[CONTRIBUTION FROM
THE
STERLING CHEMISTRY LABORATORY, YALEUNIVERSITY ]
T H E CHEMISTRY OF UNSATURATED STEROIDS. IV. THE PREPARATION AND PHOTOCHEMICAL OXIDATION O F 2,4-CHOLESTADIENE* EVALD L. SKAU
AND
WERNER BERGMANN
Received June $0, 1938
I n order to investigate the photochemical oxidation of 2,4-cholestadiene (I), it was necessary to prepare considerable quantities of this compound in a pure state. The procedure for the preparation of this hydrocarbon by the dehydration of cholesterol with aluminum oxide was described in a previous communication'. It was there pointed out that this method gave inconsistent results. In some instances the yield was very poor, and occasionally only cholesterol or a hydrocarbon of a negative rotation was obtained. An improved method has now been found by which 2,4-cholestadiene can always be obtained in good yields. The crude hydrocarbon prepared by the improved method invariably For the purification of the shows a specific rotation of +90 t o +100'. diene it was found necessary to modify the method of recrystallization recommended in our previous communication. The tendency of the partially purified hydrocarbon to revert to an oily material which would not crystallize indicated the possibility of a rearrangement or oxidation in solution. It was found that the purification of the hydrocarbon was best carried out by recrystallization from very small amounts of ether. This procedure was greatly facilitated by the use of an improved type of the centrifugal filtration tube.2 On repeated recrystallization from ether the specific rotation of the diene first rose rapidly to about +160' and then slowly to +168.5'. This value was not exceeded by further recrystallizations. The pure 2,4-cholestadiene crystallized in well-shaped, heavy blocks and melted sharply a t 68.5'. The absorption spectrum of the pure hydrocarbon showed maxima at 267mp and 275mp. It has thus been demonstrated that the sample of diene (m.p. 63O, [CY]:' = +114O) previously reported' was not pure. The comparison of the absorption spectrum of such an impure sample with that of the pure 2,4-cholestadiene
* Aided by grants from the International Cancer Research Foundation and The Jane Coffin Childs Memorial Fund for Medical Research. STAVELY AND BERGMANN, J. ORG. CHEM.,1, 576 (1936). SRAU,J. Phys. Chem., 33, 951 (1929); SKAVAND ROWE,I n d . Eng. Chem., Anal. Ed., 3, 147 (1931). 166
/I
0
OH
GIJ OH
167
vl~l
168
EVALD L. SKAU AND WERNER BERGMANN
(see Fig. 1) showed the presence of an impurity having an absorption maximum in the region of 230-240mp. The position of this maximum indicated that the impurity was a hydrocarbon possessing a pair of conjugated double bonds extending over two rings. Such a hydrocarbon has been isolated. (See Fig. 1). It seemed to be the main product when
4.c
2 i
p
3.c
L
4)
2
4
2.0
1.0 i '0
FIG.I. I. 2,4-Cholestadiene. 11. Impure 2,4-Cholestadiene, [a]== 111. Cholestadiene, [& = - 51".
l3SO
+ 114".
the dehydration of cho1este:ol was carried out a t a higher temperature and pressure than recommended for the preparation of 2 ,Ccholestadiene. The hydrocarbon melted a t 80.0-80.5", showed an optical rotation of [a]iO= -51.3' and had a maximum of absorption at 234mp.t
t The authors are greatly indebted t o the Misses Marshall and Paddock, Mount Holyoke College, South Hadley, Mass., for their measurements of the absorption spectra.
