A time-dependent inactivation of aromatase by 19-oxygenated androst-4-ene-3,6,17-triones

19-Hydroxyandrost-4-ene-3,6,17-trione (19-OHAT), its 19-oxo derivative (19-oxo AT) and 4β,5β-epoxyandrostane-3,6,17-trione (5) were synthesized as possible intermediates involved in a mechanism-based inactivation of aromatase caused by androst-4-ene-3,6,17-trione (AT). These compounds inhibited the enzyme in a competitive manner with K_i's of 0.61, 7.5 and 5.1 μM for 19-OHAT, 19-oxo AT, and compound 5. The two 19-oxygenated steroids showed a time-dependent, pseudo-first order rate of inactivation of aromatase with k_inact's of 0.222 and 0.076 min⁻¹ for 19-OHAT and 19-oxo AT, respectively, while compound 5 did not. NADPH and oxygen were required for the inactivation. Androstenedione blocked the inactivation, while -cycteine partially prevented that of 19-OHAT and almost completely that of 19-oxo AT. When the 19-oxygenated steroids were separately subjected to reaction with , these rapidly disappeared from the reaction mixture with of 25 min (19-OHAT) and 20 s (19-oxo AT). This finding indicates that -cysteine prevents inactivation by a chemical dependent elimination of the inhibitors from the incubate. These results suggest that the 19-oxygenation rather than the 4,5-epoxidation may be involved in the time-dependent inactivation by AT.     

6β,19-Bridged androstenedione analogs as aromatase inhibitors

Inhibition of aromatase is an efficient approach for the prevention and treatment of breast cancer. New 6β,19-bridged steroid analogs of androstenedione, 6β,19-epithio- and 6β,19-methano compounds 11 and 17, were synthesized starting from 19-hydroxyandrostenedione (6) and 19-formylandrost-5-ene-3β,17β-yl diacetate (12), respectively, as aromatase inhibitors. All of the compounds including known steroids 6β,19-epoxyandrostenedione (4) and 6β,19-cycloandrostenedione (5) tested were weak to poor competitive inhibitors of aromatase and, among them, 6β,19-epoxy steroid 4 provided only moderate inhibition (K_i: 2.2 μM). These results show that the 6β,19-bridged groups of the inhibitors interfere with binding in active site of aromatase.     

Aromatization of androstenedione and 16alpha-hydroxyandrostenedione in human placental microsomes. Kinetic analysis of inhibition by the 19-oxygenated and 3-deoxy analogs

Inhibition of aromatase activity in human placental microsomes with androstenedione (AD) (1a) and its 19-oxygenated derivatives 1b and 1c, their 16alpha-hydroxy compounds 2 and 3, and 3-deoxyandrost-4-ene compounds 5 and 6 was studied using [1beta-(3)H]AD as a substrate and compared to that with [1beta-(3)H]16alpha-hydroxyandrostenedione (16-OHAD). AD series of steroids, compounds 1, inhibited competitively [1beta-(3)H]AD aromatization whereas other 16alpha-hydroxy steroids 2, 3, 5, and 6 inhibited AD aromatization in a non-competitive manner. On the other hand, all of 16-OHAD series, compounds 2, blocked the [1beta-(3)H]16-OHAD aromatization in a competitive manner whereas the AD series steroids 1 as well as the 3-deoxy-16alpha-hydroxy-17-one steroids 5 and 3-deoxy-16alpha,17beta-diol steroids 6 inhibited 16-OHAD aromatization non-competitively. 3-Carbonyl and 16alpha-hydroxy functions of 16-OHAD play a critical role of selection of the 16-OHAD binding site. The results suggest that the AD derivatives 1 are kinetically aromatized at a different site from the 16-OHAD derivatives 2. Physical and/or chemical environments around the aromatase protein in the microsomal membrane may play a significant role in the expression of the substrate specificity, and the present results do not exclude the idea that the placental microsomes have a single binding site.     

Synthesis of 19-oxygenated derivatives of the competitive inhibitor of aromatase, 5-androstene-4,17-dione.

