Identification and quantification of synthetic cannabinoids in 'spice-like' herbal mixtures: Update of the German situation in 2018.

In June 2018, 15 'Spice-like' herbal products from German language internet shops were analyzed. In total, three different synthetic cannabinoids (SCs) were identified by gas chromatography-mass spectrometry (GC-MS). Two of the active ingredients were identified as the recently described 5F-ADB and Cumyl-PeGaClone. The third compound was identified as the so far unknown SC 5F-Cumyl-PeGaClone. 5F-Cumyl-PeGaClone was subject to an in-depth characterization by nuclear magnetic resonance spectroscopy (NMR), electron ionization mass spectrometry (EI-MS), electrospray ionization tandem mass spectrometry (ESI-MS/MS), infrared and uItraviolet-visible spectroscopy (IR and UV/Vis). In addition, all SCs in all products were quantified by a GC-MS method using JWH-018 as internal standard and corresponding response factors. While Cumyl-PeGaClone and 5F-ADB were detected in one, respectively two products, the newly identified 5F-Cumyl-PeGaClone was detected as the only active ingredient in the remaining twelve products. The SC content ranged from 14.7 to 76.2mg/g (average: 32.1mg/g).

Determination of cross-reactivity of poly- and monoclonal antibodies for synthetic cannabinoids by direct SPR and ELISA.

One of the main reasons for the rise in popularity of synthetic cannabinoids (SCs) is their ability to remain unrecognized in conventional drug screenings. Due to their structural diversity, caused by the constant introduction of new substances to circumvent legal regulation, antibodies with a wide range of cross-reactivity are necessary for the establishment of a reliable immunological based drug test. Therefore, high-quality binding data are needed to select promising antibody candidates for further development. In this study, we carried out a direct surface plasmon resonance (SPR) method and evaluated its suitability for the characterization of antibody-SC interactions. The cross-reactivity of 22 SCs with three polyclonal antibodies, raised against JWH-018 haptens with different attachment positions of the linker, and two commercial available monoclonal antibodies were determined. These results were compared with the commonly used competitive enzyme-linked immunosorbent assay (ELISA). It could be demonstrated, that direct SPR and competitive ELISA show comparable specificity results for the majority of the measured compounds. However, the reduced manual labor, the real-time analysis and the high information content about the binding events of SPR compared to ELISA, showed that SPR is a valuable tool during the development of antibodies against synthetic cannabinoids, currently the largest group of new psychoactive substances.

Identification and quantification of synthetic cannabinoids in 'spice-like' herbal mixtures: Update of the German situation in early 2017.

In February 2017, eleven "Spice-like" products (14 individual packages including replicates) from German language internet shops were analyzed. In total, three different synthetic cannabinoids (SCs) were identified by gas-chromatography-mass spectrometry (GC-MS), namely MDMB-CHMICA and two, so far only partially described compounds, 5F-Cumyl-P7AICA and Cumyl-PeGACLONE. All analyzed products contained only one synthetic cannabinoid as active ingredient. 5F-Cumyl-P7AICA and Cumyl-PeGACLONE were subject to an in-depth characterization by nuclear magnetic resonance spectroscopy (NMR), electron ionization mass spectrometry (EI-MS), electrospray ionization tandem mass spectrometry (ESI-MS/MS), infrared and ultraviolet-visible spectroscopy (IR and UV/Vis). Cumyl-PeGACLONE shows a rather unexpected structure compared to conventional SCs of the past. Hence a global minima calculation was conducted to demonstrate structural similarity of Cumyl-PeGACLONE to JWH-018, a classical SC. In addition, all SCs were quantified by a GC-MS method using JWH-018 as internal standard and corresponding response factors. While MDMB-CHMICA was detected in six out of 14 tested products (ranging from 6 to 20mg/g; average 10mg/g), 5F-Cumyl-P7AICA and Cumyl-PeGACLONE were detected in three (109-153mg/g; average 131mg/g) and five products (15-74mg/g; average 39mg/g), respectively.

