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A short and efficient synthesis of steroid synthons, di(tert-butyldimethylsilyl) ethers of 3,21-dihydroxy-24-nor-chol-5-en-23-al (8 and 10) and of ethyl 3,21-dihydroxy-25-homo-chola-5,23-dien-25-oate (9 and 11), having natural (20R) and unnatural (20S) configuration from 3β-(tert-butyldimethylsilyloxy)-14α,20ξ-card-5-enolide (2) is reported. Further elongation of the side chain of these synthons provides a new method for the synthesis of (20R) and (20S)-21-hydroxy steroids. The utility of the method was exemplified by the synthesis of a natural marine sterol - 21-hydroxycholesterol (18)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We report the biochemical characterization of sulfated polyhydroxysterols isolated from marine invertebrates as potent antagonists of farnesoid-X-receptor (FXR), a ligand-regulated transcription factor involved in the regulation of lipid and glucose homeostasis in mammals. Molecular characterization of a library of sulfated polyhydroxysteroids resulted in the identification of a first FXR antagonist. In contrast to partial antagonists, this compound was endowed with an antagonistic activity on the expression of a subset of FXR-regulated genes in liver cells and abrogated the release of nuclear coreceptor from the promoter of these genes. The putative binding mode to FXR, obtained through docking calculations, suggested the crucial role played by the bent shape of the molecule as well as the presence of one hydroxyl group in its side chain. This compound is a major tool to explore the effect of FXR inhibition in pharmacological settings.

The use of anionic polycyclization was investigated in an effort to develop a versatile and convergent synthesis of advanced tetracyclic intermediates of batrachotoxin and ouabain analogues. Two new 5-(trialkylsilyl)-2-cyclohexenones as A ring precursors and a new Nazarov intermediate (D ring precursor) were prepared for this purpose. The reaction of the unsaturated beta-keto aldehyde A ring precursor with the enolate of the Nazarov intermediate afforded, after subsequent transformations, a 14-beta-hydroxysteroid with complete control of stereochemistry.

The syntheses of (24S)-24,25-epoxycholesterol, (24S)-hydroxycholesterol, and 24-ketocholesterol are described. The compounds belong to oxysterols, which can be considered to be the modulators of cholesterol metabolism. The asymmetric hydroxylation of desmosterol acetate according to Sharpless was used as the key reaction in the stereoselective introduction of functionality in position 24.

Four different types of hydroxysteroids (primary alcohol, secondary alcohols, and phenol), bearing either an oxirane or an azide as a precursor of molecular diversity, were linked in good yields to solid support using the butyldiethylsilane polystyrene (PS-DES) resin. These molecules were then used as scaffolds to generate hydroxysteroid derivatives containing two levels of diversity. The proposed libraries were tested by running steroidal alcohols through a model sequence of reactions (solid-phase coupling, aminolysis of oxirane or reduction of azide, amidation, and final cleavage). As a result, two linked secondary alcohols (17beta-hydroxy-spiro-3(R)-oxirane-5alpha-androstane and 3beta-hydroxy-spiro- 17(S)-oxirane-5alpha-androstane) and a primary alcohol (spiro-17(S)-oxirane-3-(hydroxymethyl)-1,3,5(10)-estratriene) afforded good overall yields (>45%) and high HPLC purities (>90%) of hydroxysteroids derivatized as alkylamides without purification. One limitation was noted for the fourth library: the phenolic steroid linked by the diethylsilyloxy linker gave a poor overall yield of 8% of the desired model compound. Finally, the diethylsilyloxy linker was used successfully for a rapid solid-phase synthesis of a model library of twenty C19-steroid derivatives (3beta-amido-3alpha-hydroxy-5alpha-androstane-17-ones), with an average yield of 53% and average HPLC purity of 97% without purification steps.

We report the synthesis of 10 novel steroids obtained from 3 beta, 15 beta-diacetoxy-17 alpha-hydroxy-5-pregnen-20-one (1c) as intermediates in the synthesis of 15 beta-acetoxy-20,20-ethylenedioxy-17 alpha-hydroxy-4-pregnen-3-one (6a) and its tritiated tracer 15 beta-acetoxy-20,20-ethylenedioxy-17 alpha-hydroxy-1,2,6,7-3H-pregn-4-en-3-one (6d). The one pot interconversion of intermediate (6a) to 3 beta, 15 beta, 17 alpha-trihydroxy-5-pregnen-20-one (1a) and 3 alpha, 15 beta, 17 alpha-trihydroxy-5 beta-pregnan-20-one (2a) provides a new and efficient approach to the synthesis of diagnostically important metabolites of the human neonate and a possible route in the synthesis of the tritated tracers 3 beta, 15 beta, 17 alpha-trihydroxy-1,2,7-3H-pregn-5-en-20-one (1d) and 3 alpha, 15 beta, 17 alpha-trihydroxy-1,2,6,7-3H-5 beta-pregnan-20-one (2b) for the development of new immunoassays. We also report in this investigation an alternative route in the synthesis of 15 beta, 17 alpha-dihydroxy-4-pregnen-3,20-dione (7a) an intermediate in the synthesis of human 15 beta-hydroxysteroid metabolites.

