N-pyrrolidino (PYR) nitazenes are a subset of 2-benzylbenzimidazole opioids appearing in illicit drug markets, but there is limited information about the pharmacology of these compounds. Here, we examined the opioid-like effects of PYR nitazenes with varying alkoxy chain lengths (ie, methoxy, ethoxy, propoxy, and isopropoxy) compared with fentanyl. Opioid receptor binding and functional assays were employed to determine affinities and potencies of the compounds in vitro. Acute opioid-like effects were examined in mice, using hot plate antinociception, motor activity, and body temperature measures. PYR nitazenes displayed high affinity at the μ-opioid receptor (Ki = 1-12 nM), with weaker affinities at δ and κ sites. The compounds were potent and efficacious agonists in assays measuring μ-opioid receptor-mediated inhibition of cAMP formation (EC50 = 0.03-0.91 nM, Emax = 104%-106%) compared with fentanyl (EC50 = 0.23 nM, Emax = 100%). The nitazenes also induced β-arrestin 2 recruitment, but with weaker potencies. In mouse studies, the compounds induced dose-dependent antinociception, hyperactivity, and hypothermia. Across in vivo measures, the ethoxy, propoxy, and isopropoxy compounds (ED50 = 0.002-0.033 mg/kg) were more potent than fentanyl (ED50 = 0.081-0.178 mg/kg). The acute in vivo effects of ethoxy and isopropoxy analogs were fully or partially reversed by naloxone (0.3 mg/kg). PYR nitazenes with intermediate alkoxy chain lengths are most potent for eliciting opioid-like effects in vitro and in mice. In particular, the ethoxy, propoxy, and isopropoxy analogs are more potent than fentanyl, highlighting an elevated risk for overdose with these compounds. SIGNIFICANCE STATEMENT: The present study characterized the pharmacology of N-pyrrolidino nitazene opioids that vary by alkoxy chain length and are associated with overdose cases in humans. The ethoxy, isopropoxy, and propoxy N-pyrrolidino nitazenes were found to be more potent than fentanyl in both in vitro and in vivo assays, suggesting that these compounds pose a serious overdose risk to humans who are unknowingly exposed to the compounds in the illicit opioid supply.

The ongoing psychedelic renaissance has prompted a renewed search for novel drugs based on modifying existing psychedelic motifs. In this study, a synthesis of 4-bromo-N,N-dimethyltryptamine (4-Br-DMT) was achieved and used as a late-stage intermediate for analog development. Specifically, we were able to synthesize and access novel molecules by forming carbon-carbon bonds at the C4 position using palladium cross-coupling reactions. Using the synthetic route described here will facilitate the rapid development of a library of tryptamines with novel 4-position substitutions to investigate structure-activity relationships (SARs) with serotonergic targets. Furthermore, the pharmacological target profile and biological effects of 4-Br-DMT were compared to the 4-hydroxy-N,N-dimethyltryptamine (psilocin) and nonring-substituted analog, N,N-dimethyltryptamine (DMT). Overall, the data indicate that 4-Br-DMT has a serotonergic profile without psychedelic-like effects in mice but has a reduced safety profile compared to psilocin and DMT.

Tryptamine psychedelics induce psychoactive effects via agonist actions at serotonin 2A receptors (5-HT2A), but the compounds are generally nonselective. 4-Methoxy-N-methyl-N-isopropyltryptamine (4-MeO-MiPT) is a 5-HT2A agonist which also blocks the 5-HT transporter (SERT) and has blunted visual and other psychedelic effects in humans. Here, we compared the pharmacology of 4-MeO-MiPT, its 4-hydroxy derivative (4-HO-MiPT), and related analogs with N-alkyl or 4-alkoxy variations. We hypothesized that compounds with more potent SERT uptake inhibition would display reduced 5-HT2A-mediated psychedelic-like effects in the mouse head twitch response (HTR) model. In vitro target profiling revealed potent and efficacious 5-HT receptor activities for most of the compounds, including 5-HT2A receptor agonism (EC50 = 10-118 nM, Emax = 72-97% 5-HT). Importantly, 4-MeO-MiPT and its N,N-diisopropyl (4-MeO-DiPT) and N-methyl-N-cyclopropyl (4-MeO-McPT) analogs displayed more potent uptake inhibition at SERT (IC50 = 17-107 nM) than their 4-OH counterparts (IC50 = 280-423 nM). Studies administering the drugs subcutaneously to C57BL/6J mice revealed that 4-HO- and 4-MeO-MiPT (0.03-30 mg/kg) had similar potencies for inducing HTRs (ED50 = 0.75 vs 0.97 mg/kg), but 4-MeO-MiPT had reduced efficacy (Emax = 77 vs 34 HTRs/30 min). A similar trend for decreased HTRs was observed for 4-MeO-DiPT and 4-MeO-McPT. Pretreatment with the SERT inhibitor fluoxetine (10 mg/kg) prior to 4-HO-MiPT, 4-HO-DiPT, or 4-HO-McPT reduced the maximal number of HTRs to levels observed for their respective 4-MeO analogs. Overall, our data indicate that 4-MeO-MiPT interacts with 5-HT2A and other 5-HT receptors, but the drug also inhibits SERT to reduce the efficacy of psychedelic-like effects in mice. Therefore, 4-MeO-MiPT and other dual 5-HT2A/SERT ligands may be therapeutically relevant compounds with reduced potential for traditional acute psychedelic effects.

