INVESTIGATIONS ON STEROIDS I. 6-OXOPROGESTERONE AND

[CONTRIBUTION FROM THE GEORQE 5. Cox MEDICALRESEARCH INSTITUTE, UNIVERSITY OF PENNSYLYANIA, PHILADELPHIA, PA.]

INVESTIGATIONS ON STEROIDS I. 6-OXOPROGESTERONE AND T H E STEREOCHEMICAL CONFIGURATION OF SEVERAL 3,5 ,&TRIOLS* MAXIMILIAN EHRENSTEIN Received July $4 1999

The physiologically active compounds of the adrenal cortex are chemically derived from progesterone (I). Recently progesterone has also been found in the adrenal cortex‘. It has been stated and confirmed in the recent physiological literature that it possesses, at least to a limited extent, the life-maintaining property of the “cortin” compounds. Desoxycorticosterone (11) is a t present in many respects the most potent crystalline hormone2with “cortin” activity. It manifests to a certain degree also a progestational actiona. It therefore follows that the introduction in the side-chain of progesterone (I) of an alcoholic hydroxyl group, such as appears in the structure of desoxycorticosterone (21-hydroxyprogesterone) (11)) is connected with a great reduction of progestational activity and a considerable increase of “cortin” action. 20

20

21

21

CO-CH,

// 0

/ 4

5

v 6

I. Progesterone

0

4

6

11. Desoxycorticosterone (21-Hydr oxypr ogesterone)

* Presented before the Division of Medicinal Chemistry a t the Boston meeting of the American Chemical Society, September 12, 1939. Aided by grants from the Smith, Kline, and French Laboratories in Philadelphia, the Rockefeller Foundation in New York, and the van’t Hoff-Fonds of the Royal Academy of Sciences in Amsterdam. 1 BEALL AND REICHSTEIN, Nature, 143,479 (1938). x REICHSTEIN AND V. Euw, Helv. Chim.Act., 21, 1197 (1938). a VAN HEUVERSWYN, COLLINS,WILLIAMS,AND GARDNER, Proc. SOC.Exptl. Biol. Medicine, 41, 552 (1939) (contains other references). 506

INVESTIQATIONS ON STEROIDS

507

There occur other active compounds in the adrenal cortex which contain, besides the fundamental structure of desoxycorticosterone, additional oxygen atoms in the nucleus at carbon atom 11 or carbon atoms 11 and 17. I n all these compounds) however, the characteristic keto1 arrangement of the side-chain is present. No systematic attempts have been made to investigate compounds which represent progesterones oxygenated only in the ring system and therefore containing an intact methyl ketone side-chain. Reference was recently made in a brief report4 of 12-hydroxyprogesterone, which was obtained from desoxycholic acid. This compound is said to possess no remarkable progestational activity. Apparently i t has not been examined for “cortin” action. With the idea of preparing for physiological examination a series of progesterones which are oxygenated in the nucleus, it was decided to undertake the preparation of 6-oxoprogesterone (XIV) which has not yet been described in the literature. Analogous experiments have been carried out, however, in the androstane series. Utilizing a method which was developed by Mauthner and Suidab on cholesterol, Butenandt and Riege16prepared 6-oxotestosterone acetate and 4-androstene-3 ,6,17-trione (V) by oxidizing 5-androstene-3,17-dioI 17-monoacetate and dehydroisoandrosterone (111) respectively with chromic acid in glacial acetic acid. Their yields of these compounds, which crystallize in yellow prisms, were 20 per cent. and 25 per cent. respectively. Both yellow compounds show an absorption maximum a t 252 mp which agrees with that of 4-cholestene3,6-dione. The physiological examination of these two substances with oxo groups a t carbon atom 6 indicated oestrogenic activity, whereas they possessed practically no androgenic property. This demonstrated that the introduction of a second oxo group in conjugation with the double bond may greatly change the physiological character of compounds of this type. Besides these yellow substances, Butenandt and Riegel obtained in each instance traces of a white crystalline substance which they considered to be a hydrate of the respective 6-oxo compound (as in formula VI). A definite proof for the structure of these hydrates was not given. Ouchakov and Lutenberg’ subjected dehydroisoandrosterone (111) to a seriee of transformations a t the end of which they arrived a t a compound which is presumably identical with Butenandt and Riegel’s 4-androstene3 6 ,3 7-trione (V). The melting point of one of the intermediates of this Preparation indicated that it was probably identical with the by-product (VI) which was obtained by Butenandt and Riegel when they subjected )

