Synthesis and molecular structure of 14,15-pyrrolidino-and 14,16-ethano derivatives of estrone

Hydrolysis of 3-methoxy-16alpha-nitro-14,17-ethenoestra-1,3,5(10)-trien-17beta-yl acetate under weakly basic conditions leads to formation of 3-methoxy-2'-oxopyrrolidino-[4',5':14beta,15beta]-estra-1,3,5 (10)-trien-17-one, the structure of which has been confirmed by X-ray analysis and some chemical transformations. The reactivity of 3-methoxy-16alpha-nitro-14,17-ethanoestra-1,3,5(10)-trien-17beta-yl acetate under various conditions of basic hydrolysis has been investigated. The derived compounds have been identified by means of NMR spectroscopy and X-ray analysis.     

Neighboring group participation. Part 16. Stereoselective synthesis and receptor-binding examination of the four stereoisomers of 16-bromomethyl-3,17-estradiols

The four possible isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol (1a-4a) with proven configurations were converted into the corresponding 3-benzyloxy-16-bromomethylestra-1,3,5(10)-triene-3,17-diols (5e-8e). Depending on the reaction conditions the cis isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol (1a and 2a) were transformed into 3-benzyloxy-16-bromomethylestra-1,3,5(10)-trien-17-yl acetate (5b and 6b) or 16-bromomethyl-3-hydroxyestra-1,3,5(10)-trien-17-yl acetate (5c and 6c) on treatment with HBr and acetic acid. The mechanism of the process can be interpreted as involving front-side neighboring group participation. Under similar experimental conditions, the trans isomers (3a and 4a) yielded only 3-benzyloxy-16-acetoxymethylestra-1,3,5(10)-trien-17-yl acetates (3b and 4b) or 16-acetoxymethylestra-1,3,5(10)-triene-3,17-diyl diacetates (3d and 4d). Both the cis (1a and 2a) and the trans (3a, and 4a) isomers were transformed into 16-bromomethylestra-1,3,5(10)-trien-17-ol (5a-8a) by the Appel reaction on treatment with CBr4/Ph3P. Debenzylation of 5a-8a was carried out with HBr and acetic acid to yield 5e-8e. The debenzylation process in the presence of acetic anhydride produces the diacetates 5d-8d. The structures of the compounds were determined by means of MS, 1H NMR and 13C NMR spectroscopic methods. Compounds 5c-8c and 5e-8e were tested in a radioligand-binding assay. Except for the affinity of 7e for the estrogen receptor (Ki=2.55 nM), the affinities of the eight compounds (5c-8c and 5e-8e) for the estrogen, androgen and progesterone receptors are low (Ki > 0.55, 0.52 and 0.21 microM, respectively).     

Heterocyclic steroids-Part-IV. Studies on the total synthesis of 3-methoxy-7beta-phenyl-6-oxaestra-1,3,5(10)-trien-17beta-ol.

7-Methoxyflavanone(I) has been converted, into dl-3-methoxy-7β-phenyl-6-oxaestra-1,3,5 (10)-trien-17β-ol (IX) in fairly good yield.     

Preparation of 9-alpha,11-xi-tritiated 17-alpha-ethynylestradiol, mestranol, estradiol-alpha-17-beta, and norethindrone

The preparation of 9alpha, 11xi-tritiated 17alpha-ethinyl estradiol, mestranol, estradiol-17beta, and norethindrone are described. Estrone-3-methyl ether was employed as starting material, and ethinylation with lithium acetylide-ethylene diamine resulted in 95% mestranol. Demethylation of mestranol with boron tribromide at 0 degrees resulted in 92% 17alpha-ethinyl estradiol. Dimethylsulfoxide was the choice of reagent for the condensation reaction which was complete at room temperature in about 4 hours. The usually less than 3% of unreacted 17-oxo product was removed by Girard separation. Demethylation of methyl ether with boran tribromide in methylene chloride resulted in an excellent yield of 17alpha-ethinyl estradiol-9alpha, 11xi-tritium. 3-methoxyestra-1,3,5-trien-17-one-9alpha, 11xi-tritium was reduced with sodium bis(2-methoxyethoxy) aluminum hydride to the 17beta-hydroxy compound and subsequent demethylation resulted in estradiol-9alpha, 11xi-tritium. The general method of Ringold et al was employed for the preparation of 17beta-hydroxy-17alpha-ethinylestr-4-en-3-one. Improvements for small scale radiosynthesis are also presented.     

