Chemical Constituents of Ailanthus triphysa

Two new compounds,8(14),15-isopimaradiene-2α，３α，19-triol(1),and 6α，７β－dihydroxy-17(20)-cis-5α－pregna-16-one(2),together with four known copounds,a oxygenated rare phyllocladane,phyllocladan-16α，19-diol(3),kaempferol-3-0-β－Ｄ-galactopyranosied,kaempferol-3-0-α－Ｌ－rhamnopyranoside and scopoletin, were isolated from the leaves of Ailanthus tripysa.Structures of 1-3 were elucidated on the basis of spectroscopic data as compared with related compounds.     

Stereoselective Synthesis of Androstane‐Based Steroidal Phosphine Oxides Possessing the 16α‐Diphenylphosphinyl Moiety

New steroidal phosphine oxides were obtained by base‐catalysed addition of HPPh₂ to the C[dbnd]C double bond of α,β‐unsaturated steroidal esters. The presence of Pd(OAc)₂ has been shown to accelerate the reaction rate. 16α‐Phosphinyl‐17β‐metoxycarbonyl‐androstane derivatives were formed stereoselectively and isolated in moderate to high yields. The exact structure of the products were determined by various spectroscopic methods including two-dimensional NMR techniques.     

Comparison of sulfo‐conjugated and gluco‐conjugated urinary metabolites for detection of methenolone misuse in doping control by LC‐HRMS,GC‐MS and GC‐HRMS

Methenolone (17β‐hydroxy‐1‐methyl‐5α‐androst‐1‐en‐3‐one) misuse in doping control is commonly detected by monitoring the parent molecule and its metabolite (1‐methylene‐5α‐androstan‐3α‐ol‐17‐one) excreted conjugated with glucuronic acid using gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography mass spectrometry (LC‐MS) for the parent molecule, after hydrolysis with β‐glucuronidase. The aim of the present study was the evaluation of the sulfate fraction of methenolone metabolism by LC‐high resolution (HR)MS and the estimation of the long‐term detectability of its sulfate metabolites analyzed by liquid chromatography tandem mass spectrometry (LC‐HRMSMS) compared with the current practice for the detection of methenolone misuse used by the anti‐doping laboratories. Methenolone was administered to two healthy male volunteers, and urine samples were collected up to 12 and 26 days, respectively. Ethyl acetate extraction at weak alkaline pH was performed and then the sulfate conjugates were analyzed by LC‐HRMS using electrospray ionization in negative mode searching for [M‐H]^? ions corresponding to potential sulfate structures (comprising structure alterations such as hydroxylations, oxidations, reductions and combinations of them). Eight sulfate metabolites were finally detected, but four of them were considered important as the most abundant and long term detectable. LC clean up followed by solvolysis and GC/MS analysis of trimethylsilylated (TMS) derivatives reveal that the sulfate analogs of methenolone as well as of 1‐methylene‐5α‐androstan‐3α‐ol‐17‐one, 3z‐hydroxy‐1β‐methyl‐5α‐androstan‐17‐one and 16β‐hydroxy‐1‐methyl‐5α‐androst‐1‐ene‐3,17‐dione were the major metabolites in the sulfate fraction. The results of the present study also document for the first time the methenolone sulfate as well as the 3z‐hydroxy‐1β‐methyl‐5α‐androstan‐17‐one sulfate as metabolites of methenolone in human urine. The time window for the detectability of methenolone sulfate metabolites by LC‐HRMS is comparable with that of their hydrolyzed glucuronide analogs analyzed by GC‐MS. The results of the study demonstrate the importance of sulfation as a phase II metabolic pathway for methenolone metabolism, proposing four metabolites as significant components of the sulfate fraction. Copyright © 2015 John Wiley & Sons, Ltd.     

