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Gitoside—A New Digitalis Glycoside*
170
JOURNAL OF THE
AMERICANPHARMACEUTICAL ASSOCIATION
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0
7 -
a
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abilities to produce motor paralysis in rabbits when given by subarachnoid injection. 3. &Diethylaminoethyl cumate HC1 was found to have a lower acute toxicity to mice than procaine HCl. Their respective LDSo’s by intraperitoneal injection are 4T0 13.4 mg./Kg. and 189 f 10.0 mg./Kg. 4. Irritation tests showed procaine HC1 to be definitely the less irritant of the two compounds. In intradermal injections in humans, P-diethylaminoethyl cumate HC1 in concentrations less than l/z per cent commonly produced erythema while procaine HCl rarely gave this reaction. In tests on the rabbit eye, 2 per cent solutions of the former compound were found to be definitely irritating. Procaine HCI, on the other hand, gave no evidence of irritation in concentrations up to 10 per cent. The trypan blue test confirmed the fact that 0-diethylaminoethyl cumate HCI was actually considerably more irritating than procaine HC1 since i t required approximately five times the concentration of the latter drug to produce a degree of irritation corresponding to that for the cumic acid ester.
*
V v)
Vvl. XLVI, No. 3
I -
1 0.25
0.5
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2.0
4.0
C 0 N C E NTRAT10N ( O d Fig. 1.-Trypan blue test for irritation. 1-p-Diethylaminoethyl cumate HC1. 2-Procaine HC1. Trypan Blue Test for Irritation.-From Fig. 1, the TIC4 for &diethylaminoethyl cumate HC1 was found to be 0.43% as compared with 2.1% for procaine. Thus, again, P-diethylaminoethyl cumate gave evidence of being considerably more irritating. SUMMARY
1. The local anesthetic potencies, acute toxicities, and irritant properties of P-diethylaminoethyl cumate HCI and procaine HCI have been compared. 2. 0-Diethylaminoethyl cumate HC1 appeared to be the more potent of the two compounds as a local anesthetic when compared on the basis of their minimal effective concentrations as determined in human intradermal tests. Little, if any, difference in potency was observed in their
Gitoside-A
REFERENCES (1) Williams, R. B., and Voss, E.. THISJOURNAL, 40, 449
(1951).
(2) Bryan, J. T., and Foote, P. A., i b i d . , 39, 644(1950). (3) McIntyre, A. R., and Severs, K. F., J . Pharmarol. Ezpll. Therap., 61, 107 (1937). (4) Sollmann, T.,i b i d . , 11. f i Q l l Q l 8 l (5) Bieter, R. N . , Cuni McNearney, J. 1 (A) Miller, L. Biol. M e d . . 57, 2
New Digitalis Glycoside* By JAMES E. MURPHY
A new glycoside, the monodigitoxoside of gitoxigenin, has been isolated from D. lanata. Procedures for i.ts isolation and characterization are given. RESULTS of paper chromatographic analyses of extracts of digitalis run in our laboratories as well as those published by others, ROM
*
Received August 3, 1956, from the Analytical Research Laboratory, Burroughs Wellcome & Co. ( U S A . ) Inc., Tuckahoe., N. Y __ The author wishes to express his gratitude t o Dr. A. Elek for the microanalyses to Miss W. Holden for the bioassay and t o Dr. C. W. Ferry for his advice and encouragemen; during this work. ~~~~~~~~~~
it became evident that besides the known glycosides of digitalis there are many other glycosidal substances present which have not been isolated and characterized. During the past few years, several new glycosides, gitorin (l), strospeside (2, 3), digipurpurin (4), digifolein (4), glucogitofucoside (4), gitaloxin ( 5 ) , verodoxin (F), digiproside (7), and diginatin (S), have been isolated. In this paper we shall describe another glycoside which has not been previously isolated and which we have obtained from extracts of D.Zunatu.
