Synthesis of halogenated pregnanes, mechanistic probes of steroid hydroxylases CYP17A1 and CYP21A2.

The human steroidogenic cytochromes P450 CYP17A1 (P450c17, 17α-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) are required for the biosynthesis of androgens, glucocorticoids, and mineralocorticoids. Both enzymes hydroxylate progesterone at adjacent, distal carbon atoms and show limited tolerance for substrate modification. Halogenated substrate analogs have been employed for many years to probe cytochrome P450 catalysis and to block sites of reactivity, particularly for potential drugs. Consequently, we developed efficient synthetic approaches to introducing one or more halogen atom to the 17- and 21-positions of progesterone and pregnenolone. In particular, novel 21,21,21-tribromoprogesterone and 21,21,21-trichloroprogesterone were synthesized using the nucleophilic addition of either bromoform or chloroform anion onto an aldehyde precursor as the key step to introduce the trihalomethyl moieties. When incubated with microsomes from yeast expressing human CYP21A2 or CYP17A1 with P450-oxidoreductase, CYP21A2 metabolized 17-fluoroprogesterone to a single product, whereas incubations with CYP17A1 gave no products. Halogenated steroids provide a robust system for exploring the substrate tolerance and catalytic plasticity of human steroid hydroxylases.

Inhibition by bromoestrogens of the effects of estradiol on apomorphine-induced climbing behavior.

The chronic administration of estrogens to mice or rats will result in antidopaminergic effects. Apomorphine-induced climbing behavior in mice, the result of direct stimulation of dopamine receptors in the striatal and mesolimbic regions, is a simple animal model for examining these antidopaminergic effects of estrogens. Bromoestrogens, inhibitors of catechol estrogen formation, have been utilized in order to examine the role of estrogen metabolism in dopaminergic antagonism. Mice were pretreated for 3 days with 2-bromoestradiol, 4-bromoestradiol, or 2,4-dibromoestradiol dibenzoates alone or in combination with estradiol benzoate prior to apomorphine administration. The haloestrogens did not alter the climbing-induced responses elicited by apomorphine, whereas estradiol benzoate clearly attentuated the actions of apomorphine. Furthermore, the bromoestradiol dibenzoates were effective in reversing the effects of estradiol benzoate when the two steroids (estradiol benzoate and a bromoestrogen dibenzoate) were administered simultaneously during pretreatment. Thus, the bromoestrogens are able to inhibit the antidopaminergic effects of estradiol exhibited in the apomorphine-induced mouse climbing model.

Stereoselective hydrolysis of 16 alpha-halo-17-keto steroids and long-range substitution effects on the hydrolysis of 16 alpha-bromo-17-ketones and 2 alpha-bromo-3-ketones.

Epimerizations of 16 alpha-chloro- (1a), bromo- (1b), and iodo-3 beta-hydroxy-5-androsten-17-one (1c) by a brief treatment with 0.2 equiv NaOH in aqueous pyridine reached equilibrium between 16 alpha- and 16 beta-halo ketones. 16 alpha-/16 beta-Halo ketone ratios at equilibrium were 1.5 for Cl, 1.25 for Br, and 1.0 for I. Kinetic analysis showed that compounds 1a-c were stereoselectively converted to the corresponding 16 alpha-hydroxy derivative 3 by an SN2 mechanism, in which the order of the apparent reactivity was Br greater than I greater than Cl. The hydrolysis of a number of 16 alpha-bromo-17-ketones and 2 alpha-bromo-3-ketones was carried out. The yields of the corresponding alcohols were found to depend on remote structural features in the steroids.

Further characterization of membrane proteins involved in the transport of organic anions in hepatocytes. Comparison of two different affinity labels: 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid and brominated taurodehydrocholic acid.

