Steroid derivatives

Hydroxylation of l7α-acetoxy-6-chloro-16-methylene-4,6-pregnadiene-3,20-dione (Chlorosuperlutin, I) byCunninghamella blakesleeana yielded a 15β-hydroxyderivative II. Analogous transformation of 17α-acetoxy-16-methylene-4,6-pregnadiene-3,20-dione (Superlutin, IV) included a hydroxylation in position 15β and probably also in 11β with a concomitant reduction of the 6,7-double bond.     

D-homo isomers of 17-acetoxy-6-methylpregna-4,6-diene-3,20-dione

The three most readily accessible D-homo isomers of 17-acetoxy-6-methylpregna-4,6-diene-3,20-dione (Ib) have been prepared and characterized in detail. The 17-keto isomers IIb and IIIb were obtained by base-catalyzed rearrangement of Ia followed by reacetylation, and the 17a-ketone IVb by Lewis-Acid treatment of Ia.     

Microbial conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione acetates by Nocardioides simplex VKM Ac-2033D

The conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione 21-acetate (I) and 17,21-diacetate (VI) by Nocardioides simplex VKM Ac-2033D was studied. The major metabolites formed from I were identified as pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV). Pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione (III) and pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V) were formed in minorities. Biotransformation products formed from VI were pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17,21-diacetate (VII), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII), pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V). The conversion pathways were proposed including 1(2)-dehydrogenation, deacetylation, 20beta-reduction and non-enzymatic migration of acyl group from position 17 to 21. The conditions providing predominant accumulation of pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) from I and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII) from VI in a short-term biotransformation were determined.     

Binding of [3H] methyltrienolone (R 1881) in rat prostate and human benign prostatic hypertrophy (BPH).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) binding to rat ventral prostate cytosol has a specificity typical of an androgen receptor. In human benign prostatic hypertrophy (BPH) tissue, the specificity of [3H] R 1881 binding is different from that measured in rat prostate: progesterone and R 5020 (17, 21-dimethyl-19-nor-4, 9-pregnadiene-3, 20-dione) being more potent while 19-nortestosterone is less potent competitor. Moreover, the synthetic progestin [3H] R 5020 binds to BPH tissue with a similar specificity. These data suggest the presence of progestin binding components or of an atypical androgen receptor in human BPH cytosol.     

The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 under anaerobic conditions.

The bacterial degradation of cholic acid under anaerobic conditions by Pseudomonas sp. N.C.I.B. 10590 was studied. The major unsaturated neutral compound was identified as 12 beta-hydroxyandrosta-4,6-diene-3,17-dione, and the major unsaturated acidic metabolite was identified as 12 alpha-hydroxy-3-oxochola-4,6-dien-24-oic acid. Eight minor unsaturated metabolites were isolated and evidence is given for the following structures: 12 alpha-hydroxyandrosta-4,6-diene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-4,6-dien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, 3,12-dioxochola-4,6-dien-24-oic acid and 12 alpha-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid. In addition, a major saturated neutral compound was isolated and identified as 3 beta,12 beta-dihydroxy-5 beta-androstan-17-one, and the only saturated acidic metabolite was 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholan-24-oic acid. Nine minor saturated neutral compounds were also isolated, and evidence is presented for the following structures: 12 beta-hydroxy-5 beta-androstane-3,17-dione, 12 alpha-hydroxy-5 beta-androstane-3,17-dione, 3 beta,12 alpha-dihydroxy-5 beta-androstan-17-one, 3 alpha,12 beta-androstan-17-one, 3 alpha,12 alpha-dihydroxy-5 beta-androstan-17-one, 5 beta-androstane-3 beta,12 beta,17 beta-triol, 5 beta-androstane-3 beta,12 alpha,17 beta-triol, 5 beta-androstane-3 alpha,12 beta,17 beta-triol and 5 beta-androstane-3 alpha,12 alpha,17 beta-triol. The induction of 7 alpha-dehydroxylase and 12 alpha-dehydroxylase enzymes is discussed, together with the significance of dehydrogenation and ring fission under anaerobic conditions.     

The oxidation of Delta2, Delta2,4 and Delta4,6 steroids with RuO4

In order to find new ways for the functionalization of the A and B rings of the steroid nucleus, the reaction of 5alpha-androst-2-en-17beta-ol 17-acetate (1), cholesta-2,4-diene (4) and cholesta-4,6-dien-3beta-ol 3-acetate (7) was examined using stoichiometric amounts of ruthenium tetraoxide to yield 1,2-cis diols and/or alpha-hydroxy ketones. The reaction of 5alpha-cholest-2-en-3-ol 3-acetate (9) with ruthenium tetraoxide was also carried out and afforded, apart from an alpha-hydroxy ketone, also a diketone and a seco-dicarboxylic acid. The structures of all new steroids, including stereochemical details, were deduced by analysis of spectral data.     

