STROPHANTHIN.

IX. ON CRYSTALLINE KOMBE STROPHANTHIN.

BY WALTER A. JACOBS AND ALEXANDER HOFFMANN.

(From the Laboratories of The Rockefeller Institute for Medical Research, New York.)

(Received for publication, January 28, 1926.)

A crystalline strophanthin which accompanies the so called amorphous strophanthin in alcoholic extracts of Strophanthus kombe has been observed by a number of investigators. This substance has been studied comparatively recently and almost simultaneously by Heffter and Sachs,1 and by Brauns and Closson.2 The excellent critical, historical review of the chemistry of the different strophanthins with which Heffter and Sachs have pre- ceded their own report has accomplished the good service of plac- ing a proper interpretation on a number of conflicting previous investigations which were performed at a time when botanical classification of the various Strophanthus plants was uncertain. From this review it appears that Arnaud3 was the only other chemist who had previously made any study of the properties and composition of crystalline Kombe strophanthin, although before him Fraser and Catillon had probably already noted its occurrence. Arnaud prepared the substance by clearing an aqueous solution of the concentrated alcoholic extract of the seeds with basic lead acetate and by subsequent concentration to crys- tallization of the mother liquor from which excess lead had been removed. After several recrystallizations from water, sparingly soluble rosettes of crystals were obtained which softened at 165” and showed a composition of C, 60.46; H, 8.07; and [aID = +30”

1 Heffter, A., and Sachs, F., Biochem. Z., 1912, xl, 83. * Brauns, D. H., and Closson, 0. E., J. Am. Pharmaceut. Assn., 1913,

ii, 715. 8 Amaud, M., Compt. rend. Acad., 1888, cvii, 179.

609

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Strophanthin. IX

in water. Thorns4 later found that this sample gave a green colora- tion with sulfuric acid. Heffter and Sachs worked with com- mercial Kombe seeds of recognized botanical purity and employed a method essentially similar to that of Arnaud. They described their substance as neutral, and sparingly soluble, and one which crystallized in voluminous aggregates of fine, long, radially grouped needles. When dried, the substance softened to a paste at 177-181” and gave a deep green color with sulfuric acid (8:2). Their analytical results showed C, 61.93; H, 7.64; OCHB, 4.73; and [a], = t-28.72” in water. On hydrolysis strophanthidin was produced and also a reducing sugar solution which could not be crystallized and from which no osazone could be obtained. Brauns and Closson employed carefully identified Kombe seeds and again used essentially the method of Arnaud. From their description, crystalline strophanthin consists of microscopic fine needles, or long plates which, when air-dried, contain 6 to 7 per cent of water and melt to a turbid mass at 15%165’, or at 17%179”, when air-dry or anhydrous respectively. Their analyses showed C, 61.97; H, 7.98; and [cr], = +28.7” in water. In con- centrated sulfuric acid a dark green color was first obtained which changed on standing to a brownish color.

In a preliminary note6 from this laboratory, several years ago, we have described a crystalline strophanthin obtained from com- mercial Kombe seeds by an essentially similar method which agreed in properties very closely with the above descriptions. It formed sparingly soluble delicate needles, or platelets, which, when air-dried, melted at 180-183“. Analysis showed the presence of 7.6 per cent of water and C, 62.15; H, 7.55; OCH,, 5.26; and [crE = +30.5” in 95 per cent alcohol.

It is thus seen that the so called crystalline Kombe strophan- thins which have all been obtained and purified by similar methods agree, except for minor variations, so closely in properties that there is little question that the same substance has been the sub- ject of study in each case. The variations in melting points observed are not significant, and such variations are of no im- portance in view of what follows.

