[CONTRIBUTION FROM
THE
STERLING CHEMISTRY LABORATORY, YALE UNIVERSITY]
CONTRIBUTIONS TO T H E STUDY OF MARINE PRODUCTS. XV. T H E STEROLS OF STARFISH. 111 WERNER BERGMANN
AND
HARRY A. STANSBURY, JB.
Received March 5, 1944
The name stellasterol was assigned in 1915 by Kossel and Edlbacher (1) to a di-unsaturated sterol of the probable formula CZ,H440, which occurs together with astrol in the unsaponifiable matter of the starfish Asterius rubens. The suggestion has been made in a previous communication of this series (2), that asteriasterol (3), the sterol of Asterius forbesi, is a mixture of astrol, stellasterol, and other ill-identified sterols. Since then it has been shown by the present authors (4) that astrol is identical with batyl alcohol. A resumption of the investigation of starfish sterols was made possible through the generous cooperation of Mr. Charles F. Lee, Wildlife Service, U. S. Department of Interior, who supplied a large quantity of acetone-soluble material from Asterius forbesi. A preliminary study of this material led to results similar to those described previously ( 2 ) . The sterol fraction proved to be a complex mixture of a t least two sterols, the complete separation of which has not yet been accomplished. In the course of twelve recrystallizations the melting point of the sterol mixture rose from 120-133" to 149-150°, which is the melting point of stellasterol. The substance, hon-ever, was not yet pure, for upon further recrystallizations the melting point went up to 156-158". After twenty recrystallizations of a mixture of steryl acetates a fraction was obtained which like stellasteryl acetate melted at 177", and which upon further recrystallization gave a product melting a t 180". These preliminary observations suggest the identity of stellasterol with the least soluble sterol of the present sterol mixture. The probable identity between the two sterols, however, appears to be discounted by the marked difference in the melting points of the respective sterols. Thus stellasteryl benzoate has been reported to melt a t 100" to a turbid liquid n-hich turns clear a t 125". In contrast, the various fractions of steryl benzoates obtained during the present investigation melted considerably higher, the lowest at 129-134", and the highest a t 194-197". ilrational explanation for this discrepancy of results was eventually found in the observation, to be described beloIT, that starfish sterols possess a cyclic double bond which readily isomerizes under the influence of hydrochloric acid. The method used by Kossel for the preparation of stellasteryl benzoate consisted in the heating to 160" of a mixture of stellasterol and benzoyl chloride. It is certain that the hydrochloric acid formed during the reaction led to a rearrangement of the double bond, and that the benzoate obtained was not the benzoate of stellasterol, but the derivative of one or more of its isomers. In contrast, the present benzoates ivere prepared in the absence of free hydrochloric 1 This communication describes work done by Harry -4. Stansbury, Jr. in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry, Yale University, 1944.
