Identification and quantitation of two cannabimimetic phenylacetylindoles JWH-251 and JWH-250, and four cannabimimetic naphthoylindoles JWH-081, JWH-015, JWH-200, and JWH-073 as designer drugs in illegal products

Six cannabimimetic indoles have been identified as adulterants in herbal or chemical products being sold illegally in Japan, with four of the compounds being new as adulterants to our knowledge. The identifications were based on analyses using gas chromatography–mass spectrometry, liquid chromatography–mass spectrometry, high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy. The first two compounds were identified as phenylacetyl indoles JWH-251 (2-(2-methylphenyl)-1-(1-pentyl-1H-indol-3-yl)ethanone; 1) and its demethyl-methoxylated analog JWH-250 (2-(2-methoxyphenyl)-1-(1-pentyl-1H-indol-3-yl)ethanone; 2). Compound 2 was identical to that found as an adulterant in the UK and in Germany in 2009. The third compound was naphthoylindole JWH-081 (1-(4-methoxynaphthalenyl)-(1-pentyl-1H-indol-3-yl)methanone; 3), and the fourth was JWH-073 (1-naphthalenyl(1-butyl-1H-indol-3-yl)methanone; 4), which had been identified as an adulterant in our previous study. Two additional compounds were JWH-015 (1-naphthalenyl(2-methyl-1-propyl-1H-indol-3-yl)methanone; 5) and JWH-200 (1-naphthalenyl(1-(2-(4-morpholinyl)ethyl)-1H-indol-3-yl)methanone; 6). Compounds 1–4 and 6 were reported to be synthetic cannabinoids with selective affinity for cannabinoid CB₁ receptors, while compound 5 was reported to be a selective CB₂ receptor agonist causing immunosuppressive effects without psychotropic affects. One product contained both CB₁ and CB₂ receptor agonists in our collection. Quantitative analyses of the six cannabimimetic compounds in 20 products revealed that there was large variation in concentrations of the detected compounds among products; for herbal cutting products, the total amounts of these cannabinoids ranged from 26 to 100 mg.     

Identification and quantitation of two new naphthoylindole drugs-of-abuse, (1-(5-hydroxypentyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone (AM-2202) and (1-(4-pentenyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone, with other synthetic cannabinoids in unregulated “herbal” products circulated in the Tokyo area

During our continual surveillance of unregulated drugs in May–June 2011, we found two new compounds as adulterants in herbal products obtained at shops in the Tokyo area. These compounds were identified by liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry, accurate mass spectrometry, and nuclear magnetic resonance spectroscopy. The first compound identified was a naphthoylindole (1-(5-hydroxypentyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone (AM-2202, 1), which is a side-chain hydroxyl analogue of JWH-018. The second compound was (1-(4-pentenyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone (2), which is side-chain double bond analogue of JWH-018. This is the first report to identify 1 and 2 in a commercial “herbal” product to our knowledge. For quantitation of the above compounds 1 and 2, and chemical analysis for previously reported compounds (AM-2201, 3; JWH-203, 4; JWH-019, 7; JWH-210, 8; mitragynine, 9), each product was extracted with methanol under ultrasonication to prepare solutions for analysis by liquid chromatography with ultraviolet detection. For the sake of identifying JWH-203 (4) and its positional isomers [JWH-203-3-chloroisomer (5) and 4-chloroisomer (6)] correctly, simultaneous liquid chromatography analysis on fluorocarbon-bonded silica gel column was performed. And a case report of commercially available products containing synthetic cannabinoids 7 and 8, and a natural occurring alkaloid 9, was also shown. Each of 6 commercially circulated products contained compounds 1–4 and 7–9; the amounts of the compounds ranged from 4.1 to 222 mg per pack.     