Following our preliminary note on crystalline Kombe strophan- thin, we have gradually accumulated more material with the inten-

4 Thorns, H., Ber. pharm. Ges., 1904, xiv, 112. 6 Jacobs, W. A., J. Biol. Chem., 1923, Ivii, 569.

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W. A. Jacobs and A. Hoffmann 611

tion of giving more thorough study to the carbohydrate moiety of the molecule which we had found to belong, in all likelihood, to the group of desoxy sugars and which gave a purple reaction in acetic acid with iron and sulfuric acid. Further work has given a rather surprising turn to these studies, since it has now been found that the so called crystalline Kombe strophanthin is, in reality, a mixture. In the dry state this glucoside is but very sparingly soluble in chloroform and forms a pasty mass under this solvent. If, however, water is added to the mixture, the pasty mass dissolves and investigation showed that this operation separated the substance into a chloroform-soluble and a water- soluble portion. On working up the chloroform extract by con- centration and precipitation with ligroin, a copious amorphous precipitate was formed in a yield of about 50 per cent of the start- ing material. This was found t,o cryst,allize beautifully from methyl alcohol forming long prisms which melted at 143”. From dilute alcohol the substance could be obtained in another crystal- line form which melted at 185-187”. Analysis of the anhydrdus substance gave C, 65.9; H, 8.07; and OCHI, 5.73. In alcoholic solution [a], = +37.5”. By the Keller-Kiliani reaction, the substance yielded the deep blue color which has been described by KilianP as characteristic for digitoxose and which was also observed in the case of cymarin and cymarose by Windaus and Hermanns.? The analytical figures, color reactions, and other properties of this substance were such as to suggest its identity with cymarin, the crystalline glucoside first isolated by Taub and Fickewirth of the Farbenfabriken vorm. Friedr. Bayer and Co. from various species of Apocynum, and which Windaus and Hermanns have demonstrated to be a glucoside of strophanthidin and cymarose, the latter possibly a methyl ether of digitoxose. Since in a few minor points (melting points and rotation) our observations differed from the propert,ies recorded by the latter workers, cymarin was prepared directly from Canadian hemp for comparison. This comparison established definitely the identit,y nf the chloroform-soluble portion of “cryst,alline Kombe strophan- thin” with the cymarin of Canadian hemp. The Kombe “cymarin” yielded not only strophanthidin, but the cymarose

B Kiliani, H., Arch. Pharm., 1896, ccxxxiv, 275. ’ Windaus, A., and Hermanns, L., Ber. them. Ges., 1915, xlviii, 979.

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612 Strophanthin. IX

described by these workers. It is thus seen that the intimate relationship of the cardiac poison of Apocynum with that of Stro- phanthus kombe also of the family ApocynaceE, already demon- strated by the identification of the strophanthidins obtained from both sources, is made more striking by the demonstration of the common occurrence of the identical glucoside in each of these groups of plants.

An investigation of the water-soluble portion of crystalline K- strophanthin, after separation of the chloroform fraction, has shown it to be still a mixture of substances. On concentration? about 13 per cent of a second crystalline glucoside was obtained which proved to be a neutral substance, sparingly soluble in water, which melted with effervescence at 150-151”, and gave a pure, emerald green color with sulfuric acid (4 : l), untinged with brown. On analysis, it gave figures which agreed with the formula C36H5.+014 and showed the presence of one methoxyl group. On gentle hydrolysis it yielded strophanthidin and a strongly reducing sugar solution which, however, on further hydrolysis, was enhanced in reducing power by about 70 per cent. The sugar in the origi- nal glucoside, therefore, is a biose, probably with the formula C13H2d09, and since it contains one methoxyl, this would agree with a biose consisting of a hexose and cymarose. With the material available, it has not been possible to make a definite decision in this regard, but our conclusions, we believe, are sup- ported by the preparation of a crystalline tetracetyl derivative of the new crystalline strophanthin. No crystalline osazone could be obtained from the completely hydrolyzed sugar solution. In its color reactions, the new glucoside has failed to give a certain indication of the presence of cymarose. The Keller-Kiliani test was practically negative, but when the test was performed as given by one of us, and under conditions which, with the original gluco- side, gave a purple color, and with cymarin a deep green color, the new glucoside developed, at first, a green, followed by a pur- ple; but the color tones were not as clear as in the case of the former substances. The presence of cymarose in this gIucoside is sug- gested by these reactions and the failure of the Keller-Kiliani test may be due to the character of the union with the other sugar which can be broken only under conditions which cause decom- position of the unstable desoxy sugar.

