[ c O S T R I B U r I O S F R O M THG
E S R E A U O F ENTOMOLOGY AND P L A N T QUARANTIKE, ADMIXISTRATIOX, ET. s. DEPARTMENT OF AGRICULTURE]
A G R I C U L T U R 4 L RESE.4ltCH
AN AMIDE POSSESSING INSECTICIDAL PROPERTIES FROM THE ROOTS OF ERIGERON AFFINIS DC. FRED .4CREE, JR.,MARTIN JACOBSOX,
AND
H. L. HALLER
Received Marcki 10, 1945
Investigations of foreign and domestic plants are continually in progress in the Bureau of Entomology and Plant Quarantine for the discovery and development of new insecticides to replace those the supply of which has been curtailed by the war. In the course of these investigations attention was directed to the roots of the Mexican plant Erigeron ufinis DC.1 (family Compositae), commonly called “peritre del pais)’ and “chilcuan.” It grows in the vicinity of Mexico City, where the roots are employed in the preparation of native insecticides. That the petroleum ether extractive (3.30j0, dry roots) prepared in this laboratory from the ground roots contained the toxic constituents was indicated by tests against codling moth larvae, adult mosquitoes, and several leafeating insects, and it proved to have the same order of paralyzing action and toxicity to housefies as the pyrethrins.2 The toxic fraction (1.9%, dry roots) was isolated from the petroleum ether extractive by the method that Barthel, Haller, and LaForge (1) employed for the preparation of pyrethrin concentrates for use in aerosols. Briefly, the nitromethane-soluble portion extracted from the petroleum ether extractive was passed through a column of activated carbon. When the solvent was removed from the purified nitromethane solution at reduced pressure, an oily residue possessing insecticidal activity was obtained, which was distilled a t reduced pressure, and yielded 1.08% (dry-root basis) of light yellow distillate (b.p. 160-165”, p = 0.3-0.5 mm.), which crystallized when cooled but melted on standing a t room temperature. The substance produced a burning, paralytic effect on the tongue similar to that caused by pyrethrin concentrates. It mas soluble in organic solvents and contained nitrogen, but it was insoluble in aqueous alkali and acid. It rapidly decolorized a dilute chloroform solution of bromine and, on acid hydrolysis, yielded an acid and a base. Although the free acid was not identified, the base was found to be isobutylamkie by determination of the melting point and the chlorine content of the base-hydrochloride and the melting point of the basechloroplatinate, and by comparison with authentic materials. The substance 1 The first specimen of roots was submitted under this name by Agencias &electas, S..k.) Mexico City. Subsequent samples were obtained through the courtesy of the Division of Fruitfly Investigations, Bureau of Entomology and Plant Quarantine, Mexico City. Attempts are being made to obtain a botanical specimen for confirmation of this identification. * The tests against the codling moth were made by E. H. Siegler; againat mosquitoes by J. H. Fales and A . E. Routson; against several leaf-eating insects by Clemence Levin, and against the housefly by W. A . Gersdorff, all of this Bureau. Detailed results of their tests will be published elsewhere by them. 236
INSECTICIDE FROM ERIGEROK AFFINIS
23i
was thus characterized as an unsaturated isobutylamide, for which the name “affinin” is proposed. On the assumption that only one nitrogen was present, a molecular weight of about 221 was calculated for affinin from the analytical values for nitrogen. After hydrogenation in the presence of platinum oxide catalyst, the purified reaction product melted a t 37.5-38“. Calculations based on the analytical values for nitrogen (one nitrogen assumed) indicated that this product was hexahydroaffinin with a molecular weight of about 227. However, calculations based on the quantity of