July 5 , 3052
ISOLATION OF
A
CONVULSANT PRINCIPLE : ANISATIN
[CONTRIBUTION FROM THE SCHOOL O F CHEMISTRY,
321 1
RUTCERS UNIVERSITY ]
On the Toxin of Illicium Anisaturn. I. The Isolation and Characterization of a Convulsant Principle : Anisatin’ BY JOHN F. LANE,WALTERT. KOCH,NORMA S. LEEDSAND GEORGEG O R ~ N RECEIVED DECEMBER 26, 1951 From the dried, powdered seeds and carpels of the Japanese star anise there has been isolated a pure, crystalline convulsant principle, anisatin, ClaHpoOs. A study of the reactions of this compound and of its infrared absorption spectrum indicates that it is the anhydride of a pentahydroxydicarboxylic acid. Associated with anisatin are two physiologically inactive substances : co-anisatin and $-anisatin to which the tentative formulas C1aH1805 and C21H3208, respectively, have been assigned.
The convulsant activity of the fruit of the Japa- tinic acid, which is isomeric with anisatin. This nese star anise2 has been known3 for several centu- acid, which has been characterized by preparation ries, but attempts to isolate the active principle have and analysis of its methyl ester, brucine salt and met with little success. In 1881, Eykman4 re- silver salt, is most probably a y-lactonic acid. ported isolating a few milligrams of impure, crystal- Thus, when the acid was dissolved in excess of alline, toxic material (“sikimin” or “shikimin”) kali and the solution titrated rapidly with hydrowhich melted “at about 175’” but which he did not chloric acid an inflection was reached ca. p H 8 further characterize. More recently Siersch5 re- which corresponded to consumption of nearly two ported a repetition of Eykman’s work, which gave moles of alkali. This was not a stable end-point, minute quantities of “shikimin,” again not further however, the @H tending to rise as the solution characterized. Other worker^^,^ obtained only stood, a phenomenon consistent with the transition of the mono-sodium salt of a dibasic acid to the salt highly toxic concentrates. The present paper describes the isolation of a of a monobasic acid. If, on the other hand, the pure, well-defined, crystalline substance of high solution was acidified and back-titrated with base, a smooth titration curve resulted which indicated toxicity for which the name anisatin is proposed. By methods described in the Experimental part the presence of only one titratable carboxyl group. the convulsant principle was first isolated as the 2- The acid further exhibits, cf. Fig. 1, absorption propanolate, C I ~ H Z D O ~ . ~ / Z[cu]~‘D C ~ H S-~25’ , (c 2, maxima in the infrared a t 5.67 and 5.79 p which are ~ ~ ’ ~its description as a y-lactonic ethyl acetate). The isolation was complicated by in h a r m ~ n y with the presence of an impurity, co-anisatin, C13H1805, acid. [ ( r I z 3 ~-24’ ( c 1, dioxane), which had approxit mately one-fourth the solubility of anisatin in isopropyl alcohol. In general, the progress of purification was followed in the last stages by determinations of purity according to the solubility method of Webb,8with isopropyl alcohol as the solvent. Pure anisatin, [ L U ] -28’ ~ ~ D (c 2, dioxane), wasobtained on recrystallization of the 2-propanolate from water. Recrystallization from the solvents tetrachloroethane and nitromethane gave the welldefined crystalline complexes C ~ & O O ~ . ~ / & H ~ C ~ ~ and ClaH~~O~.1/2CH~N02, respectively. Anisatin is a neutral substance, sparingly soluble . 100 I in.water, from which solvent it may be recovered unchanged a t PH’s up to 6. It dissolves readily in dilute alkali, however, with the consumption of two moles of base. When the basic solution is acidified and extracted with ethyl acetate, there is obtained a physiologically inactive, monobasic acid, anisa(1) Presented in part at the Cleveland Meeting of the American Chemical Society, April 10, 1951. (2) This plant, commonly known in Japan as shikimi-no-ki DC ashikimi (evil fruit) and in China as mang tsoo (mad herb), has often been termed Illicium Rcligiosum (Siebold or Zuccarini), but according to the recent taxonomical review of A. C. Smith ( S a v g c n f h . No. 7, 1 (1947)) should be designated Illicium Anisolum, Linnaeus. The material (dried seeds and carpels) used here was supplied by S. B. Pesick and Co., New York, N. Y . , and was collected by their agents in the Shikoku and Kyushu districts of Japan. (3) Cf. S. Y. Chen, A m . J . Pharm., 101, 676 (1929), for a review of the early literature. (4) J. F. Eykman, Phorm. J . and Trans., 11, 1046 (1881). ( 5 ) E. Siersch, Pharm. ZenlroZhallc, 69, 587, 601 (1928). (6) K. K. Chen, J . A m . Phorm. Assoc., 16, 861 (1926). (7) T. Q Chou, Chincse J. Physiol., 1, 213 (1927). (8) T J Webb. A?ial Chem , 30, 100 (1948).
