Neighboring group participation Part 17 Stereoselective synthesis of some steroidal 2-oxazolidones, as novel potential inhibitors of 17alpha-hydroxylase-C(17,20)-lyase

During the alkaline methanolysis of 3beta-acetoxy-21-chloropregn-5-ene-20beta-N-phenylurethane (4a), and its 4-monosubstituted (4b-e) and 3,5-disubstituted (4f) phenyl derivatives, cyclization occurs, in the course of which 17beta-[3-(N-phenyl)-2-oxazolidon-5-yl]androst-5-en-3beta-ol (5a) and its substituted phenyl derivatives (5b-f) are formed. The cyclization takes place with (N(-)-5) neighboring group participation. The reaction of 3beta-acetoxy-21-azidopregn-5-en-20beta-ol (3d) with triphenylphosphine gave 3beta-acetoxy-21-phosphiniminopregn-5-en-20beta-ol, which reacted in situ with carbon dioxide with the participation of the sterically favored 20beta-OH to give the unsubstituted steroidal cyclic carbamate (8). Oppenauer oxidation of the 3beta-hydroxy-exo-heterocyclic steroids (5a-f, 9) yielded the corresponding Delta(4)-3-ketosteroids (7a-f, 10). The inhibitory effects (IC(50)) of these compounds on rat testicular C(17,20)-lyase were investigated with an in vitro radioligand incubation technique. The N-unsubstituted 17beta-(2-oxazolidon-5-yl)-androst-4-en-3-one derivative (10) was found to be a potent inhibitor (IC(50)=3.0 microM).     

5alpha-reduced C21 steroids are substrates for human cytochrome P450c17

The 5alpha-reduction of testosterone in target tissues is a key step in androgen physiology; however, 5alpha-reduced C(19) steroids are sometimes synthesized in testis via a pathway that does not involve testosterone as an intermediate. We studied the metabolism of 5alpha-reduced C(21) steroids by human cytochrome P450c17 (hCYP17), the enzyme responsible for conversion of C(21) steroids to C(19) steroids via its 17alpha-hydroxylase and 17,20-lyase activities. hCYP17 17alpha-hydroxylates 5alpha-pregnan-3,20-dione, but little androstanedione is formed by 17,20-lyase activity. hCYP17 also 17alpha-hydroxylates 5alpha-pregnan-3alpha-ol-20-one and the 5alpha-pregnan-3alpha,17alpha-diol-20-one intermediate is rapidly converted to androsterone by 17,20-lyase activity. Furthermore, 5alpha-pregnan-3alpha,17alpha-diol-20-one is a better substrate for the 17,20-lyase reaction than the preferred substrate 17alpha-hydroxypregnenolone and cytochrome b(5) stimulates androsterone formation only 3-fold. Both 5alpha-pregnan-3alpha-ol-20-one and 5alpha-pregnan-3alpha,17alpha-diol-20-one bind to hCYP17 with higher affinity than does progesterone. We conclude that 5alpha-reduced, 3alpha-hydroxy-C(21) steroids are excellent, high-affinity substrates for hCYP17. The brisk metabolism of 5alpha-pregnan-3alpha,17alpha-diol-20-one to androsterone by CYP17 explains how, when 5alpha-reductases are present, the testis can produce C(19) steroids androsterone and androstanediol from 17alpha-hydroxyprogesterone without the intermediacy of androstenedione and testosterone.     

Design, synthesis, and biological evaluation of substituted-N-(thieno[2,3-b]pyridin-3-yl)-guanidines, N-(1H-pyrrolo[2,3-b]pyridin-3-yl)-guanidines, and N-(1H-indol-3-yl)-guanidines

