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.     

Aromatase inhibition by 7-substituted steroids in human choriocarcinoma cell culture.

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, 7-substituted 4,6-androstadiene-3,17-diones were synthesized and demonstrated competitive inhibition of aromatase activity in human placental microsomes. 7-Substituted 1,4,6-androstatriene-3,17-diones demonstrated mechanism-based inhibition of placental aromatase activity. These agents were evaluated for inhibition of aromatase activity in the JAr human choriocarcinoma line. The 7-substituted 4,6-androstadiene-3,17-diones produced dose dependent inhibition of aromatase activity in the cell cultures, with IC50 values ranging from 490 nM to 4.5 microM. However, these agents are less effective when compared to other steroidal inhibitors, such as 7 alpha-thiosubstituted androstenediones. These results on the 7-substituted 4,6-androstadiene-3,17-diones are consistent with the data from biochemical enzyme inhibition studies using human placental aromatase. On the other hand, 7-phenethyl-1,4,6-androstatriene-3,17-dione exhibits greater inhibitory activity, with an IC50 value of 80 nM. Other mechanism-based inhibitors, 7 alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione and 4-hydroxyandrostenedione, also exhibited potent inhibition of aromatase activity in JAr cells. In summary, the most effective B-ring modified steroidal aromatase inhibitors are those derivatives that can project the 7-aryl substituent into the 7 alpha-position.     

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.     

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.     

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.     

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.     

Covalent modification of aromatase by a radiolabeled irreversible inhibitor

7 alpha-Substituted 4-androstene-3,17-diones are effective inhibitors of aromatase. The microsomal enzyme complex has a greater affinity for several of these inhibitors than for the substrate androstenedione, with 7 alpha-(4'amino)phenylthio-4-androstene-3,17-dione being the most potent competitive inhibitor of the series. A potential affinity analog, the bromoacetamide derivative of the amino compound, has been synthesized in both unlabeled and 14C-labeled forms via a condensation of bromoacetic acid with the amino compound using DCC. Inactivation studies with the unlabeled inhibitor showed a time-dependent, first-order inactivation of aromatase enzymatic activity. Androstenedione, when incubated in varying concentrations with the irreversible inhibitor, provided protection from inactivation. Binding studies with radiolabeled inhibitor and microsomal aromatase preparations showed that irreversible binding had occurred. SDS-electrophoresis, followed by fluorography, identified four major microsomal proteins that were radiolabeled, with the protein band at 52,000 mol. wt predominating. Similar studies with a solubilized aromatase preparation decreased the amount of nonspecific binding. Thus, covalent bonds between the irreversible inhibitor and the aromatase cytochrome P450 molecule were formed.     

Effects of aromatase inhibitors on in vitro steroidogenesis by Atlantic salmon (Salmo salar) gonadal and brain tissue

In order to assess the efficacy of selected aromatase inhibitors on Atlantic salmon (Salmo salar) ovarian and brain tissue, in vitro systems were developed for measuring 17beta-estradiol (E(2)) production by these tissues. Isolated vitellogenic follicles, or homogenised whole brains were incubated at 10 degrees C in complete Cortlands solution for 18 or 42 h respectively, and E(2) levels in the medium were determined by RIA. The addition of testosterone to the medium increased E(2) production in all preparations. E(2) production by whole brain homogenate was reduced by co-incubation with the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD), 4-androstene-4-ol-3,17-dione (OHA), aminoglutethimide, fadrozole or miconazole. Fadrozole, ATD, and OHA reduced E(2) production by vitellogenic follicles at a medium concentration of 0.1 microg mL(-1), whereas miconazole was only effective at 10 microg mL(-1). This study demonstrates a simple and rapid screening method for assessing the efficacy of aromatase inhibitors on fish tissues, and that the aromatase inhibitors ATD, OHA and fadrozole are potent inhibitors of both brain and gonadal aromatase in vitro, in Atlantic salmon.     

Synthesis and evaluation of analogues of 4-androstene-3,17-dione as aromatase inhibitors

Twenty-three synthetic analogues of 4-androstene-3,17-dione (androstenedione) have been evaluated as inhibitors of human placental microsomal aromatase enzyme. Among the most potent of these compounds were the 4-hydroxy, 6 alpha-fluoro, 6 beta-fluoro, and 4-fluoroandrostenediones and 4-fluoro-19-nor-4-androstene-3,17-dione. 4-Hydroxy-4-androstene-3,17-dione (4HAD) is an irreversible inhibitor of aromatase in vitro, whereas the four fluoro analogues are reversible inhibitors. 4HAD and 4-fluoro-4-androstene-3,17-dione caused significant regression of the nitrosomethylurea-induced mammary tumor in rats, but the other fluoro derivatives were inactive.     

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.     

Tritium release from [19-3H]-19,19-difluoroandrost-4-ene-3,17-dione during inactivation of aromatase

Aromatase is a cytochrome P-450 enzyme involved in the conversion of androst-4-ene-3,17-dione to estrogen via sequential oxidations at the 19-methyl group. Previous studies from this laboratory showed that 19,19-difluoroandrost-4-ene-3,17-dione (5) is a mechanism-based inactivator of aromatase. The mechanism of inactivation was postulated to involve enzymic oxidation at, and hydrogen loss from, the 19-carbon. The deuteriated analogue 5b has now been synthesized and shown to inactivate aromatase at the same rate as the nondeuteriated parent (5). We conclude that C19-H bond cleavage is not the rate-limiting step in the overall inactivation process caused by 5. [19-3H]-19,19-Difluoroandrost-4-ene-3,17-dione (5b) with specific activity of 31 mCi/mmol was also synthesized to study the release of tritium into solution during the enzyme inactivation process. Incubation of [19-3H]19,19-difluoroandrost-4-ene-3,17-dione with human placental microsomal aromatase at differing protein concentrations resulted in time-dependent NADPH-dependent, and protein-dependent release of tritium. This tritium release is not observed in the presence of (19R)-10 beta-oxiranylestr-4-ene-3,17-dione, a powerful competitive inhibitor of aromatase. We conclude that aromatase attacks the 19-carbon of 19,19-difluoroandrost-4-ene-3,17-dione, as originally postulated.     

