Progesterone biotransformation by plant cell suspension cultures.

Progesterone was converted to 5alpha-pregnane-3alpha-ol-20-one, delta4-pregnene-20alpha-ol-3-one, delta4-pregnene-14alpha-ol-3,20-dione, delta4-pregnene-7beta,14alpha-diol-3,20-dione, and delta4-pregnene-6beta,11alpha-diol-3,20-dione by cell cultures of Lycopersicon esculentum. Cell cultures of Capsicum frutescens (green) metabolized progesterone to delta4-pregnene-20alpha-ol-3-one in very high yield, and Vinca rosea yielded delta4-pregnene-20beta-ol-3-one and delta4-pregnene-14alpha-ol-3,20-dione. A stereospecific reduction of the keto groups and a double bond and stereospecific introduction of hydroxyl groups at the 6, 11, and 14 positions have been observed. The mono- and dihydroxylated progesterones have not previously been reported as metabolic products of progesterone by plant cell systems and represent de novo hydroxylation of a nonglycosylated steroid.     

NMR studies of the stereospecificity of 20 -hydroxysteroid dehydrogenase

We (3,4) previously observed the reduction of 21-dehydrocorticosteroids in the presence of 20β-hydroxysteroid dehydrogenase proceeded at a faster rate than the reduction of the corresponding corticosteroids. The presence of adjacent carbonyl groups suggested the possibility that the increased rate of reduction of the 20-one,21-a1 steroid analogs resulted from a lack of specificity of the enzyme 20β-hydroxysteroid dehydrogenase for either the aldehyde or ketone group. Nuclear magnetic resonance spectroscopy indicated that the angular methyl groups of the steroid were sensitive probes for the constituents on the basic steroid skeleton. The C₁₈ methyl resonance of 17,21-dihydroxy-4-pregnene-3,20-dione and 17-hydroxy-3,20-dioxo-4-pregnene-21-a1 were 0.722 ppm and 0.728 ppm respectively. The magnitude and sign of the change in chemical shift of the C₁₈ methyl resonance for the enzymatic products of 17,21-dihydroxy-4-pregnene-3,20-dione and 17-hydroxy-3,20-dioxo-4-pregnene-21-a1 (+0.135 ppm and +0.144 ppm respectively) were consistent with a stereochemical assignment of 20β-hydroxyl.     

Synthesis of 21-amino-5-pregnene-3,20-dione bysethylene ketal

21-Amino-5-pregnene-3,20-dione bisethylene ketal was obtained in good yield by lithium aluminum hydride reduction of 21-azido-5-pregnene-3,20-dione bisethylene ketal. The azido bisethylene ketal was synthesized by the sequence: deoxycorticosterone → deoxycorticosterone 21-p-toluene-sulfonate → 21-azidoprogesterone → 21-azido-5-pregnene-3, 20-dione bisethylene ketal. The structure of the title compound was confirmed by its conversion to the known 21-acetylaminoprogesterone. 21-Amino-5-pregnene-3,20-dione bisethylene ketal is a stable aminosteroid which is a useful intermediate for the synthesis of C-21 nitrogen derivatives of progesterone.     

Metabolism of 11-deoxycortisol by a Bacillus species

Microbial transformation by a Bacillus species was employed for the preparation of potentially important derivatives of 11-deoxycortisol. Each microbial metabolite was characterised by the application of various spectroscopic methods. The five metabolites of 11-deoxycortisol were characterised as 4-androstene-3,17-dione (2), 14-hydroxy-4-androstene-3,17-dione (3), 14,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (4), 6 beta,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (5) and 15 alpha,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (6). The availability of the metabolites enabled complete elucidation of their [13C]NMR spectra.     

The in vitro metabolism of progesterone, 4-androstene-3,17-dione, testosterone and 17 beta-hydroxy-5 alpha-androstan-3-one by fetal rhesus monkey testes.

Homogenates prepared from fetal rhesus monkey testes were incubated with progesterone, 4-androstene-3,17-dione, testosterone and 17 beta-hydroxy-5 alpha-androstan-3-one. The major progesterone metabolite was 17-hydroxy-4-pregnene-3,20-dione. Testosterone also accumulated in the progesterone incubations. 4-Androstene-3,17-dione was converted chiefly to testosterone. Testosterone was not actively metabolized by the fetal monkey testis. 17 beta-Hydroxy-5 alpha-androstan-3-one was actively converted primarily to 5 alpha-androstane-3 beta,17 beta-diol.     

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.     