Serotonergic psychedelics induce the head twitch response (HTR) in mice, an index of serotonin (5-HT) 2A receptor (5-HT2A) agonism and a behavioral proxy for psychedelic effects in humans. Existing methods for detecting HTRs include time-consuming visual scoring, magnetometer-based approaches, and analysis of videos using semi-automated commercial software. Here, we present a new automated approach for quantifying HTRs from experimental videos using the open-source machine learning-based toolkits, DeepLabCut (DLC) and Simple Behavioral Analysis (SimBA). Pose estimation DLC models were trained to predict X,Y coordinates of 13 body parts of C57BL/6J mice using historical experimental videos of HTRs induced by various psychedelic drugs. Next, a nonoverlapping set of historical experimental videos was analyzed and used to train SimBA random forest behavioral classifiers to predict the presence of the HTR. The DLC + SimBA approach was then validated using a separate subset of visually scored videos. DLC + SimBA model performance was assessed at different video resolutions (50%, 25%, 12.5%) and frame rates (120, 60, 30 frames per second or fps). Our results indicate that HTRs can be quantified accurately at 50% resolution and 120 fps (precision = 95.45, recall = 95.56, F 1 = 95.51) or at lower frame rates and resolutions (i.e., 50% resolution and 60 fps). The best performing DLC + SimBA model combination was deployed to evaluate the effects of bufotenine, a tryptamine derivative with uncharacterized potency and efficacy in the modern HTR paradigm. Interestingly, bufotenine only induced elevated HTRs (ED50 = 0.99 mg/kg, max counts = 24) when serotonin 1A receptors (5-HT1A) were pharmacologically blocked and activity at other sites of action may also impact its pharmacological effects (e.g., serotonin transporter). HTR counts for a subset of 21 videos from bufotenine experiments were strongly correlated for DLC + SimBA vs visual scoring and semi-automated software detection methods (r = 0.98 and 0.99). Finally, the DLC + SimBA approach displayed high accuracy when compared to visual scoring of HTRs for three serotonergic psychedelic drugs with variable HTR frequencies (r = 0.99 vs mean visual scores from 3 blinded raters). In summary, the DLC + SimBA approach represents a modular, noninvasive, and open-source method of HTR detection from experimental videos with accuracy comparable to magnetometer-based approaches and greater speed than visual scoring.

The human serotonin transporter (SERT) is a pertinent target for many psychiatric therapeutics and recreational drugs including new psychoactive substances. Current techniques to assess SERT inhibition typically monitor the uptake of radiolabeled or fluorescent substrates but face limitations regarding the use of nonendogenous substrates and/or low throughput. Inspired by a previously reported principle, coined 'TRACT' or 'transporter activity through receptor activation', a new bioassay to measure SERT inhibition was developed. The assay principle relies on SERT-mediated uptake of supplied serotonin (5-HT), precluding 5-HT-induced activation of a coexpressed modified serotonin 2A receptor (5-HT2AR). Upon SERT inhibition, more extracellular 5-HT is available for 5-HT2AR-activation, which is monitored via β-arrestin 2 recruitment, using functional complementation of a split-nanoluciferase and a luminescent read-out. Following optimization, the assay was successfully applied to three classes of antidepressants and the designer cathinone naphyrone. Obtained inhibition potencies ranged from 2.61 nM for paroxetine to 348 nM for naphyrone, and good agreement in rank order was obtained when compared with the outcome of a fluorescence-based and a radioactivity-based uptake assay. Our findings demonstrate that the TRACT assay principle is translatable to SERT, allowing the assessment of SERT inhibition via 5-HT2AR activation. Additionally, for amitriptyline and amoxapine, the TRACT assay indicated direct (non-SERT mediated) 5-HT2AR-modulating effects, caused by inverse agonism, as confirmed by the 5-HT2AR βarr2 recruitment assay. We also demonstrate a proof-of-principle for applying this system to detect SERT-mediated efflux. Overall, our findings support the utility of the TRACT assay for the characterization of SERT ligands.