Hydroxysteroid dehydrogenases of Pseudomonas testosteroni. Separation of a 17 beta-hydroxysteroid dehydrogenase from the 3(17) beta-hydroxysteroid dehydrogenase and comparison of the two enzymes.

When a crude extract of Pseudomonas testosteroni induced with testosterone was subjected to polyacrylamide gel electrophoresis, six bands that stained for 17 beta-hydroxysteroid dehydrogenase activity was observed. A protein fraction containing the enzyme corresponding to the fastest migrating band and devoid of the other hydroxysteroid dehydrogenase activities has been obtained. This preparation appears to be distinct from the previously isolated 3(17) beta-hydroxysteroid dehydrogenase (EC 1.1.1.51) in its chromatography properties on DEAE-cellulose, substrate and cofactor specificity, immunological properties and heat stability. The preparation appears devoid of 3alpha-, 3beta-, 11beta-, 17alpha-, 20alpha-, and 20beta-hydroxysteroid dehydrogenase activities. The enzyme transfers th 4-pro-S-hydrogen of NADH from estradiol-17beta (1,3,5(10)estratriene-3,17beta-diol) to estrone (3-hydroxy-1,3,5(10)-estratriene-17-one).     

Identifications of radioactive steroid estrogen conjugates in blood plasma of laying hens after intramuscular injection of (4--14C)-estrone.

[4--14C] Estrone was injected intramuscularly into six laying hens. Fifty minutes later the hens were exsanguinated. The plasmas were examined for conjugates of radioactive phenolic steroids by recovery on columns of Amberlite XAD-2 or by extraction with tetrahydrofuran followed by chromatography on a column of DEAE-Sephadex A-25 in a gradient of NaCl. The biggest Sephadex chromatographic fraction (50,4% of total) contained about 42% of its radioactivity as estradiol-17alpha-3-sulfate and 18% as estradiol-17beta-3-sulfate and the remaining 40% was identified tentatively as estradiol-17alpha-17-sulfate plus a small proportion of estradiol-17beta-17-sulfate. The second biggest Sephadex chromatographic fraction (12.7% of total) was a mixture of conjugates not further identified. Minor fractions identified comprised estrone-beta-glucuronide (2.8%), estradiol-17alpha-3-beta-glucuronide (2.8%), estradiol-17beta-3-beta-glucuronide (2.3%) and estrone sulfate (6.0%). Evidence was obtained for the presence of small proportions of estradiol-17alpha disulfate and estradiol-17beta disulfate.     

Lumi-mestranol and epi-lumi-mestranol.

Treatment of lumi-estrone 3-methyl ether (I) with acetylene gave the C-17-epimeric compounds lumi-mestranol (3-methoxy-17 alpha-ethynyl-13 alpha-estra-1,3,5(10)-trien-17 beta-ol, III ) and epi-lumi-mestranol (3-methoxy-17 beta-ethynyl-13 alpha-estra-1,3,5(10)-trien-17 alpha-ol, IV). The structures of the two isomers were assigned on the basis of their molecular rotations and shift-reagent experiments in the NMR. The irradiation of estrone 3-methyl ether (II) to provide compound I was investigated in two solvent systems. Minor products of these reactions were the seco-steroids VII, VIII and X.     

Metabolism of estrogens by rabbit blastocysts: formation of estrogen glucosides and preferential conversion of estrone to estradiol-17 beta

When day 6 rabbit blastocysts were cultured (3 embryos/mL) in medium 199 containing 3.68 microM estradiol-17 beta (E2), 40% of E2 was metabolized in 24 h, at a rate of 18 pmol/embryo(b)/h, yielding 4 major metabolite fractions. Two of them were identified to be estrogen glucosides: 17 beta-hydroxyestra-1,3,5(10)-trien-3-yl beta-D-glucopyranoside (E(2)3G) (12 pmol/b/h) and 17-oxoestra-1,3,5(10)-trien-3-yl beta-D-glucopyranoside (E(1)3G) (0.5 pmol/b/h). If the blastocysts were cultured in 3.68 microM E1 medium, 75% of E1 was metabolized in 24 h (34.1 pmol/b/h); most of it appears as E2 (8 pmol/b/h), E(1)3G (16 pmol/b/h), and E(2)3G (6 pmol/b/h). Thus, the 17 beta-hydroxysteroid dehydrogenase activity in the rabbit blastocysts catalyzes mainly in the direction of the E1----E2 conversion, with little or no E2----E1. This may be responsible in part for the faster metabolism of E1 than E2 by the rabbit blastocyst. In comparison with the rat, mouse, and hamster blastocyst, the rabbit embryo shows an additional capability to conjugate large amounts of estrogens into glucosides by steroid glucosyltransferase.     