Identification and quantification of synthetic cannabinoids in "spice-like" herbal mixtures: Update of the German situation for the spring of 2016.

In February 2016, nine "spice-like" products from German language internet shops were analyzed. In total, eight different synthetic cannabinoids were identified by gas chromatography-mass spectrometry (GC-MS), namely THJ-018, THJ-2201, MAB-CHMINACA, 5F-ADB, 5Cl-AKB48 (syn.: 5C-AKB48), 4-pentenyl-AKB48, MDMB-CHMICA and 5F-AB-PINACA. For the majority of products only one synthetic cannabinoid was identified as the active ingredient, while two products contained 2 and 5 compounds, respectively. For some of the identified cannabinoids (MAB-CHMINACA, 5Cl-AKB48 and 4-pentenyl-AKB48) no or only insufficient physico-chemical data were available in literature. To our knowledge 5Cl-AKB48 and 4-pentenyl-AKB48 were found for the first time in commercially available products, hence an in-depth characterization of these compounds by NMR, EI-MS, ESI-MS/MS, IR- and UV spectroscopy was conducted. In addition, all synthetic cannabinoids were quantified by a GC-MS method using JWH-018 as internal standard and the corresponding response factors to calculate the total amount of all synthetic cannabinoids in the commercial smoking mixtures. The content of synthetic cannabinoids in the investigated products ranged from 23 to 120mg/g (average: 57mg/g), while individual compounds ranged from 1 to 120mg/g.

Molecular Cloning and Characterization of a Xanthone Prenyltransferase from Hypericum calycinum Cell Cultures.

In plants, prenylation of metabolites is widely distributed to generate compounds with efficient defense potential and distinct pharmacological activities profitable to human health. Prenylated compounds are formed by members of the prenyltransferase (PT) superfamily, which catalyze the addition of prenyl moieties to a variety of acceptor molecules. Cell cultures of Hypericum calycinum respond to elicitor treatment with the accumulation of the prenylated xanthone hyperxanthone E. A cDNA encoding a membrane-bound PT (HcPT) was isolated from a subtracted cDNA library and transcript preparations of H. calycinum. An increase in the HcPT transcript level preceded hyperxanthone E accumulation in cell cultures of H. calycinum treated with elicitor. The HcPT cDNA was functionally characterized by expression in baculovirus-infected insect cells. The recombinant enzyme catalyzed biosynthesis of 1,3,6,7-tetrahydroxy-8-prenylxanthone through regiospecific C-8 prenylation of 1,3,6,7-tetrahydroxyxanthone, indicating its involvement in hyperxanthone E formation. The enzymatic product shared significant structural features with the previously reported cholinesterase inhibitor γ-mangostin. Thus, our findings may offer a chance for semisynthesis of new active agents to be involved in the treatment of Alzheimer's disease.

Structural and quantitative analysis of Equisetum alkaloids.

Equisetum palustre L. is known for its toxicity for livestock. Several studies in the past addressed the isolation and identification of the responsible alkaloids. So far, palustrine (1) and N(5)-formylpalustrine (2) are known alkaloids of E. palustre. A HPLC-ESI-MS/MS method in combination with simple sample work-up was developed to identify and quantitate Equisetum alkaloids. Besides the two known alkaloids six related alkaloids were detected in different Equisetum samples. The structure of the alkaloid palustridiene (3) was derived by comprehensive 1D and 2D NMR experiments. N(5)-Acetylpalustrine (4) was also thoroughly characterized by NMR for the first time. The structure of N(5)-formylpalustridiene (5) is proposed based on mass spectrometry results. Twenty-two E. palustre samples were screened by a HPLC-ESI-MS/MS method after development of a simple sample work-up and in most cases the set of all eight alkaloids were detected in all parts of the plant. A high variability of the alkaloid content and distribution was found depending on plant organ, plant origin and season ranging from 88 to 597mg/kg dried weight. However, palustrine (1) and the alkaloid palustridiene (3) always represented the main alkaloids. For the first time, a comprehensive identification, quantitation and distribution of Equisetum alkaloids was achieved.

