Communications to the Editor, The Tumor-Enhancing and Irritant Principles from Croton tiglium L . Sir:
Croton oil is obtained from Croton tiyliuin I,. (Euphorhiaceae) by pressing of the seed and has long been known for its vesicant, toxic, and purgative activity.' In 1941 Berenblum2 showed that this material is also a potent tumor-enhancing agent, i.e., it stimulates the appearance and rapid growth of tumors on mouse epidermis pretreated with a minute dose of a carcinogenic hydrocarbon. At the low concentration used the hydrocarbon, when applied alone, is inactive and crotoin oil it8selfis not carcinogenic. This unique activity of croton oil has important iniplicat'ions in st,udies of caiicer causation and many attempts have been made to isolate and determine the structure of the active ~omponents.1.3-~Tumor-enhancing agent,s of knonn chemical constitut,ion are all much less active tliaii croton The present report deals with the isolation of tn-o active materials and the structure elucidation of a pur? crystalline material obtained by catalytic hydrogeiiation of a biologically actire fraction of croton seed extract. The shelled seed of Croton tiglium L. was extracted with methyl alcohol to give a 417', yield of ail oily extract. The vesicant fraction of the extract, croton resin (I), was prepared by solvent partition between aqueous methyl alcohol (1 :9) and hexane.' This polar mixture of compounds (3% of whole extract') has shon.11 potent tumor-enhancing activity2 and it, was therefore chosen for further chemical studies and biological assay. Thin layer chromatography of I on silica gel showed the presence of at least 14 components. Collinin chromatography of I on acid-washed florid gare an amorphous solid (11, 1.0% of whole extract). Both I and I1 showed a pronounced increase in tumor-eiiliaricing activity compared to that of t,he whole croton seed extract or comnlercial croton T h i n layer chromatography of I1 showed t'he presence of four components (A, B, C, and 11). This material ivas further fractionated by countercurrent distribution and thin layer chronlatography to give two biologically active amorphous mat,erials, A and C, which showed ( I ) IC. Clierhnliea, I?. Ehninger, and R. &ernhard, l l p 1 1 , . Chim. d d u , 16, fj.58 (1932).
(2) I . Berenblu~n.Brit. J . Canre?, 1, 41 (liJ41). (3) N. I.. Drake and J. R. Spies, J . A m . Chem. Sor., 6'7, 184 !1933). 1 4 ) W. S.Lijinsky, Biochem. J., 7 0 , 5P (1958). (3) E. Hecker. Angeu,. Chem. Intern. E d . E n g i . , 1, 602 (1962). (6) R. K. Boutwell and D. K.Bosch, Cancer Res., 19, 413 (1959). ( 7 ) Biological tests were carried out using established test procedures: 1. Berenhlum and P. Shubik, Brit. J . Cancer, 1, 379 (1947).
siiigltl spots on thiii 1ayc.r chrornatogranis. Both and ( " arr irritant and shox tumor-enhancing activity. Compoiiiid C was tested on 20 female Swiss Millerton mice iiritiated with 300 y of 7,12-dimethylbeiiz [ a ] anthraccile. The promotor (C) was applied three times weekly, ,i y per applicatioii. First tumors were obserired a t 41 days after initiation and at 68 days 1 i niiinials bole tumors. Tumors were riot observed j i i appropriate coritrol groups. *Ill the componeiits of I1 are unsaturated and can he hydrogenated T! ith palladium black as catalyst iii methyl alcohol solution. Catalytic hydrogenation of I1 follon-cd by countercurrent distribution (lower phase : aqueous methyl alcohol, 0.5 : 9.5, containing 0.47; glacial acetic acid; upper phase: hexane; 200 tubes, 10 ml. per pliasc, 400 transfers) gave a 50% yield of' liydrogenation product which TI as purified by chromatography oil silica gel plates (2 111111.thickness.j8 The Imid with the highe,*tRfmas eluted uith ether and gave :L crystalliiie product froiri ethyl alcohol-water; colorless needles, 11i.p. %", Rf 0.25 (hexane-ether -acetic acid, 9: 1.? :0.3, silica gel). The analytical data agree I\ ith a molecular foriiiula, C3d1600s, molecular 1% eight ( 2 0 , x i t h three ester functions, two free hydroxyl groups. and six carbon-methyl groups. The coiii~~ ( I = 1 dni., pouiid is optically actiw, [ C Y ] I I+124.G0 c O . i i , chlorofoivi) The ultraviolet absorption spectrum showed two l o - ~ iiitensity maxima i i i cyclohexane and in methyl alcohol (cyclohexane: A,,,,, 248 nip; elnax 410; A,,, 302 m p ; E,,,&, 56) Trliicli huggested the presence of a polysubstituted benzene ring. The iiifrai*ed absorptioii spectrum was examined in various media atid revealed the folloTving features : intiamolecularly bonded hydroxyl (0.001 X in carbon tetrachloride * a n eak band a t 3370 cm. --I and a strong hharp barid a t 3440 cm. - l ) ; intense aliphatic stretrhiiig arid hendjng ab5orptions (IiBr pellet, methyl arid niethyleiie r?t 296:. 2935, 2860, 1435, and 1370 cm. I ; tertiary ('13 ut 288.5 (>in.- 1 ) ; intense ester carbonyl absorption (KBr pellet, 1742, 1720 cm.-I; 0.001 J I in carbon tetrachloride, l i 3 5 cm.-') ; weak aromatic C=C absorption (0.03 &' in chloroform, 1600 cm.-'). illiphatic C-A' absorption is present in 11,A, and C a t 1650 and 1G30 cm, but does riot appear in the infrared spectrum of the crystalline hydrogenated compound. However, the other bands in the infrared spectra of A arid c' arr ~ ~ similar r y to that of the hydrogenated compound The 1i.ni.r. spectrum showed a series of four peaks from 0.9 to 1.25 p.p.rn. ascribed to aliphatic CH, CII, (8'1 I. BekerskJ, Anal. Chern., 36, 261 (1963).
