2- and 3-[(Aryl)(azolyl)methyl]indoles as Potential Non-steroidal Aromatase Inhibitors

The present study was designed to follow our pharmacomodulation work in the field of non-steroidal aromatase inhibitors. All target compounds 12a–h and 28a–h were tested in vitro for human placental aromatase inhibition, using testosterone or androstenedione as the substrate for the aromatase enzyme and the IC₅₀ and relative potency to aminoglutethimide data are included. A SAR study indicated that 3-[(4-fluorophenyl)(1H-imidazol-1-yl)methyl]-1-ethyl-2-methyl-1H-indole (28?g) was a highly potent and selective aromatase inhibitor with IC₅₀ value of 0.025?μM. 28?g was also a weak inhibitor of androstenedione synthesis.     

Kinetic properties of aromatase mutants Pro308Phe, Asp309Asn, and Asp309Ala and their interactions with aromatase inhibitors.

Mutant forms of aromatase cytochrome P-450 bearing modifications of amino acid residues Pro308 and Asp309 and expressed in transfected Chinese hamster ovary cells were subjected to kinetic analysis and inhibition studies. The Km for androstenedione for expressed wild type (11.0 +/- 0.3 nM SEM, n = 3) increased 4-, 25- and 31-fold for mutants Pro308Phe, Asp309Asn and Asp309Ala, respectively. There were significant differences in sensitivity among wild type and mutants to highly selective inhibitors of estrogen biosynthesis. 4-Hydroxyandrostenedione (4-OHA) a strong inhibitor of wild type aromatase activity (IC50 = 21 nM and Ki = 10 nM), was even more effective against mutant Pro308Phe (IC50 = 13 nM and Ki = 2.8 nM), but inhibition of mutants Asp309Asn and Asp309Ala was considerably less (IC50 = 345 and 330 nM and Ki = 55 and 79 nM, respectively). Expressed wild type aromatase and Pro308Phe aromatase were strongly inhibited by CGS 16949A (IC50 = 4.0 and 4.6 nM, respectively) whereas mutants Asp309Asn and Asp309Ala were markedly less sensitive (IC50 = 140 and 150 nM, respectively). CGS 18320B produced similar inhibition. Kinetic analyses produced Ki = 0.4 nM for CGS 16949A inhibition of wild type versus 1.1, 37 and 58 nM, respectively, against Pro308Phe, Asp309Asn and Asp309Ala. The results demonstrate significant changes in function resulting from single amino acid modifications of the aromatase enzyme. Our data indicate that mutation in Asp309 creates a major distortion in the substrate binding site, rendering the enzyme much less efficient for androstenedione aromatization. The substitution of Pro308 with Phe produces weaker affinity for androstenedione in the substrate pocket, but this alteration favors 4-OHA binding. Similarly, mutant Pro308Phe exhibits a slightly greater sensitivity to inhibition by CGS 18320B than does the wild type. These results indicate that residues Pro308 and Asp309 play critical roles in determining substrate specificity and catalytic capability in aromatase.     

Synthesis and biological evaluation of 3-(azolylmethyl)-1H-indoles and 3-(alpha-azolylbenzyl)-1H-indoles as selective aromatase inhibitors

This present study identifies a number of azolyl-substituted indoles as potent inhibitors of aromatase. In the sub-series of 3-(azolylmethyl)-1H-indoles, four imidazole derivatives and their triazole analogues were tested. Imidazole derivatives 11 and 14 in which the benzyl moiety was substituted by 2-chloro and 4-cyano groups, respectively, were the most active, with IC50 values ranging between 0.054 and 0.050 microM. In the other sub-series, eight 3-(alpha-azolylbenzyl)-1H-indoles were prepared and tested. Compound 30, the N-ethyl imidazole derivative, proved to be an aromatase inhibitor, showing an IC50 value of 0.052 microM. All target compounds were further evaluated against 17alpha-hydroxylase/C17,20-lyase to determine their selectivity profile.     

