INVESTIGATIONS ON STEROIDS. IX. SOME NEW ... - ACS Publications

[CONTRIBUTION OM THE GEORGE S. Cox MEDICA^ RESEARCH INBTITUTB, UNIVERSITY OF PENNSYLVANIA]

INVESTIGATIONS ON STEROIDS. IX. SOME NEW POLYHYDROXYETIOCHOLANIC ACIDS1 MAXIMILIAN EHRENSTEIN

AND

ALDA RUTH JOHNSON

Received July $8, 1946

\In a previous publication (1) 3 5 14-trihydroxyestrane-l0,17dicarboxylic acid served as an intermediate in the preparation of compounds structurally related to hormones of the pregnane series. The dicarboxylic acid was prepared from strophanthidin (I) according t o procedures known from the literature. The stepwise degradation of the unsaturated lactone ring is a time-consuming and uneconomical procedure which, in addition, is associated with an undesired inversion of the configuration a t carbon atom 17. Doubts have recently arisen regarding the presently accepted stereochemical structure of strophanthidin.2 Ruzicka (2) states for instance that certain reactions of strophanthidin are not in agreement with the configuration of a derivative of 3(a) 5-dihydroxycoprostane. Very recent work from the same laboratory (3) supports the assumption that in the normal, naturally-occurring, sterids the side chain at carbon atom 17 is attached in @-position?" According to this postulation, the part of ring D in the cardiac aglycons has to be formulated

Because of these uncertainties regarding the configuration of strophanthidin, no stereochemical relationships will be indicated in the chemical structures of this publication though certain aspects of configuration will be discussed. A number of years ago Steiger and Reichstein (4)published a new approach to the problem of degrading the unsaturated lactone ring of cardiac aglycons. By treating the diacetate of digoxigenin with less than the stoichiometrically ].Aided by a joint grant from the Smith, Kline, and French Laboratories and Sharp & Dohme, Inc. in Philadelphia. Additional support was provided through a grant from the American Cancer Society on the recommendation of the Committee on Growth of the National Research Council. *For conventional stereochemical structure c f . e . g . (1, p. 436, formula I) In this country, Gallagher and Long (12) had previously arrived at the same conclusion. While this paper was in press, Reichstein's laboratory (13) also reported d a t a supporting this configuration a t carbon atom 17. 823

824

MAXIMILIAN EHRENSTEIN AND ALDA RUTH JOHNSON

required amount of potassium permanganate in a solution of acetone they were able to remove three carbon atoms of the unsaturated lactone ring in one operation, and thus succeeded in obtaining a 3 ,12-diacetoxy-14-hydroxyetiocholanic acid. The neutral fraction of this oxidation consisted almost exclusively of unchanged crystalline starting material. It did not appear promising to apply the same method to strophanthidin acetate because the formation of an additional carboxyl group a t carbon atom 10would complicate the separation of the reaction products. The question arose, however, whether this type of oxidation could be successfully applied to strophanthidol diacetate (111). Strophanthidin (I) can be reduced to strophanthidol (11) either with aluminum isopropoxide and isopropyl alcohol (Meerwein-Ponndorf reaction) or by the action of aluminum amalgam in a solution of 95% alcohol (5, 6). We found that the latter method furnishes very satisfactory results provided a suitable brand of aluminum is available for the preparation of the aluminum amalgam. From the recrystallized strophanthidol the 3,lg-diacetate (111)can be obtained in good yields. Whereas the melting point of pure strophanthidol is uncharacteristic, that of the diacetate is well defined(191-192'). When strophanthidol 3,194iacetate was treated in acetone solution with three moles of potassium permanganate, furnishing the equivalent of 4.5 atoms of oxygen, a fair amount of an amorphous acid oxidation product was obtained which represented the crude 3,19-diacetoxyd,14-dihydroxyetiocholanic acid (IV) -3 The neutral oxidation product, likewise amorphous, consisted only partly of unchanged starting material (111), as was proved by chromatographic separation. It was found that a purification of this amorphous neutral material is not necessary. Wh\en it was subjected to a renewed oxidation with potassium permanganate, more of the crude 3,19-diacetoxyd,14-dihydroxyetiocholanic acid could be secured, although in a somewhat smaller yield. Saponification of the crude diacetoxy acid (IV) yielded the crystalline 3,5 ,14 ,lg-tetrahydroxyetiocholanic acid (V), melting a t 217-218.5'. By means of diazomethane it was converted into the crystalline methyl 3,5 ,14,194etrahydroxyetiocholanate (VI) whose melting point was a t 168-169'. The non-crystalline part of the product resulting from the saponification of the crude IV was likewise subjected to treatment with diazomethane which yielded, after chromatographic purification of the reaction product, a certain amount of pure crystalline VI. An attempt was made to secure the diacetoxy acid (IV) in a crystalline form. For this purpose pure 3,5 ,14,19-tetrahydroxyetiocholanic acid (V) was subjected to acetylation which obviously furnished the pure 3,19-diacetoxy-5, 14-dihydroxyetiocholanic acid (IV). It resisted all attempts at crystallization. When this compound was treated with diazomethane the likewise amorphous methyl 3 ,19-diacetoxy-5,14-dihydroxyetiocholanate (VII) resulted. 3In a conversation with Professor T. Reichstein in Atlantic City on March 14,1946, the senior author disclosed the essential findings described in this paper. Professor Reichstein mentioned that he had found strophanthidol to be the aglycon of a new cardiac glycoside from Strophanthus Komb6 (cymarol, hydrolyzable t o strophanthidol and cymarose). H e has also subjected strophanthidol diacetate to an oxidation with potassium permanganate.

INVESTIGATIONS ON STEROIDS.

IX

825

1 0 U-0

t

v

x

x

M

826


Since the conclusion of this work two publications have appeared in which digitoxigenin acetate (7) and gitoxigenin diacetate (8) were subjected to the same oxidation procedure. The yields of the resulting etio acids seem to differ, depending on the structure of the aglycon. The next object of this investigation was the elimination of the tertiary hydroxyl group a t carbon atom 14 by dehydration and subsequent hydrogenation of the resulting double bond. I n our last publication (1) an example of such a selective dehydration was pre~ented.~Depending on the method used, two different dehydration products were obtained, the one probably being a As. 14andthe other a A14-compound. As is known, a double bond in As, 14-position is resistant t o catalytic hydrogenation, whereas a double bond in A14-positioncan easily be saturated. Since it was our aim t o obtain the double bond in the reducible, i.e. in the A14-position,we selected the same method which we had formerly utilized in an analogous instance (l).4 Hence 3,5,14,14tetrahydroxyetiocholanic acid (V) was treated with alcoholic hydrogen chloride. The two experiments described in the experimental part differ only in that the second experiment was carried out under slightly more vigorous conditions than the first. Using this procedure V is not only dehydrated but also largely transformed into ethyl esters and possibly also to a minor extent into lactones. Approximately 85% of the reaction product consisted of neutral material. This is in contrast to the example of our previous publication in which, under the same experimental conditions, the carboxyl group a t carbon atom 17 remained largely intact. Such a different behavior can only be explained on the basis of the difference in the stereochemical configuration of the carboxyl group in the two compounds. Whereas in the previous instance the carboxyl group is attached to carbon atom 17 in the is0 position, it is fairly certain that in the present example we are dealing with a normal configuration. The reaction product consisted mainly of non-crystalline material. A very small amount of a crystalline neutral substance could be secured, whose melting point was between 90 and 100". Its solution in chloroform gives a yellow color with tetranitromethane and the analysis is in agreement with the empirical formula CzoHzs04. The substance may therefore represent the unsaturated lactone X. The final structure cannot be given without further investigation. I n particular, attempts a t hydrogenation and saponification will be undertaken as soon as a sufficient amount of this substance has accumulated. The first of the two dehydration experiments was carried out under rather mild conditions and from this another crystalline substance was secured in small amounts from the neutral fraction. It proved t o be a simple esterification product of the starting material. Immediately after recrystallization the melting point of this ethyl 3,5,14,19-tetrahydroxyetiocholanate (VIII) is 186-188.5°.5 I n a preliminary dehydration experiment the neutral reaction product had been subjected to a chromatographic separation. A very small amount of 4 3,5,14-Trihydroxyestrane-10,l7-dicarboxylic acid was dehydrated t o AI4-estrene-3,5diol-l0,17-dicarboxylic acid. 6For variations of this melting point cf. experimental part.

827

INVESTIGATIONS ON STEROIDS. IX

x

0

A Y

\ L

Y

x

0

x

I I

x 0

\ 2 /

0

x

0

11

I4 W

x

U

P

828


another crystalline substance, melting a t 187-192", was isolated. Its structure is probably that of ethyl 3,5,19-trihydroxy-A8.*4-etiocholenate (XI). The compound will be discussed more fully below. The main product of the neutral fraction of the dehydration is ethyl 3,5, 19-trihydroxy-A14-etiocholenate (IX) which remained amorphous even after chromatographic purification.6a No purification of the acid fractions of the dehydration products was carried out. The amorphous acid material was subjected directly to catalytic hydrogenation. After the identification of the hydrogenation products, conclusions were drawn as to the composition of the original acid fraction of the dehydration, as will be discussed below. Usually, after the isolation of the crystalline substance which possibly represents the lactone X the dehydrated material was separated into neutral and acid fractions.e As mentioned, we were able in one instance' to isolate from the neutral fraction a certain amount of crystalline ethyl 3,5,14,14tetrahydroxyetiocholanate (VIII) which was separated by filtration. Otherwise, the crude neutral and acid fractions were subjected directly to catalytic hydrogenation. The main reaction product of the neutral fraction was in each instance the crystalline ethyl 3,5,19-trihydroxyetiocholanate(XV), the purest sample of which melted a t 188-190". It must have resulted from the amorphous ethyl 3,5,19-trihydr0xy-A~~-etiocholenate (IX). I n the saturated compound the hydrogen atom a t carbon atom 14 can be assumed to possess the normal con(Addition t o proof, October 29, 1946.) On repeating the dehydration experiments under conditions resembling those of Expt. I1 (cf. expt. part), treatment of the neutral material with acetone yielded substantial quantities of flat prismatic crystals. The melting points of recrystallized samples from three experiments were 187-189", 189-191' and 190-191.5". In a solution of chloroform, all substances gave a distinct yellow color with tetranitromethane. There were pronounced depressions of the melting points when the samples were mixed with compounds VI11 or XI. The material is probably the crystalline modification of ethyl 3,5,19-trihydro~y-A~~-etiocholenate (IX). Oddly enough there was no depression of the melting point when mixed with an authentic sample of compound XV. [a]: 48.9" (20.0 mg. in 2.0 cc. of chloroform; second sample). Anal. Calc'd for C ~ ~ H U C, O ~69.79; : H, 9.06 Found: C,69.68, 69.64,69.66;H,8.98,9.03,9.08. The noncrystalline part was subjected to chromatographic adsorption. From some of the early eluates crystalline residues were obtained which, on recrystallization from ether, furnished needles; negative reaction with tetranitromethane; map.205-206"; [a]: 64.8" (10.0mg. in 2.0 cc. of chloroform); this substance is obviously pure ethyl 3,5,lg-trihydroxyA8J4-etiocholenate (XI)(cf. expt. part; Expt. and Flow Sheet 11). Anal. Calc'd for CnH140s: C, 69.79;H, 9.06. Found: C, 69.41,69.79; H, 9.05,8.99. The residues obtained from some of the late eluates were recrystallized from acetone; There was no long flat prisms; positive reaction with tetranitromethane; m.p. 184-186'. depression of the melting point with the compound believed t o be the crystalline modification of IX. 6111 one experiment the crude dehydration product was hydrogenated without previous separation into neutral and acid fractions. In this instance the separation was performed after the hydrogenation; cf. experimental part, C, Exp. 1.2. 7Cf. experimental part, C, Exp. 1.1.

