Antibacterial Δ(1) -3-ketosteroids from the South China Sea gorgonian coral Subergorgia rubra.

Three new Δ(1) -3-ketosteroids characterized with a 9-OH, subergosterones A-C (1-3), together with five known analogs 4-8, were obtained from the gorgonian coral Subergorgia rubra collected from the South China Sea. The structures of 1-3, including their absolute configurations, were determined by comprehensive spectroscopic methods and electronic circular dichroism (ECD) experiments. Compounds 2 and 3 exhibited inhibitory antibacterial activities against Bacillus cereus with MIC values of 1.56 µM.

Determination of ketosteroid hormones in meat by liquid chromatography tandem mass spectrometry and derivatization chemistry.

A method for the determination and quantification of ketosteroid hormones in meat by mass spectrometry, based on the derivatization of the carbonyl moiety of steroids by O-methylhydroxylamine, is presented. The quantitative assay is performed by means of multiple-reaction-monitoring (MRM) scan mode and using the corresponding labelled species, obtained by reaction with d 3-methoxylamine, as internal standard. The accuracy of the method was established by evaluating artificially spiked samples, obtaining values in the range 90-110%. Recovery tests were performed on blank matrix samples spiked with non-natural steroids including trenbolone and melengestrol acetate. The latter experiment revealed that the yield of the extraction processes was approximately 60%. Good values of LOQ and LOD were achieved, making this method competitive with current hormone assay methods.

Steroids with inhibitory activity against the prostate cancer cells and chemical diversity of marine alga Tydemania expeditionis.

A new diketosteroid, (E)-stigmasta-24(28)-en-3,6-dione (1), along with three known steroids (2-4) was isolated from marine alga Tydemania expeditionis collected in China Sea. Their structures were elucidated by extensive spectroscopic methods. Comparison of the chemical constituents revealed significant diversity among different locations. The biological activities of 1, 3 and 4 were evaluated on the prostate cancer cell lines and androgen receptor. Compound 1 exhibited moderate inhibitory activities against the prostate cancer cells DU145, PC3 and LNCaP with IC(50) values of 31.27±1.50, 40.59±3.10 and 19.80±3.84 µM, respectively. Compound 3 showed more potent activities with IC(50) values of 12.38±2.47, 2.14±0.33 and 1.38±0.07 µM, respectively. However, compound 4 showed only weak inhibitory activities on LNCaP cells and was inactive on DU145 and PC3 cells. A competitive binding assay showed that compound 1 exhibited significant affinity to the androgen receptor with an IC(50) value of 7.19±0.45 µM, while 3 and 4 were inactive. The fact that the inhibitory properties of 1 and 3 against the prostate cancer cells were inconsistent with their affinities to the androgen receptor suggested that there might be other mechanism of action involved in the cytotoxic activity.

Antigenotoxic ketosteroid from the red algae Jania adhaerens.

A new ketosteroid, 6ß,16ß-dihydroxycholest-4-en-3-one (1), in addition to the known 6ß-hydroxycholest-4-en-3-one (2), 6ß-hydroxycholest-4,22-dien-3-one (3) and 16ß-hydroxy-5α-cholestan-3,6-dione (4), was isolated from the red alga Jania adhaerens. The structures were assigned on the basis of (1)H- and (13)C-NMR experiments. The new compound (1) was evaluated for its genotoxic and cytotoxic activities and found to possess protective antigenotoxicity in human peripheral blood cells.

Ketosteroids and hydroxyketosteroids, minor metabolites of sugarcane wax.

Besides beta-sitosterol and stigmasterol, the major steroids of sugarcane, the following minor steroids have been isolated and identified from sugarcane wax: 3,6-diketosteroids, Delta(4)-3-keto steroids, and Delta(4)-6-hydroxy-3-keto steroids. Their structures were established by spectroscopic techniques and chemical correlations.

3-Keto steroids from the marine organisms Dendrophyllia cornigera and Cymodocea nodosa.

The new (20R)-22E-cholesta-4,22-diene-3,6-dione (1), along with three known 3-keto steroids were isolated from the deep-water Mediterranean scleractinian coral Dendrophyllia cornigera (2-4). Moreover, four known related 3-keto steroids were isolated from the sea grass Cymodocea nodosa (5-8). The structure elucidation of steroid 1 and the full NMR resonance assignments of all isolated metabolites were based on interpretation of their spectral data. All compounds are reported for the first time as metabolites of the investigated organisms. Compounds 2 and 3 showed significant cytotoxicity against lung cancer NSCLC-N6 cell line.

Chemical constituents of Piper betle Linn. (Piperaceae) roots.

Column chromatography of the alcoholic extract of Piper betle roots furnished aristololactam A-II and a new phenyl propene, characterized as 4-allyl resorcinol, while the petroleum-ether extract yielded a diketosteroid, viz. stigmast-4-en-3,6-dione. All these compounds were characterized by spectroscopic means. Isolation of these compounds from this source is being reported here for the first time.

New ketosteroids from the red alga Hypnea musciformis.

