Dark Classics in Chemical Neuroscience: NBOMes

HISTORY

Psychedelics are compounds that induce perceptual alterations as well as effects on mood and cognition.¹ In scientific literature, psychedelics have been classified according to a variety of different names. Psychotomimetics, one of the earliest terms, was coined to indicate that these compounds are capable of inducing a mental state similar to psychosis. Later, these compounds were classified as hallucinogens, a term still in widespread use² also encompassing a wide range of mechanistically distinct drugs, such as the κ-opioid receptor agonist salvinorin A³ and the N-methyl-d-aspartate (NMDA) receptor antagonist phencyclidine (PCP).⁴ The term psychedelic, which was coined by Dr. Humphrey Osmond in 1956 and means “mind manifesting”. This term is usually reserved for compounds that are also referred to as serotonergic or classical hallucinogens.

The classical psychedelics exert their main pharmacological effects through activation of the serotonin 2A receptor (5-HT_2AR); they can be subdivided into two main families based on their structural properties: indoleamines and phenylalkylamines. The indoleamine scaffold is present in both the lysergamide and tryptamine psychedelics, as exemplified by lysergic acid diethylamide (LSD)⁵ and psilocybin,⁶ respectively (Figure 1). The phenylalkylamine family can be subdivided into phenylisopropylamines (“psychedelic amphetamines”, including 2,5-dimethoxy-4-iodoamphetamine (DOI) and other members of the DOX class), the phenethylamines (most notably mescaline and 2C-X compounds such as 2C-B), and last the N-benzylphenethylamines (NBOMes) (which are the main focus of this Review) (Figure 1). The respective nomenclatures for members of these three groups are rooted in different principles. The “2C” code name refers to the two ethylene carbons that separate the phenyl ring from the primary amine, with the last letter in the acronym identifying the substituent in the 4-position, for example, bromine in the case of 2C-B (Figure 1). In the case of the NBOMes, the 25B in the name 25B-NBOMe refers to the position and identity of the substituents on the phenethylamine moiety (i.e., methoxy groups in the 2- and 5-positions and a bromine substituent in the 4-position), the NB stands for N-benzyl, and the OMe represents the methoxy substituent on the ortho-position of the benzyl moiety (Figure 1).

The history of the phenethylamine family of hallucinogens has spanned a period of at least 5500 years (Table 1), with the first evidence for the ceremonial use of the sacred cactus Lophophora williamsii, better known as peyote, dating back to 3780–3660 BCE.⁷ L. williamsii is a small bulbous and spineless cactus, containing mescaline as its primary alkaloidal constituent. In 1896, mescaline was isolated from peyote by Arthur Heffter,⁸ who was also the first to recognize the psychedelic properties of mescaline based on self-experiments, thus making mescaline the first chemically pure psychedelic substance.⁹ In the following years, a large amount of scientific research was conducted with mescaline, including the first reported synthesis of the compound by Ernst Späth, proving irrefutably that the compound is a β-phenethylamine,¹⁰ and the publication of Der Meskalinrausch (“The Mescaline Rush”) by Kurt Beringer, which provided a detailed description of its psychedelic effects.¹¹ However, it was mainly the publication of The Doors of Perception by Aldous Huxley in 1954 that introduced the notion of mescaline as a psychedelic to the general public.¹²

Explorations into the putative therapeutic potential of mescaline and other psychedelics came to an abrupt halt when President Nixon signed “The Comprehensive Drug Abuse Prevention and Control Act” into law on October 27, 1970. Title II of this act, the “Controlled Substance Act” (CSA), classified many of the best-known psychedelics, including LSD, psilocybin, mescaline, and peyote, as Schedule I substances, thus severely restricting the use of these compounds in scientific research.¹³

