Chart aShown are 25B-NBOMe/CIMBI-36 ( 1 ), 25I-NBOMe ( 2 ), and 25C-NBOMe ( 3 ). In its [ 11 C]-labelled form, 1 is currently used as a PET tracer to map 5-HT 2A R in humans under the designation [ 11 C]Cimbi-36.

Since their discovery by Ralph Heim in 2003, (1) the 2,5-dimethoxy--benzylphenethylamines (NBOMes; Chart 1 ) have attracted considerable attention from the scientific community as tools to investigate the pharmacology (2-5) and localization in the brain (6, 7) of serotonin 2A receptors (5-HTR). The NBOMes were recently proposed to undergo extensive first-pass metabolism by the liver due to their high intrinsic clearance. (8) Additionally, recent reports detail severe and in some cases fatal toxicity, related to widespread illicit use of several NBOMes due to their hallucinogenic effects, which are similar to those of LSD. (9-12) The ability of, and Chart 1 ) to precipitate severe symptoms of rhabdomyolosis in some individuals has led to their placement as schedule one drugs by the DEA in 2013. This apparent toxicity is puzzling in light of the compounds high selectivity for 5-HTR coupled with the low toxicity of known nonselective 5-HTR agonists such as psilocybin, mescaline, and LSD. (13-15) These observations led us to hypothesize that the unpredictable toxicity of NBOMes may be idiosyncratic in nature and caused by the formation of toxic metabolites. Our previous pharmacokinetic studies on the NBOMe class included screening of several closely related structural analogues, including primary phenethylamines such as mescaline and 2C–B that, in contrast to their NBOMe congeners, were very stable to human liver microsomes. Thus, we wanted to identify the metabolite(s) of NBOMe to be able to address its toxic potential.

The identification of the metabolite(s) ofwas approached using the following techniques: In vitro degradation was evaluated via incubation with porcine and human liver microsomes. The identities of the produced phase I metabolites were identified via LC-MS and by comparison with authentic samples that were synthesized as described in the Supporting Information . Subsequently, the in vivo metabolism in pigs was investigated via injection of a pharmacological dose offollowed by LC-MS/MS analysis of blood samples to identify phase II metabolites. Structural assignment was again verified by comparison with authentic samples synthesized as detailed in the Supporting Information . Finally,C labeling ofin various positions and administering theseC-ligands to pigs and humans, followed by HPLC analysis of blood samples using a radiodetector, corroborated our structural assignment.

Results and Discussion ARTICLE SECTIONS Jump To

1 was incubated with human liver microsomes (HLMs) and samples were analyzed by LC-MS. All compounds with a bromine moiety, identified by spectra displaying the typical isotopic pattern of bromine (79Br (50.7%) and 81Br (49.3%)), were assigned as metabolites of 1 and initially designated as M1, M2, etc. The most abundant metabolites were three products with m/z = 366.072 (i.e., three demethylated metabolites; m/z = 396.084 (i.e., hydroxylation; First,was incubated with human liver microsomes (HLMs) and samples were analyzed by LC-MS. All compounds with a bromine moiety, identified by spectra displaying the typical isotopic pattern of bromine (Br (50.7%) andBr (49.3%)), were assigned as metabolites ofand initially designated as M1, M2, etc. The most abundant metabolites were three products with= 366.072 (i.e., three demethylated metabolites; Figure 1 , M10, M12, and M14) and one with= 396.084 (i.e., hydroxylation; Figure 2 , M13). Although it was less abundant, N-debenzylation was also observed ( Figure 1 , M1; see the Supporting Information for full details).

Figure 1 Figure 1. Relative abundance of phase I metabolites from degradation studies with porcine liver microsomes (PLMs) and human liver microsomes (HLMs).

