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THE AUSTRALIAN NATIONAL UNIVERSITY

JOHN CURTIN SCHOOL OF MEDICAL RESEARCH

~ EOICAL CHE MISTRY GROUP

ANNUAL REPORT 1974

Staff

Reader and Head of Group:

D.J. Brown, M.Sc.(Syd.): Ph.O. sD.Sc.(Lond.), F.R.iLC.I.

Professorial Fellow :

D.D. Perrin, M.Sc.(N.Z.), Ph.D.,D.Sc.{Lond.)1 F.R.f\.C.I.~ F.R.i.C.

Senior Fellows :

E. Spinner9 M.Sc.Tech .• Ph .D .,D.Sc.( tl anc.). F.R.A.C.I., H.L.F. Armarego, Ph.DqD.Sc.{Lond.), F.R.A.C.In F.R.I.COj G.13. Barlin, M.Sc.{Syd.), Ph.D., F.R.A.C.I.

Visiting Fellows :

L.G. Stephanson, B.S.P'9 Ph.D.(Br.Col.), F . Yon e d a , P h • D • { Ky o to ) { J u 1 y - Se p t . 9 i n c 1 . )

Senior Research Fellow:

J.H. Lister ~ B.Sc.) Ph.D.(Lond.L F.R.I.C. , F.P.A.C.I.

Research Fellows :

~L Pendergast) Ph.D. {Sal f.), R. P. Agarwal 9 M.Sc. (Luck.), Ph.D. (Gorak.).

Postdoctoral Fellow ;

J.E. Rowe, B.Sc. {Adel.), Ph.D. (Alta.).

Research Officer :

r1.D . Fenn, B.Sc.(Q'ld.), M.Sc., Ph.d.

Head Technician :

D.i\. Maguire.

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RESEARCH WORK

Introduction

As the name suggests9 members of the Medical c,,emistry Group undertake research in chemistry relevant to medicine. However9 the degree of iuch relevance varies widely. Thus the spectroscopic studies of fine structure in carboxylate salts (Dr Spinner)~ the wide-ranging project on the synthesis and reactivity of substituted nitrogenous heterocycles towards nuc1eophiles (Dr Barlin)9 and the exploration of Dimroth rearrangement in depth (Dr Brown). all involve the accumulation and correlation of important fundamental data which will be applied~ probably by others 9 in the design and preparation of useful drugs.

More immediately relevant to biology or medicine9 though no less fundamental 9 are the studies of metal binding by cell constituents (Dr Perrin), syntheses of tetrodotoxin and saxitoxin analogues (Dr Armarego), and the f-8-alkylation of purines in connexion with chemical carcinogenesis (Dr Lister).

Finally9 close relevance to medicine is evident in the use of chelating agents for influenza prophylaxis (Dr Perrin w i t h Dr J . S . 0 x f o r d 9 ;H MR 9 M i 1 l H i 1 1 ) a n d i n t he u s e o f phleomycin plus purine amplifiers against urinary tract infections (Dr Brown with Dr G.W. Grigg) CSIRO, Sydney}9 both of which projects have reached in viva testing ; also in the dissolution of kidney stones by buffered chelating agents (Dr Perrin with Dr G.J. Heap, Canberra)j a project already in clinical trial.

Biolo ical as ects of metal-corn lex formation Agarwal, Perrin

In view of the importance of histidyl residues in metal-binding by proteins~ stability constants of cobalt(!!) and zinc complexes with the histidyl peptides (glycyl­histidine. glycylhistidylglycine . histidylglycine~ and carnosine) have been determined at 370. The two metals form similar complexes , except that zinc has a strong tendency to form binuclear species with glycylhistidine. Three new peptides {glycylglycylhistidine. glycylglycyl ­histidylglycine . and glycylglycylhistidylglycylglycine) have been synthesised.

Metal corn lexes as electron microsco ic "stains" Allen " Perrin

Further 11 robust 11 metal complexes bearing selectively reactive groups have been prp~ared for use in electron microscopy. These include ferrocene derivatives. for making visible the reaction sites of oxidation - reduction enzyme systems 9 and some inert platinum complexes based on a number of biologically active dyes. The latter complexes are being examined as staini ng agents for sites with acidic or basic character and also as labels for sites with hydrolytic enzyme activity.

