[CONTRIBUTION FROM TEE LABORATORIO DE QU~MICA O R Q ~ I C AFACULTAD , DE CIENCIASEXACTAS,F~SICAS Y NATURALES AND INSTITUTO DE FISIOLOQ~A, FACULTAD DE CIENCIAS M&DICAS]
STUDIES ON ARGENTINE PLANTS. VIII. THE ALKALOIDS OF E R Y T H R I N A C R I S T A G A LLI. CHROMATOGRAPHIC SEPARATION OF ERYTHRATINE AND ERYSODINE V. DEULOFEU, R. LABRIOLA, E . HUG, M. FONDOVILA, A. KAUFFMANN
AND
Received January 16, 1947
Erythrina crista galli is the most common species of Erythrina in Argentina. Cicardo and Hug (1) found that, as with other Erythrina species, extractsof its seeds had curare-like activity, and Deulofeu, Hug, and Mazzocco (2) isolated hypaphorine from them. An examination of the seeds for alkaloids by Folkers, Shavel, and Koniuszy (3) showed the existence of free alkaloids that were not further identified, and of the liberated alkaloids erysodine and erysopine. Gentile and Labriola (4) isolated from them a mixture of crystalline hydriodides of the free alkaloids, and among the liberated ones erysovine as well as erysodine and erysopine. New work on both fractions starting with larger quantities of seeds demonstrated the presence of erythramine, erythratine, and erythraline among the free alkaloids by separation as hydriodides, and of erythratine by isolation of the free base and preparation of some derivatives. Erythraline seems to be the major alkaloid in the free state, followed by erythratine and erythramine. The presence of erysopine, erysodine, and erysovine was confirmed in the liberated alkaloid fraction, erysopine being the major alkaloid and erysovine that present in the smallest amount. The hydrochlorides of erysopine and erysovine could be prepared and were found to be fairly stable in water solution. We found that sometimes mixtures of erythratine and erysodine were produced that melted at 157-160' and were rather difficult to separate by crystalization. The alkaloids can be separated by chromatographic analysis ; erysodine passed with the first fractions of solvent and erythratine with the last ones. Hypaphorine was isolated in 1.25% yield. E XPERIMEN'I'AL
Erythratine. Seven kg. of Erythrina crista galli seeds were worked up according to the method described by Folkers and Koniuszy (5) as the preferred procedure. Eighteen and five-tenths grams (0.26%) of a syrup corresponding to the free alkaloid fraction was obtained. It was dissolved in 45 cc. of absolute ethanol and kept at 5". A crystalline precipitate formed with time, and after some days it was filtered and recrystallized from ethanol. Eight hundred sixty milligrams of crystals melting at 171-172' was obtained, that waa characterized as erythratine; [CY], +138.6" (ethanol, c = 0.39). Anal. Calc'd for ClsHtlNOr +0.5 H20: N, 4.32. Found: N, 4.67. Erythratine hydriodide melted at 240"; [a],, $110" (water, c = 0.209), and erythratine hydrobromide melted a t 250"; [CY],+149" (water, c = 0.288). Erythratine hydrochloride. A small amount of erythratine suspended in absolute ethanol waa dissolved by the addition of the required amount of hydrochloric acid. Ether was 486
ALKALOIDS OF ERYTHRINA CRISTA GALL1
487
