, Zh. Obshch. Biol., 1960, 21, 28 G. Enhorning, L. Huldt and B. Melin., Amer. J. Obstet. Gynecol., 1970, 108, 532 E. C. Mann, W. D. McLarn and D. B. Hayt, Amer. J. Obstet. Gynecol., 1961,81, 209 W. H. Perloff and E. Steinberger, Amer. J. Obstet. Gynecol., 1964,88,439 K. S. Moghissi, Fert. Steril., 1966, 17, 663 H. W. Rude1 and J. Martinez-Manautou, Fert. Steril., 1967, 18, 219 P. E. Lebech, P. A. Svendsen and E. Qstergaard, Int. J. Fert., 1970, 1 5 6 5 H. Fields, Amer. J. Obstet. Gynecol., 1945, 49, 385 A. P. Satterthwaite and C. J. Gamble, J. Amer. Med. Women’s Ass., 1962, 17, 797 E. Rice-Wray, J. W. Goldzieher and A. Aranda-Rosell, Fert. Steril., 1963, 14, 402 S. Chinnatamby, Proc. VIII Con$ I.P.P.F.. Santiago, 1967 G. H. Miller and L. R. Hughes, Obstet. Gynecol., 1970, 35, 44 T. Kaern, Brit. Med. J., 1967, 3, 644 K. Semm, Social and Medical Aspects of Oral Contraception. Round table conference

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V. PETROW

227

219. C. W. Burke, Brit. Med. J., 1969, 2, 798 220. D. J. Ingle, Amer. J . Physiol., 1953, 172, 115 221. D. H. Nelson, H. Tanney, G. Mestman. V. Gieschan and L. P. Wilson, J. Clin. Endocrinol. Metab., 1963, 23, 261 222. R. H. Furman, P. Alaupovic and R. P. Howard, Progr. Biochem. Pharmacol., 1967, 2, 215 223. E. M. Russ, H. A. Eder and D. P. Barr, Amer. J . Med., 1955, 19, 4 224. M. F. Oliver and G. S . Boyd, Circulation, 1956,13,82 225. R. H. Furman, R. P. Howard, L. N. Norcia and E. C . Keaty, Amer. J . Med., 1958, 24, 80 226. A. Svanborg and 0. Vikrot, Acta Med. Scand., 1966, 179,615 227. M. F. Oliver and G. S . Boyd, Cfin. Sci., 1953, 12, 217 228. J. P. Peters, M. Heinemann and E. B. Man, J. Clin. Invest., 1951, 30,388 229. H. Adlercreutz, J. Kerstell and A. Svanborg, Ann. Med. Exp. Biol. Fenn., 1967, 45, 285 230. B. Hood and K. Cramer, Acta. Med. Scand., 1959,165,459 231. E. L. Bierman, ref 202, p. 207 232. M. Aurell, K. Cramer and G. Rybo, Lancet, 1966, 1, 291 233. S. Brody, J. Kerstell, L. Nilsson and A. Svanborg, Acta Med. Scand., 1968, 183, 1 234. H. Gershberg, M. Hulse and Z. Javier, Obstet. Gynecol., 1968, 31, 186 235. V. Wynn and J. W. H. Doar, ref. 202, p. 219 236. W. R. Hazzard, M. J. Spiger, J. D. Bagdade and E. L. Bierman, New Engl. J. Med., 1969, 280,471 237. W. R. Hazzard, M. J. Spiger and E. L. Bierman, ref. 202, p. 232 238. F. Sandhofer, S. Sailer, H. Braunsteiner and H. Braitenberg, Wien. Klin. Wochenschr., 1961, 73, 392 239. V. Wvnn. J. W. H. Doar. G. L. Mills and T. Stokes. Lancet, 1969,2. 756 240. H. JiEk, D. Slone, B. Westerholm, W. H. Inman, M. P. Vessey, S. Shapiro, G. P. Lewis and J. Worcester, Lancet, 1969, 1, 539 24 1 J. H. Medalie, C. Levine, H. Neufeld, E. Riss, F. Dreyfus, C. Papier, V. Goldbourt, H. Kahn and D. Oron, Lancet, 1970, 2, 723 242. Final Report on Enovid by the Ad HOCCommittee for the Evaluation of a Possible Etiologic Relation with Thromboembolic Conditions. Food & Drug Administration, Department of Health, Education and Welfare, U.S.A., Washington, 1963 243. Preliminary Communications, Brit. Med. J . , 1967, 2, 355 244. W. H. W. Inman and M. P. Vessey, Brit. Med. J., 1968, 2, 193 245. M. P. Vessey and R. Doll, Brit. Med. J . , 1968, 2, 199 246. R. Doll, Brit. Med. J . , 1969, 2, 69 247. V. A. Drill and D. W. Calhoun, J. Amer. Med. Ass., 1968, 206, 77 248. H. Frederiksen and R. T. Ravenholt, J . Amer. Med. Ass., 1969,207, 1150 249. H. Frederiksen and R. T. Ravenholt, Pub. Health Rep., 1970, 85, 197 250. D. M. Pott, I.P.P.F. Med. Bull., 1968, 2(2), 3 251. C. R. Kay, A. Smith and B. Richards, Lancet, 1969, 2, 1228 252. M. P. Vessey and R. Doll, Brit. Med. J., 1969, 4, 651 253. E. F. Scowen, Lancet, 1969, 2, 1369 254. W. H. W. Inman, M. P. Vessey, B. Westerholm and A. Engelund, Brit. Med. J., 1970, 2, 203 255. F. J. Saunders and V. A. Drill, Ann. N . Y . Acad. Sci., 1958, 71, 516 256. M. F. Oliver, Brit. Med. J . , 1970, 2, 210 257. F.D.A. Curr. Drug Inform. 24 Ap. 1970, No. 19 258. Comments, Med. J. Ausr., 1970, 1, 1025 259. L. F. Nanni, Brit. Med. J . , 1970, 3, 644 260. F. Duckert and F. Streuli, Role of Coagulation in Thrombosis, in Pathogenesis and Treatment of Thromboembolic Disease, International Symposium, Basle, Switzerland, 1966,

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Ed. F. Duckert, p. 185 0. D. Ratnoff, R. J. Busse, Jr. and R. P. Sheon, New Engl. J . Med., 1968,279, 760 W. E. Connor, J. C. Hoak, and E. D. Warner, J . Clin. Med., 1963,42, 860 J. C. F. Poole, Brit. Med. Bull., 1958, 14, 253 V. Petrow, Chem. Ind. (London), 1970, 1105 R. Doll and M. P. Vessey, Brit. Med. Bull., 1970, 26, 33 D. M. Potts and G. I. M. Swyer, Brit. Med. Bull., 1970, 26, 26 C. A. Olven, Jr., J. H. Thompson, Jr., E. J. W. Bowie, P. Didisheim and J. A. Spittell, Jr., Blood Coagulation, Thrombosis and Female Hormones. (Ed. T. Astrup and I . S. Wright.) James H. Mitchell Foundation, Washington, D.C., 1968, p. 24 P. Brakman, ref. 267, p. 24 See refs. [268, 270 and 2741 P. Brakman, ref. 202, p. 619 G. R. Fearnley, Fibrinolysis, Edward Arnold (Publishers) Ltd., London, 1965 G. R. Fearnley, R. Chakrabarti, E. D. Hocking and J. F. Evans, Lancet, 1967, 2, 1008 H. Sise, ref. 267, p. 19 L. Poller, A. Tabiowa and J. M. Thompson, Brit. Med. J., 1968, 2, 218 R. Bernbaum and A. Osten, Arch. Gynuek, 1906, 80, 373 D. G . Daniel, H. Campbell and A. C. Turnbull, Lancet, 1967, 2, 287 D. G. Daniel, Amer. Heart J., 1969, 78, 720 T. N. A. Jeffcoate, J. Miller, R. F. Roos and V. B. Tindall, Brit. Med. J., 1968, 4, 19 Veterans Administration Co-operative Urological Research Group, Surg. Gynecol. Obstet., 1967, 124, 1011 T. Astrup, ref. 202, p. 597 R. J. Haslam. ref. 202, p. 635 R. S . Elkeles, Lancet, 1968, 2, 315 V. P. Hammerti, Acta Med. Scand., 1966, 179, Suppl. 444, 1 S . Sherlock, Brit. Med. J., 1968, 1, 227 R. Doll and M. P. Vessey, ref. 265, p. 34 I. M. Arias, ref. 202, p. 30 J. R. Plager, K. G. Schmidt and W. J. Stanbitz, J . Clin. Invest., 1964, 43, 1066 H. E. Rosenthal, W. R. Slaunwhite, Jr., and A. A. Sandberg, J . Clin. Endocrinol. Metab.,

1969, 29, 352 A. A . Sandberg, H. E. Rosenthal and W. R. Slaunwhite, Jr., ref. 202, p. 367 A. Wiseman, Brit. Med. J . , 1968, 3, 619 R. Fogelholm and E. V. Narva, Acta Neurol. Scand., 1970,46, Suppl. 43,252 E. Mears and E. C. G. Grant, Brit. Med. J., 1962, 2, 75 E. C. G. Grant, Brit. Med. J., 1968, 3, 402 B. W. Somerville and H. M. Carey, Med. J . Aust., 1970, 1, 1043 E. Mears and E. C. G . Grant, Brit. Med. J . , 1962, 2, 75 M. P. Wearing, Canad. Med. Ass. J., 1963, 89, 239 A. Nilsson, L. Jacobson and C.-A. Ingemanson, Acta Psvch. Scand., 1968, Suppl. 203, 259 Editorial, Brit. Med. J., 1969, 4, 380 Editorial, Lancet. 1970, 1, 1378 E. C. G. Grant and J. Pryse-Davies, Brit. Med. J . , 1968, 3, 777 E. C. G . Grant, Brit. Med. J . , 1970, 3,403 V. Huffer, L. Levin and H. Aronson, J . Nerv. Ment. Dis., 1970,151, 35 I.P.P.F. Med. Bull., 1969,3( l), 1 J. R. Udry and N. M. Morris, Nature (London), 1970, 227, 502 D. P. Rose, Nature (London), 1966, 210, 196 M. Wachstein, M. J. Thornton and J. M. Price, J . Clin. Invesr., 1961, 40,617 W. G . Dewhurst, Nature (London), 1968, 218, 1130

V. PETROW

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6 Recent Advances in the Chemotherapy of Malaria R. M. PINDER, B.Sc., Ph.D. Chemical Defence Establishment, Porton Down, Wiltshire

INTRODUCTION

232

MALARIA PARASITES The life cycle The malarial infection

233 233 236

EVALUATION AND TESTING OF ANTIMALARIALS Terminology Methods

237 -~ 237 238

THE CHEMOTHERAPY OF MALARIA The present status of antimalarial drugs Newer methods of therapy Other antimalarial compounds The current treatment of malaria

243 243 25 1 269 280

THE MODE OF ACTION OF ANTIMALARIAL DRUGS Plasmodial synthesis of purines and pyrimidines Plasmodial utilisation of purines and pyrimidines Plasmodial synthesis of proteins Carbohydrate metabolism in plasmodia Drug resistance

282 283 286 289 295 299

SUMMARY AND CONCLUSIONS

304

REFERENCES

306

23 I

232

RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA

INTRODUCTION Malaria research may be compared to the malaria parasite itself. Both undergo a cyclical development and both are currently undergoing resurgences after periods of low activity. Indeed, with three major symposia in 1969 [l-31 the present scientific programme on malaria seems likely to rival even the massive effort during World War I1 by chemists, pharmacologists, and clinicians [4, 51, which resulted in an armamentarium of drugs that was regarded as the complete answer to problems of therapy and prophylaxis. All that remained for the total eradication of the disease was to eliminate the carrier mosquitoes from those areas of the world where malaria is endemic, and a global programme of eradication was duly initiated in 1955 by the World Health Organisation [6]. The euphoric mood of the post-war period, characterised by rapidly dwindling support for further research into malaria [7], prompted that body to remark in 1961 that ‘the clinician has at his disposal a complete series of effective drugs for the treatment of all stages of the disease . . .’ [8]. Yet today the mood is pessimistic, new compounds are being synthesised, tested, and discarded, and the cycle is complete with the re-emergence in popularity of the oldest remedy, quinine [9, lo]. The reasons for this change in outlook are diverse, and involve both eradication and therapeutic procedures. Thus, while about 38 per cent of the world population is now totally free of malaria and a further 40 per cent have been freed from endemic malaria, or are partly protected, there remains about 380 million people living in areas where the disease is prevalent. The whole of tropical Africa, much of Central and South America, and most of the countries of southern Asia and the south-west Pacific are still malarious, and represent large sources and reservoirs of infection [ll, 121. Indeed, malaria is recognised as the paramount health problem in Africa, and the failure of eradication programmes is due not only to socio-economic problems, intensified by the lack of a coordinated programme over the whole continent, but also to a number of technical factors that are becoming increasingly important on a global scale [ 11, 13, 141. These include the development of behavioural and physiological resistance to insecticides by the mosquitoes, a factor likely to be further aggravated by the current unfavourable climate of opinion regarding the use of residual insecticides [15]. A second, more ominous, note is sounded by the appearance of drug resistance in malaria parasites, including some strains from south-east Asia which show multiple resistance to most of the known antimalarial drugs [16]. The rapid growth of air travel has led to an increasing number of cases of imported malaria in countries normally regarded as being free of the disease [12], a potentially dangerous situation since the anopheline mosquito is widespread throughout North America and most of Europe. Outbreaks of mosquitoborne malaria have occurred as a result of transmission from an infected person coming from a malarious area [ 171. Recent epidemics in Ceylon and

R. M. PINDER 233 Paraguay after apparently successful eradication programmes illustrate the dangers of complacency, and the disease still claims a million lives each year as well as debilitating over a hundred times that number. Malaria remains a formidable global problem. A previous review of the chemotherapy of malaria [18] surveyed the development of antimalarial drugs up to the end of 1966.The present review is intended to cover advances in our knowledge of the malaria parasite, the testing of antimalarial drugs, the mode of action of known antimalarials, and the possibility of new methods of therapy, in the period, 19661970. It will be necessary, however, to describe earlier material in order to provide a complete understanding of the problem.

MALARIA PARASITES THE LIFE CYCLE

Malaria is a protozoan infection in which the causative agent, a member of the genus Plasmodium, undergoes a cyclical development both in the mammalian host and in the vector, the Anopheles mosquito. The expression ‘malaria parasites’ used to include a wide group of organisms which were characterised by being pigmented parasites inhabiting red blood cells, and in particular included the whole of the family Haemoproteidae. However, the term is now intended to apply only to parasites causing malaria, and the genus Plasmodium is defined as containing those parasites which reproduce sexually and by sporogony in an anopheline mosquito and asexually by schizogony in two cycles in the vertebrate host, one producing pigment in non-nucleated red blood cells, and the other in parenchymal cells of the liver [19]. The haemoproteids are more successful in nature, and therefore more common, than the plasmodiids, because they cause little destruction of tissue and do not multiply in the blood stream. The true malaria parasites, on the other hand, undergo an apparently pointless asexual multiplication in the blood stream, thereby destroying the host erythrocytes. Garnham [191 has pointed out that it is difficult to understand how this dangerous (for both parasite and host, since the former depends upon the integrity of the latter) process ever became introduced into the cycle, but it has evolved so far in some cases, for example Plasmodium falciparum, that asexual multiplication is no longer possible except in the erythrocytes. The variety of malaria parasites is almost as confusing as their nomenclature; since the first observation of the living parasite by Laveran in 1880, there have been described over 100 different types, and the number of anopheline vectors is of the same order. Fortunately, only four plasmodia1 species infect man, namely Plasmodium fulcipurum, P. malariue, P. ovule and P. vivux, but numerous others parasitise various species of mammals,

234

RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA

birds and reptiles [19]. An understanding of the life cycle of the malaria parasite is important not only because the symptoms of the disease are related to certain phases in the life cycle but also because antimalarial drugs act at different points in the cycle and may therefore vary in effectiveness. The life cycle of the human malaria parasites is illustrated in Figure 6.1. Parasites of the human species undergo a sexual development, called sporogony, in the mosquito and ultimately produce infective sporozoites Blood meal /and

J~

M o S o U I T o \

9 GAMETOCYTES

ISexual differentiation

4 . t cfand 9 GAMETES

Vocuolotion

Conjugation ZYGOTE

/

MERU~OITE

OOKINETE

i

Erythrocytic stag.s (BLOOD) SCfflZONT

Migration through stomach wall

OOCYST Cell division a n d enlargement

\

Rupiure of oocysi

BODY CAVITY

\

Migration

SALIVARY GLANDS

\ \ \

'.

/Nuclear division a n d enlargement

Figure 6.1. The life cycle of the malaria parasites. Piasmodium malariae, P. ovale, and P. vivax (solid and dashed lines), and P. falciparum (solid line)

which are injected into the vertebrate host when the female anopheline mosquito takes a blood meal. The sporozoites rapidly disappear from the systemic circulation and enter the parenchymal cells of the liver, where they commence asexual division, called schizogony. Each sporozoite grows within a liver cell and undergoes repeated nuclear division to form a schizont. A layer of protoplasm surrounds each such daughter nucleus and the parasite grows to many times its original size, eventually forming mature tissue schizonts called merozoites. This process, which constitutes the preerythrocytic or primary tissue stage, takes from eight to twelve days and during this time no symptoms of the disease are apparent. The liver cell finally bursts and the merozoites are released into the blood

R. M. PINDER 235 stream where they invade red cells and initiate cycles of erythrocytic schizogony. Others reinvade fresh liver cells and undergo further cycles of asexual division ; these forms, called exo-erythrocytic or secondary tissue forms, are believed to be responsibie for the relapses that occur even after the blood cycle has been suppressed by either drugs or by natural immunity. However, re-invasion of the liver does not occur with P . falciparum, and infections due to this parasite do not therefore show relapses although recrudescences can appear due to renewed activity of the primary asexual phase. The young merozoites grow within the erythrocytes to form rounded, vacuolated cells or trophozoites. The vacuole enlarges, the nucleus migrates to one side to form the so-called signet ring stage, and nuclear division takes place. The fully grown erythrocytic schizonts become surrounded by cytoplasm and become merozoites, which are liberated when the erythrocytes rupture and enter fresh erythrocytes, perpetuating cycles of asexual multiplication. The production and release of asexual erythrocytic forms into the blood stream are responsible for the fever and other symptoms of malaria, but several additional days elapse after the first release of merozoites before levels of parasitemia increase to the point at which parasites are detectable in blood smears and before symptoms appear. Some of the daughter merozoites of the red cells eventually fail to reproduce asexually and differentiate into male and female forms, known as gametocytes, which are ingested by the mosquito when it again takes a blood meal. Any accompanying asexual forms are destroyed in the stomach of the insect, but the gametocytes survive and change rapidly. The male gametocyte undergoes nuclear division to form about eight daughter nuclei which migrate to the periphery of the cell. Flagellae are extruded from each nucleus, the gametocyte disintegrates, and the male microgametes are set free as motile flagellated nucleated organisms. The female gametocyte changes into the female macrogamete by a process of maturation division, and conjugation takes place between the sexual gametes with subsequent fusion of the nuclei to form a zygote. The zygote alters in shape, becoming worm-shaped and motile, and this so-called ookinete enters the stomach wall of the mosquito to form a rounded cyst, the oocyst, on the outer surface. The oocyst enlarges, undergoes nuclear division, and thousands of motile sporozoites are released when the oocyst ruptures, enter the body cavity of the mosquito, and migrate to the salivary glands. The infective sporozoites then enter the vertebrate host when next the insect takes a blood meal, and the whole cycle begins again. Although the mosquito is regarded as the definitive host and man as the intermediate host, this is open to question since the sexual gametocytes are produced in the human circulation and reach maturity while still in contact with the blood although now in the gut of the insecl. Moreover, sexual union takes place in the same medium, and only later does the zygote move into the tissue of the arthropod and become a true parasite. The whole cycle in the mosquito takes from seven to eleven days.

236

RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA

THE MALARIAL INFECTION

Periodicity in malaria was recognised in ancient Greece, and a standard duration of growth of the parasite is a notable feature at all stages of its life cycle. This inherent character of each species is almost incapable of alteration except by one factor, the temperature of its environment [19]. However, the classical symptoms of periodicity of fever are not adequate to establish diagnosis since they occur in other conditions, and the malarial fever may even be irregular or remittant, rather than intermittent, in the early clinical course of infections due to P . fakiparum or P . vivax. Nevertheless, malaria is a disease characterised by periodic bouts of fever, accompanied by anaemia and a typical enlargement of the spleen and liver. The symptoms of malaria are caused by the release into the blood stream of merozoites accompanied by pigment and the residual body and fragments of the erythrocytic envelope. The parasite apparently produces a lecithinase which is released at this time and which damages mitochondria, resulting in inhibition of cell respiration, constriction of the small veins, and finally in a condition of profound shock. The period of time which elapses between attacks of fever differs according to the time taken for completion of cycles of erythrocytic schizogony. In tertian malaria, due to P . falciparum, P . ovale, or P . vivax, the attacks occur at intervals of 48 hours, while in the quartan form, caused by P . malariae, the interval is 72 hours. Immunity plays an important part in the course of the malarial infection. Although it cannot be considered as potent a protection as in some other infectious diseases, immunity nevertheless decreases the severity of the attack. Malaria attacks are less severe and more prone to chemotherapeutic treatment in persons who live in endemic areas. Natural or innate immunity may be due to a variety of causes [20], some of which are discussed later in this review, and is quite different from acquired immunity. The former often disappears when the spleen of the naturally resistant animal is removed, while the mechanism for the latter can only be set in motion by the presence of an erythrocytic infection. Indeed, the two types of immunity operate on different stages, acquired immunity acting on the asexual stages in the blood but not on the exo-erythrocytic or sporogonic stages, while natural immunity is effective against all stages except the zygote [20]. The parasite is most susceptible to the effects of immunity when it is in contact with the body fluids, as with the sporozoite in the mosquito or the merozoite in the human plasma. Immunity does not significantly alter the course of the infection due to P . fakiparum, which often causes death in untreated cases, earning it the name malignant tertian malaria. Delay in treatment after the demonstration of parasites in the blood may lead to an irreversible state of shock, and death may occur even after the peripheral blood has been cleared of parasites. Parasites of this species multiply rapidly, forming clumps of blood cells and cytoplasm, which block the internal organs. Many secondary changes occur,

R. M. PINDER 237 such as haemorrhage and necrosis of the affected area, the brain can become involved, and pernicious malaria is the result. However, the infection responds readily to prompt chemotherapy and relapses do not occur if the infection is cleared from the blood when the host leaves the malarious areas. The distribution of the parasite is restricted to the tropics and sub-tropics. Plasmodium vivax, the cause of benign tertian malaria, produces milder clinical attacks than those of P . falciparum, and death is uncommon even in untreated cases. The build-up of immunity in the host rapidly controls the infection and schizonts disappear from the blood stream. The exo-erythrocytic forms in the liver, unaffected by immunity, continue asexual division and reinvade the circulation when immunity has fallen once more; these relapses are characteristic of vivax malaria and occur for at least two years after the primary infection. Infections due to P. ovule also follow a tertian pattern, but are much milder and more responsive to therapy than those due to P. vivax and relapses are less frequent. Infections due to both these parasites often display a prolonged incubation period between the primary infection and the development of malarial symptoms. P. vivax is widely distributed north and south of the equator, extending from the tropics to the temperate zones, while P. ovule is restricted to tropical Africa and the western Pacific. Quartan malaria is caused by infection with P . malariae. Although the mildest form of the disease, it is the most persistent, and relapses may take place years after the primary infection. Indeed, the parasite can produce cases of transfusion-induced malaria in non-immune subjects receiving infected blood. The geographical distribution of P . malariae is similar to that of P.falciparum.

EVALUATION AND TESTING OF ANTIMALARIALS TERMINOLOGY

The different stages of the life cycle of the malaria parasite, like the different species themselves, display varying susceptibilities to chemotherapeutic attack. Antimalarial drugs may be classified according to the stage of the life cycle upon which they act [21], and the terminology used herein will follow that of the World Health Organisation [22]. No drugs are known that will kill the sporozoites before they enter liver cells, in concentrations attainable after therapeutic doses at least. This is not surprising in view of the resting metabolic state of these forms of the parasite [ 191, since successful chemotherapy depends upon exploitation of differences between the host and parasite metabolisms. Drugs are known which will prevent erythrocytic infection, and are called causal prophylactics or primary tissue schizontocides, but they act on the asexual parasites in the liver during the pre-erythrocytic stages and before infection of reticuloendothelial cells.

238 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA Inasmuch as man is his own reservoir of infection, causal prophylaxis also prevents any further transmission to mosquitoes, but such therapy must be continued as long as the individual remains exposed to endemic malaria. Nevertheless, the lack of an ideal causal prophylactic agent against all forms of human malaria, necessitates the use of suppressive therapy which is aimed at the prevention of clinical symptoms. Infection is not prevented, the preand exo-erythrocytic stages are unaffected, and clinical attacks may recur if suppressive medication is withdrawn. Suppressive drugs destroy the asexual erythrocytic forms of the parasite, and are therefore known as blood schizontocides. A suppressive cure can be obtained if the effect of the suppressive medication is longer than the life span of the infection, and such medication is therefore employed during periods of exposure to infected mosquitoes and for some weeks thereafter. Blood schizontocidal drugs are also used to treat overt clinical attacks and thereby achieve a clinical cure. Complete eradication of all forms of the parasite from the body, termed radical cure, is attainable with drugs which destroy the exoerythrocytic forms as well as the erythrocytic forms, and such agents are called secondary tissue schizontocides. Radical cure is fairly readily obtained in falciparum malaria by proper treatment of the initial attack, and continuation of suppressive therapy will ensure that no further clinical symptoms will be manifest. Individuals living in endemic areas are poor candidates for radically curative therapy because of the high risk of reinfection. Finally, the parasite may be attacked when it is in the gametocyte stage, or even during its existence in the mosquito. Drugs in the former category are known as gametocytocides, and most antimalarials have such properties although such action is difficult to distinguish since causal prophylaxis suppressive treatment, or adequate treatment of the clinical attack, will all prevent the development of gametocytes. Drugs which act upon stages in the mosquito are known as sporontocides, and apparently prevent the development of oocysts in the stomach of the arthropod.

METHODS

A casual perusal of the numerous textbooks of pharmacological testing methods published in the last decade will demonstrate the lack of any discussion of methods for evaluating antimalarials, which reflects the decline of interest in the disease. Nevertheless, the methods described in the literature are almost as numerous as the species of parasite, and the criteria governing the choice of a particular test are reviewed by Davey [23]. Historically, the development of antimalarial evaluation techniques began in 1911, with the study by Kopinaris of the effects of quinine upon malarial infections in canaries [24]. However, the first method allowing a quantitative assessment of relative activities to be made was due to Roehl [25]. Roehl’s

R. M. PINDER 239 test, now of only historic interest, also employed canaries infected with p. relictum or P . cathemerium, and the delay in appearance of blood parasitemia in treated birds as compared to untreated controls was taken as a relative measure of activity. Improvements to the technique have been made [23] but the test is little used today, chiefly because of the scarcity of canaries and the vagaries of the results. Other avian plasmodia were studied following Roehl’s lead, and two in particular, P . gallinaceum in chicks and P . lophurae in ducklings, were widely used for mass screening of potential antimalarials in the World War I1 programme [4, 5, 231. All these tests detect blood schizontocidal activity since they involve the erythrocytic forms of the parasites, but the P . gullinaceum infection in chicks has been adapted to provide a test for causal prophylaxis using Aedes aegyptii as the insect vector [23]. Although avian parasites have the advantage of adaptability and can utilise different invertebrate and vertebrate hosts, the pattern of their life cycle is very different from that of the human parasites [19]; for example, the inoculation of birds with erythrocytic forms of the parasite can produce infection with the secondary tissue forms, unlike human plasmodia. while many avian parasites also lack an exoerythrocytic stage. The use of avian malarias in screening procedures has therefore fallen into disfavour in recent years, and the most widely employed method utilises P . bcrghei in rodents, particularly in mice. The parasite was first described in 1948 [26] from wild rodents in the Congo, and has since been successfully transmitted to small laboratory animals such as mice, rats, and hamsters [19]. The difficulty of rearing the insect vector, Anophefes dureni, in the laboratory had restricted the use of the test to detection of suppressive actions, but the work of Yoeli and Most finally demonstrated that A . sfephensi was a suitable experimental vector for serving as a source of sporozoites [27]. Indeed, A . stephensi is ideal since it combines a rapid life cycle with a readiness to feed on a variety of invertebrate hosts and a striking ability to transmit infection, and mass rearing procedures have now been described [28]. The test [29) usually involves a subcutaneous injection of a single dose of the drug 72 hours .after the mice have been infected. At this time the disease is well established but has not produced sufficient debility to alter the response of the host to any toxic effects of the drug. Active compounds produce increases in survival times of treated animals as compared to the normal survival times of untreated controls (6.5 & 0 5 days), while deaths occurring between drug administration and the fifth day are regarded as toxic deaths due to the drug. This procedure forms the basis of the primary screen operated by the U.S. Army Malaria Research Program [30, 311, which has examined more than 100000 compounds since its inception in 1961. Variations in the standard procedure have been reported by Peters [32], who uses daily doses of the drug for up to three days after the primary infection in his test for suppressive activity and measures erythrocytic infection rates in blood films to assess antimalarial potency. Peters also describes a causal prophylactic test using the same in-

240 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA fection but with both pre- and post-infection drug treatment [33]. Two other test procedures complete the triad of primary screens operated by the U.S. Army programme, one involving the administration of drugs to infected mosquitoes and the other using infected erythrocytes incubated in vitro. The former method was first investigated in detail by Terzian and his colleagues [34-371, who showed that there was a specific relation between the vertebrate and invertebrate phases of the malaria parasite, in that drugs with prophylactic effects on the schizogonous phase of the parasite had comparable effects on the sporogonous cycle of the same parasite by arresting the development of the oocyst and preventing the formation of infective sporozoites. Conversely, if a drug was found to be oocystocidal in the mosquito phase then it was prophylactically effective against the vertebrate phase of the same parasite. Further work by Terzian [38] established that P . gallinaceum in Aedes aegyptii was the ideal system for this method, and it is now the standard screening technique for causal prophylactic properties [30, 391. In the test, the mosquitoes are allowed to feed on infected chicks under controlled conditions and the drug is subsequently administered in their food. The insects are kept for seven days and then dissected, and the number of oocysts in the gut are counted. Finally, the absence of viable sporozoites in the salivary glands of the mosquito is taken as the ultimate criterion of the effectiveness of the test drug. Terzian [40] has now reported a somewhat similar procedure, but using Anopheles stephensi as the invertebrate host, which allows a measure of both prophylactic and curative properties. It is postulated that specific developmental stages of the sporogonous cycle of the malaria parasite have their physiological counterparts in specific developmental stages of the schizogonous cycle of the parasite; thus the oocysts resemble the primary pre-erythrocytic stages while the sporozoites resemble the secondary tissue stages of the parasite. Terzian’s method, also involving oocyst counts and sporozoite determinations, provides an efficient and reliable system for determining the two types of antimalarial activity in the same test. Malaria parasites have proved difficult to culture, and only recently have in vitro techniques been mastered sufficiently to allow their use in screening procedures [41]. However, progress with the development of a method for the culture of the mosquito stages of the parasite has been disappointingly slow [42]. Ball and his associates [43, 441 were able to grow the sporogonic stages of P . relictum in vitro, but were unable to induce ookinete formation and were obliged to remove the oocyst from the mosquito gut by microsurgery. It was presumed that some substance from the mosquito stomach was required for ookinete development and, indeed, it was subsequently demonstrated that mouse blood infected with P . berghei readily gave ookinetes when incubated with extracts of mosquito stomachs [45]. More recent work shows that such extracts are unnecessary and the ookinete stage can be obtained under appropriate culture conditions [46]. More success has

R. M. PINDER 24 1 attended the cultivation of the sporozoite stages of the malaria parasites, and much of the early work and the different culture media is reviewed comprehensively by Garnham [19]. For example, eight to nine day-old oocysts of p. gallinaceum, obtained from Aedes aegyptii, have been successfully cultured in vitro to the infective sporozoites [47], and the same technique has been used to culture P . berghei sporozoites from oocysts obtained from Anopheles stephensi [48]. Although such methods are of potential value for future developments, they have not reached the stage of introduction for screening purposes. Nevertheless, in vitro techniques are in active use for such purposes, but they utilise the vertebrate rather than the invertebrate stages of the parasite. Much of the success is due to Trager [49], who first demonstrated that P . lophurae could be grown extracellularly, .but such preparations are recommended more for the study of the mode of action of individual drugs than for screening purposes [23,49,50]. Such methods have, however, proved useful in showing that the gametocytes of P . berghei and P . gallinaceum can result by direct growth from merozoites without an intervening erythrocytic stage [51]. The erythrocytic stages of the parasite can be obtained totally free of host cell materials by a process of nitrogen cavitation, which involves high pressure treatment of infected erythrocytes with nitrogen followed by an adiabatic expansion [52], but most of the in vitro work on such stages has been carried out on parasites within the red cell [19,23]. The standard method, which uses a rocker-dilution technique in which boats containing parasitised blood and culture medium are rocked to prevent red cell sedimentation, enables high volume screens to be carried out for the effect of drugs on protein, lipid, and amino acid synthesis, and consumption of carbohydrate, in the parasite [41, 53, 541. However, this method does suffer from the fall of pH in the medium as lactic acid is produced by the growing parasites. Trigg [55-571 has described an improved method using continuous perfusion to eliminate waste products and maintain pH, and has successfully cultivated increased numbers of parasites of P . falciparum, and the two simian species, P . cynomolgi and P . knowlesi. Finally, it is worth noting that the erythrocytic forms of P . berghei can be grown free from host erythrocytic components within the reticulocyte of the rat, and the system is apparently ideal for studying the mode of action of antimalarial drugs [58, 591. Tests in man are, of course, the pinnacle of antimalarial evaluation procedures, and the factors governing such tests are similar to any organised clinical trial [23]. However, the search continues for a perfect animal model of human malarias, and the similarity between human and simian malarias has prompted a number of investigations of their interrelationships. P. cynomolgi has long been used as a secondary screen for prophylactic activity to test drugs which showed activity in the primary screens {23], since the course and therapeutic response of this infection are directly parallel to those of human vivax malaria. Moreover, human infections with the simian

242 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA parasite P. knowlesi have been used for the fever treatment of syphilis, employing inoculated blood from infected monkeys. It is apparent that man is also a natural host for simian malarias and a number of natural infections acquired from infected mosquitoes have been reported [60]. Although these findings add a zoonotic dimension to malaria and may be of significance in malaria eradication programmes, the most interesting feature from the point of view of drug evaluation is the possibility of infecting monkeys with human malarias. The first reports of the susceptibility of splenectomised gibbons and macaque monkeys to blood-induced infections of P. falciparum were made in 1965 [61-631, and it was subsequently shown that the gibbon was also susceptible to sporozoite-induced falciparum infections [64]. However, a more extensive investigation by the same group [65] concluded that the gibbon was not a suitable model for a natural reservoir of P. falciparum. Indeed, infective sporozoites of both P. fulciparum and P. vivax failed to establish themselves in gibbons, macaque monkeys, or tree shrews, in almost all cases; even in the successful trials, the microgametocytes of gibbons infected with P. falciparum, although capable of exflagellation, did not form zygotes and infection of mosquitoes was therefore not achieved. Nevertheless, some success has been achieved with both P. vivax and P. fakiparum in the owl monkey, Aoius trivirgatus [66]. More recent work by a number of laboratories has now confirmed that infections with the human malarias P. falciparum, P. malariae, and P. vivax, can be established in owl monkeys [67-691, white-handed gibbons [70,7 11, macaque monkeys [72], chimpanzees [73], and black spider and howler monkeys [74]. It seems only a matter of time before the techniques are sufficiently established to allow the use of such animal models of human infections for the evaluation of antimalarial drugs in vivo as well as providing a source of parasitised cells for in v i m studies [30]. Much of the early work on evaluation of antimalarial potential expressed the results in the form of the chemotherapeutic index (CI), which is defined as the ratio of the maximum tolerated dose to the minimum effective dose. However, there is little correlation between toxicities in man and in the avian hosts used in the early work, while even in tests using small mammals, where toxicities are more meaningful, P. berghei is the only infection which allows the calculation of an accurate Chemotherapeutic index [18]. Perhaps the most serious criticism of the use of the latter is that it is only valid when comparing drugs of common structural origin or having similar metabolic fates, and its value is limited when comparing drugs of diverse structure or markedly different modes of action. This has led to extensive use of the quinine coefficient (Q), which is defined as the ratio of the minimal effective dose of quinine to the minimal effective dose of the drug, the toxicity being quoted as a separate figure [29]. Other coefficients may be used based upon other standard antimalarial drugs, since some agents are many times more active than quinine. Pctcrs [32] quotes his P. berghei results as ED5,, values, where

R. M. PINDER 243 this represents the dose required Lo reduce the rate of erythrocytic infection by 50 per cent, as measured on the fourth day after infection, compared to the rate for untreated controls.

