Syntheses and analytical characterizations of novel (2‐aminopropyl)benzo[b]thiophene (APBT) based stimulants

Two groups of amphetamine‐like drugs with psychostimulant properties that were first developed during the course of scientific studies and later emerged as new psychoactive substances (NPS) are based on the (2‐aminopropyl)indole (API) and (2‐aminopropyl)benzofuran (APB) structural scaffolds. However, sulfur‐based analogs with a benzo[b]thiophene structure (resulting in (2‐aminopropyl)benzo[b]thiophene (APBT) derivatives) have received little attention. In the present investigation, all six racemic APBT positional isomers were synthesized in an effort to understand their structure–activity relationships relative to API‐ and APB‐based drugs. One lesson learned from the NPS phenomenon is that one cannot exclude the appearance of such substances on the market. Therefore, an in‐depth analytical characterization was performed, including various single‐ and tandem mass spectrometry (MS) and ionization platforms coupled to gas chromatography (GC) and liquid chromatography (LC), nuclear magnetic resonance spectroscopy (NMR), and solid phase and GC condensed phase infrared spectroscopy (GC‐sIR). Various derivatizations have also been explored; it was found that all six APBT isomers could be differentiated during GC analysis after derivatization with heptafluorobutyric anhydride and ethyl chloroformate (or heptafluorobutyric anhydride and acetic anhydride) under non‐routine conditions. Discriminating analytical features can also be derived from NMR, GC‐EI/CI‐ single‐ and tandem mass spectrometry, LC (pentafluorophenyl stationary phase), and various infrared spectroscopy approaches (including GC‐sIR). Availability of detailed analytical data obtained from these novel APBT‐type stimulants may be useful to researchers and scientists in cases where forensic and clinical investigations are warranted.     

Identification and characterization of 2,5‐dimethoxy‐3,4‐dimethyl‐β‐phenethylamine (2C‐G) – A new designer drug

This study presents and discusses the mass spectrometric, infrared spectroscopic and nuclear magnetic resonance spectroscopic data of 2,5‐dimethoxy‐3,4‐dimethyl‐β‐phenethylamine (2C‐G), a new designer drug. A powder sample containing 2C‐G was seized in Poland in 2011. The paper focuses on a comparison of the analytical features of 2C‐G and other members of the 2C‐series, in order to assess the possibility of unequivocal identification. The occurrence of intense peak at m/z = 178 and different intensities of the ions at m/z = 165 and 180 in the gas chromatography‐electron impact‐mass spectrometry (GC‐EI/MS) spectrum of 2C‐G made it possible to distinguish it from 2C‐E. Differences in relative intensities of the ions at m/z = 192, 179 and 177 were observed for GC‐EI/MS spectra of TFAA derivatives of 2C‐G and 2C‐E. An identical set of ions was recorded for these substances using the liquid chromatography‐electrospray ionization/quadrupole time of flight mass spectrometry (LC‐ESI/QTOFMS) method in both MS and tandem mass spectrometry (MS/MS) mode, but the distinction was possible based on differences in the ion intensities at m/z = 193.1223 and 178.0988. The Fourier transform infrared (FTIR) spectrum of 2C‐G was significantly different from other members of the 2C‐series, with a characteristic doublets at 993–1014 cm^‐1 and 1099–1124 cm^‐1, and the ratio of bands at higher wavenumbers. Final elucidation of the structure of 2C‐G was carried out by ¹H and ¹³C NMR spectroscopy. The study indicated that the marketing of analogues of controlled substances poses a real analytical challenge for forensic laboratories, and the application of sophisticated methods is often required for unequivocal identification of a new substance. Copyright © 2012 John Wiley & Sons, Ltd.     

