1268
C. H. BOATNER, R. T. O’CONNOR, M. C. CURETAND C. S. SAMUELS
specific amine or general base catalyzedl0 in which case variation of reaction rate with buffer concentration (at p H 7.4) may be expected.
Summary Hydrolytic stability of theephysostigmine (10) L. P. Hanunett, “Physical Organic Chemistry,” 1st ed., McGraw-Hill Book Company, Inc., New York, N. Y., 1940, pp. 344345 and 215-218.
[CONTRIBUTION FROM
THE
Vol. 69
analogs a t 38’ and p H 7.4 was investigated by measurement of light absorption of the phenolic hydrolysis product a t a wave length of 275 mp. The two N,N-dialkyl carbamates were stable under these conditions during a four day observation period. The N-methyl carbamate was found to be unstable and to have a half life of 52 minutes and a velocity of 0.0133. CHICAGO, ILLISOIS
RECEIVED DECEMBER 12, 1946
SOUTHERN REGIONAL RESEARCH LABORATORY‘ ]
The Pigments of Cottonseed. 111.2 Gossyfulvin, a Native Cottonseed Pigment Related to Gossypol3 B Y CHARLOTTE H. BOATNER,ROBERTT. O’CONNOR, MAIZIE
An orange colored pigment, gossyfulvin, has previously been detected4 in cottonseed. Although gossyfulvin has been shown to differ from gossypol in many of its properties i t can be readily converted into the latter pigment. Since the work on gossyfulvin was reported, larger quantities of the pigment have been prepared by the procedure outlined in the accompanying diagram and i t has therefore been possible to obtain further insight into its structure and its relationship to gossypol. Gossyfulvin forms rather large orange-colored rhombohedra (Fig. l), changing a t 212’ to a more deeply colored form which melts with decomposition a t 238-239’ (cor.). The crystalline form and habit of gossyfulvin differentiate i t sharply from gossypol, which latter pigment, upon recrystallization from diethyl ether and petroleum naphtha, forms clusters of dog-toothed prisms (Fig. a>,m. p. 182.5-183.5O. Gossyfulvin reacts with strong mineral acids when its chloroform solutions are treated with concentrated aqueous solutions of these acids yielding gossypol in amounts equal to as much as 86.87, (of the weight of gossyfulvin treated. On the basis of the experimentally determined elementary composition, C34H34N208,the molecular weight of gossyfulvin is 398. Gossypol obtained after recrystallization from diethyl ether and petroleum naphtha a t low temperatures and dried without elevation of temperature yields analytical values which agree with those calculated for C&3209. Comparison of gossyfulvin with such nitrogen derivatives of gossypol as diarnino- and dianilinogossypol, reveals several significant differences, (1) One of the Laboratorles of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, United States Deliartment of Agriculture. Article not copyrighted. (2) For previous paper of this series see Boatner, Samuels, Hall and Curet, THISJOURNAL, 69, 668-673 (1947). (3) Presented before the 109th Meeting of the American Chemical Society, Atlantic City, New Jersey, April 8 to 12, 1946. (4) Boatner, Caravella and Samuels, T H I S J O U R N A L , 66, 838 (1944).
c. CURET AND CAROLYN s. SAMUELS
ISOLATION OF GOSSYFULVIN FROM COTTONSEED Flaked cottonseed Diethyl ether
.
i Oil, F. F. A . , pigments
Aq. iTaOH (in $ Oil (in E t 2 0 )
,
Meal
7,)
(Na2S204) I
$ iYa gossypolate, orange pigments, soaps (in aq.) Et20 HCl
1
J.
j. Soaps fin aq.i
Gossypol, orange pigments (in E t 2 0 )
I
HOAc I
4 Orange pigments Et20 evapd. Hot acetone (in r o j
,
I
$ Gossypol acetic acid (PP.1 1
$
9
Orange C (residue)
Gossyfulvin, orange B cooling
: I ,
Gossyfulvin
Orange B
(ppt.)
(dissolved)
e. g., the melting point of gossyfulvin lies between those of the other nitrogen derivatives. The compounds differ with respect to solubility and stability. Diaminogossypol dissolved in diethyl ether or warmed in acetic acid is reported5 to evolve ammonia and revert to gossypol. Dianilinogossypol, on the other hand, is one of the most stable of the compounds formed from gossypol, and is hydrolyzed to gossypol only upon re(5) Miller and Adams, {bid., 1736-1738 (1937).
