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.     

In vitro and in vivo pharmacokinetics and metabolism of synthetic cannabinoids CUMYL-PICA and 5F-CUMYL-PICA

CUMYL-PICA [1-pentyl-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide] and 5F-CUMYL-PICA [1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide] are recently identified recreationally used/abused synthetic cannabinoids, but have uncharacterized pharmacokinetic profiles and metabolic processes. This study characterized clearance and metabolism of these compounds by human and rat liver microsomes and hepatocytes, and then compared these parameters with in vivo rat plasma and urine sampling. It also evaluated hypothermia, a characteristic cannabimimetic effect. Incubation of CUMYL-PICA and 5F-CUMYL-PICA with rat and human liver microsomes suggested rapid metabolic clearance, but in vivo metabolism was prolonged, such that parent compounds remained detectable in rat plasma 24 h post-dosing. At 3 mg/kg (intraperitoneally), both compounds produced moderate hypothermic effects. Twenty-eight metabolites were tentatively identified for CUMYL-PICA and, coincidentally, 28 metabolites for 5F-CUMYL-PICA, primarily consisting of phase I oxidative transformations and phase II glucuronidation. The primary metabolic pathways for both compounds resulted in the formation of identical metabolites following terminal hydroxylation or dealkylation of the N-pentyl chain for CUMYL-PICA or of the 5-fluoropentyl chain for 5F-CUMYL-PICA. These data provide evidence that in vivo elimination of CUMYL-PICA, 5F-CUMYL-PICA and other synthetic cannabinoids is delayed compared to in vitro modeling, possibly due to sequestration into adipose tissue. Additionally, the present data underscore the need for careful selection of metabolites as analytical targets to distinguish between closely related synthetic cannabinoids in forensic settings.     

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.     

Evaluation of carboxamide-type synthetic cannabinoids as CB<Subscript>1</Subscript>/CB<Subscript>2</Subscript> receptor agonists: difference between the enantiomers

Recently, carboxamide-type synthetic cannabinoids have been distributed globally as new psychoactive substances (NPS). Some of these compounds possess asymmetric carbon, which is derived from an amide moiety composed of amino acid derivatives (i.e., amides or esters of amino acids). In this study, we synthesized both enantiomers of synthetic cannabinoids, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(2-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA 2-fluorobenzyl isomer), N-(1-amino-1-oxo-3-phenylpropan-2-yl)-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (APP-CHMINACA), ethyl [1-(5-fluoropentyl)-1H-indazole-3-carbonyl]valinate (5F-EMB-PINACA), ethyl [1-(4-fluorobenzyl)-1H-indazole-3-carbonyl]valinate (EMB-FUBINACA), and methyl 2-[1-(4-fluorobenzyl)-1H-indole-3-carboxamido]-3,3-dimethylbutanoate (MDMB-FUBICA), which were reported as NPS found in Europe from 2014 to 2015, to evaluate their activities as CB₁/CB₂ receptor agonists. With the exception of (R) MDMB-FUBICA, all of the tested enantiomers were assumed to be agonists of both CB₁ and CB₂ receptors, and the EC₅₀ values of the (S)-enantiomers for the CB₁ receptors were about five times lower than those of (R)-enantiomers. (R) MDMB-FUBICA was shown to function as an agonist of the CB₂ receptor, but lacks CB₁ receptor activity. To the best of our knowledge, this is the first report to show that the (R)-enantiomers of the carboxamide-type synthetic cannabinoids have the potency to activate CB₁ and CB₂ receptors. The findings presented here shed light on the pharmacological properties of these carboxamide-type synthetic cannabinoids in forensic cases.     

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.     

Sensitive identification and quantitation of parent forms of six synthetic cannabinoids in urine samples of human cadavers by liquid chromatography–tandem mass spectrometry