SCIENTIFIC EDITION
March 1957
171
tography of some residues from digoxin processing, were made by treating the residues with acetone, in which it appeared quite soluble. This freed it from most of the digoxin and a concentrate of this solution was put on a partition column consisting of Celite containing water and fotmamide as the stationary phase and developed with chloroform-benzene 3: 1. ally called Unknown "B" i n a n earlier paper from The fractions containing the unknown glycoside were worked up and yielded a white compound, this laboratory. Its position on paper chromatom. p. 212". Paper chromatographic analyses indigrams was indicated,in Fig. 1 of that publication cated that it was a single substance and other data suggested that it was composed of two moles of (8). T h e position of gitoside on paper chromato- digitoxose and 1 mole of gitoxigenin. However, grams corresponds very well with that of the after hydrolysis, considerable digoxigenin showed on chromatograms of the hydrolysate indicating that unknown glycoside (b) detected b y Jensen (9), the separation from digoxin had not been complete. and i t may be the same substance. H e reported After development of a more sensitive spray reagent t h a t it gave a fluorescent reaction when sprayed for digoxin,' this was definitely proved to be the case. with trichloroacetic acid i n alcohol, like the Attempts a t separating digoxin from the unknown by rechromatographing with the same or similar gitoxigenin-containing glycosides, and a green solvent systems was not successful. color given b y glycosides containing 2-desoxy I t was decided t o use paper chromatography as sugars, such as digitoxose. In a later publica- a screening method for testing other solvent systems. tion (1 0) , Jensen redesignated this as glycoside Formamide saturated strips were used. Mixtures (Bd), isolated it i n microgram amounts b y paper of methylene chloride and acetone or ethylene chloride and acetone showed proinisc and instead chromatography and showed that on heating it of having a lower Rf value than digoxin as was the with dilute sulfuric acid, gitoxigenin was formed. case with chloroform and chloroform and benzene, Gitoside was isolated in our laboratory b y the unknown had a higher Rfvalue. The Rr values means of partition chromatography on Celite@ were quite high and it was found that by adding benzene the development was slowed to a rate which columns from residues obtained in the processing would warrant using a mixture of this composition of digoxin. T h e D. Zanata from which they were for a column separation. obtained was first year growth, oven dried leaf. Using a system of ethylene chloride 10 vol., benAnalytical data before and after hydrolysis showed zene 10 vol., acetone 2 vol., and aratio of formamidethat the glycoside consisted of a mole of gitoxige- water 2 : l for the stationary phase, digoxin could be completely separated from the unknown. Parnin and a mole of digitoxose. Other monosides titioning the residue on this column yielded the unof gitoxigenin have been found in digitalis, e. g., known contaminated with small amounts of gitoxin strospeside is the monodigitaloside of gitoxigenin and gitoxigenin. These were removed by rechromatographing on a column using the same stationary and gitorin is the monogluooside of gitoxigenin. A t first, results indicated t h a t t h e substance phase but a mobile phase of chloroform containing 10% n-heptane. The proper fractions were concencould be the gitalin described by Kraft and trated, the concentrate poured into cold water, Cloetta (11, 12), because assays indicated a ratio filtered off, and dried. The dry precipitate was of approximately 2 : 1 digitoxose-gitoxigenh. It refluxed with a small amount of acetone to remove was found, however, that the sample was not much of the color and then dissolved in hot 80% alcohol, treated with charcoal, and allowed to purified sufficiently and the presence of digoxin crystallize slowly. Filtration and drying yielded a increased the digitoxose result considerably. white, crystalline powder, individual particles From results of our work on both D. Zanata and D. appearing as light yellow rosettes under the micropurpurea, there does not appear to be a glycoside scope, ni. p. 215-217'. Anal.-Calcd. for CzrHarOp: C, 66.90; H, 8.52. of the composition of gitalin in digitalis unless Found C, 66.60; H, 8.73. Specific rotations were it is formed as an artifact because of the methods determined in a 2 dm. tube: [a]*;+7.8", [a15:~1 used for the extraction and isolation. +10.4" ( C = 4.1, methyl alcohol), [a]': -18.9". [a],::, -21.8' ( C = 8.9 pyridine). Using a modification of the quantitative assay It gave a positive reaction for a 2-desoxy sugar procedure worked out b y Jensen for gitoxin (13), with Keller-Kiliani reagent. The ring test for digithe amount of gitoside in a sample of dried D . talis glycosides gave a bright red color a t the juncZanata leaves was found t o be 0.015 per cent. tion of the two layers of acid as well as in the sulfuric Bioassay showed i t t o be cardioactive; a acid layer and the acetic acid layer changed to indigo U. S. P. XV pigeon assay giving a lethal dose of on standing. When assayed against digoxin using a modified 1.12 mg./Kg. Keller-Kiliani reagent, it assayed 25.0% digitoxose.
