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 ($\text{6a}$)     

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.     

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.     

Synthesis of 3,4-13C2 steroids

A-ring enollactones $\text{1a}$, $\text{1b}$ or $\text{9}$ derived from 4-cholesten-3-one, testosterone benzoate or 3-oxo-4-estren-17β-yl benzoate were condensed with [1,2-¹³C₂]acetyl chloride to give intermediates $\text{2a}$, $\text{2b}$ or $\text{10}$. $\text{2a}$ and $\text{2b}$ were cyclized by acid or base to give 3,4-¹³C₂-labeled 4-cholesten-3-one and testosterone, respectively. [3,4-¹³C₂]4-Cholesten-3-one was converted via reduction of its trimethylsilyl enol ether to [3,4-¹³C₂]cholesterol. Acetyl enollactone $\text{10}$ was cyclized in acetic acid to [3,4-¹³C₂]3-oxo-4-estren-17β-yl benzoate followed by aromatization and hydrolysis to produce [3,4-¹³C₂]estradiol-17β. Alternatively, cyclization of $\text{10}$ with base afforded [3,4-¹³C₂]3-oxo-4-estren-17β-ol directly, which was then oxidized and aromatized to yield [3,4-¹³C₂]estrone. Ozonolysis of progesterone, conversion to the diketal ester $\text{16}$ and acylation followed by acid hydrolysis furnished [3,4-¹³C₂]progesterone.     

Incorporation of [1-14C]-acetate into testosterone,androstenediol, dehydroepiandrosterone and progestogens by ram testis following human chorionic gonadotropin administration

Testicular steroidogenesis in rams was examined by constant infusion (3 hr) of [1-¹⁴C]-acetate into the testicular artery of four conscious standing animals.The following steroids (in order of decreasing levels of [¹⁴C] labeling) were secreted by the testis and found in testicular tissue: testosterone, dehydroepiandrosterone, 3β-hydroxy-5-androsten-17-one, androstenediol, 5-androsten-3β,17β-diol and 17-hydroxy-4-pregnene-3,20-dione. In addition, [¹⁴C] labeling of 17,20α-dihydroxy-4-pregnen-3-one occurred in testicular tissue but not in blood. This $\text{in vivo}$ system with the conscious standing ram demonstrated an operative Δ⁵ steroidal pathway to testosterone. The physiological significance of 17,20α-dihydroxy-4-pregnen-3-one is not yet explained in this species.     

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.     

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.     

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.     

New method for the synthesis of radioactive 19-idocholest-5-en-3beta-OL.

In 1970, ¹³¹I-19-iodocholest-5-en-3β-ol (VIII-¹³¹I) was synthesized and shown to concentrate in the adrenal cortex of dogs by Counsell $\text{et al}$. [1]. Beierwaltes and his colleagues at the university of Michigan have shown this radiopharmaceutical to be an effective adrenocortical scanning agent for the diagnosis of 5 types of adrenal disease in humans [2]. Radioactive 19-iodocholesterol is not available from commercial sources although it may be purchased from the University of Michigan Nuclear Pharmacy, Ann Arbor, as a radiochemical and converted to a radiopharmaceutical [3]. For this reason we undertook to synthesize it $\text{de novo}$ as described by Counsell $\text{et al}$. [1]. We found that 19-iodocholesterol prepared by this route contained an impurity, the amount of which varied from 20% to 60% as estimated by ¹³C nuclear magnetic resonance spectroscopy (CMR). We now describe a modification of this synthesis to yield VIII-¹³¹I of greater than 98% chemical purity.     

Metabolism and conjugation of [4-14C]progesterone by bovine liver and adipose tissues, in vitro

The ability of bovine liver and fat to metabolize progesterone and also to form glucuronide conjugates with these progestins $\text{in vitro}$ was investigated. Tissue supernatants were incubated with [4-¹⁴C] progesterone, UDP-glucuronic acid, and a NADPH generating system for 5 hr, at 37°C. Steroids were identified by thin-layer chromatography, high performance liquid chromatography, and recrystallization to a constant specific activity. The total original radioactivity which could not be removed by exhaustive ether extraction (presumptive conjugates) was 44.7 ± 14.2% in liver, 5.0 ± 3.6% in subcutaneous fat, and 3.7 ± 2.2% in kidney fat samples. Progestins identified in liver samples include 5β-pregnane-3α, 20α-diol (free and conjugate), 5β-pregnane-3α, 20β-diol (free and conjugate), 3α-hydroxy-5sB-pregnan-20-one (free and conjugate), 3β-hydroxy-5β-pregnan-20-one (free), 5β-pregnane-3, 20-dione (free), and progesterone (conjugate). Progestins identified in both the free and conjugate fractions of subcutaneous fat and kidney fat samples include progesterone, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-4-pregnen-3-one, and 20α-hydroxy-4-pregnen-3-one. Differences due to sex of bovine used were noted. These results confirm the ability of bovine liver to readily metabolize progesterone and form glucuronide conjugates of these compounds and suggest that adipose tissues take an active role in these actions in cattle.     