Steroids hydroxylated at C-15 have long provided useful information about the well-being of the fetus and feto-placental unit in human pregnancy. In an attempt to develop a new and reliable immunoassay method for use in newborn screening programs for congenital adrenal hyperplasia, we report the chemical synthesis of 3 alpha,15 beta,17 alpha-trihydroxy-5 beta-pregnan-20-one (2) from 3 alpha-hydroxy-5 beta-androstan-17-one (4) in 9 steps. In brief, 3 alpha-hydroxy-5 beta-androst-15-en-17-one (6), was obtained from 4 by phenylselenation yielding 3 alpha-hydroxy-16 alpha-phenylseleno-5 beta- androstan-17-one (5a) which on dehydroselenation gave 6. Introduction of the 15 beta-hydroxy group and the side-chain was achieved by the addition of 2-lithio-2-methyl-1,3- dithiane followed by an acid-catalyzed rearrangement to give 20,20-trimethylenedithio-5 beta-pregn-16-en- 3 alpha,15 beta-diol (8a). Acetylation then cleavage of the dithioacetal gave 3 alpha,15 beta-diacetoxy-5 beta-pregn-16-en- 20-one (9) which on hydrogenation gave 3 alpha,15 beta-diacetoxy-5 beta-pregnan-20-one (10). Reaction of base and oxygenation of 10 gave a mixture of products which on basic hydrolysis gave 3 alpha,15 beta,17 alpha-trihydroxy-5 beta- pregnan-20-one (2) in an overall yield of 8.8%.

15-Hydroxysteroids have long provided information about fetal well-being and fetal steroidogenesis. 3 beta,15 beta,-17 alpha-Trihydroxy-5-pregnen-20-one (1a) is a major 15 beta-hydroxylated metabolite unique to the human perinatal period. The synthesis of 3 beta, 15 beta, 17 alpha-trihydroxy-5-pregnen-20-one (1a) is reported here in the first of a series of publications on the chemical synthesis of 15 beta-hydroxylated steroids for use in the (a) development of new immunoassay techniques for application to newborn screening programs and fetal well-being; (b) development of new anti-androgenic drugs; and (c) study of androgen/estrogen interaction in late pregnancy. To this end, a method for the introduction of the 15 beta-hydroxy group onto the steroid nucleus was developed resulting in a nine-step stereoselective synthesis of 1a with an overall yield of 26%. A high yielding selenation-dehydroselenation procedure was developed for the synthesis of 3 beta-hydroxy-5,15-androstadien-17-one (8) which avoided the previously reported Baeyer-Villiger rearrangement. The introduction of the 15 beta-hydroxy group and the side chain was achieved by the addition of 2-lithio-2-methyl-1,3-dithiane to give 20,20-trimethylenedithio-5,15-pregnadien-3 beta, 17 beta-diol (9a) followed by its acid-catalyzed rearrangement to give 20,20-trimethylenedithio-5, 16-pregnadien-3 beta,15 beta-diol (10a). Acetylation and cleavage of the dithioacetal gave 3 beta,15 beta-diacetoxy-5,16-pregnadien-20-one (11b) which was hydrogenated to give 3 beta,15 beta-diacetoxy-5-pregnen-20-one (12b). Reaction of the ketone (12b) with oxygen and then basic hydrolysis gave the desired product 1a.

In recent years several 15 beta-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3 alpha,15 beta,17 alpha-trihydroxy-5 beta-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3 xi,5 xi-isomers, namely 3 alpha,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one (3), 3 beta,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one (7) and 3 beta,15 beta,17 alpha-trihydroxy-5 beta-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3 beta,15 beta-Diacetoxy-17 alpha-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15 beta,17 alpha-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15 beta-acetoxy-3 beta,17 alpha-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15 beta-acetoxy-3 beta,17 alpha-dihydroxy-5 alpha-pregnan-20-one (13) a common intermediate for the synthesis of the 3 beta(and alpha),5 alpha-isomers. Hydrolysis of the 15 beta-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15 beta-acetoxy-17 alpha-hydroxy-5 alpha-pregnan-3,20-dione (14) which on reduction with L-Selectride and hydrolysis of the 15 beta-acetate gave 3. Finally, hydrogenation of 4 gave 15 beta, 17 alpha-dihydroxy-5 beta-pregnan-3,20-dione (10) which on reduction with L-Selectride gave 8.

