7α Position Research Articles

Crystal structure of an apo 7α-hydroxysteroid dehydrogenase reveals key structural changes induced by substrate and co-factor binding

7α-Hydroxysteroid dehydrogenase (7α-HSDH) catalyzes the dehydrogenation of a hydroxyl group at the 7α position in steroid substrates using NAD+ or NADP+ as a co-factor. Although studies have determined the binary and ternary complex structures, detailed structural changes induced by ligand and co-factor binding remain unclear, because ligand-free structures are not yet available. Here, we present the crystal structure of apo 7α-HSDH from Escherichia coli (Eco-7α-HSDH) at 2.7 Å resolution. We found that the apo form undergoes substantial conformational changes in the β4-α4 loop, α7-α8 helices, and C-terminus loop among the four subunits comprising the tetramer. Furthermore, a comparison of the apo structure with the binary (NAD+)-complex and ternary (NADH and 7-oxoglycochenodeoxycholic acid)-complex Eco-7α-HSDH structures revealed that only the ternary-complex structure has a fully closed conformation, whereas the binary-complex and apo structures have a semi-closed or open conformation. This open-to-closed transition forces several catalytically important residues (S146, Y159, and K163) into correct positions for catalysis. To confirm the catalytic activity, we used alcohol dehydrogenase for NAD+ regeneration to allow efficient conversion of chenodeoxycholic acid to 7-ketolithocholic acid by Eco-7α-HSDH. These findings demonstrate that apo Eco-7α-HSDH exhibits intrinsically flexible characteristics with an open conformation. This structural information provides novel insight into the 7α-HSDH reaction mechanism.

Identification of iridoid synthases from Nepeta species: Iridoid cyclization does not determine nepetalactone stereochemistry

Nepetalactones are iridoid monoterpenes with a broad range of biological activities produced by plants in the Nepeta genus. However, none of the genes for nepetalactone biosynthesis have been discovered. Here we report the transcriptomes of two Nepeta species, each with distinctive profiles of nepetalactone stereoisomers. As a starting point for investigation of nepetalactone biosynthesis in Nepeta, these transcriptomes were used to identify candidate genes for iridoid synthase homologs, an enzyme that has been shown to form the core iridoid skeleton in several iridoid producing plant species. Iridoid synthase homologs identified from the transcriptomes were cloned, heterologously expressed, and then assayed with the 8-oxogeranial substrate. These experiments revealed that catalytically active iridoid synthase enzymes are present in Nepeta, though there are unusual mutations in key active site residues. Nevertheless, these enzymes exhibit similar catalytic activity and product profile compared to previously reported iridoid synthases from other plants. Notably, four nepetalactone stereoisomers with differing stereochemistry at the 4α and 7α positions – which are generated during the iridoid synthase reaction – are observed at different ratios in various Nepeta species. This work strongly suggests that the variable stereochemistry at these 4α and 7α positions of nepetalactone diastereomers is established further downstream in the iridoid pathway in Nepeta. Overall, this work provides a gateway into the biosynthesis of nepetalactones in Nepeta.

Octyl Gallate Markedly Promotes Anti-Amyloidogenic Processing of APP through Estrogen Receptor-Mediated ADAM10 Activation

Our previous studies showed that the green tea-derived polyphenolic compound (−)-epigallocatechin-3 gallate (EGCG) reduces amyloid-β (Aβ) production in both neuronal and mouse Alzheimer’s disease (AD) models in concert with activation of estrogen receptor-α/phosphatidylinositide 3-kinase/protein kinase B (ERα/PI3K/Akt) signaling and anti-amyloidogenic amyloid precursor protein (APP) α-secretase (a disintegrin and metallopeptidase domain-10, ADAM10) processing. Since the gallate moiety in EGCG may correspond to the 7α position of estrogen, thereby facilitating ER binding, we extensively screened the effect of other gallate containing phenolic compounds on APP anti-amyloidogenic processing. Octyl gallate (OG; 10 µM), drastically decreased Aβ generation, in concert with increased APP α-proteolysis, in murine neuron-like cells transfected with human wild-type APP or “Swedish” mutant APP. OG markedly increased production of the neuroprotective amino-terminal APP cleavage product, soluble APP-α (sAPPα). In accord with our previous study, these cleavage events were associated with increased ADAM10 maturation and reduced by blockade of ERα/PI3k/Akt signaling. To validate these findings in vivo, we treated Aβ-overproducing Tg2576 mice with OG daily for one week by intracerebroventricular injection and found decreased Aβ levels associated with increased sAPPα. These data indicate that OG increases anti-amyloidogenic APP α-secretase processing by activation of ERα/PI3k/Akt signaling and ADAM10, suggesting that this compound may be an effective treatment for AD.