Microbiologic oxidation of estratrienes and estratetraenes by Streptomyces roseochromogenes ATCC 13400

Incubation of estrone (1a) with Streptomyces roseochromogenes ATCC 13400 yielded a mixture of 3,16 alpha-dihydroxyestra-1,3,5(10)-trien-17-one (3a) and 3,17 beta-dihydroxyestra-1,3,5(10)-trien-16-one (4a). Transformation of 3-methoxyestra-1,3,5(10)-trien-17-one (1b), 3-hydroxyestra-1,3,5(10),9(11)-tetraen-17-one (2a), and 3-methoxyestra-1,3,5(10),9(11)-tetraen-17-one (2b) with the same microorganism gave the corresponding mixtures of 16 alpha-hydroxy-17-ketones and 17 beta-hydroxy-16-ketones (3b and 4b, 6a and 7a, 6b and 7b, respectively). In addition, in these three last experiments, the 16 beta-17 beta-dihydroxy derivatives 5b, 8a, and 8b, respectively, were also isolated. The complete assignments of the 13C nuclear magnetic resonance spectra of these compounds are given.     

The aromatization of (7 -3H) androstenedione by human placental mitochondria

A metabolite of 2,3-dihydroxyestra-1,3,5(10)-trien-17-one-6, 7-³H isolated from rat bile, was partially characterized by mass spectrometry as a methyl ether of 2,3,16-trihydroxyestra-1,3,5(10)-trien-17-one. The α configuration of the 16-hydroxy function was established by chromatographic comparison of the sodium borohydride reduced metabolite with synthetic 2-methoxy-estra-1, 3,5(10)-triene-3,16α,17β-triol and 2-methoxy-estra-1, 3,5(10)-triene-3,16β,17β-triol. The methyl group was located on the C-2 position by comparison with authentic 2- and 3- monomethyl ethers of 2,3-dihydroxy-estra-1, 3,5(10)-trien-17-one following pyrolytic removal of the 16α-hydroxyl group.3,16α-dihydroxy-2-methoxyestra-1,3,5(10)-trien-17-one was found to constitute 2% and 15% of the biliary radioactivity following administration of estrone-6,7-³H and 2,3-dihydroxyestra-1,3,5(10)-trien-17-one-6,7-³H respectively.     

Identification of radioactive 17beta-estradiol-17-beta-D-glucuronide and 17alpha-estradiol-17-beta-D-glucuronide in the urine of the domestic fowl after intramuscular injection of [4-14C]estrone.

Two previously uncharacterized radioactive estrogen conjugates, 17beta-estradiol-17-beta-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17beta-D-glucopyranosiduronate) and 17alpha-estradiol-17beta-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17alpha-yl-beta-D-glucopyranosiduronate), have been identified in small but significant amounts in avian urine and in a ratio of approximately 2:1 after intramuscular injection of [4-14C]estrone.     

Radioimmunoassay of contraceptive steroids. I. Synthesis of 6-oxomestranol 6-(O-carboxymethyl) oxime

A synthesis of 6-(O-carboxymethyl) oxime of 6-oxo-mestranol (3-methoxy-17-ethinyl-17β -hydroxy-1,3,5 (10)-estratrien-6-one) which was required for coupling with bovine serum albumin in order to produce a specific anti-sera for mestranol (3-methoxy-17-ethinyl-1,3,5 (10)-estratrien-17β-ol) has been described. 6-Oxoestradiol-17β 3-methyl ether was prepared from estradiol-17β 3,17-diacetate by chromic acid oxidation, followed by hydrolysis and methylation. It was converted to its O-carboxymethyloxime derivative which was smoothly oxidized by Jones reagent to the corresponding es estrone derivative. This was easily ethinylated with lithium acetylide-ethylenediamine complex to the desired compound. In an alternate approach to the desired compound, it was found that 6-oxoestradiol-17β 3- methyl ether could not be converted to its ketal under any of a variety of conditions. Ethinylation of 6-oxoestrone 3-methyl ether with limited amount of lithium acetylide reagent probably gave the 17α -ethinyl derivative as was indicated from IR and UV spectra, but its identity could not be further confirmed due to its extremely poor yield.     

