Dinoflagellate sterols IV: Isolation and structure of 4α,23ξ,24ξ-trimethylcholestanol from the dinoflagellate Glenodiniumhallii

The dinoflagellate $\text{Glenodinium}$$\text{hallii}$ was investigated for its sterol composition. Five of the six sterols were isolated and identified as cholest-5-en-3β-ol, (24ξ)-24-methylcholest-5-en-3β-ol, stigmasta-5,22-dien-3β-ol, (22E,24R)-4α,23,24-trimethyl-5α-cholest-22-en-3β-ol, and 4α,23ξ,24ξ-trimethyl-5α-cholestan-3β-ol.     

Minor sterols of marine invertebrates 37. Isolation of novel coprostanols and 4α-methyl sterols from the tunicate Ascidia nigra

The complex sterol mixture isolated from $\text{A, nigra}$ was found to contain a low level of Δ⁴-3-keto steroids, 5β-stanols and 4α-methyl sterols in addition to regular (4-demethyl) sterols. The following new marine sterols were isolated and identified using MS and 360 MHz NMR: 5β-cholest-22E-en-3β-ol, 24S-methyl-5β-cholest-22E-en-3β-ol, 24-methylene-5β-cholestan-3β-ol, both epimers at C-24 of 4α-methyl-24-ethyl-5α-cholest-22E-en-3β-ol, 4α, 22ξ, 23ξ-(or 24ξ-)trimethyl-5α-cholest-8(14)-en-3β-ol and (22S, 23S, 24S)-4α-24-dimethyl-22, 23-methylene-5α-cholestan-3β-ol. The latter sterol and 23-demethylgorqosterol have opposite configurations at C-22, C-23, and C-24; the Δ⁸⁽¹⁴⁾ sterol has an unprecedented side chain.     

A modified method for the estimation of pregnanediol, pregnanetriol and seven common 17-ketosteroids in urine by gas-liquid chromatography

A method is described for simultaneous determination of seven common urinary 17-ketosteroids, pregnanediol (5β-pregnane- 3α, 20α-diol) and pregnanetriol (5β-pregnane-3α,17α,20α-triol) by gas-liquid chromatography. The essential steps include hydrolysis of conjugates by an extract from $\text{Helix}$$\text{pomatia}$, use of 5β-cholestan-3α-ol as an internal standard, silylation and gas chromatography using 3% XE-60 as a stationary phase. The chromatograms were clear and the peaks easily identifiable. Laborious purification procedures were not necessary. 5β-pregnane-3α,20α-diol and 5β-pregnane-3α, 17α,20α-triol were measured in 22 adult males and 17 adult females. The subjects were trained hospital personnel, healthy and without medications. The results, in general, were in agreement with those reported by others.     

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.     

Synthesis and bioassay of 3-deoxy-1α-hydroxyvitamin D3, an active analog of 1α,25-dihydroxyvitamin D3

Two synthetic routes to 3-deoxy-1α-hydroxyvitamin D₃, an analog of 1α,25-dihydroxyvitamin D₃, are described. One involved the six-step conversion of 1α,2α-epoxy-6,6-ethylenedioxy-5α-cholestan-3- one to 1α-acetoxycholest-5-ene, whereas, in the second, the same intermediate was prepared from 1α-hydroxycholesterol. Conversion of the Δ⁵-sterol to the required 5,7-diene was accomplished most efficiently via 7-keto and 7-tosylhydrazone intermediates. Bioassay of 3-deoxy-1α-hydroxyvitamin D₃ in the rat establishes that the analog can fulfill all common vitamin D functions including stimulation of intestinal calcium transport, mobilization of calcium and phosphate from bone, stimulation of growth, and calcification of bone. Direct comparison indicates the compound to have $\text{1}\text{20}$ to $\text{1}\text{50}$ of the activity of 1α-hydroxyvitamin D₃, but it acts with a time course indistinguishable from the latter.     

Synthesis of 16α-bromoacetoxy androgens and 17 β-bromoacetylamino-4-androsten-3-one: Potential affinity labels of human placental aromatase