By that point, several analogues of mescaline had been prepared and evaluated in man (Scheme 1), including the amphetamine analogues 3,4,5-trimethoxyamphetamine (TMA)^14,15 and 2,4,5-trimethoxyamphetamine (TMA-2).¹⁶ The potency increase observed with TMA and especially TMA-2 relative to mescaline led Alexander T. Shulgin to synthesize the more potent and long-acting compound 2,5-dimethoxy-4-methylamphetamine (DOM).¹⁷ The psychedelic potency of DOM was increased even further by replacing the para-methyl group with an iodine or bromine atom, yielding 2,5-dimethoxy-4-iodoamphetamine (DOI) and 2,5-dimethoxy-4-bromoamphetamine (DOB), respectively.¹⁸ In 1974, Shulgin translated this strategy back to the phenethylamine family with the synthesis of 2,5-dimethoxy-4-bromophenethylamine (2C-B),¹⁹ which he found to be a strong hallucinogen in a series of self-experiments conducted during 1974 and 1975 (the drug was described as “beautifully effective”).²⁰ During the late 1970s and early 1980s, 2,5-dimethoxy-4-methylphenethylamine (2C-D), another compound from this class, received considerable attention from psychiatrists as a psychotherapeutic adjunct, most notably Hanscarl Leuner, who worked with 2C-D extensively under the code name LE-25 and pioneered the concept of psychedelic therapy.²¹ However, 2C-B was emergency scheduled by the Drug Enforcement Administration (DEA) in 1994, due to its appearance on the recreational drug market as a replacement for 3,4-methyl enedioxy methamphetamine (MDMA) (which had been scheduled in 1985). At that time, 2C-B was still being legally manufactured by the German company Drittewelle under the trade name of Erox and sold in Dutch “head-shops” under the name Nexus. In March 2001, the UN Commission on Narcotic Drugs added 2C-B to Schedule II of the Convention on Psychotropic Substances.

The same year that 2C-B was sch in the United States, Richard A. Glennon and co-workers reported that substitution of the primary amine of 2C-B with a benzyl moiety yielded an significant increase in 5-HT_2AR binding affinity.²² Further structural optimization as part of the doctoral thesis of Ralf Heim²³ and by David E. Nichols and co-workers²⁴ revealed that the addition of an ortho-methoxy or -hydroxy group to the N-benzyl moiety increased 5-HT_2AR affinity even further. In 2010 the first reports of online vendors distributing compounds from this class as designer drugs or New Psychoactive Substances (NPS) emerged, with 25I-NBOMe and 25B-NBOMe being the first compounds from this class to appear.²⁵ Before long, accounts of toxicity and lethal overdoses from these drugs appeared, which resulted in the scheduling of these compounds by the DEA in November 2013.

SCIENTIFIC RELEVANCE OF NBOMes

In recent years, there has been a resurgence of scientific research into serotonergic psychedelics. Although a large amount of clinical research was conducted with LSD and other hallucinogens in the 1950s and 1960s, virtually all clinical research with these substances ceased during the early 1970s due to legal restrictions codified in the Controlled Substance Act; only recently has research with these drugs resumed. Most of this new work has focused on psilocybin, which has shown promising effects in pilot studies focusing on several therapeutic indications, including depression and anxiety,^26–30 substance abuse,^31–33 and obsessive-compulsive disorder.³⁴ Notably, the U.S. Food and Drug Administration (FDA) has designated psilocybin as a Breakthrough Therapy for treatment-resistant depression. There has also been renewed interest in the use of LSD as a potential therapeutic agent.^35,36 The reader is referred to other review articles for a more in-depth discussion of the remarkable findings on the therapeutic potential of psychedelic compounds in general.^27,37–45 Psilocybin has high affinity for a number of serotonergic receptors besides 5-HT_2AR,⁴⁶ and LSD possesses high affinity for an even wider range of monoaminergic receptors (see Table 2).⁴⁷ However, the activation of 5-HT_2ARs is considered essential for the psychedelic effects, as well as the apparent therapeutic potential of both of these drugs. Thus, ligands capable of activating the 5-HT_2AR selectively would be important pharmacological tools. Given the inherent selectivity exhibited by many of the NBOMes for 5-HT₂R over other serotonergic and monoaminergic receptors, they are obvious leads in the search for novel agents that could be applicable in investigations of the in vivo effects arising from selective activation of the 5-HT_2AR.

CHEMICAL SYNTHESIS

2,5-Dimethoxybenzaldehyde (1) is a common commercial starting point for the synthesis of compounds from the 2C-X and NBOMe classes (Scheme 2).^19,51 The β-nitrostyrene (2) is prepared by the condensation of nitromethane with benzaldehyde 1. The transformation to the β-phenethylamine 3 is typically accomplished by reduction, using hydrogen and palladium on activated charcoal or another suitable reducing agent. β-Phenethylamine 3 is typically brominated at this stage. Alternatively, the 4-bromo group can be introduced at the benzaldehyde stage to avoid performing a late stage bromination (not shown).⁵⁰ All NBOMe-class compounds are usually prepared from the corresponding 2C-X compound by reductive amination of the appropriate benzaldehyde.^49,50

STRUCTURE–ACTIVITY RELATIONSHIPS OF THE NBOMes

PHARMACOLOGY

It has been recognized that most of the psychological and therapeutic effects produced by serotoninergic psychedelics are mediated by activation of 5-HT_2AR.^42,43 Compounds from the tryptamine class are usually considered to be selective for serotonin receptors over other monoaminergic receptors, but they are fairly promiscuous agonists within the serotonin receptor family (Table 2).⁵⁶ Ergolines are generally non-selective and target a broad range of monoaminergic receptors with high binding affinities (Table 2).⁵⁷ In general, the most selective agonists for 5-HT_2AR and 5-HT_2CR come from the phenylalkylamine class, with compounds from the amphetamine and phenethylamine class usually displaying substantially higher binding affinities for these receptors compared to other receptors for serotonin and other monoamines.