Chlorodecarboxylation of 17β-acetoxy-3-methoxy-9-oxo-9, 11-secoestra-1,3,5(10)-trien-11-oic acid with lead tetraacetate and trityl chloride1

In order to synthesise 9,11-seco-C-norestradiol (9,11-seco-C-norestra-1,3,5(10)-triene-3,17β-diol), decarboxylation of 17β-acetoxy-3-methoxy-9-oxo-9,11-secoestra-1,3,5(10)-trien-11-oic acid was investigated. It was found that the desired alkyl chloride could be best prepared by irradiating Pb (IV) salt of the acid with 300W tungsten lamp in the presence of trityl chloride as the source of chlorine radical supplier.     

Synthesis and mechanism of hydrolysis of estrogen 6-sulfates: model compounds for demonstrating the carcinogenesis of estrogen

To investigate the carcinogenesis of estrogen with respect to the chemical behavior of estrogen 6-sulfates, two epimeric 6-sulfates, pyridinium 3-methoxyestra-1,3,5(10)-trien-6 alpha-yl (8) and -6 beta-yl (11) sulfates, were synthesized as the model compounds, and their chemical reactivities were examined. These sulfates were shown to be highly reactive: in water, they were readily and quantitatively converted to a common product mixture, composed of 3-methoxyestra-1,3,5(10)-trien-6 alpha-ol (6) and -6 beta-ol (9) in an almost constant product ratio, with a predominant yield of the latter. The hydrolysis of both sulfates 8 and 11 proceeded in first-order kinetics with half-lives of 1.1 and 1.5 minutes, respectively. When the sulfates were hydrolyzed in 18O-water, the heavy-oxygen atom was shown to be incorporated quantitatively into the C-6 position of the products. These results demonstrate that estrogen 6-sulfates generate a highly reactive benzylic (C-6) carbocation in an aqueous solution, suggesting that the sulfates can act as carcinogens.     

Binding, X-ray and NMR studies of the three A-ring isomers of natural estradiol

The effect of the position of the phenolic hydroxyl on the conformations of the three A-ring isomers of estradiol, namely, estra-1,3,5(10)-trien-1,17 beta-diol (10), estra-1,3,5(10)-trien-2,17 beta-diol (3), and estra-1,3,5(10)-trien-4,17 beta-diol (6), has been analyzed by X-ray crystallography. The results of these analyses were correlated with the absorptions of the angular methyl groups in the [1H]NMR spectra of these isomers and natural estradiol (E2). It was observed that the changes in chemical shift of protons at C18 corresponded to skeletal modifications in the steroid structure which changed the anisotropic effect of the hydroxyl group at C17. Examination of the affinity of these A-ring isomers of E2 for the estrogen receptor has shown the 2-hydroxylated isomer 3 to retain 1/5th the affinity of E2 for its binding protein. The 1- and 4-hydroxylated derivatives (10 and 6, respectively) bound to a much lesser extent. The receptor affinities of these estrogen analogues may be related to the angle between the 18-methyl and the 17 beta-hydroxyl groups (or the dihedral angle between the planar A-ring and the angular C18 methyl) as well as the position of the A-ring hydroxyl group.     

Stereoselective halogenation of the 16-hydroxymethyl-3-methoxy-13alpha-estra-1,3,5(10)-trien-17-ols and their solvolytic investigation

The primary hydroxy functions of 16alpha-hydroxymethyl-3-methoxy-13alpha-estra-1,3,5(10)-trien-17beta-ol (3a) and 16beta-hydroxymethyl-3-methoxy-13alpha-estra-1,3,5(10)-trien-17alpha-ol (4a) were stereoselectively transformed into good leaving groups. On alkaline methanolysis of the 16-halomethyl or 16-tolylsulfonyloxymethyl derivatives, a new D-seco-13alpha-estrone derivative was obtained in high yield.     

