Concepts for the syntheses of biotinylated steroids. Part I: testosterone derivatives as immunochemical probes

We describe synthetic strategies for the biotinylation of testosterone (T) at positions 3, 7alpha, 17alpha, and 19. These T probes are able to mimic ligand binding and may provide for a better understanding of the biospecific interaction with steroid-binding proteins such as the androgen receptor, anti-steroid antibodies, or steroid-binding serum globulins. For the 7alpha- and 17alpha-derivatives, biotinyl-N-hydroxy-succinimide esters with different types of spacer chains were used. The 3-biotin hydrazone derivative was produced using N-(epsilon-biotinyl)-caproyl hydrazide, whereas for the 19-biotinylation, a biotinyl-1-N-diamino-3, 6-dioxaoctane-amide was applied. Key reaction for the biotinylation at position 3 is the oximation of the 3-oxo function. The 17alpha-position is accessible by the reaction of the 3-protected 4-androsten-17-epoxide with oxygen in the beta-position, followed by nucleophilic ring opening with cyanide which provides the 17alpha-cyanomethyl derivative. The key step is the regioselective ketal protection of the 3-oxo function of androst-4-ene-3,17-dione using a stannoxane catalyst. An alternative pathway for the insertion of biotin at the 19-position was established by the synthesis of 17beta-hydroxy-androst-4-en-3-one-19-yl carboxymethyl ether. After activation by the carbodiimide method, the compound reacts with aminoterminal biotin derivatives. The copper(I)-catalyzed 1,6 Michael addition of 17-acetoxy-6,7-dehydro-T leads to 7alpha-derivatives by use of omega-silyl protected hydroxylalkyl-modified Grignard reagents. A functional group interconversion using the Staudinger reaction transforms the azide function into a primary omega-amino group. The absolute configurations of the different biotinylated derivatives were investigated by (1)H NMR studies. For the 7alpha-biotinylated T series, additionally, an X-ray analysis proved the axial position of the spacer group. This results in a vertical orientation of the biotin moiety toward the alpha-face of the planar tetracyclic backbone. Thus, a negligible alteration of the original structure of the upper beta-face offers the feasibility of applying the 7alpha-derivatives as optimal immunochemical tracers in competitive immunoassays. Biotinylated T derivatives should be also suitable for ligand-binding studies to the androgen receptor or to sex hormone-binding globulin.     

Evidence of formation of isomeric methoximes from 20-oxosteroids

The formation and gas chromatographic behavior of syn- and anti-isomers in position 20 of the methoxime-trimethylsilyl (MO-TMS) derivatives of many 20-oxo and 3,20-dioxo-21-hydroxysteroids is reported. The existence of such isomers was established from the gas chromatographic (GC) and mass spectrometric analysis of the MO-TMS derivatives of 3 alpha,21-dihydroxy-5 beta-pregnan-20-one and its 17 alpha-epimer. The degree of separation during GC analysis of the syn- and anti-isomers in position 20, as well as those in position 3, is associated to the position of additional hydroxyl groups on the steroid ring. These data are very important for the location of oxygenated substituents such as 2 alpha/2 beta, 6 alpha/6 beta, 11 beta, 16 alpha, 17 alpha, 18, 19 or 21-hydroxyl groups during structural studies of 20-oxo and 3,20-dioxosteroids.     

Chemical synthesis and biochemical characterization of a biotinylated derivative of 17beta-estradiol with a long side chain covalently attached to its C-7alpha position

High-affinity biotinylated derivatives of 17beta-estradiol (E(2)) are of value for isolation of various estrogen-binding proteins (including estrogen receptors) and also for studying protein-protein interactions involving these proteins. In this study, we developed a simplified route for the chemical synthesis of a biotinylated derivative of E(2) (compound 7) with a side chain attached to its C-7alpha position. Compound 7, i.e., 7alpha-{7-[8-(biotinamido)-octanamido]-heptyl}-estradiol, could be readily synthesized from 6-keto-estradiol-3,17beta-di-tetrahydropyranyl ether (compound 2, which can be prepared from E(2)), with a final yield of 36%. In vitro receptor-binding assay confirmed that the synthesized affinity ligand has a high binding affinity for both human estrogen receptor alpha and beta. When the affinity ligand (compound 7) was immobilized with avidin on an affinity column, it effectively bound human estrogen receptor alpha, and the receptor protein could be selectively eluted with a biotin-containing buffer. Using the same affinity ligand, prolyl 4-hydroxylase beta-subunit (also known as protein disulfide isomerase) was identified as one of the high-affinity E(2)-binding proteins in the whole cytosolic protein mixture prepared from MCF-7 human breast cancer cells. Computational molecular modeling analysis showed that compound 7 can bind to human estrogen receptor alpha in a similar manner as ICI-182,780 and raloxifene, and their binding energy values are also similar.     

