Enzyme-assisted synthesis and structure characterization of glucuronide conjugates of methyltestosterone (17 alpha-methylandrost-4-en-17 beta-ol-3-one) and nandrolone (estr-4-en-17 beta-ol-3-one) metabolites

A new and useful method based on enzyme-assisted synthesis was developed for producing 3 alpha-O-beta-D-glucuronide conjugates from synthetic phase I metabolites of methyltestosterone and nandrolone. The formed glucuronide conjugates of 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol (I), 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (II), 5 alpha-estran-3 alpha-ol-17-one (III), and 5 beta-estran-3 alpha-ol-17-one (IV) are urinary metabolites, indicating the human misuse of the above-mentioned anabolic androgenic steroids (AAS). The common lack of reference material precludes the use and validation of these biomarkers in human doping control. Liver microsomes from Aroclor 1254-induced rats were used as a highly active source of mammalian UDP-glucuronosyltransferases (UGT, EC 2.4.1.17). After purification by protein precipitation, liquid-liquid extraction (dichloromethane), C-18 solid-phase extraction, and lyophilization, the steroid glucuronide structures were characterized by (1)H and (13)C NMR spectroscopy and tandem mass spectrometry. The enzymatic method was highly stereoselective, producing a single major conjugate from the parent steroids I-IV. The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (1.0-2.8 mg, yield 12-29%), which is sufficient for veterinary and human doping control analyses; for pharmaco-, toxico-, and enzyme kinetic studies in the pharmaceutical industry; for clinical laboratories; and for forensic medicine. A new sensitive LC-MS method was developed for controlling the product purity in syntheses, as well as for enzyme kinetic characterization of AAS-metabolizing UGT activities in rat liver toward the aglycones I-IV. In this study, the UGT enzymes responsible for the formation of 3 alpha-O-linked glucuronides from the substrates I, II, III, and IV exhibited the specific enzyme activity values: 25, 124, 48, and 212 nmol/mg microsomal protein in a 2-h incubation, respectively.     

The molecular structure of 3-methoxy-9 (10 leads to 19) abeo- 1,3, 5, (10)-estratrien-17-one.

The crystal and molecular structure of 3-methoxy-9(10 leads to 19)abeo-1,3,5(10)-estratrien-17-one has been determined by X-ray analysis in order to ascertain the configuration at C(9). The seven-membered B-ring has a chair conformation and this causes the molecule to bow towards the alpha-face rather than towards the beta-face as in estradiol.     

Cyclization of 13beta-ethyl-3-methoxy-17beta-ol-8,14-seco-1,3,5(10),9-gonatetraen-14-one and its 17 acetate derivative.

13beta-Ethyl-3-methoxy-17beta-ol-8,14-seco-1,3,5(10),8-gonatetraen-14-one (IIIa) was isolated and its participation in the well-known acidic cyclization process was established.     

Synthesis of two new haptens of 16alpha-hydroxydehydroepiandrosterone (3beta,16alpha-dihydroxyandrost-5-en-17-one)

Synthetic routes leading to 19E and 7Z O-(carboxymethyl)oximes derived from 16alpha-hydroxydehydroepiandrosterone were developed using two independent methods for introduction of the 16alpha-hydroxy group. Firstly, the oxime moiety was built, and then, either epoxidation of the enol acetate followed by the boron trifluoride mediated rearrangement or alkaline hydrolysis of the corresponding alpha-bromide in aqueous N,N-dimethylformamide were employed. The last step in both methods was removal of the protecting groups, which consisted of acid deprotection of the acetates and gentle alkaline hydrolysis of the methyl ester. Final haptens were designed as components for immunoanalytical kits.     

Radioimmunoassay of norethindrone (17 alpha-ethynyl-17 beta-hydroxy-4-estren-3-one) and ethynyl-estradiol (17 alpha-ethynyl-1,3,5, (10)-estratien-3, 17 beta-diol). Application to human plasma determination of norethindrone after oral administration of this steroid.

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.     

Age-related changes in conversion of 5 alpha-androstan-17 beta-ol-3-one to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol by rat testicular cells in vitro

Conversion of labelled 5 alpha-androstane-17 beta-ol-3-one (DHT) by isolated testicular cells from rats of different ages was examined under saturating substrate conditions in vitro (5--10 micrograms DHT/ml in a 24 h incubation). Two detectable metabolites of DHT were produced by testicular cells in vitro. 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol). Production of these diols during a 24 h period was linear, and the amounts formed were directly related to the cell number. The amount of 3 alpha- and 3 beta-diols formed by testicular cells of rats of different ages increased from Day 10 to Day 25, then declined. Testicular cells from rats 10 to 20 days of age converted DHT mainly to 3 alpha-diol, but thereafter 3 beta-diol was the predominant testicular metabolite of DHT.     

Synthesis of 3-methoxy-5,6-secoestra-1,3,5(10),8,14-pentaen-17-one

A facile synthesis of 3-methoxy-5,6-secoestra-1, 3,5(10),8,14-pentaen-17-one from the readily available p-anisaldehyde is reported.     