A novel synthesis of 16α-hydroxy-4-androstene-3,17-dione ($\text{3}$), 16α-hydroxy-4-androstene-3, 6,17-trione ($\text{4}$), 17β-amino-5-androsten-3β-ol ($\text{10}$) and 17β-amino-4-androsten-3-one (14) is described. 16α-Bromoacetoxy-4-androstene-3, 17-dione ($\text{5}$), 16α-bromoacetoxy-4-androstene-3, 6,17-trione ($\text{6}$) and 17β-bromoacetylamino-4-androsten-3-one ($\text{15}$) were synthesized as potentially selective irreversible inhibitors of androgen aromatases. 16α-Bromo-4-androstene-3,17-dione ($\text{1}$) and 16α-bromo-4-androstene-3, 6,17-trione ($\text{2}$) were converted to compounds $\text{3}$ and $\text{4}$ in 80–90% yield by controlled stereospecific hydrolysis using sodium hydroxide in aqueous pyridine. Reductive amination of 3β-hydroxy-5-androsten-17-one and 3-methoxy-3,5-androstadien-17-one ($\text{11}$) using ammonium acetate and sodium cyanohydridoborate (NaBH₃CN) and a subsequent treatment with acid gave the amines $\text{10}$ and $\text{14}$ respectively, as a salt. The corresponding 17-imino compounds $\text{9}$ and $\text{13}$ were also isolated from the reaction mixtures when methanol was used as a solvent for the reaction. The 16α-hydroxyl compounds $\text{3}$ and $\text{4}$ and the 17β-amino compound $\text{14}$ were con- verted to the corresponding bromoacetyl derivatives, $\text{5}$, $\text{6}$, and $\text{15}$, with bromoacetic acid and N,N'-dicyclohexylcarbodiimide.     

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.     

Specific fragmentation of natural inhibitor of mitochondrial ATPase by thrombin

The ability of cholestanol (5α-cholestan-3β-ol) to support growth of two independently derived sterol auxotrophs, FY3 and GL7, has been examined. Growth on this stanol was precluded unless minute quantities of sterol were also available. Contaminating sterol in most cholestanol preparations or excess sterol in the inoculum used in growth studies could provide the required sterol in quantities capable of sustaining growth through an entire culture cycle. Evidence is presented for multiple functions of sterols in $\text{Saccharomyces}\text{cerevisiae}$.     

Controlled alkaline hydrolysis of steroidal α-bromoketones: New conditions and synthesis of 2α-hydroxy-3-ones

Controlled alkaline hydrolysis of 16α-bromo-17-keto steroids $\text{1}$, $\text{5}$ and $\text{7}$ with potassium carbonate and tetra-n-butylammonium hydroxide (n-Bu₄NOH) and synthesis of 2α-hydroxy-3-ones $\text{11}$, $\text{13}$ and $\text{16}$ by the controlled hydrolysis of the corresponding 2α-bromo-3-ones $\text{9}$, $\text{12}$ and $\text{15}$ are described. Treatm carbonate in aqueous acetone or with n-Bu₄NOH in aqueous dimethylformamide (DMF) gave 16α-hydroxy-17-ones $\text{3}$, $\text{6}$ and $\text{8}$ in 85–90% yield, respectively. 2α-Hydroxy-3-ones $\text{11}$, $\text{13}$ and $\text{16}$ were obtained by hydrolysis of the corresponding bromoketones $\text{9}$, $\text{12}$ and $\text{15}$ in high yields using the above conditions or sodium hydroxide in pyridine or DMF, respectively. Deuterium labeling experiments suggested that equilibration between the 2α-bromoketone $\text{9}$ and the 2β-bromo isomer $\text{10}$ precedes the formation of the ketol $\text{11}$ in which the true intermediate might be the 2β-isomer $\text{10}$. However, rearranged androstane derivatives, 3β-hydroxy-2-ones $\text{18}$ and $\text{20}$, were stereoselectively obtained by treatment of the bromoketones $\text{12}$ and $\text{15}$ with an excess amount of sodium hydroxide.     

Reactions of 4β,5-epoxy-5β-androstan-3-ones with hydrogen fluoride in pyridine

4β,5-Epoxy-5β-androstane-3,17-dione ($\text{1a}$), 17β-hydroxy-4β,5-epoxy-5β-androstan-3-one ($\text{1b}$) and 17β-acetoxy-4β,5-epoxy-5β-androstan-3-one ($\text{1c}$) were treated with anhydrous hydrogen fluoride in pyridine (70% solution) at 55° and yielded the corresponding 4-en-4-ols e.g. 4-hydroxy-4-androstene-3, 17-dione ($\text{2a}$).As the reaction temperature was lowered each epoxide formed a second product which, at ?75°, was the major component of the reaction mixture and was identified as the 5α-fluoro-4α-ol derivative of the parent enone, e.g. 4α-hydroxy-5-fluoro-5α-androstane-3,17-dione ($\text{3a}$). These fluorohydrins are thermally unstable, losing hydrogen fluoride.The acetates of the fluorohydrins were also prepared, characterized, and shown to be more stable than the parent alcohols.     