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W. A. Jacobs and A. Hoffmann 613

On distillation with hydrochloric acid, no furfural or methyl- furfural was obtained from the glucoside, so that pentoses and methyl pentoses are excluded. A similar observation was made by Brauns and Closson in their examination of the so called crys- talline K-strophanthin for rhamnose, which led them to question the results of Feist,* who had previously worked with a supposedly crystalline Kombe strophanthin, from which a so called strophan- thobiose methyl ether was isolated. This sugar Feist concluded to be a methyl ether of a mannose-rhamnose biose. As already dis- cussed by Heffter and Sachs, it is greatly to be doubted that Feist, in reality, worked with a crystalline substance. Feist stated that it was prepared by Fraser’s method, in accordance with which the purified alcoholic solution of the crude glucosides obtained from the seeds was precipitated by ether. Such a method should eliminate most of the cymarin, since this is soluble in such an alcohol-ether mixture. We are convinced that the results of Feist were obtained with a mixture of amorphous glucosides and have no bearing on the question of “crystalline Kombe strophan- thin.” We are continuing the study of the sugars jn the new glu- coside. As a continuation of the suggestion of Thorns for the nomenclature of the strophanthins, we propose the designation K- strophanthin-p for this new strophanthidin bioside, since, in all likelihood, still others may be found. Although, in accordance with this scheme, it would be logical to call cymarin K-strophan- thin-a!, the former name has already been introduced and there appears no reason to change it to the less wieldy designation.

The aqueous mother liquor from K-strophanthin-p yielded an easily soluble, amorphous substance which was, apparently, a mixture of a number of glucosides and which gave reactions identi- cal with those given by the former.

Since our experiments were performed with commercial Kombe seeds which might have caused uncertainty, we have been ex- tremely fortunate to receive from Parke, Davis and Company a sample of crystalline K-strophanthin in answer to our request for some of the material prepared by Brauns and Closson from identified seed. The examination of this material confirmed in all respects the results obtained with the substances of our own

8 Feist, F., Ber. them. Ges., 1898, xxxi, 534; 1900, xxxiii, 2069.

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614 Strophanthin. IX

preparation with the exception that the proportions of crystalline glucosides obtained differed somewhat. It is questionable whether “crystalline Kombe strophanthin” represents a constant mixture and it is probable that the substances studied by the various workers have been of somewhat different composition.

From these experiments it is obvious that extracts of Xtrophan- thus kombe seeds contain a mixture of strophanthidin glucosides and it appears that of all the strophanthins from this source which have been examined, either chemically or pharmacologically in the past, in no case can there be certainty that a homogeneous substance has been employed. In their very careful investigations with identified Xtrophanthus hispidus seeds, Heffter and Sachs were unable to obtain evidence of the presence of a crystalline glucoside, so that it is doubtful whether cymarin can be isolated from this source, or at least only in negligible amounts. It is very likely that their amorphous hispidus strophanthin was also a mix- ture. Cymarin and K-strophanthin-/3 are the only glucosides of strophanthidin at present known which we can be certain are chemical individuals. It is, therefore, of importance to make further attempts to isolate as far as this is possible other individual glucosides which occur in these plants and to submit them to pharmacological study.

Finally, the preparation of a monoacetyl cymarin from cymarin in pyridine solution with acetic anhydride is of interest in suggest- ing the mode of linkage of the sugar on strophanthidin. If we accept what is most likely, that acetylation has occurred on the free hydroxyl of cymarose, then strophanthidin has remained unacetylated. Since strophanthidin itself forms only a monoacyl compound under such conditions, the conclusion appears war- ranted that the hydroxylg in strophanthidin which is ordinarily acylatable and which is presumably y- to the aldehyde group is here the point of glucosidic union with the sugar, cymarose.

EXPERIMENTAL.

The so called crystalline K-strophanthin employed in the follow- ing experiments was prepared by the method5 previously described

QJacobs, W. A., and Heidelberger, M., J. Biol. Chem., 1922, liv, 253. Jacobs, W. A., and Collins, A. M., J. Bid. Chem., 1924, lix, 718.

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W. A. Jacobs and A. Hoffmann 615

from commercial Kombe seeds which gave uniformly a green color with sulfuric acid. The well washed crude crystalline glucoside as first obtained by the careful addition of ammonium sulfate to the aqueous solution of the crude glucoside mixture was recrystal- lized once as previously given. For further recrystallization we have adopted a somewhat different procedure.

10 gm. of the glucoside were dissolved in 6 cc. of warm 95 per cent alcohol and the solution was then treated with 50 cc. of water. The clear solution on standing gradually deposited needles which possessed the properties already recorded.