September, 1963
COMMUNICATIONS TO THE EDITOR
and CH3 protons (type 1). Four weaker peaks a t 2.0, 2.15, 2.25, and 2.34 p.p.m. are characteristic for methyl or methylene protons next to oxygen-bearing carbon and/or aromatic ring (type 2). A peak a t 4.65 p.p.m. is ascribed to a hydroxyl proton. Integration indicates 46 protons of type 1, 12 of type 2, and 2 hydroxyl groups. The compound does not react with diazomethane and does not give a coloration with ferric chloride. Hydrolysis with 10% aqueous ethanolic sodium hydroxide (1 :1) resulted in darkening of the solution. The acidified hydrolysate gave a persistent purple color with ferric chloride. Three acids were obtained in equal molar ratios from the hydrolysate and were identified by gas-liquid chromatography of the methyl esters: 1-methylbutyric, capric, and lauric acids. This is consistent with the experimentally determined saponification equivalent for three ester functions. Subtracting the various functions indicated, a C7H residue is left. This residue can be accommodated reasonably as a fully substituted benzene ring, and this is in agreement with the ultraviolet absorption and infrared spectra. The CH residue left constitutes then the center for optical activity. The high specific rotation suggests also that this optically active center contains a strongly hydrogen-bonded function.9 The instability of the watersoluble phenolic residue indicated either a pyrogallol or a 1,2,3,5-tetrahydroxybenzene nucleus. From the n.m.r. data the two remaining carbonmethyl groups must be attached either to a carbon atom which also carries oxygen or directly to an aromatic ring. The available information leads to a partial structure
where each R is one of the following: -COCH(CHs)CHzCH3; CO(CHz)&H,; CO(CH,)&H,; H ( t ~ 0 ) . Other closely related hydrogenation products have been isolated in crystalline form and these differ from the compound described in the nature of the acids and possibly also their relative positions. Compounds B and D and the catalytic hydrogenation product (map. 96') described here have not yet been tested for cocarcinogenic activity. However, the close similarity in infrared absorption spectra and chemical properties of A, B, C, D, and the hydrogenated compound leaves little doubt that they are all chemically closely related. The exact nature of the structural features required for activity are currently under examination. It is expected that the biologically active material and the hydrogenation product described will exhibit both hydrophilic and lipophilic behavior which may play an important role in the biological activity of the material. It is of interest also that the partial structure proposed is similar to the long chain olefinic catechols which constitute the vesicant principles of poison ivy.*O Acknowledgment.-This work was supported by (9) C. Robinson a n d M.J. Bott, "JVatzlre, 168, 328 (1951). (10) W. F. Symes a n d C. R Dawson, J. 4 m . Chem. S o c , 7 6 , 2959 (1954).
617
Grant C-5946 from the Department of Health, Education and Welfare, U.S. Public Health Service, National Cancer Institute. IKSTITUTE OF IKDUSTRIAL MEDICINE B. L. VANDUUREN E. ARROYO NEWYORKUNIVERSITY MEDICAL CENTER L. ORRIS XEW YORE,N. Y. RECEIVED JUNE11. 1963
Aldosterone Antagonists.
2'3'a-Tetrahydrofuran-2'-spiro-17-(4-androsten-3-one) and Related Compounds
Sir: The spirolactones, e.g., XVII, prepared by Cella and associates, are effective aldosterone antagonists in animals2 and in human^.^ One series of spirolactone
HO A
P d
111, R = -CHzCHZCH*OTHP IV, R = -CHzCHzCHZOH V, R = -CH&H*CH20TS
w-.,
0 VI, R ' Y I I , R' XVI, R' = XT'II, R ' = XXIX, R' =
CH3, R H, X = Hz CH3, R SCOCHZ, X = Hz H, R = H, X = HP CH,, R = H, X = 0 CH3, R = SCOCH,, X = 0
analogs, t'he spirolactams,4 have marginal activity. Cella and co-workerslbshowed that 3-keto-A4system in the A-ring of spirolactones was an activity-enhancing, although not an essential, function. Introduction of methyl groups Id and increased unsaturation, lb effective activity-enhancing devices in the antiinflammatory steroid series, have had relatively little effect on spirolactone activity. The 9-fluoro-11-oxygenated derivatives, l o however, are more active. (1) (a) J. A. Cella, E. A. Brown, and R. R. Burtner, J . O w . Chem., 24. 743 (1959); (b) J. A. Cellaand R. C. Tweit, ibid.,24, 1109 (1959); (c) E. A. Brown, R . D. Muir, and J. A. Cella, ibid., 26, 96 (1960); (d) N. A. Atwater, R. H. Bible, E. A. Brown, R. R. Burtner, J. S . Mihina, L. N. Nysted, and P. B. Sollman, ibid., 26, 3077 (1961). (2) C. M. Kagawa, J. A. Cella, and C. C. Van Arman, Science, 126; I015 (1957). (3) W. S . Coppage, J r . , and G. W. Liddle, Ann. .V. Y . Acad. Sci., 88, 815 (1980). (4) A. A. Patchett, F. Hoffman, F. F. Giarrueso, H. Schwam, and G. E. S r t h , J. Ore. Chem., 2 7 , 3822 (1962); R. R . Burtner and L. N. Kysted, U. S . Patent 3,001,986 (Sept. 2 8 , 19ti1); L. S. S y s t e d and E. R . Burtner,
J. Ore. Chem., 27, 3175 (1962).