Sulfonylureas stimulate insulin secretion independent of the blood glucose concentration and therefore cause hypoglycemia in type 2 diabetic patients. Over the last years, a number of aryl-imidazoline derivatives have been identified that stimulate insulin secretion in a glucose-dependent manner. In the present study, we have developed three series of substituted N-(thieno[2,3-b]pyridin-3-yl)-guanidine (2a-l), N-(1H-pyrrolo[2,3-b]pyridin-3-yl)-guanidine (3a-l), and N-(1H-indol-3-yl)-guanidine (4a-l) as new class of antidiabetic agents. In vitro glucose-dependent insulinotropic activity of test compounds 2a-l, 3a-l, and 4a-l was evaluated using RIN5F (Rat Insulinoma cell) based assay. All the test compounds showed concentration-dependent insulin secretion, only in presence of glucose load (16.7mmol). Some of the test compounds (2c, 3c, and 4c) from each series were found to be equipotent to BL 11282 (standard aryl-imidazoline), which indicated that the guanidine group acts as a bioisostere of imidazoline ring system.     

Synthesis of some epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives and evaluation of their biological activity

Steroidal epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives have been prepared using 3beta,17beta-dihydroxy-17alpha-picolyl-androst-5-ene (1), 3beta-acetoxy-17-picolinylidene-androst-5-ene (2), and 3beta-hydroxy-17-picolinylidene-androst-5-ene (3) as synthetic precursors. The compounds 2 and/or 3 were reacted with m-chloroperoxybenzoic acid (MCPBA). The compounds synthesized from 2 were 17-picolinylidene-N-oxide 4, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 5 and 6, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 7 and 8. Starting from compound 3, a mixture of 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene 9 and 10, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 11 and 12, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 13 and 14 were obtained. From compounds 15 and 18, obtained from 1 and 3 by the Oppenauer oxidation, the 4alpha,5alpha-epoxy and 4beta,5beta-epoxy derivatives 16, 17 and 20, 21 were prepared by oxidation with 30% H(2)O(2). Oxidation of 18 with MCPBA yielded only the N-oxide 19. The structures of compounds 15 and 18 were proved by the X-ray analysis. Compounds 1-6, 9, 15, 17, 18, and 21 were tested on activity against the enzyme aromatase. Antitumor activity against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER-, MDA-MB-231, and prostate cancer PC3) was evaluated. Three tested compounds (1, 4, and 19) showed strong activity against PC3, the IC(50) values being in the range 0.55-10microM, whereas compound 17 showed strong activity against MDA-MB-231 (IC(50) 10.4microM).     

白细胞介素－2第17和20位残基在结构功能中的重要性研究

用基因定位突变法,将白细胞介素－２（ＩＬ－２）分子中１７Ｌｅｕ和２０Ａｓｐ进行一系列突变,并测定各突变体生物活性与空间结构的变化。分析结果表明１７Ｌｅｕ突变为Ａｓｐ时,ＩＬ－２的空间结构无明显变化。生物活性却显著下降;２０Ａｓｐ突变为Ｌｅｕ,以及１７Ｌｅｕ与２０Ａｓｐ对调后,均导致ＩＬ－２的空间结构发生变化,并严重影响其生物活性。上述结果说明１７Ｌｅｕ突变为Ａｓｐ后对活性的影响并非由空间结构变化所引起,而与残基本身性质有关：１７Ｌｅｕ与２０Ａｓｐ这两个重要的残基,必须位于各自特定的空间位置,才能发挥其生物作用。     

Isolation of 20 alpha and 20 beta 5 beta-pregnane-3 alpha,17 alpha,20,21-tetrol (hexahydro-Reichstein's compound-S) in a case of congenital adrenal hyperplasia

5β-Pregnane-3α, 17α, 20α, 21-tetrol (l) and 5β-pregnane-3α, 17α 20β, 21-tetrol (II) have been isolated and identified from the urine of a girl with congenital adrenal hyperplasia. The total 5β-pregnane-3α, 17α, 20(α+β),21-tetrol consisted of 60% of I and 40% of II. The final identity of the compounds was established by gas chromatography — mass spectrometry. The mass spectra of the two trimethylsilyl isomers were closely related to each other in contrast to the spectra of five other pairs of C₂₁-C-20(α and β)-hydroxy steroid-trimethylsilyl-ethers. The mass spectra of free I and II also exhibited many common features, but were less similar to each other than their trimethylsilyl derivatives.     

