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.     

Novel 7α-alkoxy-17α-(4'-halophenylethynyl)estradiols as potential SPECT/PET imaging agents for estrogen receptor expressing tumours: Synthesis and binding affinity evaluation

In order to develop potential radiolabelled probes for imaging estrogen receptor (ER) positive tumours, we have synthesized and characterized a series of novel 7α-alkoxy-17α-(4'-iodophenylethynyl)estra-1,3,5(10)-triene-3,17β-diols and 7α-alkoxy-17α-(4'-fluorophenylethynyl)estra-1,3,5(10)-triene-3,17β-diols. The fluoro-substituted compounds showed a higher ER binding affinity than the corresponding iodo-derivatives, where 7α-methoxy- and 17α-(4'-fluorophenylethynyl)estra-1,3,5(10)-triene-3,17β-diol showed the highest ER binding affinities (RBA=80.9% and 78.9%, respectively), among the halophenylethynyl compounds studied and should be further explored as potential PET biomarkers for imaging of ER expressing tumours.     

Precursor role of 15alpha-hydroxyestradiol and 15alpha-hydroxyandrostenedione in the formation of estetrol

Studies were designed to elucidate the origin of estetrol (15alpha-hydroxyestriol (estra-1,3,5(10)triene-3,15alpha,17beta-tetrol) or E4) during late human pregnancy. 3H-Labelled 15alpha-hydroxyestradiol (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E2) and 14C-labelled 17beta-estradiol (estra-1,3,5(10)-triene-3,17beta-diol or E2) were infused into the fetus during transfusion in utero for erythroblastosis fetalis, and in another study the same substrates were injected intravenously into the maternal circulation. In a third study, 3H-labelled 15alpha-hydroxyandrostenedion (15alpha-hydroxyandrost-4-ene-3,17-dione or 15delta4) and 14C-labelled E2 were infused into the fetus. Maternal urine was collected for 5--6 days, and after Glusulase hydrolysis, the following metabolites were isolated: estriol (estra-1,3,5(10)-triene-3,16alpha,17beta-triol or E3) containing 14C only and 15alpha-hydroxyestrone (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E1), 15E2, and E4, all containing both labels. From the isotope content of these metabolites, it was concluded that E4 was derived from both fetal E2 and 15delta4 and only partially via 15E2. When administered to the fetus E2 and 15delta4 contributed approximately equal amounts to urinary E4. The yield of 15alpha-hydroxylated estrogens from E2 injected into the mother was very low indicating the predominantly fetal origin of the 15alpha-hydroxylase. 15delta4 was a better precursor than E2 for urinary 15E2.     

Synthesis and receptor-binding examination of 16-hydroxymethyl-3,17-estradiol stereoisomers

The four 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers were synthesized and tested in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities are lower than 2.0% relative to that of the reference molecule estra-1,3,5(10)-triene-3,17beta-diol. The affinities of the tested compounds for the androgen and progesterone receptors are very low (K(i)> 100 microm and 1 microM, respectively). The prepared 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers are therefore estrogen receptor-selective molecules.     

Synthesis and receptor-binding examination of 16-hydroxymethyl-3,17-estradiol stereoisomers

The four 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers were synthesized and tested in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities are lower than 2.0% relative to that of the reference molecule estra-1,3,5(10)-triene-3,17beta-diol. The affinities of the tested compounds for the androgen and progesterone receptors are very low (K(i)> 100 microm and 1 microM, respectively). The prepared 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers are therefore estrogen receptor-selective molecules.     

Inactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase by 16-methylene estrone, an affinity alkylator enzymatically generated from 16-methylene estradiol-17 beta