Human urine samples are easier to obtain than human blood samples due to noninvasiveness. The urine levels of synthetic cannabinoids (SCs) in unchanged forms, however, are usually much lower than their blood and tissue levels and cannot be detected in most cases. Therefore, in the present work a sensitive analytical method was devised for the determination of urine levels of six SCs in unchanged forms such as N-(1-amino-3-methy-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA), N-(1-amino-3-methy-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA), N-[(1S)-1-(1-aminocarbonyl)-2-methyl-propyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (AB-CHMINACA), N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (MAB-CHMINACA), methyl-2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamido]-3-methylbutanoate (5F-AMB) and methyl-2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamido]-3,3-dimethylbutanoate (5F-ADB). These SCs were extracted from urine via liquid–liquid extraction. The identification and quantitation were performed by a relatively new type of an instrument for liquid chromatography–tandem mass spectrometry. The limits of detection were as low as 3–8 pg/mL, and the quantitation range was 10–1000 pg/mL using 400 μL of urine. The urine levels of AB-PINACA and AB-FUBINACA of victim 1 were 23 and 10 pg/mL, those of AB-CHMINACA and 5F-AMB of victim 2 were 239 and 19 pg/mL, and those of MAB-CHMINACA and 5F-ADB of victim 3 were 229 and 19 pg/mL, respectively. To our knowledge, this is the first report dealing with successful analysis of low levels of parent synthetic cannabinoids in authentic human urine specimens.     

Cannabimimetic activities of cumyl carboxamide-type synthetic cannabinoids

1-Pentyl-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide (CUMYL-PINACA) is a carboxamide-type synthetic cannabinoid comprising a cumylamine moiety. Recently, the detection of CUMYL-PINACA and some analogs in illicit drug products has been reported by the European Monitoring Centre for Drugs and Drug Addiction. In this study, we synthesized seven cumyl carboxamide-type synthetic cannabinoids (CUMYL-PINACA, CUMYL-5F-PINACA, CUMYL-PICA, CUMYL-5F-PICA, CUMYL-THPINACA, CUMYL-BICA, and CUMYL-5F-P7AICA) and evaluated their activities as CB₁ and CB₂ receptor agonists. We also showed the analytical characterization of these compounds using gas chromatography–electron ionization-mass spectrometry. All of the evaluated compounds exhibited moderate to strong activities as agonists acting on both CB₁ and CB₂ receptors with EC₅₀ values in the range of 8.1 × 10^?10–7.8 × 10^?7 mol/L for CB₁ and from 2.5 × 10^?10 to 9.1 × 10^?6 mol/L for CB₂. The EC₅₀ data presented will be helpful to understand the effects of these compounds in the forensic cases. Furthermore, other new cumyl carboxamide-type synthetic cannabinoids, which will be potentially distributed in the future, will probably have the activities as agonists acting on both CB₁ and CB₂ receptors.     

Identification of new synthetic cannabinoid analogue APINAC (adamantan-1-yl 1-pentyl-1H-indazole-3-carboxylate) with other synthetic cannabinoid MDMB(N)-Bz-F in illegal products

Two synthetic cannabinoid analogues were detected using high-performance liquid chromatography (HPLC)–diode array detector, and gas chromatography–time-of-flight-mass spectrometry during the inspection of illegal products in an airmail package. The analogues were separated by semi-preparative HPLC, and their structures were determined by performing liquid chromatography–high-resolution-mass spectrometry, infrared analysis, and nuclear magnetic resonance spectroscopy. Compound 1 was MDMB(N)-Bz-F, which has been reported previously. Compound 2 was elucidated as adamantan-1-yl 1-pentyl-1H-indazole-3-carboxylate (APINAC), in which the amide group of APINACA was replaced with an ester group. Because there has been no chemical or pharmacological data about this compound until now, this is the first report of its detection in illegal products.     

Identification of the cannabimimetic AM-1220 and its azepane isomer (N-methylazepan-3-yl)-3-(1-naphthoyl)indole in a research chemical and several herbal mixtures

Recently, a large number of synthetic cannabinoids have been identified in herbal mixtures. Moreover, an even higher number of cannabimimetic compounds are currently distributed as research chemicals on a gram to kilogram scale via several online trading platforms. As this situation leads to a large number of new cannabimimetics and the occurrence of isobaric substances, the analysis of such compounds using mass spectroscopy (MS) involves the risk of incorrect assignments of mass spectra. In certain cases, this leads to considerable analytical challenges. In the majority of cases, these challenges can only be mastered by combining multiple analytical techniques. We purchased a so-called research chemical advertised as the cannabimimetic compound [(N-methylpiperidin-2-yl)methyl]-3-(1-naphthoyl)indole (AM-1220) via an Internet platform. Analysis of the microcrystalline substance using gas chromatography (GC)–MS indicated the presence of pure AM-1220. However, after further purity testing utilizing thin-layer chromatography we were surprised to see an additional spot indicating a mixture of two substances with highly similar physicochemical properties. After isolation, high-resolution mass spectroscopy (HR-MS) revealed an elemental composition of C₂₆H₂₆N₂O for both substances, proving the presence of two isobaric substances. Moreover, GC–MS and LC-HR-MS/MS experiments indicated two naphthoylindoles featuring different heterocyclic substituents at the indole nitrogen. Nuclear magnetic resonance spectroscopy verified the presence of the highly potent cannabimimetic AM-1220 and its azepane isomer. Interestingly, only a few weeks after purchasing the powder we also detected both substances in a similar proportion in several herbal mixtures for the first time.     