Large-Scale Transformation of Steroids by Fungal Spores

Spores of Aspergillus ochraceus and Septomyxa affinis were produced on a large scale by surface sporulation on moist wheat bran and barley. 11alpha-Hydroxylation of progesterone and Reichstein's compound S by spores of A. ochraceus and 1-dehydrogenation of compound S by spores of S. affinis were carried out in 5-liter fermentors. It was shown that, above a certain minimum, increase in aeration and agitation did not significantly affect steroid conversion. The industrial feasibility of the spore process was further demonstrated by 11alpha-hydroxylation of 6alpha-fluoro-16alpha,17alpha-dihydroxypregn-4-ene-3,20-dione in a modified 200-gal stainless-steel vessel with spores of A. ochraceus. Strict aseptic conditions are not necessary, either during harvesting of spores or during steroid transformation.     

Activation of human placental 5-pregnene-3,20-dione isomerase activity by pyridine nucleotides

Isomerization of 5-pregnene-3,20-dione to progesterone by human placental microsomes was stimulated by NAD and NADH. Concomitant oxidation or reduction of nucleotide was not detected based on absorbance at 340 nm. Concentrations giving half-maximum activity were 0.76 microM for NADH and 24.0 microM for NAD. Vmax values with 9.28 microM 5-pregnene-3,20-dione were 22.0 nmol/min/mg protein with NADH and 65.8 nmol/min/mg protein with NAD. When isomerase was assayed as a function of 5-pregnene-3,20-dione concentration, NAD increased Vmax but had no effect on the Km value for steroid. NADP, NADPH, acetylpyridine NAD and deamino NAD did not activate nor did they compete with NAD. Exposure of microsomes to trypsin, phospholipase A2 or phospholipase C resulted in the loss of isomerase activity. Approximately 30% of the initial activity was recovered after detergent solubilization of microsomes. Hydrogen peroxide did not affect activation by NAD. The data are consistent with nucleotide enhancement of a step in the isomerization reaction other than substrate binding.     

Various 18-substituted progesterones

In order to test the potential biological activity of 18-substituted progesterones, 3,20-dioxo-4-pregene-18-carbonitrile (ld approximately) was converted to 3,20-dioxo-4-pregnene-18-carboxylic acid (lb approximately) and methyl 3,20-dioxo-4-pregnene-18-carboxylate (ld approximately) via a sequence of reactions involving an intramolecular hydrolysis of the 18--arbonitrile. Lithium aluminum hydride reduction of the bisethylene ketal derived from la approximately furnished 18-aminomethyl-5-pregnene-3,20-dione 3,20-bisethylene ketal (8 approximately). Acetylation and hydrolysis furnished 18-acetamidomethyl-4-pregnene-3,20-dione (lf approximately) and simple hydrolysis of 8 approximately furnished 3'alpha H-5' 6'-dihydro-2',19 beta-dimethyl-3-oxo-4-goneno [13,17-c]pyridine (9 approximately). None of the compounds exhibited any activity in Clauberg or anti-Clauberg tests.     

Metabolism of progesterone and testosterone by a Bacillus sp.

Microbial transformations by a Bacillus sp. were employed as a means of preparing potentially important derivatives of progesterone and testosterone. Each microbial metabolite was subjected to structure elucidation employing ¹H and ¹³C nmr, mass spectral and cd analysis. Hplc was used for the determination of the percentages of the metabolites formed. The progesterone metabolites were characterised as 14-hydroxy-4-pregnene-3,20-dione (II), 14-hydroxy-5 α -pregnane-3,6,20-trione (III)., 11 α — hydroxy-5 α — pregnane-3, 6,20-trione (IV) and 11 α, 14-dihydroxy-4-pregnene-3,20-dione (V). The testosterone analogs were identified as 4-androstene-3,17-dione (VII), 17 β-hydroxy-5 α -androstene-3,6-dione (VIII), 14-hydroxy-4-androstene-3,17-dione (IX) and 14, 17 β-dihydroxy-4-androsten -3-one (X)₁. The availability of the metabolites enabled complete elucidation of their ¹³C nmr spectra.     

Studies on the Transformation of Steroids by Microorganisms

Microbiological conversions of Reichstein’s substance S (4-pregnene-17α,21-diol-3,20-dione) and hydrocortisone to their corresponding 20β-hydroxy derivatives were achieved by means of numerous strains of Streptomyces such as S. diastaticus (ATCC 3315), S. flavogriseus (H-4449), S. albus (ATCC 3351) etc., and it became apparent that 20-carbonyl reduction is the, wide-spread type of transformation in the Streptomyces species.

Moreover, several interesting strains having both l-dehydrogenating and 20-carbonyl reducing activities were detected. For instance, when Reichstcin’s substance S was used as substrate 1,4-pregnadiene-17α,21-diol-3,20-dione, 4-pregnene-17α,20β,21-triol-3-one and 1,4-pregnadiene-17α,20β,21-triol-3-one were isolated simultaneously using S. flaveolus (D-551), s. roseochromogenes (O-36) etc. These strains also exhibited similar transformation patterns in the use of hydrocortisone.     

