In vivo metabolism study of vaccarin in rat using HPLC‐LTQ‐MSn

In order to illustrate the main biotransformation pathways of vaccarin in vivo, metabolites of vaccarin in rats were identified using a specific and sensitive high‐performance liquid chromatography–electrospray ionization linear ion trap mass spectrometry (LTQ XL?) method. The rats were administered a single dose (200 mg/kg) of vaccarin by oral gavage. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent drug, the parent compound and six metabolites were found in rat urine after oral administration of vaccarin. The parent compound and five metabolites were detected in rat plasma. In heart, liver and kidney samples, respectively, one, four and three metabolites were identified, in addition to the parent compound. Three metabolites, but no trace of parent drug, were found in the rat feces. This is the first systematic metabolism study of vaccarin in vivo. The biotransformation pathways of vaccarin involved methylation, hydroxylation, glycosylation and deglycosylation. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of metabolites of Helicid in vivo using ultra‐high performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry

Helicid is an active natural aromatic phenolic glycoside ingredient originating from a well‐known traditional Chinese herbal medicine and has the significant effects of sedative hypnosis, anti‐inflammatory analgesia and antidepressant. In this study, we analyzed the potential metabolites of Helicid in rats by multiple mass defect filter and dynamic background subtraction in ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF‐MS). Moreover, we used a novel data processing method, ‘key product ions’, to rapidly detect and identify metabolites as an assistant tool. MetabolitePilot™ 2.0 software and PeakView™ 2.2 software were used for analyzing metabolites. Twenty metabolites of Helicid (including 15 phase I metabolites and five phase II metabolites) were detected by comparison with the blank samples. The biotransformation route of Helicid was identified as demethylation, oxidation, dehydroxylation, hydrogenation, decarbonylation, glucuronide conjugation and methylation. This is the first study simultaneously detecting and identifying Helicid metabolism in rats employing UHPLC‐Q‐TOF‐MS technology. This experiment not only proposed a method for rapidly detecting and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of Helicid in vivo. Furthermore, it provided an effective method for the analysis of other aromatic phenolic glycosides metabolic components in vivo.     

Pharmacokinetics of 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐D‐glucoside in rat using ultra‐performance LC‐quadrupole TOF‐MS

2,3,5,4′‐Tetrahydroxystilbene‐2‐O‐β‐D‐glucoside (THSG) from Polygoni multiflori has been demonstrated to possess a variety of pharmacological activities, including antioxidant, anti‐inflammatory and hepatoprotective activities. Ultra‐performance LC‐quadrupole TOF‐MS with MS Elevated Energy data collection technique and rapid resolution LC with diode array detection and ESI multistage MSⁿ methods were developed for the pharmacokinetics, tissue distribution, metabolism, and excretion studies of THSG in rats following a single intravenous or oral dose. The three metabolites were identified by rapid resolution LC‐MSⁿ. The concentrations of the THSG in rat plasma, bile, urine, feces, or tissue samples were determined by ultra‐performance LC‐MS. The results showed that THSG was rapidly distributed and eliminated from rat plasma. After the intravenous administration, THSG was mainly distributing in the liver, heart, and lung. For the rat, the major distribution tissues after oral administration were heart, kidney, liver, and lung. There was no long‐term storage of THSG in rat tissues. Total recoveries of THSG within 24 h were low (0.1% in bile, 0.007% in urine, and 0.063% in feces) and THSG was excreted mainly in the forms of metabolites, which may resulted from biotransformation in the liver.     