17 beta-hydroxysteroid and 20 alpha-hydroxysteroid dehydrogenase activities of human placental microsomes: kinetic evidence for two enzymes differing in substrate specificity

During storage at 4 degrees C, the 17 beta-hydroxysteroid dehydrogenase activity of human placental microsomes with estradiol-17 beta was more stable than that with testosterone. In order to evaluate the basis for this difference, kinetics with C18-, C19-, and C21- steroids as substrates and/or inhibitors was studied in conjunction with an analysis of the effects of detergents. Both 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activities were detected. At pH 9.0, apparent Michaelis constants were 0.8, 1.3, and 2.3 microM for estradiol-17 beta, testosterone, and 20 alpha-dihydroprogesterone, respectively, 17 beta-HSD activity with testosterone was inhibited by estradiol-17 beta, 5 alpha-dihydrotestosterone, 5 beta-dihydrotestosterone, 20 alpha-dihydroprogesterone, and progesterone. In each case 90 to 100% inhibition was observed at 50 to 200 microM steroid. Activity with 20 alpha-dihydroprogesterone was similarly sensitive to inhibition by C19-steroids. By contrast, 25 to 45% of the activity with estradiol-17 beta was not inhibited by high concentrations of C19- or C21-steroids and differed from the 17 beta-HSD activity with testosterone and the major fraction of that with estradiol-17 beta by being insensitive to solubilization by detergent. These results are consistent with an association of two dehydrogenase activities with human placental microsomes. One recognizes C18-, C19-, and C21-steroids as substrates with comparable affinities. The second appears to be highly specific for estradiol-17 beta. The former activity may account for most if not all of the oxidation-reduction at C-17 of C19-steroids and at C-20 of C21-compounds at physiological concentrations by term placental tissue.     

Synthesis and biological evaluation of substituted pyrazoles as blockers of divalent metal transporter 1 (DMT1)

Three distinct series of substituted pyrazole blockers of divalent metal transporter 1 (DMT1) were elaborated from the high-throughput screening pyrazolone hit 1. Preliminary hit-to-lead efforts revealed a preference for electron-withdrawing substituents in the 4-amido-5-hydroxypyrazole series 6a-l. In turn, this preference was more pronounced in a series of 4-aryl-5-hydroxypyrazoles 8a-j. The representative analogs 6f and 12f were found to be efficacious in a rodent model of acute iron hyperabsorption. These three series represent promising starting points for lead optimization efforts aimed at the discovery of DMT1 blockers as iron overload therapeutics.     

Discriminatory inhibition of adrenocortical 17 alpha-hydroxylase activity by inhibitors of cholesterol side chain cleavage cytochrome P-450

Two inhibitors of the cholesterol side chain cleavage reaction were tested for their ability to inhibit bovine adrenocortical 17 alpha-hydroxylase and 21-hydroxylase activities. One inhibitor, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), was found to be a potent inhibitor of 17 alpha-hydroxylation of either progesterone or pregnenolone but was inactive on 21-hydroxylase activity. 22-ABC was found to be a competitive inhibitor of 17 alpha-hydroxylase (cytochrome P-45017 alpha) activity, having an apparent inhibitor constant of 29 nM when using pregnenolone as the substrate. Spectral binding studies showed that 22-ABC produces a type II difference spectrum when added to a bovine adrenocortical microsomal preparation, due presumably to a coordination of its amine nitrogen atom to the heme-iron of cytochrome P-45017 alpha. The second cholesterol side chain cleavage inhibitor tested, (20R)-20-phenyl-5-pregnene-3 beta,20-diol (20-PPD), was found not to inhibit either the 21- or 17 alpha-hydroxylase activities. It is proposed that the phenyl group projecting from C-20 of 20-PPD prevents this steroid from binding to cytochrome P-45017 alpha. The discriminatory interaction of these two steroids with adrenocortical cytochromes P-450 provides some insight with respect to possible structural features of the active-site regions of these enzymes.     

