US2666016A - Biochemical processes for the oxygenation of steroids - Google Patents

Description

Patented Jan. 12, 1954 BIOCHEMICAL PROCESSES FOR rHE OXYGENATION Ov r-mews .Osear. Ii Worc Rob rt- P-- .Jac senl .Shmws urt,

and Gregory P ncus, w M Schenker, Shrewshury, Mass, and Charles Marshall, Chicago, 111., assignors to G. D., Searle Ro J 12 Harold Levy,

1e QQ-z cheese 11 a rtfl sti 9f i hw i N Drawing .Ann icationJanuary"25,1351,

fie ial- 2,411,826 11 s am -119 .4

lCHeOH- H s GO nt orti sterve he r v la le. on y in amq mits,i man ma 'ad ens gl ds-j 7 Cbrticostergne omen the eby i r fx e .l'I-Qvdmmcofl co tmm 9 the which isv one of themost important and pharmacologically useful hormones of the adrenal gland, but-likewise heretofore available in only very minute amountsfrom natural sources.

Certain othersteroidscanbe bio-oxygenated by the methods of invention, such as androsterone which forms 1lfi-hydroxyandrosterone; 4-

androstene-3-,17 -d-ione' similarly treated forms the corresponding llp-hydroxy derivative. Pro gesterone by-themethods of our invention is convertedinto a mixture of l7-hydroxycorticosterone, corticosterone, Ii 8-hydroxyprogesterone, 1 7- hydroxycorticosterone and other sterols. Other steroids which can be bio-oxygenated include 17- hydroxyprogesterone, 1-1"-desoxycorticosterone -acetate, -11 -desoxy 1 7 --hydroxycorticosterone 21- acetate, isoandrosterone, .dehydroisoandrosterone, 1 6-dehydropregnenoione, M-androstehe-B, '11- dione, 'A pregnene 17,20,21 triol 3- one, A pregnene-3 ol-20-one and related pregnanediols and pregnanolones; Solubilized derivatives of these steroids, such as glucosides, hemisuccinates; and-the l-ike, can also'be-used.

' By the-methodsof this invention it is also possible to l l-bio-oxygenate functional derivatives of the alcove-mentioned steroids suchas the acetate .or propionate. Thus -1-1-desoxycorticosterone -21-acetate andits corresponding 17a.- hydroxy derivative are converted bythe methods of this invention into corticosterone and 17- hydroxy corticostercne respectively, the acetoxy group-beinghydrolyzed during-the perfusion and isolation processes.

It willheapparent to one skilled in the art that .th i I "'hY. Q s e p dsi qrni b the of this inventionmay 'bereadily convertedby lgnovvn methods to the corresponding phar- Inacol'ogically useful l l-oxo (oxidation) 'or 11,I 2 or 9) anhy clro (dehydration) compounds whereasth'e reverse transformations, reduction of '11- oxo or'hydration of 11,-(12 or 9)"-anhydro to 11poxy compounds are extremely diflic ult or imposbible to carry out.

aceaoic Thus it can be seen that the processes or this invention for introducing the llB-hydroxyl group into steroid hormones of the adrenal cortical type are of unique importance. llfi-hydroxy steroidal hormones like corticosterone and 17- hydroxycorticosterone, and related hormones such as 17 hydroxy ll dehydrocorticosterone and ll-dehydrocortioosterone afiect the carbohydrate and protein metabolism by increasing the livers store of glycogen. These hormones also increase the working capacity of isolated muscle. A most important use for l'i-hydroxyll-dehydrocorticosterone has been in the treatment of rheumatoid arthritis. The use of this substance in rheumatoid arthritis has resulted in improvement within a few days. Pain, stiffness and limitation of movement were reduced. and tenderness and muscular pain relieved. Appetite and weight improved and toxicity was replaced by a sense of well-being. The role of the hormone in other conditions such as myasthenia gravis and rheumatic fever is being studied.

It is the object of this invention to provide simple and relatively inexpensive methods for obtaining these llc-hydroxy steroids and their derivatives from accessible starting materials. It is further an object to provide methods for producing l'Y-hydroxycorticosterone and related llp-hydroxy compounds in a state of purity by simple and efficient biochemical methods. Another object is to provide methods of producing llfi-hydroxy steroids, such as 17-hydroxycorticosterone, which can be readily oxidized to ll-keto steroids, such as cortisone, by aeetylationof the Zl-hydroxyl radical followed by mild chromic oxide oxidation of the ll-hydroxyl radical. Other objects will be apparent to those skilled in the art.

According to our invention, excised mammalian adrenal glands are maintained in a functioning state by perfusion with an appropriate medium, natural or synthetic, at temperatures in the range of -40 C. and preferably at 3637.5 C., and

to the perfusion medium is added a quantity of steroid to be hydroxylated. After the medium has circulated through the gland at least once, the perfusate is removed and the hormones separated by suitable procedures. The perfusion medium can comprise blood, blood plasma with U saline, or saline solutions of gelatin, technical blood albumin, synthetic plasma substitutes, and the like or mixtures thereof. For most successful operation, homologous plasma or whole blood or mixtures thereof constitute the most desirable perfusion media. Whole blood, before or after hemolysis or defibrination, or saline-diluted mixtures thereof are further preferably treated by being circulated through a liver preparation before use as a perfusion medium, though for short periods of perfusion this may not be necessary. This is desirable both in order to remove and to prevent formation of clots, and to remove vasoconstrictor substances present in these media which might interfere'with the free flow of the perfusate through the organ. The perfusion medium may be supplied to a single gland under either pulsating or non-pulsating pressure or to a parallel bank of several glands under nonpulsating hydrostatic pressure. The pressures used have been in the range between 20 mm. of mercury and 200 mm. Generally pressures in the range of 20-100 mm. are preferred. The pulsations used have been at a rateof about 24 to 180 per minute. Whereas pulsatingpress ures 4 may be more physiological for the perfusion, steady hydrostatic pressures are also satisfactory. The length of the period of perfusion may vary from a fraction of an hour to several hours depending upon the rate of flow and the volume to be perfused. It is only necessary that the fluid containing the steroid pass through the functioning gland at least once. Additional cycles are useful for producing more of the oxygenated steroids particularly at the higher input concentrations of precursor. In practice we have found that the more rapid perfusions (0.5 to 7 liters/ gland/ hour) are quite satisfactory both as to conversion rate and purity of product; with faster flow rates the number of cycles may be increased. The concentration of the steroids in the perfusion medium may vary from 50 to 1000 mg. per 1000 ml. of perfusion medium. The preferred'concentration is about 50-300 mg. per

liter.

