Masao Nagaoka

Metabolic aspects of the 1β-proton and the 19-methyl group of androst-4-ene-3,6,17-trione during aromatization by placental microsomes and inactivation of aromatase

Aromatase catalyzes the conversion of androst-4-ene-3,17-dione to estrogen through sequential oxygenations at the 19-methyl group. Androst-4-ene-3,6,17-trione (AT) is a suicide substrate of aromatase, and the mechanism of inactivation of aromatase has been postulated to involve enzymatic oxygenation at the 19-position. [1 beta-3H,4-14C]-, [19-3H3,4-14C]-, and [1 beta-3H,19-14C]ATs, with high specific activities, were synthesized to study metabolic aspects and the inactivation mechanism. Incubation of the labeled AT with human placental microsomes yielded the 19-oxygenated derivatives, 19-hydroxy-AT and 19-oxo-AT, as well as the aromatization products, 6-oxoestrone and 6-oxoestradiol. A stereospecific 1 beta-proton elimination occurred during the aromatization of [1 beta-3H,4-14C]AT, and a marked tritium isotope effect was observed in the first hydroxylation at C-19 of [19-3H3,4-14C]AT. After incubation of the three double-labeled ATs, the solubilized proteins were subjected to SDS-PAGE and the 3H/14C ratio of the aromatase-bound metabolite in a 46-69 kDa fraction was analyzed. A marked decrease of the 3H/14C ratio of the metabolite was observed in the experiment using [19-3H3,4-14C]AT, compared with that of the labeled AT used, but there were no significant changes in the other experiments, indicating that the adduct retains the 1 beta-proton, the 19-carbon, and one of the three 19-methyl protons of AT. Thus, we conclude that further oxygenation of 19-oxo-AT produced by the two initial hydroxylations of AT at C-19 yields not only 6-oxoestrogen (by a mechanism similar to that involved in the aromatization of the natural substrate) but also a reactive electrophile that immediately binds to the active site in an irreversible manner, resulting in inactivation of aromatase.

A short efficient synthesis of 16-oxygenated estratriene 3-sulfates.

A novel synthesis of sodium 17-oxo-16 alpha-hydroxy-1,3,5(10)-estratrien-3-yl sulfate (4), sodium 16 alpha, 16 beta-dihydroxy-1,3,5(10)-estratrien-3-yl sulfate (5) and sodium 16-oxo-17 beta-hydroxy-1,3,5(10)-estratrien-3-yl sulfate (6) is described. 16 alpha-Bromo-3-hydroxy-1,3,5(10)-estratrien-17-one (1) was efficiently synthesized in one step with 70-97% yield by bromination of 3-hydroxy-1,3,5(10)-estratrien-17-one with cupric bromide. 3,16 alpha-Dihydroxy-1,3,5(10)-estratrien-17-one (3) was quantitatively obtained by controlled stereospecific hydrolysis of the bromoketone 1 with sodium hydroxide in aqueous pyridine. The bromoketone 1 was converted to the 16 alpha-hydroxy-17-ketone 3-sulfate 4 by sulfation with chlorosulfonic acid in pyridine and a subsequent controlled hydrolysis in a high yield without formation of the other ketols. Treatment of the sulfate 4 with sodium borohydride have the triol sulfate 5. The sulfate 4 was also rearranged to the 17 beta-hydroxy-16-ketone 6 with sodium hydroxide in water in a quantitative yield.

Structure-activity relationships of 3-deoxy androgens as aromatase inhibitors. Synthesis and biochemical studies of 4-substituted 4-ene and 5-ene steroids.

As part of our investigation into the structure-activity relationship of a novel class of aromatase inhibitors, two series of 3-deoxy androgens, androst-5-en-17-ones with a non-polar alkoxy (5 and 6), alkyl (20-22), or phenylalkyl (23 and 24) group at C-4beta and 4-acyloxyandrost-4-en-17-ones (29-32, and 34) were synthesized and evaluated. The 4beta-alkyl and 4beta-phenylalkyl compounds were obtained through reaction of 4alpha,5alpha-epoxy steroid (8) with RMgBr (R: alkyl and phenylalkyl) followed by dehydration of the 4beta-substituted 5alpha-hydroxy products (15-19) with SOCl(2) as key reactions. Acylation of 4alpha,5alpha-diol (25) with (RCO)(2)O in pyridine and subsequent dehydration with SOCl(2) gave the 4-acyloxy steroids. All of the steroids studied, except for 4-acetoxy-19-ol (34) that was a non-competitive inhibitor of human placental aromatase, blocked aromatase activity in a competitive manner. 4-Benzoyloxy- and 4-acetoxy steroids (31) and (32) were the most powerful inhibitors of aromatase (K(i)=70 and 60nM, respectively). Elongation of an acetoxy group in a series of 4-acyloxy steroids or a methyl group in a series of 4beta-alkyl steroids decreased affinity for aromatase principally in relation to carbon number of the acyl or alkyl function. The present findings are potentially useful for understanding the spatial and electronic nature of the binding site of aromatase as well as for developing effective aromatase inhibitors.