Nimonol and 6‐oxonimonol

The crystal structures of two intact limonoids, nimonol,7α‐­acetyl‐17α‐(3‐furyl)‐6α‐hydroxy‐4α,4β,8β‐trimethyl‐5α,18α‐androsta‐1,14‐dien‐3‐one (C₂₈H₃₆O₅), and 6‐oxonimonol,7α‐acetyl‐17α‐(3‐furyl)‐4α,4β,8β‐trimethyl‐5α,18α‐androsta‐1,14‐diene‐3,6‐dione (C₂₈H₃₄O₅), are reported. The molecular features are mostly the same in the two structures; however the orientations of the acetoxy group are different in the two structures. The packing in nimonol is due to O—H?O hydrogen bonds while in 6‐oxonimonol it is due to C—H?O hydrogen bonds.     

Chemical Constituents from Annona Glabra

A new kaurane diterpenoid, annoglabasin G (16α‐hydro‐19‐acetoxy‐ent‐kauran‐17‐al) ( 1 ), along with 27 compounds including 18 kaurane diterpenoids, 16β‐hydro‐ent‐kauran‐17‐oic acid ( 2 ), 16α‐hydro‐ent‐kauran‐17‐oic acid ( 3 ), 19‐nor‐ent‐kauran‐4α‐ol‐17‐oic acid ( 4 ), 16α‐hydro‐19‐ol‐ent‐kauran‐17‐oic acid ( 5 ), ent‐kaur‐16‐en‐19‐oic acid ( 6 ), 16α‐hydroxy‐ent‐kauran‐19‐oic acid ( 7 ), 16α,17‐dihydroxy‐ent‐kauran‐19‐oic acid ( 8 ), 16β,17‐dihydroxy‐ent‐kauran‐19‐oic acid ( 9 ), 16α‐hydro‐ent‐kauran‐17,19‐dioic acid ( 10 ), 16β‐hydroxy‐17‐acetoxy‐ent‐kauran‐19‐oic acid ( 11 ), 16β‐hydro‐17‐hydroxy‐ent‐kauran‐19‐al ( 12 ), 16α‐hydro‐17‐hydroxy‐ent‐kauran‐19‐al ( 13 ), 16β,17‐dihydroxy‐ent‐kauran‐19‐al ( 14 ), 16α‐hydro‐19‐al‐ent‐kauran‐17‐oic acid ( 15 ), 16α‐hydro‐17‐acetoxy‐ent‐kauran‐19‐al ( 16 ), 16α‐hydro‐19‐acetoxy‐ent‐kauran‐17‐oic acid ( 17 ), ent‐kaur‐15‐en‐19‐oic acid ( 18 ) and ent‐kaur‐15‐en‐17‐ol‐19‐oic acid ( 19 ); four acetogenins, annomontacin ( 20 ), annonacin ( 21 ), isoannonacinone ( 22 ) and squamocin ( 23 ); four steroids, β‐sitosterol ( 24 ), stigmasterol ( 25 ), β‐sitosteryl‐D‐glucoside ( 26 ) and stigmasteryl‐D‐glucoside ( 27 ) and one oxoaporphine, liriodenine ( 28 ), were isolated from the fresh fruits of Annona glabra. These compounds were characterized and identified by physical and spectral evidence.     

Determination of the deuterium/hydrogen ratio of endogenous urinary steroids for doping control purposes

The development and application of a combined gas chromatography/thermal conversion/isotope ratio mass spectrometry (GC/TC/IRMS) method for D/H ratio determination of endogenous urinary steroids are presented. The key element in sample preparation was the consecutive cleanup with high‐performance liquid chromatography of initially native and subsequently acetylated steroids. This strategy enabled sufficient cleanup off all target analytes for determination of their respective D/H values. Ten steroids (11β‐hydroxyandrosterone, 5α‐androst‐16‐en‐3α‐ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5α‐androstan‐3α,17β‐diol, 5β‐androstan‐3α,17β‐diol and dehydroepiandrosterone) were measured from a single urine specimen. Depending on the biological background, the determination limit for all steroids ranged from 10 to 15 ng/mL for a 20 mL specimen. The method was validated by application of linear mixing models on each steroid and covered repeatability and reproducibility. The specificity of the procedure was ensured by gas chromatography/mass spectrometry (GC/MS) analysis of the sample using equivalent chromatographic conditions to those employed in the GC/TC/IRMS measurement. Within the sample preparation, no isotopic fractionation was observed, and no amount‐dependent shift of the D/H ratios during the measurement was noticed. Possible memory effects occurring during IRMS measurements were corrected by applying a simple rule of proportion. In order to determine the naturally occurring D/H ratios of all implemented steroids, a population of 18 male subjects was analyzed. Relevant mean Δ values among selected steroids were calculated which allowed us to study the metabolic pathways and production sites of all the implemented steroids with additional consideration of the corresponding ¹³C/¹²C ratios. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural and Dynamic Basis of Human Cytochrome P450 7B1: A Survey of Substrate Selectivity and Major Active Site Access Channels

