74G
Bottromycin. Separation of Biologically Active Compounds aiicl Preparation and Testing of Amide Derivatives
Five biologicdly :tctive cumporitid~were epurat ed f r o m the hot i i,oriiyc,iii cwniplex. .1 iirirn1)er of derivxi iwi IVheii iest cd ngnitiat S/aph!/locorci(s nureus the simple amides are all more active in vitio ntid less ariive in tii/ro th:iii t h e parent ester. Cot~rel:itioririf K , value arid bioactivity appears to give broad peaks which are different for. in t i i ~ o:itid i'n ri/ro tesii;. Qrvera1 of the compoiirids are acstive when tested against . l l ~ / c o p l n s v ~ r . of the termind carhosyl grolip of hotti~omyc~iti were prepared and teif ed.
Early workers with bottromycin1$2 reported that the antibiotic contained a. unique dipeptide. @-methylphenylalanyl-p-(2-thiazolyl)-&alanine methyl ester (I). llild hydrolysis of the antibiotic removed the terminal methyl group and produced the corresponding acid which had low antibiotic activity. .\lore recently3 the :intibiotic was shown to consiht of two substances. hottromycin AI which contains cis-3-methyl-l-proliiic : i t i d hottromycin R . which contain- /-proline.
of A, I), :iitd E and 1:irger cluantitie- of 13 :ind ('. T I i ( ~ properties of theye compounds :ire listed in l':iblc~ 1. *\Iost of the bioactivity is due to component C. 'l'liiince yields pivalic :tcid (identified by gas c1irom:itography) arid methylprolinc. (identified by paper itrip chromatography arid 1Ioore- Stein amino acid sri:tl!-i+) :uid i i apparently identical with hottrom>cin -Il. Component €3 yields proline :md is :~ppnrentl).idciitica:iI \r-ith l)ottroni\.ciii T3. Thc rcm:iiriing conipoiiiitli nliiclt
I I
I
I1
I
I
(1) J. 11. \\.'aisvisz, R I . G. v a n der Iiorven. J. van Pegpen, a n d \V. C . 11. Zwennis, J . 4 m . Chem. Soc., 79, 4520 (19.57). (2) .I. M . Waisvisz, A I . G. van der Horven, a n d B. Te Nijenhuis, i b i d . , 79, 1524 (1957). ( 3 ) S. N a k a m u r a , T . Chikaike, Iiwti) preparing compounds resistant t(J citera i n v(+ tigated. Amide\ were -ynthesized by trmtiiig tliv met 11) 1 eiter in inethanol or dimethylformamide solutioii with the desired amine. The reaction was followed re:dil> :ind conveniently ti) paper chromatogr:iphy of the reaction mixture. The disappearance of the p:irc~il compourid arid concurrent formation of :L WIT hioactivt' zone was observed. d t 50-GOo, the reaction is complete in about 24 hr u4ng primary amines. SOappreciable reaction mas obtained with secondary amines. 1r tpectroscopy indicated loss of the ester band at 1260 cni-' but no other major change was observed. Elemental anal3 sib, obtained on x few compounds, \%-as yatisfactory. Although the ester was not crystalline, heveral aniidez were obtained crybtalline. The properties of these compounds :Ire listed in Table TI. Tertiary arriidea 'it ere obtained by reactiou of secotid:try amine? with the intermediate 2,3-dinitrophenyl ester. Thii v-tcr WLS prepared from t h r free :icid h j
BOTTROMTCIN
,July 1908
747
TABLE I SEPAR;\TION OF BOTTROMYCINS Bio-
Component
R,a
tvt,
MIC,
g
pg/mlb
activity To of total
A B (bottromycin B) C (bottromycin A1)
4.0 0,130 1.0 0.10 1.6 0.800 0.04 12.4 1.0 1.410 0.01 87.0 D 0.89 0.016 0.04 0.25 E 0.82 0.016 0.04 0.25 R, = log [( 1 - Rf),/Rf]. Ratio of peak fraction number for hottromycin .41 aiid other compound. JIinimum inhibitory conceritration (LIIC), an in z~itrotest performed u-ing Sfaphyloc o c m s a w e m and a 6-hr illcubation.