added to faint turbidity and the solution kept overnight at 5". Crystals were obtained which were purified from ethanol, m.p. 250", long prisms; [a],$165.1" (water, c = 0.333). A n a l . Calc'd for Cl~HzlNOI.HC1:C, 61.45; H, 5.97. Found: C, 62.03; H , 6.03. Folkera and Koniuszy (6) give for erythratine base hemihydrate m.p. 170-170.5", [&ID +144.9"; for the hydriodide, m.p. 242", [a],+log"; and for the hydrobromide m.p. 241', talD +158.7". Erythraline hydriodide. The free alkaloid fraction corresponding t o 5 kg. of seeds, from which erythratine base had been separated, was treated in absolute ethanol solution with 6.11 g. of sodium iodide and 3.86 g. of acetic acid. One cubic centimeter of water was added, and by long standing at 5" a crystalline precipitate appeared, yield 1.98 g., m.p. 230". Recrystallized four times from water, 1.1 g. of yellow needles was obtained, m.p. 252", [a],$176" (water, c = 0.229). Anal. Calc'd for ClsHloNOa-HI: N, 3.29. Found: N, 3.38. Erythraline hydriodide melts at 252-253" and has [all, +177". Erythramine hydriodide. The mother liquors from the preparation of erythraline hydriodide w-ere concentrated to one-half, and the crystals appearing after cooling collected. By another concentration, crystals were produced again, combined weight, 180 mg. They were recrystallized from water, the first crystals separated, and the mother liquor concentrated; 18 mg. of yellow needles melting a t 242-243", [CY],t-222.8" (water, c = 0.166). Erythramine hydriodide melts a t 242" and has [CY],+221". Hypaphorine and liberated alkaloids. The aqueous solution corresponding to 5 kg. of seeds, that had been extracted with chloroform for the free alkaloids, was acidified to Congo Red and kept a week at 5". Sixty-two and five-tenths grams of crude hypaphorine hydrochloride was collected. The remaining solution was then boiled for 5 minutes, made alkaline with sodium bicarbonate, and extracted 20 times with 50 cc. of chloroform. The chloroform was evaporated, and from the residue after solution in alcohol and crystallization, 2.69 g. of crystals melting at 197" was isolated. Repetition of the hydrolysis for 30 minutes in acid medium (Congo Red) and extraction under the same conditions gave 7.5 g. of crystals melting at 198-200". A new acid hydrolysis m s made lasting for one hour. When the solution was alkalified with sodium bicarbonate, a white precipitate was produced, which was filtered. Each time that chloroform was added, a precipitate formed; total 27 g. of the precipitated crystals, m.p. 230-235". The chloroform extracts yielded 580 mg. of crystals melting at 198-200'. A new acid hydrolysis of one hour gave only 3 g. of precipitate melting at 230-235" and no appreciable residue in the chloroform. Erysodine. The 10.8 g. of crystals melting around 200" was combined and recrystallized from ethanol, using decolorizing charcoal the first time. Six and two-tenths grams of pure erysodine melting a t 202-204" with [a],i-250" in ethanol was obtained. Eryeopine. The 30 g. of precipitated crystals melting at 230-235" was purified by suspending in a large amount of ethanol, adding acetic acid to solution, boiling with charcoal, and after filtering, adding dilute ammonia to neutralization. Seventeen grams of erysopine melting a t 240-241" was obtained. Recrystallization from ethanol gave a product melting at 242-243" and with [a],+261" (40% glycerol: 60% ethanol); intense green color with ferric chloride. Erysopine hydrochloride. Two grams of erysopine was suspended in a small amount of water and concentrated hydrochloric acid added drop by drop. The erysopine dissolved, and on further addition, the hydrochloride crystallized out, yield 1.3 g. of prisms that can be recrystallized from water. It melted at 260-265' (slow heating) or 270" (rapid heating) with decomposition and previous darkening. The solution gave an intense green color reaction with ferric chloride; [a],+%So (water, c = 0.327); calculated on the basis of the erysopine content, [a],+279". Folkers, Shavel, and Koniuszy (3) found [a],+276" for the same alkaloid dissolved in 0.5% hydrochloric acid.