甾体1，4-脱氢和11α-羟基化反应的两种不同微生物转化

Two kinds of micro-organism, Arthrobacter sp. AX86(1,4-dehdrogenator)and Absidia sp. A28(11α-hydroxylator) were used in this experiment. Two different fermentation techniques were performed to accomplish the multiple conversional reactions for producing 16β-methyl-11α, 17α, 21-trihydroxy-1,4-pregnadiene-3,20-dione (Ⅲ) from 16β-methyl-3β, 17α,21-trihydroxy-5α-pregnane-20-one-21-acetate(1) 1)To produce product(Ⅲ)by means of a two-step fermentation method which were independently performed first by Arthrobacter and next by Abslaia, and 2)the product was obtained by a sequential fermentation system of aforesaid two micro-organisms in a single fermentor without isolation of the intermediates from the mixture. Our results showed that in both fermentation systems high yield of product was obtained. However, according to the technical simplicity, shorter duration of fermentation cycle and efficient yield of product, the second method is better than the first one.     

梨头霉11α—羟基化制备16β—甲基—11α,17α21—三烃基孕甾—1, …

选育到一株对１６β－甲基－１７α,２１－二羟基孕甾－１,４＝－二烯－３,２０－二酮（Ⅱａ）１１α－羟基化活性强的梨头霉Ａ２８菌株,并发现底物２１－乙酰化（Ⅱｂ）可明显提高１１α－羟工 能力。在适宜的转化条件下,１１ｂ投料浓度０．５％,产物１６β－基１１α,１７α,２１－三羟基孕甾－１,４－二烯－３,２０－二酮（Ⅲ）收率为７３％,结构经波谱分析确认。     

Effect of benzoannulation on tautomeric preferences of 4,6-di(pyridin-2-yl)cyclohexane-1,3-dione

Density functional theory (DFT) calculations at the B3LYP/6-311+G(d,p) level show that 4,6-di(pyridin-2-yl)cyclohexane-1,3-dione is a labile compound. On the other hand, its dienolimine tautomer (4,6-di(pyridin-2-yl)cyclohaxa-1,3-diene-1,3-diol) seems stable enough to be present in vacuum. Alternatively the equilibriated species are (i) dienolimine and enolimine-enaminone ((6Z)-3-hydroxy-6-(pyridin-2(1H)-ylidene)-4-(pyridine-2-yl)cyclohex-3-enone) or (ii) dienolimine, enolimine-enaminone and dienaminone ((4Z,6Z)-4,6-di(pyridin-2(1H)-ylidene)cyclohexane-1,3-dione). Benzoannulation of the pyridine ring at position 5,6 was found to increase the contribution of the tautomers which contain the enaminone moiety. Energies of the transition states between the stable tautomers were also calculated in order to estimate activation energy of the proton transfer. Values of the geometry based harmonic oscillator model of aromaticity (HOMA) index and Laplacian of the electron density in the hydrogen bond critical point (based on quantum theory of atom in molecules) shows that the enaminone moiety in the tautomers studied are stabilized by stronger intramolecular hydrogen bond than this present in the enolimine moiety.     

Metabolism of osaterone acetate in dogs and humans

Osaterone acetate (17 alpha-acetoxy-6-chloro-2-oxa-4,6-pregnadiene-3,20-dione; OA) is a steroidal antiandrogen. In order to clarify the species differences, metabolites of OA were examined in plasma, urine, and feces of dogs and humans after oral administration of OA. Eleven metabolites in plasma, urine, and feces were identified by their spectral properties and comparison to appropriate standards. The primary routes of OA metabolism involve 11 beta-, 15 beta- and 21-hydroxylation, 17 alpha-deacetylation, and dechlorination. Other metabolites arise from combinations of these pathways to form multiple oxidized metabolites. All metabolites observed in humans occurred in dogs. 11 beta-Hydroxylated metabolites (11 beta-OH OA and 11-oxo OA) were found in the plasma and urine of dogs, but there was no evidence of their presence in humans. 11 beta-Hydroxylation of exogenous steroids represents a distinctive biotransformation pathway.     

Metabolism of progesterone by Aspergillus fumigatus

Metabolism of progesterone by a typical strain of Aspergillus fumigatus was studied. The four metabolites isolated were characterized as 5α-pregnane-3ß-ol-20-one, 15ß-hydroxy-1,4-pregnadiene-3,20-dione, 7ß,15ß-dihydroxy-4-pregnene-3,20-dione and 11α,15ß-dihydroxy-4-pregnene-3,20-dione by the application of various spectrometric techniques.     

