Biosynthesis of [7-3H]16 alpha-hydroxy-dehydroepiandrosterone having high specific activity.

Biosynthesis of [7-3H]16alpha-hydroxy-dehydroepiandrosterone in high specific activity has been studied. [7-3H] dehydroepiandrosterone (13.9 C/mM) in trace quantity was oxidized by Streptomyces roseochromogenes (NRRLB-1233) for 5 min at 27 degrees C. The radioactive products were chromatographically separated, identified and their radiochemical purity established by isotopic dilution analysis. [7-3H]16alpha-hydroxy-dehydroepiandrosterone (2.5 x 10(7) dpm) was obtained by microbial hydroxylation of substrate (1.9 X 10(9) dpm). In some cases [7-3H])5-androstene-3beta, 16alpha, 17beta-triol in a small amount of radioactivity could be found at the prolonged reaction for 30 hr.     

Conversion of sterols and triterpenes by mycobacteria. II. Transformation of 7-oxygenated sterols into androstane derivatives via a 7-deoxygenation

The bioconversion of 7-oxygenated sterols by Mycobacterium aurum was studied in a preliminary investigation of the microbial conversion of wool wax. 7-Oxocholesterol was found to be transformed mainly into 3,17-dioxygenated androstane derivatives. 7 xi-Hydroxylated sterols were formed in an initial reduction step, and the C-7 hydroxyl group was then eliminated in a dehydration reaction. This was thought to take place during the isomerisation of cholest-4-en-3-one to cholest-5-en-3-one. Deuterium labelling experiments showed that this elimination proceeded faster for the C-7 alpha isomer, although it was not stereospecific. The C-7 alpha and C-7 beta-hydroxy isomers were weakly interconverted via the 7-oxo derivatives. Cholest-4-en-3-one, cholest-1,4-dien-3-one and cholest-4,6-dien-3-one all lost their side chains following a hydrogenation/dehydrogenation reaction. The resulting 3,17-dioxoandrostene or 3,17-androstadiene derivatives were mainly hydrogenated into 5 alpha-androstane-3,17-dione and 5 alpha-androstane-3 beta-ol-17-one. Elimination of the 3 beta-hydroxyl groups giving cholesta-3,5-dien-7-one, and subsequent microbial degradation of the side chain was not observed to any significant extent. The convergence of the bioconversion pathways of cholesterol and the 7-oxygenated cholesterols enabled crude, partially auto-oxidised cholesterol to be used as a substrate for the production of 3,17-dioxygenated androstane derivatives by M. aurum.     

1H, (13)C, and (15)N NMR stereochemical study of cis-fused 7a(8a)-methyl and 6-phenyl octa(hexa)hydrocyclopenta[d][1,3]oxazines and [3,1]benzoxazines

Four 7a-methyl octa(or hexa)hydrocyclopenta[d][1,3]oxazines, five 8a-methyl octa(or hexa)hydro[3,1]benzoxazines, two 6-phenyl hexahydro[3,1]benzoxazinones, and 8a-methyl hexahydro[1,3]benzoxazinone, all cis-fused, were prepared and their stereostructures studied by various one- and two-dimensional (1)H, (13)C, and (15)N NMR spectroscopic methods. In solution, the cyclopentane-fused 2-oxo derivatives and the 1,3-benzoxazinone were found to attain exclusively the N-in/O-in conformation, whereas the 6-phenyl 2-oxo/thioxo derivatives were found to be present predominantly in the N-out conformation. The C-2 unsubstituted and the 2-oxo/thioxo 7a/8a-methyl derivatives were all present in solution as a rapidly interconverting equilibrium of the N-in and N-out conformations. The C-2 methyl derivatives were each found to be interconvertable mixtures of epimers (at C-2) with the N-in conformer predominating for one epimer and the N-out conformer predominating for the other, with both predominating conformers having the C-2 methyl group equatorially orientated. The substituent on the nitrogen (H or Me) was found to be always predominantly equatorial with respect to the heteroring, except for the epimeric 2-methyl derivatives with N-out conformations where steric constraints and the generalized anomeric effect resulted in the axial orientation of the C-2 methyl being favored.     

Synthesis of 7alpha-hydroxy-dehydroepiandrosterone and 7beta-hydroxy-dehydroepiandrosterone

The fermentation of dehydroepiandrosterone synthesized from the starting material diosgenin using Mucor racemosus produced 7alpha-hydroxy-dehydroepiandrosterone and 7beta-hydroxy-dehydroepiandrosterone. The bioactivity of the microbial metabolites is also discussed. The species M. racemosus was isolated by screening among stains from soil samples collected from various parts of China.     

