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The biosynthesis of non-aromatic 19-norsteroids has been studied using primary cultures of porcine granulosa cells. Formation of 5(10)-estrene-3 beta,17 beta-diol, a novel 19-norsteroid, from androstenedione and 19-hydroxyandrostenedione by porcine granulosa cells is reported for the first time. The structure was deduced from (i) comparison of its elution times on C18 reverse phase HPLC with authentic 5(10)-estrene-3 beta,17 beta-diol (ii) identification with 5(10)-estrene-3 beta,17 beta-diol-diacetate after acetylation (iii) oxidation/acid catalysed isomerization to 19-norandrostenedione. Serum or serum plus FSH significantly stimulated (seven fold increase) formation of 5(10)-estrene-3 beta,17 beta-diol from androstenedione and 19-hydroxyandrostenedione. Formation of 5(10)-estrene-3 beta,17 beta-diol from both substrates was significantly (p less than 0.01) reduced by the aromatase inhibitors 4-hydroxyandrostenedione (15 microM) and aminoglutethimide phosphate (10(-4)M). These results suggest that 5(10)-estrene-3 beta,17 beta-diol (and 19-norandrostenedione) may be formed by enzymes similar to the aromatase complex required for estradiol-17 beta biosynthesis. 5(10)-Estrene-3 beta,17 beta-diol is converted by granulosa cells to four metabolites. 19-Norandrostenedione was identified by crystallization to constant specific activity; 19-nortestosterone is a minor product. Production of 19-norandrostenedione and 19-nortestosterone indicates that granulosa cells possess the enzymes necessary for the transformation of 5(10)-estrene-3 beta,17 beta-diol and other 3-hydroxy-5(10)-estrenes to 19-nor-4-ene-3-ketosteroids. The formation of 5(10)-estrene-3 beta,17 beta-diol and 19-norandrostenedione as substantial metabolites of androstenedione suggest a physiological role for these 19-norsteroids in ovarian follicular development.
The crystal structure of 10 beta-hydroperoxy-4-estrene-3,17-dione (10 beta-OOH) was determined, and its inhibition of human placental aromatase was investigated. In the absence of added NADPH, 10 beta-OOH caused a time-dependent loss of aromatase activity (e.g., 50% loss after 90 s with 2.16 microM 10 beta-OOH). Protection against this loss of activity was provided when a substrate, androstenedione, was included in the incubation. Centrifugation and resuspension of the 10 beta-OOH-treated microsomes in fresh buffer failed to restore the activity, but partial recovery could be effected by dithiothreitol. Experiments to detect destruction of aromatase protoheme were done but were inconclusive. In the presence of NADPH, 10 beta-OOH did not cause a time-dependent loss of activity but was instead a competitive inhibitor (Ki = 330 nM) of androstenedione (Km = 21 nM) aromatization. The added NADPH was not utilized for the aromatization of 10 beta-OOH to estrogens, and enhanced reduction of 10 beta-OOH to 10 beta-hydroxy-4-estrene-3,17-dione could not be detected. In addition, microsomes alone were incapable of using 10 beta-OOH to support the aromatization of androstenedione. Cumene hydroperoxide and H2O2 were also investigated as inactivators of aromatase. Losses of activity comparable to those found for 10 beta-OOH could only be observed at 500-1000-fold higher concentrations of these agents, and no protection was provided by either androstenedione or NADPH. Extensive destruction of microsomal protoheme was found with these nonsteroidal agents.
Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity-labeled at pH 6.3 by 3-bromo[2'-14C]acetoxyestrone and 12 beta-bromo-[2'-14C] acetoxy-4-estrene-3,17-dione (both are substrates) in separate incubations. The affinity-alkylated enzyme samples were then treated separately as described below. Amino acid compositions of both samples revealed radioactive 3-carboxymethylhistidine. Tryptic digests of each sample were prepared, applied to Sephadex G-50, and 3-carboxymethylhistidine-bearing fractions identified. These peptides were further purified by cation exchange chromatography, gel filtration, and paper electrophoresis. The purified, 3-carboxymethylhistidine-bearing peptides labeled by the two steroids had identical electrophoretic mobilities at pH 6.5, 3.5, and 1.9. The amino acid sequence of the radioactive peptide alkylated by 3-bromo[2'-14C]acetoxyesterone was determined as: Leu-Ala-3-[14C]CmHis-Ser-Lys. The smaller quantity of peptide obtained from the inactivation with 12 beta-bromo[2'-14C]acetoxy-4-estrene-3,17-dione precluded the determination of its complete sequence. However, the first 3 residues were found to be Leu-Ala-3-[14C]CmHis and the amino acid composition showed that serine and lysine were also present. It is concluded that the steroid-binding site of human placental estradiol 17 beta-dehydrogenase contains a histidine residue which proximates the upper A-ring region of the steroid as it undergoes the reversible binding step.