Identification and quantitation of two benzoylindoles AM-694 and (4-methoxyphenyl)(1-pentyl-1<Emphasis Type="Italic">H</Emphasis>-indol-3-yl)methanone,and three cannabimimetic naphthoylindoles JWH-210, JWH-122, and JWH-019 as adulterants in illegal products obtained via the Internet

During our careful surveillance of unregulated drugs, we found five new compounds used as adulterants in herbal and drug-like products obtained via the Internet. 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 benzoylindole AM-694, which is 1-[(5-fluoropentyl)-1H-indol-3-yl]-(2-iodophenyl)methanone (1). The second compound was (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone (2), which was also classified as a benzoylindole. The three other compounds were identified as naphthoylindoles JWH-210 (4-ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone; 3), JWH-122 (4-methylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone; 4), and JWH-019 (1-hexyl-3-(naphthalen-1-oyl)indole; 5). All compounds except compound 2 had been reported to be cannabinoid receptor agonists. For quantitation of the five compounds and previously reported compounds, each product was extracted with methanol under ultrasonication to prepare a test solution for analysis by liquid chromatography with ultraviolet detection. Each compound detected in 43 commercial products showed large variation in content ranging from 4.0 to 359 mg per pack.     

Identification of the synthetic cannabinoid <Emphasis Type="Italic">N</Emphasis>-(2-phenyl-propan-2-yl)-1-(4-cyanobutyl)-1<Emphasis Type="Italic">H</Emphasis>-indazole-3-carboxamide (CUMYL-4CN-BINACA) in a herbal mixture product

We were the first to detect N-(2-phenylpropan-2-yl)-1-(4-cyanobutyl)-1H-indazole-3-carboxamide (common name CUMYL-4CN-BINACA) as a new synthetic cannabinoid, on the illegal market in Bursa, Turkey. To elucidate the chemical structure, the dried herbal mixture was extracted with methanol. The extract was purified by column chromatography. Pure compound was analyzed by gas chromatography–mass spectrometry (GC–MS), attenuated total reflection Fourier-transform infrared spectroscopy (FT-IR), and one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The GC–MS, FT-IR and ¹H and ¹³C NMR spectra of the compound coincided well with the reference data. All protons and carbons were assigned by their couplings and correlations observed in ¹H-¹H correlation spectroscopy, ¹H-¹³C heteronuclear multiple bond correlation, and ¹H-¹³C heteronuclear single quantum coherence spectra. On the basis of the spectral data, the compound was identified as CUMYL-4CN-BINACA. Herewith, we report analytical characteristics of CUMYL-4CN-BINACA enabling its (and possible analogues thereof) determination in criminal seizures.     

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.     

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.     

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.

     

Phase I-metabolism studies of the synthetic cannabinoids PX-1 and PX-2 using three different in vitro models

Purpose

Synthetic cannabinoids (SCs), highly metabolized substances, are rarely found unmodified in urine samples. Urine screening relies on SC metabolite detection, requiring metabolism knowledge. Metabolism data can be acquired via in vitro assays, e.g., human hepatocytes, pooled human liver microsomes (pHLM), cytochrome P450 isoforms and a fungal model; or in vivo by screening, e.g., authentic human samples or rat urine. This work describes the comprehensive study of PX-1 and PX-2 in vitro metabolism using three in vitro models. 5F-APP-PICA (PX-1) and 5F-APP-PINACA (PX-2) were studied as they share structural similarity with AM-2201, THJ-2201 and 5F-AB-PINACA, the metabolism of which was described in the literature.

Methods

For SC incubation, pHLM, cytochrome P450 isoenzymes and the fungal model Cunninghamella elegans LENDNER (C. elegans) were used. PX-1 and PX-2 in vitro metabolites were revealed comprehensively by liquid chromatography–high-resolution mass spectrometry measurements.

