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.     

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.     

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.     

Formation of 5α-dihydrotestosterone from 5α-androstane-3α,17β-diol in prostate cancer LAPC-4 cells – Identifying inhibitors of non-classical pathways producing the most potent androgen

5α-Dihydrotestosterone (5α-DHT) possesses a great affinity for the androgen receptor (AR), and its binding to AR promotes the proliferation of prostate cancer (PC) cells in androgen-dependent PC. Primarily synthesized from testosterone (T) in testis, 5α-DHT could also be produced from 5α-androstane-3α,17β-diol (3α-diol), an almost inactive androgen, following non-classical pathways. We reported the chemical synthesis of non-commercially available [4-¹⁴C]-3α-diol from [4-¹⁴C]-T, and the development of a biological assay to identify inhibitors of the 5α-DHT formation from radiolabeled 3α-diol in LAPC-4 cell PC model. We measured the inhibitory potency of 5α-androstane derivatives against the formation of 5α-DHT, and inhibition curves were obtained for the most potent compounds (IC₅₀ = 1.2–14.1 μM). The most potent inhibitor 25 (IC₅₀ = 1.2 μM) possesses a 4-(4-CF₃-3-CH₃O-benzyl)piperazinyl methyl side chain at C3β and 17β-OH/17α-CCH functionalities at C17 of a 5α-androstane core.     

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.     

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.     

In vitro conversion of 5alpha-reduced metabolites of testosterone by the seminal vesicles, ventral prostate glands and testes of adult rats

In order to ascertain the ability of rat seminal vesicles, testes and ventral prostate glands to interconvert 5α-reduced androgens, these three organs were incubated with either tritiated 17β-hydroxy-5αandrostan-3-one (5α-dihydrotestosterone,DHT), 5α-androstane-3α, 17βdiol (3α-diol) or 5α-androstane-3β, 17β-diol (3β-diol). The incubation environment utilized (Krebs-Ringer bicarbonate glucose buffer) was selected because the histologic appearance of the tissue at the conclusion of the incubation was indistinguishable from tissue fixed immediately after sacrifice of the animal, thereby approximating the physiologic conditions as closely as possible. In incubations of rat seminal vesicles, ³H.-3β-diol was not metabolized while 26.7 ± 3.8% of ³H-3α-diol appeared as DHT and 17.2 ± 1.5% of ³H-DHT was metabolized to 3α-diol. A small amount (7.5 ± 0.8%) of ³H-DHT was, however, converted to 3β-diol. In incubations of rat testes, the major metabolite, regardless of substrate, was 3α-diol. The conversion of 75.7 ± 2.1% of ³H-3β-diol to 3α-diol has demonstrated, for the first time, that this steroid can be metabolized by the rat testis. Rat ventral prostate glands metabolized $\text{18.5 ± 2.5\%}\text{of}{}^3\text{H-3β-}\text{diol}$ to DHT and 61± 2.9% of ³H-3α-diol to DHT. When ³H-DHT served as the substrate, 83.2 ± 1.5% remained unmetabolized. The prostate glands are, therefore, capable of metabolizing 3β-diol to DHT.     

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.     

Comparative placental steroid synthesis. II. C19 steroid metabolism by guinea-pig placentas and fetal adrenals in vitro

Placental homogenates from guinea-pigs at 16, 20, 35 and 55 days gestation were incubated with 7α-³H-dehydroepiandrosterone and 4-¹⁴C-androstenedione and analyzed for conversion products by reverse isotope dilution methods. ¹⁴C-3α-Hydroxy-5α-androstan-17-one, ¹⁴C-androstane-3α, 17β-diol and ³Handrost-5-ene-3β, 17β-diol were isolated from homogenates incubated with substrates for 2 hours. ³H, ¹⁴C-Testosterone was isolated from preparations incubated for 15 minutes or with high substrate: tissue ratios. Androst-4-ene-3, 17-dione, 5α-androstane-3, 17-dione, 5β-androstanedione derivative and C₁₈ steroid formation could not be demonstrated. These results demonstrate the capacity of guinea-pig placentas to convert dehydroepiandrosterone and androstenedione to testosterone and to derivatives reduced in ring A (5α) and at carbon 17. The activity of the Δ⁵-3β-hydroxysteroid dehydrogenase enzyme system appears to have been rate limiting.Homogenates of adrenals from 44–55 day old fetuses converted 4-¹⁴C-pregnenolone to androst-4-ene-3, 17-dione and 6β- and 11β-hydroxyandrostenedione. A guineapig fetal-placental unit is postulated, with steroid metabolic characteristics different from the human unit. Both permit reduction of fetal adrenal cortisol production and placental removal of C₁₉ steroids.     