The psychedelic phenethylamines typically exhibit less than 5–10-fold selectivity for 5-HT_2A over 5-HT_2C receptors.^53,54 Notable exceptions include (S,S)-DMBMPP and 25CN-NBOH (Figure 3), which are the most selective 5-HT_2AR agonists published to date.^49,58–61 (S,S)-DMBMPP exhibits more than 100-fold higher binding affinity for the 5-HT_2AR over 5-HT_2CR, and 25CN-NBOH has substantially higher agonist potencies at 5-HT_2AR compared to 5-HT_2CR.

The availability of 25CN-NBOH, DMPMPP, and other 5-HT_2AR-selective agonists with good pharmacokinetic properties will be instrumental for the future delineation of the role played by the receptor in the therapeutic effects of classical psychedelics. The hallucinogenic properties are also believed to be inherently linked to augmentation of 5-HT_2AR signaling, which constitutes a major potential obstacle to the realization of the therapeutic potential of 5-HT_2AR agonists. Thus, an important new frontier in this field will be needed to establish whether or not the psychedelic properties and the therapeutic effects arising from 5-HT_2AR activation can be separated in specific agonists. In this respect, it is interesting to note that in contrast to most other 5-HT_2AR agonists, lisuride, a close structural analogue of LSD and a potent 5-HT_2AR agonist, does not induce a head twitch response in rodents or produce psychedelic effects in man.^66–68 Over the past decade, research has focused on the fact that GPCRs can potentially signal through multiple intracellular pathways and that some compounds (biased agonists) can selectively activate a specific subset of these pathways.⁶⁹ If it is possible to develop biased 5-HT_2AR agonists with pathway-specific effects, then it will be interesting to probe the effects of these compounds in vivo.

EFFECT OF NBOMes IN IN VIVO BEHAVIORAL MODELS

Administration of 5-HT_2AR agonists to rodents induces a rapid paroxysmal head rotation known as the head twitch response (HTR).^70,71 In rats, the HTR often involves both the head and trunk, hence in that species the behavior is also called a wet-dog shake (WDS).⁷² The HTR is widely used as a behavioral proxy in rodents for hallucinogen effects in humans because it is one of the few behavioral effects that can reliably distinguish hallucinogenic and nonhallucinogenic 5-HT_2AR agonists, such as LSD and lisuride, respectively.^70,73

Various compounds from the NBOMe class have been tested in the HTR paradigm. After subcutaneous administration, 25I-NBOMe induces the HTR in C57BL/6J mice with an ED₅₀ of 0.17 μmol/kg.⁷⁴ In comparison to 25I-NBOMe, 2C-I has 18-fold lower potency (ED₅₀ = 2.42 μmol/kg SC), which is consistent with their relative binding affinities and agonist potencies at 5-HT_2AR.⁷⁴ Similar findings with 25I-NBOMe and 2C-I have been reported in Sprague–Dawley (SD) rats.⁷⁴ Likewise, 25C-NBOMe induces WDS in SD rats with 26-fold higher potency than 2C-C.⁷⁵ 25B-NBOMe induces the HTR in NMRI mice when administered at 0.5 mg/kg IP but not at 0.05 mg/kg, meaning it has about the same potency as 25I-NBOMe.⁷⁶ 25CN-NBOH (ED₅₀ = 1.03 μmol/kg SC)⁵⁹ also induces the HTR in C57BL/6J mice but has relatively lower potency compared to other NBOMe compounds. Importantly, experiments have confirmed that 5-HT_2A antagonists block the HTR induced by 25I-NBOMe and other N-benzyl-substituted phenethylamines.^58,61,74,75

