Microbiological Transformations of Steroids. IJI ... - ACS Publications

corticosterone (or its acetate) is converted to FB-hydroxy-11-desoxycorticosterone by Rhizopus arrhizus. Discussion. With our discovery that molds of ...
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408

E P P S T E I N , AfEISTER, PETERSON, ~ ~ U l i l i A Y LEIGH, , LYTTLE,1 i E I N E K E AND hTEINlXAU13

(b) For the Rearrangement of 2-Phenyl-20.868 pc. as the average molar radioactivity of a-naphthoiccarbo~y-C'~ acid, and 1.66 PC. as the average molar radioactivity of IV (as determined for phenyl-( anaphthyl)-acetonitrile-I-C" and for 2-phenyl-2(a-naphthyl)-acetic-]Xi*),(0.868 f I .66) X 100 ( a-naphthyl)-ethanol-1-C14(IV).-Using

[CONTRIBUTION FROM THE

VOl. i 5

= 52.3% a-naphthyl migration (and 47.7% phenyl migration). Acknowledgment-The authors are indebted to Drs. J. D. Roberts and W. M, Lauer for inany helpful suggestions during the course of this research. OAK

RIDGE,TENNESSEE

RESEARCH LABORATORIES, THEUPJOHN

COMPANY]

Microbiological Transformations of Steroids. IJI. Preparation of 11-Epi-corticosterone and of 6@-Hydroxy-ll-desoxycorticosterone I ~ S. Y H. EPPSTEIN, P. D. MEISTER,D. H. PETERSON, H. C. MURRAY, H. MARIANLEIGH,D. A. LYTTLE, L. M. REINEKEAND A. WEINTRAUB RECEIVED SEPTEMBER 29, 1952 1 I-1)esoxycorticostcrolle (or its dcetate) is coiiverted t o 11-epicorticosteroneby the mold Rhizopus mgvicans corticosterone (or its acetate) is converted to FB-hydroxy-11-desoxycorticosteroneby Rhizopus arrhizus.

Discussion With our discovery that molds of the genus Rhizopus (order of Mucorales) can oxygenate progesterone to hydroxylated steroids and especially to 11a-hydroxyprogesterone,* a systematic study was made of the ability of these organisms to introduce oxygen into various steroids. This paper reports on the biooxygenation of 1l-desoxycorticosterone and its acetate by Rhizeus nigricans Ehrb. (A.T.C.C. 6227b) and Rhizopus nrrhizus Fischer (A.T.C.C. 11145). Our general procedures aiid techniques have been reported in the preceding publications. Medium H a t a PH of 4.5-5.0 was employed together with a growth to conversion cycle of usually 24 hr.j21 hr. Ii concentration of I 1 -desoxycorticosterone acetate (I) of 0.23 g./liter of medium was optimal under the fermentation conditions used, whereas somewhat lower concentrations of 11-desoxycorticosterone, free alcohol, (11) were employed because of its greater inhibitory effect upon the course of the bioconversion. Since the fermentations with these two steroid substrates are otherwise identical, only the experiments on I with the two species of Rhizopus will be reported in detail. The major product of the biooxygenation of I or I1 by Rhizopus nigricans can be isolated most conveniently from the steroid-containing solvent-free extractives in one of several ways----directcrystallization by addition of ether, crystallization from ethyl acetate after removal of extraneous oily material with nhexane, or column chromatography over Florisil. ( 1 ) Paper I1 by I'.*D. lfeister, D. X i . Peterson, 13. C .IIurray, S. H.

Eppstein, L. >I. Reineke, A. IVeintrnub and I t . Marian I x i g h , THIS 7 6 , 55 (1953:. I ? ! i;i) n. H. Peterson a n d H C. 3Ii:rray, i b i d . , 74, 1871 (1952); ( b j 11. H. Peterson, H. C. X u r r a y , S. 13. Eppstein, I.. I f . Reineke, .?. \T-eintraub, P. D. RIeister and H. Marian Leigh, ibid., 74, 5933 (1952); (c) U. S. Patent 2,602,769, filed Feh. 23, 1952, issued July 8, 1952, based on an original application filed Aug. 19, 1950. The biooxygenations described in detail in the present communication were disclosed in this pateut. In THISJ O U R N A L , 74, 3962 (1952), J. Fried, e i a1 , m a n Couimunication to the Editor describe the bioaxygenation o f desoxgcorticonterone t o 11-epicortirosterone b y A rpevgillirs niger. (4: A synthetic magnesia-silica gel made hy the Floridin Co., W a r r e n , Pa. JOURNAL,