19-oxygenations of 3-deoxy androgens, potent competitive inhibitors of estrogen biosynthesis, with human placental aromatase

Aromatase is a cytochrome P450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione (AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the ability of 3-deoxy derivative of AD, compound 1, and its 5-ene isomer 4, which are potent competitive inhibitors of aromatase, to serve as a substrate, we studied their 19-oxygenation by human placental aromatase and the metabolites isolated were analyzed by gas chromatography-mass spectrometry. Inhibitors 1 and 4 were found to be oxygenated with aromatase to produce the corresponding 19-hydroxy derivatives 2 and 5 and 19-oxo derivatives 3 and 6 as well as the 17beta-reduced 19-hydroxy compounds 7 and 8. Kinetic studies indicated that the 5-ene steroid 4 was surprisingly a good substrate for the aromatase-catalyzing 19-oxygenation with the V(max) value of 45 pmol/min per mg prot which was approx. four times higher than that of the other. The relative K(m) value for steroids 1 and 4 obtained in this study is opposite from the relative K(i) value obtained previously in the inhibition study. The results reveal that there is a difference between a binding suitable for serving as an inhibitor of aromatase and a binding suitable for serving as a substrate of the enzyme in the 3-deoxy steroid series and the C-3 carbonyl group of AD is essential for a proper binding as a substrate to the active site of aromatase.

Novel and efficient synthesis of estriol and its 16-glucuronide via 2,4,16α-tribromoestrone

A novel synthesis of estra-1,3,5(10)-triene-3,16α, 17β-triol (5), sodium 3,17β-dihydroxyestra-1,3,5(10)-trien-16α-yl-β-D-glucopyranosuronate (12b) and sodium 3-hydroxyestra-1,3,5(10)-trien-17β-yl-β-D-glucopyranosuronate (10b) is described. 2,4,16α-Tribromo-3-hydroxyestra-1,3,5(10)-trien-17-one (2a) was efficiently synthesized in one step with quantitative yield by bromination of 3-hydroxyestra-1,3,5(10)-trien-17-one (1) with cupric bromide. Treatment of (2a) with NaOH in aqueous pyridine under the controlled conditions gave the 16α-hydroxy-17-ketone (4a) without ketol rearrangement. The ketol (4a) was converted in quantitative yield into the triol (5)via a sodium borohydride reduction in the presence of palladium chloride. Reaction of (4a) with methyl 1-bromo-1-deoxy-2,3,4-tri-O-acetyl-α-D-glucopyranosuronate using silver carbonate as a catalyst yielded the 16-monoglucuronide acetate methyl ester (11). The reductive removal of the bromines of (11) with sodium borohydride followed by NaOH hydrolysis gave the glucuronide (12b). A direct glucuronidation of 2,4-dibromoestra-1,3,5(10)-triene-3,17β-diol (8) and a subsequent hydrolysis of the 17-glucuronide (9) gave the glucuronide (10b).

Determination of estradiol 2- and 16α-hydroxylase activities in rat liver microsomes using high-performance liquid chromatography

We have developed a sensitive and nonradiometric assay of estradiol 2- and 16 alpha-hydroxylase activities using reverse-phase high-performance liquid chromatography with voltametric detector. The 2- and 16 alpha-hydroxylated estrogens produced by the incubation of estradiol with rat liver microsomes were initially separated into the catechol and phenolic fractions using a QAE-Sephadex A-25 borate column. The metabolites were detected in quantities as low as 0.5-1 ng using 3-methoxy-1,3,5(10)-estratriene-2,16 alpha,17 beta-triol or 4-hydroxyestrone 17-oxime as an internal standard. Apparent Km and Vmax of the 2- and 16 alpha-hydroxylases were 41.9 microM and 1.3 nmol/mg protein/min, and 82 microM and 480 pmol/mg protein/min, respectively.

