Proton magnetic resonance spectra of 17ξ-Hydroxy-17ξ-methyl-5ξ-androstane C-3 ketone and c-3ξ alcohol isomers in chloroform-d and pyridine-d5

17α-Hydroxy-17β-methyl-5β-androstan-3-one, 17μ-methyl-5α-androstane-3α, 17α-diol, 17β-methyl-5α-androstane-3β, 17α-diol, 17α-methyl-5β-androstane-3β, 17β-diol, 17β-methyl-5β-androstane-3α, 17α-diol and 17β-methy1–5β-androstane-3β, 17α-diol were synthesized for the first time. ¹H NMR spectra of all four 17ξ-hydroxy/17ξ-methyl C-3 ketones and all eight C-3 alcohols were recorded in chloroform-d and pyridine-d₅. Pyridine-induced chemical shifts are discussed. Thin-layer Chromatographic data are given.     

Sertoli cells from immature rats : in vitro stimulation of steroid metabolism by FSH.

Sertoli cells from 10 day old rats convert androstenedione to testosterone and 5α-androstane-3α,17β-diol, testosterone to 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α,17β-diol, and 17β-hydroxy-5α-androstan-3-one to 5α-andro-stane-3α,17β-diol after 72 hours $\text{in vitro}$. Conversions of androstenedione to testosterone and 5α-androstane-3α,17β-diol, and testosterone to 5α-androstane-3α,17β-diol were 2 to 3 times greater in FSH treated cultures. Steroid conversion was not stimulated significantly by LH or TSH. The results are interpreted as evidence that in young rats Sertoli steroid metabolism is stimulated by FSH, that Sertoli cells are an androgen target and that FSH may induce or facilitate Sertoli androgen responsiveness.     

Metabolism of 17α-methyl-5α-dihydrotestosterone in the rabbit

17α-Methyl-5α-dihydrotestosterone and the reduced metabolites, 17α-methyl-5α-androstane-3α, 17β-diol and -3β, 17β-diol together with two hydroxylated metabolites, 17α-methyl-5α-androstane-3β, 15α, 17β-triol and 17α-methyl-5α-androstane-3α, 6α, 17β-triol were isolated and identified in the urine of rabbits orally dosed with 17α-methyl-5α-dihydrotestosterone. Formation of the C-6 hydroxylated derivative demonstrates that the 4,6-enolization of a 4-en-3-one is not a necessary requirement for hydroxylation at C-6 of the androstane nucleus in the rabbit. No evidence was obtained for the presence of 17α-methyl/17β-hydroxyl epimerization.     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

Synthesis of new steroid haptens for radioimmunoassay. Part III. 15β-carboxyethylmercaptosteroid-bovine serum albumin conjugates. Specific antisera for radioimmunoassay of 5α-dihydrotestosterone, 5α-androstane-3β, 17β-diol and 5α-androstane-3α, 17β-diol

The syntheses of 15β-carboxyethylmercapto-5α-dihydrotestosterone, 15β-carboxy-ethylmercapto-5α-androstane-3β, 17β-diol and 15β-carboxyethylmercapto-5α-androstane-3α, 17β-diol and the preparation of their bovine serum albumin (BSA) conjugates are described. These conjugates were employed for the generation of specific antisera suitable for radioimmunoassay (RIA) of 5α-dihydrotestosterone (5α-DHT), 5α-androstane-3β, 17β-diol (3β3-diol) and 5α-androstane-3α, 17β-diol (3α-diol).     

Steroid metabolism by mouse submaxillary glands. I. in vitro metabolism of testosterone and 4-androstene-3,17-dione

Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of 6 month old male mice. In 15 and 180 minute incubations fortified with NADPH, submaxillary tissue converted 4-androstene-3,17-dione predominantly to androsterone and, to a lesser extent, testosterone, 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α, 17β-diol. Testosterone was converted primarily to 5α-androstane-3α, 17β-diol when exogenous NADPH was available; trace amounts of 4-androstene-3,17-dione, 17β-hydroxy-5α-androstan-3-one and androsterone were also formed. When a NADPH-generating system was omitted from the incubation medium both 4-androstene-3,17-dione and testosterone were poorly metabolized by submaxillary tissue; the amounts of reduced metabolites accumulating were markedly reduced.     