Identification of a cannabimimetic indole as a designer drug in a herbal product

A cannabimimetic indole has been identified as a new adulterant in a herbal product being sold illegally in Japan for its expected narcotic effect. Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analyses indicated that the product contained two major compounds. One was identified as a cannabinoid analog (1RS,3SR)-3-[4-(1,1-dimethyloctyl)-2-hydroxyphenyl]cyclohexan-1-ol (1) by direct comparison with the authentic compound, which we reported previously. The other compound (2) showed a molecular weight of 341 daltons, and accurate mass spectral measurements showed its elemental composition to be C₂₄H₂₃NO. Both mass and nuclear magnetic resonance spectrometric data revealed that 2 was 1-pentyl-3-(1-naphthoyl)indole [or naphthalen-1-yl-(1-pentylindol-3-yl)methanone] being identical to JWH-018, which was synthesized by Wiley and coworkers in 1998. This compound was reported as a potent cannabinoid receptor agonist possessing a pharmacological cannabimimetic activity.     

Identification and quantitation of JWH-213, a cannabimimetic indole, as a designer drug in a herbal product

In our survey of designer drugs in the Japanese market, a cannabimimetic indole was identified as a new active compound in a herbal product. The structure of this compound was elucidated by liquid chromatography–photodiode array–mass spectrometry (LC–PDA–MS), gas chromatography–mass spectrometry (GC–MS), high-resolution MS, and nuclear magnetic resonance (NMR) analyses. The compound was finally identified as (4-ethyl-1-naphthalenyl)(2-methyl-1-pentyl-1H-indol-3-yl)methanone (JWH-213), an indole-based cannabinoid receptor ligand. To our knowledge, this is the first finding of JWH-213 as a designer drug in a herbal product. The quantitative LC–PDA analysis showed that the JWH-213 content in the product was 252 mg/pack.     

Identification and quantitation of a benzoylindole (2-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone and a naphthoylindole 1-(5-fluoropentyl-1H-indol-3-yl)-(naphthalene-1-yl)methanone (AM-2201) found in illegal products obtained via the Internet and their cannabimimetic effects evaluated by in vitro [35S]GTP��S binding assays

During our careful surveillance of unregulated drugs in January to February 2011, we found two new compounds used as adulterants in herbal products obtained via the Internet. These compounds were identified by liquid chromatography?Cmass spectrometry, gas chromatography-mass spectrometry, accurate mass spectrometry, and nuclear magnetic resonance spectroscopy. The first compound identified was a benzoylindole (2-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone (1), which is a positional isomer of (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone (RCS-4, 4). The second compound was 1-(5-fluoropentyl-1H-indol-3-yl)-(naphthalene-1-yl)methanone (AM-2201, 2). The compound 2 has been reported to be a cannabinoid receptor agonist. Because the cannabimimetic effects of compounds 1 and 4 have not been reported to date, their biological activities were evaluated by measuring the activation of [³⁵S] guanosine-5??-O-(3-thio)-triphosphate binding to guanine nucleotide-binding proteins, together with those of other synthetic cannabimimetic compounds. For quantitation of the above two compounds (1 and 2) and previously identified compounds (AM-694, 3; JWH-122, 5; RCS-4, 4), each product was extracted with methanol under ultrasonication to prepare a sample solution for analysis by liquid chromatography with ultraviolet detection. Each of four commercial products contained some of cannabimimetic indoles 1?C5; their contents ranged from 14.8 to 185 mg per pack.     

Analysis of azepane isomers of AM-2233 and AM-1220, and detection of an inhibitor of fatty acid amide hydrolase [3′-(aminocarbonyl)(1,1′-biphenyl)-3-yl]-cyclohexylcarbamate (URB597) obtained as designer drugs in the Tokyo area

During our careful survey of unregulated drugs from November 2011 to January 2012 in the Tokyo area, we found two new compounds in commercial products. The first was identified as the benzoylindole (2-iodophenyl)[1-(1-methylazepan-3-yl)-1H-indol-3-yl]methanone (2), which is the azepane isomer of AM-2233 (1). Compound 2 was isolated by silica gel column chromatography, and was identified through a combination of liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry, accurate mass spectrometry, and nuclear magnetic resonance spectroscopy. The second compound was identified as [3′-(aminocarbonyl)(1,1′-biphenyl)-3-yl]-cyclohexylcarbamate (URB597, 5) by comparing analytical data with that of the authentic compound. For quantitation of these three compounds, each commercial product was extracted with methanol under ultrasonication to prepare the solution for analysis by liquid chromatography with ultraviolet detection. The occurrence of compounds 1 and 2, and AM-1220 (3) and its azepane isomer (4) in 29 commercial products found in the Tokyo area are also shown in this report.     