High affinity progesterone binding sites of human uterine microsomal membranes

Microsomal membranes sedimented at 40 000 g were prepared from human myometrium samples. The progesterone binding properties of microsomal suspensions were determined by incubating microsomes and [3H]progesterone at 4 degrees C. Dextran-coated charcoal was used for the separation of bound and free steroids. Membrane-associated progesterone binding sites of high affinity were identified in microsomes prepared from pregnant and nonpregnant uteri. The binding was saturable (Kd approximately 4 X 10(-9) M, concentration of binding sites 400-900 fmol/mg microsomal protein) and specific for natural progesterone. Of 21 steroids tested only 21-hydroxy-4-pregnene-3,20-dione, 17 alpha-hydroxyprogesterone and testosterone showed moderate competition against progesterone with relative affinities between 7.0-20.0% (R.A. of progesterone 100%). 5 alpha-Dihydroprogesterone and 5 alpha-dihydrotestosterone showed weak cross reaction (relative affinities 2.5 and 2.0%, respectively). Corticosteroids, estrogens and the 5 synthetic progestins tested showed only weak competition with relative affinities lower than 1.0%. These microsomal progesterone binding sites of high affinity and limited capacity resemble steroid hormone receptors but they are different from the soluble cytosolic progesterone receptor of human uterus in terms of steroid specificity. The physiological function of this microsomal progesterone receptor is unknown.     

6-Oxygenation of 3-oxo-4-ene-steroids in high yields after 3-imine-formation

3-Imine formation between primary amines and 3-oxo-4-ene-steroids, followed by hydrolysis of the imines (either spontaneously during work up or induced by acetic acid) has been shown to cause 6-oxygenation of the steroids tested (17 beta-hydroxy-4-androsten-3-one, 4-androstene-3,17-dione, 4-pregnene-3,20-dione and 4-cholesten-3-one). The main products are the 6 beta-hydroxy- and the 6-oxo-derivatives of the respective steroid. These derivatives were identified by chromatographic mobilities and by gas chromatography-mass spectrometry. The formation of 6 beta-hydroperoxy-derivatives is suggested and these derivatives were tentatively identified. The highest yields of 6-oxygenated products (30-50%) were found when cadaverine and spermine were reacted with the steroids. The addition of reduced glutathione during hydrolysis of the steroid 3-imines of cadaverine, hexylamine and ethanolamine as well as addition of ascorbic acid during the hydrolysis of the steroid 3-imines of cadaverine substantially reduced the 6-oxygenation. Steroid 3-imine formation and hydrolysis which yields 6-oxygenated derivatives has also been shown to occur during work up (evaporation) of organic solvent extracts of rat liver microsomes (105,000 g sediments) to which 17 beta-hydroxy-4-androsten-3-one, 4-androstene-3,17-dione, 4-pregnene-3,20-dione or 4-cholesten-3-one respectively had been added. It is concluded that there is a risk that these organic reactions are mistaken for enzymatic conversions during in vitro investigations of 3-oxo-4-ene-steroids.     

Biotransformations of steroid compounds by Chaetomium sp. KCH 6651

Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorganism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted to 14α-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6β-hydroxyandrost-4-en-3,17-dione (in low yield), while testosterone underwent regioselective hydroxylation at 6β position. Progesterone was transformed to a single product—6β,14α-dihydroxypregnan-4-en-3,20-dione in high yield, whereas biotransformation of DHEA resulted in the formation of 7α-hydroxy derivative, which was subsequently converted to 7α-hydroxyandrost-4-en-3,17-dione.     

Production and characterisation of antisera to 18-hydroxy-11-deoxycorticosterone.

The production of highly sensitive and specific antisera to 18-hydroxy-11-deoxycorticosterone (18,21-dihydroxy-4-pregnene-3,20-dione) is reported. The antisera were generated in rabbits and guinea pigs with a 3-carboxymethoxime derivative of the steroid coupled to rabbit serum albumin. Antibody characteristics were determined by a radioimmunoassay procedure. Only minor differences between the two animal species were observed. Antibody titers ranged from 10 to 8000. Association constants were in the order of 10(8) to 10(10) 1/mole. A minimal amount of 40 pg unlabeled steroid was necessary to displace 50% of the tritiated steroid. Cross reaction with cortisol was 0.0002% to 0.031%, with aldosterone 0.0007% to 1.09%, with corticosterone 0.0025% to 1%, with 18-hydroxy-corticosterone 0.05% to 1% and with progesterone 0.0048% to 1.5%.     

Human glutathione transferase A3-3, a highly efficient catalyst of double-bond isomerization in the biosynthetic pathway of steroid hormones.

