Biochemical and pharmacological development of steroidal inhibitors of aromatase

Androstenedione analogs containing 7 alpha-substituents have proven to be potent inhibitors of aromatase both in vitro and in vivo. Several of these agents have exhibited higher affinity for the enzyme complex than the substrate. In order to examine further the interaction(s) of 7-substituted steroids with aromatase, biochemical and pharmacological studies were performed on 7 alpha-thiosubstituted androstenediones and 7-substituted 4,6-androstadiene-3,17-diones. Potent inhibition of aromatase activity in human placental microsomes has been observed with several new 7 alpha-thiosubstituted androstenediones. 7-Benzyl- and 7-phenethyl-4,6-androstadiene-3,17-diones effectively inhibited microsomal aromatase, with apparent Kis ranging from 61 to 174 nM. On the other hand, 7-phenyl-4,6-androstadiene-3,17-dione exhibited poor activity, with an apparent Ki of 1.42 microM. Similar inhibitory activity was observed with reconstituted, purified cytochrome P450Arom preparations. Additionally, these agents were evaluated for inhibition of aromatase activity in two human carcinoma cell lines, the MCF-7 human mammary cancer line and the JAr choriocarcinoma line. The 7 alpha-thiosubstituted androstenediones and 7-substituted 4,6-androstadiene-3,17-diones produced dose-dependent inhibitions of aromatase activity in the cell cultures. The most effective inhibitors were the 7 alpha-substituted androstenediones, with EC50 values ranging from 7.3 to 105 nM. Finally, the JAr cell culture system exhibited prolonged inhibition of aromatase activity following exposure to 7 alpha-APTADD, suggesting enzyme inactivation by this inhibitor. Thus, these agents are effective aromatase inhibitors, and the results encourage further development of this group of medicinal agents for the treatment of estrogen-dependent mammary carcinoma.     

7-substituted 1,4,6-androstatriene-3,17-diones as enzyme-activated irreversible inhibitors of aromatase

7-Phenyl-1,4,6-androstatriene-3,17-dione (4), 7-benzyl-1,4,6-androstatriene-3,17-dione (5) and 7-phenethyl-1,4,6-androstatriene-3,17-dione (6) were synthesized and evaluated in vitro in human placental microsomes as enzyme-activated irreversible inhibitors of aromatase. The compounds were synthesized from appropriate 7-substituted 4,6-androstadiene-3,17-diones by reaction with DDQ under neutral conditions. All the compounds produced a first order inactivation of aromatase in the presence of NADPH but not in the absence of NADPH. Substrate 4-androstene-3,17-dione protected the enzyme from inactivation by the inhibitors. Furthermore, cysteine failed to protect aromatase from inactivation by compounds 5 and 6. In contrast, cysteine partially protected aromatase from inactivation by compound 4. Irreversibility studies illustrated the covalent nature of the inactivation by 4, 5 and 6. The above experimental evidence demonstrated that compounds 5 and 6 are effective enzyme-activated irreversible inhibitors of aromatase.     

7-substituted steroidal aromatase inhibitors: structure-activity relationships and molecular modeling

Androstenedione analogs containing 7 alpha-substituents have proven to be potent inhibitors of aromatase both in vitro and in vivo. Several of these agents have exhibited higher affinity for the enzyme complex than the substrate does. In order to examine further the interaction(s) of 7-substituted steroids with aromatase, biochemical and molecular modeling studies were performed on 7-substituted 4,6-androstadiene-3,17-diones. 7-Benzyl- and 7-phenethyl-4,6-androstadiene-3,17-diones effectively inhibited microsomal aromatase, with apparent Kis ranging from 61 to 174 nM. On the other hand, 7-phenyl-4,6-androstadiene-3,17-dione exhibited poor activity, with an apparent Ki of 1.42 microM. Energy minimization calculations and molecular modeling indicated that the 7-substituent is perpendicular to the steroid nucleus in the 7-phenyl analog and can only adopt a pseudo beta position. The 7-benzyl- and 7-phenethyl- groups of 4,6-androstadiene-3,17-diones orient themselves in the minimized structure in a way that the phenyl rings can protrude into the 7 alpha pocket. These orientations are similar to those observed in minimized structures for potent 7 alpha-substituted androstenediones.     