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.     

Biotransformation of testosterone and other androgens by suspension cultures of Nicotiana tabacum “Bright yellow”

It has been shown that the cultured cells of Nicotiana tabacum “Bright Yellow” are capable of transforming testosterone to Δ⁴-androstene-3, 17-dione, 5α-androstan-17β-ol-3-one, 5α-androstane-3β, 17β-diol, its dipalmitate and 3- and 17-monoglucosides, epiandrosterone, its palmitate and glucoside, testosterone glucoside. 5α-Androstane-3β, 17β-diol dipalmitate and 3- and 17-monoglucosides, epiandrosterone palmitate and glucoside, and testosterone glucoside have been found for the first time as metabolites of testosterone in plant systems. Δ⁴-Androstene-3,17-dione was converted to testosterone. 5α-Androstan-17β-ol-3-one, which has been recognized as an active form of testosterone in mammals, was also detected. It has also been demonstrated that [4-¹⁴C]testosterone is actively incorporated in these transformations.     

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 of 5α-Androstane-3α,16α,17β-triol from 3β-hydroxy-5-androsten-17-one

5α-Androstane-3α, 16α 17β-triol was synthesized from 3β-hy-droxy-5-androsten-17-one. The procedure Involved catalytic hydrogenation of 3β-hydroxy-5-androsten-17-one to 3β-hydroxy-5α-androstan-17-one. This was followed by conversion of the 3β-hydroxy group to 3α-benzoyloxy group by the Mitsunobu reaction. Further treatment with isopropenyl acetate yielded 5α-androsten-16-ene-3α, 17-diol 3-benzoate 17-acetate. This was then converted to 3α, 17-dihydroxy-5α-androstan-16-one 3-benzoate 17-acetate via the unstable epoxide intermediate after treatment with $\text{m}$-cloroperoxybenzoic acid. LiAlH₄ reduction of this compound formed 5α-androstane-3α, 16α, 17β-trlol. ¹H and ¹³C NMR of various steroids are presented to confirm the structure of this compound.     

Androgen metabolism by rat epididymis 4. The formation of conjugates

The ability to form androgen conjugates and the hormone dependency of the conjugating enzymes have been studied in the rat epididymis.Following the in vitro incubation of ³H-testosterone with epididymal slices from intact and castrated rats, the radioactivity recovered was partitioned between water and ether. Examination of the water soluble radioactivity demonstrated the presence of glucuronides and sulfates. The total radioactivity in the conjugate fraction was the same for both intact and castrated animals. However, castrated rats showed a 3-fold increase in the glucuronide fraction with a corresponding decrease in the formation of sulfates. Characterization of the ether soluble radioactivity after solvolysis of the conjugate fraction from castrated animals, showed DHT (17β-hydroxy-5α-androstan-3-one) and 3α-diol (5α-androstane-3α,17β-diol) to be the main metabolites. After β-glucuronidase hydrolysis of the same, only 3α-diol could be demonstrated at a significant level, although traces of DHT and δ¹⁶ compounds were present.Corresponding hydrolysis of the water phase from incubation of epididymis from intact rats, demonstrated a marked quantitative difference. Here approximately 40% of the conjugated aglycones consisted of Δ¹⁶ compounds, whilst only abot 12% was comprised of 3α-diol. The preferential conjugation of DHT and 3α-diol to a sulfate radical was demonstrated in both intact and castrated rats. Since the conjugated Δ¹⁶ compounds were detected only in the epididymis from intact animals, it is possible that these are formed by the spermatozoa.     