Prolintane is a synthetic stimulant that acts by inhibiting the uptake of dopamine and norepinephrine into neurons. Initially prescribed for attention deficit hyperactivity disorder and narcolepsy, its medical use was discontinued due to concerns about abuse liability. Here, we explored structure-activity relationships for novel fluoro and methyl-ring-substituted prolintanes synthesized via a modified one-pot Mannich Barbier reaction. Radiotracer flux assays in transfected human embryonic kidney 293 (HEK293) cells and rat brain synaptosomes revealed that prolintane analogs display potent uptake inhibition at the dopamine transporter (DAT) and norepinephrine transporter (NET), with weaker effects at the serotonin transporter (SERT). Across all compounds, SERT inhibitory potencies were at least 10-fold weaker at human SERT (hSERT) compared to rat SERT (rSERT). Methyl substitution at the 2-, 3-, or 4-ring position enhanced SERT inhibition potency relative to DAT, lowering the DAT/SERT ratio and suggesting reduced abuse liability. Fluorine substitution also enhanced SERT potency relative to DAT, however, to a lesser extent. Interestingly, prolintane and its analogs induced hSERT-mediated ionic currents and [3H]serotonin efflux, which was not seen in rat brain synaptosomes. Overall, these findings indicate that prolintane analogs act as potent DAT and NET inhibitors, but they can also act as substrates and evoke serotonin release in cells expressing hSERT, classifying them as hybrid compounds at human monoamine transporters. Our study demonstrates how ring modifications alter prolintane pharmacology, emphasizing the need for future investigations into therapeutic and adverse effects of these compounds. Moreover, the species difference in SERT-releasing activity for prolintane analogs warrants further research.

Ibogaine is the main psychoactive alkaloid produced by the iboga tree (Tabernanthe iboga) that has a unique therapeutic potential across multiple indications, including opioid dependence, substance use disorders, depression, anxiety, posttraumatic stress disorder (PTSD), and traumatic brain injury (TBI). We systematically examined the effects of ibogaine, its main metabolite noribogaine, and a series of iboga analogs at monoamine neurotransmitter transporters, some of which have been linked to the therapeutic effects of these substances. We report that ibogaine and noribogaine inhibit the transport function of the vesicular monoamine transporter 2 (VMAT2) with submicromolar potency in cell-based fluorometry assays and at individual synaptic vesicle clusters in mouse brain as demonstrated via two-photon microscopy. Examining the uptake and release of radiolabeled serotonin in isolated brain synaptic vesicles, we confirmed that ibogaine and noribogaine act as inhibitors of VMAT2 and showed that noribogaine induces partial serotonin release from synaptic vesicles. Noribogaine does not compete with the binding of dihydrotetrabenazine, an established VMAT2 inhibitor, indicating that noribogaine engages the VMAT2 transporter at a distinct binding site or conformational state. The iboga compounds also inhibit the plasma membrane monoamine transporters (MATs), prominently including the serotonin transporter (SERT), and a novel iboga target, the organic cation transporter 2 (OCT2). SERT transport inhibition was demonstrated in serotonin axons and somata in mouse brain slices and in rat brain synaptosomes, where ibogaine and its analogs did not act as substrate-type serotonin releasers. Noribogaine, oxa-noribogaine, and several analogs displayed dual inhibition of VMAT2 and SERT with comparable potencies, a relatively uncommon activity in the pharmacopoeia of monoamine transporter inhibitors, and were hence labeled as "Synaptic Reuptake Inhibitors" ("SynRIs"). The SynRI profile provides an explanatory model for previously reported neurochemical effects of ibogaine in rodents. Together, the updated profile of the monoamine transporter modulation offers insight into the grand complexity of the iboga pharmacology, which we termed "matrix pharmacology". The matrix pharmacology hypothesis is outlined and used to conceptualize why ibogaine and noribogaine do not induce catalepsy, as demonstrated in our study, in contrast to other VMAT2 inhibitors.