Antialgal ent-labdane diterpenes from Ruppia maritima

Seven ent-labdane diterpenes have been isolated from Ruppia maritima. The structures 15,16-epoxy-ent-labda-8(17),13(16),14-trien-19-al; 15,16-epoxy-ent-labda-8(17),13(16),14-trien-19-ol acetate; methyl 15,16-epoxy-12-oxo-ent-labda-8(17),13(16),14-trien-19-oate; 15,16-epoxy-ent-labd-8(17),13E-dien-15-ol and 13-oxo-15,16-bis-nor-ent-labd-8(17)-ene have been assigned to the five new compounds by spectroscopic means and chemical correlations. The phytotoxicity of the diterpenes has been assessed using the alga Selenastrum capricornutum as organism test.     

The transfer of estrone glucuronide to bile in an isolated perfused rat liver system.

An isolated rat liver perfusion system has been utilized in a study of the biliary excretion of estrone glucuronide. The estrogen was initially shown to be excreted without prior metabolism. Disappearance from the medium was rapid and biliary concentrations exceeded that in the medium by more than a thousand-fold. Disappearance rates were decreased when medium estrone glucuronide concentrations exceeded 0.29 mM. Inhibition by other steroidal conjugates, testosterone glucuronide, 2-methoxyestrone (3-hydroxy-2-methoxy-estra-1,3,5(10)-trien-17-one glucuronide and 2-hydroxyestrone (2,3-dihydroxyestra-1,3,5(10)-trien-17-one) glutathione, was also demonstrated. Phenolphthalein glucuronide, at 10 times the molar concentration of estrone glucuronide, did not affect the medium clearance of the latter compound. These findings indicate the possibility of utilizing this system for further studies of possible interactions by other organic compounds for excretion via the biliary route.     

Detection of a transient enzyme-steroid complex during active-site-directed irreversible inhibition of 3-oxo-delta 5-steroid isomerase

The reaction of the active-site-directed irreversible inhibitor (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol (5 beta) with 3-oxo-delta 5-steroid isomerase has been monitored by repetitive scanning ultraviolet spectroscopy of a solution of 5 beta plus isomerase against a blank containing only 5 beta. Upon initial mixing of 5 beta with the isomerase an absorbance maximum at ca. 250 nm appears. With time, this peak decreases and is replaced with a new peak near 280 nm. These results directly demonstrate the existence of a transient enzyme-steroid intermediate in the inactivation reaction. The ultraviolet spectrum suggests that the steroid in the transient complex resembles the ionized phenol, while the phenolic group in the irreversibly bound complex is un-ionized. These spectral studies support our previous proposal that there are two enzyme-steroid complexes that are related by a 180 degree rotation about an axis perpendicular to the plane of the steroid nucleus. This hypothesis offers an explanation for the reaction of 17 beta-oxiranes with the same residue (Asp-38) that is thought to be involved in the catalytic mechanism. Two new oxiranes, (17S)-spiro[estra-1,3,5(10)-triene-17,2'-oxiran]-3 beta-ol (6 beta) and (17S)-spiro[5 alpha-androstane-17,2'-oxiran]-3-one (8 beta), were also found to be potent active-site-directed irreversible inhibitors of the isomerase (k3/KI = 31 M-1 s-1 and 340 M-1 s-1, respectively). The relationship of these results to the nature of the active site of the isomerase is discussed.     

Efficient syntheses of 17-β-amino steroids

17β-Amino steroids such as 17β-amino-1,3,5(10)-estratrien-3-ol (1), 17β-amino-5α-androstan-3β-ol (2) and, 17β-amino-3β-hydroxyandrost-5-ene (3) have been widely used as a key intermediates in the synthesis of a variety of biologically active steroid derivatives though concise, high yielding syntheses of these compounds has yet to be reported. 17β-Amino-1,3,5(10)-estratrien-3-ol (1) and 17β-amino-5α-androstan-3β-ol (2) were prepared in high yield by reductive amination of estrone and epiandrosterone using benzylamine and sodium triacetoxyborohydride followed by catalytic hydrogenolysis of the resulting 17β-benzylamino derivatives. Attempts to prepare 17β-amino-3β-hydroxyandrost-5-ene (3) from dehydroepiandosterone using a similar approach resulted in partial reduction of the double bond. 17β-Amino-3β-hydroxyandrost-5-ene (3) was ultimately obtained in high yield by reductive amination of dehydroepiandosterone using allylamine and sodium triacetoxyborohydride followed by removal of the allyl group from the resulting 17β-allylamino derivative with dimethylbarbituric acid and Pd(PPh(3))(4) as catalyst.