1 was also incubated with pig liver microsomes (PLMs) in order to compare the results with the HLM data. Qualitatively, the metabolites in human and pig liver microsomes were similar, but the relative quantity of the metabolites differed, with M12 being the major metabolite in the PLM experiments ( Since we had access to in vivo studies in pigs,was also incubated with pig liver microsomes (PLMs) in order to compare the results with the HLM data. Qualitatively, the metabolites in human and pig liver microsomes were similar, but the relative quantity of the metabolites differed, with M12 being the major metabolite in the PLM experiments ( Figure 1 ). All of the detected metabolites were further analyzed by LC-MS/MS to gain additional structural information. Because it is more sensitive, LC-MS/MS analysis resulted in the detection of metabolites that were not observed by LC-MS analysis. Table S1 in the Supporting Information summarizes the accurate mass and fragment ion data for all metabolites detected in PLMs and HLMs along with proposed metabolite structures.

In order to determine the in vivo metabolic profile in pig, 2 mg of 1 HCl was administered intravenously to a fully anaesthetized female Danish landrace pig (21 kg). Blood samples were drawn over 2 h and analyzed by LC-MS/MS. In addition to the parent compound 1, two major metabolites were detected in the plasma. The primary metabolite had an m/z = 542.101, corresponding to demethylation of 1 followed by conjugation to glucuronic acid, a metabolic pathway often seen for anisole derivatives.

m/z = 542.101 metabolite from the in vivo pig experiment was determined to be glucuronide 9. In order to unequivocally establish the identity of this metabolite, we synthesized authentic samples of M10, M12, and M14 and their corresponding glucuronides ( Figure 2 and Supporting Information ). Upon comparison, the identity of the= 542.101 metabolite from the in vivo pig experiment was determined to be glucuronide

1 and its two primary metabolites in the pig (6 and 9) showed very fast clearance of the parent compound from plasma (6 present at any time point, whereas glucuronide 9 is eliminated much slower from plasma. At the 30 min mark, there is more than twice as much glucuronide 9 present in plasma as there is 1 ( Quantification ofand its two primary metabolites in the pig (and) showed very fast clearance of the parent compound from plasma ( Figure 3 ). Both metabolic steps (demethylation and glucuronidation) are very fast, with only minute levels of intermediate phenolpresent at any time point, whereas glucuronideis eliminated much slower from plasma. At the 30 min mark, there is more than twice as much glucuronidepresent in plasma as there is Supporting Information and Figure 2 ).

Figure 2 Figure 2. Structures of the demethylated phase I and derived glucuronated phase II metabolites of 1. Reagents and conditions: (a) (1) 1,2,3,4-tetra-O-acetyl-β,d-glucuronic acid methyl ester, BF 3 , CH 2 Cl 2 , rt; (2) KCN, H 2 O, MeOH, rt. See the Supporting Information for full experimental details.

Figure 3 Figure 3. LC-MS/MS chromatograms of pig plasma samples showing the relative abundance of 1, 6, and 9 over time after IV injection of 1.

1 produces a hitherto unidentified radioactive metabolite that does not cross the blood–brain barrier in the pig.11C]9. To confirm this assumption, we produced [11C]-labeled 1, where the label is placed sequentially on all three possible methoxy positions via alkylation of suitable precursors with either [11C]CH 3 I or [11C]CH 3 OTf (see the 11 and 12) should lead to the same radioactive metabolite, whereas the last (13) should not (14, which is the presumed phase I metabolite of 1, was also included in the study as this should lead to the same phase II metabolite. All of these [11C]-labeled ligands were injected into Danish landrace pigs in separate experiments, and blood samples were collected and analyzed by HPLC using a radiodetector. When used as a PET tracer in pigs and humans,produces a hitherto unidentified radioactive metabolite that does not cross the blood–brain barrier in the pig. (6) On the basis of the data presented above, we hypothesized that this metabolite is [C]. To confirm this assumption, we produced [C]-labeled, where the label is placed sequentially on all three possible methoxy positions via alkylation of suitable precursors with either [C]CHI or [C]CHOTf (see the Supporting Information for details). If our structural assignment is correct, then two of them (and) should lead to the same radioactive metabolite, whereas the last () should not ( Figure 4 ). Compound, which is the presumed phase I metabolite of, was also included in the study as this should lead to the same phase II metabolite. All of these [C]-labeled ligands were injected into Danish landrace pigs in separate experiments, and blood samples were collected and analyzed by HPLC using a radiodetector.

