[CONTRIBUTION F R O M THE
DEPARTMENT O F CHEMISTRY
O F COLUMBIA UKIVERSITY]
INVESTIGATIONS ON LOCO WEEDS. I. T H E ISOLATIOX OF a- AND 0-EARLEINE FROM ASTRAGALUS EARLEI DOSALD C . PEASE1
AND
ROBERT C. ELDERFIELD
Received October 18, 1939
The occurrence of so-called “loco weeds” in the range country of the Western states has been the cause of rather serious economic losses resulting from the consumption of such weeds by grazing live stock. At the present time, knowledge of the toxic agent or agents in the weeds is meager. The general group of “loco weeds” may be conveniently divided into two classes: those containing selenium, in which parf, if not all, of the toxicity is attributed to the presence of compounds of this element, and the nonseleniferous weeds, in which the toxicity is due to substances other than selenium compounds. The more extensive investigations of the seleniferous “loco weeds” have been reviewed by Trelease and Martin (l),and the less detailed work on the non-seleniferous weeds is summarized in various Government publications (2). Investigations of both classes of weed have been largely in the pharmacological and veterinary field. We have been able to find but two records of recent chemical investigations of non-seleniferous “loco weeds.” Couch (3) succeeded in freeing the toxic principle of Oxytropis lambertii, or white loco, from many harmless substances, but did not obtain it in crystalline form. Fraps (4) reported the isolation of an admittedly impure, although highly toxic, crystalline tartrate of an alkaloid from Astragalus earlei, or Big Bend loco. To the alkaloid he gave the name “locoine.” To these studies may be added the earlier work of Prescott ( 5 ) , Power and Cambier (6)) and Crawford (7). Through the kind cooperation of Dr. Frank P. Mathews, of the United States Department of Agriculture Loco Weed Laboratory a t Alpine, Texas, we have secured considerable quantities of Astragalus earlei. This material served in the present investigation. At the outset, careful quantitative selenium determinations on the whole weed by the method of Williams and Lakin (8) showed that this element, if present a t all, was present to the extent of less than one part per million of the dried weed. Since concentrations of selenium in excess of this amount appear to be necessary to produce selenium poisoning in animals (9)) complications from this source appear to be excluded. Du Pont fellow in chemistry, 1938-1939. 192
EARLEINES FROM LOCO WEED
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Ait first, when the general method of Fraps (4)was applied to the extraction of the weed, the impure tartrate described by him was apparently obtained. The finely ground, dried weed was extracted with alcohol and the extract was clarified with basic lead acetate. The toxic principle was then dissolved from much resinous material by boiling absolute alcohol. Precipitation with phosphotungstic acid, followed by decomposition of the precipitate with barium hydroxide, gave an aqueous solution suitable for the preparation of salts. Further recrystallization of the crude tartrate prepared from such a solution resulted in the isolation of pure potassium tartrate. The toxic material accumulated in the mother liquors. We therefore conclude that Fraps’ crude “locoine tartrate” contained potassium tartrate mixed with toxic material. The problem of the isolation of nitrogenous organic substances was thus complicated by the presence of relatively large amounts of potassium in the weed, a difficulty rendered more annoying by the fact that the solubilities of various salts of the organic bases closely approximated those of the corresponding salts of potassium. Elimination of the troublesome potassium ions by the use of various zeolites was explored. The aqueous extrrtct of the dried weed, after clarification with basic lead acetate, was passed through a column of “Zeo Karb H” (a zeolite manufactured specifically for exchanging hydrogen ions for alkali or alkaline earth ions), which had been activated prior to use by two per cent hydrochloric acid. The anticipated exchange of potassium ions for hydrogen ions took place, but the effluent from the column contained no detectable nitrogen, nor did it contain material