Analytical characterization of three hallucinogenic N‐(2‐methoxy)benzyl derivatives of the 2C‐series of phenethylamine drugs

This publication reports analytical properties of three new hallucinogenic substances identified in blotter papers seized from the drug market, namely 25D‐NBOMe [2‐(2,5‐dimethoxy‐4‐methylphenyl)‐N‐(2‐methoxybenzyl)ethanamine], 25E‐NBOMe [2‐(4‐ethyl‐2,5‐dimethoxyphenyl)‐N‐(2‐methoxybenzyl)ethanamine] and 25G‐NBOMe [2‐(2,5‐dimethoxy‐3,4‐dimethylphenyl)‐N‐(2‐methoxybenzyl)ethanamine]. These substances are N‐(2‐methoxy)benzyl derivatives of the 2C‐series of phenethylamine drugs. The applied procedure covered a variety of analytical methods, including gas chromatography with electron impact mass spectrometry (GC‐EI‐MS; without derivatization and after derivatization with trifluoroacetic anhydride (TFAA)), liquid chromatography‐electrospray ionization‐quadrupole time of flight mass spectrometry (LC‐ESI‐QTOF‐MS), Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR), which made it possible to identify the active components unequivocally. The GC‐MS spectra of analyzed compounds were very similar, with dominant ions observed at m/z = 150, 121, and 91. The remaining ions were analogous to those observed for parent substances, namely 2C‐D, 2C‐E, 2C‐G, but their intensities were low. Derivatization allowed determination of molecular masses of the investigated substances. Their exact masses and chemical formulas were confirmed by LC‐QTOF‐MS experiments and the fragmentation patterns of these compounds following ESI were determined. The tandem mass spectrometry (MS/MS) experiments confirmed that the studied substances were N‐(2‐methoxy)benzyl derivatives of the 2C‐series compounds. Final elucidation of the structures was performed by NMR spectroscopy. The substances were also characterized by FTIR spectroscopy to corroborate the identity of the compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Return of the lysergamides. Part II: Analytical and behavioural characterization of N6‐allyl‐6‐norlysergic acid diethylamide (AL‐LAD) and (2’S,4’S)‐lysergic acid 2,4‐dimethylazetidide (LSZ)

Lysergic acid N ,N ‐diethylamide (LSD) is perhaps one of the most intriguing psychoactive substances known and numerous analogs have been explored to varying extents in previous decades. In 2013, N ⁶‐allyl‐6‐norlysergic acid diethylamide (AL‐LAD) and (2’S ,4’S )‐lysergic acid 2,4‐dimethylazetidide (LSZ) appeared on the ‘research chemicals’/new psychoactive substances (NPS) market in both powdered and blotter form. This study reports the analytical characterization of powdered AL‐LAD and LSZ tartrate samples and their semi‐quantitative determination on blotter paper. Included in this study was the use of nuclear magnetic resonance (NMR) spectroscopy, gas chromatography‐mass spectrometry (GC‐MS), low and high mass accuracy electrospray MS(/MS), high performance liquid chromatography diode array detection and GC solid‐state infrared analysis. One feature shared by serotonergic psychedelics, such as LSD, is the ability to mediate behavioural responses via activation of 5‐HT_2A receptors. Both AL‐LAD and LSZ displayed LSD‐like responses in male C57BL/6 J mice when employing the head‐twitch response (HTR) assay. AL‐LAD and LSZ produced nearly identical inverted‐U‐shaped dose‐dependent effects, with the maximal responses occurring at 200 µg/kg. Analysis of the dose responses by nonlinear regression confirmed that LSZ (ED₅₀ = 114.2 nmol/kg) was equipotent to LSD (ED₅₀ = 132.8 nmol/kg) in mice, whereas AL‐LAD was slightly less potent (ED₅₀ = 174.9 nmol/kg). The extent to which a comparison in potency can be translated directly to humans requires further investigation. Chemical and pharmacological data obtained from NPS may assist research communities that are interested in various aspects related to substance use and forensic identification. Copyright © 2016 John Wiley & Sons, Ltd.     