Chromosomes 17 and 22 involved in marker formation in neurofibrosarcoma in von Recklinghausen disease

Summary We describe the cytogenetic findings in a recurrent neurofibrosarcoma in a patient with nonfamilial von Recklinghausen disease. The composite karyotype was: 40,Y,-X,+dic r(X;20)(:Xp22.2q26::20p13 q13:), -1, +der(1)t(1;3) (p21;p24),-3,-4,-5,+der(5) t(5;?)(q31;?),-9,-9,+der(9)t(3;9)(q21 or q13;p24 or p22), -11,+der(11)t(11;?)(q22.2;?), -17,+der(17)t(17; 22;?)(q21;q13.1;?), -20, -21, -22, -22, +der(22)t(17; 22;?)(q21;q13.1;?),t(2;10)(q37;q22). The derivative chromosomes were demonstrated at the 500 band level. Chromosomes 17 and 22 were shown to be involved in an unbalanced three-way translocation: t(17;22;?)(q21;q13.1;?). This event was confirmed by in situ hybridization, using two probes mapped to chromosome 17. Hill H is a probe derived from the novel oncogene TRE and is located at 17q12–22. The second probe, derived from the granulocyte colony-stimulating factor (G-CSF), is located at 17q11–q21. The rearrangement between chromosomes 17 and 22 showed breakpoints similar or close to the gene loci for neurofibromatosis 1 (NF-1) and NF-2. Based on our observations we recommend that genetic studies on NF-1 tumors include both gene sites (NF-1 and NF-2) rather than focus on one gene locus.     

[17(20)E]- and [17(20)Z]-pregna-5,17(20)-dien-21-oylamides. Facile synthesis and primary evaluation for cancer cells proliferation

Reaction of 17α-bromo-21-iodo-3β-acetoxypregn-5-en-20-one with ammonia, primary, and secondary amines is simple and convenient method for preparation of [17(20)E]- and [17(20)Z]-pregna-5,17(20)-dien-21-oylamides. Synthesis and characteristics of 12 related amides are presented. Primary testing on cells proliferation indicated differing effects of synthesized compounds on androgen insensitive MCF-7 cells and androgen sensitive LNCaP cells.     

17 beta-(cyclopropylamino)-androst-5-en-3 beta-ol, a selective mechanism-based inhibitor of cytochrome P450(17 alpha) (steroid 17 alpha-hydroxylase/C17-20 lyase)

A new compound, 17 beta-(cyclopropylamino)-androst-5-en-3 beta-ol, MDL 27,302, has been designed and synthesized as a mechanism-based inhibitor of cytochrome P450(17 alpha). The time-dependent inactivation of human testicular P450(17 alpha) is irreversible by dialysis and requires the cofactor, NADPH; Kiapp. 90 nM (determined on cynomolgous monkey testis enzyme). Inactivation was not affected by the nucleophile DTT, suggesting retention of the inhibitor in the enzyme active site during the inactivation process. Inhibition is specific to the cyclopropylamino compound, since the isopropylamino- and cyclobutylamino-analogs were not inhibitory. Enzymatic specificity of MDL 27,302 for P450(17 alpha) was demonstrated by its failure to inhibit steroid 21-hydroxylase and the cholesterol side chain cleavage enzyme (P450scc). Both the 17 alpha-hydroxylase and C17-20 lyase activities of cytochrome P450(17 alpha) of human testis microsomes were inhibited by MDL 27,302.     

Production of 17-hydroxypregnenolone and 17-hydroxyprogesterone by the immature rat ovary

The concentration of 17-hydroxyprogesterone (17-OH-prog), but not 17-hydroxypregnenolone (17-OH-preg), was detectable in the sera of 5 day old female rats. The level of 17-OH-preg increased dramatically between day 5 and 10, remained high for 5 days and then decreased to low levels by day 25; a second increase was found on day 35. 17-OH-prog began to increase in the serum after day 10, reached a peak by day 20 and then decreased by day 25 and remained the same through day 35. Stimulation of the ovaries of intact females with 20 IU of pregnant mare's serum gonadotropin resulted in a prompt increase in both progestins, but a much larger increase in 17-OH-preg than in 17-OH-prog. Increases were similar, but quantitatively less, in hypophysectomized females. The results demonstrate that the ovaries of immature rats contain an active 17-hydroxylase system.     