Ergosteroids V: preparation and biological activity of various D-ring derivatives in the 7-oxo-dehydroepiandrosterone series

Our previous finding that D-ring seco derivatives of dehydroepiandrosterone retained biologic activity (Reich et al., Steroids 1998;63:542-53) motivated us to synthesize and test a number of steroids in which the D-ring is retained but altered in various ways. Several new steroids were synthesized and characterized by (1)H and (13)C NMR spectroscopy. The availability of a number of closely related compounds allowed detailed (13)C chemical shift correlations. Using the induction of two thermogenic enzymes in rats, liver mitochondrial glycerophosphate dehydrogenase (GPDH) and cytosolic malic enzyme, as criteria of biologic activity some 30 compounds were assayed. Hydroxylation of dehydroepiandrosterone (DHEA) at the 16 alpha position was previously shown to diminish activity (Lardy et al., Steroids 1998;63:158-65); the corresponding 7-oxo compound is fully active. Hydroxylation at the 15 beta position of DHEA, 7-oxo-DHEA, or 16 alpha-hydroxy-7-oxo-DHEA greatly diminished the induction of GPDH but induction of malic enzyme was retained. Most 5,15 diene steroids tested had 2 weak, or no, ability to enhance the formation of GPDH but did increase malic enzyme.     

Cytotoxic heterocyclic triterpenoids derived from betulin and betulinic acid

The aim of this work was to synthesize a set of heterocyclic derivatives of lupane, lup-20(29)-ene, and 18α-oleanane, and to investigate their cytotoxic activities. Some of those heterocycles were previously known in the oleanane (allobetulin) group; however, to our knowledge the syntheses and biological activities of lupane heterocycles have not been reported before. Starting from betulin (1) and betulinic acid (2), we prepared 3-oxo compounds and 2-bromo-3-oxo compounds 3-10, 2-hydroxymethylene-3-oxo compounds 11-13 and β-oxo esters 14-16. Condensation of these intermediates with hydrazine, phenylhydrazine, hydroxylamine, or thiourea yielded the pyrazole and phenylpyrazole derivatives 17-22, pyrazolones 23-25, isoxazoles 26 and 27, and thiazoles 28-31. Fifteen compounds (14-16, 18-25, and 29-32) have not been reported before. The cytotoxicity was measured using panel of seven cancer cell lines with/without MDR phenotype and non tumor MRC-5 and BJ fibroblasts. The preferential cytotoxicity to cancer cell lines, particularly to hematological tumors was observed, the bromo acids 5, 6 showed highest activity and selectivity against tumor cells.     

A convenient synthesis of 3 beta,12 alpha-, 3 beta,7 alpha-, and 3 beta,7 beta-dihydroxy-5-cholen-24-oic acids: unusual bile acids in human biological fluids

The unusual bile acids 3 beta,12 alpha- (V), 3 beta,7 alpha- (XIIIa), and 3 beta,7 beta- (XIIIb) dihydroxy-5-cholen-24-oic acids were synthesized conveniently from the 3-oxo derivatives of deoxycholic (I) and lithocholic (VI) acids, respectively, to provide authentic samples for the gas chromatography-mass spectrometric determination of these bile acids in the abnormal metabolism of bile acids.     

Synthesis of deuterium labeled NMDA receptor inhibitor - 20-Oxo-5β-[9,12,12-(2)H(3)]pregnan-3α-yl-L-glutamyl 1-ester

20-Oxo-5β-[9,12,12-(2)H(3)]pregnan-3α-yl-l-glutamyl 1-ester 11 was synthesized as an internal standard for quantification of a neuroprotective NMDA receptor ligand, 20-oxo-5β-pregnan-3α-yl-l-glutamyl 1-ester 18 and its metabolites, in plasma and tissue. 11α-Hydroxy-progesterone (1) was reduced under basic conditions to yield the corresponding 5β-steroid. Protection of the 3- and 20-oxo groups and oxidation of the 11α-hydroxy group was then followed by a deuterium exchange, conducted under basic conditions using deuterated methanol. Next, the carbonyl moiety at C-11 was reduced and the 11α-hydroxyl group removed through utilization of the Barton-McCombie reaction. Subsequent deprotection of the 3- and 20-acetals and stereoselective reduction of the 3-oxo group gave the desired trideuterated pregnanolone (8). This was coupled with protected glutamic acid, which was then deprotected to yield [9,12,12-(2)H(3)]-pregnanolone glutamate (11) with >99% isotopic purity.     