11-lhoxy-

The major biooxygenation product of I or I1 by Rhizopus arrhizus can be obtained by column chromatography over Florisil. Under our conditions Rhizopus nigricans and Rhizopus arrhizus convert I or I1 completely in a 24-hour period. With Rhizopus nigricans one main component (111) is formed which by paper chromatography2bs2chas a mobility slower than that of corticosterone. X small amount of a material (IV) more polar than I11 is invariably formed (about 370 by paper chromatographic estimation). Its chemistry will be discussed at a future date. By selective acetylation of I11 the C-21 acetate (V) was formed. Reaction with two equivalents of acetic anhydride yielded the diacetate (VII). Oxidation of V with chromic anhydride yielded a compound (VI) identical to 11-dehydrocorticosterone-21-acetate (compound A acetate) by the following physical criteria : melting point, mixed melting point, optical rotation, infrared, paper chromatography and elementary analysis. An authentic sample of corticosterone was carried through the same reactions. A comparison of the constants of the two preparations with those found in the literature for compound A acetate is given in Table T. TABLE I COMPARISOK OF CONSTANTS OF COMPOUND A ACETA~B DERIVEDFROM EPICORTICOSTEROSE AND CORTICOSTERONE VIa (from 111)

V I b (from corticosterone)

Mp., "C. 179-181 179-181 Mixed m.p , OC. 179-181 [ a ](dioxane) ~ 239 O 235" Infrared spectra identical for VIa and VIb.

VI (literature)'

179-181 234'

This sequence of reactions establishes I11 as 11epicorticosterone or 11a,21-dihydroxy-4-pregnene3,20-dione (since by paper chromatography and other physical properties as well as by its ability to form a diacetate readily, IT1 is different from corticosterone). (4) T. Reichstein and C W Shoppee, T'cfamrns and Houinones, I, 363 (lQP5).

1 ~-EPI-CORTICOSTERONE AND BP-HYDROXY-~ ~-DESOXYCORTICOSTERONR

Jan. 20, 1953

409

ticosterone acetate. Our compound has a melting c=o point of 165.5-166.5' in contrast to that of the disputed material of 122Rhizopus nigricans 125'. Indeed, if epimerization should occur upder the Shoppee and Reichstein reaction cono// 0 ditions, i t is doubtful that I11 IV I, R = Ac a free 11-hydroxy com11, R = H CHZOAC pound could be isolated. The well-known chemiI C=O I C=O cal reactivity of the l l a hydroxyl group can be illustrated by the followRhizopus ing experiment. On subarrhizus jecting l l a - hydroxyprogesterone to the hydrochloric acid-acetic acid dehydration condiV,R = H VI tions employed by ShopVII, R = Ac CHzOAc CHzOH pee and Reichstein we I isolated 11a-acetoxyprogc=o c=o esterone in good yield. I Only about 20% of the 11a-hydroxy progesterone had not reacted.l Inasmuch as Shoppee and Reichstein re-acetylated (acetic anhydride in pyridine) their reaction mod/ ucts,' the isolation of the IX, R = H VI11 ' 11a-hydroxysteroid is virX, R = Ac tually precluded. When Shoppee and Reichstein5 treated corticoCompound I11 is practically the sole' steroid sterone acetate with hydrochloric-acetic acid formed in this biological transformation with (1:9) they recovered, beside dehydration products, Rhizopus nigricuns. Steroid yields of about 50 an alcoholic substance which they considered to be t o 60% of crystalline product are obtained and the 11-epicorticosterone acetate. This assignment of total steroid balance is 60-7570 (by paper chromstructure has been seriously questioned by Gal- atography). If the developed chromatogram of the lagher.6 Our work shows that Shoppee and Reich- original crude fermentation extract is treated with stein were in error. 2,4-dinitro~henvlhvdrazine or with modified Tollens no components are detected TABLE I1 other than those shown by ultraviolet absorpMOLECULAR ROTATION DIFFERENCES FOR 1 EPIMERS tion. Hence no appreciable amount of reduction [alD MD AMD in ring A or destruction of the keto1 side chain lla-Hydroxyprogesterone +179' (CHC13 $591' +143O occurs. 11s-Hydroxyprogesterone + 2 2 2 , 5 (CHCla) +734" +dog0 +1970a When the biooxvaenation of 1 or 11 is similarh 11-EDi-F f 1 1 3 (MeOH) + I 6 7 (EtOH) +606O Compound F (Kendall) carried out with -&hizopus arrhizus, one majdr 11-Epicorticosterone +165 (EtOH) +571° +20lo transformation product (VIII) is formed which has Corticosterone +223 (EtOH) +772" a mobility, by paper chromatography, just slower 11-Epicorticosterone acetate + 159 (AcetIb +618O + 139' than that of Reichstein's Compound S. Several (This paper) 11-Epicorticosterone acetate +I89 (Acet)b +726O minor transformation products are detectable by (Shoppee and Reichstein) this technique, one of which has a mobility identical Corticosterone acetate +195 (Acet)b 4-757' + 31' to that of 111. The infrared spectrum of the isoa The solvent effect of methanol was assumed t o be neglilated VI11 was identical to that of 6/3-hydroxy-11gible. b Acet = acetone. desoxycorticosterone. The melting point and opThe molecular rotation data (Table 11) strengthen tical rotation of this material showed rather large the contention of Gallagher'j that the A M ~ l l P - l l ~ discrepancies with the values reported by other^.^^^ of the substance of Shoppee and Reichstein was Indeed the values in the literature are also a t varitoo small to allow the substance to be ll-epicor- ance with each other. These differences are indicated below (Table 111). ( 5 ) C. W. Shoppee and T. Reichstein, Ilelv. Chim. Acta, 26, 1316 CHiOR

