THE JOURNAL OF BIOLOGICAL C~mrsmu Vol. 241, No. 22, Issue of November 25, pp. 53136324, 1966

Printed in U.S.A.

I@-Hydroxylation of 3a, 17a , ZOP , Zl-Tetrahydroxy-5P- pregnan-U-one and of Other 5P-Steroids in a Man

and by Surviving Liver Slices of the Guinea Pig*

(Received for publication, December 8, 1965)

JOHN J. SCHNEIDER AND NORMAN S. BHACCA

From the Department of Medicine, Jeferson Medical College, Philadelphia, Pennsylvania 19107, and the Spectroscopy Applications Laboratories, Varian Associates, Palo Alto, California 94301

SUMMARY

The Ifi-hydroxylation of 3cu,17a,20/3,21-tetrahydroxy-5/?- pregnan-11-one was shown to occur in vivo in a man and in vitro by surviving liver slices of the guinea pig. The position and configuration of the metabolically introduced hydroxyl group were determined by chemical degradation and by nuclear magnetic resonance spectroscopy. This hydroxyla- tion also was observed, but to a much smaller extent, when 3a, 17~z,21- trihydroxy-5P - pregnane - 11,20 - dione and 50 - pregnane-3a, 17a,20a-trio1 were administered to the same subject.

Seven years ago (2), we described briefly the isolation from urine of a new metabolite of 1 lp ,17a, 21-trihydroxypregn-4-ene- 3,20-dione after the oral administration of 3a! ,17a!, 20/?,21- tetrahydroxy-5/3-pregnan-11-one (3) to the senior author. Evidence, in the form of elementary analyses and paper chroma- tographic characteristics of the metabolite and the 17-ketosteroid derived from it, supported the view that the new metabolite differed from p-cortolonel only in possessing one additional hydroxyl group.

In the present paper, we detail the isolation of this metabolite and present proof, obtained from its chemical degradation and from nuclear magnetic resonance (NMR) spectroscopy studies, that the metabolically introduced hydroxyl group occupies position 1 and is in the axial (/3) configuration. The l&hydroxyl- ation of 3a!, 17a, 21-trihydroxy-5/%pregnane-ll , 28dione (4) and

* This research was supported largely by Research Grant AM - _ 01255 from the National-Institute of Arthritis and Metabolic Diseases. National Institutes of Health. United States Public Health Service. A preliminary account of some aspects of this work has appeared (1).

1 The systematic designations for the trivial names used in this report are: p-cortolone, 3cr,17cr,20P,21-tetrahydroxy-5p-pregnan- 11-one; tetrahydrocortisone, 3,~,17~~,21-trihydroxydp-pregnane- 11,20-dione; pregnanetriol, 5p-pregnane3a, 1701,20cu-trioli cortisol, 11~.17~~,21-trihvdroxvnregn-4-ene-3.20-dione: deoxvcorticoster- one; 21-hydroxypregn”-&en;-3,20-dione; and i&cortisone, 17a,21-di- hydroxy-5&pregn-1-ene-3,11,20-trione.

of 5/3-pregnane-3cr, 17a, 2Ocr-trio1 (5) in a man and of ,&cortolone by surviving liver slices of the guinea pig also are described.

RESULTS AND DISCUSSION

The reactions used to establish the location of the metabolically introduced hydroxyl group in the &cortolone metabolite (Com- pound I) are outlined in Fig. 1. Sodium periodate oxidation of I gave the 17-ketosteroid (Compound II) in good yield. Our aim then was to oxidize Compound II to both possible monohydroxy- triketones (Compounds III and VI, Fig. 1) and to dehydrate each as a means of obtaining a known a#-unsaturated ketone. Chromic anhydride in pyridine (6) was found to be the best oxidant for this purpose. Treatment of Compound II with thii reagent gave two neutral products in unequal amounts. The minor product (III) was readily dehydrated in the presence of methanolic sodium hydroxide to the known unsaturated triketone (IV), which was further characterized as the known saturated triketone (V). The reaction sequence II -+ III + IV establishes that the metabolically introduced hydroxyl group is 0 to the C-3 carbonyl group in Compound III, that is, at C-l. Additional examples of this method of characterizing 1-hydroxylated steroids are to be found in earlier papers from other laboratories (7-9).

The monohydroxytriketone (VI) was recovered largely un- changed after treatment with methanolic sodium hydroxide, but the elements of water could be removed indirectly by an alternate method of p elimination (10). Percolation of the acetate of Compound VI through a column of neutial alumina gave the unsaturated triketone (Compound VII) which furnished the saturated triketone (VIII) on catalytic reduction. As an alter- native method of preparing Compound VII, it was observed that the 3-monoacetate Compound IX could be prepared in good yield from Compound II; oxidation of Compound IX followed by percolation of the product through neutral alumina gave VII. Finally the relationship between Compounds II, III, and VI was shown in another way by further oxidizing all three to a common product, namely the P-diketone 5@-androstane-1,3,11,17-tet- rone. The preparation and characterization of this substance, as well as the corresponding 11-deoxy-@diketone, will be de- scribed in a later paper.

The configuration of the hydroxyl group at C-l in Compound I was established as axial (p) from the NMR spectrum of Com- pound IX. The predominant oxidation at C-l and the selective

5313

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5314 Steroid I@-Hydroxylutim Vol. 241, No. 22

HO-C-H

B-Cortolone

HO .’ VI

H* I 1

0

O\

ih?P Ii

VIII

1) G-4 , 2)&o,

CH,OH

c: HO- -H

_.’ HO“’

I

NaIO, )

H2

0 0

\ cl!P OH H V

FIG. 1. Metabolic origin and degradation of Metabolite I. M, a metabolic reaction.

acetylation at C-3 in Compound II, as well as the reactions in- volved in the degradation of Compound XI (Fig. 3), all clearly indicate that the C-l hydroxyl group of Compound I is in the axial configuration and thus are in agreement with the NMR assignment.

Evidence confirming the configuration of the hydroxyl group at C-20 in Compound I was secured by relating it to I@, 3ar, li’a, 21-tetrahydroxy-5/%pregnane-11,20-dione (Compound X, Fig. 2) which was isolated in very small amounts after the oral adminis- tration of tetrahydrocortisone to the senior author. Sodium borohydride reduction of Metabolite X gave Metabolite I as the only product. Since this reagent is known to reduce steroidal 20-ketones almost exclusively to the 2Op-01s (12), this evidence confirms the configuration of the C-20 hydroxyl group of Com- pound I and establishes the structure of Compound X.

In order to prepare certain 17-ketones in the 11-deoxy series, Compound I was reduced by the Barton modification of the Wolff-Kishner method (13) to 5@pregnane-lb, 3a!, 17ar, 2Op ,21- pent01 (Compound XI, Fig. 3). Its further degradation differed from that of Compound I chiefly to the extent that the chromic anhydride-pyridine oxidation of XII gave a single product (the 1,17-diketone, Compound XIII), rather than two as in the case

of Compound II.* Acetylation of Compound XIII followed by percolation of the acetate through neutral alumina gave the unsaturated diketone (XIV) which also was prepared from Compound XII through the intermediate 3-acetate (XV). Partial hydrolysis of the diacetate (XVII) gave a monoacetate (not crystallized but chromatographically homogeneous) which was assigned the structure XVIII, since oxidation followed by percolation through neutral alumina converted it to the known unsaturated diketone (XIX). As in the case of Compound II, more vigorous oxidation of XII or XIII gave a /3-diketone as- signed the structure S/3-androstane-l , 3,17-trione.