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Dissolution of insoluble calcium salts by chelating agents (Andres~ Perrin)

16/1975

The pH-dependence for dissolution of hydroxyapatite by citrate buffer differs significa~tly from the pH-profile for ethylenediamine-tetraacetic acid (EDTA). To provide infor­mation on factors governing the rate of removal of urinary calculi. the effect of pP on the dissolution of calcium oxalate was also studied.

Oxidation of flavine co-enzyme in aqueous media (Andrews 1 , Fenn)

The 1H n.m.r. spectrum of f1avine co-enzyme in alkaline deuterium oxide changes proqressively after an initial induction period dependent on temperature and concentratior. Although the exact nature of tile change is yet unknown, it appears to involve oxidation by dissolved oxygen and to require the presence of both the neut r al and anionic species of the co-enzyme as a catalyst.

Synthesis of chiral glycines (Armarego, Milloy, Pendergast)

Specifically labelled chiral 2- 2H1-glycines, prepared enzymitally using serine hydroxymethyl transferase, were shown by 1H n.m.r. spectroscopy to contain only 60% of one enantiomer. This new n.m.r. method for estimating oDtical ourity has made the task of seeki~g a chemical synthesis06f (R) ana (S) 2- 2H1- and 2- 3H1-glycines worthwhile. Starting from 0-benzylserines a synthesis of olycine has been accom~lished in which ea~h step occurred with kno~n absolute stereospecificity. This synthesis is no~ bein0 adapted for the preparation of the pure chiral isomers for use in our biological systems.

Analogues of tetrodotoxin (Armarego~ qeece)

Syntheses of ?-amin0rerhydroquinazoline analoques of tetrodoxtoxin9 with and without the Ba-carboxymethyl substituent present in the natural toxin9 have been completed. The preparation of such substances was facilitated by the discovery that 2-oxoperhydroquinazolines may be directly converted intc their 2-amino~analogues. Toxicity tests showed that these compounds affected the respiration of animals in a manner similar to tetrodotoxin, but much more of each was required to cause death. The effect of these analogues on blood pressure are being investigated.

1 A member of the Department of Physical Biochemistry. JCSMR.

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Reduced purines related to saxitoxin (Armarego, Reece)

16/1975

Saxitoxin fror. poisonous shell-fish has properties very similar to those of tetrodotoxin although it has a cis-2,8-diaminoperhydropurine nucleus. Several related structure and analogues of this nucleus were prepared for biological evaluation. During this work, unusual and facile c4-N 9 bond cleavage in alkylated 2,8~diamino-and 2,8-dioxo-hydropurines has been observed.

S ntheses and reactivities of substituted 1-, 3-9 and 7-mcthylpurines Badger. Barlin

Syntheses of 2-, 6-, an d B-methylsulphonyl-7-methylpurine have permitted the kinetics of their reactions with hydroxide ions to be examined. At 20°~ the 7-methylpurines were more reactive than their known 9-methyl isomers but 2-methylsulphonyl-7-methylpurine was exceptional. being a little less reactive than its 9-methyl isomer. The relative rates for 2-, 6-, and 8-methylsulphonyl-7-methylpurine were 1 : 840:29,000 compared to 1 : 110 :830 for their 9-methyl isomers.

The preparations of 2-, 6-, and 8-methylthio-1~ methylpurine and of 6- and 8-methylthio-3-methylpurine have been completed with a view to measurement of their reactivities and those of their derived methylsulphonyl analogues, with nucleophiles. Syntheses of the monochloro-l(and 3)-methylpurines are in hand.

Pyridazines (Barlin)

Studies in the chemistry of pyridazines were continued by preparing derivatives of 3,495- and 3,4,6-trimercaptopyridazine but isolation and purification of the free mercapto compounds are incomplete. The products from methylation of aminopyridazines are being further investigated.

Reactivitv of nitro en heteroc clic methosalts Barlin, Benbow

Studies on the preparation and reactivity of the derivatives of heterocycles with a quaternized ring-nitroge n have been extended to the quinoline 9 isoquinoline, pyridazine~ pyrimidine, and pyrazine series ; reagents included hydroxide ions in water, piperidine in ~ater, and piperidine in ethanol.