The HTR potency of NBOMes derived from 2C-I is dependent on the identity and position of the substituent on the N-benzyl ring. According to HTR studies performed in C57BL/6J mice, rearranging the ortho-methoxy group of 25I-NBOMe to the meta-position reduced its potency 55-fold (25I-NB3OMe: ED₅₀ = 9.36 μmol/kg SC), whereas the paramethoxy analogue 25I-NB4OMe showed no evidence of activity at doses up to 30 mg/kg.⁷⁷ The presence of a halogen substituent is also detrimental for activity; replacing the ortho-methoxy moiety with bromine produced a 26-fold potency reduction (25I-NBBr: ED₅₀ = 4.50 μmol/kg SC). Rearrangement of the bromine atom to either the meta- or para-position was not tolerated, and no HTR was observed with 25I-NB3Br or 25I-NB4Br at doses up to 30 mg/kg. The N-(2,3-methylenedioxybenzyl) analogue 25I-NBMD (ED₅₀ = 2.36 μmol/kg SC)⁷⁴ is significantly less potent than 25I-NBOMe and shows the same potency as the nonbenzylated parent compound 2C-I. Therefore, for N-benzylphenethylamines, the highest potency in the HTR assay is associated with an ortho-substituent on the benzyl ring, especially if the substituent contains an oxygen atom.

Drug discrimination is another behavioral paradigm used to study hallucinogen effects. It was first shown in 1971 that rats can be trained to discriminate the interoceptive stimulus effects of mescaline and LSD from saline.⁷⁸ Many other serotonergic hallucinogens have been used as training drugs, including DOM, DOB, DOI, psilocybin, 5-MeO–DMT, and DPT.^79–84 The interoceptive stimulus effects produced by serotonergic hallucinogens appear to be rather uniform in nature because they reliably produce cross-generalization in rats. Recently, 25B-NBOMe, 25C-NBOMe, and 25I-NBOMe were evaluated in SD rats trained to discriminate 0.5 mg/kg DOM (administered by the IP route).⁸⁵ 25B-NBOMe and 25C-NBOMe produced full substitution, indicating they have DOM-like stimulus effects. Evaluation of the response to 25I-NBOMe was confounded by the fact that higher doses disrupted operant responding, but it did produce a high degree of partial substitution (a maximum of 74% drug-appropriate responding occurred at 2.5 mg/kg IP).

Most drug discrimination studies with serotonergic hallucinogens have been conducted in rats, although mice have also been used in some studies. For example, mice have been trained to discriminate LSD,^86–88 DOI,⁸⁹ R-(−)-DOI,⁵⁸ and R-(−)-DOB.⁹⁰ Interestingly, there are apparently significant differences between the interoceptive stimulus effects of hallucinogens in rats vs mice.^87–89 For example, although the discriminative stimulus induced by DOI in rats appears to be exclusively mediated by 5-HT_2A,^91,92 the 5-HT_2C receptor may contribute to the stimulus effects of the drug in mice.⁸⁹ In light of these species-specific differences, it is interesting to note that 25CN-NBOH did not fully substitute when tested in NIH Swiss mice trained to discriminate 0.3 mg/kg IP R-(−)-DOI, producing a maximum of 55% drug-appropriate responding.⁵⁸ Although it is possible that 25CN-NBOH does not produce hallucinogen-like interoceptive stimulus effects, the absence of full substitution in mice could also potentially reflect the relatively high selectivity of 25CN-NBOH for 5-HT_2AR. The reported selectivity of 25CN-NBOH for 5-HT_2A over 5-HT_2C varies depending on the experimental conditions,^49,59,60 but 25CN-NBOH is clearly more selective than DOI, which is only ~5-fold and 12-fold selective for 5-HT_2A over 5-HT_2C at the human and murine receptors, respectively.^93–95 If, as noted above, the 5-HT_2C receptor contributes to the DOI stimulus in mice, then a 5-HT_2A-selective agonist such as 25CN-NBOH may not produce full substitution in that species. Importantly, 25CN-NBOH is anticipated to fully substitute for DOI in rats, but the prediction needs to be tested experimentally.

NBOMes AS NEUROIMAGING TOOLS

Positron emission tomography (PET) is a highly sensitive imaging tool that finds widespread use in basic research and drug discovery, where it can be used to determine receptor availability and to measure occupancy by drug molecules.⁹⁶ The requirements for PET ligands and therapeutic drugs are very different:⁹⁷ High affinity and selectivity, low nonspecific binding, and reversible kinetics are key requirements for imaging purposes. The NBOMes seem to possess many of these characteristics, and thus they have been investigated as PET ligands for 5-HT_2AR. After screening several members of the NBOMe class, [¹¹C]25B-NBOMe was found to have very good properties as a 5-HT_2AR PET ligand, despite having limited (~15-fold) selectivity for 5-HT_2AR over 5-HT_2CR. [¹¹C]25B-NBOMe has been evaluated as a PET ligand in pigs,^65,76,98 primates (Macaca mulatta),^99,100 and humans;^101,102 several PET centers around the world are currently using this ligand, under the code name [¹¹C]-CIMBI-36, to investigate the clinical relevance of 5-HT_2AR. Several attempts have been made to develop an ¹⁸F-based PET tracer from the NBOMe-class, since having a PET ligand based on this radionuclide would broaden its clinical applicability, but so far these attempts have been unsuccessful.^103–105 These results highlight the fact that even very small changes to the structure of a molecule can have dramatic effects on its behavior in vivo.

