Solid-state NMR study of fluorinated steroids.

Solid-state {(1)H}(13)C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy was performed to analyze two fluorinated steroids, i.e., betamethasone (BMS) and fludrocortisone acetate (FCA), that have fluorine attached to C9, as well as two non-fluorinated analogs, i.e., prednisolone (PRD) and hydrocortisone 21-acetate (HCA). The (13)C signals of BMS revealed multiplet patterns with splittings of 16-215Hz, indicating multiple ring conformations, whereas the (13)C signals of FCA, HCA, and PRD exhibited only singlet patterns, implying a unique conformation. In addition, BMS and FCA exhibited substantial deviation (>3.5ppm) in approximately half of the (13)C signals and significant deviation (>45ppm) in the (13)C9 signal compared to PRD and HCA, respectively. In this study, we demonstrate that fluorinated steroids, such as BMS and FCA, have steroidal ring conformation(s) that are distinct from non-fluorinated analogs, such as PRD and HCA.

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.

3alpha-6alpha-Dihydroxy-7alpha-fluoro-5beta-cholanoate (UPF-680), physicochemical and physiological properties of a new fluorinated bile acid that prevents 17alpha-ethynyl-estradiol-induced cholestasis in rats.

3alpha-6alpha-Dihydroxy-7alpha-fluoro-5beta-cholanoate (UPF-680), the 7alpha-fluorine analog of hyodeoxycholic acid (HDCA), was synthesized to improve bioavailability and stability of ursodeoxycholic acid (UDCA). Acute rat biliary fistula and chronic cholestasis induced by 17alpha-ethynyl-estradiol (17EE) models were used to study and compare the effects of UPF-680 (dose range 0.6-6.0 micromol/kg min) with UDCA on bile flow, biliary bicarbonate (HCO(3)(-)), lipid output, biliary bile acid composition, hepatic enzymes and organic anion pumps. In acute infusion, UPF-680 increased bile flow in a dose-related manner, by up to 40.9%. Biliary HCO(3)(-) output was similarly increased. Changes were observed in phospholipid secretion only at the highest doses. Treatment with UDCA and UPF-680 reversed chronic cholestasis induced by 17EE; in this model, UDCA had no effect on bile flow in contrast to UPF-680, which significantly increased bile flow. With acute administration of UPF-680, the biliary bile acid pool became enriched with unconjugated and conjugated UPF-680 (71.7%) at the expense of endogenous cholic acid and muricholic isomers. With chronic administration of UPF-680 or UDCA, the main biliary bile acids were tauro conjugates, but modification of biliary bile acid pool was greater with UPF-680. UPF-680 increased the mRNA for cytochrome P450 7A1 (CYP7A1) and cytochrome P450 8B (CYP8B). Both UDCA and UPF-680 increased the mRNA for Na(+) taurocholate co-transporting polypeptide (NCTP). In conclusion, UPF-680 prevented 17EE-induced cholestasis and enriched the biliary bile acid pool with less detergent and cytotoxic bile acids. This novel fluorinated bile acid may have potential in the treatment of cholestatic liver disease.

Synthesis of 21,21-difluoro-3 beta-hydroxy-20-methylpregna-5,20-diene and 5,16,20-triene as potential inhibitors of steroid C17(20) lyase.

Novel 21,21-difluorovinyl steroids, designed as difluorinated C20(21) enol mimics of pregnenolone, were targeted as potential mechanism-based inhibitors of C17(20) lyase, a crucial enzyme in the biosynthesis of testosterone. Addition of (difluoromethyl)diphenylphosphine oxide reagent to 17-acetyl steroids was the approach chosen for the construction of these compounds. Of particular interest were the abnormal Wittig products which formed during attempted preparation of the triene 9. The target difluoroolefin 3 was found to be a moderately potent, time-dependent inhibitor of the enzyme.

Synthesis of C-6 fluoroandrogens: evaluation of ligands for tumor receptor imaging.