Steroidal inhibitors as chemical probes of the active site of aromatase

Androstenedione analogs containing 7-substituents have proven to be potent inhibitors of aromatase in human placental microsomes, in MCF-7 mammary cell cultures, and in JAr choriocarcinoma cells. Recent investigations have focused on the use of mechanism-based inhibitors, such as 7-substituted 1,4-androstadienediones, to biochemically probe the active site of aromatase. Inhibition kinetics were determined under initial velocity conditions using purified human placental cytochrome P450_arom protein in a reconstituted system. Derivatives of 1,4-androstadiene-3,17-dione and 1,4,6-androstatriene-3,17-dione exhibited high affinity in the purified enzyme system. 7-(4′-Amino)phenylthio-1,4-androstadiene-3,17-dione, abbreviated 7-APTADD, demonstrated rapid time-dependent, first-order inactivation of reconstituted aromatase activity only in the presence of NADPH. The apparent K_inact for 7-APTADD is 11.8 nM, the first-order rate of inactivation is 2.72 × 10⁻³ sec⁻¹, and the half-time of inactivation at infinite inhibitor concentration is 4.25 min. The values for the rate constant and half-time of inactivation are similar to those observed in the placental microsomal assay system. Further studies were performed with radioiodinated 7-(4′-iodo)phenylthio-1,4-androstadienedione, 7-IPTADD, and the reconstituted aromatase system. Incubations with [¹²⁵I]7-IPTADD were followed by protein precipitation, solvent extraction, and column chromatography. Analysis of the isolated cytochrome P450_arom by gel elctrophoresis and autoradiography demonstrated the presence of only one radioactive band, which corresponded to the protein staining band for cytochrome P450_arom. HPLC radiochromatographic analysis of the isolated cytochrome P50_arom confirmed the presence of only one radioactive peak coeluting with the u.v. peak for cytochrome P50_arom. Peptide mapping analysis by reverse-phase HPLC of digested inhibitor-cytochrome P450_arom complex demonstrates that the radioactive inhibitor is covalently bound to a lipophilic fragment. In summary, these inhibitors produced enzyme-catalyzed inactivation of reconstituted aromatase activity, and radioiodinated 7-IPTAPP binds covalently to the cytochrome P450_arom.     

Chemoselective reduction of 1,4,6-cholestatrien-3-one and 1,4,6-androstatriene-3,17-dione by various hydride reagents

The chemoselectivity of rigid cyclic alpha,beta-unsaturated carbonyl group on the reducing agents was influenced by the ring size and steric factor. Cholesterol (cholest-5-en-3beta-ol) and dehydroepiandrosterone (DHEA) were oxidized with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to form 1,4,6-cholestatrien-3-one and 1,4,6-androstatriene-3,17-dione. They were reduced with NaBH(4), lithium tri-sec-butylborohydride (l-Selectride), LiAlH(4), 9-borabicyclo[3.3.1]nonane (9-BBN), lithium triethylborohydride (Super-hydride), and BH(3) x (CH(3))(2)S in various conditions, respectively. Reduction of 1,4,6-cholestatrien-3-one and 1,4,6-androstatriene-3,17-dione by NaBH(4) (4 equiv.) produced 4,6-cholestadien-3beta-ol and 4,6-androstadiene-3beta,17beta-diol, respectively. Reduction by l-Selectride (12 equiv.) afforded 4,6-cholestadien-3alpha-ol and 4,6-androstadiene-3alpha,17beta-diol, chemoselectively. Reaction with Super-hydride (12 equiv.) produced 4,6-cholestadien-3-one and 3-oxo-4,6-androstadien-17beta-ol. Reduction of 1,4,6-cholestatrien-3-one by 9-BBN (14 equiv.) produced 1,4,6-cholestatrien-3alpha-ol, but 1,4,6-androstatriene-3,17-dione was not reacted with 9-BBN in the reaction conditions. Reaction of LiAlH(4) (6 equiv.) formed 4,6-cholestadien-3beta-ol and 3-oxo-1,4,6-androstatrien-17beta-ol. Reduction of 1,4,6-cholestatrien-3-one by BH(3) x (CH(3))(2)S (11 equiv.) gave cholestane as major compound and unlike reactivity of cholesterol, 1,4,6-androstatriene-3,17-dione by 8 equiv. of BH(3) x (CH(3))(2)S formed 3-oxo-1,4,6-androstatrien-17beta-ol. LiAlH(4) and BH(3) x (CH(3))(2)S showed relatively low chemoselectivity.     