Aromatase in the human choriocarcinoma JEG-3: inhibition by R 76 713 in cultured cells and in tumors grown in nude mice

The aromatase enzyme and its inhibition by R 76 713 were characterized in the JEG-3 choriocarcinoma cell line in culture and in JEG-3 tumors grown in nude mice. Optimal cell culture parameters and enzyme reaction conditions for the determination of aromatase activity were established. Under these conditions, in vitro JEG-3 aromatase was inhibited by R 76 713 with IC50-values of 7.6 +/- 0.5 nM and 2.7 +/- 1.1 nM using 500 nM of androstenedione and testosterone as substrate respectively. The Km-value of the aromatase enzyme with androstenedione as substrate was 62 +/- 19 nM; with testosterone as substrate, a value of 166 +/- 27 nM was found. In the presence of increasing concentrations of R 76 713, the Km-values increased while the Vmax remained unchanged. Using androstenedione and testosterone as substrate Lineweaver-Burk analysis of the data showed Ki-values for R 76 713 of 0.43 +/- 0.06 nM and 0.47 +/- 0.39 nM respectively. R 76 713 appeared to competitively inhibit the JEG-3 aromatase. Aromatase could easily be measured in homogenates of JEG-3 tumors grown in nude mice and showed Km-values similar to those found for JEG-3 cells in vitro. IC50-values for inhibition of tumor aromatase by R 76 713 were also similar to those found in cultured cells. Tumor aromatase measured ex vivo, 2 h after a single oral administration of R 76 713 was dose-dependently inhibited. An ED50-value of 0.05 mg/kg was calculated. The JEG-3 choriocarcinoma proved to be a useful aromatase model enabling the comparative study of aromatase inhibition in vitro and in vivo.     

Effects of 10-(2-propynyl)-estr-4-ene-3,17-dione (MDL 18,962)--a mechanism-based irreversible inhibitor of aromatase--in cultured human foreskin fibroblasts

In male subjects, peripheral aromatization of androgens accounts for most of the estrogen production, and skin is an important site of such enzymatic activity. We have studied the effects of a mechanism-based, irreversible aromatase inhibitor, 10-(2-propynyl)-estr-4-ene-3,17-dione (MDL 18,962) on androgen action and metabolism in cultured human foreskin fibroblasts. Cells were incubated simultaneously in the presence of substrate, androstenedione, and inhibitor, MDL 18,962. Aromatase activity was linear with time up to 3 h of incubation at 37 degrees C in the absence and presence of 1.0-10 nM inhibitor. The IC50 for four different cell strains ranged from 4.0 to 8.6 nM MDL 18,962. Kinetic analysis of competitive inhibition by the Eadie-Hofstee method yielded an apparent Ki of 2.75 nM for the inhibitor. Preincubation of cells with MDL 18,962 resulted in irreversible inhibition of aromatase activity which was time- and concentration-dependent. We calculated a Ki of 7.6 nM for MDL 18,962. Preincubation of cells with 25 nM MDL 18,962 suppressed enzyme activity for up to 6 h following removal of the inhibitor, before a return of enzyme activity due to synthesis of new enzyme. MDL 18,962 (0.2-20 microM) did not influence the 5 alpha-reduction of testosterone (200 nM). In addition, binding of dihydrotestosterone (2 nM) to androgen receptors was not affected by MDL 18,962 (25-1000 nM). In summary, MDL 18,962 is a specific, high potency inhibitor of aromatase. By virtue of its high binding affinity to the enzyme active site, it competes very effectively with substrate, resulting in irreversible inactivation of aromatase.     