Return of the lysergamides. Part III: Analytical characterization of N6‐ethyl‐6‐norlysergic acid diethylamide (ETH‐LAD) and 1‐propionyl ETH‐LAD (1P–ETH‐LAD)

The psychoactive properties of lysergic acid diethylamide (LSD) have fascinated scientists across disciplines and the exploration of other analogues and derivatives has been motivated by deepening the understanding of ligand‐receptor interactions at the molecular level as well as by the search for new therapeutics. Several LSD congeners have appeared on the new psychoactive substances (NPS) market in the form of blotters or powders. Examples include 1‐propionyl‐LSD (1P–LSD), AL‐LAD, and LSZ. The absence of analytical data for novel compounds is a frequent challenge encountered in clinical and toxicological investigations. Two newly emerging lysergamides, namely N⁶‐ethyl‐6‐norlysergic acid diethylamide (ETH‐LAD) and 1P–ETH‐LAD, were characterized by gas chromatography–mass spectrometry (GC–MS), low and high mass accuracy electrospray MS(/MS), GC solid‐state infrared analysis, high performance liquid chromatography diode array detection as well as nuclear magnetic resonance spectroscopy. Limited analytical data for ETH‐LAD were previously available, whereas information about 1P–ETH‐LAD has not previously been encountered in the scientific literature. This study extends the characterization of lysergamides distributed on the NPS market, which will help to make analytical data available to clinicians, toxicologists, and other stakeholders who are likely to encounter these substances. The analysis of a test incubation of 1P–ETH‐LAD with human serum at 37°C by LC single quadrupole MS at various time points (0–6 h, once per hour and one measurement after 24 h) revealed the formation of ETH‐LAD, suggesting that 1P–ETH‐LAD might serve as a pro‐drug. 1P–ETH‐LAD was still detectable in serum after 24 h. Copyright © 2017 John Wiley & Sons, Ltd.     

Identification of the designer steroid Androsta‐3,5‐diene‐7,17‐dione in a dietary supplement

A liquid chromatography‐mass spectrometry (LC–MS) screen for known anabolic‐androgenic steroids in a dietary supplement product marketed for “performance enhancement” detected an unknown compound having steroid‐like spectral characteristics. The compound was isolated using high performance liquid chromatography with ultraviolet detection (HPLC–UV) coupled with an analytical scale fraction collector. After the compound was isolated, it was then characterized using gas chromatography with simultaneous Fourier Transform infrared detection and mass spectrometry (GC–FT–IR–MS), liquid chromatography–high resolution accurate mass–mass spectrometry (LC–HRAM–MS) and nuclear magnetic resonance (NMR). The steroid had an accurate mass of m/z 285.1847 (error?0.57 ppm) for the protonated species [M + H]⁺, corresponding to a molecular formula of C₁₉H₂₄O₂. Based on the GC–FT–IR–MS data, NMR data, and accurate mass, the compound was identified as androsta‐3,5‐diene‐7,17‐dione. Although this is not the first reported identification of this designer steroid in a dietary supplement, the data provided adds information for identification of this compound not previously reported. This compound was subsequently detected in another dietary supplement product, which contained three additional active ingredients.     

Structural characterization and pharmacological evaluation of the new synthetic cannabinoid CUMYL‐PEGACLONE

The number of new psychoactive substances (NPS) that have emerged on the European market has been rapidly growing in recent years, with a particularly high number of new compounds from the group of synthetic cannabinoid receptor agonists. There have been various political efforts to control the trade and the use of NPS worldwide. In Germany, the Act to control the distribution of new psychoactive substances (NpSG) came into force in November 2016. In this new act, two groups of substances were defined, the group “cannabimimetics/synthetic cannabinoids” covering indole, indazole, and benzimidazole core structures, and a second group named “compounds derived from 2‐phenethylamine.” Shortly after, the first retailers of “herbal blends” promoted new products allegedly not violating the German NpSG. We describe the identification and structural elucidation of one of the first synthetic cannabinoids not being covered by the NpSG, 5‐pentyl‐2‐(2‐phenylpropan‐2‐yl)‐2,5‐dihydro‐1H‐pyrido[4,3‐b]indol‐1‐one. For isolation of the substance a flash chromatography separation was applied. The structure elucidation was performed using gas chromatography–mass spectrometry (GC–MS), gas chromatography‐solid state infrared spectroscopy (GC–sIR), liquid chromatography–electrospray ionization–quadrupole time of flight–mass spectrometry (LC–ESI–qToF–MS) and nuclear magnetic resonance (NMR) analysis. Additionally, binding affinity towards the cannabinoid receptors CB₁ and CB₂ and efficacy in a cAMP accumulation assay were measured, showing full agonistic activity and high potency at both receptors. The new compound bears a γ‐carboline core structure circumventing the German NpSG and the generic definitions in other national laws. As a semi‐systematic name for 2‐cumyl‐5‐pe ntyl‐ga mma‐c arbol in‐1‐one CUMYL‐PEGACLONE is suggested.     