Cytochrome P450 (CYP) 7B1 is a steroid cytochrome P450 7α‐hydroxylase that has been linked directly with bile salt synthesis and hereditary spastic paraplegia type 5 (SPG5). The enzyme provides the primary metabolic route for neurosteroids dehydroepiandrosterone (DHEA), cholesterol derivatives 25‐hydroxycholesterol (25‐HOChol), and other steroids such as 5α‐androstane‐3β,17β‐diol (anediol), and 5α‐androstene‐3β,17β‐diol (enediol). A series of investigations including homology modeling, molecular dynamics (MD), and automatic docking, combined with the results of previous experimental site‐directed mutagenesis studies and access channels analysis, have identified the structural features relevant to the substrate selectivity of CYP7B1. The results clearly identify the dominant access channels and critical residues responsible for ligand binding. Both binding free energy analysis and total interaction energy analysis are consistent with the experimental conclusion that 25‐HOChol is the best substrate. According to 20 ns MD simulations, the Phe cluster residues that lie above the active site, particularly Phe489, are proposed to merge the active site with the adjacent channel to the surface and accommodate substrate binding in a reasonable orientation. The investigation of CYP7B1–substrate binding modes provides detailed insights into the poorly understood structural features of human CYP7B1 at the atomic level, and will be valuable information for drug development and protein engineering.     

New Sesquiterpenes from Ligulariopsis Shichuana

A thorough investigation of Ligulariopsis shichuana afforded five new sesquiterpenes, including 1β,10β‐epoxy‐3α‐angeloyloxy‐9β‐acetoxy‐8α,11β‐dihydroxybakkenolide ( 1 ), 1β,7‐dihydroxy‐3β‐acetoxy‐noreremophil‐6(7),9(10)‐dien‐8‐one ( 2 ), 8α‐hydroxy‐3‐oxoeremophil‐1(2),7(11),9(10)‐trien‐8β(12)‐olide ( 3 ), 1β,10β‐dihydroxy‐3β‐acetoxyeremophil‐7(11),8(9)‐dien‐8(12)‐olide ( 4 ) and 1β,10β‐epoxy‐8,12‐dihydroxy‐3β‐acetoxy‐9β‐angeloyloxyeremophil‐7(11)‐en‐8,12‐disemiketal ( 5 ). Their structures were established by spectroscopic methods and 2D NMR techniques. In addition, bakkenolide ( 1 ) and eremophilenolides ( 5 ) showed antibacterial and cytotoxic activities.     

Two isomers of 2,4‐di­benzyl‐8‐azabicyclo­[3.2.1]­octan‐3‐ol

The crystal structures of the title compounds, 2α,4α‐di­benzyl‐3α‐tropanol (2α,4α‐di­benzyl‐8‐methyl‐8‐aza­bi­cyclo­[3.2.1]­octan‐3α‐ol), C₂₂H₂₇NO, (I), and 2α,4α‐di­benzyl‐3β‐tropanol (2α,4α‐di­benzyl‐8‐methyl‐8‐aza­bi­cyclo­[3.2.1]­octan‐3β‐ol), C₂₂H₂₇NO, (II), show that both compounds have a piperidine ring in a chair conformation and a pyrrolidine ring in an envelope conformation. Isomer (I) is asymmetric, the benzyl groups having different orientations, whereas isomer (II) is mirror symmetric, and the N and O atoms, the C atom attached to the hydroxy group, and the methyl C atom attached to the N atom lie on the mirror plane. In the crystal structures of both (I) and (II), the mol­ecules are linked together by intermolecular O—H⋯N hydrogen bonds to form chains that run parallel to the a direction in (I) and parallel to b in (II).     