488
DEULOFEU, LABRIOLB, HUG, FONDOVILS, KAUFFMANT
A n a l . Calc'd for C1,HI9NOa.HC1:C, 63.45; H, 6.22; N, 4.35. Found: C, 63.10; H, 6.04; N , 4.83. Erysovine. The ethanolic mother liquors from the purification of erysodine yielded on evaporation a crop of crystals melting a t 163-165". By chromatographic analysis as described by Folkers and Shave1 ( 7 ) , 370 mg. of crystals melting at 179' was obtained; [a], +239" (ethanol, c = 0.416). Erysovine hydrochloride. Two grams of erysovine was suspended in 4 cc. of ethanol and concentrated hydrochloric acid ( d , 1.19) added to solution. Ether was then added drop by drop, until crystals formed. More ether was added and, after 24 hours a t 5', the crystals were recrystallized from ethanol; obtained as needles melting at 237-238'. This hydrochloride retains water and had to be dried at 120" in a vacuum for 8 hours to obtain an analytical sample. A n a l . Calc'd for C18H2,NOa.HC1:C, 64.28; H, 6.25. Found: C, 64.12; H, 6.95. [a],+233.8' (water, c = 0.686); calculated on the basis of the erysovine content, [& +262". Erysovine in 0.5% hydrochloric acid has [a]=$264.9" (c = 0.609). Chromatographic analysis of mixtures of erythratine and erysodine. I n some cases, when the chloroform extraction of the free alkaloids was done with some delay, it was found that after dissolving the residue from the chloroform extract in ethanol, crystals were ob-
TABLE I CHROMATOGRAPHIC SEPARATION OF ERYSODINE A N D ERYTHRATINE FXACTION
I I1
I11 IV V
I
UiLOROFOBY, CC.
I
70 50 50 50 50
RESIDUE, MG.
124 559 234 110 80
1
M.P.
"c.
200 157 172 170 170
tained melting at 152-157', that by recrystallization from the same solvent could not be purified to a higher melting point than 160". They were found to be a mixture of erythratine and erysodine that could be separated by chromatographic analysis on aluminum oxide with chloroform as solvent. As an example, one fraction of 1.3 g. melting at 152-157" was dissolved without further purification in 100 cc. of chloroform and chromatographed through a column of aluminum oxide of 200 x 15 mm., and developed with chloroform. The fractions shown in Table I were collected. Fractions I and I1 crystallized on evaporation. Fractions 111-V gave a syrupy residue that was induced to crystallize by addition of ether. By recrystallization from ethanol, fraction I yielded pure erysodine melting at 202-204"; [eln+248" (ethanol). Fractions 111-V were united and recrystallized from ethanol, whereby pure erythratine base was obtained, melting at 172-173'; [CY].+134.1' (ethanol). For further identification the hydriodide was prepared, m.p. 241'; [elo+111" (ethanol). Fraction I11 was separated again into erysodine and erythratine by a new chromatograph. SUMMARY
,4reinvestigation of the seeds of Erythrina crista galli confirmed the presence of hypaphorine and of the liberated alkaloids erysodine, erysopine, and erysovine.
489
ALKALOIDS OF ERYTHRINA CRISTA GALL1
Erythratine, erythraline, and erythramine were characterized among the free alkaloids. The hydrochlorides of the liberated alkaloids erysopine and erysovine are described. Mixtures of erythratine and erysodine difficult to separate by crystallization can be separated by chromatographic analysis. BUENOSAIRES,ARGENTINA REFERENCES
(1) CICARDO AND HUG,Rev. SOC. urgentinu biol., 13, 121 (1937); Compt. rend. 154 (1937). (2) DEULOFEU, HUQ,AND MAZZOCCO, J. Chem. SOC.,1841 (1939). AND KONIUSZY,J. Am. Chem. SOC., 63,1544 (1941). (3) FOLKERS,SHAVEL, (4) GENTILE AND LABRIOLA, J. Org. Chem., 7,136 (1942). (5) FOLEERS AND KONIUSZY, J. Am. Chem. SOC.,62, 1672 (1940). (6) FOLKERS AND KONIUSZY, J. Am. Chem. Soc., 62, 436 (1940). AND SHAVEL,J. Am. Chem. SOC.,64, 1892 (1942). (7) FOLKERS
SOC.
bioZ., 126,