+

+


IX

829

figuration, i.e. "trans" to the methyl group at carbon atom 13.8 A fair amount of moderately pure XV can usually be obtained by direct crystalli~ation.~ Additional quantities of this substance can be secured by subjecting the residue obtained from the mother liquor to a separation by chromatographic adsorption. This procedure provided a t the same time some other compounds in a pure crystalline form. From some of the first eluates of the chromatogram there was isolated a small amount of a substance which probably represents ethyl 3,5,19-trihydroxyA8s 14-etiocholenate(XI) ; the highest melting point observed was 185', though it is possible that it was not that of a completely pure sample. The formation of a certain amount of such a compound during the process of dehydration appears plausible. It is understandable that such a compound goes through the process of catalytic hydrogenation unchanged. It is furthermore not surprising that a solution of this substance in chloroform does not give a yellow color with tetranitromethane in spite of the presence of the double bond. It should be pointed out that the fractions of the chromatogram from which this substance was obtained contain some other material which still has to be identified. I n particular one mould have to examine these fractions for the presence of the hydrogenation product of the substance which has been tentatively assigned formula X. From several of the followingeluates there was isolated XV, which has been discussed above. Near the last eluate of the chromatogram it was usually possible to identify a small amount of ethyl 3,5,14,19-tetrahydroxyetiocholanate (VIII). Saponificationlo of the main hydrogenation product, XV, furnished the free 3,5,19-trihydroxyetiocholanicacid (XIV) which melts a t 259-260'. Esterification of the latter compound by means of diazomethane yielded methyl 3,5,19trihydroxyetiocholanate (XVII) with the melting point 220-222.5". By means of acetylation, XV was transformed into ethyl 3,19-diacetoxy-5-hydroxyetiocholanate (XVII) whose melting point is 108-109.5". The saponification of ethyl 3,5, 19-trihydroxy-A8s14-etiocholenate (XI) yielded 3,5, 19-trihydroxy-A8.14-etiocholenicacid (XII), melting a t 286-288", aIn our last publication (1) Al4-estrene-3,5-diol-1O,17-dicarboxylic acid (with the carboxyl group a t Cl7 in is0 position) was hydrogenated to estrane-3,5-diol-lO,l7-dicarboxylic acid. In the latter compound the hydrogen atom at carbon atom 14 had been assigned the normal configuration, i.e. "trans" to the methyl group at carbon atom 13. According t o observations made in Ruzicka's laboratory (3) it appears possible that only with the normal A14-unsaturated etio acids does hydrogenation lead to a normal configuration at carbon atom 14 (rings C/D trans), whereas with the carboxyl group a t Ci7 in the is0 position, a compound with the hydrogen atom at CICin the epi position (rings C/D cis) results. It is possible, therefore, that the assumption made in our previous publication (1) will have to be revised. 9Cf. in particular experimental part, C , exp. 11. I n this instance the over-all yield of this substance (XIII) from 3,5,1.1,19-tetrahydroxyetiocholanicacid (V) was at least 30%. 'OThe saponification was carried out with potassium hydroxide in methanol. The neutral reaction product was found to be a mixture of the ethyl and methyl esters, a sign that transesterification had taken place.

830


which in turn, by treatment with diazomethane, was converted into methyl 3,5,19-trihydroxy-A8'14-etiocholenate(XVI) with the melting point 270-274'. The acid dehydration products were identified by implication. The resinous acid material was subjected to catalytic hydrogenation. In each such instance XII:

XIV.

I

/CHIN,

CHiNn

L OH

I

CH3

OH

CHj

COO CHI

OH

OH

XVII. Methyl 3,5,19-trihydroxy-

XVI. Methyl 3,5,19-trihydroxyA8~14-etiocholenat e

etiocholanate

xv.

XVIII. Ethyl 3,lg-diecetoxy5-hydroxyetiocholsnate

it was possible to isolate a certain amount of 3,5,19-trihydroxyetiocholanic acid (XIV)which can only have resulted from 3,5,19-trihydroxy-A14-etiocholenic acid (XIII). The material contained in the mother liquor of compound XIV was transformed into the methyl ester with diazomethane. The resulting product was subjected t o chromatographic adsorption which yielded a small amount of a substance which was apparently identical with XVI. Hence it is fairly certain that a small quantity of 3,5,19-trihydroxy-A8*14-etiocholenic acid (XII) was present in the acid dehydration product. As was pointed out before it should be inert to catalytic hydrogenation.


IX

831

As is known, certain steroids with a hydroxyl group at carbon atom 14 are dehydrated to A14-unsaturated compounds by pyridine-phosphorus oxychloride a t room temperature (7, 8) whereas others are inert under these experimental conditions (9). When ethyl 3,19-diacetoxyd-hydroxyetiocholanate(XVIII) was treated according to this procedure a t room temperature for a period of twenty hours, a fair amount of unchanged starting material could be recovered and there was no indication of the formation of unsaturated material (tested with tetranitromethane).l' This indicates that under these experimental conditions the tertiary hydroxyl group a t carbon atom 5 appears to be inert. The question arises therefore, whether methyl 3,19-diacetoxy-5 ,14-dihydroxyetiocholanate (VII), when treated under these mild conditions, will yield to a selective dehydration with the formation of methyl 3 ,19-diacetoxy-5-hydroxyA14-etiocholenate. Treatment a t a higher temperature may possibly involve the tertiary hydroxyl groups both at carbon atoms 5 and 14. Experiments along these lines will be undertaken. The compounds described in this publication will serve as intermediates for further chemical transformations. EXPERIMENTAL

The melting points were determined with the Fisher-Johns melting point apparatus. The readings are sufficiently near the true melting points so that no corrections have been made. The microanalyses were carried out by Mr. William Saschek, Department of Biochemistry, Columbia University, New York. A. Preparation of strophanthidol 9,19-diacetate (ZZZ). The procedures used were essentially those of Rabald and Kraus (5, 6 ) . Strophunthidin (I). Hydrolysis of k-strophanthin with 0.5% hydrochloric acid (10) furnished the crude strophanthidin which was recrystallized from aqueous methanol. The melting points of the recrystallized, frequently somewhat brownish, material were usually between 145 and 160". On heating above the melting point, solidification occurred andeventually renewed melting at a temperature above 200'. The yields of strophanthidin, expressed as per cent fractions of the invested strophanthin (i.e. not theoretical yields) were as follows; from about 1170 g. of Strophanthin Penick U.S.P.XI: crude 26.6'%, recrystallized 24.5%; from about 270 g. of Strophanthin Merck U.S.P.XI1: crude 42.6%, recrystallized 3o.2Y0. Two samples of strophanthin which had not been standardized according to pharmacopoeial requirements were kindly provided by S. B. Penick & Co. and furnished the following yields of strophanthidin; crude 40.8Q/0, recrystallized 36.0% and crude 43.9%, recrystallized 36.6% respectively. Strophanthidol ( I Z ) . The reduction of strophanthidin (I) was carried out with active aluminum amalgam which was prepared as follows:1a The surface of 10 g. of Aluminum Metal, Foil (thickness approx. 0.025 mm.) of the J. T. Baker Chemical Co. was thoroughly cleansed with petroleum ether and cut into pieces of about 2x2 cm. T o the aluminum was added enough 2% sodium hydroxide to cover the whole metal (about 100 cc.). As soon as a lively evolution of hydrogen had set in, the alkali solution was decanted and the aluminum washed quickly and repeatedly with water. Thereupon 100 cc. of a solution of O.5y0 mercuric chloride was allowed to act upon the aluminum for a period of two minutes. The metal was then washed quickly and repeatedly with water and eventually with %yoalcohol. "Because of the negative result of this experiment it is not recorded in the experimental part. IeCf. also Houben-Weyl, 3rd. edit. 11, 256. Weygand, Organic Preparations, Interscience Publishers, p. 9.