C20H24O6, Mr = 360.41 lambda (Cu K alpha) = 1.54056 A, room temperature. (I) (5 beta,10 beta,13 alpha,14 alpha)-Methyl 14-hydroxy-1,7,17-trioxoandrost-8-ene-19-oate, triclinic, P1, a = 7.9514 (5), b = 9.2892 (5), c = 12.8534 (12) A, alpha = 81.256 (6), beta = 75.796 (6), gamma = 77.908 (5) degrees, V = 894.85 (11) A3, Z = 2, Dx = 1.338 Mg m-3, mu = 0.77 mm-1, F(000) = 383.96, final R = 0.043 for 2912 observed reflections. (II) (5 beta, 10 beta, 13 beta,14 beta)-Methyl 14-hydroxy-1,7,17-trioxoandrost-8-ene-19-oate, monoclinic, P21/n, a = 12.8704 (9), b = 10.4481 (9), c = 13.1482 (5) A, beta = 104.103 (5) degrees, V = 1714.77 (20) A3, Z = 4, Dx = 1.396 Mg m-3, mu = 0.81 mm-1, F(000) = 767.92, final R = 0.055 for 2516 observed reflections. These two non-natural steroids bear a methoxycarbonyl group at C(10). In both molecules the relative stereo-chemistry is cis for the A/B ring junction and cis for the C/D ring junction. The relative orientations of MeO2C--C(10) and HO--C(14) are anti for (I) and syn for (II). The methoxycarbonyl group lies at the axial position for (I) and equatorial for (II), relative to ring A. The energies of possible conformations for (I) and (II) are evaluated, wherein the A rings adopt a chair conformation.

The chemical stabilities of the adrenal-scanning agents, 6beta-iodo-methyl-19-norcholest-5(10)-en-3beta-ol (6-iodomethylnorcholesterol) and 19-iodocholest-5-en-3beta-ol (19-iodocholesterol), and several of their derivatives were examined by 13C nuclear magnetic resonance. Neat 6-iodomethylnorcholesterol, sealed in glass under nitrogen and stored at 0 degrees C, remains 98 mole% chemically pure for 3 months. Neat 19-iodocholesterol, stored in the dark at 25 degrees C, remains 98 mole% chemically pure for 3 months. Either 6-iodomethylnorcholesterol-125I or-131I, informulation and stored at 5 degrees C, will remain greater than 97% radiochemically pure for at least 15 days. Labelled 19-iodocholesterol, formulated and stored under the same conditions, shows 20% decomposition after 3 weeks and 40% after 6 weeks.

The first convenient chemical synthesis of 7alpha-hydroxyandrost-4-ene-3,17-dione is reported. Androsta-4,6-diene3, 17-dione was converted into its 6alpha, 7alpha-epoxy-derivative; reduction of the epoxide with aluminum amalgam gave 7alpha-hydroxyandrost-4-ene-3,17-dione. This reducing agent is more efficient than chromous acetate for the purpose.

Practical routes to 9 alpha-hydroxypregnenes were developed by epimerization and hydration of 17 alpha-ethynyl-9 alpha,17 beta-dihydroxyandrost-4-en-3-one. In the three different methods of epimerization which were used, the C-9 alpha hydroxy group was not susceptible to rearrangement or other side reactions. C-21 functionalized 9 alpha-hydroxypregnenes were obtained by introducing a 17 alpha-halogenated ethynyl group into 9 alpha-hydroxyandrost-4-ene-3,17-dione. Epimerization and hydration by the 17 beta-nitrooxy method produced 21-halogenated 9 alpha-hydroxypregnenes, which were further converted into 21-acetoxy-9 alpha-hydroxypregn-4-ene-3,20-dione.

The reactions of 3 beta-hydroxy-20-oxo-5-pregnene-16 alpha-carbonitrile, 3 beta-hydroxy-5-androsten-17-one, 3 beta-hydroxy-5-pregnen-20-one, and 5-cholesten-3 beta-ol with thioacetic acid in dioxane afford mainly 6 beta-acetylthio derivatives which were characterized by IR, NMR (1H, 13C), and mass spectroscopy. A similar reaction of 17 beta-hydroxy-1,4-androstadien-3-one yields chiefly the known 1 alpha-SCOCH3 derivative.