THE CHEMOTHERAPY OF MALARIA THE PRESENT STATUS OF ANTIMALARIAL DRUGS

The armamentarium of antimalarial drugs available for current clinical use [9, 751 has changed little from what it was four years ago [18, 76791, but a number of new compounds and types of therapy are under development and have been introduced into therapeutic practice in some cases. These will be discussed under separate headings and this section is therefore devoted to those drugs which are in active use by the majority of clinicians today. The most important drugs in order of chronological introduction are quinine (sixteenth century), pamaquine (1932), quinacrine (1933), chloroquine (1943, amodiaquine (1 943, primaquine (1950), and pyrimethamine (1950). The classification of antimalarial drugs according to their mode of action [18], that is whether they act upon plasmodial synthesis of folinic acid or upon plasmodial synthesis of nucleic acids, will be used in this review. A further distinguishing feature is that the former group, the antifolics comprising chlorguanide, pyrimethamine, and related compounds, provoke drug resistance readily and are too slow in their blood schizontocidal action to be of use in the treatment of acute malarial attacks, while the nucleic acid inhibitors, comprising the remaining drugs listed above, are used to treat such attacks and only provoke drug resistance with difficulty.

ChIorguun ide and pyr in1ethanz ine Pyrimethamine (Daraprim, Malocide, 1) bears a formal structural relationship to proguanil (chlorguanide, Paludrine, 2), and indeed this was the rationale for testing the 2,4-diaminopyrimidines [79a], originally developed as antibacterials, for antimalarial activity [80]. The structural analogy between the two is even closer than it first appears, since proguanil is biotransformed to the active metabolite, Cycloguanil (3), having a cyclic triazine structure [18, 78,791. Proguanil and pyrimethamine are the clinically available representatives of two distinct classes of antimalarials, the biguanides and the 2,4-diaminopyrimidines, and the structure-activity relationships for each group have been reviewed [8 1, 821. Pyrimethamine is the only 2,4-diaminopyrimidine in current use, but two other biguanides apart from proguanil have found clinical application. Both the 4-bromo (bromoguanide, 4) and the 3,4-dichloro (chlorproguanil, 5) analogues of

244

RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA

(2) R=L-Cl ( L ) R=L-Br

(5) R = 3,L-CL2

proguanil have useful antimalarial properties comparable to the parent drug, the latter analogue having a more persistent action because of its less rapid excretion. Proguanil and pyrimethamine exert qualitatively similar effects against different stages in the life cycle of the parasites which cause human malarias, but they differ markedly in duration of action and in certain other properties [77-791. They are highly active against the asexual blood forms of all the human parasites, but their blood schizontocidal action is too slow for them to be used to treat acute malarial attacks. Both drugs display causal prophylactic properties against susceptible strains of P . falciparwn and some effects against tissue schizonts of P. vivax. Although proguanil and pyrimethamine have little effect upon gametocytes, they have pronounced oocystocidal actions and the cyclic transmission of infection through the mosquito is effectively prevented [40].Against experimental malarias, the activity of pyrimethamine is much higher than that of proguanil; values of 4.64 and 1 2 4 against P . berghei and P . gallinacewn for proguanil compare with lO0OQ and 10664 for pyrimethamine [79]. The main uses of proguanil and pyrimethamine are as suppressive and prophylactic agents. Pyrimethamine is, dose for dose, the most powerful suppressive agent known and will achieve a suppressive cure in infections due to P . falciparwn and certain strains of P. vivax. Proguanil, however, is eliminated rapidly from the body and daily administration of the drug is necessary; the usual dosage is 100 mg daily but acute attacks of vivax malaria can be treated with the same dose every eight hours for five days. Its active metabolite, cycloguanil, is apparently less active than its parent upon oral administration, but the lack of activity is due to its even more rapid excretion [77]. Pyrimethamine, in contrast to proguanil, is eliminated quite slowly from the body and daily administration is unnecessary. The usual dose regimen is 25 mg weekly, which has proved particularly valuable in treating

R. M. PINDER 245 partially immune subjects in areas where only susceptible strains of parasite exist [76]. Pyrimethamine is becoming increasingly important as a constituent of combination therapy, in which drugs acting sequentially on different points in the plasmodia1 total synthesis of nucleic acids are administered concurrently. The chief drawback to the use of these antifolic compounds is that they both tend to provoke the appearance of drug resistant strains of malaria parasites, particularly when they are used in suboptimal doses. Indeed, this property has been responsible for the decline in importance of proguanil, but the emergence of pyrimethamine-resistant strains can be prevented if the drug is administered in conjunction with some other antimalarial drug, preferably a quick-acting schizontocide, since such strains arise when it is used alone to treat persons with established infections and parasitemia. Nevertheless, proguanil remains the most innocuous of the currently employed antimalarials and causes practically no untoward effects in therapeutic doses. Excessive doses may cause vomiting, abdominal pain, and diarrhoea, but as much as 700 mg daily can be given for two weeks without serious toxic effects and accidental overdosage of 143 g has been followed by complete recovery [78]. Pyrimethamine is more toxic and can cause macrocytic anaemia, with manifestations of folic acid deficiency, but these effects are readily reversed by discontinuation of medication or by administration of folinic acid. Recent studies of the toxicological actions of proguanil [83] and pyrimethamine [84] indicate that their most serious side-effect in mice, rats, rabbits, and dogs, is depression of cardiac function, although the doses used were higher than therapeutic doses.

8-Aminoquinolines

The 8-aminoquinolines are the only antimalarial drugs in current therapeutic practice which have sufficient activity against secondary tissue schizonts to be of practical value in achieving radical cures of relapsing malarias such as mosquito-induced infections with I! vivax. Pamaquine (6), the first 8aminoquinoline with appreciable antimalarial activity, was also the first synthetic antimalarial drug, and was developed in Germany because of the shortage of quinine during World War I [ 181. Many 8-aminoquinolines were

246 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA subsequently synthesised during the World War I1 programme and several found clinical application. However, rhodoquine (7), pentaquine (8), and isopentaquine (9), are not in current clinical use, quinocide (10) is used widely in Eastern Europe and the U.S.S.R., but the most useful of this group of drugs is primaquine (1 1) which was chosen because of its more favourable chemotherapeutic index [79]. Certainly, their toxicity is the main drawback to the clinical use of the 8-aminoquinolines and they regularly produce toxic symptoms at therapeutic doses, including anorexia, nausea, cyanosis, epigastric distress, abdominal pain and cramps, chest pain, and muscular fatigue [78]. The most serious side-effect, however, is acute haemolytic anaemia in persons whose red cells are unusually susceptible to drug-induced haemolysis. This latter effect is particularly prevalent in dark-skinned races and its geographical distribution parallels that of falciparum malaria; the mechanism involved in this toxic effect is intimately related to the mode of antimalarial action of the 8-aminoquinolines, and is discussed in a later section. The degree of toxicity is associated with the degree of substitution of the terminal amino moiety of the molecules possibly due to metabolic factors. Thus, primaquine, with a primary amino function, is much less toxic than pamaquine with a tertiary amino group, while compounds with secondary amino groups, such as pentaquine or isopentaquine, have intermediate toxicity [79]. Nevertheless, despite their relatively high toxicity, the unique action of the 8-aminoquinolines against secondary tissue schizonts has ensured their continuing place in malaria therapy in the face of more effective drugs for suppressive and prophylactic purposes. Furthermore, plasmodia1 resistance to the 8-aminoquinolines does not appear to be a problem and resistant strains are obtained only with difficulty. It has been pointed out [8, 781 that these drugs should be used with great care to ensure that resistant strains do not develop in the field, since no other drugs presently available can eliminate the tissue stages of relapsing malarias. Primaquine is the drug of choice for the radically curative treatment of vivax malaria, but it is rapidly adsorbed from the gastrointestinal tract and quickly degraded and excreted. Daily dosage is therefore necessary, and the recommended regimen is 15 mg daily for 14 days to achieve radical cure [9]. However, the 8-aminoquinolines, although very active against gametocytes of all human malaria parasites, are ineffective against the asexual blood forms and are not normally used alone for treating acute attacks of malaria or, indeed, for the prevention of malaria symptoms. In the former case they are usually administered together with a fast-acting schizontocide such as chloroquine, while in the latter the weekly administration of 45 mg primaquine together with 300 mg chloroquine may be used as a prophylactic regimen in certain areas where P mulariae, P.ovule, and I? vivux are known to exist [76]. Concurrent administration of 4-aminoquinolines such as chloroquine with primaquine is standard practice. but acridine antimalarials, like quinacrine, potentiate the toxic effects

R. M. PINDER 247 of primaquine by inhibiting its metabolic degradation, and the two should never be administered together [78].

Acridines

Dissatisfaction with the performance of pamaquine as a synthetic substitute for quinine led to further synthetic work in the acridine series, and ultimately to the discovery of the antimalarial properties of quinacrine (Atebrin, Mepacrine, 12). Other acridines to find clinical use are acriquine (13), aminoacrichin (14), and azacrin (15), the latter being designed to combine the features of a 4-aminoquinoline and an 8-aminoquinoline. Quinacrine was much less active than pamaquine in early tests against avian malaria, but it was four times as potent against P. berghei in mice [79]. It was widely used to treat Allied troops during World War 11, but has now been wholly superseded by other compounds such as chloroquine. Nevertheless, it is (12) R’= H ; R 2 = N H . C H M e . ( C H 2 ) 3 . N E t 2 (13) R ’ = H , R 2 = N H . ( C H , ) 4 . N E t ,

(lL )

R’= N H, ; R 2 = N H . C H M e , (CH,),.NEt2

y H . C H M e . (CH, I3.N E t 2

vH.CH M e . ( C H 2 ) 3 . N Et 2

CLO -M‘ CL

c 0

(151

(16)

cheap to produce and still finds use in many parts of the world. Quinacrine may be of increasing value for treating drug-resistant malarias because plasmodia1 resistance to quinacrine is relatively rare [I 61. Quinacrine and its congeners display substantial activity against the asexual erythrocytic forms of plasmodia that cause human malaria, but they are neither causally prophylactic nor radically curative and have no action on the gametocytes. The acridines are therefore used for suppressive therapy and they resemble the 4-aminoquinolines and quinine in this respect; quinacrine is less toxic and more effective than quinine but more toxic and less effective than chloroquine. The recommended regimen for quinacrine to terminate acute malarial attacks is 2800 mg given over seven days, with initial administration of five ‘loading doses’ of 200 mg every six hours to

248 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA saturate tissue reservoirs followed by subsequent administration of smaller maintenance doses [9, 771. The use of quinacrine fell into disfavour chiefly because of its toxic effects and because it cannot be administered concurrently with primaquine. Thus, it was difficult to treat acute attacks of relapsing malarias, since by the time quinacrine had controlled the blood infection the parasites were well established in their secondary tissue forms and liable to produce relapses before primaquine treatment could begin. The situation is exacerbated by the pronounced affinity of quinacrine for the liver, spleen, and leukocytes, where the drug persists for several weeks after medication is discontinued, effectively precluding prompt radically curative treatment with primaquine. Toxic effects of quinacrine are chiefly manifested as cutaneous, gastrointestinal, or neurological symptoms [78], but most, such as abdominal distress, diarrhoea, vomiting, and toxic psychoses, disappear rapidly on withdrawal of medication. Quinacrine has the unpleasant, but physiologically harmless, property of staining the skin and eyes yellow, the colour persisting long after withdrawal of the drug. Skin lesions of various sorts are a particular problem in tropical areas, and are often difficult to heal. Other disadvantages of quinacrine include the necessity of thrice daily dosage when used as a suppressive, and in non-immune subjects it may take up to seven days to control the acute attack. Acriquine and aminoacrichin have been little used in malaria chemotherapy, but azacrin is apparently more effective than quinacrine and acts more rapidly upon blood parasitemia in falciparum malaria than do even the 4-aminoquinolines. However, it is less effective than quinacrine against P. vivux and has no gametocytocidal action, and frequent dosage is still necessary [85]. It therefore offers little advantage over the 4-aminoquinolines and is unlikely to enter wide therapeutic practice. A more recent acridine derivative, 3-chloro-9-(4-diethylamino- 1-methylbutylamino)acridine- 10oxide (16), offers more promise, showing high activity against I? lophurue in ducks and I? knowlesi in monkeys [86]. Clinical trials seem promising, the compound bas activity in man comparable to chloroquine and does not stain the skin [87], but unless it is much superior to presently available suppressive drugs it is unlikely to reach therapeutic prescription.

4-Aminoquinolines In general, a 4-aminoquinoline should be used for treating an acute attack of malaria, and this group of drugs remains the most useful of all antimalarials despite the recent emergence of strains of plasmodia resistant to them [76]. The working rationale behind their original synthesis was that quinacrine consisted essentially of two 4-aminoquinoline moieties, one containing a 6methoxyl and the other a 7-chloro substituent [ 181. Structure-activity

R. M. PINDER 249 relationships in this series [4, 51 indicate that high activity is associated with a 7-chloro substituent, and this structure is evident in all of the clinically useful 4-aminoquinolines, chloroquine (Resochin, Nivaquine, Avochlor, 17), sontoquine ( 18), hydroxychloroquine ( 19), oxychloroquine (20), amodiaquine (Camoquin, 21), cycloquine (22), and amopyroquine (23). Of these (17)

R’=H;

R Z =NH.CHMe.(CH2)3.NEt2

(18) R’=Me; R 2 = N H - C H M e - ( C H 2 ) J - N E t 2 CHzCH20H ( 1 9 ) R’=H ; R2=NH.CHMe.(CH2),.N(Et) (20) R’=H ; R2=NH.CH2*CHOH.CHz-NEtz ,CH,

N Et 2

drugs, chloroquine and amodiaquine are the agents of choice and there is little to choose in their efficacy, chloroquine being more widely used because of its prior discovery and development. Sontoquine, the first 4-aminoquinoline to receive a clinical trial in man, appears to be equal in performance to chloroquine and amodiaquine being somewhat less active and less toxic, but its production is more costly and it is little used clinically [8]. Oxychloroquine and cycloquine offer no advantages over chloroquine and have not found widespread clinical use, while hydroxychloroquine is used mainly in the treatment of rheumatoid arthritis where its low chronic toxicity is of value [8]. The most recently introduced 4-aminoquinoline, amopyroquine, is reported to be less toxic than chloroquine, but its use may again be limited by its lack of any distinct advantage over currently employed 4-aminoquinolines particularly since its value seems to lie in administration by intramuscular injection [76, 881. All of the clinically available 4-aminoquinolines exhibit outstanding activity against the asexual blood forms of all the species of plasmodia that cause human malaria. They rapidly control the acute malarial attack and effect a clinical cure, while doses of 30@400 mg weekly for three to four

250 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA weeks will give suppressive cure in infections with susceptible strains of P falciparum. They are not active against the exoerythrocytic forms and although gametocytocidal in infections with I? malariae or I? vivax, they are not active against gametocytes of l? falciparum. Thus they are not used for causal prophylaxis or for effecting radical cures of relapsing malarias, but are the drugs of choice as suppressive agents in both the acute attack and the prevention of symptoms. The toxicity of the 4-aminoquinolines is very low, but mild and transient headache, visual disturbance, gastrointestinal upset, and pruritis, have been reported after therapeutic doses. These symptoms rapidly disappear upon withdrawal of medication, and these antimalarials do not stain the skin in the manner of quinacrine. The most serious sideeffect of chloroquine and its congeners seems to be their depression of cardiac rhythm and contractility [89], and chloroquine has in fact been used to treat cardiac arhythmias [90]. Chloroquine can also damage the retina since it concentrates specifically in the eye [91]. Cinchona alkaloids The cinchona alkaloids have been used to treat malaria since at least the sixteenth century, but quinine was not isolated from cinchona bark until 1820. The four main constituents of cinchona are two pairs of diastereoisomers, quinine (24) and cinchonine (25), and quinidine (26) and cinchonidine (27), each pair differing in the stereochemistry of the carbinol m

C

H = CH,

function [ 181. Although the four display different activities against different malarial infections, they appear to be of equal value in treating acute attacks of malaria, and quinine owes its favoured position in malaria chemotherapy with cinchona alkaloids to its prior isolation. The spectrum of antimalarial activity associated with quinine is closely akin to that of chloroquine and quinacrine, except that quinine is the least persistent of the three agents often requiring frequent dosing every six to eight hours. Plasma levels of quinine decrease rapidly after cessation of therapy, the drug is localised to a great extent in the parenchymatous organs, particularly the liver, and it undergoes rapid metabolic oxidation to an inactive carbostyril derivative. Quinine has considerable activity against asexual blood forms of all human plasmodia, but employed alone it is not effective against the exoerythrocytic forms, or against mature gametocytes of

R . M. PINDER 251 faleiparum. Quinine is therefore an effective suppressive agent but has no prophylactic or radically curative properties. It is, however, becoming increasingly important as a last resort for treating infections with strains of f?fakiparum resistant to all other antimalarial drugs [101. Nevertheless, quinine-resistant strains of I? f~Zciparumhave now been reported in the field, from Vietnam [92]. In addition to frequent dosage, quinine suffers from the disadvantage of its toxicity; the alkaloid has been called a ‘general protoplasmic poison’ [78], and certainly causes a wide variety of toxic effects collectively termed cinchonism. These include tinnitus, vertigo, blurred vision, deafness, and nausea, which are often severe during the first several days of therapy when plasma levels of quinine are high. Quinine has also been implicated as a factor contributing to blackwater fever, a severe condition characterised by abrupt and massive haemolytic episodes and the incidence of which dropped sharply after the introduction of synthetic antimalarial drugs, which is possibly due to an autoimmune response to red cells that have been altered by the drug [60]. Quinine has one great advantage in its cost, and it will continue to be used in those areas of the world where the economics of its use are favourable.

NEWER METHODS OF THERAPY

Sulphonamides, sulphones, and drug combinations Since the sulphonamides act as antimetabolites in bacterial utilisation of paminobenzoic acid, it might be expected that they would exert effects upon plasmodia1 metabolism. Indeed, many sulphonamides, and the isomeric metanilamides, show considerable activity against a variety of non-human plasmodia [79, 8 11. Sulphadiazine, for example, has quinine coefficients of 80&2000 against II berghei in mice [3 11 and 19 against II knowlesi in monkeys [79], two infections that are particularly susceptible to sulphonamides because of enhanced absorption of the drugs in these species. Nevertheless, despite the very favourable chemotherapeutic index of such drugs as sulphadiazine (CI = lOOO), the sulphonamides have not found widespread use in malaria therapy, both because of their rapid excretion necessitating frequent large doses and because they have not shown similar outstanding activity against human as they do against non-human plasmodia. The introduction in the last decade of longer-acting sulphonamides for antibacterial chemotherapy [93] has facilitated the use of this group of drugs in malaria chemotherapy, particularly in the treatment of chloroquine-resistant falciparum malaria. A number of long-acting sulphonamides have proved to be highly active against both I? berghei in mice and against the various species of human plasmodia.

2 52

RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA

4-Sulphanilamido-5,6-dimethoxypyrimidine(sulphormethoxine, sulphorthomidine, Fanasil, 28), which has a remarkably long half-life of 1W200 hours in human plasma [93, 941, is curative in mice at doses of 1 mg/kg [95] with an ED,, of 0-8 mg/kg [96], and a single oral dose of 13-37 mg/kg cured falciparum malaria in initial clinical trials in man [97,98]. Although relatively ineffective against P vivax, sulphormethoxine has been confirmed as a suppressive agent against both pyrimethamine- and chloroquine-resistant strains of f!falciparum [99-1051. A series of experiments by Peters and his colleagues [106-1081 has established that sulphormethoxine has a triple mode of action, acting against both the oocysts and the blood forms of plasmodia and also being toxic by ingestion to the usual mosquito vectors. Like other long-acting sulphonamides, sulphormethoxine is rapidly absorbed from the gastrointestinal tract, and reaches its peak plasma levels after four hours with slow excretion to the extent of 8 per cent in the first 24 hours. Toxic effects have not been encountered in the above human studies, but it is reasonable to assume that sulphormethoxine shares the toxic potential of other sulphonamides. An isomer of sulphormethoxine, 2,6-dimethoxy-4-sulphanilamidopyrimidine (sulphadimethoxine, Madribon, 29), has also attracted some interest. Sulphadimethoxine appears to be as active as its isomer against P berghei in mice [95, 96, 1091 and against avian plasmodia [95], although Peters [lo71 states that it is four times as potent against P berghei but acts only on the blood forms with no insecticidal effects. Against mixed infections with I? falciparum, I? vivax, and I? malariae, the drug considerably reduced asexual parasitemia within seven days after a single dose of 62-5 mg while (28)

R

OMe

OMe

Me0

R. M. PINDER

253

250 mg apparently suppressed the trophozoites, but asexual parasitemia reappeared within fourteen days of treatment [ 1011. Sulphadimethoxine has a

shorter plasma half-life than sulphordimethoxine, 3 0 hours as compared to up to 200 hours, and is much less effective as a suppressive agent with rates of parasite reappearance twofold greater. Mention may also be made of a third pyrimidine sulphonamide, 4-sulphanilamido-6-methoxypyrimidine (sulphamonomethoxine, 30), which is apparently superior to sulphormethoxine in comparative trials against I! berghei in mice and chloroquineresistant falciparum malaria in man [110]. Both oral and intravenous administration of sulphamonomethoxine in total doses of 40 mg/kg were curative in man while similar doses of sulphormethoxine were ineffective. However, sulphamonomethoxine needs daily administration for at least four days, possibly because of its much shorter plasma half-life of 3 W O hours in man [93]. Two other long-acting sulphonamides have received clinical trials in man. 3-Sulphanilamido-6-methoxypyridazine (sulphamethoxypyridazine, Midicel, 31) was even less effective than sulphadimethoxine against mixed infections with I! fakiparum, 19 vivax, and €? malariae, with an effective dose of 250 mg but reappearance of trophozoites after 12 days [loll. Another study revealed that the drug was active but not curative against multi-resistant falciparum malaria when given in an initial dose of 2 g followed by 1 g daily for five days [99]. However, the sulphonamide to receive the most attention is 2sulphanilamido-3-methoxypyrazine (sulphalene, sulphamethoxypyrazine, sulphapyrazinemethoxine,Kelfizina, 32), which has a plasma half-life in man of 65 hours but is far less protein-bound than other long-acting sulphanilamides [11 I]. Subcutaneous doses of 0 6 mg/kg, or oral doses of 5 mg/kg, effectively suppressed blood parasitemia in mouse infections due to multiresistant strains of I! berghei [31, 1091, and oral doses of 0.5 mg/kg cured trophozoite-induced infections of I? knowlesi in monkeys [112]. Initial human studies by Baruffa [ 1131 showed that sulphalene was a good schizontocide and suppressive against chloroquine-resistant I! falciparum in weekly oral doses of 1.5-2.5 g, and this report has now been confirmed in a number of studies [I 14-1 191. It is interesting that sulphalene cured 100 per cent of all cases of multiresistant falciparum malaria in single oral doses of 1 g [I 18-1 191 but was relatively ineffective against strains of I? falciparum which were sensitive to chloroquine or pyrimethamine [117-1191. These results are discussed in more detail in the section on mode of action, and it is sufficient to point out here that the resistant malaria parasites have changed their metabolism to a need to synthesise their own folic acid. Sulphalene, being an inhibitor of the enzyme folk acid synthetase, effectively blocks this step in the resistant parasites but is not so effective against normal strains whose metabolism is not so vulnerable to inhibition of this enzyme [119]. Although the antimalarial activity of sulphones against avian parasites has been known since 1941 [4] and against E! berghei in mice since the early

254 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA 1950s [31], the clinical use of these drugs did not become widespread until reports from leprosariums indicated that they reduced the rate of malarial infection in treated lepers as compared to those treated with other drugs [ 1201. The sulphone to receive extensive study is 4,4'-diaminodiphenylsulphone (diaphenylsulphone, dapsone, DDS, 33), and its activity against the blood forms of avian [121-1231, simian [121, 1221, and rodent [95, 961 malarias has been confirmed, particularly against chloroquine-resistant strains [124]. Dapsone is an effective blood schizontocide against both chloroquine-sensitive [ 125-1 271 and chloroquine-resistant [ 128, 1291 strains of Z? falciparum, but it was not rapidly effective in terminating acute attacks in non-immune persons and, although preventing patency of mosquitoinduced infections, it was not a causally prophylactic drug but rather a clinical prophylactic or suppressive. Nevertheless, daily doses of 25-50 mg per day have been used for prophylactic purposes [128-13 I] against falciparum

malaria with rates of infection being reduced by tenfold. However, the use of such regimens is put into serious doubt by the report of severe agranulocytosis after a three-month course of dapsone, causing death in 50 per cent of those affected [132], and the drug also produces haemolytic effects in susceptible subjects [133]. The drug is also effective against P malariae [126] and multiple infections with Z? malariae, P.falciparum and I! vivax [loll, 1041, but it failed to protect against infection with Z? vivax alone, when given in the usual therapeutic doses [134, 1351. A derivative of dapsone, 2-sulphamoyl-4,4-diaminodiphenylsulphone (SDDS, 34), is apparently much less toxic El361 and shows high activity against rodent and avian plasmodia [137]. SDDS maintains high plasma levels for a relatively long period in rodents and the acute lethal dose in rats was. as high as 6 g/kg. Five human patients suffering from toxoplasmosis were given oral doses of 0.5 g of SDDS daily for four weeks, without apparent toxic effects, and it will be interesting to see the results of clinical trials of this promising new agent against malaria. A number of amino- and nitrosulphones (35) proved to be inactive against P berghd and were also toxic to the mice producing haemolysis [138], while activity was also abolished by replacement of the sulphone moiety with sulphilimine or sulphoximine functions [139]. Reports by Bhattacharya and Sen [140] upon the activity of 4-ethyl- and 4-propylamino derivatives of dapsone have been confirmed in Z? berghei [141], but even the most active compounds, containing morpholino, ethanolamino, and n-pentylamino functions, were not as effective as the parent sulphone.

R. M. PINDER 255 It is apparent, therefore, that many long-acting sulphonamides and a number of sulphones have a schizontocidal effect upon the blood forms of human malaria parasites. However, it is equally apparent that response to these drugs is characterised by a relatively slow antimalarial action, greater efficacy against l? falciparum than l? vivax, suppressive action against multiresistant I!falciparum but little action against sensitive strains, and reasonably good tolerance. The slowness of their action when used alone, ineffectiveness against l? vivax, and the reluctance to use sulphonamides with very prolonged half-lives for general use with attendant dangers of toxic reactions, has militated against their widespread use. Furthermore, drugresistance is implicit in the use of any sulphonamide, and strains of l? berghei, for example, are readily made resistant to sulphalene and to other sulphonamides [142]. The importance of the sulphonamides and sulphones in antimalarial chemotherapy lies in their use in drug combinations, particularly against resistant strains of l? falciparum. The philosophy of using such combinations is based on the belief that if the parasite is attacked on several fronts by drugs of diverse modes of action then it will be less likely to develop resistance to each of the constituent drugs. Although concurrent administration of primaquine with standard suppressive drugs such as quinine or chloroquine has been used as a suppressive-prophylactic regimen for some time [75, 761, neither this combination nor the equally effective mixing of pyrimethamine with quinine [92, 143, 1441 offer any synergistic advantages tilt merely an additive effect for each drug. Synergism of antimalarial actions was first noted between sulphonamides and pyrimethamine or proguanil against P gaNinaceum [ 1451, and subsequently also in human malarial infections [146]. Since that time, extensive studies have borne out the value of synergistic combinations of drugs such as the sulphonamides and sulphones, which inhibit folic acid synthetase, and the classic antifolic antimalarials such as pyrimethamine, which inhibit dihydrofolate reductase, and the review by Herrero should be consulted for a complete list [94]. The synergism arises, of course, because of sequential blockade by the drugs of different steps in plasmodia1 synthesis of the enzyme co-factor required for utilisation of purines and pyrimidines. Peters [I471 has recently reported synergism between proguanil and napthoquinone antimalarials, whose action on the parasites is similar to primaquine by inhibition of mitochondria1 function, but the mechanism of this synergistic effect is not clear at present. It has been reported that the response to pyrimethamine depends upon the size of the parasite population in the patient under treatment, and that failure to cure is due to the presence before treatment of resistant mutants which are more likely to be present in large than small populations [148]. If such mutants are present they are selected by drug treatment, so that the relapse strain will be drug-resistant. Although the numbers of patients in these studies are two few to permit reliable conclusions to be drawn, the results do agree with extensive animal experiments and they indicate the usefulness

256 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA of sulphonamides and sulphones in attacking the resistant parasite at a more vulnerable point in its metabolism. Indeed, we have seen already that sulphalene, for example, is more effective against P falciparwn when the parasite is resistant to pyrimethamine and chloroquine [1191. Nevertheless, combinations of sulphalene and pyrimethamine are superior in both efficacy and acceptability to either sulphalene alone or to combinations of pyrimethamine with other sulphonamides or with chloroquine [I 16, 1491511, and doses of 500 mg of sulphalene combined with 25 mg pyrimethamine given twice monthly proved an effective prophylactic-suppressive regimen. This combination was particularly effective against I! maluriae, upon which it acted more quickly than chloroquine [117]. A combination almost as effective as sulphalene-pyrimethamine is that using the very longacting sulphonamide, sulphormethoxine, which is particularly valuable since it exerts an insecticidal action on the mosquito as well as its actions against the oocysts and erythrocytic forms of the parasite [106]. Sulphormethoxine and pyrimethamine are a particularly well-matched pair because of the similarity of their prolonged activity in man when given alone, and a single weekly administration of the combination has been found effective for the suppression and treatment of falciparum malaria, in both semi- and non-immune individuals [99, 100, 103-105, 149, 150, 152-1571. The combination has a poor effect upon infections with I! vivux, with a slow response compared to chloroquine and a high rate of relapse. However, against pyrimethamine- or chloroquine-resistant falciparum malaria, the combination is very effective in a variety of dose regimens. The most acceptable combination seems to be 1 g of sulphormethoxine given as a single oral dose with 50 mg of pyrimethamine, and little advantage seems to be gained by increasing these doses [154, 1551 although lower dose levels are almost as effective in the treatment of acute attacks [103-1051. Few toxic effects have been noted but the combination should be used with caution in patients with deficient glucose-6-phosphate dehydrogenase, who are particularly vulnerable to haemolytic episodes [ 1561. Finally, two other sulphonamidepyrimethamine combinations have received trials in man, utiiising the longacting sulphamethoxypyridazine and the short-acting sulphadiazine. Concurrent daily administration of 2 g sulphadiazine and 50 g pyrimethamine for three days proved effective against the asexual forms of chloroquine-resistant P fakiparum, but the combination had no effect on the gametocytes [158]. Nevertheless, this combination was effective in both clearing the fever and parasitemia, and in preventing recrudescences in falciparum malaria [ 153, 1591, but is more effective if the sulphadiazine is given every six hours in doses of 0.5 g [99]. Pyrimethamine is more effective against P fakiparum when it is combined with sulphamethoxypyridazine than with sulphadiazine, possibly because of the less rapid excretion of the former sulphonamide, and the recommended regimen includes a single dose of 50 mg of pyrimethamine, combined with 1 g of sulphamethoxypyridazine on the first day followed

R. M. PINDER 257 by 0 5 g of the sulphonamide daily for four days [99]. Dapsone inhibits the same step of plasmodial metabolism as do the sulphonamides, and it is to be expected that it would also exert a synergistic effect when combined with pyrimethamine. However, this combination does not seem to be so effective against multi-resistant falciparum malaria as the sulphormethoxine-pyrimethamine combination [ 104, 156, 1601, and it is not recommended as a suppressive therapy because of the greater danger of producing drug resistance [160]. The combination produced resistance in 25 per cent of the cases treated with 50 mg of pyrimethamine and either 200 mg of dapsone in a single dose or 100 mg repeated daily for five days, and these cases did not subsequently respond to the normally more effective combination of sulphormethoxine-pyrimethaminebut were only cured by quinine. Furthermore, the toxic effects associated with the long-term use of dapsone have already been mentioned, and combination of it with pyrimethamine seems unlikely to achieve widespread clinical practice. The synergism between pyrimethamine and the sulphonamides or sulphones is due to their sequential blockade of two steps in plasmodial synthesis of folinic acid. Since pyrimethamine inhibits dihydrofolate reductase it might be expected that other inhibitors of this enzyme might also provide a synergistic combination with the sulphonamides. The obvious choice, proguanil, has not yet been used in drug combinations to treat man, but Peters [ l o 1 has shown a distinct synergistic effect against El berghei with combinations of proguanil and sulphadimethoxine, which have a similar period of activity in vivo, and trials of this combination in man are awaited with interest. However, another potent inhibitor of dihydrofolate reductase, 2,4-diamino-5-(3,4,5-trimethoxybenzyl)-pyrimidine (trimethoprim, 36), originally designed as an antibacterial [161] has been shown to possess activity Me0

Med

against multi-resistant I!fulcipurum [162].Trimethoprim is the only compound so far reported that has effects in human malarias but not on El berghei infections in rodents, where it lacks both a synergistic effect upon sulphonamides and antimalarial activity per se [ 1091. Nevertheless, the synergism between trimethoprim and sulphalene, which have similar periods of activity in vivo, has been confirmed in simian and human malarias. Trophozoite-induced infections of I? knowlesi in the rhesus monkey were cured readily by administration of 25 mg/kg trimethoprim and 0 5 mg/kg sulphalene, doses which were hardly suppressive when used alone [163]. The dihydrofolate reductase of human protozoa seems to be peculiarly sensitive to trimethoprim and the use of trimethoprim-sulphalene combinations is a

258 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA laudable addition to the antimalarial armamentarium. Nevertheless, although effective against chloroquine-resistant P fakiparum in doses of 1 g of sulphalene to 10 mg of trimethoprim [162, 164-1661, this combination is ineffective against other strains of P falciparum [ 1671. Furthermore, mixed infections with P falciparum, P malariae, and P ovale, seem to respond less easily to trimethoprim-sulphalene than to other combinations such as sulphormethoxine-pyrimethamine[ 149, 1511, although another report [ 1661 does not confirm this. The most interesting feature of trimethoprim is its activity when used alone against pyrimethamine-resistant P falciparwn [ 1621, which must be attributed to a different mechanism of inhibition of dihydrofolate reductase. The drug shows little tendency to provoke resistance and further use of it in synergistic combinations seems likely. The ability of human malaria parasites to become resistant to drugs and even to combinations of drugs, has led to an ever-increasing complexity of such combinations. The addition of dapsone to the recommended prophylactic regimen of chloroquine and primaquine led to a dramatic decrease in the rate of infection with P falciparum in non-immune individuals [130, 1311, but both this, and the equally effective combination of chloroquine-pyrimethamine-dapsone [ 168, 1691, are endangered by the chronic toxicity of the drug [ 132, 1331. Nevertheless, the ‘blunderbuss’ approach has achieved success against multi-resistant falciparum malaria, by the addition of quinine to the sulphormethoxine-pyrimethamine combination. Quinine sulphate, given in 650 mg doses every eight hours for 14 days, together with a single dose of 1 g of sulphormethoxine and 50 mg of pyrimethamine, achieved virtually 100 per cent clinical cure [149, 151, 153, 1701. Mention may be made of one final combination, using a short-acting sulphonamide, sulphisoxazole, combined with chloroquine and pyrimethamine [ 1711. Total doses of 3.5 g of chloroquine and 300 mg of pyrimethamine were given over three days, concurrently with 1 g of sulphisoxazole every six hours for six days. These massive doses proved extremely effective, but the regimen has been severely criticized both because of the serious incidence of neutropenia, abdominal cramps and diarrhoea, and because it seems to offer no advantage over the less toxic regimen of quinine and pyrimethamine [172]. The use of drug combinations over the next few years is likely to see an increasing inclusion of primaquine as one of the constituents; primaquine exerts a powerful and surprisingly long gametocytocidal and sporontocidal effect on the sexual forms of human malaria parasites, particularly of drug-resistant I( falciparum, and will effectively prevent transmission of the resistant mutants in single doses of 45 mg [I 581. Repository drugs Quinacrine is one of the most persistent of the current anti-malarial drugs, while chloroquine and its congeners have been reported to be retained in

R. M. PINDER 259 the body for several weeks [91] despite reports of plasma half-lives of less than three days in rodents [I 731. Nevertheless, these suppressive drugs are not active against the tissue forms of plasmodia, and the concept of designing a prophylactic drug with repository properties, which would localise in fat depots or in host tissues, slowly release the active moiety over many weeks, and require only periodic administration, owes much to the desire to increase the effectiveness of such useful but short-acting prophylactics as proguanil. Although the long-acting sulphonamides can be regarded as examples of repository drugs, the time-scale with reference to drugs in this section is months rather than days. The first drug designed as a repository antimalarial was the pamoate salt of the active metabolite of proguanil, 1-(4-chlorophenyl)-4,6-diamino-l,2dihydro-2,2-dimethyl-s-triazine pamoate (cycloguanil pamoate or embonate, Camolar, CI-501, 37), single intramuscular doses of which protected mice for weeks against blood-induced infections with €? berghei [174] and monkeys

L

(37)

for months against both blood-induced [ 1741 and sporozoite-induced infections with €? cynomofgi [175]. Clinical investigations in man demonstrated the remarkably prolonged action of a single well-tolerated intramuscular dose of cycloguanil pamoate in a lipid vehicle [176]. Even when experimental exposures to infected mosquitoes were made three to six months after administration of the drug, parasitemia was generally suppressed for at least 200 days after the last exposure. Cumulative urinary excretion studies showed a good correlation between the presence of drug and protection against patent malarial infection, but drug persistence was reduced if a small particle size of the crystalline drug was used or if local reactions occurred at the site of injection [177]. The prolonged activity of the drug is undoubtedly due to diffusion of the active metabolite of proguanil from a depot at the site of injection [174, 175, 177-1791, Further clinical studies in man indicated that cycloguanil pamoate protected for months against

260 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA challenges with susceptible strains of P . v i v a [176, 1801, P. fulciparum [1811831, P. ovule [182] and P. muluriue [182]. However, plasmodia are readily made resistant to this repository drug in the laboratory [184, 1851 and such strains show cross-resistance to the anti-folic group of antimalarials. Indeed, plasmodia1 strains that are resistant to chlorguanide in the field are less sensitive to cycloguanil pamoate [ 180, 186-1881, and under conditions of natural exposure, as opposed to the initial trials in prison volunteers where inactivity slows absorption and excretion of the drug, it afforded protection for only three months when given as a single intramuscular injection of 2.5 mg/kg [182, 1891. Clyde [190], in summarising experience with the drug, reports little effect against I? vivax but states that 5 mg/kg for adults to 16 mg/kg for infants provides protection for 4-5 months against susceptible strains of P falcipurum. Development of resistance is common, and local discomfort, reactions, and abscesses, can occur unless the injections are made deep into the gluteal muscles. The drug has not lived up to earlier expectations but it may be of value in treating migrant labourers or nonimmune visitors to malarious areas, and has also shown promise as a singledose treatment for cutaneous leishmaniasis [ 1911. The success with cycloguanil pamoate prompted attempts to design repository vehicles for dapsone. The diglycine derivative (38) was as active against f? berghei as the parent sulphone [31], but little repository effect was noted and the compound has not received trials in man. The diacetyl derivative, 4,4'-diacetylaminodiphenylsulphone(acedapsone, DADDS, 391, protected mice for 6-14 weeks against challenge with I? berghei after single subW O O mg/kg [187, 192-1941, while a dose of 50 mg/kg cutaneous doses of 1 protected monkeys against I? cynomolgi for as long as 268 days [192, 1931. Acedapsone undergoes slow enzymatic hydrolysis in the host to the active parent sulphone [95, 1931, but host specificity plays a significant part since acedapsone does not protect rats against I? berghei because of inefficient deacetylation to the active compound [195]. In initial trials in man, acedapsone was effective in clearing patent infections with P fulciparum and I! muluriae after a single intramuscular dose of 6.9 mg/kg, but it acted much more slowly than cycloguanil pamoate and was effective for less than 30 days [196]. The diformyl analogue (DFDDS, 40) has only recently received a clinical trial [197] but it shows antimalarial activity equal to, but more prolonged than, dapsone when administered orally to non-immune subjects infected with chloroquine- or pyrimethamine-resistant P fulciparum. The drug is a useful prophylactic when given in weekly doses of 800-2000 mg, but has no action on the gametocytes or on transmission through the mosquito and its repository properties last only four weeks in mice [198].