Total body neutron activation analysis was employed to measure total calcium in C3H/St (Ha) female mice. Ninety-nine percent of total body calcium is in bone and loss of calcium was used as an index of osteoporosis. Heparin (500 U/kg B.I.D.) treatment for three months resulted in significant osteoporosis. 17 beta-Hydroxy-4-estren-3-one 17-decanoate 1.5 mg/kg or 4 mg/kg twice monthly prevented this heparin accelerated osteoporosis. The results suggest that 17 beta-hydroxy-4-estren-3-one 17-decanoate may be capable of preventing bone loss and partially increasing bone mass in patients exposed to osteoporosis inducing regimens.
On the basis of the large range of kinetic constants of their substrates, beta-lactamases seem to be interesting enzymes for the development of homogeneous immunoassays. For this purpose, hapten-penicillin or -cephalosporin conjugates have to be prepared. The aim of this work is to couple the anabolizing steroid nandrolone to several penicillins characterized by extremely low K(m) and kcat values: ticarcillin, carbenicillin, and oxacillin. The easy decarboxylation of derivatives of phenylmalonic acid (carbenicillin) and thienylmalonic acid (ticarcillin) imposes the choice of very mild procedures which have been specifically adapted to each substance investigated. 4-Estren-17 beta-ol-3-one hemiphenylmalonate is conjugated to 6-aminopenicillanic acid after 1,1'-carbonyldiimidazole activation, while 4-estren-17 beta-ol-3-one hemi(3-thiophene)malonate is coupled to 6-aminopenicillanic acid after activation using methanesulfonyl chloride. Before conjugation of oxacillin, a carboxylated analogue of its side chain has been prepared. A procedure resorting to tert-butyl ester protection of the carboxyl group present on the isoxazole ring allows the binding of nandrolone to the remaining carboxyl followed, after specific deprotection, by the conjugation to 6-aminopenicillanic acid giving the oxacillin derivative. In this way, conjugates retaining immunological properties of nandrolone and high inhibiting power of beta-lactamases should be obtained.
We describe a 4-month-old girl with Estren-Dameshek variant of Fanconi's anemia (FA) presenting with a pancytopenia secondary to extranodal bone marrow malignant lymphoma as the first hematologic manifestation of the syndrome. Although she was phenotypically normal, her cultured lymphocytes showed an increased spontaneous chromosome breakage, which was enhanced by diepoxybutane (DEB). Cytogenetic studies showed that 7 of her 12 relatives, including both parents, were heterozygote carriers of the FA gene. This confirms the diagnosis of FA of the Estren-Dameshek variant. A complete hematologic remission was attained within 28 days, and she remained in continuous bone marrow remission till the age of 14.5 months, when she developed acute gastroenteritis and died. No unusual sensitivity to chemotherapy was experienced, though leukopenia and recurrent infections proved troublesome. FA has an incidence many times higher in the Orange Free State region of South Africa than reported elsewhere in the world.
Guided by antiprogestational screening results, 4-substituted(4-OH, 4-Cl, 4-Br, and 4-OMe) 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one and their 17-acetate were synthesized via 4 beta, 5 beta-epoxy-17 beta-hydroxy-7 alpha-methylestran-3-one from 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one. Some of these compounds possess strong affinity to human decidual progesterone receptor, inhibit in vitro the growth of decidual cells, and prevent the implantation in rats at the dose of 1 mumol/kg.