Synthesis of some epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives and evaluation of their biological activity

Steroidal epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives have been prepared using 3beta,17beta-dihydroxy-17alpha-picolyl-androst-5-ene (1), 3beta-acetoxy-17-picolinylidene-androst-5-ene (2), and 3beta-hydroxy-17-picolinylidene-androst-5-ene (3) as synthetic precursors. The compounds 2 and/or 3 were reacted with m-chloroperoxybenzoic acid (MCPBA). The compounds synthesized from 2 were 17-picolinylidene-N-oxide 4, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 5 and 6, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 7 and 8. Starting from compound 3, a mixture of 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene 9 and 10, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 11 and 12, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 13 and 14 were obtained. From compounds 15 and 18, obtained from 1 and 3 by the Oppenauer oxidation, the 4alpha,5alpha-epoxy and 4beta,5beta-epoxy derivatives 16, 17 and 20, 21 were prepared by oxidation with 30% H(2)O(2). Oxidation of 18 with MCPBA yielded only the N-oxide 19. The structures of compounds 15 and 18 were proved by the X-ray analysis. Compounds 1-6, 9, 15, 17, 18, and 21 were tested on activity against the enzyme aromatase. Antitumor activity against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER-, MDA-MB-231, and prostate cancer PC3) was evaluated. Three tested compounds (1, 4, and 19) showed strong activity against PC3, the IC(50) values being in the range 0.55-10microM, whereas compound 17 showed strong activity against MDA-MB-231 (IC(50) 10.4microM).     

Potential bile acid metabolites. XVIII. Synthesis of stereoisomeric 3,6,12 alpha-trihydroxy-5 beta-cholanoic acids.

Two new 6-hydroxylated bile acids, 3 beta, 6 alpha, 12 alpha- and 3 beta, 6 beta, 12 alpha-trihydroxy-5 beta-cholanoic acids, were synthesized from deoxycholic acid. In addition, their C-3 epimers, 3 alpha, 6 alpha, 12 alpha- and 3 alpha, 6 beta, 12 alpha-trihydroxy acids, were prepared by a new route. The principal reactions used were 1) 6 beta-hydroxylation of 3-methoxy-3,5-dienes with m-chloroperbenzoic acid in aqueous dioxane; 2) catalytic hydrogenation of the resulting 6 beta-hydroxy-3-oxo-4-enes to the 6 beta-hydroxy-3-oxo-5 beta compounds with palladium on calcium carbonate catalyst in ethanol; and 3) stereoselective reduction of appropriate 3-oxo derivatives with potassium tri-sec-butylborohydride and tert-butylamine-borane complex. The thin-layer chromatographic, gas-liquid chromatographic, and high performance liquid chromatographic mobilities, and 1H- and 13C-nuclear magnetic resonance spectroscopic data of the four stereoisomers are presented. With this work all the 6-hydroxylated derivatives of lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and cholic acids in the 5 beta series are now known and have been synthesized.     

Hydroxylation of progesterone by some<Emphasis Type="Italic">Trichoderma</Emphasis> species

Thirty-three isolates belonging to six species of the genus Trichoderma were tested for the ability to hydroxylate progesterone to 11alpha-, 11beta-, 11alpha,17alpha- and 6beta, 17alpha-derivatives, and epicortisol. T. aureoviride, T. harzianum, T. polysporum and T. pseudokoningii produced 11alpha-hydroxyprogesterone. T. harzianum and T. hamatum can form only the 11beta-isomer. T. koningii and T. hamatum produced 11alpha-, 11beta-, 11alpha,17alpha- and 6beta,11alpha-hydroxy derivatives. 11alpha, 11beta, 6beta,11alpha- and 11alpha,17alpha-hydroxyprogesterones and epicortisol are produced by T. aureoviride and T. pseudokoningii. Cortisol was produced only when the medium was fortified by 10 g/L peptone. This is the first record of conversion of progesterone to mono-, di- and trihydroxyprogesterones by these Trichoderma species.     