Comparison between human liver microsomes and the fungus Cunninghamella elegans for biotransformation of the synthetic cannabinoid JWH-424 having a bromo-naphthyl moiety analysed by high-resolution mass spectrometry

Purpose

JWH-424, (8-bromo-1-naphthyl)(1-pentyl-1H-indol-3-yl)methanone, is a synthetic cannabinoid, which is a brominated analogue of JWH-018, one of the best-known synthetic cannabinoids. Despite the structural similarity to JWH-018, little is known about JWH-424 including its metabolism. The aim of the study was to compare human liver microsomes (HLM) and the fungus Cunninghamella elegans as the metabolism catalysts for JWH-424 to better understand the characteristic actions of the fungus in the synthetic cannabinoid metabolism.

Methods

JWH-424 was incubated with HLM for 1 h and Cunninghamella elegans for up to 72 h. The HLM incubation mixtures were diluted with methanol and fungal incubation mixtures were extracted with dichloromethane and reconstituted in methanol before analyses by liquid chromatography–high-resolution mass spectrometry (LC-HRMS).

Results

HLM incubation resulted in production of ten metabolites through dihydrodiol formation, hydroxylation, and/or ipso substitution of the bromine with a hydroxy group. Fungal incubation led to production of 23 metabolites through carboxylation, dihydrodiol formation, hydroxylation, ketone formation, glucosidation and/or sulfation.

Conclusions

Generally, HLM models give good predictions of human metabolites and structural analogues are metabolised in a similar fashion. However, major hydroxy metabolites produced by HLM were those hydroxylated at naphthalene instead of pentyl moiety, the major site of hydroxylation for JWH-018. Fungal metabolites, on the other hand, had undergone hydroxylation mainly at pentyl moiety. The metabolic disagreement suggests the necessity to verify the human metabolites in authentic urine samples, while H9 and H10 (hydroxynaphthalene), H8 (ipso substitution), F22 (hydroxypentyl), and F17 (dihydroxypentyl) are recommended for monitoring of JWH-424 in urinalysis.

     

Two new synthetic cannabinoids, AM-2201 benzimidazole analog (FUBIMINA) and (4-methylpiperazin-1-yl)(1-pentyl-1H-indol-3-yl)methanone (MEPIRAPIM), and three phenethylamine derivatives, 25H-NBOMe 3,4,5-trimethoxybenzyl analog, 25B-NBOMe, and 2C-N-NBOMe, identified in illegal products

Two new types of synthetic cannabinoids, an AM-2201 benzimidazole analog (FUBIMINA, 1) and (4-methylpiperazin-1-yl)(1-pentyl-1H-indol-3-yl)methanone (MEPIRAPIM, 2), and three newly emerged phenethylamine derivatives, 25B-NBOMe (3), 2C-N-NBOMe (4), and a 25H-NBOMe 3,4,5-trimethoxybenzyl analog (5), were detected in illegal products distributed in Japan. The identification was based on liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS), high-resolution MS, and nuclear magnetic resonance analyses. Different from the representative synthetic cannabinoids, such as JWH-018, which have a naphthoylindole moiety, compounds 1 and 2 were completely new types of synthetic cannabinoids; compound 1 had a benzimidazole group in place of an indole group, and compound 2 had a 4-methylpiperazine group in place of the naphthyl group. Compounds 3 and 4 were N-o-methoxybenzyl derivatives of 2,5-dimethoxyphenethylamines (25-NBOMe series), which had been previously detected in European countries, but have newly emerged in Japan. Compound 5 had an N-trimethoxybenzyl group in place of an N-o-methoxybenzyl group. Data on the chemistry and pharmacology of compounds 1, 2, and 5 have never been reported to our knowledge.     