The effect of a synthetic steroidal compound TSAA-291 (16 beta-ethyl-17 beta-hydroxy-4-estren-3-one) on the binding of methyltrienolone (R1881) and promegestone (R5020) to hyperplastic and neoplastic human prostate was investigated. TSAA-291 inhibits both androgen and progestogen binding to hyperplastic and neoplastic human prostate. Glycerol density gradient analysis revealed that the inhibition of promegestone (R5020) binding by TSAA-291 was significantly greater than that of methyltrienolone (R1881) in both hyperplastic and neoplastic human prostate. The nature of the inhibition was competitive as determined by Scatchard analysis and double reciprocal plots. Comparison of the Ki values for the inhibition by TSAA-291 of R1881 binding (3.2 X 10(-7) M) and of R5020 binding (2.0 X 10(-8) M) suggests that TSAA-291 binds to progesterone receptor with a greater affinity than to androgen receptor. Our results suggest that the effectiveness of the drug in the treatment of benign hyperplasia might be due not only to its anti-androgenic properties but also due to its ability to inhibit progesterone receptor.
The reaction of 3 beta, 17 beta-diacetoxy-4-estrene with N-bromoacetamide in a two phase ether/water solvent mixture gave 5-bromo-4 beta, 17 beta-diacetoxy-5 alpha-estran-3 beta-ol as the major product (75%). Four minor products were also isolated and identified. These were: 4 alpha-bromo-3 beta, 17 beta-diacetoxy-5 alpha-estran-5-ol (5%), 5-bromo-3 beta, 17 beta-diacetoxy-5 alpha-estran-4 beta-ol (6%), 5-bromo-4 alpha, 17 beta-diacetoxy-5 alpha-estran-3 beta-ol (3%), and 4 beta-bromo-3 beta, 17 beta-diacetoxy-5 alpha-estran-5-ol (4%). The 5-bromo-4 beta, 17 beta-diacetoxy-5 alpha-estran-3 beta-ol was equilibrated by heating with oxalic acid in refluxing benzene for ca. 16 h to give a mixture of it and 5-bromo-3 beta, 17 beta-diacetoxy-5 alpha-estran-4 beta-ol in the ratio of 16:84 respectively. A similar equilibration mixture (14:86) was obtained under identical conditions when 5-bromo-3 beta, 17 beta-diacetoxy-5 alpha-estran-4 beta-ol was the starting material.
A combined oral application of 30 micrograms ethinylestradiol with 50 micrograms 17 alpha-ethinyl-18-methyl-11-methylene-4-estren-17-ol (a newly developed progestagen) -- for 21 days resulted in a distinct antigonadotrophic effect, shown by hormone analysis (LH, FSH, estradiol and progesterone) in serum samples of three healthy and fertile women. A final recommendation on the daily dosage, however, depends on the results of clinical experimental investigation still in progress.
The chemical change responsible for the 3-oxo-4-estren-17 beta-yl acetate-dependent photoinactivation of delta 5-3-ketosteroid isomerase has been identified by amino acid analysis and amino acid sequencing. Amino acid analysis of the enzyme and its photoinactivated derivative shows that photoinactivation is accompanied by loss of nearly 1 residue of aspartic acid/polypeptide chain and an increase in nearly 1 residue of alanine. Edman degradation of a peptide comprising residues 31 to 48 from native isomerase showed the presence of aspartic acid at residue 38. When the corresponding peptide from photoinactivated enzyme was sequenced, residue 38 was revealed to be alanine.
Using the strategy based on the Hansch method which analyses effects of substituents on biological activity in terms of their hydrophobic, electronic and steric effects we selectively synthesised a series of 11beta-substituted-17alpha-ethynyl-4-estren-17beta-ols that combine ease of synthesis with good discrimination between these factors aiming at finding the compounds with optimum biological activity in that series. The compounds were tested quantitatively in the Clauberg test (rabbit) and the ovulation inhibition test (rat). The differences in biological activity could reasonably be correlated with two steric effects introduced by the 11beta-substituent. These were a change in the overall shape of the 11beta-substituent and the angular methyl group, and direct steric hindrance of the steroid-receptor protein binding. Some exceptions were found possibly due to metabolic conversion of these compounds to the corresponding 11beta-substituted-17alpha-ethynyl-1,3,5(10)-estra-triene-3,17beta-diols.