[CY]~ = f30.2” (c = 1.000 in 95 per cent alcohol). For the substance dried at 100’ at 20 mm. over H$Oa.

Found. C 62.52, H 7.88.

This substance when treated according to Heffter and Sachs’ with sulfuric acid which was diluted with one-quarter volume of water developed a deep brownish green color.

A small amount of substance when dissolved in acetic acid and then treated with a few crystals of ferrous sulfate followed by a few drops of sulfuric acid produced a deep purple color as previously described. If, however, the Keller-Kiliani reaction was performed as described by Kiliani,6 a slowly developing characteristic deep blue color was obtained due to the presence of cymarin (cymarose) in the glucoside.

Preparation of Cymarin from Crystalline K-Xtrophanthin.

20 gm. of crystalline K-strophanthin were shaken several hours in a mixture of 200 cc. of chloroform and 200 cc. of water. Although the starting material is apparently very little soluble in chloroform alone, by this procedure almost complete solution occurred. After separation of the layers the aqueous portion was extracted several times with small additional portions of chloro- form. The united chloroform extracts were shaken again with a little water, dried over CaCL and then concentrated to 50 cc. On addition of 700 cc. of petrolic ether a flocculent precipitate formed which after standing at 0” was collected. The dry sub- stance weighed 11 gm. For further purification the crude cymarin was dissolved in 25 cc. of hot methyl alcohol and the solution

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616 Strophanthin. IX

was treated with 120 cc. of warm water. Cymarin crystallized in long well formed crystals which were collected with cold water. The yield of the air-dry substance was 7.5 gm. The mother liquor yielded an additional 2.5 gm. of crystalline material after removal of the methyl alcohol.

From aqueous methyl alcohol the substance formed long prisms which sintered on rapid heating at 138” and melted with efferves- cence at 148” (Windaus and Hermanns give 130-13%‘). Cymarin prepared from Canadian hemp and recrystallized under the same conditions melted at the same point as did also a mixture of the two. The substance obtained by the above method of recrystal- lization separates apparently with 1 mol of methyl alcohol which is partly lost when the substance is air-dried since the loss on dry- ing for analysis as well as the methoxyl determinations gave figures which were somewhat lower than those required by the theory.

Air-Dry Substance. Dried at 78” at 15 mm. over H$Oh. C&HUO~.CH,OH. Calculated. CH80H 5.52. Found, CHIOH 4.40.

Zeisel. “ 2(OCH,) 10.69. “ 2(OCH3) S.95. Anhydrous Substance.

C50H4409. Calculated. C 65.66, H 8.09, OCHH 5.65. Found. ‘I 65.90, “ 8.07, “ 5.73.

When recrystallized from dilute ethyl alcohol the substance formed hexagonal leaflets which contained 1.5 mols of water and melted at 185-187” after slight preliminary sintering.

Air-Dry Substance. Dried at 78” and 15 mm. over HsSOd. CLOH~~OS-L~H~O. Calculated. H20 4.93. Found. Hz0 4.59.

I‘ OCHa 5.39. “ OCHI 5.23.

Occasionally by this method of recrystallization a practically anhydrous cymarin was obtained which melted at 204-205”.

FOT the Anhydrous Substance. [a]‘,” = +37.8” (c = 4.94 in chloroform). [a]: = +37.5O (c = 1.76 in 95 per cent alcohol).

For comparison the following observations were made with anhydrous cymarin prepared from Canadian hemp.

[LY]: = +35.0 (c = 4.80 in chloroform). [a}: = +34.9 (c = 1.32 in 95 per cent alcohol).

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W. A. Jacobs and A. Hoffmann

In its color reactions the cymarin from K-strophanthin proved identical with that from Canadian hemp. It gave the typical deep blue color with the Keller-Kiliani reagent but with the test as previously used by Jacobs for K-strophanthin only a dark green color was obtained with no suggestion of a purple. In sulfuric acid diluted with one-quarter volume of water only a brown color was obtained and no trace of a green color such as given by the starting material could be observed.

For further identification we have hydrolyzed the glucoside by the method given by Windaus and Hermanns. 7 gm. of cymarin yielded 5.1 gm. of strophanthidin or 97 per cent of the theory. This was characterized by the melting point 172”, rotation [LY]: = + 41.9 (c = 1.98 in methyl alcohol), and analysis, GH3206. +H,O.