Identification of two new-type synthetic cannabinoids, N-(1-adamantyl)-1-pentyl-1H-indole-3-carboxamide (APICA) and N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (APINACA), and detection of five synthetic cannabinoids, AM-1220, AM-2233, AM-1241, CB-13 (CRA-13), and AM-1248, as designer drugs in illegal products

Two new-type synthetic cannabinoids, N-(1-adamantyl)-1-pentyl-1H-indole-3-carboxamide (APICA, 1) and N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (APINACA, 2), have been identified as designer drugs in illegal products being sold in Japan. The identification was based on liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high-resolution MS and nuclear magnetic resonance (NMR) analyses. Both mass and NMR spectrometric data revealed that 1 was 1-pentyl-N-tricyclo[3.3.1.1^3,7]dec-1-yl-1H-indole-3-carboxamide, and 2 was 1-pentyl-N-tricyclo[3.3.3.1.^3,7]dec-1-yl)-1H-indazole-3-carboxamide. Although many of the synthetic cannabinoids detected in illegal products, such as JWH-018, have a 3-carbonyl indole moiety, compounds 1 and 2 are a new type of synthetic cannabinoid having an amide and an adamantyl group, and 2 also has an indazole group in place of an indole group. There has been no synthetic, chemical, or biological information about 1 or 2 until now, making this the first report of these cannabimimetic compounds (1 and 2) as designer drugs. In addition, five synthetic cannabinoids, AM-1220, AM-2233, AM-1241, CB-13 (CRA-13), and AM-1248, are also described herein as newly distributed designer drugs in Japan.     

[1-(Tetrahydropyran-4-ylmethyl)-1H-indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)methanone: a new synthetic cannabinoid identified on the drug market

A new synthetic cannabinoid, [1-(tetrahydropyran-4-ylmethyl)-1H-indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)methanone, was identified in several resinous samples seized by law enforcement officers in Poland. Its identification was based on liquid chromatography–electrospray ionization–quadrupole time-of-flight–mass spectrometry, gas chromatography–electron ionization–mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, and Fourier-transform infrared spectroscopy. The reported substance was first developed by Abbott Laboratories and patented under the name “A-834,735”. It is a potent agonist of both CB₁ and CB₂ receptors. Although A-834,735 shows moderate selectivity to CB₂ receptor, it exhibits a CB₁ affinity similar to that of ?⁹-tetrahydrocannabinol. The drug has recently become available in online shops. To our knowledge, this is the first report to disclose a synthetic cannabinoid containing a (tetrahydropyran-4-yl)methyl structure in products seized from the drug market.     

Identification and quantitation of cannabimimetic compound JWH-250 as an adulterant in products obtained via the Internet

During our careful survey of unregulated drugs in Tokyo, a new compound was disclosed as an adulterant in herbal and powder products. This compound was found to have a molecular weight of 335 by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry, and the accurate mass measurement suggested an elementary composition of C₂₂H₂₆NO₂. Using these mass data together with those obtained by nuclear magnetic resonance analysis, the compound was identified as 1-pentyl-3-(2-methoxyphenylacetyl)indole (JWH-250), which had been reported by Huffman and coworkers in 2005. This compound was classified as a phenylacetylindole and a cannabinoid receptor agonist. For quantitation of the compound in herbal and powder products, each product was extracted with methanol under ultrasonication to prepare the solution for analysis by liquid chromatography with ultraviolet detection. The contents of JWH-250 in five products ranged from 77.4 to 165 mg per pack.     

Identification of (1-pentylindol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone and its 5-pentyl fluorinated analog in herbal incense seized for drug trafficking

