the digitalis glucosides. - journal of biological chemistry · digitoxigenin would go could be only...

THE DIGITALIS GLUCOSIDES.

IV. THE CORRELATION OF GITOXIGENIN WITH DIGITOXIGENIN.

BY WALTER A. JACOBS AND EDWIN L. GUSTUS.

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

(Received for publication, December 26, 1929.)

Digitoxigenin and gitoxigenin in glucosidic combination form the principal portion of the cardiac glucosides contained in the digitalis plant. Studies with both of these substances had recently reached the point where there was reason to assume a very close structural relationship between them. The analytical studies of Windaus and coworkers’ and of the present writers2 have shown digitoxigenin and gitoxigenin to be respectively C&H3404 and C&H3405. These substances contain only one double bond and are therefore derivatives of saturated tetracyclic structures. An additional close similarity between these substances has been found by the present writers in the demonstration that they are both AB,r-la&ones and, like the other aglucones of the strophanthidin series, may be isomerized to iso compounds.2 The study of the mechanism of this rearrangement has shown that the portion of the molecule involved in the isomerization may be pictured as in the case of strophanthidin as follows?

1 Windaus, A., Westphal, K., and Stein, G., Ber. them. Ges., 61, 1847 (1928). Windaus, A., and Stein, G., Ber. them. Ges., 61, 2436 (1928).

2 Jacobs, W. A., and Gustus, E. L., J. Biol. Chem., 78, 573 (1928); 79, 553 (1928); 82,403 (1929).

3 In view of observations which have been made since this manuscript was sent to press, the statement that gitoxigenin like digitoxigenin is isomerized to an iso compound must be qualified in the following manner. Results which will be published shortly now show that the oxidic ring of isogitoxigenin does not involve the tertiary hydroxyl group which is bridged in the case of isodigitoxigenin. Instead, the additional hydroxyl group is

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200 Digitalis Glucosides. IV

/ / CH2pCpCH CH2-- CH-CH I II I + A0 I I

CQcH /,... .“T \o/cYo/y\

In our previous communications certain pronounced abnormali- ties which were encountered in the study of isogitoxigenin and its derivatives, especially the unusual stability towards alkali of the lactone acid, isogitoxigenic acid, appeared to be explainable pos- sibly on the assumption of the proximity to the lactone group of the extra hydroxyl of isogitoxigenin. After its removal as water, the resulting anhydroisogitoxigenic acid exhibited a normal sapon- ifiability and resembled that of isodigitoxigenic acid. But the extent to which the structural resemblance of gitoxigenin to digitoxigenin would go could be only a matter of inference.

In order to determine this point, Windaus and coworkers have already pursued a definite program of work. This consisted in the formation by removal of the tertiary hydroxyl groups from each aglucone of a monoanhydro derivative from digitoxigenin and of a dianhydro derivative (digitaligenin) from gitoxigenin.

involved by which gitoxigenin differs from digitoxigenin. Whereas digitoxigenin forms only a monobenxoate and gitoxigenin forms a dibenxoate, isogitoxigenin forms only a monobenzoate. Furthermore, whereas digitoxigenin, isodigitoxigenin, and isogitoxigenin form mono-ketones on oxidation with chromic acid of the identical secondary hydroxyl group in each to CO, in the case of gitoxigenin the extra hydroxyl group by which the latter differs from digitoxigenin is also oxidized to carbonyl so that this group cannot be tertiary as previously assumed. The so calledgitoxigenon previously described (Jacobs, W. A., and Gustus, E. L., J. Biol. Chem., 79, 557 (1928)), is a dicarbonyl compound, C23H3005. In addition the substance as previously obtained has been found to be a mixture of stereoiso- mers. Its behavior suggests the possibility that one of these CO groups may be aldehydic, in which case the extra hydroxyl group of gitoxigenin would, therefore, be of primary character.

The difference between the two oxidic rings of isodigitoxigenin and isogitoxigenin was not realized when the work here presented on correlation was begun. Although this difference may now affect the logic of a portion of the program presented in this paper, in particular that part in which the attempt was made to convert anhydroisogitoxigenin into isodigitoxigenin by hydrogenation, the final conclusions are correct as to the structural relationship between the two aglucones.