-04
00
04
08 Rrn
12
16
20
Figure l.-Plotb of the logarit,hm of the number of effective TIRLE I1 doses per unit weight us. R, for a series of compounds: 0, SUMMARY OF PROPERTIES A N D TESTRESULTSOF BOTTROMYCINS esters in vitro; 0 , esters in vivo; A, amides in vitro; A; amides in vivo (these four are all bottromycin A1 derivatives); 0, A N D AMIDEDERIVATIVES OF BOTTROMYCIN A, bottromycin B methylamide in vitro; . , bottromycin B methylRatio In uitroa I n viaob amide in vivo. MIC, E D ~ Q , zn vivo: Component
-4 BC Cd
n
E B methylamide C ethyl ester
Rf
0.5 0.28 0.19 0.13 0.11 0.60 0.09 0.01 0.62 0.52 0.33 0.13 0.15 0.06 0.05 0.02
pg/ml
1.0 0.04 0.01 0.04 0.04 0.20 0.03 0.04 0.10 0.05 0.05 0.5 1.0 0.25 0.2 e
pgldose
>500 200 50 100 100 50 100 e 25 10 15 10 10 10 e e
zn uatio
5000 5000 2500 2500 250 5000
C benzyl ester 250 C amidef 200 C methylamide 300 C ethylamide 20 C n-propylamide 10 C isopropylamide 40 C t-butylamide C cyclohexylamide C 0-phenethylamide C a-naphthylmethyl0.02 amide e e 0.02 C benzylamide 36 0.5 18 C 2,3-propanediol-125 0.79 amide 1.0 25 0.69 C 2-ethanolamide 36 0.5 18 a Standard test condition using Staphylococcus aweus test organisms, 6-hr incubation. b Test conditions, intraperitoneal infection, intraperitoneal treatment, two doses 0 and 6 hr postinfection. Bottromycin B. d Bottromycin A,. e Not tested. f All compounds are terminal amides of the type RCONHR.
reaction with 2,4-dinitrophenyl carbonate in the presence of trieth~lamine.~The product amides8 were isolated by preparative tlc and the purity was demonstrated by tlc. The properties of compounds prepared by this method are lkted in Table 111. The structure of the amides was verified in many cases by mass spectrometry. At moderate temperatures a characteristic peak arises from the C-terminal dipeptide and corresponds to the structure
Since the R group is in t'his case the variable position, the presence of this fragment' serves as a confirmation of ~ t r u c t u r e . ~ ( 7 ) R. Glatthard a n d 11. >\latter, Hela. Chim. Acta, 40, 795 (1963). (8) We are indebted t o hIr. K. W.Kelly for technical assistance. (9) G . Albers-Schonberg (submitted for publication) has further data on t h e mass spectrum of bottromycin AI.
Ten of the compounds were analyzed by mass spectrometry. lo The K-methylhydroxamate and the hydrazide did not give the indicated parent peak, presumably because of the low volatility (and stability?) of the resulting dipeptide fragment. TABLE I11 PROPERTIES OF ADDITIOXAL CARBOXYL DERIV-~TIVES OF BOTTROMYCIN Ai Compd (RCO-)
NHC(CH3)a h"CH(CH3)z "(a-naphthyl) NHCHsCsHj NHCsH6 NHCeHaF-p
Rr tlc"
In aiuo act. EDao. pg/dose
1.0 0.82 1.1 0.88 0.65 1 . 7S
> 100
0.95
...
100 ... 60
> 100 ... ...
82 95
...
NHNHz 0.25 46* NHiT(CH3)z 0.48 35 N(OH)CH3 0.24 28 a Due to some variation in absolute movement the Rr relative to bottromycin Ai is listed. Tlc was carried out using silica gel G plates and developed with XleOH-CHC13 (6:94). The Ri of bottromycin Ai is usually about 0.45. The zones were locat,ed by spraying wit,h 0.01 % bromophenol blue or by placing the dried plate i n an iodine vapor chamber. * N. Fanaka, T. Xishimura, S. Nakamura, and H. Umezxwa, J . dntibiotfcs (Tokyo), 19, 149 (1966).
Biological Tests.-The new compounds were tested for inhibition of multiplication of Staphylococcus a w e u s both in vitro and in z h o . The in vitro tests were carried out using a rapid tube dilution test." Mouse in vivo (10) TTe are indebted t o G. Albers-Schonherg, J. Beck, and K.R. Trenner for running and interpreting t h e mass spectra. (11) W e are indebted t o E. 0. Stapley a n d Rose > f . Miller for in vitro tests.