Four herbal incense products were seized from suspected drug abusers in Korea. The major ingredients in the herbal incense samples were purified, and their structures were elucidated using gas chromatography–electron ionization–mass spectrometry (GC–EI–MS), liquid chromatography–time-of-flight–mass spectrometry (LC–TOF–MS), and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. As a result, ingredients in the herbal incense were identified as (1-pentylindol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone and its 5-pentyl fluorinated analog [1-(5-fluoropentyl)indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)methanone. The former is being sold via the Internet as a "research chemical" named UR-144, and the latter is sold as 5F-UR-144. UR-144 is a selective full agonist of CB₂ cannabinoid receptor, and was first developed by Abbott Laboratories as an analgesic. It exhibits analgesic activity against both neuropathic and inflammatory pain associated mainly with the CB₂ receptor, but shows less psychotropic effects associated with the CB₁ receptor. Fluorination of the N-pentyl side chain of cannabimimetic compounds increases their cannabinoid receptor affinity such as with AM-2201, which shows both increased analgesic and psychotropic effects simultaneously. UR-144 and 5F-UR-144 can be classified as research chemicals based on their analgesic effects, but in practice are abused as psychotropic agents and can cause unexpected toxic effects. Thus, the trade and diversion of these chemicals should be monitored carefully for possible abuse. To our knowledge, this is the first report disclosing cyclopropylcarbonylindoles in herbal products.     

Identification of (1<Emphasis Type="Italic">H</Emphasis>-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone (DP-UR-144) in a herbal drug product that was commercially available in the Tokyo metropolitan area

We encountered during our investigation a case of herbal drug products commercially available in the Tokyo metropolitan area in 2014, in which a small unknown peak was detected, along with the intense peak of FUB-144, by liquid chromatography–ultraviolet detection. The present study was conducted to identify and clarify the pharmacological characteristics of the compound present in this small peak. We isolated a compound using a silica gel column from the peak, which was then identified to have a molecular weight of 241 Da by liquid chromatography–mass spectrometry and gas chromatography–mass spectrometry. The accurate mass measurement suggested an elementary composition of C₁₆H₁₉NO. Using these mass data together with those obtained by the nuclear magnetic resonance analysis, the compound was finally identified as (1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone (despentyl-UR-144; DP-UR-144). In addition, this compound was revealed to have affinities for cannabinoid receptors CB₁ and CB₂ with EC₅₀s of 2.36 × 10^?6 and 2.79 × 10^?8 M, respectively. To our knowledge, there is no information in the scientific literature on structural or pharmacological properties of this chemical. These results suggest that the components present in small amounts can contribute to the effects of a major component in their mother product, if they have sufficient pharmacological activities, and, therefore, even such small amounts of components should be precisely characterized and well evaluated to control illegal and potentially illegal drug products.     

In vitro and in vivo metabolisms of 1-pentyl-3-(4-methyl-1-naphthoyl)indole (JWH-122)

1-Pentyl-3-(4-methyl-1-naphthoyl)indole (JWH-122) is an agonist of the cannabinoid receptors CB₁ and CB₂. In this study, the phase I and phase II metabolisms of JWH-122 were investigated using two models. In vitro studies using incubations of JWH-122 with human liver microsomes were performed to obtain metabolites of the drug at the initial step; 11 classes of metabolites were found and analyzed by liquid chromatography–mass spectrometry (LC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS–MS). Hydroxylation(s) on the naphthalene moiety and/or the indole moiety of the molecule took place as such or in combination with dehydrogenation or cleavage of the N-pentyl side chain. Furthermore, dihydrodiol metabolites were formed probably via epoxide formation on the naphthalene moiety, irrespective of the combination with hydroxylation(s). A metabolite carrying a carboxyl group on the N-pentyl side chain was also detected. As the second step of the study, in vivo experiments using chimeric mice were performed; the mice were orally administered JWH-122, and their urine samples were collected, subjected to enzymatic hydrolysis, and analyzed by LC–MS and LC–MS–MS. The urine samples without hydrolysis were also analyzed for their molecular formulae in the conjugated forms by LC–high resolution MS. The in vivo model using chimeric mice confirmed most metabolite classes and clarified the phase II metabolism of JWH-122. It was concluded that all metabolites formed in vivo were excreted conjugated as glucuronide or sulfate, with conjugation rates above 50 %.     

New cannabimimetic indazole derivatives, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA) and N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA) identified as designer drugs in illegal products

Two new cannabimimetic indazole derivatives, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA, 1) and N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA, 2), have been identified as designer drugs in illegal products. These identifications were based on liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry, high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy. Because there have been neither chemical nor pharmacological data about compound 1 until now, this is the first report of this compound. Compound 2 was reported as a potent cannabinoid CB₁ receptor modulator when synthesized by Pfizer in 2009; but this is the first report of its detection in illegal products.     