Biochemistry and pharmacology of 7α-substituted androstenediones as aromatase inhibitors

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7α-substituted androstenediones. Numerous 7α-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7α-(4′-aminophenylthio)-androsta-1,4-diene-3,17-dione (7α-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7α-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7α-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7α position with a carbon-carbon linkage. Several 7α-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent K_is ranging between 13 and 19 nM. Extension of the research on these 7α-arylaliphatic androgens includes the introduction of a C₁---C₂ double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7α-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7α-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k_inacts ranging from 4.4 × 10⁻⁴ to 1.90 x 10⁻³ s⁻¹. The best inactivator of the series was 7α-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T_1/2 of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC₅₀ values of 64-328 nM. The 7α-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7α-position of the steroid in the active site of aromatase. The size of the 7α-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.     

The constitutive 7-ethoxycoumarin O-deethylase activity of human placental microsomes: relationship to aromatase

The constitutive 7-ethoxycoumarin deethylase activity of human placental microsomes from non-smokers was acutely inhibited by a number of androgens which serve as substrates for and/or competitive inhibitors of estrogen synthesis by the aromatase activity of these preparations. 10 beta-(2-Propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione, androgen derivatives which produce a mechanism-based, time-dependent inactivation of placental aromatase caused a cofactor-dependent decay in deethylase activity which paralleled the loss of aromatase activity caused by these agents and which was antagonized by aromatase substrates. Conversely, 7-ethoxycoumarin antagonized the time-dependent action of 10 beta-(2-propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione on aromatase and inhibited competitively the aromatization of 4-androstene-3,17-dione. The Ki for 7-ethoxycoumarin was equivalent to its Km as substrate for deethylation. It is concluded that a common oxidase species is responsible for both the aromatase and constitutive 7-ethoxycoumarin deethylase activities of human placental microsomes.     

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.     

Induction of implantation by aromatase inhibitors in ovariectomized mice

The ability of aromatase inhibitors to induce implantation in mice was tested in animals in which implantation was delayed by ovariectomy and progesterone treatment. Implantation was consistently induced by 7 mg 4-hydroxyandrostene-3,17-dione (4-OH-A), 7 X 5 mg 1,4,6-androstatriene-3,17-dione (ATD) or 15 mg 4-acetoxyandrostene-3,17-dione, an activity comparable to that of 1 mg testosterone. In intact mice treated with 2 or 10 mg 4-OH-A or ATD/day from Day 2 of pregnancy (Day 1 = vaginal plug), the number and size of implantation sites were not affected. These results may not be necessarily due to inhibitory effects of the compounds on aromatase.     

7 alpha-substituted androstenediones as effective in vitro and in vivo inhibitors of aromatase

Research efforts over the past several years have focused on the synthesis of competitive and irreversible aromatase inhibitors and examination of these inhibitors in microsomal preparations, in cell culture, and in vivo. Several 7 alpha-substituted androstenediones have demonstrated high affinity for placental aromatase, with apparent Ki's ranging from 1 to 30 nM. Inactivation of aromatase occurred following incubation with alkylating and enzyme-activated irreversible inhibitors. 7 alpha-(4'-Amino)phenylthio-4-androstene-3,17-dione (7 alpha-APTA) exhibits potent inhibitory activity of aromatase in the MCF-7 human mammary carcinoma cell line with an ED50 of approximately 25 nM. The inhibitor did not bind to the estrogen receptor of the cells in vitro nor induce levels of progesterone receptors in intact cells. In vivo studies of 7 alpha-APTA in the DMBA-induced rat mammary carcinoma model resulted in 80% of the tumors responding completely or partially at doses of 25 and 50 mg/kg body wt/day. Thus, these 7 alpha-substituted steroidal aromatase inhibitors are effective medicinal agents and may be useful for the treatment of estrogen-dependent breast cancer.     

Synthesis and structure-activity relationships of 6-phenylaliphatic-substituted C19 steroids having a 1,4-diene, 4,6-diene, or 1,4,6-triene structure as aromatase inhibitors.

