[CONTRIBUTION FROM THE BUREAU OF ENTOMOLOGY AND PLANT QCARANTIXE, u. DEPARTMENT OF AGRICULTURE, WASHINGTON, D. C.]
s.
CONSTITUENTS O F PYRETHRUM FLOWERS. X. IDENTIFICATION OF THE FATTY ACIDS COMBINED WITH PYRETHROLONE FRED ACREE, JR.,
.4ND
Received August
F. B. LAFORGE
4, 1937
The active principles of pyrethrum flowers are usually isolated as semicarbazones from concentrates obtained from petroleum ether extractives that have been subjected to certain purifications and separations as described in previous publications1, 2. The semicarbazone of pyrethrin I1 can be obtained as a chemical individual with definite physical and chemical properties. The seinicarbazone of pyrethrin I, on the other hand, never has been prepared in a state of similar purity, and preparations of this derivative always exhibit an unsharp melting point, even after repeated recrystallizations, and they always contain methoxyl. It is evident that such preparations consist of mixtures which cannot be separated by ordinary mcms. Pyrethrin I semicarbazone has already been studied, and the results have been r e p ~ r t e d . ~ The procedure consisted in examining the products of the hydrolysis of a purified semicarbazone preparation. By saponification carried out in the usual manner, only pyrethrolone semicarbazone was obtained as the neutral alcoholic component. The acid products, however, were found to consist of a mixture in which, as was to be expected, chrysanthemum monocarboxylic acid strongly predominated. The remaining acid products not volatile with steam comprised chrysanthemum dicarboxylic acid and one or more acids of the fatty series. The fatty acids could be separated from the dicarboxylic acid and, after distillation, consisted of a semi-solid product which by analysis and titration seemed to correspond to the formula C18H3002. Its methyl ester (b. p. a,bout 155" at 1 mm.) was also prepared, and its analysis and methoxyl content were found to agree with the formula C17H3202. The acid product absorbed one mol of bromine and, on hydrogenation, took up one mol of hydrogen; therefore it was unsaturated. When this product was treated with thionyl chloride, an acid chloride was LAFORGE AND HALLER, J . Am. Chem. Soc., 67, 1893 (1935). HALLERAND LAFORGE, J. ORG.CHEM.,1, 38 (1936). 3 LAFORGE AND HALLER, i b i d . 2, 56 (1937). 308
1 2
CONSTITUENTS O F PYRETHRUM FLOWERS
309
obtained which, when treated with ,p-bromoaniline, gave a small yield of a bronioanilide melting at 109-110". On analysis this derivative agreed with the corresponding derivative of an acid of the above formula. The results described led to the conclusion that the acid had the formula C1e€I3002. A discordant fact was observed in the case of the hydrogenated material of m. p. 53', the titration of which indicated it to have a molecular weight of about 290. The data so far reported show that preparations of pyrethrin I semicarbazone consist of mixtures containing more or less of pyrethrin I1 semicarbazone together with the semicarbazone of at least one pyrethrolone ester in which the acid component is an unsaturated fatty acid. Owing to the uncertainty of the nature of the fatty acid component, further study of the subject was indicated. The petroleum ether extractives of pyrethrum flowers always contain free fatty acids, which are removed in the course of the purification of the concentrates. It was surmised that these fatty acids might contain larger quantities of the acid which was combined with pyrethrolone. Therefore, a considerable quantity of the crude acid mixture was esterified with methanol, and the esters were subjected to repeated fractionation to obtain that fraction boiling around 130-150" at 0.5 mm. This material had about the same properties as the fatty acid ester obtained from pyrethrin I semicarbaeone. It was observed, however, that a part separated in crystalline form from a methanol solution on cooling in a freezing mixture. By this means a sharp separation into two fractions could be made. The crystalline ester, boiling at 130-135' at 0.5 mm., m. p. 27-28', was identified, by the mixture melting-point method, as the methyl ester of palmitic acid. The ester that was obtained from the methanol solution was subjected to fractionation at 0.5 mm. About 80 per cent. of it boiled at 140-146'. Analysis and methoxyl determination indicated the formula C19H3402. The ester was unsaturated, and absjorbed two moles of hydrogen. The free acid was prepared by saponification of the ester. It boiled at 170175" at 0.5 mm. The molecular weight was found by titration to be 282. The crystalline tetrabromide was prepared and melted at 112-113". These properties of the acid and its derivatives are in agreement with those recorded for linoleic acid and j ts corresponding derivatives. On hydrogenation of the free acid, two moles of hydrogen were absorbed and the resulting saturated acid melting at 69" was identified as stearic acid. The methyl ester of the unsaturated acid on hydrogenation yielded methyl stearate, which was identified as such by the mixture meltingpoint method. The original fraction therefore consisted of a mixture of palmitic and linoleic acids.