CHzOH

CHzOH

I

I

1 c=o

___f

Gv '

.1 FoAc

(1943). (6) L. F. Fieser and Mary Fieser, "Natural Products Related to Phenanthrene," 3rd Edition, Reinhold Publ. Corp., New York, N. Y., p. 409.

(7) Unpublished data. 18) P. T. Herzig and M . Ehrenstein, J . Ovg. Chcm., 16, 1050 (1951). (9) W. J. Haines, "Recent Progress in Hormone Research," Vol. V I I , Chap. 111, 1952, p. 282.

410

EPPST@IN, hfEISTBR, J'ETERSON,

~ I U R R , \ Y , LEIGII, LYTTLE, REINEKE AND WEINTRAUB

TABLE I11 PHYSICAL

CONSTANTS FOR 66-HYDROXY-11-DESOXYCORTICOSTERONE A N D DERIVATIVES IVLP., o c . [UlD Ir:

VOl. 'ij

An error on the specific rotation of 1I-ketoprogesteronc appeared in our first paper in this series: the rotation of +227" as given watdone in acetone, not chloroform, in which tlic rotation is 275 .

Infrared

6@-Hydroxy-l l -desoxycorticosterone (VI I I )

Experimental

IIerzig and Ehrenstein* Hainesg This paper

A . Fermentation of 11-Desoxycorticosterone Acetate 190-192 + 6 2 , 6 (CHC13 13,730' Identicald (2 l-Acetoxy4pregneneb,2O-dione)(I) by Rhiaopus nigri14,500)} 181-183 +1LO (EtOH) cans and Isolation of 11-Epicorticosterone (lla,2l-Dihy198-202 + l o 1 (CHCIa) 13,700 Identical droxy4pregnene-3,2O-dione(111).-This fermentation was carried out in the stir bottle assembly.lb The concentration 6P,21-Diacetoxy-4-pregnene-3,20-dione (X) of I used was 3 g. in 12 liters of medium. The fermentationHerzig and conversion cycle was 24 hr./30 hr. Paper chromatography Bhrenstein* 125-127 + 1 0 9 . 5 (CHCIa) 15,950 Identical of crude extract showed a trace of 11, a large amount of 111 This paper 127-129 4-103 (CHCls) 13,150 and a very small amount of IV. The weight of the extract was 4.66 g. This was fractionated over 240 g. of Florisil 6~-Hydroxy-2l-acetory-4-pregnene-3,20-dione (IX) with 390-ml. fractions of solvents as follows: ethylene T h i s paper 196-198 + I 1 3 (CHCIa) 13,900 dichloride (8 fractions); ethylene dichloride-acetone mixtures 25:1, 15:1, 12:l and 1 O : l (2 fractions in each ina Compared as the diacetates.$ stance); ethylene dichloride-acetone 8: 1 (3 fractions); Isolation of the minor transformation product ethylene dichloride-acetone 5: 1 (4 fractions); ethylene having the niobility of I11 allowed the infrared dichloride-acetone 2: 1 ( 3 fractions); acetone (4 fractions). fractions were analyzed by paper chromatography. identification of this transformation product as The Compound I1 appeared in a broad peak centering about being, in fact, 11-epicorticosterone. fraction 8. Compound IV was present in fractions 21-23. The specificity of the enzyme systems involved Fractions 14-20 (2.06 9.) were combined and crystallized in these microbiological oxygenations and the from 15 ml. of ethyl acetate t o give 1.46 g. of nicely crystalline product, m.p. 15C-159°.11 This is a 65% yield (taking profound effects of substituents in the steroid into account the loss of the 21-acetate). Paper chromatognucleus used as substrate are worthy of special raphy showed only I11 with a trace of IV. Recrystallizanotice. Thus with progesterone as a substrate tion from ethyl acetate yielded pure 111, m.p. 153-155", Rhizopus arrhizus forms 11a-hydroxyprogesterone [a]*'D +166' (c 0.764 in chloroform) and +165' (c 0.711 in fair yields (up t o 50% as crystals) and in addi- in 95% ethanol). Calcd. for GlH3004: C, 72.80; H, 8.73. Found: tion forms appreciable amounts of 6p-11a-di- C,Anal. 72.88; H, 8.69. hydroxyprogesterone (up to 15% yield of crystals) : The infrared spectrum was different from that of the Rhizopus nigricans, however, forms the 11CY- then known steroids and indicated the addition of probably hydroxyprogesterone in essentially quantitative one new hydroxyl group. This was verified by the elemen.