Seven androgens, substituted with fluorine at C-6, were prepared as potential imaging agents for androgen receptor-positive prostate tumors and were evaluated in vitro in terms of their lipophilicity and their relative binding affinities (RBA, relative to R 1881 = 100) for the androgen receptor and for sex steroid binding protein. Introduction of a fluorine atom into the C-6 position of an androgen generally decreases binding affinity to the androgen receptor, except in the two cases: 6 alpha-fluoro-19-nor-testosterone (RBA = 41.6 versus 30.6 for the unsubstituted steroid) and 6 alpha-fluorotestosterone (RBA = 8.9 versus 6.6). Receptor binding of the C-6 fluoro-androgens is also stereospecific, showing higher binding affinities for the alpha-epimers compared to the corresponding beta-epimers (4:1-15:1). Binding affinity to sex steroid binding protein is the lowest with 19-nor-testosterone, which is also the least lipophilic androgen studied. Based on the binding properties of compounds in this series, 6 alpha-fluoro-19-nor-testosterone appears to have the most promise as a tumor imaging agent.

Synthesis of A-ring fluorinated derivatives of (17 alpha,20E/Z)-[125I]iodovinylestradiols: effect on receptor binding and receptor-mediated target tissue uptake.

We have prepared a series of 2- and 4-fluoro derivatives of the isomeric (17 alpha,20E)- and (17 alpha, 20Z)iodovinylestradiols (IVE2) and also the analogs substituted with either a 7 alpha-methyl (7 alpha-Me-IVE2) or 11 beta-methoxy group (11 beta-OMe-IVE2) and evaluated their in vitro and in vivo properties. Electrophilic substitution of the estrone derivatives with N-fluoropyridinium salt gave the 2- and 4-fluoro analogs which were subsequently converted to the 17 alpha-ethynyl derivatives. The tributylstannyl intermediates were obtained from the corresponding 17 alpha-ethynyl analogs using azobisisobutyronitrile or triethylborane as catalyst. All 12 products were also prepared as their no-carrier-added [125I]iodovinyl analogs via destannylation of the tributylstannyl precursors. Binding affinity for the estrogen receptor (ER) was in general higher for the 4-F derivatives as compared to the 2-F derivatives, while the 20Z isomers of the same compounds showed somewhat higher ER binding affinity as compared to the 20E isomers. The combination of an A-ring fluoro and 7 alpha- or 11 beta-substituent decreased ER binding affinity. Substitution of a fluoro atom at C-4 on either the 17 alpha-ethynylestradiol or isomeric 17 alpha-IVE2 enhanced the affinity of the parent molecule for the ER. A-ring fluorination of all other analogues tested had no effect or depressed ER binding affinity. Varying incubation conditions showed substantial differences in ER binding kinetics between the 20E and 20Z isomers. Tissue distribution in immature female rats showed that the highest uterus uptake and uterus to blood/nontarget ratios in the IVE2 series were obtained with the 4-F-(17 alpha,20Z)IVE2 isomer. The combination of A-ring fluoro and 7 alpha- or 11 beta-substitution decreased uterus uptake but had little or no effect on uterus to blood/nontarget ratios. The highest uterus to blood ratios were observed for the 4-F-(17 alpha,20E)11 beta-OMe-IVE2 (75 at 6 h and 125 at 12 h pi) reflecting rapid blood clearance and in vivo stability, as confirmed by the low levels of thyroid radioactivity. The lack of correlation between ER binding affinities and uterus uptake, and/or uptake ratios, suggests that other factors, including nonspecific binding and metabolic processes, also are involved in the tissue localization process. Our data suggest that 4-F substitution onto (17 alpha,20Z)IVE2 and (17 alpha,20E)11 beta-OMe-IVE2 enhances the potential of these compounds to function as SPECT imaging agents of ER-rich tissues.

Liposomes containing fluorinated steroids: an analysis based on photon correlation and fluorine-19 nuclear magnetic resonance spectroscopy.

Small unilamellar liposomes containing fluorinated steroids (flumethasone and dexamethasone) were obtained. A physicochemical evaluation was conducted based on photon correlation spectroscopy (PCS) and fluorine-19 (19F) nuclear magnetic resonance (NMR) spectroscopy compared with standard biochemical methods (HPLC). The PCS method provides a fast and adequate evaluation of some critical features of liposomes (size, physical stability). In addition, 19F NMR spectroscopy gives substantial information, in a nondestructive manner, on steroid behavior in the membrane upon encapsulation and also when the temperature of liposomes is increased. The combined spectroscopic approach proposed here might prove useful in (1) the management of liposomal formulation, especially in the documentation of physicochemical properties, (2) pharmaceutical control in the industrial production line, and (3) control preceding injection at the clinical site. Spectroscopic techniques might offer a complementary approach to classical biochemical methods in the evaluation of the properties of a liposomal formulation and could be usefully integrated into quality control procedures.