Synthesis and biological evaluation of 5-[(aryl)(1H-imidazol-1-yl)methyl]-1H-indoles: potent and selective aromatase inhibitors

The synthesis and the aromatase (CYP19) inhibitory activity of 5-[(aryl)(imidazol-1-yl)methyl]-1H-indoles were reported. Among the tested racemate compounds, 5-[(4-chlorophenyl)(1H-imidazol-1-yl)methyl]-1H-indole 8b emerged as a potent CYP19 inhibitor (IC(50)=15.3 nM). Chiral chromatography allowed isolation of the (+) enantiomer 8b2, which was about twice as active as the racemate (IC(50)=9 nM).     

Enzyme inactivation by potential metabolites of an aromatase-activated inhibitor (MDL 18,962)

MDL 18,962, 19-acetylenic androstenedione, is an enzyme-activated inhibitor of estrogen biosynthesis which is in Phase I clinical evaluations as a potential therapeutic agent for estrogen-dependent cancers. 19-Acetylenic analogs corresponding to the major metabolites of androstenedione were synthesized as potential metabolites of MDL 18,962. These compounds were 19-acetylenic testosterone, the product of 17 beta-hydroxy steroid oxidoreductase, 6 beta-hydroxy- and 6-oxo-19-acetylenic androstenedione, products of P450 steroid 6 beta-hydroxylase and alcohol dehydrogenase, respectively. All of these analogs showed time-dependent inactivation of human placental aromatase activity. The time-dependent Ki and t1/2 at infinite inhibitor concentration (tau 50) were 4.3 nM, 12.0 min for MDL 18,962; 28 nM, 7.8 min for 17-hydroxy analog; 13 nM, 37 min for 6 beta-hydroxy analog; and 167 nM, 6.1 min for the 6-oxo analog. The 19-acetylenic testosterone, a confirmed metabolite from primate studies, was 25% as efficient as MDL 18,962 for aromatase inactivation, while 6 beta-hydroxy- and 6-oxo analogs were 11% and 5%, respectively as efficient as their parent compound. These data indicate that first-pass metabolism of MDL 18,962 does not cause an obligatory loss of time-dependent inhibition of human aromatase activity.     

Imidazole antimycotics: selective inhibitors of steroid aromatization and progesterone hydroxylation

Econazole, imazalil, and prochloraz, which have broad spectrum antimycotic activity, are shown to be potent inhibitors of steroid aromatase activity of human placental microsomes. The IC50 values for the inhibition of aromatase activity by econazole, imazalil, miconazole, prochloraz, clotrimazole, ketoconazole, and aminoglutethimide are 0.03, 0.15, 0.6, 0.7, 1.8, 60, and 45 microM, respectively. Econazole and 4-hydroxyandrostenedione also inhibit the steroid aromatase activity of human fetal liver, a finding which suggests that extraplacental aromatase may have many similarities to the placental enzyme. Econazole is a more effective inhibitor of placental aromatization of 19-hydroxyandrostenedione than of androstenedione. This observation is consistent with the competitive nature of the inhibition of aromatase by imidazole antimycotic agents and the reduced affinity of the placental aromatase enzyme for 19-hydroxyandrostenedione compared to androstenedione. The effectiveness of these imidazole antimycotic agents to inhibit the multiple hydroxylations of progesterone which are catalyzed by human fetal adrenal microsomes is also defined. While all of the imidazole antimycotic agents are potent inhibitors of the 16 alpha-, 17 alpha-, and 21-hydroxylations of progesterone, selective inhibitory profiles are apparent. Ketoconazole is a most potent inhibitor of human fetal adrenal progesterone 16 alpha- and 17 alpha-hydroxylases while clotrimazole and imazalil are the most potent inhibitors of progesterone 21-hydroxylase. These results are strongly supportive that imidazole drugs are selective inhibitors not only of steroid aromatase but also of other microsomal steroid hydroxylases.     