Tetra- and hexahydro derivatives of aldosterone and 18-hydroxycorticosterone by chemical and microbial reductions

Preparative methods were developed for reduction with NaBH4 at 0 of 3 beta, 5 alpha- and 3 alpha, 5 beta-tetrahydroaldosterone (1) and (12) to their respective 20 alpha-ol derivatives 2a and 13a. Corroboration of structures was obtained by periodate oxidations to the lactols 3b and 14b and thence, by further oxidation, to the lactones 4 and 15 respectively; these lactones were also independently obtained from 1 and 12. Reduction with NaBH4 at 80 degrees C converted 1 and 12 into 18-hydroxy-3 beta, 5 alpha, 20- and 18-hydroxy-3 alpha, 5 beta, 20-hexahydrocorticosterone 6a and 17a respectively, which were mixtures of epimers at C-20. Compound 17a could also be prepared by reduction of the lactone 21 with sodium aluminum bis-(methoxyethoxy) hydride. Again, periodate oxidations of 6a and 17a gave the lactols 7b and 22b and thence, by Jones oxidation, the diketolactones 8 and 23, which were also prepared from 18-hydroxy-11-dehydrocorticosterone (10) and 18-hydroxycorticosterone (24) respectively. Improved conditions for reduction with Clostridium paraputrificum permitted convenient conversion of aldosterone (11), the corresponding 18 leads to 11 lactone 18a and 18-hydroxycorticosterone (24) into their 3 alpha, 5 beta-tetrahydro derivatives.     

Synthesis of 7alpha-substituted derivatives of 17beta-estradiol

Estrogen receptor (ER) pure antagonists such as ICI-182,780 (fulvestrant) are effective alternatives to tamoxifen (an ER antagonist/weak partial agonist) in the treatment of postmenopausal, receptor-positive human breast cancers. Structurally, these pure antagonists contain the basic core structure of 17beta-estradiol (E(2)) with a long side chain attached to its C-7alpha position. We explored and compared in this study various synthetic routes for preparing a number of C-7alpha-substituted derivatives of E(2), which are highly useful for the design and synthesis of high-affinity ER antagonists, ER-based imaging ligands, and other ER-based multi-functional agents. Using E(2) as the starting material and 1-iodo-6-benzyloxyhexane as a precursor for the C-7alpha side chain, a seven-step synthetic procedure afforded 3,17beta-bis(acetoxy)-7alpha-(6-hydroxyhexanyl)-estra-1,3,5(10)-triene (one of the derivatives prepared) in an overall yield of approximately 45% as compared to other known procedures that afforded substantially lower overall yield (8-27%). The synthetic steps for this representative compound include: (1) protection of the C-3 and C-17beta hydroxyls of E(2) using methoxymethyl groups; (2) hydroxylation of the C-6 position of the bismethoxymethyl ether of E(2); (3) Swern oxidation of the C-6 hydroxy to the ketone group; (4) C-7alpha alkylation of the C-6 ketone derivative of E(2); (5) deprotection of the two methoxymethyl groups; (6) reprotection of the C-3 and C-6 free hydroxyls with acetyl groups; (7) removal of the C-6 ketone and the benzyl group on the side chain by catalytic hydrogenation in acetic acid. As predicted, two of the representative C-7alpha-substituted derivatives of E(2) synthesized in the present study retained strong binding affinities (close to those of E(2) and ICI-182,780) for the human ERalpha and ERbeta subtypes as determined using the radioligand-receptor binding assays.     

