7-substituted 1,4,6-androstatriene-3,17-diones as enzyme-activated irreversible inhibitors of aromatase

7-Phenyl-1,4,6-androstatriene-3,17-dione (4), 7-benzyl-1,4,6-androstatriene-3,17-dione (5) and 7-phenethyl-1,4,6-androstatriene-3,17-dione (6) were synthesized and evaluated in vitro in human placental microsomes as enzyme-activated irreversible inhibitors of aromatase. The compounds were synthesized from appropriate 7-substituted 4,6-androstadiene-3,17-diones by reaction with DDQ under neutral conditions. All the compounds produced a first order inactivation of aromatase in the presence of NADPH but not in the absence of NADPH. Substrate 4-androstene-3,17-dione protected the enzyme from inactivation by the inhibitors. Furthermore, cysteine failed to protect aromatase from inactivation by compounds 5 and 6. In contrast, cysteine partially protected aromatase from inactivation by compound 4. Irreversibility studies illustrated the covalent nature of the inactivation by 4, 5 and 6. The above experimental evidence demonstrated that compounds 5 and 6 are effective enzyme-activated irreversible inhibitors of aromatase.     

The constitutive 7-ethoxycoumarin O-deethylase activity of human placental microsomes: relationship to aromatase

The constitutive 7-ethoxycoumarin deethylase activity of human placental microsomes from non-smokers was acutely inhibited by a number of androgens which serve as substrates for and/or competitive inhibitors of estrogen synthesis by the aromatase activity of these preparations. 10 beta-(2-Propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione, androgen derivatives which produce a mechanism-based, time-dependent inactivation of placental aromatase caused a cofactor-dependent decay in deethylase activity which paralleled the loss of aromatase activity caused by these agents and which was antagonized by aromatase substrates. Conversely, 7-ethoxycoumarin antagonized the time-dependent action of 10 beta-(2-propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione on aromatase and inhibited competitively the aromatization of 4-androstene-3,17-dione. The Ki for 7-ethoxycoumarin was equivalent to its Km as substrate for deethylation. It is concluded that a common oxidase species is responsible for both the aromatase and constitutive 7-ethoxycoumarin deethylase activities of human placental microsomes.     

Inactivation of aromatase in vitro by 4-hydroxy-4-androstene-3,17-dione and 4-acetoxy-4-androstene-3,17-dione and sustained effects in vivo

4-Hydroxy-4-androstene-3,17-dione (4-OHA) and 4-acetoxy-4-androstene-3,17-dione (4-AcA), in addition to being competitive inhibitors of aromatase, cause time-dependent, irreversible, loss of enzyme activity in both human placental and rat ovarian microsomes. $\text{In vivo}$, treatment of rats with 4-OHA also causes loss of ovarian aromatase activity. To test whether this loss of activity could have $\text{in vivo}$ significance, rats with hormone-dependent, mammary tumors were treated with 4-OHA on alternate weeks. Tumor regression continued to occur during the weeks without treatment. These findings suggest that inactivation of aromatase is important in the mechanism of action of the compounds $\text{in vivo}$.     

Synthesis and some reactions of 6-bromoandrogens: potential affinity ligand and inactivator of estrogen synthetase.

The synthesis of epimeric 6-bromo-4-androstene-3,17-dione (1a and 1b), 6-bromotestosterone (2a and 2b) and its acetate (3a and 3b), and 6-bromo-16 alpha-acetoxy-4-androstene-3,17-dione (5a and 5b), and 6 beta-bromo-16 alpha-hydroxy-4-androstene-3,17-dione (4) is described. The interconversions among compounds 1, 2, and 3 are also studied. The 6 beta-isomer (1b, 2b, and 3b) was epimerized to the 6 alpha-isomer (1a, 2a and 3a) in carbon tetrachloride or chloroform-methanol (9:1) and the 6 alpha-isomer was isolated by fractional crystallization from the epimeric mixture. 6 alpha-Bromo isomer 1a was also epimerized back to 6 beta-bromo isomer 1b in chloroform-methanol (9:1). Two polymorphic forms of 6 beta-bromotestosterone acetate (3b) were isolated (mp. 114--117 degrees and 138--141 degrees). The 6 beta-bromo isomers were found to be unstable in methanol and decomposed to give 5 alpha-androstane-3,6-dione derivative (6). The results of irreversible inactivation of human placental androgen aromatase with some of these 6-bromoandrogens are discussed.     

Rainbow trout, Oncorhynchus mykiss, as a model for aromatase inhibition.