4,4'-Diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (H2DIDS) known as an irreversible inhibitor of the anion transport in red blood cells (Cabantchik, Z.I. and Rothstein, A. (1972) J. Membrane Biol. 10, 311-330) blocks also the uptake of bile acids and of some foreign substrates in isolated hepatocytes (Petzinger, E. and Frimmer, M. (1980) Arch. Toxicol. 44, 127-135). [3H]H2DIDS was used for labeling of membrane proteins probably involved in anion transport of rat liver cells. The membrane proteins modified in vitro by [3H]H2DIDS were compared with those labeled by brominated taurodehydrocholic acid. The latter is one of a series of suitable taurocholate derivatives, all able to bind to defined membrane proteins of hepatocytes and also known to block the uptake of bile acids as well as of phallotoxins and of cholecystographic agents (Ziegler, K., Frimmer, M., Möller, W. and Fasold, H. (1982) Naunyn-Schmiedeberg's Arch. Pharmacol. 319, 254-261). The radiolabeled proteins were compared after SDS-electrophoresis with and without reducing agent present, solubilization by detergents, two-dimensional electrophoresis and after separation of integral and peripheral proteins. Our results suggest that the anion transport system of liver cells cannot distinguish between bile acids and the anionic stilbene derivative (DIDS). The labeling pattern for both kinds of affinity labels was very similar. Various combinations of separation techniques gave evidence that the radiolabeled membrane proteins are not subunits of a single native channel protein.

Chemical modification of membrane proteins by brominated taurodehydrocholate in isolated hepatocytes; relationship to the uptake of cholate and of phalloidin and to the sensitivity of hepatocytes to phalloidin.

In vitro treatment of isolated rat hepatocytes with brominated taurodehydrocholic acid (BTC) reduced their sensitivity against phalloidin and inhibited the uptake of phalloidin as well as of cholate in an irreversible and concentration dependent manner. BTC was taken up itself by liver cells; this process was inhibited by 4,4'-diisothiocyano 2,2'-stilbene disulfonate (DIDS). When hepatocytes were incubated with 35S-BTC their plasma membranes contained five labeled protein species with molecular weights of 67,000, 49,000, 38,000, 32,000 and 24,000 as shown by SDS-electrophoresis. No marked difference was observed when isolated plasma membranes from livers were directly treated with the affinity label. DIDS suppressed covalent binding of 35S-BTC to membrane components drastically. Incubation of phalloidin insensitive AS-30D ascites hepatoma cells with 35S-BTC did not result in a chemical modification of the above five proteins. This agrees with an earlier observation that hepatoma cells are unable to take up phalloidin and bile acids (Petzinger et al. 1979; Rufeger and Grundmann 1977; Kroker et al. 1978).

Controlled alkaline hydrolysis of steroidal alpha-bromoketones: new conditions and synthesis of 2 alpha-hydroxy-3-ones.

Controlled alkaline hydrolysis of 16 alpha-bromo-17-keto steroids 1, 5 and 7 with potassium carbonate and tetra-n-butylammonium hydroxide (n-Bu4NOH) and synthesis of 2 alpha-hydroxy-3-ones 11, 13 and 16 by the controlled hydrolysis of the corresponding 2 alpha-bromo-3-ones 9, 12 and 15 are described. Treatment of the bromoketones 1,5 and 7 with potassium carbonate in aqueous acetone or with n-Bu4NOH in aqueous dimethylformamide (DMF) gave 16 alpha-hydroxy-17-ones 3m 6 and 8 in 85-90% yield, respectively. 2 alpha-Hydroxy-3-ones 11, 13 and 16 were obtained by hydrolysis of the corresponding bromoketones 9, 12 and 15 in high yields using the above conditions or sodium hydroxide in pyridine or DMF, respectively. Deuterium labeling experiments suggested that equilibration between the 2 alpha-bromoketone 9 and the 2 beta-bromo isomer 10 precedes the formation of the ketol 11 in which the true intermediate might be the 2 beta-isomer 10. However, rearranged androstane derivatives, 3 beta-hydroxy-2-one 18 and 20, were stereoselectively obtained by treatment of the bromoketones 12 and 15 with an excess amount of sodium hydroxide.

Synthesis of dehydroepiandrosterone sulfatide and 16 alpha-halogenated steroids.

Dehydroepiandrosterone sulfatide was prepared in a 68% yield by the reaction of 5-androstene-3 beta-ol-17-one 3 sulfate (silver salt) with dipalmitoyl alpha-iodopropylene glycol. The sulfatide was found to be a more potent inhibitor of human glucose-6-phosphate dehydrogenase than dehydroepiandrosterone. 16 alpha-Halogenated steroids also were prepared by direct halogenation of the steroid or indirect halogenation of an appropriate steroidal intermediate. Among various halogenated steroids, 16 alpha-bromoepiandrosterone was 50 times as potent as dehydroepiandrosterone as an inhibitor of glucose-6-phosphate dehydrogenase.