Metabolic profiles of dioscin in rats revealed by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Dioscin (DIS), one of the most abundant bioactive steroidal saponins in Dioscorea sp., is used as a complementary medicine to treat coronary disease and angina pectoris in China. Although the pharmacological activities and pharmacokinetics of DIS have been well demonstrated, information regarding the final metabolic fates is very limited. This study investigated the in vivo metabolic profiles of DIS after oral administration by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry method. The structures of the metabolites were identified and tentatively characterized by means of comparing the molecular mass, retention time and fragmentation pattern of the analytes with those of the parent compound. A total of eight metabolites, including seven phase I and one phase II metabolites, were detected and tentatively identified for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. In addition, a possible metabolic pathway on the biotransformation of DIS in vivo was proposed. This study provides valuable and new information on the metabolism of DIS, which will be helpful for further understanding its mechanism of action. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of bio‐active metabolites of gentiopicroside by UPLC/Q‐TOF MS and NMR

Gentiopicroside (GPS), the main bioactive component in Gentiana scabra Bge., has attracted our attention owing to its high bioactivity, especially the treatment of hepatobiliary disorders. The aglycone form of GPS, a typical secoiridoid glycoside, is considered to be more readily absorbed than its parent drug. This study aimed to identify and characterize the metabolites after GPS incubated with β‐glucosidase in buffer solution at 37°C. Samples of biotransformed solution were collected and analyzed by ultraperformance liquid chromatography (UPLC)/quadrupole–time‐of‐flight mass spectrometry (Q‐TOF MS). A total of four metabolites were detected: two were isolated and elucidated by preparative‐HPLC and NMR techniques, and one of those four is reported for the first time. The mass spectral fragmentation pattern and accurate masses of metabolites were established on the basis of UPLC/Q‐TOF MS analysis. Structure elucidation of metabolites was achieved by comparing their fragmentation pattern with that of the parent drug. A fairly possible metabolic pathway of GPS by β‐glucosidase was proposed. The hepatoprotective activities of metabolites M1 and M2 were investigated and the results showed that their hepatoprotective activities were higher than that of parent drug. Our results provided a meaningful basis for discovering lead compounds from biotransformation related to G. scabra Bge. in traditional Chinese medicine. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of forsythoside A metabolites in rats by a combination of UHPLC‐LTQ‐Orbitrap mass spectrometer with multiple data processing techniques

Forsythoside A (FTA), the main active constituent isolated from Fructus Forsythiae, has various biological functions including anti‐oxidant, anti‐viral and anti‐microbial activities. However, while research on FTA has been mainly focused on the treatment of diseases on a material basis, FTA metabolites in vivo have not been comprehensively evaluated. Here, a rapid and sensitive method using a UHPLC‐LTQ‐Orbitrap mass spectrometer with multiple data processing techniques including high‐resolution extracted ion chromatograms, multiple mass defect filters and diagnostic product ions was developed for the screening and identification of FTA metabolites in rats. As the result, a total of 43 metabolites were identified in biological samples including 42 metabolites in urine, 22 metabolites in plasma and 15 metabolites in feces. These results demonstrated that FTA underwent a series of in vivo metabolic reactions including methylation, dimethylation, sulfation, glucuronidation, diglucuronidation, cysteine conjugation and their composite reactions. The research enhanced our understanding of FTA metabolism and built a foundation for further toxicity and safety studies.     

Identification of the metabolites of anti‐inflammatory compound clematichinenoside AR in rat intestinal microflora

Clematichinenoside AR (C‐AR), a pentacyclic triterpenoid saponin with anti‐inflammatory and anti‐rheumatoid activities, is the main active component of the traditional Chinese medicine Clematidis Radix et Rhizoma. However, its poor oral absorption indicated that not only the parent compound C‐AR itself, but also its metabolites could be responsible for the pharmacological effects in rats. The present study aimed to investigate the metabolism of C‐AR in rat intestinal microflora, where C‐AR was extensively metabolized. C‐AR was incubated with the content of the large intestine. The culture solution was collected at different time points and analyzed for the metabolites of C‐AR. Eight metabolites were identified by liquid chromatography/quadrupole time‐of‐flight mass spectrometry. M1, M2 and M5 were the major metabolites. In addition, it was proposed that deglycosylation was the only pathway contributing to the biotransformation of C‐AR in rat intestinal microflora. Copyright © 2013 John Wiley & Sons, Ltd.     