Abstract 2474: Synthesis and antiproliferative activity of novel 17β-acetyl-testosterone-7α-platinum(II) hybrids designed for the treatment of human prostate cancer.

Abstract Prostate cancer is a major public health problem worldwide. In the United-States, prostate cancer is ranked first of all new cancer cases diagnosed in men and it is the second most lethal cancer. Antiandrogens are normally used in clinic to slow down the progression of androgen-sensitive (AR+) prostate cancer inducing prolonged remission without definite cure. In general, 10 years after the initial treatment of early prostate cancers, 19% of patients will experience local tumor progression, 15% distant metastases and 10% will die. Furthermore, advanced prostate cancers quickly (18 to 24 months) become androgen-insensitive (AR-) and antihormone therapy becomes ineffective. At this point, the median survival of approved drug combination treatments is then under 20 months. Consequently, the search for new, more selective and efficient treatments is still a subject of intense research. To that end, we used the natural tropism of testosterone for prostate tissues to target platinum(II) complexes to prostate cancer cells. The platinum complexes were linked to the 7α position on testosterone as it is located between the 3-carbonyl and 17-hydroxyl groups involved in key hydrogen bonds essential for androgen receptor interactions. L- and D-2-pyridylalanine, L-histidine and L-4-thiazolylalanine derivatives were linked to 17β-acetyl-testosterone selected to chelate platinum(II). Four new amino acid platinum(II) complexes were prepared (compounds 1a-1d), characterized and evaluated for their antiproliferative activity on LNCaP (AR+), PC3 (AR-) and DU145 (AR-) human prostate adenocarcinoma cancer cell lines. The new testosterone-platinum(II) complexes exhibited antiproliferative activities at the micromolar level on all prostate cancer cell lines tested. Structure-activity relationships show that the stereochemistry of amino acids (compounds 1a, b) does not affect the antiproliferative activity. Moreover, the replacement of a pyridinyl moiety by an imidazolyl group (compound 1c) decreased the activity by up to 10 fold whereas the replacement by a thiazolyl group (compound 1d) showed an antiproliferative activity between 2.5 to 8.0 times higher than that of cisplatin. The innovative design and the significant activity of our testosterone-platinum(II) complexes toward AR+ and AR- prostate cancer cells suggest that they might be useful for treatment of advanced prostate cancers. Citation Format: Sébastien Fortin, Kevin Brasseur, Nathalie Morin, Éric Asselin, Gervais Bérubé. Synthesis and antiproliferative activity of novel 17β-acetyl-testosterone-7α-platinum(II) hybrids designed for the treatment of human prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2474. doi:10.1158/1538-7445.AM2013-2474

Novel metabolites of dehydroepiandrosterone and progesterone obtained in Didymosphearia igniaria KCH 6670 culture