The feasibility of utilizing rainbow trout, Oncorhynchus mykiss, as an alternative model for studying the inhibition of aromatase (CYP 19) was investigated. The suppression of estrogen-dependent tumors by aromatase inhibitors has been important in the treatment of breast cancer. Estrogens, estrogen precursors and xenoestrogens have been found to promote liver cancer in the trout model. A steroid, 4-hydroxy-4-androstene-3,17-dione (4-OHA), and non-steroids, aminoglutethimide (AG) and Letrozole (CGS 20267), all of which are known aromatase inhibitors in rats and humans, were examined in vitro for activity in trout ovarian microsomes. Aromatase activity was quantified as the release of 3H2O from the conversion of [3H]-4-androstene-3,17-dione to 17beta-estradiol and estrone. Trout ovarian microsomes exhibited activity between 39-60 fmol mg(-1) min(-1) with a calculated Vmax of 71.1 fmol mg(-1) min(-1) when incubated at 25 degrees C with 200 nM 4-androstene-3,17-dione (K(M) = 435 nM). Significant inhibition by 4-OHA up to 80% was seen at 1.5 microM. At 2000 microM, AG decreased aromatase activity by up to 82%. Letrozole reduced aromatase activity a maximum of 90% in a dose-dependent manner, but the Ki (2.3 microM) was 1000-fold higher than reported in human trials. Indole-3-carbinol and some of its derivatives, two DDE isomers and four flavones (except alpha-naphthoflavone) at 1000 microM did not significantly inhibit aromatase in vitro. Letrozole and clotrimazole, fed to juvenile rainbow trout at doses up to 1000 ppm for 2 weeks, were not effective in suppressing dehydroepiandrosterone (DHEA) induced increases in vitellogenin and 17beta-estradiol levels. These results document that trout aromatase is sensitive to inhibition in vitro by known inhibitors of the mammalian enzyme. The mechanism(s) for lack of inhibition in vivo is currently unknown and must be further investigated in order to develop a trout model for studying the role of aromatase in carcinogenesis.     

Covalent modification of aromatase by a radiolabeled irreversible inhibitor

7 alpha-Substituted 4-androstene-3,17-diones are effective inhibitors of aromatase. The microsomal enzyme complex has a greater affinity for several of these inhibitors than for the substrate androstenedione, with 7 alpha-(4'amino)phenylthio-4-androstene-3,17-dione being the most potent competitive inhibitor of the series. A potential affinity analog, the bromoacetamide derivative of the amino compound, has been synthesized in both unlabeled and 14C-labeled forms via a condensation of bromoacetic acid with the amino compound using DCC. Inactivation studies with the unlabeled inhibitor showed a time-dependent, first-order inactivation of aromatase enzymatic activity. Androstenedione, when incubated in varying concentrations with the irreversible inhibitor, provided protection from inactivation. Binding studies with radiolabeled inhibitor and microsomal aromatase preparations showed that irreversible binding had occurred. SDS-electrophoresis, followed by fluorography, identified four major microsomal proteins that were radiolabeled, with the protein band at 52,000 mol. wt predominating. Similar studies with a solubilized aromatase preparation decreased the amount of nonspecific binding. Thus, covalent bonds between the irreversible inhibitor and the aromatase cytochrome P450 molecule were formed.     

Effects of aromatase inhibitors on in vitro steroidogenesis by Atlantic salmon (Salmo salar) gonadal and brain tissue

In order to assess the efficacy of selected aromatase inhibitors on Atlantic salmon (Salmo salar) ovarian and brain tissue, in vitro systems were developed for measuring 17beta-estradiol (E(2)) production by these tissues. Isolated vitellogenic follicles, or homogenised whole brains were incubated at 10 degrees C in complete Cortlands solution for 18 or 42 h respectively, and E(2) levels in the medium were determined by RIA. The addition of testosterone to the medium increased E(2) production in all preparations. E(2) production by whole brain homogenate was reduced by co-incubation with the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD), 4-androstene-4-ol-3,17-dione (OHA), aminoglutethimide, fadrozole or miconazole. Fadrozole, ATD, and OHA reduced E(2) production by vitellogenic follicles at a medium concentration of 0.1 microg mL(-1), whereas miconazole was only effective at 10 microg mL(-1). This study demonstrates a simple and rapid screening method for assessing the efficacy of aromatase inhibitors on fish tissues, and that the aromatase inhibitors ATD, OHA and fadrozole are potent inhibitors of both brain and gonadal aromatase in vitro, in Atlantic salmon.     