This new glycoside has also been detected in extracts of D. purpurea, D. thapsi, D. sibirica, D. lutea, D. ambigua, D. mertonensis, and D. ferruginea. It is also one of the substances present i n commercial amorphous gitalin. It has been given the name gitoside and was origin-
EXPERIMENTAL First attempts at isolation of the unknown substance. which had been discovered by paper chroma-
1 Twenty per cent trichloroacetic acid in chloroform to which 4 drops of 30% hydrogen peroxide was added just before use.
172
JOURNAL OF
THE
AMERICAN PHARMACEUTICAL ASSOCIATION
Assayed against gitoxigenin by a fluorescence assay (14), it assayed 75.9% gitouigenin. Theory for gitoxigenin and 1 mole of digitoxose is digitoxose 25% and gitoxigenin 75%. The ultraviolet spectrum was determined in 507, alcohol and is shown in Fig. 1.
2.0
.
i' 1.5
.
220
250
280 Mu
310
340
Fig. 1.-Ultraviolet Spectra. . . . . . . Gitoside in 50'% alcohol. - - - - Gitoxigenin from gitoside in alcohol. -Dianhydro gitoxigenin from gitoside in alcohol. Hydrolysis of Gitoside.-One-half gram was refluxed with 25 ml of alcohol, 25 ml. of water, and 0.2 ml. of hydrochloric acid for thirty minutes. The mixture was then cooled, neutralized with sodium hydroxide and concentrated under reduced pressure to about 10 ml. A large amount of aglycone separated. The mixture was filtered and the dried precipitate amounted t o 0.32 Gni. The filtrate was extracted with two 10-ml. portions of chloroform. The chloroform was evaporated and the residue was dissolved in acetone, diluted with water, heated to remove the acetone, cooled, and filtered. This gave 0.04 Gm. more aglycone for a combined yield of 97.3y0 of theory. The aglycone was recrystallized from methanol, m. p. 227-228". A mixed melting point with a known sample of gitoxigenin showed no depression. Paper chromatography also suggested it was gitoxigenin. The ultraviolet spectrum is given in Fig. 1. And-Calculated for C23H3aOa: C, 70.74; H, 8.78. Found: C, 70.51; H, 8.70. For further identification the dianhydro derivative was formed. Fifty milligrams of the aglycone was stirred with 10 ml. of propylene glycol-hydrochloric acid 1 : 1 until dissolved and kept a t 15-20" for one hour. Forty milliliters of water was added; the mixture was cooled and filtered. The precipitate was recrystallized from 50% alcohol. Yield, 28.5 mg., m. p. 215'. A mixed melting point with a known sample of dianhydrogitoxigenin showed no depression. [a]': +589" (C = 0.26, methyl alcohol). Reported for diarihydrogitoxigenin, +573" (methyl alcohol). The ultraviolet spectrum is given in Fig. 1.
Vol. XLVI, No. 3
The chloroform extracted filtrate from the above hydrolysis was evaporated to dryness under reduced pressure, the residue was extracted with 10 ml. of pyridine, and the pyridine was removed under reduced pressure. Paper chromatography of the residue, using a solvent system of n-butyl alcohol-acetic acid-water 4:1:5, gave two spots for 2-desoxy sugars when sprayed with trichloroacetic acid in alcohol. One a t Rf 0.59 corresponded to digitoxose. The other was a t RfO.83.It was found that when the hydrolysis and paper chromatography were repeated on the aqueous portion of the hydrolysate, the ratio of the intensity of the two spots differed. This suggested that the extra spot was caused by degradation of part of the digitoxose or by formation of an a or 6 pyranose form of digitoxose. To check this, a sample of pure digitoxose, giving only one spot on paper chromatograms was subjected to the hydrolysis procedure. The same two spots were obtained after the treatment. Similar results were obtained when digoxin was hydrolyzed by the same procedure. Isolation of this second hydrolysis product is being attempted and it is hoped to report on it at a later date. The benzimidazole derivative of the residue from the hydrolysis was made using the procedure of Dimler and Link (15). m. p. 210-210.5". A mixed melting point with a known sample of the benzimidazole of digitoxose showed n o depression. SUMMARY
1. A new cardioactive glycoside which has been named gitoside has been isolated from D. Zunatu. It has been detected in several other species of digitalis including D.purpurea. 2. Analyses and hydrolysis of this glycoside show i t to consist of 1 mole of gitoxigenin and 1 mole of digitoxose. 3. On hydrolyzing glycosides containing digitoxose part of the digitoxose forms another compound which still gives the reaction for a 2desoxy sugar on paper chromatograms. REFERENCES (1) Tschesch Chem. Ber. 85 I (2) Ritiel. 'V Chim: Acla (3) Sat1 Pharm. BI&, 1 (4) Tschesch
m ,-"--,.