R. M. PINDEX 261 The results of extensive investigations into repository sulphones have now been reported by the Parke, Davis workers [198-2011 to whom we owe most of the inspiration and initiative for the development of repository antimalarial drugs. The chief concern was to develop a repository sulphone which would undergo slow, non-enzymatic hydrolysis upon contact with body tissue or fluids, because acedapsone was enzymatically deacetylated too slowly for really effective plasma levels of the active drug to be attained. It has already been stated that replacement of the acetamido by formamido functions diminished repository properties, and such activity was abolished when (a) both acetamido functions were replaced by amido functions containing more than two carbon atoms, (6) one acetamido function was Nalkylated, (c) chlorine atoms were introduced into the nucleus, or (d)the sulphone moiety was replaced by thio, sulphinyl, oxalyl, or 2,2,2-trichlorethylidene linkages. However, seven other compounds (41-47) were found to have repository activity against trophozoite-induced infections with /

R3

(41) R 1 = M e ( C H 2 b ;R2=R3=H

(42) R'=R3=Me : Rz=COMe (43) R'=Me : R Z = C O E t ; R 3 = H 144) R ' = Me ; R2=C0.(CH2),,Me (15) R ' = M e

:

: R3= H

R2=CH0 ; R 3 = H

(46)R ' = M e ; R2=CO*[CHzl,,,Me

(47)R'=Me(CHz)5

; R3 = H

; R2=R3=H

l? berghei in mice, but all were inferior to (39) giving protection for periods of seven to ten (4144) and four to seven weeks (4547) [198]. Of a series of Schiff bases prepared by condensation of the appropriate aldehyde with the free amino groups of a number of 4,4'-diaminophenylsulphones,four compounds, (48-51), all containing the monoacetamido function, displayed noteworthy repository activity against P berghei [200]. These compounds

(48)R = Ph

262

a

RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA OH

(19) R = O N H . O , ,

(50) R =

3

(51) R =

Cl

Br

all afforded protection for up to nine weeks, while two derivatives containing allyl- and propyl-amino functions in place of acetamido protected for six weeks. A seventh compound (52) in this series lacked appreciable repository properties and did not protect mice for even one week, but it did display strong therapeutic effects against fl berghei when given daily as a prophylactic, with a quinine coefficient of 190. Nevertheless, in view of the inherent cross resistance between (52) and dapsone, trials in man are not planned. Finally, a study of a series of bis-Schiff bases demonstrated repository activities of over nine weeks and 5-7 weeks for 4,4lp-phenylene-bis-(methylidyneiminop-phenylenesulphonyl)]bis-formanilide (PSBF, 53) and the corresponding bis-acetanilide (PSBA, 54) respectively [199]. The period of activity of PSBA

R co .N

-

o so,

0 -

( 5 L ) R = Me

N= cH

was dependent upon particle size, and is intermediate between the shortacting dapsone and the very long acting acedapsone. A single subcutaneous dose of PSBA protected rats against challenge with fl berghei for one week with strong suppressive action at five weeks; it is interesting to note that acedapsone does not provide protection in this species because of the slow rate of mobilisation of the active drug from the depot site and because of the deficiency of the necessary hydrolytic enzymes in the rat [ 1951. The reason for the rapid effect in all species is the extreme lability of PSBA in aqueous media, the compound having a half-life of less than 30 minutes in 50 per cent water-dimethylformamide at room temperature [ 1991. PSBA also protected monkeys against I? cynomolgi for up to 13 weeks, after single lo0 mg/kg doses, and was effective against strains of f? berghei resistant to cycloguanil but not against those resistant to dapsone. It seems unlikely that PSBA will be used in humans, since injection of the drug in laboratory animals consistently produces a chronic granulomatous response with vigorous foreign body cell proliferation and final fibrosis of the site of injection, and crystals

R. M. PINDER 263 of the drug were still evident at these sites at least 12 weeks after injection [199]. Early attempts [202, 2031 to obtain repository activity by co-polymerising sulphonamides or sulphones with formaldehyde had indicated that such polymers ( 5 5 , 5 6 ) , although curative at high doses against P bergheiinfections in mice, were effective for relatively short periods. However, a further study

(55)

(56)

by Elslager and his colleagues [201] demonstrated significant repository activity in a series of polymers prepared from 4,4'-diaminophenylsulphones and a variety of suitable aldehydes. Polymeric N-allylidene-4,4-diaminodiphenylsulphone (57) protected 50 per cent of mice for 8.5 weeks after a single subcutaneous dose of 400 mg/kg, while the polymers of N-allylidene2-methyl-4,4'-diaminodiphenylsulphone(58) and N-benzylidene-N'-methylene-4,4'-diaminodiphenylsulphone(59), and the dimeric compound (60), (57) R'=N=CH.CH=CH.NH;

R~=H

(513) R ' = N = C H . C H = C H . N H ;

RLM~

(59) R'=N=CH

o

w =N

Rz=H

n

r

1

afforded protection for approximately 3 . 5 4 weeks. The same authors have provided a comparison of the repository sulphones on the basis of the parameters important for antimalarial activity [201].It is evident that acedapsone exceeds the other repository sulphones in repository activity by several orders, but that PSBA, compounds (48-51 ), and the sulphone polymers,

264 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA provide a relatively more intense, albeit less prolonged, chemotherapeutic effect than equivalent doses of acedapsone. Moreover, these drugs are safer than dapsone itself in terms of relative production of methaemoglobulinaemia. The more intense effect arises, of course, from the relatively facile nonenzymatic hydrolysis of these compounds as opposed to the enzyme-dependent hydrolysis of acedapsone. It is evident that a number of sulphones have repository properties against I-! berghei, but, nevertheless, their use alone in man seems unlikely. The current interest in synergistic combinations of antimalarials has prompted the use of acedapsone in combination with cycloguanil pamoate, since the parents, dapsone and chlorguanide, inhibit sequential steps of plasmodia1 metabolism. It seems likely that the other repository sulphones will also be tested in combination with cycloguanil pamoate, if they ever reach the stage of clinical trials in man. Synergism between acedapsone and cycloguanil pamoate was evident in laboratory animals and a I : 1 mixture of the two drugs (CI-564, Dapolar) afforded broader repository action against strains of I! berghei resistant to either drug alone [187]. Extensive human studies with the mixture have confirmed these results [190, 196, 204-2081. The general consensus of the clinical results is that Dapolar is less effective against l? vivux than I? fakiparum, since strains of the former parasite that are already resistant to proguanil are unaffected by the repository mixture although susceptible strains are suppressed for as long as five months. Against I-! fakiparum the mixture offers protection for four months if the parasites are susceptible to proguanil and pyrimethamine but less than two months if they are resistant to the antifolic drugs and to the 4-aminoquinolines. It is concluded that in intramuscular doses of 5 mg/kg given every four months Dapolar is particularly suitable for the protection of migrants, other nonimmune visitors to malarious areas, and for use in the consolidation phase of malaria eradication programmes or in the attack phase in conjunction with insecticidal spraying. Some additional prolongation of repository effects can be obtained, and the emergence of resistant strains diminished, when a single oral dose of a 4-aminoquinoline is given concurrently with the repository injection [190, 208, 2091. Thus amodiaquine enhanced the protection afforded by cycloguanil pamoate against I!fakiparum, but had relatively little enhancement against P.vivux, the exoerythrocytic stages of which are unaffected by 4aminoquinolines. Similar effects were observed when amodiaquine was combined with Dapolar. It is apparent that a radically curative agent with repository properties is required for the long-term treatment of vivax malaria, since combination of the repository anti-folic compounds together with a long-acting primary tissue schizontocide would provide an ideal prophylactic. It is also apparent that administration of a repository 4-aminoquinoline together with repository anti-folics might be more satisfactory than the use of a normal 4aminoquinoline in the combination, in order that periods of

265

R. M. PINDER

anti-malarial activity are better matched. The relatively short-acting' repository sulphone, DFDDS, has been combined with chloroquine and primaquine, which have similar durations of activity, and this regimen provides better protection against P.falciparum than any other combination of the threeconstituents [ 1971.Nevertheless, a number of repository vehicleshave been designed for the 4-aminoquinolines and it seems only a matter of time before they are tested by concurrent administration with repository antifolics. Several large molecules containing two 4-aminoquinoline moieties linked by a piperazine ring were synthesised in France [210], and the most interesting compound, bis-(7-chloro-4-quinoly l-Zaminopropy1)- 1,4-piperazine (61), gave complete protection to mice for two months against challenges with Z? berghei after single injections of 500 mg/kg [89, 21 1, 2121. The

/7

N HCHMe.CHi N

QI2

CI

N .CHiCHMe,NH

u b (61)

CL

compound has a marked affinity for the liver and kidney, and a delay in the release of the drug from liver parenchymal and Kupffer cells is apparently responsible for its striking repository properties. It is active per se against plasmodia, and there seems no evidence to suggest a possible metabolic degradation to two discrete 4-aminoquinoline molecules. The original reports that chloroquine-resistant strains of Z? berghei were sensitive to the piperazine derivative have not been substantiated, and the drug is not effective .against such strains [2 131. Comparative trials with chloroquine against Z? berghei in mice [89] showed that chloroquine was fifty times more potent than the repository drug, but although the acute LDS0of the latter was higher than that of chloroquine it did not depress cardiac function in therapeutic doses. It therefore seems a likely candidate for inclusion in a multi-repository combination for the therapy of human malaria, particularly since oral doses are more acceptable than other 4-aminoquinolines [13, 871, but the drug does depress respiratory functions in therapeutic doses [213]. Attempts to obtain repository 8-aminoquinolines and 9-aminoacridines by incorporating labile ester or amide functions into high molecular weight salts of the active drugs failed to provide drugs with repository properties, but similar efforts with the oxychloroquine and amodiaquine molecules afforded eight compounds [62-691 which protected mice against Z? berghei for two to four weeks after a single dose of 400 mg/kg [215]. None of these compounds seems likely to achieve clinical use in view of the increasing resistance of Z?falciparum to the 4-aminoquinolines.

266

RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA NH.CHi CH (OCOMebCH; NEt,

-

~

Parnoic acid

(6'4

CL

r

1

L

J

( 6 3 ) R1= Me ; R2= 1,5-naphthalenedisulphonic acid ( N D S ) 1

2

(61) R = Me ; R = L,~-brphenyld,sulphonic acid (80s) (65) R'=(CH2)5Me ; R 2 = Parnoic acid (66) R'= (CH2)1LMei R 2 = 2 HCl (67) - (69) R'= (CH2),LMe : R2= NOS, BDS,

OT

Pamoic acid

Immunolog?, The possibility of immunisation against malaria [l, 20, 216, 2171 is not strictly within the context of this review of chemotherapy, but it deserves mention because of the potential value in the eventual conquest of the disease just as in other communicable diseases. Immunity to plasmodia shares many features in common with other microbiological infections but, owing to the complicated life cycles of the parasites, the problem is not a simple reaction of the host to a single stage of the parasite, and the factors involved in resistance to plasmodia1 infection are multiple and varied. Moreover, sterile immunity is frequently not achieved, relatively small numbers of organisms may continue to survive in the immune host, and splenectomy during this state of premunition may lead to recrudescences of infection. There are at least two varieties of natural or innate immunity [217], both based upon genetically inherited haemoglobinopathies [20]. Sickle-cell anaemia provides a partial barrier to extreme proliferation of the parasites in red cells, and, although the likelihood of infection is as great among individuals with this trait as among normals, they do not show the very high parasitemia associated with mortality in infections with P.,fulciparum.

267 It has been suggested that selective removal of sickled parasitised cells from the circulation, impaired development of parasites in sickled cells, or suboptimal utilisation of sickle haemoglobin by parasites, may be responsible for the phenomenon, but the mechanism is still unknown. Other haemoglobinopathies, either singly or in combination, may also protect against malarial infections [20, 2171. The innate resistance of the Negro to infection, particularly with II vivax, has prompted the speculation that glucose-6phosphate dehydrogenase (G6PD) is a vital enzyme for parasite development, since this enzyme is deficient in dark-skinned races. However, from the point of view of the genetically normal individual, acquired immunity is of more importance than natural immunity. Evidence of acquired immunity, that is an antibody response to an antigenic stimulus, is provided by the elevated globulin levels exhibited by malarious populations. Malarial infections in both man and in other mammals is followed by increased immunoglobulin synthesis and the production of antimalarial antibodies, detectable by a number of serological techniques [217]. The antibodies increase in number until the crisis but then slowly decline, but are usually still apparent many months after the infection and may return in greater density after relapses; for example, antibodies against II falciparum may persist for eight years and in II malariae for as long as 26 years [216]. Although these antibodies may cross-react with various plasmodial species, immunity to malarial infection is largely species specific, while the correlation between the immune status of an individual and the observed levels of antibodies is poor, which indicates that much of the antimalarial immunoglobulin formed has no protective effect against plasmodial infection. Indeed, figures of less than one per cent have been reported for the concentration of antimalarial immunoglobulin that is actually absorbed by mature parasites or by parasitised erythrocytes [2161. Such studies are enhanced by the application of in vitro methods, to distinguish between protective and non-protective antibodies and to indicate which phase of the erythrocytic cycle is affected or which antigenic component of the parasite evokes the immune response. Experiments with P . knowlesi in the presence of immune serum suggest that leucine uptake is inhibited during schizogony, and that the antibodies combine with free merozoites and thereby prevent re-invasion of red cells [218]. Complementary to such investigation is the necessity for mass isolation of plasmodia, and procedures have been described for the collection of parasite antigens free from erythrocytic and leucocytic material for a variety of plasmodial species [217, 2191. However, in vitro methods are not totally reliable since the immunogenicity of sporozoites under experimental conditions is in sharp contrast to the absence of an effective immune response to sporozoites under conditions of natural infection. The short life of injected viable sporozoites is apparently responsible, while the relative isolation of the exoerythrocytic stages in the liver is a possible reason for the ability of these stages to develop in hosts immune to the blood phase [2 171. The spleen R. M. PINDER

268 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA plays an important role in removing erythrocytes damaged by infection with malaria parasites, and splenectomy can result in a lowered resistance due to a weakened antibody response to new antigenic variants. Immunity can in fact be transferred by means of spleen cells from immune donors [217]; in P. berghei infections, for example, passive transfer of donor spleen cells produces high levels of antibodies in the sera of recipient mice, who are able to effect complete cures within 11-13 days after infection [220, 2211. Immunisation against malaria is faced with very difficult problems because of the specificity of the immune response for individual parasites, particularly since P. falciparum and P . vivax, for example, can comprise a multiplicity of strains each with its own antibody-antigen characteristics. Certainly, it is apparent that the chronic course of malarial infections may result from variabilities in the erythrocytic forms of the parasite, and serologically distinct variants have been detected in simian [222-2241 and rodent [225,226] malarias. Nevertheless, immunity in experimental animals is readily transferred to non-immune hosts by passive transfer in spleen cells or serum, and transfer of immunity seems to involve both cell and humoral components [217].Tissue culture has been used to attenuate P. berghei, and such parasites were able to protect mice from challenge with virulent P. berghei while the vaccine did not induce more than a transient parasitemia [227]. Similar effects have been reported using cell-free extracts of erythrocytic forms of P . berghei [228], and with intact erythrocytes [229]. Killed erythrocytic parasitised cells proved very effective in monkeys subsequently challenged with P . knowlesi, and unlike immunity following chronic infections the protection was effective against a number of strains [224]. In both P. gallinaceum in chicks and P . berghei in mice, immunisation with killed sporozoites suppressed development of sporozoites but not of the erythrocytic stages, while vaccination with killed erythrocytic forms did not affect sporozoite development but prevented the proper growth of the subsequent erythrocytic stages [230]. Similar observations have been made using repeated immunising injections of X-irradiated erythrocytes and sporozoites of P . berghei, and protection of 60 per cent [231] and 100 per cent [232] respectively have been claimed for subsequent challenges with lethal erythrocytic or sporozoite inocula. Immunity to malaria in man seems to be similar to that evoked by other organisms, for malaria-immune gamma globulin drastically reduces parasitemia in falciparum malaria [20, 233, 2341, and a vaccine for the disease is therefore a theoretical possibility albeit some way from practical realisation 12351. Promising results have been obtained from studies of human malarias in other species; thus, multiple reinfection of gibbons with a strain of P . falciparum confers immunity not only against that strain but also to a smaller extent against heterologous strains [236]. Moreover, four weekly immunisations of owl monkeys with X-irradiated parasitised erythrocytes afforded significant protection against otherwise lethal challenges with non-

R. M. PINDER 269 irradiated P . fakiparum [237]. Further interesting results can be expected in these animal models of human malarias, but possibly the most significant area as far as chemotherapy is concerned involves attempts to stimulate the production of interferon, the body’s natural defence mechanism against virus infections [238]. Although bacterial endotoxins confer added resistance to mice against P . berghei, this action does not seem to be associated with interferon production but with a non-specific stimulation of humoral or cellular factors of immunity [239, 2401. Nevertheless, several inducers of interferon, including Newcastle disease virus, statolon and polycytidylicpolyinosinic acids, have protected mice against P . berghei, and the protection was far greater against sporozoite than blood-induced infections [241-2431. Furthermore, prolonged incubation in vitro of parasitised red blood cells with serum containing interferon decreased or abolished the capacity of these cells to initiate lethal malarial infections in mice [244]. Simple polycarboxylate polymers, such as polyacrylic acid-pyran copolymer, also protect mice against sporozoite- but not merozoite-induced infections with P . berghei, and it is suggested that interferon inducers either inhibit a specific host cell function necessary for sporozoite maturation or they affect liver cells in a way which does not exist in the case of erythrocytes [245]. The role of interferon in this mechanism is confirmed by recent observations that exogenously administered serum containing interferon protects mice against P . berghei, and the extent of antimalarial effects was directly related to the titre of interferon [246]. OTHER ANTIMALARIAL COMPOUNDS

Many different types of structure have been shown to possess antimalarial activity, and much of the current synthetic work is based upon leads from the World War I1 programme [4, 51. In general, most of the compounds in this section have been tested only against non-human plasmodia although human data are mentioned where appropriate. The selection is very much a personal one of compounds which we feel offer promise for future development, and readers who desire a comprehensive listing are directed to the review by Aviado [31] and the yearly reviews by Cain [247]. Alkaloids and related compounds

One of the oldest remedies for malaria is the Chinese drug Ch’ang Shun, which consists of the powdered roots of Dichroa febrifuga and whose active constituent is the alkaloid, febrifugine (70). Although effective against avian, simian, and rodent malarias, febrifugine has poor activity in man against P . fakiparum or P . vivax and is also a powerful emetic [79]. Synthetic manipulation of the side-chain has not proved fruitful, but substitution in the 5 6 ,

270

RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA H

or 7 positions of the quinazoline ring system marginally improved the chemotherapeutic index. Methylation of the piperidyl hydroxyl group had little effect, but toxicity was drastically reduced with methylenedioxyl substitution in the above positions; nevertheless, such compounds displayed similar chemotherapeutic indices to the parent alkaloid. This group of compounds remains of interest, however, because plasmodia1 resistance does not seem to be a problem [248]. Attempts to delineate the active moiety of quinine [18]revealed the activity of piperid-2-yl-6-methoxyquinol-4-yl methanol (71). This knowledge, coupled with the discovery that the ease of biotransformation of quinine to inactive carbostyrils could be reduced by 2-phenyl substitution, led to the synthesis of a large number of quinolinemethanols [4,5]. The most interesting compound, piperid-2-y1-6,8-dichloro-2-phenylquinol-4-y1methanol (72), was eighty times as potent as quinine against P. cathemerium in the duck but produced severe photosensitivity in man that persisted long after withdrawal of the drug [249]. This compound is of particular interest since it is firmly bound to host tissues and therefore has repository properties, and because it shows one of the highest activities against P. berghei in mice [31]. A variety of analogous compounds has been synthesised in efforts to delineate the antimalarial activity from the phototoxicity. Numerous permutations of substituents in both phenyl -rj4gs of piperid-2-yl-2-phenyIquinol-4-yl methanol (73) [250], and of the corresponding compounds with. other (71)

R’=G-OMe

; R2=H

(72)

R’=6,8-C12

; R2=Ph

d$) ( 7 3 ) R’:H

R1

; R2:Ph

R2

heterocyclic or dial kylaminomethyl groups in place of the piperidyl function [250-254], failed to abolish phototoxic effects although several compounds were very active against P. berghei. Antimalarial activity was reduced and phototoxicity retained in the 5- [255], 7- [256], and 8-quinolinemethanols [257], and when the secondary carbinol was converted into tertiary [258]. NO effects against P. berghei were evident when the quinoline moiety was replaced by the isosteric benzothiazole and such compounds were highly toxic [259], while replacement by quinoxaline produced non-toxic, inactive compounds [260]. Phototoxicity seems to be associated with functional

R. M. PINDER 271 substitution of the 2-position, the methanol side-chain, and halogen substitution of the quinoline ring [261]. Attempts to reduce conjugation from the 2-aryl group by replacement with trifluoromethyl merely reduced phototoxicity [262], which was, however, drastically reduced with hydroxy or iodo substitution in the 2-phenyl ring [263]. The phototoxic effects are apparently related to the relative electronegativities of the 2-substituents [261], and molecular orbital calculations confirm that the 2-position is the most electropositive in the quinolinemethanols in contrast to the 4-aminoquinolines [264]. The former are photodynamically active, independent of biological metabolism, undergoing a non-specific reaction with a number of substrates 52651, and the mechanism remains to be elucidated.

Acridines and 4- and 8-aminoquinolines Parke, Davis workers have reported on the effects of introducing distal and proximal hydrazine moieties into the structures of active acridines and 4-aminoquinolines [266, 2671. Substitution of a hydrazine moiety for the amine function at the distal position of compounds such as azacrin, quinacrine, and chloroquine, had a deleterious effect on antimalarial activity. Several such compounds containing proximal hydrazino functions, however, showed activity greater than chloroquine; 4,4-( 1,4-piperazinediyldi-irnino) bis (7-chloroquinoline) (74) and 7-chloro-4-[(4-methyl- 1-piperazinyl)amino] quinoline (75) had quinine coefficients of 27 and 28 respectively against P . berghei compared to chloroquine’s 11, but these compounds were highly

n

d (nC[ cLb NH-NWN-NH

CL

A NH-NWN.M

(71)

(75)

cross-resistant with the latter. Other workers [268] have reported similar results for a number of 1-(7-chloro-4-quinolyl)-2-dialkylaminoalkylhydrazines related to (79, which were as active as chloroquine but much less toxic. It is concluded that substitution of a hydrazine moiety for a proximal amine function has a deleterious effect in quinacrine analogues but a beneficial effect in chloroquine analogues, as far as activity against P . berghei is concerned. Similarly, introduction of unsaturation into the side chain of chloroquine, in the form of acetylenic or cis- or trans-ethylenic linkages, produced more potent, less toxic compounds [269]. The chloroquine structure is obviously still open to successful manipulation, and activity was also

272 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA increased when the side chain was ‘folded’ as in the piperidyl (76) and tetrahydropyridyl (77) derivatives [270, 27 I]. Conversely, ring closed analogues NH.CH Me.R ( 7 6 ) R = l -et h y l piperrd - L - y l

CL

( 7 7 ) R = 1 - e t h y l (1,2,3,6 tetrahydro) pyrid - L - y l

of chloroquine in which the dialkylaminoalkylamino side chain was part of a tricyclic system showed negligible activity against P . berghei [272]. Little work has been done on the 8-aminoquinolines, but one report gives evidence for the activity associated with 6-ethoxy and 6-methoxy-2benzyloxy-8-aminoquinolines,which are regarded as the immediate precursors of carbostyril-5,6-quinones [273]. It will be apparent later that this work is based upon the known in vivo conversion of primaquine and its congeners into 5,6-quinone derivatives, which are the active metabolites of this oxidative group of drugs. Finally, a number of 4,8-diaminoquinolines, designed to incorporate both active moieties, had little activity [274], but the design can be criticised since the 6-position was not substituted with methoxyl nor was the 8-amino group anything but primary.

Anti-folics and related compounds A series of amidino ureas synthesised in Poland, and exemplified by nitroguanil (78), were introduced as substitutes for proguanil because of their more favourable chemotherapeutic index [275, 2761. Although proguanil is

more potent against avian, rodent, and human malarias, the toxicity is higher in both acute tests and in terms of the cardiac depression and reduction in fertility associated with these compounds [83]. Nitroguanil seems likely to find clinical use [79], but it is probable that plasmodia1 resistance will be a problem in view of its similar mode of action to proguanil. Other proguanil analogues to receive attention are the 5-aryl-2,4-pentadienamides, following reports of the activity of 5-(4-chlorophenyl)-N-isopropyl-2,4-pentadienamide (79) against P . gallinaceum in chicks [5]. However, extensive investigations [277, 2781 of this series has failed to reveal any activity against either P . gallinaceurn or P . berghei, but several 5,5-diaryl derivatives (80) were curative at high doses in mice [279].

R. M. PINDER

(79) Ar = 4-ClC6H4, R’ = H, A r c ( R’)=CH.CH=CH.COR’ (80) Ar = R’ = 4-ClC.5H4,

273 RZ = NHPr’ R2 = NEtz

The success with trimethoprim (36) in the treatment of falciparum malaria [ 1651 has prompted the investigation of other inhibitors of dihydrofolate

reductase. In view of the wide range of compounds which are effective in this respect [280], it is apparent that antimalarial properties will be found for many more compounds than those discussed below. 5-Piperonyl-2,4diaminopyrimidine (81); closely related in structure to both trimethoprim and pyrimethamine, has been selected for clinical trials because it combines the therapeutic effect of the former against chloroquine-resistant strains of P.fulcipurum with the prophylactic and therapeutic effects of the latter [84]. Certainly this compound is effective against P. berghei at dose levels where trimethoprim is without effect, and it does not depress cardiac function like the two parent compounds. Another 2,4-diaminopyrimidine which looks promising in initial clinical trials is (82), which combines the structural features of pyrimethamines and proguanils [2811, but similar compounds lacking the 4-amino function are inactive [276]. An attempt to combine the structural features of trimethoprim with those of dapsone led to the inactive 3,4,5-trimethoxy-4’-aminophenylsulphone (83) [282], which is hardly surprising in view of the importance of the pyrimidine ring for the antimalarial activity of such compounds. Other well-known classes of dihydrofolate reductase inhibitors to receive attention include the 2,4,7-triamino-6-arylpteridines [29]. Several compounds

(81 1

Me

/

Me0

274 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA containing ortho-substituents in the aryl moiety were highly active against both P. berghei in mice and P . gallinaceurn in chicks, and 2,4,7-triamino-6-0tolypteridine (84) has been selected for human trials because of its very low toxicity in rodents and dogs when given in therapeutic doses [30].A series of 1,2,4-benzotriazines, quinazolines, and quinoxalines, related to known inhibitors of the enzyme, were ineffective against P. berghei [283, 2841, but another related compound, 2,4-diamino-6-(3,4-dichlorobenzylamino)quinazoline (85) was active orally against blood forms of P . berghei in mice and of P . cynomolgi and P . knowdesi in monkeys [285]. Against rodent malarias, this drug was 4-11 times as potent as quinine depending on the route of administration, acted rapidly in clearing parasitemia after one oral dose of 200 mg/kg, and was effective against strains resistant to chlorguanide and pyrimethamine. The action of the drug is potentiated by sulphadiazine, and in view of the known synergism between dihydrofolate reductase inhibitors and sulphonamides and the structural relationship of (85) to the former, it is

NH

likely that it acts in a manner similar to the pteridines. The low toxicity of the compound, coupled with its activity against three different parasites in two hosts, augers well for its use in man particularly as a component of synergistic combinations. Antimalarial activity has recently been detected in a series of substituted tetrahydrofurans, which appear to inhibit dihydrofolate reductase but are totally different in structure from other such compounds [286]. The most active compound, 2-(4-chlorophenyl)-2-(piperid-4-yl)tetrahydrofuran(86), was as active as chloroquine as a suppressive agent against P. berghei and was effective against strains resistant to cycloguanil, dapsone, quinacrine, and chloroquine, although a slight degree of cross-resistance was evident with the antifolics. Further interest in these compounds with a view to their clinical application depends upon the results of toxicity studies in various animal species. Interest has also been shown in a series of compounds closely related in structure to natural metabolites of the folic acid type. Thus,

R . M. PINDER 275 methotrexate (87), an inhibitor of dihydrofolate reductase related to folic acid itself (88), is particularly effective against P. vivux in man and produces clinical cures within 48 hours after total doses of 7.5-17-5 mg [287]. Toxic effects with this drug are related to its prolonged use, as in its original role as an antineoplastic, but effects such as gastrointestinal haemorrhage or mucosal ulceration are readily reversed by folinic acid or by withdrawal of methotrexate. The drug is so effective because it penetrates the reticulocytes which are preferentially parasitised by P. vivux, but it also enters new red blood cells and mature parasitised erythrocytes, irreversibly inactivating any dihydrofolate reductase that is present. Although tetrahydrohomofolic acid (89) is without antimalarial activity, the analogous tetrahydrohomopteroic acid (90) is effective against both normal and pyrimethamine-resistant strains

( 8 7 ) 7,8-Dihydropteridine ; n-I

2

R =Me;

R’=NH,;

i

3

R = NH.CH (CO,H)~(CH,)yCO,H

(881 Pteridinej n = l 3

R

=

1 ; R=OH j

2

R =H;

NH.CH (C02H)-(CH2)2.C02H

(89) 5,6,7,8-Tetrahydropteridine

i n=2

2

j

R1=OH

i

R = R = O H ; R2= H

i

R=H

i

R3’ NH.CH (C0,H j.(CH,)i C 0 2 H

190) 5,6,7,8-Tetrahydropteridine

;

n=2

1

3

of P. cynomolgi [288]. Intravenous administration of tetrahydrohomopteroic acid in daily doses of 80 mg/kg for five days eradicated parasitemia in rhesus monkeys, with no recrudescences. The monkeysdeveloped high blood urea and loss of weight, symptoms which were unaffected by concomitant addition of tetrahydrofolic acid; the latter did, however, prevent the antimalarial actions. The drug apparently does not inhibit dihydrofolate reductase, and represents a new type of antimalarial which may prove useful in synergistic combinations since it acts at sites not attacked by pyrimethamine or sulphonamides. Nuphthoquinones

A number of 2-hydroxy-3-alkyl- 1,4-naphthoquinones were very active against avian malarias but showed little activity against human infections [5,289]. These compounds are powerful inhibitors of plasmodia1 respiration because

RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA 276 of their effects on the enzyme, succinic dehydrogenase. In man, however, they are rapidly metabolised by oxidation of the terminal methyl group, and compound (91), for example, with a quinine coefficient of 2.1 against P . lophurae in ducks, is oxidised in man to the corresponding inactive w-carboxylic acid. Introduction of a hydroxyl group into the side chain renders the drugs resistant to metabolic degradation in this manner but reduces potency. A detailed study of the distribution characteristics of this series of compounds has established that this loss of potency can be compensated by the lengthening of the hydrocarbon chain, and lapinone (92) (911 R = CH2.CHMe.(CH2),Me

(921 R = (CH,I,C(OHl

(931 R = (CH,),.

OH

R J @

0

(C5H,,l,

Cyclohexyl

( 9 L ) R = NH.C,H,.SO,NH,

(95) R = NH . NH.COR’

proved to be effective against P . vivax. Furthermore, biological potency could be retained and metabolic oxidation inhibited by introduction of a terminal cycloalkyl group, and menoctone (93) was curative in P. berghei infections at oral doses of 25 mg/kg [29&292]. Structure-activity relationships in this series [291] indicate that peak activity is associated with heptyl, octyl, or nonyl side chains adjacent to the hydroxyl or the terminal cycloalkyl group. A number of w-adamantylalkyl derivatives were also active at similar doses, however, with only ethyl or propyl side chains but the bulk of the group probably inhibits oxidation [290]. Attempts to combine the structural features of a sulphonamide with those of the naphthoquinones led to inactive compounds such as (94) [293], and similar deleterious effects on activity were observed with a series of 2-acylhydrazino-I ,4-naphthoquinones (95) [294]. Several quinone imines related to menoctone showed some activity against P . berghei, compound (96) doubling the survival time of infected mice in subcutaneous doses of 640 mg/kg [295]. Activity was also evident in a series of 4-amino- 1,2-naphthoquinones, related to both the

lapinone series and the active metabolites of the 8-aminoquinolines [296]. Lipophilicity again seemed to be related to antimalarial activity, but even the most potent compound (97) was less active than quinine against avian and rodent malarias.

R. M. PINDER 277 It is apparent that the naphthoquinones contain a number of active antimalarials, and that the problems of rapid bioinactivation in man have been overcome. Lapinone and menoctone are currently undergoing clinical trials, and it is particularly relevant that the latter drug seems to potentiate the aktion of proguanil [147] suggesting its use in synergistic combinations. However, the drugs do suffer from the disadvantage of parented administration but may offer advantages in other areas. For example, they reduce pulmonary oedema, both in mice infected with P . berghei and in mice treated with large doses of adrenaline or with carbon dioxide [297], and pulmonary oedema is one of the more serious complications of falciparum malaria in man [298]. Furthermore, lapinone is relatively non-toxic, and against P . berghei it is equally effective when given orally, intraperitoneally, or subcutaneously [299]. If such results can be found in man, then the naphthoquinones represent a welcome addition to the list of clinically useful antimalarial drugs.