Results

In total, 30 metabolites for PX 1 and 15 for PX-2 were detected. The main metabolites for PX-1 and PX-2 were the amide hydrolyzed metabolites, along with an indole monohydroxylated (for PX-1) and a defluorinated pentyl-monohydroxylated metabolite (for PX-2).

Conclusions

CYP isoforms along with fungal incubation results were in good agreement to those obtained with pHLM incubation. CYP2E1 was responsible for many of the metabolic pathways; particularly for PX-1. This study shows that all three in vitro assays are suitable for predicting metabolic pathways of synthetic cannabinoids. To establish completeness of the PX-1 and PX-2 metabolic pathways, it is not only recommended but also necessary to use different assays.

     

Chemical analysis of a benzofuran derivative, 2-(2-ethylaminopropyl)benzofuran (2-EAPB), eight synthetic cannabinoids,five cathinone derivatives,and five other designer drugs newly detected in illegal products

From November 2013 to May 2014, 19 newly distributed designer drugs were identified in 104 products in our ongoing survey of illegal products in Japan. The identified compounds included 8 synthetic cannabinoids, FUB-PB-22 (1), 5-fluoro-NNEI indazole analog (5-fluoro-MN-18, 2), AM-2201 indazole analog (THJ-2201, 3), XLR-12 (4), 5-fluoro-AB-PINACA (5), 5-chloro-AB-PINACA (6), AB-CHMINACA (7), and 5-fluoro-AMB (8); 5 cathinone derivatives, DL-4662 (9), α-PHP (10), 4-methoxy-α-POP (11), 4-methoxy-α-PHPP (12), and 4-fluoro-α-PHPP (13); and 6 other substances, namely, the benzofuran derivative 2-(2-ethylaminopropyl)benzofuran (2-EAPB, 14), nitracaine (15), diclofensine (16), diphenidine (17), 1-benzylpiperidine (18), and acetylfentanyl (19). To our knowledge, this is the first report on the chemical properties of compounds 9–11 and 14. A total of 33 designer drugs, including compounds 1–19, were detected in the 104 illegal products, in 60 different combination patterns. The numbers of detected compounds per product ranged from 1 to 7. In addition, several products contained three different types of compounds, such as synthetic cannabinoids, cathinone derivatives, and phenethylamine derivatives per product. It is apparent that the types of compounds emerging as illegal products are becoming more diverse, as are their combinations.     

5F-Cumyl-PINACA in ‘e-liquids’ for electronic cigarettes: comprehensive characterization of a new type of synthetic cannabinoid in a trendy product including investigations on the in vitro and in vivo phase I metabolism of 5F-Cumyl-PINACA and its non-fluorinated analog Cumyl-PINACA

Purpose

In recent years e-liquids used in electronic cigarettes have become an attractive alternative to smoking tobacco. A new trend is the use of e-liquids containing synthetic cannabinoids (SCs) instead of smoking cannabis or herbal mixtures laced with SCs. In the frame of a systematic monitoring of the online market of ‘legal high’ products, e-liquids from online retailers who also sell herbal blends were bought.

Methods

The products were analyzed by gas chromatography-mass spectrometry. In some of the e-liquids an unknown compound was detected which was identified as the SC 5F-Cumyl-PINACA (1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide) by nuclear magnetic resonance analysis. To investigate the phase I metabolism of this new class of compounds, 5F-Cumyl-PINACA and its non-fluorinated analog Cumyl-PINACA were incubated with pooled human liver microsomes (pHLM). Cumyl-PINACA was additionally ingested orally (0.6 mg) by a volunteer in a controlled self-experiment. To assess the relative potency of Cumyl-PINACA a set of SCs were characterized using a cAMP assay.

Results

Metabolism of 5F-Cumyl-PINACA and Cumyl-PINACA showed similarities with AM-2201 and JWH-018. The main metabolites were formed by hydroxylation at the N-pentyl side chain. The main metabolites detected in the volunteer’s urine sample were the same as in the pHLM assay. All SCs tested with the cAMP assay were full agonists at the CB₁ receptor. Cumyl-PINACA was the most potent SC among the tested compounds and showed an EC₅₀ value of 0.06 nM.

Conclusions

The increasing popularity of e-liquids particularly among young people, and the extreme potency of the added SCs, pose a serious threat to public health. To our knowledge, this is the first report describing the tentative identification of human in vivo metabolites of Cumyl-PINACA and 5F-Cumyl-PINACA.