precipitable with phosphotungstic acid. Elution of the column with dilute hydrochloric acid gave no better results. Better success attended the use of “Decalso” (a zeolite which exchanges alkaline earth ions for those of the alkali metals). Passage of the clarified aqueous extract of the weed over “Decalso,” which had been activated previously with barium chloride solution, gave a solution which contained nitrogen and which also contained bases precipitable with phosphotungstic acid. From this solution we have succeeded in isolating two presumably pure nitrogenous bases in the form of their salts, and a considerable amount of d-pinite. The details of the purification of the latter, and its isolation from white loco are described in the following paper. As more information concerning the properties of the two bases accumulated, it was possible to eliminate the use of “Decalso,” although it appears that this material will be helpful in the isolation of other constituents of the weed. We suggest the names a- and p-earleine for the two nitrogenous substances. The bases are strikingly similar in their general properties, and their separation was accomplished only by chromatographing a solution of their picrates over aluminum oxide. The empirical formula of a-earleine,
194
DONALD C. PEASE ANI) ROBERT C. ELDERFIELD
as derived from analyses of the picrate, styphnate, and hydrobromide, corresponds to that of a triacidic base, (Cl~Hs7Na07),.p-Earleine similarly appears to be a triacidic base, (ClaHs7N304),. With the amount of material available it has not been possible to determine exactly the molecular sizes of the two bases. Both substances resemble quarternary ammonium hydroxides in their general behavior, and apparently contain a methyl carbinol group, and at least one primary amine group. They are optically inactive. In the isolation of a- and p-earleine we have used cats to follow the course of the toxic activity of the weed. Our observations in general parallel those of Fraps (4). It is necessary to feed a crude extract of from one to two kilos of dried weed in order to produce definite symptoms of locoism. The disease manifests itself by lack of muscular coordination which may result in collapse when the cat jumps voluntarily from a height as low as six inches. The hind legs seem stiff and the animal walks with a crouching gait. The eyes assume a staring appearance and the head shakes continuously. Drs. Abner Wolf and Homer Kesten, of the College of Physicians and Surgeons, have examined the gross and micropathological symptoms of a number of locoed cats. Their findings will be reported elsewhere. Xeither a- nor /3-earleine appears to be toxic to cats. However, we have secured several other fractions of material from the weed which are highly active and from which impure crystalline material has been isolated. Work on these fractions as well as structural studies on the two bases here described is being continued. We wish to express our appreciation for the kind cooperation of Dr. F. P. Mathews in collecting the weed used in this investigation, and of the Permutit Company, who generously contributed the special zeolites used. EXPERIMENTAL
All melting points are corrected. Extraction and isolation of a- and 8-earleine. As 6nally developed, the procedure for the isolation of the two bases as picrates is as follows. One hundred pounds of finely ground Astragalus earlei was soaked overnight in 20 gal. of 70% alcohol and was then pressed out in a hydraulic press. The process was repeated twice more, the third extract being used for the first extraction of a subsequent batch. The combined extracts were concentrated at reduced pressure at 40" t o a volume of about 5 1. Fifteen liters of water was added to the concentrate, and the clear, supernatant liquor was decanted from the water-insoluble portion. Ten liters more of water was added t o the residue and, after agitation, the supernatant liquor was again decanted. The combined aqueous extracts were treated with excess basic lead acetate solution and the precipitate was filtered through a layer of diatomaceous earth. After removal of the excess lead with hydrogen sulfide, the solution retained practically all of the toxic activity of the original weed.