Conversion of cholic acid and chenodeoxycholic acid into their 7-oxo derivatives by Bacteroides intestinalis AM-1 isolated from human feces

Secondary bile acid-producing bacteria were isolated from human feces to improve our appreciation of the functional diversity and redundancy of the intestinal microbiota. In total, 619 bacterial colonies were isolated using a nutrient-poor agar medium and the level of secondary bile acid formation was examined in each by a liquid culture, followed by thin-layer chromatography. Of five strains analyzed by 16S rRNA gene sequencing and biochemical testing, one was identified as Bacteroides intestinalis AM-1, which was not previously recognized as a secondary bile-acid producer. GC-MS revealed that B. intestinalis AM-1 converts cholic acid (CA) and chenodeoxycholic acid into their 7-oxo derivatives, 7-oxo-deoxycholic acid (7-oxo-DCA) and 7-oxo-lithocholic acid, respectively. Thus, B. intestinalis AM-1 possesses 7α-hydroxysteroid dehydrogenase (7α-HSDH) activity. In liquid culture, B. intestinalis AM-1 showed a relatively higher productivity of 7-oxo-DCA than Escherichia coli HB101 and Bacteroides fragilis JCM11019^T, which are known to possess 7α-HSDH activity. The level of 7α-HSDH activity was higher in B. intestinalis AM-1 than in the other two strains under the conditions tested. The 7α-HSDH activity in each of the three strains is not induced by CA; instead, it is regulated in a growth phase-dependent manner.     

Syntheses of the methyl glycosides of the repeating units of chondroitin 4- and 6-sulfate

3,4,6-Tri-O-acetyl-D-galactal was transformed into methyl 6-O-acetyl-2-azido-4-O-benzyl-2-deoxy-beta-D-galactopyranoside and its 4-O-acetyl-6-O-benzyl analogue, each of which was glycosylated with activated, O-acetylated derivatives of methyl D-glucopyranosyluronate. The resulting beta-(1----3)-linked disaccharide derivatives were each reductively N-acetylated, hydrogenolysed, O-sulfated, and saponified to afford the disodium salts of methyl 2-acetamido-2-deoxy-3-O-(beta-D-glucopyranosyluronic acid)-4-O-sulfo-beta-D-galactopyranoside and the 6-O-sulfo analogue. D-Galactal was also transformed into activated derivatives of 2-azido-3,6-di-O-benzyl-2-deoxy-D-galactopyranose and their 3,4-di-O-benzyl analogues with various substituents at O-4 and O-6. These glycosyl donors were condensed with 6-O-protected derivatives of methyl 2,3-di-O-benzyl-beta-D-glucopyranoside to give the beta-(1----4)-linked disaccharide derivatives, which were selectively deprotected, then oxidised at C-6 of the gluco unit, reductively N-acetylated, selectively deprotected, O-sulfated at C-4 or C-6 of the galacto unit, and hydrogenolysed to give the disodium salts of methyl 4-O-(2-acetamido-2-deoxy-4-O-sulfo-beta-D-galactopyranosyl)-beta-D- glucopyranosiduronic acid and the 6-O-sulfo analogue.     

Selective reduction of oxo bile acids: synthesis of 3 beta-, 7 beta-, and 12 beta-hydroxy bile acids

Preparation of some biologically important keto bile acids is described. Advantage is taken of the preferential ketalization of 3-oxo group in bile acids over 7- and 12-oxo groups for the selective reduction of these keto groups. The method was found to be specially useful for preparation of 7 beta-, 12 alpha, and 12 beta-[3H]-3-oxo bile acids. Improved methods are also described for the preparation of epimers of naturally occurring bile acids at C-3, C-7, and C-12. 3 beta-Hydroxy bile acids (iso-bile acids) were prepared with the use of diethylazodicarboxylate/triphenylphosphine/formic acid. Iso-bile acids were obtained in excellent yields (80-95%) except during synthesis of isoursodeoxycholic acid (yield, 50%). Isoursodeoxycholic acid was, however, prepared in very good yield via epimerization of 3 alpha-hydroxyl group in 7-oxolithocholic acid followed by stereoselective reduction of 7-oxo group. A highly efficient method for the reduction of 7-oxo and 12-oxo groups was developed. Thus, 7-oxolithocholic acid and 7-oxoisolithocholic acid on reduction with potassium/tertiary amyl alcohol yielded ursodeoxycholic acid and isoursodeoxycholic acid in yields of 96% and 94%, respectively, while reduction of 7-oxodeoxycholic acid resulted in ursocholic acid in 93% yield. In a similar manner, reduction of 12-oxolithocholic acid and 12-oxochenodeoxycholic acid yielded 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acid (lagodeoxycholic acid; 92% yield) and 3 alpha, 7 alpha, 12 beta-trihydroxy-5 beta-cholanoic acid (lagocholic acid, 86% yield).     