Experimental Section Crude Bottromycin: --The ireaklj- I):i.4v, .~i~lveni-sr~Iiiblc l)oitroniyr.iti was prepared by solvenr exi ract,ioii of fernieiit at i o r i broths at pII 8.5 with CHCI,. The CHCI, extracts were c ~ ~ n c r i i (rated and percolated through a column of Florosil using :t 30: 1 ratio uf atlsorberrt to solvent-soluble solitis. The cwlumn w:i* t t i e r i elriietl w i t h lle2('O-('JTC'l~ 11 : I ). T h e active cants \vert' c~)nil)inetl :ind CI)IIII:II~ i~ntetli o clryiies.. The hydrocliloi~ided i c i f the : i n t i l ~ i o t i vn i i ~ r u i ~ w:ic c ~ pwpiiiwl liy :tdclitioii t J f inrthariolic~ i l x~luiioii of t11v c:xtr:ic*t. .Iliei~n:itc~ly,t11tl mi \)I! :itl.;r)rhed froni widified I,rotli on .iO-S2 :tiid eluted with ~1 niistiire of 7OI'i 1IeOlI :ind :10 STIrOII. .iftei reriroval uf the lleOII, the :ictivity \v:w (as illto CII('Id foIliJwet1 b y chruniatugraph~ 111:ij- be followd l)y :intibacterial d' assa>- a n t l pnpc'r chroniat o g r a p l i ~ . Paper Strip Chromatography.----The paper strip systein iis:etl is reversed pliase. \Vhat.mari ]io. 1 paper was spotted with I-: pg of aniihiotii* aiid clipped in a 20''; iiiethanolic cnpryl iilfOh,Jl sjlutioii i i p t i l I lie oi,igiii, IlllJtted o i i :il)sorhent p:iper, :iir drietl : i l i o i i t , .i inin, : i i d tkYd(Jpet1 wit11 0.01 .I[, p1I 6.0, phosphatp 1)uf'l'er. 'The -1 rip' itrt: iwiioved w l i t ~ r ithe SlJlVent front renc~hes tlie r n t i Of I hc *trip, tlrird ill :I v:icuiini oven t o reniove the c:ipryl : i l i w l i o l , :ind i3io:~iiti~gr:i~111ttd o n agar .wtled witli Ilnciil71,s s i h ~ Separation of Components.-To .i..ikg of acid-washed Celitc. .i4.i (Johiis 1I:iriville) in z: Pattersoii-Kelly doiible-cone blentler war nddeti 0 I. of :t 2057, mprj-1 :dcohol solution i n l l e 2 C 0 , After ihoroiigh inixiiig, the Celite w t s dried in l i m ( o i o ~ e n i o v c ~ the LKY'ttJTlO. T I i c ~cnpryl alcohol impregnated Celite w:is thrr) i r l :L i * o I i i i i 1 i i (15 x I20 cm) iirider slight v : i r i i i i r n i r i with 1:inipiiig. T h e coliirriii was rharged w i t h 2.8 g Ol' ct~iicleIii~ttri~i~~ycit: i i i IS00 n i l of u 0.10 '11, pTi (j.0, pliospl~at(. I N X ~ I ~ . 'The (~II~ILIIIII LWS dcvclopetl w i t h pII 6.O hiiITer. Fr:lrttioils of .iOO n i l w.ei'(~c~ollected. 'The reyiilts :ire s h o w n i n Tril~le1. Component C (Bottromycin A~).--Frsc+ioiis 190-215 frc)m t h c l :il)ove part it iori criliinin w e r ~( ~ ~ n i l ) i n eand d concentrated fiveflJld. Tlic, cToriceritr:ite TKH adjiisted t c i pTI 8.3 arid cast racfed siiccessively with thrcc e q i d volumes of CHCI,. The extracts were evaporated tlo dryness zn mcuo : ~ n dthe residue was dissolved in Et.0 (300 1111i. AIethaiicrlic IICI was added t o precipitate the component (1 liy(lroi~Ii11~ride (1.40 g). &tnal. Cdcd for C,,H,?. 2 f 1 2 0 : (?, 57.6; 11. 7.G: X, 12.70; S,3 . 5 7 ; Cl. 4.02. o t , t i i l ( b