Lefetamine‐derived designer drugs N‐ethyl‐1,2‐diphenylethylamine (NEDPA) and N‐iso‐propyl‐1,2‐diphenylethylamine (NPDPA): Metabolism and detectability in rat urine using GC‐MS,LC‐MSn and LC‐HR‐MS/MS

N‐Ethyl‐1,2‐diphenylethylamine (NEDPA) and N‐iso‐propyl‐1,2‐diphenylethylamine (NPDPA) are two designer drugs, which were confiscated in Germany in 2008. Lefetamine (N,N‐dimethyl‐1,2‐diphenylethylamine, also named L‐SPA), the pharmaceutical lead of these designer drugs, is a controlled substance in many countries. The aim of the present work was to study the phase I and phase II metabolism of these drugs in rats and to check for their detectability in urine using the authors’ standard urine screening approaches (SUSA). For the elucidation of the metabolism, rat urine samples were worked up with and without enzymatic cleavage, separated and analyzed by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). According to the identified metabolites, the following metabolic pathways for NEDPA and NPDPA could be proposed: N‐dealkylation, mono‐ and bis‐hydroxylation of the benzyl ring followed by methylation of one of the two hydroxy groups, combinations of these steps, hydroxylation of the phenyl ring after N‐dealkylation, glucuronidation and sulfation of all hydroxylated metabolites. Application of a 0.3 mg/kg BW dose of NEDPA or NPDPA, corresponding to a common lefetamine single dose, could be monitored in rat urine using the authors’ GC‐MS and LC‐MSⁿ SUSA. However, only the metabolites could be detected, namely N‐deethyl‐NEDPA, N‐deethyl‐hydroxy‐NEDPA, hydroxy‐NEDPA, and hydroxy‐methoxy‐NEDPA or N‐de‐iso‐propyl‐NPDPA, N‐de‐iso‐propyl‐hydroxy‐NPDPA, and hydroxy‐NPDPA. Assuming similar kinetics, an intake of these drugs should also be detectable in human urine. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolism of the tryptamine‐derived new psychoactive substances 5‐MeO‐2‐Me‐DALT, 5‐MeO‐2‐Me‐ALCHT,and 5‐MeO‐2‐Me‐DIPT and their detectability in urine studied by GC–MS,LC–MSn,and LC‐HR‐MS/MS

Many N,N‐dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5‐methoxy‐2‐methyl‐N,N‐diallyltryptamine (5‐MeO‐2‐Me‐DALT), 5‐methoxy‐2‐methyl‐N‐allyl‐N‐cyclohexyltryptamine (5‐MeO‐2‐Me‐ALCHT), and 5‐methoxy‐2‐methyl‐N,N‐diisopropyltryptamine (5‐MeO‐2‐Me‐DIPT) using gas chromatography–mass spectrometry (GC–MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC–MSⁿ), and liquid chromatography‐high‐resolution tandem mass spectrometry (LC‐HR‐MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC‐HR‐MS/MS. 5‐MeO‐2‐Me‐DALT (24 phase I and 12 phase II metabolites), 5‐MeO‐2‐Me‐ALCHT (24 phase I and 14 phase II metabolites), and 5‐MeO‐2‐Me‐DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O‐demethylation, hydroxylation, N‐dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O‐demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N‐dealkylation. For SUSAs, GC–MS, LC‐MSⁿ, and LC‐HR‐MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC–MS SUSA, both LC–MS SUSAs were able to detect an intake of 5‐MeO‐2‐Me‐ALCHT and 5‐MeO‐2‐Me‐DIPT via their metabolites following 1 mg/kg BW administrations and 5‐MeO‐2‐Me‐DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.     