N-Methylation of iodoquinolines and iodoisoquinolines increased their reactivity towards hydroxide io .. s at 115° by 1.3-3.4 x 107-fold for the a-iodo-compounds and by

6.1 x io 5-fold for the y-iodoquinoline. In the reactions

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of haloaeno-N-methylpyridinium and halogeno-N-methyl­quinol inium salts towards half-neutralized piperidine in water, smaller differences in reactivity between a- and y-isomers were observed than with hydroxide ions: energies of a~tivation and frequency factors for the reactions of the halogenopyridinium salts with piperidine were lower than for the reactions with hydroxide ions.

The reactions between 2- and 4-methylthiopyrimidine methi dides and hydroxide io~s in waters ethanolic piperidine~ or ethanolic morpholine, each occurred in two distinct stages~ possibly involving a Meisenheimer type intermediate.

Alkylation of purines at the 8-position (Bhushan , Lister)

Current theories sunaest that chemical carcinogens act by C-8-alkylation of the purine bases in ONA. To define conditions which may favour a·kylation at this unusual site, studies using simple alkylating agents on naturally-occurring purines nave been undertaken. To date , theophylline has been shown to undergo, not only an expected N- 7-benzylation; but also C- 8-benzylation under remarkably mild conditions. -

Purine amplifiers of phleomycin (Brown, Grigg 1• Lister, Yoneda)

The synthesis of new purines for testing in vitro as amplifiers of phleomycin against E.coli ~has been directed towards analogues of the (previously reported) active carbamoylmethylthio- and dimethylamino-purines: several N-alkyl(and aryl)carbamoylmethylthiopurines proved disappointing; in contrast. 6,9-dimethyl-2-piperidino­pyrimidine was quite active alt~ough the closely related morpholino-analogue showed fittle activity. About 80 purines have now been tested with the kind co-operation of the Animal Genetics Division. CSIRO, Epping, NSW.

Dimroth rearran ement of 3,4-dih dro-4-imino­~:3-polymet 1ylenepyrimidines Brown~ Ienaga

5-Cyano - 3,4-dihydro-4-irnino-2 : 3-hexamethylenepyrim-idine, prepared by a new general synthesis from a cyclic iminoether and aminomethylenemalononitrile, underwent a unique type of Dirnroth rearrangement. The product was a 4-amino-5-cyanopyrimidine , B- bridged by a hexamethylene chain attached to the amino ~roup at one end and the 2-position at the other. Ho~ologous tetra- or penta­methyleneimines did not rearrange because their poly­methylene chains were too short to form the required bridge but the heptamethylene homologue could not be isolated on account of its extremely facile rearrangement into a bridged isomer.

1 Not a member of this University.

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3-Aminopyrimido[S,4-e]-as-triazin-5-ones (Brown, Lynn)

With a view to anti-leukaemia activity, two synthetic routes to 3-aminopyrimidotriazin-5-oncs have been developed. The first was selective hydrolysis of di- or tri-alkoxypyrimidotriazines followed by aminolysis of the remai ning 3- alkoxy group, the other was the addition of a pyrimidine ring to an existing 1,2 ?4-triazine, e.g. by converting methyl 6-amino-3-ethoxytriazine-5-carboxylate into the correspondina amide and thence with triethyl orthoformate~acetic anhydride into 3-ethoxypyrimido­triazin-5-one, which gave the required 3-amino-analogue on aminolysis.

Metabolism of purines (Brown, Stephanson)

With a view to the treatment of urinary tract infections with phleomycin + a purine amplifier , the attainment of a high level of the unchanged amplifier in urine by oral administration is desirabl e . A troublesome 8-hydroxylation of the amplifier) 2- carbamoyl ­methylthio-6~9 -d imethylpurine ? in mice or rats has now been overcome by addition of a~ in ert 8 methyl group during the initial synthesis. A dose of the resulting 2-carbamoylmethylthio- 6"8,9- trimethylpurine was recovered unchanged (apart from a small percentage converted into the correspondinq sulphoxide) from the urine during the first 12 hcurs after administration.

S·nthesis and rcactivit of 2~ and 4-halogenomethylpyriw.idines Brow ; Waring)

The previously unknown simple derivatives, 2(and 4)-bromomethyl-~ dibromom e thyl -, and tribromo­methyl-pyrimidine) as well as their chloro-analcgues , were made by direct halogenation of 2- or 4- methyl­pyrimidine with the appropriat e N-halogenosuccinimide. The monohalogenomethyl derivativis underwe nt pipcridino­lysi s and alkaline hydrolysis very much more rapidly than the correspondin g 2- or 4- halogenopyrimidines.

