Sept. 5, 193%
COMMUNICATIONS TO THE EDITOR
Studies were initiated in these laboratories to isolate the active ingredient from cottonseed. During the course of this investigation a compound was obtained in crystalline form which was found to be partially effective in preventing the SSTinduced growth retardation in mice. This compound was identified as the purine, guanine.
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acids or their degradation products present in the extracted liver residue. FROM THE DEPARTMENT OF BIOCHEMISTRY RESEARCH DIVISION JESSE W. HUFF SHARP & DOHME,INC. DAVIDI(.BOSSHARDT WEST POINT,PENNSYLVANIA RECEIVED JULY 28, 1952
TARLE I KFFECT OF PURINES AND PURINE RIBOSIDESON A SSTINDUCED GROWTH RETARDATION IN MICE"
HYPOTENSIVE ALKALOIDS OF VERATRUM FIMBRIATUM G U Y Sir : Av. 12-day Two new hypotensively active ester alkaloids, wt. gain, germanitrine and germinitrine, have been isolated J)iet g. from Veratrumjimbriatum Gray. Control 9. 1 The extraction procedure employed was essen2% SST 4.6 tially the same as the one reported in our previous 2% SST + 5% Cottonseed meal" 8.6 investigation of Veratrum viride Ait.' The crude 10.8 2y0 SST + 15Y0 Cottonseed meal amorphous fraction thus obtained was subjected to 2% SST + 0.02% Adenine 8.3 an eight plate countercurrent distribution usin? 2% SST + 0.08% Adenine 8.8 benzene-2M acetate buffer PH 5.5 as the solvent 2'30 SST + 0.027, Guanine 7.8 system with the lower phase moving. This 27'0 SST + 0.0870 Guanine 7.4 yielded two main fractions, A (tubes 0-1) and B 27'0 SST + 0.02% Xanthine 8.1 (tubes 2-5). 2% SST + O.OSyOXanthine 8.9 Fraction A was distributed on a 24-plate counter2% SST + 0.0270 Hypoxanthine 9.5 current machine with 0.5 M sodium acetate buffer 27, SST + 0.0870 Hypoxanthine 8.2 40 : 60. Careful p H 5.0-benzene-cyclohexane 2y0 SST + Adenosine equiv. t o 0.0270 adenine 7.8 of the material recovered fractional crystallization 2% SST + Adenosine equiv. t o 0.08% adenine 9.0 from tubes 4-14 from acetone-water gave ger2y0SST + Guanosine equiv. t o 0.2% guanine 7.6 manitrine, and germinitrine. 2% SST + Guanosine equiv. t o 0.08% guanine 8.1 Germanitrine crystallized as heavy needles; 2% SST Inosine equiv. t o 0.02% hypoxanthine 8.2 [aIz4"n-61 k 2' (C 1.0 in pyr.); m.p. 228-229'; 2% SST 1% Yeast Ribonucleic acid (GBI) 9.0 0.0 =k 2' (C 1.15 in CHC13). Analytical data indiEight male mice per group. Proflo, Traders Oil Mill cate the empirical formula C39H58011N; calcd. C, Co., Fort Worth, Texas. 65.25; H, 8.28; N, 1.95; eq. wt., 717.87; found: C, 65.30; H, 8.26; N, 1.99; eq. wt., 721; picrate, The purines adenine, guanine, xanthine and m.p. 240-241 O (dec.), C3gHsgOllN"OC6H,j(NOz)a: hypoxanthine as well as the ribosides adenosine, C, 57.07; H, 6.60; found: C, 56.68; H, 6.52. guanosine and inosine were tested with growing Volatile acid determination, found : 2.66 equivamice using the SST containing low fat basal diet. lents of acid. Alkaline hydrolpsis of germanitrine The results obtained are shown in Table 1. A11 