GC‐MS and GC‐IRD studies on the six‐ring regioisomeric dimethoxybenzylpiperazines (DMBPs)

Gas chromatography with infrared detection (GC‐IRD) provides direct confirmatory data for the differentiation between the six regioisomeric aromatic ring substituted dimethoxybenzylpiperazines (DMBPs). These regioisomeric substances are resolved by GC and the vapour‐phase infrared spectra clearly differentiate among the six dimethoxybenzyl substitution patterns. The mass spectra for these regioisomeric substances are almost identical. With only the 2,3‐dimethoxy isomer showing one unique major fragment ion at m/z 136. Thus mass spectrometry does not provide for the confirmation of identity of any one of these compounds to the exclusion of the other isomers. Perfluoroacylation of the secondary amine nitrogen for each of the six regioisomers gave mass spectra showing some differences in the relative abundance of fragment ions without the appearance of any unique fragments for specific confirmation of structure. Gas chromatography coupled with time‐of‐flight mass spectrometric detection (GC‐TOF) provided an additional means of confirmation of the elemental composition of the major fragment ions in the mass spectra of these compounds. Copyright © 2013 John Wiley & Sons, Ltd.     

Power of Orbitrap‐based LC‐high resolution‐MS/MS for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on‐spot cleavage in comparison to established LC–MSn or GC–MS procedures

Reliable, sensitive, and comprehensive urine screening procedures by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) with low or high resolution (HR) are of high importance for drug testing, adherence monitoring, or detection of toxic compounds. Besides conventional urine sampling, dried urine spots are of increasing interest. In the present study, the power of LC–HR–MS/MS was investigated for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on‐spot cleavage in comparison to established LC–MSⁿ or GC–MS procedures. Authentic human urine samples (n = 103) were split in 4 parts. One aliquot was prepared by precipitation (UP), one by UP with conjugate cleavage (UglucP), one spot on filter paper cards and prepared by on‐spot cleavage followed by liquid extraction (DUSglucE), and one worked‐up by acid hydrolysis, liquid–liquid extraction, and acetylation for GC–MS analysis. The 3 series of LC–HR–MS/MS results were compared among themselves, to corresponding published LC–MSⁿ data, and to screening results obtained by conventional GC–MS. The reference libraries used for the 3 techniques contained over 4500 spectra of parent compounds and their metabolites. The number of all detected hits (770 drug intakes) was set to 100%. The LC–HR–MS/MS approach detected 80% of the hits after UP, 89% after UglucP, and 77% after DUSglucE, which meant over one‐third more hits in comparison to the corresponding published LC–MSⁿ results with ≤49% detected hits. The GC–MS approach identified 56% of all detected hits. In conclusion, LC–HR–MS/MS provided the best screening results after conjugate cleavage and precipitation.     

Analytical characterization of N,N‐diallyltryptamine (DALT) and 16 ring‐substituted derivatives