Identification of N,N-dimethylpentylone (DMP) in counterfeit "Ecstasy" and "Molly" tablets poses risk to public health due to its adverse effects. Little information is available regarding the pharmacological activity or relevant blood or tissue concentrations of DMP, and even less is known about other structurally related beta-keto methylenedioxyamphetamine analogs on recreational drug markets, such as N-propyl butylone. Here, a novel toxicological assay utilizing liquid chromatography-tandem quadrupole mass spectrometry was developed and validated for the quantitation of DMP and five related synthetic cathinones [eutylone, pentylone, N-ethyl pentylone (NEP), N-propyl butylone, and N-cyclohexyl butylone], with chromatographic resolution from isomeric variants and quantitation performed by standard addition. A forensic series of 125 cases is presented for DMP and related analogs, along with pharmacological activity assessments using monoamine transporter and mouse behavioral assays. The blood concentration range for DMP in postmortem forensic cases was 3.3-4600 ng/mL (mean: 320 ± 570 ng/mL, median: 150 ng/mL), whereas pentylone, the primary N-desmethyl metabolite of DMP, was identified in 98% of cases with a concentration range 1.3-710 ng/mL (mean ± SD: 105 ± 120 ng/mL, median: 71 ng/mL). N-Propyl butylone, a newly identified synthetic cathinone, was quantitated in seven cases (mean ± SD: 82 ± 75 ng/mL, median: 50 ng/mL, range: 1.7-200 ng/mL). DMP displayed potent uptake inhibition at the dopamine transporter [half maximal inhibitory concentration (IC50) of 49 nM], with 100-fold weaker potency at the serotonin transporter (IC50 = 4990 nM). DMP was a locomotor stimulant in mice [medium effective dose (ED50) of 3.5 mg/kg] exhibiting potency relatively similar to eutylone, NEP, and pentylone. Our results show that DMP is a psychomotor stimulant associated with adverse clinical outcomes leading to death. Forensic laboratories must continue to update testing methods to capture emerging drugs, with specific emphasis on resolution and identification of isomeric species. Following the scheduling of DMP in early 2024, there could be an anticipated market shift toward a new unregulated synthetic stimulant to replace DMP.

2-Benzylbenzimidazole derivatives or 'nitazenes' are increasingly present on the recreational drug market. Here, we report the synthesis and pharmacological characterization of 15 structurally diverse nitazenes that might be predicted to emerge or grow in popularity. This work expands the existing knowledge about 2-benzylbenzimidazole structure-activity relationships (SARs), while also helping stakeholders (e.g., forensic toxicologists, clinicians, policymakers) in their risk assessment and preparedness for the potential next generation of nitazenes. In vitro µ-opioid receptor (MOR) affinity was determined via competition radioligand (3[H]DAMGO) binding assays in rat brain tissue. MOR activation (potency and efficacy) was studied by means of a cell-based β-arrestin 2 recruitment assay. For seven nitazenes, including etonitazene, opioid-like pharmacodynamic effects (antinociception, locomotor activity, body temperature changes) were evaluated after subcutaneous administration in male C57BL/6 J mice. The results showed that all nitazenes bound to MOR with nanomolar affinities, and the functional potency of several of them was comparable to or exceeded that of fentanyl. In vivo, dose-dependent effects were observed for antinociception, locomotor activity, and body temperature changes in mice. SAR insights included the high opioid-like activity of methionitazene, iso-butonitazene, sec-butonitazene, and the etonitazene analogues 1-ethyl-pyrrolidinylmethyl N-desalkyl etonitazene and ethylene etonitazene. The most potent analogue of the panel across all functional assays was α'-methyl etonitazene. Taken together, through critical pharmacological evaluation, this work provides a framework for strengthened preparedness and risk assessments of current and future nitazenes that have the potential to cause harm to users.

3,4-Methylenedioxymethamphetamine (MDMA) has shown efficacy as a medication adjunct for treating post-traumatic stress disorder (PTSD). However, MDMA is also used in nonmedical contexts that pose risk for cardiovascular and neurologic complications. It is well established that MDMA exerts its effects by stimulating transporter-mediated release of the monoamines 5-hydroxytryptamine (5-HT), norepinephrine, and dopamine. Current research efforts are aimed at developing MDMA-like monoamine releasers with better efficacy and safety profiles. To this end, we investigated neurochemical and behavioral effects of novel analogs of the designer drug 5-(2-methylaminopropyl)benzofuran (5-MAPB). We used in vitro transporter assays in rat brain synaptosomes to examine transmitter uptake inhibition and releasing properties for enantiomers of 5-(2-methylaminobutyl)benzofuran (5-MABB) and 6-(2-methylaminobutyl)benzofuran (6-MABB) compared with MDMA. We then tested these same compounds in male Sprague-Dawley rats trained to discriminate MDMA (1.5 mg/kg) from saline. In vitro results revealed that S isomers of 5- and 6-MABB are efficacious releasing agents at transporters for 5-HT (SERT), norepinephrine (NET), and dopamine (DAT). By contrast, R isomers are efficacious releasers at SERT and partial releasers at NET but lack releasing activity at DAT. In vivo results showed that all compounds produce dose-dependent increases in MDMA-lever responding and full substitution at the highest dose tested. The diminished NET and DAT releasing activities for R isomers of 5- and 6-MABB are associated with reduced potency for inducing behavioral effects. Collectively, these findings indicate that the aminoalkyl benzofuran scaffold may be a viable template for developing compounds with MDMA-like properties. SIGNIFICANCE STATEMENT: Despite the clinical utility of 3,4-methylenedioxymethamphetamine (MDMA), the drug is associated with certain cardiovascular risks and metabolic side effects. Developing a therapeutic alternative with MDMA-like monoamine releasing activity is of interest. Our in vitro and in vivo findings indicate that the aminoalkyl benzofuran scaffold may be useful for developing compounds with MDMA-like properties.