Antibiotics and antineoplastics Antibiotics and antineoplastics are expected to exert effects against plasmodia since their mode of action is similar to that of many more familiar antimalarials, but even the most effective ones such as aureomycin, terramycin, and chloramphenicol, are active only at high doses with very slow actions [300]. However, prodigiosin at doses of 40 mg/kg doubled the survival time of mice infected with P . berghei [301],while rifampicin proved even more effective in oral doses of 10-100 mg/kg or in subcutaneous doses of 1.0 mg/kg [302]. The antibiotic to receive the most interest is lincomycin (98), which has no antimalarial effects until the 7(R)-hydroxyl group is replaced by chlorine [303-3051. These halogenated lincomycins (99-101) showed activity against P . berghei of the order of chloroquine and dapsone, as well as being R3

I

Me

01 1 1

R-C-R

2

CO-NH-CH

2

1

L

3

(98) R = O H ; R = H ; R = M e ; R = P r 1

3

2

1

3

2

L

(99) R = R = H i R = C L ; R=C,H,,

(100) R = R =H ; R =CL ; RL= Pr 1

2

(101) R = H ; R = CL i R3=Me ;RL=Pr OH

effective against strains resistant to these drugs [303, 3041. The chlorinated lincomycins are more rapidly absorbed, give higher blood concentrations, and penetrate tissues more readily, than lincomycin itself, and this probably accounts for their effects on plasmodia. Tests in monkeys infected with P . cynomolgi [305] showed that (99) and (101) were curative in oral doses of

278 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA 50 mgikg, daily for five days, while (100) was effective at 100 mg/kg. The first and last named were also curative when given subcutaneously in doses of 25 and 50 mg/kg respectively. All three compounds were relatively slow in clearing blood parasitemia, requiring 3 4 days, which would limit their use in falciparum malaria. Nevertheless, the chlorinated lincomycins seem to be effective against multi-resistant strains of plasmodia and their mode of action may be different from presently employed antimalarials. Their low toxicity in man encourages clinical trials against malaria particularly in infections with chloroquine-resistant P. falciparum. 3-Piperonylsydnone ( 102) was originally synthesised as an antineoplastic, but showed activity against P. berghei comparable to chloroquine [306]. Structure-activity relationships in this series have demonstrated the importance of the piperonyl group [307, 3081, while replacement of the sydnone moiety by hydantoin, thiohydantoin, and related systems, abolished the activity although such compounds suppressed sporozoite development in the mosquito [309]. The mode of action of 3-piperonylsydnone remains unknown. The promising activity against P. gallinaceum of a series of dithiosemicarbazones of glyoxal[310] has not been confirmed in recent studies [18]. However, a number of guanylhydrazones of substituted benzophenones (103) were curative in mice, and this series of compounds may well provide leads

X C,H

*

C [ = N .N H .C ( N H ) *

N H2]. C6HLX

(103)

for further successful development [3111. Two more familiar antineoplastics, actinomycin D and cyclophosphamide, appeared to be more active than chloroquine against P. berghei, and have prompted investigations of the efficacy of other such agents as antimalarials [312]. Finally, 1-aminocyclopentane-1-carboxylic acid (104) is effective against the tissue forms of P. berghei, but has relatively poor activity against the blood forms. This compound nevertheless suppresses blood parasitemia in subcutaneous doses of 100 mg/kg, which causes very few toxic effects [313], and is active against both chloroquine-sensitive and resistant strains. It does not replace natural amino acids in protein biosynthesis but apparently inhibits their transport into plasmodia1 cells, possibly through its actions on methionine adenosyl transferase [3141.

R. M. PINDER

279

Miscellaneous drugs

Numerous series of compounds have shown activity against P. berghei in the present U. S. Army programme, including 6,8-disubstitutedpyrido [2,3-b]pyrazines, mono- and di-sydnone derivatives of dapsone, 3-phenylrhodanines, 4-aminobenzo[g]quinolines, 2-aminoalkylamino-4-trichloromethyl-s-triazines, and p-sulphamoylphenylazo compounds [247], and 3-amino-1 -halo-4-methylisoquinolines [3151. A series of phenanthrenemethanols, many of which were synthesised during World War 11, have recently proved to be active in man [281]. Although several of the initial compounds (105) were relatively ineffective against P. berghei [316] it is evident that the isomeric compounds (106) are very effective. The interest in

(105) R ‘ = H or B r ; R 3 = H ; R 2 = C H O H * C H 2 . N R 2

I

( 1 0 6 ) R’= B r ; R2= H ; R 3 = C H O H * C H 2 * N (C7H,5)2

I

R’

R’

radically curative agents led to the development of 6-(4-diethylamino-1 methylbutyIamino)-5,8-dimethoxy-quinaldine( 107), which had a profound effect on the exo-erythrocytic forms of P . cathemerium and P . cynomolgi, but was much too toxic to test in man [317]. A related compound, 4-bromoS[bis(diethylaminoethyl)] aminoveratrole (RC- 12, 108), shows more promise, and although a poor schizontocide in P. cynomolgi infections it does act as a casaul prophylactic and radically curative agent [318]. The drug may well be useful in combination with a quick-acting schizontocide like chloroquine, and is currently undergoing clinical evaluation. Substitution of the bromo group by a variety of other groups or replacement of the diethylamino by other terminal amino functions abolished the antimalarial activity [3191.

BrQ

OMe

OMe

280 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA Another compound whose antimalarial activity has been revealed in screening programmes is the broad spectrum antiparasitic drug, cr,cr,cr,cr‘,cr‘,cr‘hexachloro-p-xylene (109), the most active of a series of halogenated hydrocarbons tested against P . berghei in mice [320]. This drug localises in fat depots and maintains therapeutic levels in the infected erythrocytes for at least 14 days after a single subcutaneous dose of 640 mg/kg. It may also be given in the diet for six days, being twice as potent as quinine in this respect, and is effective orally against blood forms of P . cynomolgi in monkeys in doses of 32-1 58 mg/ kg. The compound was active against chloroquine- and dapsone-resistant strains, while cycloguanil-resistant strains seemed to be hypersensitive to its action. The mode of action of these drugs is unknown and is not related to that of known antimalarials; speculation that the active metabolite is a terephthalic acid formed by oxidation of the trichloromethyl groups is not substantiated by observations of the lack of activity of such acids [320]. cc,a,cr,cr’,cr’,a’-Hexachloro-p-xylene has a favourable chemotherapeutic index, since relatively low activity is matched by very low toxicity, but it does potentiate the depression of liver respiratory enzymes produced during malarial infections and may damage liver function [321]. Several members of a series of 350 quinolones that were originally synthesised as coccidiostatics [322] have displayed antimalarial properties [323]. The two most active compounds ( 1 10) and ( 1 1 1) suppressed erythrocytic infections of P. berghei in mice at daily doses of 1 mg/kg (s.c.), with causal prophylactic action against (110) R = PhO*CH,.CH,*O(111) R=Me(CH2)7.0-

both P . berghei and P . cynomolgi. The quinolones are poorly absorbed from the gut and are relatively ineffective when given orally, but they retain good suppressive activity against multi-resistant strains of P . berghei. The mode of action of these drugs resembles that of primaquine, but resistance develops relatively easily in P. berghei when they are used alone. Marked potentiation was demonstrated between the quinolones and sulphonamides, and the former show great promise for use in synergistic combinations. Certainly, the discovery of radically curative properties in a series of compounds unrelated to primaquine and of low toxicity gives rise to hope for their eventual use in man. THE CURRENT TREATMENT OF MALARIA

The treatment of acute malarial infections caused by sensitive strains of all four types of human plasmodia, and prophylactic therapy against the same,

R. M. PINDER 28 1 still depends upon the same drugs as were used in the last two decades [9, 10, 75, 324, 3251. Chloroquine and amodiaquine remain the drugs of choice for treating the acute attack, together with primaquine when radical cure of a relapsing malaria is required. Proguanil and pyrimethamine are still the best causal prophylactics for non-immunes if they are taken regularly, and the former in doses of 100 mg offers a greater margin of safety because of the more dependable regularity of its daily intake than the once weekly regimen of 25-50 mg of pyrimethamine. Drug combinations, such as chloroquine-pyrimethamine or chlorproguanil-chloroquine, are better prophylactics than single compounds, and a protective regimen of daily proguanil combined with a weekly dose of 300 mg of chloroquine may be of advantage [325]. The use of sulphones and sulphonamides is falling into some disfavour because of the ease of induction of cross-resistance [160, 325-3271, and the use of repository anti-folic drugs looks as though it will suffer the same fate. Administration of chloroquine or amodiaquine at a weekly dose of 300 mg is still recommended for suppressive purposes in those areas where there is a low possibility of selecting 4-aminoquinoline-resistant strains of P.fulcipurum, and higher doses may be used in areas of high endemicity [75, 3251. Chloroquine and pyrimethamine are often added to common salt for suppressive purposes in areas where other modes of administration are difficult from social and administrative viewpoints. This practice can be criticised since there is no way of controlling dose levels and resistant strains are more likely to emerge in the face of suboptimal doses. The most serious problem in current malaria therapy, and indeed the reason for the present resurgence of interest in malariology, is the treatment of chloroquine-resistant falciparum malaria. Several prophylactic regimens have been used in areas where resistant strains are prevalent, but the original regimen of 300 mg chloroquine and 45 mg primaquine, given weekly, used for chemoprophylaxis of U.S. troops in Vietnam is not satisfactory for preventing the development of falciparum malaria [lo]. Subsequent regimens combining chloroquine, with or without primaquine, with a sulphonamide or a sulphone [lo, 751 are apparently successful, but the east of induction of resistance combined with the toxic effects associated with long-term usage of sulphonamides or sulphones militates against their use. The treatment of acute attacks of chloroquine-resistant falciparum malaria is a most urgent problem in view of the possible fatal consequences of inadequate treatment. In heavily endemic areas of Vietnam, as many as 50 per cent of U.S. troops exposed to P.fulcipurum developed clinical malaria, and chloroquine therapy proved to be of universal ineffectiveness in bringing about radical cure [328]. Two recent regimens, pyrimethamine with a sulphonamide or trimethoprimsulphalene, are proving of increasing value in this respect [325]. The onset of action of these combinations has been claimed to be as rapid as that of quinine [328], but, nevertheless, most workers emphasise the need to initiate therapy with quinine in any patient with a heavy parasitemia or in whom there

282 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA is evidence of any cerebral or renal involvement. Blount [lo] has reported that 600 mg quinine given every eight hours for 14 days, parenterally in severe cases, and 25 mg pyrimethamine every 12 hours for three days, has given good clinical results in over 2000 cases of acute falciparum malaria. Higher cure rates, up to 98 per cent, are obtained when patients were treated with pyrimethamine plus a long-acting sulphonamide such as sulphormethoxine together with quinine, or with quinine and pyrimethamine followed by diaphenylsulphone [9, 3281. Despite the success of these regimens, many workers still prefer to use quinine alone for treating acute attacks of suspected chloroquine-resistant falciparum malaria in non-immune individuals, and to keep the alternative combinations for patients whose infections recrudesce after adequate quinine therapy [9]. Even this procedure is in doubt, however, because of the emergence of quinine-resistant strains of P. falciparum [92, 3281. Such strains are suppressed by the administration of sulphalene in four doses of 250 mg given on the first day of a 14-day course of quinine (18.9 g total dose). However, the continually broadening spectrum of drug resistance on the part of P. falciparum suggests that this may be only a temporary solution, and the need for new compounds is further emphasised by the present uncertainty of obtaining a 100 per cent radical cure in falciparum malaria with any of the present drugs, either singly or in combinations.

THE MODE OF ACTION OF ANTIMALARIAL DRUGS Successful chemotherapy depends upon exploitation of biochemical differences between the metabolism of the parasite and that of the host. Like all protozoa, plasmodia are unicellular animals in which all functions of the animal have to be carried out by the single cell, and the bioche’micalprocesses are likely to be different in the vertebrate and invertebrate hosts where the parasite is exposed to vastly different conditions. The subject of plasmodial biochemistry is still in its infancy [19,329] and direct attempts to study it have practically been limited to the blood phase of a few species of parasites. The principal biochemical reactions may be summarised as follows, (a) phosphorylation of glucose, which finally produces the energy required for the parasite’s metabolism, ( b ) oxidative processes, which are maintained by erythrocytic haemoglobin, (c) enzymic breakdown of the globin portion of haemoglobin into amino acids and peptides, which are built up into parasite protein, and ( 6 ) synthesis of large quantities of lipids. Antimalarial drugs have two general modes of action, the anti-folics inhibiting some step in the metabolic conversion of p-aminobenzoic acid into the enzyme co-factors required for plasmodial synthesis of purines and pyrimidines, while the antinucleic acids inhibit the synthesis of proteins at the DNA and RNA level. Nevertheless, these drugs do have other actions on plasmodial metabolism

R . M. PINDER 283 which may be of significance, and in addition there are a number of antimalarials which fit into neither group and have distinct modes of action. Indeed, it is obvious that the developing knowledge of plasmodial biochemistry will encourage the use of a rational approach to the design of new antimalarial drugs.

PLASMODIAL SYNTHESIS OF PURINES A N D PYRIMIDINES

Sulphonamides, sulphones, pyrimethamine, chlorguanide, and the dihydrofolate reductase inhibitors, all act at points in the synthesis of those enzymes that are concerned in nuclear division. Parasites appear to develop normally in the presence of these drugs until it is time for the nucleic material (chromatin) to divide, when morphological abnormalities become apparent [330]. P. fakiparum [331] or P. knowlesi [322], for examples, undergo uninhibited maturation in vitro up to the pre-schizont stage, when the early schizonts are attacked by drugs such as proguanil, pyrimethamine, or trimethoprim, the chromatin does not segment properly and becomes finely divided, and the parasite degenerates. Indeed, if nuclear division is slowed down or halted, pyrimethamine and proguanil no longer have an effect, and this may well explain their failure to cure long relapse strains as opposed to short term strains of P. vivax; in the latter there isa continuous production of exoerythrocytic schizonts with rapid nuclear proliferation, while in the long relapse strains there is latency with cessation of nuclear division. The effects of the anti-folic antimalarials on nuclear division is entirely consistent with their interference in the production of nucleotides, required for plasmodial synthesis of nucleic acids. Their chief mode of action seems to be to inhibit the production of the enzyme co-factors required to allow the parasite to synthesise essential metabolites such as adenines, guanines, and thymines, from simple purine and pyrimidine precursors (Figure 6.2). The growth of plasmodia is stimulated in vivo by the folate precursor, p-aminobenzoic acid (PABA), and infections in hosts fed on a milk diet, which is deficient in PABA, were completely suppressed; addition of PABA or folic acid to the milk diet abolished the suppressive effects [333-3371. However, it has been suggested [336] that the growth of the parasite is determined not only by the PABA content of the gut, but that host reactions play a part so that the availability of PABA for erythrocytes is affected. Certainly, P. berghei, for example, completely suppresses the ability of rodent reticulocytes to acetylate ingested PABA that is excess to host requirements [338], and this may be the mechanism by which plasmodia successfully compete with the host cells for the available PABA. The growth of plasmodia both in vivo and in vitro is inhibited by antimetabolites of PABA, such as the sulphonamides and sulphones, which have a highly favourable chemotherapeutic index because the blocked reaction is not operative in the host who can

2 84

RECENT ADVANCES I N THE CHEMOTHERAPY OF MALARIA

(Man only) -

FOLIC ACID

FOLINIC ACID

PURINE AND PYRIMIDINE PRECURSORS

I

:B I

Proguah, Tetrahydrohomopteroic acid, sulphonamides, - - - + Methotrexate, sulphones Pyrimethamine, PABA Trimethoprim

"20Fp

I

I

/'

'

Tetraiydro- , homopteroik acid

4

PURINES AND PYRIMIDINES

(Plasmodia only) Figure 6.2. Scheme of the mechanism of action of anti-folic antimalarials. PABA represents p-arninobenzoic acid, A and B the intracellular forms of dihydrofolic and tetrahydrofolic acids, and CoF is the enzyme co-factor for purine and pyrimidine synthesis

utilise exogenous supplies of preformed folic and folinic acids. The dihydrofolate reductase inhibitors act further along the same chain, blocking the reduction of dihydrofolate to tetrahydrofolate; this reaction is operative in the host, but it can be by-passed by the availability of exogenous supplies of folinic acid in the diet. The antimalarial action of sulphonamides and sulphones is reversed competitively by PABA and non-competitively by folic acid [18, 79, 1451, higher doses of which are required to reverse the effects of pyrimethamine and proguanil, and similar effects are apparent with a number of folic acid analogues. The mode of action of the sulphonamides and sulphones as antimetabolites of PABA by inhibition of its reaction with the pyrophosphate ester of 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine has been so extensively documented [93] that no further discussion is necessary here. It is pertinent to note, however, that sulphones may compete with the zwitterionic form of PABA, which may have implications for the design and use of anti-folic antimalarials [339]. The mode of action of proguanil and pyrimethamine was originally designated by reference to potentiation and cross-resistance studies [79]. Thus, their action was potentiated by PABA competitors which were active antimalarials but not by some sulphonamides which are not PABA competitors, and they did not potentiate each other. It is apparent from the variety of synergistic combinations already discussed that the sulphonamides and sulphones must act at a different point in the' same metabolic chain from proguanil and pyrimethamine. That this is a similar, sequential point for both of the latter is shown from cross-resistance studies, where (a) strains resistant to proguanil are usually resistant to pyrimethamine, and vice-versa, ( b ) sulphonamideresistant strains are usually resistant to proguanil and pyrimethamine, and (c) strains resistant to proguanil and pyrimethamine are not normally resistant to sulphonamides or sulphones. The mode of action of proguanil

R. M. PINDER 285 and pyrimethamine has now been put firmly on an enzymological basis [340,341]. It is evident that plasmodia synthesise folate-containing co-factors de novo, and the established pathway involves the enzyme dihydrofolate reductase. Early studies indicated that R berghei, for example, could convert added dihydrofolate to tetrahydrofolate in vitro but could not utilise added folk acid, suggesting that it resembled other micro-organisms in possessing a dihydrofolate reductase but not a folate reductase [342]. The former enzyme has now been isolated and characterised as distinctly different from host erythrocytic enzyme, both for I? berghei and I? knowlesi [332, 3411, and there appears to be a close correlation between the concentrations of antimalarial drugs required to inhibit the enzyme in a cell-free system, the concentrations required to inhibit parasite growth in vitro, and the activity of the drugs in vivo. Pyrimethamine was a thousand times more effective in binding to plasmodial dihydrofolate reductase than to the host enzyme, and was more effective than dihydrotriazines or compounds like methotrexate. The exact mode of binding in vivo is still to be elucidated, and is complicated by the lack of knowledge about plasmodial concentrations of dihydrofolate. There have been suggestions that plasmodia depend upon the erythrocyte for folic acid, and monkeys infected with I? knowlesi, for example, show significant falls in plasma folate levels at a time when the concentrations of folic and folinic acids are increasing within parasitised erythrocytes [343]. Trager [344] has suggested that this is a possible explanation of the apparent lack of antimalarial effects of sulphadiazine, proguanil, and pyrimethamine, on I? lophurae developing extracellularly, since the drugs might interfere with a cellular process which produces products required by the parasite, namely folic and folinic acids. Alternatively, it is possible that the parasites meet their requirements for these vitamins by concentrating plasma folic acid intracellularly [345]. Nevertheless, it is well established that plasmodia require PABA for growth in vitro and in vivo [345, 3461 and the explanation of Trager’s results probably lies in the ability of parasitised red cells to concentrate the drugs, rather than the vitamins, with consequent higher intracellular concentrations and greater inhibition of intracellular than extracellular growth. Certainly, chloroquine had the same effects in Trager’s experiments, and it is known to be selectively toxic to plasmodia because of its higher concentrations in parasitised rather than normal erythrocytes [347]. It is evident, therefore, that the 2,4-diaminopyrimidines, like pyrimethamine and trimethoprim, the dihydrotriazines like proguanil (in its active metabolite form), the 2,4-diaminopteridines, and methotrexate, inhibit dihydrofolate reductase. Their selective toxicity to plasmodia may be due to a combination of greater binding to the parasite enzyme and to their selective uptake by parasitised erythrocytes. However, a new antifolic mode of action has recently been proposed for compounds like tetrahydrohomopteroic acid [288], which may inhibit folate metabolism by an action on the feedback

286 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA control of the system by tetrahydrofolate or by competing with the latter for coenzyme sites involved in purine and pyrimidine synthesis. Alternatively, it may act earlier on the enzyme that catalyses the addition of glutamate to dihydropteroate, or on that which catalyses the pyrophosphorylation of 2amino-4-hydroxy-6-hydroxymethyldihydropteridine. In this context, it is pertinent to note that malaria parasites may depend upon exogenouslysupplied preformed purines but have to synthesise pyrimidines de novo [348, 3491, which has considerable chemotherapeutic significance. Many of the anti-folic compounds which have been discussed are thought to act on purine synthesis at the two places where one-carbon transfer is important, by preventing the formation of the required co-factors. However, if the parasite can utilise exogenous purine nucleosides from the red cell, or from the serum via the red cell, then the blockade of purine synthesis is probably not important in terms of supply of purine nucleosides. Z? Zophurae synthesises significant quantities of pyrimidines but little purines [350], and the pyrimidine biosynthetic enzyme, dihydro-orotic acid dehydrogenase, has been isolated from berghei and I! vinckei [351]. Moreover, activity of the initial enzyme of pyrimidine biosynthesis, aspartate transcarbamylase, shows a linear correlation with l? berghei parasitemia in vitro and high activity is associated with the free parasite and not red cell fractions [349]. In pyrimidine synthesis, however, one-carbon transfer occurs only in methylation of deoxyuridine to form deoxythymidine, and it seems possible that the antifolic antimalarials may therefore act on chain initiation of protein synthesis where folate co-factors are known to take part [352]. Much more information is desirable, particularly about the effects of pyrimidine antimetabolites on the biosynthesis of parasite pyrimidines, before the exact mechanism of action of the antifoiics is firmly established. What was regarded as a closed book over a decade ago is only now beginning to look like Pandora’s box! PLASMODIAL UTILISATION OF PURINES AND PYRIMIDINES

Evidence is accumulating from radioactive labelling and other experiments that pyrimidine nucleosides supplied exogenously are not incorporated into parasite nucleic acids in contrast to purine nucleosides [58, 346, 348-351, 353-3551. In particular, the parasite in vitro utilises a variety of preformed purines, especially adenosine, and there is now evidence that it depends upon the host for supplies of purines in vivo, such material coming from the adenosine nucleotide pool of the red cells [356]. The plasmodia1 necessity for de novo synthesis of pyrimidines is apparently due both to a lack of penetration of pyrimidines through the red cell membrane and to a similar transport deficiency through the parasite membrane, rather than to an inability to polymerise them into nucleic acids [349]. Five possible points of attack have therefore been suggested for an antimalarial to exert its effects, ( a ) com-

R. M. PINDER 287 plexation with adenosine, thereby limiting the amount taken up by the red cells, ( b )competition at the sites of uptake of adenosine on the membrane of parasitised cells, (c) and (d)similar actions within the red cell or at the parasite membrane respectively, and ( e ) inhibition of the phosphorylation steps of adenosine within or without the parasite [354] (Figure 6.3). Dapsone markedly inhibits the utilisation of labelled adenosine by intraerythrocytic I? berghei for nucleic acid synthesis [354], by a mechanism involving interference with transport at the level of the host erythrocytic Chlorguanide, Chloroqui ne, Pyrirnetharnine,

Figure 6.3. Possible points of attack of antimalarial drugs on plasmodia1 synthesis of nucleic acids, AMP, ADP, and ATP, represent the mono-, di-, and tri-phosphates ofadenosine

membrane. The drug does not affect the incorporation of labelled adenosine by equivalent free parasites even though these possessed at least as much ability to incorporate adenosine into nucleic acids as did the intraerythrocytic parasite. This action is partly explained by the ability of the red cell, be it parasitised or not, to selectively concentrate dapsone at the membrane 13571. Quinacrine has similar effects upon the transport of adenosine, and it is probable that such drugs act by inhibition of the membrane-bound enzyme, adenosine kinase [358]. Imipramine also has the same effect, although it does not appear to have antimalarial properties, and this action may not be lethal per se to plasmodia but probably plays a part in the overall effect. Nevertheless, it is clear that other blockers of purine nucleoside uptake, which may starve the red cells of essential metabolites for the parasites, should be tested as antimalarial agents [349]. Similar experiments on the incorporation of labelled adenosine into f! berghei have indicated that

288 RECENT ADVANCES I N THE CHEMOTHERAPY OF MALARIA chloroquine, quinacrine, and quinine, inhibit direct incorporation into nucleic acids, although the first-named is less active possibly explaining why 19 berghei is relatively insensitive to it [58]. Quinacrine is apparently the only member of the group of drugs formerly regarded as antinucleic acids which has definitely been shown to block uptake of adenosine at the level of the host red cell membrane, but preliminary reports indicate a similar action for chloroquine [359]. It appears that the importance of the classic DNAtemplate intercalation of quinacrine may be secondary to that of the magnitude of its inhibition of uptake mechanisms at the parasite level, since neither the purine antimetabolites, 2-amino-6-mercaptopurine or 5-fluorouracil, nor the intercalating agents, actinomycin D or daunomycin, have antimalarial effects in vivo [354]. It is not clear whether the parasite takes up free adenosine and then phosphorylates it in situ or whether it takes up phosphorylated adenosine directly from the erythrocyte. Labelling experiments indicate that both processes can take place, but the suggestion has been made that ATP could be dephosphorylated before transport of the resultant adenosine through the parasite membrane, followed by rephosphorylation and incorporation into nucleic acids [354]. This point is still to be elucidated, but it is significant that adenine kinase is an active concentrating enzyme concerned with both transport and phosphorylation of adenosine to AMP [360, 3611, and it therefore seems likely that phosphorylation takes place before entry into the parasite. The work of Schellenberg and Coatney [362] has shown that proguanil and pyrimethamine inhibit the uptake of 32P-labelled phosphorus into parasite DNA, while chloroquine, quinacrine, and quinine, inhibit uptake into both DNA and RNA, indicating blockade of enzymatic phosphorylation of nucleosides. More recently, quinacrine has been shown to exert a selective effect on the incorporation of ATP into parasite RNA at the level of the parasite membrane [358], possibly indicating some differences in the nucleic acid synthesising systems of parasite and host. Certainly, the base compositions of DNA from different malaria parasites varies widely, rodent parasites having 20 per cent guanine and cytosine while primate parasites have 40 per cent guanine and cytosine [363, 3641. Interference with phosphorus metabolism may well play a vital part in the antimalarial action of both the anti-folics and the anti-nucleic acids, and there is a strong correlation between the level of erythrocytic ATP and parasitemia in infections with E! falciparum [365]. Relatively low levels of ATP may confer a biological advantage against falciparum malaria, since the red cells may have a diminished ability to remain intact for the time normally required for maturation of the parasites; lysis of such cells may occur before mature schizonts are formed with release of immature parasites into the circulation [366]. Alternatively, ATP may be used by the parasite in the course of maturation so that its concentration in the red cell is the rate-limiting factor governing the development of intraerythrocytic parasites, and in respect of the mode of action of antimalarials

R. M. PINDER 289 this seems to be the more likely possibility. Brewer and Coan [367] have proposed that red cells are better able when parasitised to accumulate purines needed for ATP synthesis, indicating the susceptibility of the parasite to drug interference with any of the transport or synthetic mechanisms leading to its obtention of purines. These workers have gone further to show that hyperoxia has a dramatic beneficial effect on P.berghei infections in rats; three atmospheres of compressed air completely suppressed otherwise lethal infections, and produced a concomitant decrease in ATP levels in the host red cells. It is possible that hyperoxia may be an extremely valuable adjunct to treatment of malaria with drugs, particularly oxidants like primaquine, and clinical evaluation of the technique is awaited with interest. PLASMODIAL SYNTHESIS OF PROTEINS

Protein synthesis is essentially a translational process of messenger RNA which has been transcribed from the DNA template. The first reaction is the activation of amino acids by amino-acyl transfer RNA synthetases making use of ATP as an energy source, and the enzymes also attach the amino acid to transfer RNA to form amino-acyl transfer RNA. We have already seen that protein synthesis in plasmodia can be inhibited much further back along the chain, either at the folate co-factor level or during nucleic acid synthesis. It is obvious that interference with RNA transcription from DNA (or with DNA replication), or interference with the availability of essential amino acids, will also affect plasmodial synthesis of protein. Morphological studies of the effects of antimalarials on plasmodia support the occurrence of both modes of action for chloroquine, quinacrine, and quinine. Effects of antimalarials on plasniodial utilisation of nucleic acids

It is evident from a number of studies [331, 368-3701 that chloroquine produces a progressive degeneration of parasite ribosomal RNA as a result of ribonuclease action in and around the autophagic vacuole. The most likely mechanism for this action is that the drug causes a lesion in cytoplasmic ribosomes by interruption of synthesis of messenger RNA in the nucleus, which is totally in accord with the effects to be described upon plasmodia1 utilisation of DNA and RNA. The morphological changes in the presence of chloroquine also support the contention [371] that the selective concentration of the drug by parasitised red cells involves accumulation into lysosomes by a process of vesiculation of the cell membrane [372-3741. Whether such effects are involved in the actions of quinine and quinacrine is not clear, but chloroquine certainly stimulates the hydrolysis of RNA by ribonuclease by a mechanism which does not involve the tertiary structure of the nucleic acid [375].

290 RECENT ADVANCES I N THE CHEMOTHERAPY OF MALARIA It has long been known that chloroquine interacts with nucleoproteins [374], and it is now evident that in vilro chloroquine [376-3831, quinacrine [377, 381, 382, 384-3861, and quinine [381, 382, 3871, all form molecular complexes with DNA and inhibit DNA and RNA polymerases. Much of the work has been done with mammalian or bacterial nucleoproteins, which is rather unsatisfactory from the viewpoint of chemotherapy, which tries to attack plasmodia1 rather than host structures. Nevertheless, other studies have shown that chloroquine, for example, interacts rapidly with the DNA of P.berghei [388] and of P.knowlesi [389]. Chloroquine [381, 3821 and quinacrine [384] form complexes in which it is proposed that the quinoline or acridine ring is intercalated between base pairs of double stranded DNA involving electrostatic forces, while the 1,4-diarninopentane side chain falls outside the contour of the base pairs and bridges the minor groove of the double helix by binding electrostatically to DNA phosphates (Figure 6.6).In this way, the electropositive 2-amino group of guanine is brought into close contact with the 7-chlorine atom of the quinoline ring of chloroquine, the former being critical for interaction with DNA; no such base specificity determines the interaction between DNA and quinacrine. The important structural features which determine both binding to DNA and antimalarial

Figure 6.4. Hypothetical structure of the DNA-chloroquine complex. In the upper Jigure the quinoline ring of chloroquine is horizontally inserted between bases of double-helical D N A und the dianrino side chain diagonally spans the minor groove beiween D N A phosphates. In the lower figure the quinoline ring is shown superiniposed over a D N A base pair with lhe 7-CI in proximity to the 2-amino group ofguunine

R. M. PINDER 29 1 activity are indicated by the inactivity of the strong DNA-complexing agents, 1,4-diaminobutane, 1,5-diaminopentane, and spermine. Although these molecules have the correct geometry of two secondary amino groups 7.5 A apart to span the distance between DNA phosphates across the minor groove, it is evident that a quinoline ring substituted in the 7-position with an electronegative group is a prerequisite for antimalarial activity of this type. The binding of chloroquine and quinacrine to DNA therefore seems to involve both electrostatic attraction between the positively charged diaminopentane moiety and the anionic phosphates of DNA (weakly reacting sites), and a more specific interaction involving the ring portions of the antimalarials and the DNA nucleotide bases (strongly reacting sites). The experimentally observed specific interaction of chloroquine with the guanine of DNA is explained satisfactorily by the electron-donating or accepting characteristics of the molecules [390], and is confirmed by nuclear magnetic resonance studies of complexes of chloroquine with a variety of nucleotides [391]. Indeed, the integrity of the ring interaction is apparently unchanged even when the diaminopentane side chain interacts with the phosphates, indicating the importance of the former. Some doubt has been cast on the interpretation by recent X-ray crystallographic data for chloroquine diphosphate, where it appears that the favoured conformation has aliphatic nitrogen atoms 5.54 A apart interacting with phosphate oxygen atoms 5-75 A apart, and where it is impossible for the N-N separation to exceed 6.5 A in the side chain in any conformation [392]. Nevertheless, the proposed model seems satisfactory on present evidence. In contrast to complex formation with chloroquine and quinacrine, which is strongly influenced by an ionic environment, that of DNA with quinine chiefly involves hydrogen bonds [387]. The proposed model is one in which the quinoline ring of quinine is intercalated between bases in double helical DNA and the alcoholic hydroxyl group engages in formation of a hydrogen bond, while the bulky quinuclidine moiety projects into one of the grooves of DNA and is electrostatically attracted with its tertiary aliphatic amino group to phosphates of the sugar phosphate backbones of the double helix. Quinine can apparently bind equally effectively to adenine as to guanine, since only hydrogen bonds are involved, in contrast to the electronic interaction involved in the DNA-chloroquine complex. Replication of DNA requires the separation of the two companion strands of the parental double-helical structure, and substances that interfere with strand separation should inhibit DNA replication. Moreover, RNA transcription from DNA requires the availability of certain base pairs, and constraint of these pairs will hinder the process. Thus, one of the modes of action of chloroquine, quinacrine, and quinine, and their congeners, is an inhibition of DNA replication primarily, but also of RNA transcription from DNA secondarily, mediated through inhibition of the respective DNA and RNA polymerase reactions. Whether these processes take place in vivo

292 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA is still to be proven, but in vitro studies have shown that they do play some part in mediating the antimalarial actions of the three drugs, although they may be secondary to the importance of inhibition of adenosine uptake [354]. In knowlesi, at least, chloroquine and quinine seem to inhibit DNA replication to a much greater extent than they do RNA or protein synthesis [389]; both drugs owe their selective effects against plasmodial DNA to their selective concentration by parasitised red cells. A number of 8-aminoquinolines, including primaquine, pentaquine, and pamaquine, have recently been shown to bind to DNA by a process involving electrostatic interaction of the protonated terminal nitrogen, an interaction between the quinoline ring and the rings of the nucleotide bases, and possibly hydrogen bonding [393]. Thus, these drugs apparently bind to DNA by a process which resembles both chloroquine and quinine, and it is proposed that inhibition of DNA function is involved in their mode of action. A subsequent study [394] has further demonstrated that the active metabolites of the 8-aminoquinolines, the 5,6-quinone or 5,6-dihydroxy derivatives, bind to a lesser extent than the parent compounds. Primaquine and its congeners do not seem to intercalate into DNA in the manner of chloroquine, but interact with the nucleic acid bases through the quinoline ring system. In contrast to the 4-aminoquinolines, the 8-aminoquinolines bind strongly to polyribonucleotides suggesting that they may alter the functions of various RNA fractions in protein synthesis, although chloroquine also showed some effects in this respect and is known to stimulate ribonucleases by a mechanism not involving the tertiary structure of the nucleoprotein [375]. The suggestion that antimalarials may affect protein synthesis as well as nucleic acid polymerases as a result of interaction of their structures with nucleic acids of plasmodia is also in accord with recent observations regarding quinacrine [395]. Efects of antinialarials on plasmodia1 utilisation of amino acids

Plasmodia derive adequate supplies of essential amino acids by digestion of haemoglobin and enzymic breakdown of the globin portion [19]. P berghei [368, 396, 3971, P. knowlesi [370, 398, 3991, and avian parasites [369, 400, 4011, ingest haemoglobin and degrade it in the phagosomes so formed, which are finally recognised as the pigment vesicles. Although an isolated plasmodial system capable of cleaving haemoglobin into haematin and globin is still to be demonstrated, it is apparent that malaria parasites can hydrolyse globin and that this hydrolytic activity is associated with parasite, and not host, enzymes. Certainly, parasitised cells produce large quantities of haematin, a known constituent of the malaria pigment haemazoin and believed to be the insoluble residue from plasmodial digestion of haemoglobin, and there is a large increase of free amino acids in such cells as compared to

R. M. PINDER 293 normal red cells. Furthermore, in general the amino acids produced in highest concentrations by parasitised cells are those found to be the most abundant in host haemoglobin, although the parasite apparently produces more amino acids than it can utilise [402]. Chloroquine [359, 368, 4031, and quinacrine and quinine [404], affect malaria parasites at those stages of their life cycle when the parasites are actively metabolising haemoglobin. These drugs rapidly cause clumping of the phagosomes and later their expulsion from the parasite, probably by a direct action on the pigment- and digestivevesicles involving membrane changes. Cessation of haemoglobin digestion must be related to the clumping and expulsion of the phagosomes, and it seems likely that amino acid starvation, resulting from inhibition of haemoglobin degradation, may be the primary means whereby chloroquine, quinacrine, and quinine, are effective against the trophozoite stages of malaria parasites. It is rapidly becoming evident that plasmodia, in addition to meeting most of their requirements for amino acids via haemoglobin degradation, also need to obtain some from without the host cell. In vitro studies of the growth of P berghei indicate that it produces more isoleucine than can be accounted for by proteolysis of the relatively small amount present in mouse haemoglobin [402]. Furthermore, isoleucine is absent from rhesus haemoglobin [405] but it is evident that it is incorporated into the protein of P knowlesi by concentration from the plasma [346,406-409]. Other work on the uptake of various amino acids, both radioactively labelled and unlabelled, shows that 19 berghei and I? knowlesi have a requirement for both isoleucine and methionine [409, 4101, and the similarity of rates of utilisation of the two amino acids suggest that the dilution of methionine from globin degradation by methionine from extracellular sources occurs at a constant rate throughout the parasitic growth cycle [54]. Isoleucine and methionine appear to be the two amino acids that plasmodia must obtain from without the host cell, and the absence of the former in primate haemoglobins, including man, and the presence of only small amounts of the latter, suggests that inhibition of their uptake may be a possible point of attack for antimalarial drugs. Certainly, N-acetyl-L-isoleucine and L-alloisoleucine compete effectively with isoleucine for incorporation into parasite protein, while L-0-methylthreonine acted differently in inhibiting incorporation of orotic acid into DNA by an isoleucine-reversible mechanism; all three compounds inhibited in vitro growth of E! knowlesi [406, 4071. The conventional antimalarials, pyrimethamine and quinacrine, inhibit the formation of various aminoacyl transfer RNA synthetases, which control attachment of the amino acid to RNA, and their effects on plasmodia may therefore be mediated through inhibition of amino acid activation for protein synthesis [411]. L-0-methylthreonine may act similarly by being activated by isoleucyl-RNA synthetase, thereby effectively preventing the enzyme transferring isoleucine to isoleucine transfer RNA [412]. Parasite requirements for methionine in

294 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA protein synthesis necessitate obtention from without the host cell since methyl groups cannot be recycled through methionine and the parasite needs a continuing source of this amino acid [409]. Z? berghei, for example, does not have the ability to store labile methyl groups in the choline portion of lecithin and cannot retrieve them by oxidation to betaine, and therefore depends upon de novo synthesis of the methyl group of methionine. The cofactor requirements for this synthesis include tetrahydrofolates, and this may well be another point of attack for antimalarials of the antifolic type on plasmodial metabolism. De novo synthesis also plays an important part in plasmodial requirements for aspartate and glutamate. Uptake studies with Z? knowlesi show that while isoleucine and methionine are preferentially absorbed into parasitised erythrocytes, other amino acids are incorporated to lesser degrees, but aspartic and glutamic acids are not taken up at all [40&408]. Sherman [413,414] had earlier shown that the production of amino acids might reflect, at least in part, an ability of the parasite to synthesise amino acids de novo by fixation of carbon dioxide. In particular, increased intraerythrocytic levels of aspartate and glutamate in parasitised cells were due to parasite and not to host cell activity, and 14C from labelled bicarbonate was incorporated preferentially into these two amino acids and alanine. I? knowlesi behaves similarly to avian malarias in this respect, and aspartic and glutamic acids, and alanine, were the only amino acids to become 14C-labelled in the presence of '4C-glucose, pyruvate, or acetate; it is evident that plasmodia can synthesise these three amino acids de novo from glucose and ultimately from carbon dioxide [406]. Nevertheless, although this mechanism is operative in most malarias it is not present in the parasite used in the primary screen of new antimalarial compounds; I! berghei lacks a citric acid cycle and therefore cannot convert pyruvate obtained via glucose into aspartate or glutamate by transamination of the intermediate oxalacetic and cc-ketoglutaric acids respectively [4 151. Antimalarial interference with glucose metabolism is discussed later, but it is pertinent to note here the role of coenzyme A, which controls the conversion of pyruvate to citrate and which is derived from pantothenic acid. Plasmodia do not utilise extracellular sources of the latter since successful growth is achieved in vitro in media lacking it [346], but I! lophurae, I! coatneyi, and Z? fakiparum, require preformed coenzyme A supplied to them by the host erythrocyte [416]. A number of antipantothenates have shown moderate activity against avian, simian, and human malarias but they are apparently inactive in tests with Z? berghei [417, 4181. It is evident that such compounds cannot affect the growth of Z? berghei since it lacks the system upon which they act, and such factors must be kept in mind when interpreting screening results in mice. Plasmodia1 synthesis of protein seems little affected by variations in the composition of the extracellular material [419]. Cells, both normal and parasitised erythrocytes, as well as free parasites, accumulate amino acids

R . M. PINDER 295 in like fashion whether the medium is rich in sodium or potassium ions; indeed, with the exceptions of isoleucine and methionine, the plasma amino acids can be totally replaced by stearic acid without affecting plasmodial growth [346]. However, the absence of an energy source, glucose, diminishes amino acid accumulation and incorporation. Parasitised erythrocytes incorporate three times as much total amino acid as normal red cells, and free parasites accumulate 1.5-34 times as much as do infected cells, an overall concentration gradient of 1 to 9 from plasma to parasite. The amino acid metabolism for plasmodia is summarised in Figure 6.5, and an attempt is made to indicate where some antimalarials act although much of the evidence is still very scanty. Nevertheless, it will be interesting to see whether new antimalarials can be designed by study of the inhibition of vital steps in plasmodial synthesis or utilisation of amino acids.

