Steroid derivatives

Mycobacterium flavum was used to effect the transformation of 16β-methyl-16,17-oxido-7β,11α-dihydroxypregn-4-ene-3,20-dione (I) and the final products were isolated and identified as 16β-methyl-16,17-oxido-7β,11α-dihydroxypregna-1,4-diene-3,20-dione (II) and 16β-methyl-16,17-oxido-11α-hydroxypregna-1,4,6-triene-3,20-dione (IV), and the intermediate product as 16β-methyl-16,17-oxido-11α-hydroxypregna-4,6-diene-3,20-dione (III).     

Steroid derivatives

Hydroxylation of l7α-acetoxy-6-chloro-16-methylene-4,6-pregnadiene-3,20-dione (Chlorosuperlutin, I) byCunninghamella blakesleeana yielded a 15β-hydroxyderivative II. Analogous transformation of 17α-acetoxy-16-methylene-4,6-pregnadiene-3,20-dione (Superlutin, IV) included a hydroxylation in position 15β and probably also in 11β with a concomitant reduction of the 6,7-double bond.     

Isolation and partial synthesis of a new metabolite of medroxyrogesterone acetate.

3 alpha-Hydroxy-17-acetoxy-6 alpha-methyl-5 beta-pregnan-20-one (IIIa) has been isolated from urine of patients receiving medroxyprogesterone acetate (MPA). It was characterized by partial synthesis from MPA by catalytic reduction with palladium-charcoal to 17-acetoxy-6 alpha-methyl-5 beta-pregnan-3,20-dione (IV) and reduction of the latter with sodium borohydride. The isolation of 6 beta, 17,21-trihydroxy-6 alpha-methyl-pregn-4-ene-3,20-dione (IIc) is reported for the first time. The 17- and 21-monoacetates of this compound have been isolated and characterized earlier by other investigators. 7 alpha-3H-Medroxyprogesterone acetate was administered to 4 subjects by intravenous and intramuscular injections and by mouth. The ring A saturated metabolite IIIa was excreted in 0.1% to 4.0% of the administered dose; the highest excretion was after the intravenous dose and lowest after oral ingestion. 6 beta, 17,21-Trihydroxy-6 alpha-methylpregn-4-ene-3,20-dione (IIc) and its 17- and 21-monoacetates were excreted in about 5% of the doses in all subjects. No increase in 6 beta-hydroxylation was observed in the patient treated with o,p'-DDD,2,2-bis(2-chlorophenyl, 4'-chlorophenyl)-l,1-dichloroethane.     

Synthesis of 18-hydroxy-19-norcorticosterone and 18-deoxy-19-noraldosterone. Structure determination of related 19-nor steroids by means of 2-D 1H nmr

The compounds named in the title have been synthesized from the di-(ethylene ketal) of 21-hydroxy-3,20-dioxo-19-norpregn-5-ene-18, 11 beta-lactone and its 5(10)-ene isomer. Reduction of this mixture 1 with sodium aluminum bis-(methoxyethoxy)hydride furnished the 11 beta, 18, 21-triol 2a. Conversion to the 18,21-diacetate 2b, followed by deketalization to the free dione 3 and hydrolysis, afforded 18-hydroxy-19-norcorticosterone 4a which, in the solid state and probably in solution, has the 18,20-hemiacetal structure. Periodate oxidation of 4a gave 11 beta-hydroxy-3-oxo-19-norandrost-4-ene-17 beta, 18-carbolactone 5a, and acid treatment of 4a or its precursor 2a yielded 18-deoxy-19-noraldosterone 6a. The structure of 5a was confirmed by mass spectrometry and 1H nmr, and compared with that of its C-19 methyl homolog 5b and 19-noraldosterone-gamma-etiolactone 8. In particular, 2-D nmr COSY 45 experiments, affording full 1H line assignments, have rigorously established the "natural" beta (axial) configuration of the C-10 hydrogen in the 19-nor lactones 5a and 8, and therefore also in the related 4a, 6a and 19-noraldosterone 7.     

Studies on the Microbiological Transformation of Steroids

The microbiological reduction of the 20-carbonyl group of steroids has been investigated. Candida pulcherrima IFO 0964 and Sporotrichum gougeroti IFO 5982 converted the following substrates into the corresponding 20β-hydroxy derivatives (yields of the products are indicated in parentheses): Reichstein’s Compound S (60~70%) and 17α,21-dihydroxypregna-l,4-diene- 3,20-dione (40~80%). Rhodotorula glutinis IFO 0395 converted the following substrates into the corresponding 20α-hydroxy derivatives: Reichstein’s Compound S (65%), 17 α,21-dihydroxy- pregna-l,4-diene-3,20-dione (80%), llβ,l7α-dihydroxypregn-4-ene-3,20-dione (45%) and 17α, 19,21 -trihydroxypregn-4-ene-3,20-dione (10%).     