Ionization of tubocurarine (Fenn9 Johnston 1)

1H N.rn.r. spectra have been used to measure the three ionization constants of the alkaloid, tubocurarine , and to assign each constant to a particular grouping in the molecule.

1 A member of the Department of Pharmacology, JCS 'l R.

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Ac l derivativ2s of 4~5-diamino rimidines Fenn, Lister

Acylated diaminopyrimidines are essential inter~ mediates in many syntheses of purines. The structures and conformations of a variety of mono-, di-. and tri­acylated derivatives of 4~5-diamino-6-~ethyl-2-methyl­thiopurine have been elucidated by usinq lu n.m . r. spectra as the main to ·il. Among the results were the first clear demonstration of cis- and trans-formamido­pyrimidines9 an indication of--rhe different ratios of such isomers in sevcra solvents. and unequivocal evidence against literature claims that th~se ac 1 1 derivatives really existed as the isomeric dihydroourines.

Chemolithiasis (Heap 1 ~ Perrin)

Most kidney stones contain calcium oxalate or calcium phosphate so that irrigation with appropriate metal-cheluting agents mi9ht be expected to lead to their dissolution. Clinical trials have been i~ progress using buffered solutions of EOTA and sodium citrate to establish the suitability of the treatment for the non-surgical removal of stones lodged in the ureter.

Chelating agents in influenza prophylaxis (Oxford 1 , Perrin)

Solutions of calcium disodium ethylenediaminetetra­acetate (CaNa 2EOTA) and calcium trisodium diethylcne-triaminepenta-acetate (CaNa 3DTPA) inhibit activity of the RNA polymerase of influenza virus in vitro by chelcting free zinc ions. Attempts to applythis observation to prevent replication of influenza virus in viva were limited by the difficulty of mai taining-adequate concentrations of the chelating aqents in the body to deplete sufficiently the zinc ion level in those cells invaded by the virus. To overcome this problem, experiments have been in progress using liposome-e~cap­sulated chelating agents to stimulate passage of Ca Na 2 E 0 TA an d re 1 a t e d c o r!I po u n d s t h r o u g h t h e c e l l w a l 1 s by pinacytosis and to encourage retention in the body by phagocytosis.

Metal-binding by bovine serum albumin (Perrin 1 Ryall)

_U~ing an improved competitive dialysis technique, condi~ion~l con~tants ~ave been determined for copper(II) and z1nc ions with bovine scrum albumin in the presence of glycine and histidine to serve as metal-ion buffers.

1 Not ~ member of this University.

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Albumin formed mono- and bis-complexes with copper as well as mixed complexes of the type al»umin-copper-aminoacid. In contrast9 zinc (studied as its radioisotope~ 65 zn) formed only 1:1 and 2:1 zinc-albumin complexes. A theoretical treatment of the technique has been formulated.

Vibration s ectra of carboxy1ate salts

Previous spectral studies of bond structure in carboxylate ions usin0 deuteration are being extended by 180-substitution, initially in the formates of sodium and potassium. Specimens of high isotopic purity have been prepared for the first time so that the resulting spectra would be more meaningful than those obtained by others.

Studies of carboxylate salts (Spinner)

Results obtained with the bisformates of sodium and potassium necessitated work on potassium formate. When pressed into potassium bromide discs under mild pressure. potassium formate was transformed into a second modification. This became evident in shifts of frequencies to unusual values and in the sharpening of a normally broad intense infrared band, indicating a large change in intermolecular environment.

Substituent effects i l acid strengths (Spinner)

The large acid-weakeninp effect of fluorine in a-fluoronitromethanes9 and the acid-strengthening effect of a-methyl groups in nitroalkanes, which are relevant to nitro~-aci-nitro tautomcrism9 have not been explained satisfactorily. New calculations of sur.h substituent effects, based on intramolecular London~van der Waals attractions, explained the Gbovc acid-weakenin~ by halogen but not yet the acid-strengthening by methyl groups.