Ring‐opening polymerization of benzylated 1,6‐anhydro‐β‐D‐lactose and specific biological activities of sulfated (1→6)‐α‐D‐lactopyranans

A new anhydro disaccharide monomer, 1,6‐anhydro‐2,3‐di‐o‐benzyl‐4‐o‐(2′,3′,4′,6′‐tetra‐o‐benzyl‐β‐D ‐galactopyranosyl)‐β‐D ‐glucopyranose (benzylated 1,6‐anhydro lactose (LSHBE)), was synthesized from D ‐lactose to investigate the polymerizability and biological activities of the resulting branched polysaccharides. The ring‐opening polymerization of LSHBE was carried out with phosphorus pentafluoride as a catalyst under high vacuum to give a stereoregular benzylated (1 → 6)‐α‐D ‐lactopyranan. The molecular weights of poly(LSHBE)s increased with an increase in the amount of CH₂Cl₂ solvent, and polymerization temperatures were affected in both molecular weights and yields of the polymers. The copolymerization of LSHBE with benzylated 1,6‐anhydro‐β‐D ‐glucopyranose (LGTBE) gave the corresponding copolysacchrides having different proportions of lactose and glucose units in good yields. After debenzylation to recover hydroxyl groups and then sulfation, sulfated homopoly(lactose)s and copoly(lactose and glucose)s were obtained. Sulfated homopoly(lactose)s had moderate anti‐HIV (EC₅₀ = 5.9 and 1.3 μg/mL) and blood anticoagulant activities (AA = 18 and 13 unit/mg), respectively. Sulfated copoly(lactose and glucose) having 15 mol % lactose units gave high anti‐HIV and blood anticoagulant activities of 0.3 μg/mL and 54 unit/mg, respectively. These biological results suggest that the distance between branched units on the main chain plays an important role in the anti‐HIV and blood anticoagulant activities. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 913–924, 2009     

Chemical UPLC‐ESI‐MS/MS profiling of aconitum alkaloids and their metabolites in rat plasma and urine after oral administration of Aconitum carmichaelii Debx. Root extract

In this paper, an ultra high performance liquid chromatography tandem mass spectrometric (UPLC‐ESI‐MS/MS) method in positive ion mode was established to systematically identify and to compare the major aconitum alkaloids and their metabolites in rat plasma and urine after oral administration of Fuzi extract. A total twenty‐nine components including twenty‐five C19‐diterpenoid alkaloids and four C20‐diterpenoid alkaloids were identified in Fuzi extract. Thirteen of the parent components and five metabolites were detected in rat plasma and sixteen parent compounds and six metabolites in urine. These parent components found in rat plasma and urine were mainly C19‐diterpenoid alkaloids. All of the metabolites in vivo were demethylated metabolites (phase I metabolites), which suggested that demethylation was the major metabolic pathway of aconitum alkaloids in vivo. A comparison of the parent components in rat plasma and urine revealed that 3‐deoxyacontine was found in plasma but not in urine, while kalacolidine, senbusine and 16‐β‐hydroxycardiopetaline existed in urine but not in plasma, which indicated that most alkaloids components were disposed and excreted in prototype form. This research provides some important information for further metabolic investigations of Fuzi in vivo.     

Biotransformation and metabolic profile of caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐cymaropyranoside with human intestinal microflora by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In our previous studies, caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐ cymaropyranoside (CDMC) was for the first time isolated from Cynanchum auriculatum Royle ex Wightand and was reported to possess a wide range of biological activities. However, the routes and metabolites of CDMC produced by intestinal bacteria are not well understood. In this study, ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) technique combined with Metabolynx^TMsoftware was applied to analyze metabolites of CDMC by human intestinal bacteria. The incubated samples collected for 48 h in an anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC‐Q‐TOF‐MS within 12 min. Eight metabolites were identified based on MS and MS/MS data. The results indicated that hydrolysis, hydrogenation, demethylation and hydroxylation were the major metabolic pathways of CDMC in vitro. Seven strains of bacteria including Bacillus sp. 46, Enterococcus sp. 30 and sp. 45, Escherichia sp. 49A, sp. 64, sp. 68 and sp. 75 were further identified using 16S rRNA gene sequencing owing to their relatively strong metabolic capacity toward CDMC. The present study provides important information about metabolic routes of CDMC and the roles of different intestinal bacteria in the metabolism of CDMC. Moreover, those metabolites might influence the biological effect of CDMC in vivo, which affects the clinical effects of this medicinal plant. Copyright © 2015 John Wiley & Sons, Ltd.     