Many N ,N ‐dialkylated tryptamines show psychoactive properties in humans and the number of derivatives involved in multidisciplinary areas of research has grown over the last few decades. Whereas some derivatives form the basis of a range of medicinal products, others are predominantly encountered as recreational drugs, and in some cases, the areas of therapeutic and recreational use can overlap. In recent years, 5‐methoxy‐N ,N ‐diallyltryptamine (5‐MeO‐DALT) has appeared as a new psychoactive substance (NPS) and ‘research chemical’ whereas 4‐acetoxy‐DALT and the ring‐unsubstituted DALT have only been detected very recently. Strategies pursued in the authors' laboratories included the preparation and biological evaluation of previously unreported N ,N ‐diallyltryptamines (DALTs). This report describes the analytical characterization of 17 DALTs. Fifteen DALTs were prepared by a microwave‐accelerated Speeter and Anthony procedure following established procedures developed previously in the authors' laboratories. In addition to DALT, the substances included in this study were 2‐phenyl‐, 4‐acetoxy‐, 4‐hydroxy‐, 4,5‐ethylenedioxy‐, 5‐methyl‐, 5‐methoxy‐, 5‐methoxy‐2‐methyl‐, 5‐ethoxy‐, 5‐fluoro‐, 5‐fluoro‐2‐methyl‐, 5‐chloro‐, 5‐bromo‐, 5,6‐methylenedioxy‐, 6‐fluoro‐, 7‐methyl, and 7‐ethyl‐DALT, respectively. The DALTs were characterized by nuclear magnetic resonance spectroscopy (NMR), gas chromatography (GC) quadrupole and ion trap (EI/CI) mass spectrometry (MS), low and high mass accuracy MS/MS, photodiode array detection, and GC solid‐state infrared analysis, respectively. A comprehensive collection of spectral data was obtained that are provided to research communities who face the challenge of encountering newly emerging substances where analytical data are not available. These data are also relevant to researchers who might wish to explore the clinical and non‐clinical uses of these substances. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis,radio‐LC–MS analysis and biodistribution in mice of 99mTc–NIM–BAT

S,S′‐bis‐trityl‐N‐BOC‐1,2‐ethylenedicysteamine (S,S′‐bis‐trityl‐N‐BOC–BAT) was conjugated to 2‐nitroimidazole (NIM) through a propylene spacer in order to provide a precursor for a potential technetium‐99 m labelled hypoxia tracer. For labelling with technetium‐99 m, a two‐step one‐pot procedure was developed consisting of deprotection of the ligand by heating in mild acidic conditions and subsequent exchange labelling in the presence of SnCl₂, tartrate and ^99mTcO. The labelling reaction mixture was analyzed using electrospray radio‐LC–MS and the observed mass spectrum corresponding to the main radiometric peak was in accordance with the predicted structure of oxo–Tc(V)–NIM–BAT. ^99mTc–NIM–BAT was purified using RP–HPLC and its biodistribution was evaluated in normal mice at 10 min and 4 h p.i. ^99mTc–NIM–BAT was cleared from plasma mainly by hepatobiliary excretion. Copyright © 2003 John Wiley & Sons, Ltd.     

Synthesis of [1,3, NH2‐15N3] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine

To facilitate NMR studies and low‐level detection in biological samples by mass spectrometry, [1,3, NH₂‐¹⁵N₃] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine was synthesized from imidazole‐4,5‐dicarboxylic acid in 21 steps. The three ¹⁵N isotopes were introduced during the chemo‐enzymatic preparation of [1,3, NH₂‐¹⁵N₃]‐2′‐deoxyguanosine using an established procedure. The ¹⁵N‐labeled 2′‐deoxyguanosine was converted to a 5′‐phenylthio derivative, which allowed the 8‐5′ covalent bond formation via photochemical homolytic cleavage of the C–SPh bond. SeO₂ oxidation of C‐5′ followed by sodium borohydride reduction and deprotection gave the desired product in good yield. The isotopic purity of the [1,3, NH₂‐¹⁵N₃] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine was in excess of 99.94 atom% based on liquid chromatography–mass spectrometry measurements. Copyright © 2013 John Wiley & Sons, Ltd.     

The chemistry and pharmacology of synthetic cannabinoid SDB‐006 and its regioisomeric fluorinated and methoxylated analogs

Synthetic cannabinoids are the largest and most structurally diverse class of new psychoactive substances, with manufacturers often using isomerism to evade detection and circumvent legal restriction. The regioisomeric methoxy‐ and fluorine‐substituted analogs of SDB‐006 (N‐benzyl‐1‐pentyl‐1H‐indole‐3‐carboxamide) were synthesized and could not be differentiated by gas chromatography–mass spectrometry (GC–MS), but were distinguishable by liquid chromatography–quadrupole time‐of‐flight–MS (LC–QTOF–MS). In a fluorescence‐based plate reader membrane potential assay, SDB‐006 acted as a potent agonist at human cannabinoid receptors (CB₁ EC₅₀ = 19 nM). All methoxy‐ and fluorine‐substituted analogs showed reduced potency compared to SDB‐006, although the 2‐fluorinated analog (EC₅₀ = 166 nM) was comparable to known synthetic cannabinoid RCS‐4 (EC₅₀ = 146 nM). Using biotelemetry in rats, SDB‐006 and RCS‐4 evoked comparable reduction in body temperature (~0.7°C at a dose of 10 mg/kg), suggesting lower potency than the recent synthetic cannabinoid AB‐CHMINACA (>2°C, 3 mg/kg).     