Microbial conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione acetates by Nocardioides simplex VKM Ac-2033D

The conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione 21-acetate (I) and 17,21-diacetate (VI) by Nocardioides simplex VKM Ac-2033D was studied. The major metabolites formed from I were identified as pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV). Pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione (III) and pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V) were formed in minorities. Biotransformation products formed from VI were pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17,21-diacetate (VII), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII), pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V). The conversion pathways were proposed including 1(2)-dehydrogenation, deacetylation, 20beta-reduction and non-enzymatic migration of acyl group from position 17 to 21. The conditions providing predominant accumulation of pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) from I and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII) from VI in a short-term biotransformation were determined.     

The chemistry of 9 alpha-hydroxysteroids. 1. Preparation of 9 alpha,17 beta-dihydroxy-17 alpha-ethynylandrost-4-en-3-one

9 alpha-Hydroxyandrost-4-ene-3,17-dione 1, when allowed to react with dipotassium acetylide in tetrahydrofuran, resulted, after chromatographic separation, in 4-methyl-19-norandrosta-4,9-diene-1,17-dione 2, 4 xi-methyl-19-norandrosta-5(10),9(11)-diene-1,17-dione 3, 4-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-4,9-dien-1-one 4, 4 xi-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-5(10),9(11)-dien- 1-one 5, and 17 alpha-ethynyl-17 beta-hydroxy-9,10-secoandrost-4-ene-3,9-dione 6. Selective protection of delta 4-3-ketone of 9 alpha-hydroxyandrost-4-ene-3,17-dione 1 as its dienol methyl ether 7, and subsequent reaction with lithium acetylide-ethylenediamine followed by acidic hydrolysis, afforded 9 alpha,17 beta-dihydroxy-17 alpha- ethynylandrost-4-en-3-one 8.     

Androgenic and anti-androgenic effects of progesterone derivatives with different halogens as substituents at the C-6 position.

The pharmacological activities of four pregnane derivatives: 17alpha-hydroxy-16beta-methylpregna-4,6-diene-3,20-dio ne (7), 17alpha-acetoxy-16beta-methylpregna-4,6-diene-3,20-dio ne (8), 17alpha-acetoxy-6-bromo-16beta-methylpregna-4,6-diene- 3,20-dione (10), and 17alpha-acetoxy-6-chloro-16beta-methylpregna-4,6-diene -3,20-dione (11), were determined. The derivatives were evaluated on gonadectomized male hamster flank organs and seminal vesicles. The results indicate that topical applications of testosterone (T) on the flank organs increased the diameter of the pigmented spot. Similarly, the same phenomenon occurred on the glands treated with compound 11, whereas compound 10 decreased the size of the spot significantly. In this study, we determined the effects of several new steroids on the conversion of T to DHT in flank organs and seminal vesicles. The results show that compound 10 inhibited T conversion to DHT, but compound 11, at a dose of 200 microg, stimulated T conversion in both flank organs and seminal vesicles. However, when 2 mg of compound 11 was applied, it inhibited the conversion of T to DHT, suggesting that this compound also represses gonadotropin release. The difference between compounds 10 and 11 involves the electronegativity of the halogen at the C-6 position of the progesterone skeleton. These data clearly indicate that by decreasing the electronegativity of the halogen at C-6 (compound 10), 5alpha-reductase is inhibited in both tissues and at different pHs. On the other hand, when the electronegativity of the halogen atom was increased (11), there was a much lower inhibitory effect on the conversion of T to DHT.     

Synthesis of 3 beta,16 beta,19-trihydroxyandrost-5-en-17-one and 16 beta,19-dihydroxyandrost-4-ene-3,17-dione and their 19-oxo derivatives

3 beta,16 beta,19-Trihydroxyandrost-5-en-17-one (12) was synthesized from 5 alpha-bromo-3 beta-acetoxy-6 beta,19-epoxyandrostan-17-one (2) through acetoxylation at C-16 beta of the enol acetate 4 with lead tetraacetate and reductive cleavage of the epoxide ring with zinc dust yielding the 3 beta,16 beta-diacetoxy-19-hydroxy steroid 11, followed by hydrolysis of the acetoxy groups with sulfuric acid. Jones oxidation of compound 11 followed by the acid hydrolysis gave the 19-oxo steroid 15. 5 alpha-Bromo-3 beta-hydroxy-16 beta-acetoxy-6 beta,19-epoxyandrostan-17-one (8), obtained by selective hydrolysis of the 3-formate 5 with ammonium hydroxide, was oxidized with Jones reagent to afford the 3-oxo steroid 16, which was converted into the 19-hydroxy derivative 17 by treatment with zinc dust. 16 beta,19-Dihydroxyandrost-4-ene-3,17-dione (18) and its 19-oxo derivative 21 were obtained from compound 17 through a similar reaction sequence.     