832

MAXIMILYAN EHRENSTEIN AND ALDA RUTH JOHNSON

T o the still moist aluminum amalgam was added at once a solution of 10 g. of recryst. strophanthidin in 380 cc. of 95% alcohol. A lively reaction set in immediately and i t was sometimes necessary to cool the reaction mixture for a moment. The mixture was allowed to stand a t room temperature for a period of several days during which there was added a total of 30 cc. of water in installments of 5 cc. each. Usually after about four days the aluminum had almost completely disintegrated, save for a few shreds of metal. The grey sludge of aluminum hydroxide was filtered by suction and subsequently extracted by refluxing i t three times for one hour each with 400 cc. of 95% alcohol. The combined colorless alcoholic solutions were concentrated to a smaller volume in vacuo, which caused the separation of white crystals. After the filtration of the latter and renewed concentration, etc., more crystalline crops could be secured. The crystalline material usually totalled about 9.0-9.2 g. From the final mother liquor there was obtained a dry residue weighing0.5-0.6 g. The melting points of the various crystalline fractions were usually between 135 and 150', occasionally slightly higher. The combined crystalline fractions were recrystalized by dissolving them in about 200 parts of ethyl acetate and concentrating to a smaller volume in a partial vacuum. Several crops of large colorless transparent prisms were obtained. The melting points of the major parts of the crystalline material were between 140 and 155'. Lower-melting crops of several experiments were pooled and the melting points raised by renewed crystallization from ethyl acetate. The total yield of such purified strophanthidol (11), obtained in 25 reduction experiments (starting material: 250 g. of recryst. strophanthidin) was 212 g. The Legal test, with alcohol as a solvent (II), was positive. Anal. Calc'd for C & t & . : C, 67.94; H , 8.43. C&3,0a &CH,COOCJ3,: C, 66.62; H, 8.50. Found: C, 66.32; H, 8.55. Strophanthidol 9,Ig-diacetate ( I I I ) . To a solution of 5.145 g. of purified strophanthidol in 20.6 cc. of pyridine was added 10.3 cc. of acetic anhydride and the mivture allowed t o stand at room temperature for 24 hours. After pouring i t into 103 cc. of watt-r and permit ting the precipitate to stand overnight, the diacetate separated in white platelets. They were filtered, washed with water and dried; wt: 3.81 g. The filtrate was extracted with 250 cc. of ethyl acetate and the latter phase washed neutral by treating i t twice with 60 cc. of N hydrochloric acid, once with 60 cc. of N sodium bicarbonate solution and twice with 60 cc. of water. After drying overnight with sodium sulfate and removal of the solvent, a dry residue of 1.77 g. was obtained. The crystalline material (3.81) g.) was recrystallized by dissolving i t in about 25 cc. of acetone and gradually adding to this solution petroleum ether over a period of several hours. Too rapid addition produces an oily separation. After standing overnight, the crystals were filtered and washed with an acetone-petroleum ether mixture (3:7). The filtrate was concentrated to a smaller volume on a water-bath and the addition of petroleum ether continued. First crop; wt. 2.132 g.; m.p. 186-185'. Second crop; wt. 1.094 g.; m.p. 187-188.5'. Third crop; wt. 0.237 g.; m.p. 162-166". Similar treatment of the residue from the ethyl acetate extraction (1.77 g.) furnished crystalline material; wt. 0.734 g.; m.p. 184-186". I t should be pointed out, that in several experiments of this type the residue from the ethyl acetate extract did not yield crystals when subjected to the same handling. Such resinous material can be partially transformed into pure crystalline diacetate by chromatographing its solution in benzene over a column of aluminum oxide. The adsorbed material is eluted by washing with benzene, benzeneether, ether, various ratios of ether-acetone, acetone, and various ratios of acetonemethanol. The crystalline diacetate can be secured from the ether-acetone eluates by bringing them to dryness and recrystallizing the residues from acetone-petroleum ether. Altogether 202.7 g. of purified strophanthidol were subjected to acetylation in small lots (30 experiments). A total of 178.2 g. of crystalline fractions was obtained, more than 90% of which consisted of strophanthidol diacetate (111)with melting points above 185". Since only a small sample of resinous acetylation product has been subjected to chromatographic separation, similar treatment of the remainder of such material should increase the above yield substantially.

+


833

The optical rotations and microanalyses are recorded for two crystalline samples melting +49.4" (30.0 mg. in 2.0 cc. of chloroform), I c Y ] ~ +54.7" '~ (30.0 mg. in 2.0 cc. of chloroform). Anal. Calc'd for CgrHtsOs: C, 66.08; H, 7.81. Found: C, 66.11,66.02; H, 7.95, 7.80. B. Preparation of 5,6,l4,19-tetrahydroxyetiocholanic acid and derivatives. Crude 8.19-diacetoxy-6,14-dihydrozyetiochoZanic acid (ZV). Strophanthidol diacetate (If I) was oxidized in a solut,ion of acetone with three moles of potassium permanganate. T o 5.0 g. of 111dissolved in 294 cc. of purest dry acetone was added 4.84 g. of finely ground potassium permanganate. The mixture was shaken in a bottle with glass stopper for about two hours, after which the permanganate color had completely disappeared. The reaction mixtures originating from three oxidations of a total of 16.1 g. of I11 were transferred into a distilling flask and the acetone removed from the brown suspension by distilling first in a partial and eventually in a full vacuum. T o the residue was added about 50 cc. of water to produce a sludge which was transferred into a separatory funnel. It was then acidified to Congo paper by slowly adding about 100 cc. of approx. 10% sulfuric acid. The brown suspension was extracted once with 200 cc.of ether and seventimes with 150 cc.of ether. The etherextracts were eventually combined (combined ether extracts). Thereafter t h e brown BUSpension was extracted twice with 150 cc. of ethyl acetate and six times with 100 cc. of ethyl acetate. The ethyl acetate extracts were eventually combined (combined ethyl acetate extracts). The combined ether and ethyl acetate extracts were each separated into acid and neutral niaterial as follows: A. Combined ether extracts. They were extracted with 50 cc. and 25 cc. of a 5% solution of sodium carbonate and then washed three times with 10 cc. of water. The ether phase (neutral ether) was dried with sodium sulfate overnight, filtered, and eventually brought completely to dryness in vacuo. The residue was a brittle foam; weight after drying in a vacuum desiccator, 5.976 g. The above mentioned carbonate phases and aqueous washings were combined. This solution was made acid to Congo paper by slowly adding t o i t in a separatory funnel about 40 cc. of 10% sulfuric acid, which caused a sticky precipitate t o appear. The suspension was thoroughly extracted with 200 cc., 150 cc., and five times with 1OOcc.of ether.'" Tnese ether extracts were combined, washed eight times with8cc. of water and finally dried overnight with sodium sulfate. The ether phase (acid ether) was then filtered, concentrated, and eventually brought completely to dryness in a vacuum. The residue was a brittle foam; weight after drying in a vacuum desiccator over KOH, 7.1872 g. B. Combined ethyl acetate extracts. They were extracted with 30 cc. and 15 cc. of a solution of 5y0 sodium carbonate and subsequently washed three times with 10 cc. of water. The ethyl acetate phase (neutral ethyl acetate) was dried n-ith sodium sulfate, filtered and eventually brought completely to dryness in vacuo. The residue was a brittle foam; weight after drying in a vacuum desiccator over KOH, 0.558 g. The above mentioned carbonate phases and aqueous washings were combined. The resulting solution was acidified to Congo paper by slowing adding to i t in a separatory funnel about 35 cc. of 10% sulfuric acid. The acidified material was very thoroughly extracted five times with 100 cc. of ethyl acetate. The combined ethyl acetate extracts were washed twice with 8 cc. of water and six times with 5 cc. of water and eventually dried with sodium sulfate. The ethyl acetate phase (acid ethyl acetate) was filtered, concentrated, and finally brought to dryness in vacuo; weight after drying over KOH in a vacuum desiccator, 0.227 g. The acid material as obtained from the ether extract or the ethyl acetate extract resisted all attempts a t crystallization. The pooled neutral material as recovered from the oxidation likewise resisted all attempts at crystallization. A sample of 100 mg. was subjected t o chromatographic adsorp-

at 185-188' and 191-192' respectively.

(v)

~~

~

I n some experiments a subsequent extraction was carried out with 75-100 cc. of ethyl acetate. The residues obtained from these extracts weighed slightly over 1% of the material invested in the oxidations. I**

834

MAXIMILUN EHRENSTEIN AND ALDA RUTH JOEINSON

tion. For this purpose i t was dissolved in a mixture of 10 cc. of benzene and 1.5 cc. of petroleum ether which was filtered through a column of 4.0 g. of aluminum oxide (Brockmann). The column was washed successively with 15-cc. portions of benzene, benzene, benzeneether ( l : l ) , ether, ether-acetone ( l : l ) , acetone, acetone, methanol. The residues obtained from the ether-acetone and the first acetone eluate represented the main fractions weighing 36.0 mg. and 8.5 mg. respectively. They were combined (44.5 mg.) and dissolved in acetone t o which petroleum ether was added gradually. On seeding with strophanthidol diacetate (111), crystallization began at once. The crystalline material had the melting point of 185188" and there was no depression of the melting point when admixed to an authentic sample of 111. The pooled crude neutral material, as recovered from various experiments, was subjected to reoxidations with varying ratios of potassium permanganate. When treated with the same ratio (ratio 1) of the oxidant as the original 111, the color of the permanganate disappeared only very slowly, and hence shaking had t o be extended overnight. When the amount of the permanganate was cut down t o two-thirds or one-half of the ratio of the original oxidation, the color disappeared within 4 to 5 hours or 2&hours respectively. From the following tabulation (Tables A and B) of yields i t can be concluded that in the reoxidation experiments optimal yields of acid material are obtained when the amount of permanganate s cut down to about two-thirds of the ratio of the oxidation of 111.

Tabulation of yields (Figures in parentheses are the numbers of oxidations into which each experiment was divided. The ratio of KMnO, used in the original experiment is taken as 1. The yields are expressed as per cent of the invested starting material. In all experiments a t least 2s of the acid material was obtained from the ether fraction.) A. OXIDATION OF STROPHANTRIDOL DIACETATE (111) EXPEPIMENT NO.

.................................

Invested g . . . ........................ Ratio of IZMnO,. .................... Total acid % ......................... Total neutral %. ..................... Total recovered yo...................

15.59 (3) 1 48 35 83

16.1 (3) 1 46.0 40.6 86.6

15.56 (3) 1 52 36 88

15.83 (3) 1 48.6 41.6 90.2

B. REOXIDATION OF NEUTRAL MATERIAL (recovered from oxidation of strophanthidol diacetate) EXPEBIXENT NO..

.........................

I

Invested g.. .................... Ratio of KMnOc.. . . . . . . . . . . . . . . Total acid %.. . . . . . . . . . . . . . . . . . Total neutral %... . . . . . . . . . . . . . Total recovered yo. . . . . . . . . . . . . .