The dichloromethane/methanol extract from the red alga Hypnea musciformis exhibited PPE elastase inhibition. A diketosteroid, the 20-hydroxy-5alpha-cholest-22-ene-3,6-dione was responsible for this activity. Two new steroids were isolated, 2 was assigned as the 6alpha-hydroxy-cholest-4-ene-3-one and 3 as the 6alpha-hydroxy-cholest-4,22-diene-3-one. The structures were assigned mainly on the basis of 1H and 13C NMR experiments.

Analysis of steroids Part 50. Derivatization of ketosteroids for their separation and determination by capillary electrophoresis.

4-Ene-3-ketosteroids and 17-ketosteroids were quantitatively transformed into the corresponding hydrazones using Girard P and T reagents, respectively. The positively charged derivatives were separated by capillary electrophoresis. The spectrophotometric characteristics of the derivatives permitted their sensitive detection in the 230-280 nm range. The steroids investigated included nortestosterone and its phenylpropionate, norethisterone and its oenanthate, d,l-norgestrel, dehydroepiandrosterone, androstenedione and ethisterone.

Isolation of five new 5 alpha-hydroxy-6-keto-delta 7 sterols from the marine sponge Oscarella lobularis.

Five novel sterols isolated from the marine sponge Oscarella lobularis have been identified on the basis of spectral arguments: cholest-7-ene-3beta,5alpha-diol-6-one (1), cholesta-7,22E-diene-3beta, 5alpha-diol-6-one (2), 24-methylcholesta-7,22E-diene-3beta,5alpha-diol-6-one (3), 24-methylcholesta-7,24(28)-diene-3beta,5alpha-diol-6-one (4), and 24-ethylcholest-7-ene-3beta,5alpha-diol-6-one (5).

Solid-phase reagents for the isolation and protection of carbonyl compounds.

Resins which can serve as solid-phase Girard reagents have been prepared. These consist of porous cross-linked polystyrene-divinylbenzene matrices functionalized either with acid hydrazide groups, 1 or with O-alkylhydroxylamine groups, 2. Both resins react rapidly, under acid catalysis, with steroidal ketones and with glucose to form resin bound carbonyl derivatives. The carbonyl compound can be recovered by exchange with acetone under mildly acidic conditions. Resin 2 was used to extract steroidal ketones from an extract of bovine adrenals. By protecting the C-20 ketone group of allopregnanolone, resin 2 was used to prepare the counterpart of the steroid bearing tritium in the 3 alpha-position.

Simultaneous measurement of four testicular delta 4-3-ketosteroids by isocratic high-performance liquid chromatography with on-line ultraviolet absorbance detection.

A method is described for the simultaneous measurement of testosterone, androstenedione, 17 alpha-hydroxyprogesterone and progesterone in venous effluent from in vitro perfused rat testes. The assay uses a non-radioisotopic internal standard (11 beta-hydroxyandrostenedione), isocratic high-performance liquid chromatography (HPLC) and UV absorbance detection at 240 nm. Either of two isocratic HPLC systems described in this report (tetrahydrofuran-methanol-water, 16:28:56; methanol-acetonitrile-water, 9:36:55; v/v/v) may be used, and assay specificity is the same in each. The separation and measurement of all four steroids are completed in 25 min. Sensitivity of the method is 10 ng for testosterone, androstenedione and 17 alpha-hydroxyprogesterone and 25 ng for progesterone. The linear range of the assay extends through 1600 ng which is the upper amount of each steroid tested. Average inter-assay coefficient of variation was 3.3% and average intra-assay coefficient of variation was 3.6%. This rapid, specific and reliable method requires minimal sample preparation and may be performed by inexperienced personnel.

Formation and isolation of delta 5-3-ketosteroids using a purified rat liver alcohol dehydrogenase.

3 beta-Hydroxy-5-androsten-17-one is converted to 5-androstene-3, 17-dione by rat liver alcohol dehydrogenase (ADH). We have reported on the purity of the enzyme which is eluted with pyrazole as a single homogeneous protein using an AMP-agarose affinity column. Rat liver ADH can oxidize hydroxyl groups not only at 3 beta-, but also at 3 alpha-, and 17 beta-positions to a lesser extent; thus it is a pure mammalian enzyme with multifunctional activity for steroids. Since it does not contain delta 5-isomerase activity, the reaction of the dehydrogenase to form the delta 5-ketosteroid intermediate can be observed at pH 7.0, 25 degrees C. Similarly, intermediary product, 5-pregnene-3,20-dione, can be isolated in the conversion of pregnenolone by ADH to progesterone. With buffer alone in a cuvette, a non-enzymatic isomerization of the delta 5-3-ketone occurs at a slow rate (t 1/2 = 6 hrs) but occurs rapidly during isolation procedures. The delta 5-3-ketosteroid intermediates were identified by their behavior on TLC plates with UV light and by their characteristic spectra in the NMR.

Application of over-run thin-layer and gas-liquid chromatography in the separation of closely related C19O2 steroids.

An improved method for the separation of epimeric C19O2 steroids and their related allylic alcohols is described. In this method, the steroids are first separated by over-run thin-layer chromatography, and the unresolved groups are further analysed as free or as trimethylsilyl ether derivatives by gas-liquid chromatography. The behaviour of twenty-one C19O2 steroids was investigated by thin-layer chromatography in four systems and by gas-liquid chromatography in four liquid phases. All steroid pairs of similar polarity were resolved by the combination of these two fractionation procedures.