3
5
7
9
11 13 15 Wave length in microns. Fig. 1.-Infrared absorption curves (nujol mulls): A, anisatin (2-propanolate) ; B, anisatinic acid ; C, anisatin triacetate.
Anisatin is quite stable in alcoholic solutions under ordinary conditions. However, prolonged treatment with anhydrous methanol in a sealed tube a t 125’ converted it to the methyl ester of anisatinic acid. At room temperature in a sealed tube with (9) J. F. Grove and H. A. WiIIis, J . Chcm. Soc., 877 (1951). (10) R. S. Rasmussen and R. R . Brattain. THISJOURNAL,71, 1073
(1949).
liquid ammonia it reacted slowly to give a salt from which anisatinic acid could be obtained. This behavior suggests the presence in anisatin of a stable anhydride function arid a t least one hydroxyl group so placed that it can readily enter into lactone formation with one of the carboxyl groups produced by opening of the anhydride linkage: e. g.
I--v
Confirmation for this hypothesis was obtained in two ways. First, the infrared absorption curves of anisatin (cj. Fig. 1) showed maxima in the carbonyl region a t 5.51 and 5.87 p which is characteristic of the anhydride linkage.” Second, on treatment with sodium niethoxide in warm, anhydrous methanol, anisatin consumed one mole of sodium methoxide, behavior which is again characteristic of anhydrides,I2but not of esters or lactones. The relatively great stability of this linkage toward hydroxylic solvents (and even cold sodium rnethoxide) is somewhat surprising and suggests that it must be so situated as to present considerable steric hindrance to the approach of bases which normally cleave anhydrides. I t is of interest to note that similar stability is exhibited by a number of the anhydrides of bicyclic dicarboxylic acids, e.g., cantharidin,13 camphoric anhydrideI4and apocamphoric anhydride. l5 The remaining oxygens in anisatin appear to be hydroxylic. Thus, it forms a triacetate which gives a positive test for active hydrogen (2 moles) by the method of Zerewitinoff and which displays absorption maxims (cf. Fig. 1) a t 2.81, 7.34 and 8.5G 1.1, characteristic of tertiary hydroxyl. The two non-acylable hydroxyl groups must be so located as to be singularly unreactive, for even under rigorous conditions anisatin triacetate is indifferent to the actioii of phosphorous oxychloride in pyridine and to mixtures of acetyl chloride and acetic anhydride. In conclusion it is to be noted that the KuhnRoth C-methyl determination on (solvent-free) anisatin indicated two (1.5) such groups to be present. Accordingly, anisatin i s tll(JSt p r o b a b l y to be forinu1:zted as 0
0
/I
I,
(CH,),( CitI-To)(CJH):,(-C---O-C-)
This leads to the formulation of the “nucleus” as CuHls which, since anisatin displays no unsaturation, must contain three rings. Studies on the na( 1 1 ) N. B. Colthup, J . Optical SOL.A m . ,40, 397 (1960). (12) W. &I. D. Bryant and D. M . Smith, THISJOIJRNAL, 58, 2452
(1936). (13) G. Marchiolo. Burl. SOT. chiin. Farm., 62, 6 5 (1623); C. A , . 17, 2166 (1923). (14) A . T , a ~ m t i t A , n , ! , 2 2 , 141 (1837); 0. Aschaii. Ber., 26, 1639 ( 1 8!K3) ( 1 5 ) J l i ~ T . I I ~ I :Ia i i c l I A ( h r ~ l t i t , rJ, . (‘/if)>! . ? s i ( , 69, C.;O (1891).