Calculated. C 66.79, H 8.05. Found. ‘I 66.62, “ 8.05.

From the aqueous mother liquor of strophanthidin the sugar, cymarose, was obtained as given by Windaus and Hermanns and was found to agree in all properties with the description of these workers. 1.2 gm. of needles were obtained which melted after recrystallization from anhydrous ether, petrolic ether at 91” @So Windaus and Hermanns) .

CJhOa. Calculated. C 51.81, H 8.70. Found. “ 52.02, “ 8.59.

Cymarose shows in aqueous solution a slight mutarotation. For the final reading [a]: = +53.4” (c = 2.245 in HzO).

Acetylcymarin.-0.5 gm. of anhydrous cymarin was dissolved in 3 cc. of dry pyridine and the solution was treated with 1 cc. of acetic anhydride. The next day on dilution with water a milky emulsion was formed from which the acetyl compound slowly separated as rosettes. On dilution of the solution in methyl alco- hol acetylcymarin forms silky needles which melted at 160-161°, depending somewhat on the rate of heating. An acetylcymarin prepared from hemp cymarin showed the same properties.

0.1234 gm. of substance was refluxed for 1 hour in 15 cc. of alcohol and 14.1 cc. of 0.1 N NaOH and the mixture was titrated back against phenolphthalein. Calculated for two equivalents for monoacetyl cymarin, 4.15 cc. Found, 4.05 cc. Since one

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618 Strophanthin. IX

equivalent is required for the lactone group of strophanthidin the substance is a monoacetyl compound and the acetyl group must be situated on the one free hydroxyl of cymarose.

Air-Dry Substance. Dried at 100’ and 15 mm. over H&O,. C32H4601~-+H20. Calculated, Hz0 1.53. Found, 1.57.

Anhydrous Substance. CdLo0m Calculated. C 65.04, H 7.85.

Found. “ 65.15, ‘I 7.78.

The “Water-Soluble” Glucosides.

K-Strophanthin-/L-The aqueous fractions obtained by the treat- ment of crystalline K-strophanthin with water and chloroform were united and concentrated under diminished pressure below 40” to small bulk, when the contents of the flask became a uniform crystalline mass which under the microscope appeared as long, thin, silky needles. These were collected with small portions of water and then carefully washed with cold water in which it appeared now to be but very sparingly soluble. The yield was 2.65 gm. It was found very difficult to recrystallize the substance.lO It slowly dissolved on shaking in about 100 parts of water at ordinary tem- perature but on warming but little difference was noted in its solubility. However, the addition of small amounts of alcohol greatly increased the solubility but on cooling the substance did not separate again.

The new crystalline glucoside is neutral to litmus and melted, when air-dried, at 150-151” with effervescence. It is easily soluble in ethyl and methyl alcohol and practically insoluble in chloroform, acetic ether, ether, and petrolic ether. With sulfuric acid diluted with one-quarter volume of water it gives a pure emerald green color. The Keller-Kiliani reaction was negative but if the test is performed as described by Jacobs, under which conditions K-strophanthin gives a deep purple color, this glucoside gives at first a dirty green which changes to a violet. When distilled with hydrochloric acid, as in the determination of furfural and methyl furfural, the distillate gave no precipitate

10 More recently the substance has been recrystallized by dissolving in two volumes of alcohol, by adding an equal volume of water and then by removing the alcohol under diminished pressure. When obtained under these conditions the substance showed a Hz0 content of 1.65 and melted at 176’ after preliminary sintering.

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W. A. Jacobs and A. Hoffmann 619

with phloroglucin which should definitely exclude pentoses or methyl pentoses. Rotation for the anhydrous substance:

[a]; = +33.6’ (c = 0.97 in water). Air-Dry Xubstance. Dried at 78” and 15 mm. over HzSOJ.

C H 0 36 54 14 -2iHzO. Calculated, Hz0 5.96. Found, H20 5.92. Anhydrous Substance.

CdL~Ow Calculated. C 60.81, H 7.66. Found. “ 60.66, “ 7.62.

