New approach to 3-oxo-4-aza-5alpha-androst-1-ene-17beta-(N-tert-butylcarboxamide)

We describe the synthesis of 3-oxo-4-aza-5alpha-androst-1-ene-17beta-(N-tert-butylcarboxamide) (finasteride) from 4-androstene-3,17-dione (AD) in seven steps in an overall yield of 18.6% via oxidation, ammoniumation, dehydration, and dehydrogenation.     

A theoretical model of the catalytic mechanism of the Delta5-3-ketosteroid isomerase reaction

The present paper describes a theoretical approach to the catalytic reaction mechanism involved in the conversion of 5-androstene-3,17-dione to 4-androstene-3,17-dione. The model incorporates the side chains of the residues tyrosine (Tyr(14)), aspartate (Asp(38)) and aspartic acid (Asp(99)) of the enzyme Delta(5)-3-ketosteroid isomerase (KSI; EC 5.3.3.1). The reaction involves two steps: first, Asp(38) acts as a base, abstracting the 4beta-H atom (proton) from C-4 of the steroid to form a dienolate as the intermediate; next, the intermediate is reketonized by proton transfer to the 6beta-position. Each step goes through its own transition state. Functional groups of the Tyr(14) and Asp(99) side chains act as hydrogen bond donors to the O1 atom of the steroid, providing stability along the reaction coordinate. Calculations were assessed at high level Hartree-Fock theory, using the 6-31G(*) basis set and the most important physicochemical properties involved in each step of the reaction, such as total energy, hardness, and dipole moment. Likewise, to explain the mechanism of reaction, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), atomic orbital contributions to frontier orbitals formation, encoded electrostatic potentials, and atomic charges were used. Energy minima and transition state geometries were confirmed by vibrational frequency analysis. The mechanism described herein accounts for all of the properties, as well as the flow of atomic charges, explaining both catalytic mechanism and proficiency of KSI.     

Estrogen metabolism in neural tissues of rabbits: 17 beta - hydroxysteroid oxidoreductase activity.

Incubation of adult rabbit neural tissue homogenates with (3H)-estrone and (3H)-estradiol revealed that the conversion of estrone to estradiol is higher in both male and female animals than estradiol to estrone. Both 17 oxidation and reduction are higher in male animals than in females. However, it is observed that the quotient of estrone leads to estradiol/estradiol leads to estrone for pituitary tissue and hypothalamus are higher in females than in males. There is no such dimorphism in cerebral cortex. The overall metabolism in pituitary is higher than in hypothalamus and cortex in both sexes. These results suggest that 17 beta hydroxysteroid oxidoreductase activity may play an important role in the regulation of estrogen function in neuroendocrine tissues.     

Radioimmunoassay for plasma betamethasone 17-benzoate.

A sensitive radioimmunoassay for plasma betamethasone 17-benzoate has been developed. The antiserum used was obtained by immunizing rabbits with betamethasone 17-benzoate-21-hemisuccinate-bovine-serum-albumin conjugate. All of the endogenous steroids tested cross reacted less than 0.10%. A standard curve was established with a useful range from 0.05-5 ng. Reliability criteria were satisfactory. Measurement of plasma concentrations of betamethasone 17-benzoate was performed in patients and in rabbits following occlusive dressing of betamethasone 17-benzoate cream and gel base.     

Steroids 36: synthesis of 16,16-dimethyl-17-ketosteroids and 16,16-dimethyl-17 beta-hydroxysteroids

Direct conversion of 17-ketosteroids (Ia-f) into 16,16-dimethyl-17 beta-hydroxysteroids (IIa-f) and 16,16-dimethyl-17-ketosteroids (IIIa-f) was achieved with methyl iodide in the presence of NaH.     

Steroid specificity of human placental 5-ene-3β-hydroxysteroid oxidoreductase

The properties of 5-ene-3β-hydroxysteroid oxidoreductase (3β-HSD) from human placental homogenates were studied invitro. The apparent Michaelis constants for 3β-HSD with the substrates pregnenolone (Δ⁵P) and dehydroepiandrosterone (DHA) were 170 nM and 40 nM respectively. The optimal pH for both these substrates was between 10 and 12. With NAD as the substrate, the Km for pregnenolone was 20 μM and for DHA, 17 μM. The activity of 3β-HSD was inhibited by various steroids. Competitive inhibitors (pregnenolone substrate) included: ethynylestradiol (inhibition constant Ki=7.3 nM), DHA (Ki=46 nM), estradiol-17β (Ki=46 nM), cholesterol (Ki=0.68 μM) and 16α-hydroxydehydroepiandrosterone (16αOHDHA) (Ki=2.2 μM). When the substrate was DHA, competitive inhibition occurred with the following steroids: ethynylestradiol (Ki=6.4 nM), estradiol-17β (Ki=69 nM), pregnenolone (Ki=91 μM), cholesterol (Ki=1.3 μM) and 16αOHDHA (Ki=1.9 μM). 4-Ene-3-ketosteroids such as androstenedione, progesterone (Δ⁴P), norethindrone and chlormadinone acetate acted as noncompetitive inhibitors towards both substrates.     