The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Delta(5)-androstene-3,17-dione and Delta(5)-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (k(cat)/K(m)) with Delta(5)-androstene-3,17-dione was determined as 5 x 10(6) m(-1) s(-1), which is considerably higher than with any other GST substrate tested. The rate of acceleration afforded by GST A3-3 is 6 x 10(8) based on the ratio between k(cat) and the rate constant for the nonenzymatic isomerization of Delta(5)-androstene-3,17-dione. Besides being high in absolute numbers, the k(cat)/K(m) value of GST A3-3 exceeds by a factor of approximately 230 that of 3beta-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Delta(5)-Delta(4) double-bond isomerization. Furthermore, GSTA3-specific polymerase chain reaction analysis of cDNA libraries from various tissues showed a message only in those characterized by active steroid hormone biosynthesis, indicating a selective expression of GST A3-3 in these tissues. Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones.     

New aromatic esters of progesterone as antiandrogens

The in vivo and in vitro antiandrogenic activity of four new progesterone derivatives: 4-bromo-17alpha-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione 1,4-bromo-17alpha-(pchlorobenzoyloxy)-4-pregnene-3,20-dione 2, 4-bromo-17alpha-(p-bromobenzoyloxy)-4-pregnene-3,20-dione 3 and 4-bromo-17alpha-(p-toluoyloxy)-4-pregnene-3, 20-dione 4 was determined. These compounds were evaluated as antiandrogens on gonadectomized hamster prostate and reduced the weight of the prostate glands in gonadectomized hamsters treated with testosterone 5 (T) or dihydrotestosterone 6 (DHT) in a similar manner to that of commercially available finasteride, thus indicating a potent in vivo effect. The in vitro studies showed that steroids 1-4 have a weak inhibitory activity on 5alpha-reductase with IC50 values of: 280 (1), 2.6 (2), 1.6 (3) and 114 microM (4). The presence of Cl and Br atoms in the C-17 benzoyloxy group tends to increase the inhibitory potency of the compounds. The binding efficiency of the synthesized steroids 1-4 to the androgen receptor of the prostate gland is also evaluated. All compounds form a complex with the receptor and this explains the weight reduction of the seminal vesicles in the animals treated with DHT plus steroids 1-4.     

Identification of 2 alpha-hydroxy-4-pregnene-3,20-dione in human pregnancy urine.

2 alpha-Hydroxyprogesterone (2 alpha-hydroxy-4-pregnene-3,20-dione) was identified in human late pregnancy urine by liquid-gel chromatography, GLC and GC-MS. In addition, the following 2-hydroxylated C21 steroids were found and identified as 2 zeta-hydroxy-5 zeta-pregnane-3,20-dione, 2 zeta,20 zeta-dihydroxy-4-pregnen-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha- (and 5 beta)-pregnan-20-one, two isomers of pregnane-2,3,20-triol and 2 zeta,3 zeta,16 zeta-trihydroxy-5 zeta-pregnan-20-one.     

11β-Hydroxysteroid dehydrogenase activity in progesterone biotransformation by the filamentous fungus Cochliobolus lunatus

Progesterone biotransformation was examined in relation to hydroxylating and dehydrogenating enzymes of Cochliobolus lunatus. 11β-hydroxysteroid dehydrogenase activity (11β-HSD) was located in cytosolic fraction and was NADP-dependent, inducible by progesterone and apparently unidirectional. Several inhibitors of 11β-hydroxysteroid dehydrogenase were tested; furosemide, glycyrrhizic-acid and carbenoxolone did not influence the dehydrogenation of 11β-hydroxy-4-pregnene-3,20-dione to 4-pregnene-3,11,20-trione, although grapefruit juice significantly reduced the rate of progesterone hydroxylation.     

Synthesis of the allylic gonadal steroids, 3 alpha-hydroxy-4-pregnen-20-one and 3 alpha-hydroxy-4-androsten-17-one, and of 3 alpha-hydroxy-5 alpha-pregnan-20-one

A method for the convenient synthesis of the recently isolated allylic gonadal steroids, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha-dihydroprogesterone; 3 alpha-DHP) and 3 alpha-hydroxy-4-androsten-17-one (3 alpha-HA), was developed using 4-pregnene-3,20-dione (progesterone) and 4-androstene-3,17-dione as substrates and potassium trisiamylborohydride (KS-Selectride) as reducing agent. Similar reactions were also used for the reduction of 5 alpha-pregnane-3,20-dione to 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-HP). The yields were about 15%, 50%, and greater than 90% for 3 alpha-DHP, 3 alpha-HA and 3 alpha-HP, respectively. Structures of the products, including the 3 beta-isomers and the 17 alpha-epimer, formed in these reactions were determined by NMR and mass spectroscopic methods.