Study on the pharmacokinetics and metabolism of costunolide and dehydrocostus lactone in rats by HPLC‐UV and UPLC‐Q‐TOF/MS

A method based on high‐performance liquid chromatography coupled with ultraviolet detection was developed for studying the pharmacokinetics of costunolide (Cos) and dehydrocostus lactone (Dehy) in rats after intravenous (i.v.) administration. Following i.v. administration, the maximum plasma concentrations of Cos and Dehy were observed to be 12.29 ± 1.47 and 5.79 ± 0.13 µg/mL, respectively. The bioavailability of Cos was larger than that of Dehy; however, the clearance and the volume of distribution of Dehy were much larger than those of Cos. An ultraperformance liquid chromatography/quadrupole time‐of‐flight mass spectrometry system with automated MS^E (E represents collision energy) data analysis software (MetaboLynx^TM) was used to analyze and identify the metabolites of Cos and Dehy in vivo. Four metabolites of Cos and six metabolites of Dehy were discovered from the plasma, urine and feces of rats. The main metabolic pathway of Cos was phase II biotransformation, but the main metabolic pathways of Dehy was phase ? biotransformation. Two sequential desaturations and N‐acetylcysteine conjugation were the common metabolic pathways of Cos and Dehy in rats. This information may be useful for the further development of the two drug candidates. Copyright © 2014 John Wiley & Sons, Ltd.     

Design,Synthesis and Biological Evaluation of Novel N‐(4‐([2,2′:5′,2′′‐Terthiophen]‐5‐yl)‐2‐methylbut‐3‐yn‐2‐yl) Benzamide Derivatives

On the basis of the principle of combination of active groups, a series of novel N‐(4‐([2,2′:5′,2′′‐terthiophen]‐5‐yl)‐2‐methylbut‐3‐yn‐2‐yl) benzamide derivatives were designed, synthesized and systematically evaluated for their antiviral activity against tobacco mosaic virus (TMV). The bioassay results showed that most of these compounds displayed good anti‐TMV activity, and some of them exhibited higher antiviral activity than commercial Ningnanmycin. Especially, compound 8e with excellent anti‐TMV activity (inactivation activity, 92.3%/500 µg·mL^?1; curative activity, 85.7%/500 µg·mL^?1 and protection activity, 64.7%/500 µg·mL^?1) emerged as a potential inhibitor of plant virus TMV. Quantitative structure‐activity relationship studies proved that the van der Waals volume (V) and electronic parameter (∑(∑σ_o+σ_p) and ∑σ_m) for the substituent R¹ were very important for antiviral activities in this class of compounds.     

Two‐injection workflow for a liquid chromatography/LTQ‐Orbitrap system to complete in vivo biotransformation characterization: demonstration with buspirone metabolite identification