19-Hydroxy- and 19-oxo-steroids 13 and 15, respectively, which are potential metabolites of the aromatase inhibitor 5-androstene-4,17-dione (3), were synthesized from 19-(tert-butyldimethylsilyloxy)androst-5-en-17-one (5) or 4beta-acetoxyandrost-5-en-17-one (16), respectively, through 5alpha-bromo-4beta-hydroxy-6beta,19-epoxyandrostan+ ++-17-one (10) as a key intermediate in each sequence. Reaction of the 19-siloxy compound 5 with Br2 gave 5alpha-bromo-6beta,19-epoxide 8, which was treated with N,N'-dimethylacetamide followed by reaction with N-bromoacetamide and 0.28 M HCIO4, to yield compound 10. On the other hand, treatment of the 4beta-acetoxy steroid 16 with N-bromoacetamide-HCI04 followed by oxidation with Pb (IV) acetic acid and I2 under irradiation and subsequent hydrolysis with K2CO3 also produced compound 10 and in better yield than that in the above synthesis. Jones oxidation of the 4beta-ol 10 followed by reductive debromination with zinc dust yielded the 19-ol 13 in low yield as well as 6beta,19-epoxy-4-one 12 as the major product. Furthermore, the major product 12 was converted into the 19-ol 13 in moderate yield from compound 12 through acetolysis and subsequent alkaline hydrolysis. The 19-oxo steroid 15 was obtained after treatment of compound 13 with pyridinium dichromate. Compounds 13 and 15 were analyzed as the methoxime-trimethylsilyl and methoxime-dimethylisopropylsilyl derivatives and the methoxime derivative, respectively, using gas chromatography-mass spectrometry.     

Probing the active site of aromatase with 2-methyl-substituted androstenedione analogs

To 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. A series of 2alpha-methyl steroids were good substrates for aromatase, whereas 2beta-methyl steroid 5 was an extremely poor substrate, and a series of 2,2-dimethyl steroids did not serve as substrate, suggesting that a 2beta-methyl moiety of the 2,2-dimethyl and 2beta-methyl steroids would prevent the aromatase reaction probably due to steric hindrance in each case. The 2-methylene compound 17 was also aromatized to produce 2-methylestrogen with a low conversion rate where the 1,4-diene structure may have been created before the C(10)-C(19) bond cleavage. Kinetic analysis of the aromatization of androgens revealed that a good substrate was not essentially a good inhibitor for aromatase.     

Reversible inactivation of rat liver inorganic pyrophosphatase by substrate and its analogs.

Dissociation of Mg2+ from one of the two metal-binding sites whose occupancy is absolutely required for catalysis by rat liver inorganic pyrophosphatase is a slow reaction (tau 1/2 = 3 h). Polycarboxylic Mg2+ complexons markedly accelerate this process due to their binding with Mg2+ on the enzyme. PPi, ATP and a number of diphosphonate analogs of PPi also bind with Mg2+ on the enzyme with concomitant decrease in enzyme activity by 75% but do not release the bound Mg2+. The resulting ternary complex rapidly (tau 1/2 of several seconds) dissociates upon dilution into substrate-free medium. PPi and imidodiphosphate, which are substrates for pyrophosphatase, decrease the rate of reactivation by at least two orders of magnitude. The results can be explained by existence of two interconvertible forms of the enzyme, of which one is inactive and is stabilized by substrate or its analogs.     

Structure-activity relationships of 2alpha-substituted androstenedione analogs as aromatase inhibitors and their aromatization reactions

Aromatase 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. 2alpha-Halogeno (F, Cl, and Br) and 2alpha-alkyl (CH3 and CH2CH3) steroids 1b-1f were powerful to good inhibitors (Ki=45-171 nM) whereas steroids 1g-1i, having an oxygen function (hydroxy or alkoxy) at C-2alpha, were poor inhibitors (Ki=670-1150 nM). Aromatization of some of the steroids with placental microsomes was analyzed by gas chromatography-mass spectrometry, indicating that the aromatization rate of the bromide 1d was about two-fold that of the natural substrate AD and that of 2alpha-methoxide 1h was similar to that of AD. Kinetic analysis of the aromatization of androgens revealed that a good substrate was not essentially a good inhibitor for aromatase.     

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.     