Characterization of the major steroids present in amniotic fluid obtained between the 15th and 17th weeks of gestation

In pooled amniotic fluid obtained between the 15th and 17th weeks of gestation the concentration of free steroids and steroid glucuronides was found to be 40 micrograms/dl. The concentration of steroid monosulfates and disulfates was 19 micrograms/dl. About half of the characterized steroids are progesterone metabolites. The "fetal type" 3 beta-hydroxy-5-ene steroids were found exclusively in the sulfoconjugated form. Their concentration represents 20% of the total steroid content. The identification of two 15 beta-hydroxylated C21 steroids, 3 beta,15 beta,17 alpha-tridoxy-5-pregnen-20-one and 5-pregnene-3 beta,15 beta,17 alpha,20 alpha-tetrol isolated from mid-pregnancy amniotic fluid is reported here. Metabolites of cortisol and 17-deoxycorticosteroid metabolites had similar quantitative importance, 8.6 and 9.4%, respectively.     

Expression of cytochrome P450c17 and other steroid-converting enzymes in the rat kidney throughout the life-span

We have investigated the metabolism of [14C]-labelled progesterone (P4) and dehydroepiandrosterone (DHEA) by kidney tissues of newborn and 7-, 15-, 30-, 60- and 365-day-old rats of both sexes. The following enzymes were revealed at all ages by radiochemical identification of the corresponding products: 5alpha-reductase, cytochromes P450c17 and P450c21, 3beta-hydroxysteroid dehydrogenase (HSD)/delta5-delta4 isomerase, and 17beta- and 20alpha-HSDs, catalyzing reductive reactions. The major P4 metabolites were 5alpha-reduced C21 steroids, whose formation was almost completely suppressed by the 5alpha-reductase 4-azasteroid inhibitor, PNU 156765. Androstenedione and testosterone were also formed via 17alpha-hydroxyprogesterone, together with 11-deoxycorticosterone and 20alpha-dihydroprogesterone. DHEA was mainly converted to androst-5-ene-3beta,17beta-diol, with smaller amounts of the above androgens. Cytochrome P450c17 mRNA and protein were demonstrated by Northern blotting and Western blotting analyses, respectively. P450c17 mRNA, assessed by Northern blotting, protein and catalytic activity all peaked in the kidney samples at 15 days of life and declined thereafter. Cytochrome P450arom was below the level of detection of semi-quantitative RT-PCR. Since the rat kidney has been previously shown to contain cytochrome P450scc as well as androgen and estrogen receptors, it is suggested that it is capable of autonomous hormonal steroidogenesis and that renal steroids, or nephrosteroids, may act locally, in a paracrine or autocrine fashion.     

Mechanism-based inactivation of bovine adrenal cytochromes P450 C-21 and P450 17 alpha by 17 beta-substituted steroids