PLasma

Figure 6.5. Possible points of attack for antininlarials on plasniodial synthesis and utilisation of aniino acids

CARBOHYDRATE METABOLISM IN PLASMODIA

It has long been appreciated that glucose is an essential energy source for intracellular growth of plasmodia, particularly for the de n o w synthesis of folates which depends upon an active glycolytic mechanism. Sulphones [41], and dapsone in particular [357], have now been shown to inhibit the transport of glucose through the host red cell membrane when administered in v i m

296 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA in therapeutic doses. This inhibition is antagonised by increasing medium concentration of glucose but not of p-aminobenzoic acid, and sulphones inhibit neither glucose metabolism of free parasites nor glucose uptake by unparasitised red cells. Thus, the sulphones have a twofold mode of action on folic acid metabolism in plasmodia, and their action against glucose transport at the cell membrane may well be related to the demonstrated ability of red cells to concentrate the drugs at the membrane; such action is also involved in the inhibition of adenosine uptake by sulphones [354]. Although such effects may not be lethal per se to the parasites, they must surely play some part in the overall antimalarial effect of the sulphones. Investigations of the pathways of carbohydrate metabolism in different species of plasmodia have indicated that all three of the major routes, namely the Embden-Meyerhof pathway, the pentose shunt, and the Krebs tricarboxylic acid cycle, may be utilised. There is still considerable disagreement, however, as to which are involved in individual species, particularly since cytochrome oxidase activity and Krebs cycle activity are present in white blood cells and platelets which are often contaminants of in vitro preparations of parasitised red cells or free parasites [420]. It is increasingly apparent that most plasmodia contain cytochrome oxidase activity and require oxygen for survival, although the Krebs cycle seems to be absent in many of them. Certainly, P. berghei, P. cynomolgi, and P. knowlesi, contain ubiquinones, and it is likely that inhibition of either the function of these in electron transport or of their biosynthesis may be the basis for an antimalarial action [421]. Indeed, oxidative processes in the parasitised red cell are intimately involved with the actions of the naphthoquinones, chloroquine, and quinacrine. Therapeutic doses of chloroquine or the naphthoquinones produced significant inhibition of mitochondrial oxidation by the parasite of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and of succinate [422]. Both these oxidations involve coenzyme Q (CoQ) and it is suggested that the similarity of structure between the naphthoquinones and the benzoquinone moiety of CoQ leads to inhibition of its function by competition at the sites of action. The mode of action of chloroquine in this respect is not clear, but it may interfere with biosynthesis of CoQ. Quinacrine has little effect on the oxidation of NADPH, but is very effective in inhibiting oxidation of succinate; its action here, however, is probably not against CoQ but against the flavoprotein, succinic dehydrogenase, since quinacrine is effective generally against enzymes which have flavin as a prosthetic group [329]. Certainly, CoQ reverses the actions of chloroquine and the naphthoquinones against mitochondrial oxidation in plasmodia, but has no effect against inhibition by quinacrine [422]. Pathological lesions in the mitochondria of plasmodia treated with primaquine are virtually identical to those in plasmodia treated with naphthoquinones [50, 423, 4241, but it is apparent that the mode of action of these drugs is different albeit at the same locus. Thus, primaquine has no effect on

R. M. PINDER 297 NADPH oxidation and little effect on succinic oxidation, but it markedly inhibits oxygen uptake by plasmodia, even in concentrations well below its therapeutic level; this effect is not reversed by CoQ [422, 4241. Moreover, intraerythrocytic survivors of primaquine treatment show an enlarged system of the organelles which have been considered to be mitochondrial-like in function and which possess cytochrome oxidase activity. The relatively poor effect of primaquine against the erythrocytic stages of malaria parasites as compared to their striking effects against the exoerythrocytic stages is attributed to this active synthesis of additional mitochondria1 organelles [424]. Indeed, damaged erythrocytes may overcome the effects of both primaquine and menoctone by actively increasing their respiratory capacity through biogenesis of mitochondria. Phospholipid and lipoprotein synthesis must be an integral part of this biogenesis, and it is likely that methionine metabolism is intimately associated, not only in protein synthesis but also in the transmethylation reactions concerned with phospholipid anabolism. Since P berghei trophozoites lack a Krebs cycle but are still susceptible to menoctone, it is probable that the drug affects methionine utilisation in this species, and this suggestion is supported by the synergism between it and proguanil [1471. Thus, the latter inhibits the synthesis of folate co-factors required for the de novo plasmodia1 synthesis of the transferable methyl group of methionine [409], and the potentiation is to be expected since sequential steps of a metabolic chain are inhibited by the two drugs. Although interference with methionine metabolism is possibly involved in the action of menoctone it is probably not involved directly in that of primaquine, which shows no synergismwith proguanil. Little is known about the lipid metabolism of malaria parasites except that their growth is characterised by a pronounced increase in the lipid content of parasitised cells [19]. Intraerythrocytic P . berghei actively synthesises phospholipids from glucose carbon [425], but depends upon its environment for the bulk of its fatty acids required for that synthesis; glucose carbon does not serve as a substrate for de novo fatty acid synthesis but enters phospholipids by way of a-glycerol phosphate. Furthermore, P. fullux utilises added fatty acids in the growth medium [426], and P . berghei satisfies some of its fatty acid requirements through hydrolysis of host blood cell lipids since parasitised red cells produce large quantities of free fatty acids when incubated in vitro and free parasites possess appreciable phospholipase A activity [427]. More recent evidence indicates that intraerythrocytic P . berghei readily utilises plasma glucose and free oleic acid for the synthesis of phospholipids, particularly lecithins and cephalins, and the latter substrate supplies 1620 per cent of the total requirements for fatty acids [428]. Although chloroquine inhibits lipid synthesis in P . fullux [426], other known inhibitors of lipid metabolism are ineffective against P . berghei [428]. Moreover, the apparent inability of P . berghei to synthesise fatty acids de novo may be related to an inability to form sufficient coenzyme A, because acetate and lactate are the final products of pyruvate oxidation in this para-

298 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA site. More studies with human malarias are obviously required before the effects of antimalarials on lipid metabolism in plasmodia can be evaluated. The haemolytic and antimalarial effects of primaquine appear to be related to the formation of reversible reduction-oxidation intermediates during the biotransformation of the 8-aminoquinolines in the host. Thus, the 5,6quinolinequinone derivatives of these drugs, formed by demethylation, ortho-hydroxylation, and oxidation, are undoubtedly the active metabolites of the therapeutically useful 6-methoxy-8-aminoquinolines [ 181. Erythrocytes of individuals susceptible to primaquine haemolysis are deficient in glucose6-phosphate dehydrogenase (G6PD) activity, the enzyme which controls the initial oxidative step in the pentose phosphate pathway [429]. This pathway provides the only source in mature human red cells for the regeneration of NADPH, which is important in many reductive processes including the reduction of oxidised glutathione and methaemoglobin via the respective reductase reactions. Reduced glutathione (GSH) serves to protect the sulphydryl groups of haemoglobin and the sulphydryl-containing enzymes against oxidative destruction. G6PD-deficient cells have a low GSH content which decreases further prior to a haemolytic episode, while the 8-aminoquinolines accelerate the transfer of hydrogen from NADPH, GSH, haemoglobin, the free sulphydryl groups of proteins, and other donors. Under the stress of oxidation by such drugs, normal erythrocytes can raise the rate of NADPH regeneration by increasing the amount of glucose metabolised by the pentose phosphate pathway, but G6PD-deficient cells are incapable of sufficiently rapid regeneration making them unusually susceptible to oxidative damage. It has been suggested [430] that plasmodia resemble the G6PD-deficient cell in that they lack a pentose phosphate pathway and therefore are susceptible to oxidative drugs. Certainly, plasmodia do not prosper in the G6PD-deficient cell because they need what the host has in only short supply, and this deficiency seems to bestow a biological advantage against P . fulciparum infections [4311. However, it is becoming increasingly evident that a number of plasmodia possess the pentose shunt in their metabolism, and this may be one way by which the erythrocytic forms resist the effects of 8-aminoquinolines. P . berghei contains most of the enzymes of the pentose shunt [432] and P . knowlesi metabolises 10 times more glucose than unparasitised cells by this route 14331 Methylene blue, a haemolytic antimalarial like primaquine acting through redox intermediates, stimulates the use of the pentose shunt by P . knowlesi. Thus, primaquine and other oxidative drugs may unfavourably affect NADPH-linked reductive processes in those liver cells that are already under stress by the tissue schizonts which they contain, and the infected parenchymal cells may, under the additional stress of the drugs, be unable to furnish the parasite with essential nutrients. Alternatively, tissue schizonts, compared to blood schizonts, may yet have deficiencies of enzymes of the pentose shunt or involving the related cofactors, so that those forms are unusually vulnerable to oxidative damage.

R. M. PINDER 299 It has already been mentioned that plasmodia synthesise their own aspartic and glutamic acids de novo by fixation of carbon dioxide, in P. lophurae [413, 4141, P . berghei [434, 4351, and P. knowlesi [436]. The process involves the irreversible formation of oxaloacetic acid by reaction of carbon dioxide with phosphoenolpyruvic acid, catalysed by phosphoenolpyruvic carboxylase, and a similar reversible formation requiring phosphoenolpyruvic carboxykinase and a nucleoside diphosphate as a phosphate acceptor. Both enzymes are inhibited by the antimalarial drugs chloroquine and quinine, but not by spermine [434], by a mechanism involving competition with the phosphorylated substrates for available ehzyme. The fixation process utilises glucose as an energy source, but the sugar also plays another controlling role in contributing to the formation of pyruvate, the intracellular concentration of which must increase before plasmodia fix carbon dioxide. Sherman [437], in a general overview of glucose metabolism in plasmodia, confirms that pyruvate is a key regulatory intermediate. The major fraction of glucose is metabolised by the parasitised cell and by the plasmodium itself to pyruvate and then to lactate. A smaller fraction of the pyruvate is probably carboxylated via the C0,-fixing enzymes to form oxaloacetate, the latter yielding either malate via malate dehydrogenase or aspartate via transamination. Pyruvate may also be the precursor of alanine, or alanine could be formed by decarboxylation of aspartate. By entry into the Krebs cycle, oxaloacetate could form a-ketoglutarate and then glutamate ; it is not clear how plasmodia lacking a Krebs cycle in their respiratory chain form glutamate, but possibly only the dicarboxylic acid part of the cycle is present in such parasites [435]. Free parasites differ from intraceliular ones in forming large quantities of acetate which is not further metabolised, and the basis for this shift in carbohydrate metabolism is probably due to plasmodial requirements for preformed coenzyme A from the host. This requirement is suggested as a possible basis for the obligate intracellular nature of malaria parasites [437]. It is clear that plasmodial requirements for aspartate, glutamate, and alanine, have to be met by a de novo synthetic process utiiising glucose, and any interference with glucose metabolism in the parasite or its host cell will provide an antimalarial effect.

DRUG RESISTANCE

Resistance to proguanil, pyrimethamine, sulghonamides, and sulphones, is readily produced in the laboratory and the field, and has seriously compromised the use of these drugs, particularly proguanil which has been withdrawn from the U.S. Pharmacopoeia [16, 324, 3301. Resistance to chloroquine, quinacrine, and quinine, is more difficult to produce both in the laboratory and the field, but it is rapidly becoming a serious problem in the therapy and prophylaxis of falciparum malaria [16, 324, 3251. It is evident that all four

300 RECENT ADVANCES I N THE CHEMOTHERAPY OF MALARIA of the possible mechanisms for the emergence of drug-resistant strains [438] exist in plasmodia. Thus, the sudden appearance of resistance, the variation in the rate of its development in different strains, and its relative stability, all support the selection of mutant strains. However, plasmodia can transfer nonchromosomal genetic information between themselves, and factors governing resistance are carried over during conjugation of resistant and susceptible strains by transfer of units of DNA called episomes. Moreover, malaria parasites possess the ability to induce adaptive enzymes, in the way that penicillin-resistant staphylococci direct the synthesis of penicillinase. Finally, superinfection by environmental strains of a resistant nature, which take advantage of the selective effect of the antimalarial drug on the sensitive strains, is an obvious possibility in the cyclical transmission of malaria. An ti-folic antimalarials In bacteria, resistance to sulphonamides or sulphones can arise by genetic mutation or by the action of infective agents, the episomal resistance transfer factors. In malaria, little is known about the mechanisms by which resistance to these drugs is expressed or of the origin of the resistance, but it is likely that resistant mutants are present and are selected by drug pressure [330, 439, 4401. Nevertheless, episomal transfer also plays a part since the development of sulphonamide-resistant strains of P . berghei is inhibited by quinacrine [441], which is known to interact with, and inactivate, the episomes of bacteria [442] and presumably of plasmodia. The susceptibility of multi-resistant strains of plasmodia to long-acting sulphonamides like sulphalene is possibly due to an increased vulnerability of the folic acid synthetase system, this adaptional disadvantage being produced by the metabolic adjustments which have accompanied the development of resistance elsewhere in the folate metabolism of the parasite [118]. There is now voluminous evidence to indicate that resistance to pyrimethamine and proguanil by human plasmodia develops because of the selection of resistant mutants under drug pressure [324, 330, 3391. Certainly, the two drugs are endowed with qualities that should encourage this process ; both have extremely flat dose-response curves, they exhibit significant antimalarial activity at pg or smaller doses but are not 100 per cent effective in curing apparently susceptibleinfectionseven at multi-milligramme levels, and both are intrinsically slow in action allowing at least one full developmental cycle of the parasite before the infection is controlled. Pyrimethamine and proguanil are parasitostatic rather than parasitocidal, and arrest develop mental processes of the parasite at the point where the merozoites are formed from unichromatinated stages leaving ultimate responsibility for eliminating the arrested forms to natural or acquired immune forces in the host. However, there is also evidence that both enzyme induction and episomal transfer

R. M. PINDER 301 play roles in the development of resistance to the dihydrofolate reductase inhibitors, represented by pyrimethamine and chlorguanide (in its dihydrotriazine form). Dihydrofolate reductase isolated from resistant strains of P. berghei [ 4 4 3 4 5 ] and P . vinckei [444] showed marked differences from normal parasite enzyme. In particular, the specific activity for dihydrofolate was 1 1-fold higher than the normal enzyme because of the increased number of catalytic sites on the protein chain, effectively increasing the enzyme levels in the parasite, while there was a similar decrease in the enzyme’s affinity for the inhibitors. The two factors combine to increase the parasite’s ability to synthesise tetrahydrofolate even in the presence of dihydrofolate reductase inhibitors. This inductive mechanism can also be genetically transferred to sensitive strains of P. berghei [443,444]or P . vinckei [444,446]. The properties of the enzyme from the resistant P. berghei recipient were intermediate in character between those of the resistant P. vinckei donor and the sensitive P . berghei recipient, and the marked differences among the enzymes suggests that their protein structures are not identical. In the resistant strains, it is likely that mutant genes code for altered dihydrofolate reductase, selected during the development of resistance, and these genes, in whole or in part, are responsible for coding the enzyme of the recipient by genetic transfer.

0 ther an timalarials

It is well established that chloroquine-resistant parasites are often crossresistant with quinine and quinacrine as well as with other 4-aminoquinolines like amodiaquine, and the majority of work on resistant strains has been done with chloroquine [ 1241. Nevertheless, it must be pointed out that much of the earlier work has been performed with P . berghei which may have little relevance to the problems of chloroquine resistance in other species [324]. Hawking [447]has designated two types of chloroquine-resistance in P. berghei, acquired and spontaneous resistance. The latter is illustrated by the strain of P . berghei isolated by Warhurst and Killick-Kendrick [448], and is apparently restricted to the yoelii sub-species; this resistance is stable even when exposure to chloroquine is stopped and occurs in strains not previously exposed to the drug. Acquired resistance is produced in previously sensitive strains by prolonged exposure to subeffectivedoses of chloroquine, although a shorter exposure to such a regimen apparently enhances the effect of subsequent therapeutic doses of chloroquine [449]. This type of resistance takes several months to produce, the subeffective doses have to be graded within narrow limits, and when exposure to chloroquine is stopped the resistance is rapidly lost [450, 4511. It may be acquired and lost in a series of discontinuous steps, and changes in drug sensitivity and parasite virulence are not always synchronous; several different mechanisms of resistance

302 RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA may be functioning, some nuclear and some cytoplasmic, and the heterogenous wild populations contain mixtures, in different proportions, of mutants containing one or more of the factors responsible for different resistance levels. Resistance can be cyclically transmitted without loss through Anopheles stephensi [451, 4521, and chloroquine seems to enhance the infectivity of resistant strains of P. berghei to this vector [452,453]. It has been shown that chloroquine is retained in the tissues of A . stephensi after it has fed on chloroquine-treated monkeys, and it is possible that pharmacological pressure by such retained drug in the sporogonic phase of parasite development could contribute to the acquisition of resistance [454]. One of the early attempts to explain chloroquine resistance was that resistant plasmodia might have an abnormally high rate of conversion of haemin to ferrihaemic acid, an intermediate in the conversion of haemin to haematin by malaria parasites and which is known to antagonise the antimalarial actions of chloroquine and quinine [455, 4561. This was attributed to the formation of a complex [457], and chloroquine-resistant plasmodia would therefore be extensively complex and inactivate the antimalarial by virtue of their excessive accumulation of ferrihaemic acid. Certainly, Peters [456] showed that chloroquine-resistant P. berghei had apparently lost the power to form pigment because of an excessive liberation of an intermediate soluble porphyrin, ferrihaemic acid. However, it has now been shown that the reduced production of haemozoin in the resistant P. berghei is due not to drug resistance in the parasite but to its presence in immature red cells [397, 4581. Indeed, it is probable that reticulocyte-dwelling trophozoites of P. berghei largely utilise substrates other than haemoglobin, and P. fakiparum produces pigment whether chloroquine-resistant or sensitive. Peters [4511 has now withdrawn his assertion that chloroquine-resistant P. berghei does not produce haemozoin, and his evidence is supported by other workers [459]; the most virulent strain of P. berghei, which displays an innate resistance to chloroquine, produces abundant haemozoin. Nevertheless, ferrihaemic acid may still play a part in chloroquine resistance but in entirely the opposite sense to that expressed above. Thus, the formation of this porphyrin may serve to concentrate the drug within the parasite, particularly in the digestive vesicles, where it will interfere with digestion and utilisation of haemoglobin [368]. This hypothesis is attractive since chloroquine does not bind to malaria pigment in vitro and therefore only malaria parasites actively forming pigment will concentrate the drug, and it explains the ineffectiveness of chloroquine (and quinacrine and quinine) against gametocytes. However, it does not explain why certain very virulent strains of P. berghei produce large amounts of haemozoin but are still resistant to chloroquine [45 11. Deficiencies of chloroquine binding by infected erythrocytes have been implicated in the development of resistance to chloroquine, but it is likely that they are not related to binding to haemoglobin degradation products.

R . M. PINDER 303 Thus, mouse erythrocytes infected with chloroquine-sensitive P. berghei showed a 600: 1 (cells: medium) concentration gradient when exposed to 10-’M chloroquine at 22°C and physiological pH, while similar cells infected with resistant strains showed only 100: 1 gradients; controls had gradients of only 14: 1 [459, 4601. The processes involved in these gradients were saturable in agreement with the proposal of chloroquine binding to cellular constituents, and no degradation of the drug was detected. Since therapeutic plasma concentrations of the drug are relatively low in the mouse as well as man, the deficiency of high-affinity binding is a plausible explanation for the reduced ability of chloroquine-resistant parasites to concentratechloroquine in vivo.The large gradients and fast rates of uptake argue against involvement of membrane transport processes and suggest interaction with cellular constituents such as protein or DNA, a proposal supported by the similarity of the binding constants to those obtained in experiments with DNA-chloroquine complexes. Chloroquine resistance of plasmodia is therefore attributed to a decrease in number, affinity, or accessibility, of chloroquine receptor sites on cellular constituents of the infected erythrocyte. Similar effects have been observed in erythrocytes of the owl monkey infected with P .fakiparum [461], suggesting that deficiencies in binding might also play a part in the development of resistance in human malarias. Recent work [403] indicates that these binding changes may be secondary to a more fundamental change in the parasite’s physiology, at least as far as P. berghei is concerned. Resistant strains may utilise the citric acid cycle in contrast to the sensitive strains; the former contain succinic dehydrogenase which is produced during a respiratory switch associated with gametocytogenesis. Support is provided by the chloroquine treatment of non-gametocyte producing strains, which rapidly develop resistance and produce gametocytes, and by the enhancement of infectivity of chloroquine-resistant strains to Anopheles stephensi. Furthermore, the action of menoctone and lapinone against chloroquine-resistant P . berghei is readily explained by their inhibitory effects on the induced enzyme. Whether these switches in respiratory metabolism play any part in human malarias is unclear. In contrast to the anti-folics, chloroquine and associated drugs are parasitocidal, rapid in action, act at many points in the parasite’s development, and have a steep dose-response curve [16]. Thus, small increases in dosage can vastly increase lethality to the parasite, the chances of a rare resistant organism surviving are very small, and consequently chloroquine-resistant strains have taken much longer to emerge in the field. Furthermore, immunity will rapidly overcome any resistant mutant that survives the initial chloroquine treatment, and the relative lack of chloroquine-resistant P . fakiparum in Africa is probably due to this factor [16, 3251. The poor activity of primaquine if used alone against erythrocytic forms of plasmodia is an obvious incentive to the development of resistant strains, both in the

RECENT ADVANCES IN THE CHEMOTHERAPY OF MALARIA 304 laboratory and the field, but they are unlikely to emerge in the latter both because primaquine is usually administered together with a quick-acting schizontocide and because of the pronounced gametocytocidal effects of the drug which prohibit transmission to the mosquito. The possible enhancement of infectivity of gametocytes of P . faleiparum by chloroquine, as was demonstrated for P . berghei [452, 4531, emphasises the importance of the gametocytocidal properties of primaquine. Nevertheless, resistant strains of P . gallinaceum can be successfully transmitted to Aedes aegyptii with subsequent stable passage through to infective sporozoites [462]. Primaquine resistance in P . berghei probably involves the ability of the parasite to actively increase its respiratory capacity by biogenesis of mitochondria [397, 4241, and in this respect they resemble the chloroquine-resistant strains in switching their respiratory metabolism. Resistant strains of rodent malarias certainly take up twice as much oxygen as sensitive strains [463], possibly indicating their increased respiration via mitochondria1 oxidative processes.

SUMMARY AND CONCLUSIONS Since Laveran’s discovery of Plasmodium in 1880, research in malariology has progressed like British economic policy since 1945. The present ‘go’ represents the most active period in the history of the subject, and this review has attempted to provide a brief, yet comprehensive, account of important advances in the chemotherapy of malaria during this period. Readers who desire a fuller account should consult the admirable volume by Peters [464], which came to our notice after completion of this manuscript. The search for the ideal antimalarial drug continues. Such a drug will combine the virtues of causal prophylaxis, suppression, rapid and complete curative action, and sporontocidal activity, together with no liability to. produce resistance ;it should have low toxicity, prolonged action, palatability, and low cost. This euphoric compound is unlikely to be forthcoming, but, nevertheless, new drugs are needed, particularly a good, safe anti-relapse drug to replace primaquine in the treatment of P . vivax and P . malariae infections, and a potent and safe blood schizontocide that will maintain its effect for 3 4 months after a single dose. The need for new drugs of protracted action is especially urgent if the aim of global eradication of malaria is to be pursued. This is not always obvious to the clinician who has a number of dependable drugs and drug combinations with which to treat a particular patient, but the public health worker faces problems of a different magnitude in eradicating the disease from whole communities. The promise shown by repository drugs like cycloguanil pamoate is being clouded by the ease with which plasmodia become resistant to them. Other compounds of all types are also required to form a stockpile of antimalarials that can be called upon when needed, and ‘me-too’ drugs are not to be denigrated in this respect if

R. M. PINDER 305 they offer a different spectrum of activity. Resistance to antimalarial drugs by P . falciparum is the most urgent problem in malaria therapy, and the appearance of resistance to 4-aminoquinolines has revealed the paucity of our antimalarial arsenal. Methods of testing potential antimalarials have improved tremendously, and the present P . berghei infection in mice can test up to 800 new compounds per week; the Walter Reed Army Institute of Research has, to date, tested over 100OOO compounds [30]. In vitro techniques allow an assessment to be made of the effects of drugs on a variety of metabolic parameters in the parasite, and the increasing knowledge of parasite biochemistry may at last allow development of a rationale for the design of new drugs. Nevertheless, most of our present antimalarials were discovered by empirical methods and are likely to continue so when the problem of therapy is so urgent, and the large-scale screening procedures are therefore of immense value. Structural refinements of existing drugs seem to hold little hope in solving the problem of drug-refractory malaria because most of the present drugs act by inhibition of nucleic acid function, albeit at different loci. Perhaps more attention should be turned to new or unusual modes of action, many of which have been discussed here, such as the inhibition of glucose uptake by sulphones; indeed, drugs known to inhibit processes vital to plasmodia1 metabolism, but so far untested as antimalarials, should be investigated. Our prediction about the rapid development of synergistic drug combinations [ 181 has been amply fulfilled in the last four years, and the number of such regimens can be expected to increase still further unless new single drugs become available. Malaria remains the major threat to life and one of the most serious public health problems in the underdeveloped nations. It seems only a matter of time before chloroquine-resistant P . fakiparum emerges in Africa, where, until now, no proved foci of such strains have been recorded. The main predisposing conditions for its emergence seem to be the influx of people with low levels of immunity into endemic areas, the presence there of resistant mutant parasites, and free availability of antimalarial drugs. These conditions prevail in Vietnam, and the increasing European investment in Africa in terms of technical aid is setting the scene for similar influxes of non-immunes. Residual spraying with insecticides is the main weapon against drug resistance by interruption of malaria transmission, but in areas where thiq is not possible little can be done when multi-resistant strains are present iintil better prophylactic drugs are available. The wily Plasmodium remains extraordinarily adaptable and still presents the medicinal chemist with one of his greatest challenges.

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R. M. PINDER 387. 388. 389. 390. 391. 392. 393. 394. 395. 396. 397. 398. 399. 400. 401. 402. 403. 404. 405. 406. 407. 408. 409. 410. 41 1. 412. 413. 414. 415. 416. 417. 418. 419. 420. 421. 422. 423. 424. 425. 426. 427. 428. 429. 430. 431. 432.

315

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7 The Prostaglandins M.P. L. CATON, B.Sc., Ph.D., The Research Laboratories, May & Baker Ltd., Dagenham, Essex, RMlO 7 X S .

INTRODUCTION

318

STRUCTURE AND NOMENCLATURE

318

OCCURRENCE AND RELEASE

321

DETECTION AND ANALYSIS

324

BIOSYNTHESIS

325

TOTAL CHEMICAL SYNTHESIS Prostaglandins E, F, A and B Dihydroprostaglandins 1 1-Deoxyprostaglandins 15-Dehydroprostaglandins 7-Oxaprostaglandins

329 329 339 341 342 343

CHEMICAL PROPERTIES AND TRANSFORMATIONS

344

METABOLISM

345

BIOLOGICAL ACTIONS Gastrointestinal smooth muscle Reproductive smooth muscle Respiratory smooth muscle Cardiovascular system Blood platelet aggregation Metabolic effects Gastric secretion and ulcer formation Diuretic and natriuretic effects Luteolytic effects Nervous system Ocular effects

341 348 351 353 355 358 359 360 361 362 363 365

CONCLUSION

365

REFERENCES

366

317

318

THE PROSTAGLANDINS

INTRODUCTION The varied and potent biological effects exhibited by the prostaglandins and the possibility of their therapeutic application in several important fields has aroused widespread interest in this group of natural products. Although most of the now extensive literature on these substances has arisen from research conducted within the past decade, their biological properties were first noted in the 1930s from observations on the activity of a then unknown factor found in seminal plasma and sheep vesicular glands [ 1-51. This factor, which in 1935 was named prostaglandin by von Euler, is now known to consist of a mixture of several closely related compounds which occur widely in mammalian tissues. The isolation and purification of the prostaglandins by Bergstrom in the 1950s [6-71, and the subsequent structure determination and development of laboratory scale biosynthesis led to the extensive studies of their effects on numerous biological systems. More recently, total chemical syntheses have provided further sources of the natural prostaglandins as well as unnatural structural variants and stereoisomers. The main object of this chapter is to review the biosynthesis, chemical synthesis and metabolism of the prostaglandins and to summarise their principal types of biological activity. In the biological sections, attempts have been made to draw attention to the variation of the type of activity with structure, a feature of particular interest in this series which is of fundamental relevance to consideration of clinical utility. A number of other reviews [8-131 and publications based on symposia [1&17] give much material which receives only brief or incidental reference here and these are quoted in the appropriate sections below. An attempt has been made to include key and representative references to original papers; a full bibliography of prostaglandins is published by the Upjohn Co. (Kalamazoo, Michigan) which is kept up to date by the issue of supplements. STRUCTURE AND NOMENCLATURE Prostaglandins are fatty acids of 20 carbon atoms having a cyclopentane ring with two adjacent side chains. Their chemical structure was elucidated in the early 1960s as a result of ultramicroanalysis and mass spectrometry together with various degradations of the small amounts of material then available [18]. A detailed review of the methods used has been published by Samuelsson [19]. The absolute stereochemistry was finally decided upon in 1966 [20]; some papers published before then show the side chains in the positions now known to represent the mirror image of the natural forms. The skeleton on which the prostaglandins (PG) are based has been named

M. P. L. CATON 319 prostanoic acid (Figure 7.1) and different members of the series are distinguished by the presence of double bonds, hydroxyl or carbonyl groups. Those derived from natural sources are of four basic types which are designated by the letters E, Fa, A and B (Figure 7.1). Subscript numerals after the 7

5

13

15

17

19

Prostanoic acid

PGE

PGE

PGE,

kbk OH

bH

OH

PGA,

PGB,

19- OH-PGB,

OH

Figure 7.1. Prostanoic acid and some members of the prostaglandin group

letters denote the number of double bonds in the side chains; thus PGE, has one (trans) double bond at C-13, 14, PGEz has in addition a (cis)double bond at C-5, 6 and PGE3 a third (cis) double bond at C-17, 18. The E and F series are often known as the primary prostaglandins. The E's are characterised by the presence of a keto group at C-9 and a hydroxyl group at C-1 1, the latter being in the a configuration, i.e. on the same side of the ring as the carboxyhexyl chain. In the F series the C-9 keto is replaced by another hydroxyl. Natural PGFs have this C-9 hydroxyl in the a configuration and are referred to as PGFa to distinguish them from the PGFB series

320 THE PROSTAGLANDINS with the opposite configuration. The latter do not occur in nature but are obtained in addition to the PGFcr series, by chemical reduction of the PGEs. The PGAs (e.g. PGA1, Figure 7.1) lack the hydroxyl substituent at C-11 and have an additional double bond at C-10, 11 ; they are readily formed on treatment of the corresponding PGEs with weak alkali. PGBs (e.g. PGBl, Figure 7.1) have a double bond in the C-8,12 position and are obtainable by rearrangement of the PGAs. The A and B series show U.V.absorption maxima at 217 and 278 nm respectively and for this reason were formerly referred to as PGE 217 and PGE 278. Members of these two series are also known which have an additional hydroxyl group at C-19, e.g. 19-hydroxy-PGB1, where the 19-OH has been shown to possess the D-configuration [21]. All four classes have an a hydroxyl group at C-15. Changes in configuration at both C-1 1 and C-15 are indicated by the term ‘epi’, alternatively they may be designated ‘8’ to distinguish them from the above a forms. The side chain hydroxyls may also be described by the Cahn-Ingold-Prelog convention, i.e. S for the normal and R for the epi forms. The mirror images of the natural prostaglandins are denoted by the prefix ‘ent’. Prostaglandins where the two carbon side chains are in the cis relationship to each other, rather than trans as in prostanoic acid, are named 8-isoprostaglandins. Examples of these as applied to the E series are shown in Figure 7.2. Although the above abbreviated terms suffice for most practical purposes, prostaglandins can also be given thdr full systematic nomenclature [20]. W

OH

OH 11.15-epi -PGE, or PG (E/?,81,

OH

C

O

Y

P

OH

enf-PGE,

OH

8-iso-PGE,

Figure 7.2. Some prostaglandins of the E-series

They are then described in terms of their relationship to prostanoic acid (Figure 7.1). Here the word ‘trans’applies to the side chain double bond, ring hydroxyls are all stated as a or B and 15-hydroxyl groups referred to as S or R. Thus PGEl which is laevorotatory is designated ( - )1 la, 1S(S)-dihydroxy9-0xo-l3-trans-prostenoicacid and PGFI, as ( - )9a, 1 la, 15(S)-trihydroxy13-trans-prostenoic acid [20]. Structural variants of these basic prostaglandins are usually named after the most closely related natural type. Thus variants having side chains one or more methylene groups shorter than normal are referred to by the terms nor, dinor etc. and longer chains are similarly denoted as homo. In this context the letters a and o are sometimes added to signify whether the

M. P. L. CATON 321 carboxyhexyl ( a ) or hydroxyoctyl ( w ) chain is affected; the use of the term a here must on no account be confused with its significance in connection with the configuration as described above.

