July 1965 and evaporated on a steam bat,h. The residue was fractionated under reduced pressure. The bases gave crystalline salts with difficuky; those which were obtained are described in Table I, after the respective base. Pharmacology.-For all compounds, we evaluated (using codeine as reference substance) the antitussive activity on cats,5.6 observing whether this was accompanied by hypotension and respiratory impairment, the acute LD,, in mice and albino rats,' and the local anesthetic* and analgesic acti~ity.~n~O The occurence of central sedation was measured through variations in the spontaneous motility and in the performance of the grip test, and t,hrough the influence exerted by the compounds on the duration of the barbit,urate narcosis.11 The spasmoly-tic activity was tested on the isolated rat and guinea pig ileum against histamine, acetylcholine, BaC12, and nicotine contractions.12 Compound 15 was studied as antitussive also on unanesthetized rats and guinea pig~138~~ and further as an inhibitor of pentylenetetrazole seizures," for its effect on peristalsis,'5 and on the bronchial and tracheal muscles in vivo and in u ~ L T o ~ finally ~ , ~ ~ ; its influence upon the bronchial and tracheal secretion was examined.I8 I n Table I11 only the compounds appreciably active as antitussives are recorded with t,he results concerning their antitussive, local anesthetic, analgetic, narcosis-enhancing, hypotensive, and respiratory-depressing activities and the data about their acute toxicity. A few observations relating the structure to the pharniacological activities are to be made. The antitussive as well as the other pharmacological activities listed are generally more pronounced in the thymol than in the guaiacol derivatives. The antitussive effect lasted for a time roughly proportional to the activity index (from 30 min. to 3-4 hr.) and was sometimes accompanied by a depression of the respiratory function. The compounds of both series containing a diethyl radical were inactive, whatever the length of the chain. I n agreement with the literature,'g the piperidine group seems to enhance this activity, as clearly shown by 15 (though a guaiacol derivative) and 18, both being the most active of all our antitussives. As spasmolytics the guaiacol derivat,ives were completely inactive, while t,he thymol derivatives were very weak inhibitors of acetylcholine contractions (less than 1% of atropine), and of about the same activity as papaverine against BaC12contractions and hexamethonium against nicotine spasms. The behavioral pattern,2o body temperature, motility, and muscular tonus and force of the treated animals as well as the response to the hot plate test, for analgesia were unaffected; the only signs (more evident in the thymol derivatives) of a certain t,rend to affect CXS functions were the analgesia detected in the phenylquinone test and t,he moderate prolongation of the barbiturate narcosis. These mild and unspecific effects on the CNS are in contrast with the marked sedative properties of some thymol derivatives described in literature.2 Therefore, the observed antitussive effect does not seem t,o be bound to general analgesia or sedation, or to the intensity of the local anesthetic eflect ; in fact for some active compounds hypotensive and rejpiratory-depressing effects were lacking. (,j)R. Domeiijoz. A r c h . ezptl. Path. Pharmakol.. 215, 19 (1952). (6) 21. Carissimi. A . Cattaneo, R. D'Aiiibrosio, E. Grunielli, E. Milla. and F. Ravenna. Farmaeo (Pavia), Ed. Sci., 18, 315 (1963). (7) J. T. Litclifield and F. Wilcoxon, J . Pharmacol. Ezptl. Therap., 96, 99 (1949). (8) E. Rulbring and I. Wayda, i b i d . , 85, 7 8 (1945). (9) J. .Jacob and AI. Blozovski, Arch. Intern. Pharmacodyn.. 122, 287 (1959). (IO) L. Hendershot and J. Forsaith. J . Pharmacol. E x p t l . Therap., 125, 237 (1959). i l l ) XI. Carissiini, I. Grssso, E. Griiiiielli, E. llilla. and F. Ravenna, Farmaco (Pavia), Ed. Sci., 17, 390 (1962). (12) E. Grunielli and A . Cattaneo, i b i d . , 16, 773 (1061). (13) P.Zamboni and G. Siro-Hrigiani, drch. ita/. Sci. Farmacol., [ 3 ] 12, 152 (1062). (14) 13. Silvestrini and G. Maffii, Farmaco ( P a r i a ) , Ed. Sci.. 14, 440 (195!1). (15) P. Janssen and A . H. Jaaeneau Pharm. Pharmacol., 9, 381 (1957). (16) H. Konzett and R . Rossler, Arch. e z p t l . . Pathol. Pharmakol., 196, 71 (1940). (17) C. J. Castillo and E. Beer, J . Pharmacol. Ezpt/. Therap., 90, 104 (1947). (18) 31. Iloyd and -4. Roman, A m . .I. Phusiol., 136, 383 (1041). (19) T.Kas6, T. Yuirono, and 11. M u t o , J . Y e d . Ckem.. 6, 118 (1963). (20) S. Irwin, AI. Slabok. P. L. Debiase, and W. 11. Govier, Arch. Intern. Pharmacodyn., 118, 3 5 8 (1959).