Results and Discussion .I11 coinpound' were active i l l the tert systems employed. When comp:ircd by 211 vzt/o tests the amide derivatives were all l e ~ sactive t1i:in tlie esters. Honever, by in vino tests all :Lniiciei: obt:iined from primary aniirics were more activr thaii tlic c4terh. Although +,tiihc:uitl\ (lift c l ~ ~ i i 5pccific t 217 vitw wtivity i- o 1 ) t : u i i c d TI ith thr iiidividual compounds, inclutling ester.. only \light differences in the in z i v o tcyt. were obherwd. :I group, the :~niidesare about ten times as active 211 v i r o :IS the eqters while qpecihc activity difference. ljy the I ) / r ~ t i ot w t s rniige froni 11: to l'loo a 5 acti\c a. the So *at iifactory esp1an:t r t l w e rehultr C:LII tw ibly wnie controlliiig f:ictor which i. not influenced by tlifferciice, iii polurity at the c:irbox> I termhi1 of thv mole cult^ h i \\hic.h 1 1 1 : ~ ~rcflcct :it1 inherent tliffcwiice hctv w t i cbtor :ind :imide groups ovcrridei re1:ttiwIy niitior t1iffc.rciicc.i twtxvccii the amides themqelves. The gencr:tl hypothesis',l th:it activity relatioriships of :L series of compounds c:m be corre1:ited with the logirithrn of tlw partition r:itio of the individual compound:, c : ~ n he tcsted u h g thc potcncy :ind the (log [(l - Rf)/Rf])in the paper chromatogtern for esters and :Linidcs of primary :tmiiics. each srriei of coinpouuds and with each type of test the reiulti :ire in gciicr:il agreement with the :hove hypothesii. For each icxrici I: broad maxima of :ictivity is oht:iined by plotting the logarithm of the iiurnber of ef'fective daws pvr unit weight 1's. T l i c w d;itn :ire showi in Figure 1. -1lthough t h r U L r w o :uid 771 vitio potcriciei : i w m:isinium in the saiiie f?,,, rcgiori for the wterb, cxmiplc5 are limited. It appe:irs that different m:axini:t for the two tests are ohtaiiied with the :tniidcs, although the in vzuo tests are all nitliin :t f:ictor of -1. \\'it11 thti biological tests report ed, the prediction of in L ' / L o I'E\POIIV 1):tsed on tlici in uit)o potency does riot :tppe:ir positive. 7 ' 1 1 ~ large difference in the 112 v i z ~ ii, 7 ~v i t ~ oratio between the ester berics and the :miidc hcries c m be due to instability of the. cster W L v / v o or to different tissue di:,tribution. Because the distribution properties \voultl tw affcctcd by the partition ratio and bince ester nriti amitlc tlcrivatives with ncnrlj- the Same partition ruiio ~veretcsted, it Teems likely that the greater in L'IW potcticy is in fact diw I ( J grwter stability of the :trnidr5. ( 1 2 ) 4 I\ Miller "Antimicroliial Agents a n d Chemotherap), 1963 " \merican Society for Alicrobiology, Ann Arbor Mich., 1964, p 33. ( 1 3) B. 11 Miller, C 0 Stttpley, €1 R. Woodruff, 7th Interscience ('onfercnce on Antimicrobial Agents and Chemotherapy, Chicano, Ill., 1967. ( 1 1) These tests were carrled o u t h v Dr. I.. +orensen. Alerrk Institiitp f o r 1 herapeutic Researcli. R a h n a y (15, I3 < € 3 o j r e a n d 13 \ Uillw~lcin \&ire 208 577 (IOh5).
I i y d r ~ ~ c h l ~ ~of r i d('Ic : r i , i d :it :I ~ ~ ~ r i c ~ e n t r : iof ( i o10 n rng/nil and the 1'11 carefully wiii :tdjuit,cd to 5.7 wit,h S a O l I . The frcLe h s e preti !IOf'; yicld; nip 157-170" dec, [a]% -13' ( wt, (by titration) SOS (pITi,~,8.2). :lritrl. H 2 0 : Cy, 59.9: IT, 7.6: pr', 18.:32: S, 1 1 , T . G ; 5 , 1 2 3 ; s, 4.3. Component B (Bottromycin B).---Ciits 110--11:3from t lie parfree base, [,]% -25" wed t,hefollowing :tiialysi,s. c', ,38.2; IT, 7.58; K, IT, 7 . 3 5 ; S , 13.47: S, 3.9s. 'l'lle
I J L - ~ art:iiic
dissolved in :30 nil of toC; IIeOI tube, antl 1ie:ited :it X" f l J Y 20 h
l.i,:Z. Fiiiin(1: C', 61.0; I i ,
lie hydrochloride was prepared by precipitation from ether by the addition IJf rnethanolic IIC1. r i n d . C d c d for C4?TT,&&S. 2 H L O : C', >ti,.-): TI, 7.i: 5 , 14.l.i. Found: C, 56.20: TT, 7.6:
s,14.00.