hydrogen absorbed indicated a molecular weight of about 233 for the unsaturated substance in contradiction t o the value 221 previously calculated from the nitrogen content. A choice appeared to exist, therefore, between two formulas for a h i n that differed from each other by CH2. Evidence presented below, however, shows that a formula corresponding t o the higher molecular weight is untenable. The data presented above, together with the similarity of the common names for the respective plant sources of the two substances, indicated that affinin might be related to pellitorine, C14H26N0, m.p. 72” (corr.), b.p. 162-165”, p = 0 . 3 4 . 5 mm., a constituent of Anacyclus pyrethrum. DC. (“pelitre”), isolated from the roots and characterized by Gullartd and Hopton (2) as the ?J-isolbutylamide of a, decadienoic acid. Pellitorine i,j an isomer of spilanthol (3), the N-isobutylamide of 4,6-decadienoic acid, b.p. 165’, p = 1 mm. Both pellitorine and spilanthol yield the same saturated tetrahydro derivative on hydrogenationnamely, N-isobutylcapramide, m.p. 37-38’. Hexahydroafbin was hydrolyzed in a sealed tube with ethanolic hydrochloric acid. The acid-soluble reaction product yielded isobutylamine hydrochloride equivalent t o approximately 1 mole. The alkali-soluble reaction product consisted of a small quantity of capric acid, which was identified by titration and by the properties of the p-bromophenacyl ester. Almost all of the acid fraction was esterifiedduring hydrolysis, and the neutral reaction product yielded an additional quantity of capric acid after having been hydrolyzed with ethanolic potassium hydroxide. The combined portions of capric acid were equivalent to approximately 1 mole. No other products of hydrolysis could be detected. Further proof that hexahydroafiin is identical with N-isobutylcapramide (and therefore with tetrahydropellitorine and tetr:thydrospilanthol) was gained when its melting point, mixture melting point, and refractive index were found to be the same as the corrcsponding constants of the synthetic compound and different from those of synthetic N-isobutylhendecanoamide. It remained then to determine the three points of unsaturation in affinin. Attempts t o establish the presence of a conjugated system of double bonds through its reaction with a-naphthoquinone and maleic anhydride were unsuoccessful. The spectrographic data3 p i g . 1) show an absorption maximum 2285 A. (E = 31,500). Froom a comparison of these constants withothose of conjugated diene imax. 2340 A, E = 25,000) and triene (max. 2680 A systems of double 3 These data were obtained and interpreted by R. E. Davis and Harry Bastron, U. P. Rure,iu of Animal Industry.
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ACREE, JR., JACOBSON, AND HALLER
bonds (4),it was concluded that a f h i n contained a conjugated diene system. Since the B value for the substance is much higher than that for the compound known to contain a single conjugated diene system, it was further concluded that a&in probably contained two isolated conjugated diene systems of double bonds. In keeping with this hypothesis and in view of the fact that analysis
3500
3000
h FIG.1.
(A)
2500
h S O R P T I O N SPECTRUM OF
2000
AFFININ
showed the presence of two terminal methyl groups, a f i i n could be represented only by, formula I or formula 11. CHI
CHI
0
\ II CH-CH2-NH-C-CH=CH-CH2-CH2-CHZ-CH=CH-CH=CH-CHa / I
INSECTICIDE FROM ERIGERON AFFINIS
CHs
239
0
n \ CH-CH~-NH-C-CH=C€E-CH~-CH=CH-CH=CH-CH~-CH~ /
CHs
I1
