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Query: UMLS:C0338671 (Steroids)
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Reactions of androst-4-ene-3,6,17-trione (1) and its 19-hydroxy or 19-oxo derivative (2 or 3), suicide substrates of aromatase, with thiols were initially studied. Treatment of 4-ene-3,6-diones 1-3 with benzyl-mercaptan in MeOH at room temperature gave the corresponding 4 alpha-benzylthio-5 alpha-androstane-3,6-diones (4-6) as the major products in 24-80% yields. The C18 steroid, estr-5(10)-ene-3,6,17-trione (7), was also isolated on the treatment of 19-oxo steroid 3. Oxidation with NaIO4 and reduction with Raney Ni of the adducts gave the corresponding 4-ene-3,6-dione and desulfurized products, respectively. The results show that 19-oxygenated steroids 2 and 3 react with a thiol in a 1,4-addition manner. By means of PM3 molecular orbital calculations, the conformational features of the 19-oxygen functions of 4-ene-3,6-diones 2 and 3, 5 alpha-3,6-diones 10 and 11, and their 4 alpha-methylthio derivatives 14 and 15, model compounds of 1,4-adducts 5 and 6, were determined. In the compounds examined, the 19-hydroxy steroids favor a conformation having the hydroxyl group above the A-ring, whereas the 19-oxo substituent is oriented in the out-of-ring position (not above either A- or B-ring). These calculations suggest that compound 1 would inactivate aromatase by the same steric course of the oxygenation at C-19 as that of the natural substrate, androstenedione.
Steroids 1993 Sep
PMID:1,4-addition reaction with thiols and conformational analysis with PM3 molecular orbital calculations of 19-oxygenated androst-4-ene-3,6,17-triones. 823 28

Exemestane (6-methylenandrosta-1,4-diene-3,17-dione; FCE 24304) is an orally active irreversible aromatase inhibitor which is in phase II clinical evaluation for the potential therapy of postmenopausal breast cancer. A series of exemestane analogs, with modifications at the 6-methylene group and with additional reduction at the 17-keto group, were synthesized as potential metabolites and tested in vitro for their effect on human placental aromatase. All these new analogs were found to be less potent in inhibiting aromatase than exemestane. The most effective compound was the 17 beta-hydroxy-derivative (compound 2), which is 2.6-fold less potent than exemestane [50% inhibitory concentration (IC50) 69 and 27 nM, respectively]. The various C-6 modified derivatives of the 17-oxo series were found to inhibit the aromatase enzyme in the following descending order: 6-methylene (exemestane) > 6-spirooxirane (6) > 6 beta-hydroxymethyl (11) > 6-hydroxymethyl (7) > 6 beta-carboxy (13), showing IC50 values of 27, 206, 295, 2,300, and 7,200 nM, respectively. The 17 beta-hydroxy analogs of some of the above mentioned compounds were also synthesized (3,4,12) and found to be 3-8-fold less potent than the corresponding 17-keto analogs.
Steroids 1993 Nov
PMID:Synthesis and aromatase inhibition by potential metabolites of exemestane (6-methylenandrosta-1,4-diene-3,17-dione). 827 15

Diastereomeric (19S)- and (19R)-19-ethynyl-19-acetoxy derivatives of androst-4-ene-3,6,17-trione (AT) (9 and 10) and 19,19-difluoro AT (12) were synthesized. The 19,19-difluoro compound (12) was an effective competitive inhibitor of human placental aromatase with an inhibition constant (ki) of 1.8 microM but the acetylenic 9 and 10 were poor inhibitors of the enzyme with k(is) of 75 and 67 microM, respectively. Inhibitor 12 caused a time-dependent, biphasic loss of aromatase activity in the presence of reduced nicotinamide-adenine-dinucleotide phosphate (NADPH) in air, whereas the other two caused a time-dependent, pseudo-first-order inactivation of the activity with rate constants for inactivation of 0.250, 0.077, and 0.065 min-1 for steroids 12, 9, and 10. NADPH was required for the time-dependent inactivation, and the substrate androst-4-ene-3,17-dione prevented it. L-Cysteine did not protect aromatase from the inactivation.
Steroids 1993 Jan
PMID:A time-dependent inactivation of aromatase by 19-substituted androst-4-ene-3,6,17-triones. 843 Apr 44