In vitro and in vivo studies of androst‐4‐ene‐3,6,17‐trione in horses by gas chromatography–mass spectrometry

This paper describes the application of gas chromatography–mass spectrometry (GC‐MS) for in vitro and in vivo studies of 6‐OXO in horses, with a special aim to identify the most appropriate target metabolite to be monitored for controlling the administration of 6‐OXO in racehorses. In vitro studies of 6‐OXO were performed using horse liver microsomes. The major biotransformation observed was reduction of one keto group at the C3 or C6 positions. Three in vitro metabolites, namely 6α‐hydroxyandrost‐4‐ene‐3,17‐dione (M1), 3α‐hydroxyandrost‐4‐ene‐6,17‐dione (M2a) and 3β‐hydroxyandrost‐4‐ene‐6,17‐dione (M2b) were identified. For the in vivo studies, two thoroughbred geldings were each administered orally with 500 mg of androst‐4‐ene‐3,6,17‐trione (5 capsules of 6‐OXO_®) by stomach tubing. The results revealed that 6‐OXO was extensively metabolized. The three in vitro metabolites (M1, M2a and M2b) identified earlier were all detected in post‐administration urine samples. In addition, seven other urinary metabolites, derived from a further reduction of either one of the remaining keto groups or one of the remaining keto groups and the olefin group, were identified. These metabolites included 6α,17β‐dihydroxyandrost‐4‐en‐3‐one (M3a), 6,17‐dihydroxyandrost‐4‐en‐3‐one (M3b and M3c), 3β,6β‐dihydroxyandrost‐4‐en‐17‐one (M4a), 3,6‐dihydroxyandrost‐4‐en‐17‐one (M4b), 3,6‐dihydroxyandrostan‐17‐one (M5) and 3,17‐dihydroxyandrostan‐6‐one (M6). The longest detection time observed in urine was up to 46 h for the M6 metabolite. For blood samples, the peak 6‐OXO plasma concentration was observed 1 h post administration. Plasma 6‐OXO decreased rapidly and was not detectable 12 h post administration. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolism of androsta‐1,4,6‐triene‐3,17‐dione and detection by gas chromatography/mass spectrometry in doping control

The urinary metabolism of the irreversible aromatase inhibitor androsta‐1,4,6‐triene‐3,17‐dione was investigated. It is mainly excreted unchanged and as its 17β‐hydroxy analogue. For confirmation, 17β‐hydroxyandrosta‐1,4,6‐trien‐3‐one was synthesized and characterized by nuclear magnetic resonance (NMR) in addition to the parent compound. In addition, several reduced metabolites were detected in the post‐administration urines, namely 17β‐hydroxyandrosta‐1,4‐dien‐3‐one (boldenone), 17β‐hydroxy‐5β‐androst‐1‐en‐3‐one (boldenone metabolite), 17β‐hydroxyandrosta‐4,6‐dien‐3‐one, and androsta‐4,6‐diene‐3,17‐dione. The identification was performed by comparison of the metabolites with reference material utilizing gas chromatography/mass spectrometry (GC/MS) of the underivatized compounds and GC/MS and GC/tandem mass spectrometry (MS/MS) of their trimethylsilyl (TMS) derivatives. Alterations in the steroid profile were also observed, most obviously in the androsterone/testosterone ratio. Even if not explicitly listed, androsta‐1,4,6‐triene‐3,17‐dione is classified as a prohibited substance in sports by the World Anti‐Doping Agency (WADA) due to its aromatase‐inhibiting properties. In 2006 three samples from human routine sports doping control tested positive for metabolites of androsta‐1,4,6‐triene‐3,17‐dione. The samples were initially found suspicious for the boldenone metabolite 17β‐hydroxy‐5β‐androst‐1‐en‐3‐one. Since metabolites of androst‐4‐ene‐3,6,17‐trione were also present in the urine samples, it is presumed that these findings were due to the administration of a product like ‘Novedex Xtreme’, which could be easily obtained from the sport supplement market. Copyright © 2008 John Wiley & Sons, Ltd.     