~ yields.2b With 11-desoxycorticosterone (or its tary analysis. Fraction 21. mainlv I11 with about 4% of IV. was acetate) Rhizopus arrhiaus forms predominantly crystallized from 2 ml: of ethyl acetate t o Seld 135 mg. of 6P-hydroxy-l l -desoxycorticosterone and only small crystals, m.p. 140-155', which by paper chromatographic amounts of 11-epicorticosterone, whereas Rhizopus analysis consisted of I11 with about 9% of IV. This nigricuns produces essentially the latter compound ; brings the steroid balance of crystalline material to about with some remaining in the mother liquors. none of the 60-hydroxy compound has been de- 70% Formation of C-2 1 Monoacetate ( 1la-Hydroxy-2 1tected. Other cases of the influence of the steroid acetoxy4pregnene-3,2O-dione) (V) from 1II.-To 101.2 molecule on the course of the fermentation will be mg. of I11 in 2 ml. of dry pyridine was added 1 ml. of a solureported in future communications. l o Perhaps tion of 0.304 ml. of redistilled acetic anhydride diluted t o 10 nil. with pyridine. After 16 hours a t room temperature, the qualitative and quantitative differences herein the reactioii mixture was diluted with 50 ml. of water and, described are the result of competition of different after an hour a t room temperature, was refrigerated. enzymes, one steroid structure being more Suitable Crystallization did not occur. The aqueous solution was for a given enzyme. I t is also possible that the five times extracted with 25-ml. portions of ether, the conibined ether fractions washed with 2% hydrochloric acid substituents. affect the spatial relations of the until the washings were acid, then twice with 2% bicarbonenzyme-substrate complex so that different posi- ate solution and once with water. The extract was dried tions of the steroid nucleus are activated. If this with anhydrous sodium sulfate and the solvent allowed to is the case, then one must postulate that consider- evaporate a t room temperature leaving 106.5 mg. of a oil. This was fractionated over 5.3 g. of alumina able difference exists iii the structures of the colorless (acid washed, activated a t 120') with 10-ml. portions of enzymes in question in these two species of Rhiz- solvents in the following manner: ether-chloroform 1: 1 opus. ( 3 fractions); chloroform (3 fractions); chloroform-acetone Another characteristic of the Rhizopus molds is 18:l ( 2 fractions). Fraction 5 , the second chloroform fraction, showed a very sharp peak representing a 62% the presence of very active esterase. In general yield of product V. Attempts to crystallize this material these organisms hydrolyze the 21-acetates with failed and so i t was used as such for the subsequent oxidasufficient rapidity that only non-esfevified steroids tion with chroinic anhydride. Fraction 0 (6.4 nig.) was saved. This crystallizc~l :ire fouiitl a t the cnd of the fcrineritatioiis. Prespoiitaiieotisly tluritig the cnurse of 6 months. When it W;L\ sumtbly the oxygeiiatiiig eiizyiries x t c i l 011 the rccrystallizetl froiii 0.2 1 1 1 1 . of methaciol a t -.j" ailti wa.;hr(l Irrc strroid a.lcolin1. Thus, if the Eernic~itatioii tlirce tiines with 0.3 1111. o f cold methanol ( - 5 ' ) the product with a 2 1-acetate is interrupted long before coiii- weighed 3 tiig. and melted 139-163'. Itifrared indicated pietion of the steroid oxygenation oiie finds non- thc presence of the 21-acetoxy, the lla-hydroxy and the Purified 21-acetoxy-1l-epicorticoacetylated oxygenated steroid, the free alcohol of :~-keto-A4-configuration. sterone of identical infrared spectrum prepared in a subsethe steroid substrate and traces of the original quent experiment melted a t 163-165', and had an [ a I z 3 D substrate (ester) ; never has oxygenated steroid of fl59" ( c 0.7324 in acetone), and of +168" ( c 0.588 in chloroform). ester been foun (1. (10) T h c interesting trariafuriiiati,,~~aBf should lic recalled in this conuection.