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W. A. Jacobs and E. L. Gustus

The plan was then to hydrogenate the two double bonds in the former case and the three double bonds in the case of digitaligenin to form perhaps the identical saturated hydroxy la&one. Failing identity at this point, the remaining secondary hydroxyl group in each substance could be oxidized to carbonyl which could then in turn be replaced by CH2 with the formation of the saturated lactones, C23H3602. In a series of articles4 the results of these efforts have been recorded and although the desired chemical transformations were successfully accomplished the exact relationship of gitoxigenin to digitoxigenin was not disclosed by these experiments since the saturated lactones obtained from each substance proved to be not identical but isomeric. The possibility remained that the failure of the direct correlation of the two aglucones by this method was caused by the opportunity given for the formation of a number of isomers during the hydrogenation of so many double bonds, and that the substances which predominated in each case were different.

It appeared to us that the iso compounds might prove to be a more useful starting point for studies on correlation. Having once obtained evidence that isogitoxigenin is a normal iso compound,3 the possibility appeared that its structural difference from isodigitoxigenin might consist solely in the possession of an extra hydroxyl group. In such a case the formation of an anhydroisogitoxigenin by removal of this hydroxyl group as water and subsequent hydrogenation should yield isodigitoxigenin. Unfortunately, only a portion of this program dould be accomplished. When isogitoxigenin was dissolved in concentrated hydrochloric acid at low temperature (O”), it dissolved and was replaced by a crystalline chloro compound, chloroisogitoxigenin, in which the hydrogyl group in question3 has been substituted by chlorine. When, however, the reaction ‘was performed at 20”, although the chloro compound was formed as an intermediate stage, the final substance isolated was halogen-free, and on analysis proved to be anhydroisogitoxigenin. Unfortunately, all attempts to hydrogenate this substance after repeated recrystallization were unsuccessful, a result which is very difficult to understand in view of the work

4 Windaus, A., and Bandte, G., Ber. them. Ges., 66, 2001 (1923). Win- daus, A., Bohne, A., and Schwieger, A., Ber. &em. Ges., 6’7, 1388 (1924). Windaus, A., and Freese, C., Ber. them. Ges., 68, 2503 (1925).


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Digitalis Glucosides. IV

which will be described below. Apparently anhydroisogitoxigenin possesses a double bond which is very resistant to hydrogenation by the usual procedure. The formation of a new ring during the loss of water is greatly to be doubted. The attempt was also made to replace the chlorine in the chloro compound by hydrogen but with a similar lack of success. However, our main purpose was brought to a successful conclusion by a different procedure in which use was made of another isogitoxigenin derivative, isogitoxigenic acid.

When isogitoxigenic acid is treated with concentrated hydrochloric acid at ordinary temperature it dissolves with the formation of a crystalline chloro-y-isogitoxigenic acid in which the tertiary hydroxyl group is replaced by the chlorine atom. Contrary to the experience with isogitoxigenin itself, the reaction appeared to stop at this point and no evidence of the subsequent formation of an anhydro acid was obtained. The latter was prepared by another step. The chlorine in the chloro acid proved to be labile towards alkali and depending upon the conditions employed could be replaced by hydroxyl or removed to form the unsaturated anhydro acid. Both of these reactions appeared to occur simultaneously but conditions were finally found in which either the one or the other reaction predominated. When the chloro acid was dissolved at 0” in a slight excess of very dilute ammonia, the chlorine was gradually replaced by hydroxyl. The resulting acid, however, in its optical behavior proved to be isomeric -with the parent isogitoxigenic acid. Isomerization on a center of asym- metry has occurred under the influence of the strong acid which was used for the preparation of the chloro acid. This is apparently analogous to the observations which we have made in the case of the isomerization of cu-isostrophanthic acid to y-isostrophanthic acid,5 and, as will be later shown, occurs also in the case of isodigitoxigenic acid. We have adopted, therefore, the desig- nation y to indicate the analogy of these isomerization products in the digitalis series with those obtained by similar means in the isostrophanthidin series.