Canine testicular 17beta-hydroxysteroid oxidoreductase activity. I. Substrate specificity.

The substrate specificity of 17beta-hydroxysteroid oxidoreductase (17beta-HOR) activity was investigated in microsomal preparations of canine testes. Enzyme activity was measured by quantitating the conversion of radioactive substrates to products. The apparent Michaelis constants were determined to be 1.3 x 10(-6)M for androstenendione, 3--10 x 10(-6)M for dehydroepiandrosterone and 25 x 10(-6)M for estrone. These data are similar to those reported for human testicular 17beta-HOR activity and suggest that the canine activity may serve as an animal model for the study of testicular 17beta-HOR.     

Factors affecting the adrenocorticotropic hormone stimulation of rabbit adrenal 17α-hydroxylase activity

Adrenocorticotropic hormone (ACTH)-stimulated 17α-hydroxylase activity of rabbit adrenal tissue has been shown to be associated with the subcellular fractions sedimented from 0.25 M sucrose at 33 000 × g for 60 min and at 105 000 × g for 60 min. The fraction sedimenting at 9000 × g for 20 min (mitochondria) contained the majority of the 11β-hydroxylase activity but also had a significant amount of 17α-hydroxylase activity. All subcellular 17α-hydroxylase activity showed an apparent preference for pregnenolone over progesterone. A 1 : 1 mixture of wholehomogenates of adrenal tissue from control and ACTH-stimulated rabbits incubated with[4-¹⁴C]pregnenolone synthesized as much 17α-hydroxylated corticosteroids as homogenate from the ACTH-stimulated tissue alone. However, the mixed homogenate synthesized only 1/4th–1/5th as much 17-deoxycorticosteroids as control, non-stimulated tissue, suggesting that the control tissue contained no inhibitor of 17α-hydroxylation, whereas ACTH-stimulated tissue may contain an inhibitor of 17-deoxycorticoid formation. 24-h dialysis of whole homogenates and subcellular fractions of adrenal tissue from control and ACTH-stimulated animals showed that 17α-hydroxylation was not activated in control tissue and somewhat inactivated in ACTH-stimulated tissue by this treatment. On the other hand, dialysis activated 17-deoxycorticoid formation by whole homogenates, but not in subcellular fractions, of both ACTH-stimulated and control adrenal tissue. Injection of 5 mg/kg cycloheximide prior to the first of 2 daily ACTH injections caused an average of 270 g body weight loss while not affecting the increase in adrenal weight effected by the ACTH. Adrenal tissue homogenates from cycloheximide injected animals produced only 50% as much 17α-hydroxycorticosteroids as homogenates of tissue from animals injected with ACTH alone and produced an amount of17-deoxycorticoids intermediate between homogenates of control and ACTH-stimulated tissue, suggesting the requirement of protein synthesis for 17α-hydroxylation stimulating activity of ACTH.     

Natural oils are better than organic solvents for the conversion of soybean sterols to 17-ketosteroids by Mycobacterium fortuitum

Conversion of soybean sterols to 17-ketosteroids by thawed cells of Mycobacterium fortuitum is strongly inhibited by organic solvents. Use of natural oils instead of the solvents leads to enhanced activity, even after 4 h.The authors are with the School of Life Sciences, Devi Ahilya Vishwavidyalaya, Vigyan Bhawan, Khandwa Road Campus, Indore-452 001, India.     

Screening of mycelial fungi for 7α- and 7β-hydroxylase activity towards dehydroepiandrosterone

In total, 481 fungal strains were screened for the ability to carry out 7(α/β)-hydroxylation of dehydroepiandrosterone (DHEA, 3β-hydroxy-5-androsten-17-one). Representatives of 31 genera of 15 families and nine orders of ascomycetes, 17 genera of nine families and two orders of zygomycetes, two genera of two families and two orders of basidiomycetes, and 14 genera of mitosporic fungi expressed 7(α/β)-hydroxylase activity. The majority of strains were able to introduce a hydroxyl group to position 7α. Active strains selectively producing 3β,7α-dihydroxy-5-androsten-17-one were found among Actinomucor, Backusella, Benjaminiella, Epicoccum, Fusarium, Phycomyces and Trichothecium, with the highest yield of 1.25 and 1.9 g L^?1 from 2 and 5 g L^?1 DHEA, respectively, reached with F. oxysporum. Representatives of Acremonium, Bipolaris, Conidiobolus and Curvularia formed 3β,7β-dihydroxy-5-androsten-17-one as a major product from DHEA. The structures of the major steroid products were confirmed by TLC, gas chromatography (GC), mass spectra (MS), and ¹H-NMR analyses.     