Small amounts of a third l-oxygenated metabolite were re- covered following the administration of pregnanetriol to the senior author. Although the metabolite was not recovered in crystalline form, it appeared homogeneous in several paper chromatographic systems and had a mobility relative to pregnane- trio1 consistent with the introduction of one unhindered hy-

2 This is one of several examples of interaction between func- tional groups at C-l and C-11 noted in the course of this investiga- tion. In this case, the increased resistance of the axial hydroxyl group at C-l in Compound II to oxidative attack is ascribed to hydrogen bonding.

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Issue of November 25, 1966 J. J. Schneider and N. S. Bhacca

F H,OH FH,OH

5315

Ho ,,,,_, &p HO _,,,,,, -:1)-H M ,

Tetrahydrocortisone

M I

? H,OH

x

NaBH, I

TKOH

8-Cortolone I FIG. 2. Metabolic origin and reduction of Metabolite X. While p-cortolone is a major metabolite of tetrahydrocortisone in man

(3, II), it is not implied that the recovered metabolite, I, is derived solely from the former. 2K, a metabolic reaction.

droxyl group. Its mobility was unaltered when chromato- graphed on paper pretreated with boric acid and borate buffers with a pH range of 7.6 to 10; this method clearly differentiates between 17~~) 2Ocr-glycols and their 20-epimers since the mobility of the latter is markedly increased at low pH values and par- ticularly in the presence of boric acid itself (14). Sodium perio- date oxidation of the metabolite gave a single, easily crystallized 17-ketosteroid identical with Compound XII (Figs. 3 and 4). On the basis of this evidence, the metabolite was assigned the structure 5/?-pregnane-lP, 3a, 17a!, 20a!-tetrol (Compound XXI, Fig. 4).

Naturally occurring 1-hydroxylated steroids are found only in plants and include both cardiac-active glycosides and sapogenins (15). In all cases, the hydroxyl group at C-l is axial in con- figuration and, in those members lacking nuclear double bonds, the A:B ring juncture is cis. The introduction of both axially and equatorially oriented hydroxyl groups into position 1, chiefly of steroids bearing the 3-keto-4-ene system, has been effected microbiologically; specific examples are provided in a report of Dodson et al. (16).3 The several chemical approaches for effecting l-hydroxylation, all limited to A:B truns steroids, are listed in a recent paper of Djerassi, Williams, and Berkoz (19).

Our results show that, under what may be regarded as extreme conditions of administration, l/3-hydroxylation occurs to a demonstrable extent in three different 5&pregnane precursors in a man. We have been unable to show the excretion of Com- pound I normally or subsequent to adrenocorticotropic hormone

3 We have been unable to I-hydroxylate 3a-hydroxy-5p-andro- Stan-17-one or 3@-h.ydroxy-58-androstane-11,17-dione with a Peni- cillium species (ATCC i1598), Aspergillus niger (ATCC 6275), Rhizopus arrhizus (ATCC 11145), Aspergillus ochraceus (ATCC 12337), a Penicillium species (ATCC 12556), Penicillium chryso- genum (ATCC 12687), Penicillium notatum (ATCC 9478), or Peni- cillium spinulosum (ATCC 9123) as the enzyme source. Since the only examples of 1-hydroxylation of saturated 17-ketosteroids are the la-hydroxylation of 3@-hydroxy-5a-androstan-17-one (17) and the la-hydroxylation of 5a-androstane-3,17-dione (18), both by the fifth organism of this group, it seems likely that the type of A:B ring juncture is a limiting steric factor.

administration to the senior author, but we regard our isolation and detection methods as unsuited for this purpose. It seems reasonable to predict that steroid 1-hydroxylation in man will prove to be a generally occurring, if minor, metabolic reaction.

The result in vitro described in this paper extends the I@- hydroxylation of ,&cortolone to another species, the guinea pig, and shows that an organ site of this reaction is the liver. This result serves also as evidence against the possibility that the hydroxylation observed in the senior author was in fact effected by intestinal microorganisms. Other steroid hydroxylations effected by guinea pig liver in vitro include the 2cu-hydroxylation of cortisol (20), the 6@-hydroxylation of cortisol (21), and the 7ol-hydroxylation of 21-hydroxypregn-4-ene-3,20-dione (22) .4 It is to be noted that all the metabolites described in this paper conform to the rule for corresponding steroids of plant origin, namely that the hydroxyl group at position 1 is in the axial configuration.

We feel certain, from a consideration of the isolation procedure, that Metabolite I recovered after /?-cortolone administration is excreted in the free form rather than liberated from a conjugate in the course of the several manipulations. But we have been unable definitely to exclude the possibility that this metabolite also is excreted in a conjugated form. Since excreted p-cortolone is wholly conjugated with glucuronic acid (2), it seemed reason- able to expect that its metabolite, which differs only slightly from it, also would be excreted in this form. Such a conjugate would be extraordinarily polar (water-soluble), and difficult not only to extract from urine but to dissociate from nonsteroidal polar material. In support of the assumption that Metabolite I is excreted solely as the free compound, it can be argued that its high water solubility (12.8 mg per ml at 38”, whereas that of /3- cortolone is 0.72 mg per ml at this temperature) makes any further gain in water solubility unnecessary. This assumes, with Williams (23), that the main utility of conjugating a bio- logically inactive metabolite is to increase its water solubility.

4 Paper chromatographic evidence; metabolite was not formally identified.

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5316 Steroid Ifi-Hydroxylation

? HzOH CH,OH

HO-&H HO-&-H

XI

NaIO,

Aco . . . . . . . @ iAc20 Hb ./.. :&9 Acpo’ xv XII

Act ,/... -:;::--

XVII

OH-

0

AC?

-.: . ..’

HO’.’ H XVIII

1) croa 2) ALO, I

0

@

/

OH H XIX

Vol. 241, No. 22

FIG. 3. Preparation and degradation of 5~-pregnane-lp,3~,17cu,20~,2l-pentol (Compound XI). W:K:B, Wolff-Kishner-Barton reduction.

H-C-OH

Pregnanetriol XXI XII FIG. 4. Metabolic origin and oxidation of Metabolite XXI. M, a metabolic reaction.

The method used to recover the 1-hydroxylated tetrahydro- cortisone (Compound X) was based on the assumption that it

EXPERIMENTAL PROCEDURE’

was excreted solely in the free form and no attempt was made to investigate its possible conjugation. But in the case of the

Isolation and Degradation of 1 B , Scr ,17a:, SOP, 21 - Pentahydroxy-6&vegnan-II-one

tetrol (XXI) derived from the administered pregnanetriol, it is clear from its position relative to the pregnanetriol glucuronoside

Preparation of P-Cortolone-Reduction of tetrahydrocortisone

at the conclusion of the countercurrent distribution that it was in dry methanol with an excess of sodium borohydride for 1 hour

excreted as a conjugate, presumably a glucuronoside. Hy- 5 Melting points were determined with a Fisher-Johns apparatus

droxylation at C-l is therefore not of itself a bar to conjugation. and are recorded as read. Optical rotations were obtained with a Zeiss 0.005” photoelectric polarimeter. Measurements were

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Issue of November 25, 1966 J. J. Schneider and N. S. Bhacca 53 17

at O-5” gave, following three crystallizations from methanol, @-cortolone in 70% yield. Constants: m.p. 262-263”; [a]= $43.3”. In the literature (3) : m.p. 260-261.5”; [aID +40” (ethanol).