The adrenal glands which are used in our processes may be obtained from any of the conimon animals including the horse, sheep, pig, cattle, dog, rabbit, cat and monkey; those from the larger species are most convenient to use. The following illustrate the me'thods'of preparing mammalian adrenal glands for use in our procedure: A. Intact gland arterial flow.--A laparotomy is performed on an anesthetized or freshly killed animal and the kidney region exposed. Theen tire region of the paired adrenal glands is dissected. Two glands are removed from the kidneys of each animal, care being taken not to slash the gland. The gland is dropped at once into cold saline solution and used as soon as possible. Extraneous fat and tissue is removed from the gland and small venal entries near the main large vena cava are tied oif. A cannula is inserted into the aorta just below the renal arterial branch supplying the adrenals or into any direct aortic branch to the adrenal region, and all of the extra-adrenal arteries are ligated. A buffered balanced salt solution may be washed through the cannulated preparation to remove all blood remaining in the vessels of the preparation but this is not absolutely necessary. The cannula is then affixed to the arterial entry of a perfusion pump designed to circulate the perfusion medium continually under either pulsating or nonpulsating hydrostatic pressure as specified above, and circulation of this medium is promptly started. It is essential that transfer of the cannulated preparation to the perfusion apparatus be as rapid as possible and that prompt perfusion be started with the medium at a pressure sufficient to insure active circulation through the glandular tissue. The above operations and perfusion are best conducted under completely sterile conditions in order to insure maximal survival of the glands, though for shorter periods of perfusion absolute sterility is not required. Practical sterility can be maintained by the addition to the medium of suitable concentrations of antibiotics such as penicillin and streptomycin.

B. Intact gland venous flow-The dissected adrenal obtained as in A is trimmed to remove all of the extra-glandular tissue. A suture is looped around the neck of the adrenal vein and a cannula is pressed in and tied. By flooding the cannula with saline solution while massaging the gland, the system is freed of air. Saline supply under low pressure is passed through to flush out the gland. During this time all fat unemmanousaissue and the major residueof the wall oii the vena cava are 'trirnmed oil. The g land is tii'enstored 1 in l cold-saline solution. The cannulat'ed preparation', is then washed with saline and theicannulaamxed to a perfusionapparatus as above.

C. Lacerated gland venous flow.The trimmed 'g-lan'dwith venous cannulation as in B-is-su-bjected ='--to multiple cortical laceration =ove'r vir tually its'entiresurface, the cutsbein'g spaced m5to' 3 -mm.'-=apart and*made to a depth-of about Mite /g' the' thicknessof the-cortical layer. Careis taken to lacerate the cortical layer only and toavoid' the 'deeper modular-layer. When lacer ation' 'is' complete-saline solution under a-pres-- sure of zo scmmpis applied to the cannula to chech adequacy 'off fiow. "Following laceration the gland is attached to :the perfusion apparatus, care being taken to avoid air bubbles. 'lheperfusion medium is passedthrough the gland and thasolution of thesteroid is added to the: supply reservoir of.fluid. During the perfusion pressuresof 2'0'to 70 mm. are maintained throughout andoxygenis continuously fedv into the perfusion liquid. Thistype of preparation is particularly usefulin banks of several glands each attached to. a branchof a manifold type of perfusion apparatus. utilizing .nonpulsating flow under hydrostatic .pressure.

"j'lhesteroidi hormones which are produced by our, processescan be isolated from the perfusion medium by a variety of methods. The simplest is'b'ydir'ect adsorption with charcoal followed by elution of the charcoal with suitable solvents such asmethylene chloride, ethylene chloride, benzene, chloroform, acetone, Q methanol, ethyl acetate. and thelike. .Upon removal of the sol vent, .preferab ly 'by evaporation under vacuum atjlowtemperature. in an inert atmosphere, there isobtained .a residue of the steroids. This residue'is contaminated with a small fraction of the lipoidalmaterial normally present in blood. Alternativelythe perfusion medium may be dial'yzedxagainst saline solution and the latter treated. with charcoal as above (or during the dialysis) or extracted directly with a solvent such-as chloroform, methylene chloride, ethylene chloride'andthe' like. The steroids which are in solution in the perfusate pass through the membrane and are about equally distributed between themedium andthesaline solution. With charco'aliinthe salineduringdialysis the-steroids are rapidlyadsorbed' and the dialysis period shortened; This method has the advantage-or avoidingccontaminationwith lipoidal material, but is time-consuming. In the most convenient isolation procedure, the hemolyzed perfusate is stirred each'day with about 5 g. per liter of activated charcoal for 3 to 6 days, the blood being decantedafter. settling of the charcoal. In small scale'experimentsthe charcoal portions are combined andstored in saline at about 5 C. during the treating period, while on a larger scale the individual charcoal portions may be collected at once, washed with water or saline and dried. Thedriedcharcoal is then exhaustively extracted with warm solvent disclosed above, such-as methylene chloride, and the desired .ll-oxygenated steroidslare. isolated in a relatively pure, semicrystalline form. Further extraction of the charcoal with-warm benzene, or other suitable solventsmentioned above, maybe resortedto if necessary,- to remove any remaining material. Theyproducti'may-be further purified by direct crystallization or by chromatographic resolution:

The preferred adsorbant for 'the latter :jmet-hod is silica gel in finely divided fform.

As used herein, the term oxygenation refers to the introduction of an atom-oi oxygen-into an organic compound, resulting in the formationcof a hydroxylradical. A tormaldehydogenic steroid is one which contains a'n-a-ketolio'r a-glycolqgroup' at the 20-21-position, which on-pei'ioliieacid oxidationgives r ise -to formaldehyde. Our invention is illustrated in":detail b z-means of thefollo'wingexamples but is 'notto Ice-construed as limited thereto. In' these examples; volumes are reported as liters (1.) and milliliters (ml.), weights as grams (g.) and milligrams (-mg.) pressures as millimeters of mercury, v(mm), and meltingpoints as degreescentigrade. 0.). The term .citrated-blood means volumesrof whole-blood mixed with 15 volumes of citrate solution (an aqueous solution containing 1.6% sodium citrate and 5% glucose). The term .citrated saline solution means an aqueous solution containing 0.8% sodium chloride and 0.35% sodium citrate. Saline solution means a solution of sodium chloride of 0.8 to 0.85% concentration.

