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.

Novel steroidal vinyl fluorides as inhibitors of steroid C17(20) lyase.

20-fluoro-17(20)-pregnenolone derivatives were designed as enol mimics of pregnenolone. All of the targeted, novel fluoroolefins were potent inhibitors of C17(20) lyase.

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.

Side chain modified sterols as probes into insect molting hormone metabolism. II: synthesis of monofluorocholesterols.

The hydroxylations of the cholesterol side chain at C-20, 22, and 25 are key terminal events in ecdysone biogenesis. We have prepared the C-20, C-22, C-24, and C-25 monofluorinated cholesterols as potential inhibitors of these hydroxylation events, and preliminary bioassay results in Manduca sexta are reported. The synthesis of [26(14)C]-20-fluorocholesterol is also described. Although the 20-, 22-, and 25-monofluorocholesterols do not appear to affect larval growth and development, the 24-fluoro isomer shows a moderate retardation of growth and a modest increase in mortality.

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.

Synthesis of fluorine-18 labeled 21-fluoroprogesterone.

21-Fluoroprogesterone has been synthesized by the nucleophilic displacement of the mesyl group of the 21-hydroxypregn-4-ene-3,20-dione 21-methanesulfonate by fluoride ion, as formed from the solvolysis of potassium fluoride in acetonitrile, using 18-Crown-6 as the catalyst. In a similar manner, the synthesis of 21-fluoroprogesterone labeled with fluorine-18 has been accomplished, producing approximately 800 muCi of product from 6 mCi of K18F in one hour. This demonstrates that 18F labeled molecules of potential values as readiopharmaceuticals can be prepared rapidly in sufficient yield to allow animal investigation.

Synthesis and topical antiinflammatory properties of 17,21-bis(acetyloxy)-6beta,9-difluoro-11beta-hydroxypregna-1,4-diene-3,20-dione and related 2-halogenated compounds.

Introduction of a halogen atom at C-2 of steroid 3-ketofluorohydrins, obtained from the corresponding 5alpha,6alpha-epoxides by trans-diaxial opening with hydrofluoric acid, prevents the 6beta-fluorine atom from undergoing rearrangement to the more stable 6alpha configuration when the 5-tert-hydroxyl is split off to yield to yield a conjugated double bond. Two processes were investigated for the synthesis of 17,21-bis(acetyloxy)-6beta-fluoro-1,4,9(11)-triene-3,20-dione (24a) and the related 2-bromo compound 24b starting from the known 21-(acetyloxy)-6beta-fluoro-5alpha,11alpha,17-trihydroxypregnane-3,20-dione (13). Successive reaction with hypobromous acid, epoxidation, and fluorination converted 24a and 24b into the title compound 27a and the analogue 2-bromo compound 27b. In addition, a synthesis of 17,21-bis(acetyloxy)-2-chloro-6beta,9-difluoropregna-1,4-diene-3,20-dione (27c) is reported. The antiinflammatory activity of 17,21-bis-(acetyloxy)-6beta,9-difluoropregna-1,4-diene-3,20-dione (27a) and its 2-halogenated analogues 27b and 27c in comparison with the corresponding 6alpha,9-difluoro epimers was studied. Some 6beta-fluoro compounds displayed high topical antiinflammatory activity without systemic effects.

Synthesis of fluorinated 3beta-hydroxypregn-5-en-20-one derivatives.

Treatment of the unstable 3beta-hydroxy-20, 20-dimethoxypregn-5-ene 3-acetate with acetic anhydride at reflux temperature gave a mixture of 3beta-hydroxy-20-methoxypregna-5, 17(20)-diene and 3beta-hydroxy-20-methoxypregna-5, 20-diene 3-acetates. Fluorination of this mixture with perchloryl fluoride afforded after fractionated crystallization 3beta-hydroxy-17-fluoro-20-methoxypregna-5, 20-diene 3-acetate. Acid hydrolysis of the reaction mixture and subsequent chromatographic separation led to 3beta-hydroxy-17-fluoropregn-5-en-20-one 3-acetate and 3beta-hydroxy-21-fluoropregn-5-en-20-one 3-acetate. 3beta-Hydroxy-17-fluoro-20-methoxy-pregna-5, 20-diene 3-acetate did not react further with perchloryl fluoride even under forcing conditions. Fluorination of 3beta-hydroxy-20-(N-ethyl benzylamino)-pregna-5, 17(20)-diene gave 3beta-hydroxy-17, 21-difluoro-pregn-5-en-20-one, exclusively.