Studies directed towards a mechanistic evaluation of inactivation of aromatase by the suicide substrates androsta-1,4-diene-3,17-diones and its 6-ene derivatives aromatase inactivation by the 19-substituted derivatives and their enzymic aromatization.

To gain insight into the mechanistic features for aromatase inactivation by the typical suicide substrates, androsta-1,4-diene-3,17-dione (ADD, 1) and its 6-ene derivative 2, we synthesized 19-substituted (methyl and halogeno) ADD and 1,4,6-triene derivatives 8 and 10 along with 4,6-diene derivatives 9 and tested for their ability to inhibit aromatase in human placental microsomes as well as their ability to serve as a substrate for the enzyme. 19-Methyl-substituted steroids were the most powerful competitive inhibitors of aromatase (K(i): 8.2-40 nM) in each series. Among the 19-substituted inhibitors examined, 19-chloro-ADD and its 6-ene derivatives (7b and 9b) inactivated aromatase in a time-dependent manner in the presence of NADPH in air while the other ones did not. The time-dependent inactivation was blocked by the substrate AD and required NADPH. Only the time-dependent inactivators 7b and 9b in series of 1,4-diene and 1,4,6-triene steroids as well as all of 4,6-diene steroids 9, except for the methyl compound 9a, served as a substrate for aromatase to yield estradiol and/or its 6-ene estradiol with lower conversion rates compared to the corresponding parent steroids 1,4-diene, 1,4,6-triene and 4,6-diene derivatives. The present findings strongly suggest that the aromatase reaction, 19-oxygenation, at least in part, would be involved in the time-dependent inactivation of aromatase by the suicide substrates 1 and 2, where the 19-substitutent would play a critical role in the aromatase reaction probably though steric and electronic reasons.

Stereoselective hydrolysis of 16α-halo-17-keto steroids and long-range substitution effects on the hydrolysis of 16α-bromo-17-ketones and 2α-bromo-3-ketones

Abstract Epimerizations of 16α-chloro- ( 1a ), bromo- ( 1b ), and iodo-3β-hydroxy-5-androsten-17-one ( 1c ) by a brief treatment with 0.2 equiv NaOH in aqueous pyridine reached equilibrium between 16α- and 16β-halo ketones. 16α-/16β-Halo ketone ratios at equilibrium were 1.5 for Cl, 1.25 for Br, and 1.0 for I. Kinetic analysis showed that compounds 1a-c were stereoselectively converted to the corresponding 16α-hydroxy derivative 3 by an SN2 mechanism, in which the order of the apparent reactivity was Br > I > Cl. The hydrolysis of a number of 16α-bromo-17-ketones and 2α-bromo-3-ketones was carried out. The yields of the corresponding alcohols were found to depend on remote structural features in the steroids.

Controlled alkaline hydrolysis of steroidal α-bromoketones: New conditions and synthesis of 2α-hydroxy-3-ones

Abstract Controlled alkaline hydrolysis of 16α-bromo-17-keto steroids 1 , 5 and 7 with potassium carbonate and tetra-n-butylammonium hydroxide (n-Bu4NOH) and synthesis of 2α-hydroxy-3-ones 11 , 13 and 16 by the controlled hydrolysis of the corresponding 2α-bromo-3-ones 9 , 12 and 15 are described. Treatm carbonate in aqueous acetone or with n-Bu4NOH in aqueous dimethylformamide (DMF) gave 16α-hydroxy-17-ones 3 , 6 and 8 in 85–90% yield, respectively. 2α-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 2α-bromoketone 9 and the 2β-bromo isomer 10 precedes the formation of the ketol 11 in which the true intermediate might be the 2β-isomer 10 . However, rearranged androstane derivatives, 3β-hydroxy-2-ones 18 and 20 , were stereoselectively obtained by treatment of the bromoketones 12 and 15 with an excess amount of sodium hydroxide.

Efficient synthesis of estriol 16-glucuronide via 2,4,16α-tribromoestrone

A novel synthesis of estriol 16-glucuronide, a major estrogen involved in human pregnancy, has been accomplished using the controlled stereospecific alkaline hydrolysis of 2,4,16α-tribromoestrone and the selective glucuronidation of its hydrolysed product as key reactions.