Abstract Dehydroepiandrosterone (DHEA) (10) and its five derivatives: testosterone (1), androstenedione (2), 17α-methyltestosterone (6), progesterone (13) and pregnenolone (14) were subjected to microbial transformation by the filamentous fungus Didymosphaeria igniaria KCH 6670. The predominant metabolism of the incubated 5-ene steroids (10 and 14) occurred through 3β-hydroxy-steroid dehydrogenase/5,4-en isomerase pathways resulting in the generation of a 4-en-3-oxo system on ring-A. The transformations of C19 steroids (1, 2, and 10) included a hydroxylation at 7α position, ketone–alcohol interconversion at C-17 and reduction of the double bond at C-4 and 3-keto group to the 3β-alcohol with 5α-stereochemistry at A/B ring. D. igniaria also carried out 6(7)-dehydrogenation and 6,7β-epoxidation during transformation of DHEA. Under these conditions transformation of DHEA (10) gave four products: 7α-hydroxyandrost-4-en-3,17-dione (4), 17β-hydroxyandrost-4,6-dien-3-one (11), 17β-hydroxyandrost-6β-epoxy-4-en-3-one (12) and 3β,17β-dihydroxy-5α-androstane (5). The compounds 11 and 12 are identified as DHEA metabolites for the first time. The transformation of C21 steroids (13 and 14) led to the mixture of mono- (mainly 11α- and 15β-) and dihydroxy- (7α,15β-; 14α,15β-; 11α,15β-; 11α,14α-) products. 7α,15β-Dihydroxypregnan-4-en-3,20-dione (18) and 14α,15β-dihydroxypregnan-4-en-3,20-dione (19) were found to be new compounds. The main product of transformation of 17α-methyltestosterone (6) was 12β-hydroxy-17α-methyltestosterone (7). The results of these transformations demonstrate the dependence of hydroxylation position on the structure of steroid nucleus.

Selective recognition of [formula omitted] by a cholestane-imidazole-zinc ensemble

A new facile amphiphile cholestane-based zinc complex 4 containing a 3-aminopropylimidazole moiety at the 3α and 7α positions of cholestane was designed and synthesized. Recognition selectivity of the new receptor 4 with various anions was assessed by 1H NMR titration. Dihydrogen phosphate showed the highest binding affinity among all the tested anions (Ka=4.4×105M−1).

New 2-aminoethylimidazole-based dicarboxylic acid receptor derived from cholestane

A new facial amphiphile cholestane-based receptor 1 containing a 2-imidazolylethylamino moiety at the 3α and 7α positions of cholestane was synthesized. Recognition selectivity of the new receptor 1 with various dicarboxylic acids was assessed by 1H NMR titration. Maleic acid showed the highest binding constant among all the tested acids ( K a = 9.36 × 10 4 M −1).

New insights into the protective effects of DHEA1).

Numerous studies investigated the effects of pharmacological doses of DHEA in animals. Among protective effects, antiglucocorticoid potencies, triggering and modulation of immunity and anticancerous effects were reported. Because DHEA levels decrease in aging humans, this steroid has been assayed as replacement therapy in elderly volunteers without striking evidence for beneficial effects. Examination of the investigations carried out in animals lead to suspect that, rather than DHEA, its metabolites produced in tissues could be responsible for some of the observed effects. Known as the "mother steroid", DHEA is a precursor for androgenic and estrogenic steroid hormones. In addition, DHEA is hydroxylated at the 7α position by the cytochrome P450 7B1 (CYP7B1), and the 7α-hydroxy-DHEA produced is a substrate for the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which converts it into 7β-hydroxy-DHEA. Both 7-hydroxylated metabolites were shown to favor the onset of immunity in mice and the activation of memory T cells in humans. Other DHEA and testosterone-derived metabolites, namely epiandrosterone and 5α-androstane-3β,17β-diol, are also substrates for the CYP7B1 and their 7α-hydroxylated products were also converted into the 7β epimer by the 11β-HSD1. When assayed at doses 104 lower than DHEA, 7β-hydroxy-epiandrosterone was shown to shift the prostaglandin metabolism patterns from prostaglandin E2 (PGE2) to PGD2 production, thus triggering the resolution of inflammation. In addition, 7β-hydroxy-epiandrosterone (1nM) exerted the same effects as tamoxifen (1μM) on the proliferation of MCF-7 and MDA-231 human breast cancer cells. These findings suggest that the observed effects of 7β-hydroxy-epiandrosterone could be mediated by estrogen receptors. This overview of recent research implies that DHEA does not act directly and that its effects are due to its metabolites when produced in tissues. Treatments with DHEA should take into account the target tissue abilities to produce the desired metabolites through the two key enzymes, CYP7B1 and 11β-HSD1.