A series of 6alpha- and 6beta-phenylaliphatic-substituted androsta-1,4-diene-3,17-diones [9b-f and 10b-f; (CH2)nPh, n = 1-5] and their 4,6-diene and 1,4,6-triene analogs (11b-f and 12b-f) along with their respective phenyl analogs 9a-12a were synthesized and tested as aromatase inhibitors. All of the steroids examined were very powerful competitive inhibitors of aromatase in human placental microsomes with apparent Ki values ranging from 8.5 to 80 nM. The inhibitory activities of the benzyl- and phenethyl-4,6-dienes 11b and 11c (Ki, 9.0 and 10 nM) as well as the 6-phenethyl-1,4,6-triene 12c (Ki, 8.5 nM) were extremely high among them. All of the phenylaliphatic steroids, except for the 6beta-phenethyl compound 10c, and the 6-phenyl-4,6-diene 11a had higher affinity for aromatase than the corresponding parent 1,4-diene, 4,6-diene, and 1,4,6-triene steroids 9g, 11g, and 12g. All of the 6alpha-substituted 1,4-dienes (9a-9g) and the 6-substituted 1,4,6-trienes (12a-12g) caused a time-dependent inactivation of aromatase. On the other hand, only the 6beta-substituted 1,4-dienes (10a-10d) having no or less than four carbon atoms between the steroid nucleus and the phenyl group also caused a time-dependent inactivation of aromatase. Their inactivation rates (k(inact) 0.076-0.156 min(-1)) were higher than the respective parent steroids, 9g and 12g. In contrast, in the 4,6-diene series, only the 6-phenpropyl steroids 11d inactivated aromatase in a time-dependent manner with 0.155 min(-1) of k(inact) value. The inactivation was prevented by the substrate androstenedione, and no significant effect of L-cysteine on the inactivation was observed in each case. These results indicate that length and/or stereochemistry of the C-6 substituent of steroids 9-12 as well as a terminal phenyl group incorporated in the C-6 substituent play a critical role not only in tight binding to the active site of aromatase but also in the cause of a time-dependent inactivation of the enzyme.     

Probing the binding pocket of the active site of aromatase with 6-ether or 6-ester substituted androst-4-ene-3,17-diones and their diene and triene analogs

A series of 6-ester- (3 and 4) and 6-ether- (7 and 8) substituted androst-4-ene-3,17-diones (androstenediones) and their 1,4-diene analogs (5 and 6, and 9 and 10) as well as C6-substituted 4,6-diene and 1,4,6-triene steroids 11 and 12 were synthesized as aromatase inhibitors to gain insight into the structure-activity relationship between various substituents and inhibitory activity. All of the inhibitors synthesized blocked aromatase in a competitive manner. The inhibitory activities of all of the steroids, except for the 6beta-benzoates 4g and 6h and the 6beta-acetate 6a, were fairly effective to very powerful (K(i): 7.0-320 nM). The 6alpha-n-hexanoyloxy- and 6alpha-benzyloxyandrostenediones (3e and 7e) were the most potent inhibitors (K(i): 7.0 nM each). In the series of 4-ene and 1,4-diene steroids, the 6alpha-substituted steroids had higher affinity for the enzyme than the corresponding 6beta-isomers. In the 1,4-diene steroid series, 6beta-substituted steroids 6a, e, g, and 10a, b, e caused a time-dependent inactivation of aromatase, whereas their 6alpha-isomers 5 and 9 essentially did not. The ether-substituted 1,4,6-trienes 12 inactivated the enzyme in a time-dependent manner; in contrast, their 4,6-diene analogs 11 did not. The substrate androstenedione blocked the inactivation, but no significant effect of L-cysteine was observed. Based on molecular modeling with the PM3 method, along with the present inhibition and inactivation results, it is thought that both the steric effects of the 6-substituents as well as the electronic effects of the C-6 oxygen functions play a critical role in the binding of inhibitors to the active site of aromatase.     

Synthesis and evaluation of novel 4-amino-4,6-androstadiene-3,17-dione: an analog of formestane

Synthesis of 4-amino-4,6-androstadiene-3,17-dione 7, an analog of formestane used in breast cancer therapy as an aromatase inhibitor, from 4-acetoxy-4-androstene-3,17-dione 2 is described. This is the first report of a 4-amino diene (4,6) system in this series of molecules. The new (7) and reported molecules were screened by the National Cancer Institute (NCI, Bethesda, USA) for in vitro antitumor activity against 60 human cancer cell lines. Molecule 7 showed best activity against breast cancer cell line (MCF-7).     

Catalytic differences between porcine blastocyst and placental aromatase isozymes.

Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO cells using the mammalian cell transfection method. Using an 'in-cell' assay (a 3H-water release method), catalytic parameters of the porcine placental aromatase were found to be very similar to those of the human enzyme; however, the activity of the blastocyst isozyme was found to be one-thirtieth that of the placental isozyme. Product isolation assay (using testosterone as the substrate) revealed that the major steroid products were 17beta-estradiol and 19-nortestosterone. The product ratio of estradiol/19-nortestosterone was found to be 94 : 6 for the porcine placental form, 6 : 94 for the porcine blastocyst form, and 92 : 8 for the human wild-type aromatase. Therefore, the porcine blastocyst aromatase isozyme catalyzes mainly androgen 19-desmethylation rather than aromatization. In addition, inhibition profile analyses on the placental and blastocyst isozymes were performed using three steroidal inhibitors [4-hydroxyandro-stenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1, 4-androstandiene-3,17-dione (7alpha-APTADD), and bridge (2, 19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)]. While the two isozymes of porcine aromatase share 93% amino-acid sequence identity, our results indicate that the two porcine aromatase isozymes have distinct responses to various aromatase inhibitors.     

In vitro potency and selectivity of the non-steroidal androgen aromatase inhibitor CGS 16949A compared to steroidal inhibitors in the brain.

A sensitive in vitro 3H2O microassay for aromatase activity was used to evaluate the potency and selectivity of three aromatase inhibitors in mammalian (gerbil) and avian (ring dove) hypothalamus. The steroidal inhibitors, 1,4,6-androstatrien-3,17-dione (ATD) and 4-hydroxy-androstenedione (4-OH-A) were compared with a new non-steroidal imidazole inhibitor, CGS 16949A [4-(5,6,7,8-tetrahydroimidazo-[1,5-a]-pyridin-5-yl)benzonitrile HCl]. Adult male dove hypothalamic aromatase is highly active [Vmax = 5.3 pmol testosterone (T) converted/h/mg protein], has high substrate binding affinity (Km = 4.0 nM), and direct involvement in control of sexual behaviour. With [1 beta-3H]T or [1 beta-3H]A as substrate, male dove preoptic aromatase activity was inhibited more effectively and selectively by CGS 16949A. Thus, Kis and IC50s for aromatization were approximately 50 times lower for the non-steroidal inhibitor, and inhibition of the other major androgen-metabolizing enzymes (5 alpha/beta-reductase) occurred at concentrations at least one order of magnitude greater than for ATD and 4-OH-A. Neonatal male gerbil hypothalamic aromatase activity (Vmax = 1.3 pmol T converted/h/mg protein) was lower than in the dove. Aromatase inhibition by CGS 16949A is more potent in the neonatal gerbil than in the dove (Kis of 0.03 and 0.60 nM, respectively, with A as substrate). We conclude that the imidazole is an effective aromatase inhibitor in both the adult and developing brain.     

Dissociation of 19-hydroxy- 19-oxo-, and aromatizing-activities in human placental microsomes through the use of suicide substrates to aromatase

Suicide substrates of aromatase were used as chemical probes to determine if free 19-hydroxyandrost-4-ene-3,17-dione (19-OHA) and 19-oxoandrost-4-ene-3,17-dione (19-oxoA) are obligatory intermediates in the aromatization of androst-4-ene-3,17-dione (androstenedione) to oestrone by human placental aromatase. A radiometric-HPLC assay was used to monitor 19-hydroxy, 19-oxo-, and aromatized products formed in incubations of [14C]androstenedione and human placental microsomes. When microsomes were preincubated with the suicide substrates 10 beta-mercapto-estr-4-ene-3,17-dione (10 beta-SHnorA), or 17 beta-hydroxy-10 beta-mercaptoestr-4-ene-3-one (10 beta-SHnorT), it was found that 19-hydroxy-, 19-oxo- and aromatase activities were inhibited in parallel. However, when the suicide substrates 4-hydroxyandrost-4-ene-3,17-dione (4-OHA) and 19-mercaptoandrost-4-ene-3,17-dione (19-SHA) were preincubated with placental microsomes, significantly greater inhibition of formation of oestrogens was observed in comparison to the inhibition of formation of 19-hydroxy- and 19-oxo-metabolites. Furthermore, significantly more time-dependent inhibition of 19-oxoA formation was observed in comparison to inhibition of 19-OHA formation with these same inhibitors. These results suggest that 19-hydroxy- and 19-oxo-androstenediones are not free, obligatory intermediates in the aromatization of androstenedione by human placental aromatase, but rather are products of their own autonomous cytochrome P-450-dependent, microsomal enzymatic activities.     