f ia
(5) Haack, E., Kaiser, F., and Spingler, H., ibid., 89, 87 (1956). (6) Haack, E.,Kaiser, F., and Spingler, H., Nalrvwiss., 43. 130(1956\. ' (7) Satod, D., Tsbii, H., Oyama, Y., and Oknmura, T . , J . Pharm. SOC.Japan 75 1573(1955). (8) Murphy J. E.' T&s JOURNAL44 719(1955). (9) Jensen K B. k c l a Phaumacol.' Tokicol., 9, 275(1953). (10) Jensen' K: B.' i b i d . 12 ll(1966). (11) Kraft.'F.. Arch. Phhvm:. 250: 118(1912\. (12) Cloetta, M.,Arch. e z p t l : Paihol. Phavkakol, 112, 261 ~
f\ ' 1m 0a "9, C I
(13) Jensen K. B. Acla Phavmacol. Tozicol. 10,69(1954). (14) Murphy J. E'. THIS JOURNAL 43 659(1954). (15) Dimler, R . J.,'and Link, K. P., Biol. Chem., 150, 345(1943).
.!.
JOURNAL OF THE
AMERICANPHARMACEUTICAL ASSOCIATION
I ¶ -
w
8 -
0
7 -
a
6 -
z 0 F
4--
T I
32 -
5 -
8 0:
abilities to produce motor paralysis in rabbits when given by subarachnoid injection. 3. &Diethylaminoethyl cumate HC1 was found to have a lower acute toxicity to mice than procaine HCl. Their respective LDSo’s by intraperitoneal injection are 4T0 13.4 mg./Kg. and 189 f 10.0 mg./Kg. 4. Irritation tests showed procaine HC1 to be definitely the less irritant of the two compounds. In intradermal injections in humans, P-diethylaminoethyl cumate HC1 in concentrations less than l/z per cent commonly produced erythema while procaine HCl rarely gave this reaction. In tests on the rabbit eye, 2 per cent solutions of the former compound were found to be definitely irritating. Procaine HCI, on the other hand, gave no evidence of irritation in concentrations up to 10 per cent. The trypan blue test confirmed the fact that 0-diethylaminoethyl cumate HCI was actually considerably more irritating than procaine HC1 since i t required approximately five times the concentration of the latter drug to produce a degree of irritation corresponding to that for the cumic acid ester.
*
V v)
Vvl. XLVI, No. 3
I -
1 0.25
0.5
1.0
2.0
4.0
C 0 N C E NTRAT10N ( O d Fig. 1.-Trypan blue test for irritation. 1-p-Diethylaminoethyl cumate HC1. 2-Procaine HC1. Trypan Blue Test for Irritation.-From Fig. 1, the TIC4 for &diethylaminoethyl cumate HC1 was found to be 0.43% as compared with 2.1% for procaine. Thus, again, P-diethylaminoethyl cumate gave evidence of being considerably more irritating. SUMMARY
1. The local anesthetic potencies, acute toxicities, and irritant properties of P-diethylaminoethyl cumate HCI and procaine HCI have been compared. 2. 0-Diethylaminoethyl cumate HC1 appeared to be the more potent of the two compounds as a local anesthetic when compared on the basis of their minimal effective concentrations as determined in human intradermal tests. Little, if any, difference in potency was observed in their
Gitoside-A
REFERENCES (1) Williams, R. B., and Voss, E.. THISJOURNAL, 40, 449
(1951).