Synthesis and investigation of the spatial arrangement of the 17β-acetoxy-7α,18-dimethyl-3-methoxy-6-oxaestra-1,3,5(10),8(9)-tetraene

A synthesis was developed of 17β-acetoxy-7α,18-dimethyl-3-methoxy-6-oxaestra-1,3,5(10),8(9)-tetraene. The spatial arrangement of the compound in a crystal and in solution was investigated by means of X-ray diffraction analysis and NMR spectroscopy. The conformation prevailing in solution with the 7α-methyl group in a quasiaxial position corresponds to the structure of the compound in the crystalline state. The presence of a methyl in the 7α position is an important factor governing the osteoprotecting activity of steroids with the similar structure.

Estradiol derivatives bearing sulfur-containing substituents at the 11β or 7α positions: versatile reagents for the preparation of estrogen conjugates

Estradiol derivatives bearing HS-, HSCH 2-, HSCH 2CH 2-, MeS-, MeSCH 2-, MeSCH 2CH 2-, or PhCH 2SCH 2CH 2-groups at the 11β position or an HS-group at the 7α position have been synthesized, and their binding affinity to the estrogen receptor (ER) determined. Nearly all of these substituted estrogens retain high binding affinity, and at the 11β position, the sulfur atom has an effect on ER binding that is similar to that of a carbon atom. These thiol derivatives are promising intermediates for the preparation of a variety of estradiol conjugates. The methyl sulfides, in particular, might potentially be developed as 11C-labeled agents for imaging ER-positive tumors by positron emission tomography.

Dehydroepiandrosterone 7-hydroxylase cyp7b: predominant expression in primate hippocampus and reduced expression in alzheimer's disease ☆

Neurosteroids such as dehydroepiandrosterone (DHEA), pregnenolone and 17β-estradiol are synthesized by cytochrome P450s from endogenous cholesterol. We previously reported a new cytochrome P450 enzyme, CYP7B, highly expressed in rat and mouse brain that metabolizes DHEA and related steroids by hydroxylation at the 7α position. Such 7-hydroxylation can enhance DHEA bioactivity in vivo. Here we show that the reaction is conserved across mammalian species: in addition to mouse and rat, DHEA hydroxylation activity was present in brain extracts from sheep, marmoset and human. Northern blotting using a human CYP7B complementary deoxyribonucleic acid (cDNA) probe confirmed the presence of CYP7B mRNA in marmoset and human hippocampus; CYP7B mRNA was present in marmoset cerebellum and brainstem, with lower levels in hypothalamus and cortex. In situ hybridization to human brain revealed higher levels of CYP7B mRNA in the hippocampus than in cerebellum, cortex, or other brain regions. We also measured CYP7B expression in Alzheimer's disease (AD). CYP7B mRNA was significantly decreased (approximately 50% decline; P<0.05) in dentate neurons from AD subjects compared with controls. A decline in CYP7B activity may contribute the loss of effects of DHEA with ageing and perhaps to the pathophysiology of AD.

Radiochemical synthesis and tissue distribution of Tc-99m-labeled 7α-substituted estradiol complexes

The diagnosis and staging of breast cancer could be improved by the development of radiopharmaceutical imaging agents that provide a noninvasive determination of the estrogen receptor (ER) status of tumor cells. Agents labeled with 99mTc would be especially valuable in this regard. In attempting to achieve this goal, we synthesized four 99mTc-labeled 7α-substituted estradiol complexes. One complex utilizes the “3+1” mixed ligand design to introduce the Tc metal, whereas the other three took advantage of the cyclopentadienyltricarbonylmetal (CpTM) design. The Tc moieties were attached to the 7α position of estradiol with a hexyl tether, a monoether tether, or a polyether tether. The corresponding rhenium compounds have binding affinities for the ER of 20–45% compared with estradiol. Radiochemical yields of the 99mTc-labeled compounds ranged from approximately 15% for the CpT-Tc complexes to 95% for the 3 + 1 inorganic complex. Tissue distribution studies in immature female rats showed low nonreceptor-mediated uptake in the target organs and high uptake in nontarget organs such as the liver and fat. These complexes represent the first time that estradiol has been labeled at the 7α position with 99mTc and provide a further refinement of our understanding of ligand structure-binding affinity correlations for the ER.