‘&H,aO~

Calculated: C 68.81, H 9.35 Found : C 68.70, H 9.20

Examination of the product by a paper chromatographic method which separates p-cortolone from its C-20 epimer (14), and which utilizes periodic acid oxidation in situ followed by application of the Zimmermann reagent as the method of detection (24), confirmed its homogeneity.

Administration of p-Cortolone and Extraction of Urine--In a representative experiment, a total of 15 g of p-cortolone in aqueous ethanol was administered orally to the senior author over a 4-day period, as hourly 250-mg doses, from 6 a.m. to 8 p.m. daily. The urine was collected from the initiation of administra- tion to a point 12 hours following its cessation and was stored at 10” prior to extraction.

The methods of extraction and subsequent fractionation are based on the assumption that the metabolite is excreted solely as the free substance. The pH of the pooled urine was adjusted to approximately 8 (to reduce emulsion formation and limit ex- traction of the ,&cortolone glucuronoside), and it was extracted 25 times with wet n-butyl alcohol. The combined extracts were evaporated6 to dryness without prior washing. The neutral n-butyl alcohol extract, obtained as indicated previously (Refer- ence 2, p. 206), weighed 10.5 g.

Countercurrent Distribution of Neutral n-Butyl Alcohol Extract- The use of countercurrent distribution as an initial fractionating step illustrates the ability of this method to handle weighty ex- tracts without distortion and to concentrate substances of un- known stability without hazard. The neutral n-butyl alcohol extract was dissolved in some of the lower phase of System 1, placed in the first 11 tubes of a 200-tube Craig-Post (25) all glass countercurrent distribution apparatus, and submitted to 150 transfers. The metabolite has a partition coefficient, K (25), of approximately 2.1 in this system, and was well separated from the greater part of the extraneous material which moved but slowly. The limits of the metabolite band were defined by paper chroma-

made in methanol at 26-27” and at a concentration of around 1.5%. The absorption spectra of all compounds were determined in methanol over the range of 200 to 350 rnp with a Zeiss PM& II spectrophotometer, but values are recorded only for those sub- stances which showed specific absorption due to a particular chro- mophore. Infrared spectra were determined as KBr pellets with the use of a Beckman IR-8 infrared spectrophotometer. Our paper chromatographic methods have been outlined in recent pub- lications (14, 22). The composition of systems referred to by number in the text appears in Table I. Our method for preparing partition type columns is indicated in the first example under “Experimental Procedure.” Particular care was taken to ensure even packing. These columns were prepared and developed at 25 =t 1” and at generally low rates of flow. Most of the elementary analyses were those of E. Thommen, Basel, Switzerland.

6 The limited use of n-butyl alcohol as an agent for extracting polar or conjugated steroids from urine appears to center on diffi- culties in concentrating the extract. We use a large (Rinco) evaporator closely connected to a large trap immersed in a Dry Ice-ethanol mixture and evacuate the system with a two-stage mechanical (oil) pump. With such an apparatus, extracts can be concentrated at temperatures under 30” and at rates approaching 2 liters per hour. It is essential to employ only the best grade of n-butyl alcohol and to distill it prior to use.

TABLE I Composition of paper and column chromatographic systems

-

n

-_

Systen

1

2

3

4 5

6

7

8

9 10 11 12

13

14

15

16

17 18

19

20 21

22

23

24

25

Composition

NH,OH (2&30y0 NH,), 1.2 ml, diluted with water to 180 ml; n-butyl alcohol, 80 ml; ethyl acetate, 100 ml

Ethyl acetate, 150 ml; isooctane,a 50 ml; methanol, 100 ml; water, 100 ml

Ethyl acetate, 200 ml; methanol, 20 ml; water, 180 ml (alternate, simpler system is ethyl acetate-water)

Toluene, 200 ml; methanol, 140 ml; water, 60 ml Toluene, 150 ml; isooctane,a 50 ml; methanol, 140 ml;

water, 60 ml Toluene, 60 ml; isooctane,a 140 ml; methanol, 140 ml;

water, 60 ml Ethyl acetate, 30 ml; isooctane,” 170 ml; methanol, 100

ml; water, 100 ml Toluene, 65 ml; isooctane,” 135 ml; methanol, 150 ml;

water, 50 ml Ethanol, 20 ml; diluted with ethyl acetate to 100 ml Toluene, 125 ml; tert-butyl alcohol, 75 ml; water, 150 ml Toluene, 130 ml; tert-butyl alcohol, 70 ml; water, 150 ml Toluene, 70 ml; isooctane,a 130 ml; tert-butyl alcohol, 50

ml; methanol, 50 ml; water, 150 ml NHIOH (28-30% NH,), 1.2 ml, diluted with water to 180

ml; n-butyl alcohol, 60 ml; ethyl acetate, 120 ml Ethyl acetate, 140 ml; isooctane,a 60 ml; methanol, 100

ml; water, 100 ml Isooctane,a 110 ml; tert-butyl alcohol, 90 ml; water, 150

ml Toluene, 145 ml; tert-butyl alcohol, 55 ml; methanol, 70

ml; water, 80 ml Toluene, 200 ml; methanol, 130 ml; water, 70 ml Toluene, 160 ml; isooctane,a 40 ml; methanol, 120 ml;

water, 80 ml Toluene, 100 ml; isooctane,a 100 ml; methanol, 150 ml;

water, 50 ml Isooctane,a 200 ml; methanol, 160 ml; water, 40 ml Toluene, 60 ml; isooctane,” 140 ml; methanol, 150 ml;

water, 50 ml Toluene, 25 ml; isooctane,a 175 ml; methanol, 150 ml;

water, 50 ml Ethyl acetate, 200 ml; glacial acetic acid, 20 ml; water,

180 ml

I. Toluene, 170 ml; tert-butyl alcohol, 30 ml; methanol,

50 ml; water, 100 ml Toluene, 150 ml; ethyl acetate, 50 ml; methanol, 100 ml;

water, 100 ml -

a 2,2,4-Trimethylpentane.

tography of small aliquots of the residues from tubes 70 through 90 and 115 through 130 with the use of System 2 and detection of the metabolite (RF = 0.18) by the method indicated above (24). The residue obtained by combining the contents of tubes 75 through 130 weighed 1.3 g.

Column Chromatography of Distributed Extract-Separation of Metabolite I from the remaining extraneous material and from small amounts of two closely associated steroids7 was effected by partition column chromatography. Four hundred grams of Celite 545, suspended in the mobile phase, were stirred with 200

’ These are the 3a,6cu(and GP),17cu,20p,2l-pentahydroxy-5fl- pregnan-U-ones. Their purification and identification are de- tailed in the accompanying paper (26).

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5318 Steroid l&Hydroxylution Vol. 241, No. 22

FIG. 5. Infrared spectrum of lp,301,1701,20@,21-pentahydroxy-5fi-pregnan-11-one (Metabolite I). Upper curve, from reference com- pound; lower cww, from specimen isolated in guinea pig experiments in vitro. In each case, approximately 1.2 mg of steroid were dis-

I I I I I I 3500 3ooo 2500 1500 1cJoo 600

-1 wavenumber cm

persed in 400 mg of KBr.

ml of the stationary phase of System 3 and packed into a glass column, 51 X 640 mm. The above residue, dispersed on Celite, was placed at the top of the column which then was developed at a rate of 40 ml per hour, with indexing adjusted to give lo-ml fractions of effluent. The efficiency of fractionation and the limits of the band of interest were established by paper chroma- tography with the use of System 2. In this case, the metabolite occupied tubes 226 through 310 and contained only traces of the noted companions.