The oxidation of a steroidal bromohydrin revisited.

A comparative study was made of the reactions of 5-bromo-3 beta, 6 beta-dihydroxy-5 alpha-androstan-17-one 3-acetate (1) with lead tetraacetate alone and in the presence of iodine in both high intensity visible light and in total darkness using a variety of solvents. Markedly different product profiles were obtained under the different reaction conditions, making our results of both practical importance and theoretical interest.

16 alpha-[77Br]bromoestradiol-17 beta: a high specific-activity, gamma-emitting tracer with uptake in rat uterus and uterus and induced mammary tumors.

16 alpha-[77Br]Bromoestradiol-17 beta (Compound 1) has been synthesized by radiobromination of estrone enoldiacetate. Tissue uptake studies performed 1 hr after administration of Compound 1 to immature or mature female rats showed uterus-to-blood ratios of 13, with nontarget issue-to-blood ratios ranging from 0.6 to 2. Co-administration of unlabelled estradiol caused a selective depression in the uterine uptake with no effect on nontarget tissue uptake. In adult animals bearing adenocarcinomas induced by DMBA (7,12-dimethylbenz(a)anthracene), tumor-to-blood ratios of 6.3 were obtained, this uptake also being depressed in animals treated with unlabeled estradiol. The studies demonstrate that Compound 1 has suitable binding properties and sufficiently high specific activity so that its uptake in estrogen target tissues in vivo is mediated primarily by the estrogen receptor. Furthermore, they suggest that this compound may be suitable for imaging human breast tumors that contain estrogen receptors.

Bifunctional enzyme activity at the same active site: competitive inhibition kinetics with 3 alpha/20 beta-hydroxysteroid dehydrogenase.

20 beta-Hydroxy-5 alpha-pregnan-3-one (HPO) is a competitive inhibitor of reduction by 3 alpha/20 beta-hydroxysteroid dehydrogenase (3 alpha/20 beta-HSD; E.C.1.1.1.53) of 17 beta-hydroxy-5 alpha-androstan-3-one (DHT; 3 alpha-activity; Ki = 4.6x10(-5)M), and of 6 beta-acetoxyprogesterone (6 beta-AP; 20 beta-activity; Ki = 4.34x10(-5)M). HPO and DHT inhibit affinity alkylation of 3 alpha/20 beta-HSD by 6 beta-bromoacetoxyprogesterone (6 beta-BAP). The facts that 1) enzyme 3 alpha-activity and 20 beta-activity are both competitively inhibited by HPO with practically identical Ki-values, 2) 6 beta-BAP is solely a 20 beta-activity substrate for 3 alpha/20 beta-HSD, 3) one mole of 6 beta-BAP reacts with one mole of 3 alpha/20 beta-HSD to simultaneously inactivate 3 alpha- and 20 beta-activity, and 4) inactivation of 3 alpha/20 beta-HSD by 6 beta-BAP is inhibited by DHT (a C19-steroid) or HPO (a C21-steroid), support the view that the same active site of 3 alpha/20 beta-HSD possesses both 3 alpha- and 20 beta-activity. Bifunctional activity at the same active site is considered for other steroid-specific enzymes in female mammalian reproductive systems.

Synthesis and some reactions of 6-bromoandrogens: potential affinity ligand and inactivator of estrogen synthetase.

The synthesis of epimeric 6-bromo-4-androstene-3,17-dione (1a and 1b), 6-bromotestosterone (2a and 2b) and its acetate (3a and 3b), and 6-bromo-16 alpha-acetoxy-4-androstene-3,17-dione (5a and 5b), and 6 beta-bromo-16 alpha-hydroxy-4-androstene-3,17-dione (4) is described. The interconversions among compounds 1, 2, and 3 are also studied. The 6 beta-isomer (1b, 2b, and 3b) was epimerized to the 6 alpha-isomer (1a, 2a and 3a) in carbon tetrachloride or chloroform-methanol (9:1) and the 6 alpha-isomer was isolated by fractional crystallization from the epimeric mixture. 6 alpha-Bromo isomer 1a was also epimerized back to 6 beta-bromo isomer 1b in chloroform-methanol (9:1). Two polymorphic forms of 6 beta-bromotestosterone acetate (3b) were isolated (mp. 114--117 degrees and 138--141 degrees). The 6 beta-bromo isomers were found to be unstable in methanol and decomposed to give 5 alpha-androstane-3,6-dione derivative (6). The results of irreversible inactivation of human placental androgen aromatase with some of these 6-bromoandrogens are discussed.