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The solution was divided into four equal parts and concentrated at reduced pressure a t 40" to a thick syrup. The residual water was removed by repeated concentration with absolute alcohol and benzene. Each of the resinous residues was extracted with 3 1. of commercial absolute alcohol with vigorous agitation a t 55". Sfter standing overnight in the refrigerator, the supernatant alcoholic extract was decanted and the residual resin was again extracted with 1 1. of absolute alcohol per portion. The combined alcoholic extracts from the four portions were concentrated to about 5 1. and refrigerated. On standing, with occasional scratching, d'-pinite gradually crystallized. After the pinite was filtered, the solution was concentrated to about 3 1. and again refrigerated. After 2 or 3 days, crystallization of pinite was substantially complete. The filtrate from the pinite was concentrated to a resin, 2 1. of water was added, and about 1 1. of solvent was removed under reduced pressure in order to remove traces of alcohol. The volume was then made up to about 7 1. with water and the solution was filtered from a slight amount of insoluble material. This solution contained the major portion of the toxic material present in the original weed. A 24% solution of phosphotungstic acid in 4% sulfuric acid was added t o the aqueous solution obtained above until precipitation was complete. The supernatant liquid was immediately decanted from the heavy curdy precipitate, which was quickly washed with five 500-cc. portions of water before i t congealed. The precipitate was combined with any that had come down in the mother liquor and washings, suspended in a mixture of 1.5 1. of water and 1 1. of acetone, and then decomposed as usual in the cold with barium hydroxide. About one-half of the original toxic material was present in the solution of the decomposed phosphotungstates. This solution was concentrated to dryness under reduced pressure and the residue was dissolved in 1400 cc. of warm 95% alcohol. Saturated alcoholic picric acid solution was added until the solution was just acid to bromphenol blue. Two and eight-tenths liters of benzene was added and the solution was cooled. This solution of picrates was passed by gravity through an adsorption column of aluminum oxide (Baker and Adamson, reagent grade) 120 cm. long and 3.3 cm. in diameter. The column gradually became completely colored and the liquid running out the bottom was yellow. After passage of all the solution, the column was sucked as dry as possible a t the water-pump. a-E:arZeine. The upper two-thirds of the aluminum oxide column was eluted with hot 95% alcohol. On concentration of the eluate, a picrate crystallized. The yield was about 1.5 g. per 100 lb. of dried weed. After five recrystallizations from alcohol, the picrate formed long, silky, yellow needles and melted constantly a t 250". It wa3 optically inactive. Although the picrate appeared to be homogeneous, i t apparently contained sodium and deflagrated on attempted combustion. A sodiumfree picrate was obtained by dissolving the substance melting a t 250" in water, acidifging the solution with sulfuric acid, extracting the picric acid with benzene, and reprecipitating the base as the phosphotungstate. a-Earleine picrate, obtained as before by decomposition of this phosphotungstate, melted constantly with some decomposition at 184" after recrystallization from alcohol. A n d . Calc'd for (ClaH37N307.3CeHsNa07),: C, 38.1; H, 4.3; N, 15.7. Found: C,38.3;H, 4.1; N, 15.6. a-Earleine styphnate melted with decomposition a t 186-188" after recrystallization firom alcohol. A n d . Caic'd for (C1eHsrNsO?.3CsHsNsOs).:C, 36.5; H, 4.2; N, 15.0. Found: C, 36.8;H, 4.1; N,15.0.
196
DONALD C. PEASE AND ROBERT C. ELDERFIELD
