[CONTRIBUTION FROM THE INSECTICIDE DIVIBION,BUREAUOF ENTOMOLOGY AND P L A N T QUARANTINE, s. DEPARTMENT O F AGRICULTURE]
u.
CROTON RESIN. IV. THE PETROLEUM-ETHER-INSOLUBLE ACIDS* JOSEPH R. SPIESt
Received February 1,1857
The saponification products of the complex mixture of esters known as croton resin may be separated into three groups: a mixture of fatty acids, the composition of which has been previously reported,* 30 per cent.; an amorphorus brown acidic mixture insoluble in petroleum ether, 40 per cent.; and water-soluble phenolic constituents, 30 per cent. The percentage composition of the fractions is approximate. This paper is primarily a report of a study of the petroleum-ether-insoluble constituents, which have been shown to consist mainly of amorphous unsaturated polyhydroxy acids. The saponification products of a croton resin were first separated by Boehm,2 who recognized the acidic character of the petroleum-etherinsoluble fraction. Wagne9 found a large percentage of these acids in the saponification products of the resin and suggested that they may be auto-oxidation or polymerization products of linoleic acid known to be present. Flaschentrager and Wolffersdorffl reported 6.93 per cent. of petroleum-ether-insoluble acids in the saponification products of croton In the present study a large quantity of the petroleum-ether-insoluble acids was prepared by careful saponification of croton resin. Precautions
* Since it appears improbable that the author will have further opportunity for the study of croton resin, it seems worth while to publish the information accumulated up to the present time. The experimental work recorded in this paper was carried out in the Chemistry Department of the University of Maryland. For articles I, 11, and I11 of this series see J. A m . Chem. SOC.,67,180-187 (1935). t Present address, Allergen Investigations, Bureau of Chemistry and Soils, U. S. Department of Agriculture, Washington, D. C. 1 DRAKE AND SPIES, J. Am. Chem. SOC.,67,184-187 (1935). 2 BOEHM, Arch. E x p t . Path. Pharmakol., 79,138-153 (1915). 8 WAGNER, Inaugural Dissertation, Leipzig, 1929. 4 FLASCHENTRXGER, AND WOLFFERSDORFF, Helv. Chim. Acta, 17, 1444-1452 (1934). 8 For a review of the recent chemistry of the toxic constituent of croton oil refer to the following articles: FLASCHENTRXGER, in Festschrift Heinrich Zsngger 11. Teil, pp. 857-873, Zurich, Leipzig, and Stuttgart, 1934; BOEHM,FLASCHENTR~LGER, AND LENDLE, Arch. E x p t . Path. Pharmakol., 177,212-220 (1935). 62
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were taken to make the separation from the fatty acids as nearly complete as possible. The substance resisted efforts to obtain crystalline derivatives, and consequently oxidation studies were considered the most likely means of obtaining crystalline degradation products. With this purpose in view the substance was methylated to protect hydroxyl and acid groups. The methylation was carried out in dry dioxane solution with the use of methyl iodide and silver oxide. The first product contained 8.4 per cent. methoxyl. This partially methylated material was further methylated twice with methyl iodide (in which it was now soluble) and silver oxide. The methoxyl content rose to 11.2 and 12.5 per cent., respectively. Fractional precipitation of the methylated substance was made by dissolving it in dry acetone and effecting partial precipitation by adding petroleum ether to the solution, redissolving the precipitated fraction, and repeating the process twice. In three precipitations 74, 17.6, and 7.8 grams remained in the acetone-petroleum-ether solution. These three fractions were combined, yielding a dark-colored viscous oil (I). The fraction insoluble in acetone-petroleum-ether was freed from solvent, and a light-brown amorphous powder (11), insoluble in cold aqueous alkali and containing 10 per cent. methoxyl, was obtained. Eighty-nine grams of the liquid (I) was subjected to fractional distillation at 1 mm. pressure in a Podbielniak column, and 34.6 ml. of distillate, representing several fractions, was collected. The undistilled residue was obtained as a dark-colored glass-like substance. From the distilled fractions were identified the methyl esters of caprylic, capric, and lauric acid which have been previously reported.' These acids were identified by determination of the saponification equivalents of the esters, as well as by preparation of the corresponding toluidides according to the method of Koelsch and Tenenbaum! In the distillate were also obtained some high-boiling, dark-colored, viscous fractions possessing a green fluorescence which did not yield crystalline toluidides. In the preparation of toluidides from the fractions consisting of the methyl esters of capric and lauric acids, small quantities of an etherinsoluble crystalline substance were obtained which analysis and molecularweight determinations showed to have the formula C ~ S H ~ O N ~This O~. corresponds to the toluidide of azelaic acid. Determination of the melting point of a mixture with the toluidide of authentic azelaic acid showed this to be the case. Azelaic acid has not been previously reported in the saponification products of the resin. It may not be present as such, however, but may be formed by the oxidizing action of silver oxide, during
* KOELSCH AND TENENBAUY, J . A m . Chem. Soc., 66,3049-3050 (1933). THE JOURNAL OF ORQANIC CHEMISTRY, VOL.