Bottromycin A, Amide.-13ottromyciii -41 (200 nip) W:IS (lis;;olved i i i 10 nil of l0yoMeOH-NH3 and heated in a sealed tube fur 20 Iir at, *jO". The :imide was isolated by partition chromatography in the rapryl alcohol-pII 6.0 buffer syst'em, yield 120 nig, nip 173-179" dec, [ a l 2 5 ~ -32" (C 1, 9570 EtOH). .4nul. Calcd for c4I&1x90ss'I~&: C, 59.5; H, 7.6; N, 15.2; S, 3.9. Finind: (2, 59.0; IT, 7 . 7 ; E, 15.0; S,4.1. Bottromycin Al Ethy1amide.-Bottromycin AI (0.5 g) was prepsred in the same way as the methylamide; mp 174-180" der, I . [ 2511 - 16" (C 0.1, :I,?(',; RtOH). Anal. Calcd for C4sH~N90aS. ; N, 14.41; R, 3.7. Found: C, 59.20;
Bottromycin A1 benzylamide was prepared similarly using benzylamine crystallized from EtOAc; mp 160-168", [ a ]25D -.56O (c 1, Otji'% EtOII). Anal. Calcd for C48TI67N90&.1120: C, 62.9; H , 7.5; N, 13.7; S, 3.5. Found: C, 83.0, H, 7.6; N, 13.4; S, 3.5. Bottromycin AI Ethyl Ester.-A 10-mg/ml solution of bottromycin -41 free base in anhydrous EtOH containing l5YO E t & was heated a t 50" in a sealed tube for 20 hr. T h e res:ultant mixture of bot,tromycin A1 and the ethyl ester of bottromyciri A1 was separated by partition chromatography. A nul. Calcd for C ~ Z H ~ ~ N ~ O M C, . H 60.4; ~ O ; IT, 7 . 5 ; N, 13.1; R, 3.7. Found: C, 60.1; H , 7.4; N, 12.7; S,3.4. Bottromycin t-Butylamide.-Bottromyrin carboxylate16 (480 mg) was dissolved in 5 ml of anhydrous IIMF and was treated successively a t 0" with 219 mg of bis(2,4-diriitrophenyl) carbonate and 0.14 ml of Eta?;. The solution was stirred a t 0" for 75 min (16) J. A I . Waisvisz and 11. G. van der H o r r r n , J . A m . C h ~ mS. O C . 80, , 383 (1958).
and then a t room temperature for 10 min. T o this mixture was added 2 ml of t-BuNHz. A precipitate formed in a few minut,es. The mixture was heated at 5(1-60° for 30 min, diluted with 20 ml of CHCl3, and filtered to remove the yellow by-product. The filtrate was concentrated to near dryness under reduced pressure. The residue was dissolved in CHCl, and then washed (saturated until the aqueous layer was colorless. NaCl containing 570"3) The organic solution was dried (Na2S04)and concentrated to dryness. A yellow glass (390 mg) was obtained. Tlc ( 1 0 5 MeOH in CHC13, silica gel) gave two zones when sprayed with bromophenol blue. The desired amide was located at Iff 0 . 7 0 , and a slower moving subst,ance IZf 0.13 was observed. These components were separated by preparative tlc yielding 107 mg of the t-butylamide as an off-white glass. This general procedure was used to prepare the derivatives listed in Table 111. In some cases the thin layer chromatograms were developed with XIeOH-CHC13 of different composition to improve resolution of the components. The zones were sometimes developed by staining with iodine vapor or located l)y spraying the dried plate with H20.