Synthesis of 19-noraldosterone, a potent mineralocorticoid

19-Noraldosterone has been prepared for biological re-evaluation through an extension of a recent synthesis of 19-hydroxyaldosterone: 21-hydroxy-6 beta,19-epoxy-4-pregnene-3,20-dion-20-ethylene ketal-18,11 beta-lactone (1a) was acetylated and then reduced with zinc-acetic acid-isopropanol to the 19-ol 2b. Treatment with sodium acetate transposed the double bond into conjugation, and 2a thus obtained was oxidized with pyridinium chlorochromate to the 19-oxo compound 3. Decarbonylation to the 19-nor lactone 4 was effected by heating with alkali. Protection of the C-3 carbonyl was achieved by ketalization and the resulting mixture of the 5-ene and 5(10)-ene ketals 5 was reduced with DIBAH to the corresponding mixture of the hemiacetals 6. Acid hydrolysis of the latter afforded 19-noraldosterone (7), accompanied by the 18,21-anhydro ketal 8. 19-Noraldosterone in the solid state exists in the cyclic form 7b, which appears to be also the predominant isomer present under conditions of mass spectrometry. [1H]NMR indicates that in chloroform 19-noraldosterone exists mostly as an equilibrium mixture of structures 7a and 7b. Sodium periodate oxidation furnished the gamma-etiolactone 9, confirming the 17 beta configuration in 7.     

Recognition of D-homoannulation by proton and carbon NMR spectra

One-and two dimensional proton and carbon NMR spectra of the D-homoannulated rearrangement product of triamcinolone (9 alpha-fluoro-11 beta,16 alpha,17 alpha, 21-tetrahydroxy-pregna-1,4-diene-3,20-dione) establish its structure as that of 9 alpha-fluoro-11 beta,16 alpha,17 alpha-trihydroxy-17 beta-hydroxy-methyl-D-homoandrosta-1,4-diene-3,17 alpha-dione. These methods accord ready recognition of D-homoannulation of C21-17-hydroxy-20-ketosteroids.     

ORG-2058 as a ligand in the assay of progesterone receptor in breast cancer

Tritiated [(16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione)-6,7-3H] (ORG-2058) and 17,21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020) were compared as ligands in the assay of progesterone receptor in human and rat breast tumors. We found that ORG-2058 is a better ligand because of its low nonspecific binding. Most of the nonspecific binding of the other ligand R5020, is to proteins which bind corticosteroids. In cancerous tissue ORG-2058 binds to progesterone receptor linearly in a range of protein concentrations which are normally used in the receptor assay. On the other hand, R5020 exhibits binding linearity over a narrower protein concentration in many tumor biopsies, which may cause severe limitation in the assay procedure or frequent underestimation of receptor content.     

Microbial Transformation of 19-Hydroxypregnanes

Nocardia species aromatized 19-hydroxyprogesterone, 3beta, 19-dihydroxy-pregn-5-en-20-one 3-acetate and pregn-5-ene-3beta, 19, 20beta-triol 3-acetate, without cleavage of the side chain, into 3-hydroxy-19-norpregna-1,3,5 (10)-trien-20-one. Septomyxa affinis aromatized the ring A and cleaved the side chain of 19-hydroxyprogesterone to yield estrone. With 19-hydroxypregna-4, 7-diene-3, 20-dione as substrate, the transformation was more complex and many products were formed.     

Synthesis of 18,19-dihydroxycorticosterone

A four-step synthesis of 18,19-dihydroxycorticosterone 5c, starting with 19,21-dihydroxy-3,20-dioxopregn-5-ene-18,11 beta-lactone-di-(ethylene ketal) 2, is presented. Reduction of 2 with sodium aluminum bis-(methoxyethoxy)hydride gave 11 beta,18,19,21-tetrahydroxy-pregn-5-ene-3,20-dione-di-(ethylene ketal) 3a. Acetylation furnished the corresponding 18,19,21-triacetate 3b, which on treatment with a mixture of perchloric and acetic acids gave 18,19-dihydroxycorticosterone 18,19,21-triacetate 4b. Mild saponification yielded the title compound which, on the basis of ir and nmr spectra, exists as one C-20 isomer of the hemiacetal structure 5c. Periodate oxidation of 5c gave the expected 11 beta, 19-dihydroxy-3-oxoandrost-4-ene-17 beta, 18-carbolactone 6b.     