KOTES
545
Compound 15 was studied particularly &ce it was the only one which showed a good degree of activity also by the oral route. Its antitussive activity is of same order of magnitude as that of codeine in the experiments on cats and seems to be fairly specific because of lack of side effects (respiratory and circulatory implications, inhibition of intestinal peristalsis, bronchoconstriction, and general sedation). N o protection against pentylenetetrazole seizures and no change in the mucous tracheal secretion were observed. I t did not produce tolerance: in cats injected dailyfor 4weekswith lOmg./kg.i.p., the inhibitory effect on the experimental cough536 was obtained by the same doses which were active in cats not pretreated. It does not appear that the hypotensive activity shown by several compounds is cause for concern. It is presumably bound to myorardic impairment as seen on the isolated rabbit heart, and to an intense and persistent peripheral vasodilatory effect, as also seen in the isolated rabbit ear vessela,*I together with an adrenolytic effect: in any event, thi. was not observed o n the blood pressure response to epinephrine. (21) -1.Fleckenstein, Brit. J . I'harmncol.. 7, 5.53 ( 1 0 5 2 ) .
Insect Chemosterilants. 11. N-Carbamoylaziridines ALEXEJ B. B&KOVECASD CHARLES IT.
\~OODS
Entomology Research Division,
c'.5'. Department of Agriculture, Beltsville, .2laryland Received February 23, 1965
Certain S-substituted aziridines are outstanding in their ability to interfere with insect reproductioii. This type of biological activity can be found among other alkylating agents (e.g., nitrogen mustards, alkyl alkanesulfonates), but the aziridiiies proved to be particularly useful because of their effectiveness in sterilizing male insects and their often wide toxicity-sterility margin. In general, the aziridine bases with no substituent on the ring nitrogen and the N-alkylaziridines have little or no activity as insect sterilants. Nost of the screening and syrit hesis of aziridinyl cheniosterilants centered on the compounds which had an unsaturated group attached to the ring nitrogen. The S-acyl and Saroylaziridines exhibited the sterilizing activity in various degrees, but none of the compounds tested approached the high potency of tepa or other phosphoruscontaining aziridines. It appeared desirable to increase the electron deficiency of the carbonyl carbon of S-acylaziridines by replacing the acyl group with a carbamoyl group. Several diaxiridinyl compounds of this type were conimercially available, and their testing and comparison with similar S-acylaziridines indicated substantially increased Sterilizing properties. In our previous coinniunication,' we have reported the synthesis of 1,l'suberoylbisaziridirie (I) and 1,l'-isophthaloylbisaziridine (11). The first coinpourid exhibited no appreciable sterilizing properties when fed to house flies (Musca doinestica L.) at 1% concentration and the second produced a 75% reduction in egg hatch when fed a t 5% concentration. On the other hand, the two similar conipounds N,N'-hexaniethylenebis-1-aziridine(1) P a r t I in the series' C. W. Woods, A. B. Boikoiec, and F. M. Hart, J . .lied. C h e m . , 7 , 3 7 1 (1964). (2) A. B. BoikoLec, Reszdue Rea., 6, 87 (1964). (3) S. C. Chang and A. B. Boikovec, J . Econ. Cntumol., 57, 488 (1964).
s O"h
c$
cH
1-01,x
,I
tI
:;50 dec.
('H,
c,arl)osat I I idc (11I ) a tid 5 ;S '-(J-iiict Iiyl-iit-phvi iylci I C ) l)i~-l-aziridiiiccarb~xaiiiide (IT-)4provcd t o be rat hct, potent house fly sterilaiits whivh conipletely cliiiiiiiated thv egg hatch at O.lTcc*oiic*ciitratioii. Wc wish to ivport t h e syrit hesis of t w ~ i i i yS-cari,atiioylaziridiIic,s which ~verc' prepared as raildidat cl c*lit>iiiostcrilant s. All of thew roiiipounds were s y i t h sized froiii ethyletiimine or %-iiiethylaziridiiie aiid roiiiiiicwially availahlr isoryariatcs ; ihcir physical rharacntc3ristic.s are suiiiiiiarized in Table I . The last caoluiiin i t i Tahlr I contains preliuiinary house fly screening data;' obtained by Dr. G . C'. LaBrecque arid his assori-
hli
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NOTES
July 1965
Jl
lv diriecarboxaiiiide (Table I) has been found to produce the same effect at 0.01% concentration. Although other coiinparisons with difuiictiorial conipounds in Table I are riot available, it appears that in the diaziridinyl series the acyl-carbanioyl exchange is highly significant. Experimental General Method for Carbamoy1aziridines.-A solution or suspension containing 0.1 equiv. of the isocyanate in 100 ml. of solvent (ether or benzene) mas added over a 10-min. period to 0.2 eyuiv. of the aziridine in 100 ml. of solvent. The reaction mixture was cooled in an ice bath during the addition and then kept a t rooin temperature for 1 hr. The carbanioylaziridine usually started to cp-stallize a t this point, and the reaction niisture was rooled for 1 hr. in an ice bath. I n a few instances it was necessary to remove most of the solvent before crystallization occurred. The products were collected by filtration and recrystallized from anhydroris ether or benzene.