OCCURRENCE AND RELEASE Prostaglandins occur in a wide variety of animal tissues and there are a number of instances where they are released on tissue stimulation or activation. Human seminal fluid is the richest source where thirteen different members of the series have been identified (El, E,, EJ, F1,, F,,, Al, Az, B,, B, and 19-OH A,, A,, B, and B,) with a total concentration of about 300 pg/ml [22, 231. Considerable species variation is exhibited both in the number and concentration; thus only five of these thirteen [24] are found in ram semen and that of some other species contains none at all [5]. The ram vesicular gland is an important source of prostaglandins and the enzyme system from this organ is used for laboratory synthesis starting from added fatty acid precursors [25]. Prostaglandins are also present in human endometrium and menstrual fluid where, in contrast to the seminal fluid, much smaller amounts of the PGEs are found compared to the F series [26, 271. PGEs and Fs have also been identified in the human amniotic fluid and the blood vessels of the umbilical cord [28-301. Concentrations outside the reproductive system are generally much lower. Karim, Hillier and Devlin have carried out systematic surveys of the distribution of PGs El, El, FIX,F2, in a wide variety of tissues in man [31] (Table 7.1), and in six widely used laboratory animals [32] (Table 7.2). In the majority of cases one or more of these prostaglandins was present in varying amounts, with PGE, and PGFz, occurring most commonly. PGFz, appears to predominate in the lungs, with some species differences [32, 331 and both PGF,, and PGE, are released into the pulmonary circulation when sensitised guinea-pig lungs are challenged [34, 351. The smooth muscle stimulating factor formerly known as irin, found in the iris of certain animals, has been shown to contain prostaglandins PGFz, and PGE, [36, 371. PGF,, is also the most abundant [38-41] in the central nervous system. Studies of the distribution of PGs in the dog C.N.S. showed that El, E2, F1, and F,, are widespread and that there is no large difference in PG concentration between the different regions [39]. Homogenates of rat cerebral cortex have been shown to contain PGEs and Fs which are concentrated mainly in the light microsomal and mitochondria1 fractions and it is concluded that they are stored in the nerve endings [41]. Prostaglandins are also released on nerve stimulation [42]. They also occur in the gastrointestinal tract [43] and in the kidney 144-481.

Table 7.1 DISTRIBUTION OF PROSTAGLANDINS I N H U M A N TISSUES (From Karim, Sandler and Williams3'. by courtesy of British Journal of Pharmacology and Chemotherapy.)

Prostaglandins Tissue

Thyroid

Pancreas

Adrenal cortex Adrenal medulla Thymus Infant Infant Adult Parotid gland Submandibular salivary gland Cardiac muscle Rectus abdominis muscle

Subjecr N o . 1

2 3 4 5 1 2 3 4 1 2 1 2 3 1 2 3 1 2 1

2 3 1 2 1

2 Psoas muscle Cervical sympathetic chain Vagus nerve Phrenic nerve Brachial plexus

1

2 1 2 3 1 2 1 1

2 Bronchi

1

Lung parenchyma

2 1 2

' El

A

E2

4.5

500

-

1000

-

25.0 4.0 2.0 1.5 0.8 3.0 6.3 255 64.0 34.0

-

-

-

-

-

-

24.5 12.5 3.5 075 -

1.5 03 2.5 3.0 45-0 22.5

05 5.0 5.5 10.0 1.O 2.25 3.50 1.o 105 1.9 1.3 1.9 3.0 15.0 5.3 3.1

2.5 0.5 I .o -

-

4.0 3.9 -

5.0 9.4

-

-

2.0 1.6 4.5 7.8 2.4 1.3

4.2 3.9

The figures refer to concentrations In ngrg tissue. A dash indicates that prostaglandm were not detected

1 .o

2.5 500 12.4

M. P. L. CATON 323 Table 7.2 DISTRIBUTION(ng/g OF TISSUE) OF PROSTAGLANDINS I N ANIMAL TISSUE (From Karim, Hillier and D e ~ l i n by ~ ~courtesy , of Journal of Pharmacy and Pharmacology.) Animal Tissue

Dog

Heart Thymus Skeletal muscle Submaxillary salivary glands Spleen Adrenals

ND 2.5 El 2.0 E2 4.0 F2a 1.0 E2 2.7 F2a 1.9 E2 1.9 Fza 0 4 El 18.7 E2 2.3 F2a 5.0 E2

cur

Rat

Guinea-pig

Rabbit

Chicken

3.7 F2a

3.8 E2

ND

ND

4.0 El 5.1 E2 3.3 F2a 1.0 El

17.5 El

6.0 F2a

1.0 F2a

3.5 F2a 51.0 E2 254 EL

4.4 E2 2.1 F2a 9.2 Ez 6.9 F2a 37.5 E2 8.0 F2a

1 . 1 El 1 . 1 Fza 4.7 E2 7.8 F2a 4.0 EZ 6.1 F2a

ND

0.75 E2 1.1 F2a 8.75 E2

Thyroid

31.7 E2 57.5 F2a 1.45 E2 1 . 1 F2a ND

Kidney

ND

8 0 0 E2 101.7 F2a 37.5 E2 25.6 FZa 3.3 E2 3.2 F2a 2 0 0 E2 6.0 F2a ND

Liver

ND

ND

Blood Vagus

ND 45-3 E2 304 F2a ND

ND 90.9 E2 45.0 F2a 65 E2 15.5 F2a

Sympathetic chain Pancreas

Lungs

17.5 E2 - NE 2.7 FZU 23.2 E2 180.2 E2 20.0 F2a 6 0 6 F2a

ND

ND

42.0 E2

NE

NE

NE

18.2 E2 9.7 F2a 154.4 E2 162.1 F,a 50.4 E2 9.0 Fza 18.2 E2 9.4 F2a ND NE

ND

16.6 EZ 90.4 F2a

4808 E2 1609 F2a 28.5 E2 2 0 2 F2a 12.0 F2a ND NE 2 5 E2 375.0 F2a

1.8 F2a

40.1 E2 2509 F2a NE

5.5 E2 12.5 E2 2.4 F2a 10.0 Fza 133.0 E2 192.3 F2a 1.83 F2a 160.4 E2 32.0 E2 9.2 F2a 18.2 F2a 1.75 E2 ND 8.50 F2a ND ND NE NE 5.4 E2 8F2a

7.7 E2 30'4FZa

ND-Prostaglandins E , , E2,Flu. Flu not detected NE-Tissue not extracted

In addition to PGE2 and F2a,a substance known as medullin has been derived from the rabbit kidney medulla. This has been identified as PGA2 although it has been suggested that it was an artifact derived by dehydration of PGEz during the isolation procedure. Further details of tissue distribution are reviewed by Pickles [lo], and Horton [13] has tabulated some other circumstances in which prostaglandins are released. More recently it has been shown that they are also released by the isolated dog spleen in response to the infusion of particles [49] and during carrageenin-induced inflammation in the rat [5&51]. PGs (F, and E2) occur in a wide variety of amine-peptide secreting tumours [52, 531. A particularly interesting finding has been the discovery of 15-R PGA2 in the Gorgonian (Plexaura homomalla), a species of coral found in the

324 THE PROSTAGLANDINS Caribbean. This prostaglandin, the 15-epimer of those found in vertebrates, and the 15-acetyl derivative of its methyl ester are present in the air-dried cortex of this species to the extent of 0.2 and 1.3 per cent respectively [54]. DETECTION AND ANALYSIS The occurrence of a number of closely related structures in minute amounts has necessitated the development of sensitive techniques for the detection and separation of prostaglandins in tissue extracts. Reviews are available of the methods used together with spectral data and physical constants for the principal prostaglandins, and this subject will not be discussed in detail here [ll, 55, 561. Chromatographic methods have been evolved whereby the different prostaglandins can be separated and identified [l 1, 22, 23, 55, 57-62). Thin layer chromatography has been used extensively and a number of different solvent systems and adsorbents have been employed to separate various mixtures of prostaglandins, either as the free acids or after conversion to suitable derivatives. Gas-liquid chromatography of trimethylsilyl ethers, methyl ethers, and acetylated derivatives of prostaglandin esters has also been widely used [l 1,23,59-61,63-661. Recently a method has been reported whereby prostaglandins may be identified and estimated in nanogramme quantities by combined gas chromatography-mass spectrometry (651. Thin layer and gas liquid chromatography have also been employed in combination ; thus a very effective separation of prostaglandins in human semen resulted from preliminary chromatography on silicic acid to divide PGEs from the PGFs, after which individual members of the E group were further separated by T.L.C. on silver nitrate-impregnated silica gel, and the PGFs by G.L.C. of their trimethylsilyl ethers [23]. Another recent method employs group separation of the methyl esters using the lipophilic dextran derivative Sephadex LH-20 [67]. For the detection of very small amounts of material, isotope dilution methods have been employed [68]. Bioassays based on the stimulatory action of prostaglandins on smooth muscle preparations have also been widely employed, and these are particularly useful where minute quantities x e ir- 'ved [12]. Tissues used here include rat and guinea-pig intestine, rat stomach fundus, rat uterus and colon of hamster and jird. A very sensitive method uses thin layer chromatography in conjunction with a micro-modification of this smooth muscle assay [69]. Methods based on vasodepressor responses have also been employed [12, 701 and a blood-bathed organ technique has been developed to assay prostaglandins in circulating blood [34, 711. An enzymatic method of analysis which employs a specific nicotinamideadenine dinucleotide-dependent prostaglandin dehydrogenase from swine lung permits analysis of prostaglandins with a lower limit of 10 -I2 mole [72].

325

M. P. L. CATON

BIOSYNTHESIS Prostaglandins are biosynthesised from certain C2,, straight chain fatty acids which have a system of methylene-interrupted double bonds. Thus PGE,, PGE, and PGEJ are derived from all-cis-8,11,14-eicosatrienoic (dihomo-y-linolenic); 5,8,11,14-eicosatetraenoic(arachidonic) and 5,8,11,14, 17-eicosapentaenoic acids respectively (Figure 7.3) [62, 73-75]. This conversion was first demonstrated when the acids were incubated with homogenates of sheep vesicular glands, but it also occurs with acetone G

+e

H

4

8,11,14-Eicosatrienoic acid (dihomo-y-Linolenic acid)

OH

OH

GH -L PGEl

C -,'-02H

1 I

I

OH 5.8.11,

OH

1L-Eicosatetraenoic acid (arachidonic acid)

PGE;,

-

k

OH

5,8, l l , l L , 17 -Eicosapentaenoic acid Figure 7.3.

H

1

OH

PGE3

Biosyntheiic origin of some prostaglandins

powders of bovine seminal vesicles [76] as well as in a number of tissues outside the reproductive system, e.g. lung, iris, brain, stomach, kidney medulla and intestinal mucosa [12, 25, 77-82]. Biosyntheses with guinea-pig lung homogenates also give rise to prostaglandins of the F series as well as certain prostaglandin metabolites [77, 83-85]. PGAl has been isolated during some experiments with sheep vesicular glands although it may have arisen by dehydration of PGEl during the isolation procedure [86]. The biosynthetic process can be used to effect the preparation of certain homologues [62, 73, 751 and isomers of prostaglandins. The success of the

326 THE PROSTAGLANDINS conversion depends upon the chain length, the position of the double bond system and the total number of double bonds in the starting acid [87, 881. The substrate specificity of the enzyme from sheep vesicular glands has been studied for a series of acids, and it has been found that the PGEl and PGE, precursors referred to above gave the highest rate and maximum yield of conversion, but with the PGEJ precursor which has an additional double bond at C-I 7, 18, these values were much lower. The effect of shifting the double bond system was studied in a series of eicosatrienoic acids, but only the 7, 10, 13 (w7)* acid, i.e. where the system is shifted one carbon atom nearer to the carboxyl group, gave a prostaglandin in good yield. The w6 tri-and tetraenoic acids of 19 and 21 carbon atoms gave good yields of a-nor and a-homo prostaglandins, but the yields of corresponding dinor and dihomo prostaglandins from the Cl.8 and C , , acids were rather lower. The C1905 and Czlw7 tri-and tetraenoic acids were converted into w-nor and o-homo prostaglandins 1891. A free carboxyl group was found to be essential for conversion, alcohols and methyl esters being unaffected by the enzyme 1871. The requirement of adequate amounts of endogenous prostaglandin precursors may explain why certain of these acids are essential to the diet [73, 87, 891. The essential fatty acid (E.F.A.) activity of a series of acids, as measured by the ability of their methyl esters to produce a change in weight in E.F.A. deficient rats, has been shown to correlate approximately with their conversion rates into prostaglandins. Only those fatty acids which yielded biologically active prostaglandins showed considerable activity [89]. Several other prostaglandins and related compounds have been isolated from the products of these biosyntheses. Thus 8-iso-PGEl (Figure 7.2) [90, 911 and 1 1-dehydro-PGF1, (i.e. 9a,15-dihydroxy-ll-oxoprost-13-enoic acid) (I, Figure 7.4) [92] have been obtained from the dihomo-y-linolenic acid incubation, the former having probably arisen by isomerisation of the PGE, . The prostanoic acid derivative (11) and the tetrahydropyran (111) (Figure 7.4) as well as certain tetrahydrofuran derivatives, have been isolated from the arachidonic acid incubation after the PGE, had been removed [93,94]. Prostaglandins oxygenated at C-18 and C - 19 have been biosynthesised by an initial microbiological hydroxylation of 5,8,11,1Ceicosatetraenoic acid (Figure 7.3) followed by cyclisation of the resulting oxygenated unsaturated fatty acids or their derivatives by exposure to bull seminal vesicle microsomes [95]. C-19 and C-20 hydroxylated PGAl derivatives have been obtained from PGA, on incubation with a preparation of guinea-pig liver [961. The enzyme system responsible for effecting prostaglandin biosyntheses has been partially purified [97]. It is associated with the microsome fraction

* The term 0 7 is a means of stating that the double bond from the terminal methyl group of the fatty acid.

system commences 7 carbon atoms

327

M. P. L. CATON

0

I

H

0

C

HO

11

H

OH

OH

OH

z

111 Figure 7.4. Biosyntheric products

and a heat stable factor in the supernatant and the co-factor requirement can be provided by reduced glutathione, tetrahydrofolate or 67-dimethyltetrahydropteridine [98-1001. The yields of PGE and F can be varied by the addition of glutathione. Certain acids such as 8 4 s -12-truns-l4-cis-eicosatrienoic acid, 5-cis-84s-12-trans-14-cis-eicosatetraenoic acid and eicosa5,8,11,14-tetraynoic acid act as inhibitors to prostaglandin biosynthesis [ 10 1-1031. Extensive studies have been carried out to determine the mechanism of biosynthesis. Experiments with 1802have shown that the three oxygen atoms at C-9, C-11 and C-15 are derived from molecular oxygen and that the two ring oxygens are derived from the same oxygen molecule [78, 97, 104-1 111. The reaction mechanism which has been proposed as a result of these studies is shown for dihomo-y-linolenic acid in Figure 7.5. Using tritium labelled acids, it has been established that the initial step of the transformation involves a stereospecific removal of the C-13 L hydrogen. This is followed by an allylic shift of the unpaired electron and introduction of oxygen at C-1 1, in a lipoxidase type of reaction, to give a 1 1-peroxy acid. The latter is cyclised to an endoperoxide (IV) by a concerted reaction involving addition of oxygen at C-15, isomerisation of the 12,13 double bond, formation of a new carboncarbon bond at C-8-C-12 and attack by oxygen at C-9. The endoperoxide can then be transformed into PGEl without change in the overall oxidation state or into PGF1, by reductive cleavage. Besides the evidence from experiments with isotopic oxygen, the existence of the endoperoxide is supported by the fact that PGEs and PGFs are formed by independent steps and are not interconvertible [97]. Additional evidence

328

THE PROSTAGLANDINS

PGF,,

PGE,

Figure 7.5. Mechanism of prostaglandin biosynthesis

is also provided from experiments which have shown that a fragmentation is able to occur with the formation of a C I 7hydroxy acid and malondialdehyde which can arise from the endoperoxide as shown in Figure 7.6 [78, 107, 1lo]. 1 1-Dehydro PGFI, would arise by yet another path whereby the hydrogen at C-11 of the endoperoxide is removed instead of that at C-9 as in the formation of PGE, [92]. That neither 1 1-dehydro-PGF,, nor PGEl can be intermediates in the formation of PGFI, has been demonstrated by establishing that hydrogens at both C-9 and C-1 1 are retained during conversion to PGF1, [11 11. Until chemical syntheses became available, procedures based on these bioconversions provided the only source of prostaglandins. The original

T q e c 0

P

i l

+

O C H H

-----

"

OH

0

OH

Figure 7.6. Mechanism Ofbio.~j.nthesis

W

C

0

2

H

329 procedures have been developed together with suitable isolation procedures in order to provide adequate amounts of material for the various laboratory studies [ 1 12-1 141. Nevertheless, biosynthesis suffers from the severe disadvantage of relying on an enzyme which is only available in very limited amounts and substrate specificity restricts the range of prostaglandin analogues obtainable by this means. M. P. L. CATON

TOTAL CHEMICAL SYNTHESIS Following the publication in 1966 of the first route to a biologically active prostaglandin [115], many total chemical syntheses of the natural prostaglandins and their structural variants have been reported. Here syntheses of the four main groups-PGE, F, A and b a n d their unnatural stereoisomers are discussed first, followed by an account of routes to dihydroprostaglandins and novel derivatives of prostanoic acid. See also note on p. 366. ROUTES TO PROSTAGLANDINS E, F, A A N D B

(a) The elegant work of Corey at Harvard University has provided syntheses of the natural primary prostaglandins as well as several of their unnatural stereoisomers. Members of the E and F series have both been synthesised from the common intermediate (1x1which was prepared from the anti-bicyclic ketone (V), obtained by alkaline hydrolysis of the DielsAlder adduct of 5-methoxymethyl-l,3-cyclopentadieneand 2-chloroacrylonitrile [116]. Baeyer-Villiger oxidation of (V) to the lactone (VI), saponification and treatment with potassium tri-iodide gave the iodo-lactone (VII) which was converted into the aldehyde (IV) via acetylation, deiodination with tributyltin hydride, demethylation and oxidation of the resulting hydroxymethyl group with Collins reagent. Wittig reaction with sodio dimethyl-2oxoheptylphosphonate gave a trans-enone which on reduction with zinc borohydride gave a (c. 1 :1) mixture of the alcohol (VIII) and its l5g-epimer. Deacetylation of (VIII) and conversion of the resulting diol to the bistetrahydropyranyl ether was followed by reduction of the lactone to the corresponding lactol with di-isobutylaluminium hydride. The tetrahydropyranyl ether (IX) then followed as a result of reaction with the Wittig reagent from 5-triphenylphosphoniopentanoic acid and sodio methylsulphinylcarbanide. ( f)-PGF,, was obtained from (IX) by hydrolysis with aqueous acetic acid and oxidation of the 9-hydroxyl group with chromic (two phase) reagent followed by a similar hydrolysis gave ( & )-PGE2. Two modifications of this route have also been reported [117, 1181. The first of these [ 1171 started from 6-rnethoxybicyclo[3,l,0]hex-Zenewhich on

330 THE PROSTAGLANDINS reaction with dichloroacetyl chloride and triethylamine underwent positionspecific and stereospecific addition of the elements of dichloroketene to form the tricyclic ketone (X). Dechlorination and Baeyer-Villiger oxidation gave the methoxylactone (XI), which, after demethylation and oxidative cleavage of the cyclopropyl ring using chromic acid gave the alcohol (XII) corresponding to (IV). The synthesis was then completed essentially as before. In the second modification [118] the epoxide (XIII), prepared in four steps from bicyclo[3,2,0]hept-5-en-2-one, reacted with 1,3-bis(methylthio)allyllithium to give a coupling product which on hydrolysis using mercuric chloride-calcium carbonate afforded the aldehyde (XIV) together with an unwanted positional isomer. Reaction of the aldehyde group of (XIV) with n-pentyl-lithium then gave two epimeric side chain alcohols. One of these on hydrolysis to the lactol and Wittig reaction as before, yielded ( 5 )-PGFz, and the other likewise gave ( f)-15-epi-PGFz,. In order to obtain the naturally occurring optically active prostaglandins, the lactone (VI) was hydrolysed to the ($-)-hydroxy acid (XV). After resolution with ( + )-ephedrine, the dextrorotatory acid was converted with potassium tri-iodide to the required antipode of the iodolactone (VIII) and the synthesis completed, essentially as for the racemic mixtures to give natural PGFz, and PGEz [119]. Finally, natural PGEl and PGF1, have been synthesised by selective hydrogenation of the double bond in the acid side chain of the resolved tetrahydropyranyl ether (IX) and treatment of the product in the manner used to convert (IX) to PGEz and Fz, [120]. (b) In an earlier and completely different approach by the Harvard group 11211, Diels-Alder addition of the diene (XVI) and the nitro-alkene (XVII) afforded the adduct (XVIII). The latter, after reduction of the nitro group, MeOCH,

CHiOMe

,

OH

VI I

VI

V

1

,

OTHP

CHO

OTHP

IX

OAc

OH

VlII

OAc

IV

33 1

M. P. L. CATON

0

0-Q A

Cr,,, OH

X

0’‘0

XII

XI

G OH C

,CH,CO,H

H

O

CHiOMe

OH

xv Xlll XIV formylation of the resulting amine and exchange of the dithiane for a dioxolane ketal, was treated with osmium tetroxide to oxidise the double bond to the diol. Cleavage with lead tetra-acetate then gave the aldehyde (XIX) which on treatment with 1,5-diazabicyclo[4,3,O]-5-nonenefollowed by acetylation furnished the cyclopentanol derivative (XX). A further series of transformations-borohydride reduction of the ketone, ketal hydrolysis and dehydration of the resulting hydroxy ketone using dicyclohexylcarbodiimide and cupric chloride as catalyst-led to the enone (XXI). Reduction of the unsaturated carbonyl group of (XXI) with zinc borohydride produced a mixture of C- 15 epimers which was converted to an epimer mixture of the amino acid (XXII) by successive deacetylation, reaction with dihydropyran, hydrolysis of cyano to carboxyl and deformylation. The N-bromo derivative of (XXII), prepared by the action of N-bromosuccinimide, underwent basecatalysed dehydrobromination and hydrolysis of the resulting imine at pH 2 affording a mixture of ( -t )-PGE, and ( f)-15-epi-PGE1which were separated by chromatography. The ( i-)-PGE, was converted with 0.5 N hydrochloric acid in 1 : 1 water-tetrahydrofuran to ( +)-PGA, and by reduction with sodium borohydride to ( +)-PGF,, and ( f)-PGF,, . Another route which converges with the above sequence could also be adapted to provide the C-11 epimer [122]. The Michael adduct (XXIII) of 3-nitropropanal dimethyl acetal and 9-cyano-2-nonenal underwent Wittig reaction with sodio dimethyl 2-oxoheptylphosphonate to give an unsaturated ketone which was converted with ethylene glycol into the nitro bisdioxolane (XXIV). After reduction and formylation to the corresponding formylamino compound, treatment with p-toluenesulphonic acid in acetone led to a mixture of four stereoisomeric cyclisation products (XXV a-d). These were separated by chromatography of the free alcohols or the corresponding acetates; the acetate of (XXVa) is identical to (XXI) above and

332 THE PROSTAGLANDINS was converted to (f)-PGE1 as already described. Similar treatment of (XXVb) gave ( 5)-PGE, together with ( f)-15-epi-PGEl, whereas (XXVc) and (XXVd) each afforded ( f)- 1 1-epi-PGEl and ( f)- 11,15-epi-PGEl. Prostaglandins have also been obtained from the mixture of stereoisomers of structure (XXVI), which was obtained by direct acid-catalysed cyclisation of the nitroketal (XXIV). Chromatography of these gave a pair of C - l l normal alcohols epimeric at C-9 and a second pair of C-1 I-epi alcohols also epimeric at C-9. Borohydride reduction of the carbonyl function of the first pair gave a pair of C-15-normal alcohols epimeric at C-9, which was converted to ( f)-PGE1 by the previously described sequence, together with a pair of C-9 epimeric C-15-epi alcohols, which on similar treatment yielded ( +)-15-epi-PGEl. Similar treatment of the C-9-epimeric pair of nitro alcohols in the C-1 1-epi series yielded ( )- 1 1 -epi-PGE, and ( f)-11,15-epiPGEl. In order to prepare the naturally occurring optically active prostaglandins by this approach, the cyclisation of the nitro ketal (XXIV) was carried out with stannic chloride in acetone when the C-1 1-normal alcohol was obtained essentially free of the C- 1 1-epimer [1231. Reduction with zinc borohydride followed by mild base treatment to place the 9-nitro substituent in the more stable p orientation led to a mixture of two nitro-diols epimeric at C-15. These were separated chromatographically and reduced with aluminium amalgam to give the racemic amine (XXVII) which was resolved with (-)a-bromocamphor-nsulphonic acid to give the laevo-amine. Natural PGEl was then obtained by application of the procedure for the final stages described above for the racemic mixture. Resolution of the amine (XXVII) with ( + )-a-bromocamphor-n-sulphonic acid gave the dextro amine which by a similar process afforded ent-PGEl. (c) Syntheses of racemic prostaglandins by workers at McGill University and the Upjohn Laboratories are based upon the cleavage of bicyclo[3,1,0] hexane intermediates. In the first paper of this series [124], the exo-bicyclic ester (XXIX), obtainable by an ethyl diazoacetate-copper reaction on the tetrahydropyranyl ether of cyclopenten-3-01, was converted into the ketone (XXVIII) via the sequence-lithium aluminium hydride reduction, Jones oxidation to the aldehyde, Wittig reaction with hexyltriphenylphosphonium bromide, removal of tetrahydropyranyl and oxidation of the ring hydroxyl. Alkylation of (XXVIII) with methyl 7-iodoheptanoate gave the ester (XXX) which was originally converted to ( f)-PGF1,, after borohydride reduction and ester hydrolysis, by oxidative formolysis with 30 per cent hydrogen peroxide in formic acid buffered with sodium carbonate. However, other workers [ 1251 found this procedure unsatisfactory although they reported that the ketone (XXX), on reaction with sodium carbonate, afforded a facile route to ( f)-PGB,. More recently this approach has been developed by several modifications [ 126-1 3 11. The olefinic ketone (XXVIII) was first separated chromatographically into cis and frans isomers

+

333

M. P. L. CATON

1

HCO.NH

-

[CH,),.CN

H

xx I

0

THPO

(')-PGEl+(')-15-ep/

OTHP

XXII

XXIlI X ,y

[CH,),.CN

xxv

, ,Hq - n r , , p+$ OH 0 c X=HCONH, Y=H d X=H Y=HCONH

XXVl

XXVll

PGE,

334 THE PROSTAGLANDINS and the cis isomer alkylated with methyl o-iodoheptanoate to give a mixture of the cis isomers of the two keto esters of formulae (XXX) and (XXXI) [126, 1271. Sodium borohydride reduction of the cis-isomer of (XXX) afforded a mixture of alcohols (XXXII) and (XXXIII), epimeric at C-9, which were hydrolysed to the corresponding acids. The latter were reoxidised with Jones reagent to the !%ox0 compounds and the side chain double bond hydroxylated with performic acid to yield a mixture of glycol acids of formula (XXXIV). This underwent cleavage to give ( k)-PGE1 via bismesylation and solvolysis of the 2,2,2-trichloroethyl esters and a similar sequence starting with the ketone (XXXI) yielded ( f)-8-iso-PGE,. Methyl esters of (i-)-PGE, and ( f)-15-epi-PGEl could be obtained more directly via the glycols from the ketone (XXX) or the methyl w-iodoheptanoate alkylation product of the trans isomer of (XXVIII). Prostaglandins of the F series were obtaineg by converting the alcohols (XXXII) and (XXXIII) into their epoxides, treating with formic or trifluoroacetic acids and hydrolysing with sodium carbonate [126, 1281. Thus (XXXII) led to a mixture of the methyl esters of ( Jr_)-PGF1,and (+)-15-epiPGF,, and XXXIII gave the corresponding F,, compounds. A similar series of 8-iso-PGFs were obtained by the same procedure using the two alcohols derived from borohydride reduction of the ketone (XXXI). Further work showed that higher yields of ( f)-PGE1 are obtainable if the glycols used have the endo configuration a t C-13 (XXXV) rather than the exo form as in (XXXIV) [ 1291. These endo-glycols were derived from the ketone (XXXVI) which was prepared, using essentially the same procedure as for the exo-compounds, from the bicyclo ester (XXXVII). The latter was obtained via hydroboration and oxidation of the ester (XXXVIII), readily available from norbornadiene. The endo-bicyclohexane intermediates have also provided routes to the PGEz and PGE3s [130, 1311. The acetonide of the vic-glycol from osmium tetroxide hydroxylation of the olefin (XXXVI) was alkylated with l-bromo7-tetrahydropyranyloxyhept-2-yneto give the ketone (XXXIX) [ 1301. After selective removal of the tetrahydropyranyl group with oxalic acid in methanol and Jones oxidation to the carboxylic acid, the triple bond was partially reduced to a cis olefin. Hydrolysis of the acetonide and solvolysis of the bismesylates of trichloroethyl esters as before led to ( f)-PGE2. The solvolysis also afforded the trichloroethyl ester of ( f ) 15-epi-P-GE2. The ester (XL) 0

OTHP

Q,

I=cH.c~H,,

COZEt

XXIX

XXvIlI

335

C,

&,

_---(CH2)6 C02H

,-(CH,),.CO,Me CHOH.CHOHC5H,,

~ c H . c 5 H l 1

'\

H

CHOH-CHOH.C,H,,

XXXIV

xxxv

XXXVI

OTHP

H

xxxvm

XXXVI I

CH,),.CH,-OTHP

( CH,), 'H

(XXXIX)

.CH2.C=C-( CH,),.CO,

Me

CH I CH*CHI.CzCEt

Me

H XL

336 THE PROSTAGLANDINS was prepared by Wittig reaction of the phosphonium salt of I-bromohex-3yne and the aldehyde from the ester (XXXVII), removal of the tetrahydropyranyl group, oxidation to the ring ketone and alkylation with methyl 7bromohept-5-ynoate [131]. Selective hydroxylation of the double bond in XL, mesylation, solvolysis and partial hydrogenation of the two triple bonds gave ( +)-PGE3 methyl ester. (d) In an approach by the Ciba group [132, 1331, the ketone (XLI) obtained by cyclisation of the ester (XLII) was converted into the enol acetate, brominated and the bromo-derivative treated with triethylamine to give the enone (XLIII). Allylic bromination and reaction with silver acetate followed by treatment with methanolic hydrogen chloride then gave the hydroxy enone (XLIV) of which the trimethylsilyl ether underwent hydrogenation to give the silyloxy-compound (XLV), having the all-cis configuration. The ketone (XLV), after methoxime formation and simultaneous removal of the trimethylsilyl group, was heated with potassium carbonate in methanol to give a diacid which on re-esterification yielded the diester (XLVI), having the all trans configuration. Selective sodium borohydride reduction of the ring ester group of the tetrahydropyranyl ether of (XLVI),

XLI

XLlIl

( ? ) PGE M e t h o x i m e

-

M. P. L. CATON 337 Moffatt-type oxidation of the resulting carbinol to the aldehyde and Wittig reaction with 1-tributylphosphoranylideneheptan-2-oneled to the enone (XLVII). Borohydride reduction, removal of the tetrahydropyranyl group and ester hydrolysis then gave ( f)-PGE1 methoxime. (e) A route by the Merck, Sharp and Dohme group, which incorporates a high degree of stereoselectivity in the generation of the nuclear asymmetric centres, is based upon the hydrindanone intermediate (XLVIII), obtained in seven steps from 6-methoxy-3-indanol [ 1341. Birch reduction of (XLVIII) followed by esterification, hydrolysis and methylation afforded the ketone (XLIX). A further sequence-ketone reduction with lithium tri-t-butoxy aluminium hydride, deacetalization, sodium methoxide isomerisation to the A -ketone, hydrogenation, mesylation and elimination led to the ketone (L). The ethylene glycol ketal from (L) was then oxidised with permanganateperiodate to cleave the cyclohexene ring and the acetyl function of the product epimerised with sodium methoxide to give the seco-acid (LI) which was converted into its benzyl ester. Baeyer-Villiger oxidation to replace the acetyl function by acetoxyl, hydrogenolysis and oxidative decarboxylation led to the olefin (LII). Osmium tetroxide-sodium iodate oxidation of the double bond followed by Wittig coupling of the resulting aldehyde with dimethyl 2-oxoheptylphosphonate yielded the derivative (LIII) of 15dehydro-PGE, which was converted to ( +)-PGE, on reduction and removal of protecting groups.

I

Me

Me

XLVIII

L

XLIX

LI

LII

LllI

338

THE PROSTAGLANDINS

(f) It has been shown that prostaglandins can be synthesised by pro-

cedures which follow the enzymatic conversion of polyunsaturated carboxylic acids. Thus ( +)-PGE1 has been obtained from autoxidation of allcis-S,ll, 14-eicosatrienoic acid, followed by reduction with alcoholic stannous chloride; however, the yields were low (c. 0.1 %) owing to the occurrence of many side reactions and the complex stereochemistry of the products [ 1351. A recent patent [ 1361claims the preparation of primary prostaglandins by singlet oxygen oxidation of the unsaturated acids to form peroxide intermediates, followed by a reduction to give the PGF series or disproportionation to give the PGEs; no yields are stated. (g) Although PGBl may be obtained by alkali treatment of PGEl or PGA1, prepared by the above schemes, several independent routes are also available. Thus the carbinol (LIV) from 3-t-butoxyoctynylmagnesium bromide and the ethyl ester of 2-(6-carboxyhexyl)cyclopent-2-en-1-one, underwent allylic rearrangement to the alcohol (LV) which led to ( -E)-PGB1 after oxidation to the ketone, partial hydrogenation of the triple bond and removal of protecting groups [137]. A related sequence using the Grignard OH

I

LIV

LV

R = H, E l or But

LVI

reagent of 3-tetrahydropyranyloxyoct-1-yne has also been reported [ 1381. The latter has also been reacted with 2-(6-carboxyhexyl)-3-alkoxy-2cyclopenten-1-ones or their ethyl or t-butyl esters to give the ketone (LVI) which afforded ( f)-PGB1 or its 13,144s analogue on partial reduction and hydrolysis [139-1411. Other workers [142]claim to have obtained (f)-PGBl methyl ester in low yield in an approach to prostaglandin synthesis based on the Birch reduction of an appropriately substituted benzene ring and subsequent conversion of the resulting cyclohexene to the cyclopentane system by ozonolysis of the double bond and recyclisation of the resulting aldehyde.

M. P. L. CATON

339

DIHYDROPROSTAGLANDINS

Several syntheses are available to the 13,14-dihydroprostaglandins, some of which are metabolites of the E and F series. The first of these routes [143, 1441 started from the formyl derivative (LVII) of the enol ether of cyclopentan-l,3-dione which on reaction with ethyl 6-bromosorbate and triphenylphosphine followed by selective catalytic reduction afforded the ester (LVIII). A second formylation followed by elaboration with n-hexanoylmethylenetriphenylphosphonium chloride led to the ketone (LIX) which on reduction of the exocyclic double bond and acid-catalysed solvolysis in benzyl alcohol afforded the benzyl ether (LX) and its isomeric enol ether. Reduction with lithium tri-t-butoxyaluminium hydride to the corresponding 15-hydroxy-compound and palladium-charcoal catalysed hydrogenolysis followed by prolonged catalytic hydrogenation with rhodium-charcoal led to (&)-dihydro-PGE, ethyl ester. A synthesis of the ethyl ester of the cis (i.e. S-iso) analogue of dihydroPGF,, (LXI) is based upon the tetrahydroindanedione (LXII) prepared by Diels-Alder reaction of cyclopentene-l,3-dione and butadiene [ 144, 1451. Ketone reduction afforded the cis-diol of which the dibenzoate was subjected to hydroboration and chromic acid oxidatiw to give the ketone (LXIII). Reaction with n-pentyl Grignard reagent and p-nitrobenzaldehyde then afforded the benzylidene derivative (LXIV). Formation of the vicinal cis diols by osmium tetroxide oxidation of the cyclohexene ring and cleavage with lead tetra-acetate gave the aldehyde (LXV) which led to the dihydroPGF (LXI) on Wittig reaction with the ylid from ethyl w-bromosorbate, removal of the p-nitrobenzylidene group, hydrogenation and sodium borohydride reduction. Dihydroprostaglandins and related compounds have been synthesised by a sequence based on the ring closure of keto-aldehydes of the type (LXVI) with sodium hydroxide to give cyclopentenones for example (LXVII) [146]. Removal of the protecting groups then gave dihydro-PGAs and epoxidation of the ring double bond followed by cleavage of the epoxides by catalytic hydrogenation led to dihydro-PGEs. The aldehyde (LXVI) was prepared by a multistage process of which a key step was condensation of the intermediates (LXVIII) and (LXIX) to give the acetylene (LXX), followed by an eight stage sequence involving partial reduction of the triple bond to olefin and cleavage by ozonolysis. Dihydro-PGE, has also been obtained via the introduction of an acetoxy group with N-bromosuccinimide followed by silver acetate, into the allylic (i.e. 11) position of the 15-acetoxy derivative of 13,14-dihydro-PGB1 and hydrolysis and hydrogenation of the product [ 1471.