Preparation of 9-alpha,11-xi-tritiated 17-alpha-ethynylestradiol, mestranol, estradiol-alpha-17-beta, and norethindrone

The preparation of 9alpha, 11xi-tritiated 17alpha-ethinyl estradiol, mestranol, estradiol-17beta, and norethindrone are described. Estrone-3-methyl ether was employed as starting material, and ethinylation with lithium acetylide-ethylene diamine resulted in 95% mestranol. Demethylation of mestranol with boron tribromide at 0 degrees resulted in 92% 17alpha-ethinyl estradiol. Dimethylsulfoxide was the choice of reagent for the condensation reaction which was complete at room temperature in about 4 hours. The usually less than 3% of unreacted 17-oxo product was removed by Girard separation. Demethylation of methyl ether with boran tribromide in methylene chloride resulted in an excellent yield of 17alpha-ethinyl estradiol-9alpha, 11xi-tritium. 3-methoxyestra-1,3,5-trien-17-one-9alpha, 11xi-tritium was reduced with sodium bis(2-methoxyethoxy) aluminum hydride to the 17beta-hydroxy compound and subsequent demethylation resulted in estradiol-9alpha, 11xi-tritium. The general method of Ringold et al was employed for the preparation of 17beta-hydroxy-17alpha-ethinylestr-4-en-3-one. Improvements for small scale radiosynthesis are also presented.     

Synthesis and characterization by 1H and 13C nuclear magnetic resonance spectroscopy of 17 alpha-hexanoic derivatives of 5 alpha-dihydrotestosterone and testosterone.

The synthesis and characterization of 17 alpha-(6'-hexanoic acid) derivatives of 5 alpha-dihydrotestosterone and testosterone, useful as ligands for affinity chromatography purification or as precursors for affinity-labeling of androgen-binding proteins, is described. Alkynylation of 3-ethylenedioxy-, 3 beta-hydroxy-, and 3 beta,5-dihydroxy-5 alpha-androstan-17-one precursors with the potassium derivative of 5-hexyn-1-ol led to the corresponding 17 alpha-(6'-hydroxyhex-1'-ynyl) derivatives, which were hydrogenated over 10% Pt-C catalyst to give 17 alpha-(6'-hydroxyhexyl) derivatives. Chromic acid oxidation of the primary hydroxy group of the 3-ethylenedioxy-17-hexyl intermediate into carboxylic acid followed by acid cleavage of the 3-ketal group gave 17 alpha-(5'-carboxypentyl)-5 alpha-dihydrotestosterone, which was also obtained directly by chromic acid oxidation of the 3 beta-hydroxy intermediate. Chromic acid oxidation of the primary hydroxy group of the 3 beta,5 alpha-dihydroxy precursor resulted in a 5 alpha-hydroxy-3-oxo intermediate, which was dehydrated to give 17 alpha-(5'-carboxypentyl)testosterone. The 17 alpha configuration of these derivatives and of synthetic precursors was established by comparing their molecular rotations and their 1H and 13C nuclear magnetic resonance (NMR) spectra including solvent effects, with data reported for 17 alpha- or 17 beta-substituted steroid analogs as well as with 1H and 13C NMR reference data recorded in this work for 17 alpha-ethynyltestosterone, 17 alpha-ethynyl-19-nortestosterone, 17 alpha-ethyl-19-nortestosterone, 17 alpha-methyltestosterone, and 17 alpha-methyl-5 alpha-dihydrotestosterone.     

Alternate hydroxylating activities in newborn rat adrenal cells in primary culture

During the course of a study to produce reference compounds, the metabolism of tetrahydrogenated derivatives (ring A reduced) of progesterone, 6 alpha-hydroxyprogesterone, 11-deoxycorticosterone and corticosterone in newborn rat adrenal cells in primary culture was studied. Analysis of the metabolites was made by gas chromatography-mass spectrometry. Most products resulted from the enzymatic reactions of 11 beta-, 18- and 21-hydroxylation, reduction of the 20-oxo group and oxidoreduction of the 3-hydroxyl group. However, unexpected metabolites were produced from the incubation of 3 beta, 5 alpha-tetrahydroprogesterone and 6 alpha-hydroxy-3 alpha, 5 beta-tetrahydroprogesterone. They resulted from the 16 alpha-hydroxylation of the precursors and probably from the 15 alpha-, 16 beta- and 17 alpha-hydroxylation of 6 alpha-hydroxy-3 alpha, 5 beta-tetrahydroprogesterone. These hydroxylating activities are weak and were not detected from the endogenous steroidogenesis. They were not detected either from the incubation of exogenous steroids with a 3-oxo-4-ene structure or from steroids with a 21-hydroxyl substituent. They result only from substrates showing diminished or no affinity towards the 11 beta/18- and 21-steroid hydroxylase systems. These unusual hydroxylations could be catalyzed by monooxygenase systems in the endoplasmic reticulum similar to those present in the liver or by the monooxygenase systems specific to steroidogenesis. In particular, the reaction specificity of cytochrome P-450(11) beta could be altered by the presence of a 6 alpha-hydroxyl group in a tetrahydrogenated steroid.     