Androgen metabolism by rat epididymis: 5. Metabolic conversion and nuclear binding after injection of 5α-androstane-3α,17β-diol, in vivo

The pattern of androgenic metabolites in blood, muscle, caput and cauda epididymidis has been investigated in functionally hepatectomized 24 hours castrated rats, 3 hours after the intra-muscular injection of 200 μCi of ³H -3α-diol. Identification of the radioactive metabolites showed only negligible differences between the epididymal regions. In both caput and cauda the main metabolite was DHT (17β-hydroxy-5α-androstane-3-one); 3α- and 3β-diol, androsterone (3α-hydroxy-5α-androstane-17-one), 5-A-dione (5α-androstane-3,17-dione), Δ¹⁶-3α-ol (5α-androst-l6-en-3α-ol), Δ¹⁶-3β-ol (5α-androst-l6-en-3α-ol) and Δ¹⁶-3-one (5α-androst-l6-en-3-one) were also present.$\text{Androsterone}$ and $\text{3α-diol}$ were the predominant metabolites in blood and muscle. No Δ¹⁶ compounds could be detected and in constrast to epididymis, more than 50% of the radioactivity was associated with polar compounds. From determination of total radioactivity, it was seen that retention by epididymis varied from two to four times that of muscle. Purification and identification of the radioactivity associated with the nuclear fraction demonstrated that DHT was the only nuclear bound androgen.It is suggested from these results that at least one effect of 3α-diol on the rat epididymis is exerted through its conversion to DHT.     

Metabolism of 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one in the rabbit

An acidic metabolite, 2α-carboxy-5α-androstane-3α, 16α, 17αtriol and two neutral metabolites, 2α-hydroxymethyl-5α-androstane-3α, 17α-diol, and 2α-hydroxymethyl-5α-androstane-3α, 16α, 17α-triol have been identified in the urine of rabbits orally dosed with 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one. 2α-Hydroxymethyl-5α-androstane-3α, 16α, 17α-triol was previously obtained from the urine of rabbits dosed with 17β-hydroxy-2α-methyl-5α-androstan-3-one. The acidic metabolite was the major urinary excretion product.     

Androgen metabolism by rat epididymis. 2. Metabolic conversion of 3H-testosterone in vitro

The epididymis of adult rats metabolizes ³H-testosterone by experiments $\text{in}$$\text{vitro}$. After incubation of slices from epididymal tissue for 2 hrs at 37°C, 8% of the total radioactivity was found in the water-soluble fraction, whereas 92% in the ether soluble fraction (free steroids). The free steroids were examined further and the following metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 10,4%, $\text{androstendione}$ (4-androstene-3,17-dione) 6,2%, $\text{5α-A-dione}$ (5α-androstane-3,17-dione) 7,3%, $\text{DHT}$ (17β-hydroxy-5α-androstane-3-one) 39,3%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 22,7%, $\text{3β-diol}$ (5α-androstane-3β,17β-diol) 4,6% and androsterone(3α-hydroxy-5α-androstan-17-one) 8,9%. The relative amount of each metabolite is given in per cent of the total radioactivity in the ether soluble fraction. When segments (caput, corpus, cauda) of epididymis were incubated in the same way, differences in steroid metabolism were demonstrated. Characteristic for caput epididymidis was high formation of DHT (58,4%) and 3α-diol (23,5%). Corpus epididymidis showed lower formation of DHT (50,6%) and 3α-diol (12,7%), but an approximately 3 times higher formation of 5α-A-dione (12,0%) than caput (3,4%) and cauda (3,5%). Cauda epididymis showed the lowest formation of DHT (38,3%), whereas 3α-diol (29,1%) and androsterone (11,4%) formation were relatively high. The ratio between 17β-hydroxy metabolites (DHT and androstanediols) and 17-keto metabolites were much higher in the caput (8,8) than in the corpus (3,2) and cauda (3,6), indicating a higher 5α-reductase activity in this segment.     