340

LXVlIl

THE PROSTAGLANDINS

J Lxx

LXlX

OTHP

OTHP

LXVIl

LXVI

34 1

M. P. L. CATON

1 1-DEOXYPROSTAGLANDINS

The 11-deoxyprostaglandins, a group not found in nature, have been synthesised by workers at the Ayerst Laboratories [115, 148-1511. 11Deoxy-PGFIB (LXXI) has been prepared starting from the enone (LXXII), obtained by the action of sulphuric acid on the monobromo derivative of the condensation product of ethyl 2-cyclopentanone carboxylate and obromoethylheptanoate [115, 148, 1491. Reaction of (LXXII) with acetone cyanohydrin, hydrolysis of the ester-nitrile to the dicarboxylic acid and reaction with methanol and p-toluenesulphonic acid gave the mono ester (LXXIII) of which the acid chloride was converted with heptyne and aluminium chloride into the chlorovinylketone (LXXIV). The sequence was then completed by replacement of chloro with methoxyl, ester hydrolysis and borohydride reduction to the unsaturated ketone (LXXV) followed by borohydride reduction of the side chain carbonyl group. In an earlier, longer, route [I 15, 1491, the acid chloride of (LXXIII) with acetylene and aluminium chloride followed by the action of methanolic sodium hydroxide gave the acetal (LXXVI) which on borohydride reduction and treatment with 2N-sulphuric acid gave a mixture of two epimeric unsaturated aldehydes. The predominant isomer of these then led to (LXXI) on reaction with pentylmagnesium bromide followed by ester hydrolysis. &H,);COL

&

ICHZ)&02Me &Hzl~COzMe ~

COzH

LXXII

LXXIII

C*CH=C-ICH,),

a

I

Me

CL

LXXIV

I

6 LXXVI

0

OH 11-deoxy PGFlg

LXXI

LXXV

0

LXXVll

L XXVl I1

LXXIX

342 THE PROSTAGLANDINS (+I-11-Deoxy-PGE, has been obtained by an eight stage route from the acid (LXXV) involving reversal of the positions of carbonyl and hydroxyl groups [ 1501. ( & )-Dihydro-1 1-deoxyprostaglandins have been synthesised by an interesting route based on the photochemical addition of the methyl ester of the enone (LXXII) and the chlorovinyl ketone (LXXVII) [151]. Reaction of the adduct (LXXVIII) with zinc and acetic acid gave the diketone (LXXIX) which on borohydride reduction afforded a mixture of the methyl ester of (+)-dihydro-l1-deoxy-PGFlB and a smaller amount of the corresponding Fa compound which were separated by chromatography. Alkaline hydrolysis of the latter afforded ( )-dihydro-11-deoxy-PGF,.. 15-DEHYDROPROSTAGLANDINS

Workers at the Searle Laboratories have synthesised certain 15-dehydroprostaglandins [ 152-1 541. 15-Dehydro-PGB1 has been prepared by a sequence based on the cyclisation of a bicyclic ketone (LXXX) where the double bond destined to occupy the 13-14 position in the prostaglandin is protected by a cyclopentenyl group [152, 1531. The triketone (LXXX) was prepared by condensation of the bicyclo[2,2,l]hept-5-ene(LXXXI) with the sodium enolate of dimethyl 3-oxoundecanoate and the cyclisation accomplished with alkali to give the diketo acid (LXXXII). Ring decarboxylation, esterification and pyrolysis to remove the cyclopentyl group then led to 15dehydro-PGB, methyl ester (LXXXIII). Hydrogenation of (LXXXIII) afforded a mixture of the 13,14-dihydro derivative of (LXXXIII) and the methyl ester of dihydro-PGB,.

jCH21LMe

\

H

L xxx

LXXX I

-

+

H02C

(CH21LMe

0 LXXXIII

LXXXII

343

M. P. L . CATON (CHz)6COzH OH m

P

h

b OH C H = C H Ph

L xxxv

LXXXVI

Gc @I%; 2) Me

0

OH

COP

OH

LXXXV I I

LXXXIV

15-Dehydro-PGE1 (LXXXIV) has been synthesised by cyclisation with alkali of the diketone (LXXXV), prepared by condensation of 3-0x0undecan-1,ll-dioic acid with styryl glyoxal, to give the cyclopentenone (LXXXVI), followed by Pappo-Allen oxidation to the aldehyde (LXXXVII), zinc-acetic acid reduction and Wittig condensation with n-hexanoylmethylene triphenylphosphorane [154]. The product was mixed with its 11epimer which was separated chromatographically. 7-OXAPROSTAGLANDINS

A series of biologically active 7-oxaprostaglandins has been synthesised [ 155-1 581. A key step in this work is the reaction of the epoxide (LXXXVIII), OCH2Ph

?.CHZPh

*o--

O'CHzPh

C = C j C H2)5M e

ALE 17 C=C(CH2)5Me

-0*-

. , O ~ C O Z B U '

-*--OH

Q::I.o

I

A! LXXXI x

LXXXVIII

c 7 G0

OH

0.C HzPh

O.CHzPh

0-CH2 Ph

bH

xc I

LXXXlX a

I

OH

OH

xc

344 THE PROSTAGLANDINS prepared from cis-cyclopentene-3,5-diol,with the dialkylalkynyl aluminium reagent diethyl octynyl alanane to give the alcohol (LXXXIX) which on 0alkylation with t-butyl o-iodohexanoate gave the ether (LXXXIXa). Removal of the protecting groups and partial reduction of the triple bond was followed by insertion of the 15-hydroxyl by selenium dioxide oxidation to yield (f)-7-oxa-PGF1. (XC) and its 15-epimer [155]. A mixture of (f)7-oxa-PGE1 (XCI) and its 15-epimer was obtained via selective removal of the 9-benzyl group in the t-butyl ester (LXXXIXa) and Jones oxidation to the 15-ketone [156]. Some 9,ll-deoxyoxaprostaglandinsand their cyclohexyl analogues have also been prepared in the course of this work [157, 1581. CHEMICAL PROPERTIES AND TRANSFORMATIONS In addition to direct synthesis, several prostaglandins and their derivatives have been obtained by chemical modification of other members of the series. Some of this work has been referred to in preceding sections; attention is drawn here to some other transformations which have been carried out on prostaglandins derived from either natural or chemical sources. Early work on the chemistry of prostaglandins is included in the review on structure determination by Samuelsson [ 191. A particularly useful paper from the Upjohn laboratories describes a number of transformations on a preparative scale [159]. Thus PGA’s can be prepared from PGE’s by dehydration with 90 per cent acetic acid-water, whereas with base the dehydration is followed by isomerisation to PGB’s. Selective oxidation of PGF,, or PGE, with manganese dioxide gave the corresponding 15-ketones and borohydride reduction of 15-oxo-PGFl, afforded a mixture of PGF1, and the 15epi-compound. A particularly interesting transformation is the formation of 15-epi-prostaglandins by the treatment of the natural compounds with formic acid-sodium formate at room temperature followed by base treatment. 8-Iso-PGEl can be isomerised under mild basic conditions to PGE, in high yield. Preparation of the 9-oxime of PGEl offers a suitable means of protecting this ketonic function, the free ketone being regenerated with nitrous acid. Reaction of the methyl ester of PGEl with lithium tetrahydridoaluminate led to simultaneous reduction of ester and carbonyl functions to give a mixture of the 1,9a,1 la, 15s-tetrahydroxyprostene and the corresponding 9B-isomer [ 1601. PGA’s on hydrogenation gave rise to 11-deoxyprostaglandins [ 161, 1621. 9-Thiosemicarbazones and carbodi-imides have been prepared and are a useful means of isolating prostaglandins from crude mixtures 1114, 163, 1641. The stability of prostaglandins (El, E2, F1,, Fz,) in dilute solutions, a factor of major importance in connection with their therapeutic application, has been studied in detail a t a wide range of pH values by measuring the loss

M. P. L. CATON 345 with time of smooth muscle stimulating activity [165]. At alkaline pH values the E series-awing to their ready dehydration to PGAs and PGBs-are less stable than the Fs, thus at pH 8 a 100 ng/ml solution of PGE, had lost 80 per cent of its activity after nine days at room temperature, whereas PGFI, at pH 5-11 showed no reduction in activity even after 182 days. However, the PGEs appeared to be the more stable at pH 1 4 .

METABOLISM The metabolism of prostaglandins in the lung has been extensively studied by a Swedish group [77,83, 84, 1661691. Early work showed that in guinea-pig lung homogenates PGE’s undergo reduction of the 13, 14 double bond and dehydrogenation to 15-0x0 compounds whereas in swine lung only the latter transformation takes place (Figure 7.7). The dehydrogenase from swine lung has been purified and studied with respect to substrate specificity [169-1721. PGEl and PGEz were found to be the best substrates, PGE3 and members of the F series were about 60% as effective and lower rates of oxidation were noted for dihydro-PGE,, PGA,, PGAz and the 19-hydroxy-PGA’s. In all of these compounds it could be shown that the enzyme specifically oxidised the hydroxyl group at C-15. Prostaglandins where the planarity of the configuration of the carboxyl side chain relative to the ring has been altered (for example PGB’s, 8-iso-PGE,) are poor substrates for the enzyme [170]. e

HGuinea I OH

pig

H= I

OH

OH

I

OH

D i h y d r o PGE,

PGE,

I

OH 0 15-Oxodihydro

PGE

1 5 - 0 x 0 PGE,

Figure 7.7.

Metabolism of PGE, in guinea-pig and swine lung

346 THE PROSTAGLANDINS Experiments with nor- and homo-prostaglandins show that the natural length of side chains is not necessarily required for conversion and some of these compounds (e.g. some homo-, nor-PGE’s) show rates similar to that for PGE, [171]. Some other prostaglandins with shortened side chains (for example, dinor-PGF,,) are poor substrates [ 1701. The stereospecificity of the dehydrogenase is also markedly dependent upon the stereochemistry of the prostaglandin substrate [170, 1721. A 15-ahydroxyl group appears to be a major requirement, since ruc-1 5-epi-PGEl is not dehydrogenated by the enzyme, but the conversion is not prevented by a change in configuration at C-11. Racemic mixtures (for example, rucPGE1) are only dehydrogenated at half molar amounts of the natural isomers; with excess enzyme the reaction does not proceed beyond 50 %, indicating that only one antipode is used and thus the ent forms are not affected. However, experiments with ruc- 11,15-epi-PGEl indicate that the ent- 11,15epi-PGE, which has the unnatural back-bone configuration (i.e. inversion at C-8 and C-12) but the proper C-15 (S) configuration is in fact consumed by the enzyme and in preference to the component with the natural configuration. The work indicates, however, that the rate of inactivation of this ent form is only about 15 % that of PGEl whereas the biological activity on several preparations should be equal to or even greater than for PGE,. This is of considerable relevance to the possible therapeutic employment of prostaglandins, since metabolism to the relatively inactive 15-keto compounds is probably a principal mode of biological deactivation [169]. Any synthetic stereoisomer where this loss can be minimised and yet the desired biological response preserved thus clearly has a potential therapeutic advantage over the natural forms. Studies in the intact lung [173-1791 have shown that prostaglandins El, E2 and Fz, are almost completely inactivated on one passage through the pulmonary circulation but that PG’s A l and Az are unaffected. Besides its presence in the lung, prostaglandin dehydrogenase activity has been located in the kidney [180- 1821 and the liver [1 831. Another means whereby prostaglandins undergo metabolic inactivation is by /%oxidation of the carboxylic acid side chain. This process became evident when the main urinary metabolite of PGF1, in rats was identified as the a-dinor compound [184-1951. Incubation of a series of prostaglandins with the b-oxidising enzyme system of rat liver showed that all those investigated were degraded by one or two two-carbon units [187]. PG’s El, B1, F,, and F,, were converted into the C-18 homologues; PGA,, 1Ia,l5-dihydroxy-9-0~0- and 1 lahydroxy-9,15-dioxoprostanoicacids give C18 and C16 mixtures and norand homo-PGF,, both afford trinor-PGF,,. Studies in the guinea-pig [I881 showed that the main metabolite of PGEz is Sfl,7a-dihydroxy-l1-oxotetranorprostanoic acid, p-oxidation, 13,14 reduction and dehydrogenation having been accompanied by reduction of the ring

M. P. L. CATON 347 ketone to hydroxyl. It was found that the main urinary metabolite of PGFz, in this species was the tetranor-I I-oxodihydro compound (i.e. 5cr,7crdihydroxy- 1 1-0xotetranor-prostanoic acid) [ 1891. Experiments with blood-perfused isolated rat liver using labelled PGEl and PGF1, indicate that circulating prostaglandins are rapidly taken up by the liver where they undergo decarboxylation resulting in pharmacological inactivation, the products then being excreted into the bile and venous effluent [190, 1911. Carbon dioxide from fragments lost during metabolic degradation of PGEl has been measured in expired air [192, 1931. Studies in man have shown that the major urinary metabolites of PGF,, and PGEz are the tetranor-9,lO-dihydro- 11-ox0 compounds (XCII, XCIII) [194, 1951.These arise as a result of C-13-C-14 reduction, 15-dehydrogenation

Gco2

H9

COZH

0

Hd

XCII

0

OH

X C III

and two steps of 6-oxidation together with w-oxidation to a terminal carboxyl group. Another transformation which has recently been reported is the conversion of PGAl to PGBl by an isomerase found in the cat [196]. Since the PGB's have very weak biological activity this may provide yet another means of prostaglandin metabolic inactivation. BIOLOGICAL ACTIONS

A major proportion of the literature on prostaglandins has been devoted to studies of their effects in various biological systems and this has received detailed treatment in several other reviews [8-171. Here an attempt has been made to summarise the main fields of investigation, some of which show encouraging prospects of leading to therapeutic application. Their widespread occurrence and biological properties indicate that prostaglandins have a fundamental physiological role but although this has been the subject of much speculation, as yet no clear cut picture has emerged. Much evidence has been accumulated on the mechanism of action of prostaglandins and particularly on the mediation of their effects by an influence on the levels of 3',5'-cyclic adenosine monophosphate (cyclic AMP) and some of this is briefly referred to in the appropriate sections below. For a comprehensive account of this subject and other hypotheses on the mechanisms of action, the reader is referred to a review by Horton [13].

348 THE PROSTAGLANDINS Some biological studies have been followed by investigations in man and the current status of clinical studies is indicated in the appropriate sections. A detailed survey of clinical studies has recently been published by Hinman [ 1971. Structure-activity data are given where available, although in some cases care has to be exercised in making direct comparisons especially where different test systems have been used. Nevertheless, a very interesting structureactivity pattern is emerging and the specificity of action made possible by chemical manipulation indicates that the broad spectrum of action shown by some of the natural compounds can be narrowed down to provide the more selective action normally required for therapeutic application. GASTROINTESTINAL SMOOTH MUSCLE

In general, prostaglandins contract isolated segments of longitudinal gastrointestinal smooth muscle [198-2061, an effect which has been demonstrated with a large number of different preparations including guinea-pig ileum [198, 201-2031 rat and rabbit jejunum and duodenum [20&204], guinea-pig, hamster, rat, jird and chicken colon [28, 194, 199, 201-203, 2061 and rat stomach strip [loo, 2051. Relative activities of different prostaglandins are to some extent dependent upon the preparation used but in general the E and F series are the most potent, PGEl, for example showing stimulation of guinea-pig ileum at a concentration of 10ng/ml and of some other preparations, for example guinea-pig colon, at less than 0.5 ngjml [38, 198, 199, 203, 2041. PGA’s, PGB’s and their 19-hydroxy analogues have relatively weak activity [207-2091 for example PGA, and PGB, have respectively 2-8 and 10-20 per cent of the potency of PGE, on rabbit jejunum [207]. Of prostaglandin metabolites, dihydro PGE, is less active than the parent compound and the potency is lowered again on further metabolism to the 15-keto compounds [21&211]. Studies in the PGE series have shown that variations in the length of the carboxy (tl) side chain cause a substantial reduction in activity but o-nor and o-homo compounds still display high potency [87-89, 1981. Comparative data for several prostaglandins on guinea-pig ileum relative to that of PGE, are shown in Table 7.3. 1 1-Deoxyprostaglandins possess weak activity ( < 5 % of PGE,) [211] on guinea-pig ileum and rabbit duodenum and 8-iso-PGE1 has 0.014 the potency of El on the latter preparation [90]. Racemic 7-oxa-PGF1, has about lj500 the activity of PGF,, on gerbil colon [212] and the corresponding derivative where the side chain double bond is replaced by a triple bond is of comparable potency but the related compounds lacking the side chain hydroxyl group are less active namely < ljlO00 of PGF,,. A mixture of (+)-7-oxa-PGE, and the corresponding 1 1-epicompound had 4 x 10 of the potency of El [ 1561.

Table 7.3

RELATIVE STIMULANT ACTION OF PROSTAGLANDINS ON GUINEA-PIG ILEUM

(PGE1 = 1.0) 15-Keto-

Activity

Reference ref. 208 gives 0 2

E2

E3

F1.

F2.

Al

A2

1.6 198

0.2 198

0.02 198

0.55 201

0,002* 209

00057 209

cc-Nor-E, w-Nor-El w-Homo-El Dihydro-El 15-Keto-El 01 198

0 44 89

1.24 89

0.14 210

008 210

dihydro-El

0.02 210

350 THE PROSTAGLANDINS Results for the synthetic stereoisomers of the PGE, Fa, and A series on guinea-pig ileum and rat jejunum have been compared with those of the natural forms (Table 7.4) [213]. Racemic PGEl and Flu have about half the potency of the natural stereoisomers showing that the unnatural components Table 7.4

BIOLOGICAL ACTIVITIES OF PROSTAGLANDIN STEREOISOMERS ON DIFFERENT PREPARATIONS

(From Ramwell, Shaw, Corey and Andersenz13by courtesy of Nafure.) Prostaglandins

Rat uterus 1.00

Natural PGE, Rac-PGE, Ent-PGE,

0.44 +0.06

0.001

Natural l5-epi-PGE1 Rac-15-epi-PGE Rac-l 1-epi-PGE, Rac-1 1,15-epi-PGE1

,

,

Natural PGA Rac-PGA Natural 15-epi-PGA1 Rac-15-epi-PGA Natural PGF,, Rac-PGF l a

,

0.008~0401 004 +0.007* 0.13 + O W 1.00 &0.2* 1.00 0.73 +006 0.42 1 .00 1 .00 0.45 0.09

+

Guinea-pig ileum 1 .00 0.5 -

0.02 0.06 0.036 0.1

Rabbit jejunum

Rat pressure

1 .00 0.4 00013

0.57

1.00 -

0.05 0.05 0.05

0 1 2 & 002 5.4 5 1 . 6

0.04

1.00 0.53

1.00 1.00

-

1 .00 0.56

0.16 0.16

2.00 1.00

0.4

* A log dose-response curve deviates significantly from that of natural PGE, Potency expressed relative

10

parent prostaglandln ( = I 00)within each group ( + S E when available, n

=

4)

of these mixtures (i.e. the enf forms) are less active and indeed ent-PGE, itself is shown to possess only 00013 the potency of nat-El on rat jejunum. However, some of the racemates of the epi forms for example, rac 15-epiPGA, and rac 1 1,1 5-epi-PGEl are more active than the natural isomers and this is attributed to an even higher level of potency for the ent components of these mixtures. Comparative data for PGs E and F on several other intestinal smooth muscle preparations have been tabulated elsewhere [13, 198, 199, 2111. It has been shown that the smooth muscle stimulating actions of PGFzu and E2 are reversibly antagonised by polyphloretin phosphate [214]. On the jird colon, PGFz, was antagonised more readily than E2 whereas on isolated rabbit jejunum the effect was similar for both. Some oxaprostaglandins are also antagonists of the action of natural prostaglandins on smooth muscle preparations [I 571. 1-Acetyl-2-(8-chloro- 10,ll -dihydrodibenz[b,f] [ 1,410xazepine-10-carbony1)hydrazine (S.C. 19220) has been shown to antagonise contractions produced by PGE, on guinea-pig ileum [2 151. Prostaglandins also act on gastrointestinal circular muscle. However, whereas PGFs produce contraction as in the case of the longitudinal smooth muscle, with PGEs the effect is that of relaxation [216-2181.

M. P. L. CATON 351 These potent actions on smooth muscle of the gut have led to suggestions that endogenous prostaglandins may play a part in the control of intestinal motility [218, 2191. In four human volunteers, the ingestion of 2 mg of PGEl notably increased the transit rate through the small intestine and the colon with the production of abdominal colic and the passage of fluid and faeces -per rectum It has been suggested that diarrhoea associated with prostaglandin secretion from certain tumours is a consequence of this effect [220]. The mechanism of action of prostaglandins on gastrointestinal smooth muscle has been investigated by several authors [216, 221-2251 and is reviewed by Horton [13] and Pickles [lo]. It is suggested that PGEl exerts its effect by a direct action on the cell membrane [222], but observations that certain responses are reduced by nerve-blocking drugs, for example, atropine, point to the possibility that there is some element of mediation via a nervous pathway [223].

REPRODUCTIVE SMOOTH MUSCLE

Contraction is also the general response to prostaglandins shown by isolated segments of smooth muscle of the reproductive system and preparations on which this has been observed include rat and guinea-pig uterus and rabbit oviduct [198, 199, 2W203, 2081. With PGEl a concentration of 60 ng/ml is sufficient to produce a response on rat uterus [199]. The structure-activity pattern on rat uterus for some principal members of the series is shown in Table 7.5. Of the synthetic stereoisomers (Table 7.4) [213], ent-PGEl had only 1/1OOO of the activity of natural PGEl on rat uterus, although assay of Table 7.5

Activity Reference

RELATIVE STIMULANT ACTION OF PROSTAGLANDINS ON RAT UTERUS

(PGE, = 1.0)

E2

E3

Fi.

Fzo

Ai

aNor El

1.1 198

03 198

09 198

8 198

0041 208

198

I

ruc-PGA, indicates that the antipode of PGAl (i.e. ent-PGA,) has significant activity. As with the rabbit jejunum, data from synthetic racemates indicate higher activity than the corresponding natural forms for enf- 15-epi-PGAl and ent-1 1,l 5-epi-PGE,. The latter stereoisomer has been separated to the extent of 80 per cent optical purity and preliminary assay indicates that it is 1.1-1.4 times more potent than natural PGEl. betailed studies have been carried out on the effects of prostaglandins on the human myometrium. The E series normally relax the non-pregnant human myometrium in vitro and the spontaneous motility is inhibited [226-2351. PGFson the other hand in most cases cause a stimulation, although

352 THE PROSTAGLANDINS at higher doses [226,230,232]. PGAs, PGBs and their 19-hydroxy derivatives usually cause an inhibition in motility similar to that of the PGEs but they are less potent [207,226,233]. The response is dependent upon the hormonal state of the preparation used; thus human myometrial strips are more sensitive to PGE, at about ovulation time [226]. With the pregnant myometrium, however, the usual response of PGEs is stimulation, like that of the PGFs. This has been observed in vitro [236] where it was found that while the lower segment myometrium was relatively inactive, the upper segment underwent contraction. The stimulation also occurs in vivo [237] and the uterine contractions induced by administration of prostaglandins have led to the successful induction of labour in several groups of patients [238-2441. This has been achieved with PGFza [238, 239, 2441, PGEl [241] and PGE2 [24&243]. The prostaglandins were administered by continuous i.v. infusion; PGF2,, for example, was effective in all cases in a group of ten women at or near term when infused at a rate of 0.05 pg kgmin- 12441. There were no undesirable side effects at the effective doses. It has recently been reported that labour inductions have been successfully induced following oral administration [245]. Prostaglandins have also been successfully employed in the induction of therapeutic abortion [240, 246-2501 although higher doses are required. PGF2, infused at 50 pg/min stimulated the uterus to contract and produced abortion in early pregnancy [249]. The only side effects were nausea and vomiting which were almost absent when PGE2 was used [249-2511. Recently it has been reported that the administration of PGE2 (20 mg) or PGF2, (50 mg) in the form of a pessary inserted into the vagina every 2.5 hours produced successful abortions within 15 hours in all 45 cases under trial. This remarkable result carries the implication that if prostaglandins became readily available, abortion on demand and at any time in pregnancy will become a practical possibility [252]. This uterine stimulant action and the appearance of prostaglandins in human amniotic fluid during normal labour has led to suggestions that endogenous prostaglandins are involved in the normal labour process [29, 239, 241, 2531. This is further supported by observations of the appearance of PGF2, in blood samples taken during normal spontaneous labour which reached its highest concentration at the time of the uterine contraction [253]. Prostaglandins also have an action on the Fallopian tube [199]. With some prostaglandins this varies with the segment used; PGE,, for example, contracts the proximal part of the human Fallopian tube and relaxes other sections [201, 254-2561. These potent actions on the female reproductive smooth muscle suggest that seminal prostaglandins may have a function of importance in connection with fertility, for example by facilitating sperm migration and it has been observed that males from infertile marriages have a low mean seminal PGE

M. P. L. CATON 353 concentration [233, 257-2591. As with gastrointestinal smooth muscle, the mechanism of action of prostaglandins on reproductive smooth muscle has been investigated by several workers [227, 231, 26&263] and is reviewed by Horton [13].

RESPIRATORY SMOOTH MUSCLE

By contrast with their effects on gastrointestinal and reproductive smooth muscle, prostaglandins usually relax smooth muscle of the respiratory system. This has been studied in vitro using isolated tracheal preparations of the cat, monkey, rabbit, guinea-pig and ferret where PGE, showed activity in concentrations as low as 1 ng/ml [198, 201, 2641. With species where tracheal smooth muscle does not exhibit inherent tone (for example, cat, monkey and rabbit) the effect of PGE, can be demonstrated after a sustained contraction has first been produced by a suitable agent such as acetylcholine. Relative activities of principle prostaglandins have been measured and some results on cat trachea are shown in Table 7.6 [198, 2081. In general PGFs are much less potent than the E series. PGE, produces inhibition of the inherent tone in human bronchial strips at threshold concentrations of 0.25 pg/ml [265] and a similar inhibitory Table 7.6

Activity Reference

RELATIVE INHIBITORY ACTION OF PROSTAGLANDINS ON CAT TRACHEA (PGEI = 1) [198,208] E2

E3

F,,

1 198

0.2 198

0002 198

a-Nor PGEl 0.1 198

PGA, 0034 208

action was noted when the bronchial muscle had been contracted by histamine acid phosphate. PGE2 also relaxes the inherent tone although it is rather less active than PGE, but FZacauses a contraction [266], an effect which is antagonised by fenamates [267]. The inhibitory action of prostaglandins on respiratory smooth muscle has been demonstrated in vivo by measuring the change in resistance to inflation of lungs by the Konzett-Rossler and Dixon-Brodie methods. In the anaesthetised rabbit and guinea-pig, PGE, antagonised the rise in resistance to inflation obtained after vagal stimulation or i.v. administration of histamine and it sometimes lowered the resistance to inflation in these species [264]. However, in the cat lung PGE, and PGF2, increased resistance to inflation [203, 2641. PGE2 causes hyperventilation when administered i.v. to anaesthetised dogs and PGF2. produces a sharp fall of dynamic lung compliance and a decrease in alveolar ventilation [268, 2691.

354 THE PROSTAGLANDINS PGEl also has bronchodilator activity in guinea-pigs when administered by aerosol, as assessed in terms of the antagonism of bronchoconstrictor responses elicited by the i.v. administration of a bronchoconstrictor agent such as histamine [270]. Whereas the activity of PGE, when given i.v. is slightly less than that of isoprenaline, by aerosol the potency is 10-100 times greater. Aerosol administration is not accompanied by the cardiovascular changes obtained under similar conditions with isoprenaline or with PGE, when given intravenously. The higher potency and lack of systemic effects on aerosol administration may be related to the observation that up to 95 per cent of i.v. infused PGA, in the dog, cat and rabbit is removed during a single pulmonary circulation [170-1751 suggesting that there is in the lungs a system for inactivation of prostaglandins. Aerosol-administered PGE, prevents bronchoconstriction which has been induced by histamine aerosol. The bronchodilator properties of this prostaglandin were shown to be greater in the guinea-pig and monkey compared with the dog [271,2721. Observations of relief of bronchoconstriction without cardiovascular side effects suggest that prostaglandins have a potential clinical role in conditions such as asthma. Cuthbert [273] compared the effect of aerosol-administered PGEl on airways resistance in healthy and asthmatic volunteers with reversible airways obstruction. In the healthy subjects, the forced expiratory volume in one second (FEV1) was unaffected by the prostaglandin when administered as the free acid or as the triethanolamine salt. However, in the asthmatics, inhalation of 55 pg of the triethanolamine salt produced an increase in FEVl comparable in both degree and duration with that produced by an inhalation of 550 pg of isoprenaline sulphate. The triethanolamine salt was employed in the asthmatic trials since it was better tolerated, the free acid being irritant to the upper respiratory tract. Similar results were obtained with PGEz [274]. Various studies have provided information on the mechanism of action of prostaglandins on respiratory smooth muscle. Experiments with adrenergic blocking agents indicate that the inhibitory effect is not produced by an action on sympathetic P-receptors [265, 2701. It has been found [275] that the degree of inhibition of contractions of isolated dog tracheal muscle varies with the stimulant used. Thus low concentrations of PGE, completely block the stimulant effect of 5-hydroxytryptamine, but even large concentrations do not completely antagonise the contractions caused by acetylcholine. The inhibitory effect is blocked by methysergide but not by propranolol, morphine or dihydroergotamine. From these results it is concluded that in tracheal muscle, PGEl interacts with cell membranes close to the 5-hydroxytryptamine D receptors. This causes activation of the smooth muscle sarcoplasmic reticulum, leading to accumulation of calcium ions and relaxation. The occurrence of prostaglandins in human lung has led to speculations about their role in pathological conditions involving bronchoconstriction,

M. P. L. CATON 355 which may result from an over production of the bronchoconstrictor PGFza from arachidonic acid at the expense of the relaxant PGE, [131.

CARDIOVASCULAR SYSTEM

Prostaglandins have potent effects on the cardiovascular system. PGEl lowers arterial blood pressure in a wide range of species which include the dog [276, 2771, guinea-pig [278], rat [279, 2801, cat [278, 2791, rabbit [199, 204, 2791 and chick [281] where minimally effective single intravenous doses normally range from 1 to lOpg/kg [12]. Comparative data for the principal prostaglandins on rat and rabbit blood pressure are shown in Table 7.7. The PGFs show considerable species variation, thus in the cat and rabbit they resemble the E series in having depressor activity [201, 2031, whereas in the dog and rat they are pressor [12,282]. The PGAs which have relatively weak activity on smooth muscle are here approximately equipotent to the E series, although there are some species variations 1209, 283-2851 and the B series as on other biological systems have very low potency [285]. 8-Zso-PGEl also has relatively low activity [90, 286-2881. 15-Epi-PGA2 has no significant effect on the blood pressure of dogs [289] and 15-epi-PGELand 15-epi-PGAl have low activity in the rat [213] (Table 7.4). As with smooth muscle preparations, data for synthetic racemates (for example ruc-PGA,) suggest that certain ent forms may have a high level of potency (Table 7.4) [213]. w-Nor-and o-homo-PGE, display activity of the order of that of PGEl on rat blood pressure [89] but a-nor-El is only 0 1 as active in the rabbit [198]. Dihydroprostaglandins follow the PGAs in showing a much higher level of activity relative to the PGEs compared with that on smooth muscle [210, 21 11; thus dihydro PGEl is only slightly less potent than PGEl in the rabbit and is more potent in the guinea-pig [210]. 11-Deoxy PGEl derivatives also show substantial activity but 1I-deoxy PGFs are only weakly active [211]. 15-0x0 prostaglandins show little effect, as on other biological systems [210]. PGEl has been shown to lower blood pressure when infused into normal human volunteers at 0.2-0.7 pg kg min ; studies with somewhat lower doses showed no effect [290-2941. These infusions also gave rise to an increase in cardiac output with both enhanced stroke volume and heart rate but PGF2, had no effect on blood pressure or heart rate at doses up to 2 pg kg- min- [294]. PGEl infused simultaneously with noradrenaline reduced the increase in arterial pressure found when the latter was given alone [291, 2921. PGEl also increased the forearm blood flow when infused into the brachial artery [295]. PGA, (medullin)decreased the systemic blood pressure when given i.v. to a patient with fixed essential diastolic hypertension [296], and PGAl lowered the blood pressure of several groups of hypertensive patients when given i.v. in doses of 03-2 pg kg-' min-' [297-3001. The place of prostaglandins in the therapeutic control of blood pressure is not

-'

'

-'

Table 7.7

Rat Reference Rabbit Reference * Pressor

actlwt"

R E L A T I V E DEPRESSOR ACTION OF PROSTAGLANDINS ON R A T

E2

E3

Fla

F2.

0.1 3

-

*

*

209 1 198

03 198

0.08 20 1

0.1

-

~

-

201

A1

A2

0.3 209

033 209

Y D RABBIT BLOOD PRESSURE

a-Nor

(PGE,

=

I)

IS-KetoDihydro-E, 15-Keto-E, dihydro-E,

8-iso-E, -

~

~

-

~

-

01 198

~

~

0.63 210

-

004 210

~

0.0058 90

<0.01

-

210

-

M. P. L . CATON 357 clear at present but general therapeutic use may become feasible especially if longer acting, orally-active forms can be prepared [197]. Extensive studies have been carried out to determine the mechanism of the blood pressure changes. The hypotensive effects are attributed to a decrease in peripheral resistance resulting from vasodilatation, an action which is not blocked by atropine, P-blocking agents or antihistaminic compounds and thus does not appear to involve cholinergic, adrenergic or histaminergic components [300-3061. Similarly, the pressor action as shown by the F series is attributed to vasoconstriction [307-3111. It has recently been suggested that PGAs differ from the PGEs in lowering blood pressure by a delayed indirect mechanism of action [312]. Evidence is also being accumulated in favour of some mediation of prostaglandin cardiovascular effects via nervous pathways [284]. It has been suggested that the vasodepressor lipids found in the kidney medulla which contain prostaglandins have a function in the regulation of normal blood pressure and it is of interest that certain prostaglandins are able to prevent renoprival hypertension [313-3 171. It has been proposed that an alteration in the balance of pressor substances (renin, PGF2,) and depressor substances (PGE2, PGA2) may determine the development of renal hypertension [318]. A PGE2-like lipid has been detected in the renal venous blood of hypertensive patients [319]. It has also been suggested that PGE, may serve as a local hormonal regulator of blood flow in the microcirculation by virtue of its vasodilator properties and ability to reduce vascular responsiveness to endogenous constrictor amines and polypeptides [320]. Although the PGEs usually produce vasodilation, there are certain cases where they are vasoconstrictor. This has been noted in the blood vessels of the nasal mucosa where initial studies in dogs have shown that PGE, and PGE, induced a constriction and both are equipotent (doses of 5 pg/kg) with noradrenaline but with a duration of over seven times as long [321,322]. PGA, and PGF,, were about ljl0Oth as active as PGE,. The prolonged duration of increased nasal patency suggested the possibility of clinical use as a decongestant and trials in human volunteers have been carried out [323, 3241. Topically applied doses of PGE, administered via an atomiser caused an increase of patency of the nasal airway and a visible blanching of the mucosa, effects which lasted from 30 min at a dose of 37 pg to 10-14 hour at 75 pg. It has been shown that PGE, raises pulmonary arterial pressure, and PGF2, has a similar action although it is much more potent. It is of interest that 8-iso-PGE1 here is five times more potent than PGE, [287]. Prostaglandins also show a direct action on the heart [325-3311. Thus studies in open chest, anaesthetised dogs showed that PGE,, PGAl and PGF2, all increase heart rate, cardiac output and myocontractile force [283].