Lead optimization of 7-benzyloxy 2-(4'-pyridylmethyl)thio isoflavone aromatase inhibitors

Aromatase, the enzyme responsible for estrogen biosynthesis, is a particularly attractive target in the treatment of hormone-dependent breast cancer. The synthesis and biological evaluation of a series of 2-(4'-pyridylmethyl)thio, 7-alkyl- or aryl-substituted isoflavones as potential aromatase inhibitors are described. The isoflavone derivatives demonstrate IC(50) values from 79 to 553 nM and compete with the endogenous substrate, androstenedione. Data supporting the ability of these analogs to suppress aromatase enzyme activity in the SK-BR-3 breast cancer cell line are also presented.     

Comparison of aromatase activity in human prostatic, testicular and placental tissues.

The aromatase enzyme was quantified by the release of tritiated water from [1 beta-3H] androstenedione. Tritiated water was released by the crude homogenates in 4 of 18 samples of benign prostatic hyperplasia tissue and one of 5 samples of prostate carcinoma tissue. However, this apparent aromatase activity was not inhibited by 4-hydroxyandrostenedione (0.5 and 5.0 microM), and none of the particulate fractions (100,000 g pellet) prepared from each of the prostatic tissues exhibited aromatase activity. Particulate fractions from rat ovary (n = 3) and human testes (n = 6) displayed significant aromatase activity (mean values of 9.9 and 0.033 nmol estrone formed/g protein/h, respectively). The testicular aromatase was inhibited by aminoglutethimide, 4-hydroxyandrostenedione and CGS 16949A with IC50 values of 6.4, 0.17 and 0.0017 microM, respectively. These are of a similar order to values obtained with the aromatase enzyme from human placental microsomes (14, 0.43 and 0.0075 microM, respectively).     

Inhibition of human cytochrome P450 aromatase activity by butyltins

Organotin compounds are widely used as antifouling agents and bioaccumulate in the food chain. Tributyltin chloride (TBT) has been shown to induce imposex in female gastropods. On the basis of this observation it has been suggested that TBT acts as an endocrine disrupter inhibiting the conversion of androgens to estrogens mediated by the aromatase cytochrome P450 enzyme. However, to date, the molecular basis of TBT-induced imposex and in particular its putative inhibitory effects on human aromatase cytochrome P450 activity have not been investigated. Therefore, we examined the effects of the organotin compounds tetrabutyltin (TTBT), TBT, dibutyltin dichloride (DBT) and monobutyltin trichloride (MBT) on human placental aromatase activity. TBT was found to be a partial competitive inhibitor of aromatase activity with an IC(50) value of 6.2 microM with 0.1 microM androstenedione as substrate. TBT impaired the affinity of the aromatase to androstenedione but did not affect electron transfer from NADPH to aromatase via inhibiting the NADPH reductase. DBT acted as a partial but less potent inhibitor of human aromatase activity (65% residual activity), whereas TTBT and MBT had no effect. The residual activity of TBT-saturated aromatase was 37%. In contrast, human 3beta-HSD type I activity was only moderately inhibited by TBT (80% residual activity). Moreover, neither TTBT or DBT nor MBT inhibited the 3beta-HSD type I activity. Together, these results suggest that the environmental pollutants TBT and DBT, both present in marine organisms, textile and plastic products, may have specific impacts on the metabolism of sex hormones in humans.     

Aromatase inhibition by R 76 713: a kinetic analysis in rat ovarian homogenates

Reaction kinetics of the aromatase enzyme and of a new nonsteroidal aromatase inhibitor, R 76 713 (6-[(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)-methyl]-1-methyl-1H- benzotriazole), were studied in ovarian homogenates obtained from pregnant mare's serum gonadotropin (PMSG)-injected female Wistar rats. The Km (Michaelis constant) of the aromatase enzyme with androstenedione as the substrate was 47 +/- 13 nM; for testosterone as the substrate, a value of 159 +/- 10 nM was found. In the presence of increasing concentrations of R 76 713, the Km increased while the Vmax (maximal velocity of enzyme-catalyzed reaction) remained unchanged. Using androstenedione and testosterone as the substrate, Lineweaver-Burk analysis of the data showed a Ki (dissociation constant of the enzyme-inhibitor complex) for R 76 713 of 0.7 +/- 0.3 nM and 1.6 +/- 0.4 nM, respectively. R 76 713 appeared to competitively inhibit the rat ovarian aromatase.     