~ ~ ; - ~ l c l i ) - ~ l r ( ~ ~ i ~ , ~ ~ c s ~( c1 1r )~ , All u c ' nicltinp points rcported i n this I'tuhcr J d u ~block s aurl are uncorrected.

paper werc takcn on a

Jan. 20, 1953

1 1-EPI-CORTICOSTERONE AND GB-HYDROXY-1 1-DESOXYCORTICOSTERONE

411

corticosterone.-A stir bottle assembly of 12 liters of medium was employed under fermentation conditions dissolved in 1.4 ml. of chlorobenzene, cooled in an ice-bath similar' t o those of section A, above. Three grams of I and stirred for 2 hours with a solution of 53 mg. of sodium in 100 mi. of acetone was added in a 24 hr./24 hr. cycle. dichromate in 0.8 ml. of water and 0.135 ml. of sulfuric acid. The solvent-free steroidal extractives weighed 5.727 g . Then 10 ml. of cold water was added and sufficient benzene Paper chromatography indicated the formation of a large to bring the organic phase to the top. This was separated amount of a new compound (VIII) with small amounts of and washed (twice) with cold water until colorless, twice several other compounds, one of which could not be diswith 5% sodium bicarbonate (5-ml. portions) and again tinguished from I11 by this technique. The extractives were dissolved in 200 ml. of ethylene with water. The benzene solution was dried with anhydrous sodium sulfate and the solvent allowed to evaporate dichloride and chromatographed over 300 g. of Florisil spontaneously to give a white crystalline residue, weighing with 600-ml. fractions of solvent as follows: ethylene di,50.2 ing. (71 .4y0yield), m.p. 177-180". This wasrecrystal- chloride, ethylene dichloride-acetone 25: 1 and 15: 1 ( 2 lized from 1 ml. of methanol in the cold and washed twice fractions in each instance); ethylene dichloride-acetone with cold methanol (0.5-id. portions) to give VIa, m.p. 12:1, 10:1, 8:l and 5 : l (3 fractions in each instance); ethylene dichloride-acetone 3: 1 ( 2 fractions); acetone ( 1 l79-181', [ a I z 3f239" ~ (c 1.5 in dioxane). Anal. Calcd. for Cl~HaoOb:C, 71.47; H , 7.83. Found: fraction). Fractions 10-16 (10.1 and 8:l solvent mixtures) contained compound VIII. These fractions were C, 71.47, 71.60; H , 7.94, 7.90. individually triturated with 10 ml. of ether-acetone 3 : 1 Fifty mg. of an authentic sample of corticosterone, m.p. and the solution decanted and discarded. The crystalline 177.5-180' [ a ] +220' ~ (ethanol) (identity by infrared) residues were dissolved in acetone-chloroform 1: 1. The was carried through the same series of reactions to give VIb combined solutions were concentrated to a thin sirup and of identical melting point, [ a I z 4 +235' ~ ( c 0.988 in dioxane). diluted with 10 volumes of ether t o give 501 mg. of crystals, Anal. Found: C, 71.28; H, 7.84. m.p. 198-205". Recrystallization from 5 ml. of methanol An aliquot was The mixed melting point of the two preparations showed yielded 396 mg. melting at 205-210'. again recrystallized from methanol to yield crystals melting n o depression, and the infrared spectra were identical. Infrared spectroscopy confirmed that this maSince I11 was not corticosterone this sequence of reactions 206-210'. establishes it as lla,21-dihydroxy-4-pregnene-3,20-dione terial was 6p-hydroxy-l l-desoxycorticosterone (VIII). The crystalline compound was solvated with one equivalent of or 11-epicorticosterone. 1l0,2 l-Diacetoxy-4-pregnene-3,20-dione(VII) from 111. methanol which was retained on drying a t 60' (0.01 mm.). O H69.81; : H , 9.05. -To 50 ing. of 111, dissolved in 1 ml. of dry pyridine, was Anal. Calcd. for C Z I H ~ ~ O ~ . C H ~C, ~ D (c, 0.859 chloroadded 4 nil. of a solution of 0.304 ml. of acetic anhydride Found: C, 70.26; H, 8.64; [ ( Y ] ~4-97" diluted to 10 mi. with pyridine (fourfold excess). The form). Drying for 15 hours over phosphorus pentoxide work-up was similar to that for the monoacetate (V). The (0.01 mm.) yielded the anhydrous compound, m.p. 198non-crystalline product was partitioned over 3.5 g. of 202', [ a I z 3+~l o l o (c 0.9134 in chloroform). alumina (acid washed and activated a t 120') with 7-ml. Anal. Calcd. for C Z I H ~ O OC,~ :72.80; H , 8.73. Found: portions of solvent as follows: ether (2 fractions); etherC, 72.96; H , 8.59. chloroform 19: 1 (2 fractions); ether-chloroform 9 : 1 (2 6P-Hydroxy-1I-desoxycorticosterone Acetate (60-Hyfractions); ether-chloroform 1 : 1 (2 fractions); chloroform droxy-2l-acetoxy-4-pregnene-3,20-dione)(IX).