A methanol solution of the pooled residues was treated briefly with charcoal and filtered. The filtrate was concentrated to a small volume with a nitrogen stream, diluted with acetone, and seeded with crystals from a previous lot. Recrystallization from methanol gave 577 mg of colorless prisms. Constants: m.p. 252-253”; [aID +52.2”; RF values were 0.18 (System 2) and 0.28 (System 3). Its infrared spectrum is indicated in Fig. 5. Its designation as lp, 3a, 17~~) 20@, 21-pentahydroxy-5&pregnan-ll- one (Compound I, Fig. 1) is based on the chemical and NMR spectral evidence which follow.

CdL406

Calculated: C 65.94, H 8.95, 0 25.09 Found: C 66.11, H 8.84, 0 (Unterxaucher) 25.17

Sodium Periodate Oxidation of Compound I-A concentrated solution of 457 mg of Compound I in warm ethanol was diluted with 60 ml of warm water. After cooling to room temperature, a solution of 1.14 g of H5106 in 6 ml of water adjusted to approxi- mately pH 4 was added after which the pH of the solution was adjusted to approximately 6. After 15 hours at room tempera- ture in the dark, the pH (which had decreased to around 4) was adjusted to about 7, salt was added, and the solution was ex- tracted repeatedly with ethyl acetate. The combined extracts

were washed once with neutral brine, dried with anhydrous sodium sulfate, and evaporated to dryness under reduced pres- sure. Crystallization of the product from acetone-n-hexane and from acetone gave 325 mg (84 %) of prismatic needles, designated I@, 3a-dihydroxy-5@-androstane-ll , 17-dione (Compound II in Fig. 1). Constants: m.p. 235-236”; [cu], +124.5”.

GJbOa

Calculated: C 71.21, H 8.80 Found : C 71.27, H 8.86

Paper chromatography of Compound II in System 4 (RF = 0.17) showed that no more polar or more mobile Zimmermann- positive substances were present. I f unneutralized periodic acid is used in this oxidation, or if sulfuric acid is included in the reaction mixture, the product is contaminated with small amounts of a secondary oxidation product (Compound VI, see below) which cannot be removed by simple crystallization.

Chromic Anhydride-Pyridine Oxidation of Compound II to III and VI-To a solution of 315 mg of Compound II in 40 ml of pyridine (reagent grade, distilled from potassium hydroxide), 315 mg of chromic anhydride in 78 ml of pyridine were added. The solution was kept at room temperature for 36 hours, diluted slightly with methanol, and, 2 hours later, concentrated to near dryness under reduced pressure. The residue was wet with methanol, diluted with brine, and extracted with ethyl acetate. The organic phase was washed successively with acid, alkaline, and neutral brine, dried with anhydrous sodium sulfate, and evaporated to give a 223-mg neutral fraction. Re-extraction of the combined aqueous phases following acidification gave an acidic fraction weighing 67 mg.

Paper chromatography of an aliquot of the neutral fraction in System 5 showed the presence of traces of Compound II (RF =

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Issue of November 25, 1966 J. J. Schneider and N. X. Bhacca 5319

0.07), the major product (Compound VI, Fig. 1; RF = 0.15), the pyridine (0.3 ml) for 16 hours at room temperature. The re- minor product (Compound III, Fig. 1; RF = 0.31), and traces of covered acetate, which resisted crystallization, was applied in a very mobile substance, RF approximately 0.85. The ratio of benzene to a column, 10 x 100 mm, of alumina (Camag, Grade VI:111 was judged to be about 2: 1. Partition column chroma- 507-C, Brockmann No. 1, neutral) prepared with the same tography of the remaining neutral fraction by the use of System solvent. Following slow percolation with benzene and with 5 easily separated the products, giving, from acetone, 77 mg of 0.15% ethanol in benzene, the product was eluted with 0.30% l&hydroxy-5&androstane-3,11,17-trione (Compound III, Fig. ethanol in benzene. Crystallization from acetone-n-hexane 1) and, from methanol, 107 mg of 3cu-hydroxy-5@-androstane- gave 22 mg of 5P-androst-2-ene-l , 11,17-trione (Compound VII, 1,11,17-trione (Compound VI, Fig. 1). Constants for Com- Fig. 1). Constants: m.p. 223.5-224.5”; [cr], +95.5”; X,,, = 225 pound III: m.p. 210-211”; [alI, f137.4”; RF = 0.30 (System 5). mp; e = 70508; Rp = 0.16 (System 6) and 0.08 (System 7).

CdMh G&403

Calculated: C 71.66, H 8.23 Calculated: C 75.96, H 8.05 Found : C 71.66, H 7.82 Found : C 75.71, H 7.92

Constants for Compound VI: m.p. 262-263”; [ar], +88.2”; RF = Alternate Conversion of Compound II to VII via IX-One

0.15 (System 5). hundred and fi f ty milligrams of Compound II were treated for 1

GJMh hour with a mixture of 37.5 ml each of pyridine and acetic an- hydride. The neutral product (170 mg) was chromatographed

Calculated: C 71.66, H 8.23 on a Celite column, 31 X 675 mm, prepared with System 8. Found: C 71.59, H 8.30 Seven-milliliter fractions of effluent were collected at 15-min

Dehydration of Compound III to IV-Treatment of 25 mg of intervals. What was assumed to be the diacetate of Compound

Compound III in methanol with 5 ml of 1 N sodium hydroxide for II (25 mg, not crystallized) was contained within tubes 68 to 112.

3 hours at room temperature followed by isolation of the neutral The main product, assigned the structure l&hydroxy-3cr-ace-

product in the usual fashion gave, from acetone-n-hexane, 18 mg toxy-5P-androstane-ll , 17-dione (Compound IX, Fig. l), was

of Compound IV as prisms with the following constants: m.p. eluted in tubes 261 to 350 and furnished, from ethyl acetate-n-

180-181”; [(Y]~ +222.0”; X,,, = 225 ml; e = 9380. hexane, 116 mg of colorless prisms. Constants: m.p. 156.5 157.5”; [a], +140.7”.

C~Hz403 CzJLaO~

Calculated: C 75.96, H 8.05 Found: C 75.92, H 8.06 Calculated: C 69.58, H 8.34

Found : C 69.77, H 8.26

A reference sample of 5/3-androst-1-ene-3,11,17-trione, prepared by the oxidation of 17~~) 21-dihydroxy-5&pregn-1-ene-3,11,20-

In effecting the oxidation of Compound IX, 98 mg of the pure

trione (27) with sodium bismuthate, had the constants: m.p. substance in 2 ml of glacial acetic acid were treated with 1.2 eq

177-178”; [cu], +219.2”. Recorded constants for 5/3-androst-l- of chromic anhydride in 0.1 ml of water. Since the oxidant

ene-3,11,17-trione are: m.p. 174.5-177.5”; [&, +205” (CHClJ; seemed to be reduced only slowly, the reaction mixture was

x Inax = 224 mp; E = 9425 (28). The melting point of the de- allowed to stand at room temperature for 2 hours. The re-

rived unsaturated triketone (IV) was unaltered on admixture covered neutral fraction was percolated through neutral alumina

with the reference sample of 5/3-androst-1-ene-3,11,17-trione. in the manner described above for the conversion of Compound