Effect of ketoconazole on human ovarian C17,20-desmolase and aromatase

Ketoconazole, an imidazole antimycotic drug, inhibits steroid biosynthesis in adrenal and testicular tissue by blocking cytochrome P-450 dependent enzymes. To study the effect of ketoconazole on steroid biosynthesis in the human ovary we incubated human ovarian tissue (mainly theca cells) or granulosa cells with radiolabeled precursors and increasing concentrations of ketoconazole. After incubation, steroids were extracted and separated by thin layer chromatography (TLC). Activity of C17,20-desmolase and aromatase was estimated by measuring the amount of their radioactive products with liquid scintillation counting. After incubation of ovarian tissue with [3H]17-hydroxyprogesterone the production of [3H]androstenedione was reduced by increasing concentrations of ketoconazole (0-200 microM) to a minimum of 31% of basal production. This indicates a strong inhibition of ovarian C17,20-desmolase by ketoconazole with a 50% inhibiting concentration (IC50) of 23 microM. After incubation of human granulosa cells with ketoconazole (0-2000 microM) and [3H]androstenedione the production of [3H]estrone and [3H]estradiol was suppressed to minimally 37 and 35% of basal values, indicating a significant inhibition of ovarian aromatase. IC50-values were 105 microM ketoconazole for estradiol and 130 microM for estrone. In conclusion, ketoconazole was shown to inhibit human ovarian C17,20-desmolase and aromatase in vitro. As in human adrenals and testes ovarian C17,20-desmolase seems to be most sensitive to the inhibitory effect of ketoconazole.     

Inhibition of human placental aromatase in a perfusion model. Comparison with kinetic, cell-free experiments

In vitro perfusion of human placenta was evaluated for characterization of aromatase inhibitors. The results were compared with those in kinetic experiments in cell-free system. Inhibition constants (Ki) were determined by measuring the release of tritiated water during coincubation of human placenta microsomes with varying amounts of [1 beta,2 beta 3H]androstenedione and inhibitor in the presence of NADPH-generating system. Irreversible inactivation constants (Kinact) were determined in a similar manner following preincubation of the microsomes with different amounts of inhibitor for varying times. Lineweaver-Burk plots indicated a competitive type of inhibition with Ki values of 37 nM for 4-hydroxy-androstenedione, 3,700 nM for testolactone, 15 nM for 1-methyl-androsta-1,4-diene-3,17-dione, and 7.5 nM for 19-azido-androstenedione. Additionally, irreversible enzyme inactivation by all four substances could be demonstrated with Kinact values of 3.64 x 10(-3), 0.57 x 10(-3), 0.34 x 10(-3), and 0.69 x 10(-3)sec-1, respectively. Perfusion of a single cotyledon of human term placenta was performed by infusing medium through catheters placed in a fetal artery and in the maternal intervillous space. Perfused medium was collected from a cannulated fetal vein and from the maternal basal plate. The medium was supplemented with [3H]androstenedione (4.2 nM) and inhibitor. The perfusates were analyzed for their [3H]estrone and estradiol content following phenolic partition and Sephadex-LH 20 chromatography. The main results were, (1) the recovery of labelled steroids increased rapidly after perfusion started and reached a plateau within 60 min, when 55 and 30% (mean values) of the infused radioactivity were recovered in the fetal and maternal perfusates, respectively, (2) similar amounts of estrone and estradiol were found in both effluates, whereas androgens (mainly androstenedione and lower amounts of 5 alpha-androstane-3,17-dione) were found nearly exclusively in the fetal perfusate, (3) formation of estrogens (estrone + estradiol) reached a plateau within 20 min of perfusion. (4) The percentage of estrogens formed was not changed by increasing androstenedione concentration in the perfusion medium unless this concentration exceeded 3.5 microM indicating limited capacity of aromatase. (5) The four aromatase inhibitors reduced estrogen formation by 50% at concentrations about 100-fold of their Ki determined in the cell-free system, (6) irreversible aromatase inhibition could not be demonstrated in the perfusion model. It was concluded that the human placenta perfusion model can be successfully used to evaluate aromatase inhibitors.     

