Forensic Toxicology (2018) 36:385–403 https://doi.org/10.1007/s11419-018-0415-z ORIGINAL ARTICLE Pharmacological evaluation of new constituents of “Spice”: synthetic cannabinoids based on indole, indazole, benzimidazole and carbazole scafolds Clara T. Schoeder1,3 · Cornelius Hess2 · Burkhard Madea2 · Jens Meiler4 · Christa E. Müller1,3 Received: 16 March 2018 / Accepted: 5 April 2018 / Published online: 26 April 2018 © The Author(s) 2018 Abstract Purpose In the present study we characterized a series of synthetic cannabinoids containing various heterocyclic scafolds that had been identifed as constituents of “Spice”, a preparation sold on the illicit drug market. All compounds were further investigated as potential ligands of the orphan receptors GPR18 and GPR55 that interact with some cannabinoids. Methods The compounds were studied in radioligand binding assays to determine their afnity for human cannabinoid ­CB1 and ­CB2 receptors expressed in CHO cells, and in cAMP accumulation assays to study their functionality. Results Structure-activity relationships were analyzed. The most potent ­CB1 receptor agonist of the present series MDMB- FUBINACA (12) (Ki = 98.5 pM) was docked into the human CB­ 1 receptor structure, and a plausible binding mode was identifed showing high similarity with that of the co-crystallized THC derivatives. MDMB-CHMCZCA (41) displayed a unique profle acting as a full agonist at the ­CB1 receptor subtype, but blocking the ­CB2 receptor completely. Only a few weakly potent antagonists of GPR18 and GPR55 were identifed, and thus all compounds showed high CB receptor selectiv- ity, mostly interacting with both subtypes, ­CB1 and ­CB2. Conclusions These results will be useful to assess the compounds’ toxicological risks and to guide legislation. Further stud- ies on 41 are warranted. Keywords Pharmacological evaluation of new synthetic cannabinoids · Afnities for ­CB1 and ­CB2 receptors · β-Arrestin assay at GPR18 and GPR55 · cAMP accumulation assay · Benzimidazole and carbazole · Structure-activity relationships Introduction A challenging issue for forensic toxicologists and law mak- Electronic supplementary material The online version of this ers is how to efectively respond to the constantly changing article (https​://doi.org/10.1007/s1141​9-018-0415-z) contains new psychoactive substances on the illicit drug market [1]. supplementary material, which is available to authorized users. Among these, synthetic cannabinoids feature prominently [2, 3]. Between 2008, when so-called “Spice” products [4] * Christa E. Müller christa.mueller@uni‑bonn.de containing synthetic cannabinoids began to appear on the drug market, and 2016, 169 new synthetic cannabinoids 1 PharmaCenter Bonn, Pharmaceutical Institute, were confscated and identifed [2]. Most of them were dis- Pharmaceutical Chemistry I, University of Bonn, An der covered as powders, often in bulk amounts, while others Immenburg 4, 53121 Bonn, Germany 2 were found in preparations of plant materials, e.g., minced Institute of Forensic Medicine, Forensic Toxicology, herbs, onto which solutions of the cannabinoids had been University Hospital of Bonn, Stiftsplatz 12, 53111 Bonn, Germany sprayed [5]. These substances have been shown to bind to and in many cases activate cannabinoid (CB) receptors. 3 Research Training Group 1873, University of Bonn, 53127 Bonn, Germany CB receptors are divided into two subtypes, ­CB1 and ­CB2, which belong to the large family of rhodopsin-like class A 4 Departments of Chemistry and Pharmacology, Vanderbilt University, Stevenson Center, Station B 351822, Nashville, G protein-coupled receptors (GPCRs) [6]. Both CB receptor TN 37235, USA subtypes are coupled to G­ i proteins including a reduction in Vol.:(0123456789)1 3 386 Forensic Toxicology (2018) 36:385–403 intracellular cAMP levels. The main psychoactive efects restricted. Newly discovered SARs of synthetic cannabinoids of cannabinoids are mediated by the CB 1 receptor, which is will, therefore, provide a basis for future amendments. How- predominantly expressed in the central nervous system [7], ever, in many cases, only limited information is available while CB2 receptor expression in the brain is restricted to regarding the activity of new substances. Both the afnity microglial cells [8, 9]. CB2 receptors are highly expressed in of a drug for its receptor and its ability to produce an ago- the immune system, for example in tonsils and spleen [10, nistic response are important features, and these should be 11]. Activation of the CB2 receptor is considered as a new determined according to a compound’s chemical structure. therapeutic option for the treatment of infammatory diseases For important classes of synthetic cannabinoids, at least four and pain [12, 13]. structural components, which have frstly been described by The plant-derived partial CB1 and CB2 receptor agonist Hufman et al. and were later refned by the European Moni- Δ9-tetrahydrocannabinol (Δ9-THC, 1, Fig. 1) is used in toring Centre for Drugs and Drug Addiction (EMCDDA), therapy to target muscle spasms, nausea and cachexia [14]. are of importance (see Fig. 2 [3]): (1) a heterocyclic core The synthetic compound CP55,940 (2, Fig. 1) represents a consisting of indole or indazole with diferent substitutions; potent full agonist at both receptor subtypes. A CB1 receptor (2) a linker, e.g., an ester, amide or ketone; (3) a bulky lipo- antagonist, rimonabant, had been approved for the treatment philic residue (R1), e.g., a heterocyclic or aryl substituent, of obesity but was later withdrawn from the market due to but in newer synthetic cannabinoids a lipophilic amino acid side efects resulting in depression and an increased suicide can also be found; and (4) a residue (R 2) which is a hydro- rate [15]. phobic “side chain” attached to the nitrogen atom of the The prevalence for the use of illegal psychoactive sub- indole or the indazole ring system [21, 22]. The compound stances in Europe by 15–16 year-old teenagers was estimated JWH-018 (3, see Fig. 1), a potent CB1 and CB2 receptor in 2015 to be about 4% [5]. Synthetic CB1 receptor agonists agonist, displays the basic features of this compound class are abused as an alternative to natural marijuana due to their and was one of the frst synthetic cannabinoids identifed in psychoactive and analgesic efects. For synthetic cannabi- herbal blends for abuse [23, 24]. The common features of noids more and more severe side efects and intoxications known synthetic cannabinoids are depicted in Fig. 2. are reported; they are predominantly neurologic symptoms, In a previous study [25], we had determined the phar- but acute organ toxicity has also been observed [16]. In the macological properties of 48 synthetic cannabinoids USA, the principle of enumeration is used to restrict newly collected by the Institute of Forensic Medicine of the discovered synthetic cannabinoids, and every single syn- University of Bonn. In the present study, we investi- thetic cannabinoid has to be individually listed by name in gated the affinities and functional properties of a new the US List of Schedule I drugs [17]. In Germany new syn- series of 42 synthetic cannabinoids, 16 of which have thetic cannabinoids are legally controlled since November not been reported as cannabinoid receptor ligands before. 2016 when the “Neue-Psychoaktive-Stofe-Gesetz” (NpSG, The investigated set of compounds comprises four dif- New Psychoactive Substances Act) was adopted in [18]. ferent core structures. The first three groups (A, B, C, Similar regulations exist in Austria and Switzerland [19, see Table 1) represent differently substituted indoles and 20]. All corresponding compounds, the chemical structures indazoles, which are structurally derived from the syn- of which are represented by a general formula in the statute thetic cannabinoids previously introduced by Huffman with known structure-activity relationships (SARs), were et al. and are widely distributed in illicitly sold "Spice" OH O CH3 OH OH H N H H3C CH3 O CH3 H3C H3C CH3 OH CH3 1 ∆9-THC 2 CP55,940 3 JWH-018 CB : K = 0.5 - 5.0 nM CB : K = 9 nM CB1: Ki = 5.05 - 80.3 nM [12] 1 i [12] 1 i [22] CB : K = 0.69 - 2.8 nM CB : K = 2.9 nM CB2: Ki = 3.13 - 75.3 nM [12] 2 i [12] 2 i [22] Fig. 1 Standard cannabinoid CB1/CB2 receptor agonists [12, 22] 1 3 Forensic Toxicology (2018) 36:385–403 387 Fig. 2 Common structural fea- tures of synthetic cannabinoids. The fgure was adopted from the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) [3] and modifed products. In the current study we investigated compounds Radioligand binding assays with L-valinamide (AB)/L-tert-leucinamide (ADB or MAB), methyl-3,3-dimethylbutanoate (MDMB), methyl- Radioligand binding assays were performed as described 3-methylbutanoate (MMB), and 2-methyl-2-phenylpropyl previously [25]. Membrane preparations of Chinese hamster (cumyl) moieties as substituents in the R 1 position. Fur- ovary (CHO) cells overexpressing the human CB receptor ther classes of compounds consist of carbazoles (E), sub- subtype CB1 or CB 2 were incubated in the presence of the stituted in position 3, and benzimidazole derivatives (F). test compound and the radioligand [3H]CP55,940 (0.1 nM, Radioligand binding and cAMP functional studies on see Fig. 1) (Perkin-Elmer Life Sciences, Rodgau-Jügesheim, CB1 and CB2 receptors were complemented by CB1 recep- Germany), for 2 h. Bound and unbound radioligand were tor modeling and docking of the most potent CB 1 receptor separated by rapid fltration through glass fber GF/C-flters agonist of the present series to predict its interactions. We (Perkin-Elmer, Boston, MA, USA), using a Brandel 96-well further tested all compounds for their ability to activate or Harvester (Brandel, Gaithersburg, MD, USA). Radioactivity block the two orphan GPCRs GPR18 and GPR55, both of on the flters was determined by liquid scintillation count- which are known to interact with cannabinoids [26–29]. ing. Three separate experiments were performed, each in We discuss SARs of the newly investigated compounds, duplicates.