In vitro conversion of testosterone-4-14C to androgens of the 5alpha-androstane series by a normal human ovary

In a previous communication (1) the identification of Δ⁴ -3-oxo-steroids and estrogens as metabolites of testosterone-4-¹⁴C incubated with normal post-ovulatory human ovaries was reported. Thin-layer chromatography of the extracts of those ovaries which contained no corpus luteum yielded zones of radioactivity which were not associated with any of these products. Detailed investigation of these zones from the extract of one of these glands resulted in identification of the following radiometabolites of the 5α-androstane series: 5α-androstane-3,17-dione, androsterone, 3β-hydroxy-5α-androstan-17-one, 17β-hydroxy-5α-androstan-3-one, 5α-androstane-3ga, 17β-diol and 5α-androstane-3β, 17β-diol. The capacity of a normal human ovary to produce these 5α-reduced androgens, especially the potent 17β-hydroxy-steroids, suggests a regulatory role of these compounds in ovarian function.     

The synthesis of 17-substituted 2α-chloro-5α-androstan-3-ols

This paper describes the synthesis of 2α-chloro-3α-hydroxy-5α-androstan-17-one and 2α-chloro-5α-androstane-3α,17β-diol and their 3-epimers. The epimers were characterized by nmr spectroscopy.     

Androgen metabolism by rat epididymis. 1. Metabolic conversion of 3 H-testosterone in vivo

The epididymis of adult rats metabolize ³H-testosterone by experiments $\text{in vivo}$. Thirty minutes after the injection of 100 μCi ³H-testosterone, some 10 per cent of the total radioactivity of the epididymis was found in the water-soluble fraction, whereas 90 per cent was found in the ether soluble fraction (free steroids). The free steroids were examined further and the following androgenic metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 8, 9%, $\text{androstendipne}$ (4-androstene-3, 17-dione, 2,7%,$\text{5α-A-dione}$ (5α-androstane-3, 17-dione) 6,5%, $\text{DHT}$ (17β-hydroxy-5α-androstan-3-one) 47, 2%, $\text{3β-diol}$ (5α-androstane-3β, 17β-diol) 4, 4%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 20, 8% and $\text{androsterone}$ (3α-hydroxy-5α-androstan-3-one) 3,4%. The relative amount of each metabolite is given in per cent of total radioactivity in the ether soluble fraction.     

Androgen regulation of progestin biosynthetic enzymes in FSH-treated rat granulosa cells in vitro

The influence of androgens on the FSH modulation of progestin biosynthetic enzymes was studied $\text{in vitro}$. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 3 days in a serum-free medium containing FSH (20 ng/ml) with or without increasing concentrations (10^?9?10^?6 M) of 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone; DHT), 5α-androstane-3α, 17β-diol (3α-diol), or the synthetic androgen 17β-hydroxy-17-methyl-4,9,11-estratrien-3-one (methyltrienolone; R1881). FSH treatment increased progesterone and 20α-hydroxy-4-pregnen-3-one(20α-OH-P) production by 10.2- and 11-fold, respectively. Concurrent androgen treatment augmented FSH-stimulated progesterone and 20α-OH-P production in a dose-related manner (R1881 > 3α-diol > DHT). In the presence of an inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), the FSH-stimulated pregnenolone (3β-hydroxy-5-pregnen-20-one) production (a 20-fold increase) was further enhanced by co-treatment with R1881, 3α-diol or DHT. Furthermore, FSH treatment increased 4.4-fold the activity of 3β-HSD, which converts pregnenolone to progesterone. This stimulatory action of FSH was further augmented by concurrent androgen treatment. In contrast, androgen treatment did not affect FSH-stimulated activity of a progesterone breakdown enzyme, 20α-hydroxysteroid dehydrogenase(20α-HSD). These results demonstrate that the augmenting effect of androgens upon FSH-stimulated progesterone biosynthesis is not due to changes in the conversion of progesterone to 20α-OH-P, but involves an enhancing action upon 3β-HSDΔ⁵, Δ⁴-isomerase complexes and additional enzymes prior to pregnenolone biosynthesis.     