EHRHART, RUSCHIO, AND A U M ~ L L EAngewandte R, Chemie, 62, 363 (1939). MAUTHNERAND SUIDA,Monatsh, 17,584 (1896). BUTENANDT AND RIEGEL, Ber., 69, 1163 (1936). 7 OUCHAKOV AND LUTENBERO, Bull. S O C . chim., [51, 4, 1394 (1937).

4

5

508

MAXIMILIAN EHRENSTEIN

dehydroisoandrosterone (111) to a chromic acid oxidation. A direct comparison was not made, however. Since the data of the paper of Ouchakov and Lutenberg are not complete, a re-investigation of the various oxidations of dehydroisoandrosterone was made. In agreement with Butenandt and Riegel’s statements, the white by-product (VI) of the chromic acid oxidation of dehydroisoandrosterone was easily isolated. The same compound could be obtained by first treating dehydroisoandrosterone acetate (IV) with 30 per cent. hydrogen peroxide according to Miescher and Fischer*, and then oxidizing the 3,5,6-triol (VII) with chromic acid according to Ouchakov and LutenbergS The melting point of a mixture showed no depression; in addition, the optical rotation of Ouchakov and Lutenberg’s substance was in fair agreement with that given by Butenandt and Riegel. The identity of the two substances is therefore definitely established. When two hydroxyl groups are added to an alicyclic double bond by means of Criegee’s osmic acid methodi0 the intermediary formation of a cyclic ester of osmic acid fixes the hydroxyl groups in the cis position: 0

0

\ / os / \

0

HC-CH

/

\

+

I

C-C

0 5 0 4 -+

/i

0

I

i\

H

OH

--+

1

CC -& -

/;

H

OH

1

i\

H

H

In a brief communicationi1 without experimental details Ushakov and Lutenberg stated that they subjected dehydroisoandrosterone (111) to this treatment and obtained a 3,5,6-triol (VIII) which is different from the above-mentioned compound which would therefore appear to be the 5,6-truns compound (VII). It was interesting to determine in the 5,6-cis and 5,6-truns forms the actual configuration of the hydroxyl groups at carbon atoms 5 and 6. It was established that the configuration a t carbon atom 5 is the same in both triols because the chromic acid oxidations of both substances (VI1 and VIII) furnished the identical androstane-3,6,17-triond-o1 (VI).

* MIESCI~ER AND FISCHER, Helu. Chim. Acta, 21,353 (1938). OUCHAKOV AND LUTENBERQ, Bull. S O C . chim., [5],4,1397 (1937). CRIEQEE,Ann., 632,75 (1936). 11 USHAEOV AND LUTENBERQ, Nature, 140, 466 (1937). 9

10

509

INVESTIGATIONS ON STEROIDS

0

CHs II

111. R = H , Dehydroisoandrosterone Dehydroisoandrosterone IV. R = -CO-CHs, acetate

v /E h

RO

4

6

I11

1 HC1 (Ouohakov & Lutenbere)'-)

\

\

0

-\.

0 VI. Androstane-3,6,17-trion-5-01 .p

-

V. 4-Androstene-3,6,17-trione

\

\

\

\

OH VII. Androstan-17-one3 (8),5,6(tram)-triol

0

0

OH VIII. Androstan-17-one3 ( 8 ),5,6(&)-triol

The assignment of a definite configuration to these compounds at carbon atom 5 would automatically fix the configuration a t carbon atom 6. I n this respect observations of Butenandt, Schmidt-Thorn$ and P a u P and of Westphal, Wang, and Hellmann'3 should be mentioned. In those I* 1)

BUTBNANDT, SCHMIDT-THOME, AND PAUL,Ber., 72, 1114 (1939). WESTPHAL,WANQAND HELLMAN, Ber., 72,1235 (1939).