Preparation, structural properties and thermal isomerization of hex-3-ene-1,5-diyne bridged [2.2]paracyclophanes.

The [2.2]paracyclophane moiety is used as a spacer to connect the ends of a hex-3-ene-1,5-diyne unit, a π-system that on thermolysis usually cycloaromatizes to a benzene ring (Bergman cyclization). For the preparation of the pseudo-geminally-bridged system 9, the diacetylene 3 was chain-extended to the diol 16, which after conversion to the pseudo-geminal dibromide 17 was ring-closed by treatment with LiHMDS/HMPA to the [2.2]paracyclophane enediyne 9. Whereas the McMurry coupling of the pseudo-ortho bisaldehyde 24 resulted in the formation of the hexadienyne-bridged cyclophane 27, the pseudo-ortho-bridged hydrocarbon 11 was obtained by preparing first the diol 28 from 24, converting the latter into the dioxolane 29, which in the last step furnished the olefin 11 by treatment with Tf2 O/EtN(iPr)2 . The authentic Bergman product 10 of the pseudo-gem-bridged hexenediyne 9 was synthesized by a conventional sequence starting from the ethynyl formyl substrate 18. Since the pseudo-ortho-enediyne-bridged hydrocarbon 11 is thermally labile, its benzannelated derivate 34 was prepared. No classical Bergman cyclization reactions could be observed for any of the [2.2]paracyclophane-bridged hexenediynes prepared here. In the pseudo-gem-series the fulvenes 14 and 15 were the only products that could be identified under thermal conditions (McMurry coupling); the benzannelated substrate 34 gave the benzofulvene-bridged cyclophane 36 on photolysis. Bergman cyclizations yielding fulvene derivatives are extremely rare. The mechanism of the cyclization of 9 and 34 is discussed, using compliance constants. The structure assignments of the hydrocarbons synthesized in this study are based on spectroscopic studies as well as X-ray structural analyses for 9, 10, 11, 27, and 34.

Identification and quantification of synthetic cannabinoids in 'spice-like' herbal mixtures: a snapshot of the German situation in the autumn of 2012.

Synthetic compounds mimicking cannabis-like effects are a recent trend. Currently, these so-called synthetic cannabinoids are the largest and fastest growing class of newly appearing designer drugs. Many national authorities are continuously adapting their regulations to keep pace with the permanently changing variety of compounds. We have analyzed eight herbal smoking blends containing synthetic cannabinoids. Altogether, nine compounds could be identified, namely AM-2201, AM-2201-pMe (MAM-2201), AM-1220, AM-1220-azepane, UR-144, XLR-11, JWH-122-pentenyl, AM-2232, and STS-135. Newly appearing compounds were isolated by column chromatography and their structures elucidated by 1D- and 2D-nuclear magnetic resonance (NMR) experiments. In addition, the compounds were investigated by electron ionization-mass spectrometry (EI-MS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to complete the physicochemical dataset. Based on the purified compounds a universal gas chromatography-mass spectrometry (GC-MS) method was developed for the identification and quantification of these compounds in commercial smoking blends. By applying this method, up to five different compounds could be found in such products showing total concentrations from 72 to 303 mg/g smoking blend while individual compounds ranged from 0.4 to 303 mg/g. (1)H NMR spectra of the chiral compounds AM-1220 and its azepane-isomer recorded in the presence of 1 equivalent of (R)-(+)-α-methoxy-α-trifluoromethylphenylacetic acid (MTPA, Mosher's acid) showed them to be racemic mixtures.

Pyrrolizidine alkaloids in the food chain: development, validation, and application of a new HPLC-ESI-MS/MS sum parameter method.