Development of a sensitive and specific new plasma 4-androstene-3,17- dione time-resolved fluoroimmunoassay (TR-FIA)

We describe, for the first time to our knowledge, the development of a new, non-isotopic time resolved-fluoroimmunoassay of 4-androstene-3,17-dione in plasma or serum. This steroid exhibits a key role in steroid metabolism and is often assayed in the investigation of various pathologic endocrine states. Most of the 4-androstene-3,17-dione immunoassays are performed using a radioactive tracer. We synthesized a biotinylated 4-androstene-3,17-dione tracer from 4-androstene-3,17-dione-3-carboxymethyloxime by acylation of biotinylaminopropylammonium trifluoroacetate. A specific rabbit anti 6-hemisuccinate-4-androstene-3,17-dione/BSA was indirectly bound via an anti-rabbit sheep antibody immobilized on microtiter plate wells. The amount of biotinylated-4-androstene-3,17-dione tracer was then measured by adding streptavidin-europium, and the europium fluorescence was quantified by time resolved-fluorescence (TR-FIA, Delfia System). The plasma 4-androstene-3,17-dione-levels measured with this non-isotopic assay were compared to those measured with a radioimmunoassay previously published. In both cases, the same anti-4-androstene-3,17-dione antibody was used, and the assays were performed after an extraction step and a chromatographic step. The results obtained by the two methods were virtually the same. However, the main advantages of the new plasma 4-androstenedione-3,17-dione time-resolved-fluorescence immunoassay were its greater sensitivity than radioimmunoassay and its higher precision.     

Metabolism of the aromatase inhibitor 4-hydroxyandrostenedione in vivo. Identification of the glucuronide as a major urinary metabolite in patients and biliary metabolite in the rat

4-Hydroxyandrost-4-ene-3,17-dione (HAD) is a potent and selective inhibitor of the enzyme complex aromatase, both in vitro and in vivo. The glucuronide is a major metabolite in the urine of patients and in the bile of rats given HAD and it was identified by chemical ionization-MS of the permethylated derivative. HAD glucuronide was quantified by first converting it enzymically into HAD, then determining HAD by capillary column GC-MS of the perfluorotolyl derivative using 4-hydroxyandrost-2,4-diene-3,17-dione as internal standard.     

An improved radioimmunoassay for 4-androstene-3,17-dione

A radioimmunoassay using an antiserum produced against 6β-hydroxy-4-androstene-3,17-dione-6-succinyl-BSA conjugate is described which permits the rapid determination of 4-androstene-3,17-dione in multiple serum samples that are purified by column chromatography on neutral alumina. Steroids which reacted significantly with the antiserum were found to be 5α-androstane-3,17-dione, 5β-androstane-3,17-dione and 6β-hydroxy-4-androstene-3,17-dione. After column chromatography on alumina, however, the only significantly cross-reacting steroids were the 5α and 5β-androstane-3,17-diones, while cross-reactivity from other steroids was reduced to less than 1%.     

Synthesis and evaluation of novel 4-amino-4,6-androstadiene-3,17-dione: an analog of formestane

Synthesis of 4-amino-4,6-androstadiene-3,17-dione 7, an analog of formestane used in breast cancer therapy as an aromatase inhibitor, from 4-acetoxy-4-androstene-3,17-dione 2 is described. This is the first report of a 4-amino diene (4,6) system in this series of molecules. The new (7) and reported molecules were screened by the National Cancer Institute (NCI, Bethesda, USA) for in vitro antitumor activity against 60 human cancer cell lines. Molecule 7 showed best activity against breast cancer cell line (MCF-7).     

Investigation of rat liver microsomal 6 beta-hydroxylation of 4-androstene-3,17-dione and 4-pregnene-3,20-dione using methodology which excludes steroid-3-imine induced introduction of the 6 beta-hydroxyl group

To avoid artefactual 6 beta-hydroxylation of 3-oxo-4-ene steroids due to steroid-3-imine formation and rearrangement a combined extraction and liquid chromatography purification procedure for incubated rat liver microsomes has been worked out. With this procedure no nonenzymatic 6 beta-hydroxylation could be observed. Conventional termination of incubations with male rat liver microsomes (105,000 g sediments) and 4-14 C-labelled 4-androstene-3,17-dione (or progesterone) by solvent extraction and evaporation might lead to a 30% overestimation of the formation of 6 beta-hydroxy-derivatives at substrate saturation. Furthermore this work-up procedure produces 6-oxo-derivatives which were not observed when the new procedure was used. By elimination of the artefactual 6-oxygenation some properties of the male rat liver microsomal 4-androstene-3,17-dione 6 beta-hydroxylase have been studied, and the activity has been compared to the artefact produced by solvent extraction and evaporation. Using the combined extraction-liquid chromatography purification it was demonstrated that the microsomal 6 beta-hydroxylase active on 4-androstene-3,17-dione and progesterone was inhibited to 95% by carbon monoxide. This makes previous suggestions regarding participation of a non cytochrome P450-dependent 4-androstene-3,17-dione 6 beta-hydroxylase less likely.     