The substrate 16-methylene estra-1,3,5(10)-triene-3,17 beta-diol (16-methylene estradiol-17 beta) and its enzyme-generated alkylating product, 3-hydroxy-16-methylene estra-1,3,5(10)-triene-17-one (16-methylene estrone), were synthesized to study the 17 beta- and 20 alpha-hydroxysteroid dehydrogenase activities which coexist in homogeneous enzyme purified from human placental cytosol. 16-Methylene estradiol, an excellent substrate (Km = 8.0 microM; Vmax = 2.8 mumol/mg/min) when enzymatically oxidized to 16-methylene estrone in the presence of NAD+ (256 microM), inactivates simultaneously the 17 beta- and 20 alpha-activities in a time-dependent and irreversible manner following pseudo-first order kinetics (t1/2 = 1.0 h, 100 microM, pH 9.2). 16-Methylene estradiol does not inactivate the enzyme in the absence of NAD+. 16-Methylene estrone (Km = 2.7 microM; Vmax = 2.9 mumol/mg/min) is an affinity alkylator (biomolecular rate constant k'3 = 63.3 liters/mol-s, pH 9.2; KI = 261 microM; k3 = 8.0 X 10(-4) S-1, pH 7.0) which also simultaneously inhibits both activities in an irreversible time-dependent manner (at 25 microM; t1/2 = 7.2 min, pH 9.2; t1/2 = 2.7 h, pH 7.0). Substrates (estradiol-17 beta, estrone, and progesterone) protect against inhibition of enzyme activity by 16-methylene estrone and 16-methylene estradiol. Affinity radioalkylation studies using 16-methylene [6,7-3H]estrone demonstrate that 1 mol of alkylator binds per mol of inactivated enzyme dimer. Thus, 16-methylene estradiol functions as a unique substrate for the enzymatic generation of a powerful affinity alkylator of 17 beta,20 alpha-hydroxysteroid dehydrogenase and should be a useful pharmacological tool.     

Synthesis of a steroidal A ring aromatic sulfonic acid as an inhibitor of steroid 5 alpha-reductase

The synthesis of 17 beta-(N,N-diisopropylcarbamoyl)estra-1,3,5(10)-triene-3-sulfonic acid (3) has been accomplished. Sulfonate 3 was designed as a novel inhibitor of human steroid 5 alpha-reductase based on considerations of enzyme mechanisms, and exhibits an inhibition constant in the low nanomolar range.     

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.     

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).     

Affinity alkylation of 3-oxo-delta 5-steroid isomerase by steroidal 3 beta-oxiranes: identification of the modified amino acid by reduction with hydroxyborohydride

The steroidal 3 beta-oxirane (3S)-spiro[5 alpha-androstane-3,2'-oxiran]-17 beta-ol (1 beta) is an active site directed irreversible inhibitor of the 3-oxo-delta 5-steroid isomerase from Pseudomonas testosteroni. Two steroid-bound peptides (TPS1 and TPS2) were isolated by high-performance liquid chromatography (HPLC) from the trypsin digest of enzyme inactivated with 1 beta. The modified tryptic peptides (residues 14-45 of the enzyme) were further digested with chymotrypsin, each giving rise to a single steroid-containing product (CPS1 and CPS2, respectively) derived from residues 31 to 45 of the enzyme. The modified chymotryptic peptides were isolated by HPLC, and the peptide-steroid ester linkage was reduced with sodium hydroxyborohydride. Amino acid analysis of the reduced peptides gave ca. 0.5 residue of homoserine and one less residue of aspartic acid than the corresponding unreduced peptides. Sequence analysis of both reduced chymotryptic peptides revealed that homoserine was located at position 8 in the peptide sequence, corresponding to residue 38 of the enzyme. The finding that the steroidal 3 beta-oxirane, like the 17 beta-oxiranes, inactivates the isomerase via esterification of aspartic acid-38 is strong evidence that this enzyme binds steroids in at least two orientations.     

Synthesis and biologic activities of 11 beta-substituted estradiol as potential antiestrogens

The effect of attachment of a dimethylaminoethoxy or a dimethylaminopropoxy group at the 11 beta-position of estradiol (E2) on its relative binding affinity (RBA) to estrogen receptor (ER) and intrinsic biologic activity is described. The binding of 11 beta-[2-(N,N-dimethylamino) ethoxy]estra-1,3,5(10)-triene-3,17 beta-diol (4) and 11 beta-[3-(N,N- dimethylamino)propoxy]estra-1,3,5(10)-triene-3,17 beta-diol (5) to the ER from immature rat uterine tissue was measured relative to that of [3H]E2 by a competitive binding assay. It was found that the 11 beta-substituted E2 analogs have considerably lower RBA to ER than the corresponding parent compound. The intrinsic activity of compounds 4 and 5 were studied in terms of uterotrophic and antiuterotrophic activity. It was found that the uterotrophic activity of these compounds was drastically reduced compared with E2. However, no antiuterotrophic activity was observed in these compounds at dosages ranging from 1 to 100 micrograms/rat/d.     