Metabolism of 17α-methyl-5β-dihydrotestosterone in the rabbit

17α-Methy1-5β-androstane-3α, 17β-diol together with the hydroxylated metabolites 17α-methyl-5β-androstane-1β, 3α, 17β-triol, 17α-methyl-5β-androstane-3α, 12β, 17β-triol, 17α-methyl-5β-androstane-3α, 16α, 17β-triol and 17α-methyl-5β-androstane-3α, 16β, 17β-triol were isolated and identified in the urine of rabbits orally dosed with 17α-methyl-5β-dihydrotestosterone. Biotransformations differ from the 5α-series where hydroxylation occurred at C-6 and C-15. In both series, the C-3 equatorial epimer was the major urinary excretion product among the non-hydroxylated metabolites. The 5β-compound was more resistant to metabolic hydroxylation than the 5α-compound. No evidence for epimerization at the C-17 position was observed.     

Synthesis and pyrogenic effect of 3α,7α-dihydroxy-5β-androstan-17-one and 3α-hydroxy-5β-androstane-7,17-dione

The first chemical synthesis of 3α,7α-dihydroxy-5β-androstan-17-one and 3α-hydroxy-5β-androstane-7,17-dione is reported. In this method, the 17β-side chain of commercial chenodesoxycholic acid was degraded in 6 steps after selective protection of the hydroxyl groups : 3α-OH by a $\text{tert}$-butyldimetfaylsilyl group and 7α-OH by an acetoxy group. The capacity of 3α,7α-dihydroxy-5β-androstan-17-one and 3α-hydroxy-5β-androstane-7, 17-dione to release a pyrogen by human leukocytes was investigated by two independent methods : supernatants from leukocytes incubated with a steroid are injected to rabbits whose fever is measured, or tested by the Limulus Test (a pyrogen detection technique). The 7-keto substituted etiocholanolone still possessed pyrogenic activity, while the 7α-hydroxyl substituted one did not.     

Action of endogenous steroid inhibitors of brain aromatase relative to fadrozole

To study mechanisms of aromatase inhibition in brain cells, a highly effective non-steroidal aromatase inhibitor (Fadrozole; 4-[5,6,7,8-tetra-hydroimidazo-(1,5-a)-pyridin-5-yl] benzonitrile HCl; CGS 16949A) was compared with endogenous C-19 steroids, known to be formed in the preoptic area, which inhibit oestrogen formation. Using a sensitive in vitro tritiated water assay for aromatase activity in avian (dove) preoptic tissue, the order of potency, with testosterone as substrate was: Fadrozole (K_i < 1 × 10⁻⁹ M) > 4-androstenedione 5-androstanedione > 5-dihydrotestosterone (K_i = 6 × 10⁻⁸ M) > 5β-androstanedione > 5β-dihydrotestosterone (K_i = 3.5 × 10⁻⁷ M) > 5-androstane-3, 17β-diol (K_i = 5 × 10⁻⁶ M) > 5β-androstane-3β,17β-diol. Five other steroids, 5β-androstane-3,17β-diol, 5-androstane-3β,17β-diol, progesterone, oestradiol and oestrone, showed no inhibition at 10⁻⁴ M. The kinetics indicate that endogenous C-19 steroids show similar competitive inhibition of the aromatase as Fadrozole. Mouse (BALB/c) preoptic aromatase was also inhibited by Fadrozole. We conclude that endogenous C-19 metabolites of testosterone are effective inhibitors of the brain aromatase, and suggest that they bind competitively at the same active site as Fadrozole.     

Quantitation of six androgens by combined high performance liquid chromatography and radioimmunoassay

A technique was developed to separate six androgens (testosterone, dihydrotestosterone, androstenedione, 5α-androstane-3 α, 17β-diol, 5α-androstane-3β, 17β-diol, and androsterone) by high performance liquid chromatography prior to quantitation by specific radioimmunoassay systems. Methanol:water (60:40 v:v) was used as the solvent system with a C18 reversed-phase column. The method was verified and used to quantitate the androgens in serum from adult rams bled every 20 minutes for 6 hours and yearling bulls bled every 30 minutes for 8 hours. Concentrations of all 6 androgens varied in an episodic manner with testosterone being the dominant androgen.     