Metabolic transformations of some ent-kaurenes in Gibberella fujikuroi

The conversion of ent-kaur-16-enes to gibberellic acid in Gibberella fujikuroi is blocked by A-ring modifications. Thus ent-3β-hydroxykaur-16-en-19-yl succinate gives good conversion (46%) to the 7β-hydroxy derivative.* Under the same conditions the 3β-epimer gives 7β- or 6α-hydroxylation and the former occurs for the 3-oxo analogue. The succinoyloxy function acts as a less efficient block and ent-kaur-16-en-19-yl succinate is converted to 7β-hydroxy and 6β,7β-dihydroxy derivatives along with gibberellic acid. Hydrolysis of the succinate block of the metabolities provides the 7β, 19-diol and 6β,7β, 19-triol. Of this pair only the former was effectively metabolized to gibberellic acid in G. fujikuroi.     

Determination of aromatization of 19-oxygenated 16 alpha-hydroxyandrostenedione with human placental microsomes by high-performance liquid chromatography coupled with coulometric detection

A sensitive assay of aromatization of 16 alpha-hydroxylated androgens, 16 alpha-hydroxyandrostenedione (16 alpha-OHA), 16 alpha,19-dihydroxyandrostenedione [16 alpha,19-(OH)2A], and 16 alpha-hydroxy-19-oxo androstenedione (16 alpha-OH-19-oxo A), was developed using reversed phase high-performance liquid chromatography with a coulometric detector. The estrogens, estriol and 16 alpha-hydroxyestrone, were simultaneously detected in quantities as low as 300 pg of the estrogens formed in an assay by an internal standard method. Apparent Km and Vmax of the microsomal aromatase for 16 alpha-OHA, 16 alpha,19-(OH)2A or 16 alpha-OH-19-oxo A were 1.06, 4.00 or 571 microM and 0.014, 0.087 or 1.67 pmol/min/micrograms protein, respectively. The results show that the 19-oxo steroid has extremely low affinity for aromatase relative to the other substrates.     

Kolavane and kaurane diterpenes from the stem bark of Xylopia aethiopica

The stem bark of Xylopia aethiopica has yielded four diterpenes, two of them novel. Three of the diterpenes were identified as (?)-kaur-16-en-19-oic acid and its 7-oxo and 7β-hydroxy derivatives. The fourth was the novel kolavane derivative 2-oxo-kolav-3,13-dien-15-oic acid, a type of compound not previously recorded in the Annonaceae.     

Synthesis and anti-HBV activity of thiouracils linked via S and N-1 to the 5-position of methyl beta-D-ribofuranoside

Reverse nucleoside derivatives of 2-(methylsulfanyl)uracils 6a-d were prepared by treating of the sodium salt of 2-(methylsulfanyl)uracils (5a-d) with methyl 2,3-O-isopropylidene-5-O-p-toluenesulfonyl-beta-D-ribofuranoside (2). The alkylation of 2-thiouracils 4a-d with methyl 5-deoxy-5-iodo-2,3-O-isopropylidene-D-ribofuranoside (3) afforded the corresponding S-ribofuranoside derivatives 8a-d. Deisopropylidenation of 6a-d and 8a-d afforded the corresponding deprotected derivatives 7a-d and 9a-d, respectively. The Anti-HBV activity of selected compounds was studied.     

Synthesis and antiviral activities of 1'-carbon-substituted 4'-thiothymidines

4-Thiofuranoid glycals substituted at the 1-position with methyl (5), (t-butyldimethylsilyloxy)methyl (7), and acetoxymethyl (8) groups were prepared from the 3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl) (TIPDS)-4-thiofuranoid glycal (3) by way of LDA-lithiation. N-Iodosuccimide-initiated electrophilic glycosidation between silylated thymine and these 1-carbon-substituted 4-thioglycals gave the respective beta-anomers (9, 10, and 13) stereoselectively. Tin radical-mediated removal of the 2'-iodine atom from these products provided the corresponding 1'-branched 4'-thiothymidine derivatives (11, 12, and 14) in good yields. The 1'-hydroxymethyl derivative (15) served as a precursor for the preparation of the formyl (16), cyanoethenyl (17), and cyano (19) derivatives. Among the deprotected 1'-branched 4'-thiothymidines (20-25), the 1'-methyl analogue 20 showed the most potent anti-HSV-1 activity, but it was much less active than the parent compound 4'-thiothymidine.     