5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a potent regulator of cholesterol metabolism: occurrence in rat skin

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one is a potent inhibitor of cholesterol biosynthesis which has significant hypocholesterolemic activity upon oral administration to animals. Described herein are the results of experiments that indicate the presence of the 15-ketosterol in rat skin. The 15-ketosterol was, after purification by medium pressure liquid chromatography on Lichroprep RP-8 columns, thin-layer chromatography on silica gel G, and reverse phase high performance liquid chromatography, characterized by gas-liquid chromatography-mass spectrometry in the form of its trimethylsilyl ether derivative. The use of an internal standard containing both tritium and deuterium permitted the determination of the levels of the 15-ketosterol by mass fragmentography. The results of five separate analyses of portions of the skin of a male Sprague Dawley rat showed a mean value of 84.5 +/- 4.1 (SEM) ng per g. Analyses of hair samples of ten male Sprague Dawley rats indicated a mean level of 143 +/- 19 (SEM) ng per g of hair. Most (approximately 72%) of the 15-ketosterol in hair was esterified. This report constitutes the first isolation of the 15-ketosterol from animal tissues.     

Synthesis and biological activities of new 1,2,4-triazol-3-one derivatives

New 2-phenacyl-1,2,4-triazol-3-ones were obtained by the reaction of 5-alkyl-1,2,4-triazol-3-ones with alpha-bromoacetophenone in alkaline medium. Selective reduction of the side chain carbonyl group to hydroxy group was achieved with NaBH4. The reaction of some compounds containing a phenolic hydroxyl with 4-toluenesulfonyl chloride or benzyl bromide in the presence of NaOH led to tosylated or benzylated derivatives. The tosylation or benzylation at the alcoholic hydroxyl was carried out in the presence of sodium metal. Some of the newly synthesized compounds revealed an antimicrobial activity; 6 of 14 new compounds that were studied by the National Cancer Institute were found to possess antitumor activity.     

Inhibitors of sterol synthesis. Synthesis and spectral characterization of fatty acids esters of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one

Reported herein are the chemical syntheses of 29 fatty acid esters of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one. These compounds were characterized by the results of ultraviolet, 1H nuclear magnetic resonance, and mass spectral studies.     

Glut-5(10)-en-3-one from Andrachne cordifolia

Glut-5(10)-en-3-one, a new pentacyclic triterpene ketone, was isolated from Andrachne cordifolia (aerial parts roots).     

Synthesis and biological activity of 3 beta-hydroxy-9,10-secopregna-5,7,10[19]-triene-20-one: a side chain analogue of vitamin D3

The synthesis, biological and antagonistic activity of 3 beta-hydroxy-9,10-secopregna-5,7,10[19]-triene-20-one (20-oxopregnacalciferol, 7) a shortened side chain analogue of vitamin D3, are described. At the highest dose tested the analogue was found to have small though significant bone and soft tissue mobilization activity; no significant increase in intestinal calcium transport was noted. The compound was found to possess no antagonistic activity against vitamin D3.     

Rapid chromatography for quantitation of radioimmunoassayable 5alpha-androstane-17beta-ol-3-one and testesterone in ram, bull and boar serum.

A rapid technique for the simultaneous determination of serum 5alpha-androstane-17beta-0l-3-one (DHT) and testosterone (T) was developed and validated. The procedure incorporates a chromatographic step prior to binding with a T-3BSA antiserum. Anakrom columns used to separate DHT from T also isolate other steroids quickly and efficiently. Utilizing this procedure, levels of both androgens have been measured in the intact ram, bull and boar. Concentrations of DHT and T expressed as mean +/- SE in ng/ml were respectively: ram, 0.14 +/- 0.03 and 4.96 +/- 0.80; bull, 0.10 +/- 0.01 and 3.92 +/- 0.80; boar, 0.71 +/- 0.11 and 5.58 +/- 0.64. Whether these minute quantities of serum DHT play important physiological roles, particularly in the boar, is unresolved. Monitoring DHT under varying experimental or pathological conditions may allude to its importance in the domestic male.     

Secretion of metabolites of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one by Hep G2 cells

We demonstrated that two-thirds of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one was efficiently taken up and consequently metabolized and secreted by Hep G2 cells when it was pulsed for 16 hrs followed by chasing for another 72 hrs. The metabolism was clearly reflected by the cellular secretion. Approximately 61%, 26% and 10% of uptaken [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one was metabolized to its water-soluble metabolites, polar metabolites in lipid phase and ketosteryl esters, respectively. Ninety-four percent of these metabolites was secreted into media. Interestingly, polar forms of the metabolites of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one accounted for over 95% of the cellular secretes. Limited secretion of ketosteryl esters was also detected. The data strongly suggest that Hep G2 cells have the potential to process 5 alpha-cholest-8(14)-en-3 beta-ol-15-one and could provide a good model for studying its secretion.