Synthesis of 3 beta,16 beta,19-trihydroxyandrost-5-en-17-one and 16 beta,19-dihydroxyandrost-4-ene-3,17-dione and their 19-oxo derivatives

3 beta,16 beta,19-Trihydroxyandrost-5-en-17-one (12) was synthesized from 5 alpha-bromo-3 beta-acetoxy-6 beta,19-epoxyandrostan-17-one (2) through acetoxylation at C-16 beta of the enol acetate 4 with lead tetraacetate and reductive cleavage of the epoxide ring with zinc dust yielding the 3 beta,16 beta-diacetoxy-19-hydroxy steroid 11, followed by hydrolysis of the acetoxy groups with sulfuric acid. Jones oxidation of compound 11 followed by the acid hydrolysis gave the 19-oxo steroid 15. 5 alpha-Bromo-3 beta-hydroxy-16 beta-acetoxy-6 beta,19-epoxyandrostan-17-one (8), obtained by selective hydrolysis of the 3-formate 5 with ammonium hydroxide, was oxidized with Jones reagent to afford the 3-oxo steroid 16, which was converted into the 19-hydroxy derivative 17 by treatment with zinc dust. 16 beta,19-Dihydroxyandrost-4-ene-3,17-dione (18) and its 19-oxo derivative 21 were obtained from compound 17 through a similar reaction sequence.     

Addition reactions at the 16(17) double bond of 3-methoxy-13alpha-estra-1,3,5(10),16-tetraene

The epoxidation, the addition of hypobromous acid, and the hydroboration of 3-methoxy-13alpha-estra-1,3,5(10),16-tetraene 1 with diborane, catecholborane, and 9-BBN were investigated in order to determine the stereochemical outcome and to synthesize new 13alpha-estra-1,3,5(10)-trienes for biological and conformational investigations. It was shown that the sterically demanding reagent 9-BBN participated in a preferred beta attack (53% 16betaOH 10, 34% 17betaOH 8, 13% 16alphaOH 11). This stereochemical result is in agreement with that from another cis addition reaction, the recently described OsO4 dihydroxylation of 1 [Steroids 68 (2003) 113]. With smaller reagents such as B2H6, catecholborane, or magnesium monoperoxyphthalate, a diminished stereoselectivity was observed with only a slight excess of beta attack. The ionic trans addition of hypobromous acid gave two 17-bromo-16-alcohols with 16beta,17alpha (4, 76%) and 16alpha,17beta configuration (5, 24%) formed by trans cleavage of the 16,17alpha- and beta-bromonium ion at position 16. The same regioselective and stereoselective course was found for the cleavage of the 16alpha,17alpha- and 16beta,17beta-epoxides (3 and 2) with hydrazoic acid (3-->16betaN3,17alphaOH 7, 2-->16alphaN3,17betaOH 6). The stereochemistry of the addition reactions to 1 can be explained in terms of a twist-boat conformation involving the C ring of compound 1. From a synthetic viewpoint the synthesis of the beta-epoxide 2 from the bromohydrin 4, the cleavage of this epoxide to 16alpha-substituted-17beta-hydroxy compounds, such as 6, and hydroboration/oxidation with 9-BBN to the hitherto unknown 16beta-hydroxy compound 10 are useful procedures. The bromohydrin 5 is the first 13alpha-steroid with a 17beta-bromo substituent. X-ray analysis revealed twist-boat and 16beta-envelope conformations for rings C and D, respectively.     