Selective 19-hydroxylase inhibition by an aromatase inhibitor, 4-hydroxyandrostenedione

19-Nor-deoxycorticosterone is a newly recognized mineralocorticoid which has been associated with some forms of genetic, experimental, and human hypertension. To further examine this relationship, specific inhibitors of 19-nor-deoxycorticosterone biosynthesis must be developed. Since 19-hydroxylation is the pivotal step in both 19-nor-deoxycorticosterone biosynthesis and aromatization of androgens to estrogens, we evaluated an aromatase inhibitor, 4-hydroxyandrost-4-ene-3,17-dione on the inhibition of 19-hydroxylation in both rat and human adrenal mitochondria in vitro and 19-nor-deoxycorticosterone production and blood pressure in spontaneously hypertensive rats in vivo. Adrenal mitochondria from 48 male Sprague-Dawley rats and 1 patient with an aldosterone-producing adenoma were incubated in the presence of deoxycorticosterone substrate both with and without 4-hydroxyandrost-4-ene-3,17-dione. 4-Hydroxyandrost-4-ene-3,17-dione produced significant inhibition of 19-hydroxy-deoxycorticosterone production in both rat and human adrenal mitochondria, with a smaller and not significant inhibition of corticosterone and 18-hydroxy-corticosterone. 4-Hydroxyandrost-4-ene-3,17-dione given subcutaneously to spontaneously hypertensive rats lowered 19-nor-deoxycorticosterone by 69% and completely abolished hypertension compared to Wistar-Kyoto controls. These data demonstrate that 4-hydroxyandrost-4-ene-3,17-dione is a specific inhibitor of 19-hydroxylase, that it lowers 19-nor-deoxycorticosterone production and prevents hypertension in the spontaneously hypertensive rat. These studies reinforce the possible pathogenic significance of 19-nor-deoxycorticosterone in hypertension in spontaneously hypertensive rats.     

Synthesis of 3 beta,16 beta,19-trihydroxyandrost-5-en-17-one and 16 beta,19-dihydroxyandrost-4-ene-3,17-dione and their 19-oxo derivatives

3 beta,16 beta,19-Trihydroxyandrost-5-en-17-one (12) was synthesized from 5 alpha-bromo-3 beta-acetoxy-6 beta,19-epoxyandrostan-17-one (2) through acetoxylation at C-16 beta of the enol acetate 4 with lead tetraacetate and reductive cleavage of the epoxide ring with zinc dust yielding the 3 beta,16 beta-diacetoxy-19-hydroxy steroid 11, followed by hydrolysis of the acetoxy groups with sulfuric acid. Jones oxidation of compound 11 followed by the acid hydrolysis gave the 19-oxo steroid 15. 5 alpha-Bromo-3 beta-hydroxy-16 beta-acetoxy-6 beta,19-epoxyandrostan-17-one (8), obtained by selective hydrolysis of the 3-formate 5 with ammonium hydroxide, was oxidized with Jones reagent to afford the 3-oxo steroid 16, which was converted into the 19-hydroxy derivative 17 by treatment with zinc dust. 16 beta,19-Dihydroxyandrost-4-ene-3,17-dione (18) and its 19-oxo derivative 21 were obtained from compound 17 through a similar reaction sequence.     

Inhibition of mevalonate 5-diphosphate decarboxylase by fluorinated substrate analogs

Mevalonate 5-diphosphate decarboxylase (MDD) is a peroxisomal enzyme in the cholesterol biosynthetic pathway, which plays an important role in regulating cholesterol biosynthesis. In the present study, rat MDD was cloned and purified to apparent homogeneity. Two fluorinated MDD substrate analogs, P'-geranyl 2-fluoromevalonate 5-diphosphate (4) and 2-fluoromevalonate 5-diphosphate (6), were synthesized, and both were found to be irreversible inhibitors of rat MDD. These two inhibitors were characterized, and mechanisms of the inactivation process were proposed. Kinetic studies indicate both analogs only bind into mevalonate binding-site of MDD. Compound 4 shows competitive inhibition on mevalonate kinase (MVK), and its IC(50) value was determined to be comparable with that of geranyl diphosphate. Further kinetic studies indicate compound 4 only bind into ATP binding-site of MVK. These studies provide an example for a single inhibitor to carry out sequential blocking of two enzymes in cholesterol biosynthesis, which may provide useful information for drug discovery for the purpose of treating cardiovascular disease and cancer or for pest control.     