A comparison of three methods of hydrolysis for estrogen conjugates

The efficiencies for estrogen conjugate hydrolysis were compared between enzyme hydrolysis, acid solvolysis and a new method, ammonolysis. Samples included: 1) crystalline 1,3,5(10)-estratriene-3, 17 beta-diol disulfate (estradiol 3,17-disulfate), 2) squirrel monkey urine collected following an intravenous injection of [2,4,6,7-H] 1,3,5(10)-estratriene-3,17 beta-diol (estradiol) and 3) a pool of human pregnancy urine. Ammonolysis demonstrated a significant increase over the other techniques in "free" estrogen yields, specifically, from estradiol 3,17-disulfate.     

Biliary metabolites of estriol in the rat

Following the subcutaneous administration of estriol-6,7-³H to rats, biliary metabolites were identified and quantitated. Approximately 70% of the metabolites were excreted in the form of “glucosiduronate” conjugates. 3, 17β-Dihydroxy-2-methoxy-1,3,5(10)-estratrien-16-one was the major metabolite in this conjugate fraction. Significant amounts of 3,17β-dihydroxy-1,3,5(10)-estratrien-16-one and 2,3,17β-trihydroxy-1,3,5(10)-estratrien-16-one, as well as smaller quantities of 1,3,5(10)-estratriene-2,3,16α,17β-tetrol and 2-methoxy-1,3,5(10)-estratriene-3,16α, 17β-triol, were also found. In 17α-ethinylestradiol - treated animals, the rate of excretion of radioactivity and the proportion of 16-oxo-17β-ol metabolites found in the “glucosiduronate” fraction were reduced.     

Synthesis of C-2 and C-4 deuterium-labeled estradiol-17 beta

Estradiol-17 beta labeled with deuterium in the positions 2 or 4 can be prepared from 2-chloromercurio-1,3,5(10)-estratriene-3,17 beta-diol 3-methyl ether 17-acetate or 4-chloromercurio-1,3,5(10)-estratriene-3,17 beta-diol, respectively, in refluxing CH3COO(2)H/(2)H2O. The same reaction performed on 4-acetoxymercurio-1,3,5(10)-estratriene-3,17 beta-diol afforded 2,4-dideuterio-estradiol-17 beta in good yields.     

The altered specificity of cortisone reductase with certain retroandrostan-3-one substrates.

The retro steroids 17beta-hydroxy-5beta,9beta,10alpha-androstan-3-one and 5beta,9beta,10alpha-androstane-3,17-dione were good substrates for cortisone reductase in the presence of NADH, and the products corresponded to the respective 3beta-hydroxy compounds, in which the 3beta-hydroxyl group is axial and the absolute configuration is 3S. The analogous natural steroids 17beta-hydroxy-5beta,9alpha,10beta-androstan-3-one and 5beta,9alpha,10beta-androstane-3,17-dione were very poor substrates, and gave the corresponding 3alpha(equatorial,3R)-hydroxy compounds, and, in the latter case, also an appreciable amount of 3beta(axial, 3S)-hydroxy-5beta,9alpha,10beta-androstan-17-one. The natural steroids 17beta-hydroxy-5alpha,9alpha,10beta-androstan-3-one and 5alpha,9alpha,10beta-androstane-3,17-dione were better substrates than the retro steroid 17beta-hydroxy-5alpha,9beta,10alpha-androstan-3-one, but were not such good substrates as the retro steroids 17beta-hydroxy-5beta,9beta,10alpha-androstan-3-one and 5beta,9beta,10alpha-androstane-3,17-dione. Unlike these retro steroid 5beta,9beta,10alpha-androstan-3-ones, the natural steroids 17beta-hydroxy-5alpha,9alpha,10beta-androstan-3-one and 5alpha,9alpha,10beta-androstane-3,17-dione gave the corresponding 3alpha(axial,3R)-hydroxy compounds. The retro steroid 17beta-hydroxy-5alpha,9beta,10alpha-androstan-3-one was not a good substrate, and the product of reaction corresponded to the 3alpha(axial,3R)-hydroxy compound. The nature of substrate recognition by this enzyme is discussed in the light of these structure-activity relationships.     