By the use of stronger alkali, hydrochloric acid is removed from the chloro acid with the formation of an unsaturated anhydro-y-

5 Jacobs, W. A., and Gustus, E. L., J. Biol. Chem., 74,835 (1927).


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W. A. Jacobs and E. L. Gustus 203

isogitoxigenic acid6 It was hoped that this substance, contrary to anhydroisogitoxigenin, might yield to catalytic hydrogenation with the formation of y-isodigitoxigenic acid.3 As the methyl ester this anhydro derivative indeed absorbed hydrogen readily, but an unexpected complication was encountered in that the reaction did not stop at the normal 1 mol stage required by the one double bond assumed to be present. Instead 2 mols of hydrogen were absorbed and in accordance with this observation the resulting substance was found to possess the formula CkH3R05. This substance on investigation proved to our great surprise to be the half ester (monomethyl ester) of a dibasic acid, C13H360S, which was readily obtained from it by saponification. Titration experiments showed that the lactone group of the parent anhydro acid had disappeared during the hydrogenation. In other words, while 1 mol of hydrogen was required to saturate the double bond the other was used for the cleavage of the la&one group by hydrogenation to the desoxy acid. This may be represented as follows:

’ CH2---CH /

CHZ- C- C -CH I I I+I I

COOCHa CO C COOCHs COOH CH \o/l\ /\

At the present time we have under consideration as an alter- native possibility the cleavage of an unsaturated Mactone which may be represented as follows:

/ / CHz-CH -CH CH2--CH--- CH I I I I I

COOCHI CO -+ COOCH3 COOH CH I i\ /\ O- CJ-5

I /

The latter formulation has been suggested by certain observations which will be discussed in a later communication.3 However, at the moment it may be stated that this type of cleavage appears to be attributable to the association of the double bond with the lactone ring since all attempts to cause a similar cleavage with the

6 Jacobs, W. A., and Gustus, E. L., J. Biol. Chem., 82,408 (1929).


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Digitalis Glucosides. IV

saturated isogitoxigenic methyl ester were unsuccessful. A nega- tive result was obtained also in the case of the saturated isodigitoxigenic methyl ester as well as its y-isomer. A study of the factors involved in this type of cleavage is now in progress in this laboratory (W. A. Jacobs and A. B. Scott). During the present year, observations on a similar cleavage of lactone groups to the desoxy acid during catalytic hydrogenation have been reported by Mannich and Butz7 in the case of certain unsaturated 6- lactones, by Borsche and Peitzschs in the case of methysticin, and by LaForge and Smith9 in the case of rotenone. On the other hand, although chloro-y-isogitoxigenic acid is a saturated substance, when shaken with hydrogen and platinum oxide catalyst it yielded the above described half ester, so that the chlorine appeared to be replaced by hydrogen with simultaneous lactone cleavage. In this case, however, there is reason to believe that the chloro ester was first converted into the anhydro ester by removal of hydrochloric acid before hydrogenation occurred. The chloro ester has shown in general a distinct lability in this regard. On attempting to replace the halogen with hydrogen by the use of hydrochloric acid and amalgamated zinc, the ester of anhydro- y-isogitoxigenic acid was obtained.

As previously discussed, the fact that the stability towards alkali of the lactone group of isogitoxigenic acid is greatly reduced on passing to the anhydro acid is probably referable to t’he proximity of the extra hydroxyl group. And now the observation that the unsaturated anhydroisogitoxigenic acid shows a behavior on catalytic hydrogenation which is not shown by the saturated substances is in agreement with the inferred proximity of the hydroxyl group and consequently of the double bond to the lactone group. It appeared for the moment that we were blocked in the attempt to correlate isogitoxigenin with isbdigitoxigenin because of this abnormal course of the hydrogenation experiments. Fortunately, however, it has been possible to prepare the above dibasic acid also from isodigitoxigenin through the following series of substances.

Isodigitoxigenic acid on treatment wit,h concentrated hydro-

7 Mannich, C., and Butz, A., Ber. them. Ges., 62,461 (1929). * Borsche, W., and Peitzsch, W., Ber. them. Ges., 62,360 (1929). Q L&Forge, F. B., and Smith, L. E., J. Bm. Chem. Sot., 61,2574 (1929).