547
epidermoid carcinonia.3 It also showed a correspondingly high activity (8000 Bu./mg.) in a disk-plate assay against KB cells in agar.4 The discovery of the caytotoxicaity of convallatoxiii led us to test soiiie available cardiac aglycones and glycosides for the saiiie activity. The results, presented in Table I,deiiioristrate that all of the coiiipounds tested had significant activity arid iiiost of theiii had activity of a high order. The data presented in Table I, in conjunction with that available i n the literature,'S2 suggest that cytotoxicity is associated with an unsaturated lactone either attached to position 17 by a carbon-carbon bond or fused to ring D across the 16,17-position. Thus, the cardiac principles have either a cardenolide ring (as in I) or a bufadienolide ring (as in 11) attached to position 17, while the iiiost active of 150 steroids tested in these laboratories have an a$-unsaturated y-lactone fused to the 16,l'i-positions.' The data in Table I also indicate that, in our assay, the glycosides are iiiore active than the corresponding aglycones mid that the cardenolide and bufadienolide rings are equally effective in conferring activity. The two most potent coinpounds iii Table I, coiivallatoxiti and hellebrin, have the follou-ing structural features in coiiiiiioii: (a) they are both rhaiiinosides, (b) they both have p-hydroxy1 groups at positions 5 and 13, and (c) they both have the C-19 carbon atom represented by an aldehyde function. However, the forinier has a carderiolide ring at position 17 while the latter has a bufadieiiolide ring at the saiiie position.
R10 I
1Zeseurch Luborutories, ?'he Upjohn Covipanu,Kalamazoo, Michigan
Cytotoxicity associated with cardiac principles aiid cwtain other steroidal derivatives has been reported it1 recent publicatioiis.l~2 We are proiiipted by these reports to publish soiiie of our observations in this respect. During ail iiivestigatioii of the cytotoxicity of extracts of the bulbs of Oixithoyaluiia uiiibellatuiu, we isolated ail active principle as a crystalline compound and identified it as convallatoxin. This cardiac glycoside is a potent caytotoxic agent having an IDb0of 0.002 y / 1111. when assayed against Eagle's KB strain of human (1) .J. P2. Pike, .J. E. Grtidy, .I. S. Lvaiis, and C . G. Sinitli, J . M e d . Chem., 7, 348 (19641. (2) (8) S. AI. I i u w l l a n , R. J. IIeiningway, tind R. W. Doskotcli, iiiid., 7 , 80:3 ( 1 9 6 4 ) ; (b) S. 31. K u p c h a n , J. R. Knox, J. E. Kelsey, a n d J. A. 6 . Renauld. Science, 146, 1683 (1964).
J II
O-C-CHZ
BAY.4RU SPALLDISG
lieceived February 1, 1966
I1
0 0
Cytotoxicity of Cardiac Principles It. B. KELLY,E D W A R D G. DAYIELS, Ah'D L.
OH
HO
R
I11
Experimental Cytotoxicit'y of the extracts and purified frac'tions is determined hy a disk-plate assay against, KB cells in agar.* Isolation of Convallatoxin.-O,nithoyalcLnL umbeilaturn bulbs ( 2 i kg., air dried a t room t,eniperature) were ground and stirred in 130 1. of water at 85" for 1 hr. The solids were removed by centrifugation and re-extracted as above with 95 1. of water. The aqueous extracts were extracted in a continuous extractor with 1.5 vol. of 1-butanol. The butanol extracts were evaporated in uucuo to a dark brown tar weighing 270 g. and assaying 56 Bu./mg. A portion of the tar (87 g., 4.9 x lo6 Bu.) was subjected to a 25transfer countercurrent distribution between the two phases of (3) For details of this assay see C. G. Smith, IV. L. Luminiu, and .J. li. G r a d y , Cancer R e s . , 19, 843 (1959). ( 4 ) This assay was a modification of l l i y a m u r a ' s techniflue (Y. AliyaInura, Antibiot. Chemotherapy, 6 , 280 (195611; details are t o be publislied later. Bu means biological units.