281
282
TT'ERNER BEZGMANN AND H. A . STASSBURY
acid, by treating the sterols with benzoyl chloride in the presence of pyridine. When the benzoates thus obtained were exposed to the action of anhydrous hydrochloric acid, a mixture of compounds was obtained which melted a t 103' or below. It soon became apparent that the available starting material was insufficient for the isolation of pure compounds by fractional crystallization of the sterols, their acetates or benzoates. Attempts to separate the acetate mixture by adsorption on alumina were abandoned because of inconclusive results. In one experiment the principal fraction of the eluate, which represented about seventy per cent of the starting material, melted forty degrees below the melting point of the original mixture. This observation suggested that adsorption was accompanied by some chemical reaction, such as isomerization. Titration of various fraction of sterols and their acetates with perbenzoic acid gave values which suggested the presence of a mixture of mono- and di-unsaturated sterols. In previous instances ( 5 ) the separation of such mixtures mas effected by means of bromine addition products. The application of this method to the separation of the starfish sterols, however, did not appear promising, because it had been shown previously (2) that these sterols and their derivatives react with bromine with the formation of green-colored decomposition products. Nevertheless a sample of acetate was brominated, principally in order to test for the presence of cholesteryl acetate, which mould form a difficultly soluble dibromide of melting point 117'. The only crystalline product obtained in this reaction was a very small amount of an unknown dibromide melting a t 184-185'. Because of its small yield, and the extensive destruction of the bulk of the material, this method of separation was abandoned. After the failure of the methods listed above to bring about the isolation of pure compounds, it was decided to obtain evidence concerning the structure of the starfish sterols by an investigation of the chemical behavior of their mixture. The selection of suitable methods was guided by a working hypothesis formulated on the basis of certain properties of the mixture which seemed to be of particular significance. As has been mentioned before, the degree of unsaturation of the sterol mixture suggests the presence of mono- and di-unsaturated sterols. According to Kossel, stellasterol is a di-unsaturated compound, and in agreement n-ith this formulation the present sterol fractions most closely resembling stellasterol show the presence of two double bonds ( 2 ) . It was now assumed as part of the working hypothesis that like the co-occurring sponge sterols, poriferasterol and clionasterol ( 5 ) , the starfish sterols are closely related compounds differing from each other only in the presence of a double bond in the side chain. For the simplification of the discussion the name stellasterol shall be retained to designate the di-unsaturated sterol, and the mono-unsaturated sterol shall be referred to 2s stellastenol. All fractions of the starfish sterols and their derivatives were found to be slightly dextro-rotatory . These observations a t once suggested that unlike all other known unsaturated animal sterols, the starfish sterols were devoid of a 5,6-ethenoid linkage v-hich is known to confer a strong negative contribution to
283
STEROLS O F STARFISH
the rotation of steroids. A11 fractions of the starfish sterols and their derivatives give a green color reaction with bromine, which had already been observed by Kossel (1). This coloration which is now known under the name of TortelliJaffB reaction (6) is generally believed to be typical for steroids possessing a double bond joined to the C-8 atom of the ring system. It was therefore assumed as a further part of the working hypothesis that both stellasterol and stellastenol possessed a double bond in the y-(7 ,8), 6-(8,9) or a-(8,14)- position. ?'he presence of an ethenoid linkage in the side chain of one of the sterols, pro visionally named stellasterol, was readily shown by the ozonization of the steIol mixture. An oily, water-insoluble, steam-distillable aldehyde was isolated in the form of its 2,4-dinitrophenylhydrazone,m.p. 119-120', [a]:*+14.5", which gave analytical values for the derivative of an aldehyde of the formula CsH&HO. When mixed with a sample of the corresponding derivative of me1 hylisopropylacetaldehyde, m.p. 124-124.5', [a]i6 - 37.7', which had been prepared from ergosterol, the melting point was 119-122.5'. Bearing in mind that partial recemization of the aldehydes is difficult to prevent, these data a t once suggest that the aldehydes from stellasterol and ergosterol are optical antipodes. Methyl-n-propylacetaldehyde is the only other asymmetric aldehyde of the formula C6HnCH0. Since this aldehyde lacks the usual isopropyl group of 1he sterol side chain, its identity with the aldehyde from stellasterol seems highly improbable. It is therefore reasonable to assume that stellasterol carries a side chain of the structure I in which the optical configuration at C-24 is the opposite of that of ergosterol. The occurrence of this type of isomerism among 24
-CH-CH=CH-CH-CH( (1)
I
CHa
I
CH8)z
CHe
natural sterols was first described by Fernholz and Ruigh (7) who proved convincingly that campesterol and 22,23-dihydrobrassicasterol differ only in the optical configuration on C-23. The present case represents the second example of this type of isomerism, and the first of its kind t o be found among animal sterols. It had been assumed for reasons discussed above that stellasterol and stellastenol possess double bonds a t C-8 in the positions shown in formulas II-IV. Such double bonds are known to be resistant to hydrogenation. When treated with platinum and hydrogen in acetic acid, y- (11) and 6- (111) unsaturated steroids rearrange t o the a-isomers (IV). The latter are very resistant to catalytic hydrogenation under ordinary conditions but can be isomerized by anhydrous hydrochloric acid to the 0-isomers (V), which absorb hydrogen readily to give the corresponding saturated compounds (VI). Preliminary studies on the hydrogenation of starfish sterols indicated the presence of inert double bonds, in support of the assumptions made above. If then both stellasterol and stellastenol were t o possess double bonds in they-,&, or a-position, and if an additional double bond mere present in the 22,23-position of the side chain of stellasterol, the catalytic hydrogenation of a mixture of such sterols should give a uniform
284
WERNER BERGMANS- AND H. A. STANSBURY
product, a-stellastenol. Accordingly a mixture of acetates, which titrated for 1.4 double bonds, was hydrogenated with a platinum black catalyst in acetic acid at room temperature and atmospheric pressure. The reaction stopped after about one-half mole of hydrogen had been consumed. The reaction product, which will be named a-stellastenyl acetate, proved to be a uniform com12.5'; it showed the presence pound of constant melting point 105-106', of one double bond on titration with perbenzoic acid. Hydrolysis of the acetate
+
CI" HOC[)
PI-]
''
11. y-Stenol
SU"
HO 111. b-Stenol
VI. Stanol
+
gave a-stellastenol, m.p. 123-125", [a]k6 19.8', the m-dinitrobensoate of which gave analysis for a sterol of the molecular formula CzsHr7OH. As expected, a-stellastenyl acetate isomerized in a chloroform solution with dry hydrogen chloride at 0'. A p-stellasteryl acetate, m.p. 94-96', [a]:6 19.8',was thus obtained, which on hydrolysis gave p-stellastenol, m.p. 122-124', [a1;' +29.4". On hydrogenation at room temperature the p-acetate gave the comStellastanol, pletely saturated stellastanyl acetate, m.p. 138-139', [a]:' f13.5'. m.p. 143O, [a]:' +23O gave a m-dinitrobenzoate which gave values for the derivative of a sterol of the molecular formula C28Hd90H. The optical activities of
+
285
STEROLS OF STARFISH
the two stellastenols and stellastanol agree with the general rule (8) that a-unsaturated steroids have a less positive, and p-unsaturated steroids a more positive rotation than the corresponding saturated sterols. Stellastanol is isomeric with ergostanol and campestanol (7), and like the latter it differs from ergostanol in the configuration a t C-24. A comparison of the data shown in Table I also shows that stellastanol is different from campestanol. The reimlts of the analyses of the m-dinitrobenzoates mentioned above, and the isolation of methylisopropylacetaldehyde from the products of ozonization, prove tha,t the starfish sterols are Czs-sterols. They are the first principal sterols of this order to be found to occur in animal tissue. Up to the present the only C*s.-sterolknown to occur in animals has been ergosterol, which constitutes the provitamin-D of certain snails and the earthworm, Lumbricus terrestris (9). The fact that the starfish sterols are of the order Czs is difficult if not impossible to reconcile vith the hypothesis of the esogenous origin of the sterols of marine invertebrates, which has been tentatively advanced by one of the present authors (10). The starfish Asterias forbesi feeds principally on bivalves, crustaceans, TABLE I COMPARISON OF SATURATED STEROLS OF THE FORMULA C~~H~oO STEROL
ACETATE
O(-DINITBOBENZOATE
NAKE
-Stellastanol . . . . . . . . . . . . . . . . . . . . . . . . . Ergostanol . . . . . . . . . . . . . . . . . . . . . . . . . . Campestanol (7). . . . . . . . . . . . . . . . . . . .
m.p."C
m.p.*C
aD
aD
m.p.0~
____________
143 144 147
+22 $15 $31
,
139 145 147
$13 $6 $18
i:t
198
I
I
aD
$13 $22
and fish, animals which contain sterols of the order CZ9like ostreasterol (11) or of the order C27 like cholesterol. On the basis of the hypothesis one should expect sterols of these orders to occur in starfish, but no evidence for their presence has as yet been discovered. The structures of all natural, unsaturated sterols which were known before 1940 included a double bond in the 5 ,6-position, and it appeared a t that time that the presence of this double bond was indeed typical for allunsaturated, naturally occurring sterols. That this is by no means true was first shown in the cases of zymosterol from yeast (12) and a-spinasterol from higher plants (13) which are both unsaturated sterols devoid of the 5,6-d0uble bond. The starfish sterols represent the first example of this type of unsaturated sterols to be found t o occur in animal tissues. As far as the cyclic double bond of the starfish sterols is concerned, the available evidence only shows its attachment t o C-8, but does not establish it as a y-,6-, or a-double bond. The possibility may well be envisaged that the original sterol mixture consists of mono- and di-unsaturated with double bonds in any one of these three positions. A mixture of such complexity might well account for the unusual difficulties encountered in the attempts to bring about a separa-
286
WERNER B E R G M A N N A N D H. A . S T . 4 N S B U R Y
tion, and also for the ease with which it is converted into the uniform stellastenol on catalytic hydrogenation.
cy-
EXPERIMENTAL
All melting points are corrected. All optical rotations were taken in a 1-dm. tube, the sample being dissolved in 3 cc. of chloroform. Isolation of the starfish sterols. The unsaponifiable matter from the oil of the starfish Asterias forbesi was dissolved in the minimum amount of hot methanol, and the brownish crystalline material which separated upon cooling was removed by filtration. After two recrystallizations of this material from low-boiling petroleum ether, there was obtained a colorless mixture of compounds of m.p. 129-135", [a];$9.4". Sixteen recrystallizations of this mixture from acetone in a Skau-tube brought about a slow but steady rise of the melting point t o 155-158", [a];+5.9". Anal. Calc'd for C2gHdoO 0.5 HzO: C, 82.90; H, 11.28. C2gH180 0.5 HzO: C , 82.50; H, 11.62. Found: C, 82.12; H, 11.77. The sample m.p. 155-158" gave an acetate of m.p. 162-166", which showed the presence of 1.6 double bonds upon titration with perbenzoic acid. Preparaton of the steryl acetates. Refluxing the crude sterol mixture with acetic anhydride gave a mixture of steryl acetates of m.p. 137-146", [a]: +3.8". It was recrystallized sixteen times from absolute ethanol in a Skau-tube. During the last three recrystallizations the melting point seemingly remained constant a t 170-173°. Subsequent recrystallizations from acetone, however, brought about a further rise of the melting point, and eventually a very small amount of a product was obtained which melted at 178-181", [a];0'. Anal. Calc'd for C80H4g02: C, 81.76; H, 10.97. CaoHsoOz: C, 81.39; H, 11.38. Found: C , 81.12; H, 11.50. Attempted separation by chromatography. A solution of 1.25 g. of a n acetate of m.p. 130-133" in 45 cc. of hexane was passed through a column (19 by 1.5 cm.) of activated alumina. It was followed by 40 cc. of hexane. The percolates were collected in ten fractions of 5 cc. each and evaporated to dryness. No weighable residues were obtained. The column was then eluted with a saturated solution of dry methanol in hexane. Five fractions of 10 cc. each gave a total of less than 10 mg. residue of m.p. 118-131". The column was then eluted with a saturated solution of hexane in dry methanol. The Srst fraction, 10 cc., gave 0.23 g. of residue which melted at 101-115" after one recrystallization from methanol. The second fraction, 5 cc., contained the bulk of the material, 0.84 g., which melted a t 89-94' after one recrystallization. Three of the following fractions, 20 cc., gave a total of 0.14 g. of a product of m.p. 94100". The wide difference between the melting points of the original acetate and of the bulk of the recovered material indicated that some chemical reaction had accompanied the adsorption. Bromination of the steryl acetates. A solution of 122 mg. of acetates of m.p. 135-137" in 1.2 cc. of ether was treated dropwise with a 596 solution of bromine in glacial acetic acid. During the addition, the solution was cooled in an icebath. Some crystalline precipitate formed in the solution, which rapidly turned green and then black. After standing in the refrigerator overnight, the precipitate was filtered and washed with glacial acetic acid. There was obtained 8 mg. of a bromide of m.p. 184-185". Anal. Calc'd for C31H50Br202:Br, 26.0. Found: Br, 23.24. Preparation ofthe steryl benzoates. A solution of 4.7 g. of sterols of m.p. 127-130" in 50 cc. of dry pyridine was treated with 15 cc. of benzoyl chloride. The mixture was left standing at room temperature for twenty-four hours and then poured slowly with stirring into 300 cc. of ice-cold 3 N sulfuric acid. The precipitated material was filtered, washed with water, and recrystallized twice from absolute ethanol. -4total of 4.4 g. of colorless needles was thus obtained, which melted a t 159-166". During fourteen recrystallizations of this
+ +
287
STEROLS O F STARFISH
material from acetone the melting point rose continually and eventually a very small amount of a product of m.p. 194-197" was obtained. A n d . of a fraction of map.176-179', [a]:$9.0". Calc'd for C36Hs002:C , 83.61; H, 10.02. C86H3202: C, 83.28;H, 10.38. Found: C, 82.98; H, 10.12. Anal. of fraction of m.p. 194-197". Calc'd for CS3H6001: C, 83.61; H, 10.02. CssH5zOa: C, 83.28; H, 10.38. Found: C, 83.15; H, 10.30. Action of hydrochloric acid o n the steryl benzoates. A stream of dry hydrochloric acid was passed at 0" for three and one-half hours through a solution of 1 g. of benzoate, m.p. 162-164", in 50 CC. of chloroform. The solvent was then removed in vacuo, and the residue recrystallized from ethyl acetate and absolute ethanol. The product (140 mg.) appeared to be unchanged starting material. Concentration of the mother liquor gave 580 mg. of a crystalline product melting at about 60". After numerous recrystallizations from ethyl acetate and ethanol it melted at 91-92". Ozonization of the steryl acetate mixture. One gram of acetate, m.p. 137-146", was suspended in 12 cc. of glacial acetic acid, and an excess of ozone was passed through the vigorously stirred suspension at 20'. The ozonization was discontinued fifteen minutes after all suspended material had gone into solution. Zinc dust was added to the solution, which was stirred vigorously for twenty minutes. A few drops of silver nitrate solution were then added, and the stirring continued until the mixture no longer gave a positive test for peroxides. The liquid filtered from the zinc dust was poured into water, and the resulting milky suspension was distilled until oily droplets no longer passed over with the distillate. The distillate, about 80 cc., was mixed with 30 cc. of a warm solution of 2,4-dinitrophenylhydrazine (Brady's reagent) (14). The crystalline precipitate, 121 mg. was dissolved in dry benzene, and the solution waB passed through a 1 cm. by 11 cm. column of activated alumina. 13y washing the column with dry benzene a yellow percolate was obtained, which was evaporated to dryness in zlacuo. The residue, after several recrystallizations from ethanol, melted a t 119-12Qo,[ C Y ] : +14.1" (25.5 mg., a 0.12'). When mixed with the 2,4-dinitrophenylhydrazone of 1-methylisopropylacetaldehyde,m.p. 124-124.5", prepared from ergosterol, i t melted a t 119.5-122.5", and when mixed with the 2,4-dinitrophenylhydrazone of I-ethylisopropylacetaldehyde from stigmasterol, i t melted at 107-122'. S r d . Calc'd for C12H16N404: C, 51.42; H, 5.76. CisHisN4Or: C, 53.05; H, 6.16. Found: C , 51.66; H, 5.97. 1,4-Dinifrophenylhydrazone of I-methylisopropylacetaldehyde. Ergosteryl acetate was ozonized, and the aldehyde isolated as the 2,4-dinitrophenylhydrazonein the manner described above. -4fter several recrystallizations from ethanol the compound melted at 124-124.5", [a]: -37.7" (20.7 mg., CY -0.26"). BnaI. Calc'd for ClzHlsXrO,: C, 51.42; H, 5.76. Found: C, 51.83; H , 5.90. w-Stellastenyl acetate. h solution of 1 g. of steryl acetates m.p. 149-154", 1.4 double bonds, in 100 cc. of glacial acetic acid was hydrogenated at room temperature and atmospheric pressure with a platinum black catalyst. The reaction stopped completely after about 0.5 moles of hydrogen had been absorbed. The solution was filtered from the catalyst and concentrated to a small volume. Methanol was then added until a copious precipitate appeared, which was then filtered and recrystallized several times from ethanol. The acetate gave a strong Liebermann test, and shoR7ed the presence of one double bond upon titrrhtion with perbenzoic acid. It melted at 105-106", [a],"+12.5" (24.0 mg., a 0.10'). -4nal. Calc'd for C80H3002: C, 81.39; H , 11.38. Found: C, 81.13; H , 11.48. a-Stellastenol. The saponification of a-stellastenyl acetate with a hot 5% solution of potassium hydroxide in 90% ethanol yielded a-stellastenol, which after several recrystallizations from methanol melted at 123-125', [a]: +19.8" (31.9 mg., 0.21').
+
+
+
288
WERNER BERGMANE AND H. A. STANSBURY
Anal. Calc'd for C28Hd~O: C , 83.92; H, 12.08. Found: C, 84.05; H, 12.08. a-Stellastenyl 3,6-dinitrobenzoate. A solution of 300 mg. of a-stellastenol and 1.2 g. of 3,5-dinitrobenzoyl chloride in 4 cc. of anhydrous pyridine was heated for three hours at 80-90". It was then poured into 20 cc. of ice-cold, 6 N sulfuric acid. The precipitate was filtered, washed with water and acetone, and recrystallized several times from a mixture of equal parts of ethyl acetate and absolute ethanol. The compound crystallized in plates and melted at 196.5-197.5'. Anal. Calc'd for Csd&8Nd&: C, 70.31; H, 8.33. CzsHroN,Oe: C , 70.67; H, 8.40. CssHseNzOe: C, 71.02; H, 8.61. Found: C, 70.70; H, 8.49. P-Stellastenyl acetate. A rapid stream of anhydrous hydrogen chloride was passed through an ice-cold solution of 1.7 g. of a-stellastenyl acetate in 100 cc. of chloroform. After eight hours the solution was washed thoroughly with a solution of sodium bicarbonate and water, dried over potassium carbonate and evaporated t o dryness in vacuo a t room temperature. The residual oil solidified upon rubbing with methanol. After several recrystallizations from absolute ethanol, the acetate melted a t 94-96', [a]: +19" (40mg., CY 0.25'). Anal. Calc'd for C ~ O H W O C,~81.39; : H , 11.38. Found: C, 80.95; H, 11.43. fi-Stellastenol. The saponification of the 0-acetate in the usual manner gave p-stellastenol, which crystallized from absolute ethanol in the form of short needles, m.p. 122-124", [ C$29.4' Y ] : (48.8 mg., a $0.48'). Stellastanyl acetate. A solution of 6-stellastenyl acetate in glacial acetic acid was hydrogenated with a platinum black catalyst at room temperature and atmospheric pressure until hydrogen was no longer absorbed. The reaction product which was isolated in the usual manner, melted a t 132-134", and gave a faint Liebermann test. A solution of 200 mg. of this material in 3 cc. of chloroform was treated with a mixture of 0.3 cc. of chloroform containing one drop of concentrated sulfuric acid and 0.3 cc. of acetic anhydride. After thirty minutes the solution had turned green. It was then washed successively with water, sodium bicarbonate solution, and water, decolorized with Norit and dried with sodium carbonate. The residue remaining after evaporation of the solvent was recrystallized several times from methanol. The acetate no longer gave a Liebermann reaction. It crystallized in the form of small needles and melted at 13&139", [ a ] : $13.5' (42.3 mg., a f0.19O). Anal. Calc'd for C ~ O H S ZC, O ~81.02; : H, 11.79. Found: C, 81.26; H, 11.96. Stellastanol. The saponification of the acetate described above gave a sterol which after several recrystallizations from methanol melted a t 1 4 3 O , [a]:: $22.0" (39.6 mg., a $0.29'). Anal. Calc'd for CZ8H600: C, 83.51; H, 12.53. Found: C , 82.21; H, 12.48. Like most other saturated sterols, stellastanol contains solvent of crystallization which is difficult to remove. Stellastanyl 3,6-dinitrobenzoate. This derivative was prepared in the usual manner. After three recrystallizations from a mixture of equal parts of ethyl acetate and absolute ethanol i t melted at 204-205'. Anal. Calc'd for C&5sN&: c, 70.44; H, 8.78. Found: C, 70.61; H, 8.88.
+
SUMMARY
The mixture of sterols which is obtained from the starfish, Asterias forbesi, has been reinvestigated. It wm found in accordance with the results of a pre-
STEBOLS OF STARFISH
289
vious investigation that the least soluble, highest-melting sterol shows close resemblance to the di-unsaturated stellasterol. It has been proposed to retain the name stellasterol for the di-unsaturated sterol present in the mixture, and to assign the name stellastenol t o the mono-unsaturated sterol. A complete separation of the sterols has not yet been effected. An explanation has been given for the discrepancies between the reported melting points of stellasteryl benzoate. It has been shown that the starfish sterols, unlike all other unsaturated principal sterols of animal origin which so far have been discovered, are of the order C2*,possess a slight positive rotation and are devoid of the usual 5,6double bond. It has been shown that the side chain of stellasterol from C-22 on is identical with that of ergosterol but for an opposite configuration at (3-24. It has been shown by indirect evidence that the starfish sterols possess a cyclic double bond a t C-8. Catalytic hydrogenation of a mixture of the steryl acetates gave the uniform a-stellastenyl acetate, which has a double bond in the 8,14position. Treatment of a-stellastenyl acetate with hydrochloric acid gave ,f-stellastenyl acetate, which upon catalytic hydrogenation gave stellastanyl acetate. Stellastertol is isomeric with ergostanol and campestanol. The significance of these observations has been discussed. NEW HAVEN,CONN. REFERENCES (1) KOSSEL AND EDLBACHER, 2. physiol. Chem., 94,277 (1915). (2) ]3ERGY.4NN, J . BiOl. Chem., 117, 777 (1937). (3) PAGE, J. B i d . Chem., 67, 471 (19%). (4) BERGNANN AND STANSBURY, J . OTg. Chem., 8, 283 (1943). (5) VALENTINEAND BERGMANN, J . Org. Chem., 6,452 (1941). (6) HAEUSSLER AND BRAUCHLI, Helv. Chim. Acta., 12, 187 (1929). (7) FERNBQLZ AND RUIGH,J. Am. Clacm. Soc., 63,1157 (1941). (8) BERNSTEIN AND WALLIS,J . Am. Chem. Soc., 61,2311 (1939). AND WETTER,2.physiol. Chem., 266, 33 (1938). (9) BOCK (10) BERGNANN, MCLEAN,AND LESTER,J. Org. Chem., 8,271 (1943). (11) BERGMANN, J. Biol. Chem., 104,553 (1934). (12) HEATH-BROWN, HEILBRON, AND JONES,J . Chem. Soc., 1482 (1940). AND RUIGH, J . Ant. Chem. Soc., 62, 2341 (1940). (13) FERNBOLZ (14) BRADY,J . Chem. Soc., 756 (1931).