(2) Bryan, J. T., and Foote, P. A., i b i d . , 39, 644(1950). (3) McIntyre, A. R., and Severs, K. F., J . Pharmarol. Ezpll. Therap., 61, 107 (1937). (4) Sollmann, T.,i b i d . , 11. f i Q l l Q l 8 l (5) Bieter, R. N . , Cuni McNearney, J. 1 (A) Miller, L. Biol. M e d . . 57, 2
New Digitalis Glycoside* By JAMES E. MURPHY
A new glycoside, the monodigitoxoside of gitoxigenin, has been isolated from D. lanata. Procedures for i.ts isolation and characterization are given. RESULTS of paper chromatographic analyses of extracts of digitalis run in our laboratories as well as those published by others, ROM
*
Received August 3, 1956, from the Analytical Research Laboratory, Burroughs Wellcome & Co. ( U S A . ) Inc., Tuckahoe., N. Y __ The author wishes to express his gratitude t o Dr. A. Elek for the microanalyses to Miss W. Holden for the bioassay and t o Dr. C. W. Ferry for his advice and encouragemen; during this work. ~~~~~~~~~~
it became evident that besides the known glycosides of digitalis there are many other glycosidal substances present which have not been isolated and characterized. During the past few years, several new glycosides, gitorin (l), strospeside (2, 3), digipurpurin (4), digifolein (4), glucogitofucoside (4), gitaloxin ( 5 ) , verodoxin (F), digiproside (7), and diginatin (S), have been isolated. In this paper we shall describe another glycoside which has not been previously isolated and which we have obtained from extracts of D.Zunatu.
SCIENTIFIC EDITION
March 1957
171
tography of some residues from digoxin processing, were made by treating the residues with acetone, in which it appeared quite soluble. This freed it from most of the digoxin and a concentrate of this solution was put on a partition column consisting of Celite containing water and fotmamide as the stationary phase and developed with chloroform-benzene 3: 1. ally called Unknown "B" i n a n earlier paper from The fractions containing the unknown glycoside were worked up and yielded a white compound, this laboratory. Its position on paper chromatom. p. 212". Paper chromatographic analyses indigrams was indicated,in Fig. 1 of that publication cated that it was a single substance and other data suggested that it was composed of two moles of (8). T h e position of gitoside on paper chromato- digitoxose and 1 mole of gitoxigenin. However, grams corresponds very well with that of the after hydrolysis, considerable digoxigenin showed on chromatograms of the hydrolysate indicating that unknown glycoside (b) detected b y Jensen (9), the separation from digoxin had not been complete. and i t may be the same substance. H e reported After development of a more sensitive spray reagent t h a t it gave a fluorescent reaction when sprayed for digoxin,' this was definitely proved to be the case. with trichloroacetic acid i n alcohol, like the Attempts a t separating digoxin from the unknown by rechromatographing with the same or similar gitoxigenin-containing glycosides, and a green solvent systems was not successful. color given b y glycosides containing 2-desoxy I t was decided t o use paper chromatography as sugars, such as digitoxose. In a later publica- a screening method for testing other solvent systems. tion (1 0) , Jensen redesignated this as glycoside Formamide saturated strips were used. Mixtures (Bd), isolated it i n microgram amounts b y paper of methylene chloride and acetone or ethylene chloride and acetone showed proinisc and instead chromatography and showed that on heating it of having a lower Rf value than digoxin as was the with dilute sulfuric acid, gitoxigenin was formed. case with chloroform and chloroform and benzene, Gitoside was isolated in our laboratory b y the unknown had a higher Rfvalue. The Rr values means of partition chromatography on Celite@ were quite high