Determination of AM-2201 metabolites in urine and comparison with JWH-018 abuse

With respect to the continuous emergence of new synthetic cannabinoids on the market since 2008, evaluation of the metabolism of these compounds and the development of analytical methods for the detection of these drugs including their respective metabolites in biological fluids have become essential. Other than JWH-018 or JWH-073, AM-2201 is one of the frequently identified synthetic cannabinoids in Korea. Recently, in our laboratory, several JWH-018 metabolites have been detected in some urine samples obtained from subjects who were arrested for the possession of herbal mixtures containing only AM-2201 or from those who confessed AM-2201 abuse. In the present study, we identified major urinary metabolites of AM-2201 and several metabolites of JWH-018, i.e., N-5-hydroxylated and carboxylated metabolites from rats administered AM-2201 and found that the metabolic profile in rats was similar to those in human subjects in this study. Analytical results of the urine samples from suspects who had a considerable possibility of AM-2201 or JWH-018 intake were also compared to distinguish between AM-2201 and JWH-018 abuse. The presence of 6-indole hydroxylated metabolites of each drug and N-4-hydroxy metabolite of AM-2201 was found to contribute to the decisive differences in the metabolic patterns of the two drugs. In addition, the concentration ratio of the N-(5-hydroxypentyl) metabolite to the N-(4-hydroxypentyl) metabolite of JWH-018 may be used as a criterion to differentiate between AM-2201 and JWH-018 abuse.     

In vitro metabolism of synthetic cannabinoid AM1220 by human liver microsomes and <Emphasis Type="Italic">Cunninghamella elegans</Emphasis> using liquid chromatography coupled with high resolution mass spectrometry

Purpose

Identifying intake of synthetic cannabinoids generally requires the metabolism data of the drugs so that appropriate metabolite markers can be targeted in urine testing. However, the continuous appearance of new cannabinoids during the last decade has made it difficult to keep up with all the compounds including {1-[(1-methylpiperidin-2-yl)methyl]-1H-indol-3-yl}(naphthalen-1-yl)methanone (AM1220). In this study, metabolism of AM1220 was investigated with human liver microsomes and the fungus Cunninghamella elegans.

Methods

Metabolic stability of AM1220 was analysed by liquid chromatography–tandem mass spectrometry in multiple reaction monitoring mode after 1 µM incubation in human liver microsomes for 30 min. Tentative structure elucidation of metabolites was performed on both human liver microsome and fungal incubation samples using liquid chromatography–high-resolution mass spectrometry.

Results

Half-life of AM1220 was estimated to be 3.7 min, indicating a high clearance drug. Nine metabolites were detected after incubating human liver microsomes while seven were found after incubating Cunninghamella elegans, leading to 11 metabolites in total (five metabolites were common to both systems). Demethylation, dihydrodiol formation, combination of the two, hydroxylation and dihydroxylation were the observed biotransformations.

Conclusions

Three most abundant metabolites in both human liver microsomes and Cunninghamella elegans were desmethyl, dihydrodiol and hydroxy metabolites, despite different isomers of dihydrodiol and hydroxy metabolites in each model. These abundant metabolites can potentially be useful markers in urinalysis for AM1220 intake.

     

Comprehensive analytical and structural characteristics of methyl 3,3-dimethyl-2-(1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido)butanoate (MDMB-4en-PINACA)

Purpose

The purpose of the study was to evaluate a complete analytical and structural characterization of methyl 3,3-dimethyl-2-(1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido)butanoate (MDMB-4en-PINACA), a novel synthetic cannabinoid being the analogue of 5F-ADB.

Methods

The compound was analyzed by gas chromatography–mass spectrometry (GC–MS), high-resolution liquid chromatography–mass spectrometry (LC–MS), X-ray diffraction and spectroscopic methods, such as nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) spectroscopies. To derive MDMB-4en-PINACA molecular geometry and to assign infrared absorption bands, quantum calculations with the employment of density functional theory were also used.

Results

We present a wide range of chromatographic and spectroscopic data supported with theoretical calculations allowing to identify MDMB-4en-PINACA.