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chloric acid was isomerized to y-isodigitoxigenic acid. On heating this acid with a mixture of acetic anhydride and acetyl chloride the la&one group was opened with 6he formation of a substituted succinic anhydride and simultaneously the exposed tertiary hydroxyl group was removed as water. These transformations may be represented as follows:

/ / / F-CH-CH CH,-CH-CH CHt----CH--C

I I I I I I I II COOH CO C -+ co co COH --) co co c

\o/l\ \o/ / \ \o/ / \

At the same time, the secondary hydroxyl group was acetylated. The formation of this substance, the acetate of y-digitoxenoldiacid anhydride, paralleled exactly that of the unsaturated anhydride obtained from ,&isostrophanthic lactone acid.‘O Contrary to our experience with the latter, however, the attempt to accomplish cleavage of the lactone group of the isodigitoxigenic acid by the use of methyl alcoholic hydrochloric acid instead of the above reagents was unsuccessful, due to the formation of obscure non- crystalline alteration products. Similarly, saponification experiments with alkali on the above unsaturated anhydride resulted in non-crystalline products which are probably referable to the instability of the unsaturated dibasic acid towards alkaline reagents. It was possible, however, by gentle treatment with methyl alcoholic hydrochloric acid to open the anhydride group with the formation of a half ester, the acetate of y-digitoxenoldiacid monomethyl ester.

/ CHr---- CH- C I I II

COOCHs COOH C

/\

On hydrogenation this substance was readily converted into the acetate of y-digitoxanoldiacid monomethyl ester. This saturated substance was now readily saponified by alkali with simultaneous removal of the acetyl group to the dibasic y-digitoxanoldiacid. The

lo Jacoba, W. A., and Gustus, E. L., J. Biol. Chem., 84,183 (1929).


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latter proved to be identical in all respects with the dibasic acid which resulted on saponification of the half ester obtained on hydrogenation of anhydro-y-isogitoxigenic methyl ester. This was confirmed by the preparation of the identical dimethyl ester from both sources. Likewise, partial saponification of the dimethyl ester in each case gave rise to identical substances, the stable monomethyl ester. This stable half ester is isomeric with the half ester which had been obtained by hydrogenation of anhydroisogitoxigenic methyl ester. Since the latter is more readily saponified by alkali we have designated it as the labile half ester.

/ / CHz- CH- CH CH,-CH- CH I I I I I I

COOCHI COOH CH COOH COOCHI CH /\ /\

Labile half ester. Stable half ester.

From these results it may be definitely concluded that gitoxigenin is hydroxydigitoxigenin. Digitoxigenin may be represented in partial formula as:

-cm I

CH

CHOH

Cd& or

/\ Y-c-CH II I CTO/CH /““\”

For gitoxigenin we have under consideration the following partial formulse

/H /H / / CHI~C-CH CHI~C-CH CHOH CHOH CH2-- C-CH CH2-- C-CH

II I II I $,CHsC~ $,CHsC~

II I II I


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-CHOH

I

/““\ 1 CHr-C-CH I II I

CY/CH 7y

in which the extra hydroxyl group of gitoxigenin may be secondary or primary. In the latter case the corresponding group of digitoxigenin would be methyl.

We are especially indebted to IX. Merck, Darmstadt, and Merck and Company, Inc., of Rahway for their generous gift of the “digitoxin insoluble by-product” from which the material used in this research was prepared.

EXPERIMENTAL.

Derivatives of Isogitoxigenin.

Chloroisogitoxigenin.-0.4 gm. of isogitoxigenin was treated with 4 cc. of hydrochloric acid (1.19) at 0”. Solution rapidly occurred. On maintaining this temperature, a thick paste of crystals gradually formed. After 2 hours the mixture was diluted with ice water and the precipitate was collected with water. After washing free of acid, the dried substance was recrystallized by the addition of dry ether to the chloroform solution. It formed lustrous needles which melted at 167”. The melting point remained unchanged after repeated recrystallization from 95 per cent alcohol. Since the substance decomposed when maintained at an elevated temperature, the analyses were made on the desic- cator-dried substance.

The results obtained were not satisfactory after repeated trials. This is probably attributable to the tenacious retention of solvent. However, since the exhaustive study of the substance would have caused a profitless waste of costly material, we have remained content. with the data obtained.

4.633 mg. substance: 3.493 mg. H20, 11.300 mg. COz. 7.928 “ “ : 2.535 “ AgCl.

ChH330&1. Calculated. C 67.53, H 8.14, Cl 8.68. Found. “ 66.51, “ 8.43.

“ Cl 7.90.


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Anhydroisogitoxigenin.-0.5 gm. of isogitoxigenin was dissolved at 20” in 20 cc. of hydrochloric acid (1.19). Within a few minutes crystallization of the chloro compound began. This, however, on continued shaking redissolved and about 15 minutes later a new crystalline deposit began to separate. After several hours the mixture was carefully diluted and extracted with chloroform. The washed and dried extract on concentration gave a crystalline residue which was collected with 50 per cent alcohol.