Antiapoptotic effects of dietary antioxidants towards N‐nitrosopiperidine and N‐nitrosodibutylamine‐induced apoptosis in HL‐60 and HepG2 cells

The aim of this work was to determine the effect of vitamin C, diallyl disulfide (DADS) and dipropyl disulfide (DPDS) towards N‐nitrosopiperidine (NPIP) and N‐nitrosodibutylamine (NDBA)‐induced apoptosis in human leukemia (HL‐60) and hepatoma (HepG2) cell lines using the terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling assay. None of the vitamin C (5–50 µm ), DADS and DPDS (1–5 µm ) concentrations selected induced a significant percentage of apoptosis. In simultaneous treatments, vitamin C, DADS and DPDS reduced the apoptosis induced by NPIP and NDBA in HL‐60 and HepG2 cells (around 70% of reduction). We also investigated its scavenging activities towards reactive oxygen species (ROS) produced by NPIP and NDBA using 2',7'‐dichlorodihydrofluorescein diacetate in both cell lines. ROS production induced by both N‐nitrosamine was reduced to control levels by vitamin C (5–50 µm ) in a dose‐dependent manner. However, DADS (5 µm ) increased ROS levels induced by NPIP and NDBA in HL‐60 (40 and 20% increase, respectively) and HepG2 cells (18% increase), whereas DPDS was more efficient scavenger of ROS at the lowest concentration (1 µm ) in both HL‐60 (52 and 25% reduction, respectively) and HepG2 cells (24% reduction). The data demonstrated that the scavenging ability of vitamin C and DPDS could contribute to inhibition of the NPIP‐ and NDBA‐induced apoptosis. However, more than one mechanism, such as inhibition of phase I and/or induction of phase II enzymes, could be implicated in the protective effect of dietary antioxidants towards NPIP‐ and NDBA‐induced apoptosis in HL‐60 and HepG2 cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of As‐ and Hg‐Species in Metal‐Resistant Oral Bacteria,by Imaging ToF‐SIMS

Bacterial strains were isolated from the oral cavity of healthy volunteers and grown in the presence of Hg‐ions (1–10 ppm) or arsenate ions at concentrations of 0.1–1.0%. To elucidate how bacteria take up and transform toxic metals inside the cells, we performed ion imaging and depth profiling with time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). This analysis relies on the use of a pulsed ion beam to ionize surface molecules that can be extracted into a time‐of‐flight mass spectrometer. By combining the pulsed ion beam with another ion beam in direct current (DC) mode, depth profiles are obtained as a result of consecutive removal of surface layers. The spatial resolution of the depth profile along the y‐axis is in the range of a few nanometres, and the spatial resolution along the x‐ and z‐axes is in the μm range. The ToF‐SIMS analysis was performed on crude biofilms of bacteria air‐dried at aluminium foil surfaces, allowing subcellular resolution along the y‐axis. The mercury ions were found transformed to methylmercury preferably in the periplasmic space, and the arsenate ions were found reduced to arsenite inside the cells, close to the cell membrane. The data are discussed in relation to current concepts in bacterial resistance to metals and antibiotics.     