Effect of 5 alpha-dihydrocorticoids on enzymes of gluconeogenesis in rat liver

5 alpha-Dihydrocortisol (11 beta, 17, 21-trihydroxy-5 alpha-pregnane-3,20-dione), 5 alpha-dihydrocorticosterone (11 beta, 21-dihydroxy-5 alpha-pregnane-3,20-dione) as well as cortisol (11 beta, 17, 21-trihydroxy-4-pregnene-3,20-dione) and corticosterone (11 beta, 21-dihydroxy-4-pregnene-3,20-dione) were administered for seven days to male rats. Blood glucose increased in cortisol- and corticosterone-treated rats and blood insulin decreased after 5 alpha-dihydrocorticosteroid treatment. In the liver, total protein was elevated after cortisol, corticosterone and 5 alpha-dihydrocorticosterone application. Phosphoenolpyruvate carboxykinase and fructose-1,6-diphosphatase activities in liver were significantly lowered after treatment with 5 alpha-dihydrocortisol and 5 alpha-dihydrocorticosterone.     

Convenient synthesis of 18-hydroxylated cortisol and prednisolone.

18,20-Epoxy-11 beta,17 alpha,20 beta,21-tetrahydroxypregn-4-en-3-one was synthesized by the application of hypoiodite reaction to the cortisol acetonide. The intermediary 18-iodo derivative was converted to the 11-oxo steroid by chromic acid prior to silver ion-assisted solvolysis. Removal of the protective group with hydrochloric acid was finally carried out to give the desired 11 beta,17 alpha,18,21-tetrahydroxypregn-4-ene-3,20-dione as the hemiacetal form. 18,20-Epoxy-11 beta-17 alpha,20 beta,21- tetrahydroxypregna-1,4-dien-3-one was also prepared from prednisolone through a similar reaction sequence.     

D-homo isomers of 17-acetoxy-6-methylpregna-4,6-diene-3,20-dione

The three most readily accessible D-homo isomers of 17-acetoxy-6-methylpregna-4,6-diene-3,20-dione (Ib) have been prepared and characterized in detail. The 17-keto isomers IIb and IIIb were obtained by base-catalyzed rearrangement of Ia followed by reacetylation, and the 17a-ketone IVb by Lewis-Acid treatment of Ia.     

Studies on the Microbiological Transformation of Steroids

An attempt was made to clarify how Pellicularia filamentosa f. sp. microsclerotia IFO 6298 capable of hydroxylating C₂₁-steroids at the C-19 position converts C₁₉-steroids, especially monohydroxyderivatives of androst-4-ene-3, 17-dione. Such substrates as 11β-hydroxyandrost-4-ene-3,17-dione (I), androst-4-ene-3, 11, 17-trione (II), androsta-1,4-diene-3, 17-dione (III), 11β-hydroxyandrosta-1,4-diene-3,17-dione (IV), 14α-hydroxyandrost-4-ene-3, 17-dione (V), 15α-hydroxyandrost-4-ene-3, 17-dione (VI) and 9α-hydroxyandrost-4-ene-3, 17-dione (VII) were converted by the organism. All the main and several minor products were then isolated and identified. As a result it is concluded that this organism converts I and II into 14α-hydroxyandrost-4-ene-3,11,17-trione, III and IV into 14α-hydroxyandrosta-1,4-diene-3,1l,17-trione, V into 11α 14α dihydroxyandrost-4-ene-3, 17-dione (main) and 11β, 14α-dihydroxyandrost-4-ene-3, 17-dione (minor, a tentative structure), VI into 11β, 15α-dihydroxyandrost-4-ene-3,17-dione (main) and 15α-hydroxyandrost-4-ene-3,11,17-trione (minor, a tentative structure) and VII into 9α, 14α-dihydroxyandrost-4-ene-3, 17-dione (main) and 6β, 9α-dihydroxyandrost-4-ene-3,17-dione (minor).

In addition, the structural requirement of substrate for the 19-hydroxylation catalyzed by the organism and the influence of a hydroxyl group on steroid nucleus upon the 11β- and 14α-hydroxylations and the 11β-OH-dehydrogenation was discussed.     

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.     

A practical large-scale synthesis of 17alpha-acetoxy-11beta-(4-N, N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione (CDB-2914)

A new practical synthesis of 17alpha-acetoxy-11beta-(4-N, N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione (CDB-2914) is described. The synthesis gives easily isolable solids at all steps and is amenable to large-scale process.