Unraveling the In Vitro Toxicity Profile of Psychedelic 2C Phenethylamines and Their N-Benzylphenethylamine (NBOMe) Analogues

Abstract

Mescaline derivative (2C phenethylamines) drugs have been modified by the introduction of a N-2-methoxybenzyl group to originate a new series of compounds with recognized and potent psychedelic effects, the NBOMe-drugs. Although they are prevalent in unregulated drug markets, their toxicity profile is still poorly understood, despite several reports highlighting cases of acute intoxication, with brain and liver toxicity. Thus, in this study, mescaline, 2C-N (insertion of a nitro in the para position of the 2C phenethylamines aromatic ring) and 2C-B (insertion of a bromide in the para position of the 2C phenethylamines aromatic ring) and their corresponding NBOMe counterparts, mescaline-NBOMe, 25N-NBOMe and 25B-NBOMe, were synthetized and the in vitro neuro- and hepatocytotoxicity evaluated in differentiated SH-SY5Y and HepG2 cell lines, respectively. Cytotoxicity, oxidative stress, metabolic and energetic studies were performed to evaluate the main pathways involved in their toxicity. Our results demonstrated that the presence of the N-2-methoxybenzyl group significantly increased the in vitro cytotoxicity of 2C phenethylamines drugs in both cell lines, with the NBOMe drugs presenting lower EC50 values when compared to their counterparts. Consistently, our data showed a correlation between the drug’s lipophilicity and the EC50 values, except for 2C-B. The 2C-B presented higher cytotoxic effects in both cell lines than mescaline-NBOMe, a result that can be explained by its higher passive permeability. All the NBOMe derivatives were able to cross the blood–brain barrier. Considering metabolic studies, the cytotoxicity of these drugs was shown to be influenced by inhibition of cytochrome P450 (CYP), which suggests a potential role of this enzyme complex, especially CYP3A4 and CYP2D6 isoenzymes in SH-SY5Y cells, in their detoxification or bioactivation. Furthermore, in differentiated SH-SY5Y cells, the drugs were able to induce mitochondrial membrane depolarization, and to disrupt GSH and ATP intracellular levels, these effects being concentration dependent and more pronounced for the NBOMe derivatives. No ROS overproduction was detected for any of the drugs in the tested experimental conditions. A correlation between a drug’s lipophilicity and the EC50 values in both cell lines, except for 2C-B, was also obtained. In summary, the introduction of a NBOMe moiety to the parent drugs significantly increases their lipophilicity, brain permeability and cytotoxic effects, with GSH and ATP homeostasis disruption. The inhibition of CYP3A4 and CYP2D6 emphasized that CYP-mediated metabolism impacts the toxicity of these drugs.

Mechanistic Insights into the Neurotoxicity of 2,5-Dimethoxyphenethylamines (2C) and Corresponding N-(2-methoxybenzyl)phenethylamine (NBOMe) Drugs

“…Similar results were described for other NPS, including piperazines (BZP, TFMPP, MeOPP, and MDBP) and phenethylamines (mescaline, 2C-B, 2C-N, mescaline-NBOMe, 25B-NBOMe, and 25C-NBOMe). In agreement with our results, and using the same in vitro model, both studies showed a significant decrease in the tGSH content with no ROS production [21,63].…”

“…We observed that after 24 h of exposure, all the tested drugs (EC 20 and EC 50 ) induced a significant mitochondrial membrane depolarization s and decreased the intracellular ATP levels (Figures 6 and 7), evidencing mitochondrial impairment. Similar results have been reported for other drugs [50,51], including drugs from the 2C and NBOMe families [21].…”

“…Since the brain is a target organ of these drugs, understanding their neurotoxic profile is of utmost importance, specially to minimize their potential harm. Previous studies, in vivo and in vitro, demonstrated the neurotoxicity of synthetic phenethylamines, with acute intoxications and fatalities being reported [ 10 , 11 , 12 , 21 , 42 , 43 ]. Nonetheless, to the best of our knowledge, there is only one published study regarding specifically the neurotoxicity of 2C-T-2, 2C-T-4, and 2C-T-7 [ 42 ], and no studies regarding 25T2-NBOMe, 25T4-NBOMe, and 25T7-NBOMe.…”

“…In brief, after 24 h of exposure to the tested drugs, the differentiated SH-SY5Y cells and primary rat cortical cultures were incubated with NR (50 µg/mL in complete medium) for 90 or 210 min, respectively, at 37 °C, in a 5% CO 2 -95% air atmosphere. To extract the absorbed dye, an acidified ethanol solution was used, and the absorbance was then measured at 540 nm in a multi-well plate reader (PowerWaveX, BioTek Instruments, Winooski, VT, USA) [ 21 ]. The cytotoxicity was estimated by the percentage of NR uptake relative to the control cells, and a minimum of 4 independent experiments were conducted in triplicate.…”

Bonding to Psychedelics: Synthesis of Molecularly Imprinted Polymers Targeting 4-Bromo-2,5-dimethoxyphenethylamine (2C-B)

“…4-Bromo-2,5-dimethoxyphenethylamine (2C-B) and all psychoactive compounds tested in this study were prepared following synthetic procedures published by our group [22,23]. 2C-B was prepared by a straightforward synthetic route.…”