Mechanism of inactivation of alanine racemase by beta, beta, beta-trifluoroalanine

The alanine racemases are a group of PLP-dependent bacterial enzymes that catalyze the racemization of alanine, providing D-alanine for cell wall synthesis. Inactivation of the alanine racemases from the Gram-negative organism Salmonella typhimurium and Gram-positive organism Bacillus stearothermophilus with beta, beta, beta-trifluoroalanine has been studied. The inactivation occurs with the same rate constant as that for formation of a broad 460-490-nm chromophore. Loss of two fluoride ions per mole of inactivated enzyme and retention of [1-14C]trifluoroalanine label accompany inhibition, suggesting a monofluoro enzyme adduct. Partial denaturation (1 M guanidine) leads to rapid return of the initial 420-nm chromophore, followed by a slower (t1/2 approximately 30 min-1 h) loss of the fluoride ion and 14CO2 release. At this point, reduction by NaB3H4 and tryptic digestion yield a single radiolabeled peptide. Purification and sequencing of the peptide reveals that lysine-38 is covalently attached to the PLP cofactor. A mechanism for enzyme inactivation by trifluoroalanine is proposed and contrasted with earlier results on monohaloalanines, in which nucleophilic attack of released aminoacrylate on the PLP aldimine leads to enzyme inactivation. For trifluoroalanine inactivation, nucleophilic attack of lysine-38 on the electrophilic beta-difluoro-alpha, beta-unsaturated imine provides an alternative mode of inhibition for these enzymes.     

New 4-methylated and 19-oxygenated steroids from the Formosan soft coral Nephthea erecta

Two new 4-methylated steroids, erectasteroids A and B (1 and 2), six new 19-oxygenated steroids, erectasteroids C-H (3-8) and two known 19-oxygenated steroids (9 and 10) were isolated from the acetone solubles of the Formosan soft coral Nephthea erecta. The structures were elucidated by extensive NMR spectroscopic analysis and their cytotoxicity against selected cancer cells was measured in vitro.     

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.     

Synthesis of c-2, 3, 17 and 19-oxygenated androgens

The synthesis of C19-androgens oxygenated at carbons 2, 3, 17 and 19 and of their deuterium or deuterium-tritium labeled analogs is described.     

In vitro inhibition of aromatase by the enantiomers of aminoglutethimide and analogs

The in vitro aromatase activity in microsomal fractions from rat ovary and its inhibition by enantiomers of aminoglutethimide (AG), rogletimide (RG), and cyclohexylaminoglutethimide (ChAG) were studied by analysing the [³H]H₂O released when [1β-³H]androstenedione was converted to estrone. Maximum velocity (V_max) and the Michaelis-Menten constant (K_m) of the microsomal aromatase enzyme were 17.40 ± 0.45 pmol/ml/mg protein/min and 1.02 ± 0.06 μM, respectively. The IC₅₀s for the enantiomers were similar for (+)-R-AG and (?)-R-ChAG (0.86 ± 0.06 and 0.89 ± 0.15 μM, respectively). (+)S-ChA'G was most potent with IC₅₀ of 0.075 ± 0.003 μM. The IC₅₀s for (?)-S-AG, (+)-R-RG, and (?)-S-RG were in the same range (23.15 ± 2.74, 24.58 ± 2.46, and 24.43 ± 2.20 μM, respectively). © 1994 Wiley-Liss, Inc.     

An investigation into the 19-hydroxylation of androstenedione,cortexolone and progesterone by selected fungi

Summary An investigation was performed to recognise a fungus capable of 19-hydroxylating the steroids androstenedione, cortexolone and progesterone, in high yield with little or no side reactions. The fungi selected for study belong to a group which has been previously reported to possess 19-hydroxylation ability. Pellicularia filamentasa f. sp. microsclerotia IFO 6298 and P. filamentosa f.sp. sasakii IFO 5254 both 19-hydroxylated cortexolone, but 19-hydroxylated products were not detected using either androstenedione or progesterone as substrate. When 19-hydroxylating cortexolone, both strains of P. filamentosa produced 11-hydroxycortexolone as a by-product, but P. filamentosa f.sp. microsclerotia IFO 6298 was superior in terms of 19-hydroxylation and the relative amounts of the two products.