Biosynthesis of androgens by the sesterterpene pathway via 23,24-dinor-4-cholen-3-one

Rat testicular and adrenal gland microsomal preparations were incubated with 23,24-dinor-5-cholen-3β-ol (Guneribol) a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by thin layer and high-performance liquid chromatography and crystallized to constant specific radioactivity. These preparations converted the substrate to 23,24-dinor-4-cholen-3-one. Radioactive 23,24-dinor-4-cholen-3-one was synthesized and incubated with further tissue preparations and shown to be converted to steroid hormones. These findings suggest that 23,24-dinor-4-cholen-3-one is an intermediate on the sesterterpene pathway for steroidogenesis.     

Biosynthesis of adrenal corticosteroids by the sesterterpene pathway via 23,24-dinor-4-cholen-3-one

Rat adrenal gland preparations were incubated with 23,24-dinor-5-cholen-3 beta-ol (Guneribol), a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by thin layer and high-performance liquid chromatographies and crystallized to constant specific activity. These preparations converted the substrate to 23,24-dinor-4-cholen-3-one. Radioactive 23,24-dinor-4-cholen-3-one was synthesised and incubated with further tissue preparations and shown to be converted to corticosteroids. These findings suggest that 23,24-dinor-4-cholen-3-one is an intermediate on the sesterterpene pathway for steroidogenesis.     

The conversion of 23,24-dinor-5-cholen-3β-ol to cortisol by the canine adrenal

An alternative pathway for steroidogenesis, via a sesterterpene, has been proposed. This communication presents evidence that the canine adrenal can utilise 23,24-dinor-5-cholen-3β-ol to biosynthesise cortisol.It has been proposed that steroid hormones could be biosynthesised by a pathway other than that through cholesterol, possibly by a sesterterpene pathway (1,2).The previous studies were carried out using bovine adrenal tissue. This communication extends these studies to include the canine adrenal gland.     

Biosynthesis of cholest-5-ene-3beta, 24-diol (cerebrosterol) by bovine cerebral cortical microsomes

The presence of cholest-5-ene-3β, 24-diol (cerebrosterol) in samples of human, bovine, and rabbit brains has been established by isolation of the sterol therefrom and identification by comparison of physical properties. Cholest-5-ene-3β, 24-diol was present at the level of 66.5 sg/g of dried tissue in human brain, 42.9 μg/g in cattle brain, and 89.5 pg/g in rabbit brain. Cholest-5-ene-3β, 24-diol was the only readily detectable hydroxycholesterol derivative in these brain tissues and was concentrated in the 105,000 g pellet (microsomal fraction) of both human and bovine cerebral cortex, with no demonstrable amounts of the sterol present in nuclear or mitochondrial fractions. Incubation of [1,2-³H]- or of [4-¹⁴C]-cholesterol with the 105,000 g microsomal pellet from bovine cerebral cortical homogenates demonstrated 0.1-0.38 per cent conversions to radioactive cholest-5-ene-3β, 24-diol, isolated and purified as the 3β, 24-dibenzoate. The bioconversion required oxygen, and a stimulation of hydroxylation by added NADPH₂ was demonstrated. Our observations establish that a sterol 24-hydroxylase system is present in bovine cerebral cortex.     

Transfer of cholesterol and a fluorescent cholesterol analog, 3'-pyrenylmethyl-23,24-dinor-5-cholen-22-oate-3 beta-ol, between human plasma high density lipoproteins

A fluorescent cholesterol analog, 3'-pyrenylmethyl-23,24-dinor-5-cholen-22-oate-3 beta-ol (PMCA), has been synthesized as a spectroscopic probe of cholesterol function. The substrate activity of PMCA, about two-thirds that of cholesterol, with lecithin:cholesterol acyltransferase indicates that PMCA is a reasonable cholesterol analog and that the orientation of the substituted sterol in the phospholipid interface is similar to that of cholesterol. The fluorescence properties of PMCA are similar to those of other pyrene-containing compounds that exhibit concentration-dependent excimer fluorescence. The rate of transfer of [3H]PMCA between HDL is about six times faster than cholesterol. These results indicate that the analog will be useful in studies of cholesterol function.     