I

0.94 (1) 1 21.7 31.5 53.2

I

I1

12.04 (2)

I

I11

12.20 (2)

3

B

35 49 84

29 56 85

3,6,14,19-Tetrcshydrozyetiocholanicacid (V). To a solution of 3.0 g. of the above acid (from ether extract) in 15 cc. ofmethanol was added a solution of 4.5 g. of potassium hydroxide in 60 cc. of methanol and the mixture then refluxed on a water-bath for a period of thirLy minutes. After the addition of 150 cc. of water the solution was immediately concentrated i n Vacuo (45-50") t o a volume of about 65 cc. The solution was cooled with ice and made acid to Congo paper by slowly adding 7.5 cc. of conc'd hydrochloric acid which caused a precipitate to appear. The reaction mixture was thoroughly extracted eight times, each


835

time with"!@ cc. of ethyl acetate. It was found that more numerous extractions will increase the yield of the saponification product too slightly to be practical. Thecombined ethyl acetate extracts were washed eight times with 5-6 cc. of water and then dried with sodium sulfate. After filtering, the solution was concentrated t o a small volume (about 4045 cc.) in vacuo (40-50'). In this concentration the deacetylated acid apparently forms a supersaturated solution. The reaction product separated in a microcrystalline form by heating the solution briefly on a water-bath and scratching the walls of the container. After standing at room temperature overnight, the almost white microcrystalline material was filtered and thoroughly washed with ethyl acetate; yield 1.42 g.; m.p. 208-210' (subseq. effervescence). On concentrating, the filtrate occasionally furnished small amounts of further crystalline material. I n the present instance the filtrate was brought t o dryness in vacuo (40-50') and furnished a brittle foam which weighed 0.89 g. after drying over KOH in a vacuum desiccator. This amorphous material still contains some of V, because on esterifying i t with diazomethane and subjecting the reaction product to chromatographic adsorption, an appreciable amount of the crystalline methyl ester of the acid (VI) can be obtained. The preparation of the methyl ester from the crystalline as well as amorphous acid will be described below. The yields of the crystalline acid were approximately the same in thirteen different saponification experiments; a total of 35.52 g. of acid ether extract furnished 16.36 g. of crystalline V. It made no difference whether the starting material (acid ether extract) had originated from an oxidation of strophanthidol diacetate (111) or from a reoxidation of "neutral material". The melting points were sometimes slightly lower than recorded above. It should be pointed out that several attempts t o saponify acid ethyl acetate extracts furnished only resinous products. Although i t was not tried, i t is considered possible that some Crystalline methyl ester of the 3,5,14,19-tetrahydroxyetiocholanic acid (VI) could be obtained from such saponified material by treatment with diazomethane and subsequent chromatographic purification. For analysis the crystalline acid as obtained from ethyl acetate was recrystallized by dissolving i t (reflux) in the required amount of acetone and concentrating t o a smaller volume on a water-bath. On subsequent standing a t room temperature the separation of glistening platelets began rather early. They were filtered after standing overnight. The melting point of the recrystallized V was 217-218.5' (subseq. effervescence). [a]:" +38.9" (15.0 mg. in 2.0 cc. of acetone). Anal. Calc'd for CZoHa~06:C, 65.17; H, 8.76. Found: C, 65.31, 65.04; H, 8.50, 8.47. Methyl 9,5,14,19-tetrahydroxyetioeholanate( V I ) . a. From cryst. 5,6,1.6,19-tetrahydroxyetiocholanic acid ( V ) . About 0.12 g. of pure acid (V) which had been recrystallized from acetone was dissolved (reflux) in the required amount of acetone (14 cc.) and the solution concentrated t o about two-thirds of this volume on a water-bath. T o this solution was added at 0" an amount of an ethereal solution of diazomethane sufficient t o produce a persistent yellow color. After brief standing at room temperature the excess of diazomethane was evaporated on a water-bath and the solution then brought t o dryness in vacuo (40-45') The residue was a brittle foam which was transferred into a separatory funnel by means of a total of 40 cc. of ether. The ether solution was washed successively with 3 cc. of N hydrochloric acid, 3 cc. of water, 3 cc. of a solution of 5% sodium carbonate, and three times with 3 cc. of water. After drying with sodium sulfate, the solution waa filtered and brought t o dryness, eventually in vacuo. The residue was a colorless resin which became almost completely crystalline after a few days standing in a vacuum desiccator; yield 0.091 g. This material was recrystallized by dissolving i t in acetone and slowly adding at room temperature some petroleum ether. The separation of spear-shaped crystals began very soon. The crystalline material was filtered on the following day; yield 0.053 g.; m.p. 168-169". More crystalline ester could be secured from the mother liquor. [a]$" $59.6' (20.0 mg. in 2.0 cc. of chloroform). Anal. Calc'd for CntHt,O6: C, 65.92; H, 8.96. Found: C, 65.87; H, 8.86.

836


6. From amorphous acid (u. supra). T o a solution of 2.46 g. of amorphous acid (noncrystalline fraction, obtained by saponifying "acid ether extract") in 20.0 cc. of acetone was added at 0' an excess of an ethereal solution of diazomethane. The reaction mixture was kept in ice for about 3 minutes and then at room temperature for 15 more minutes. Thereaft.er the excess of the diaeomethane was removed on a water-bath and the solution brought to dryness in uacuo (40-51)'). The residue was a foamy mass which proved to be very sparingly soluble in ether. It was dissolved in 15 cc. of acetone and this solution poured in a separatory funnel into 750 cc. of ether. The resulting solution was washed successively twice with 15 cc. of N hydrochloric acid, once with 10 cc. of water, once with 15 cc. of a solution of 5% sodium carbonate, and four times with 8 cc. of water. After drying with sodium sulfate and filtering, the solution was brought completely t o dryness, first on a water-bath, eventually in vacuo. The residue was a brittle foam; wt: 1.857 g. This residue was subjected to chromatographic adsorption for which purpose it was dissolved in a mixture of 180 cc. of benzene and 20 cc. of petroleum ether. The solution was filtered through a column (diam. 1.8 cm.) of 55 g. of aluminum oxide (standardized acc. t o Brockmann, Merck & CO., Rahway). The original solution was passed through within six hours, the following eluate within five hours and all other eluates within about thirty minutes each.

CHROMATOGRAPHIC FRACTIONATION NO. OF SOLVENT

FPACTION

I

+

WEIGHT OF BESIDUE G.

180 cc. benzene 20 cc. petr. ether 0.0092 (original solution) 200 cc. benzene .0673 .1339 150 cc. benzene 50 cc. ether .I426 50 cc. benzene 150 cc. ether 200 cc. ether .0141 150 cc. ether 50 cc. acetone .0132 .0244 50 cc. ether 150 cc. acetone 200 cc. acetone .0823 199.5 cc. acetone 0.5 cc. methanol .1715 .2712 198 cc. acetone 2 cc. methanol .3184 195 cc. acetone 5 cc. methanol ,3302 180 cc. acetone 20 cc. methanol ,1174 150 cc. acetone 50 cc. methanol 200 cc. methanol .1512