The relatively high background matrix in in vivo samples typically poses difficulties in drug metabolite identification, and causes repeated analytical runs on unit resolution liquid chromatography/mass spectrometry (LC/MS) systems before the completion of biotransformation characterization. Ballpark parameter settings for the LTQ‐Orbitrap are reported herein that enable complete in vivo metabolite identification within two HPLC/MS injections on the hybrid LTQ‐Orbitrap data collection system. By setting the FT survey full scan at 60K resolution to trigger five dependent LTQ MS² scans, and proper parameters of Repeat Duration, Exclusion Duration and Repeat Count for the first run (exploratory), the Orbitrap achieved the optimal parallel data acquisition capability and collected maximum number of product ion scans. Biotransformation knowledge based prediction played the key role in exact mass ion extraction and multiple mass defect filtration when the initial data was processed. Meanwhile, product ion extraction and neutral loss extraction of the initial dependent data provided additional bonus in identifying metabolites. With updated parent mass list and the data‐dependent setting to let only the ions on the parent mass list trigger dependent scans, the second run (confirmatory) ensures that all precursor ions of identified metabolites trigger not only dependent product ion scans, but also at or close to the highest concentration of the eluted metabolite peaks. This workflow has been developed for metabolite identification of in vivo or ADME studies, of which the samples typically contain a high level of complex matrix. However, due to the proprietary nature of the in vivo studies, this workflow is presented herein with in vitro buspirone sample incubated with human liver microsomes (HLM). The major HLM‐mediated biotransformation on buspirone was identified as oxidation or hydroxylation since five mono‐ (+16 Da), seven di‐ (+32 Da) and at least three tri‐oxygenated (+48 Da) metabolites were identified. Besides the metabolites 1‐pyrimidinylpiperazine (1‐PP) and hydroxylated 1‐PP that formed by N‐dealkylation, a new metabolite M308 was identified as the result of a second N‐dealkylation of the pyrimidine unit. Two new metabolites containing the 8‐butyl‐8‐azaspiro[4,5]decane‐7,9‐dione partial structure, M240 and M254, were also identified that were formed apparently due to the first N‐dealkylation of the 1‐PP moiety. Copyright © 2009 John Wiley & Sons, Ltd.     

Design,Synthesis and Biological Evaluation of Isothiazole Based 1,2,4‐Trizaole Derivatives

A series of novel 3,4‐dichloroisothiazole based 1,2,4‐triazole derivatives were rationally designed and synthesized. Their structures were confirmed by ¹H NMR, ¹³C NMR, HRMS or elemental analysis; the typical crystal structure was determined by X‐ray diffraction for validation. All target compounds were evaluated for their in vitro fungicidal and in vivo anti‐TMV activities. The bioassay results indicated that compound 6b , namely 1‐(3,4‐dichloroisothiazol‐5‐yl)‐1‐(4‐fluorophenyl)‐2‐(1H‐1,2,4‐triazol‐1‐yl)ethanol, exhibited excellent growth inhibition against B. cinerea, C. arachidicola and P. piricola with median effective concentrations (EC₅₀) of 6.98, 2.73 and 3.07 μg/mL, respectively, and good in vivo anti‐TMV activity of over 60% of inactivation and induction activity at 100 μg/mL. These data demonstrate that compound 6b is both a fungicide and an anti‐TMV lead, deserving further studies.     

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.     

Identification of metabolites of gardenin A in rats by combination of high‐performance liquid chromatography with linear ion trap–Orbitrap mass spectrometer based on multiple data processing techniques

Gardenin A is one of the less abundant hydroxylated polymethoxyflavonoids (OH‐PMFs) in nature, and has many potential significant health benefits. In the present study, an efficient strategy was established using high‐performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometer to profile the in vivo metabolic fate of gardenin A in rat plasma and various tissues. First, an online LC‐MSⁿ data acquisition method was developed to trace all the probable metabolites. Second, a combination of offline data processing methods including extracted ion chromatography and multiple mass defect filters was employed to screen the common and uncommon metabolites from the background noise and endogenous components. Finally, structures of the metabolites were elucidated based on an accurate mass measurement, the diagnostic product ions of PMFs, and relevant drug biotransformation knowledge. Based on the proposed strategy, a total of 26 metabolites were observed and characterized. The results indicate that some biotransformations, such as methylation, demethoxylation, demethylation, glucuronide conjugation, sulfate conjugation and their composite reactions, have been discovered for OH‐PMFs. Moreover, some diagnostic biotransformation pathways are summarized. Overall, this study gives us a first insight into the in vivo metabolism of gardenin A. The study also provides a practical strategy for rapidly screening and identifying metabolites, which can be widely applied for the other biotransformations. Copyright © 2014 John Wiley & Sons, Ltd.     