Growth inhibition of estrogen receptor-positive and aromatase-positive human breast cancer cells in monolayer and spheroid cultures by letrozole, anastrozole, and tamoxifen

Two third-generation aromatase inhibitors, letrozole and anastrozole, and the antiestrogen tamoxifen, were compared for growth-inhibiting activity in two estrogen receptor (ER)-positive aromatase-overexpressing human breast cancer cell lines, MCF-7aro and T-47Daro. Inhibition of hormone (1 nM testosterone)-stimulated proliferation was evaluated in both monolayer cultures and in three-dimensional spheroid cultures. Letrozole and anastrozole were also compared for effectiveness of aromatase inhibition, and relative affinity for aromatase, under both monolayer and spheroid growth conditions. Letrozole was an effective inhibitor of MCF-7aro monolayer cell proliferation, with an estimated 50% inhibitory concentration (IC50) of 50-100 nM, whereas an IC50 was not reached with anastrozole at any concentration tested (100-500 nM). An IC50 of tamoxifen was 1000 nM. Proliferation of T-47Daro monolayer cells was more sensitive to inhibition by all three agents; as with MCF-7aro cells, letrozole was the most effective inhibitor. MCF-7aro spheroids were slightly less sensitive than monolayer cells proliferation-inhibiting effects of letrozole (IC50 about 200 nM), and there was no significant inhibition with 100-200 nM anastrozole or 200-1000 nM tamoxifen. Letrozole and anastrozole significantly inhibited T-47Daro spheroid cell proliferation, at 15-25 and 50 nM, respectively, consistent with the greater sensitivity of T-47Daro monolayer cells to inhibition of proliferation by these agents. Tamoxifen failed to significantly inhibit T-47Daro spheroid cell proliferation over a 100-500 nM concentration range. Determination of aromatase inhibition in monolayers of both cell lines by a direct-access microsomal assay and an intact-cell assay revealed that letrozole was more active than anastrozole in monolayers of both cell lines and in both assays. In MCF-7aro spheroids following cell lysis, only letrozole significantly inhibited aromatase activity, supporting the conclusion that letrozole binds stronger to aromatase than anastrozole does. Our results demonstrate that MCF-7aro and T-47Daro spheroids could be a suitable model for evaluation of growth-inhibitory effects of agents used in hormonal therapy of breast cancer.     

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.     

Biological characterization of A-ring steroids

Hydroxylated 2,19-methylene-bridged androstenediones were designed as potential mimics of enzyme oxidized intermediates of androstenedione. These compounds exhibited competitive inhibition with low micromolar affinities for aromatase. These inhibitory constants (K_i values) were 10 times greater than the 2,19-methylene-bridged androstenedione constant (K_i = 35–70 nM). However, expansion of the 2,19-carbon bridge to ethylene increased aromatase affinity by 10-fold (K_i = 2 nM). Substitution pf a methylene group with oxygen and sulfur in this expanded bridge resulted in K_i values of 7 and 20 nM, respectively. When the substituent was an NH group, the apparent inhibitory kinetics changed from competitive to uncompetitive. All of these analogs exhibited time-dependent inhibition of aromatase activity following preincubation of the inhibitor with human placental microsomes prior to measuring residual enzyme activity. Part of this inhibition was NADPH cofactor-dependent for the 2,19-methyleneoxy- but not for the 2,19-ethylene-bridged androstenedione. The time-dependent inhibition for these four analogs was very rapid since they exhibited τ₅₀ values, the t_1/2 for enzyme inhibition at infinite inhibitor concentration, of 1 to 3 min. These A-ring-bridged androstenedione analogs represent a novel series of potent steroidal aromatase inhibitors. The restrained A-ring bridge containing CH₂, O, S, or NH could effectively coordinate with the heme of the P450 aromatase to allow the tight-binding affinities reflected by their nanomolar K_i values.     