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FRED ACREE, JR., AND F. B. LA FORGE
Since the acid mixture obtained from the methyl ester fraction of b.p. 130-150" resembled so closely the fatty acid component obtained from pyrethrin I semicarbazone, it seemed probable that the latter also might consist of a mixture of the same fatty acids. Therefore, more of this material was prepared froni pyrethrin I semicarbazone. The method employed for the separation of the acid components differed from that recorded in the first article3 on this subject. The total acid material obtained on saponification of the semicarbazone was converted completely into the methyl esters. The methyl ester of chrysanthemum monocarboxylic acid and the dimethyl ester of chrysanthemum dicarboxylic acid have much lower boiling points than the esters of the fatty acids. Therefore, a separation could be made readily by fractionation of the total esters. The fraction boiling at 130-150" and 0.5 mm. was isolated in this manner. It was found that a methanol solution of this fraction also crystallized partly on cooling, and could be separated into solid and liquid components. The liquid ester was saponified to the free acid. On hydrogenation of the acid, two moles of hydrogen were absorbed, and the resulting saturated acid was identified as stearic acid by its melting point and titration. Finally the p-bromoanilide* was prepared from palmitic acid and found to agree as to properties and mixture melting point with the original pbromoanilide obtained from the acid mixture as described in the previous article3. Attempts to prepare the p-bromoanilide of linoleic acid have been unsuccessful. Therefore, the acid products other than the chrysanthemum acids obtained from the pyrethrin I semicarbazone consist mainly of a mixture of palmitic and linoleic acids, and these are present combined with pyrethrolone semicarbazone in all preparations of pyrethrin I semicarbazone. EXPERIMENTAL
Preparation of acids.-The potassium carbonate solutions, obtained during the purification of 8 pounds of py:rethrum oleoresin, were extracted with petroleum ether. The carbonate solutions were then acidified, and the free fatty acids were removed by extraction with the same solvent. The petroleum ether solution was washed with water, dried over anhydrous sodium sulfate, and the solvent was removed, yielding 105 grams of acid mixture. The acids thus obtained were converted into their methyl esters by boiling under reflux for two hours in 20 parts of methanol and 1 part of concentrated sulfuric acid. The methanolic solution was concentrated to a small volume under reduced pressure pressure, diluted with several parts of water, and extracted with petroleum ether. The petroleum ether solution was washed with sodium carbonate solution, then with water, and dried over anhydrous sodium sulfate. On removal of the sol-
* No reference to this compound has been found in the literature.