Synthesis of 2,2-dimethyl-4-hydroxy-4-androstene-3,17-dione as an inhibitor of aromatase

2,2-Dimethyl-4-hydroxy-4-androstene-3,17-dione (4) has been synthesized and has been shown to be a powerful competitive inhibitor of aromatase (Ki = 11.4 nM). However, compound 4 does not cause time-dependent loss of enzyme activity, in contrast to the unmethylated parent compound, 4-OHA.     

Comparison of the effect of 4-hydroxy-4-androstene-3,17-dione on aromatase activity in granulosa cells from preovulatory follicles of rats, rabbits, and humans

The effect of the aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione (4-OH-A) on the synthesis of estradiol (1,3,5 (10)-estratriene-3,17 beta-diol) by granulosa cells from preovulatory follicles of rats, rabbits and humans was examined. Granulosa cells from all three species were incubated for 4 h without treatment (control) or in the presence of androstenedione (4-androstene-3,17-dione, 0.5 microM), 4-OH-A (5 microM), or both compounds together. Estradiol levels were determined in the medium and cells by radioimmunoassay. In all three species, estradiol synthesis was markedly increased by androstenedione and this increase was blocked by 4-OH-A. In the rabbit, however, 4-OH-A alone caused a small but significant increase in radioimmunoassayable estradiol. The apparent increase seen with 4-OH-A alone may be due to a metabolite of 4-OH-A that cross-reacts in the estradiol radioimmunoassay. With granulosa cells from humans, in which 4-OH-A is of potential therapeutic importance, no similar effect of 4-OH-A alone was observed.     

Hydroxylation of testosterone at carbons 1, 2, 6, 7, 15 and 16 by the hepatic microsomal fraction from adult female C57BL/6J mice.

The metabolism of a mixture of [4-14C]- and [7 beta-2H]testosterone by the hepatic microsomal fraction from adult femal C57BL/6J mice has been investigated. The following metabolites were identified by their mass spectra and by their retention times on gas chromatography on one or two phases: 1epsilon-, 2beta-, 6alpha-, 6beta-, 7alpha-, 15alpha-, 15beta-, 16alpha- and 16beta-hydroxytestosterone; 6alpha-, 6beta- and 7alpha-hydroxy-4-androstene-3,17-dione; and 4-androstene-3,17-dione. A compound tentatively identified as 6- or 7-oxotestosterone was also isolated. 17beta-Hydroxy-4,6-androstadien-3-one, 17beta-hydroxy-1,4-androstadien-3-one and 4,6-androstadiene-3,17-dione were identified but are considered to arise non-enzymatically from 7alpha-hydroxytestosterone, 1epsilon-hydroxytestosterone and 7alpha-hydroxy-4-androstene-3,17-dione, respectively.     

Steroid metabolism by mouse submaxillary glands. I. in vitro metabolism of testosterone and 4-androstene-3,17-dione

Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of 6 month old male mice. In 15 and 180 minute incubations fortified with NADPH, submaxillary tissue converted 4-androstene-3,17-dione predominantly to androsterone and, to a lesser extent, testosterone, 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α, 17β-diol. Testosterone was converted primarily to 5α-androstane-3α, 17β-diol when exogenous NADPH was available; trace amounts of 4-androstene-3,17-dione, 17β-hydroxy-5α-androstan-3-one and androsterone were also formed. When a NADPH-generating system was omitted from the incubation medium both 4-androstene-3,17-dione and testosterone were poorly metabolized by submaxillary tissue; the amounts of reduced metabolites accumulating were markedly reduced.     

Metabolism of 11-deoxycortisol by a Bacillus species

Microbial transformation by a Bacillus species was employed for the preparation of potentially important derivatives of 11-deoxycortisol. Each microbial metabolite was characterised by the application of various spectroscopic methods. The five metabolites of 11-deoxycortisol were characterised as 4-androstene-3,17-dione (2), 14-hydroxy-4-androstene-3,17-dione (3), 14,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (4), 6 beta,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (5) and 15 alpha,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (6). The availability of the metabolites enabled complete elucidation of their [13C]NMR spectra.     