Sitosterol bioconversion with resting cells in liquid polymer based systems

The use of a biocompatible water-immiscible organic phase as a substrate and product pool has been acknowledged as an effective tool to overcome the low volumetric productivity of aqueous bioconversion systems involving hydrophobic compounds. The growing environmental and public health awareness is nevertheless leading to restrictions in the use of organic solvents in industrial processes, in order to render these more environmentally friendly. Different approaches are hence being assessed for the design of alternative bioconversion media, involving the use of supercritical fluids, ionic liquids and natural oils and liquid polymers, among others.     

Guinea pig liver aromatic aldehyde-ketone reductases identical with 17 beta-hydroxysteroid dehydrogenase isozymes.

Two NADPH-dependent aromatic aldehyde-ketone reductases purified from guinea pig liver catalyzed oxidoreduction of 17 beta-hydroxysteroids and 17-ketosteroids. One enzyme efficiently oxidized 5 beta-androstanes and reduced 17-ketosteroids of A/B cis configuration, whereas the other enzyme efficiently oxidized 5 alpha-androstanes and equally reduced both 5 alpha-and 5 beta-androstanes of 17-ketosteroids. However, aromatic aldehydes and ketones, and 3-ketosteroids were irreversibly reduced by the two enzymes. The two enzymes utilized NADP+ or NADPH as cofactor, but little activity with NAD+ or NADH was found. Phosphate ions enhanced the NAD+-dependent dehydrogenase activity and NADH-dependent reductase activity of the two enzymes, whereas the activities with NADP+ and NADPH were not affected. The ratios of the two activities of ketone reduction and 17 beta-hydroxysteroid oxidation of the two enzymes were almost constant during the purification steps after the two enzymes had been separated by DEAE-cellulose chromatography. By kinetic studies and electrophoresis and isoelectric focusing experiments it was confirmed that both of the two enzymes were responsile for the reduction aldehydes, ketones, and ketosteroids and for the oxidation of 17 beta-hydroxysteroids. These results indicate that 17 beta-hydroxysteroid dehydrogenases may play important roles in the metabolism of exogeneous aldehydes and ketones as well as steroids.     

Sites of in vivo extraction and interconversion of estrone and estradiol in the dog

The interconversion and extraction of estrone and estradiol-17β across and within different tissues or areas have been studied in the dog by the constant infusion technique. The results were calculated using the ³H/¹⁴C ratios and radioactive concentrations of estrone and estradiol obtained from afferent and efferent blood and tissues at equilibrium. From these results it is concluded that: (1) there is no significant difference between metabolic clearance rates of estrone and estradiol, (2) blood transfer constants indicate a higher conversion of estradiol to estrone than of estrone to estradiol, (3) the transtissue interconversion favors the formation of estrone while the intratissue interconversion favors the formation of estradiol, (4) no interconversion of the two estrogens is observed in adipose tissue, (5) the extraction of estradiol entering a tissue was lower than the extraction of estradiol formed in these tissues, (6) calculation of the tissue metabolic clearance rates show that 63% and 61% of the total metabolism of estrone and estradiol, respectively, occurs in the splanchnic bed, and (7) the contribution of each tissue to the total interconversion of estrone and estradiol show that more than 90% of this interconversion occurs extrahepatically.     

A study in twins of the urinary excretion of vanilmandelic acid, 17-keto-steroids, 17-hydroxycorticosteroids, and creatinine]

The 24-hour urinary excretion rates of creatinine, vanilmandelic acid (VMA), 17-hydroxycorticosteroids, 17-ketosteroids were analysed in 30 female twin pairs. For each of these variables, a comparative study based on the breakdown of total variance into interpair and intrapair variance was undertaken, first on identical (MZ) and fraternal (DZ) twins and then on twins who live together or separately. The comparison of test results shows a strong likeness in the MZ group and in the living together group with respect to the urinary excretion of 17-OH. The variability of VMA excretion can be explained by genetic factors while the variability of 17-ketosteroids and creatinine can be explained with reference to environmental factors.     

Conversion of 4-androstene-3,17-dione to testosterone by Pisum sativum

4-Androstene-3,17-dione-[4-¹⁴C] was applied to the leaves of growing pea plants, Pisum sativum. Within a week, 28% of the administered steroid was specifically reduced to testosterone. Part of the testosterone was present in esterified form, and 5α-androstane-3β,17β-diol was also identified as a metabolite, but neither epitestosterone nor estrogens were detected.     