1

2 3 4 5 6 7 8 9 10 11 12 13 14 ~

I

+ + + +

+ + + + +

~

Total

~~

~

~~~~~

~

1

APPEABANCE OF BESmUE

I

Colorlessgrease Colorless resin Colorless, partly foamy resin Colorless, partly foamy resin Slightly yellowish grease Yellow resin Slightly yellow resin Yellow resin Slightly yellow resin Colorless resin, partly cryst Colorless brittle foam Colorless brittle foam Colorless brittle foam Slightly yellowish mass

~

......... ....... ......... .... .. ... . . .

The residues of fractions 8 t o 12 were selected for examination. They were separately dissolved in small m o u n t s of acetone and petroleum ether was added gradually t o these solutions. Fraction 8 yielded only oily material. Fraction 9 yielded two crystalline fractions: 9a. 0.0417 g., m.p. 169-171'; 9b. 0.0016 g., m.p. 166-169'. Fraction 10 also yielded two crystalline fractions: loa. 0.1240 g., m.p. 16&170°; lob. 0.0154 g., m.p. 169-171". Fraction11 likewise yielded two crystalline fractions: lla. 0.1535 g., map.163-165'; Ilb. 0.0123 g., m.p. 167-170'. All these crystalline fractions (total: 0.3485 g.) did not give a depression of the melting point when mixed with the analytical sample of the methyl ester (u. supra). From fraction 12 only traces of crystals were obtained; the major part of the material came out oily. It is believed that the yield of crystalline ester can be increased by combining the non-crystalline parts of fractions 9-11 and all of fraction 12 and subjecting this material to a renewed chromatographic separation.


IX

837

3,19-Diacetoxy-5,l4-dihydroxyetiocho2anicacid (ZV), T o a solution of 50 mg. of pure crystalline 3,5,14,19-tetrahydroxyetiocholanicacid (V) in 0.4 cc. of pyridine was added 0.2 cc. of acetic anhydride and the mixture allowed t o stand at room temperature overnight. It was then brought to dryness in vacuo (50°), the viscous residue taken up in 15 cc. of ether and this solution shaken with 4 cc. of N hydrochloric acid and a few times with 2 cc. of water. After extracting the ether solution twice with a few cc. of a cold solution of 5% sodium carbonate, the combined carbonate phases were immediately made acid t o Congo paper by adding cold N hydrochloric acid. After thoroughly extracting with ether, washing the combined ether phases a few times with water, and drying with sodium sulfate, the ether solution was brought to dryness, leaving 32.7 mg. of a colorless resin. All attempts at crystallization failed. Methyl 3,19-diacetoxy-5, 14-dihydroxyetiocholanate (VZZ) . The above resinous IV was treated in an ethereal solution with diazomethane and the reaction product was isolated in the usual way. The yield was 27 mg. of a colorless resin. This material was dissolved in a mixture of benzene and petroleum ether and chromatographed over 1.0 g. of alumina. The adsorbate was successively eluted with 5-cc. portions of benzene-petroleum ether (benzene content gradually increasing), benzene, benzene-ether (ether content gradually increasing), ether, ether-acetone (1:l), acetone, acetone-methanol (methanol content gradually increasing), and methanol. All residues obtained from these eluates were resinous. The main fraction (10 mg.) was secured from the ether-acetone phase. It resisted all attempts a t crystallization. C . Dehydration of 3 , 6 ,14,19-tetrahydroxyetiochoZanicacid (V) and subsequent hydrogenation of the unsaturated reaction product; preparation of 3,5,19-trihydroxyetiocholanicacid (XZV) and 3,5, 19-trihydroxy-A8J4-etiocholenic acid (XZZ) and their derivatives. The dehydration was carried out by the action of a 0.1 N solution of hydrogen chloride in absolute alcohol. This process transforms at the same time about 8540% of the acid material into the ethyl ester. Separation into acid and neutral fractions was carried out either before or after the hydrogenation. For the sake of clarity the experiments will be described with the aid of flow sheets. Experiment Z (Flow Sheet I ) . A total of 2.5088 g. of crystalline V was dissolved in 240 CC. of 0.1 N hydrogen chloride in absolute alcohol. This solutionwas kept at room temperature for a period of 35 minutes, after which a slow distillation was begun a t atmospheric pressure and under anhydrous conditions. The bath temperature was kept between 103 and 105' throughout the whole distillation which was carried out over a period of 95 minutes. The residue was 75-80 cc. of a light golden solution, to which was added 50 cc. of water and the distillation then continued in Vacuo (45-47') until a distinct turbidity appeared. The latter was brought into solution by briefly heating on a water-bath and the solution was then allowed t o stand a t room temperature overnight which caused the separation of some oily material and also macrocrystalline needles. The crystals were partly imbedded in the oil and hence frequent leaching with aqueous alcohol (1:l) was necessary t o obtain them in a colorless form; dry weight: 42.7 mg.; m.p. about 97-99.5'. This substance, which is POSsibly the lactone X, proved t o be essentially insoluble in a solution of sodium carbonate. A solution in chloroform gave with tetranitromethane a distinct yellow color, indicating its unsaturated character. I t s recrystallization proved to be difficult.la All decantates and filtrates were combined and the alcohol completely removed in vacuo. This produced a light yellow precipitate which was taken up with 175 cc. of ether. The aqueous phase was thereafter extracted four times with 75-cc. portions of ether. The combined ether extracts were washed six times, each with 5 cc. of water. After the drying of the solution with sodium sulfate and filtering, the ether was removed, eventually in vacuo. The residue was a brittle foam; weight after drying, 2.3531 g . For further experiments this material was divided into two parts. 1. Experiments withjirst part of the crude dehydration product. A solution of 1.2273 g. of the crude moduct in 140 cc. of ether was seDarated into neutral and acid material as follows. For microanalysis cf. second dehydration experiment.

838


It was extracted successively with 10 cc. and 5 cc. of 5% sodium carbonate and then five times with 2-cc. portions of water. The ether solution was dried with sodium sulfate, filtered and brought completely to dryness. The neutral residue was a colorless brittle foam; dry weight: 1.0177 g. The above mentioned carbonate phases and aqueous washings were combined and made acid to Congo by the addition (ice-cooling) of 2.5 cc. of conc'd hydrochloric acid, which produced a cheesy precipitate. It was extracted successively with 100 cc., 50 cc., and three times with40-cc. portions of ether. The combined ether extracts were washed six times each with 3 cc. of water and then dried with sodium sulfate. After filtering, the solution was brought completely to dryness. The acid residue was a slightly yellowish brittle foam; dry weight 0.1865 g. It was kept for further experiments. Treatment of the neutral residue (1.0177 9.) with about 5 cc. of ether yielded a clear yellowish solution to which some petroleum ether was added very slowly over the period of a whole day. Very gradually some hard white crystalline material deposited on thewalls, which increased on standing overnight. The crystals were filtered the following day and washed with ether containing petroleum ether; dry weight 0.0724 g.; m.p. about 164469.5'. These crystals were identified as ethyl 3,5,14,19-tetrahydroxyetiocholanate (VIII) (u. infra). T o the mother liquor was added more petroleum ether over a period of several hours which resulted in the separation of only resinous material. This precipitate was not isolated but brought to dryness together with the supernatant solution. The residue (wt. 0.9473 9.) was a brittle foam, consisting probably mainly of ethyl 3,5,19-trihydroxy-A14+46.4" (20.0mg. in 2.0 cc. of chloroform). I n asolution of chloroetiocholenate (IX); form the substance gave a yellow color with tetranitromethane. The analytical figures indicate the presence of a certain amount of VIII. Anal. Calc'd for C22Haa06:C, 69.79; H, 9.06. Found: C, 68.97; H, 9.01. Ethyl 8,6,t 4,19-tetrahydroryetiochoZuna~e(VZZZ). The crystalline material mentioned above (wt. 72.4 mg.; m.p. 164-169.5") was dissolved in acetone. The solution was concentrated to a small volume on a water-bath and then some petroleum ether added at room temperature. The separation of sheaves of spear-shaped crystals began a t once. The sub+45.7" (20.0mg. stance was filtered the following day; wt. 44.2 mg.; map.186-188.5'; in 2.0 cc. of chloroform). A n d . Calc'd for CBH,,O~: C, 66.62; H, 9.16. Found: C, 66.22; H, 8.98. When the filtrate was concentrated and petroleum ether was added, a second crop of crystalline material was obtained; wt. 6.2 mg.; m.p. 184.5-187.5". It should be mentioned that the two samples gave melting points about ten degrees lower after having been'kept in as desiccator for a period of several weeks. Hydrogenation. A suspension of 350 mg. of platinum oxide in 4.5 cc. of glacial acetic acid was reduced and after the addition of a solution of 0.8505 g. of the previously mentioned amorphous unsaturated ethyl ester in 10 cc. of glacial acetic acid, shaking was continued at room temperature (23") for a period of 34 hours. The total hydrogen absorption was 55.2 cc.; of this amount about 50 cc. was absorbed during the first hour. Calc'd for ethyl 3,5.19trihydr~xy-A~~-etiocholenate (IX), 54.5 cc. (23"). The solution was filtered from the catalyst and quickly brought t o dryness i n vacuo (45-50'). T o the syrupy residue 5 CC. of water was added a t once. After standing overnight the material was extracted twice with 60-cc. portions of ethyl acetate, The combined extracts were washed neutral by treating them three times with 3-cc. portions of a solution of 5% sodium carbonate and six times with 2.5 cc. of water each. After drying with sodium sulfate and filtering, the solution was brought t o dryness. The residue was 0.8278 g. of a resin, a part of which was purified by chromatographic adsorption. For this purpose 0.6937 g. of the residue was dissolved in 70 cc. of ether. The solution was filtered through a column (diam. 20 mm.) of 19 g. of aluminum oxide. The original solution was passed through within one hour and the following eluates within 25 minutes each.

CY^^:'


839

IX

CHROMATOGRAPHIC FRACTIONATION NO. OF PPACIION

1 3

3 4 .5 6 7 8 9 10 11 12

--

SOLVENT

WEIGHT OX RESIDUE G

70 cc. ether (original solution) 0.0201 40 cc. ether 30 cc. acetone .0912 20 cc. ether 50 cc. acetone ,0103 70 cc. acetone .ON2 70 cc. acetone 0.2 cc. methanol .0451 70 cc. acetone 0.3 cc. methanol .0689 TO cc. acetone 0.5 cc. methanol .0733 69 cc. acetone 1 cc. methanol .0775 68 cc. acetone 2 cc. methanol .0895 65 cc. acetone 5 cc. methanol ,0958 50 cc. acetone 20 cc. methanol .0407 70 cc. methanol .0235

+ +

+ + + + + + +

Total.. , , , , . . . . , . . . . . . . . . . , . , . . . . . . . . , . . .

A

P

m

or~msmm ~