CHEMISTRY OF UNSATURATED STEROIDS
169
The carcinogenic activity of the 2 ,4-cholestadiene has been tested by Dr. Leone11 C. Strong of the Yale Medical School. Twenty mice of the Strong CBA strain were given six subcutaneous injections of a 1 per cent solution of 2,4-cholestadiene in sesame oil at weekly intervals. Each injection consisted of 0.2 cc. of the solution, so that each mouse received a total of 12 mg. of the hydrocarbon. The tests were begun twelve months ago. No obvious tumors have yet been observed, and all the mice are still in excellent health. Like ergosterol3 (11), 2,4-cholestadiene is easily photo-oxidized by bubbling oxygen through an alcoholic solution containing eosin during exposure to the light of a 200-watt bulb. The oxidation product is a crystalline peroxide of the melting point 113-114" and specific rotation 48.3'. If this compound is repeatedly recrystallized from ethyl acetate a slow rise of the melting point and of the specific rotation is observed. This phenomenon is due to the tendency of the peroxide to undergo a gradual rearrangement, the exact nature of which has not yet been fully established. The previously reported sample of the peroxide4, melting over a range of 118.5-120.5" and having a specific rotation of +52.8", had already undergone partial transformation of that type. The peroxidic nature of the pure 2 ,Pcholestadiene peroxide of the m.p. 113-114' is evidenced by the fact that it liberates an exactly equimolecular quantity of iodine from a solution of potassium iodide in glacial acetic acid. The readiness with which 2,4-cholestadiene adds one mole of oxygen indicates that the reactive system of two conjugated double bonds in one ring is very favorable to the formation of peroxides. It also shows that a pair of conjugated double bonds, each of which is connected with a quaternary carbon atom, as in ergosterol (11)) is not a prerequisite for the formation of peroxides in the steroid series. The addition of one mole of oxygen to 2,4-cholestadiene might conceivably lead to one of three peroxides (structures 111-V). On catalytic hydrogenation the peroxide adds two moles of hydrogen to give a saturated diol, C2,H4802,having one of the structures VI-VIII. On acetylation with acetic anhydride the diol gives a monoacetate, indicating the presence of an unreactive tertiary hydroxyl group a t Cg. This observation eliminates structure V I 1 for the diol and structure IV for the peroxide. Of the remaining two possible structures for the diol one (VI) represents a glycol. According to Criegee5 such a substance would be expected to react with lead tetraacetate. The diol obtained on hydrogenation of the peroxide, however, gives absolutely no reaction with this reagent. Conse-
+
3
WINDAUS AND BRUNKEN, Ann., 460, 225 (1928).
* SKAUAND BERGMANN, J . Am. 6
CRIEGEE, Ber., 64, 264 (1931).
Chem. Xoc., 60, 986 (1938).
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EVALD L. SKAU AND WERNER BERGMANN
quently structures V I for the diol and I11 for the peroxide can also be eliminated. The peroxide of 2 ,Pcholestadiene therefore must be 2,5peroxidocholestene-3 (V), and its hydrogenation product a 2,5-dihydroxycompound having structure VIII. An interesting rearrangement takes place when 2,4-cholestadiene peroxide of m.p. 113-114" in alcoholic solution is exposed to sunlight. The irradiation product melts a t 166-168" and has a specific rotation of +141". The same product can be obtained directly from 2,4-cholestadiene if the photo-oxidation of this substance is carried out in sunlight instead of in the light of a 200-watt bulb. This compound seems to be identical with the "2,4-cholest,adiene peroxide" recently described by Butenandt and Kudssus6. It is uncertain, however, that this compound is a true peroxide, because it does not liberate iodine from a solution of potassium iodide in glacial acetic acid. Further studies on the irradiation products of 2,4-cholestadiene are in progress. The carcinogenic activity of the various compounds is being tested by Dr. Leone11 C. Strong of the Yale Medical School. EXPERIMENTAL
