Mii Tachibana

Mechanism for aromatase inactivation by a suicide substrate, androst-4-ene-3,6,17-trione: The 4β,5β-epoxy-19-oxo derivative as a reactive electrophile irreversibly binding to the active site☆

Aromatase is a cytochrome P450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione to estrone through three sequential oxygenations of the 19-methyl group. Androst-4-ene-3,6,17-trione (1) is a suicide substrate of aromatase. The inactivation mechanism for steroid 1 has been studied to show that the inactivation reaction proceeds through the 19-oxo intermediate 3. To further clarify the mechanism, 4 beta, 5 beta-epoxyandrosta-3,6,17,19-tetraone (6) was synthesized as a candidate for a reactive electrophile involved in irreversible binding to the active site of aromatase, upon treatment of compound 3 with hydrogen peroxide in the presence of NaHCO3. The epoxide 6 inhibited human placental aromatase in a competitive manner (Ki = 30 microM); moreover, it inactivated the enzyme in an active-site-directed manner in the absence of NADPH (K1 = 88 microM, kinact = 0.071 min-1). NADPH and BSA both stimulated the inactivation rate without a significant change of the K1 in either case (kinact: 0.133 or 0.091 min-1, in the presence of NADPH or BSA, respectively). The substrate androst-4-ene-3,17-dione protected the inactivation, but a nucleophile, L-cysteine, did not. When both the epoxide 6 and its 19-methyl analog 4 were subjected separately to reaction with N-acetyl-L-cysteine in the presence of NaHCO3, the 19-oxo steroid 6 disappeared from the reaction mixture more rapidly (T1/2 = 40 sec) than the 19-methyl analog 4 (T1/2 = 3.0 min). The results clearly indicate that the 4 beta, 5 beta-epoxy-19-oxo compound 6, which is possibly produced from 19-oxo-4-ene steroid 3 through the 19-hydroxy-19-hydroperoxide intermediate, is a reactive electrophile that irreversibly binds to the active site of aromatase.

4-Oxygenated androst-5-en-17-ones and their 7-oxo derivatives as aromatase inhibitors

A series of androst-5-ene-4,7-diones and 4-oxygenated androst-5-enes were synthesized and tested for their ability to inhibit aromatase in human placental microsomes. All of the steroids examined inhibited the enzyme in a competitive manner. The inhibitory activity of 4beta-hydroxy-5-ene steroid 7 (Ki = 25 nM) was much more powerful than that of the parent 5-ene steroid 11 (Ki = 78 nM), whereas 4beta-acetate 8 and 4-oxo analog 5 (Ki = 90 and 120 nM, respectively) were less potent than compound 11. This indicates that a hydrogen bonding between a hydroxy group of the 4beta-ol 7 and a residue of the active site of aromatase plays an important role in its binding. The 5-en-4-one steroid 5 did not cause a time-dependent inactivation of aromatase. In contrast, 5-ene-4,7-dione 13 as well as its 19-hydroxy and 19-oxo analogs 19 and 20 caused the time-dependent inactivation only in the presence of NADPH in air with the k(inact) values ranging from 0.057 to 0.192 min(-1), although their affinities for the enzyme were not high (Ki = 430-6300 nM). The inactivation was prevented by androstenedione, and no significant effect of L-cysteine on the inactivation was observed in each case. These results suggest that oxygenation at C-19 would be at least in part involved in the inactivation caused by the inhibitor 13.