Resinous Slightly yellow, partly cryst. Yellow, crystalline Microcrystalline Partly cryst. resin Crystalline Crystalline Crystalline Partly cryst. resin Resinous Brittle foam Amorphous

.6761

The residues of the eluates were separately dissolved in acetone t o which, in some instances, petroleum ether was added to induce crystallization. Fractions 2 to 4. No uniform crystalline substance could be secured from fraction 3. Fractions 2 and 4 furnished a total of 9.2 mg. (2.9 mg. and 6.3 mg. respectively) of needleshaped crystals melting at 189-194'. Fraction 2 furnished an additional crop of 6.9 mg. of somewhat less pure material, melting a t 184-188". These substances (total 16.1 mg.) were identified as ethyl 3,5, 19-trihydroxy-A8J4-etiocholenate (XI). They gave no depressions of the melting points when mixed with authentic samples of this substance (u. infra.). Fraction 5. The crystalline material (total 10.2 mg.) obviously represented a mixture. Fractions 6 to 10. Four fractions of needle-shaped crystals, totalling 0.1255 g., had melting points between 178 and 182'. Two fractions (total 0.0457 g.) of almost the same purity had melting points between 175 and 178". By mixed melting points all these fractions were identified as ethyl 3,5,19-trihydroxyetiocholsnate(XV) ( u . infra.). The total yield of this substance was therefore 0.1712 g. The yield can perhaps be somewhat increased by subjecting the material contained in the mother liquors of these fractions t o a renewed chromatographic separation. Fraction 11. No crystalline material could be obtained. 2. Experiments with second part of the crude dehydration product. Chronologically these experiments preceded those carried out with the first part of the crude dehydration product. I n retrospect the following procedure appears less convenient than the one described above. It IS recorded because i t yielded a number of chemically pure compounds which served aa reference samples. The product was subjected t o a catalytic hydrogenation without subjecting i t first to a separation into acid and neutral material. A suspension of 455 mg. of platinum oxide in 7 cc. of glacial acetic acid was reduced and after the addition of a solution of 1.1258 g. of the crude dehydration product in 12 cc. of glacial acetic acid, shaking was continued a t room temperature (23") for a period of 34 hours, when the hydrogenation came to a standstill. The total hydrogen absorption was 65.8 cc. of which 62.5 cc. were absorbed during the first I+ hours. Calc'd for ethyl 3,5,19-trihydroxy-A14-etiocholenate (IX), 72.3 cc. (23"). As may be concluded from the preceding experiment, the deficiency of the hydrogen absorption is probably mainly attributable t o the presence of a fair amount of VIII. The solution was filtered from the platinum and concentrated in uucuo (about 52") t o a slightly turbid colorless syrup t o which 5 cc. of water was added immediately. The following day the sticky material was taken up in 100 cc. of ethyl acetate and this solution washed with 6 cc. and with two 2-cc. portions of water. There-

840

MAXIMILIAN ERRENSTEIN AND ALDA RUTH JOHNSON

after it was separated into neutral and acid material by extracting it successively with 10 cc. and 5 cc. of 5% sodium carbonate and then five times with 2-cc. portions of water. The ethyl acetate solution was dried with sodium sulfate, filtered, and brought t o dryness, eventually in vacuo. The neutral residue was a white, largely crystalline cake; weight 0.962 g. The above-mentioned carbonate phases and aqueous washings were combined and made acid to Congo by adding 2.5 cc. of conc'd hydrochloric arid (ice-cooling), which caused a cheesy precipitate. It was thoroughly extracted with 75 cc., 50 cc., and three times with 40-cc. portions of ethyl acetate. The combined ethyl acetate extracts were washed six times each with 2 cc. of water and finally dried with sodium sulfate. After filtering, the solution was brought to dryness. The acid residue was a colorless resin; weight 0.1831 g. By treating the neutral residue (0.962 9.) with ether, altogether 0.603 g. of crystals and 0.318 g. of resinous material (brittle foam) was obtained. The crystalline fraction was subjected to numerous crystallizations from which, however, no uniform substances could be obtained; altogether 0.512 g. of non-uniform crystalline material and 0.082 g. of an amorphous product (brittle foam) resulted. The crystalline material (0.512 9.) was subjected t o an elaborate purification by chromatographic adsorption. It was dissolved in 60 cc. of benzene and this solution passed through a column (diam. 19 mm.) of 15 g. of aluminum oxide within a period of three hours. The following four eluates were passed through within an hour each and the rest within about 35 minutes each. CHROMATOGRAPHIC FRACTIONATION NO. OF FRACTION

1 2 3 4 5 6 7 8 9 10 11

12 13 14 15

SOLVENT

60 cc.benzene (original solution) 45 cc. benzene 15 cc. ether 15 cc. benzene 45 cc. ether 60 cc. ether 40 cc. ether 20 cc. acetone 20 cc. ether 40 cc. acetone 60 cc. acetone 60 cc. acetone 0.15 cc. methanol 60 cc. acetone 0.20 cc. methanol 60 cc. acetone 0.30 cc. methanol 60 cc. acetone 0.50 cc. methanol 58 cc. acetone 4- 2 cc. methanol 55 cc. acetone 5 cc. methanol 45 cc. acetone 15 cc. methanol 60 cc. methanol

+ +

+ +

+ + + + + +

Total. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

KEIGET OF LESIDVE 0.

0.0006 * 0020 .0073 .0161 .0127 .0067 .0102 .0275 .OM4 .0414

.0464 .1134 .0944 .0409 .0235

APPEARANCE OF BESIUUE

Greasy Resinous Partly cryst. resin Pt. cryst. yellowish resin Yellowish crystals Resinous Crystalline Crystalline Crystalline Large crystals Large crystals Crystalline Brittle foam Resinous Essentially crystalline

.4878

The residues of the above chromatographic fractionation were subjected t o a thorough examination : Fractions 4 and 5. These residues were combined and dissolved in a very small volume of acetone to which a little petroleum ether was added. After standing for a few days some stout, slightly yellowish prismatic crystals had separated; wt. 3.9 mg.; m.p. 17.1-176'. This substance was obviously identical with ethyl 3,5,19-trihydroxy-A88 14-etiocholenate (XI) to be described in a subsequent experiment. Anal. Calc'd for CZSH,,O~:C, 69.79; H, 9.06. Found: C, 69.72; H, 9.06. Ethyl S , b ,19-trihydroxyetiocholanate ( X V ) . Fractions 8 to 11. The residues were separately dissolved in acetone and concentrated to a small volume on a water-bath. Long,

841

IX


flat, rather stout prismatic crystals separated on standing at room temperature, sometimes after the addition of a little petroleum ether. After filtering, additional crystslline separaFLOW SHEET ( E X P E R I h 5 N T

I)

Cryst. 3,5,14,19-tetrahydroxyetiocholanio add a.5ossg.

(V).

Resinous Material 23531 g.

Second Part 1.1268 g.

First Part 1.2273 g.

r--i j Neutral

I7 Acid

Resinous 0.1886 K . (not investigated)

Resinous 1.0177 K.

I + I

1 Crystalline 0,071 g.

Resinous 0.9471 g.

E,, pa H

Hz ,Pt 1

I Neutral

\Acid

1

1

Resinous 0.1881 g.

Semicrystalline 0.9620 g.

1

I

etiiohclanic mid (XIV)

Recrystsllized

At l a s t 0.0114 g.

Resinous 0.3171 g. (not 1nvBO tigated)

Y enated ~

o.&

Resinous 0.8278 g.

1

Crystalline 0.6030 g.

Fairly pure 3,6,19-trihydroxy-

8.

1

Pureethyi3 6 14 19tetmhydmx;etio-' cholsnata (VIII) 0.0501g.

Chromatographed 0.6937 g.

I

Crystalline

1

Resinoua 0.08!7 g. (not mvB& tigated

Chromatographed

Ethyl 3 5 19-trihydroxyA&L%ti&holenate (XI), (Trace) Pure ethyl 3 5 19-trihydroxyetiochoLAate (XV), at least 0.1712 g.

Pure ethyl 3.5,19-trihydroxy etiocholanata (XV), 0.2287 g.

Pure ethyl 3 5 14 19-tetrahydroxyetidhhaAate (VIII) , 0.011 g.

tions were obtained by analogous treatment of the concentrated mother liquors. Nine crystdline fractions, totalling 0.1228 g., represented identical material, with melting points

842

MAXIMILrAN EHRENSTEIN AND ALDA RUTH JOHNSON

above 185". The carbon-hydrogen determinations were carried out with the first crops obtained from fractions 10 (25 mg.; map. 18&190') and 11 (27 mg.; m.p. 188.5-190") respectively. The optical rotation refers to the first crop derived from fraction 11. +62.9" (20.0mg. in 2.0 cc. of chloroform), Anal. Calc'd for CezHasOs:C, 69.42;H, 9.54. Found: C, 69.43,69.26;H, 9.57,9.82. Fractions 12 and 13. When the residues were separately recrystallized from acetone (the addition of petroleum ether produced mixtures) one crop of crystalline material was secured in each instance; weights 0.0516 g. and 0.0170 g. respectively; m.p. 184-187' and 184-189.5' respectively. These substances were identical with ethyl 3,5,19-trihydroxyetiocholanate (XV). Material obtained from the mother liquors of fractions 8-11 (0.0369 9.) and fractions 1213 (0.13959.) was combined and subjected t o a renewed chromatographic separation. Such treatment yielded a number of crystalline fractions totalling 0.0373g., with melting points above 185". This material was likewise identical with XV. The total yields of pure samples of this substance, as derived from the original chromatogram, was therefore 0.2287 g. Fraction 14. When the residue was recrystallized from a small volume of acetone to which a n equal amount of petroleum ether was added, about 11 milligrams separated as crystalline scales; m.p. 1%-189'. There was no depression of the melting point when mixed with an authentic sample ,of ethyl 3,5,14,19-tetrahydroxyetiocholanate(XVIII). T h e analysis was further proof for the identity. Anal. Calc'd for CtsH,,Oa: C, 66.62; H, 9.16. Found: C, 66.39;H, 9.24. The acid part of the hydrogenation product (0.1881 g.) was dissolved in a little acetone. Crystalline material separated on standing overnight; wt. 11.4mg.; m.p. 240-247" (melting to light brown liquid, subseq. effervescence) ; no depression of melting point when mixed with authentic sample (v. infra.) of 3,5,19-trihydroxyetiocholanic acid (XIV). A second crystalline crop separated from the mother liquor: wt. 26.0 mg.; m.p. between 210 and 230' (melting to yellow liquid; subseq. effervescence). Experiment ZI (Flow Sheet II). Since the first dehydration experiment had yielded a certain amount of VII1,indicating that the reaction had not gone t o completion,it was decided to perform the dehydration under slightly more vigorous conditions. A total of 1.2716 g. of recrystallized V was dissolved in 130 cc. of 0.1 N hydrogen chloride in absolute alcohol. It was kept a t 87" (re5ux) for one hour. It was then distilled at atmospheric pressure at a slow rate (bath temp. 92-95")for a period of ninety minutes and finally at a more rapid rate (bath temp. gradually raised t o 100') for a period of 45 minutes. T o the residue, representing approximately 40 cc. of a yellowish solution, was added 30 cc. of water and the distillation was then continued in YQCUO (45-50")until a distinct turbidity appeared. The turbidity was brought into solution by brie%yheating on a water-bathand the solution then allowed to stand a t room temperature overnight which caused the separation of some resinous material and also of yellowish needles. Compound C n o H e ~ O(Lactone ~ X 9 ) . The crystals were separated from the resin by frequent leaching with aqueous alcohol (1:l); pale yellow needles; dry weight 45.8 mg.; m.p. 91-93'. A solution of this substance in chloroform gave with tetranitromethane a distinct yellow color. Anal. Calc'd for CsoHzsOa: C, 72.24;H, 8.49. Found: C, 72.68,72.60;H, 8.37,8.35. From the mother liquor the alcohol was completely removed in vacuo. This produced an oily precipitate which was taken up with 100 cc. of ether. The aqueous phase was thereafter extracted five times with 50-cc. portions of ether. The combined ether extracts were washed five times with 3-cc. portions of water, extracted successively with 10 cc. and 5 cc. of 5% sodium carbonate and finally washed seven times with 3 cc. of water. The ether solution was dried with sodium sulfate, filtered, and brought to dryness. The neutral residue was a colorless brittle foam; weight 1.085g. T o a solution of this neutral material in 4 cc. of