510


derivatives of dehydroisoandrosterone in which the hydroxyl group at carbon atom 3 is esterified (for instance acetylated), the 5:g-double bond is inert to the addition of osmic acid. Since the hydroxyl group at carbon atom 3 in these compounds is in the B position, esterification of this hydroxyl group should sterically hinder the addition of osmic acid provided the latter leads to a cis linkage of rings A and B.

A

/ A \C& / \I/

/

/ A \CH3/

B \

\/

I

--+

”\ 4

/5\

\/ 4

i6

H

/

\

OH / :6

H

I n the case of the 5,6-trans compound the following configuration might be assigned:

4

:I3

OH Since the configuration of these compounds at carbon atom 5 cannot be considered finally proven, their present nomenclature as androstane derivatives will not be changed. If the configuration is actually that of the above scheme, all compounds of this series ought to be labeled as etiocholane derivatives. Analogous experiments, as described above, have been performed on 4-pregnen-20-on-3-01 (IX). This compound gave with chromic acid in glacial acetic acid a fair yield of a white substance which proved to be pregnane-3,6 ,20-trion-501 (XIII)**. The 6-oxoprogesterone may per-

** This name is used for a cis linkage of rings A and B. A proof of a different configuration a t carbon atom 5 would change the names of the compounds of this group only in that the prefix “allo” would have to be added.

511


haps be present in the yellow mother liquors; thus far it has not been possible to secure from the latter the desired substance. The hydrogen peroxide oxidation of the acetate of 5-pregnen-20-on-3-01 (X) leads to a 20

21

IX. R = H, 5-Pregnen-20-on-3-01 X. R = -CO-CH8, 5-Pregnen-20-on-3-01acetate

RO IX

20

21

20

\

\

0 XIII. Pregnane-3 ,6,20-trion-5-01

% '

CO-CHs

X

21

0

\

XIV. 4-Pregnene-3,6,2O-trione [6-Oxoprogesterone]

\\

20

a1

CO-CH,

HlOz Saponifiwtio?

HO

4OHi

OH XI. Pregnan-20-one-3(,9) ,5,6(trans)-triol

HO

40H OH

XII. Pregnan-LO-one-3@) ,5,6(cis)-triol

pregnan-20-one-3,5,6-triol(XI) with the hydroxyl groups a t carbon atoms 5 and 6 in trans position. On the other hand, when 5-pregnen-20-on-3-01 (IX) was treated with osmic acid the corresponding pregnan-20-one-3,5,6triol (XII) with the hydroxyl groups a t carbon atoms 5 and 6 in cis position

512


was obtained. Both 3 ,5 ,6-triols yielded the same pregnane3 ,6 ,20-trion5-01 (XIII) when they were subjected to a chromic acid oxidation. The trione (XIII) also proved to be identical with the compound obtained by directly oxidizing 5-pregnen-20-on-3-01 (IX) with chromic acid. The production of an identical trione from either X I or XI1 proves that the two triols have the identical configuration at carbon atom 5. Since 5pregnen-20-on-3-01 (IX) has the same p configuration at carbon atom 3 as dehydroisoandrosterone it was interesting to learn whether its acetate was also inert towards osmic acid. It was, indeed, not possible to isolate any pregnan-20-one-3,5 ,6-triol; the reaction product seemed to consist

xA x

10-1

FIQ.1. ABSORPTION CURvE: OF 6-OXOPROGESTERONE log

(F)

(IN

ABSOLUTEALCOHOL)

= Kcl

io,incident light intensity; i transmitted light intensity; coefficient; c, concentration; I , cell length.

K,specific extinction

to a large extent of unchanged starting material. This is another instancel2,l3 in which the esterification of a hydroxyl group in the p position at carbon atom 3 makes the double bond between carbon atoms 5 and 6 inert to the addition of osmic acid. The same steric considerations outlined above with the androstan-17-one-3,5 ,6-triols (VI1 and VIII) can therefore also be applied here. If there actually is a cis linkage of rings A and B the labeling of these compounds as pregnane rather than allopregnane derivatives would be justified.*** *** When this paper was in press ELLIS AND PETROW, J . Chem. Soe., 1939,1078, reported on the stereochemical configurations of the cholestane-3,5,6-triols. The stereochemical considerations of the present paper are not a t variance with the conclusions of the English authors concerning the cholestane series.