Elucidation of the metabolites of the novel psychoactive substance 4‐methyl‐N‐ethyl‐cathinone (4‐MEC) in human urine and pooled liver microsomes by GC‐MS and LC‐HR‐MS/MS techniques and of its detectability by GC‐MS or LC‐MSn standard screening approaches

4‐methyl‐N‐ethcathinone (4‐MEC), the N‐ethyl homologue of mephedrone, is a novel psychoactive substance of the beta‐keto amphetamine (cathinone) group. The aim of the present work was to study the phase I and phase II metabolism of 4‐MEC in human urine as well as in pooled human liver microsome (pHLM) incubations. The urine samples were worked up with and without enzymatic cleavage, the pHLM incubations by simple deproteinization. The metabolites were separated and identified by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). Based on the metabolites identified in urine and/or pHLM, the following metabolic pathways could be proposed: reduction of the keto group, N‐deethylation, hydroxylation of the 4‐methyl group followed by further oxidation to the corresponding 4‐carboxy metabolite, and combinations of these steps. Glucuronidation could only be observed for the hydroxy metabolite. These pathways were similar to those described for the N‐methyl homologue mephedrone and other related drugs. In pHLM, all phase I metabolites with the exception of the N‐deethyl‐dihydro isomers and the 4‐carboxy‐dihydro metabolite could be confirmed. Glucuronides could not be formed under the applied conditions. Although the taken dose was not clear, an intake of 4‐MEC should be detectable in urine by the GC‐MS and LC‐MSⁿ standard urine screening approaches at least after overdose. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolism of (‐)‐cis‐ and (‐)‐trans‐rose oxide by cytochrome P450 enzymes in human liver microsomes

The in vitro metabolism of (‐)‐cis‐ and (‐)‐trans‐rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9‐oxidized metabolite were determined using gas chromatography‐mass spectrometry (GC‐MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (‐)‐cis‐ and (‐)‐trans‐rose oxide. CYP1A2 also efficiently oxidized (‐)‐cis‐rose oxide at the 9‐position but not (‐)‐trans‐rose oxide. α‐Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti‐CYP2B6 antibody inhibited (‐)‐cis‐rose oxide 9‐hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (‐)‐trans‐rose oxide was suppressed by thioTEPA and anti‐CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (‐)‐9‐hydroxy‐cis‐ and (‐)‐9‐hydroxy‐trans‐rose oxide formations correlated with (S)‐mephenytoin N‐demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (‐)‐cis‐ and (‐)‐trans‐rose oxide in human liver microsomes. Copyright © 2015 John Wiley & Sons, Ltd.     

Gas chromatography−mass spectrometry analysis of non‐hydrolyzed sulfated steroids by degradation product formation

Steroid detection and identification remain key issues in toxicology, drug testing, medical diagnostics, food safety control, and doping control. In this study, we evaluate the capabilities and usefulness of analyzing non‐hydrolyzed sulfated steroids with gas chromatography?mass spectrometry (GC–MS) instead of the conventionally applied liquid chromatography?mass spectrometry (LC–MS) approach. Sulfates of 31 steroids were synthesized and their MS and chromatographic behavior studied by chemical ionization?GC?triple quadrupole MS (CI?GC‐TQMS) and low energy?electron ionization?GC?quadrupole time‐of‐flight?MS (LE?EI?GC?QTOF?MS). The collected data shows that the sulfate group is cleaved off in the injection port of the GC–MS, forming two isomers. In CI, the dominant species (ie, [MH – H₂SO₄]⁺ or [MH – H₄S₂O₈]⁺ for bis‐sulfates) is very abundant due to the limited amount of fragmentation, making it an ideal precursor ion for MS/MS. In LE?EI, [M – H₂SO₄]^.+ and/or [M – H₂SO₄ – CH₃]^.+ are the dominant species in most cases. Based on the common GC–MS behavior of non‐hydrolyzed sulfated steroids, two applications were evaluated and compared with the conventionally applied LC–MS approach; (a) discovery of (new) sulfated steroid metabolites of mesterolone and (b) expanding anabolic androgenic steroid abuse detection windows. GC–MS and LC–MS analysis of non‐hydrolyzed sulfated steroids offered comparable sensitivities, superseding these of GC–MS after hydrolysis. For non‐hydrolyzed sulfated steroids, GC–MS offers a higher structural elucidating power and a more straightforward inclusion in screening methods than LC–MS.