Example 1 To a mixture of 500 cc. of plasma and 500 cc. of whole blood was addedZcc. of propylene. glycol containing mg. of ll-de'soxycorticosterone and the resulting solution was perfused with arterial fiOW through an intact beef adrenal-gland at an average flow rate of about. 0.5 liter per hour and a pulsing pressure of about-100 mm. orm'ercury. l"l1e perfusate was .then hemolyzed pby freezing and thawing, stirred for.30 minuteswith 5 g. of activated carbon at room teinperature'and allowed to settle for several hours. The blood was decanted and the charcoal-treatment repeated twice during-a period of two days. The combined charcoal was collected, washed .-with saline solution and dried-by washing with a little acetone. The air-dried material was then-exe tracted in a Soxhlet apparatus with methylene chloride for twenty hours and the extract concentrated under diminished pressure undernit rogen. The residue consisted offal mg. of semicrystalline material containing 23 mg. ot-formahdehydogenic steroids (The Hormones, by Pincus and Thimann, vol. I, 1948, p.613). The combined residue from 5 liters of perfusateprocessed as above was chromatographed on silicagelby -elu-, ting with benzene-ethyl acetate mixtures providing 64 mg. of crude crystalline corticosterone and 70 mg. of unaltered desoxycorticosterone.

The corticosterone fraction, after purification byrecrystallization from ethyl acetate-neohexane or :ether-acetone-hexane, melted at. 1'73.3-180 C. (powder) or 178-186.? C. (intactcrystals). The rotationin ethanol was [a] =+227 (c. '-0.*240).; Mixed withauthentic corticosterone isolatedfromnatural sources, M. P. 173-179". C. (powder) or. 176.5-181 C. (intact crystals), the perfusion product mixture-melted at 173-l81" C Acetylation of the perfusion product provided an acetate melting at 1505-1525 (3., .whose mix-- ture I with authentic corticosterone 2l-aoetate,- M. P..l51-.5-152.5Q., meltedat- 150.51152 ;C.

Example 2 A solutionof 100: mg .of' desoxyco'rtico'sterone in 400. cc. of whole blood was perfused with arterial 'flow through an-intact beef adrenal gland The saline solution was then extracted with chloroform and the solvent layer washed, dried and concentrated under diminished pressure under nitrogen. Analysis of the semi-crystalline residue by the periodic acid method indicated the presence of 21 mg. of formaldehydogenic steroid from the 100 mg. of desoxycorticosterone used. This material was assayed by the glycogen deposition test (Pabst et al., Endocrinology, 41: 55-65, July 1947) and was found active at the 500 microgram dosage level. This material was therefore approximately equivalent to 10 mg. of corticosterone.

Ezrample 3 A solution of 150 mg. of desoxycorticosterone in 1500 cc. of plasma was perfused as in Example 1 for about 3 hours. The perfusate was then placed in viscose tubing and allowed to stand at 2 C., with gentle rocking, for about 2.5 days with 500 cc. of citrated isotonic saline (0.80% sodium chloride, 0.45% hydrated sodium citrate) containing 1.5 g. of activated charcoal. The charcoal was collected, washed with. water, and extracted with acetone and benzene. The solvent solutionswere evaporated under reduced pressure in a nitrogen atmosphere and the residue extracted with chloroform. The chloroform solution was dried with anhydrous sodium sulfate and evaporated under vacuum. The residue of 83 mg. of semi-crystalline material contained 61 mg. of formaldehydogenic material. This was chromatographed on silica gel as in Example 1 providing 25 mg. of crystalline corticosterone of practical purity melting above 160 C. and 25 mg. of unaltered desoxycorticosterone melting above 130 C.

Example 4 A solution of 250 mg. of desoxycorticosterone acetate in 2500 cc. of plasma was perfused as in Example 1. The perfusate was treated with 25 g. of activated carbon in two lots at room temperature during about ten hours. The charcoal was washed, dried, and extracted with acetone, methylene chloride and benzene. The bulk of extracted residue (193 mg. out of 256 mg.) was obtained from the methylene chloride extract and was found to contain 164 mg. of formaldehydogenie material. This was chromatographed on silica gel providing 24 mg. of crude crystalline corticosterone and 51 mg. of unaltered desoxycorticosterone.

Example 5 A solution of 100 mg. of 17-hydroxy-11-desoxycorticosterone in 1000 cc. of citrated isotonic saline containing hemolyzed blood was perfused with venous flow through a laceratedbeef adrenal gland using pulsating pressure for 1.5 hours (8 cycles). The perfusate from two such perfusions was treated at room temperature with 40 g. of activated charcoal over a three-day period. The combined charcoal was collected, washed, dried, and exhaustively extracted with methylene chloride. The methylene chloride extract was evaporated under nitrogen at reduced pressure. The extract residue was dissolved in warm ethyl acetate and chilled. Crystalline 1'7- hydroxycorticosterone was thus obtained. The mother liquors were evaporated under nitrogen at reduced pressure and the residue was taken up in benzene and chromatographed over silica gel. The l'l-hydroxycorticosterone was eluted with benzene-ethyl acetate. There was obtained a total of 32.5 mg. of l'l-hydroxycorticosterone of practical purity melting above 200 C. together 8. .with a trace of unaltered starting material. The, product after further recrystallization fromethyk acetate melted at 207-209 C. The mixed melting point with authentic 17hydroxycorticosterone: isolated from natural sources was not depressed.

Emmple 6 A solution of 600 mg. of desoxycorticosterone in 3000 cc. of whole blood was perfused with venous flow through a lacerated beef adrenal using non-pulsatile hydrostatic pressure for three hours (1 cycle). The perfusate after hemolysis was treated over a three-day period at roomv temperature with 60 g. of activated charcoal. Extraction of the charcoal and chromatographyof the extracted residue provided 241 mg. of corticosterone of practical purity melting above 160". C. together with mg. of unaltered starting material.

Example 7 A solution of 2'70 mg. of androsterone in 2700 cc. of plasma was perfused with arterial now through an intact beef adrenal gland for 6 hours using pulsating pressure. The perfusate was. treated with 40 g. of activated charcoalover a three-day period. The charcoals were'combined, washed with saline solution and then extracted with methylene chloride for 16 hours in a Soxhlet; apparatus. Evaporation of the extract under nitrogen at reduced pressure provided 405 mg. of semi-crystalline material. This residue was dissolved in benzene and chromatographed on silica gel. The column was developed with a benzeneethyl acetate mixture (20:1) and then the androsterone fraction mg.) was removed by ben zene-ethyl acetate (4:1). The column was then eluted with benzene-ethyl acetate (2:1) and this eluate was evaporated giving a residue of 18' mg. of ll-hydroxyandrosterone in ,non-crystalline form. This residue was dissolved in pyridine and treated with acetic anhydride to form ll-j hydroxyandrosterone 3-acetate, which is a crystalline ester. This material was recrystallizedfrom ether and melted at 238240 C. It. was found to be identical with an authentic. sample of ll-hydroxyandrosterone 3-acetate by mixed melting point and by infrared absorption.

spectra.