When recrystallized from 95 per cent alcohol, it formed lustrous rectangular platelets which melted at 212”. It is soluble in alcohol, chloroform, and acetone, and but sparingly soluble in ether.

For analysis the substance was dried at 100” and 15 mm.

4.550 mg. substance: 3.534 mg. H20, 12.342 mg. COZ. C23H3204. Calculated. C 74.14, H 8.67.

Found. “ 73.98, “ 8.69.

Chloro-rdsogitoxigenic Acid.-2 gm. of isogit.oxigenic acid were dissolved at 20” in 20 cc. of hydrochloric acid (1.19) as previously described.‘j The chloro ‘acid which separated was collected after 2 hours with additional reagent and washed with water. The dried substance was recrystallized by solution in dry acetone and subsequent concentration to smaller volume. The acid crystallized as long, broad platelets which melted with decomposition at 255” after preliminary softening. It is appreciably soluble in methyl and ethyl alcohols and acetone, and butsparingly soluble in chloroform or ether. The alcoholic solution gradually deposits silver chloride when boiled with silver nitrate.

[LY]~’ = -101’ (c = 0.650 in 95 per cent alcohol).

When dried at 75” under diminished pressure the substance did not lose appreciably in weight.

3.725 mg. substance: 2.530 mg. HzO, 8.880 mg. CO,. 9.275 “ “ : 3.010 “ AgCI.

G3HS306C1. Calculated. C 64.99, H 7.83, Cl 8.35. Found. “ 65.01, “ 7.60.

“ Cl 8.03.

Chloro-r-Isogitoxigenic Methyl Ester.-A suspension of the acid in acetone was esterified with diazomethane. The ester formed


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needles from dilute methyl alcohol which effervesced at 155” after preliminary softening and contained solvent. It is easily soluble in methyl and ethyl alcohols, acetone, and chloroform, and dissolves fairly readily in ether and benzene.

For analysis the substance was dried at 55-75” and 15 mm. When heated higher it gradually melted with decomposition.

4.577 mg. substance: 3.433 mg. H,O, Il.030 mg. COZ. 6.003 “ “ : 1.901 “ AgCl.

C24H350sC1. Calculated. C 65.64, H 8.04, Cl 8.08. Found. “ 65.71, “ 8.38.

“ Cl 7.84.

y-Isogitoxigenic Acid.-0.7 gm. of chloroisogitoxigenic acid was suspended in 175 cc. of water and the mixture was treated with 0.5 cc. of concentrated ammonia. After being shaken to dissolve, the clear solution was allowed to stand for 2 days at 20”. Acidifi- cation with acetic acid caused the precipitation of a rapidly crystallizing acid which was collected with water and proved to be halogen-free. It was recrystallized by solution in a few cc. of 50 per cent alcohol with the aid of a drop of ammonia and subsequent acidification with acetic acid. The acid separated as broad, flat needles which were anhydrous and effervesced at 260”. It is soluble in descending order in methyl and ethyl alcohol and acetone, and very sparingly soluble in the other usual solvents.

[01]:” = -27” (c = 0.637 in 95 per cent alcohol). 3.378 mg. substance: 2.556 mg. HxO, 8.455 mg. COZ.

CBHS~O.S Calculated. C 67.99, H 8.44. Found. “ 68.25, “ 8.46.

Anhydro-plsogitoxigenic Acid.-1 gm. of chloroisogitoxigenic acid was treated with 25 cc. of a 5 per cent solution of sodium hydrqxide in 50 per cent alcohol. A gelatinous mass of the salt formed. On being shaken at room temperature a clear solution gradually formed which t,ended to deposit a crystalline sodium salt. After 2 hours the mixture was carefully acidified with acetic acid and cautiously diluted. A copious crystallization of broad, flat prisms slowly deposited, which possessed the properties previously reported6 except that now the substance after recrystallization was found to melt at 210” instead of 215”.


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[a];” = $61” (c = 0.673 in 95 per cent alcohol). 3.900 mg. substance: 2.965 mg. HzO, 10.182 mg. CO,.

CdL~O~. Calculated. C 71.08, H 8.31. Found. “ 71.19, ‘( 8.50.