513

When a chloroform solution of pregnane-3,6,2O-trion-5-01(XIII) was subjected to a stream of dry hydrochloric acid gas it was dehydrated to 4-pregnene-3,6,2O-trione [6-oxoprogesterone] (XIV). The absorption cuwe (Figure 1) of this compound was kindly furnished by Dr. G. 0. Langstroth of the Department of Physics of the Massachusetts Institute of Technology. I n preliminary testst on two mice, each given 4 X 500 y, there was a moderate oestrogenic reaction. In comparison, Korenchevsky and Hall" obtained no oestrogenic action upon the vagina with 500 y of pure progesterone per day. The substance waa tested for progestational activity by the CornerAllen technique. A total dose of 5 milligrams failed to produce any reaction, whereas a total dosage of 1 milligram of progesterone produces a strong response by the same method. EXPERIMENTAL$

Androstane-S,d, 17-trion-6-01 ( V I ) from dehydroisoandrosterone (III).-A. B y direct chromic acid ozidation of dehydroisoandrosterone (111). l L T w o hundred milligrams of dehydroisoandrosterone (111) was dissolved in 6 cc. of glacial acetic acid; thereafter a solution of 280 mg. of chromium trioxide dissolved in a few drops of water was added; 3 cc. of glacial acetic acid was used for rinsing. The mixture was shaken for seven hours a t room temperature (29'). Thereafter it was diluted with water and extracted several times with ample quantities of ether. After the washing of the combined ether phases with 2N sodium carbonate and water, followed by drying over anhydrous sodium sulfate, the ether was removed. The yellow, partly resinous and partly crystalline residue was extracted twice with benzene a t room temperature and once on the water bath. After the removal of the solvent i n vacuo a yellow, largely resinous, but somewhat crystalline residue was obtained. It waa treated with some 95% alcohol a t room temperature, whereby some white material remained undissolved. The white precipitate was washed several times by decantation with fresh alcohol. On concentrating the combined alcoholic extracts a white crystalline substance (8.1 mg.) was secured. All the white material was combined and recrystallized from 95% alcohol. It crystallized as flat prisms in rosette arrangement. A total yield of 4.8 mg. of this desired substance was obtained. The melting point was 248-249' which is in agreement with the statements of Butenandt and Riegel". The latter give as optical rotation [a]: 54.6' (in acetone).

+

t The author is grateful to Dr. Franklin Payne and Dr. Carl Bachman for conducting the oestrin and progestin assays respectively. 14 KQRIBNCBEVSKY AND HALL,Nature, 140,154 (1937). 1The dehydro-iso-androsterone and pregnenonol were kindly furnished by Dr. Erwin Schwenk of the Schering Corporation in Bloomfield, N. J. All melting points were determined with the Fisher-Johns Melting Point apparatus of the Fisher Scientific Company. The readings are sufficiently near the true melting points so that no corrections have been made. All micro-analyses were carried out by Dr. Ing. A. Schoeller, Berlin-Schmargendorf. 1s See BUTENANDT AND RIEGEL,Ber., 69,1167 (1936).