Structure-activity relationships of 2-, 4-, or 6-substituted estrogens as aromatase inhibitors

Aromatase, which is responsible for the conversion of androgens to estrogens, is a potential therapeutic target for the selective lowering of estrogen levels in patients with estrogen-dependent breast cancer. To develop a novel class of aromatase inhibitors, we tested series of 2- and 4-substituted (halogeno, methyl, formyl, methoxy, nitro, and amino) estrones (7 and 9), as well as series of 6alpha- and 6beta-substituted (alkyl, phenalkyl, and alkoxy) estrones (13 and 14), and their estradiol analogs (8, 10, 11, and 12) as aromatase inhibitors. All of the inhibitors examined blocked the androstenedione aromatization in a competitive manner. Introduction of halogeno and methyl functions at C-2 of estrone as well as that of a phenalkyl or methyl function at the C-6alpha or C-6beta position markedly increased affinity to aromatase (apparent K(i) value=0.10-0.66 microM for the inhibitors versus 2.5 microM for estrone). 6alpha-Phenylestrone (13c) was the most powerful inhibitor among the estrogens studied, and its affinity was comparable to that of the androgen substrate androstenedione. Estradiol analogs were much weaker inhibitors than the corresponding estrone compounds in each series, indicating that the 17-carbonyl group plays a critical role in the formation of a thermodynamically stable enzyme-inhibitor complex.     

Pseudoaromatase in circulating lymphocytes

A variety of data suggesting a relationship between estrogens and the immune system prompted a study of aromatase activity in blood lymphocytes. A tritiated water aromatase assay detected activity of 1.9 to 25.1 pmol/g protein/h in 14 samples of human lymphocytes. To confirm these results, additional tritiated water and product isolation assays were performed on a large pool of lymphocytes obtained from 4 U of blood. An assay using [1β-³H]androstenedione generated high apparent aromatase activity, 943 pmol/g protein/h, but this activity could not be blocked by the aromatase inhibitor, CGS 16949A. More direct methods of evaluation yielded the following results: (1) PCR demonstrated no aromatase mRNA production in lymphocytes; (2) direct product isolation using [1,2,6,7-³H]androstenedione yielded insignificant production of estrone and estradiol; (3) immunostaining of fixed lymphocyte smears with a polyclonal antibody to aromatase yielded equivocal results. These data suggest the presence of pseudoaromatase in blood lymphocytes. Since circulating lymphocyte pseudoaromatase levels can be correlated with various factors in patients, such as age, menopausal status, and glucose ingestion, further studies of this activity are warranted.     

Enzyme interactions of 2,10-ethanoandrostene-3,17-dione: aromatase and 17 beta-hydroxysteroid dehydrogenase

An analogue of androstenedione containing an ethano bridge between carbons 2 and 10 of the A ring of the steroid, 1, has been evaluated as an inhibitor and a possible substrate of human placental aromatase. This compound was found to be a competitive inhibitor versus androstenedione (Kis = 25 +/- 2 nM) of the aromatase activity. Analyses of the incubation mixtures of 1 with human placental microsomes and NADPH by GC-MS indicated the formation of a new compound having an increase in molecular weight of 2 mass units (300 m.u.) from that of the parent steroid (298 m.u.). Subsequent analyses of incubations of 1 with an isolated 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) from Pseudomonas testosteronii in the presence of NADPH resulted in the formation of a new compound having the same retention time and molecular mass as that found for the product from the placental microsome incubation. Consequently, steroid 1 is both an inhibitor of human placental aromatase and a substrate for 17 beta-HSD.