Bioconversion of Stemodia maritima diterpenes and derivatives by Cunninghamella echinulata var. elegans and Phanerochaete chrysosporium

Stemodane and stemarane diterpenes isolated from the plant Stemodia maritima and their dimethylcarbamate derivatives were fed to growing cultures of the fungi Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725. C. echinulata transformed stemodin (1) to its 7alpha-hydroxy- (2), 7beta-hydroxy- (3) and 3beta-hydroxy- (4) analogues. 2alpha-(N,N-Dimethylcarbamoxy)-13-hydroxystemodane (6) gave 2alpha-(N,N-dimethylcarbamoxy)-6alpha,13-dihydroxystemodane (7) and 2alpha-(N,N-dimethylcarbamoxy)-7alpha,13-dihydroxystemodane (8). Stemodinone (9) yielded 14-hydroxy-(10) and 7beta-hydroxy- (11) congeners along with 1, 2 and 3. Stemarin (13) was converted to the hitherto unreported 6alpha,13-dihydroxystemaran-19-oic acid (18). 19-(N,N-Dimethylcarbamoxy)-13-hydroxystemarane (14) yielded 13-hydroxystemaran-19-oic acid (17) along with the two metabolites: 19-(N,N-dimethylcarbamoxy)-2beta,13-dihydroxystemarane (15) and 19-(N,N-dimethylcarbamoxy)-2beta,8,13-trihydroxystemarane (16). P. chrysosporium converted 1 into 3, 4 and 2alpha,11beta,13-trihydroxystemodane (5). The dimethylcarbamate (6) was not transformed by this microorganism. Stemodinone (9) was hydroxylated at C-19 to give 12. Both stemarin (13) and its dimethylcarbamate (14) were recovered unchanged after incubation with Phanerochaete.     

Fluorescent derivatives of bile salts. II. Suitability of NBD-amino derivatives of bile salts for the study of biological transport.

Interaction of unconjugated and taurine-conjugated NBD-amino-dihydroxy-5 beta-cholan-24-oic acids bearing the fluorophor in the 3 alpha, 3 beta, 7 alpha, 7 beta, 12 alpha, or 12 beta position with albumin results in a small hypsochromic shift of the emission maximum and an increase in quantum yield, suggesting binding by hydrophobic interactions. The different unconjugated fluorescent bile salt derivatives are metabolized by intact rat liver in different ways. The unconjugated 3 beta-NBD-amino derivative is completely transformed to its taurine conjugate and secreted as such, whereas the 3 alpha-NBD-amino derivative is completely transformed to a polar fluorescent compound not identical with its taurine conjugate. The unconjugated 7 alpha- and 7 beta-NBD-amino derivatives are only partially conjugated with taurine and mainly secreted in unmetabolized form. The unconjugated 12 alpha- and 12 beta-NBD-amino derivatives are not at all transformed to their taurine conjugates, but are partially metabolized to unidentified compounds. They are predominantly secreted as the unmetabolized compounds. In contrast to the unconjugated derivatives, all taurine-conjugated fluorescent bile salt derivatives are secreted into bile unmetabolized. With the exception of the 3 alpha-compound, all synthesized taurine-conjugated fluorescent derivatives interfere with the secretion of cholyltaurine. Differential photoaffinity labeling studies using (7,7-azo-3 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2'-[2'-3H(N)]aminoethanesulfonate as a photolabile derivative revealed that in liver cells all fluorescent bile salt derivatives interact with the same polypeptides as the physiological bile salts. The hepatobiliary transport of taurine-conjugated NBD-amino bile salt derivatives is, due to hydrophobic interactions, accompanied by an increase in fluorescence intensity which is favorable for the study of biological bile salt transport by fluorescence microscopy.     