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JOSEPH R. SPIES
methylation, on the oleic acid known to be present. Its derivation from oleic acid would denote the presence of the A9Jo isomer. Saponification of the amorphous petroleum-ether-insoluble ester (11) with 10 per cent. alcoholic alkali yielded a dark-colored noncrystalline acid (111) containing 7.3 per cent. methoxyl, together with some ethersoluble resin. This shows the presence of hydroxyl groups in the molecule. The acid (111) is completely soluble in dilute, but not in concentrated, alkali. Ebulliscopic molecular-weight determinations in methanol gave values of 533 and 524 for I1 and 420 and 437 for 111. A molecular-weight determination by electrometric titration of I11 gave a value of 512 f 5. The equivalence point was a t pH 8.14, indicating a dissociation equal to that of the usual organic acid.1 The unsaturated character of the acid (111) was demonstrated by its reaction with alkaline permanganate and the precipitation of a bromide when its acetic acid solution was treated with bromine. This material has not been studied further. Boehm, Flaschentrager, and co-workers6 have reported the isolation of phorbol, a crystalline nontoxic polyhydroxy compound obtained by carefully controlled hydrolysis of a toxic constituent of croton oil. This interesting substance reacquires almost the original toxicity of the parent material when acetylated. While the author has not repeated this work, he has observed7 that methylation of croton resin yields a resinous product containing 11.7 per cent. OCHa, which is no longer toxic to goldfish or vesicant to man. Since this observation was first reported, an experiment has been carried out to show that this loss of activity was due to masking of the free hydroxyls of the resin and not to any oxidizing action of the silver oxide used in methylation. These facts suggest the possibility of the presence of more than one toxic substance in the resin mixture. An analogy to this loss of toxicity due to masking of hydroxyl groups is furnished by urushiol, the active constituent of both Japan lac. from Rhus vernicifera D c . and ~ poison ivy (Rhus toxicodendron) $, whose vesicant action closely resembles that of croton resin. Urushiol consists of a mixture of unsaturated isomers, which upon hydrogenation yields the single compound, hydrourushiol, or l-n-pentadecyl-2,3-dihydroxybenzene. Toyama'O first showed that urushiol dimethyl ether is not vesicant, and while hydrourushiol is less toxic than the unsaturated parent cam$ The author is indebted to s. A. Shrader for the ebulliscopic molecular-weight determinations and to D. H. Wheeler for the electrometric titration. 7 SPIES,J . A m . Chem. SOC., 67,182-184(1935). * MAJIMA,Ber., 66B, 172-191 (1922). HILL, MATTACOTTI, AND GRAHAM, J . Am. Chem. SOC.,66,2736-2738 (1934). 1 0 TOYAYA, J . Cutaneous Diseases, SO, 157-165 (1918).
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pounds, it also becomes inactive upon methylation. Hill and co-workersv verified the observations of Toyama regarding the important relation of the hydroxyl groups to toxicity. This relationship of hydroxyl groups to toxicity indicates the possibility in the case of croton resin, of isolating from the complex mixture the compound or compounds responsible for its physiological activity. Hydrolysis of the methylated resin (11.7 per cent. OCH3) by alcoholic alkali should leave the methoxyl groups intact, and the component containing them should be more stable and could then possibly be isolated from the saponification products. A preliminary experiment was conducted with this in view. The saponification products of some methylated resin were separated, and the methoxyl content of the fractions was determined as follows: Fatty acids, 5.5 per cent.; petroleum-ether-insoluble acids, 5.5 per cent. ; water-soluble constitutents, 13.1 per cent. ; an ethereal extract of the aqueous solution of the water-soluble constituents, 8.1 per cent. The acidified aqueous solution of the saponification products of the methylated resin, after removal of the acids, still gives a deep red-violet color with ferric chloride. No crystalline products were obtained. EXPERIMENTAL
Isolation of croton resin.-The resin used in this study was isolated by the method of Cherbuliez, Ehninger, and Bernhard” as modified by the author.” Anal. Found: C,68.3;H,8.90;OCH~,131.3,1.4. Saponification of the resin.-Four hundred eighty grams of resin was refluxed for two hours in 2 1. of 8 per cent. alcoholic potassium hydroxide under an atmosphere of hydrogen gas. The alcohol was evaporated, and the dark-colored residue waa dissolved in 1,200 ml. of water over which was stratified 700 ml. of petroleum ether (b.p. 50-70”). The mixture was vigorously stirred mechanically while it was slowly made acid t o Congo red with dilute sulfuric acid. The water layer was separated from the petroleum-ether layer, which contained the insoluble acids in suspension. These acids were separated and thoroughly washed with water. “Purification” of the petroleum-ether-insoluble acids.-For further separation of the fatty acids, the petroleum-ether-insoluble acids were dissolved in about 3 1. of 95 per cent. methanol, and the solution was thoroughly extracted with petroleum ether (b.p. 50-70”) the final portion of which removed 0.2 g. of substance. The methanol-water was evaporated, and the residue was dried by distilling absolute ethanol-benzene from it. The benzene was finally removed by vacuum distillation on the steam bath in a current of hydrogen; yield, 225 g. When thoroughly freed from solvent this substance is obtained as a brown amorphous powder. Methylation of the petroleum-ether-insoluble acids.-Two hundred twenty-five grams of the dried acids was dissolvedin 850ml. of anhydrous dioxane to which was added 220 CHERBULIEZ, EHNINGER, AND BERNHARD, Helv. Chim. Acta, 16,658-670 (1932). SPIEB,Ph.D. Dissertation, University of Maryland, 1934. l a Found by method described by CLARE,J . Assoc. 08.Agr. Chem., 16, 136-140 (1932). 11 12