A series of progesterone derivatives has been studied as potential inactivators of the bovine adrenocortical cytochromes P450, P450 17 alpha, and P450 C-21. Replacement of the 21-methyl group of progesterone with a difluoromethyl group resulted in a selective inactivator of P450 C-21 in a reconstituted system. The loss of 21-hydroxylase activity caused by this compound exhibits a number of characteristics of mechanism-based inactivation including NADPH dependence, pseudo-first-order kinetics, saturability, irreversibility, and protection by substrate. In addition to the difluoro compound, 21,21-dichloroprogesterone, the acetylenic compound pregn-4-en-20-yn-3-one, and the olefinic compound pregna-4,20-dien-3-one all inactivate P450 C-21. In contrast, the only compound to inactivate the rabbit adrenal progesterone 21-hydroxylase is 21,21-dichloroprogesterone. In binding studies, the 21,21-dihalo steroids produce a greater maximal type I spectral shift of P450 C-21 than the two 17 beta-unsaturated steroids. The dihalo compounds inactivate P450 C-21 by both heme destruction and protein modification as shown by significant decreases in residual 21-hydroxylase activity and spectrally detectable P450 after incubation with P450 C-21 in a reconstituted system. Liquid chromatographic and mass spectral analyses of the organic extracts from these incubations showed that 21-pregnenoic acid is a major metabolite of the dihalo compounds with a partition ratio of 5 nmol of acid produced/nmol of P450 C-21 inactivated. This supports the hypothesis that inactivation proceeds in part through an acyl halide intermediate. In contrast, the acetylenic compound pregn-4-en-20-yn-3-one inactivates P450 C-21 mainly by protein modification, producing an NADPH-dependent irreversible type I spectral shift. The stoichiometry of inactivation is approximately 1.5 nmol of compound bound/nmol of enzyme inactivated, indicating selective modification of the enzyme at or near the substrate binding site.     

Microbial conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione acetates by Nocardioides simplex VKM Ac-2033D

The conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione 21-acetate (I) and 17,21-diacetate (VI) by Nocardioides simplex VKM Ac-2033D was studied. The major metabolites formed from I were identified as pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV). Pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione (III) and pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V) were formed in minorities. Biotransformation products formed from VI were pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17,21-diacetate (VII), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII), pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V). The conversion pathways were proposed including 1(2)-dehydrogenation, deacetylation, 20beta-reduction and non-enzymatic migration of acyl group from position 17 to 21. The conditions providing predominant accumulation of pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) from I and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII) from VI in a short-term biotransformation were determined.     

Properties of antisera to progesterone and to 17-hydroxy-progesterone elicited by immunization with the steroids attached to protein through position 7

Antibodies to progesterone (P) and to 17-hydroxyprogesterone (17-OHP) were raised by immunization of rabbits with progesterone-7α-carboxyethyl thioether--bovine serum albumin (P-7—BSA) or with 17-OHP-7α-carboxyethyl thioether--BSA (17-OHP-7--BSA). The antisera produced were of high affinity: K_a towards the homologous hapten was 3. 7 × 10¹⁰ 1./mol for the anti-P serum and 5. 9 × 10⁹ 1/mol for the anti-17-OHP serum. The antiserum to P-7—BSA displayed little or no cross reaction (?= 2%) with the 20α-, 20β- or 5β-dihydro-derivatives of progesterone, moderate cross-reaction with pregnenolone (4%), but considerable cross-reaction with 11-deoxycorticosterone (7%), 5α-dihydro-progesterone (11%) and 17-OHP (15%). The antiserum to 17-OHP-7--BSA showed very little cross-reaction (?= 2%) with progesterone and other steroids lacking a 17α-hydroxyl group, such as pregnenolone or 11-deoxycorticosterone, but reacted significantly with 17α, 21-dihydroxy-4-pregnene-3, 20-dione (8%) and 3β, 17-dihydroxy-5-pregnen-20-one (13%). None of the sera reacted with testosterone, cortisol or estradiol-17β. It appears that conjugation of progesterone to protein through carbon-7 affords antisera comparable in specificity to those raised with 11α-conjugates and superior to those raised with 3-, 6- and 20-conjugates. The antiserum to 17-hydroxyprogesterone described is the first one that specifically recognizes this metabolite.     