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

The compounds named in the title were prepared by routes which included the reduction of suitable 16 alpha,17-epoxypregnan-20-ones with aluminium amalgam to give 16 alpha-hydroxypregnan-20-ones, and reduction of the 20-oxo function either with sodium borohydride to obtain the 3,16 alpha,20 beta-triols or with lithium-liquid ammonia to obtain the 3,16 alpha,20 alpha-triols.

The reaction of 16-methylene-17-ketosteroids (Ia), (Ic) and (Ie) with methyl magnesium iodide yields 16-methylene-17 alpha-methyl-17 beta-hydroxysteroids (IIa), (IIb) and (IId). These are subjected to the addition of hypobromous acid and the subsequent anionotropic rearrangement to convert them into 16 alpha-methyl-16 beta-bromomethyl-17-ketosteroids (Va), (Vb) and (Vd). These were reduced with LiA1H4 to obtain 16,16-dimethyl-17 beta-hydroxysteroids (VIa), (VIb) and (VId). Compounds (VIIa) and (VIIe) were selectively deacetylated yielding (VIIb) and (VIId); these were then oxidized and hydrolyzed to convert them into (VIf) and (VIg).

Certain structural similarities between prostaglandins with close-packed side chains and the perhydrocyclopentanophenanthrene nucleus of steroids prompted the synthesis and biological evaluation of 6beta, 17 beta-dihydroxy-5alpha-androstane-2alpha-carboxylic acid (30), its 6-deoxy derivative 28, and the corresponding 6-deoxy-2beta derivative 29 in an attempt to evaluate carbocyclic acids as potential prostaglandin analogs. Preliminary in vitro studies on isolated guinea pig ileum have shown weakly specific, prostaglandin-stimulated smooth muscle antagonism for 28 when compared with antagonism of bradykinin- and acetylcholine-induced contractions. Complete dose-response curves for 28 on prostaglandin-stimulated guinea pig ileum have shown a reduction in the maxium response and a decrease in the slope of the curve, indicating a noncompetitive type of inhibition for this type of derivative.

A series of 17alpha-furylestradiol and dihydroequilin derivatives was synthesized by reacting the appropriate 3-substituted estrone and equilin with 2- or 3-furyllithium. The oral estrogenic activity of the compounds was compared with that of mestranol. In the Allen-Doisy test, the 17alpha-(3-furyl) analogs were 4-19 times as potent orally as the standard in rats but they were less active in mice. Acetylation of the 17-alcohol or replacement of the 3-furyl by a 2-furyl group produced a decrease in activity. In the mouse uterotrophic assay in mice the compounds were less effective than mestranol and exhibited very shallow dose-response curves.

Described herein are chemical syntheses of the following compounds: 4-methyl-(24S)-24-ethyl-cholesta-4,22-dien-3-one, 4,4-dimethyl-(24S)-24-ethyl-cholesta-5,22-dien-3-one, 4beta-methyl-(24R)-24-ethyl-5alpha-cholestan-3beta-ol, 4alpha-methyl-(24R)-24-ethyl-5alpha-cholestan-3beta-ol, 4alpha-methyl-(24S)-24-ethyl-5alpha-cholest-22-en-3beta-ol, 4-methyl-6beta-bromo-(24S)-24-ethyl-cholesta-4,22-dien-3-one, 4alpha-methyl-(24S)-24-ethyl-cholesta-5,22-dien-3beta-ol, 4alpha,5alpha-epoxy-(24S)-24-ethyl-cholesta-4,22-dien-3beta-yl acetate, 4beta-methyl-(24S)-24-ethyl-cholest-22-en-3beta,5alpha-diol, 4beta-methyl-5alpha-hydroxy-(24S)-24-ethyl-cholest-22-en-3beta-yl acetate, 4beta-methyl-(24S)-24-ethyl-cholesta-5,22-dien-3beta-yl acetate and 4beta-methyl-(24S)-24-ethyl-cholesta-5,22-dien-3beta-ol. Chromatographic, nuclear magnetic resonance, and mass spectral data are presented for the compounds under consideration.

The synthesis of 7alpha-[12alpha, 12beta-2 H2]hydroxy-4-cholesten-3-one is described. It was shown with different techniques that this compound was 12alpha-hydroxylated by the microsomal fraction of a rat liver homogenate without marked isotope effect, indicating that cleavage of the C--H bond is not the rate-limiting step in this hydroxylation. The rate of 12alpha-hydroxylation was decreased by about 20% when performed in a medium containing deuterated water. The findings were discussed with reference to the specific properties of the 12alpha-hydroxylating system and to the results of previous studies on rate-limiting step in microsomal hydroxylation of steroids.