A compound of formula I would yield succinic acid on oxidation, while malonic acid would be obtained from one of formula 11. When a sample of a5nin was oxidized with alkaline permanganate, 8 crystalline acid was isolated from the reaction mixture, and it was identified as succinic acid. The yield was 36% based on a compound of formula I. It therefore appears that afEnin, a t least in part, is N-isobutyl-2,6,8decatrienoamide, represented by formula I. However, the steam-volatile acids isolated from the oxidation have not been identified. Thus there is no evidence available as yet to eliminate the possibility that affinin might be a constant-boiling mixture composed of the compounds represented by formulas I and 11. Further work to clarify this point is in progress a t the present time. EXPERIMENTAL'
Isolation of a l n i n . As a typical example, 1,976 g. of finely ground roots was extracted exhaustively by percolation with petroleum ether (b.p. 30-60°), and the marc was then extracted with several solvents. None of the extracts of the marc showed any insecticidal action, but when the solvent was removed from the petroleum ether extract the residue (65 g., 3.3% of dry roots) was toxic to several species of insects, and it was especially effective against houseflies. [A refined-kerosene (Deobase) solution of the petroleum ether extractive containing the equivalent of 62 mg. of roots per milliliter showed knockdown and toxicity t o houseflies comparable to that produced by a Deobase solution containing the equivalent of 94 mg. of pyrethrum powder (1.5% total pyrethrins) per milliliter.] The petroleum ether extractive (65 9.) was dissolved in 500 ml. of petroleum ether, and the toxic fraction was extracted (1) with five 100-ml. portions of nitromethane. The separated nitromethane solution was passed through an 8 by l .5 inch column of activated carbon. After elution with 300 ml. of nitromethane, the active material was recovered from the solution on removal of the solvent a t 15 mm. pressure in an atmosphere of nitrogen, and it weighed 37.8 g. (1.9% of roots). It partly polymerized during slow distillation in nitrogen and yielded 26.6 g. of distillate, b.p. 155-160°, p = 0.3-0.5 mm., that contained nitrogen. (Anal. Found: N , 6.45, 6.55.) When this fraction was redistilled in nitrogen a t a faster rate, less polymerization occurred and 21.3 g. (1.08%) of affinin was obtained, b.p. 160-165", p = 0.3-0.5 mm.; nz 1.5128; X max., 2285 A; e = 31,500. The substance was soluble in organic solvents but insoluble in aqueous alkali and acid, and i t rapidly decolorized a chloroform solution of bromine. tinal. Calc'd for C,rH&O: N, 6.32; 2CH3, 13.6; Mol. wt. 221. Found: N, 6.34, 6.30; CHs, 11.5, l l . l ; s Mol. wt. (calc'd for 1 N) 221. Acid hydrolysis of afinin. A mixture of 2 g. of affinin, 15 ml. of ethanol, and 5 ml. of concentrated hydrochloric acid was heated in a sealed tube at 100" for 91 hours and then cooled. The reaction mixture was diluted with 3 volumes of water and then extracted with ether. The separated acid solution of basic material is described below. The ether solution was washed free of mineral acid and dried, and the solvent was ermoved. The residue was boiled on reflux for 2 hours with 1 g. of potassium hydroxide
4
All melting points reported are corrected. These analyses were made by W. F. Barthel of this Bureau.
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ACREE, JR., JACOBSON, AND HALLER
dissolved in 50 ml. of ethanol; the reaction mixture, after being cooled and acidified, wa8 extracted with ether. The ether solution was washed free of mineral acid and dried, and the solvent removed. The oily acid residue (1.3 g.) obtained was completely soluble in an aqueous solution of sodium bicarbonate, but the acid was not identified. The original hydrochloric acid solution of basic material was evaporated t o dryness, yielding 0.78 g. of crystalline product. After two recrystallizations from ethyl acetate, the separated product (0.5 g.) melted a t 174-175'. It was identified as isobutylamine hydrochloride by the mixture melting point determination with authentic material, m.p. 174175".