1 gm. of the crystalline glucoside was dissolved in a mixture of 8 cc. of 50 per cent alcohol and 2 cc. of concentrated hydrochloric acid, and allowed to stand for 6 hours, at 20’. On dilution, strophanthidin crystallized and additional amounts were ob- tained after removal of Cl ions with Ag&JOs and concentration of the mother liquor. 0.4 gm. was obtained, which possessed the usual properties. The mother liquor was concentrated under diminished pressure and finally, in a desiccator, to dryness. The resulting syrup showed no tendency to crystallize, but proved to be mostly a disaccharide, since, when a portion of the syrup was warmed in the water bath, for 15 minutes, with 2 per cent HCI, its reducing power for Fehling’s solution increased by 70 per cent. Up to the present, with the limited material available, it has not been possible to identify the sugars. Rhamnose is certainly not present. The Keller-Kiliani test was practically negative. When boiled with strong hydrochloric acid, a yellow coloration is obtained, followed by the rapid deposition of brown flocks. After further hydrolysis, it did not yield a crystalline osazone. With orcinol and ar-naphthol it gave color reactions identical with those given by cymarose and in all likelihood the sugar is a biose of cyma- rose and a hexose.

Tetracetate of K-Xtrophanthin-/S--O.5 gm. of the anhydrous glucoside was acetylated in pyridine solution with acetic anhy- dride. On dilution with water, 0.7 gm. of insoluble substance was obtained which was recrystallized by careful dilution of the alco- holic solution. It formed thin, microscopic needles which melted at about 167” to a vitreous mass. Titration after saponification gave figures in agreement with those required for four acetyl groups.

0.1 gm. was refluxed in 15 cc. of alcohol and 14.2 cc. of 0.1 N NaOH and titrated against phenolphthalein. Calculated for five equivalents (four acetyl and one lactone groups), 5.69 cc. Found, 5.55 cc.

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620 Strophanthin. IX

Air-Dry Substance. Dried at 78’ and 15 mm. over H&04. CUHG~O~~.H~O. Calculated. Hz0 2.01.

Found. “ 1.55. Anhydrous Substance.

C44H62018. Calculated. C 60.10, H 7.12. Found. “ 59.69, “ 6.96.

Water-Soluble Amorphous Fraction.

The aqueous mother liquor from K-strophanthin-p, when allowed to evaporate at ordinary temperature, yielded a non- crystalline, glassy residue, which weighed 5.35 gm. Although this substance was, in all probability, a mixture of glucosides, we shall record the few observations which were made. The melting point depended upon the rate of heating and varied from 160 to 170’. It was very soluble in water and alcohol, and insoluble in chloro- form and acetic ether. The color reactions were the same as those given by K-strophanthin-P.

[a]‘,” = +20.4” (c = 5.27 in water for the anhydrous substance). Anhydrous substance (dried at 100’ and 15 mm. over H#JO~). C 59.07,

H 7.39.

Crystalline K-Strophanthin (Parke, Davis and Company).

A sample of crystalline K-strophanthin, which was generously sent to us by Parke, Davis and Company in answer to our request for a sample of the material prepared in their laboratories by Brauns and Closson, from identified Strophanthus kombe seeds, was submitted to similar study. This distinctly microcrystalline substance sintered at 157’ and melted at 177-179”, and gave the usual reactions for crystalline K-strophanthin. When 1 gm. was submitted to the treatment with water and chloroform, as previ- ously described, 0.32 gm. of cymarin, 0.27 of K-strophanthin-p, and 0.36 gm. of amorphous glucoside were obtained. The cymarin possessed the usual properties and melted at 148”.

The sample of K-strophanthin-/3 melted at 154-156” with effer- vescence, and agreed in all other properties with our K-strophan- thin-p.

[a]: = +32.7’ (c = 0.67 in water for the anhydrous substance). Air-Dry Substance. Dried at 78’ and 15 mm. over H&Sod.

CssH&~.2Hto. Calculated. Hz0 4.82. Found. “ 4.31.

Anhydrous Xubstance. CS6H64014. CalcuIated. C 60.81, H 7.66.

Found. ‘I 60.71, “ 7.54.

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Walter A. Jacobs and Alexander HoffmannSTROPHANTHIN

CRYSTALLINE KOMBE STROPHANTHIN: IX. ON

1926, 67:609-620.J. Biol. Chem. 

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