     

Evaluation of carboxamide-type synthetic cannabinoids on the functional activities at cannabinoid receptors and biological effects via inhalation exposure test

Purpose

Three synthetic carboxamide-type cannabinoids (5F-MDMB-PICA, 5F-EMB-PINACA, and AMB-CHMICA) were evaluated in terms of their in vitro activities at the cannabinoid receptors CB₁ and CB₂ and in vivo biological effects when smoking the synthetic cannabinoids to assess their biological effects.

Methods

[³⁵S]Guanosine-5′-O-(3-thio)-triphosphate binding assays were performed to investigate the half maximal effective concentration values of the test compounds at the CB₁ and CB₂ receptors. Additionally, the biological effects were evaluated by observing and scoring the behavior of mice with equipment in which they inhaled smoke from a herbal mixture containing the test compounds.

Results

All three synthetic cannabinoids tested in this study activated the CB₁ and CB₂ receptors in vitro. 5F-MDMB-PICA showed less than 1 nM of the half maximal effective concentration value for both receptors. Therefore, it was suggested that 5F-MDMB-PICA was the strongest CB₁ and CB₂ receptor agonist in comparison with synthetic cannabinoids evaluated in the past. The degree of the various biological effects, specifically passivity, spontaneous activity, abnormal gait, abnormal position, and grip strength, when smoking the synthetic cannabinoids corresponded to the functional activity at the CB₁ receptor. However, some biological effects differed between 5F-MDMB-PICA and 5F-MDMB-PINACA, used as a positive control, and AMB-CHMICA induced some biological effects in contrast to the other tested synthetic cannabinoids.

Conclusion

This study provides information regarding the biological effects when smoking synthetic cannabinoids from the functional activities at the CB₁ and CB₂ receptors, considering their way of inhalation and thermal degradation.

     

In vivo metabolism of the new synthetic cannabinoid APINAC in rats by GC–MS and LC–QTOF-MS

The recent appearance of APINAC (AKB-57, ACBL(N)-018, adamantan-1-yl 1-pentyl-1H-indazole-3-carboxylate) in the market of the so-called novel psychoactive substances resulted in the need of defining its characteristics and searching its metabolites for subsequent detection in biological samples. The structure of the APINAC molecule has great similarity to the molecules of other synthetic cannabinoids. Here we report on the in vivo metabolism of APINAC using rats as an experimental model. Rat urine samples were analyzed by using gas chromatography–mass spectrometry and liquid chromatography–high resolution mass spectrometry. Data were acquired via time-of-flight mass scan, followed by Auto MS and triggered product ion scans. The predominant metabolic pathway for APINAC was ester hydrolysis yielding a wide variety of N-pentylindazole-3-carboxylic acid metabolites and 1-adamantanol metabolites. Ten metabolites for APINAC were identified, with the majority generated by hydroxylation, carbonylation, and carboxylation with or without glucuronidation. Therefore, in vivo metabolic profiles in rats were generated for APINAC. N-Pentylindazole-3-carboxylic acid, hydroxylated N-pentylindazole-3-carboxylic acid, and 1-adamantanol are likely the best targets to incorporate into analytical screening methods for drugs analysis. The presented mass spectra and retention time data may be useful for detection of these compounds in human urine.     

New-generation azaindole-adamantyl-derived synthetic cannabinoids

Purpose

This work reports the synthesis and pharmacological and analytical data for a new series of recently identified azaindole-adamantyl-derived synthetic cannabinoids (SCs).

Methods

Each SC was synthesised using an efficient and divergent synthesis, and assessed by electron ionisation mass spectrometry (EIMS). The cannabimimetic activity of each compound was conducted using a fluorometric imaging plate reader (FLIPR) assay.

Results

The described EIMS method and retention time by gas chromatography were able to effectively differentiate each of the analogues regardless of the bicyclic core. For the first time in these SC structures, the bicyclic ring system was shown to have an impact on the cannabimimetic activities in the fluorometric assay of membrane potential. Analogues ranged from moderately potent at both CB₁ and CB₂ (e.g., AP4AIC EC₅₀?=?160 nM and EC₅₀?=?64 nM, respectively) to not active at either cannabinoid receptor (AP4AICA, AP5AICA, and APIC).

Conclusions

Further investigation into receptor selectivity surrounding these bicyclic cores could prove useful for future therapeutic applications.