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vent, 112 grams of mixed methyl esters was obtained, which was distilled a t 0.5 mm. The fraction boiling at 135-150" was collected; i t weighed 42 grams. Palmitic acid methyl ester.-This ester fraction was dissolved in 3 parts of methanol and cooled for 4 hours at -6". The 14.5 grams of material that crystallized was removed by filtration in the cold. This crystalline material partly melted a t room temperature. By distilling 6.7 grams of the material at 0.5 mm., 6.3 grams was obtained, boiling a t 130-135". It again crystallized on cooling and was found to melt sharply at 28". Anal. Calc'd for ClrHadh: CHaO, 11.48; m. w., 270. Found: C&O, 11.4, 11.2; m. w. 271, 276. Palmitic acid.-One gram of the crystalline ester was saponified by dissolving in 10 cc. of alcohol and boiling under reflux for one and one-half hours with 12 cc. of 0.5 N alcoholic potassium hydroxide. The alcoholic solution was diluted m'ith water, and most of the alcohol was removed by boiling. After cooling, the alkaline solution was extracted with ether and then acidified. The free fatty acid was removed with ether, which was washed w.ith water and dried over anhydrous sodium sulfate. The solvent was then removed and gave 0.9 gram of crystalline material which, when recrystallized from methanol, melted at 62-63'. A mixture melting point made with an authentic sample of palmitic acid showed no depression. Anal. Calc'd for CMHSZO~: m. w., 2!i. Found: Equiv. wt. (titration) 256, 261. Lmoleic acid methyl ester.-The methanol solution, from which the palmitic acid methyl ester had been removed, was freed from solvent by evaporation and the 27 grams of residue was subjected to fractional distillation at 0.5 mm. Twenty grams distilled at 140-146'; ng = 1.4621; ng - n t = 0.009. Anal. Calc'd for C19Ht402:CHsO, 10.53. Found: CH30, 10.54, 10.73. One gram of ester was hydrogenated in acetic ester solution with platinum oxide catalyst. It absorbed 155 cc. (two molen) of hydrogen and gave 1 gram of a crystalline product. On recrystallization from methanol it melted a t 38" and showed no depression in the melting point when mixed with a n equal quantity of authentic methyl stearate. Linoleic acid.-On saponification by boiling under reflux for two hours with 80 cc. of 0.5 N alcoholic potassium hydroxide and subsequently removing any unreacted ester, 5.6 grams of the ester yielded 5.5 grams of acid that boiled at 170-175" at 0.5 mm.; nz = 1.4690; n2,' - nz = 0.009. Anal. Calc'd for C18HszOz: m. w., 280. Found: Equiv. wt. (titration) 283, 281. On the addition of 4 atoms of bromine, 0.5 gram of the acid was converted into the crystalline tetrabromide, yield 0.3 gram. After recrystdlization from petroleum ether it melted a t 112-113". The recorded melting point for tetrabromdlinoleic acid is 113-114". On hydrogenation in acetic ester solution with platinum oxide catalyst, the acid absorbed two moles of hydrogen. The crystalline reidduction product, after rearystallization from alcohol, melted a t 69". There was no depression in the melting point when this was mixed with an equal quantity of authentic stearic acid. Preparation of acids from pyrethrin I ssmicarbazone.-A quantity of twice-recrystallized pyrethrin I semicarbazone was saponified in the usual manner.2 After filtration from the crystalline pyrethrolone semicarbazone, the total acids were isolated from the alcoholic alkaline mother liquor by the usual procedure.
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FRED ACREE, JR., AND F. B. LA FORGE
Ten grams of these acids was converted into their methyl esters by boiling under reflux for two hours in 200 cc. of methanol containing 10 grams of concentrated sulfuric acid. After most of the methanol had been removed under redwed pressure, the residue was strongly diluted with water and extracted with petroleum ether. The petroleum ether solution was washed with water, dilute potassium hydroxide, and again with water. After being dried over anhydrous sodium sulfate, the solvent was removed. The yield of mixed methyl esters was 10.4 grams. Separation of methyl esters.--Twenty-two grams of mixed methyl esters was distilled a t 19 mm. Eighteen grams distilled a t 90-100" and consisted essentially of chrysanthemum monocarboxylic acid methyl ester together with some chrysanthemum dicarboxylic acid dimethyl ester. The remainder of the material was distilled at 0.5 mm. Of this 1.8 grams boiled at 110-135' and 0.6 gram boiled at 135-145". There was some material that would not distill readily. Palmitic acid methyl ester.