Intramolecular catalysis. VII: The nature of side chain shielding of the 12alpha-hydroxyl group of steroids

A series of 12α-hydroxy steroids with varying side chains was prepared, and their 24-hour acetylation yields were compared, l2α-Hydroxy-5β-pregnan-20-one ($\text{lb}$) was prepared from 3α, 12α-diacetoxy-5β~pregnan-20-one ($\text{2}$) and also by side chain degradation of 12α-acetoxy-5β-cholanoic acid ($\text{5d}$). 21-Benzyl-5β-pregnan-12α-ol ($\text{1g}$) was synthesized by hydrogenation of the 21-benzylidine derivative of ketone $\text{1b}$. 23-Pheny1-5β-norcholan-12α-ol ($\text{1k}$) was obtained by the Grignard reaction of 2-phenyl-ethylmagnesium bromide and ketone $\text{1b}$, dehydration, hydrogenation and hydride reduction; a similar sequence produced 20-methyl-5β-pregnan-12α-ol ($\text{lm}$). The acetylation results (Table 11) imply that branching at C-20 may be more significant for 12α-hydroxyl reactivity than side chain length or type. An additional compound with an unbranched side chain, 21-nor-5β-cholan-12α-ol ($\text{14}$), was synthesized by a Grignard reaction on the 21-bromo intermediate $\text{11b}$. Acetylation rates determined by glc indicate (Table 111) That compounds with unbranched side chains have 12α-hydroxyl groups about ten times as reactive as their analogs with 20-methyl groups.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

Sterols of a diatomaceous ooze from walvis bay

Almost an of the solvent-extractable sterols and their nuclearsaturated analogues in a sample of Walvis Bay surface sediment have been analysed by capillary GLC and GC-MS, and by coinjection with a variety of standards. The presence in sediments of 22-$\text{trans}$-24-nor-5α-cholest-22-en-3β-ol, 24-methylene-5α-cholestan-3β-ol, and components tentatively assigned as 23,24-dimethylcholesta-5,22-dien-3β-ol and 23,24-dimethyl-5α-cholest-22-en-3β-ol has been demonstrated for the first time. A novel sterol and its saturated analogue have also been found. The sterol distribution cannot be related solely to the reported major input of phytoplankton; the presence of 22,23-methylene-23,24-dimethylcholest-5-en-3β-ol and its saturated analogue indicates a coelenterate contribution. The analysis emphasises the necessity of glass capillary columns and coinjection of standards.     

Quantitative studies on prostaglandin synthesis in man. II. Determination of the major urinary metabolite of prostaglandins F1 alpha and F2 alpha

A method was developed for quantitative determination of 5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins F_1α and F_2α in man. The method was based on the use of the O-methyloxime derivative of [5β-³H; 10,10,12,12-²H₄]5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid as internal standard and determination of ratios between unlabeled and deuterium-labeled molecules by multiple-ion analysis. Excretion values found for healthy human subjects were: males, $\text{10.8–59.0 μ}\text{g}\text{24 hr}$ (n = 10, mean value, 24.0 ± 17.2 (SD) μg) and females, $\text{7.6–13.6 μ}\text{g}\text{24 hr}$ (n = 10, mean value, 10.5 ± 2.1 (SD) μg).     

5α-reduction of an anti-androgen TSAA-291, 16β-ethyl-17β-hydroxy-4-estren-3-one,by nuclear 5α-reductase in rat prostates

Inhibition of 5α-reduction of testosterone by an anti-androgen TSAA-291 (16β-ethyl-17β-hydroxy-4-estren-3-one) was studied in rat ventral prostates and the metabolic conversion of ³H-TSAA-291 was examined both $\text{in vitro}$ and $\text{in vivo}$. In the $\text{in vitro}$ experiment using nuclear 5α-reductase of the prostate, 5α-dihydrotestosterone formation from ³H-testosterone was inhibited in a competitive manner by the anti-androgen. In the $\text{in vitro}$ experiment using ³H-TSAA-291, 5α-reduction of the anti-androgen occurred. One, 2 and 4 hr after an intravenous administration of 140 μCi/rat of ³H-TSAA-291 to castrated rats, the unchanged TSAA-291 accumulated in higher amounts in the ventral prostate than in the plasma, skeletal muscle and levator ani muscle, thereby indicating the selective uptake of the anti-androgen by the androgen target organ. No appreciable amounts of the 5α-reduced metabolite of TSAA-291 were detected in the prostate, thus suggesting that TSAA-291 itself may be responsible for the anti-androgenic properties. The inhibitory potency on the 5α-reductase activity of several other 16β-substituted androstane and estrane analogues was also examined.     