Combining pharmacophore, docking and substructure search approaches to identify and optimize novel B-Raf(V600E) inhibitors

In this study for searching novel B-Raf(V600E) inhibitors, pharmacophore-based virtual screening identified 1 as a hit bearing 5-benzylidene-2-thioxodihydropyrimidine-4,6(1H,5H)-dione. Based on 1, scaffold hopping inspired by molecular docking discovered 5-(furan-2-ylmethylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione as a new and better scaffold. Substructure search with the new scaffold identified 28 active compounds, among which 12 compounds (42.9%) showed IC(50) less than 1μM. Especially, compound 3o, which is 10-fold more potent than the hit 1, is a potent inhibitor comparable to that of the marketed drug vemurafenib.     

Synthesis, spectral analysis, antibacterial and antifungal activities of some 4,6-diaryl-4,5-dihydro-3-hydroxy-2[H]-indazole-a novel fused indazole derivative

A novel class of 4,6-diaryl-4,5-dihydro-3-hydroxy-2[H]-indazoles 25-32 were synthesized and evaluated for their in vitro antibacterial and antifungal activities. Four Compounds, which all possessed electron withdrawing functional groups (-Cl, -NO(2), -Br) 27, 28, 30 and 32 were more potent against the tested bacterial/fungal strains than the standard bacterial and fungal drugs ciprofloxacin and fluconazole respectively.     

Biological activity of some steroidal compounds in the adult and larval frogs.

Four steroids were tested for their biological activity, using the sex-steroid-dependent redevelopment of the secondary sex characteristics in adult frogs and gonadal sex differentiation in larval frogs as end points. In adult frogs, 19-norprogesterone and 6-chloro-17alpha-hydroxy-4, 6-pregnadiene-3,20-dione had antiandrogenic and antiestrogenic effects. 2alpha, 17alpha-Dimethyl-DHT and 2alpha-methyl-DHT were potent androgens and effective antiestrogens. In the larval frogs, all four compounds had a masculinizing effect upon the undifferentiated gonads.     

Pregnanes in the root bark of Nerium odorum

Neridienone-A (12β-hydroxy-pregna-4,6,16-triene-3,20-dione), neridienone-B (20β,21-dihydroxy-pregna-4,6-diene-3,12-dione), 12β-hydroxy-pregna-4,6-diene-3,20-dione, 12β-hydroxy-pregn-4-ene-3,20-dione and 12β-hydroxy-16α-methoxy-pregna-4,6-diene-3,20- dione were obtained from the root bark of Nerium odorum.     

Progestin binding in mammary tissue of prepartum,nonlactating and postpartum,lactating cows

Binding of [³H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3, 20-dione) to bovine mammary cytosol indicated the presence of progestin binding sites of high-affinity and low-capacity in tissue from prepartum, nonlactating and from postpartum, lactating cows. To prevent binding of [³H]R5020 to glucocorticoid binding sites, a 200-fold molar excess of nonradioactive cortisol was included during all incubations, thus specific binding was limited to progestin binding sites. Nonradioactive R5020 and progesterone effectively inhibited [³H]R5020 binding to progestin binding sites, while estradiol-17β, dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), dexamethasone (9-fluoro-11β, 17, 21-trihydroxy-16α-methyl-1,4-pregnadiene-3,20-dione) or additional cortisol were ineffective. Dissociation constants for specifically bound [³H]R5020 in cytosol from mammary tissue of nonlactating and lactating cows were nearly identical, averaging 1.9 ( ± 0.3) and 0.8( ± 0.2) × 10?⁹M, respectively. However, binding capacities (fmol/mg cytosolic protein) were greater in cytosol from prepartum, nonlactating (179 ± 53) than postpartum, lactating (41 ± 15) cows. Specific binding components in cytosol from lactating cows sedimented in the 6-7S region on linear sucrose density gradients. When subjected to isoelectric focusing, specific binders with isoelectric points (pI) of approximately 6.1, 7.9 and 8.3 were resolved. The decrease in number of binding sites during lactation was due to the virtual absence of the anionic binding species, suggesting that their presence is necessary for progesterone to inhibit milk secretion.     

Relative binding affinity of novel steroids to androgen receptors in hamster prostate

The in vivo and in vitro antiandrogenic activity of four aromatic esters 10a-10d, one aliphatic ester 10e based on the pregna-4,16-diene-6, 20-dione structure and two aromatic 17c, 17d and two aliphatic valeroyloxy esters 17a, 17b based on the more saturated 4-pregnene-6,20-dione skeleton was examined. The biological activity of steroids 9, 10a-10e and 17a-17d, was determined using prostate glands from gonadectomized adult male golden hamsters. In the in vitro studies, the relative binding affinity of these steroids to cytoplasmic androgen receptor (AR) of hamster prostate was determined from, the corresponding IC50 values obtained from the competitive binding plots. The standards dihydrotestosterone (DHT) and cyproterone (CA) acetate used have displaced [3H]DHT from the AR with an IC50 value of 3.2 and 4.4 nM respectively. All steroidal compounds synthesized in this study showed a binding affinity for the androgen receptor, present in the cytosol from prostate hamster; compounds 10a-10c showed the highest affinities for this receptor. The in vivo experiments showed that all steroidal derivatives were subcutaneously active, since they decreased the weight of the prostate gland in gonadectomized hamsters treated with DHT, and are antagonists for the androgen receptor since they block the DHT-induced prostate weight gain. The derivatives having the more conjugated 4,16-pregnadiene-6, 20-dione system (10a-10c) exhibited a higher antiandrogenic activity than the corresponding steroids (17a-17d) based on the more saturated 4-pregnene-6,20-dione system.     