Inhibition of 5alpha-reduction of testosterone by an anti-androgen TSAA-291 (16beta-ethyl-17beta-hydroxy-4-estren-3-one) was studied in rat ventral prostates and the metabolic conversion of 3H-TSAA-291 was examined both in vitro and in vivo. In the in vitro experiment using nuclear 5alpha-reductase of the prostate, 5alpha-dihydrostestosterone formation from 3H-testosterone was inhibited in a competitive manner by the anti-androgen. In the in vitro experiment using 3H-TSAA-291, 5alpha-reduction of the anti-androgen occurred. One, 2 and 4 hr after an intravenous administration of 140 muCi/rat of 3H-TSAA-291 to castrated rats, the unchanged TSAA-291 accumulated in higher amounts in the ventral prostate than in the plasma, skeletal muscle and levator ani muscle, thereby indicating the selective uptake of the anti-androgen by the androgen target organ. No appreciable amounts of the 5alpha-reduced metabolite of TSAA-291 were detected in the prostate, thus suggesting that TSAA-291 itself may be responsible for the anti-androgenic properties. The inhibitory potency of the 5alpha-reductase activity of several other 16beta-substituted androstane and estrane analogues was also examined.
Four ring A steroidal epoxyenones as probable intermediate in the formation of catechol estrogens were synthesized. The isomeric 1 alpha,2 alpha-epoxy-17 beta-hydroxyestr-4-en-3-one (9) and 1 beta,2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (8) were synthesized from 17 beta-hydroxy-5 alpha-estra-3-one. The isomeric 4 alpha,5 alpha-epoxy-17 beta-hydroxyestr-1-en-3-one (11) and 4 beta,5 beta-epoxy-17 beta-hydroxyestr-1-en-3-one (10) were prepared from 19-nortestosterone. The reaction of 9 and 10 with sodium/ethanethiol resulted in the formation of three types of reactions leading to multiple products: 1,4-addition, opening of epoxide, and epoxide opening followed by dehydration. Reaction of 8 with ethanethiol gave only one compound identified as 2-ethanethio-1,4-estradien-17 beta-ol-3-one, while reaction of 9 with ethanethiol gave an unusual product identified as 4-estren-1 alpha,17 beta-diol-3-one. Unlike reaction of ethanethiol with 9 and 10, reaction with N-acetylecysteine or glutathione results in epoxide opening followed by dehydration leading to the formation of estradiol-4-thioethers.
Pharmacological tests on rats and dogs showed that a novel steroid substance, ORG OI-65 (code name for 17alpha-ethinyl-6-estrene-5alpha, 7beta-diol) has both progestational and estrogenic properties. The effect of this substance on menstrual cycle was studied on 13 healthy fertile women, who received 2 mg of ORG OI-65 daily for 20 consecutive days from the 5th day of cycle. The following parameters were studied during pretreatment and treatment cycles: bleeding pattern, plasma progesterone, urinary excretion of FSH, LH and estrone, vaginal cytology, cervical mucus, endometrium biopsy and basal body temperature. Treatment with ORG OI-65 did not change the bleeding pattern or inhibit ovulation: all 12 cases with ovulatory pretreatment cycles showed evidence of ovulation--timely or delayed--also during ORG OI-65 treatment. The vaginal cytology and cervical mucus remained almost unaffected by the treatment. No serious side-effects were found. The authors conclude that ORG OI-65 2 mg daily is not effective enough for oral contraception.
The experiment conditions for the evaluation of Norethindrone (17 alpha-Ethynyl-17 beta-hydroxy-4-estren-3-one, NET) and Ethynyl-estradiol (17 alpha-ethynyl-1, 3, 5 (10) estratrien-3, 17 beta-diol, EE) by radioimmunoassay are described. A minimal quantity of 25 pg of these two steroids could be evaluated using different reduced metabolites of NET, very little cross reaction is observed with 200 pg of these metabolites. No effect was observed with estradiol for the EE-antiserum. The NET-antiserum was used to evaluate this steroid and ethynodiol diacetate after oral administration to female volunteers. Maximal values in the plasma (2-3% of the administered dose) was found between 1-3 h after administration and at 24 h a concentration of 0.1-0.3% still remained in the plasma.
An iodine-125 labeled ligand for progesterone receptor determination was synthesized: (Z)-17 beta-hydroxy-17 alpha-(2-[125I]iodovinyl)-4-estren-3-one ([125I]SH-D 510). The ligand is stable chemically as well as under the conditions of a receptor assay. The relative binding affinity of the nonradioactive compound towards human uterine progesterone receptor was 7.0 for the Z-isomer (promegestone (R5020) 1.0) and 0.95 for the E-isomer. 4 S and 8 S receptor forms were obtained on sucrose density gradient analysis. Progesterone receptors were assayed in 103 human mammary tumour cytosols, using either [3H]promegestone or [125I]SH-D 510. The coefficient of correlation was r = 0.951.