4-ethenylidene steroids as mechanism-based inactivators of 3β-hydroxysteroid dehydrogenases

The synthesis of 4-ethenylidene-5α-androstane-3β, 17β-diol ($\text{5}$) and of 4-ethenylidene-5α-androstane-3,17-dione ($\text{4}$) is described. Compound $\text{5}$ is a competitive inhibitor of solubilized bovine microsomal adrenal Δ⁵-3β-hydroxysteroid dehydrogenase, with Ki =2.7μM, and is converted by the enzyme to the corresponding 3-ketone. Compound $\text{4}$ shown to irreversibly inactivate the enzyme in a time-dependent manner ($\text{t}\text{1}\text{2}\text{=31}\text{min}\text{; 55μ}\text{M}\text{;}\text{pH}\text{=7.0}$). The substrate, dehydroepiandrosterone, protects against inactivation by compound $\text{4}$. In contrast, compound $\text{5}$ is not oxidized at the 3-position by the 3β-(and 17β)-hydroxysteroid dehydrogenase from $\text{P. testosteroni}$, but is oxidized at the 17-position. Nevertheless, the 4-ethenylidene-3,17-diketone ($\text{4}$) causes irreversible time-dependent inactivation ($\text{t}\text{1}\text{2}\text{=28}\text{min}\text{; 64μ}\text{M}\text{;}\text{pH}\text{=7.0}$) when incubated directly with this bacterial enzyme, acting as an affinity label.     

Conversion of 20alpha-hydroxy-4-pregnen-3-one to 20alpha-hydroxy-5alpha-pregnan-3-one and 5alpha-pregnane-3alpha, 20alpha-diol by rat anterior pituitary

³H-20α-hydroxy-4-pregnen-3-one was incubated with anterior pituitaries from proestrous rats. The $\text{in vitro}$ metabolic products, identified by reverse isotopic dilution and purification to constant specific activity, were 20α-hydroxy-5α-pregnan-3-one (23.0%) and 5α-pregnane-3α,20α-diol (11.4%). These are qualitatively the same metabolites which result from $\text{in vitro}$ incubation of 20α-hydroxy-4-pregnen-3-one with medial basal hypothalamus. 68.8% of the recovered radioactivity remained as 20α-hydroxy-4-pregnen-3-one. These three compounds accounted for all of the recovered radioactivity.     

Marine sterols. XIII.★ Isolation and synthesis of 1β,3β,5,6β-tetrahydroxy-5α-androstan-17-one from the soft coral Sarcophyton glaucum

A new polyhydroxysterol 1β,3β,5,6β-tetrahydroxy-5α-androstan-17-one ($\text{1}$) was isolated from the soft coral $\text{Sarcophyton glaucum}$. The structure of $\text{1}$ was deduced from comparison of the spectral data with those of known 1β,3β,5α,6β-tetrahydroxysterols and confirmed by the synthesis starting from 1β,3β-dihydroxy-5,16-pregnadien-20-one ($\text{6a}$)     

Synthesis and biological activity of brassinolide and its 22β,23β-isomer: Novel plant growth-promoting steroids

Brassinolide (2α,3α,22α, 23α-tetrahydroxy-24α-methyl-B-homo-7-oxa-5α-cholestan-6-one), a novel plant growth-promoting steroid isolated from rape pollen, and its hitherto unknown 22β, 23β-isomer were synthesized from a C-24 epimeric 60:40 mixture of 22-dehydrocampesterol (24α-methyl) and brassicasterol (24β-methyl) from oysters. The method of synthesis favored the formation of the 22β, 23β-isomer by better than 4:1. Comparative plant growth-promoting capabilities of brassinolide, both natural and synthetic, and its three side chain $\text{cis}$-glycolic isomers in the bean second internode bioassay showed that the natural and synthetic brassinolides were equally active and caused splitting of the internode at the 0.1 μg level. The least active was the 22β,23β-isomer of brassinolide. The isomers with the 22α, 23α and 24β, and the 22β, 23β and 24β configurations were highly active and were required at about 10 times the concentration of brassinolide to cause the same physiological response. In the bean first internode bioassay, an auxin-induced growth test system which employs isolated bean plant segments, the isomer with 22β, 23β and 24β configuration caused a greater response than brassinolide. Two of the four tetrahydroxy ketones obtained in the synthesis of the isomers were also active in both assays.     

5α,6α- AND 5β,6β-dichloromethylene adducts of 3β-acetoxy-5-androsten-17-one

Both the 5α, 6α- and 5β, 6β-dichloromethylene adducts ($\text{2a}$ and $\text{2b}$) of 3β-acetoxy-5-androsten-17-one ($\text{1}$) are produced when the latter is exposed to dichlorocarbene generated from chloroform and base by Phase Transfer Catalysis using ultrasound as a means of agitation. The ¹H NMR substituent effects of 5α, 6α- and 5β, 6β-dichloromethylene on the angular methyl groups (Zürcher values) are given. The ¹³C NMR spectra for both compounds are presented and discussed.