514

MAXIMILIAN ERRENSTEIN

No attempt was made t o isolate the main product of the chromic acid oxidation, 4-androstene-3,6,17-trione(V). B . From dehydroisoandrosterone acetate (IV) by way of androstan-l7-0ne-d(j3),6,6(trans)-triol ( V I I ) . The acetate of dehydroisoandrosterone (IV) was obtained by treating 250 mg. ol dehydroisoandrosterone (111) with 1 cc. of pyridine and 1 cc. of acetic anhydride overnight at room temperature. After the mixture has been poured into water the crude acetate was recrystallized from aqueous alcohol; yield, 239.6 mg. ; melting point, 171-172". Androstan-17-one-d@) ,6,6(trans)-triol (VII)n.-To a solution of 200 mg. of dehydroisoandrosterone acetate (IV) in l cc. of glacial acetic acid was added 0.2 cc. of 30% hydrogen peroxide. The mixture was heated on the water bath for two hours. The major part of the acetic acid was removed i n vacuo (45'). The residue was a light-colored resin which was saponified with 5 cc. of 5% methanolic potassium hydroxide for 7$ hours on the water bath. Thereafter water was added, and the methanol was removed in vacuo. To the aqueous residue was added sodium chloride; this mixture was extracted several times with ample quantities of hot chloroform. The combined chloroform extracts were brought to dryness in vacuo; they left a preponderantly white residue which was recrystallized from acetone, yielding colorless prisms. Melting point of the first crop (48.7mg.):295298"with decomp. Later crops (18.0 mg.) of somewhat lower melting points could be secured. Androstane-S,6,17-trion-6-oZ ( V I )9.-Fifty milligrams of androstan-17-one-3@),5,6 (trans)-triol (VII) was dissolved in 4 cc. of glacial'acetic acid on a water bath. After the solution had reached room temperature (29") 37.5 mg. of chromium trioxide, dissolved in a mixture of a trace of water and 1.5cc. of glacial acetic acid, was added. After standing about 22 hours at room temperature, 2 cc. of ethyl alcohol was added, and the mixture was then concentrated to a low volume in vacuo (45'). During concentration a precipitate of glistening crystals appeared. After the addition of water, the crystals were collected by filtration and washed with water; yield of crude crystalline material: 29.2 mg., melting point 238.5242'. After recrystallizing from 95% alcohol 20.1 mg. of flat crystals i n rosettes were obtained. The melting point was 249.5-250.5" (decomp.). The melting point of a mixture with the substance obtained by directly oxidizing dehydroisoandrosterone with chromium trioxide" was 249.5251' (decomp.). From the mother liquors was secured 3.9 mg. of crystals withamelting point of 247-248.5'; CY]^^+ 62.2' (4.5mg. in2.0cc. acetone). (The experimental error of this determination is rather large.) A n a l . Calc'd for ClaHzeOc:C, 71.65;H, 8.23. Found: C, 71.33,71.30; H, 8.11,8.03. C . From dehydroisoandrosterone (111)by way of androstan-l7-one-d@) ,6,6(cis)triol (VIII).-Androstan-l7-one-d(j$ ,6, G(cis)-triol (VIII)ll.-To a solution of 350 mg. of dehydroisoandrosterone (111) in 18 cc. of absolute ether was added a solution of 350 mg. of osmic acid in 35 cc. of absolute ether. After three days' standing at room temperature (28-30.5")the ether was removed from this mixture in vacuo. To the black residue was added a solution of 2.5 g. of sodium sulfite in dilute alcohol (25cc. 95% alcohol, 50 cc. water), and the mixture was refluxed on a water bath for 2f hours. After filtration, the black precipitate was refluxed three times with 95% alcohol. The combined filtrates were brought to a low volume in vacuo, causing needles t o settle out of the concentrated aqueous solution. The concentrate was saturated with sodium chloride and extracted four times with chloroform. The combined chloroform extracts were washed with a little water, dried with anhydrous sodium sulfate and brought to dryness. The residue consisted of 385 mg. of a resin which manifested a tendency t o crystallize. It was dissolved in ethyl acetate by