-Two hun(2 fractions). Fractions 7 and 8 (ether-chloroform 1: 1) dred fifty mg. of VI11 was dissolved in 2.6 ml. of a pyridineshowed a sharp peak and were combined (39.7 mg.). First acetic anhydride mixture (1 ml. of this solution contained attempts to crystallize this material failed. As in the case 30 mg. of acetic anhydride. One equivalent was calculated of V it crystallized spontaneously during the course of six a t 73.8 mg.; the amount used was 78 mg.). After 18 months standing. I t was recrystallized from 0.3 ml. of hours a t room temperature the reaction mixture was cooled methanol a t -5' and washed with 0.5-ml. portions of cold with ice and diluted with ice water to give a crystalline methanol. The recrystallized product (long needles) precipitate which was filtered off. The crystals (164 mg., weighed 21.3 mg., 1n.p. 146-149', [aIz3D+152" (c 0.979 in m.D. 184-188") were recrvstallized from 2 ml. of acetone. chloroforn~). ' After an additional acetone recrystallization the compound 24nnl. Calctl. for C2jFh06: C, 69.74; H, 7.96.. Found: melted a t 196-198'. C, 69.46; H, 8.32. Anal. Calcd. for CzaH32Oa: C, 71.10; H , 8.30. Found: Infrared indicated no hydroxyl and the presence of two c, 71.12; H. 8.36; 237 mp ( E 13,900), [ C Y ] ~ f113" ~D acctoxy groups. (c 1.185 in chloroform). I t is interesting to note that the acetates of ll-epicorticosThe aqueous filtrate (above) was extracted twice with ether terone crystallize with difficulty in contrast tb the 21(50 ml.). The ether extracts were washed once with 10 acetate of corticosterone.'Z B. Isolation of I11 by Direct Crystallization (Rhizopus ml. of 50/, hydrochloric acid, twice with 10-id. volumes nigricans Fermentation) .-The oxygenation of 60 g. of I of 5% sodium carbonate and four times with 10-ml. volumes was carried out in a 100-gallon fermenter containing 240 of water. After drying over sodium sulfate and concentraliters of medium. The growth/conversion cycle was 24 tion, an additional crop of 103.5 mg. of crystals, m.p. 160-173', was obtained. hr ./24 hr . 6p-Hydroxy-1I-desoxycorticosterone Diacetate (6p,21The tarry, steroid-coutairling, solvent-free extractives were dissolved in 180 rnl. of methylene chloride and 900 ml. of Diacetoxy4-pregnene-3,20-one) (X).-Forty-seven mg. of ether added. The oily precipitate crystallized on refrigera- VI11 was dissolved in 3 ml. of pyridine and 2 ml. of acetic tion for 24 hours. The brown 'crystals were separated by anhydride. After standing a t room temperature overnight decantation and washed with 200-1111, portions of ether until the solution was diluted portionwise with ice-water. Some amorphous gray material which precipitated a t the beginno more appreciable color was extracted. The yield of crude product was 24.4 g. This material was decolorized ning of the dilution process was separated mechanically. in methylenc chloride solution with 20 g. of Magnesol,2b~2cOn further dilution a milky amorphous suspension was obtained. This was extracted twice with 50-ml. portions evaporated to dryness and crystallized from 300 ml. of ethyl acetate to yield 15.32 g. of 111, m.p. 156-162'. A of ether. The ether extracts were washed with hydrosecond crop of crystals from th: ethyl acetate mother liquor chloric acid, sodium carbonate solution and water and dried weighed 2.74 g.. m.p. 156-161 . The total yield of purified in the same manner as described for the monoacetate (above). Evaporation of the solvent yielded an oily 111 isolated by direct crystallization was 18.06 g. or 32.4% (based on the amount of I1 equivalent to the I used). The residue of 61.2 mg. which could be crystallized from a few mother liquors of the direct ether precipitation contained drops of ethyl acetate. The crystals were dissolved in a some 111 (paper chromatography) which could only be few drops of ethyl acetate, a like amount of ether was added purified by column chromatography. and the mixture was refrigerated overnight. The resulting C. Biooxygenation of I by Rhizopus arrhizus with Isola- crystals melted a t 125-127'. Recrystallization from ethyl tion of 6p. Hydroxy-1 I-desoxycorticosterone and 1 I-Epi- acetate-ether once more yielded 19 mg. of X, m.p. 127_236 m& 129', [a]!% +103' (c 0.610 in chloroform) Xi:; (12) T. I?. Gallagher, "Recent Progress in Hormone Research, I," ( E 13,150). The infrared spectrogram was identical to Chap. 4, p. 95, reported the chemical synthesis of the 1121-diacetate that of Ehrenstein's diacetate. but obtained only amorphous product.

