Pharmacology of Valinate and tert-Leucinate Synthetic Cannabinoids 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA, MDMB-FUBINACA, MDMB-CHMICA, and Their Analogues Samuel D. Banister, †,∥,○ Mitchell Longworth, †,○ Richard Kevin, ‡ Shivani Sachdev, ⊥ Marina Santiago, ⊥ Jordyn Stuart, ⊥ James B. C. Mack, # Michelle Glass, ∇ Iain S. McGregor, ‡ Mark Connor, ⊥ and Michael Kassiou* ,†,§ † School of Chemistry, ‡ School of Psychology, and § Faculty of Health Sciences, The University of Sydney, Sydney, NSW 2006, Australia ∥ Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305, United States ⊥ Department of Biomedical Sciences, Macquarie University, Sydney, NSW 2109, Australia # Department of Chemistry, Stanford University, Stanford, California 94304, United States ∇ School of Medical Sciences, The University of Auckland, Auckland 1142, New Zealand * S Supporting Information ABSTRACT: Indole and indazole synthetic cannabinoids (SCs) featuring L-valinate or L-tert-leucinate pendant group have recently emerged as prevalent recreational drugs, and their use has been associated with serious adverse health effects. Due to the limited pharmacological data available for these compounds, 5F-AMBICA, 5F-AMB, 5F-ADB, AMB- FUBINACA, MDMB-FUBINACA, MDMB-CHMICA, and their analogues were synthesized and assessed for cannabimi- metic activity in vitro and in vivo. All SCs acted as potent, highly efficacious agonists at CB 1 (EC 50 = 0.45−36 nM) and CB 2 (EC 50 = 4.6−128 nM) receptors in a fluorometric assay of membrane potential, with a general preference for CB 1 activation. The cannabimimetic properties of two prevalent compounds with confirmed toxicity in humans, 5F-AMB and MDMB-FUBINACA, were demonstrated in vivo using biotelemetry in rats. Bradycardia and hypothermia were induced by 5F-AMB and MDMB-FUBINACA doses of 0.1−1 mg/kg (and 3 mg/kg for 5F- AMB), with MDMB-FUBINACA showing the most dramatic hypothermic response recorded in our laboratory for any SC (>3 °C at 0.3 mg/kg). Reversal of hypothermia by pretreatment with a CB 1 , but not CB 2 , antagonist was demonstrated for 5F-AMB and MDMB-FUBINACA, consistent with CB 1 -mediated effects in vivo. The in vitro and in vivo data indicate that these SCs act as highly efficacious CB receptor agonists with greater potency than Δ 9 -THC and earlier generations of SCs. KEYWORDS: Cannabinoid, THC, JWH-018, AMB, MDMB S ynthetic cannabinoids (SCs) are the most rapidly growing class of “designer drugs”, or new psychoactive substances (NPSs). 1 Consumer products available since about 2004 and intended as “legal cannabis substitutes” were found in 2008 to contain JWH-018 (1, Figure 1) and CP 47,497-C8 (2). 2,3 In 2014, 177 different SCs were reported to the United Nations Office on Drugs and Crime (UNODC) Early Warning Advisory (EWA). 4 Many novel SCs have already been discovered in 2016, and the structural diversity of these substances is increasing. 5−14 SCs are typically found to function as agonists of cannabinoid receptor type 1 (CB 1 ) and type-2 (CB 2 ), with activation of the former accounting for the psychoactivity of these substances. 15 However, many SCs are unknown prior to first detection by forensic chemists, and nothing is known of their activity in humans. The scarcity of data regarding the pharmacological and toxicological properties of emergent SCs poses an ongoing challenge for scientists, healthcare workers, and lawmakers across the globe. 16−26 We have previously described the in vitro and in vivo pharmacology of SCs based on 3-benzoylindoles (e.g., RCS-4, 3), 3-naphthoylindoles (e.g., AM-2201, 4), 3-alkanoylindoles (e.g., XLR-11, 5), indole-3-carboxylates (e.g., 5F-PB-22, 6), and indole-3-carboxamides (e.g., STS-135, 7). 27−32 One of the most prevalent, recent groups of SCs are 1-alkyl-1H-indazole-3- carboxamides featuring pendant valinamide and tert-leucina- mide groups, exemplified by AB-FUBINACA (8) and ADB- PINACA (9), respectively. Following the designation of several members of this class as Schedule I substances by the Drug Enforcement Administration (DEA) in the United States Received: May 18, 2016 Accepted: July 15, 2016 Published: July 15, 2016 Research Article pubs.acs.org/chemneuro © 2016 American Chemical Society 1241 DOI: 10.1021/acschemneuro.6b00137 ACS Chem. Neurosci. 2016, 7, 1241−1254