Example 8 A solution of 750 mg. of desoxycorticosterone in 1000 cc. of plasma containing 20% hemolyzed blood was perfused with venous flow through a lacerated beef adrenal gland using pulsating pressure for 45 minutes (2.5 cycles). The perfusate was treated at room temperature with 20 g. of activated charcoal over a three-day period. The material extracted from the charcoal with math ylene chloride weighed 732 mg. and afforded. after chromatography on silica gel, 82 mg. of corticosterone of practical purity melting above 0. together with 407 mg. of unaltered starting material. I

Example 9 300 mg. of ll-desoxycorticosterone acetate in, 16 cc. of propylene glycol and 8 liters of citrated blood as in Example 17 was perfused with arterial flow through an intact beef adrenal gland for 10 hours (one cycle). The perfusate was frozen, thawed, and treated with activated carbon. The carbon was washed with saline and acetone. then extracted in a Soxhlet apparatus for 20 hours with methylene chloride. The methylene ch10 ride extract was evaporated under nitrogen in vacuo. The: residue: was. dissolved in benzene and passed rover: 18cc; f: silica-gel: .The ,cc lumn; was developed with benzene-ethylnacetate mixtures as in Example 17. There were thus obtained 24 mg. of corticosterone melting at 168-172 C. and 95 mg. of desoxycorticosterone melting at 13.4:-1.38 C., after recrystallization from ethyl acetate.

.Excmple .10.

A solutionof 200mg. of desoxycorticosteroneiin 2000 cc. of'citratedisotonic saline eontaining6% of technical blood-albumin and-2% of hemolyzed fresh blood. was perfused. with. venous flow through a lacerated beef adrenal gland using pulsatingipressure for abouttwo hours (6' cycles). The perfusate was treated with charcoal and the later extracted afiording 377 mg. of semicrystalline residue. Thiswas chromatographed on silica gel yielding 71-. mg, of corticosterone of practical purity melting above 160- 0. together with; 39 mg. of unaltered starting material.

' Example 11 Example 12 Bliters of-"citrated; filtered, whole" bovine blood were perfused'through two rat livers to remove fibrin. 'I?o,,the ,.v blood wassadded' penicillin and streptomycirr'solution (800,000 units of penicillin G and-"800'- mg of" streptomycin sulfate in 80 ml. of saline solution) 'and'then22 ml. of apro:- pylene glycol solution, containing 551 mg. of progesterone.

The mixture was then perfusedv Each: acetone wash: or extract waszconcentrated in vacuo to remove almost all of the acetone, and the residualzliquid. whichwasmainly water was extracted with five portions'of chloroform. The, combined chloroform extract was concentrated inyacuo to a weighed residue.

Each activated carbon preparation was then washed copiously on the filter with methylene ch ri andfinal xtr ctedv with th ene chloride irra ,Soxhlet. apparatus for. 16-24 hours. The, methylene. chloride solutions, from each. per! tion of activated" carbon were combined and con.- centrated in. vacuo to Weighed residues, After the methylene chloride extractions, the first batch of. charcoalwas combined with the second, the thirdlwiththe fourth and the fifth was. combined'with the charcoallfrom the saline washes, andeach ofjthethree resulting charcoal prepara tions. was. extracted continuously with benzene. for 3-5 hours using, amodified Soxhlet apparatus. The modification consisted in stirring the charcoal during the extraction and using a plug ofglass wool asafilter.

From the. six charcoal preparations described above the following. weights. of. material .were obtained by the various extraction-procedures described above. Each of these residues was tested with concentrated. sulfuric. acid: for green fiuos rescence, which is an in'dication of llehydroxy steroids such as corticosterone' and 17-hydroxy corticosterone. The following table indicatesthe weights of materials obtained from the various charcoal fractions by extraction and theresults obtained on the concentrated-sulfuric acid test:

Acetone 3 I Batch of extract Fluorescence gg g gg Fluorescence. Benzene. FluorescencedarbO-n (viachlowvjn cone; extract th cone. extract, with cone.

rofglrgm), 112804 H2304 mg. H2SO4 112 Stron een 622 St on 12g .;.d 392 49 shghtgreen" reen 93 -'ace- 2% Tree; Neggtive 59. 5 Negative.

o V o 16 -Negative I .42 'do through four laceratedbeef adrenal glands in holllffiz allowingctha charcoal :to. settle overnight at, room; .tcmnerature and then separating the su ata t: bloods. Ira-decapitati n and 56611313101511? The. thimblesand. filter papers. used. in the extractions above'were combined and extracted by boiling with two portions of benzenaeachfor 1 hour. The-combined benzene extracts'were evaporatedin vacuo. givingaresidueof 23.5 mg. which gavenofluorescence with sulfuric acid. The total weight of: extracted:materialfromall of the fore.- going procedurestwas 283.7 mg.

The third, fourth, fifth, and sixth methylene chloride extract residues. in the above table were combined with the third benzene extractdissolved inbenzene and passed over a column containing 30 g. of silica gel passes througha IOU-mesh screen and'9 '0%..is retained on a 200- mesh screen). The column of silica gel was et Eachrcharcoal pgrtion wagthemwashed '25 thendevelopedby-treatment with'benzene-a-nd benzene ethyl acetate mixtures according to the following table:

7 7 v 1 Weight H so cum 1 a Ehmte (ml) solvent residue test 1 80 Benzene 132 2 100 Benzene-ethyl ace- 687 tote (30:1). 3 100 Benzene-ethyl ace- 182 tate (:1).

4 100 Benzene-ethyl 2100- 58 Negative.

tate (10:1 5 100 Ethyl acetate 115. 5 D0- 6 100 do 14 Green fluorescence. 7 100 do 8 Weak fluorescence. 8 100 Acetone 28 Slight fluorescence. 9 100 Methanol Each of these eluates was evaporated in vacuo and each of the residues was given the sulfuric acid test. The results of these tests are indicated in the above table.

A second chromatogram was conducted as fol- .lows. The charge consisted of the'first and secand methylene chloride extract residues. were dissolved in benzene and passed overa column containing g. of silica gel, as above. column was then eluted with benzene, benzeneethyl acetate mixtures, ethyl acetate, acetone and These The methanol according to the following table:

1 2 A fourth chromatogram was conducted using as the charge the benzene extracts and 10 g. of silica gel. The following results were obtained:

v1 weirght H53 0 um 0 2 4* Eluate (ml.) solvent residue test 16 Benzene v42 v l5 Benzene-ethyl ace- 15. 5

tote 10:1). 15 Benzene-ethyl ace- 2.5

tate (9:1). 4X15 Benzene-ethyl ace- 61 1 Y tate 2X15 Benzene-ethyl ace- 2 tote (7:1). 2X15 Benzene-ethyl ace- 1. 5 2X15 Benzene ethyl ace- 4 3X15 Betngene-ethyl ace- 5 Negative a e 2X15 Benzene-ethyl ace- 3 D0. 2X15 Bengenethyl ace- 3 Do.

a e 2X15 Benzene-ethy1 ace- 5. 5 Trace.

tate (1:1). 2X15 Benzeneethyl ace- 1. 5 Negative.

tate (1:2). 2X15 Benzeneethyl ace- 2. 5 Strong fluotate (1:3). rescence. 50 Ethyl acetate. 7 Slight fluorescence. 50 Acetone 6. 5 Negative. 50 Methanol 7 Do.