Seized designer supplement named "1-Androsterone": identification as 3β-hydroxy-5α-androst-1-en-17-one and its urinary elimination

New analogues of androgens that had never been available as approved drugs are marketed as “dietary supplement” recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids.In the present study, we report the detection of a steroid in a product seized by the State Bureau of Criminal Investigation Schleswig-Holstein, Germany. The product “1-Androsterone” of the brand name “Advanced Muscle Science” was labeled to contain 100 mg of “1-Androstene-3b-ol,17-one” per capsule. The product was analyzed underivatized and as bis-TMS derivative by GC-MS. The steroid was identified by comparison with chemically synthesized 3β-hydroxy-5α-androst-1-en-17-one, prepared by reduction of 5α-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by nuclear magnetic resonance. Semi-quantitation revealed an amount of 3β-hydroxy-5α-androst-1-en-17-one in the capsules as labeled.Following oral administration to a male volunteer, the main urinary metabolites were monitored. 1-Testosterone (17β-hydroxy-5α-androst-1-en-3-one), 1-androstenedione (5α-androst-1-ene-3,17-dione), 3α-hydroxy-5α-androst-1-en-17-one, 5α-androst-1-ene-3α,17β-diol, and 5α-androst-1-ene-3β,17β-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common steroids of the urinary steroid profile were altered after the administration of “1-Androsterone”. Especially the ratios of androsterone/etiocholanolone and 5α-/5β-androstane-3α,17β-diol and the concentration of 5α-dihydrotestosterone were influenced. 3α-Hydroxy-5α-androst-1-en-17-one appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to nine days after a single administration of one capsule.     

Selective oxidation of steroidal allylic alcohols

Pyridinium chlorochromate in CH₂Cl₂ containing pyridine (2%) at 2—3°C has been found to effect the high yield selective oxidation of the hydroxyl function of a number of steroidal allylic alcohols. Under these conditions the oxidation of cholest-4-cn-3β-ol to the corresponding ketone was effected in 92% yield. Only the allylic hydroxyl function of 5α-cholest-8(14)-ene-3β,15α-diol, 5α-cholest-8(14)ene-3β,15β-diol and 5α-cholest-8(14)-ene-3β,7β-diol was oxidized under these conditions to give the corresponding α,β-unsaturated ketones in high yields. 5α-Cholest-8(14)-ene-3β,7α,15α-triol gave 5α-cholest-8(14)-ene-3β,7α-diol-15-one in 82% yield. Attempted oxidations of the 5α-cholestan-3β,15α-diol and 5α-cholest-7-ene-3β,15α-diol, both lacking an allylic hydroxyl function, under these conditions, were unsuccessful. Selective oxidation of the allylic alcohol function of 5α-cholest-8(14)-ene-3β,15β-diol using activated manganese dioxide gave 5α-cholest-8(14)-en-3β-ol-15-one in high yield while oxidation of the corresponding 15α-hydroxy epimer using manganese dioxide was unsuccessful.     

Biotransformation of dehydroepiandrosterone with Macrophomina phaseolina and β-glucuronidase inhibitory activity of transformed products

The biotransformation of dehydroepiandrosterone (1) with Macrophomina phaseolina was investigated. A total of eight metabolites were obtained which were characterized as androstane-3,17-dione (2), androst-4-ene-3,17-dione (3), androst-4-ene-17β-ol-3-one (4), androst-4,6-diene-17β-ol-3-one (5), androst-5-ene-3β,17β-diol (6), androst-4-ene-3β-ol-6,17-dione (7), androst-4-ene-3β,7β,17β-triol (8), and androst-5-ene-3β,7α,17β-triol (9). All the transformed products were screened for enzyme inhibition, among which four were found to inhibit the β-glucuronidase enzyme, while none inhibited the α-chymotrypsin enzyme.