TEACHING AHD OTHER ACTIVITIES

Of the nine students proceeding to the degree of Doctor of Philosophys two (K.Bhushan and P.Waring) commenced, two (P.A.Reece and J.A.Benbow) held Commonwealth Postgraduate Awards, three (R.G.Ryall, K. Ienaga~ J.A.Benbow) completed theses~ and one (R.K.Lynn) was awarded the degree; P.Waring was awarded the M.Sc. degree. Two theses were examined for other Universities.

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Fortnightly seminars led by visitors or members of the Group were organized by Dr E.Spinner. Most members of the Group attended and delivered papers at an appropriate i n t e r s tat e Di v i s i on a 1 r1 e et i n ~l of the Roy a 1 Au s t r a 1 i an Chemical Institute. Dr W.L.F.Armarego delivereri an invited lecture to the University of Queensland Chemical Society, and Mr O.E. Allen spoke at the Bth International Electro~ Microscopy Conference held in Canberra.

Dr L.G.Stephanson's Canad i an Medical Research Council Fellowship has been extended for a third year to enable studies on the metabolism of pur ines to be completed.

The Group was privileaed to host two distinguishe~ visitors durin~ the year. Professor Fumio Yoneda from Kumamoto University spent three months here as the 1974 Leverhulme Fellow, during that time he joined in current work on the phleomycin project and was available for lectures and nume ro us ccnsultatio ;s. Later in the yea r , Professor H.C.van der P1as from the Landbouwho9eschool, Wageningen, who is the acknowledged authority on heterocyclic rinc transformations. was here for a shorter period devoted to lecturess consultations, and examinin~.

No ve l organic compounds prepared by Gro up memb0rs were supplied for collaborative projects with colleaguess workin~ mainly i n biclooical or medicll fields: Dr G.11. Grigg~ CSIROj Division of A~inal Genet ics (23 purines); Professor W. Pfleiderer, Fachbereich Che~ie 9 Universitat Konstanz {5 pteridines)s Or rLG. Harris 11 Uni versity of British Columbia (pyrimidi~es); Dr H.B. Wood, ~ ru q Development Brancll. NIH~ Bethesda (7 heterocycles); Professor ... Sunner, Chemical Cente r , Un iv ersity of Lund (a heterocycle); Dr ~J . S . Oxford9 NIMR. Hill Hill (supplies of thiosemicarbazid~s); and Dr G.J . Heap> Turner ~ Canberra (chelatinq a~ents) .

Services: The number of 1H n.m.r. spectra measured by Or f·l.D . Fenn and 1fr S.E. Br own increased by 40% this year following the commissioning of a new T-60A instrument in June. Of the 29500 spectra, nea rly 20% !ere for members of the School outside this Group. In addition, de.terminations of ionization constants (Mr I. Hawkins for Gr D.D. Perrin} arid of u.v. and i.r. soectr:-: (Mr D.T. Light for Dr E. Spinner) were carried out as routine services.

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PUBLICATIONS

ALLEN~ D.E. and PERRIN, D.D.

'Robust metal chelates? ferrocenylmethyl carboxy­hydrazine and 1-chloromercuriferrocene, as electron opaque stains for aldehydes and thiol groups.' The Journal of Histochemistry and Cytochemistry ~j 919-928 {1974).

ANGYAL, Ah~ette M. 1J GRIGG. G.W. 1 , BADGER~ R.J.,

BROWN, O. J. and LISTER, J.H.

'Purines as amplifiers of the antibiotic activity of phleomycin against E.coli B.' The Journal of general Microbiology 85, - (1974).

ARMAREGO, W.L.F. and MILLOY, Beverly A.

'A simple method for enhancing the optical rotation of a-amino acids.' Journal of the chem·ical Society~ Perkin Transactions I 1905-1907 {1974).

ARMAREGO, W.L.F. and REECE, P.A.

'Quinazolines. Part XX. Synthesis and stereochemistry of N-methyl-cis-perhydroquinazolin-2-ones and N-methyl-cis-perhydroquinazolines. A new conformation for cis-perhydroquinazolines.' Journal of the chemual Society, Perkin Transactionsd 2313-.;:319 (1974).

BADGER, R.J.? BROWN, D.J. and LISTER, J.H.

'Purine Studies. Part X. Further synthetic approaches to purines for the amplification of phleomycin activity against E.coli.' Journal of the chemical Society, Perkin Transactions I 152-158 (1974).