The fifth chromatograph was conducted on 17 g. of silica gel using as the charge the residues Weight fr 1 t 5 d 24 d b d Th 1' volume om e ua es an escri e above. e to Solvent ofresi- 1180 test mum (m1) due mg) 4 lowing results were obtained. 1"

10 i 80 Benzene '52 11 100 Benzene-ethyl ace- 8 12 100 state 31 119 v 1 weirght nso tate (20:1). 40 must" (1111.) 5mm residue 12...; 13 e. 100 Benzene'ethyl acc- 60 (mg mm 10:1). 14 100 Ethyl acetate 416 Green fluo- Iescellce- 61 30 Benzene 2. 5 138 D0- 62 25 Benzene-ethyl ace- 3 '18.5 Do. tate(10:1). 8 DO- 63 25 Benzene-ethyl ace- 1 41 Do. tate (91). Methanol 11 5 64-65 2X25 Benzelgeishyl ace- 20 66-70 5x25 e n zei 'e gim acees a e 2 Each of the various eluates was evaporated in 71.73 3X25 Benzerze-eghylacevacuo and the residue given the sulfuric acid test. me 6:

74-76 3 25 Ben ene-ethl ce- 15.5 Crystallization of the residue from eluate No. 15, X a. a which weighed 138 mg., led to 22 mg. of crystal- 7- 3X Benzenejethvl ace line I'Z-hydroxycorticosterone melting at 200- 0. 5X25 z n le i 201 5 C tate (3:1).

85-87 3X25 Benzene-ethyl ace- 14 A third chromatograph was carried out using me as the charge of the six acetone extracts. These 88-90 3X25 iggg i 322 332}- combined residues were dissolved in benzene and 91-97 e 2s,100 Be lngene-ezthyi ace- 41.5 See note. 2. G I passietd over 18 g. of silica gel Wllih the fOllOWmg 100 t ,fi 3 Negative,

u s: a e res 100 Ethyl acetate. 3 100 Acetone 16 100 Methano1 6 Weight H so Eluate 781F315 Solvent 5a g 4 Norm-Residues 91 and 92 gave a slight fluorescence. 93-95 gave 1 a strong fluorescence. 93-97 were negatlve.

20 Benzene Negame- Crystallization of the residues 81 to 84 which Benzene-ethyl ace- 34 Do. 21 mm (30.1), weighed 34.5 mg., from ethyl acetate led to 3.2 22 100 ggg fl mg. of crystalline steroid of unknown structure 23 100 Benzen ethyla melting at 202-205 C. The crystallization of 24 100 E1??? ice't c'e 138 Do. 0 residues 93 to 95, weighing 18.5 mg. from a mix- 25 100 igga g ture of ethyl acetate and neohexane, led to two 3.5 150. crops of crystalline materials, the first weighing 9.5 mg. and melting at res-197.5" c. and the sec- 100 Methanol 25 0nd weighing 2.9 mg. and melting at 191.5-199 C.

13 *afiDJZdC'CIECEJZ-E mg: 01: 117+hydroxycortieosterone mlting at207fl-208i5." Ci. ..A:sixthzrchromatogramzwas:conductedmsing;as .thez chargezthezresidues from eluates fi, .7, 14; '15 (except for the 222mg: :of: l7-hydroxycorticose terone obtained from residue 15), 16, 17, 25, 26,

and 27. These combined residues were dissolved -irrhenzeneand"passed over-a column:of- 37:5g; of silioa gel;

acetate, acetone, and methanol with the- 'follow= 'ingresultsr Weight 'Volume' of H550 (ml) Solvent test 4 residue Benzene:

Benzenee" tate:(10:

; Negative.

Do. Do. Do;

Dov Do. v Dot "Draco" .and f l u' o r e s cence.

Do.- Slight fiuorescence.

o. .Greenfl-uorescence.

Do. Slight .iluorescence.

Benzene-ethyl" ace-' tate ;(4:1)

F111 1' es cencc.

Benzene-cthyl ace- Do. (2:1)

Benzene-ethyl acetatc (1:1).

Slight -fluo rescencc.

I cu

, Benzene-ethyl acetote-(1:3).

,1 Benzene-ethyl aceate(154).

Benzene-ethyl race! tate 1:

Ethyl acetate Acetone ltlethanolinnnn Do; Do.

Certain of theresidues from the eluates from the ioregoing chromotogram were crystallized from appropriate solvents (ethyl acetate, acetone and-benzene, alone'or with 'neohexane) and the following 'steroidswere"obtained? The column was' 'then eluted with benzene; benzene-ethyl acetate-mixtures; ethyl Fluores- M. P.,C.,rof' Emace W 25? crystalline wgghti Identification product 107 24 f186190,recryst; 1012 13. 5 Progesterone.

2 6.2 Do. 1.5 3.9 0.4 2.2 -17. 2 17-hydroxyprogesterone. 5.1 Do.

1.4 0.8 2. 2 IIB-hydroxyprogesterone. 4. 1 v Gorticosterone. 22-41 1 Do.