A photoaffinity inhibitor of aromatase

Several 7-substituted 4-androstene-3, 17-diones are potent inhibitors of the biosynthesis of estrogens, with the most effective being 7-(4'-amino)phenylthio-4-androstene-3, 17-dione. An azide derivative of this 7-thioether compound has been prepared as a potential photoaffinity inhibitor. The enzyme kinetics of the azide analog were examined under both dark conditions and UV irradiation. In the dark, the azide was a very potent competitive inhibitor, with an apparent K_i of 1.3 nM. Under UV-irradiation, a time-dependent loss of aromatase activity was also observed. These studies indicate that the 7-substituent enhances the affinity of the steroidal analogs for the enzyme site.     

The constitutive 7-ethoxycoumarin 0-deethylase of human placental microsomes: relationship to the intermediary steps in steroid aromatization

All oxidative functions of aromatase, i.e., estrogen production, 19-oxygenated androgen production and 7-ethoxycoumarin deethylation, were inhibited in parallel in placental microsomes from non-smokers by the mechanism-based, time-dependent inactivators (suicide substrates) 10 beta-(2-propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione. In contrast, the aromatase suicide substrate androst-4-ene-3,6,17-trione had little or no effect on the conversion of androst-4-ene-3,17-dione to 19-hydroxyandrost-4-ene-3,17-dione or on the conversion of the latter to 3,17-dioxoandrost-4-en-19-al while severely limiting the capacity for estrogen production from androst-4-ene-3,17-dione and 19-hydroxyandrost-4-ene-3,17-dione in such microsomal preparations. Androst-4-ene-3,6,17-trione, therefore, appears to uncouple the 19-hydroxylation of androgens from estrogen synthesis. This agent also produced only a minimal inhibition of 7-ethoxycoumarin deethylation, indicating that this major constitutive transformation of a xenobiotic chemical is associated with the steroid 19-hydroxylating function of the aromatase system.     

Novel silylated steroids as aromatase inhibitors

Androst-4-en-3-one analogs incorporating a trimethylsilyl or a trimethylsilylmethyl group at C-1, C-2 or C-19 were prepared and evaluated as inhibitors of aromatase. Only 10-[1-hydroxy-2-(trimethylsilyl)ethyl]estr-4-ene-3,17-dione inhibited human placental aromatase. Enzyme kinetic analysis revealed competitive inhibition [apparent dissociation constant (Ki) of 562 +/- 12 nM] associated with marginal time-dependent inhibition.     

Potent enzyme-activated inhibition of aromatase by a 7 alpha-substituted C19 steroid

Enzyme-activated inhibitors of aromatase would result in effective medicinal agents for modulating estrogen-dependent processes and thus may be useful in controlling reproductive processes and in treating estrogen-dependent diseases such as breast and endometrial cancer. A potential enzyme-activated inhibitor of aromatase, 7 alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione (7 alpha-APTADD), was synthesized and examined in vitro with placental aromatase. Under initial velocity conditions, 7 alpha-APTADD exhibited high affinity for the enzyme and is a potent inhibitor of aromatase with an apparent Ki of 9.9 +/- 1.0 nM and with a Km for androstenedione of 52.5 +/- 5.9 nM. This inhibitor produced a rapid time-dependent, first-order inactivation of aromatase in the presence of NADPH, while no inactivation of aromatase activity was observed in the absence of NADPH. Protection of aromatase from inactivation was observed when the substrate, androstenedione, was included in the incubation mixture containing enzyme, inhibitor, and NADPH. On the other hand, nucleophilic trapping agents such as cysteine did not protect the enzyme from inactivation by 7 alpha-APTADD. Additionally, second enzyme pulse experiments demonstrated identical rates of inactivation, suggesting that the enzyme-activated inhibitor was not being released from the active site of the enzyme. The apparent Kinact for 7 alpha-APTADD is 159 +/- 21 nM and represents the inhibitor concentration required to produce a half-maximal rate of inactivation. The half-time of inactivation at infinite inhibitor concentration was 1.38 +/- 0.92 min and is the most rapid enzyme-activated aromatase inhibitor reported to date. Thus, 7 alpha-APTADD is a potent enzyme-activated inhibitor of aromatase, exhibiting high affinity and rapid inactivation. This inhibitor will be useful in probing the biochemistry of aromatase and should also serve as an effective medicinal agent for the treatment of estrogen-dependent cancers.