[4'tS

INVESTIGATIONS ON KCEROIDS.

IX

843

ether, petroleum ether was added very gradually (cf. Expt. 1.1). Only a resinous precipitate was obtained, indicating that probably no appreciable amount of VI11 was present. This precipitate was brought to dryness together with the supernatant solution. The above carbonate phases and final aqueous washings were combined and acidified t o Congo by adding 2.5 cc. of conc'd hydrochloric acid. The acid material was isolated by extracting once with 100 cc. and five times with 40-cc. portions of ether. The combined ether extracts were washed five times with 3 cc. of water and then dried with sodium sulfate. After filtering and bringing to dryness, the acid residue was obtained as a slightly yellow resin; weight 0.1186 g. The neutral and acid residues were separately subjected t o catalytic hydrogenation. Hydrogenation of neutral residue. A suspension of 450 mg. of platinum oxide in 7 cc. of glacial acetic acid was reduced and after the addition of a solution of 1.080 g. of t h e neutral residue in 12 cc. of glacial acetic acid, shaking was continued a t room temperature (36") for a period of about.3) hours. The total hydrogen absorption was 68.7 cc.; calc'd for ethyl 3,5,19-trihydroxy-A~~-etiocholenate (IX), 69.8 cc. The solution was filtered from the catalyst and immediately brought to dryness in vacuo (45-50"). T o the syrupy residue 5 cc. of water was added at once. The following day the wax-like, semi-crystalline material was taken up in 100 cc. of ethyl acetate. The latter solution was separated from the water and washed neutral by treating i t successively with 5 cc. and 2 cc. of 5% sodium carbonate and finally five times with 2-cc. portions of water. After drying with sodium sulfate, the ethyl acetate solution was brought completely t o dryness in vacuo; weight of the resinous residue, 1.0404 g. The residue was dissolved in a small volume of apetone and this solution seeded with an authentic sample of ethyl 3,5,19-trihydroxyetiocholanate (XV) (v. Exp. I. 2.). Separation of stout prismatic crystals began immediately. They were filtered the following day and identified as moderately pure XV; yield 0.3474g.; m.p. about 170-175"; mixed m.p. with pure authentic sample, 177". Anal. Calc'd for CBHa,Ob: C, 69.42; H, 9.54. Found: C, 69.40; H, 9.30. From the filtrate 0.0368 g. of definitely impure crystalline material (m.p. 155-158') was obtained. The final mother liquor was brought completely to dryness; weight of the resinous residue, 0.5740 g. This residue was subjected to chromatographic adsorption, for which i t was dissolved in 60 cc. of ether. The solution was filtered through a column (diam. 20 mm.) of 16 g. of aluminum oxide within a period of 80 minutes. The eluates were passed through, each within 25-40 minutes. CHROMATOGRAPHIC FRACTIONATION NO. OF PBACTXON

1

2 3 4 5 6 7 8 9 10 11

12 13 14

I

SOLVENT

60 cc. ether (original solution) 40 cc. ether 20 cc. acetone 20 cc. ether 40 cc. acetone 60 cc. acetone 60 cc. acetone 0.15 cc. methanol 60 cc. acetone 0.20 cc. methanol 60 cc. acetone 0.30 cc. methanol 60 cc. acetone 0.40 cc. methanol 60 cc. acetone 0.50 cc. methanol 1 cc. methanol 59 cc. acetone 58 cc. acetone 2 cc. methanol 55 cc. acetone 5 cc. methanol 45 cc. acetone 15 cc. methanol 60 cc. methanol

+ + + + + + + + +

Total .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

-

0.0693 .1382 .0251 .0338 .0255 .0097 .0065 .0117 .0288 ,0461

+ +

1

1

1

.a640

I

.0647 .a293 .0153 .5650

I

Resinous Partly cryst. resin Crystalline Crystalline Partly cryst. resin Crystalline Resinous Partly cryst. resin Crystalline Crystalline Foamy glass Foamy glass Foamy glass Semi-crystalline

844


The residues of the above chromatographic fractionation were separately recrystallized. Fractions 1-5 definitely represented mixtures, but no attempt was made a t this time t o subject them to a systematic. purification. Ethyl S,b, 18-trihydroxy-Au#lh-etiocholenate (XZ). When fractions 2, 3, and 4 were recrystallized from acetone, a number of crystalline crops (dagger-shaped crystals) were obtained with melting points between 185 and 190" (the substances melted first around 170" and then solidified to remelt at the stated temperature); total yield 42 mg. A sample in chloroform gave with tetranitromethane no yellow color. There were several crystalline crops with melting points below 100". The major part of the material contained in fractions 2-4 remained resinous. The analysis was performed with a crystalline sample melting a t 185". This melting point is not claimed to be that of an entirely pure sample. The mixed melting point with ethyl 3,5,19-trihydroxyetiocholanate(XV) was 156". Anal. Calc'd for CnrH840b:C, 69.79; H, 9.06. Found: C, 69.40; H, 8.85. When the residues of fractions 9 and 10 were recrystallized from acetone, a total of only 40.8 mg. of crystalline material (transparent rod-shaped crystals) with melting points between 170 and 180" was obtained. These crops represented moderately pure XV, as was also established by mixed melting point with an authentic sample. Anal. Calc'd for C I I H ~ ~ O C,~ 69.42; : H , 9.54. Found: C, 69.27; H, 9.54. On recrystallizing the residue of fraction 11 from acetone, several crystalline crops with melting points below 150" were obtained. They obviously represented mixtures. When the residue of fraction 12 was recrystallized from acetone, 17.2 mg. of flaky white crystals were obtained; m.p. 175-180". There was no depression of the melting point when mixed with an authentic sample of ethyl 3,5,14,19-tetrahydroxyetiocholanate (VIII). According to the analysis, the substance was not quite pure. Anal. Calc'd for CzeHssOa:C, 66.62; H, 9.16. Found: C, 67.55; H, 9.24. A second crop ww obtained by adding a little petroleum ether to the mother liquor; wt. 11.2 mg.; m.p. 169-174". This material was obviously less pure. No uniform crystalline material could be secured from the residue of fraction 13. Hydrogenation of acid residue. Platinum oxide (50 mg.) in glacial acetic acid (3 cc.) was reduced and a solution of the acid residue (0.1186 g.) in 4 cc. of glacial acet,ic acid was added and the shaking with hydrogen continued for a period of three hours. The total absorption of hydrogen was 8.9 cc. (26") ; calc'd for 3,5,19-trihydroxy-A~*-etiocholenic acid (XIII), 8.3 cc. Immediately thereafter the solution was filtered from the catalyst and brought to dryness in uucuo. To the resinous residue was added 3 cc. of water. By kneading the resinous material on the following day repeatedly with 0.5-cc. portions of water it eventually became crumbly and filterable; dry weight after filtering, 0.094 g. By recrystallizing this material from acetone or acetone-petroleum ether, three crystalline crops totalling 25.9 mg. were obtained; the melting points were between 230 and 255'. The mother liquor, on being brought to dryness, yielded 66.9 mg. of a resin. The combined three crystalline crops were recrystallized from acetone. The pure acid crystallized in white, broad, flat needles; wt. 12.7 mg.; m.p. 250-252'. There was no depression of the melting point when mixed with an authentic sample of 3,5,19-trihydroxyetiocholanicacid (XIV) (u. infra.). C, 68.13; H, 9.16. Anal. Calc'd for COJISZOL: Found: C, 67.93; H, 9.03. From the mother liquor 5.8 mg. of crystalline material, melting a t 245-252", was obtained. A solution in ether of the resinous part (66.9 mg.) of the hydrogenation product was treated with an excess of diazomethane. The weight of the resinous reaction product was 69.5 mg. Its chromatographic purification was inconclusive. No methyl 3,5,19-trihydroxyetiocholanate (XVII) could be isolated. About 5 mg. of crystalline material was secured which probably represents, though in an impure form, methyl 3,5,19-trihydroxyA-*.14-etiocholenate (XVI)

.


845

IX

FLOW SHEET I1 (EXPERIMENT 10 Remyst. 3,6,14,19-tetrahydmxyetiocholanicacid (V) 12716 g.

I Dehydration (0.1N HCl absol. ethanol)

Cryst. compound X 46.8 mg. (m.p. 91-B3?

I

Neutral

Acid J. Reeinoua 0.1186g.

Reainous 1.086 g. I Hr,Pt Hydrogenation 1.080 g.

I

I Hi, I

i

Pt

Fair1 pure3 6 19triKydrox hi0 cholanic acd(XfV) 0.0181 g.

Reainous 1.0404g.

Moderately pure ethvl3.6.19tr& droxy etWCho&nate (XV) 0.3474 g.

ResioOuS 0.67409.

Impure or st. m a 4 0.0368 g.

Chromatographed J. Only small amounts of pure compounds: ethyl 3 6,1%trihydroxy-A891' etioChoh8te (XI) 0.042 g. ethyl 3 6 19-trihydroxyetie ehol&&a (XV) 0.041 g. ethyl 3 6 14 19-tetrahyclroxyetiooLo8n'ate (VIII) 0.0284 g.

9,6,19-Trihydroxyetiocholanicacid ( X Z V ) . To a solution of 86.8 mg. of purest ethyl 3,5,19-trihydroxyetiocholanate(XV) (from Expt. 1.2) in 2.5 cc. of methanol was added 0.17 g. of potassium hydroxide dissolved in 1.0 cc. of methanol. The mixture was refluxed on a water-bath for two hours. After the addition of 2 cc. of water the solution was brought almost to dryness in vacuo and the residue taken up in 5-6 cc. of water. The suspended crystalline material was extracted by shaking with 10 cc. of ether. The ether phase waa washed three times with 1-cc. portions of water. After drying with sodium sulfate and filtering, the ether solution was brought t o dryness; weight of the crystalline neutral residue, 29.5 mg. The combined alkaline solution and aqueous washings were macle acid to Congo by the addition of 1 cc. of conc'd hydrochloric acid (ice-cooling), which produced an emulsion from which a fine white precipitate separated soon. It was extracted with 10 cc. of ether to which, because of the insolubility of the precipitate, 10 cc. of ethyl acetate had to be added. It had to be subsequently extracted five times each with 10 cc. of ethyl acetate to bring all of the white material into solution. The aqueous phase was then extracted once more with 10 cc. of ethyl acetate. The combined extracts were washed once with 2 cc. and four times with 1-cc. portions of water. After drying with sodium sulfate the solution was brought to dryness in vacuo and the residue dried in a vacuum desiccator over potassium hydroxide; weight of the crystalline acid residue, 53.8 mg. This material was dissolved in the required amount (10-11 cc.) of acetone and the solution concentrated