Both had Rp values of 0.54 (System 6) and 0.45 (System 7). VI to VII. The fraction eluted with 0.3% ethanol in benzene

Their infrared spectra were identical. gave, from acetone-n-hexane and from acetone, 78 mg of colorless

Catalytic Reduction of Compound IV to V-To 15 mg of Com- prisms. Constants: m.p. 223-224.5”, unchanged on admixture

pound IV in 4 ml of cyclohexane-absolute ethanol (5: 1) were with the sample of Compound VII prepared from VI; [ar],

added 45 mg of 5% palladium-carbon. After shaking for 1 hour +93.9”; A,,, = 225 mp; e = 7100.

in a hydrogen atmosphere, the product was recovered in the Catalytic Reduction of Compound VII to VIII-This was

usual manner. Crystallization from methanol-ether gave 11 mg carried out as in the above reduction of Compound IV to V.

of Compound V: m.p. 131.5-132.5”; [arID +112.2”. Similar re- From 40 mg of Compound VII, 31 mg of needles were obtained

duction of the reference sample of 5&androst-1-ene-3,11,17- from ethyl acetate-n-hexane. This product was assigned the

trione furnished an authentic specimen of B/3-androstane- structure 5/3-androstane-l , 11,17-trione (Compound VIII, Fig.

3,11,17-trione. Its melting point was 131-132“, and remained 1). Constants: m.p. 202-203”; [a&, $81.9’; Rp = 0.31 (System

unaltered when the derived and authentic samples were melted 6) and 0.23 (System 7). It showed no specific absorption over

together. Both had RF values of 0.57 (System 6) and 0.51 the range of 290 to 350 rnp.

(System 7). Neither compound showed any specific absorption Cd%&3 in the region of 200 to 350 mM. Calculated: C 75.45, H 8.66

Cdb03 Found : C 75.28, H 8.79

Calculated: C 75.45, H 8.66 Found: C 75.34, H 8.73

* It is to be noted that, although the extinction coefficients of Compounds IV and VII differ, their absorption maxima are so

Dehydration of Compound VI to VII-Thirty-three milligrams similar that differentiation on the basis of ultraviolet spectroscopy alone would be impossible. Their ready discrimination by means

of Compound VI were treated with acetic anhydride (0.2 ml) and of NMR spectroscopy is discussed at a later point in this paper.

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5320 Steroid I&Hydroxylation Vol. 241, No. 22

ID-Hydroxylation of /3-Cortolone by Surviving Liver Slices of Guinea Pig

Incubation of ,L-Cortolone and Isolation of Metabolites-One hundred and twenty milligrams of p-cortolone, in the minimum volume of warm ethanol, were diluted to 480 ml with warm Tris buffer, pH 7.4. Ten-milliliter volumes of this solution, in 300- ml Erlenmeyer flasks, were incubated for 2 hours (38”, gas phase oxygen) with liver slices prepared from adult male and female animals, corresponding to a tissue to steroid ratio of not less than 1OOO:l. Each incubate was diluted with hot methanol and filtered; the insoluble portion was washed repeatedly with hot methanol and the pooled filtrates were concentrated to near dryness under reduced pressure. tert-Butyl alcohol was added to suppress foaming. After partitioning the concentrate between 60% (v/v) aqueous methanol and n-hexane, the aqueous alcoholic phase was concentrated to a small volume, diluted with water, and extracted with wet n-butyl alcohol. The pooled extracts were concentrated to dryness under reduced pressure and the residue was dispersed on Celite.

Separation of the metabolites from the greater part of the extraneous material was effected by means of adsorption column chromatography. The dispersate was placed at the top of a column, 25 x 340 mm, of silica gel (Davison, Grade 923, 100 to 200 mesh) prepared and developed with System 9. Seven- milliliter fractions of effluent were collected at a rate of 4 per hour. On the basis of paper chromatographic evaluation of the residues from every 10th tube (System 10 with Compound I as the standard of reference), the contents of tubes 61 to 210 were pooled and evaporated to dryness under reduced pressure.

The metabolites were separated from each other and from the remaining extraneous material on a Celite partition type column, 20 x 745 mm, prepared with System 11. Five-milliliter frac- tions were collected at a rate of 4 per hour. In terms of the paper chromatographic assessment indicated above, the first metabolite was confined to tubes 85 to 107 and its companion to tubes 125 to 153. Both gave near colorless residues on concentration.

The first residue gave, from acetone, 5.5 mg of prisms with the following constants: m.p. 252-253”, unaltered on admixture with the reference specimen of Compound I; [cy], +51.0”. Both the reference and isolated metabolites had an Rp value of 0.26 in System 10. The infrared spectra of both were identical (Fig. 5). This served to identify the first, more mobile, metabolite as l/3, 3a, 17a(,20P,21-pentahydroxy-5@pregnan-11-one (Compound I, Fig. 1).

The second residue yielded 3.2 mg of prisms from acetone, m.p. 277-279”. Its Rs value in System 10 was 0.19. Its in- frared spectrum differed significantly from that of Compound I in the region of 1500 to 600 cm-l. This metabolite was charac- terized further by the sequential oxidation procedure (Fig. 1) em- ployed in the degradation of Compound I. Sodium periodate oxidation, as in the conversion of Compound I to II, gave cubic crystals from methanol-acetone which melted at 294-295”. The RF value of this product in System 12 was 0.16, whereas that of Compound II was 0.25: ,both comppunds eve intense colors with the Zimmermann reagent, characteristic of 11,17-diketones. Further oxidation of the new 17-ketosteroid, under conditions known to convert Compound II to 5fl-androstane-l , 3,11,17- tetrone, gave a product which was less mobile than the tetrone and which did not absorb ultraviolet light. This evidence allows the conclusion that the second, chromatographically more polar, metabolite is not the I-epimer of the first, that is, of Compound I.

The paper chromatographic characteristics of the unidentified metabolite also differed from those of the 3cr,6ac(and 60)) 17a, ZOfi, 21-pentahydroxy-5@-pregnan-1 l-ones.

In parallel with the above experiment, the adrenal glands from the same animals were divided and incubated aerobically with &cortolone by the use of the tissue to steroid ratio and other conditions indicated above. Only traces of metabolites more polar than p-cortolone could be detected, and none of these had the paper chromatographic properties of Compound I.

Isolation and IdentQication of ifl ,Soc,lYor,dl-Tetrahydroxy- G/3-pregnane-11 ,20-dione

Administration of Tetrahydrocortisone and Fractionation of Crude Extract-Twelve grams of tetrahydrocortisone were administered to the senior author over a a-day period as in the p-cortolone experiments. The urine was extracted with wet n-butyl alcohol at pH 8 and the neutral n-butyl alcohol extract (11 g) was distributed through 150 transfers with System 13. Examination of the residues from tubes 51 through 110 by paper chromatography (System 2) showed that a substance with the mobility of 10,3a!, 17a, 200,21-pentahydroxy-5&pregnan-1 l-one (Compound I, Fig. 1) occupied tubes 61 through 105 (K = ~1.4). Partition column chromatography of the pooled residues (650 mg) with System 3 gave 198 mg of prisms with the same con- stants and infrared spectrum as the previously characterized Metabolite I.