Androsterone derivatives substituted at position 16: chemical synthesis,inhibition of type 3 17beta-hydroxysteroid dehydrogenase,binding affinity for steroid receptors and proliferative/antiproliferative activity on Shionogi (AR+) cells

A series of androsterone (ADT) derivatives substituted at position 16 were efficiently synthesized in short reaction sequences; the ether analogues were also synthesized in the case of the methyl and allyl derivatives. The aim of this study was to develop inhibitors of the steroidogenic enzyme type 3 17beta-hydroxysteroid dehydrogenase and then evaluate their ability to inhibit this activity in transfected HEK-293 cells. For each compound we measured the percentage of inhibition of the transformation of 4-androstene-3,17-dione, the natural substrate of this steroidogenic enzyme, into the active androgen testosterone. The synthesized compounds proved to be weak inhibitors of this enzyme, but interestingly, these ADT derivatives do not bind to androgen, estrogen, glucocorticoid, and progestin receptors, suggesting no unsuitable receptor-mediated effects. One exception, 16alpha-(3'-bromopropyl)-5alpha-androstane-3alpha,17beta-diol, the only compound bearing a hydroxy group at position 17beta instead of a ketone, showed a strong binding affinity for the androgen receptor (70% at 1 microM) and also exhibited an antiproliferative activity on Shionogi (AR+) cells (86% at 1 microM), which was comparable to that of hydroxyflutamide, a pure antiandrogen (100% at 1 microM).     

Syntheses of stable isotope-labeled 6 beta-hydroxycortisol, 6 beta-hydroxycortisone,and 6 beta-hydroxytestosterone

A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.     

Parallel solid-phase synthesis of a model library of 7alpha-alkylamide estradiol derivatives as potential estrogen receptor antagonists.

The C17-THP derivative of 7alpha-(11-azidoundecanyl)-estradiol (4) was synthesized and coupled to an aminomethyl resin via a photolabile o-nitrobenzyl linker. Reduction of the azide by the Staudinger reaction to its corresponding amine followed by acylation using four activated NFmoc protected amino acids gave a first level of diversity. Subsequent deprotection of the Fmoc followed by a second acylation with five activated carboxylic acids produced, after photocleavage, a model library of twenty antiestrogen-related 7alpha-alkylamide estradiol derivatives in acceptable overall yields and very good purities.     

Taxanes with C-5-amino-side chains from the needles of Taxus canadensis

Five taxanes with an amino-side chain on C-5 were identified for the first time in the needles of the Canadian yew, Taxus canadensis. Their structures were characterized as 2alpha,7beta,9alpha,10beta,13-pentaacetoxy-11beta-hydroxy-5alpha-(3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),12-diene (1), 2alpha,9alpha-dihydroxy-10beta,13alpha-diacetoxy-5alpha-(3'-methylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (2), 2alpha17-dihydroxy-9alpha,10beta,13alpha-triacetoxy-5alpha-(3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (3), 2alpha-hydroxy-7beta,9alpha,10beta,13alpha-tetraacetoxy-5alpha-(2'-hydroxy-3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (4), and 9alpha-hydroxy-2alpha,10beta,13alpha-triacetoxy-5alpha-(3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (5) on the basis of 1D-, 2D-NMR spectroscopic data and high-resolution fast atom bombardment MS analyses. Metabolite (1) was isolated from the needles of the Canadian yew for the first time but had previously been detected in the stems of the Japanese yew, whereas taxanes (2-5) are only now reported. Metabolite (3) is the first reported nitrogen-containing taxane with a 17-hydroxyl substitution.     

Influence of 1-double bond and 11 beta-hydroxy group on stereospecific microbial reductions of 4-en-3-oxo-steroids

The yeast Rhodotorula glutinis and the anaerobic bacterium Clostridium paraputrificum were used for stereospecific reductions of 4-chloro-11 beta-hydroxy-17 alpha-methyl-testosterone and the corresponding 1-dehydro compound to prepare 5 alpha- and 5 beta-H derivatives, respectively. C. paraputrificum was able to 5 beta-reduce both substances, whereas the 5 alpha-reduction by R. glutinis was inhibited by the structure elements 1-en and 11 beta-OH so that the substrate with both structure elements was not 5 alpha-reduced. The microbial conversion of the four steroids with and without 1-en and 11 beta-OH was compared in semiquantitative experiments. A number of new substances are described, 11 beta-hydroxy and 11-oxo derivatives of 5 alpha- and 5 beta-dihydro-4-chloro-17 alpha-methyltestosterone including some 3-OH compounds and characterized by NMR, mass spectrometric and further data.