Crystallization and preliminary crystal structure of the complex of 17β-hydroxysteroid dehydrogenase with a dual-site inhibitor

Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1) catalyzes the synthesis of 17β-estradiol (E 2) from estrone, in the ovary and peripheral tissues. While the structures of 17β-HSD1 alone and in complex with E 2 have been determined (D. Ghosh, V. Pletnev, D.-W. Zhu, Z. Wawrzak, W.-L. Duax, W. Pangborn, F. Labrie, S.-X. Lin, Structure of human 17β-hydroxysteroid dehydrogenase at 2.20 Å resolution, Structure 3 (1995) 503–513; A. Azzi, P.H. Rhese, D.-W. Zhu, R.L. Campbell, F. Labrie, S.-X. Lin, Crystal structure of human estrogenic 17β-hydroxysteroid dehydrogenase complexed with 17 β-estradiol, Nature Struct. Biol. 3 (1996) 665–668, no structures of inhibitor/enzyme complex, either modeled or from crystallography, have been reported before the submission of the present paper. The best available inhibitors are among the ‘dual-site inhibitors’, blocking estrogenic 17β-HSD and the estrogen receptor. These compounds belong to a family of estradiol analogues having an halogen atom at the 16α position and an extended alkyl-amide chain at the 7α position (C. Labrie, G. Martel, J.M. Dufour, G. Levesque, Y. Merand, F. Labrie, Novel compounds inhibit estrogen formation and action, Cancer Res. 52 (1992) 610–615). We now report the crystallization of this enzyme/inhibitor complex. The complex of the best available dual-site inhibitor, EM-139, with 17β-HSD1 has been crystallized using both cocrystallization and soaking methods. Crystals are isomorphous to the native crystals grown in the presence of 0.06% β-octyl-glucoside and polyethyleneglycol 4000, with a monoclinic space group C2. Data at 1.8 Å have been collected from a synchrotron source. Even though the size of the inhibitor is greater than that of the substrate, our preliminary X-ray-diffraction study shows that EM-139 fits into the active site in a position similar to that of estrogen. The availability of such structural data will help design more potent inhibitors of estrogenic 17β-HSD.

Modulation of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activities by steroids and physiological conditions in hamster

Our purpose was to examine the in vitro modulation of liver mitochondrial sterol 27-hydroxylase (S27OHase) and microsomal cholesterol 7α-hydroxylase (CH7αOHase) activities by certain drugs, sterols, oxysterols and bile acids, and to compare the influence of sex, age, diet and cholestyramine on these activities, in the hamster. In vitro, 7β-hydroxycholesterol and 5α-cholestan-3β-ol (cholestanol) were strong inhibitors (at 2 μM) of both enzyme activities, while 5β-cholestan-3α-ol (epicoprostanol, 2 μM) and cyclosporin A (20 μM) inhibited S27OHase, but not CH7αOHase. These data suggest that a hydroxyl group at the 7α position is not required to inhibit CH7αOHase and that the presence of an aliphatic CH2-CH-(CH3)2 chain appears to be structurally important for S27OHase activity. Both enzyme activities remained unchanged by hyodeoxycholic acid (40 or 80 μM) while epicoprostanol inhibited only S27OHase and chenodeoxycholic acid only CH7αOHase. Adult (9-week old) male or female hamsters displayed similar S27OHase activity but the CH7αOHase activity was lower in females than in males, suggesting that the neutral bile acid pathway has a less important role in females. In male hamsters, S27OHase activity did not change with age, while CH7αOHase activity significantly increased (one-year vs 9-week old). A semipurified sucrose-rich (lithogenic) diet significantly lowered both enzyme activities compared to the commercial diet. Cholestyramine induced a stimulation of both enzymes, slightly more vigorously however for the key enzyme involved in the neutral pathway. Taken together, these data indicate that the two enzymes are separately regulated and that certain drugs or steroid compounds can be useful for specifically inhibiting or stimulating the neutral or acidic bile acid pathway.

Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7alpha-hydroxy dehydroepiandrosterone and 7alpha-hydroxy pregnenolone.