BARLIN, G.B.

'Nucleophilic Substitution' Chap.8 in Specialist Periodical Reports~ Aromatic and heteroaromatic Chemistry~ vol.2 (ed. C.lL Bird and G.W.H. Cheeseman) The Ch~mical Society, Lendor (1974) pp.271-296.

'Tautomerism of N-heterocyc1es. Part II. 3-HydroxypyridazTn-6-one und 3-mercaptopyridazine-6-thi one.' Journal of the chemi ea 1 Society, Perkin Transactions II 1199-1202 {1974).

BARLIN~ G.B. and BENBOW~ J.A.

'Kinetics of reactions in heterocycles. Part X. Reactions of substituted N-mP.thylpyridinium salts with hydroxide ions. 1 Journal of the chemical Societ_ s

Perkin Transactions II 790-797 1974 .

1 Not a member of this University.

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BROWN, D.J. and IENAGi'\, l~.

'The Dimroth rearrangement. PJrt XVI. A new general synthesis and rearrangement of C-alkylated 1~6-dihydro-6-imino-1-methylpyrimi~ines. 1 J~urnal of the chemical Society, P0 rkin Transactic~s I 372-378 (1974).

B R 0 W N , D • J . an d LY N i~ ~ R • K •

'Purine methan to give Journal 349-252

studies. Part XI. Condensation of tetrJethoxy­and similar ort~oc~rbonates with ortho-diamincs 8-ethoxyourines anc related fused imidazoles. 1

of the chemical ~cciety, P~rkin Tr~nsactions I (1974j.

'Aza-analogues of pteridinc. VIII. 3-I'lmino·· from ~-alkoxy-pyrimido[5,4~eJ-es-triazin-5-ones prepar2d uy cyclization of 3-ethoxy-l)t'.s·~-triazincs er other means.' Australian Jourr.al of Chemistry 27, 1781-1790 (1974}.

s~owNj D.J. and STEPHANSO~, L.G.

'Purine studies. XIII. The m~tabolism of 2~9-dimethyl-8-methylthiopurine and 2-car~~moylmethylthio-6,9-dimethylpurine in mice. 1 t\llstral inn Journal of Chemistry ?2~ 1371-1375 (1974).

BROWN, D.J. and WARING P.

'Pyrimidine r~actions. Part XXV. Synthesis and piperidinolysis of some simple fluGropyrir.iidiries. 1

Journal of the ch~mical Society9 Perkin Transactions II 204-208 { 197 11).

'Simple pyrimidines. XV. The synthesis, piperidinolysis, and hydrolysis of simple 2- a .d 4-(halogenomethyl) pyrimidines.• Australian ~ournal of Chemistry 27. 2251-2259 (1974).

FENNs M.D. and LISTER9 J.H.

'Purine studies. Part XII. Derivatives resulting from acylation and subsequent ~thylation of ~95-diamino-6~ r11ethyl-2-methy1thiopyrinidine. 1 Journ2'1 of the chemical Society Perkin Trnns~ctions I 1300-1303 (1974).

OXFORD, J.s. 1 and PERRIN~ D.O.

'Inhibition of the particle associated RNA dependent polymerase activity of influenza viruses by chelating agents. 1 The Journal of general Virology ~~ 59-71 (197~).

1 Not a member of this University.

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PERRIN, D.D. and DEMPSEY, e. 1

'Buffers for pH and metal ion control. 1

Chapman and Hall, London (1974) 1 i-viii, pp.1-176.

SPINNER , E.

'Strongly hydrogen-bonded molecular solids with 11 h i g :11 y a no ma 1 o u s n i n f r a red 0 H a b s o r p t i on . T he effect of 0-deuteration. • Australian-Journal of Chemistry 27 ~ 11 49-1160 (197 1 ).

WEST, C.E. 2 • PERRIN, 0.0., SHAW, o.c. 1 , HEAP, G.J. 1 and

SOE NAtHO l

'Djenkol bean poisoning (Djenkolism) : proposals for treatment and prevention. 1 South East Asian Journ~l of tropical Me dicine and public Health 1, 56t;-570 (1973).

1 Not a member of this University.

2 A member of the Department of Experimental Pathology ) J CSMR.

3 A member of the Department of Physical Biochemistry, JC SMR .

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