Sterols from the Fungus Catathelasma imperiale

Eight ergostane-type sterols and three their derivatives (one mono-linoleate and two mono-glucosides) were isolated from the ethyl acetate soluble fraction of the fungus Catathelasma imperi.a/e. Two of them are novel compounds, namely 22E,24R-er-gosta-7,22-diene-3β ,5α-diol-6β-linoleate (1) and 22E, 24R-er-gosta-7,22-diene-3β, 5β, 6α-triol (5) with an uncommon c/s-fused A/B ring. Structures of these compounds were demon-strated on the basis of their chemical evidences and spectroscop-ic methods, especially 2D NMR techniques.     

21α‐Fluoro‐7‐norvouacapane‐17β,21α‐lactone

The crystal structure of 21α‐fluoro‐7‐norvouacapane‐17β,21α‐lactone, C₂₀H₂₅FO₃, a new synthetic derivative of the diterpenoid 6α,7β‐di­hydroxy­vouacapan‐17β‐oic acid isolated from Pterodon polygalaeflorus Benth fruits, is described.     

1H and 13C NMR signal assignments of carboxyl‐linked glucosides of bile acids

Complete ¹H and ¹³C resonance assignments were carried out for a new type of carboxyl‐linked glucosides of chenodeoxycholic (3α,7α‐dihydroxy‐5β‐cholan‐24‐oic) and hyodeoxycholic (3α,6α‐dihydroxy‐5β‐cholan‐24‐oic) acids by using several homonuclear (¹H–¹H) and heteronuclear (¹H–¹³C) 2D NMR techniques. Differences in the ¹H and ¹³C resonances between the α‐ and β‐anomers of the ester glucosides of bile acids were clarified for the first time. A comparison of the ¹H and ¹³C signal shifts induced by β‐D ‐glucosidation at the 24‐carboxyl and 3α‐hydroxyl groups in the parent 5β‐cholanoic acid was also made. Copyright © 2003 John Wiley & Sons, Ltd.     

Determination of (13)C/(12)C ratios of endogenous urinary steroids excreted as sulpho conjugates

The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry‐based method for the measurement of stable carbon isotopes of endogenous urinary steroids excreted as sulphates is presented. The key element in sample preparation is the consecutive cleanup with high‐performance liquid chromatography of underivatized and acetylated steroids, which allows the isolation of seven analytes (pregn‐5‐ene‐3β,17α,20α‐triol, etiocholanolone, androsterone, epiandrosterone, dehydroepiandrosterone (DHEA), androst‐5‐ene‐3β,17β‐diol and androst‐5‐ene‐3β,17α‐diol) from a single urine specimen. These steroids are of particular importance to doping controls as they should enable the sensitive and retrospective detection of DHEA abuse by athletes. Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers the items, repeatability and reproducibility. The specificity was further demonstrated by gas chromatography/mass spectrometry for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring ¹³C/¹²C ratios and urinary concentrations of all implemented steroids, a reference population of n = 67 subjects was measured to enable the calculation of reference limits for all relevant steroidal Δ values. The applicability of the developed method was tested by means of a DHEA excretion study. Despite the fact that orally ingested DHEA is preferentially converted into sulphated metabolites and that the renal clearance of sulphated steroids is slow, only the ¹³C/¹²C ratio of EpiA demonstrated the potential to prolong the detection time for DHEA misuse. Copyright © 2010 John Wiley & Sons, Ltd.     