Contamination of food and feed with pyrrolizidine alkaloids is currently discussed as a potential health risk. Here, we report the development of a new HPLC-ESI-MS/MS sum parameter method to quantitate the pyrrolizidine alkaloid content in complex food matrices. The procedure was validated for honey and culinary herbs. Isotopically labeled 7-O-9-O-dibutyroyl-[9,9-(2)H2]-retronecine was synthesized and utilized as an internal standard for validation and quantitation. The total pyrrolizidine alkaloid content of a sample is expressed as a single sum parameter: retronecine equivalents (RE). Ld/Lq for honey was 0.1 µg RE/kg/0.3 µg RE/kg. For culinary herbs, 1.0 µg RE/kg/3.0 µg RE/kg (dry weight, dw) and 0.1 µg RE/kg/0.3 µg RE/kg (fresh weight, fw) were determined, respectively. The new method was applied to analyze 21 herbal convenience products. Fifteen products (71%) were pyrrolizidine alkaloid positive showing pyrrolizidine alkaloid concentrations ranging from 0.9 to 74 µg RE/kg fw.

Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia cell cultures treated with Venturia inaequalis.

Aucuparin is the most widely distributed biphenyl phytoalexin in the rosaceous subtribe Pyrinae, which includes the economically important fruit trees apple and pear. The biphenyl scaffold is formed by biphenyl synthase, which catalyzes biosynthesis of 3,5-dihydroxybiphenyl. Conversion of this precursor to aucuparin (3,5-dimethoxy-4-hydroxybiphenyl) was studied in cell cultures of Sorbus aucuparia after treatment with an elicitor preparation from the scab-causing fungus Venturia inaequalis. The sequence of the biosynthetic steps detected was O-methylation - 4-hydroxylation - O-methylation. The two alkylation reactions were catalyzed by distinct methyltransferases, which differed in pH and temperature optima as well as stability. Biphenyl 4-hydroxylase was a microsomal cytochrome P450 monooxygenase, whose activity was appreciably decreased by the addition of established P450 inhibitors. When fed to V. inaequalis-treated S. aucuparia cell cultures, radioactively labeled 3,5-dihydroxybiphenyl was not only incorporated into aucuparin but also into the dibenzofuran eriobofuran, the accumulation of which paralleled that of aucuparin. However, biphenyl 2'-hydroxylase activity proposed to be involved in dibenzofuran formation was detected in neither microsomes nor cell-free extracts in the presence of NADPH and 2-oxoglutarate, respectively. Nevertheless, a basis for studying biphenyl biosynthesis at the gene level is provided.

In vitro formation of the anthranoid scaffold by cell-free extracts from yeast-extract-treated Cassia bicapsularis cell cultures.

The anthranoid skeleton is believed to be formed by octaketide synthase (OKS), a member of the type III polyketide synthase (PKS) superfamily. Recombinant OKSs catalyze stepwise condensation of eight acetyl units to form a linear octaketide intermediate which, however, is incorrectly folded and cyclized to give the shunt products SEK4 and SEK4b. Here we report in vitro formation of the anthranoid scaffold by cell-free extracts from yeast-extract-treated Cassia bicapsularis cell cultures. Unlike field- and in vitro-grown shoots which accumulate anthraquinones, cell cultures mainly contained tetrahydroanthracenes, formation of which was increased 2.5-fold by the addition of yeast extract. The elicitor-stimulated accumulation of tetrahydroanthracenes was preceded by an approx. 35-fold increase in OKS activity. Incubation of cell-free extracts from yeast-extract-treated cell cultures with acetyl-CoA and [2-(14)C]malonyl-CoA led to formation of torosachrysone (tetrahydroanthracene) and emodin anthrone, beside two yet unidentified products. No product formation occurred in the absence of acetyl-CoA as starter substrate. To confirm the identities of the enzymatic products, cell-free extracts were incubated with acetyl-CoA and [U-(13)C(3)]malonyl-CoA and (13)C incorporation was analyzed by ESI-MS/MS. Detection of anthranoid biosynthesis in cell-free extracts indicates in vitro cooperation of OKS with a yet unidentified factor or enzyme for octaketide cyclization.

Cinnamate:CoA ligase initiates the biosynthesis of a benzoate-derived xanthone phytoalexin in Hypericum calycinum cell cultures.

Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate:CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg²âº and K⁺ at optimum concentrations of 2.5 and 100 mM, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a carboxyl-terminal type 1 peroxisomal targeting signal made up by the tripeptide Ser-Arg-Leu, which directed an amino-terminal reporter fusion to the peroxisomes. Masking the targeting signal by carboxyl-terminal reporter fusion led to cytoplasmic localization. A phylogenetic tree consisted of two evolutionarily distinct clusters. One cluster was formed by CoA ligases related to benzenoid metabolism, including HcCNL. The other cluster comprised 4-coumarate:CoA ligases from spermatophytes, ferns, and mosses, indicating divergence of the two clades prior to the divergence of the higher plant lineages.

Synthetic cannabinoids in "spice-like" herbal blends: first appearance of JWH-307 and recurrence of JWH-018 on the German market.

Herbal smoking blends, available on the German market were analyzed and several known synthetic cannabinoids were identified (JWH-122 and JWH-018). In addition, we isolated a new active ingredient by silica gel column chromatography and elucidated the structure by nuclear magnetic resonance (NMR) methods. The compound was identified as JWH-307, a synthetic cannabinoid of the phenyl-pyrrole subclass with known in vitro binding affinities for cannabinoid receptors. To date, this is the first appearance of this subclass of cannabimimetics in such products. JWH-307 has been further characterized by gas chromatography accurate mass spectrometry (GC-HRMS), electrospray tandem mass spectrometry (ESI-MS/MS), ultraviolet (UV) and infrared (IR) spectroscopy. JWH-018 was among the first compounds banned by many countries world-wide including Germany. The identification of JWH-018 was striking, since this is the first report where JWH-018 recurred on the German market thus violating existing laws. A generic method was established to quantify synthetic cannabinoids in herbal smoking blends. Quantification was achieved using an isotopically labeled standard (JWH-018-D(3)). JWH-018 was found at a level of 150 mg/g while JWH-122 and JWH-307 occurred as a mixture at a total level of 232 mg/g.

Analysis of synthetic cannabinoids in "spice-like" herbal highs: snapshot of the German market in summer 2011.

In this study, seven commercial "spice-like" products available on the German market were analyzed. They all contained significant amounts of synthetic cannabinoids and had distinctly different compositions of these adulterants. All synthetic cannabinoids were extracted and purified by different chromatographic techniques from the respective product. The structures of all compounds were elucidated by nuclear magnetic resonance spectroscopy and further characterized by mass spectrometry (MS) and ultraviolet and infrared spectroscopy to generate a full data set of each compound. Altogether, eight compounds were identified, and one deuterium-labeled cannabinoid was used as internal standard. Four products contained only one individual compound, while three products contained mixtures of two compounds. Among the eight isolated compounds, six were already known from recent publications (JWH-081, JWH-210, JWH-122, AM2201, RCS-4, and JWH-203), but the published data were not always complete. In addition, two unknown compounds (AM2201-pMe, RCS-4-(N-Me)) were isolated. Overall, compounds from three distinct classes of synthetic cannabinoids could be identified, characterized, and compared. The MS data of the different subclasses allowed the postulation of some general key fragmentations to distinguish between these subclasses. In addition, we established a general method using an isotopically labeled internal standard (JWH-018-D(3)) to quantify synthetic cannabinoids in herbal mixtures. The total content of the synthetic cannabinoids ranged from 77.5 to 202 mg/g, while individual compounds were detected from 19.3 to 202 mg/g in these products. The spectroscopic data for all compounds mentioned here were collected and added en bloc as Electronic supplementary material to this manuscript.

Identification and characterization of JWH-122 used as new ingredient in "Spice-like" herbal incenses.

Recently, several cases across Germany were reported where teenagers were hospitalized showing severe side effects after consumption of a new "Spice-like" herbal incense called "Lava red". The active component of "Lava red", obtained from German internet shops, was isolated by silica gel column chromatography and the structure was elucidated by NMR methods. It is a known N-alkyl-3-(1-naphthoyl)indole (CAS No.: 619294-47-2) JWH-122 which was synthesized recently as model component for in vitro drug testing. The structure is related to compounds that were used two years ago (JWH-018, JWH-073) as synthetic cannabimimetics in similar incense products which are now banned as illegal drugs in Germany and several other European countries. The concentration of JWH-122 was estimated to be 82mg/g product. The isolated compound was further analyzed by different spectroscopic and mass spectrometry techniques to obtain a complete dataset of the physico-chemical properties of the molecule. The data presented here is useful for easy and fast detection of JWH-122 in trace amounts in complex matrices.