Anhydro-y-Isogitoxigenic,Methyl Ester. The ester was obtained in the usual manner from the acid with diazomethane. Careful dilution of the methyl alcoholic solution caused the separation of stout platelets which melted at 151”. The substance is soluble in the usual solvents except ligroin.

For analysis t,he substance was dried at 100” and 15 mm.

4.583 mg. substance: 3.432 mg. HzO, 12.005 COZ. mg. G3Haa06. Calculated. C 71.59, H 8.52.

Found. “ 71.44, “ 8.38.

Hydrogenation of Anhydro-y-Isogitoxigenic Methyl Ester. y- Digitoxanoldiacid Monomethyl Ester (the Labile Half Ester).-0.7 gm. of anhydro-y-isogitoxigenic methyl ester was hydrogenated in methyl alcoholic solution with 0.1 gm. of platinum oxide catalyst prepared according to Adams and Shriner. Following the prompt reduction of the catalyst, absorption completed itself within 5 minutes after approximately 2 mols of hydrogen had been absorbed. The filtrate from the catalyst yielded on concentration and careful dilution lustrous hexagonal leaflets which proved to be acid in character. After recrystallization by careful dilution of its solution in methyl alcohol the half ester melted constantly at 191-192” and contained solvent of crystallization. It is readily soluble in the alcohols and acetone and is appreciably soluble in chloroform, benzene, and ether. The substance dissolves readily in dilute ammonia or sodium carbonate. If, however, the 1aOter reagent is too concentrated, the acid is replaced by the sparingly soluble sodium salt which redissolves only on great dilution.


4.753 mg. substance: 3.990 mg. HSO, 12.335 mg. COZ. 5.120 “ “ : 2.780 “ AgI.

&H380h. Calculated. C 70.88, H 9.43, OCHa 7.63. Found. I‘ 70.78, “ 9.39.

“ OCH3 7.17.


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In experiments in which platinum black and colloidal palladium were used, absorption of 2 mols of hydrogen was also observed with the formation of the same half ester. In an experiment in which the absorption was interrupted at the 1 mol stage, half of the starting material was recovered and the other half was found to have been converted into the above half ester.

In an experiment on the catalytic hydrogenation of chloro-y- isogitoxigenic methyl ester the reaction was found to take the same course, which involved cleavage of the chlorine as halogen acid with hydrogenation and cleavage of the lactone group. -y-Digitoxanoldiacid monomethyl ester was obtained in excellent yield. The product melted at 191” and agreed in all properties with the substance obtained from the anhydro ester.

3.955 mg. anhydrous substance: 3.415 mg. HzO, 10.270 mg. CO*. Found. C 70.82, H 9.66.

The fact that the substance is a half ester was confirmed by titration.

For this purpose 14.800 mg. of substance were treated with 1 cc. of alcohol and titrated against phenolphthalein with 0.1 N NaOH. Found, 0.356 cc. Calculated for 1 equivalent, 0.364 cc.

2.6 cc. of 0.1 N NaOH were then added to the titration mixture. After being refluxed in an atmosphere of nitrogen for 33 hours, the mixture was titrated back. Found, 0.354 cc. Calculated for 1 equivalent, 0.364 cc.

yDigitoxanoldiacid.-When the solution of the salt obtained by saponification of the above half ester was acidified with acetic acid, a jelly formed, which slowly crystallized as needles. When recrystallized from dilute methyl alcohol the acid formed lustrous leaflets which contained solvent. It melted with effervescence at 207”, although this point depended upon the rate of heating.

[a]:” = +5.0” (c = 0.700 in95 per cent alcohol).


4.470 mg. substance: 3.660 mg. HzO, 11.515 mg. CO*. C23H360~. Calculated. C 70.35, H 9.25.

Found. “ 70.27, “ 9.16.


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y-Digitoxanoldiacid Dimethyl Ester.-The half ester was com- pletely esterified in acetone solution with diazomethane. From the concentrat,ed methyl alcoholic solution it readily crystallized as lustrous, rectangular plates or leaflets which were anhydrous and melted at 156-157” after slight preliminary softening. The substance is readily soluble in the usual solvents.

[oL]~’ = $6.5” (c = 1.997 in chloroform). 4.319 mg. substance: 3.765 mg. HzO, 11.330mg. COZ.