To gain further insight on the relationship between 6-alkylandrost-4-ene-3,17-diones and their aromatase inhibition activity, a series of alkyl steroids with long alkyl chains (n-pentyl, n-hexyl, or n-octyl) at C-6 alpha and 6 beta were synthesized. All of the steroids studied inhibited human placental aromatase in a competitive manner with apparent Ki values ranging from 2.8 to 80 nM. The 6 beta-pentyl analog 4a (Ki = 2.8 nM) was the most potent inhibitor. The inhibitory activities of the 6 beta-alkyl steroids 4 were more powerful than those of the corresponding 6 alpha-isomers 5. The addition of one methylene unit to the 6 alpha- and 6 beta-n-butyl moieties of androst-4-ene-3,17-dione markedly increased the affinity to aromatase, whereas further elongation of the n-pentyl group decreased affinity in relation to the carbon number of the alkyl chain. These results, along with molecular modeling with the PM3 method, suggest that the increased affinities of the pentyl steroids 4a and 5a may essentially depend on the formation of thermodynamically stable enzyme-inhibitor complex in the hydrophobic binding pocket.
Steroids 1995 Aug
PMID:Further studies on 6-alkylandrost-4-ene-3,17-diones as aromatase inhibitors: elongation of the 6-alkyl chain. 853 92

We produced a murine monoclonal antibody (MAb) to human placental aromatase cytochrome P450. This MAb, designated MAb3-2C2, was selected on its ability to suppress aromatase activity. The specificity of this MAb was assessed by selective immunoprecipitation of 125I-labeled aromatase cytochrome P450 as well as by the identification of a 55-kDa protein, which was enriched and purified by immunoaffinity chromatography on a MAb-coupled Sepharose 4B column. The MAb was able to suppress both human placental and ovarian microsomal aromatase. Species differences of aromatase were recognized by MAb3-2C2 on the basis of differential immunosuppression of aromatase activity. The antibody had no effect on non-aromatase cytochrome P450s. MAb3-2C2 gave negative results with human placental aromatase P450 in the Western blot analysis. The data presented indicate that MAb3-2C2 is specific for aromatase cytochrome P450 and that its epitope is located in a fragile tertiary conformation of the enzyme, thus making it capable of sensitively affecting catalysis.
Steroids 1996 Mar
PMID:Preparation of an activity-inhibiting monoclonal antibody against human placental aromatase cytochrome P450. 885 29

The novel 10 beta-(1'-azirinyl)estr-4-en-3,17-dione (10) has been synthesized from 19-methyl-19-methiodide salt intermediates 15 and 16 by a modified Neber reaction. Four other hitherto undescribed 10 beta-1'-azirinyl steroids 9, 12, 17, and 18 were also synthesized. Compound 10 inhibited human placental aromatase in vitro (IC50 value = 5.5 microM) but was not as potent as the related (19R)-10 beta-aziridine (1).
Steroids 1996 Mar
PMID:Synthesis of 10 beta-(1'-azirinyl)estr-4-en-3,17-dione as an aromatase inhibitor. 885 31

Aromatase (CYP19) mRNA is induced by follicle-stimulating hormone (FSH) in granulosa cells of preovulatory follicles and subsequently is rapidly diminished as a consequence of the luteinizing hormone (LH) surge. Primary cultures of rat granulosa cells were used to identify some of the cellular mechanisms by which FSH increases and LH decreases steady-state levels of aromatase mRNA. Induction of aromatase mRNA by FSH was increased by cycloheximide but was blocked by alpha-amanitin and the C-kinase activators gonadotropin-releasing hormone (GnRH) and phorbol 12-myristate 13-acetate (PMA). In contrast, the decrease in steady-state levels of aromatase mRNA by LH was mimicked by A-kinase (forskolin) and C-kinase (PMA or GnRH) activators. The decrease in aromatase mRNA was associated with decreased amounts of mRNA and protein for steroidogenic factor-1 (SF-1), a nuclear orphan receptor that binds and trans-activates the aromatase promoter, and with the A-kinase subunit type II (RII beta), which is required for mediating cAMP action in these cells. The down-regulation of aromatase, SF-1, and RII beta by each kinase activator and alpha-amanitin was prevented by cycloheximide when the drug was added in combination with the activator. If, however, cycloheximide was added 2 h after PMA (or LH), the drug did not prevent the rapid loss of mRNA. When granulosa cells were transfected with an aromatase CAT transgene, CAT activity was stimulated 10- to 20-fold by FSH and forskolin but not by PMA. Taken together, these results indicate that the A-kinase but not the C-kinase pathway can trans-activate the aromatase gene in immature granulosa cells, whereas the C-kinase, as well as A-kinase pathways, mimic the LH surge to decrease aromatase mRNA in preovulatory cells. By increasing degradation of aromatase mRNA and by inhibiting transcription, the LH surge rapidly terminates the granulosa cell pattern of gene expression while reprogramming the cells to express genes associated with ovulation and luteinization.
Steroids 1997 Jan
PMID:Expression of aromatase in the ovary: down-regulation of mRNA by the ovulatory luteinizing hormone surge. 902 37