Steric considerations regarding the biodegradation of cholesterol to pregnenolone.-exclusion of (22S)-22-hydroxycholesterol and 22-ketocholesterol as intermediates

(22$\text{S}$)-[22-³H₁]-Cholesterol was incubated with an adrenocortical preparation and the isolated (22$\text{R}$)-[22-³H₁]-22-hydroxycholesterol had a small loss of radioactivity, proving that direct replacement of the hydrogen from the now hydroxylated position occurred.In addition [1-³H₁]-4-methylpentanol was isolated, which also had incurred a relatively small loss of its specific activity, thereby excluding (20$\text{R}$)-3β,20-dihydroxycholest-5-en-22-one as an important metabolite in the degradation of cholesterol to pregnenolone by adrenal tissue.     

Synthesis of d1-4,5,6-trinor-3,7-inter-m-phenylene-3-oxaprostaglandins including one which inhibits platelet aggregation

The synthesis of $\text{d1}$-4,5,6-trinor-3,7-inter-$\text{m}$-phenylene-3-oxaprostaglandins oxaprostaglandins of the E₁ and F₁α series⁷ from 6-endo-(1-heptenyl)-bicyclo[3:1:0]hexan-3-one (III), is described. Preliminary biological screening data for gerbil colon smooth muscle stimulation, rat blood pressure and substrate specificity toward 15-hydroxyprostaglandin dehydrogenase is presented. Platelet function studies, both $\text{in vitro}$ and $\text{in vivo}$ of $\text{d1}$-4,5,6-trinor-3,7-inter-$\text{m}$-phenylene-3-oxa-PGE₁, methyl ester (VIII) are presented.     

A rapid,sensitive method for simultaneous measurements of tissue levels of oxygenated derivatives of cholesterol

A mass spectrometric procedure which utilizes multiple selected ion monitoring (SIM) for measuring the tissue levels of cholest-5-en-3β,7α-diol, cholest-5-en-3β,7β-diol, cholest-5-en-3β,25-diol, and cholest-5-en-3β-ol-7-one is described. Trimethylsilyl ethers (TMS) of sterols in a lipid extract are analyzed directly by focusing the ions at $\text{m}\text{e}$ 546, 472, and 443. Endogenous cholesterol serves as an internal standard and its concentration is determined by gas chromatography. The sensitivity of this method has allowed measurement of 2 ng of oxygenated sterol which corresponded to the amount present in 1 mg of rat liver.     

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.     

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.     

Steroids from starfish

Starfish $\text{Linckia multifora, Protoreaster nodosus, Protoreaster lincki, Culcita schmideliana, Nardoa variolata}$ and $\text{Acanthaster planci}$ were examined for sterols and sapogenins. All asteroids contained cholestanol in addition to C₂₇ to C₃₀ mono and diunsaturated sterols. A planci contained largest amounts of 5α-cholesta-9(11), 20(22)-diene-3β, 6α-diol-23-one and 5α-pregn-9(11)-ene-3β,6α-diol-20-one whereas $\text{P. nodosus, P. lincki and C. schmideliana}$ contained only small amounts. Neither pregnane nor cholestane genins were detected in $\text{L. Multifora}$ and $\text{N. variolata}$.     

Assay of liver microsomal cholesterol 7 alpha-hydroxylase using deuterated carrier and gas chromatography-mass spectrometry

The activity of cholesterol 7α-hydroxylase in rat liver microsomes was assayed by measuring the mass of 5-cholestene-3β, 7α-diol formed from endogenous cholesterol under standardized incubation conditions. After termination of incubations, a known amount of 5-[24,25,7β-²H₃]cholestene-3β,7α-diol was added. A chloroform extract of the incubation mixture was subjected to thin layer chromatography and the fraction containing 5-cholestene-3β,7α-diol was converted into trimethylsilyl ether. The trimethylsilyl ether was subjected to combined gas chromatography-mass spectrometry and the amount of unlabeled 5-cholestene-3β,7α-diol in the mixture was calculated from the ratio between the relative intensitics of the peaks at $\text{m}\text{e}$ 456 (M-90) and $\text{m}\text{e}$ 459 [M-(90 + 3)]. The precision of the method was ±2.2% (SD). The results with this method of assay of cholesterol 7α-hydroxylase were compared with those obtained with a method based on conversion of a trace amount of added [4-¹⁴C]cholesterol into 5-cholestene-3β,7α-diol.     

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.     

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.     

A-nor steroids. 1. The stereochemistry of A 2-carbomethoxy-A-nor-cholestan-3-one obtained via the Dieckmann condensation

The Dieckmann condensation of dimethyl 3, 4-$\text{seco}$-5α-cholestan-3, 4-dioate ($\text{1}$), using sodium methoxide in benzene under reflux for two hours, is shown to give 2α-carbomethoxy-A-nor-5α-cholestan-3-one ($\text{2}$). Confirmation of the stereochemistry of the β-keto ester $\text{2}$ was obtained through its sodium borohydride reduction product, 2α-carbomethoxy-A-nor-5α-cholestan-3β-ol ($\text{4}$).