Studies directed toward a mechanistic evaluation of aromatase inhibition by androst-5-ene-7,17-dione : Time-dependent inactivation by the 19-nor and 5β,6β-epoxy derivatives

To gain further insight into the mechanism for inactivation of aromatase by androst-5-ene-7,17-dione (1) and its 19-nor analog 4, 10 beta-oxygenated steroids 5 and 6, delta 1(10)-steroid 7, and 19-oxo-5 beta,6 beta-epoxy compound 8 were synthesized and tested for their ability to inhibit aromatase in human placental microsomes. All of the steroids studied inhibited the enzyme in a competitive manner with apparent Ki values ranging from 1.1 to 35 microM. The delta 1(10)-compound 7 was the most potent inhibitor among them. All of the inhibitors caused a time-dependent inactivation of aromatase in the presence of NADPH in air with the kinact values ranging from 0.036 to 0.190 min-1. The substrate androstenedione protected the inactivation, but a nucleophile, L-cysteine, did not, in each case. In contrast, each inhibitor did not cause the time-dependent inactivation in the absence of NADPH. These results show that the 5 beta,6 beta-epoxide 8 and/or the dienone 7 are not a reactive electrophile involved in the irreversible binding to the active site of aromatase during the mechanism-based inactivation caused by the suicide substrates 1 and/or 4.

A- or B-ring-substituted derivatives of androst-4-ene-3,6,17-trione as aromatase inhibitors. Structure-activity relationships.

2,2-Dimethylandrost-4-ene-3,6,17-trione (5) and its 4-methoxy- (7) and 4-hydroxy- (8) derivatives were synthesized. 7 alpha-Acetoxy-4-ene-3,6-dione steroid 2 was also prepared by the improved method involving the lead tetraacetate oxidation of androst-4-ene-3,6,17-trione (1). These steroids along with the 2-acetoxy-(11 and 12), 2-substituted 1-ene- (9 and 10), and 4-substituted (13-15) derivatives of compound 1 were evaluated as inhibitors of human placental aromatase. All the steroids, except the 2-acetoxy-1-ene 10 and the 2 beta-acetate 11 of which Ki values were not determined because of their poor inhibitory activities, blocked aromatase in a competitive manner. Compounds 5 and 8 as well as the 4-hydroxy steroid 15 were potent inhibitors (Ki: 25-42 nM) whereas the inhibitory activities of steroids 2, 7, 9, 13, and 14 were good to fair, respectively (Ki: 160-810 nM). Inhibitors 2 and 15 inactivated the enzyme in a time-dependent manner in the presence of NADPH but the 2,3-dimethyl derivatives 5 and 8 did not. Androstenedione blocked the inactivation but L-cysteine did not. The results suggest that the 2 beta-methyl group would prevent the aromatase-catalyzed oxygenation at C-19 of the dimethyl steroids 5 and 8 most likely through the steric reasons.

Aromatization of 16α-hydroxyandrostenedione by human placental microsomes: effect of preincubation with suicide substrates of androstenedione aromatization

Estrogen synthase (aromatase) catalyzes the aromatization of androstenedione (AD) as well as 16alpha-hydroxyandrostenedione (16alpha-OHAD) leading to estrone and estriol, respectively. We found that several steroid analogs including 4-hydroxyandrostenedione (1), 6-oxoandrostenedione (6-oxoAD, 2) and its 19-hydroxy analog (3), 10beta-acetoxyestr-5-ene-7,17-dione (4), androst-5-ene-4,7,17-trione (5), and 17alpha-ethynyl-19-norteststerone (6), which are known suicide inactivators of AD aromatization, are not effective in inactivating 16alpha-OHAD aromatization in a time-dependent manner. The compounds were tested with the use of human placental microsomes and 1beta-tritiated-16alpha-OHAD as the substrate. The results of the tritium water method of 16alpha-OHAD aromatization was confirmed by the gas chromatography-mass spectrometry (GC-MS) method of estriol formation. The 1beta-tritiated-AD was used to measure AD aromatization as a positive control for these experiments. The compounds were tested at concentrations up to 40-fold higher than the K(i)s determined for inhibition of AD aromatization. These studies suggest that differences exist in the binding site structures responsible for aromatization of 16alpha-OHAD and AD.