Inhibition of cholesterol side-chain cleavage by intermediates of an alternative steroid biosynthetic pathway

Mitochondrial preparations from endocrine tissues were incubated with radioactive cholesterol and the effect of hydroxylated metabolites of 23,24-dinor-5-cholen-3 beta-ol (23,24-dinor-5-cholene-3 beta,20-diol and 23,24-dinor-5-cholene-3 beta,21-diol) on the production of pregnenolone was measured. These compounds are intermediates in an alternative, sesterterpene pathway for steroid hormone biosynthesis. It was found that these materials, like the analogous side-chain-hydroxylated derivatives of cholesterol (20 alpha-hydroxycholesterol and 22S-hydroxycholesterol), inhibit cholesterol side-chain cleavage. The possibility that there could be a control mechanism whereby metabolites of 23,24-dinor-5-cholen-3 beta-ol inhibit steroidogenesis occurring by the cholesterol pathway is discussed.     

Acetylation and hydroxylation of 5alpha-androstane-3beta,17beta-diol by prostate and epididymis

An unknown radiometabolite, formed in the canine prostate and epididymis after intra-arterial infusion of testosterone-4-¹⁴C in physiologic saline and extraction of the organs with ethyl acetate-acetone, was identified as the 3-monoacetate of 5α-androstane-3β, 17β-diol (3β-diol). Transformation of 3β-diol-¹⁴C to its identified 3-monoacetate derivative could also be demonstrated, if the incubation of the radiosubstrate with minced canine prostate was terminated by ethyl acetate extraction. The formation of polar products in high yield was noted. Whereas minced canine prostate actively converted 5α-androstane-3α,17β-diol-¹⁴C to 17β-hydroxy-5α-androstan-3-one-¹⁴C, the same preparation hydroxylated 3β-diol-¹⁴C predominantly at the 7ξ- and, to a lesser extent, at the 6ξ-positions. Partial identification of the hydroxylated radiometabolites was by crystallization of the CrO₃-oxidation products 5α-androstane-3,6,17-trione-¹⁴C and 5α-androstane-3,7,17-trione-¹⁴C to constant SA and by GLC/MS of the latter derivative. NADPH-supplementation of the preparation enhanced the yield of hydroxylated products derived from 3β-diol-¹⁴C in a 1 hr incubation from 22% to 41%. Analogous supplemented incubations of benign hyperplastic human prostate and canine epididymis produced polar metabolites (in 12.5% and 76% yields, respectively) which gave rise to similar proportions of the same androstanetrione epimers on CrO₃-oxidation.     

The role of (25S)-5α-cholestan-3β, 26-diol and (25S)-5α-furostan-3β,26-diol in the biosynthesis of tomatidine and neotigogenin

(25S)-5α-Cholestan-3β,26-diol was incorporated into neotigogenin and tomatidine, and (25S)-5α-furostan-3β,26-diol only in neotigogenin,     

Synthesis of “glycospirostanes” via ring-closing metathesis

Syntheses of “glycospirostanes” from 3β-hydroxy-23,24-dinor-5α-cholano-22,16-lactone and 3α-hydroxy-23,24-dinor-5β-cholano-22,16-lactone were performed. The key step of these syntheses was ring-closing metathesis of the corresponding C,O-diallyl derivative. Further elaboration of the six-membered ring F consisted of allylic hydroxylation with SeO₂ followed by OsO₄ dihydroxylation of the C24–C25 double bond. The obtained final products proved to be simultaneously O- and C-l-arabinopyranosides.     

A preparation of 3alpha-methyl-5alpha-cholestane-2beta,3beta-diol.

A simple preparation of 3α-methyl-5α-cholestane-2β, 3β-diol (1a) by a four-step synthesis from 2α, 3α-epoxy-5α-cholestane is described.     

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.     

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.     

Synthesis of 25-hydroxy[23,24-3H]vitamin D3

A synthesis of 25-hydroxy[23,24-³H]vitamin D₃ leading to a radiochemically pure product with a specific acitivity of 78 Ci/mmol is described. The structure of the product was confirmed by comparison with unlabeled material and its biological activity was established by in vitro conversion to 1α,25-dihydroxy[23,24-³H]vitamin D₃ using the chick kidney 1α-hydroxylase system.     