Dehydration of cholesterol with aluminum oxide.-The aluminum oxide used for the dehydration of cholesterol was "Activated Alumina, Grade A, 40 to SO mesh," obtained from the Aluminum Ore Co. It was found t o be advantageous t o reactivate it immediately before use by heating it in a shallow pan at 200" for four hours and then cooling it in the absence of moisture. Ten grams of cholesterol were melted and allowed t o solidfy in a 125-cc. Pyrex retort. A mixture of 20 g. of cholesterol and 34 g. of alumina was then introduced, and a plug of glass wool was inserted into the curved part of the neck. After evacuation to 1 mm. pressure the retort was heated on a metal bath at 220-230". The heating was continued until most of the cholesterol had reacted. When this stage was reached the refluxing droplets remained glassy-clear when they were cooled locally by a jet of air and allowed to come back to the original temperature. Depending on the activity of the alumina, from one to six hours of heating was required. The retort was then removed from the bath, and cooled rapidly with a stream of air. After the vacuum had been broken the glass wool was pushed into the retort, and 10 g. of alumina was introduced. The heating was now continued as before for thirty minutes more. The retort was then lowered until the bath level was a few mm. above the level of the reaction mixture and covered by a metallic hood, which was heated gently from time t o time. An ordinary inverted tin can was used for this purpose. Distillation was carried out with the bath temperature as low as possible, preferably not exceeding 230". It is advantageous t o use a mercury vapor diffusion pump during distillation. The distillation product was a clear oily liquid which slowly crystallized. It was always obtained in yields from 70 to 75 per cent., and had a specific rotation of +90 t o +loo". The crude product was recrystallized from small amounts of ether with the help of a slightly modified centrifugal filtration tube designed by one of us. 6
BUTENANDT AND KUDSSUS,2. physiol. Chem., 263, I (1938).
CHEMISTRY OF UNSATURATED STEROIDS
171
Modified centrifugal filtration tube.-The centrifugal filtration tube (see Fig. 2) consists of the crystallization chamber, A , and the receiver of the mother liquor, B. The parts are connected by an interchangeable ground joint. A perforated porcelain disc, C , is ground into the ground joint of B , deep enough so as to permit a tight connection between the two chambers. In order to facilitate the removal of the disc, a nichrome wire, D , is attached to it. For the filtration the disc is covered tightly by a piece of filter paper which has been slit where the wire is fastened to the disc. Purification of I ,.&cholestadiene.-The crude distillation product was transferred into the crystallization chamber and dissolved in half its weight of ether. The sides of the chamber were washed down with a few drops of ether which thus formed a separate layer on top of the solution. The tube was then assembled and allowed to cool very slowly to 0", when the hydrocarbon crystallized out in large crystals. The tube was now inverted, placed in a centrifuge cups and centrifuged for twenty
FIG.I1 minutes a t a speed not exceeding 1100 r.p.m. The crystals were then removed by carefully withdrawing the disc. This procedure was then repeated two or three times until the specific rotation of the crystals was above +150". Eight parts of the hydrocarbon of such a degree of purity were recrystallized from ten parts of ether, and the procedure was continued until a product of a specific rotation above + E O " was obtained. This product is suitable for general use. By continuing this procedure pure 2,4-cholestadiene was prepared, which melted sharply a t 68.5"; [a]: = +168.5" (45.0 mg. in 3.04 ec. ether, 1 dm. tube). Anal. Calc'd for CP~HII:C, 87.96;H, 12.04. Found: C,88.03; H, 11.88. The mother liquors from the various recrystallizations contained considerable quantities of 2,4-cholestadiene which were recovered by one of two methods. They $ Tubular pieces of wood are used to adapt the filtration tubes to the size of the centrifuge cups.