Rapid identification of phase I and II metabolites of artemisinin antimalarials using LTQ‐Orbitrap hybrid mass spectrometer in combination with online hydrogen/deuterium exchange technique

Artemisinin drugs have become the first‐line antimalarials in areas of multi‐drug resistance. However, monotherapy with artemisinin drugs results in comparatively high recrudescence rates. Autoinduction of CYP‐mediated metabolism, resulting in reduced exposure, has been supposed to be the underlying mechanism. To better understand the autoinduction of artemisinin drugs, we evaluated the biotransformation of artemisinin, also known as Qing‐hao‐su (QHS), and its active derivative dihydroartemisinin (DHA) in vitro and in vivo, using LTQ‐Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange high‐resolution (HR)‐LC/MS (mass spectrometry) for rapid structural characterization. The LC separation was improved allowing the separation of QHS parent drugs and their metabolites from their diastereomers. Thirteen phase I metabolites of QHS have been identified in liver microsomal incubates, rat urine, bile and plasma, including six deoxyhydroxylated metabolites, five hydroxylated metabolites, one dihydroxylated metabolite and deoxyartemisinin. Twelve phase II metabolites of QHS were detected in rat bile, urine and plasma. DHA underwent similar metabolic pathways, and 13 phase I metabolites and 3 phase II metabolites were detected. Accurate mass data were obtained in both full‐scan and MS/MS mode to support assignments of metabolite structures. Online H/D exchange LC‐HR/MS experiments provided additional evidence in differentiating deoxydihydroxylated metabolites from mono‐hydroxylated metabolites. The results showed that the main phase I metabolites of artemisinin drugs are hydroxylated and deoxyl products, and they will undergo subsequent phase II glucuronidation processes. This study also demonstrated the effectiveness of online H/D exchange LC‐HR/MSⁿ technique in rapid identification of drug metabolites. Copyright © 2011 John Wiley & Sons, Ltd.     

An Efficient Synthesis of Spiro‐oxindole Derivatives by Three‐Component Reactions in Water

An efficient synthesis of (3S)‐1,1′,2,2′,3′,4′,6′,7′‐octahydro‐9′‐nitro‐2,6′‐dioxospiro[3H‐indole‐3,8′‐[8H]pyrido[1,2‐a]pyrimidine]‐7′‐carbonitrile is achieved via a three‐component reaction of isatin, ethyl cyanoacetate, and 1,2,3,4,5,6‐hexahydro‐2‐(nitromethylidene)pyrimidine. The present method does not involve any hazardous organic solvents or catalysts. Also the synthesis of ethyl 6′‐amino‐1,1′,2,2′,3′,4′‐hexahydro‐9′‐nitro‐2‐oxospiro[3H‐indole‐3,8′‐[8H]pyrido[1,2‐a]pyrimidine]‐7′‐carboxylates in high yields, at reflux, using a catalytic amount of piperidine, is described. The structures were confirmed spectroscopically (IR, ¹H‐ and ¹³C‐NMR, and EI‐MS data) and by elemental analyses. A plausible mechanism for this reaction is proposed (Scheme 2).     

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.     

Synthesis and Molecular Structure of New S‐Nucleosides of 5‐(4‐Pyridyl)‐4‐Aryl‐4H‐1,2,4‐Triazole‐3‐Thiols

The synthesis of some new S‐nucleosides of 5‐(4‐pyridyl)‐4‐aryl‐4H‐1,2,4‐triazole‐3‐thiols ( 4a‐n ) is described. Direct glycosylation of ( 4a‐n ) with tetra‐O‐acetyl‐α‐D‐glucopyranosyl bromide in the presence of potassium hydroxide followed by deacetylation using dry ammonia in methanol gave the corresponding 3‐S‐(ñ‐D‐glucopyranosyl)‐5‐(4‐pyridyl)‐4‐aryl‐4H‐1,2,4‐triazoles ( 6a‐n ) in good yields. All the compounds were fully characterized by means of ¹HNMR, ¹³C NMR spectra and elemental analyses. To assist in the interpretation of the spectroscopic data, the crystal structure of 3‐S‐(2′,3′,4′,6′‐tetra‐O‐acetyl‐β‐D‐glucopyranosyl)‐5‐(4‐pyridyl)‐4‐phenyl‐4H‐1,2,4‐triazole ( 5a ) was determined by X‐ray diffraction.