a-Earleine hydrobromide was prepared by adding cold, dilute alcoholic hydrobromic acid to an alcoholic solution of the base. The hydrobromide crystallized from its strongly chilled alcoholic solution as rectangular prisms which melted a t 225" with partial sublimation. Anal. Calc'd for (ClsHarNsOi.3HBr),: C, 30.7; H, 6.6; X, 6.7; Br, 38.4. Found: C, 31.1, 30.8; H, 6.2, 6.6; N, 7.0; Br, 40.1. The free base, a-earleine, formed colorless prisms from absolute alcohol. I t is insoluble in acetone and ethyl acetate. It is so hygroscopic that an accurate melting point determination was impossible. The analytical data are probably unreliable for the same reason. Anal. Calc'd for (CisHarNsO7),: C, 50.1; H, 9.7; N, 11.0. Found: C, 48.1; H, 10.2; N, 11.2. a-Earleine is saturated as far as immediate reaction with permanganate is concerned, but is slowly oxidized by this reagent when warmed on the steam-bath. Bromine water is not decolorized by the base. Dilute ferric chloride gives neither a color nor a precipitate. The base gave a positive iodoform test, but did not react with the usual carbonyl reagents. Its behavior with nitrous acid and with benaenesulfonyl chloride indicates the presence of a t least one primary aliphatic amine group. Its alcoholic solution gives a precipitate with alcoholic mercuric chloride solution. The Molisch test and biuret test were negative. The substance did not contain methoxyl groups. p-Earleine. The solution which passed through the aluminum oxide column was concentrated, and gave about 25 g. of crude 8-earleine picrate per 100 lb. of dried weed. After 5 recrystallizations from alcohol, the picrate formed bright yellow, rectangular prisms and melted constantly a t 247". Although this melting point is close to that of a-earleine picrate before removal of sodium, a mixture of the two melted a t 190". When either the sodium-containing or-earleine picrate or p-earleine picrate is chromatographed over aluminum oxide in 2:l benzene-alcohol solution, but a single ring is formed. A mixture of the two picrates under similar conditions gives two rings. p-Earleine picrate is optically inactive. Anal. Calc'd for ( C i e H s ~ ~ ~ 0 a . 3 C ~ H ~ N C,a39.8; O ~ )H, ~ : 4.4; N, 16.5. Found: C, 39.8, 39.6; H, 4.7, 4.7; N, 16.6. The styphnate melted with decomposition a t 209" with sintering above 180" after recrystallization from alcohol. Anal. Calc'd for (ClsHs~NaO4.3C6HaPI'sOs)r:C, 38.1; H, 4.3; N, 15.7. Found: C, 37.9; H, 3.9; N, 15.5. p-Earleine hydrobromide formed irridescent crystals on strong chilling of its alcoholic solution. It melted with decomposition a t 296" on slow heating, and at 304" on more rapid heating. It is so hygroscopic that accurate analytical data could not be obtained. The free base, p-earleine, melted with decomposition a t about 187" after recrystallization by careful addition of dry acetone to its solution in absolute alcohol. The base is so hygroscopic that analytical data were impossible to obtain. It is saturated towards permanganate in the cold but is slowly oxidized by this reagent on standing. It does not decolorize bromine water. Ferric chloride solution gives a dark precipitate, and excess of the reagent dissolves the precipitate with evolution of gas. The action of nitrous acid and of p-toluenesulfonyl chloride indicates the presence of at least one primary a1iph:ttic amine group. The base gives a very strong iodoform test but does not react with the usual carbonyl reagents. Precipitates were obtained with silicotungstic acid and with alcoholic mercuric chloride solution. The biuret and Molisch tests were negative. When 8-earleine is heated in a test
EARLEINES FROM LOCO WEED
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tube, i t decomposes and gives off a gas with the odor of a lower aliphatic amine. We have not had sufficient material to identify this amine, but the general behavior is characteristic of quarternary ammonium bases. Crystalline acetyl and benzoyl derivatives of 6-earleine have been prepared] but in insufficient quantity for purification, The micro-analyses here reported were performed by Mr. Saul Gottlieb of these laboratories.
NEWYORK,N. Y. REFERENCES (1) TRELEASE AND MARTIN, Bot. Rev., 2, 373 (1936). (2) V, S . Dept. of Agriculture Farmers Bull. 1064 (Nov. 1936); Texas Agriculture Exp. Sta. Bull. 466 (Sept. 1932). (3) COUCH, J . Pharmacol., 36, 55 (1929). (4) FRAPS,Texas Agriculture Exp. Sta. Bull. 637 (Nov. 1936). Am. J . Pharm., 60,564 (1878). (5) PRESCOTT, (6) POWER AND CAMBIER, Pharm. Rund., 9, 8 (1891). (7) CRAWFORD, Bull. 129, U.S. B. Plant Industry, (1909). (8) WILLIAMSAND LAKIN,Ind. Eng. Chem., Anal. Ed., 7, 409 (1935); Robinson, Dudley, Williams and Byers, ibid., 6, 274 (1934). (9) Chem. Agriculture Exp. Sta. South Dakota State College of Ag. and Mech., Brookings, D., Bul2. 311 (May 1937).
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