172
EVALD L. SKAU AND WERNER BERGMANN
were evaporated to dryness in a stream of nitrogen, or were centrifuged, as described above, after cooling slowly to -78" in a large test tube immersed in a carbon dioxidealcohol bath. The recovered material was then again subjected to fractional crystallizations from small amounts of ether. Inasmuch as the diene, when dissolved in ether, is prone to undergo oxidation it is advisable to work up the mother liquors immediately. Subjected t o this procedure, 180 g. of cholesterol yielded 52 g. of 2,4-cholestadiene of a specific rotation over $160". This yield could be further improved by a systematic recrystallization of the residues. Titration w'th perbenzoic acid.-By titration with perbenzoic acid in the usual manner, 71.0 mg. of diene took up 6.22 mg. of oxygen in 72 hours, an amount corresponding t o 2.02 double bonds. Crystallography and optical pvoperties of 8,4-cholestadiene [ B y W . E . Ford and J . P . Sickels].--2,4-Cholestadiene is monoclinic, the crystals being elongated parallel to the ortho axis and showing a prismatic development. The principal forms have been taken as (100) and {OOl); in addition there are smaller faces of the unit prism (1101 and pyramid (111);the orthodome (201} occurs as a very narrow truncation. The crystal faces were not of such a quality as to permit of accurate measurement of the interfacial angles. The approximate angles were as follows: (100) A (001) = 75"45',
FIG. I11 (100) A (110) = 60°, (001) A (111) = 51"30', (001) A (201) = 46"45'. From these angles the following approximate crystal constants were derived by plotting methods: a : b:c, 1.78:1:1.34; p = 75'45'. Figure 3, drawn by Charles M. Warren, represents one habit of crystal development. Another habit was observed in which the prism-like crystals were terminated by prominent faces of the unit pyramid. Two cleavages were observed, parallel to (loo} and (Ool]. The substance is biaxial, optically negative. The optical axial plane is parallel t o the 010 plane. Cleavage plates parallel to (100) show the emergence of an optic axis near the center of the microscope field, while cleavage plates parallel t o (001) show an optic axis emerging near the edge of the field. The optic angle 2V is very large. The substance shows strong dispersion, r > u. The indi&s of refraction were not measured since the material is soluble in the usual immersion media. Isolation of cholestadiene, [a]: = -bl".-The dehydration of cholesterol with activated alumina was carried out as described above. The product was then distilled at 2 mm. pressure and at 290 to 315". The distillate was recrystallized from an equal weight of ether in a centrifugal filtration tube. After one recrystallization the hydrocarbon had a specific rotation of -38" and after four additional recrystallizations it melted a t 80.0-80.5", [a]: = -51.3" (47.0 mg. in 3.04 cc. ether, 1 dm. tube).
CHEMISTRY OF UNSATURATED STEROIDS
173
Titration rdth perbenzoic acid.-By titration with perbenzoic acid in the usual manner, 95.7 mg. of diene took up 8.56 mg. of oxygen in 72 hours, an amount corresponding t o 1.99 double bonds. Photo-oxidation of 8,4-cholestadiene.-Nine grams of the diene, [ a b = +164", was dissolved in one liter of warm absolute alcohol, and 14 mg. of eosin was added to the solution. The photo-oxidation was then carried out at 25" in the apparatus described by Windaus and Brunkena. A 200-watt Mazda bulb was used as a source of light. After nine hours of irradiation the solution was evaporated to dryness in vacuo below 35". The crystalline residue after one recrystallization from dilute acetone yielded a crude peroxide of m.p. 110-112". One further recrystallization from dilute acetone and three recrystallizations from 95 per cent. alcohol brought the melting point to 113-114", [a]: = +48.3" (63.2mg. in 3.06 cc. CHCl,, 1 dm. tube). Additional quantities could be obtained from The yield of peroxide was 60-70'%. the mother liquors. C, 80.93; H, 11.08. Anal. Calc'd for C~YHUOZ: Found: C, 81.15; H, 10.94. Determination of active ozygen.