June, 1947
GOSSYFULVIN, A NATIVECOTTONSEED PIGMENT RELATED TO GossypoL
Fig. 1.-Photomicrographs of crystalline gossyfulvin, X fiR. (a) in ordiiiary light, M. E. Jeflerson and P. D. Krecgcr.
Fig. Z.-Phatolnicrographs
of crystalline gossypol, X 123, (a) in ordimiry light, M. E. JcKmon and F. B. Krceger.
action in concentrated sulfuric acicl,l alcoholic potassium hydroxide' or hot acetic Gossyfulvin is very unstable in solution, and in thc absence of strong mineral acids i t decomposes without loss of nitrogen. The absorption spectrum (Fig. 3 ) of gossyfulvin is identical, withiu the limits of expcrimeiital error, with that of ilianilinogossypol (Fig. 4). From the latter ob( 6 ) Clark. 3. B i d . Chen!., 78, 229-28.9 (1928). (7) carruth. 40,047.663 (1918). (8) Murty, Murty end Seshadri, Pior. lrrdiorr A m d . S r i . . 61 11912).
TH~SSOURM..
leA, 54-
iiml
1269
(I,] IX~L\VWII crocscrl nicols. by
m i d (1,)
l ~ l w w crossed ~ ~ nicols, by
servation it may be concluded that the basic structures of gossyfulvin and dianilinogossypol are identical. The tautoineric structures which have been proposed for dianilinogossypol' closely resemble those proposed for gos~ypol,'~ differing only in the substitution of two anilino groups for the two doubly-bound oxygen atoms of the carbonyl groups of gossypol. In view of the observation (0) Adarne. Price and Dial, 7x11s Jouarrr~, 60, 1258-1260 (1938). (IO) Adarnr, Morns. Gemman. Rutterbaugh and Kirkpatrick ibid.. 60. 2193-2204 (1938).
C. H. BOATNER,li. T . O'CONNOR, 11. C. CURETAND C. S.SXMUELS
1270 t
200
300 400 500 600 700 Wave length, m p . Fig. 3.--Absorption spectrum of gossyfulvin in chloroform. !-
Vol. 01,
range. The isomerism of these dioximes can therefore be related to their origins from closely related compounds. The formation of isomeric carbonyl derivatives requires that the nitrogencontaining groups of gossyfulvin and dianilinogossypol be removed during reaction. The existence of three isomeric dioxirnes suggests that the carbonyl groups of gossypol are more labile than has heretofore been presumed. Acetylation of gossyfulvin gave a yellow solid, ni. p. 185-1S5.25°. The color, melting point, and acetyl content, 34..Ci%, of this compound suggested that it might be identical with one of the compounds reported to be formed in small proportion upon acetylation of gossypol,11 and as the sole product of acetylation of d i a n i l i n o g o ~ s y p o l . ~ ~ ~ ~ TABLE I COMPOSITION ASD MELTING POINTSOF OXIMESFORMED FROM GOSSYPOL, GOSSYFULVIX AND DIASILISOGOSSYPOL
300 400 500 GOO 70') Wave length, mg. Fig. 4.-Absorption spectra of (A) gossypol mid (B) dianilinogossypol in chloroform. 200
'
that the absorption spectrum and, therefore, by inference, the structure of gossyfulvin is identical with that of dianilinogossypol, but very different from that of gossypol, i t seems evident that the changes occurring during the formation of both gossyfulvin and dianilinogossypol from gossypol must involve more than the simple substitution of nitrogen-containing groups for the oxygen atoms of the carbonyl groups of gossypol. The observation that the specific extinction coefficients of gossyfulvin and dianilinogossypol are identical indicates that the presence of two phenyl groups in dianilinogossypol produces enhancemeizt of its molecular absorption over that of gossyfulvin. The absorption spectra of the antimony trichloride reaction products (Fig. 3 ) of gossypol, dianilinogossypol, and gossyfulvin exhibit the same interrelation as those of the pure pigments. Gossypol and dianilinogossypol form stable reaction products with antimony trichloride, but their absorption spectra are very different. The antimony trichloride reaction product formed by gossyfulvin is very unstable; its absorption spectrum is similar in shape but less intense than that of the dianilinogossypol reaction product. Further evidence of the complexity of the interrelation of gossypol, gossyfulvin, and dianilinogossypol is observed on comparison of the oximes formed by these compounds (Table I). A11 three compounds form dioximes of identical elemental composition but they differ in melting points, and a mixture of any pair of oximes melts over a wide
Dioxime of gossypol Dioxime formed by gossyfulvin Dioxime formed by dianilinogossypol
312
CaoHinOrN? 65.70
204.5
CmHanOaK2
2211-221.5 CpoHszOiN?