Properties of antisera to progesterone and to 17-hydroxy-progesterone elicited by immunization with the steroids attached to protein through position 7

Antibodies to progesterone (P) and to 17-hydroxyprogesterone (17-OHP) were raised by immunization of rabbits with progesterone-7α-carboxyethyl thioether--bovine serum albumin (P-7—BSA) or with 17-OHP-7α-carboxyethyl thioether--BSA (17-OHP-7--BSA). The antisera produced were of high affinity: K_a towards the homologous hapten was 3. 7 × 10¹⁰ 1./mol for the anti-P serum and 5. 9 × 10⁹ 1/mol for the anti-17-OHP serum. The antiserum to P-7—BSA displayed little or no cross reaction (?= 2%) with the 20α-, 20β- or 5β-dihydro-derivatives of progesterone, moderate cross-reaction with pregnenolone (4%), but considerable cross-reaction with 11-deoxycorticosterone (7%), 5α-dihydro-progesterone (11%) and 17-OHP (15%). The antiserum to 17-OHP-7--BSA showed very little cross-reaction (?= 2%) with progesterone and other steroids lacking a 17α-hydroxyl group, such as pregnenolone or 11-deoxycorticosterone, but reacted significantly with 17α, 21-dihydroxy-4-pregnene-3, 20-dione (8%) and 3β, 17-dihydroxy-5-pregnen-20-one (13%). None of the sera reacted with testosterone, cortisol or estradiol-17β. It appears that conjugation of progesterone to protein through carbon-7 affords antisera comparable in specificity to those raised with 11α-conjugates and superior to those raised with 3-, 6- and 20-conjugates. The antiserum to 17-hydroxyprogesterone described is the first one that specifically recognizes this metabolite.     

Androgen receptor in human skin fibroblasts. Characterization of a specific 17beta-hydroxy-5alpha-androstan-3-one-protein complex in cell sonicates and nuclei.

Cultured human skin fibroblasts were shown to contain an androgen binding activity (receptor) which was heat-labile and destroyed by trypsin. Specific binding was seen after incubations of these cells with 1,2-3-H-testosterone, 1,2-3-H17beta-hydroxy-5alpha-androstan-3-one (dihydrotestosterone, DHT) and 1,2-3-H-5alpha-androstane-3alpha, 17beta-diol. This receptor had a high affinity (Kd=0,2-1.6 nM) and a high degree of specificity for DHT. It was measured as a 3-H-DHT-protein complex by gel filtration chromatography using a method which distinguishes specific from nonspecific binding. Receptor activity was distributed about equally between nuclear and extranuclear components at all times studied and was present in both compartments when cell incubations were carried out at 4 degrees and 37 degrees. Saturation analysis indicated that there were 1250-18,600 binding sites per whole cell. By sucrose gradient centrifugation the receptor had a sedimentation coefficient (S20,w) of about 4. Cells grown for 8 days without serum in the medium maintained the same levels of 3-H-DHT binding. Within 15 hours puromycin (20 mug/ml) in serum-free medium caused a 40-60 percent decrease in binding for the same cell lines. Although the highest levels of 3-H-DHT binding were observed in fibroblasts from newborn foreskin, appreciable cytosol and nuclear binding were seen in cells from forearm, neck and abdominal skin. Receptor activity was stable during prolonged culture. Fibroblasts from several skin sites from patients with the androgen insensitivity syndrome (testicular feminization) had no detectable specific DHT binding. In this study it was demonstrated that skin fibroblasts can rapidly convert testosterone to its active form, DHT, bind DHT to a specific receptor protein and transport this complex to their nuclei. Therefore this may prove to be a convenient system for studying androgen action in vitro.     

Steroids 35: synthesis of 16,16-dimethyl-17 beta-hydroxysteroids

The reaction of 16-methylene-17-ketosteroids (Ia), (Ic) and (Ie) with methyl magnesium iodide yields 16-methylene-17 alpha-methyl-17 beta-hydroxysteroids (IIa), (IIb) and (IId). These are subjected to the addition of hypobromous acid and the subsequent anionotropic rearrangement to convert them into 16 alpha-methyl-16 beta-bromomethyl-17-ketosteroids (Va), (Vb) and (Vd). These were reduced with LiA1H4 to obtain 16,16-dimethyl-17 beta-hydroxysteroids (VIa), (VIb) and (VId). Compounds (VIIa) and (VIIe) were selectively deacetylated yielding (VIIb) and (VIId); these were then oxidized and hydrolyzed to convert them into (VIf) and (VIg).     

β-环糊精对甾体微生物羟化反应的影响

考察了β-环糊精(β-cyclodextrin, CD)对雄甾-4-烯-3,17-二酮(androst-4-ene-3,17-diorle,AD)在水中的溶解度及微生物对其11а羟化反应的影响,结果表明β-环糊精能显著提高底物AD在发酵培养基中的溶解度,增溶效果优于有机溶剂.在底物投料浓度0.2%(w/v)时,与4%无...