Select Drug Category Opiates/OpioidsTopic Molecular PharmacologyAbstract Detail Preclinical - In VitroAbstract Category Original Research Aim New synthetic opioids continue to emerge on recreational drug markets. Recently, opioids with a 2-benzylbenzimidazole core (‘nitazenes’, e.g. isotonitazene) have become increasingly prevalent, the potency of some members dwarfing that of fentanyl. The aim of our work is to in vitro and in vivo characterize existing, as well as 'prophetic' nitazenes, to allow risk prioritization based on structure activity relationships. As a case example, the pharmacological characterization of ethyleneoxynitazene, which we predicted to emerge, and which was first found in January 2023, will be presented. Methods In vitro pharmacological characterization (experiments performed in quintuplicate) encompassed assessment of mu opioid receptor (MOR) activation via a β-arrestin2 recruitment assay to derive the potency and efficacy, as well as radioligand binding assays performed in rat brain tissue. Pharmacodynamic effects were evaluated in male Sprague Dawley rats and included assessment of antinociceptive, cataleptic, and thermic effects. Results Radioligand binding assays revealed a Ki of 57.9nM at MOR; only slightly higher than the Ki of etonitazene (38.4nM), the most potent nitazene. Despite a similar affinity, ethyleneoxynitazene had a >100-fold lower potency in the MOR-β-arrestin2 recruitment assay (EC50 etonitazene 0.588nM; ethyleneoxynitazene 70nM). Also its efficacy (relative to the reference hydromorphone) was lower than that of etonitazene (Emax 187% vs. 254%). The strongly reduced MOR activation potential was also evident from the in vivo antinociception (mouse hot plate) assay, with an ED50 of 0.0223mg/kg and 11.1mg/kg for etonitazene and ethyleneoxynitazene, repectively. The hypothermia and catalepsy assays revealed the same pattern. Conclusions The a priori availability of pharmacological (in vitro and in vivo) data by the time the 'prophetic' opioid ethyleneoxynitazene hit the recreational drug market allowed us to predict that, compared to several other nitazenes, this is not the opioid of highest concern. Similarly, pharmacological data for other 'prophetic' nitazenes are now readily available.

Introduction: 2-Benzylbenzimidazole opioids (‘nitazenes’) have become increasingly prevalent on the recreational drug market. We performed pharmacological characterization of various 'prophetic' nitazenes, allowing risk prioritization based on structure-activity relationships. Ethyleneoxynitazene, which we predicted to emerge and which was first found in January 2023, is presented as a case example. Methods: In vitro pharmacological characterization encompassed radioligand binding assays in rat brain tissue and a cell-based µ-opioid receptor activation (MOR-β-arrestin2) assay. Antinociception (hot plate assay), locomotor activity, and thermic effects were evaluated after subcutaneous administration in C57BL/6J mice. Results: Binding assays revealed a Ki of 57.9 nM; only slightly higher than that of etonitazene (38.4 nM). However, ethyleneoxynitazene had a >100-fold lower potency in the MOR-β-arrestin2 assay (EC50, ethyleneoxynitazene=70.0 nM; EC50, etonitazene=0.588 nM). Its efficacy (relative to the reference hydromorphone) was also lower (Emax,ethyleneoxynitazene=187% vs. Emax,etonitazene=254%). The strongly reduced activity was reflected in vivo, with an ED50, antinociception of 11.1 mg/kg and 0.0223 mg/kg for ethyleneoxynitazene and etonitazene, respectively. Hypothermia and locomotor assays revealed the same pattern. Conclusions: The a priori availability of pharmacological data upon the first emergence of ethyleneoxynitazene allowed to rapidly communicate that (compared to other nitazenes) this is likely not the opioid of highest concern. Similarly, pharmacological data for other anticipated nitazenes are readily available.