5:52 -D0.- 3" Mixture of corticosterone-and' lf-hydroxycorticosterone. 115.5 196-201; 7..5 ,17-hydrox3tcort1costerone. 141 24.5 198-206 7.5 Do. 197 .5-204 6 D0; 186-192 (from 6.5 Do. 142 16;5 motherliquol's from 140-142 recryst.) 143 11.5 186-195 13.:5 ?17.-11yd1ioxycort1costerone "(impure) Examplelt 8 liters ofcitrated, filtered;:whole'ib.ovine blood were passed through tworatlivers.to;remouewfi brin. Then a solution of'streptomycinand penicillin were added (as in Example12)*followedby 20 m1. of a propylenev glycolrsolution containing 600-mgzof M-androstene-Za;lll dione. 'll'heresulting-solution was perfused through four Iacera-ted beef adrenal glands in .parallelusing venous entry asrin. Example. 12. The perfusion wasconducted over a periodof" 6.5 hours, corresponding-- to 14 cycles of the perfusate medium. The. perfusa-te was then frozen toeffeet hemolysis, thawed,;and treated. with fourhportionsof activated charcoal (Dacro G-6O),SeachVWeighingAOt-g. Each treatment consisted in stirring the blood i'With, char- .coal for l-r-3 hours.-=at room temperature, allowing the charcoal tosettle overnight-at'roomtemperature, separatingsthe supernatant blood by-decantation and centrifugation. Each batch of activated carbon was then-individually washed by centrifugationwith 1% saline solution, repeating this-washing;generally four onfiveir'timest Each of the four charcoal fractions above was stirrediwith acetone-,-ufiltered,landthe filter cake washed copiously with acetone. The filtrate from a each 10f these: washings (called the 1 acetone extract) was concentrated: in vacuo' to dryness. Thetresiduewas 1 then extracted-with boiling-ace tone and concentrated in vacuo 'to a -weighed residue; Each batch ofcharcoal was washed copiously on the: filterwith -methylene:.chloride and! then extractedr in 1 a 1 Soxhlet; apparatus* with methylene chloride for 16424 hours; .then with vmethar-zol for-"3: 1 hours; Each: of. the extracts was: evaporated in vaeuo toia weighed-residue: The results are set'forth in the following table:

The residues from the first and second methylene chloride washes and the first and second methylene chloride extracts (weighing a total of 1400 mg.) were combined, dissolved in benzene and passed over a column containing then extracted in a Soxhlet apparatus with -methylene chloride and finally with benzene, as in Example 12. Each of the extracts was evaporated and the residues were given the sulfuric acid test with the following results:

V Charcoal 1," Charcoal 2 Charcoals 3-5nnd saline Extract 1 Hours Mg. H2504 Hours Mg. H2804 Hours Mg. H2804 Acetone (via chloroform) 103 194 140 Trace Methylene chloride wash. 134 309 2. 75 339 Slight 50 g. of silica gel, as in Example 12. The column was then-developed and eluted as follows:

f Vol- Eluate Solvent ume, Product Inl.

Benzene-hexane 200 Syrup. Benzene 300 Do. Benzeneet-her (19: 200 M. P. 52-53" C. Benzene-ether (9:1) 200 Syrup.

Benzene-ether (4:1) 400 Do.

Benzene-ether (3:1) 600 M. P. 131-132" C. Benzene-ether (2:1) 400 Do. Benzene-ether (1:1) 400 M. P. 225227 C. Benzene-ether (1:2 400 Do. Benzene-ether (1:3 200 M. P. 192-109 C.

Benzene-ether (1:4) 200 D0.

Ether 600 Do. Ether-ethyl acetate (19:1) 400 Do.

Example 14 To 8 liters of citrated, filtered, defibrinated, whole bovine blood containing penicillin and streptomycin as in Example 12 was added a solution of 200 mg. of pregnenolone (A -pregnene- 3p-o1-20-one) in 15 ml. of propylene glycol. The mixture was perfused through three lacerated .beef adrenal glands in parallel using venous entry for 200 minutes, which corresponds to eight cycles of the fluid. The perfusate fluid (7700 m1., equivalent to 192 mg. of pregnenolone) was frozen toefiect hemolysis and then thawed. The blood was then treated with five 40-g. portions of activated carbon (Darco G-GO) as in Example 12. After the second charcoal treatment 5 g. of sodium fluoride were added to suppress mold growth. Each of the five charcoal batches was washed with saline solution and with acetone as in EX- ample 12. The saline washes were combined and treated with activated charcoal (5 g. per liter). The acetone washes were evaporated, the aqueou residues extracted with chloroform, and the chloroform extract of each batch was evaporated to a weighed residue. The batches of activated carbon were washed with methylene chloride and The former was converted into androdo 34. 1 Benzene-ethyl acetate (8:1). 18. 8 Benzene-ethyl acetate 1) 20. 2 Benzene-ethyl acetate (4: 1) 67. 4 Ethyl acetate 516. 6

-do 12. 6 Acetone 19. 0

. do 36. 0 Methanol 61. 9

The total amount of solids obtained from these extracts was 5109.5 mg. This include 32 mg. of material giving a negative sulfuric acid test, which was obtained by extracting the filter papers and thimbles with benzene.

The acetone extracts of the third, fourth, and fifth charcoals and the charcoals from the saline washes and from all of the benzene extracts were combined, dissolved in benzene, and chromatographed over a column of 80 g. of silica gel as in Example 12. The column was developed and eluted with benzene, benzene-ethyl acetate mixtures, acetone and methanol with the following results:

Solvent Benzene Benenc-ethyl acetate (10:1) o...

Volume, Residue, Eluate I Solvent mg.

17 75 Benzene 8. 9 18 5O Benzene-ethyl acetate (10:1) 1. 5 19 50 Bcnzeneethyl acetate (8:1) 6. 2 20-26.. 7X50 Benzene-ethyl acetate (6:1) 313. 9 27-29.. 3X50 Benzene-ethyl acetate (4:1) 30. 0 30-35 3X50, 75, Benzene-ethyl acetate (3:1) 112. 9 36-39- 4X50 Benzene-ethyl acetate (2:1) 20. 8 4042 3X50 Benzene-ethyl acetate (1:1) 19. 2 4345 3X50 Benzene-ethyl acetate (1:2) 10.8 40-421 3X50 Benzene-ethyl acetate (1:3) 4. 7 49-51. 3X50 Ethyl acetate 5. 7 52. 5O Acetono 5. 3 53 100 Methanol 12. 0

A third chromatogram was conducted on g. of silica gel using as the charge all of the methylene chloride extracts of the first and second batches of activated carbon and residues 10 to 1? 1 ;;1 1Q 11 V of h fi s h ma qe m in hi example. The following results were obtained:'

18 all. extrac (s of the remaining batches .of, charcoal.

The following results wereobtained:

Eluate Solvent Benzene Benzene-ethyl acetate (21:1) Benzene ethyl acetate (10:1) Benzene-ethyl acetate (:1) Benzene-ethyl acetate (8:1) Benzene-ethyl acetate (7:1) Benzene-ethyl acetate (6:1) Benzene-ethyl acetate (:1) Benzene-ethyl acetate (4:1) v Benzene-ethyl acetate (3:1) Benzene-ethyl acetate (2:1)

l3c nzene-ethyl acetate (1:1). Benzene-ethyl acetate (1:2). Benzene-ethyl acetate (1:3). Ethyl acetate Acetone... Methanol Crystalline materials were obtained from vari- Weight of Eluate gf Solvent residue 175 Benzene 135 2x100 Benzene-ethyl a tate 274.5 2x100 Benzene-ethyl acetate (20:1) '11. 5 3x100 Benzene-ethyl acetate (:1) 1, .489.