Examination of the residues from tubes 80 through 145 by paper chromatography (System 14) showed that a blue tetra- zolium-positive compound, RF = 0.30, occupied tubes 115 through 140 (K = -5.8). The pooled residues (420 mg) were fractionated on a Celite column, 31 x 680 mm, prepared with System 14. The fractions of interest (totaling 51 mg) contained, as the chief contaminants, small amounts of blue tetrazolium- positive compounds both slightly more polar and more mobile than the major metabolite. Following investigation of this mixture with 17 different paper chromatographic systems, it was further fractionated on a Celite column, 24 X 690 mm, prepared with System 15. The recovered metabolite (RF = 0.36 in this system) was free from the noted contaminants and furnished, first from methanol and then from methanol-ether, 11 mg of colorless rosettes.

Identijication of Metabolite as l~,Sa,iYor,Wi-Tetrahydroxy-6& pregnane-11 ,dO-&one-Following small scale experiments in which it was shown that sodium borohydride converted the metabolite to a compound with the same paper chromatographic mobility as Compound I, 6.5 mg of the pure metabolite were treated with sodium borohydride as in the preparation of p- cortolone from tetrahydrocortisone. The neutral product gave, from acetone, 3.8 mg of prisms, m.p. 251-252”. The melting point was unaltered on admixture with an authentic specimen of Compound I, and the infrared spectra of the two compounds were identical. The I-hydroxylated metabolite of tetrahydrocorti- sone was therefore assigned the structure lb, 3a, 17a!, 21-tetra- hydroxy~5~-p.re~ane-l1,20-dione (Compound X, Fig: 2).

Preparation and Degradation of 5B-Pregnane- 10, QCY , i7a, 20@, 21 -pent01

Wol$-Kishner-Barton Reduction of Compound I to XI-In the first trial, 200 mg of Compound I were added to a cooled solution of 130 mg of sodium in 6.5 ml of dry diethylene glycol. Dry hydrazine was then distilled into the reaction mixture until the

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solution refluxed freely at around 165”. Twelve hours later, sufficient hydrazine was distilled out of the system to raise its temperature to around 210”, at which it was maintained for 24 hours. Paper chromatography of an aliquot of the recovered neutral fraction (System 16) showed that only small amounts of Compound I remained (RF = O.lO), and that the major product had an RF value of 0.26. Chromatography of the remaining neutral fraction on a Celite column, 30 X 775 mm, prepared with System 16 gave 109 mg of product. A second trial, carried out under similar circumstances, furnished 140 mg of the desired pentol. It has not been obtained in crystalline form. Con- stants: [a(]= $6.3”; RF = 0.26 (System 16). Its designation as 5/Lpregnane-lp , 301,17a, 20@, 21-pent01 (Compound XT, Fig. 3) is based on its paper chromatographic mobility relative to Compound I, on the absence of any specific absorption over the range 2000 to 1500 cm-l, and on its subsequent reactions.

Sodium Periodate Oxidation of Compound XI to XII-Two hundred milligrams of Compound XI, oxidized under the condi- tions employed in the preparation of Compound II from I, gave, from acetone, 165 mg of needles. Constants: m.p. 204-204.5’; [c& t-94.7”; RF = 0.35 (System 17) and 0.25 (System 18). This product was assigned the structure lfl,3a-dihydroxy-5/I- androstan-17-one (Compound XII, Fig. 3).

Calculated: C 74.47, H 9.87 Found : C 74.34, H 9.99

Chromic Anhydride-Pyridine Oxidation of Compound XII to XIII-Fifty milligrams of Compound XII in 17.5 ml of pyridine containing 50 mg of chromic anhydride were stored at room temperature for 44 hours. Paper chromatography of an aliquot of the recovered neutral fraction in System 19 showed that only traces of Compound XII remained (RR = O.OS), and that the only detectable Zimmermann-positive compound present had an RF value of 0.31. The neutral fraction was chromatographed on a Celite column, 20 X 615 mm, prepared with System 19. Two crystallizations of the recovered product from acetone-ether gave 27 mg of pure XIII with the following constants: m.p. M-182”; [cu], -20.8”; RF = 0.30 (System 19). The designation of this compound as 3a-hydroxy-5/?-androstane-l , 17-dione (Compound XIII, Fig. 3) is based on its further degradation and on its relation to other degradation products in this series.

Calculated: C 74.96, H 9.27 Found: C 74.96, H 9.42

Dehydration of Compound XIII to XIV-Treatment of 21 mg of Compound XIII with acetic anhydride and pyridine followed by percolation of the crude acetate through a small column of neutral alumina as in the preparation of Compound VII from VI gave, from methanol, 16 mg of needles. Constants: m.p. 197-198”; [a], +2.7”; X,,, = 226 mp; E = 7600; RF (System 20) = 0.43. Its designation as 5P-androst-2-ene-l , 17-dione (Compound XIV, Fig. 3) is supported by its ready formation from Compound XIII by @ elimination of the acetoxyl group and by its spectrum in the ultraviolet region.

CdLsO~

Calculated: C 79.67, H 9.15 Found : C 79.50, H 9.00

Catalytic Reduction of Compound XIV to XVI-Palladium- catalyzed hydrogenation of a 15-mg sample of Compound XIV as in the preparation of Compound VIII from VII gave, from n-hexane, 10 mg of plates. Constants: m.p. 155-156”; [a], -34.4”; no specific absorption in the region of 200 to 300 rnE.1; RF = 0.66 (System 20). It was assigned the struct.ure 5p- androstane-1 ,17-dione (Compound XVI, Fig. 3).

Calculated: C 79.12, H 9.79 Found : C 79.32, H 10.02

Alternate Preparation of Compound XIV from XII via XV- A solution of 60 mg of Compound XII in 6 ml each of acetic anhydride and pyridine was kept at 25” for 1 hour. Paper chromatography of an aliquot of the neutral fraction (System 21) gave the following RF values: 0.25 (l-acetate), 0.55 (a-acetate), and 0.87 (diacetate), these assignments being made on the basis of the greater retarding effect of the C-3 equatorial hydroxyl group as compared with the C-l axial hydroxyl function. Chro- matography of the neutral fraction on a Celite column, 20 X 710 mm (System 22), furnished, from acetone-n-hexane, 43 mg of I@-hydroxy-3a-acetoxy-5P-androstan-17-one (Compound XV, Fig. 3). Constants: m.p. 160-160.5”; [c& +11.9”; RF (System 21) = 0.55. Its designation as the a-acetate (XV) follows from its conversion to XIV.

CdL,Oa

Calculated: C 72.38, H 9.26 Found : C 72.59, H 9.42

To a solution of 22.8 mg of Compound XV in 1 ml of glacial acetic acid, 7.8 mg of chromic anhydride in 0.01 ml of water and 0.99 ml of glacial acetic acid were added. After 13 hours at 25”, methanol was added and the neutral fraction was recovered in the usual fashion. This was percolated through a small column of neutral alumina as in the previous examples. The product, which was eluted with 0.1% ethanol in benzene, was crystallized from methanol to give 14 mg of needles which had the same constants as those indicated above for pure XIV: m.p. 197-198”, unchanged on admixture with the sample of Compound XIV prepared from XIII; [aID $2.2”; X,,, = 225 mp; E = 7650; RF (System 20) = 0.43.

Degradation of Compound XII to XX via XVII, XVIII, and XIX-This sequence, which is outlined in Fig. 3, served to re- late the dihydroxyketone XII to the known compounds, XIX and XX. It is based on the fact that the saponification, as well as the formation, of the C-3 equatorial acetoxyl group pro- ceeds at a rate exceeding that of the C-l axial acetoxyl function.

Twenty milligrams of Compound XII were treated for 48 hours at room temperature with a mixture of 0.3 ml of pyridine and 0.2 ml of acetic anhydride. Paper chromatography of an aliquot of the neutral fraction (not crystallized) with the use of System 20 showed that the diacetate only (RF = 0.63) was present. Seventeen milligrams (0.043 mmole) of this product in 0.5 ml of methanol were treated with 50 ~1 (0.05 mmole) of aqueous 1 N sodium hydroxide for 10 min at 25”. After adding acetic acid, the neutral fraction was recovered in the usual fashion. Paper chromatography by the use of System 21 showed that a trace only of the 3-acetate (RF = 0.56) was present and that the major product was the l-acetate (RF = 0.26). Chromatography of the neutral fraction on a Celite column,

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5322 Steroid I@-Hydroxylation Vol. 241, No. 22

16 X 250 mm (System 21), gave 9 mg of a product assigned the structure 3cr-hydroxy-I@-acetoxy-5@-androstan-17-one (Com- pound XVIII, Fig. 3). No attempt was made to crystallize or otherwise characterize it. Oxidation of this substance with chromic anhydride, as in the other examples, gave a crude neutral fraction weighing 8.2 mg. Paper chromatography of an aliquot by t.he use of System 20 showed that the major product had an Rs value of 0.15 and that a mobile contaminant, RF = 0.33, was present. The remaining neut,ral fraction was percolated through neutral alumina in the usual manner. Elution with 0.1% ethanol in benzene gave a fraction from which 6.4 mg of needles were obtained from acetone-n-hexane. This compound, which was assigned the structure 5/3-androst-l-ene-3,17-dione (Com- pound XIX, Fig. 3), had the constants: m.p. 171-172”; [a~], $238.2”; A,,, = 230 mp; e = 9600; RF (System 20) = 0.33. On admixture with a reference sample of 5fi-androst-1-ene-3,17- dione, m.p. 170-171”, the melting point was unchanged. The infrared spectra of the derived and reference samples were identical.

A 4.1~mg sample of Compound XIX, recovered from the optical rotation determination, was reduced catalytically in the usual fashion. Crystallization of the product from aqueous acetone and from acetone-n-hexane gave 2.8 mg of needles which melted at 130-131”. On admixture with a reference sample of 5B-androstane-3,17-dione, m.p. 13&131”, the melting point was unaltered. The infrared spectra of the reduction product and the reference diketone were identical. The saturated diketone XX was therefore assigned the structure 5/Landrostane-3,17- dione.

Isolation and Characterization of 5P-Pregnane- lp,Sa,l~cX,do)cr-tetrol

Preparation of Pregnanetriol Bishemisuccinate-Eight grams of pregnanetriol (m.p. 252-253”; [(u], -2.4”), prepared as indi- cated earlier (14), and 60 g of succinic anhydride were main- tained at 60” for 4 hours in the minimum volume of dry pyridine. From the acid fraction there were obtained, from aqueous ethanol, 10.2 g (80%) of the bishemisuccinate as fine needles. Constants: m.p. 178180”; [cr], -0.5”.

C~L~OQ

Calculated: C 64.90, H 8.26 Found: C 65.19, H 8.41

Administration of Bishemisuccinate-To a solution of 5.56 g of the bishemisuccinate (equivalent to 3.0 g of the free triol) in 50 ml of ethanol, a solution of 1.1 g of sodium carbonate in 1200 ml of cold water was added. The clear solution had a pH of approximately 6.5; it was kept at -5” during the period of administration. One hundred-milliliter volumes of this solution were taken hourly, orally, by the senior author, and his urine was collected during the 48-hour period9 following the initiation of administration.

The urine was adjusted to pH 2.5 by the addition of dilute hydrochloric acid, after which it was extracted 20 times with wet n-butyl alcohol. The combined extracts were diluted somewhat with dry n-butyl alcohol, adjusted to a pH of approximately 5 by shaking with small volumes of aqueous sodium carbonate

Q The collection period was extended to compensate for the oli- guria which accompanied the administration of this steroid.

solution, and evaporated to dryness under reduced pressure. The neutral n-butyl alcohol extract, obtained as indicated earlier (2), weighed 13.5 g.

Ijsractionation of Neutral n-Butyl Alcohol Extract-The crude extract was dissolved in 50 ml of the lower phase of System 23, adjusted to a pH of approximately 3 by adding 5 N hydrochloric acid, diluted with additional lower phase, and divided between tubes 0 through 10 and 120 through 130 of the countercurrent distribution apparatus. After 100 transfers, the contents of tubes 0 through 90 and 120 through 210 individually were withdrawn and evaporated. Wholly on the basis of inspection, the following pooled fractions were prepared: (a) the neutral aqueous phase remaining after removal of the n-butyl alcohol- soluble fraction, (b) the contents of tubes 0 through 22 plus 120 through 142, (c) the contents of tubes 23 through 64 plus 143 through 184, and (d) the contents of tubes 65 through 90 plus 185 through 210. Fractions a, b, and c separately were dissolved in dilute acetate buffer, pH 4.5, and incubated for 36 hours with a large excess of B-glucuronidase. The incubates were extracted with ethyl acetate and the neutral fractions were recovered in the usual manner.

Fraction d was dissolved in methanol and the solution was treated briefly with charcoal and filtered. The filtrate was concentrated to near dryness, diluted slightly with water, and adjusted to a pH of about 8 by the cautious addition of dilute aqueous sodium hydroxide solution. Following concentration to a small volume with a stream of nitrogen, the solution was slowly diluted with hot acetone until crystallization began. Two additional crystallizations from aqueous acetone gave 1.85 g of sodium pregnanetriol glucuronosidate as colorless plates.

The neutral residues from Fractions a, 6, and c were examined by paper chromatography by the use of System 24 and the use of the periodate-Zimmermann test (24) as the detecting method. This showed that t,he more polar of the two metabolites present (RF = 0.19) was confined to the first two fractions and that the more mobile metabolite (RF = 0.29) was present in all three fractions. All three fractions therefore were combined, and the residue (280 mg) was chromatographed on a Celite column, 30 X 790 mm, prepared with System 25. The mobile component (33 mg, RF = 0.30 (System 24) and 0.25 (System 25)) was well separated from the polar component (12.5 mg, RF = 0.18 (Sys- tem 24) and 0.13 (System 25)).l” Both resisted crystallization.

Identification of Mobile Component as 5P-Pregnane-1 fi ,Sol ,

17(~, gOOar-tetrol-Paper chromatography of the metabolite in three systems (in which its RF value varied from 0.1 to 0.2) in parallel with pregnanetriol gave an averaged AR,,, value (29) of 1.10. When chromatographed (System 24) on paper pretreated with water only, with boric acid, and with borate buffers at pH 7.6, 8.6, and 10 (14), its Rf value remained in the range of 0.29 f 0.02.

Twenty-two milligrams of the crude metabolite were oxidized with sodium periodate under the usual conditions. The neutral fraction furnished, from acetone, 14 mg of needles with the following constants: m.p. 203.5-204.5”, unaltered on admixture with authentic 1/3,3a-dihydroxy-5/3-androstan-17-one (Com- pound XII, Figs. 3 and 4); [(Y]~ + 93”; Rp = 0.25 (System 18). The infrared spectra of the two diolones were identical. On the basis of this evidence, the mobile component was assigned the

lo The polar component proved to be 5p-pregnane-3cu,6a,17a!,2001- tetrol. Its characterization is described in the accompanying paper (26).

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. . 60 MC

220 MC (in C, D, )

C3 -H Cl-H

: 3 -0Ac

Cl943

l,,,,II,..,,,1.I,.,,,,,,,l,,,,,,,,,I,,,,,,,,,I,,..,,,,,],,,,, 6.0 5.0 4.0 3.0 PPM(6) 2.0

I , 1.0 0

FIG. 6. NMR spectrum, 60 MC, of lp-hydroxy-3a-acetoxy-5p-androstane-11, li-dione (Compound IX) in deuteriochloroform. sitions are given in parts per million relative to tetramethylsilane.

Peak po- Varian A-60 spectrometer.

C 18’

5323

structure 5/%pregnane-lb, 3a, 17a, 20ar-tetrol (Compound XXI, Fig. 4).

NMR Spectroscopy Studies

The technique of high resolution NMR spectroscopy was employed either to determine or to confirm the configurations at various centers in this series of compounds. This was accom- plished by examining the changes in the absorption patterns of methyl and other protons effected by the introduction or substi- tution of various functional groups. Two of the several applica- tions of this technique in the present work are presented in this paper.

NMR Spectrum of i fl-Hydroxy-Sar-acetoxy-5&androstane-11, i7-dione (Compound IX) (Fig. @-The broad resonance at 5.20 ppm in the 60-MC spectrum is due to the proton at C-3 adjacent to the acetoxyl group.” The -3O-cps width of the signal due to this proton indicates that it is axial in configuration and is coupled to four other protons, namely two axial (J,, = 10 cps) and two equatorial (Jae = 5 cps) (30). The spin-coupling pattern in the resonance of this proton is blurred because of the virtual coupling (31), but is clearly discernible when the spectrum is obtained at 220 MC in deuteriobenzene (Reference 32, Chap- ters 2,4, and 7). The blurred triplet (31) at 4.55 ppm is assigned to the proton at C-l adjacent to the hydroxyl group. The narrow line width (-5 cps) of this resonance indicates that H-l

11 For comparison, see the spectrum of 3a,12p-diacetoxy-5@- pregnan-20-one (No. 361 in the NMR Spectra Catalog, Varian Asso- ciaest, Palo Alto, California (1962)).

IV VII

J HlH2 = 10 cps J Ix2H3 = 10 cps

J %A4 = 1 cps J QH4u = 2.8 cps

J moH128 = 12 cps J =zmeq = 1.2 cps

J wu.x = 4.8 cps

J %Qeq = 2.8 cps

J W&W = 12 cps

FIG. 7. Coupling constants and chemical shifts for the un- saturated triketones IV and VII. Varian A-60 spectrometer.

is spin-coupled to the axial and equatorial protons at C-2 and has an equatorial configuration. This, in turn, clearly establishes that the hydroxyl group at C-l is axially oriented. The un- usually low chemical shift of 4.55 ppm for the equatorial proton at C-l (normal shift is 5.05 to 5.30 ppm) is due to the anisotropic effect of the carbonyl group at C-11 (33). The tall signals at 0.83, 1.26, and 2.01 ppm correspond to the C-18 and C-19 protons and the acetyl group at C-3, respectively, and are in harmony with the calculated values (Reference 32, Chapters 2 and 4; Reference 34).

Diflerentiation between Compounds IV and VII (Fig. 7)-It is

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well known that in a molecule containing an a,@unsaturated 8. ketone, the NMR due to an OL proton appears at a higher field than the one caused by a p proton. This is due to the

9 ’

highly electro-deficient terminus of the system. This moiety 10. occurs in both Compounds IV and VII. However, in IV, 11.

the olefinic proton which is /3 to a carbonyl group is also subjected to the anisotropic effect of the carbonyl group at C-11 and, there- 12* fore, this proton must resonate at a much lower field than its counterpart in VII. The values derived from the spectra of

13.

Compounds IV and VII are in accord with these considerations. 14. This differentiation is confirmed by the fact that only in the spectrum of VII do we see the large couplings caused by the

15,

protons at C-4. 16.

Acknowledgments-Our work has been greatly assisted by the 17’ generosity of a number of investigators. We wish to thank Dr. 18, C. Tamm, whose earlier chemical work formed the basis for ours, for his encouragement and good counsel; Dr. David Fukushima, 19. for reference samples of &cortolone and for the conditions used in its preparation from tetrahydrocortisone; Mr. C. R. Meeks, of

2. ’

the Upjohn Company, for supplies of 5&androst-l-ene-S, ll , 17- 21. trione; Dr. Howard Ringold, for a reference sample of 5/.-androst- 1-ene-3,17-dione; Dr. Vernon Mattox, for supplies of isocorti- sone; Dr. D. H. R. Barton, for several interesting suggestions;

ii. ’

Dr. R. Norman Jones, for determining and interpreting certain 24. infrared spectra; and our associate, Dr. Marvin Lewbart, for many good ideas, including the suggestion that 20,21-acetonides 25. would be useful derivatives in this work, and for the use of his improved method for their hydrolysis. 26.

REFERENCES 27.

1. SCHNEIDER, J. J., AND BH~cc.~, N. S., Federation Proc., 23, 276 28. (1964).

2. SCHNEIDER, J. J., AND LE~B~RT, M. L., Recent Progr. Hor- mone Res., 15, 201 (1959).

3. FUHUSHIMA, I). K., LEEDS, N. S., BRADLOW, H. L., KRITCHEV- 29. SKY, T. H., STOKEM, M. B., AND GALLAGHER, T. F., J. Biol. Chem., 212, 449 (1955). 30.

4. SCHNEIDER, J. J., J. Biol. Chem., 183, 365 (1950). 5. BUTLER, G. C., AND MIIRRIAN, G. F., J. Biol. Chem., 119, 565 31.

(1937). 32. 6. Poos, G. I., ARTH, G. E., BEYLER, R. E., AND SARETT, L. H.,

J. Am. Chem. Sot., 75, 422 (1953). 7. GREENSPAN, G., SCHAFFNER, C. P., CHARNEY, W., HERZOG, 33.

H. L., AND HERSHBERG, E. B., J. Am. Chem. Sot., 79, 3922 (1957). 34.

BURN, D., ELLIS, B., AND PETROW, V., J. Chem. Sot., 795 (1958).

MCALEER, W. J., KOZLO~SKI, M. A., STOUDT, T. H., AND CHEMERDA, J. M., J. Org. Chem., 23, 508 (1958).

STRIEBEL, P., AND TAMM. C.. Helv. Chim. Acta, 37.1094 (1954). SCHNEIDER, j. J., LEWB~RT,‘M. L., LEVITAN, P., ri~~ LIEBER-

MAN, S., J. Am. Chem. Sot., 77,4184 (1955). NORYMBERSKI, J. K.. AND WOODS. G. F., J. Chem. Sot., 3426

(1955). ’ BARTON, D. H. R., IVES, D. A. J., AND THOMAS, B. R., J. Chem.

SOL, 2056 (1955). SCHNEIDER, J. J., AND LEWBART, M. L., Tetrahedron, 20, 943

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John J. Schneider and Norman S. Bhacca-Steroids in a Man and by Surviving Liver Slices of the Guinea PigβOther 5

-pregnan-11-one and ofβ,21-Tetrahydroxy-5β,20α,17α-Hydroxylation of 3β1

1966, 241:5313-5324.J. Biol. Chem. 

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