J Steroid Biochem Mol Biol
November 2006
To explore a stereochemistry of hydrogen removal at C-1 of the powerful aromatase inhibitor 2-methyleneandrostenedione (1), of which the A-ring conformation is markedly different from that of the natural substrate androstenedione (AD), in the course of the aromatase-catalyzed A-ring aromatization producing 2-methylestrone (2), we synthesized [1alpha-2H]labeled steroid 1 and its [1beta-2H]stereoisomer, and the metabolic fate of the C-1 deuterium in aromatization was analyzed by gas chromatography-mass spectrometry (GC-MS) in each. Parallel experiments with the natural substrates [1alpha-2H] and [1beta-2H]ADs were also carried out. The GC-MS analysis indicated that 2-methyl estrogen 2 produced from [1alpha-2H]labeled substrate 1 retained completely the 1alpha-deuterium (1beta-H elimination), while product 2 obtained from [1beta-2H]isomer 1 lost completely the 1beta-deuterium.
View Article and Find Full Text PDFTo gain insight into the catalytic function of aromatase, we studied 19-oxygenation of 19-methyl-substituted derivative of the natural substrate androstenedione (AD), compound 1, with human placental aromatase by use of gas chromatography-mass spectrometry (GC-MS). Incubation of the 19-methyl derivative 1 with human placental microsomes in the presence of NADPH under an aerobic condition did not yield a detectable amount of [19S]19-hydroxy product 2 or its [19R]-isomer 3 when the product was analyzed as the bis-methoxime-trimethylsilyl (TMS) derivative by GC-MS; moreover, the production of estrogen was not detected as the bis-TMS derivative of estradiol (detection limit: about 3 ng and 10 pg per injection for the 19-ol and estradiol, respectively). The results reveal that the 19-methyl steroid 1 does not serve as a substrate of aromatase, although it does serve as a powerful inhibitor of the enzyme.
View Article and Find Full Text PDFReduction of a double bond at C-1 of 1,4-dien-3-one steroids 7 and 8 with LiAl2H4 in THF or NaB2H4 in MeOH and H2O gave stereospecifically [1alpha-2H]-labeled 4-en-3-one steroids 9 and 10, respectively. When the deuterated solvents, MeO2H and 2H2O, were used for the reaction of steroid 8 with NaB2H4, [1alpha,2xi-2H2]-labeled compound 10 was produced. This indicates that the reaction proceeds through the initial hydride attack at the C-1alpha position, followed by ketonization of the 2-en-3-ol intermediate.
View Article and Find Full Text PDFAromatase catalyzes the conversion of androstenedione (1a, AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the spatial nature of the AD binding (active) site of aromatase in relation to the catalytic function of the enzyme, we tested for the ability of 2alpha-substituted (halogeno, alkyl, hydroxy, and alkoxy) ADs (1b-1i) to inhibit aromatase in human placental microsomes as well as their ability to serve as a substrate for the enzyme. All of the steroids inhibited the enzyme in a competitive manner with the apparent K(i)'s ranging from 45 to 1150 nM.
View Article and Find Full Text PDFTo gain insight into the spatial nature of the androstenedione (AD) binding (active) site of aromatase in relation to the catalytic function of the enzyme, we synthesized 2,2-dimethylAD (4), 2beta- and 2alpha-methylADs (5 and 6), 19-oxygenated derivatives of compounds 4 and 6, and 2-methyleneAD (17), and we then tested their inhibitory activity as well as their aromatase reaction (aromatization for 2-methyl and 2-methylene analogs or 19-oxygenation for 2,2-dimethyl steroids) with human placental aromatase. 2-Methyl and 2-methylene steroids 5, 6, and 17 were good competitive inhibitors of aromatase (K(i)=22-68nM), but less effective compared to the 2,2-dimethyl analog 4 (K(i)=8.8nM), indicating that a combination of 2beta- and 2alpha-methyl moieties is essential for the formation of a thermodynamically stable inhibitor-aromatase complex.
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