The leading explanation of temperature-dependent sex determination (TSD) in reptiles postulates that (1) ovarian differentiation is directed by estrogen and that (2) estrogen is synthesized in the developing gonad following induction of aromatase expression. However, the source of steroid substrate for aromatization has not yet been identified. In addition, sex ratios vary as a function of clutch, but such biases are as yet unexplained. To address these issues, we measured estradiol, testosterone, and androstenedione in yolks of the American alligator (Alligator mississippiensis) before, during, and after the period of gonadal differentiation in this TSD species. Eggs were collected from a wild population in Louisiana and were incubated at male- and female-determining constant temperatures in the lab, as well as at intermediate temperatures that produced both sexes. Steroids were assayed in yolk extracts after celite column chromatography. All three steroids were found to be in the range of nanograms/gram of yolk at stage 16. Androstenedione was the predominant steroid, 2- to 3-fold higher in concentration than estradiol and 15- to 20-fold higher than testosterone. The levels of these steroids declined (5- to 30-fold) between stages 16 and 25, most markedly between stages 21 and 23, regardless of incubation temperature. The chronology of this sharp decline in steroid levels in our study coincides with the timing of gonadal differentiation in this species, between stages 21 to 23 based on previous reports. Estradiol levels in yolks differed by 3-fold in some clutches relative to others, whereas, no clutch differences were apparent for either androstenedione or testosterone. These data demonstrate that alligator yolk contains high concentrations of two steroid substrates utilized for estrogen synthesis, as well as significant quantities of estradiol itself. We hypothesize that estradiol levels in yolk provide a steroid background, variable among and within clutches, on which gonadal development is initiated and proceeds. As a consequence, we suggest that yolk provides an epigenetic maternal contribution that modulates the effect of incubation temperature on hatchling sex.
 ...
PMID:Yolk steroids decline during sexual differentiation in the alligator. 924 27

To gain further insight into the mechanism for inactivation of aromatase by androst-5-ene-7,17-dione (1) and its 19-nor analog 4, 10 beta-oxygenated steroids 5 and 6, delta 1(10)-steroid 7, and 19-oxo-5 beta,6 beta-epoxy compound 8 were synthesized and tested for their ability to inhibit aromatase in human placental microsomes. All of the steroids studied inhibited the enzyme in a competitive manner with apparent Ki values ranging from 1.1 to 35 microM. The delta 1(10)-compound 7 was the most potent inhibitor among them. All of the inhibitors caused a time-dependent inactivation of aromatase in the presence of NADPH in air with the kinact values ranging from 0.036 to 0.190 min-1. The substrate androstenedione protected the inactivation, but a nucleophile, L-cysteine, did not, in each case. In contrast, each inhibitor did not cause the time-dependent inactivation in the absence of NADPH. These results show that the 5 beta,6 beta-epoxide 8 and/or the dienone 7 are not a reactive electrophile involved in the irreversible binding to the active site of aromatase during the mechanism-based inactivation caused by the suicide substrates 1 and/or 4.
Steroids 1997 Jul
PMID:Studies directed toward a mechanistic evaluation of aromatase inhibition by androst-5-ene-7,17-dione. Time-dependent inactivation by the 19-nor and 5 beta, 6 beta-epoxy derivatives. 925 90

Two series of 6-alkylandrosta-4,6-diene-3,17-diones (5) and their 1,4,6-triene analogs 6 were synthesized as aromatase inhibitors to gain insight into the structure-activity relationship between varying the 6-n-alkyl substituents (C1-C7) and inhibitory activity. All of the steroids synthesized were extremely powerful competitive inhibitors of aromatase in human placental microsomes, with apparent Ki values for the 6-alkyl-4,6-diene steroids 5 ranging from 17 to 36 nM and with those for the 1,4,6-triene steroids 6 ranging from 2.5 to 58 nM. The 6-ethyl-1,4,6-triene compound 6b (Ki = 2.5 nM) was the most potent inhibitor among them. The 6-alkyl-1,4,6-triene steroids 6, except for the 6-methyl analog 6a, and higher affinity for aromatase than the natural substrate androstenedione (K(m) = 24 nM), and their inhibitory activities were more potent than the corresponding 4,6-diene steroids 5. In a series of the 4,6-diene steroids 5, compounds 5c-f with the n-alkyl chain substituents (C3 to C6) also had slightly higher affinity than androstenedione for dromatase. All of the 1,4,6-triene steroids 6 inactivated aromatase in a time-dependent manner, with k(inact) values ranging from 0.021 to 0.074 min-1; in contrast, the 4,6-diene analogs 5 did not. The inactivation was prevented by androstenedione, and no significant effect of L-cysteine on the inactivation was observed in each case. These results indicate that the length of the n-alkyl substituent at C-6 of androsta-1,4,6-triene-3,17-dione (6h), rather than its 4,6-diene analog 5h, plays a critical role in tight binding to the active site of aromatase. No significant correlation was observed between affinity for the enzyme and the inactivation ability of the 6-alkyl-1,4,6-trienes.
Steroids
PMID:6-Alkylandrosta-4,6-diene-3,17-diones and their 1,4,6-triene analogs as aromatase inhibitors. Structure-activity relationships. 929 34


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