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.
tlie solvent system : ~vater-ethancrl-l-butaiiol-Skell~s~~Iv~ 13 (10: 2 :
S:,;); the vohime of enrh phase was 50 nil. After distrihutioii, the contents of tubes 5-18, containing niost Of the activity, were i'otnbined m d ev:tpiir:ited in m c m . The residue (1:3.:3 g . ) w:ts dissci~vedin 20 nil. of eth)-lene dj,.hlc,ride-nieth:trii)l ( 1 : 1) :rnd applied tii :I c ~ i i l u i t i i iof 250 g. of silicic. acid. The c3olunin xvas eluted i i i 25-1111.fixcTioiis with 1250 nil. each i ~ ethj~leiie f dichloride-rrietlim o l i 9: 1 ) :md ethylene dichloride--methanol ( 3 : 1 ) . I."c.tions 41-R))w-hic~liront:tined most of the activity u-ere c-otiihined :iiitl cv:ipor:tted t o dryness in mew. The residue (3.9 x IOfiBn.) W A S subjerted to L: 600-transfer countercurrent tlistrihut ioii hetv-eeii the two ph:tses of the solvent system : c.hloroforrri-acetoiie-~-atel. ( 1 : 2 : 2 ) ; the volume of each phase was 10 nil. -4fter distrihutioii tlie contents of tubes 411-460, which cwntained the acativity, were c,ottibineti and evapomt ed in z " x o almost to dr?-ness. (hi c*ooling, t lit, caonc~rnlr:ttedeposited crystalline material n-hic,li, o i l crjWn11iz:ition froiti acetone--n-ater, yielded 432 ~ n g .01' c~c~ti~~:rllatcisiii. lenticd with an aut,hent ic smiple i j f c~oiiv:~1latosiii iiiisture melting p i i i n l , ultr:i\-iiJlet, infrnred. :illti iilr ( , ' 2 9 1 1 d ) 1 , 1 :
(I,
(i:I.2::
11. 7.6!1.
ICiiilll(1:
C',
6 2 , ! ) 5 : H, 7.04.
Acknowledgment.41-e are iiidebted to 111.. d. 1'. O\veii aiid 11. 11. Rurch for biological assays aiid to Dr.
(;corgc: Slornp and associates for arialysis and d e t w iniiiation of spectra. The authors thank J l r . J . TV. Hiiiniaii, Head of out' Satural Products Scctioti, for his int,crest and advice.
The Relationship between Structure and Anticonvulsant Activity in a Series of Benzenesulfonamides