C2SH4005. Calculated. C 71.38, H 9.59. Found. “ 71.54, “ 9.75.

y-Digitoxanoldiacid Monomethyl Ester (Stable Half Ester) .- 0.12 gm. of the dimethyl ester was suspended in 30 cc. of 50 per cent methyl alcohol and treated with 2 cc. of 10 per cent sodium hydroxide solution. On gentle warming, complete solution occurred within a fraction of a minute. On acidification with acetic acid, glistening leaflets of the stable half ester gradually separated. The substance was recrystallized by concentration of its solution in dry acetone and separated as pointed prisms and platelets. From methyl alcohol it forms characteristic, long, hexagonal platelets. It melts at 218” and is very triboelectric. It is appreciably soluble in methyl alcohol and acetic acid and but sparingly soluble in acetone, chloroform, and ether. The substance has properties which lend themselves well for the characterization of the parent acid. For analysis the substance was dried at 100” and 15 mm.

4.887mg. substance: 4.090mg. H20,12,680mg. COZ. C&H380h. Calculated. C 70.88, H 9.43.

Found. “ 70.77, “ 9.36.

Derivatives of Isodigitoxigenin.

Isodigitoxigenic Methyl Ester.-Isodigitoxigenic acid was esterified in acetone solution with diazomethane. After removal of the solvent, the residue was dissolved in a small volume of methyl alcohol. On the addition of a few drops of water the est.er crystallized. After repetition of this recrystallization the substance formed flat needles or long, rectangular plates which melted at 174” and contained no solvent.


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4.565 mg. substance: 3.715 mg. H20,11.900 mg. CO*. CS~H,,O~. Calculated. C 71.23, H 8.98.

Found. “ 71.10, “ 9.10.

All attempts at catalytic hydrogenation of carefully recrystallized samples of’ this ester resulted in recovery of unchanged material.

y-Isodigitoxigenic Acid.-2.1 gm. of isodigitoxigenic acid were dissolved at 25” in 40 cc. of hydrochloric acid (1.19). The isomer started to crystallize within a few minutes. After standing for l+ hours, the mixture was carefully diluted and the reaction product was collected with water. The acid was suspended in a small volume of 50 per cent alcohol and then brought into solution by the addition of a slight excess of ammonia. On careful reacidification with acetic acid the acid crystallized as hexagonal or pointed platelets. These were most conveniently collected with 33 per cent alcohol because of the great solubility of the substance in stronger alcohol. As so obtained, it contained 1 mol of water of crystallization and melted at 118” with decomposition.

[a]:” = $60” (c = 0.553 in 95 per cent alcohol).

For analysis the air-dry substance was dried at 100” and 15 mm.

4.609 mg. substance: 0.205 mg. HzO. C23H3406.Hz0. Calculated. Hz0 4.41. Found. Hz0 4.45.

3.735 mg. substance: 2.925 mg. HzO, 9.615 mg. COZ. C23H3406. Calculated. C 70.72, H 8.78.

Found. “ 70.21, “ 8.77.

y-Isodigitoxigenic Methyl Ester.-This was prepared in the usual manner with diazomethane. On careful dilution of its methyl alcoholic solution it formed lustrous leaflets which melted at 168” and contained no solvent.

3.683 mg. substance : 2.980 mg. HzO, 9.632 mg. COZ. CZ~HSGO~. Calculated. C 71.23, H 8.98.

Found. “ 71.32, “ 9.05.

In attempts at the catalytic hydrogenation of this substance, it was recovered unchanged. When the -attempt was made by heating the ester in 1 per cent met,hyl alcoholic hydrochloric acid to achieve lactone cleavage with the formation of an anhydro dimethyl ester only non-crystalline resin was recovered.


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Acetate of -pDigitoxenoldiacid Anhydride.-l gm. of y-isodigitoxi- genie acid was heated in a sealed tube in a mixture of 18 cc. of acetic anhydride and 2 cc. of acetyl chloride for 15 hours. The clear solution was concentrated under diminished pressure to remove all reagent. This was aided by addition of small amounts of chloroform to the glassy residue and redistillation. The residue was finally dissolved in chloroform and the resulting solution was shaken out successively with dilute sodium carbonate solution and water. The dried chloroform solution of neutral material after concentration left a glassy residue which readily crystallized under methyl alcohol.

When recrystallized carefully from methyl alcohol it formed leaflets which melted at 182-184” after preliminary softening and contained no solvent.