-A sample of the peroxide was introduced into a glass-stoppered bottle, and 20 cc. of a saturated solution of potassium iodide in glacial acetic acid was added. The mixture was kept in the dark at room temperature for 24 hours. The liberated iodine was then titrated with N/10 thiosulfate solution. A sample containing 54.1 mg. of 2,4-cholestadiene peroxide required 6.51 cc., and the blank, 3.71 cc. of 0.0990 N thiosulfate solution. The difference corresponded to 1.03 atoms of active oxygen. The corresponding values for 31 mg. of ergosterol peroxide were 5.70 CC. and 3.71 cc., or 0.96 atoms of active oxygen. Catalytic hydrogenation.-One gram of peroxide was dissolved in 75 cc. of ethyl acetate and shaken with platinum catalyst, prepared from 150 mg. of PtOz, in an atmosphere of hydrogen. After two hours, 2 moles of hydrogen had been absorbed. The solution was then filtered and evaporated to dryness i n vacuo. The residue was recrystallized several times from acetone. The diol crystallized in long silky needles, and melted a t 155O, [a]: = +19.6" (34.2mg. in 3.04 CC. CHCb, 1 dm. tube). The substance did not react with perbenzoic acid, and i t distilled without decomposition in a high vacuum. Anal. Calc'd for C~rHtsOl:C, 80.12; H, 11.97. Found: C, 80.24;H, 12.30. Titration with lead tetraacetate.-Ten cc. of a saturated solution of lead tetraacetate in glacial acetic acid was added to 26 mg. of the diol. After 20 hours the excess of lead tetraacetate was determined by the method described by Criegee6. The solution containing the sample used 14.59 cc. of N/10 thiosulfate solution, and two blanks 14.60 and 14.61 cc. respectively. No reaction had taken place. Acetylation of the diol.-The monoacetateTwas prepared by refluxing the diol with acetic anhydride for 90 minutes. It was recrystallized several times from acetone. The acetate crystallized in the form of small needles, and melted a t 141142", [a]! = -9" (27.6mg. in 3.0 cc. ether, 1 dm. tube). Anal. Calc'd for C * ~ H S ~ C, O ~77.96; : H, 11.30. Found: C, 78.04;H, 11.44. On saponification of the acetate the diol of m.p. 155" was recovered. Photochemical rearrangement of 8,4-cholestodiene peron'de.-One part of the peroxide, m.p. 113-114', was dissolved in 250 parts of absolute alcohol in a large Erlenmeyer flask, and 0.005 part of eosin was added. The solution was then exposed to sunlight in the open air. After a few hours of exposure the solution had become colorless, and after 2 days dense crystals began to separate. One week later the crystalline material was filtered off, washed with small amounts of absolute alcohol,
174
EVALD L. SKAU AND WERNER B E R G M A "
and recrystallized-from boiling absolute alcohol. The material melted a t 160-168", [a]: = +141° (30.0 mg. in 3.06 cc. CHCla, 1 dm. tube). The same substance ws8 obtained in almost quantitative yield when the irradiation was carried out in the absence of eosin and oxygen. Sunlight irradiation of %,4-cholestadiene.-2,4-Choleertrdiene, [ a ] = + l a " , was subjected to irradiation in sunlight, using the same proportions as in the case of the peroxide. After half a day the solution had become colorless, and after 3 days dense crystals began to separate in a considerable quantity. Two days later they were collected by filtration, washed with absolute alcohol, and recrystallized from boiling absolute alcohol. The material melted a t 168", [a]: = +140° (30.0 mg. in 3.04 cc. CHCla, 1 dm. tube). I t was identical with the product obtained by irradiation of the peroxide. Anal. Calc'd for C ~ ~ H U OC, , : 80.93; H, 11.08. Found: C, 81.13; H, 11.05. SUMMARY
1. An improved method for the preparation and purification of 2,4cholestadiene has been presented. Pure 2 ,4-cholestadiene melts at 68.5', and has a specific rotation of +168.5'. 2. 2 ,4-Cholestadiene is photo-oxidized in an alcoholic solution containing eosin during exposure to the light of a 200-watt bulb. The photooxidation product was identified as 2 ,5-peroxidocholestene-3. 3. 2,4-Cholestadiene peroxide rearranges during exposure to sunlight to a compound of m.p. 166-168' and specific rotation +141'.