of yielded germine, acetic acid, methylethylacetic the purines and purine ribosides that were studied acid and tiglic acid.? as well as yeast ribonucleic acid were found to be On methanolysis germanitrine was converted to a able partially to replace cottonseed in preventing di-ester, germanidine, by the loss of the labile acetyl the SST-induced growth retardation. group; m.p. 221-222'; [ a I z 4-4.1 ~ f 2' (C 1.0 in On the basis of the data presented here purines pyr.); 4- 18.1 f 2' (C 0.49 in CHClJ. Analytical may function to modify the action of succinylsulfa- data indicate the empirical formula C ~ Y H ~ ~ O : ~ON thiazole thus permitting the intestinal micro- (calcd. C, 65.75; H , 8.50; eq. wt., 675.84; found: organisms to synthesize an as yet unknown factor C, 65.66; H, 8.61; eq. wt., 672). Volatile acid which possibly plays some role in fat metabolism. determination, found: 1.97 equivalents of acid. On the other hand purines or nucleic acids may Germinitrine crystallized as irregular prisms; represent essential accessory food factors required 111.p. 175-liG'. [ a ] * *-30.0 ~ f 2' 1.12 in pyr.); for fat metabolism in the mouse. +i.8 2' ( k 1.33 in CHCIJ. Xnalytical data In other studies, to be reported later, it has been indicate the empirical formula CasHsiO1lS; calcd. found that other stress agents such as thyroid active C, 65.43; H, 8.03; S , 1.96; eq. wt., '715.83; materials or atabrine administered orally, or in- found: C, G5.35; H, S."; N, 1.61; eq. wt., 72%; jected thyroxine cause a retardation of growth in picrate, m.p. 338' (dec.), Cs9Hb,011N.HOCgH2inice fed a low fat diet. I n these cases it is also ( N 0 4 ) ~ : C, 57.19; H, 6.40; found: C, 37.17; possible to prevent this growth retardation b y the H, 6.66. Volatile acid determination, found: 2.32 feeding of cottonseed meal, fat, or a water extracted equivalents of acid. Alkaline hydrolysis of gerliver residue. It is possible that the beneficial effects minitrine yielded germine, acetic acid, tiglic acid of extracted liver residue in overcoming the toxicity and angelic acid.2 of thyroxine or atabrine in the rat that were reFraction B was distributed on a 24-plate counterported by E r ~ h o f f ~may - ~ be due to the nucleic
+ +
(c
(2) B. H. Ershhff, Arch. Bio.. 16, 365 (1947). (3) B. H. Ershoff a n d H. B. McWilliams, Scieticc, 108, 632 (1948). (4) B. H. Ershoff, J . N u f . , 36, 269 (1948).
(1) hi. W. Rlohs, R. Arons, hl. D. Draper, F. Keller, S Koster, W. Malesh a n d F. J. Petracek, Tam JOURNAL, '74, in press (1952) (2) T h e acids were identified by conversion t o their p-phenylphenacyl esters and characterized after chromatographic separation
current machine using 2 X acetate buffer pH 5.5 and benzene. The known triester, neogermitrine,3 was obtained by crystallizing the material recovered from tubes 6-10 from acetone-water. The hypotensive activity4p5of germanitrine, gerniaiiidine, and germanitrine was found to be 0.12 pg. [O.ll-O.14], 0.77 pg. [0.4G-2.3], and 0.41 pg. [0.36-0.49], respectively.