358

THE PROSTAGLANDINS

BLOOD PLATELET AGGREGATION

PGE, is a powerful inhibitor of platelet-to-glass adhesiveness in a wholeblood system and of in vitro platelet aggregation, induced by A.D.P., noradrenaline, A.T.P., 5-H.T., thrombin and collagen. Species where aggregation inhibition has been observed include the rat, pig and man [332-3371; the minimal effective concentration for activity against ADPinduced aggregation of human platelets is 0.05 pg/ml [336]. Structure activity relationships have been established for a substantial number of prostaglandins [89, 334-3361. It is noteworthy that PGE, has a very much lower potency than PGE, and in some instances has even caused a stimulation of aggregation. This marked divergence between PGEl and PGE2 is of particular interest since in most biological systems their effect is similar. Another observation of note is the high level of activity of 8-iso-PGE1 which is comparable with that of PGEl. Since the iso-compound is much less active as a spasmogen and a vasodepressor agent [286], i t may thus exhibit fewer side-effects when employed clinically as an antithrombotic agent. w-HomoPGE, shows a particularly high level of potency (c. 4 times that of El) f89, 3341, but prostaglandins of the F, A and B series have only weak activity [334-3361. The spectrum of activity of prostaglandins against blood platelet aggregation thus differs markedly from that on hypertension and may be contrasted with that of other antithrombotic agents where the level of the two types of action run closely parallel. It has been shown that topically applied or i.v. administered PGEl suppresses platelet thrombus formation in injured rabbit brain arteries [333]. In rats, experiments showed that PGE, and w-homo-PGE, inhibited ADPinduced aggregation, and transient thrombocytopenia induced by i.v. ADP was inhibited by both of these prostaglandins [338]. PGEl at 0 . 1 4 . 5 pg/min, but not PGF1,, blocked the formation of thrombi in veins on the brain sur2 kg-' min-' face [339]. PGE, infused i.v. into humans at rates of 0 0 5 ~ 0 . pg for 15 to 30 minutes showed no discernable effect on platelet aggregation but platelet adhesiveness fell from 36 per cent before the infusion to 1 1 per cent at the end and was still reduced in each patient (mean 19 %) 30 minutes later [340, 3411. Studies have suggested that the effect of prostaglandins on platelet aggregation is mediated via cyclic AMP [342-3461. Thus it has been shown that PGE, stimulates cyclic AMP synthesis of platelet membrane fractions and that cyclic AMP inhibits the aggregation. PGE,-stimulated cyclic AMP synthesis of intact platelets is decreased by noradrenaline which is in turn inhibited by the sympathetic a-receptor antagonist phentolamine but not by the @-receptorantagonist, propranolol [343].

M. P. L. CATON

359

METABOLIC EFFECTS

Prostaglandins antagonise basal lipolysis and lipolysis stimulated by a number of hormonal compounds including adrenaline, noradrenaline, ACTH, TSH and glucagon [347-36 I]. This has been studied extensively in vitro using rat epididymal fat pads by measuring the release of glycerol into the incubation medium which determines the rate of lipolysis independently of that of re-esterification. PGE, has a significant action against the effect of 100 ng/ml of noradrenaline at a dose of 3.2 ng/ml but a maximal effect (41 % inhibition) occurs at 100 ng/ml [362]. Comparative data reveal that the highest activity is shown by the E series [209, 21 1, 352, 357, 3581. In a direct comparison, PGE, was 2.9 times more potent than PGEl [209]. PGFs are much less active than their PGE counterparts [352, 3581 and PGAs are very weakly active [209]. 8-Iso-PGE1 had 0.24 the potency of PGE, [90]. With basal (non-stimulated) lipolysis, PGE, is effective in doses as low as 1 ng/ml and exerts a maximal effect at 100 ng/ml [352-356, 3631. Inhibition of lipolysis in vitro has also been demonstrated in human adipose tissue [363-3651 and in isolated white fat cells [366]. Experiments with anaesthetised dogs produced results which might have been expected from the in vitro studies, namely i.v. administration of PGEs reduced hormone-raised levels of plasma FFA [276]. However, with unstimulated lipolysis in vivo, the effects are complicated by the fact that on injection in dogs and human volunteers PGEl itself induces lipolysis in doses lower than those that have an inhibitory action on the lipolytic actions of other hormones [291, 292, 352, 367, 3681. Thus a dose of 0.2 pg kg-' min-' in man or dog increases plasma glycerol and FFA [367-3691. This stimulant action is abolished by ganglion blocking agents [368] and thus appears to be mediated via a nervous pathway. At higher doses, however, in dogs (0.41.6 pg kg min-') lipolysis is increased [368]. The results of these observations and other studies which are summarised by Bergstrom [ 121, suggest that i.v. PGEl acts by two independent mechanisms; at low doses stimulation of FFA mobilisation by way of the sympathetic nervous system, probably as a compensatory reaction to lowered blood pressure, and at higher doses reduction of FFA mobilisation by inhibition of lipolysis enough to overcome the sympathetic effect. The actions of other prostaglandins in vivo are what would be expected from their effects in vitro [276-2851. In man infusion of PGE, (0.0560 3 2 pg kg-' min-' for 30 minutes) as with PGE,, tended to raise plasma FFA levels while the same doses of PGs A,, F1,, F,, and FZahad no clear effect [ 121. Many data support the contention that the lipolytic action of prostaglandins is mediated via the adenyl cyclase system which catalyses the formation of cyclic AMP from ATP (Figure 7.8) [356, 357, 370-3861. Thus under a variety of experimental conditions (for example, in the presence of

-'

360 THE PROSTAGLANDINS lipolytic hormones, methylxanthines, adrenergic blockers etc.) the rate of lipolysis and intracellular cyclic AMP levels have been correlated temporarily, qualitatively and quantitatively. Available evidence suggests that prostaglandins affect the cyclic AMP level by influencing its formation rather than by interfering with the steps with which it activates the lipase. If the norad renaline +C

ATP

J

Phosp hod iesterase

PGE

C y c l ~ cA M -

5'-AMP

Llpoiysls

Figui e 7 6

7 he function of cyclic A M P

in

the actions of lipolytic hormones and PGE

latter were so, the prostaglandin would be expected to influence the effect of added cyclic AMP but this is not the case, even though in some experiments it had its usual effect on the action of noradrenaline on basal lipolysis 13561. PGEl does not cause increased cyclic AMP levels in isolated fat cells, suggesting that the stirnulatory action occurs in another type of cell 12851. The observation that prostaglandins are released from adipose tissue in v i m in response to nerve stimulation or to catecholamines in amounts sufficientto have an inhibitory action on the lipolytic action of catecholamines [387], supports the negative feedback hypothesis [13]. By this means hormones that act via cyclic AMP formation also release prostaglandins which have an inhibitory action on adenyl cyclase, tending to limit the hormone's action. Thus whilst adrenergic nerve stimulation of adipose tissue increases cyclic AMP formation and so stimulates lipolysis, the accompanying release of prostaglandm tends to inhibit adenyl cyclase leading to a limitation of the released catecholamine's response. This hypothesis could also apply to other tissues and hormones where action depends upon the cyclic AMP system. Prostaglandins also have an effect on carbohydrate metabolism 1121. Thus it has been shown that in isolated rat adipose tissue, PGEl influences some aspects of glucose metabolism [388]. PGEl given i.v. elevates blood sugar [353, 3681. GASTRIC SECRETION AND ULCER FORMATION

Prostaglandins inhibit basal gastric secretion as well as that which has been induced by vagal stimulation or by secretalogues such as histamine, pentagastrin, 2-deoxyglucose and food. This effect has been observed in rats, thus with PGEl given by infusion the EDs0 value was 05-1 pg kg-' min-' for four hours [389-39 I]. Studies have also been carried out in dogs [392-3941 where PGE, and PGEz each caused near total inhibition of food- and histamine-induced

M. P. L. CATON 36 1 secretion when administered i.v. at 1 pg kg-' min-' with a maximum inhibition occurring after 30-45 min. PGAl and PGF,, did not affect histamine-induced secretion at this dose, although PGAl was shown to inhibit that due to food [392]. Further studies showed that PGEl inhibited volume, acid concentration and output and pepsin output of the gastric juice which had been stimulated by various agents whether the prostaglandin was administered subcutaneously, i.v. or by constant i.v. infusion [393]. A high level of inhibitory activity against gastric secretion in the rat is shown by certain 11-deoxyprostaglandins [395, 3961. One member of this caused 52 per cent inhibition of basal series, 1 1-deoxy-l3,14-dihydro-PGE,, gastric secretion at 0 8 mg/kg and was about one-half as active as PGEl. Unlike PGEl, which is optically pure, this deoxyprostaglandin is a racemate with four possible isomers and it is suggested that one of these might prove even more active. The corresponding 13,14-unsaturated compound was less potent-l/lOth of PGE,-and related deoxy-PGFs about 1/80th of PGE1. This influence of prostaglandins on gastric secretion has aroused interest in their possible clinical role in antiulcer therapy. Activity has been demonstrated against experimental ulcers; thus PGEl inhibits the formation of pylorus ligation- or steroid (Predniso1one)-induced ulcers in rats [389, 3941. The first report of investigations of a prostaglandin on gastric secretion in man was that PGEl failed to inhibit pentagastrin-induced secretion when given orally in doses of lMOpg/kg [397]. However, recently it has been shown that PGAl infused for 30 min (05-1.25 pg kg min-') decreased both mean volume and acidity in human volunteers receiving i.v. histamine at 0015 mg kg h [398], and that PGEl gave rise to significant reduction in basal hydrochloric acid production during infusion of 300 pg over 30 min [3991. Some confusion exists over the role of cyclic AMP in the action of prostaglandins on gastric secretion [390, 391, 3994021. It has been suggested that PGEl might influence gastric secretion by diminishing tissue levels of cyclic AMP as in other systems. However, studies in the intact dog do not accord with this since it has been shown that exogenous cyclic AMP inhibits rather than stimulates gastric secretion. Mucosal blood flow is also diminished by both cyclic AMP and PGEl and it is suggested that it is this effect which leads to the gastric secretion inhibition [401]. It has been further suggested that PGEl here in fact stimulates gastric cyclic AMP. More recently it has been proposed that PGEl inhibits gastric secretion by a direct action on secreting cells rather than by blood flow restriction and that the latter appears to be the result and not the cause of the gastric secretory inhibition [403].

-'

-' -'

DIURETIC AND NATRIURETIC EFFECTS

Several studies have demonstrated diuretic and natriuretic effects of prostaglandins [40+410]. When PGEl was infused into one renal artery of the dog

362 THE PROSTAGLANDINS at doses of 001-2 pg/min, urine volume, urinary sodium excretion, free water clearance and renal plasma flow were increased [404406] on the infused side. Similar results have been observed with PGE, and PGAl [407, 408, 412, 4131. PGEl also produces diuresis and natriuresis in the cat [414]. The effects of PGA, have been studied in man [197, 415, 4161. When administered at low infusion rates of 0 .1 4 .3 pg kg- min- (a level which does not affect arterial blood pressure), renal blood flow, urinary sodium and potassium excretion, urine flow and free water clearance all rose significantly. At higher infusion rates, the effects were reversed with renal plasma flow, urine flow and electrolyte excretion falling to control values. It has been suggested that general therapeutic use may become feasible especially when longer acting, orally active prostaglandins can be prepared [197]. However, endogenous PGE's, which occur in the renal medulla, may serve as an intrarenal regulator of sodium and water excretion [404-408, 410, 411,414,4151. It has been suggested that these diuretic and natriuretic effects result from an inhibition of the action of vasopressin, an effect which has been observed in the isolated toad bladder and rabbit renal tubule, where PGEl inhibits vasopressin-induced water permeability [12, 13, 4174191. It is postulated that vasopressin activates adenyl cyclase, thus increasing cyclic AMP formation, which in turn leads to an increase in water permeability. PGE, may then inhibit the vasopressin and hence reduce the permeability by acting via the adenyl cyclase in the same way as it reduces hormone-stimulated lipolysis [13,404,407, 410,4121. LUTEOLYTIC EFFECTS

Reference has been made in the section on reproductive smooth muscle to the possibility of using prostaglandin for the termination of pregnancy at any stage. Antifertility action by prostaglandins administered shortly after conception has been observed in several laboratory animals [42@422]. Thus PGE, or PGFza when given subcutaneously to rats at a dose of 0.5 mg twice daily during the first seven days of pregnancy, resulted in a significantly smaller number of implantation sites compared to the controls. Furthermore, PGFz, has been shown to interrupt pregnancy in rabbit and hamster [422]. The termination of pregnancy at these early stages is probably related to the regression of the corpus luteum. Evidence for this has been derived from work by Duncan and Pharriss [422]; thus when PGF,, was infused into pseudopregnant rats at a rate of 1 mg kg-' day-' on days five and six of pseudopregnancy, a dramatic shift occurred in the steroid pattern of the ovaries. Progesterone levels were depressed and 20a-dihydroprogesterone

M. P. L. CATON 363 levels were elevated to such an extent that the latter was much in predominance, an effect which gives an early indication of luteal degeneration [423, 4241. Rats injected subcutaneously twice a day with PGFz, on days 1 through 7 of pseudopregnancy returned to oestrus on day 8 (range 5-13 days) while the controls remained pseudopregnant for 17 days (range 13-20 days) [422, 4241. Luteolysis has also been noted in the sheep [425], guinea-pig [426] and rhesus monkey [427]. In the guinea-pig, luteolysis was observed by histological examination of the ovaries of hysterectomised animals after intraperitoneal administration of 0.5 mg of PGF2, twice daily for seven days, after which the corpora lutea showed an advanced state of regression. In rhesus monkeys 30 mg/day of PGF2, given subcutaneously twice daily for five days commencing on day 11, 12 or 13 post-ovulation of fertile cycles, caused elevation of circulating progestin levels. Such an effect was not obtained by injection earlier in the luteal phase of the reproductive cycle which indicates that the corpus luteum of this species is more vulnerable to luteolysis at the later stages [427]. It has been proposed that this luteolytic effect of PGFz, is a consequence of constriction of the utero-ovarian vein. The resulting reducticn of blood flow through the ovary would then be insufficient to support the normal biochemical processes with consequent cessation of progesterone synthesis and the final demise of the luteal tissue [422,424]. This action does not appear to involve the pituitary [422]. The results of in vitro experiments using slices of corpora lutea from pregnant dairy cows showed that the prostaglandins tested-El, Ez, FZdand Al-all increased steroidogenesis, with E2 having the greatest effect [428]. These results are in contrast to the in vivo luteolytic effect of PGFz, which it seems, therefore, is unlikely to be due to direct inhibition of luteal steroidogenesis. The in vitro and in vivo effects here may thus represent two different modes of action.

NERVOUS SYSTEM

Prostaglandins have powerful actions on the central nervous system. PGE’s when injected into the cerebral ventricles of unanaesthetised cats produced sedation, stupor and signs of catatonia at a threshold dose of 3 pg/kg [429]. On i.v. injection in the cat slight sedation was observed at a dose of 20 pg/kg, but in the chick, which lacks a blood-brain barrier, i.v. administration (1WOOpg/kg) caused profound sedation and at the higher dose level, loss of the righting reflex. Prostaglandins have a selective excitory action on brain stem neurones [431, 4321. This was shown by applying prostaglandins by means of microiontophoresis into the medulla of unanaesthetised decerebrate cats and observing the spontaneous activity of single neurones.

364 THE PROSTAGLANDINS PGFz,, when administered i.v. in chicks increases gastrocnemius muscle tension, an effect which is attributed to an action on the spinal cord [430]. The effect was observed in the decapitated chick but was abolished by denervation of the muscle. It was elicited in the urethanised chick in which reflex contractions were blocked. PGE, also increased gastrocnemius muscle tension when administered i.v. into spinal cats [433, 4341. Prostaglandins El, F1, and Fzu injected intra-arterially in the cat produced pronounced and long lasting effects on monosynaptic spinal reflexes [435, 4361. Thus PGE, at a dose of 3.5-17.8 pg/kg reduced the amplitude of monosynaptic responses from 15 minutes until three hours after injection. It has been shown that PGEl has in the cat an action at the spinal level to facilitate the crossed extensor reflex and in addition a second spinal action inhibiting the reflex. Since facilitation was never observed with PGEl when either the brain or the brain stem was present, it seems likely that the prostaglandin also exerts an inhibitory effect via higher centres which overshadows the facilitatory action at the spinal level [433,436]. Studies in dogs have shown that PGEl may have a selective action on a group of neurones within the brain stem, the effect of which is to inhibit the irradiation of activity from respiratory to cardio-inhibitory and vasomotor pathways [437]. These potent central actions, together with the widespread natural occurrence of the prostaglandins in the central nervous system and their release from the brain and spinal cord on nerve stimulation, has led to suggestions that they fulfil a role as central transmitters or more likely as modulators of transmission. The evidence for this and a more detailed account of CNS actions is presented by Horton [ 13,4381. Microiontophoretically-administered prostaglandins antagonise the response of cerebellar Purkinje cells to noradrenaline. It has been suggested that endogenous prostaglandins function to modulate central noradrenergic junctions and that prostaglandins and noradrenaline may influence the Purkinje cells via a reciprocal interaction with adenyl cyclase [439441]. The finding that PGE, when injected into the third ventricle of conscious cats produces a rise in body temperature has led to suggestions that endogenous prostaglandins may act as modulators of temperature regulation [442]. Prostaglandins also show actions on the peripheral nervous system [44345 I]. They thus increase the responses obtained by sympathetic stimulation and sympathomimetic amines on the isolated hypogastric nerve seminal vesicle preparation in the guinea-pig [443445]. PGEl showed a response at a concentration of 0.1 pg/ml and PGs Ez, A, and Flu had I/lOth, 1/50th and 1/100th of this potency [443]. PGEl also reduces pressor response to nerve stimulation in isolated perfused cat spleen [445] and inhibits sympathetic neurotransmission in the rabbit heart [4514531. These actions and the association of prostaglandin release with nerve activity together with their presence in nervous tissue suggest that prostaglandins have a role in the

M . P. L. CATON 365 physiology of the peripheral nervous system. Much evidence supports the view that they act as modulators of sympathetic neurotransmission whereby the released prostaglandins, which are locally mobilised by the nerve stimulation, may counteract further noradrenaline release by exerting a braking effect on the sympathetic neuroeffector system. [445, 446,448, 4504531.

OCULAR EFFECTS

Several studies have demonstrated ocular effects of prostaglandins [454-458]. Thus PGEl (0.1 pg) administered into the anterior chamber of the rabbit eye caused a large elevation of intraocular pressure (I.O.P.) as well as miosis; the latter also having been observed in the cat and monkey. The order of potency here is PGEl z E2 > Fz, > Al > F1,; with all except F1, the effects are well marked. PGEs have also been known to raise I.O.P. when given intravenously, in the cat, with a threshold dose at about 0 2 5 pg [459]. These actions together with the presence in the iris tissue of certain prostaglandins-formerly referred to as irin [36, 371-suggest that prostaglandins fulfil a physiological role in eye function. The miotic effect has been interpreted as meaning some direct action on iris smooth muscle and it is suggested that the rise in I.O.P. is mediated in part by an increase in permeability of the blood-aqueous barrier [457, 4581, an effect which is indicated by an observed increase in protein content of aqueous humour which correlates with the increase in I.O.P. The rise in I.O.P. produced by PGE, is antagonised by polyphloretin phosphate and it is suggested that this antagonism arises as a result of stabilisation of the blood-aqueous barrier [457, 4581.

CONCLUSION The wide range of biological activity and high potency of action shown by the prostaglandins has justified the intense interest which these substances have generated over the past decade and the possibility of medicinal application suggested by animal studies has led to clinical trials which have shown encouraging results. Of these, the demonstration of effectiveness in the termination of pregnancy and possible use in controlling the various stages of the reproductive cycle clearly have far reaching possibilities and trials in the treatment of bronchial asthma, gastric hypersecretion, hypertension and nasal decongestion suggest a diversity of other potential applications. There are, of course, many difficulties to overcome but signs that this can be achieved are encouraging. Earlier problems of accessibility are being eased with the displacement of biosynthetic methods of preparation by total chemical synthesis and the latter is also making available prostaglandin analogues

366 THE PROSTAGLANDINS which may show greater specificity of biological activity and hence decreased side effects when employed clinically. There is also evidence that the short duration of action attributed to metabolic inactivation can be increased without adversely affecting potency. The application of the prostaglandins offers a much needed fresh approach to therapy in several important medical fields. The next few years will no doubt show whether this can be realised but at present it can be fairly said there are good grounds for optimism. Two recent papers describe several important improvements [459, 4601 to the Harvard syntheses (pp. 329-330) which provide advantages over the earlier procedures for large scale operation. Another paper [4611 describes the first stereospecific synthesis of natural PGE3 and PGF3,. A series of papers on the chemistry, biosynthesis, metabolism and mechanism of action of prostaglandins and their role in female reproductive physiology was published recently [462].

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THE PROSTAGLANDINS

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INDEX The contents list at the beginning of each chapter should be consulted first. Italicised page numbers indicate that the subject is referred to on succeeding pages.

Accothron, 4 Acedapsone and malaria, 260 Acetazolamide and reproduction, 185 Acridines and malaria, 247 Acriquine, 247, 248 Adalin, 76 1-Adamantanamine, 122 Adamantane derivatives, 124, 152 2-Adamantanone, 125 I-Adamantyl-biguanide, 124 -guanidine, 124 -thiourea, 124 -urea, 124 Albizziin, 96 Aldrin, 16 Algestone acetophenide, 181, 184 Alisobunal, 76 Allen-Doisy assay for oestrogens, 182 Allobarbital, 76 Allobarbitone, 76 Allonal, 72 Alloxan, 64 Alloxantin, 64 Allylbarbituric acid, 76 Allypropymal, 72 Alurate, 72 Amantadine, 122 Amenorrhea, 220 Amino acids and malaria, 292 Arninoacrichin, 247, 248 I-Aminoadamantane, 122, 124, 152, 154 2-Arninoadamantane, 125 p-Aminobenzoic acid and malaria, 283 I -Aminocyclopentane-1-carboxylic acid, 278 4-Aminoquinolines and malaria, 248, 264, 265,27 1,292, 301, 305 8-Aminoquinolines and malaria, 245, 247, 272, 292 Amobarbital, 73 Amodiaquine, 249 Amopyroquine, 249

Amylobarbitone, 73 Apholate, 14 Apoarantin, 159 Aprobarbital, 72 Aprobarbitone, 72 Analgesics, 76 Anaphylaxis, protection, 65 Androgens, 174 Androstenedione, I75 19-hydroxy-, 177 19-nor-, 179 I~-oxo-, 177 Anovulation, 220 Antibiotics and malaria, 277 Anticonvulsants, 72, 78 Antifungal action, 94, 100 Antihypercholesterolemicaction, 93 Anti-inflammatory action, 79 Antimalarial drugs, 243 antifolic compounds, 272 evaluation, 237 mode of action, 282 repository, 261 synergism, 255,257, 264 Antimycin A3, 158 Antineoplastics and malaria, 277 Anti-oestrogenic activity, 183 Antiprotozoal action, 98 Antitrichomonal action, 98 Antitumor action, 97, 99, 101, 150, 277 Antiviral agents, 80, 120 aliphatic, I55 amidines, 151 amino acids, 156 aromatic, 137, 153, 161 guanidines, 15 1 heterocyclic, 138, 143 multicyclic, 126 naturally occurring, 157 peptides, 156 thiosemicarbazones, 141 Antivirin, 137

378 Aranotin, 159 Ascochlorin, 157 Asuntol, 3 Atebrin, 247 Avlochlor, 249 Azacrin, 247 Azinphos-methyl, 4, 7, 16 Azodrin, 2 Baeyer-Villiger oxidation, 329, 337 Banol, 5 Barbitone, 72, 76 Barbituric acids, 66 analgesics, 76 anticholinergic, 78 anticonvulsants, 72, 78 antifungal, 94 anti-inflammatory, 79 antiviral, 80 diuretic, 80, 92 effect of lipid solubility, 75 effect of pH, 75 hydroxy-, 64 hypnotic, 72, 81 hypotensive, 76, 79 long-acting, 72 muscle-relaxant, 77 schistosomicidal, 94 short-acting, 74 tranquillising, 77, 93 Basadin, 3 Benzhexidol, 78 Benzimidazoles, antiviral, 139 Bidrin, 2 Birlane, 2 Bladan, 2 Bleomycin A2, 97 Blood-brain barrier, 16 Blood coagulation, 211 Bomyl, 2, 21 Bornane derivatives, 128 Borrelidin, 158 Brefeldin A, 158 Brevital, 74 Brietal, 74 Bromadyl, 76 Bromoguanide, 243 Busatin, 141 Butabarbital, 73 Butabital, 76 Calcium elenolate, 155

INDEX

Camolar, 259 Camoquin, 249 Canavanine, 152 Carbieron, 2 Carbohydrate metabolism, 202, 208, 295 Carbonic anhydrase assay, 184 Carbophenothion, 5, 17 Carbromal, 76 Cel: wall and antibiotics, 43 Ch’ang Shan and malaria, 269 Chetocin, I59 Chetomin, 159 Chlorfenvinphos, 3,8, 18, 19, 22, 29 Chlorguanide, 243 Chlormadinon acetate, 181, 219 Chloroquine, 246,281,287 6-Chloro-3, 4-xylyl methylcarbamate, 5 Chlorthion, 3, 16 Chlorveniphos, 23 Cholesterol, conversion to dehydroepiandrosterone, 178 Cholinesterases, 5 CI-501,259 (3-564, 264 Ciodrin, 2 Cinchona alkaloids and malaria, 250 Cinchonidine, 250 Cinchonine, 250 Combination pill, 190 Contraceptives, blood coagulation, 2 11 cutaneous effects, 220 depression, 216 hypertension, 217 liver function, 214 migraine, 2 16 neoplastic disease, 218 ophthalmological effects, 2 I7 pills, 190 respiration, 220 selection, 201 side-effects, 202 thromboembolism, 208 types, 201 Convicine, 63, 64 Corner-Allen assay, 183 Co-Ral, 3 Corpeus luteum, 174, 199 Cotnion, 4 Coumaphos, 3, 17 C-Quens, 2 15 Crotoxyphos, 2 Cycloguanil embonate, 259, 262 pamoate, 259,260 Cyclo-octylamine, 132

INDEX

Cyclophosphamide and malaria, 278 Cycloquine, 249 Cygon, 4 Cylindrochlorin, 158 Cytel, 4 Cytidine, 84 triphosphate, 84 Cytosine, 82, 83 antiviral derivatives, 149 derivatives, 86 DADDS, 260 Dalf, 4 Danathion, 4 Dapolar, 264 Dapsone, 254, 256, 263, 277,287,295 Daraprim, 243 Daunomycin, 160 DDS, 254 DDVP, 2 DFP, 3, 8, 10 Deciduoma formation, 185 Dedevap, 3 Dehydroepiandrosterone (DHA), 175, 176 from cholesterol, 178 15-Dehydroprostaglandins, 342 Deladroxate, 215 Delnav, 4 Demeton-O,4, 16 Demeton-S, 3 1 I-Deoxyprostaglandins, 261, 341, 348 Depo-Provera, 21 5 Depot preparations, antifertility, 195 Diabetes and contraceptives, 204 4, 4'-Diacetylaminodiphenylsulphone,260 Dial, 76 2,4-Diaminopyrimidines and malaria, 243, 273,285 Diaphenylsulphone and malaria, 254 Diazinon, 3, 14, 17 Dichlorvos, 2, 14, 17, 29 Dicrotophos, 2, 19, 22, 23, 29 3, 4-Dihydroisoquinolines, antiviral, 138 Dihydroprostaglandins, 339 Dimaz, 4 Dimecron, 3 Dimefox, 3 Dimethisterone, 180, 181, 184, 203 Dimethoate, 4, 9 Dioxathion, 4, I6 Disulfoton, 4, 16 Disyston, 4 Dithio-Systox, 4

Diuretics, 80, 92, 93 Divicine, 63, 64 DMP, 8 Dydrogesterone, 186, 195, 196 Dyflos, 3 E 600,2 E 838, 4, 8, 10 EE-see Ethynyloestradiol Elenolate, calcium, 155 Embathion, 4 Enovid, 2 15 EPN, 4,7, 10, 16 Ethion, 4, 17 Ethisterone, 178, 184 &-methyl-, I80 19-nor-, 179 lg-nor-, acetate, 180 Etimidin, 104 Ethymidine, 104 Ethynodiol, 180 diacetate, 180, 184, 203, 207 17a-Ethynyl-17P-hydroxyoestra-4, 9, 1 l-trien-3-one, 196 Ethynyloestradiol, 178, 183, 186, 190 3-cyclopentyl ether, 179 Fanasil, 252 Febrifugine, 269 Fenathion, 4 Fenchlorphos, 4, 18 Fenitrothion, 4, 8 Fertility, cervical factors, 197 lactation and, 200 Folidol, 4 Folidol-M, 4 Folithio, 4 Follicle, 174 Formycin, 160 Fosferno, 4 Fostion MM, 4 Funiculosin, 158 Gardona, 3 Garrathion, 5 Gliotoxin, 158 GO-560, 77 Gonadotrophin inhibition, 187 Guthion, 4 Hanane, 3 Herbicidal action, 100

379

380 Herkol, 2 Heterocycles, antiviral, 138, 143 Hooker-Forbes assay, 184 Hormones, 174 follicle stimulating, 174, 177 lutenising, 174, 177 Hydrocortisone, metabolism, 180 Hydroxychloroquine, 249 Hydroxy-ipral, 8 1 Hydroxy-probarbital, 8 1 5-Hydroxyuridine, 62 Hypertension and contraceptives, 217 Hypnotics, 72, 8 I Hypotensive action, 76, 79, 93 Interferon, 133 inducers, 133, 136, 161 Isocytosine, 82, 88 derivatives, 89 Isolan, 15 Isopentaquine, 246 Isopestox, 3 Isouramil, 63 Isowillardiine, 96 Kelfizina, 253 Kethoxal, 155 Korlan, 4 Krebs cycle and malaria, 299 Lactation and contraceptives, 200 Lathyrine, 94, 95 Leydig cells, 174 Lincomycin and malaria, 277 Lipids and contraceptives, 207 Lipoproteins and contraceptives, 205 Lynestrenol, 179, 184 Madribon, 252 Mafu, 2 Malaria, current treatment, 280 immunology, 266 life cycle, 233 resistant strains, 245, 255, 258, 264, 281, 299, 303 types of, 236 Malaoxon. 3, 22 Malathion, 14, 22 Malocide, 243 McGinty assay, 184

INDEX

Medroxyprogesterone acetate, I8 1, 184,2 19 Megesterol acetate, 181, 186, 188 Mepacrine, 247 Meprobamate, 77 Mestranol, 178, 180, 183,203, 207 Metacide, 4 Metation, 4 Metharbital, 74 Methisazone, 141 Methocarbamol, 77 Methohexital, 74 Methohexitone, 74 a-Methyl-l -adamantme methylamine, 123 Methylphosphazine, 104 Mevinphos, 3, 21, 29 Midicel, 253 Migraine and contraceptives, 218 Minipill, 190 Mintacol, 2 Mipafox, 3 Monocrotophos, 2, 19, 22 Murphos, 4 Muscatox, 3 Muscle relaxants, 77 Mucin and fertility, 198 Mycophenolic acid, 158 Nankor, 4 Naphthoquinones and malaria, 275, 296 Navadel, 4 Neoplastic disease and contraceptives, 219 Neostigmine, 16 Nialate, 4 Nifos T, 2 Niran, 4 Nitrox 80, 4 Nivaquine, 249 Nogos, 2 Norethindrone, 179, 184, 188, 190 acetate, 188 thromboembolism, 210, 215 19-Norethisterone, 187 acetate, 179, 191, 207 Norethynodrol. 179, 184,207 Norgestrel, 180, 190, 195 Norinyl I , 215 19-No>testosterone, 174, 176, 179, 219 biosynthesis, 177 progestagens from, 179 Novathion, 4 Noviose, 54 Novobiocin, adsorption, 55 antibacterial spectrum, 41

INDEX

Perfekthion, 4 cell wall synthesis, 43 Pesticides, 2 cytoplasmic membrane, 46 list of, 2 decarbamoyl-, 54, 55 metabolism, 15, 22 magnesium deficiency, 49 therapy of intoxication, 23 mechanism of action, 41 toxicity, 9 nucleic acid synthesis, 47 Pestox 3, 3 protein synthesis, 47 Pestox IS, 3 resistance to, 55 Piericidin A, 158 structure, 40 Pill, combination, 190 structure-activity, 54 minipill, 190, 192 Nucleosides, antiviral, 149 postcoital, 196 Numal, 72 precoital, 190, 196 Nuvacron, 2 sequential-serial, 190, 191 weekend, 190 PIN, 4 Obidoxime, 28 Phagicin, 136 Oestradiol, 174, 187, 189 Phenobarbital, 72 Oestriol, 176 Phenoharbitone, 72, 77 Oestrogens, 175 p H and, 75 assays, 182 Phosdrin, 3 deficiency, 201 Phosphamide, 4 discovery, 178 Phosphamidon, 3,22 excess of, 201 Phosphates, 3 plasma lipids, 206 enzyme inhibition, 7 Phosphazine, 104 thromboembolism from, 192 Oestrone, 175, 211 Phosphorothionates, 3, 16 3-methyl ether, 179 enzyme inhibition, 7 OMPA, 3 metabolism, 22 Ophthalmological effects of contraceptives, Plasma lipids, 207 217 Potasan, 4 Organophosphates and cholinesterase, 5 PPS, 1 1 Orthonovin, 215 Pregnenolone, 175 Oryzachlorin, 179 Primaquine, 246, 292, 303, 304 Ovary, 174 Prostagens, assays, 183 Ovulation, 206 6-chloro-, 182 contraceptives and, 220 deficiency of, 201 inhibition, 186 discovery, 178 7-Oxaprostaglandins, 343 excess of, 201 Oximes and organophosphorus poisoning, 27 6-methyl derivatives, 18 I Oxychloroquine, 249 plasma lipids, 206 thromhoemholism. 208 types, 201 Paludrine, 243 Progesterone, 174, 184, 206 PAM, 27 17a-acetoxy-, 180 Pamaquine, 245,292 I7-caproate-, 180 Paramidine, 79 chlorthion, effect of, 16 Paraoxon, 2, 15, 19, 30 16-dehydro-, I82 Parathion, 4 , 20 17a-hydroxy-, I74 Parathion-methyl, 4 Proguanil, 243, 272,287 Partron, 4 Prostaglandins, 221, 317 Pentaquine, 246, 292 biological actions, 347 Pentobarbital, 73 biosynthesis, 325 Pentobarbitone, 73, 80 chemical properties, 344

38 1

382 detection, 324 metabolism, 345 nomenclature, 3 18 occurrence, 321 pharmacology, 348 structure, 318 synthesis, 329 Prostanoic acid, 319 PSBA, 262, 263 PSBF, 262 Pteridines and malaria, 273, 275, 285 Purines, antiviral, 147 Pyran co-polymer, 136 Pyrazomycin, 160 Pyrimethamine, 243, 28 I , 287 Pyrimidines, amino acids, 94 aminohydroxy-, 63, 82 antiviral, 147 diamino-, 90 dihydroxy-, 63, 64, 85 5-hydroxy-, 62 nitro- and nitroso-, 98 thio-, 80, 88 trihydroxy-, 63, 66 Quinacrine, 246, 287 Quinalbarbitone, 73 Quinestrol, 178, 179 Quingestdnol acetate, 180, 184, 188 Quinidine, 250 Quinine, 250, 25 I , 282, 287, 292 Resitox, 3 Resochin, 249 Rhodoquine, 246 Rifampicin, 159 and malaria, 277 Rimantadine, 123 Rogor, 4 Ronnel, 4 Roxion, 4 Sapecron, 2 Sarin, 3 Sandoptal, 76 Schistosomicides, 94 Schradan, 3, 16, 22 SDDS, 254 Secobarbital, 73 Secobutobarbitone, 73

INDEX

Sertoli cells, 174 SKF 525A, 7 , 8 Silastic capsule, 194, 195 Soman, 3 Somonil, 2 Sonar, 4 Sontoquine, 249 Spectracide, 3 Sporidesmin, 159 Statolon, 136 Stendomycin, 97 Steroids, biogenesis, 172 Stroma, 174 Sulphadiazine in malaria, 251, 256 Sulphadimethoxine and malaria, 252, 253 Sulphamethoxypyriddzinein malaria, 253, 256 Sulphamonomethoxine, 253 2-Sulphamo yl-4, 4'-diaminodiphenylsulphone,254 Sulphapyrazinemethoxine, 253 Sulphisoxazole and malaria, 258 Sulphalone and malaria, 253, 256, 282 Sulphonamides and malaria, 251 Sulphones and malaria, 251, 253, 264, 295 Sulphormethoxine and malaria, 252, 256 Sulphorthomidine, 252 Sumithion, 4 Supona, 2 Symmetrel, 122 Synergism in antimalarial drugs, 255, 257, 264 Systox, 3, 4 Sytam, 3 Tabun, 3 Teichuronic acid biosynthesis, 44 Tempal, 3 Tepa, 14 TEPP, 2, 8, 10 Terra Sytam, 3 Testosterone, I74 blood clotting, 215 chlorthion and, 16 Tetrachlorvinphos, 3, 22, 23 Tetrax, 3 TG-see trigljceride Thiobarbituric acids, 80 antiviral, 145 Thionomevinphos, 21 Thiophos, 4 Thiosemicarbazones, antiviral, 141 Thromboembolism, and oestrogen, 192, 208

INDEX

Tilorone, 161, 162 Tingitanine, 94 TMB-4,28 TOCP, 2, 13 Tranquillisers, 77, 93 Tri-o-cresylphosphate, 13 Trithion, 5 Triazinoindoles, antiviral, 142 Trichothecin, 158 Triglyceride and contraceptives, 205 Trihexyphenidyl, 78 Trimethoprim and malaria, 257, 258, 281 Trolene, 4 Tuberactidine, 98 Uracil, amino-, 101 5-bromo-, 62 5-hydroxy, 62 mustard, 101, 102 Uridine derivatives, 147 Vapona, 2 Vapotone, 2 Verrucarin A, I58 Verthion, 4 Vinbarbital, 73 Vinbarbitone, 73 Willardiine, 95, 96, 103 Willardine, 95 Xanthocillin X monomethyl ether, 158 Xenaldial, 154 Xenylamine, 153

383

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