Steroid metabolism in gonads of turtle embryos as a function of the incubation temperature of eggs

In embryos of many reptiles, the sexual differentiation of gonads is temperature-dependent. In the turtle Emys orbicularis, all individuals become phenotypic males at 25 degrees C, whereas 100% phenotypic females are obtained at 30 degrees C. Steroid metabolism in embryonic gonads was studied at both temperatures, during and after the thermosensitive period for sexual differentiation. Pools of gonads were incubated for various times, with 3 beta-hydroxy-5-pregnen-20-one (pregnenolone), progesterone, dehydroepiandrosterone or 4-androstene-3,17- dione as substrates. The analysis of metabolites combined two successive chromatographies (HPLC and TLC) and autoradiography. Conversion of pregnenolone to progesterone and of dehydroepiandrosterone to 4-androstene-3,17-dione was more important in testes at 25 degrees C than in ovaries at 30 degrees C. In ovaries, a large amount of 5-pregnene- 3 beta,20 beta-diol was formed from pregnenolone, and 5-androstene-3 beta,17 beta-diol was produced from dehydroepiandrosterone. In both testes and ovaries, 5 alpha-pregnane and 5 alpha-androstane derivatives were the main metabolites obtained from progesterone and 4-androstene-3,17-dione, respectively. Progesterone was also converted to 20 beta-hydroxy-4-pregnen-3-one. Dehydroepiandrosterone and 4-androstene-3,17-dione were also metabolized into 11 beta-hydroxy-4-androstene-3,17-dione (only in testes), testosterone, 11 beta,17 beta-dihydroxy-4-androstene-3-one, 17 beta-hydroxy-4-androstene-3,11-dione (low amounts in testes, traces in ovaries), 17 alpha-hydroxy-4-androstene-3-one, estrone and estradiol-17 beta (traces).     

Quantitative determination of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, in human plasma, using isotope dilution mass spectrometry

An original method is described for the determination in human plasma of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, by isotope dilution mass-spectrometry using 7,7-[2H2]-4-OHA as internal standard. This compound was synthesized starting from 7,7-[2H2]-4-androstene-3,17-dione. The procedure includes an extraction step using an Extrelut 1 column and a derivatization with N,o-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The minimum detection level of the method is 0.650 pg and the coefficients of variation for the 0.5 ng/ml (plasma) and 5 ng/ml (plasma) concentrations are 3.2% (within assay) and 6.7% (between assay) and 1.86% (within assay) and 2.3% (between assay) respectively.     

Dissociation of 19-hydroxy- 19-oxo-, and aromatizing-activities in human placental microsomes through the use of suicide substrates to aromatase

Suicide substrates of aromatase were used as chemical probes to determine if free 19-hydroxyandrost-4-ene-3,17-dione (19-OHA) and 19-oxoandrost-4-ene-3,17-dione (19-oxoA) are obligatory intermediates in the aromatization of androst-4-ene-3,17-dione (androstenedione) to oestrone by human placental aromatase. A radiometric-HPLC assay was used to monitor 19-hydroxy, 19-oxo-, and aromatized products formed in incubations of [14C]androstenedione and human placental microsomes. When microsomes were preincubated with the suicide substrates 10 beta-mercapto-estr-4-ene-3,17-dione (10 beta-SHnorA), or 17 beta-hydroxy-10 beta-mercaptoestr-4-ene-3-one (10 beta-SHnorT), it was found that 19-hydroxy-, 19-oxo- and aromatase activities were inhibited in parallel. However, when the suicide substrates 4-hydroxyandrost-4-ene-3,17-dione (4-OHA) and 19-mercaptoandrost-4-ene-3,17-dione (19-SHA) were preincubated with placental microsomes, significantly greater inhibition of formation of oestrogens was observed in comparison to the inhibition of formation of 19-hydroxy- and 19-oxo-metabolites. Furthermore, significantly more time-dependent inhibition of 19-oxoA formation was observed in comparison to inhibition of 19-OHA formation with these same inhibitors. These results suggest that 19-hydroxy- and 19-oxo-androstenediones are not free, obligatory intermediates in the aromatization of androstenedione by human placental aromatase, but rather are products of their own autonomous cytochrome P-450-dependent, microsomal enzymatic activities.