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To gain insight into the mechanism for irreversible inactivation of aromatase by 6beta-bromoandrostenedione (1), one of the earliest discovered suicide substrates, in relation to the catalytic function of the enzyme, the 2,2-dimethyl derivative of compound 1, steroid 4, and its 6alpha-isomer 5, as well as 2-methyl-1,4-diene steroid 8 and its 6alpha-bromide 10, were synthesized. All of the steroids inhibited aromatase activity in human placental microsomes with apparent K(i)'s ranging between 10 and 81 nM. The 2,2-dimethyl-6beta- and 6alpha-bromo steroids 4 and 5 were extremely powerful inhibitors (K(i): 14 and 10 nM, respectively), but these two did not cause a time-dependent inactivation of aromatase in the presence of NADPH; in contrast, the 2-methyl-1,4-diene steroids 8 and 10 caused time-dependent inactivation with apparent k(inact) of 0.035 and 0.071 min(-1), respectively, in a suicide manner. These results indicate that the 2,2-dimethyl function of the 6beta-bromide 4 would prevent the inactivation of aromatase caused by inhibitor 1 in a suicide manner, probably through steric activity, whereas the 2-methyl group of steroid 8 did not significantly affect the suicidal inactivation by the parent 1,4-diene steroid, a typical suicide substrate.

To gain insight into the nature of the substrate binding site and the catalytic function of aromatase, we studied the inhibition of androstenedione aromatization by 4beta,5beta-epoxy-16alpha-hydroxyandrostenedione (4) and its 19-hydroxy and 19-oxo derivatives, 5 and 6, as well as the biochemical aromatization of these steroids in human placental microsomes. The 19-methyl and 19-oxo compounds, 4 and 6, were weak competitive inhibitors of aromatase, with apparent K(i) values of 246 microM and 270 microM, respectively, whereas the 19-hydroxy compound 5 inhibited aromatase in a non-competitive manner with the K(i) of 135 microM. The 19-methyl compound 4 inactivated aromatase in a time-dependent manner with k(inact) of 0.213 min(-1) in the presence of NADPH in air, but the other two did not cause it. The conversion of the three epoxides into estrogen, as well as 19-oxygenation of 19-methyl steroid 4 with human placental microsomes in the presence of NADPH in air, were not detected by gas chromatography-mass spectrometry. The present results are consistent with the two binding sites theory in the active site of aromatase.

To gain insight into the binding aspects of 6alpha- and 6beta-methylandrostenediones (1 and 2), potent competitive inhibitors and effective substrates of aromatase, at the active site of the enzyme, we synthesized their 19-hydroxy and 19-oxo derivatives to determine their inhibition of aromatase activity as well as their aromatization rates in human placental microsomes. The 6beta- and 6alpha-methyl-19-ols 12 and 13 were produced from 19-(tert-butyldimethylsiloxy)androstenedione (6) in 6 steps in which the Grignard reaction of 5alpha,6alpha-epoxy steroid 8 with CH3MgBr was employed as a key reaction. Oxidation of the 19-ols 12 and 13 yielded the corresponding 19-als 14 and 15. The 6alpha-methyl steroids 13 and 15 were good competitive inhibitors of aromatase (Ki< or =100 nM), and their aromatization rates obtained by gas chromatography-mass spectrometric analysis were 110 and 205 pmol/min/mg protein, respectively. In contrast, the 6beta-methyl isomers 12 and 14 were non-competitive inhibitors, with Ki values of more than 500 nM, and they were aromatized at rates of 16 and 20 pmol/min/mg protein, respectively. These results reveal that there is a marked difference in binding to the active site between the 19-oxygenated 6alpha-methyl and 6beta-methyl inhibitors where the binding manner of the 6alpha-steroids, rather than the 6beta-isomers, is suitable as a substrate for aromatase.

To gain insight into the catalytic function of aromatase and its substrate specificity, we studied reversible inhibition of 16alpha-hydroxyandrostenedione (16alpha-OHAD) aromatization in human placental microsomes by several suicide substrates of androstenedione (AD) aromatization, including 4-hydroxyAD (1), 6-oxoAD (2) and its 19-hydroxy analogue 3, androst-5-ene-4,7,17-trione (4), and 10beta-acetoxyandrost-5-en-7,17-dione (5) that, in contrast, do not cause a suicide inactivation of 16alpha-OHAD aromatization. All inhibitors examined blocked 16alpha-OHAD aromatization in a competitive manner with apparent K(i) values ranging from 0.50 to 980 nM. The relative K(i) values between inhibitors 1-5 obtained in the 16alpha-OHAD aromatization experiments were markedly different from those obtained in the AD aromatization experiments. The results predict that all inhibitors examined bind to the 16alpha-OHAD binding site in a manner that does not cause suicide inactivation of 16alpha-OHAD aromatization. These findings would be useful for understanding the active (binding) site structure as well as the catalytic function of aromatase.

Aromatization of 16alpha-hydroxyandrostenedione (16alpha-OH AD) with aromatase in human placental microsomes was studied by gas chromatography-mass spectrometry (GC-MS) using 12,4,6,6,9alpha,16beta,17alpha-2H7]estriol as an internal standard. 16alpha-OH AD was incubated with the microsomes in the presence of NADPH in air. The metabolite was extracted with ethyl acetate and treated with NaBH4. The reduced product, estriol, was isolated by Sep-Pak C18 cartridge and then analyzed as the tris(trimethylsilyl)ether by a GC-MS (EI mode). The production of estriol was dependent upon protein concentration and incubation time. Apparent Km and Vmax values of the microsomal aromatase for 16alpha-OH AD were 568 nM and 25.5 pmol/min/mg protein, respectively. In this assay, aromatase activity, estriol formation, could be determined at a level as low as 1 pmol/min/mg protein. Aromatase inhibitors, 4-hydroxy- and 6-oxo-androstenediones, prevented the estriol formation in a competitive manner with 25 and 30 nM of apparent Ki values, respectively.

To gain insight into the binding manner of androst-4-ene-6,17-dione (4) and its 5-en-4-one analog 9, good competitive inhibitors of aromatase, in the active site of the enzyme, their 19-oxygenated derivatives were evaluated as inhibitors of human placental aromatase. The 19-hydroxy steroids 6, 7, and 10 and the 19-oxo analogs 8 and 12 were much poorer competitive inhibitors than their corresponding parent 19-methyl steroids. Conversion of the 17-keto inhibitors 4, 6, and 10 to the 17beta-ols 5, 7, and 11, respectively, markedly decreased their affinity to the enzyme. The results suggest that steroids 4 and 9 would bind to the active site in a similar manner to that involved in the binding of the substrate androstenedione (1).

Conformational analysis of potent competitive inhibitors of aromatase, androst-4-enes 5, as well as 2, 4-diene steroids 3, 4, and 6 was carried out, using theoretical calculations, to determine the stereochemistry of their aromatase-catalyzed oxygenation. In the steroids examined, both the 19-alcohols and the 19-aldehydes favor the above-A ring conformation among the possible three in each. The results suggest that the 3-deoxy steroid 5a as well as the 2, 4-diene steroids 4a and 6a would be oxygenated at C-19 by aromatase through the same stereomechanism as that involved in the androstenedione aromatization.