BOMT (6 alpha-bromo-17 beta-hydroxy-17 alpha-methyl-4-oxa-5 alpha-androstan-3-one) is not an androgen antagonist within the central nervous system.

This study investigates the efficiency of BOMT as an androgen antagonist within the central nervous system. The efficiency of BOMT in suppressing neural receptor binding of testosterone, and the ability of this antiandrogen to block the feedback loop of testosterone onto the central nervous system, as evidenced by plasma testosterone levels, is reported. BOMT was found to be unable to open the feedback loop of testosterone onto the central nervous system, which was correlated with the low competing efficiency of this antiandrogen for receptor sites in vitro within the hypothalamic-preoptic area of the brain - a region known to be involved in gonadotrophin secretion. The observed divergence in the degree of antiandrogenicity of BOMT between peripheral and central target tissues of testosterone is discussed.

Investigations into the use of 77Br labelled 5alpha-dihydrotestosterone for scanning the prostate.

The potential use of a potent androgen labelled with a gamma emitting radionuclide for scanning the prostate was investigated. 2alphabromo 5alpha-dihydrotestosterone was synthesized and subsequently labelled with 77Br. The distribution of this compound was studied in rat and human tissues. The maximum concentration of radioactivity in the rat prostate at 1-4 h following the injection was found to be between 0.5-0.8% of the injected dose per gram of the tissue. In the human however, that value was 0.002-0.003%. Considering the overall results it was concluded that this compound is not appropriate for scanning the prostate.

Synthesis an crystal structure of 3beta-p-bromobenzoyloxy-14alpha, 15alpha-epoxy-5alpha-cholest-7-ene.

The chemical synthesis of 3beta-bromobenzoyloxy-14alpha, 15alpha-epoxy-5alpha-cholest-7-ene is described. Single crystal structral analysis was employed to unambiguously determine the location and absolute configuration of the epoxide moiety in the 3beta-p-bromobenzoyloxy-14alpha, 15alpha-epoxy-5alpha-cholest-7-ene. The space group of the crystal was P1, with unit cell parameters: a=10.873 A, b=13.841 A, c=11.037 A, alpha=75.19 degrees, beta=78.79 degrees, gamma=101.57 degrees, and two molecules per unit cell. Intitial phases were derived from the two bromine atoms. Least squares refinements on all non-hydrogen atoms were carried out to a final unweighted R value of 0.10 and weighted R value of 0.04. The epoxide ring was located at the 14, 15 position and was found to extend to the alpha side of the molecule. Molecular measurements for asymmetry parameters of the sterol nuceus indicate that ring A has a symmetrical chair conformation and ring B has a half chair conformation due to the double bond at C(7). Ring C has a fairly distorted chair conformation due to the trigonal C(8) on one sie and the almost planar 5-membered ring on the other. Ring D has the 17alpha-envelope conformation.

Radiobromine labeled norcholesterol analogs. Synthesis and tissue distribution study in rats of bromine-82 labeled 6beta-bromomethyl-19-norcholest-5(10)-EN-38-OL.

6beta-Bromomethyl-19-norcholest-5(10)-en-3beta-ol (NCL-6-Br) was synthesized directly from cholest-5-ene-3beta,19-diol 19-toluene-p-sulfonate via homoallylic rearrangement with ammonium bromide or sodium bromide in acetonitrile. This method was applied to the radiolabeling of NCL-6-Br with bromine-82. Tissue distribution of bromine-82 labeled NCL-6-Br (NCL-6-Br-82) in rats was determined. The mean percent dose per gram uptake in adrenal at 24 and 120 h was 98 and 80 %/gm, respectively, which indicated a higher adrenal uptake as compared to iodine-131 labeled 19-iodocholest-5-en-3beta-ol (CL-19-I-131), but was at a lower level than that achieved with iodine-131 labeled 6beta-iodomethyl-19-norcholest-5(10)-en-3beta-ol (NCL-6-I-131). The ratio of radioactivity in the adrenal-to-liver concentration was also lower than that of CL-19-I-131 or NCL-6-I 131.