The 2-alkyl-2H-indazole regioisomers of synthetic cannabinoids AB-CHMINACA, AB-FUBINACA, AB-PINACA, and 5F-AB-PINACA are possible manufacturing impurities with cannabimimetic activities

Forensic Toxicol (2016) 34:286–303 DOI 10.1007/s11419-016-0316-y ORIGINAL ARTICLE The 2-alkyl-2H-indazole regioisomers of synthetic cannabinoids AB-CHMINACA, AB-FUBINACA, AB-PINACA, and 5F-ABPINACA are possible manufacturing impurities with cannabimimetic activities Mitchell Longworth1 • Samuel D. Banister1,2 • James B. C. Mack3 • Michelle Glass4 • Mark Connor5 • Michael Kassiou1,6 Received: 25 February 2016 / Accepted: 1 April 2016 / Published online: 27 April 2016 Ó Japanese Association of Forensic Toxicology and Springer Japan 2016 Abstract Indazole-derived synthetic cannabinoids (SCs) featuring an alkyl substituent at the 1-position and L-valinamide at the 3-carboxamide position (e.g., AB-CHMINACA) have been identified by forensic chemists around the world, and are associated with serious adverse health effects. Regioisomerism is possible for indazole SCs, with the 2-alkyl-2H-indazole regioisomer of AB-CHMINACA recently identified in SC products in Japan. It is unknown whether this regiosiomer represents a manufacturing impurity arising as a synthetic byproduct, or was intentionally synthesized as a cannabimimetic agent. This study reports the synthesis, analytical characterization, and pharmacological evaluation of commonly encountered indazole SCs AB-CHMINACA, AB-FUBINACA, AB- M. Longworth and S. D. Banister contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s11419-016-0316-y) contains supplementary material, which is available to authorized users. & Michael Kassiou PINACA, 5F-AB-PINACA and their corresponding 2-alkyl-2H-indazole regioisomers. Both regioisomers of each SC were prepared from a common precursor, and the physical properties, 1H and 13C nuclear magnetic resonance spectroscopy, gas chromatography–mass spectrometry, and ultraviolet–visible spectroscopy of all SC compounds are described. Additionally, AB-CHMINACA, AB-FUBINACA, AB-PINACA, and 5F-AB-PINACA were found to act as high potency agonists at CB1 (EC50 = 2.1–11.6 nM) and CB2 (EC50 = 5.6–21.1 nM) receptors in fluorometric assays, while the corresponding 2-alkyl-2H-indazole regioisomers demonstrated low potency (micromolar) agonist activities at both receptors. Taken together, these data suggest that 2-alkyl-2H-indazole regioisomers of ABCHMINACA, AB-FUBINACA, AB-PINACA, and 5FAB-PINACA are likely to be encountered by forensic chemists and toxicologists as the result of improper purification during the clandestine synthesis of 1-alkyl-1Hindazole regioisomers, and can be distinguished by differences in gas chromatography–mass spectrometry fragmentation pattern. Keywords Indazole synthetic cannabinoid
2H-indazole regioisomer
Manufacturing impurity with cannabinoid activity
CHMINACA
FUBINACA
PINACA 1 School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia 2 Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA 3 Department of Chemistry, Stanford University, Stanford, CA, USA Introduction 4 School of Medical Sciences, The University of Auckland, Auckland, New Zealand 5 Department of Biomedical Sciences, Macquarie University, Sydney, Australia 6 Faculty of Health Sciences, The University of Sydney, Sydney, Australia Synthetic cannabinoids (SCs) are the most rapidly growing class of novel psychoactive substances (NPSs) [1]. SC ‘‘designer drugs’’ are intended to mimic the psychoactive effects of D9-tetrahydrocannabinol (D9-THC, 1, Fig. 1), the principal bioactive component of cannabis. Unlike D9THC—a partial agonist at both cannabinoid type 1 (CB1) 123 Forensic Toxicol (2016) 34:286–303 287 Fig. 1 Selected natural and synthetic cannabinoids (SCs) and type 2 (CB2) receptors—most SCs possess high efficacy agonist activities at both CB receptor subtypes. Since the discovery of CB1/CB2 agonist JWH-018 (2) in consumer products in Germany, Austria, and Japan in 2008 [2, 3], more than 130 SCs have been reported in Europe, with 30 identified in 2014 alone [4]. The identification and subsequent prohibition of individual SCs have motivated clandestine chemists to produce analogues of increasing structural diversity, intended to evade legal restriction [5, 6]. Many newer SCs have no precedent in the scientific literature and little, if anything, is known of their activity or toxicity [7–14]. One particularly prevalent class of SCs, presumably inspired by recent Pfizer patents [15, 16], is comprised of an indazole core decorated at the 1-position with various aliphatic, alicyclic, or aromatic groups, and at the 3-position with valine- or tert-leucine-derived carboxamides. Unlike cannabis itself, these newer SCs are associated with exposures resulting in hospitalization or death in Europe, the USA, Japan, and Russia [17–25]. (S)-N-(1-Amino-3-methyl-1-oxobutan-2-yl)-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (AB-CHMINACA, 3) was formally notified to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) in 2014 following identification in Latvia [4], and was also detected in Japan [26] and Germany [27]. An analogue featuring a 4-fluorobenzene group in place of the cyclohexane ring, (S)-N-(1-amino-3-methyl- 123 288 1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA, 4), was found in consumer products by Japanese researchers at the National Institute of Health Sciences in 2013 [28], and was also identified in Belgium [29] and Germany [27]. (S)-N-(1-Amino-3-methyl-1-oxobutan-2-yl)-1-(pentyl)-1H-indazole-3-carboxamide (AB-PINACA, 5), featuring the n-pentyl substituent of JWH-018, was also found alongside AB-FUBINACA in Japan in 2013 [28]. Consistent with the trend for incorporation of fluorine into newer SCs [30], the terminally fluorinated analogue of AB-PINACA, (S)-N-(1-amino-3-methyl-1-oxobutan-2yl)-1-(5-fluoropentyl)-1H-indazole-3-carboxamide (5F-ABPINACA, 6), was discovered in products originating from Sweden in 2013 [29], Japan in 2014 [26], and Germany in 2015 [27]. Recently, AB-CHMINACA, AB-FUBINACA, ABPINACA, and 5F-AB-PINACA were shown to act as potent and efficacious agonists at human CB1 and CB2 receptors in vitro [31, 32]. Moreover, AB-CHMINACA, AB-FUBINACA, and AB-PINACA exert potent cannabimimetic effects on locomotion, body temperature, heart rate, and nociception in mice and rats, as well as substituting for D9-THC in drug discrimination assays [31–33]. Taken together, these data indicate that a range of alkyl substituents are tolerated at the 1-indazole position in this class of SCs. These SCs appear more toxic than earlier examples, and multiple overdoses and fatalities in the USA have been attributed to AB-CHMINACA, AB-FUBINACA, and ABPINACA [34–36]. Due to the abuse potential and toxicity of these newer SCs, the Federal Government of the USA has used emergency scheduling laws to temporarily place AB-FUBINACA, AB-PINACA, and AB-CHMINACA into Schedule I, the most restrictive category of the Controlle (...truncated)