--The material from another experiment comparable to this higher-boiling fraction was converted to the free acid, from which the acid chloride was prepared. The latter was treated with p-bromoanaline and gave a crystalline p-bromoanilide. The compound, twice recrystallized from methanol, melted a t 109-110". It showed no depression when mixed with an equal quantity of an authentic sample of the p-bromoanilide of palmitic acid. The yield of the p bromoanilide was small, but it corresponded t o a quantity of methyl palmitate proportionate to the amount of crystalline methyl ester obtained by freezing the methanolic solution of mixed inethyl esters of b. p. 135-145" as described below. p-Bromoanilide of palmitic acid.-One gram of palmityl chloride dissolved in 10 cc. of dry benzene was slowly added, with stirring, to a solution of 1.5 grams of pbromoaniline in 10 cc. of the same solvent. After standing for 30 minutes, the crystalline material that immediately separated was filtered and washed with benzene, dilute hydrochloric acid, and water. After recrystallization from alcohol i t melted at 114". It weighed 1.3 grams. A mixture with a sample of the p-bromoanilide of melting point 109-110" obtained from the acid isolated as a pyrethrolone ester melted a t 111-112". Linoleic acid methyl ester.--The 0.6 gram of liquid esters obtained from pyrethrin I semicarbazone of b. p. 135-145" at 0.5 mm. was dissolved in 3 parts of methanol and subjected to a temperature of -6". Two-tenths of a gram of impure palmitic acid methyl ester deposited and was filtered and washed with a little cold methanol. From the methanolic filtrate, after removal of the solvent, 0.4gram of oily material was obtained. This material was shown to be linoleic acid methyl ester by its conversion in good yield to the free acid. The acid was reduced catalytically with the absorption of two moles of h,ydrogen to stearic acid. The recrystallized reduction product of melting point 68" was identified by the melting point, 68-69", of a mixture with authentic stearic acid. Anal. Calc'd for C18H3b01; m. w., 284. Found: equiv. wt. (titration), 289. Quantity of fatty acids present i n a mixture of pyrethrin semicarbazones as pyrethrolone esters.-One hundred thirty grams of partly purified pyrethrum concentrate was converted to the mixture of pyrethrin I and pyrethrin I1 semicarbazones.2 The dried material, twice recrystallized from benzene and washed with petroleum ether, weighed 31.6 grams. 1. t was saponified in the usual manner, and the crystalline pyrethrolone semicarbazone was separated by filtration. The alcoholic mother liquor was concentrated to a small volume under reduced pressure and diluted with an equal volume of water. An additional quantity of pyrethrolone semicarbazone
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313
crystallized and was removed from the solution by filtration. The filtrate was made strongly alkaline and boiled under reflux for one hour. After being cooled, the solution was acidified and the acids were removed by extraction with petroleum ether. The petroleum ether solution was washed with water, dried over anhydrous sodium sulfate, and evaporated. Ten and one-tenth grams of acids was recovered. They were dissolved in 70 cc. (10 per cent. excess) of normal sodium hydroxide and diluted to 200 cc. with water. After the addition of 1 gram of Filter-cel, 25 cc. of a 10 per cent. solution of barium chloride was added, and the solution was thoroughly mixed. The precipitated barium salts were collected by filtration, washed, suspended in dilute hydrochloric acid, and decomposed by vigorous shaking. The free acids were extracted with petroleum ether, and the solution was mashed with water and dried over anhydrous sodium sulfate. The solvent was removed, leaving 0.3 gram of free fatty acids originally combined as pyrethrolone esters. SUMMARY
The semicarbaxone of pyrethrin I as ordinarily prepared from concentrates consists of a mixture of the semicarbazones of pyrethrins I and I1 together with the semicarbazones of the esters of pyrethrolone with palmitic and linoleic acids. This has been established by the isolation and identification of chrysanthemum mono- and dicarboxylic acids and palmitic and linoleic acids from the acid products of saponification of purified preparations of pyrethrin I semicarbasone. It is practically impossible to prepare pure pyrethrin I semicarbazone by recrystallization. Palmitic and linoleic acids are contained in the free state in the oleoresins of pyrethrum flowers and may be isolated by fractional distillation of the mixture of the methyl esters of the total free acids. The recrystallized crude semicarbazone mixture obtained from pyrethrum concentrates yields fatty acids on saponification.