515

refluxing with a comparatively large amount of solvent. Glistening crystals (prisms) of a melting point of 243-245.5' separated from the concentrated solution; yield: 133.5mg. Other crops with slightly lower melting points could be secured from the mother liquors. [a]:' 79.5" (19.0 mg. in 2.0 cc. methanol). Anal. Calc'd for CL&&: C, 70.75; H, 9.38. Found: C, 71.35; H, 9.40. Androstane-8,6, i7-trion-6-02 (VI).-One hundred milligrams of androstan-17one-3@),5,6(cis)-triol (VIII) was dissolved in 8 cc. of glacial acetic acid on the water bath. To this solution was added a t room temperature (27.5') a mixture of 75 mg. of chromium trioxide in a trace of water and 3 cc. of glacial acetic acid. After standing at room temperature for 18) hours 4 cc. of ethyl alcohol was added, and the mixture was then concentrated i n Vacuo (45-50") to a green gummy residue. On addition of water, a white precipitate appeared; after standing a few hours the product was separated by filtration; yield: 38.9 mg.; m.p. 235.5-239' (decomp.). It was recrystallized from 95% alcohol from which 22.1 mg. of long, flat crystals was obtained; the melting point was 249-250.5' (decomp.). The melting point of a mixture with the compound obtained by chromic acid oxidation of androstan-17one-3(,9) ,5,6(trans)-triol (VII) was 246.5-249'. AnaZ. Calc'd for CIOH~~OI: C, 71.65; H, 8.23. Found: C, 72.17, 71.59; H, 8.49, 8.54. Pregnane-$, 6,904riond-ol (XIII) from 6-pregnen-M-on-8-01 (IX) .-A. By direct (IX).-To a solution of 396.2 mg. of chromic acid oxidation o j 6-pregnen-~OO-on-3-ol pregnenonol (IX) in 15 cc. of glacial acetic acid was added a solution of 506 mg. of chromium trioxide in a trace of water; 3 cc. of glacial acetic acid was used for rinsing. The mixture was shaken a t room temperature (27') for about 5) hours. After addition of 150 cc. of water i t was extracted four times with ample quantities of ether. The combined ether solutions were washed with 2N sodium carbonate solution, and thereafter with water. After the sodium carbonate solution had been made acid t o Congo red by the addition of dilute sulfuric acid, and extracted with ether, a n acid residue was secured; i t was a yellow resin, and was not investigated further. The neutral ether solution was dried with anhydrous sodium sulfate. On concentration of this ether solution a white crystalline precipitate appeared; the supernatant mother liquor was slightly yellow. The crystals were washed several times by decantation with fresh ether. The solvent was completely evaporated from the combined ether washings. The residue was a yellow resin which has thus far not yielded any further crystalline material. The white crystals weighed 61.2 mg. after drying; the melting point was 252-254". The product was dissolved i n about 30 cc. of hot 95% alcohol; this solution was concentrated to one-fourth to one-fifth of its original volume. On cooling, crystallization started at once, yielding rather flat prismatic crystals with various shapes, showing some rosette arrangement; yield, 38.7 mg.; melting point: 267-268' (melts to a dark brown liquid). Further crops with somewhat lower melting points were secured from the mother liquor. Anal. Calc'd for C21H8oO4: C, 72.78; H, 8.73. Found: C, 72.72, 72.78; H, 8.70, 8.77. B . From b-pregnen-9O-on-8-ol acetate (X) by way of pregnan-200-one-8@),6 , d(trans)triol (XI).-Seven hundred fifty milligrams of pregnenonol (IX) was dissolved i n 3 cc. of pyridine on the water bath; to this solution was added 3 cc. of acetic anhydride. The mixture was kept at room temperature for about 16 hours and then was poured into water. The crystalline precipitate was collected by filtration after a few hours and recrystallized from 95% alcohol; yield, 797.3 mg.; melting point, 144.5-146.5". Pregnan-9O-one-S(@),6, B(trans)-triol (XI).-To a solution of 400 mg. of pregnen-