Conclusions

To our knowledge, this is the first report presenting a comprehensive analytical and structural characterization of MDMB-4en-PINACA obtained by 1D and 2D NMR, GC–MS, LC–MS(/MS), attenuated total reflection-FTIR spectroscopy, powder X-ray diffraction and quantum chemical calculations. The presented results not only broaden the knowledge about this psychoactive substance but also are useful for forensic and clinical purposes.

     

Regioisomeric differentiation of the alkyl-substituted synthetic cannabinoids JWH-122 and JWH-210 by GC-EI-MS/MS

Synthetic cannabinoids (SCs) are known to have structural or positional isomers. While regulations on synthetic drugs like synthetic cathinones and SCs have been placed worldwide for the ever-growing variety of new designer drugs, laws may not necessarily be applicable to their isomers. Toxicological differences may also exist among isomers for which most new designer drugs are still uninvestigated; thus, isomer differentiation becomes of forensic importance. The aim of this study was to differentiate the regioisomers of alkyl-substituted naphthoylindole-type SCs JWH-122 and JWH-210. Reference standards of the two drugs and their regioisomers were analyzed by gas chromatography–electron ionization-mass spectrometry (GC–EI-MS) first in full scan mode. Isomers that produced identical EI spectra were further analyzed by GC-tandem mass spectrometry (MS/MS) by selecting appropriate precursor ions. For JWH-210, comparison of the product ion spectra and the relative ion intensity ratios obtained from precursor ions at m/z 312 and 183 enabled differentiation between all seven regioisomers. Complete isomeric differentiation by MS/MS analysis was not attainable for JWH-122; however, combining chromatographic results with MS/MS analysis results enabled differentiation for all isomers. Two basic fragmentation pathways were speculated for both SCs; for JWH-210, fragmentation pathway tendencies differed among the isomers, resulting in their distinguishability. Our results demonstrated that the difference between the methyl (JWH-122) and ethyl (JWH-210) group substituents contributed to fragmentation pathway tendency differences and further distinguishability between the regioisomers. Functional group differences, especially their stereochemistries, were indicated to be critical factors in positional isomer differentiation by GC-MS/MS.     

Two new-type cannabimimetic quinolinyl carboxylates, QUPIC and QUCHIC, two new cannabimimetic carboxamide derivatives, ADB-FUBINACA and ADBICA, and five synthetic cannabinoids detected with a thiophene derivative α-PVT and an opioid receptor agonist AH-7921 identified in illegal products

We identified two new-type cannabimimetic quinolinyl carboxylates, quinolin-8-yl 1-pentyl-(1H-indole)-3-carboxylate (QUPIC, 1) and quinolin-8-yl 1-(cyclohexylmethyl)-1H-indole-3-carboxylate (QUCHIC, 2); and two new cannabimimetic carboxamide derivatives, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (ADB-FUBINACA, 3) and N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-pentyl-1H-indole-3-carboxamide (ADBICA, 4), as designer drugs in illegal products. Compound 3 was reported to have a potent affinity for cannabinoid CB₁ receptor by Pfizer in 2009, but this is the first report of its detection in illegal products. No chemical or pharmacological data for compounds 1, 2, and 4 have appeared until now, making this the first report on these compounds. We also detected synthetic cannabinoids, APICA N-(5-fluoropentyl) analog (5), APINACA N-(5-fluoropentyl) analog (6), UR-144 N-(5-chloropentyl) analog (7), JWH-122 N-(5-chloropentyl) analog (8), and AM-2201 4-methoxynaphthyl analog (4-MeO-AM-2201, 9) herein as newly distributed designer drugs in Japan. It is of interest that compounds 1 and 2 were detected with their synthetic component, 8-quinolinol (10). A stimulant thiophene analog, α-pyrrolidinovalerothiophenone (α-PVT, 11), and an opioid receptor agonist, 3,4-dichloro-N-([1-(dimethylamino)cyclohexyl]methyl)benzamide (AH-7921, 12), were also detected as new types of designer drugs coexisting with several synthetic cannabinoids and cathinone derivatives in illegal products.