Transformation of 23,24-bisnorchol-4-en-3-one-22-ol by Rhizopus arrhizus

The transformation of 23,24-bisnorchol-4-en-3-one-22-ol into 6β,11α,22-trihydroxy-23,24-bisnorchol-4-en-3-one by the fungus Rhizopus arrhizus has been shown to be dependent on the composition of the culture medium, with respect to yield of the triol. The transformation of the 22-alcohol to 6β,11α-dihydroxy-pregn-4-ene-3,20-dione is also reported.     

Synthesis of 5 beta,17 alpha-19-norpregn-20-yne-3 beta,17-diol and of 5 beta,17 alpha-19-norpregn-20-yne-3 alpha,17-diol, human metabolites of norethindrone

A convenient synthesis of both 5 beta,17 alpha-19-norpregn-20-yne-3 beta,17-diol (1) and 5 beta,17 alpha-19-norpregn-20-yne-3 alpha,17-diol (2) in multigram quantities from estr-4-ene-3,17-dione is reported. Full characterization of these often-cited human metabolites of norethindrone is presented for the first time.     

Adrenal androgen biosynthesis by a sesterpene pathway

Rat and human adrenal gland preparations were incubated with radioactive cholesterol and 23,24-dinor-5-cholen-3 beta-ol, the latter being a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by TLC and crystallised to constant specific activity. It was found that rat and human adrenal glands can utilise 23,24-dinor-5-cholen-3 beta-ol to produce androstenedione and dehydroepiandrosterone. Also, it was found that the conversion of 23,24-dinor-5-cholen-3 beta-ol to androgens occurs in the microsomal fraction. It was concluded that the sesterterpene pathway for steroid biosynthesis can function in the rat and human adrenal glands to produce androgens and that the intermediates are converted to androgens in the microsomal fraction.     

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.     

Chemical synthesis of (22E)-3 alpha,6 beta,7 beta-trihydroxy-5 beta-chol-22-en-24-oic acid and its taurine and glycine conjugates: a major bile acid in the rat

A method for the synthesis of Delta(22)-beta-muricholic acid (Delta(22)-beta-MCA), (22E)-3 alpha,6 beta,7 beta-trihydroxy-5 beta-chol-22-en-24-oic acid, and its taurine and glycine conjugates (Delta(22)-beta-muricholyltaurine and Delta(22)-beta-muricholylglycine) is described. The key intermediate, 3 alpha,6 beta,7 beta-triformyloxy-23,24-dinor-5 beta-cholan-22-al, was prepared from beta-muricholic acid (beta-MCA) via the 24-nor-22-ene and 24-nor-22,23-diol derivatives. Wittig reaction of the aldehyde with (carbomethoxymethylene) triphenylphosphorane and subsequent hydrolysis gave (unconjugated) Delta(22)-beta-MCA. Condensation reaction of the unconjugated acid with taurine or glycine methyl ester using diethylphosphorocyanide yielded the naturally occurring taurine or glycine conjugate (N-acylamidate) of Delta(22)-beta-MCA. These synthetic reference compounds are now available for investigation of the metabolism of beta-MCA by bacterial and hepatic enzymes in the rat and should also be useful as substrates for reductive deuteration or tritiation to give the 22,23-(2)H or (3)H-beta-MCA.     

The synthesis of 23,24-14C-ecdysone

The synthesis of 23,24-¹⁴C-ecdysone, using 3,4-¹⁴C-2-methyl-3-butyn-2-yl tetrahydropyranyl ether, is described.     

Steroid hormone biosynthesis by a sesterterpene pathway in the rat and rabbit testis

Rat and rabbit testis preparations were incubated with [4-14C]cholesterol and 23,24-dinor-[7 alpha-3H]5-cholen-3 beta-ol, the latter being a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by thin-layer chromatography and crystallised to constant specific activity. It was found that rat and rabbit testis can utilise 23,24-dinor-5-cholen-3 beta-ol to produce testosterone. The tritium/carbon-14 ratios in the testosterone and androstenedione isolated indicated that these tissues differentiated between the two substrates. This finding is supported by the observation that, on stimulation with HCG, the tritium/carbon-14 ratios in the testosterone isolated were increased compared to the controls. The results of further experiments implied that, while the biosynthesis of testosterone from cholesterol occurred in the rat testis mitochondrial fraction, its biosynthesis from 23,24-dinor-5-cholen-3 beta-ol occurred in the microsomal fraction.