+

516


onol acetate (X) in 2 cc. of glacial acetic acid was added 0.4 cc. of 30% hydrogen peroxide. This mixture was heated on a water bath for two hours; thereafter the major part of the acetic acid was removed in vacuo (50"). The residue was a lightyellow resin which was refluxed with 10 cc. of 5% methanolic potassium hydroxide for 6t hours. Then two parts of water was added, and the methanol was removed in vacuo (45"). The remaining aqueous solution was saturated with sodium chloride and then extracted with chloroform three times a t room temperature and three more times a t a gentle water bath temperature. The combined chloroform phases were washed with a little water and then brought to dryness in vacuo (45") yielding 350.4 mg. of a light-yellow very sticky resin. On treating the latter with a little acetone a mass of white crystals separated almost instantaneously. The crystals were filtered, washed with acetone and dried; yield: 101.7 mg. The melting point of this crude material was between 243.5 and 251". A sample was recrystallized from acetone. It was necessary to dissolve it first in a rather large amount of solvent and to concentrate this solution to a small volume. The substance crystallized in rectangular platelets; the melting point was 256-258" (without decomp.). Anal. Calc'd for Ca~HsrOc:C, 71.94; H, 9.78. Found: C, 72.47,72.23; H, 9.81, 9.72. Prepnane-S,6,.??O-trion-6-02 (XIII).-Seventy milligrams of crude crystalline pregnan-2O-one-3@),5,6(trans)-triol (XI) was dissolved in 5 cc. of glacial acetic acid on a water bath. To this was added a t room temperature 45 mg. of chromium trioxide dissolved in a little water and 1.5 cc. of glacial acetic acid; 1 cc. of glacial acetic acid was used for rinsing. The mixture was kept a t room temperature overnight. The next morning a few glistening white crystals were visible in the green solution. Thp latter was concentrated to a low volume in vacuo (43") after 2 cc. of alcohol had been added. On diluting the concentrate with water the quantity of white crystalline material was increased. I t was filtered and thoroughly washed with water; yield of the dried material: 46.3 mg. The melting point of this crude substance was between 262 and 269". This was dissolved in 25 cc. of 95% alcohol. After concentrating to about one-fourth of its volume crystals (platelets) began to separate; yield: 36.9 mg. The substance melted and turned dark-brown a t 271". The melting point of a mixture with the compound obtained by direct chromic acid oxidation of 4-pregnen-20-011-3-01 (IX) was 268.5-269.5". ~ : 72.78; H, 8.73. Anal. Calc'd for C ~ I H N OC, Found: C, 72.50, 72.59; H, 8.51, 8.68. C. From b-pregnen-.??%on-d-ol ( I X ) by way of pregnan-.??O-one-S(@), 6,6(cis)-triol (XZI).-Pregnan-.??O-one-d@),6, 6(cis)-trioZ.-To a suspension of 340 mg. of 5-pregnen20-on-3-01 (IX) in 27 cc. of absolute ether was added a solution of 310 mg. of osmic acid in 31 cc. of absolute ether. The mixture was left a t room temperature for four days. After the removal of the ether in vacuo, the black residue was refluxed for 3 hours with a solution of 2.2 g. of anhydrous sodium sulfite in 40 cc. of water and 25 cc. of 95% alcohol. After filtration, the black residue was boiled four times with alcohol. The combined filtrates were concentrated to a low volume in vacuo, producing a crystalline precipitate. After adding sodium chloride the concentrate was extracted four times with chloroform. After the usual manipulations the combined chloroform extracts left a crystalline white residue, weighing 341.4 mg. On being recrystallized from absolute or aqueous methanol, various fractions were obtained, which apparently consisted in large part of unchanged starting material which melts a t 190". There was an indication that traces of a higher-melting substance were present. The conclusion was drawn that the reaction had taken place only to a limited extent, as a result of the slight solubility of the starting material in ether.