Preparation of highly hindered polyenes with tert-butyl groups in internal positions.

The conjugated tetraenes 3 and 4 a-c have been prepared and shown to possess an orthogonal structure. This was not only demonstrated by their spectroscopic properties and X-ray structural analysis of solid representatives (e.g., 4 a-c) but also by the resolution of these chiral compounds by GC and HPLC chromatography using various chiral selector systems. The chemical behavior of the typical tetraene 4 a has been studied using bromination, hydrogenation, epoxidation, and photo equilibration reactions.

Preparation and NMR spectra of four isomeric diformyl[2.2]paracyclophanes (cyclophanes 66).

Four isomeric dialdehydes 4, readily available from cycloaddition of propiolic aldehyde (2) to 1,2,4,5-hexatetraene (1), were separated by chromatography and recrystallization, and were characterized by their spectroscopic data. The individual isomers can now be easily identified from their ¹H NMR spectra even if only one of them is present.

Differential effect of elicitors on biphenyl and dibenzofuran formation in Sorbus aucuparia cell cultures.

The Rosaceous subtribe Pyrinae (formerly subfamily Maloideae) is well-known for its economically important fruit trees, such as apple and pear, and also includes Sorbus aucuparia. Elicitor-treated S. aucuparia cell cultures are used to study the biosynthesis of the Pyrinae-specific phytoalexins, biphenyls and dibenzofurans. Three biphenyls (aucuparin, noraucuparin, 2'-hydroxyaucuparin) and a dibenzofuran (eriobofuran) were isolated and structure elucidated using GC-MS and NMR. A second dibenzofuran of low abundance was tentatively assigned as noreriobofuran. Treatment of S. aucuparia cell cultures with yeast extract induced the formation of aucuparin as the major phytoalexin. In contrast, addition of preparations from the fire blight bacterium, Erwinia amylovora, and the scab-causing fungus, Venturia inaequalis, resulted in accumulation of eriobofuran as the major inducible constituent. Methyl jasmonate was a poor elicitor. The observations are suggestive of a biogenic relationship between biphenyls and dibenzofurans. Elicitor-treated S. aucuparia cell cultures provide an interesting in vitro system for studying biphenyl and dibenzofuran metabolism in the economically valuable Pyrinae.

Singly and doubly twisted [36]annulenes: synthesis and calculations.

A cyclophane with a [36]annulene periphery, including four anthrylene and two phenylene units, was synthesized in a four-step sequence using a McMurry cyclization. Upon crystallization from different solvents, four different conformations were determined by X-ray structure analysis. Two conformations exhibit a double twist (Hückel topology) and two structures include a single twist (Möbius topology). By using DFT calculations a conformation with a triple twist (Möbius) was located. However, our calculations and the NMR spectroscopy data do not provide evidence for aromaticity (for Möbius structures) or antiaromaticity (for Hückel structures).

Reversible intramolecular photocycloaddition of a bis(9-anthrylbutadienyl)paracyclophane--an inverse photochromic system. (Photoactive cyclophanes 5).

The title compound, 4,13-bis[(1E,3E)-4-(9-anthracenyl)buta-1,3-dienyl][2.2]paracyclophane (2), prepared in 35% overall yield from [2.2]paracyclophane, absorbs light at lambda(max) = 400 nm with a tail down to 480 nm. By irradiation into this band, 2 generates a single photoproduct, 4, whose absorption maximum is situated at 306 nm. The starting material is recovered by irradiation at 306 nm or by heating. This 'inverse' photochromic system has a potential for optical information storage, compound 4 being stable in visible light, at ambient temperature.