Studies on the effect of 5α-androstane-3α, 17α-diol in the canine prostate in vivo

Adult beagle dogs, castrated one month previously, were treated with 5α-androstane-3α, 17α-diol (total dose 300 mg) over a period of one month. Examination of the prostate after treatment showed no significant change in size, weight or histological appearance from the castrate dog prostate. Subsequent administration of 5α-androstane-3α, 17β-diol (300 mg) over the same period of time resulted in restoration of the prostate size, weight and histological appearance to that of the normal intact dog prostate. It is concluded that exogenously administered 5α-androstane-3α, 17α-diol does not promote prostatic growth in the castrate beagle dog.     

Testosterone metabolism by isolated human axillary corynebacterium spp.: A gaschromatographic mass-spectrometric study

Transformations of [4-¹⁴C]testosterone have been studied in Corynebacterium spp. isolated from the axillae of men. Metabolites have been separated by TLC and capillary gas chromatography and have been identified by gas chromatography-mass spectrometry (GC-MS). The introduction of a clean-up step using Florisil columns, prior to TLC, removed Tween-80 which co-extracted from the medium with the metabolites. This procedure greatly improved TLC resolution.Testosterone was converted enzymically to 5α- and 5β-DHT, identification being assisted by the inclusion of [3,4-¹³C]testosterone in some incubations. Other metabolites formed enzymically were 4-androstene-3,17-dione, 5β-androstane-3,17-dione, 3β-hydroxy-5β-androstan-17-one and 5β-androstane-3α.l7α-diol. Some spontaneous breakdown of [¹⁴C]testosterone occurred giving rise to 5α(β)-DHT, androstanediol and a monohydroxy-diketo-androstene, the latter being reduced enzymically to 2 monohydroxy-diketo-androstanes. Under the conditions used, no clear evidence has been obtained for the formation of 5α-androst-16-en-3-one, an odorous steroid that occurs in the axillae of men; the possible reasons why we were unable to prove the biosynthesis of this compound are discussed.     

Increased testicular 5α-androstane-3α, 17β-diol formation induced by treatment with [D-Ser(TBU)6, des-Gly-NH210]LHRH ethylamide in the rat

While the intact male adult rats respond to LH with a predominant increase of testicular and plasma testosterone levels, the response to LH stimulation in animals treated with the LHRH agoriist, [D-Ser(TBU)⁶, des-Gly-NH₂10]LHRH ethylamide is characterized by a major production of 5α-androstane-3α, 17β-diol. The marked increase of 5α-androstane-3α, 17β-diol levels in the presence of a 90% decrease of testosterone concentration strongly suggests that 5α-reductase and 3α-hydroxysteroid oxidoreductase activities are increased during testicular desensitization induced by treatment with the LHRH agonist.     

One step synthesis of 6-oxo-cholestan-3β,5α-diol

Cholesterol metabolism has been recently linked to cancer, highlighting the importance of the characterization of new metabolic pathways in the sterol series. One of these pathways is centered on cholesterol-5,6-epoxides (5,6-ECs). 5,6-ECs can either generate dendrogenin A, a tumor suppressor present in healthy mammalian tissues, or the carcinogenic cholestane-3β,5α,6β-triol (CT) and its putative metabolite 6-oxo-cholestan-3β,5α-diol (OCDO) in tumor cells. We are currently investigating the identification of the enzyme involved in OCDO biosynthesis, which would be highly facilitated by the use of commercially unavailable [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol. In the present study we report the one-step synthesis of [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol by oxidation of [¹⁴C]-cholesterol with iodide metaperiodate (HIO₄).     

Steroidal constituents of Solanum xanthocarpum

From the extract of the fruits of Solanum xanthocarpum (Solanaceae), five new steroidal compounds were isolated and characterized: 4α-methyl-24ξ-ethyl-5α-cholest-7-en-3β,22ξ-diol (1), 3β,22ξ-dihydroxy-4α-methyl-24ξ-ethyl-5α-cholest-7-en-6-one (2), 3β-benzoxy-14β,22ξ-dihydroxy-4α-methyl-24ξ-ethyl-5α-cholest-7-en-6-one (3), 3β-benzoxy-14α,22ξ-dihydroxy-4α-methyl-24ξ-ethyl-5α-cholest-7-en-6-one (4) and 3β-(p-hydroxy)-benzoxy-22ξ-hydroxy-4α-methyl-24ξ-ethyl-5α-cholest-7-en-6-one (5).