100 .B enzene etliyl acetate (2:1) 137 100 Benzene-ethyl acetate (121).. 189' 100 :Bcn'zcne-ethyl acetate (1:2).. 110 100 Benzene-ethyl acetate (1:3) 57. 4x100 Ethyl acetate. 189; 5

100 Acetone... 101 100 Methanol. ,133

A. second chromatogram was conducted psing 72 g, of silica. gel and the acetone and he methylene chlorideextracts of the first activated carbo T e fo ow n resul we obta nah ous eluates, using as crystallization media ethyl acetate or acetone, alone or with neohexane. The Eluate 8 Solvent restate following results were obtained: (mg) 2 llgienzeneufilfl1 .E..(..-..) 34.5 2 P. f enzene-c y aceta e 30:1 7 Residue We ght, Weight, Identity 2x50 Benzene-ethyl acetate (:1 4.5 v mg. tamed 0 Q 2- 3X50 Benzene-ethyl acetate (10:1). 692-5 2X75 Benzene-ethyl acetate (9:1).. 51 2X75 Benzene-ethyl acetate (8:1).. 2 5 0. 5 2X75 Benzene-ethyl acetate'(7: 1)... 11.5 16.8 167182 6.8 Oorticosterone. 3x75 Benzene-ethyl a e .(6: 10.5 3 174.131 6, 2 D0 3X75 Benzene-ethyl acetate (5:1; 13, 5 14' 2 175481 3. 2 15x75 Benzene-ethyl acetate (4:1 65619 17. 5 173-181 4 1) 11x75. Benzene-ethyl acetate (3:1) 68.8 19. 0 195496 5. 2 Impure 17-hy- 5X75 Benzene-ethyl acetate (2:1) 237, 4 droxycortico- 9X75 Benzene-ethyl acetate (1:1) --63."5 sterone. 9X75 Benzene-ethyl acetatc (l:2) -70 5 117 13.8 191-196 3.7 Do. X Benzene-ethyl e e 1 2.3 118 11.7 106-197 3.2. l7-hydroxycortico- 2X75 Benzene-ethy acetate (1P1) 5.0 t r 2X75 Benzene-ethyl acetate (1:6) 5, 2 119 9.2 19%200 2.3 Do. g y a et te I th 1 .44. 152. 5-53 1.1 cc one. ,4 N 3.521%. 7 1 3x100 Methano e2, 9 dues from 115-118. I 135. 15.1 320 varlous crystalllneproducts were obtamed 136 0 g from the foregoing eluates using ethyl acetateas the crystallization solvent. The following -re- Example 15 sults were obtained:

8 liters of citrated, defibrinated, filtered, whole i' bovine blood containing streptomycin and peni- 1M of cillin prepared as in Example 12 was treated with Eluate gg g gg iggi li Identity asolution of 934 mg. of l'l-hydroxyprogesterone 0 Q term in propylene glycol and then perfused through e) four lacerated beef adrenaliglands in parallel for 1 7 7 a 1 I 3 hours using venous entry. This corresponds i???- gg-g 7' lgio prpg sterone. to two actual cycles of the fluid. 7340 ml. of per- 50:: 17I2 20s -214v 112 D81 fusate fluid (corresponding to 852 mg. of 1'7- gg hydroxyprogesterone) was frozen, thawed and 56316.1: c812, 193 --,195 8:9 treated with 40-g. portions of activated charcoal 184 %QQ s3 8.4 186 -1s9 3.2 at room temperature. The various charcoal preparations were washed or extracted with ace- Z 16 tone, methylene chloride and benzene as in Ex: mamp ample 12 with the following results. i '7 liters of filtered, defibrinated. whole boyine Charcoal 1 2 3 4 5 Saline Acetone extract (via chloroform)-. 323 mg. 60.5 mg--.. 32 mg....- 88.5 mg.... .100 mg..-- .34 mg. Fluorescence with cone. 112804.... vStrong Positive... Slight Negative.. Negative .Negative. Methylene chloride wash 392 mg mg-- 98n 1,...- 228 mg..';. 23lmg..'.; 18 mg.

Strong Positive... Positive... Pos 1tive Negative" Negative. 463mg... 262 mg-- 337 mg..-. 404mg..- 517mg..-. 24 mg. Strong Negative. Negative;. Positive... Negative.- Negative. 44 mg...-.- 27.5 mg... 67.5 mg, 59.5 mg."

Positive... Negative Slight.

Benzene extraction of all-.thimloles and filter pa- .pers yielded 18.5, mg. of. material which gave a slight fluorescence with concentrated sulfuric acid. The total extracted material, was 4064 mg.

. A chromatogram was conducted on 140, g. of silica gel as in Example 12 using as the charge the benzene extract of thefirst bath of charcoal and .hydroxycorticosterone" blood were mixed with3 litersof citrated saline solution (0.8% sodium chloride and.0.45% sodium citrate hydrate). 'Then;1',O00,0 00.units otpenicillin G and 1 g. of streptomycin sulfate in saline solution are added.' To. this meo'iuniywas added a. solution 1of 2 000 ,mg. of ll desoxyl'7- i 1 0 f 1 of. propy ene glycol. This solution was perfused through ten lacerated beef adrenal glands in parallel using venous entry for a total period of 7 hours, which corresponds to eleven cycles of the perfusion fluid. The citrated blood was removed from the perfusion apparatus and passed through a colloid mill to hemolyze the cells. 8.60 liters of the perfusion fluid (equivalent to 1720 mg. of ll-desoxy- 17-hydroxy-corticosterone) were treated with three 50-g. portions of activated carbon (Darco G-60) as in Example 12. The three charcoal batches were combined, washed with saline solution and then with a minimum of acetone to remove water. The charcoal was then extracted in a Soxhlet apparatus with 3 liters of methylene chloride for a period of 20 hours. The methylene chloride solution was then evaporated in vacuo under nitrogen. The residue of steroidal material was crystallized from 50 cc. of ethyl acetate. There was obtained in this way 413 mg. of 17- hydroxycorticosterone which melts at 208 210 C.

The mother liquors from the foregoing crystallization were evaporated in vacuo under nitrogen and the residual steroid mixture was taken up in 150 ml. of benzene-ethyl acetate (9:1) and passed over a column of 80 g. of silica gel. The ,column was developed with 600 ml. of benzeneethyl acetate (4:1) and eluted with 550 m1. of benzene-ethyl acetate (1:1), followed by 250 ml. benzene-ethyl acetate (2:3) and then 750 ml. of ethyl acetate. The eluates were evaporated junder nitrogen at reduced pressure, and the residues were crystallized from ethyl acetate. In this way an additional crop of crystalline 17-hydroxycorticosterone, weighing 327 mg., was obtained from the ethyl acetate eluate, as well as 240 mg. of ll-desoxy-l'l-hydroxycorticosterone from the benzene-ethyl acetate (1:1). The total yield of l'l-hydroxycorticosterone was 740 mg., which is equivalent to 50% of theory based on the amount of starting material consumed.