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.     

A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm

A method is reported for the quantification of 3-oxoacyl homoserine lactones (3-oxo AHLs), a major class of quorum-sensing signals found in Gram-negative bacteria. It is based on the conversion of 3-oxo AHLs to their pentafluorobenzyloxime derivatives followed by gas chromatography-mass spectrometry (electron capture-negative ion). The method used [13C16]-N-3-oxo-dodecanoyl homoserine lactone ([13C16]-OdDHL) as the internal standard, and its validity was tested by spiking the supernatant and cell fractions with three levels of 3-oxo AHLs, i.e. 1, 10 and 100 ng per sample. These showed the method to be both sensitive (S/N ratio >10:1 for 1 ng) and accurate. The assay was applied to the biofilm and effluent of a green fluorescent protein (GFP)-expressing strain of Pseudomonas aeruginosa (6294) culture grown in flow cells. Biofilm volume was determined for three replicate flow cells by confocal scanning laser microscopy. OdDHL was detected in the biofilm at 632 +/- 381 microM and the effluent at 14 +/- 3 nM. The biofilm concentration is the highest level so far reported for an AHL in a wild-type bacterial system. The next most abundant 3-oxo AHL in the biofilm and effluent was N-3-oxo-tetradecanoyl homoserine lactone (OtDHL) at 40 +/- 15 microM and 1.5 +/- 0.7 nM respectively. OtDHL is unreported for P. aeruginosa and has an activity equivalent to OdDHL in a lasR bioassay. Two other 3-oxo AHLs were detected at lower concentrations: N3-oxo-decanoyl homoserine lactone (ODHL) in the biofilm (3 +/- 2 microM) and effluent (1 +/- 0.1 nM); and N-3-oxo-octanoyl homoserine lactone (OOHL) in the effluent (0.1 +/- 0.1 nM).     

High-performance liquid chromatographic method for profiling 2-oxo acids in urine and its application in evaluating vitamin status in rats

B-group vitamins are involved in the catabolism of 2-oxo acids. To identify the functional biomarkers of B-group vitamins, we developed a high-performance liquid chromatographic method for profiling 2-oxo acids in urine and applied this method to urine samples from rats deficient in vitamins B₁ and B₆ and pantothenic acid. 2-Oxo acids were reacted with 1,2-diamino-4,5-methylenebenzene to produce fluorescent derivatives, which were then separated using a TSKgel ODS-80Ts column with 30 mmol/L of KH₂PO₄ (pH 3.0):acetonitrile (7:3) at a flow rate of 1.0 mL/min. Vitamin B₁ deficiency increased urinary levels of all 2-oxo acids, while vitamin B₆ deficiency only increased levels of sum of 2-oxaloacetic acid and pyruvic acid, and pantothenic acid deficiency only increased levels of 2-oxoisovaleric acid. Profiles of 2-oxo acids in urine samples might be a non-invasive way of clarifying the functional biomarker of B-group vitamins.     

Steroids excreted in urine by neonates with 21-hydroxylase deficiency: Characterization, using GC-MS and GC-MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes

Steroid metabolites in urine from neonates with 21-hydroxylase deficiency are predominantly polyhydroxylated 17-hydroxyprogesterone and androgen metabolites, and most have incompletely defined structure. This study forms part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal types of steroid metabolism.Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra, obtained with constant excitation conditions, were used together to determine the structure of the D-ring and the side chain of 20-oxo and 20-hydroxy pregnane(ene)s without oxo groups on the A-, B-, and C-ring.All possible combinations of D-ring and side chain configuration were considered. Most fragmentations could be interpreted as partial or complete D-ring cleavages with loss of the side chain, aided by comparison with spectra of deuterated derivatives and of borohydride reduced metabolites. Possible rearrangement ions are also discussed. More than 140 endogenous metabolites were characterized.GC-MS/MS was especially beneficial for characterization of compounds with 16,17-dihydroxy-20-oxo structure, interpreted as markers of intra-uterine enzyme induction. It also assisted the differentiation of 16-hydroxy-20-oxo metabolites, present in urine of non-affected neonates, from the diagnostic 17-hydroxy-20-oxosteroids and enabled the detection of 15,17-dihydroxy-20-oxo compounds in low concentrations. The presence of 17,21-dihydroxylated pregnane(ene)s despite the deficit in CYP21A2 is discussed.We conclude that GC-MS combined with GC-MS/MS allows reliable identification of the structure of the D-ring and side chain of pregnane(ene)s without prior isolation, even when in low concentrations in urine.