Competing pathway involved in allylic acetoxylation of androst-5-en-17-one, and oxidation of allylic alcohols with chromium oxides

Allylic acetoxylation of androst-5-en-17-one (1) with bromine and silver acetate gave 6 alpha- and 6 beta-acetoxyandrost-4-en-17-ones [4 (3%) and 5 (12%)] and 5 alpha-bromo-6 beta-acetoxy steroid 8 (4%) along with the expected product 4 beta-acetoxy derivative 2 (45%). Treatment of 5 alpha,6 beta-bromide 12, an intermediate of the acetoxylation reaction, with silver acetate also produced the acetates 2, 4, 5, and 8 in relative yields similar to those above. These results indicate that the 6-acetates 4 and 5 are produced through a competing pathway involving formation of a bridged carbonium ion 13 followed by attack of an acetate anion. Oxidation of the axial allylic alcohol, 5-en-4 beta-ol 3, with Jones reagent yielded no trace of the previously reported androst-5-ene-4,17-dione (18) but instead gave a 1:4 mixture of 5 beta,6 beta-epoxy-4-one 16 and 4 beta,5 beta-epoxy-6-one 17 in high yield. In contrast, a 1:4 mixture of androst-4-ene-6,17-dione (10) and compound 18 was obtained upon treatment with chromium trioxide in pyridine. Similar results were also found with the oxidation of another axial allylic alcohol, 4-en-6 beta-ol 7.

Time-dependent aromatase inactivation by 4β, 5β-epoxides of the natural substrate androstenedione and its 19-oxygenated analogs

Aromatase catalyzes the conversion of androgens to estrogens through three sequential oxygenations. To gain insight into the catalytic function of aromatase and its aromatization mechanism, we studied the inhibition of human placental aromatase by 4 beta,5 beta-epoxyandrostenedione (5) as well as its 19-hydroxy and 19-oxo derivatives (6 and 7, respectively), and we also examined the biochemical aromatization of these steroids. All of the epoxides were weak competitive inhibitors of aromatase with apparent K(i) values ranging from 5.0 microM to 30 microM. The 19-methyl and 19-oxo compounds 5 and 7 inactivated aromatase in a time-dependent manner with k(inact) of 0.048 and 0.110 min(-1), respectively, in the presence of NADPH. In the absence of NADPH, only the former inhibited aromatase with a k(inact) of 0.091 min(-1). However, 19-hydroxy steroid 6 did not cause irreversible inactivation either in the presence or absence of NADPH. Gas chromatography-mass spectrometric analysis of the metabolite produced by a 5-min incubation of the three epoxides with human placental microsomes in the presence of NADPH under air revealed that all three compounds were aromatized to produce estradiol with rates of 8.82, 0.51, and 1.62 pmol/min/mg protein for 5, 6, and 7, respectively. In each case, the aromatization was efficiently prevented by 19-hydroxyandrost-4-en-17-one, a potent aromatase inhibitor. On the basis of the aromatization and inactivation results, it seems likely that the two pathways, aromatization and inactivation, may proceed, in part, through a common intermediate, 19-oxo compound 7, although they may be principally different.

The solvolytic ring opening of a 4β,5β-epoxy-3,6-dione steroid: preparation of potential aromatase inhibitors

4β,5β-Epoxyandrostane-3,6,17-trione 2 undergoes cleavage of the epoxy ring as a result of nucleophilic attack by water to give both 2-hydroxy-1,4-diene-3,6-dione 3 and 7α-hydroxy-4-ene-3,6-dione 9 upon treatment with sulfuric acid in aqueous acetone. In the similar reaction in acetic acid, 2α-acetoxy-4-ene-3,6-dione 6 and its 2β-isomer 7 are produced along with the diosphenol 3. Reaction of the epoxide 2 with sodium hydroxide in methanol affords 4-methoxy-4-ene-3,6-dione 12 the dealkylation of which with hydrochloric acid yields the 4-hydroxy derivative 14.