Example 17 3 liters of citrated, filtered, defibrinated, whole bovine blood containing penicillin and streptomycin as in Example 12 were treated with a solution of 600 mg. of ll-desoxycorticosterone in 45 ml. of propylene glycol. The resulting solution was perfused through eight lacerated beef adrenal glands for 4 hours (equivalent to 9.3 cycles). The blood was then hemolyzed by thawing and freezing. 2.88 liters of the perfusion liquid were treated with three 15-g. portions of activated carbon and the three batches of carbon were combined, washed with saline and a minimum of acetone and then extracted in a Soxhlet apparatus with methylene chloride for hours. The methylene chloride extract was evaporated in vacuo under nitrogen and the residue was taken up in '70 cc. of benzene-ethyl acetate (9:1). This solution was passed over a column containing g. of silica gel. The column was developed with 250 ml. of benzene-ethyl acetate (4:1). The column was then eluted with 250 cc. of benzene-ethyl acetate (2:1) which removed most of the desoxycorticosterone. This eluate was evaporated under vacuo in nitrogave a greenish fluorescence were crystallized from ether and material melting in the range of 160-170 C. was recovered as corticosterone. Material melting in the range of -140 C. was recovered as unchanged starting material. The column was finally eluted with 300 cc. of ethyl acetate. This eluate was evaporated in vacuo under nitrogen and the residue was crystallized from a mixture of ethyl acetate and petroleum ether. Three batches of crystalline corticosterone melting respectively at 169-1'72 0., 165168 Cs and -164 C. were obtained, weighing a total of 220 mg., which represents a yield of 41% of theory on the basis of unrecovered starting material.

Example 18 300 mg. of 11-desoxy-17-hydroxycorticosterone 21-acetate dissolved in 10 ml. of propylene glycol was added to 3 liters of citrated whole bovine blood containing 300,000 units of penicillin G and 300 mg. of streptomycin sulfate. The resulting fluid was perfused through an intact beef adrenal gland, using arterial flow for 10 hours (one cycle). The blood was frozen, thawed, and treated successively with three 15-g. portions of activated carbon. The activated carbon batches were combined, washed with saline solution and with a minimum of acetone. The carbon was then extracted exhaustively in a Soxhlet apparatus with methylene chloride for 20 hours. The methylene chloride extract was evaporated in vacuo under nitrogen. The residue was taken up in benzene-ethyl acetate (9:1) and passed over a column of 18 g. of silica gel. The column was eluted with benzene-ethyl acetate and ethyl acetate as in Example 16. From the ethyl acetate eluate was obtained crystalline 17-hydroxycorticosterone (25 mg.) melting at 208-210 C. after recrystallization from ethyl acetate. From the benzene-ethyl acetate (1:1) eluate was obtained 110 mg. of 11- desoxy-1'7-hydroxycorticosterone.

We claim:

1. The method of hydroxylating a steroid which comprises addinga member of the group of steroids having one of the following formulas:

CH: Z

and

CH; IY

gen and the residual steroid was recrystallized from ether, yielding 42 mg. of desoxycorticosterone melting at 134-137" C. The column was then eluted with '15 cc. of benzene-ethyl acetate (1:1). This eluate was evaporated in vacuo under nitrogen and the steroid residue was tested with concentrated sulfuric acid. Residues which steroid may contain a double bond at position 5, to a fluid selected from blood, blood components, blood substitutes and mixtures thereof with aqueous salt solutions, perfusing a functioning, excised, mammalian adrenal gland with said fluid, said adrenal gland being so incised as to penetrate superficial layers without cutting the medullary layer and being structurally intact, and subsequently isolating the hydroxylated steroids from said fluid.

2. The method of claim 1 wherein the steroid is ll-desoxycorticosterone.

3. The method of claim 1 wherein the steroid is 11-desoxy-17-hydroxycorticosterone.

4. The method of claim 1 wherein the steriod is progesterone.

5. The method of claim 1 wherein the steroid is A -androstene-3,17-dione.

6. The method of claim 1 wherein the steroid is l'l-hydroxyprogesterone.

7. The method of producing corticosterone which comprises perfusing a functioning, excised, mammalian adrenal gland with blood plasma containing 11-desoxycorticosterone, said adrenal gland being so incised as to penetrate superficial layers without cutting the medullary layer and being structurally intact, and subsequently isolating corticosterone from said plasma.

8. The method of producing 1'7-hydroxycorticosterone which comprises perfusing a functioning,

excised, mammalian adrenal gland with blood plasma containing 11-desoxy-l7-hydroxycorticosterone, said adrenal gland being so incised as to penetrate superficial layers without cutting the medullary layer and being structurally intact, and subsequently isolating 17-hydroxycorticosterone from said plasma.

9. The method of hydroxylating progesterone which comprises perfusing a functioning, excised, mammalian adrenal gland with blood containing progesterone, said adrenal gland being so incised as to penetrate superficial layers without cutting the medullary layer and being structurally intact,

22 and subsequently isolating the hydroxylated steroids from said blood.

10. The method of producing 11-hydroxy-A andr0stene-3,l7-dione which comprises perfusing a functioning, excised, mammalian adrenal gland with blood containing A -androstene-3,17-dione, said adrenal gland being so incised as to penetrate superficial layers without cutting the medullary layer and being structurally intact, and subsequently isolating 11-hydroxy-A -androstene-3,l7 dione from said blood.

11. The method of producing 17-hydroxycorticosterone which comprises perfusing a functioning, excised, mammalian adrenal gland with blood containing 17-hydroxyprogesterone, said adrenal gland being so incised as to penetrate superficial layers without cutting the medullary layer and being structurally intact, and subsequently isolating 17-hydroxycorticosterone from said blood.

OSCAR HECI-ITER. ROBERT P. JACOBSEN. ROGER JEANLOZ. HAROLD LEVY. GREGORY PINCUS. VICTOR SCHENKER. CHARLES W. MARSHALL.

References Cited in the file of this patent UNITED STATES PATENTS Name Date Stern Nov. 18, 1924 OTHER REFERENCES Number

Claims (1)

1. THE METHOD OF HYDROXYLATING A STEROID WHICH COMPRISES ADDING A MEMBER OF THE GROUP OF STEROIDS HAVING ONE OF THE FOLLOWING FORMULAS: