CONTRIBUTION FROM THE STERLING CHEMISTRY
LABORATORY OF
Y A L E UNIVERSITY]
T H E CHEMISTRY OF UNSATURATED STEROIDS. I. THE CONSTITUTION OF CHOLESTERILENE* HOMER E. STAVELY
AND
WERNER BERGMANN
Received December $1, 1956
I n recent years it has been 'shown that certain dehydrogenated steroids possess carcinogenic properties. For example, oestrone, a steroid containing one aromatic ring, has been found by Lacassagnel,to be definitely carcinogenic. I n order to discover whether the presence of any particular number and arrangement of double bonds will produce carcinogenicity in steroids an investigation of their stepwise dehydrogenation has been initiated. The simplest unsaturated hydrocarbons of the C27 series are cholestene and coprostene, which contain one double bond each in the A5 and A4 position respectively. At least one of them, cholestene,2 is not carcinogenic. Of the cholestadienes, several representatives have been described. The structure of all but one of these, 7-dehydro~holestene,~ is unknown. We have therefore begun our investigation with a study of the structure of the cholestadienes. In 1849 Zwenger4 found that the treatment of cholesterol with phosphoric acid yields a hydrocarbon, which however was not sufficiently characterized. I n later publications by various authors it has been shown that the elimination of water from cholesterol, either by direct or indirect methods, leads to a hydrocarbon containing two double bonds, which is generally known as cholesterilene. A summary of these methods is contained in Table I. With one exception (method 2) the melting point of cholesterilene has been found to be between 75" and 80". There is, however, no agreement as to the specific rotation. T'alues ranging from 1.45" to - 116.2' have been recorded. The elimination of one molecule of water from cholesterol might rea-
+
* Aided by a grant from the International Cancer Research Foundation. LACASSAGNE, Compt. rend., 196, 630-2 (1932); LACASSAGNE AND NYKA,Compt. rend. S O C . biol., 116, 844-5 (1934). 2 BARRY, COOKet al., Proc. Roy. SOC.(London), 117B, 345 (1935). DIMROTH AND TRAUTMANN, Ber., 69, 673 (1936). ZWENGER,Ann., 69, 347 (1849). 567 1
568
HOMER E. STAVELY AND WERNER BERGMA"
sonably lead to the formation of one or more of the three cholestadienes diagrammed below.
cholesterol
\/\ 2,5-cholestadiene
3,5-cholestadiene
2,4-cholestadiene
The formation of I11 would involve a shift of the double bond of cholesterol from the A5 to the A4 position. Such rearrangement, which involves the formation of a conjugated system of double bonds, occurs during the oxidation of cholesterol t o cholestenone. The best known cholesterilene is that obtained through dehydrogenation of cholesterol with anhydrous copper sulfate. In accord with previous investigators we have found that cholesterilene prepared by method 1 reactswith but one molecule of bromine,6 that it contains two double bonds, 8
MAUTHNER AND SUIDA, Monatsh., 17, 34 (1896).
569
CHEMISTRY O F UNSATURATED STEROIDS
NO.
__
1 2 3 4 5 6 7 8
9 10 11
12 13 14 15 16 17 18
-
METHOD
+ + +
M.P. I N
Cholesterol copper sulfate Cholesterol zinc dust (distilled) Cholesterol kieselguhr Cholesteryl chloride NaOCzHs Cholesteryl chloride calciumoxide Cholesteryl chloride quinoline Cholesteryl chloride zinc oxide Cholesteryl chloride potassium cholesterolate Cholesteryl bromide N a I in acetone Cholseteryl bromide NaI piperidine acetate in acetone Cholesteryl phenylurethane (distilled) Methylcholesterylxanthate (distilled) Cholesterol phosphoric acid Monocholesteryl phosphate (heated) Dicholesteryl phosphate (heated) Allo- or epiallocholesterol HCl Epicholesterol HCl Reduction of 7-ketocholesterilene semicarbazone
+
+ + + + + +
+
+
+
+
“c.
[alD
80 68, 75 79 79-80 79 77 79-80 79-80
- 104 +1.45--53 -47 -65.9 -61.55 -86.09 -116.2
77-78 78-79
-65.4 -103
75 79-80 79-80.5 76-78 78.2 80 76-77 78-79
-68.99 -77.53 -112 -78.3 -63.75
REFERENCE
5, 6 7 8 9, 5 10 11 11 11
12 12 13 14, 15, 16 4, 5 17 17 18 19
that it can be hydrogenated catalytically to produce cholestane as the principal product, together with a small amount of coprostane’t 16, 211 and that it is not reduced by the action of sodium in amyl The latter observation eliminates formula I11 from consideration, as it contains HEILBRON, MORTON AND SEXTON, J. Chem. Soe., 1928, 50. FANTL, Monatsh., 47, 256-7 (1926). * STEINKOPP, J. prakt. Chem., 100, 70 (1920). WALITSKY,J. russ. chem. Ges., 8 , 237. l o MAUTHNER AND SUIDA,Monatsh., 24, 666 (1903). l1 STEINKOPF AND BLUMNER, 1.prakt. Chem., 84, 466-7 (1911). n WAGNER-JAURREG . ~ N DWERNER,z. physiol. Chem., 213, 119 (1932). 1 3 B ~ 0 cBull. ~ , SOC. chim., 31, 73 (1904). l4 TSCHUGAEV AND GASTEFF, Ber., 42, 4633 (1902). TSCHUGAEV AND FOMIN, Ann., 376, 293 (1910). BOSEAND DORAN,J. Chem. soc., 1929, 2246. l i MULLER AND PAGE, J. Biol. Chem., 101, 128 (1933). SCHOENHEIMER AND EVANS, ibid., 114, 568 (1936). l g MARKER, KAMM,OAKWOOD AND LAUCIUS, J. Am. Chem. Soc., 68, 1950 (1936). zo HEILBRON, “Dictionary of Organic Compounds,” Oxford University Press, New York City, 1934, Vol. I, p, 337. 21 WINDAUSA N D SENG,Z. physiol. Chem., 117, 158 (1921). 22 HEILBRON, MORTON AND SEXTON, J. Chem. SOC.,1928, 48. THE JOURNAL OF ORQANIC CHEWISTRY. VOL.
1,
NO.
6
570
HOMER E. STAVELY AND WERNER B E R G M A "
a conjugated system of double bonds which should be easily reduced by this method. It does not, however, eliminate formula 11, whose conjugated double bonds are located in two different rings. Wagner-Jauregg12 has found that maleic anhydride does not add to cholesterilene in boiling toluene. We have found that under more drastic conditions, namely the heating of cholesterilene, maleic anhydride, and and xylene in a sealed tube for 12 hours a t 135", addition takes place. As yet we have been able to obtain the acid only as an amorphous white powder which decomposes from 240-245". Analyses indicate that this material has been formed from one molecule each of cholesterilene and maleic acid. The alkali salts of this substance are extremely insoluble, and in this respect differ from other known maleic acid addition products of steroids. For stereochemical reasons, normal 1,4-addition of maleic anhydride to a substance of formula I1 is inconceivable. If I1 is the correct structure for cholesterilene addition would have to take place in some unusual manner. Schoenheimer and Evans1*found that allo- and epiallocholesterol, when refluxed with dilute alcoholic hydrochloric acid, lose water rapidly to form a cholestadiene. This substance possessed a melting point and specific rotation similar to those of cholesterilene prepared by method 1. Since, however, the absorption spectrum was different from that given by Heilbrad for cholesterilene, the authors felt justified in denying the identity of these two dienes and in assigning structure 111to their compound. This substance seemed to be the logical dehydration product of allo- or epiallocholesterol.
-Hz0
allocholesterol
2,4-cholestadiene
However, when we studied the absorption spectrum* of cholesterilene prepared by method 1, we found it to have the same typical absorption maxima a t 229,235 and 244 mp as Schoenheimer and Evans' hydrocarbon, indicating the identity of these two cholestadienes. That this conclusion
* The authors are greatly indebted to Drs. Schoenheimer and Evans for measuring this and subsequent absorption spectra.
571
CHEMISTRY OF UNSATURATED STEROIDS
was correct we could further demonstrate by chemical evidence. Like cholesterilene, Schoenheimer and Evans' diene could not be reduced by sodium in amyl alcohol; cholestane and coprostane were the products of catalytic hydrogenation; and maleic anhydride was added in the same unusual manner. Schoenheimer and Evans' hydrocarbon therefore does not possess formula 111, but is identical with cholesterilene prepared by method 1. Of the remaining two possible formulas for cholesterilene, namely I and 11,I has been assigned to it by Heilbron,*Oalthough there is very little evidence to support this view. Because of its high absorption in the ultraviolet, which is indicative of a system of conjugated double bonds, we believe cholesterilene, prepared by method 1 or 16, to possess formula 11. I n order to obtain further evidence in support of formula I1 for cholesterilene we have prepared a cholestadiene by the Wolf-Kishner reduction of 7-ketocholesterilene1which was prepared by the oxidation of cholesteryl acetate with chromium trioxide, and subsequent loss of acetic
pi"7/ L
v
-+
/v\
AGO
cholesteryl acetate
vI/(Jy CH,I
--f
AcO
\ \
0
7-ketocholesteryl acetate
7-ketocholesterilene semicarbazone
CH,/
VII(y)(
\/ \/\
0
7-ketocholesterilene
3,5-cholestadiene
The cholestadiene obtained by this method melted a t 78-79' and showed Although the specific rotation of this substance is about 50" lower than that of cholesterilene prepared by method 1 or 16, its absorption spectrum is quite similar. On comparing the specific rotations given in Table I, one may separate the cholesterilenes into two groups. Substances prepared by the methods 4, 5 , 9, 14, and 18 have rotations between -60 and -70", while those obtained by methods 1 , 7 , 10, and 16 have a specific rotation greater than -100". Since a representative of one group possessed an absorption spectrum similar to that shown by a representative of the other, we are inclined to believe that members of [ a ]=~ -64".
P3
MAUTHNER AND SUIDA, Monatsh., 17, 496 (1896).
572
HOMER E. STAVELY AND WERNER BERGMANN
both groups contain the same 3,5-conjugated system. An investigation as to the cause of the differences in rotation is now under way. EXPERIMENTAL
Preparation of cholesteri1ene.-Cholesterol was dehydrated according to the method of Mauthner and Suida.5 After several recrystallizations from ether and ethanol the resulting hydrocarbon melted a t 78-79", and [CY]: = -97.5" (27.2 mg. in 3.04 cc. CHCl,, 1 dm. tube, CY = -0.88'). Properties of Cholesterilene Reaction with brornine.-when a solution of bromine in glacial acetic acid was added to a solution of cholesterilene in ethsr, decolorization took place until exactly one mole of bromine had been absorbed. The addition product could not be isolated in crystalline form. When two moles of bromine were added to a solution of cholesterilene a brown liquid was obtained from which even on long standing at low temperature no crystalline bromides separated. Attempts to recover cholesterilene from the bromides according to the method described by Schoenheimer24 gave only a black, tarry material. This observation is in disagreement with the statement of Marker's et al. that a tetrabromide of cholesterilene had been prepared. They gave no details concerning the preparation, properties, and composition of this tetrabromide. Titration with perbenzoic acid.-When treated with perbenzoic acid in the usual manner 0.192 gm. of cholesterilene absorbed 16.5 mg. of oxygen during 48 hours, an amount corresponding to 1.97 double bonds. Catalytic hydrogenation.-Cholesterilene was hydrogenated in ethyl acetate with platinum oxide as catalyst. The hydrogenated substance was then treated with acetic anhydride and sulfuric acid according to Anderson's26 method to remove any unsaturated material that might have escaped hydrogenation. The purified saturated material, on recrystallization from ethanol, crystallized in plates. It was identified as cholestane, m.p. 78-79", [CY]:= +22.5'. From the mother liquors the characteristic needles of coprostane could be isolated, m.p. 58-60" and [CY]:= +25.9". The yields were 80 per cent. cholestane and 20 per cent. coprostane. Reaction with maleic anhydride.-When cholesterilene and maleic anhydride were refluxed in benzene for several hours no reaction occurred. Four grams of cholesterilene and 2 g. of maleic anhydride were then dissolved in 15 CC. of xylene, and the mixture was heated in a sealed tube a t a temperature of 135" for 12 hours. After cooling, the contents of the tube were evaporated to dryness i n vacuo and the residue was refluxed with a solution of 3 g. of potassium hydroxide in 40 cc. of methanol for 3 hours. During the saponification a considerable amount of insoluble material was observed. A large volume of water failed to dissolve it. The suspension was extracted thrice with low-boiling petroleum ether to remove any uncondensed material. From the petroleum-ether extract 600 mg. of cholesterilene was recovered. The insoluble material was filtered, washed with water, and dried. It was a slightly yellow amorphous powder, insoluble in ether, alcohol, benzene, and water. On combustion an ash was obtained, indicating that the substance wasasalt. The free acid was prepared by heating the salt with glacial acetic acid a t a moderate temperature for a few minutes. Water was then added, and the precipitated acid 24 25
SCHOENHEIMER, J . Biol. Chem., 110, 461-462 (1935). ANDERSONA N D NAHENHAUER, J . Am. Chem. floc., 46, 1959 (1924).
CHEMISTRY OF UNSATURATED STEROIDS
573
was extracted with ether. After washing with water the ether extract was shaken vigorously with a concentrated aqueous solution of sodium bicarbonate, whereby the sodium salt of the acid was precipitated. The entire mixture was centrifuged, and the sodium salt, which formed a solid layer between the ether and water, was filtered, washed with water, ethanol, and ether, dried and extracted with ether in a Soxhlet apparatus. The salt was shaken vigorously with dilute hydrochloric acid and ether, until all liberated acid had been dissolved in the ether. After the ether layer had been removed, washed with water, dried and evaporated, a white amorphous material was obtained. This was redissolved in ether and was precipitated as a snow-white amorphous material by the addition of methanol. Further attempts to crystallize i t were unsuccessful. The material decomposed at 240-245'. A n d . Calc'd for CaiH4804: C, 76.80, H, 9.99. Found: C, 75.90; H, 10.11. Properties of Schoenheimer and Evans' Hydrocarbon Treatment with sodium in amyl alcohol.-To 1 g. of hydrocarbon dissolved in boiling amyl alcohol 15 g. of sodium was added over a period of four hours. The product had a m.p. of 80" and [ a ]=~ -111" (42.2 mg. in 3.04 cc. CHCla, 1dm. tube, 01 = -1.54'). No reduction had taken place. Catalytic reduction.-On hydrogenation with platinum the hydrocarbon gave approximately 85 per cent. cholestane (m.p. 76-78", and [a]: = f25.5') and 15 per cent. coprostane (m.p. 64-66" and [a]: = $24.6'). Reaction with maleic anhydride.-The hydrocarbon (4.4 9.) and maleic anhydride (2.4 g.) were treated in the manner described above. In addition to 1.9 g. of uncondensed material, 2.4 g. of an acid was obtained, which in every respect behaved like the acid obtained by the condensation of cholesterilene and maleic anhydride. It decomposed at 240-245'. rlnul. Calc'd for C ~ I H U OC, ~ : 76.80; H, 9.99. Found: C, 75.79; H, 10.14. Preparation of 3,6-Cholestadiene
7-Keto-b,6-choZestadiene.-Cholesteryl acetate was oxidized with chromium trioxide according to the directions given by Windaus.26 Five grams of 7-ketocholesteryl acetate was refluxed for one hour with 100 cc. of absolute ethanol containing 5 cc. of dilute hydrochloric acid. After cooling, the 7-ketocholesterilene was filtered, washed, and recrystallized twice from 95% ethanol. 7-Ketocholesterilene semicarbazone.-7-Ketocholesterilene was refluxed with twice the theoretical amount of semicarbaxide in alcohol for 24 hours. After cooling, the semicarbazone was filtered and recrystallized from chloroform and ethanol. It formed long soft yellow needles and melted a t 198-200". Anal. Calc'd for C2gHdsN30: C, 76.40; H, 10.56. Found: C, 76.47; H, 10.52. Reduction of the semicarbazone.-The semicarbazone was heated in a sealed tube with a solution of a n equal weight of sodium in ten times the amount of absolute ethanol for 8 hours a t 200". After cooling, the contents of the tube was dissolved in ether and water, the ether solution was washed with water, dried and evaporated to dryness. The oily residue was twice extracted with boiling absolute ethanol. A resinous substance which could not be obtained in a crystalline form remained. After addition of water to the alcoholic extracts crystals separated; these were recrystallized several times from ether and ethanol or ether and acetone; m.p. 78-79', z6
WINDAUS,LETTRI~ AND SCHENCK, Ann., 620, 102 (1935).
574
HOMER E. STAVELY AND WERNER BERGMANN
and [ C Y =]-63.75' : (0.0312 g. in 3.04 cc. CHCls, 1 dm. tube, of the hydrocarbon was rather small.
CY
= 65.03').
The yield
SUMMARY
1. A discussion of the literature on the dehydration products of cholesterol has been presented. 2. The cholestadiene of Schoenheimer and Evans has been shown to be identical with the cholesterilene of Mauthner and Suida. 3. 3,5-Cholestadiene has been prepared by the reduction of 7-ketocholesterilene. 4. Because of its great absorption in the ultraviolet, characteristic of conjugated systems, cholesterilene has been assigned the structure of 3,5-cholestadiene.