and it was found that by adding benzene the development was slowed to a rate which columns from residues obtained in the processing would warrant using a mixture of this composition of digoxin. T h e D. Zanata from which they were for a column separation. obtained was first year growth, oven dried leaf. Using a system of ethylene chloride 10 vol., benAnalytical data before and after hydrolysis showed zene 10 vol., acetone 2 vol., and aratio of formamidethat the glycoside consisted of a mole of gitoxige- water 2 : l for the stationary phase, digoxin could be completely separated from the unknown. Parnin and a mole of digitoxose. Other monosides titioning the residue on this column yielded the unof gitoxigenin have been found in digitalis, e. g., known contaminated with small amounts of gitoxin strospeside is the monodigitaloside of gitoxigenin and gitoxigenin. These were removed by rechromatographing on a column using the same stationary and gitorin is the monogluooside of gitoxigenin. A t first, results indicated t h a t t h e substance phase but a mobile phase of chloroform containing 10% n-heptane. The proper fractions were concencould be the gitalin described by Kraft and trated, the concentrate poured into cold water, Cloetta (11, 12), because assays indicated a ratio filtered off, and dried. The dry precipitate was of approximately 2 : 1 digitoxose-gitoxigenh. It refluxed with a small amount of acetone to remove was found, however, that the sample was not much of the color and then dissolved in hot 80% alcohol, treated with charcoal, and allowed to purified sufficiently and the presence of digoxin crystallize slowly. Filtration and drying yielded a increased the digitoxose result considerably. white, crystalline powder, individual particles From results of our work on both D. Zanata and D. appearing as light yellow rosettes under the micropurpurea, there does not appear to be a glycoside scope, ni. p. 215-217'. Anal.-Calcd. for CzrHarOp: C, 66.90; H, 8.52. of the composition of gitalin in digitalis unless Found C, 66.60; H, 8.73. Specific rotations were it is formed as an artifact because of the methods determined in a 2 dm. tube: [a]*;+7.8", [a15:~1 used for the extraction and isolation. +10.4" ( C = 4.1, methyl alcohol), [a]': -18.9". [a],::, -21.8' ( C = 8.9 pyridine). Using a modification of the quantitative assay It gave a positive reaction for a 2-desoxy sugar procedure worked out b y Jensen for gitoxin (13), with Keller-Kiliani reagent. The ring test for digithe amount of gitoside in a sample of dried D . talis glycosides gave a bright red color a t the juncZanata leaves was found t o be 0.015 per cent. tion of the two layers of acid as well as in the sulfuric Bioassay showed i t t o be cardioactive; a acid layer and the acetic acid layer changed to indigo U. S. P. XV pigeon assay giving a lethal dose of on standing. When assayed against digoxin using a modified 1.12 mg./Kg. Keller-Kiliani reagent, it assayed 25.0% digitoxose.
This new glycoside has also been detected in extracts of D. purpurea, D. thapsi, D. sibirica, D. lutea, D. ambigua, D. mertonensis, and D. ferruginea. It is also one of the substances present i n commercial amorphous gitalin. It has been given the name gitoside and was origin-
EXPERIMENTAL First attempts at isolation of the unknown substance. which had been discovered by paper chroma-
1 Twenty per cent trichloroacetic acid in chloroform to which 4 drops of 30% hydrogen peroxide was added just before use.
172
JOURNAL OF
THE
AMERICAN PHARMACEUTICAL ASSOCIATION
Assayed against gitoxigenin by a fluorescence assay (14), it assayed 75.9% gitouigenin. Theory for gitoxigenin and 1 mole of digitoxose is digitoxose 25% and gitoxigenin 75%. The ultraviolet spectrum was determined in 507, alcohol and is shown in Fig. 1.
2.0
.
i' 1.5
.