517

The recovered material which obviously represented a mixture proved to be easily soluble in dioxane. Therefore 286 mg. of this substance was dissolved in 10 cc. of re-purified dioxane, and then 300 mg. of osmic acid was added. After three days standing the solvent was removed i n vacuo. The black residue was treated with 2.2 g. of anhydrous sodium sulfite and the other reagents as described above. When the combined chloroform extracts were concentrated, an ample quantity of white crystals settled out. The chloroform was evaporated to dryness, yielding a white crystalline residue (277 mg.). I t was dissolved in 40 cc. of hot ethyl acetate. When this solution was concentrated t o about one-third of its volume on the water bath, the separation of needles began and greatly increased on standing a t room temperature. The first crop weighed 120.3 mg.; the melting point was 231-232.5' (no de59.8" (18.4 mg. in 2.0 cc. methanol). camp.; Eiintering a t 229"). [&]: C, 71.94; H, 9.78. Anal. Calc'd for CZIHS~O,: Found: C, 72.09, 71.67; H, 9.77, 9.65. Pregnune-.9,6,BOO-trion-6-o~ (XIII).-To a solution of 140 mg. of pregnan-20-on3@) ,5,6(cis)-triol (XII) in 10 cc. of glacial acetic acid was added a solution of 90 mg. of chromium trioxide in a trace of water and 3 cc. glacial acetic acid; 2 cc. of glacial acetic acid was used for rinsing. The mixture was left a t room temperature (25") overnight. After adding 4 cc. of alcohol the green solution was concentrated almost to dryness in vacuo (45"). Thereby a white precipitate separated, which greatly increased after the addition of water. I t was filtered after about one hour and thoroughly washed with water; weight of the dried material, 68.2mg. I t was dissolved in 20 cc. of hot 95% alcohol; this solution was then concentrated until crystallization began, and was allowed to stand a t room temperature. The first crop weighed 35.7 mg. and melted a t 267-268.5" to a dark-brown fluid. The melting point of a mixture with the compound obtained by chromic acid oxidation of pregnan-20one-3(8), 5,6(trans)-triol (XI) was 269-269.5'. Anal. Calc'd for CltHsoOI: C, 72.78; H, 8.73. Found: C, 72.50, 72.36; H, 8.79, 8.61. d-Pregnene-S,6, BO-trione [L16-ozopr~gesterone"]( X I V ) by dehydrationof pregnane3,6,$0-lrion-6-ol (XIIT).-Seventy milligrams of crude pregnane-3,6,20-trion-5-01 (XIII) (m.p. 265.5-266.5') was suspended in 16 cc. of pure chloroform which had been dried over calcium chloride. A gentle stream of dry hydrogen chloride was passed through this suspension from almost three hours a t 4-5" (ice-cooling). The suspended material went into solution within a few minutes; the solution soon turned yellow, and eventually orange. The orange solution was poured into ice-cooled N sodium carbonate solution, which caused the color to disappear. After shaking this mixture in a separatory funnel, the chloroform phase was washed with water and dried with sodium sulfate. The chloroform was distilled in vacuo; the residue was a yellow oil which crystallized completely after standing some time in a vacuum desiccator; weight: 73.0 mg. The residue was easily dissolved by 10 cc. of 95% alcohol. This solution was concentrated to a low volume; then a few drops of ether was added, and the mixture was allowed to stand in the ice-box overnight. This treatment resulted in the separation of stout lemon-yellow needles, which were washed with 95% alcohol; yield: 26.1 mg.; melting point, 185-188". From the mother liquor was secured 8.5 mg. of melting point 181-184.5'. C, 76.78; H, 8.60. Anal. Calc'd for CZIHBO~: Found: C, 75.80, 75.74; H, 8.61, 8.51. Whether or not the low carbon figure is due t o the presence of some pregnane3,6,2O-trion-5-01 is uncertain. Repeated crystallizations did not raise the melting point above 188'.

+

518


Treatment of 6-pregnen-80-on-5-01 acetate (X) with osmic acid.-One hundred flfty milligrams of 5-pregnen-20-011-3-01 acetate (X) was dissolved in 8 cc. of absolute ether in which it was easily soluble. A solution of 120 mg. of osmic acid in 12 cc. of absolute ether was added, and the mixture was left a t room temperature (28-31") for 5 days. Then the mixture was worked up as described above. The white crystalline residue was saponified, thereby yielding 132 mg. of saponified material. After recrystallization from ethyl alcohol the melting point was not very sharp; it indicated, however, mainly unchanged 5-pregnen-20-on-3-01 (IX). The recovered material was acetylated with a mixture of acetic anhydride and pyridine a t room temperature. The melting point of the acetate was somewhat lower than that of the acetate of 5-pregnen-20-on-3-01 (X). SUMMARY

(1) Dehydroisoandrosterone (111) can be hydroxylated either to androstan-17-one-3(8) ,5 ,6(trans)-triol (VII) or androstan-17-one-3@) ,5 ,6(cis)triol (VIII). Both compounds treated with chromic acid yield the same androstane-3 ,6 ,20-trione-5-01 (VI). (2) 5-Pregnen-20-on-3-01 (IX) was oxidized with chromic acid to pregnane-3,6 ,20-trion-5-01 (XIII). Furthermore, it (IX) can be hydroxylated either to pregnan-20-one-3(8), 5 , 6(trans)-triol (XI) or pregnan-2O-one-3(@),5,6(cis)-triol (XII). Both compounds (XI or XII) give with chromic acid the same pregnane-3 ,6 ,2O-trion-5-01 (XIII). The latter can be dehydrated to 4-pregnene-3,6,2O-trione(6-oxoprogesterone) (XIV). (3) The stereochemical configurations of the above-mentioned substances are discussed.

INVESTIGATIONS ON STEROIDS I. 6-OXOPROGESTERONE AND

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