Oxidation of V to Compound A Acetate (21-Acetoxy-4pregnene-3,11,20-trione).-The 70.5 mg. of fraction 5 was

Xz;

412

h T E R S O N , EPPSTEIN, AIEISTER, J I A G E R L E I N , AfCRRAY,

Isolation of 11-Epicorticosterone (Rhizopus arrhizus Fermentation) .-Fractions 1841 from the Florisil column described above contained the material which had the mobility of 11-epicorticosterone by paper chromatography. These fractions (1 g.) were dissolved in 120 ml. of ethylene dichloride and rechromatographed over 65 g. of Florisil with 120-ml. portions of solvents as follows: ethylene dichloride (2 fractions); ethylene dichloride-acetone mixtures lO:l, 8:1, 5:1, 3:1, 2 : l and 1:l (2 fractions in each instance); acetone (1 fraction). Fractions 11-11 (378 mg.) were combined and crystallized from 0.5 ml. of ethyl acetate to give 171.5 mg. of 111, m . p . l.53-158°, identified by infrared spectroscopy.

Acknowledgments.-The authentic corticosterone was furnished by Dr. IV. J. Haines of the Endo-

[CONTRIBUTION FROM

THE

LEIGH,W E I N T R A U B

AND REINEKE

VOl.

76

crinology Department of the Upjohn Research Division. We are grateful t o Dr. J. L. Johnson and Mrs. G. S. Fonken for the infrared analyses, to Mr. LV. A. Struck and his associates for all optical rotations and microanalyses, and to Miss Jennie I. Mejeur, Miss Irene N. Pratt and Mr. Glenn Staffen for technical assistance. Again the authors wish to express their profound obligation and thanks to Drs. R. H. Levin and D. I. Weisblat for their helpful suggestions, discussions and interest in this project. KALAMAZOO, MICHIGAN

RESEARCH LABORATORIES, THEUPJOHNCOMPANY]

Microbiological Transformations of Steroids. IV. The 11-Epimer of Compound F and Other New Oxygenated Derivatives of Reichstein's Compound S. A New Route to Cortisone' BY D. H. PETERSON, S. H. EPPSTEIN, P. D. MEISTER,B. J. MAGERLEIN, H. C. MURRAY, H. MARIAN LEIGH,A. WEINTRAUB AND L. M. REINEKE RECEIVED SEPTEMBER 29, 1952 Incubation of Rhizopus nigricans Ehrb. (A.T.C.C. 6227b) with Reichstein's Compound S produced two new 11-oxygenated steroids; viz., the 11-epimer of compound F which was easily converted to cortisone acetate in good yields; and a small A third new, compound, 6~,17a,21-trihydroxy-4-pregnene-3,20amount of 11~,17~,21-trihydroxypregnane-3,2O-dione, dione, was also obtained in good yield using Rhizopus arrhizus Fischer (A.T.C.C. 11145) on Reichstein's Compound S as the substrate. This new steroid was then also found as a minor transformation compound from the fermentation with R hizopm nigricans.