A n a l . Calc'd for CrHllN.HCl: (3, 32.01. Found: C1, 31.99,31.59. The chloroplatinate was prepared by the addition of 5 drops of a saturated aqueous solution of chloroplatinic acid to 50 mg. of the base-hydrochloride dissolved in 3 ml. of ethanol. The crystalline product (87 mg.) that precipitated was filtered, washed with water and ethanol, and dried. It melted a t 222-223' (dec.), and the melting point was not depressed by isobutylamine chloroplatinate, m.p. 222-223" (dec.), prepared as just described from authentic material. Preparation of hexahydroaffnin. An ethanol solution containing 2.196 g. of affinin was shaken in an atmosphere of hydrogen with reduced platinum oxide catalyst. The reaction proceeded rapidly and then stopped when 632 ml. (corr.) of hydrogen had been absorbed. (This volume corresponds to the requirement for 3 moles of hydrogen by the above weight of a substance of molecular weight 333.) The reaction mixture was separated from the catalyst, and the solvent was removed at reduced pressure. The crystalline residue (2.3 g.) was dissolved in petroleum ether and cooled in solid carbon dioxide. The separated product melted a t 37.5-38", but the yield was low. An almost quantitative yield was obtained, however, when the product was purified by distillation. The distillate, b.p. 123-125", p = 0.2-0.3 mm., was collected in three fractions, all of which had the same refractive index,
n'f;' 1.4440. A n a l . Calc'd for ClrHtgNO: X , 6.16. CISHSINO: N , 5.80. Found: N, fraction 1,6.OB, 6.11; fraction 2,6.16,6.14; fraction 3,6.10,6.11. The three fractions of hexahydroaffinin were combined, m.p. 37.538'. The product waa found to be identical with X-isobutylcapramide (mixture m.p. 37.5-38") and different from N-isobutylhendecanoamide (mixture m.p. 33.548") when mixed with authentic specimens of these two compounds. These compounds were prepared for comparison by reaction of one molar equivalent of the corresponding acid chloride with two molar equivalents of isobutylamine in dry ether solution. The reaction products, upon separation from the ether and distillation, had the following properties: N-isobutylcapramide, b.p. 123-126", p = 0.2-0.3 mm.; n4i61.4440; m.p. 38-38.5" ( A n a l . Calc'd for Ct&9KO: K, 6.16. Found: X, 6.15, 6.20). N-isobutylhendecanoamide, b.p. 136-137", p = 0.2-0.3 mm.; n'; 1.4414; m.p. 50.5-51' ( A n a l . Calc'd for CllHSINO: N , 5.80. Found: N , 5.82, 5.76). Acid hydrolysps of hexahydroaffinin. Affinin (1.599 g.) dissolved in 15 ml. of ethanol was hydrogenated as described above; 477 ml. (corr.) of hydrogen was absorbed. The reaction mixture (separated from the catalyst) was mixed with 4 ml. of concentrated hydrochloric acid in a sealed tube, heated at 100" for 118 hours, and then cooled. The contents of the tube were diluted with water and extracted with ether. The hydrochloric acid solution was separated and evaporated to dryness. It yielded 0.52 g. of crystalline residue, and on recrystallization of the residue from ethyl acetate 0.5 g. of crystalline product was separated. It melted a t 174-175", and was identified as isobutylamine hydrochloride by a mixture melting point determination with authentic material. The ether solution was extracted with a dilute solution of sodium carbonate, washed with water, and dried. On removal of the solvent, the ether solution yielded 1.1 g. of neutral residue. The carbonate solution was acidified and then extracted with ether. hfter the ether solution was washed and dried, and the solvent removed, 0.2 g. of acid residue remained, which crystallized readily when cold. The residue was recrystalliced from methanol, and the acid (0.18 e;.) that was separated melted at 29.530".