ADME

Only limited data are available on the human absorption, distribution, metabolism, and excretion (ADME) properties of NBOMes, due to the ethical and legal concerns associated with their use. Therefore, there is a great need for controlled experiments on the properties of these compounds, for both scientific as well as toxicological reasons. For instance, metabolites may have toxic pharmacological properties and/or produce dangerous drug–drug interactions with either pharmaceuticals or other illicit substances.

The microsomal stability of several NBOMes has been determined in human liver microsomes (HLMs).⁵⁰ The average intrinsic clearance (Cl_int) was 6 (1.9–14) L/kg/h, which is much higher than the corresponding Cl_int values for 2C-X compounds (average clearance = 0.51 L/kg/h) and mescaline (0.19 L/kg/h).¹⁰⁶ Thus, NBOMes are generally metabolized at a much faster rate than their parent phenethylamines, indicating that their lack of per oral bioavailability is a consequence of extensive first pass metabolism. In a follow-up study, five phase I metabolites of 25B-NBOMe were identified. All three O-demethylated metabolites (4–6) were found in approximately the same abundancies, as were the hydroxylated metabolites (7–8), albeit with relatively lower abundancy. 2C-B, the N-debenzylated metabolite, was also detected, but it also had lower abundancy.¹⁰⁷

Studies of other NBOMes have shown that there is a plentitude of overlap between the metabolites of different members of this group.¹⁰⁸ CYP2D6 is the main contributor to the hepatic clearance of most NBOMes, as exemplified by 25B-NBOMe, where the 2D6 isoenzyme is responsible for 69% of the clearance.¹⁰⁹ Other cytochrome P450s involved in the phase I metabolism of these compounds include CYP1A2, CYP2B6, CYP2C19, and CYP3A4.^109,110 Detailed studies of the phase I and phase II metabolites have found a large array of minor metabolites, ranging up to 69 unique metabolites (Scheme 3).^108–119

SAFETY

Many instances of acute toxicity due to NBOMe exposure have been reported; currently, there are over 50 confirmed cases.¹²⁰ Of these cases, at least 9 were fatalities,^121–125 of which at least 2 were direct 25B-NBOMe poisonings.^126,127 Including those 2 fatalities, a total of 18 cases of acute 25B-NBOMe toxicity have been reported. In addition to causing hallucinations, other common side effects produced by NBOMes include severe agitation, confusion, diaphoresis, hypertension, tachycardia, and hyperthermia. Less common, but more severe are seizures, rhabdomyolysis, metabolic acidosis, renal failure, multiorgan failure, and coma.^128–132 The in vitro neurotoxicity of both 25B-NBOMe and 25C-NBOMe has also been investigated, and these compounds were found to inhibit neuronal activity and cell viability, respectively.^133,134 Additionally, 25D-NBOMe and 25C-NBOMe were shown to have potential cardiotoxic effects, as both compounds prolonged QT intervals in rat ECG studies. Furthermore, 25D-NBOMe also inhibited K⁺-channels in a hERG assay.¹³⁵

CONCLUSIONS

In conclusion, numerous cases have occurred where recreational use of NBOMes caused severe toxic effects, necessitating emergency medical intervention (which unfortunately did not always prevent a fatal outcome); the fact that in some of these cases NBOMes had been taken at moderate doses should serve as warning about the recreational use of these substances. The origin of the NBOMe-induced toxicity is not well understood, and certain individuals may show idiosyncratic responses. Nonetheless, this class of psychedelic compounds, in particular 25B-NBOMe and 25CN-NBOH, have provided the scientific community with new tools to selectively investigate 5-HT_2A receptors in preclinical and clinical research. Given the recent surge in clinical research with classical psychedelics, NBOMes have emerged as an important class of compounds that will allow researchers to focus on the specific effects produced by 5-HT_2AR activation. Furthermore, certain members of the NBOMe class may have unique clinical or therapeutic effects compared to existing psychedelic compounds due to their relatively high selectivity for the 5-HT_2AR.