5.72 5.04
G5.35 6 . 0 2 65.25
5.00
6.03 5 . 3 3
Composition c:tlculatcd for gossypol dioxime, 0,s.:C, 65.66; H, 5.83; S, 5.12.
C30H32-
A4cetylationof gossypol has been variously reported to yield a colorless tetra- or p e n t a a ~ e t a t e , ~ a colorless hexaacetatePG and a mixture of a colorless and a yellow hexaacetate. l 1 The elementary composition of the colorless acetate prepared during the present investigation agreed with the values calculated for tetraacetoxygossypol, and determination of its acetyl content by the method of Elek and Harte13 for 0-acyl compounds also yielded values consistent with the formula of a tetraacetate. Attempts to obtain the yellow acetylated gossypol derivative, m. p, 185', reported by Miller, Butterbaugh and Adams" in sufficiently pure condition for reliable analytical values were no more successful than those of the aforementioned investigators. The colorless acetate was found to be very stable and retreatment with the acetylating agent provided no evidence of its convertibility to the more highly acetylated yellow form. -1yellow compound, ni. p. lS.jo, formed by acetylation of dianilinogossypol was reported by Adams, Price and Dial9 to be hexaxetoxydianilinogossypol, and by I l u r t y and Seshadri12 to be a nitrogen-free hexdacetoxygossypol, identical with thc coiripound formed in small ainounts by the direct acetylation of gossypol. Ihririg the present investigation, neither of the previously reported dcrivatives was obtained by acetylation of (I:)
Rfiller, B u t t u b a u g h a n 3 Adams. Tiirs
JULKNAI.,
59, 1729-
1731 ( 1 9 3 i j (12) A1u;ty and Seshadri, PJUOC. Iizifiun A m d . Sci., 16A, 141-145 (1912). (13) Hlek a n d Harle,lizi/. E n x . C/wni.. A n d . Kd.,8, 267-269 (1036).
J U I I ~ , 1947
ChSSYFULVIN, -4 NATIVE COTTONSEED PIGRZENT
dianilinogossypol, but mixtures were obtained whose content of acetyl indicated the introduction of only foiir acetyl groups.
Experimental Isolation and Purification of Gossypol and Gossyfuivin.-Gossyfulvin was isolated from 'an ethereal extract of cottonseed after removal of gossypol according to the following procedure. The ethereal extract obtained from 110 11). of decorticated and flaked cottonseed was extracted in 1-liter portions with 500 ml. of 0.5 lVsodium hydroxide containing 1 0 g. sodium dithionite (sodium hydrosulfite: S a p S 2 0 a ) . The ethereal layer was washed with 250 ml. water, and the wtshings added to the alkaline extract. T h e cornbined alkaline extract and washings were then washed with 250 ml. of diethyl ether. Concentrated hydrochloric acid was added slowly with stirring to the alkaline extract until the solution attained a pH of 7 to 7.5, and a yellow ethereal layer scparated from the aqueous extract. T o the separated ethereal layer there was added 25 ml. of glacial acctic arid, after which the solution was heated on a steam-bath for about one-half hour, until the ether no longer evolved rapidly. Vpon cooling, a copious precipitate of gossypolacetic acid formed. The precipitate was washed several times by decantation with large volumes of 1)etroleum naphtha until the washings became lightly colored after which it was transferred to a Buchner funnel. and dried bv asoiration: total vield of crude ECOSSVpolacetic acid, 1 ~ g . The crud