inclusive of the infrared characteristics over the whole measurable range, with the oxidation product from triacetyldihydroveratramine (m.p. 242-245", [ a ] 2 3+57.5"; ~ final. Calcd. for C33H4506N: C, 71.83; H, 8.22. Found: C, 71.94; H, 8.26) The respective N-acetates (V), prepared by hydrolysis with methanolic potassium hydroxide, were likewise identical (m.p. 2G4-266.5', [o!]'~.~D +71.7", t 6 8 . S " ; Anal. Calcd. for Cz9H4104N: C, 7'4.46: (3 J I ried 1' \umrrof and Y 11 Lus I J O L R \ % L 74, JU41 If. 8.84. Found: C, 74.57; H, 8.64). The 1932) (4) Edward I> Swiss and George I V a i w n , b ederatiun I'roceediiig\ vicinal effect of the indanone grouping on the 1 ol 11, S o 1. March, 1052 contribution to molecular rotation of Cg is evident ( 5 ) Expressed ds micrograms per kilogram of dnesthetrzed dog per in the abnormally high A [ \ I ] D for the saturation minute required for a ten-minute intravenous infusion t o lower t h e mean of the double bond (+481O for I1 --t IV, 1-493' arterial blood pressure ?(IL?, when administered according t o the method of G L Maison and J Vv Stutzman l h c bracketed numfor 0-desacetylated I1 -+ V) as compared with the hcrs express t h e %C-, confidence limits values +399" for triacetylveratramine + triacetyl11 \\ KLOHS dihydroveratramine, +404" for N-acetyheratra\I D DRAPER mine -+ S-acetyldihydroveratramine, and +243 O KIKERLABORATORIES, h c 17 KELLER 9480 BEVERLY BOULEVARD S KOSTER for normal A5-stenyl acetates5 The significance of this result lies in the fact that I os A\GELES48, CALIFORMA \! M A L E S H 1 J PETRACEKi t renders extremely remote the possibility of a R E C ~ I P EJ D L I 1 28, I%.! rearrangement of the carbon skeleton in the formation of the acetolysis product I1 from diacetyljervine, since ( 1 ) the presence in 1-eratramine of a THE STRUCTURAL CORRELATION OF JERVINE AND preformed benzenoid ring has been assured not only VERATRAMINE by ultraviolet spectrophotometry6 but also by Sir: chemical ~ n e a n s ,(2) ~ the conversion of triacetylAs reported in preliminary communications from dihydroveratramine to IV obviously cannot involve this Laboratorv.' 0.N-diacetvliervine (1) on ace- a change in the skeleton, and ( 3 )i t is reasonable on tolysis with acgtic anhydrideiLcetic acid .IC containing a catalytic amount of sulfuric 0 acid gave rise to a triacetate, CS3H4306K, (map. 239-240", [ a I z 4 D -29'9, which on the basis of its ultraviolet and infra red characteristics 251 rnp, log c 1.08; 300 mp, log E 3.30; I K : intense bands a t 5.75, 3.88 and 6.09 p, indicative, respectively, of 0-acetyl, ketonic carbonyl and N-acetyl) and other evidence was assigned the indanone structure 11. On the other hand, there has been obtained b y chromic acid oxidation of triacetyldihydroveratrarnine (C3YH4705K, now assigned structure 1113)a compound C33H4506N (rn.p.241-245", [ c r I 2 l ~+5Sc) which likewise exhibited the above spectral properties, and the new keto group I\-, K = Ac of which. like that of 11. was unreactive T', R = H to ketone reagent^.^ 'Lye have now reduced I1 catalytically with palladiuin---calciuni biogenetic grounds to accord also to jervine the carbonate in ethanol to its 3,(i-dihydro derivative abnormal ring structure which has now been shown W , 4arid found the latter ic1entic:il i i i :ill respects, to pre-exist in ver:i tr;iniine. \
( 1 j J. Fried, 0. Wiutersteinrr, A . L l i i i y s l x r p . AI h l w r e :r t x r u i i i : t i t v j 1%it ii l r i c ri
0 . \\-INTERSTEI.UEK .
RECEIVED JLTS
SORMAN HOSANSKY' 21, 1952
parently for t h e most part unchanged starting material), which could not always be completely removed by recrystallization or eliminated by rehydrogenation. so t h a t t h e yield of pure dihydro product was always iorv ( 5 ) D . €1. R. Barton, J . C / ~ P ?SOC., I L , 512 (1946). 16) TV A Jacobs and 1, C Craig, J . Biol. Chem., 160, 555 (1945). ' 7 ) This paper is part of the dissertation t o be presented by Norman 1Iosansky in partial fulfillment of t h e requirements for the Ph.D. de~ r t . ? i n tlic ( k ~ c l u : ~ tSrlim~l t, i,i R i i l p e r s University.