ture of the nuclear structure are now in progress and will be reported a t a later time. Experimental Materials.--The trichloroethylene ( h . p . 86-87’) used in the defatting experiments was a technical product obtained from the Donner and Smith Chemical Company, Kewark, N. J. The ethanol and ethyl acetate for extraction were of C.P. grade and were used without further purification. The ethyl acetate used in chromatography was of reagent gratle, dried over anhydrous magnesium sulfate and distilled. Chromatographic alumina (Fischer or Aluminum Co. of America, F-20), acid washed to pH 3 with dilute sulfnric acid and dried a t 170’ was used as an adsorbent. ?‘lie plant material, obtained* from Japan, consisted of drietl seeds and carpels which were ground to 30 mesh. The resulting meal was brown in color, oily to the touch and highly aromatic. Bioassay.-The material to be assayed was dissolved (or suspended) in water, and each of a number of white mice (15-2.5 9.) was injected intraperitoneally with a volume of solution equal in one-hundredths of a millilitei to its weight in grams. Mice which had received dosages of the order of the MLD exhibited partial paralysis of the hind limbs shortly after injection. This was followed by generalized body trembling with convulsive seizures in from 15 to 30 minutes after injection. These seizures increased rapidly in severity and duration, death usually occurring within 90-120 millutes. Mice that survived this period, however, alnio;! always recovered fully without seeming ill effects. In general the order of toxicity was first roughly estimated with the aid of a few animals. Dosages over a suitable range were then admiiiisteied (at intervals of about 10%) to larger groups (e.g., 5 to 10) of animals, and the value of the LD 50 estimated by Behrens’ method.16 This was determined to be 1.1 rt 0.1 mg. for anisatin Z-propanolate (see below) and 1.1 =k 0.1 g. for the powdered crude drug (injected in a 2% aqueous starch suspension). Defatting of the Crude Drug.-Seven hundred eighteen grains of crude, ground drug was stirred with 1440 ml. of trichloroethylene for three hours and filtered. The residue was washed thoroughlywith 1150ml. of fresh solvent. Evaporation of the combined solutions gave 73.6 g. (10.3%) of n green oil. This oil, suspended in 2% starch solution, caused no convulsions when injected in dosages up to 400 mg./kg. body weight. Alcoholic Extraction of the Defatted Drug.-Twentv ke. of defatted ground drug was stirred with two successiGe 65-1. portions of 95% ethanol for two days and filtered. The volume of solution was reduced by distillation at atmospheric pressure to about 18 1. and allowed to stand. After several days 600 g. of precipitated, crude shikimic acid separated and was removed by filtration. On complete removal of the solvent the residue weighed 2.5 kg.; LDbo 250 mg. Extraction with Ethyl Acetate.-Four hundred grams of dried alcoholic extract (LDso250 mg.), dissolved in 1200 nil. of water was neutralized to pH 6 with 110 g. of sodium bicarbonate. After filtration and washing the volume ,was 1,700 ml. This was extracted with four 1,700-ml. portions of ethyl acetate. Evaporation of the solvent gave 20 g. of a greenish gum, LDM20 mg. Chromatographic Separation and Purification of Anisatin. -Two hundred grams of ethyl acetate extract, L D ~28 o mg., dissolved in 4 1. of ethyl acetate, was added t o a column 100 cm. high which contained 3.5 kg. of alumina. Elution with 4,300 ml. of ethyl acetate removed 16.3 g. of material having negligible activity. An additional 2,300 ml. of solvent eluted the greater part of the activity in a fraction containing 24 g. (estimated by evaporation of an aliquot portion), LI).,n 5.0 mg. Evaporation of this fraction in steps yielded 8.21 g. of crystals which separated spontaneously. The rest of the solid material could be recovered by evaporating the solution almost to dryness and diluting with chloroform, but the precipitate then was amorphous. From the crystalline m a terial there was obtained, after two recrystallizations from isopropyl alcohol, 2.9 g. of anisatin 2-propanolate which, by solubility analysis (see below), was shown to be about 805’1 pure. ____I___
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July 5 , 19