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JOSEPH R. SPIES
ml. of methyl iodide and 200 g. of silver oxide in small portions. The mixture was stirred mechanically and refluxed for ten hours. The solvents were distilled off on the steam bath under reduced pressure, and the residue waa taken up in ethanol and filtered from the silver oxide. The product was again dried by distilling with benzene. The average methoxyl content was 8.43 per cent. This partially methylated product was methylated twice more, methyl iodide being used as solvent. The methoxyl content was 11.2 and 12.5 per cent., respectively. Fractional precipitation of the methylated product.-The methylated material (12.5 per cent. OCHa) was dissolved in 150 ml. of dry acetone, to which solution was added with stirring 600 ml. of petroleum ether (b.p. 50-70"). The addition of the petroleum ether caused precipitation of a dark, tarry mass which was separated from the solution. The acetone-petroleum ether solution contained 74 g. of dissolved ester. This treatment was repeated twice on the insoluble mass, and 17.2 g. and 7.8 g. of it remained in solution on successive treatments. The combined soluble fractions were obtained as a viscous dark-colored liquid (I). The solvent was removed from the fraction insoluble in petroleum-ether-acetone in a vacuum desiccator, and 108.5 g. of an amorphous brown powder (11) containing 10.0 per cent. methoxyl was obtained. Anal. (11). Found: C, 68.00,67.86; H, 7.57,7.52. Alcoholic alkaline saponification of I1 yielded a dark, amorphous acidic material. This substance was extracted with ether, in which about 20 per cent. was soluble. The insoluble amorphous powder (111) contained 7.3 per cent. methoxyl. Fraction I11 is completely soluble in dilute, but not in concentrated, aqueous alkali. Anal. (111). Found: C, 66.85, 66.91; H, 7.04, 7.04. Bromination.-To a solution of 0.5 g. of I11 in 25 ml. of glacial acetic acid bromine was added t o excess. The amorphous precipitate was filtered off, washed with water, and dried; yield, 0.1 g. Anal. Found: C, 49.18, 49.67; H, 5.20, 5.33. The mother liquor was poured into water and another bromine-containing amorphous precipitate was obtained. It was filtered, washed, and dried; yield, 0.47 g. The ester (11) also yields an amorphous bromide. Effect of silver oxide on the toxicity of croton resin.-Two and three-tenths grams of croton resin dissolved in 50 ml. of absolute ether to which 0.3 ml. of water waa added was refluxed with 2 g. of silver oxide for 46 hours. The resin was recovered and its toxicity to goldfish a t a concentration of 0.5 mg. per liter at 26.6" was determined. Four fishes were killed by this resin in an average of 20 minutes, while, as previously reported, the methylated resin did not kill fish in stronger concentration in 1740 minutes. * * Identification of azelaic acid.-Eighty-nine grams of the liquid esters (I) was fractionally distilled a t 1 mm. pressure with the use of the Podbielniak column. Several fractions were obtained, and, in the preparation of the toluidides of the fractions corresponding to the methyl esters of capric and lauric acids, about 0.7 g. of an ether-insoluble crystalline substance was obtained. When recrystallized to constant melting point from methanol, i t melted sharply at 201-202' (a standardized Anschutz thermometer was used). The melting point of the toluidide of azelaic acid is not recorded in the literature, but analysis and molecular-weight determinations corresponded to this substance. The toluidide of authentic azelaic acid melted a t 201202", and the melting point of a mixture showed no depression.
** The author wishes to thank W. A. Gersdorff for these tests.
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Anal. Calc’d for CZJH~ONPO~: C, 75.33; H,8.25; N. 7.65. Mol. wt., 366. Found: C, 74.89,75.09; H, 8.19, 8.28; N, 7.68, 7.85. Mol. wt. (Rast); 338, 335. SUMMARY
1. The petroleum-ether-insoluble substance obtained by saponification of croton resin has been shown to be a complex mixture of unsaturated polyhydroxy acids of high molecular weight. 2. Azelaic acid has been shown to be present in the saponification products, either as such or derived, by oxidation, from one of the unsaturated fatty acids known to be present. Its derivation from oleic acid would denote the presence of the A9J0 isomer. 3. Croton resin does not lose its toxicity to goldfish on prolonged boiling of its moist ethereal solution with silver oxide. 4. An analogous relationship between croton resin and urushiol from poison ivy and Japan lac is pointed out. In both substances the hydroxyl groups are intimately associated with toxicity. 5. Attention is directed to evidence of the presence of more than one toxic substance in the resin mixture.