846


to a smaller volume (about 2 cc.) on a water-bath when suddenly crystallization set in. The substance crystallized in bunches of flat spear-shaped crystals. It was filtered after a few hours standing at room temperature. Additional crops of rather pure material were obtained by concentrating the mother liquor; yields: Crop 1, wt. 28.2 mg.; m.p. 259-260". Crop 2, wt. 15.1 mg.; m.p. 256.5258". Crop 3, wt. 1.8 mg.; m.p. 254-256". All these crops melted t o a light-brown liquid and there was subsequent effervescence. The determination of the optical rotation and the microanalysis were performed with the first crop. [a]: 69.5" (8.0 mg. in 2.0 cc. of acetone). Anal. Calc'd for C ~ O H ,C,~ 68.13; ~ ~ : H, 9.16. Found: C, 67.66; H, 9.18. A solution of the neutral crystalline residue (29.5 mg.) i n ether was concentrated on a water-bath until crystallization set in. The stout, ruler-shaped crystals were filtered the following day (crop 1, wt. 17.7 mg.; m.p. 208-211"). This material obviously represented a mixture of the ethyl and methyl esters of 3,5,19-trihydroxyetiocholanic acid (XV and XVII). Additional crystalline material was obtained by concentrating the filtrate on a water bath (crop 2, wt. 4.8 mg.; m.p. 222.5-224"). On bringing the final mother liquor to dryness, a crystalline residue (6.1 mg.) was obtained. The second crop represented pure XVII. There was no depression of the melting point when it was mixed with a n authentic sample of that compound (v. infra.). The results of the microanalysis were likewise in agreement with this conclusion. Anal. Calc'd for CnHar06: C, 68.80; H, 9.36. Found: C , 68.89; H, 9.24. Methyl S , b , 19-trih?/drozyetiocholanate (XVZZ). To a solution of 26.6 mg. of 3,5,19trihydroxyetiocholanic acid (XIV) in 5 cc. of acetone was added at 0" a slight excess of an ethereal solution of diazomethane. After standing at room temperature for a period of twenty minutes, the excess of the diazomethane was removed on a water-bath and the resulting colorless solution brought to dryness in vucuo. The crystalline residue was dissolved in 15 cc. of ether and this solution washed neutral by treating i t successively with 1 cc. of N hydrochloric acid, 1 cc. of water, 1 cc. of 5% sodium carbonate, and three times with 1-cc. portions of water. After drying with sodium sulfate the solution was brought to a small volume on a water-bath. Crystallization set in spontaneously; characteristic hexagonal platelets. The material was filtered after some standing at room temperature; weight 15.2 mg.; m.p. 220-222.5". [a1;8.6+61.3O (8.0 mg. in 2.0 cc. of chloroform). Anal. Calc'd for C2~H8405:C, 68.80; H, 9.36. Found: C, 68.75; H, 9.34. A second crop of a similar looking crystalline product was secured by concentrating of the mother liquor; weight 3.8 mg.; m.p. 218-220.5'. EthyZ 3,19-diacetozy-6-hydrosyetiochoZa~a~e(XVZZI). To a solution of 108 mg. of pure ethyl 3,5,19-trihydroxyetiocholanate(XV) (from Expt. 1.2) in 0.4 cc. of pyridine was added 0.4 cc. of acetic anhydride and the mixture allowed to stand at room temperature for a period of 22 hours. It was then concentrated in vacuo (65") t o a viscous, colorless oil which was taken up in 25 cc. of ether. This solution was washed neutral by shaking it successively twice with 2-cc. portions of N hydrochloric acid, twice with 2 C C . of 5% sodium carbonate, and five times with 1 cc. of water each. After drying with sodium sulfate, the solution was brought t o dryness. The residue was a colorless resin which partly crystallized overnight in an evacuated desiccator; weight, 127.5 mg. On adding a little ether, some white crystalline material separated which was filtered after brief standing; weight of this first crop, 39.0 mg.; m.p. 108-109.5". [a]~*~6+60.3° (20.0 mg. in 2.0 CC. of chloroform). O I67.20; : H, 8.68. Anal. Calc'd for C ~ ~ H ~ OC, Found: C, 67.26; H, 8.66. By concentrating the filtrate to a smaller volume several additional crops of crystalline material were obtained; crop 2, wt. 31.3 mg.; m.p. 107-108"; crop 3, wt. 22.4 mg.; m.p. 03-105"; crop 4, wt. 12.4 mg.; m.p. 97-101.5'.

+


IX

847

S,6,19-Trihyd~oxy-A~~~~-etiocholenic acid (XIZ). T o a solution of 34.9 mg. of ethyl 3.5, 19-trihydroxy-A8J4-etiocholenate (XI) (cf.Expt. 11) in 1 cc. of methanol was added 70 mg. of potassium hydroxide, dissolved in 1 cc. of methanol. The mixture was refluxed for two hours and then kept at room temperature for about 30 minutes, 3 cc. of water was added, and the solution concentrated in vacuo t o a volume of about 1 cc. After the addition of 3 cc. of water the turbid solution was extracted four times with 5-cc. portions of ether. The combined ether phases were washed three times with 1.5 cc. of water, dried with sodium sulfate, and finally brought to dryness; weight of the crystalline neutral residue, 4.1 mg. The combined aqueous phases were made acid t o Congo by the addition of 0.5 cc. of conc'd hydrochloric acid (ice-cooling). This produced a turbidity from which a white, apparently crystalline precipitate separated promptly. It was extracted seven times with 5-cc. portions of ethyl acetate. The combined extracts were washed five times each Kith 1 cc. of water, dried with sodium sulfate, brought completely t o dryness and then kept i n a vacuum desiccator over potassium hydroxide; weight of the crystalline acid residue, 27.6 mg. This residue was dissolved in 10 cc. of acetone and the solution concentrated on a water-bath to a volume of about 3 cc. The white, scaly microcrystalline material was filtered after a few hours' standing a t room temperature. Additional crops of fairly pure material were obtained by concentrating the mother liquor. Yields: Crop 1, wt. 6.6 mg.; m.p. 286-288'. Crop 2, wt. 5.4 mg.; m.p. 285.5-287.5". Crop 3, wt. 2.9 mg.; m.p. 281-283". Crop 4, wt. 0.6 mg.; m.p. 283-286". All these crystalline crops melted t o a dark brown liquid. The final mother liquor was brought to dryness in vacuo;weight of the crystalline residue, 8.9 mg. The microanalysis was performed with the first crop and the optical rotation was determined with the second crop. The error of the latter determination is possibly larger than usual, because the scanty solubility of this substance afforded a solution of a very low concentration. [~~18,0*~+77.3" (3.6 mg. in 2.0 cc. of acetone) Anal. Calc'd for C2OH8006: C, 68.52; H, 8.63. Found: C, 68.11; H, 8.56. Methyl 5,6,19-trihydrosy-A8~~4-etiocholenate (XVZ). A solution of 8.9 mg. of 3,5,19trihydroxy-A8~14-etiocholenic acid (XII) in 6 cc. of acetone was concentrated t o about two-thirds of this volume on a water-bath. T o this was added at 0' an excess of an ethereal solution of diazomethane. The reaction mixture was allowed to stand cold for about 10 minutes and at room temperature for about 25 minutes. The solution was brought t o dryness in vacuo and the white residue taken u p in 45 cc. of ether. The ether solution was washed neutral by treating i t successively with 1 cc. of N hydrochloric acid, 1cc. of water, 1cc. of 5% sodium carbonate, and three I-cc. portions of water. After drying with Bodium sulfate and bringing t o dryness, the ether solution yielded 9.8 mg. of a white crystalline residue. It was treated with some acetone and the white crystalline material then ieparated by filtration; microscopic flat short needles; wt. 4.9 mg.; m.p. 270-274" (brown w.1. C,~ 69.18; : H, 8.83. Anal. Calc'd for C ~ I H ~ Q O Found: C, 69.03; H, 8.75. The filtrate was brought to dryness in vacuo and yielded 3.3 mg. of a white crystalline residue. The methylation was also carried out with the crystalline residue obtained from the final mother liquor of 3,5, 19-trihydroxy-A8Fld-etiocholenic acid (XII) (8.9 mg. ;cf. preceding expt.). Worked up the same way as described above there was obtained 1.3 mg. of microscopic needles; m.p. 272-276' (brown liq.). When the filtrate was brought to dryness, 11.9 mg. of a resinous residue was obtained. SUMMARY

1. Strophanthidol (11) and its diacetate (111) were prepared from strophanthidin (I)according to procedures published in the literature. Supplementary observations are recorded.

848


2. Strophanthidol diacetate (111) was oxidized to the 3 19-diacetoxy-5,14dihydroxyetiocholanic acid (IV) which in turn was saponified to 3,5,14,19tetrahydroxyetiocholanic acid (V). The methyl esters of the latter two compounds are described (VI1 and VI respectively). 3. Treatment of 3 5,14)19-tetrahydroxyetiocholanic acid (V) with alcoholic hydrogen chloride yielded predominantly neutral substances. The main reaction product of the neutral fraction was ethyl 3 , 5 )19-trihydroxyA14-etiocholenate (IX) which in turn was hydrogenated to ethyl 3,5,19-trihydroxyetiocholanate (XV). The latter compound furnished by acetylation ethyl 3,19-diacetoxy-5-hydroxyetiocholanate(XVIII) and by saponification 3 , 5 )19-trihydroxyetiocholanic acid (XIV). The methyl ester (XVII) of this acid was prepared. Minor neutral products of the reaction with alcoholic hydrogen chloride were ethyl 3 5,14,19-tetrahydroxyetiocholanate (VIII), an unsaturated compound of the possible empirical formula C20H~804(X?), and ethyl 3 5,19-trihydroxyAE*14-etiocholenate (XI). The latter compound resists catalytic hydrogenation. It was saponified to 3,5 ,19-trihydroxy A8~14-etiocholenic acid (XII) which in turn was transformed into its methyl ester (XVI). It was concluded that the acid products of the reaction with alcoholic hydrogen chloride contained 3,5,19-trihydr0xy-A~~-etiocholenic acid (XIII) and possibly also 3,5,19-trihydroxy-A8*14-etiocholenic acid (XII). Among the hydrogenation products of the whole acid fraction there wa8 identified 3 )5 19-trihydroxyetiocholanic acid (XIV) which can only have originated from the unsaturated acid XIII. )

)

)

)

)

PHILADELPHIA 4, PA. REFERENCES EHRENSTEIN, J . Org. Chem., 9, 435 (1944). RUZICBA, PLATTNER, HEUSSER,AND ERNST,Helv. Chim. Acta, 29, 248 (1946). PLATTNER, RUZICKA, HEUSSER, PATAKI, AND MEIER,Helv. Chim. Acta, 29, 942 (1946). STEIGER AND REICASTEIN, Helv. Chim. Acta, 21, 828 (1938). AND KRAUS,2. physiol. Chem., 266, 39 (1940). Chem. Zentr., 1943, I, 2495. RABALD Chem. Abstr., 36, 1408 (1941). (6) RABALD AND KRAUS, Ger. Pat. 723,224 of June 18, 1942 ( t o C. F. Boehringer & Soehne, G.m.b.H). Chem. Zentr., 1943, I , 60. Chem. Abstr., 37, 5198 (1943). (7) HUNZIKER AND REICHSTEIN, Helv. Chim. Acta, 28, 1472 (1945). (8) MEYER,Helv. Chim. Acta, 29, 718 (1946). AND HEUSSER, Helv. Chim. Acta, 29, 727 (1946). (9) PLATTNER (10) FEIST,Ber., 33, 2069 (1900). (11) PAIST,BLOUT,UHLE,AND ELDERFIELD, J . Org. Chem., 6, 287 (1941). (12) GALLAGHER AND LONG,J. Biol. Chem., 162, 495 (1946). (13) SORKIN AND REICHSTEIN, Helv. Chim. Acta, 29, 1218 (1946). (1) (2) (3) (4) (5)

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