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.

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.

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.

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.

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.

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.