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.     

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.     

The time-dependent inactivation of aromatase by 17β-hydroxy-10-methylthioestra-1,4-dien-3-one

17β-Hydroxy-10-methylthioestra-1,4-dien-3-one is an active-site irreversible inhibitor of aromatase, the cytochrome P-450 dependent enzyme responsible for the conversion of androst-4-ene-3,17-dione to estrone. Two time-dependent pathways to inactivation are observed, one of which requires NADPH activation.     

Synthesis and some reactions of 6-bromoandrogens: potential affinity ligand and inactivator of estrogen synthetase.

The synthesis of epimeric 6-bromo-4-androstene-3,17-dione (1a and 1b), 6-bromotestosterone (2a and 2b) and its acetate (3a and 3b), and 6-bromo-16 alpha-acetoxy-4-androstene-3,17-dione (5a and 5b), and 6 beta-bromo-16 alpha-hydroxy-4-androstene-3,17-dione (4) is described. The interconversions among compounds 1, 2, and 3 are also studied. The 6 beta-isomer (1b, 2b, and 3b) was epimerized to the 6 alpha-isomer (1a, 2a and 3a) in carbon tetrachloride or chloroform-methanol (9:1) and the 6 alpha-isomer was isolated by fractional crystallization from the epimeric mixture. 6 alpha-Bromo isomer 1a was also epimerized back to 6 beta-bromo isomer 1b in chloroform-methanol (9:1). Two polymorphic forms of 6 beta-bromotestosterone acetate (3b) were isolated (mp. 114--117 degrees and 138--141 degrees). The 6 beta-bromo isomers were found to be unstable in methanol and decomposed to give 5 alpha-androstane-3,6-dione derivative (6). The results of irreversible inactivation of human placental androgen aromatase with some of these 6-bromoandrogens are discussed.     

Inactivation of aromatase in vitro by 4-hydroxy-4-androstene-3,17-dione and 4-acetoxy-4-androstene-3,17-dione and sustained effects in vivo

4-Hydroxy-4-androstene-3,17-dione (4-OHA) and 4-acetoxy-4-androstene-3,17-dione (4-AcA), in addition to being competitive inhibitors of aromatase, cause time-dependent, irreversible, loss of enzyme activity in both human placental and rat ovarian microsomes. $\text{In vivo}$, treatment of rats with 4-OHA also causes loss of ovarian aromatase activity. To test whether this loss of activity could have $\text{in vivo}$ significance, rats with hormone-dependent, mammary tumors were treated with 4-OHA on alternate weeks. Tumor regression continued to occur during the weeks without treatment. These findings suggest that inactivation of aromatase is important in the mechanism of action of the compounds $\text{in vivo}$.     

Time-dependent aromatase inactivation by 4 beta,5 beta-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.     

Effects of aromatase inhibitors and different doses of testosterone on gonadotropins in one year old male Atlantic salmon (Salmo salar)

The effects of different doses of testosterone (T), the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD) and 4-hydroxy-4-androstene-3,17-dione (4OH), and the combined treatment of T and ATD on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at the onset of puberty in juvenile Atlantic salmon males were investigated. T always increased pituitary LH. Also, ATD increased pituitary LH, though to a lesser extent than T. However, ATD combined with T diminished pituitary LH levels compared to T alone, indicating an aromatase-dependent positive feedback of T on LH in immature males. 4OH, which was less effective than ATD as an aromatase inhibitor, increased LH content. ATD treatment resulted in increased pituitary FSH levels, similar to those of mature controls. Positive effects of ATD on plasma FSH were found, indicating the presence of an aromatase-dependent negative feedback. The 4OH effects on FSH levels were inconsistent. T exerted both positive and negative effects on pituitary FSH and testes growth, depending on dose and season, with the positive effects being more pronounced with the low doses and the negative effects with the high doses. The treatment of T combined with ATD did not affect the positive effect of T alone on pituitary and plasma FSH, indicating the presence of an aromatase-independent positive feedback on FSH. There was a positive correlation between FSH and gonadosomatic index, especially during summer when gonadal development occurs.     

Aromatase inhibition depresses ultrasound production and copulation in male hamsters

Rates of ultrasound production and copulatory behavior were observed in castrated male hamsters maintained on 100 micrograms/day of injected testosterone propionate (TP). Groups matched on their initial levels of behavior received either continued treatment with TP alone, or TP together with 6 mg/day injections of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD). Testing at 11-15 days after the start of these treatments revealed deficits in the sexual behaviors of the subjects in the latter group. Specifically, these males showed lower rates of ultrasound production and intromission during, as opposed to before, treatment with ATD. These results support previous work suggesting that aromatization plays significant roles in the mediation of androgenic effects on both the courtship and copulatory behaviors of male hamsters.