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (Km, 13.6 microM) and pregnenolone (Km, 4.0 microM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17beta-estradiol and 5alpha-androstane-3beta,17beta-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7alpha-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7alpha-hydroxy DHEA but not with 7beta-hydroxy DHEA; when [7alpha-3H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7alpha position. Brain extracts also efficiently liberated tritium from [7alpha-3H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7alpha-hydroxy DHEA. We conclude that Cyp7b is a 7alpha-hydroxylase participating in the synthesis, in brain, of neurosteroids 7alpha-hydroxy DHEA, and 7alpha-hydroxy pregnenolone.

Biochemistry and Pharmacology of 7α-substituted Androstenediones as Aromatase Inhibitors

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7α-substituted androstenediones. Numerous 7α-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7α-(4′-aminophenylthio)-androsta-1,4-diene-3,17-dione (7α-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7α-APTADD in vivo . In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7α-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7α position with a carbon-carbon linkage. Several 7α-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent K i s ranging between 13 and 19 nM. Extension of the research on these 7α-arylaliphatic androgens includes the introduction of a C 1 C 2 double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7α-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7α-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k inact s ranging from 4.4 × 10 −4 to 1.90 x 10 −3 s −1 . The best inactivator of the series was 7α-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T 1 2 of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC 50 values of 64-328 nM. The 7α-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7α-position of the steroid in the active site of aromatase. The size of the 7α-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.

Biochemistry and pharmacology of 7α-substituted androstenediones as aromatase inhibitors

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7α-substituted androstenediones. Numerous 7α-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7α-(4′-aminophenylthio)-androsta-1,4-diene-3,17-dione (7α-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7α-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7α-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7α position with a carbon-carbon linkage. Several 7α-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent Kis ranging between 13 and 19 nM. Extension of the research on these 7α-arylaliphatic androgens includes the introduction of a C1-C2 double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7α-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7α-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent kinacts ranging from 4.4 × 10−4 to 1.90 x 10−3 s−1. The best inactivator of the series was 7α-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T12 of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC50 values of 64-328 nM. The 7α-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7α-position of the steroid in the active site of aromatase. The size of the 7α-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.

Biotransformation XXXIX. Metabolism of testosterone, androstenedione, progesterone and testosterone derivatives in Absidia coerulea culture

The strain of Absidia coerulea was used to investigate the transformations of testosterone, androstenedione, progesterone and testosterone derivatives with additional C1–C2 double bond and/or 17α-methyl group. All the examined substrates were transformed, mainly hydroxylated. It was found that the position and stereochemistry of the introduced hydroxyl group, as well as the yield of products, depended on the structure of the substrate. The first three substrates (hormones) underwent hydroxylation at C-14, and additional hydroxylation at 7α was observed in progesterone. The presence of the double bond (C1–C2) in 1-dehydrotestosterone did not influence the position of hydroxylation, but the product with additional C14–C15 double bond (at the same site as hydroxylation) was formed. 17α-Methyltestosterone was hydroxylated at the 7α position, and also the dehydrogenated product (at the same site, with C6–C7 double bond) was obtained. The testosterone derivative with both C1–C2 double bond and 17α-methyl group underwent hydroxylation at the 7α or 11β position, and a little amount of 14α, 15α epoxide was formed.

Synthesis of mosesin-4, a naturally occurring steroid saponin with shark repellent activity, and its analog 7-β-galactosyl ethyl cholate

The first synthesis of mosesin-4 ( 13), a naturally occurring steroid saponin with shark repellent activity, and its analog 19, isdescribed. They both possess a free galactose residue attached axially at the 7α position of the steroidal aglycon. Methyl cholate 3-cathylate (2) was used as a model for exploring various several methods to glycosidate the severely hindered 7α-position. Best results were obtained with β-galactose pentacetate ( 3a), using trimethylsilyl triflate as a promoter, in 1,2-dichloroethane, at −20°C, for 14 hours.

Synthesis of new fluorescent spirolactone derivatives: Determination of their affinities for aldosterone receptors

Fluorescent spirolactone derivatives are obtained by coupling 3-carboxylic coumarins to a spirolactone bearing a 3-hydroxypropyl chain in the 7α position. The two esters prepared by this method are highly fluorescent (emission 383 and 408 nm).