S,S-Diarylpenta-2,4-c2ienoicAcid Amides as Potential Antimalarial Agents' \vILLIA1I
T. C O L W E L L , JUDY H.
LrlNGE, .4ND DAVID
\v. HEXRY
Department of Pharmnceictzcal Chantistry, Stanford Research Institute, Menlo Park, Calqornia 94025 Received February 10, 1968
A series of 5,5-diarylpenta-2,4-dienoicacids and their amides have been synthesized and evalriated as antimalarial agents. The acids were prepared from the corresponding diary1 ketones either directly by a Reformatsky procedure or through acetylenic alcohol and acrolein intermediates. The preparation of a series of 3,sbis(4-chloropheny1)acrylic acid amides is also reported. One compound, IVjPi-diethyl-5,5-bis(4-chloropheny1)penta-2,4-dienoic acid amide, provided significant antiplasmodial activity.
Among the more novel compounds revealed by the World War I1 malaria program to have interesting antiplasmodial action was KDj-isopropy1-5-(p-chlorophenyl)penta-2,4-dienoic acid amide (1).2 This compound was four times more active than quinine in the chick Plasmodium gallinaceum assay employed in that work and had a therapeutic index of 12.5. Two other amides, obtained by coupling the same acid with guanidine and cyanoguanidine, were inactive. Because of the development of resistance to chloroquine in many parts of the world by Plasmodium falciparum, there is an increasing need for new antimalarial drugs of novel structural t ~ p e . ~ This J need suggested that examination of additional chemical structures related to 1 for antiplasmodial properties would be of value. I n addition to antimalarial activity, other biological properties have been associated with pentadienoic acid derivatives. The hydrazide of 5-phenylpenta-2,4-dienoic acid has in vitro antituberculous activity,6and a series of the free acids shows inhibitory action against bacteria, yeast, and fungi.' Sorbic acid (2,4-hexadienoic acid) is widely used for its antifungal properties8 (1) This work was supported by t h e U. S. Army Medical Research and Development Command under Contract No. DA-49-193-MO-2150. This contribution is no. 331 from t h e Army Research Program on hlalaria. (2) G. R. Coatney, W. C. Cooper, N. B. E d d y , a n d J. Greenberg, "Survey of Antimalarial Agents," Public Health Service Publication No. 193, Washington, D. C., 1953, p 139. ( 3 ) Reference 2, p 131. (4) World Health Organ. Tech. R e p t . Ser., No. 296, 1 (1965). ( 5 ) P. J. Bartelloni, F. W. Sheely, and W. D. Tigertt, J. Amer. ,]fed. Ass., 199, 141 (1961), a n d references cited therein. (6) S. Kakimoto, I. Sekikawa, and K . Yamamoto, .I. Pharm. Sac. .Japan, 76, 353 (1955); Chem. Abstr., SO, 1663e (1956). (1) K. Takeichi, Hahho K O ~ QZasshi, ~ U 38, 99, 224. 313, 431, 539, 602 (1960); Chem. Abstr.. 66, 2791b (1961); 57, 2669 (1962).
C1 e C H -- - C H C H = C H C O S H - i - F r
2
/8'
C=CHC=CHCON
c1
I
n N-
W
CH:
CH3 3
and piperine (2, the pungent element of pepper) has insecticidal proper tie^.^ The use of derivatives of 5- (5-nitro-2-furyl)penta-2,4-dienoic acid as antibacterial agents has been patented.'O Of more pertinence to parasitic disease chemotherapy, it has been reported that a series of 5,s-diarylpenta2,4-dienoic acid derivatives of piperazine (e.g., 3) possess marked act'ivity against Dicrocoelium dendriticum, a liver fluke of considerable vet'erinary importance."*12 The closely related fluke, Fasciola hepatica, which infests both animals and man, is affected by these (8) Chemicals Used in Food Processing, Publication 1274, National Academy of Science, National Research Council, Washington, D. C . . 1965, P 5. (9) E. K. Haevill, A . Hartsell, and J. 31. Arthur, Contrib. Boyce Thompson Inst., 19, 81 (1943). (10) H. Saikachi and S. Ogawa, Japanese Patent 11,981 (1962); Chem. Abstr., 59, P11426c (1963). (11) M. Schorr, H. Loewe, E. JUrgens, H . Weber, a n d G. Lammler, Arzneim-Forsch., 14, 1151 (1964). (12) G. Lammler, 2. Tropenmed. Parasitol., 16, 164 (1964).