220
250
280 Mu
310
340
Fig. 1.-Ultraviolet Spectra. . . . . . . Gitoside in 50'% alcohol. - - - - Gitoxigenin from gitoside in alcohol. -Dianhydro gitoxigenin from gitoside in alcohol. Hydrolysis of Gitoside.-One-half gram was refluxed with 25 ml of alcohol, 25 ml. of water, and 0.2 ml. of hydrochloric acid for thirty minutes. The mixture was then cooled, neutralized with sodium hydroxide and concentrated under reduced pressure to about 10 ml. A large amount of aglycone separated. The mixture was filtered and the dried precipitate amounted t o 0.32 Gni. The filtrate was extracted with two 10-ml. portions of chloroform. The chloroform was evaporated and the residue was dissolved in acetone, diluted with water, heated to remove the acetone, cooled, and filtered. This gave 0.04 Gm. more aglycone for a combined yield of 97.3y0 of theory. The aglycone was recrystallized from methanol, m. p. 227-228". A mixed melting point with a known sample of gitoxigenin showed no depression. Paper chromatography also suggested it was gitoxigenin. The ultraviolet spectrum is given in Fig. 1. And-Calculated for C23H3aOa: C, 70.74; H, 8.78. Found: C, 70.51; H, 8.70. For further identification the dianhydro derivative was formed. Fifty milligrams of the aglycone was stirred with 10 ml. of propylene glycol-hydrochloric acid 1 : 1 until dissolved and kept a t 15-20" for one hour. Forty milliliters of water was added; the mixture was cooled and filtered. The precipitate was recrystallized from 50% alcohol. Yield, 28.5 mg., m. p. 215'. A mixed melting point with a known sample of dianhydrogitoxigenin showed no depression. [a]': +589" (C = 0.26, methyl alcohol). Reported for diarihydrogitoxigenin, +573" (methyl alcohol). The ultraviolet spectrum is given in Fig. 1.
Vol. XLVI, No. 3
The chloroform extracted filtrate from the above hydrolysis was evaporated to dryness under reduced pressure, the residue was extracted with 10 ml. of pyridine, and the pyridine was removed under reduced pressure. Paper chromatography of the residue, using a solvent system of n-butyl alcohol-acetic acid-water 4:1:5, gave two spots for 2-desoxy sugars when sprayed with trichloroacetic acid in alcohol. One a t Rf 0.59 corresponded to digitoxose. The other was a t RfO.83.It was found that when the hydrolysis and paper chromatography were repeated on the aqueous portion of the hydrolysate, the ratio of the intensity of the two spots differed. This suggested that the extra spot was caused by degradation of part of the digitoxose or by formation of an a or 6 pyranose form of digitoxose. To check this, a sample of pure digitoxose, giving only one spot on paper chromatograms was subjected to the hydrolysis procedure. The same two spots were obtained after the treatment. Similar results were obtained when digoxin was hydrolyzed by the same procedure. Isolation of this second hydrolysis product is being attempted and it is hoped to report on it at a later date. The benzimidazole derivative of the residue from the hydrolysis was made using the procedure of Dimler and Link (15). m. p. 210-210.5". A mixed melting point with a known sample of the benzimidazole of digitoxose showed n o depression. SUMMARY
1. A new cardioactive glycoside which has been named gitoside has been isolated from D. Zunatu. It has been detected in several other species of digitalis including D.purpurea. 2. Analyses and hydrolysis of this glycoside show i t to consist of 1 mole of gitoxigenin and 1 mole of digitoxose. 3. On hydrolyzing glycosides containing digitoxose part of the digitoxose forms another compound which still gives the reaction for a 2desoxy sugar on paper chromatograms. REFERENCES (1) Tschesch Chem. Ber. 85 I (2) Ritiel. 'V Chim: Acla (3) Sat1 Pharm. BI&, 1 (4) Tschesch
m ,-"--,.
f ia
(5) Haack, E., Kaiser, F., and Spingler, H., ibid., 89, 87 (1956). (6) Haack, E.,Kaiser, F., and Spingler, H., Nalrvwiss., 43. 130(1956\. ' (7) Satod, D., Tsbii, H., Oyama, Y., and Oknmura, T . , J . Pharm. SOC.Japan 75 1573(1955). (8) Murphy J. E.' T&s JOURNAL44 719(1955). (9) Jensen K B. k c l a Phaumacol.' Tokicol., 9, 275(1953). (10) Jensen' K: B.' i b i d . 12 ll(1966). (11) Kraft.'F.. Arch. Phhvm:. 250: 118(1912\. (12) Cloetta, M.,Arch. e z p t l : Paihol. Phavkakol, 112, 261 ~
f\ ' 1m 0a "9, C I
(13) Jensen K. B. Acla Phavmacol. Tozicol. 10,69(1954). (14) Murphy J. E'. THIS JOURNAL 43 659(1954). (15) Dimler, R . J.,'and Link, K. P., Biol. Chem., 150, 345(1943).
.!.