Mechanism-based inactivation of 17 beta,20 alpha-hydroxysteroid dehydrogenase by an acetylenic secoestradiol

14,15-Secoestra-1,3,5(10)-trien-15-yne-3,17 beta-diol (1) is a mechanism-based inactivator of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase (estradiol dehydrogenase, EC 1.1.1.62). Inactivation with alcohol 1 requires NAD-dependent enzymic oxidation and follows approximately pseudo-first-order kinetics with a limiting t1/2 of 82 min and a "Ki" of 2.0 microM at pH 9.2 and 25 degrees C. At saturating concentrations of NAD, the initial rate of inactivation is slower than in the presence of 5 microM NAD, suggesting that cofactor binding to free enzyme impedes the inactivation process. Glutathione completely protects the enzyme from inactivation at both cofactor concentrations. Inactivation with 45 microM tritiated alcohol 1 followed by dialysis and gel filtration demonstrates a covalent interaction and affords an estimated stoichiometry of 1.4 molecules of steroid per subunit (2.8 per dimer). Chemically prepared 3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one (2) rapidly inactivates estradiol dehydrogenase with biphasic kinetics. From the latter phase, a Ki of 2.8 microM and a limiting t1/2 of 12 min at pH 9.2 were determined. Estradiol, NADH, and NAD all retard this latter inactivation phase. We propose that enzymatically generated ketone 2 inactivates estradiol dehydrogenase after its release from and return to the active site of free enzyme.     

Species differences in biotransformation of 17α-cyanomethylestradiol 3-methyl ether

Following oral administration of 9,11- ³H-17α-cyano-methylestra-1,3,5(10)-triene-3,17-diol 3-methyl ether, urinary metabolites were studied in man, baboon, beagle dog, minipig and rat. The metabolite pattern revealed remarkable species differences, especially in quantitative respects. 17α-Cyanomethylestra-1,3,5(10)-triene-3,17-diol, 17α-cyanomethylestra-1,3,5(10)-triene-2,3,17-triol 2-methyl ether, 17α-hydroxymethylestra-1,3,5(10)-triene-3,17-diol and 17α-cyanomethylestra-1,3,5(10)-triene-3,16$\text{6}\text{5}$,17-triol were isolated as principal metabolites. In rat bile, a metabolite was tentatively identified as aγ-lactone of a 17α-carbozymethyl-16α-hydroxy compound.     

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.     

Syntheses of estetrol monoglucuronides.

Four possible monoglucuronides of estetrol (estra-1,3,5(10)-triene-3,15 alpha, 16 alpha, 17 beta-tetraol) have been prepared from appropriately protected estetrol by the Koenigs-Knorr reaction employing cadmium carbonate as a catalyst. Condensation of methyl acetobromoglucuronate with estetrol 15,16,17-triacetate provided the 3-glucuronide acetate-methyl ester in a satisfactory yield. Introduction of the glucuronyl residue into C-17 was similarly attained by the use of estetrol 3-benzoate 15,16-acetonide. When estetrol 3,17-diacetate and acetobromosugar were stirred in anhydrous toluene in the presence of cadmium salt, the reaction occurred at C-16 and C-15 yielding two isomeric monoglucuronide derivatives in a ratio of ca. 5 to 2. Removal of the protecting groups in the four glucuronide acetate-methyl esters gave the desired estetrol glucuronides, respectively. These synthetic substrates underwent readily enzymatic hydrolysis with beef-liver beta-glucuronidase to afford estetrol.     

Deuterium-labeled steroids for study in humans. II. Preliminary studies on estrogen production rates in pre- and post-menopausal women.

6,7-Dideuterio-3-hydroxy-1,3,5(10)-estratrien-17-one (dideuterio-estrone) and 4-deuterio-1,3,5(10)-estratriene-3,17 beta-diol (monodeuterio-17 beta-estradiol) were used for the estimation of estrogen production rates in pre- and post-menopausal women. The results are concordant with those obtained by radioisotope administration as reported in the literature. This preliminary study suggests that one or more steroids labeled with one or multiple deuterium and/or other stable isotopes may be employed for the measurement of production rates of steroid hormones which are derived from multiple precursors.