Sterols from the Stems of Momordica charantia

A new sterol, 5α,6α‐epoxy‐3β‐hydroxy‐(22E,24R)‐ergosta‐8,22‐dien‐7‐one ( 1 ), together with eight known sterols, 5α,6α‐epoxy‐(22E,24R)‐ergosta‐8,22‐diene‐3β,7α‐diol ( 2 ), 5α,6α‐epoxy‐(22E,24R)‐ergosta‐8,22‐diene‐3β,7β‐diol ( 3 ), 5α,6α‐epoxy‐(22E,24R)‐ergosta‐8(14),22‐diene‐3β,7α‐diol ( 4 ), 3β‐hydroxy‐(22E,24R)‐ergosta‐5,8,22‐trien‐7‐one ( 5 ), ergosterol peroxide ( 6 ), clerosterol ( 7 ), decortinol ( 8 ), and decortinone ( 9 ), were isolated from the stems of Momordica charantia. Their structures were elucidated by mean of extensive spectroscopic methods, including ¹H, ¹³C, 2D‐NMR and HR‐EI‐MS, as well as comparison with the literature data. Compounds 1 , 4 , 5 , 8 , and 91 were not cytotoxic against the SK‐Hep 1 cell line.     

A new tropane alkaloid from the leaves of Erythroxylum subsessile isolated by pH‐zone‐refining counter‐current chromatography

Tropane alkaloids are bioactive metabolites with great importance in the pharmaceutical industry and the most important class of natural products found in the Erythroxylum genus. However, these compounds are usually separated by traditional chromatographic techniques, in which the sample is progressively purified in multiple chromatographic steps, resulting in a time‐ and solvent‐consuming procedure. In this work we present the isolation of a novel alkaloid, 6β,7β‐dibenzoyloxytropan‐3α‐ol, together with the two known 3α‐benzoyloxynortropan‐6β‐ol and 3α,6β‐dibenzoyloxytropane alkaloids, directly from the crude alkaloid fraction from the leaves of Erythroxylum subsessile, by using a single run pH‐zone‐refining counter‐current chromatography method. The ethyl acetate/water (1:1, v/v) biphasic solvent system with triethylamine and HCl as retention and eluter agents, respectively, was used to isolate tropane alkaloids for the first time. The structures of the isolated alkaloids were elucidated by spectroscopic methods.     

Two New Diterpenoids and other Constituents from Isodon Nervosus

Two new ent‐kaurane diterpenoids, 15β‐hydroxy‐6,7‐seco‐6,11β:6,20‐diepoxy‐1α,7‐olide‐ent‐kaur‐16‐ene ( 1 ), 11α,15α‐dihydroxy‐6β‐methoxy‐6,7‐seco‐6,20‐epoxy‐1α,7‐olide‐ent‐kaur‐16‐ene ( 2 ), together with four known diterpenoids, nodosin ( 3 ), isodocarpin ( 4 ), odonicin ( 5 ) and maoyecrystal F ( 6 ) were isolated from the aerial parts of Isodon nervosus. The structures of the new compounds were elucidated on the basis of their spectral evidence, especially on 2D NMR.     

胆甾-4α-甲基-8-烯-3β，7α-二醇二乙酸酯的结构与性质研究

Lophenol, cholest-4α-methyl-7-en-3β-ol (1), obtained from Dracaena cochinchinensis (Lour.) S. C. Chen, was structurally modified. It was acetylated to protect 3β-hydroxyl group, and then oxidised by selenium dioxide in acetic acid to give cholest-4a-methyl-8-en-3β, Ta-diol diacetate (3). This compound 3 is unstable in chloroform solution or when heated and easily converted to a diene compound, cholest-4a-methyl-7,14-dien-3β-ol acetate (4). The structures of 3 and 4 were elucidated by means of IR, ^1H NMR, ^13C NMR and MS, and the absolute configuration of 3 was established by X-ray crystallography. The property of 3 was also discussed in this paper. Both 3 and 4 are new compounds and were reported for the first time.