Chemical synthesis and in vitro biological evaluation of a phosphorylated bisubstrate inhibitor of type 3 17beta-hydroxysteroid dehydrogenase

Type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD) catalyzes the last step in the biosynthesis of the potent androgen testosterone (T) by selectively reducing the C17 ketone of 4-androstene-3,17-dione (delta4-dione), with NADPH as cofactor. This enzyme is thus an interesting therapeutic target for androgen-sensitive diseases. Using an efficient convergent chemical approach we synthesized a phosphorylated version of the best delta4-dione/adenosine hybrid inhibitor of type 3 17beta-HSD previously reported. An appropriately protected C2' phosphorylated adenosine was first prepared and linked by esterification to the steroid delta4-dione bearing an alkyl spacer. After three deprotection steps, the phosphorylated bisubstrate inhibitor was obtained. The inhibitory potency of this compound was evaluated on homogenated HEK-293 cells overexpressing type 3 17beta-HSD and compared to the best non-phosphorylated bisubstrate inhibitor. Unexpectedly, the phosphorylated derivative was slightly less potent than the non-phosphorylated bisubstrate inhibitor of type 3 17beta-HSD. Two hypotheses are discussed to explain this result: 1) the phosphorylated adenosine moiety does not interact optimally with the cofactor-binding site and 2) the bisubstrate inhibitors, phosphorylated or not, interact only with the substrate-binding site of type 3 17beta-HSD.     

Synthesis and evaluation of biological and antitumor activities of 5,7-dimethyl- oxazolo[5,4-d]pyrimidine-4,6(5H,7H)-dione derivatives as novel inhibitors of FGFR1

Abstract

A series of 5,7-dimethyl-oxazolo[5,4-d]pyrimidine-4,6(5H,7H)-dione derivatives, N5a–5l, was designed, synthesized and evaluated for their FGFR1-inhibition ability as well as cytotoxicity against three cancer cell lines (H460, B16F10 and A549) in vitro. Several compounds displayed good-to-excellent potency against these cancer cell lines compared to SU5402. Structure–activity relationship analyses indicated that compounds with a rigid structure and more heteroatoms at the side chain of the parent ring were more effective than those without these substitutions. The compound N5g (37.4% FGFR1 inhibition at 1.0?μM) was identified to have the most potent antitumor activities, with IC₅₀ values of 5.472, 4.260 and 5.837?μM against H460, B16F10 and A549 cell lines, respectively. Together, our results suggest that 5,7-dimethyl-oxazolo[5,4-d]pyrimidine-4,6(5H,7H)-dione derivatives may serve as potential agents for the treatment of FGFR1-mediated cancers.     

Bioconversion of steroids by Cochliobolus lunatus--II. 11 beta-hydroxylation of 17 alpha, 21-dihydroxypregna-1,4-diene-3,20-dione 17-acetate in dependence of the inducer structure.

The 11 beta-hydroxylase of the filamentous fungus Cochliobolus lunatus m 118 was induced with the substrate 17 alpha, 21-dihydroxypregna-1,4-diene-3,20-dione 17-acetate (11 beta-deoxyprednisolone 17-acetate) itself, substrate analogues, different pregnane compounds, sterols, intermediates of microbial sterol side-chain degradation or bile acids, together with 24 different steroids in a standardized test system. The resulting 11 beta-hydroxylation rate, leading to prednisolone 17-acetate and prednisolone, respectively, was determined and compared with the hydroxylation rate of non-induced cultures. The transformation yield strongly depended on the inducer structure. The microbial sterol side-chain degradation intermediates (20S)-20-hydroxymethylpregn-4-en-3-one and the corresponding pregna-1,4-diene compound caused the highest induction effects (induction factors 5.1 and 4.9, respectively). The metabolism of (20S)-20-hydroxymethylpregna-1,4-dien-3-one during the cultivation was elucidated. The induction effect decreased with the rising oxidation of the inducer. The significant increase of the 11 beta-hydroxylation rate of 1-dehydro-pregnane substrates by specific induction allows alternative pathways to glucocorticoid partial syntheses.