3.970 mg. substance: 2.930 mg. HQO, 10.530 mg. C&. CasH,,Oa. Calculated. C 72.42, H 8.27.

Found. “ 72.34, “ 8.26.

Attempts to obtain the free acid by saponification were unsuccessful apparently due to more deep seated chemical change under the influence of the alkali employed.

Acetate of r-Digitoxenoldiacid Monomethyl Ester.-0.1 gm. of the above anhydride was treated with 2 cc. of a solution of 1 per cent hydrochloric acid in dry methyl alcohol and warmed very gently until all dissolved. This occurred fairly readily. The mixture was at once cooled and carefully diluted. On rubbing, the half ester rapidly crystallized and was collected with 70 per cent methyl alcohol. After recrystallization from dilute methyl alcohol, it formed leaflets which contained solvent and melted to a resin at 89”. It is very readily soluble in most solvents.

For analysis it was dried at 65” and finally at 75” and 15 mm.

5.115 mg. substance: 0.295 mg. HsO. C&H380~.1+ HZO. Calculated. Hz0 5.71. Found. Hz0 5.77.

4.820 mg. substance: 3.700 mg. HzO, 12.360 mg. COZ. 5.385 “ “ : 2.960 “ AgI.

CtaHJsOa. Calculated. C 69.91, H 8.58, OCH3 6.95. Found. “ 69.94, “ 8.59.

‘I OCH, 7.25.

Acetate of y-Digitoxanoldiacid Monomethyl Ester.-The above


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half ester was hydrogenated in methyl alcoholic solut.ion with platinum oxide catalyst. Approximately 1 mol of hydrogen was absorbed after saturation of the catalyst. The saturated substance readily crystallized on concentration to a few cc. Recrys- tallized from a small volume of methyl alcohol, it formed four- sided leaflets which contained solvent and exhibited a very uncer- tain melting point. After repeated recrystallization it softened to a jelly at 80” which appeared to resolidify at about 130” and then melted again at 182”. Although the melting point was not satisfactory, the presence of isomers seemed to be excluded by the fact that recrystallization made no apparent change in the optical activity of the substance recovered.

[a]$,” = +4” (c = 0.67 in methyl alcohol).


4.720mg. substance: 3.905mg. HzO, 11.988mg. COS. C&HtoOa. Calculated. C 69.59, H 8.99.

Found. “ 69.27, “ 9.26.

pDigitoxanoldiacid.-0.2 gm. of the above saturated acetate was warmed for 1 hour in 10 cc. of methyl alcohol and 25 cc. of 0.4 per cent sodium hydroxide solution. Acidification with acetic acid caused the separation of lustrous needles. After recrystallization from dilute methyl alcohol, it formed leaflets which melted with effervescence at 207”. When mixed with the dibasic acid obtained from isogitoxigenin it showed no depression. In all other properties the two substances appeared to be identical.


4.190 mg. substance: 3.413 mg. HzO, 10.783 mg. 602. C23H360~. Calculated. C 70.35, H 9.25.

Found. “ 70.19, (’ 9.12.

-y-Digitoxanoldiacid Dimethyl Ester.-The identity of the above diacid with that obtained from isogitoxigenin was further confirmed by the preparation of the dimethyl ester, which agreed in all properties with that previously described. It melted at 156-157” and showed no depression with the isogitoxigenin material.

[cy]:” = +7.5” (c = 2.003 in chloroform).

4.067 mg. substance: 3.460 mg. H,O, 10.640 mg. COz. C&LoO~. Calculated. C 71.38, H 9.59.

Found. “ 71.35, “ 9.52.


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y-Digitoxanoldiacid Monomethyl Ester (Stable Half Ester) .-On partial saponification as previously described the dimethyl ester gave the stable half ester which also proved to be identical with the analogous isogitoxigenin derivative. The stable half ester formed long, hexagonal platelets from acetone which melted at 218”.

5.320 mg. substance: 4.462 mg. HzO, 13.892 mg. COZ. 4.476 “ “ : 2.503 “ AgI.

&HS805. Calculated. C 70.88, H 9.43, OCH, 7.63. Found. “ 71.21, “ 9.38.

“ OCHa 7.38.


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Walter A. Jacobs and Edwin L. GustusWITH DIGITOXIGENIN

CORRELATION OF GITOXIGENIN THE DIGITALIS GLUCOSIDES: IV. THE

1930, 86:199-216.J. Biol. Chem.

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