INSECTICIDE FROM ERIGERON AFFINIS
241
A n a l . Calc'd for CloI-IaoOz: Mol. wt., 172. Found: Mol. wt. by titration, 1.73, 174. This substance was identified as capric acid by comparison of the p-bromophenacyl ester with authentic material. The ester was prepared according t o the method of Hann, Reid, and Jamieson (5) by the reaction, after neutralization, of 72.6 mg. of the acid with 118 mg. of p-bromophenacyl bromide. 'rhe.separat,ed crystalline ester (99 mg.) was recrystallized from dilute ethanol and melted a t 64.5-65". AnaE. Calc'd for C18H25BrOa: Br, 21.68. Found: Br, 21.54, 21.25. The ester was identified as p-bromophenacyl caprate by the mixture melting-point determination with a sample of ester prepared as just described from authentic capric acid and which melted a t 65-65.5" ( A n a l , Calc'd for ClsH2sBrOs:Br, 21.68. Found: Br, 21.67, 21.45). The 1.1g. of neutral residue isolated from the ether solution was dissolved in 20 ml. of an ethanol solution containing 5% of potassium hydroxide. The solution was boiled under reflux for 5 hours, and then dilutedwith water and extracted with ether. The ether solution was dried and, on removal of the solvent, yielded 0.3 g. of apparently unchanged hexahydroaffinin ; The alkaline solution was acidified, y.ielding an oily precipitate, which was dissolved in ether. The ether solution was washed and dried and then, on removal of the solvent, yielded 0.M g. of acid residue, which crystallized when cold. It was twice recrystallized from methanol, and the 0.79 g. of crystalline acid that was separated melted a t 29.5-30". Anal. Calc'd for CloH200e:Mol. wt., 172. Found: Mol. wt. by titration 173. The substance was identified as capric acid by the mixture melting point determination, and it was combined with the 0.18-g. portion of capric acid isolated as described above. The total quantity of capric acid isolated (0.97 8 . ) represented 0.96 mole. Ozidation o j a f i n i n . T o 1 g. of affinin continuously stirred in 200 ml. of warm water, 10.2 g . (25% excess for 8.5 moles of oxygen) of powdered potassium permanganate was added in small portions. When the reaction mixture had become colorless the manganese dioxide was filtered and washed thoroughly with warm water. The aqueous filtrate was evaporated to about 50 mi. and acidified with sulfuric acid. The acidified solution was steam-distilled t o remove the volatile acids (15.57 ml. of N NaOH) and then extracted with ether in a continuous extractor. After removal of the solvent the ether solution yielded 0.75 g. of residue, which crystallized a t once. The residue was recrystallized from ethyl acetate and 0.19 g. of crystalline product was separated, m.p. 185-186". A n d . Calc'd for CIHaO,: Mol. a t . , 113. Found: Mol. wt. by titration, 119. The substance was identified as succinic acid by the mixture melting point determination with authentic material, m.p. 185-186". A dark oily residue (0.5 9 . ) was obtained upon removal of the solvent from the combined ethyl acetate mother liquors, The residue gave a positive test for nitrogen and contained a small quantity of crystalline material. This product was not investigated. Reaction of ui$inin with maleic anhyds.de and with a-naphthoquinone. AEnin (2.2 g . ) , was heated a t 100" for 4 hours with 0.98 g. of maleic anhydride. Upon addition of water to the reaction mixture a rubbery mass was precipitated, but nothing of a definite character could be isolated. Affiriin (0.5 g.), together with 360 mg. of a-naphthoquinone dissolved in 5 ml. of ethanol, was heated at 100" for 2 hours in a sealed tube and then cooled. The contents of the tube deposit,ed 270 mg. of crystalline material, which was separated and recrystallized from ethanol. The product melted a t 125", and was identified as &-naphthoquinone by the mixture melting point determination with authentic mat,erial. SUMMARY
An iaobutylamide of an unsaturated Cloacid has been isolated from the roots of Erigeron afinis DC. The amide, for which the name "afhh" is proposed,
242
ACREE, JR., JACOBSON, AND HALLER
has the same order of paralyzing action and toxicity to houseflies as the pyrethrins, and is toxic to several other species of insects. On hydrogenation the amide was converted i o N-isobutylcapramide. BELTSVILLE, MD. REFERENCES (1) BARTHEL, HALLER,AND LAFORGE, Soup and Sanit. Chem., 20 (7), 121 (1944). (2) GULLAND AND HOPTON, J. Chsm. Soc., 6 (1930). (3) ASANOAND KANEMATSU, Ber., 66, 1602 (1932). (4) MITCHELL, KRAYBILL, AND ZSCHEILE,Ind. Eng. Chem., Anal. E d . , 1 6 , l (1943). (5) HA", REID,AND JAMIESON, J. A m . Chem. Soc., 62,818 (1930).