The production and characterisation of antisera to 17-hydroxyprogesterone

Sheep were immunised with 11-deoxycortisol-21-hemisuccinate-bovine serum albumin (11-deoxycortisol-21-HS-BSA) or with 17-hydroxyprogesterone-7 alpha-carboxyethyl thioether-keyhole limpet haemocyanin (17-OHP-7 alpha-CETE-KLH) or with 17-OHP-3-(O-carboxymethyl)oxime-KLH (17-OHP-3-CMO-KLH). The resultant antisera were assessed using [3H]17-OHP and dextran-coated charcoal to separate the antibody bound and free fractions. All sheep produced antisera with an apparent affinity constant of from 1.4 to 6.6 X 10(9) 1/mol. Those raised against 11-deoxycortisol-21-HS-BSA had titres ranging from 1:12,000 to 1:78,000 but showed significant cross-reactivity with many of the steroids tested. Sheep immunised with 17-OHP-7 alpha-CETE-KLH had antisera titres of from 1:102,000 to 1:180,000 and only 17-hydroxypregnenolone cross-reacted significantly (10-20%). The best antisera were raised in sheep immunised with 17-OHP-3-CMO-KLH. Titres ranged from 1:168,000 to 1:390,000 and there were about 8 g/l of specific antibodies which cross-reacted 5.7% or less with 17-hydroxypregnenolone, and less than 0.5% with progesterone, 11-deoxycortisol and the other steroids studied. The antisera to 17-OHP-3-CMO-KLH were further assessed using [125I]17-OHP; titres ranged from 1:5,700,000 to 1:18,000,000 with affinity constants of from 1.67 to 2.5 X 10(10) 1/mol. They showed minimal or no cross-reactivity with the steroids studied. Reimmunisation after an 8-month interval produced antisera with a higher affinity constant and even lower cross-reactivity with other steroids.     

17-Epimerization of 17 alpha-methyl anabolic steroids in humans: metabolism and synthesis of 17 alpha-hydroxy-17 beta-methyl steroids.

The 17-epimers of the anabolic steroids bolasterone (I), 4-chlorodehydromethyltestosterone (II), fluoxymesterone (III), furazabol (IV), metandienone (V), mestanolone (VI), methyltestosterone (VII), methandriol (VIII), oxandrolone (IX), oxymesterone (X), oxymetholone (XI), stanozolol (XII), and the human metabolites 7 alpha,17 alpha-dimethyl-5 beta-androstane-3 alpha,17 beta-diol (XIII) (metabolite of I), 6 beta-hydroxymetandienone (XIV) (metabolite of V), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (XV) (metabolite of V), 3'-hydroxystanozolol (XVI) (metabolite of XII), as well as the reference substances 17 beta-hydroxy-17 alpha-methyl-5 beta-androstan-3-one (XVII), 17 beta-hydroxy-17 alpha-methyl-5 beta-androst-1-en-3-one (XVIII) (also a metabolite of V), the four isomers 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol (XIX) (also a metabolite of VI, VII, and XI), 17 alpha-methyl-5 alpha-androstane-3 beta,17 beta-diol (XX), 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (XXI) (also a metabolite of V, VII, and VIII), 17 alpha-methyl-5 beta-androstane-3 beta,17 beta-diol (XXII), and 17 beta-hydroxy-7 alpha,17 alpha-dimethyl-5 beta-androstan-3-one (XXIII) were synthesized via a 17 beta-sulfate that spontaneously hydrolyzed in water to several dehydration products, and to the 17 alpha-hydroxy-17 beta-methyl epimer. The 17 beta-sulfate was prepared by reaction of the 17 beta-hydroxy-17 alpha-methyl steroid with sulfur trioxide pyridine complex. The 17 beta-methyl epimers are eluted in gas chromatography as trimethylsilyl derivatives from a capillary SE-54 or OV-1 column 70-170 methylen units before the corresponding 17 alpha-methyl epimer. The electron impact mass spectra of the underivatized and trimethylsilylated epimers are in most cases identical and only for I, II, and V was a differentiation between the 17-epimers possible. 1H nuclear magnetic resonance (NMR) spectra show for the 17 beta-methyl epimer a chemical shift for the C-18 protons (singlet) of about 0.175 ppm (in deuterochloroform) to a lower field. 13C NMR spectra display differences for the 17-epimeric steroids in shielding effects for carbons 12-18 and 20. Excretion studies with I-XII with identification and quantification of 17-epimeric metabolites indicate that the extent of 17-epimerization depends on the A-ring structure and shows a great variation for the different 17 alpha-methyl anabolic steroids.