11 and 12 lead to the formation of the same radioactive metabolite, which has a retention time identical to that of 9. When the label is moved to the last methoxy group (11C]9 is no longer produced. That is, even though 9 is still formed, it is no longer detectable because it is not [11C]-labeled. Furthermore, injection of 14 (11C]9. As seen in Figure 4 A,B,andlead to the formation of the same radioactive metabolite, which has a retention time identical to that of. When the label is moved to the last methoxy group ( Figure 4 C), [C]is no longer produced. That is, even thoughis still formed, it is no longer detectable because it is not [C]-labeled. Furthermore, injection of Figure 4 D) leads to the same radioactive metabolite as that in Figure 4 A,B, confirming that we have established the identity of the major metabolite as [C]

Figure 4 Figure 4. HPLC radiochromatograms of plasma samples from PET experiments in pigs with (A) 11, (B) 12, (C) 13, and (D) 14. The blue box highlights the presence or absence of the [11C]-labeled glucuronide [11C]9.

11C]Cimbi-36 labeled at two different positions (11 and 13) was initiated, and the conclusions from that study will be published elsewhere in due course, but some preliminary data on the metabolism will be provided here. On the basis of the PLM and HLM data presented in 11 in humans is comparable to that in pigs.11 and 13 in humans is presented. 11 leads to the formation of the same major metabolite as that in pigs: [11C]9. Switching the site of labeling (11 to 13) leads to the disappearance of this metabolite, in analogy to the data from the pig experiments. Accordingly, we suggest that the major metabolite of 1 in humans is 9. The same hitherto unidentified radioactive metabolite is also present in plasma from human investigations with 11, which confirms that 5′-demethylation followed by glucuronic acid conjugation is also the primary route of metabolism of 1 in humans. When radioactive metabolites of a CNS PET tracer are produced in significant amounts and cross the blood–brain barrier, this generates problems with correct quantification. Thus, a head-to-head comparison in humans of [C]Cimbi-36 labeled at two different positions (and) was initiated, and the conclusions from that study will be published elsewhere in due course, but some preliminary data on the metabolism will be provided here. On the basis of the PLM and HLM data presented in Figure 2 , one might expect to see several abundant metabolites in vivo in humans compared to those in pigs, but we found that the metabolism in humans follows the same pathway as that in the pig. Furthermore, the rate of metabolism at tracer doses ofin humans is comparable to that in pigs. (7) In Figure 5 , the combined data from three head-to-head comparisons ofandin humans is presented.leads to the formation of the same major metabolite as that in pigs: [C]. Switching the site of labeling (to) leads to the disappearance of this metabolite, in analogy to the data from the pig experiments. Accordingly, we suggest that the major metabolite ofin humans is. The same hitherto unidentified radioactive metabolite is also present in plasma from human investigations with, which confirms that 5′-demethylation followed by glucuronic acid conjugation is also the primary route of metabolism ofin humans.

Figure 5 Figure 5. Two graphs show the collected data from three head-to-head comparisons in humans of 11 and 13. In (A) the same metabolite as that found in the pig experiments accumulates over time, whereas in (B) this radiolabeled metabolite is absent (analogous to Figure 4C). Polar metabolites refer to fast eluting metabolites appearing within the first 2 min in the HPLC chromatograms from the plasma samples (see the Supporting Information for details).

11C]-labeled analogues of 1 were investigated as PET ligands, including 2 and 3. Upon inspection of the radiochromatograms, a large peak at approximately the same retention time as that of glucuronide 9 is present in all other NBOMe-derived ligands.1 (including 2 and 3) are transformed in a similar fashion via 5′-demethylation followed by glucuronic acid conjugation ( We previously conducted a tracer development study, where several structurally similar [C]-labeled analogues ofwere investigated as PET ligands, includingand. Upon inspection of the radiochromatograms, a large peak at approximately the same retention time as that of glucuronideis present in all other NBOMe-derived ligands. (6) Therefore, we speculate that the closest analogues of(includingand) are transformed in a similar fashion via 5′-demethylation followed by glucuronic acid conjugation ( Figure 6 ).

Figure 6 Figure 6. Proposed general metabolic fate of the NBOMe class of hallucinogens.