Discussion pound S (or the acetate) in 12 1. of medium H 3 qt The introduction of oxygen into position 11 of PH 4.3 t o 5.0 for 48-72 hours (growth cycle 24 the steroid nucleus by microorganisms in one step hours) with Rhizopus nigricans Ehrb. (A.T.C.C. has been previously reported. Earlier papers in 6227b) or Rhizopus arrhizus Fischer (A.T.C.C. this series have now recorded in detail the forma- 11143). Paper chromatography* (papergram studtion of 1la-hydroxyprogesterone, l la-hydroxy-l Ta- ies) indicated the formation of one major steroid progesterone and l l a,21-dihydroxy-4-pregnene-and two minor steroidal components on the con3,20-dione (11-epicorticosterone) from progesterone, centrate obtained from Rhizopus nigricans. Based 4,16-pregnadien-3,20-dioneand 1l-desoxycorticos- on isolation and structure studies it was found that terone, respectively. This paper reports the micro- CiO-S07c of the 11-epimer of compound F, epi biological transformation of Reichstein's Compound F or 1 1a , 1Ta,21 -trihydroxy-4-pregnene-3,20-dione S or its acetate by Rhizopus nigricans Ehrb. (A.T.- (11) could be isolated by direct crystallization or by C.C. 6227b) and Rhizopus nrrhizus Fischer (AT.- chromatography over Florisil. The crystalline concentrate contained 3-1 0y0 of C.C. 11145). (111) Methods and conditions employed for the micro- t$, 17a,21-trihydro~y-4-pregnene-3~2O-dione which cou Id be isolated by chromatography over biological conversion have been described elsewheree3 For isolation and structure studies i t was Florisil.'" Traces of a saturated steroid lla,17a,21adequate t o ferment 2.0 g. of Reichstein's Com- trihydroxypregnane-3,20-dione(IV) could also be detected by papergram analyses, and this com11 j (a) Paper 111in this series: S. H. Eppstein, P. D. Meister, D. H. pound was isolated and purified over Florisil. l'eterson, H. C. Murray, H. Marian Leigh, D. A. Lyttle, L. M. Reineke and A. Weintraub, THIS JOURNAL, 71, 408 (1953). (h) The transforCompound I11 was detected by papergram analysis mations recorded in detail in this manuscript are contained in our U. Y. and then isolated by direct crystallizations in good Patent 2,602,769, issued July 8, 1952, based on an original application yields from the fermentation of Reichstein's Comfiled Aug. 19, 1950. (c) In THIS JOURNAL, 74,3962 (1952). Fried, el n l . , pound S with Rhizopus arrhizus. reported the bioconversion of Reichstein's Compound S t o the l l a Microanalyses and conversion to a diacetate hydroxy Epimer of Compound F using Aspergillus Izigev. id) I n Cizemisfvy aiid Iizdnslvy, 31, 783 (1952), J. Romo, A. Zaffaroni. J . (1'11) showed that the new compound I1 contained Hendricks, G. Rosenkranz, C. Djerassi and F. Sondheimer have rean additional hydroxy group, otherwise retaining ported the chemical synthesis of the 11-epimer (epi F) as the diacetate. the basic structure of the starting material (ReichThe biosynthesis of epi F by adrenal brei from lla-hydroxyprogesterstein's Compound S). Infrared studies and the one on a micro scale was also accomplished. In the latter case the epi F was identified without isolation through t h e diacetate by paper absence of biological activity6 showed that Comchromatography and the ultraviolet absorption curve of the sulfuric pound I1 differed from Kendall's Compound F. acid-chromogen. (2) D

H. Peterson and H. C. XIurray. THISJ O U R N A L . 74, 1871

i1932)).

(3) D. H. Petersdn, H. C. Murray, S. H . Eppstein, L. X I . Reineke, P. D. Meister and H. Marian Leigh, paper I, ibid.,

A. U'eintraub,

(4) A Z d T u o n i . K B Burton A n d F H Keutmann 5 < 1 ? i i ~ r i l l , 0 (19iO ( 5 ) Tested by Dr K J Olson of our laboratories by the rat l i t e r glycogen arsay ( h l I, Pahst, R Sheppard and M H Kuizenga Ciidoc i & ~ i u l o g y41, ii (illt7)) in dose5 uf 4 mg per animal