1,2-Diarylethylamines including lanicemine, lefetamine, and remacemide have clinical relevance in a range of therapeutic areas including pain management, epilepsy, neurodegenerative disease and depression. More recently 1,2-diarylethylamines have been sold as ‘legal highs’ in a number of different forms including powders and tablets. These compounds are sold to circumvent governmental legislation regulating psychoactive drugs. Examples include the opioid MT-45 and the dissociative agents diphenidine (DPH) and 2-methoxy-diphenidine (2-MXP). A number of fatal and non-fatal overdoses have been linked to abuse of these compounds. As with many ‘legal highs’, little is known about their pharmacology. To obtain a better understanding, the effects of DPH, 2-MXP and its 3- and 4-MeO- isomers, and 2-Cl-diphenidine (2-Cl-DPH) were investigated using binding studies at 46 central nervous system receptors including the N-methyl-D-aspartate receptor (NMDAR), serotonin, dopamine, norepinephrine, histamine, and sigma receptors as well as the reuptake transporters for serotonin, dopamine and norepinephrine. Reuptake inhibition potencies were measured at serotonin, norepinephrine and dopamine transporters. NMDAR antagonism was established in vitro using NMDAR-induced field excitatory postsynaptic potential (fEPSP) experiments. Finally, DPH and 2-MXP were investigated using tests of pre-pulse inhibition of startle (PPI) in rats to determine whether they reduce sensorimotor gating, an effect observed with known dissociative drugs such as phencyclidine (PCP) and ketamine. The results suggest that these 1,2-diarylethylamines are relatively selective NMDAR antagonists with weak off-target inhibitory effects on dopamine and norepinephrine reuptake. DPH and 2-MXP significantly inhibited PPI. DPH showed greater potency than 2-MXP, acting with a median effective dose (ED 50 ) of 9.5 mg/kg, which is less potent than values reported for other commonly abused dissociative drugs such as PCP and ketamine.

Funding: The authors gratefully acknowledge support of the USA NIH NIMH Psychoactive Drug Screening Program for the screening of the compounds in receptors and the monoamine transporter assays. Dr. Halberstadt was supported by NIMH (MH100644) and NIDA (DA002925).

Copyright: © 2016 Wallach et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To extend earlier reports of NMDAR binding, competitive binding experiments with [ 3 H]-MK-801 were performed with DPH and 2-MXP, along with the methoxy- substituted positional isomers 3-methoxy-diphenidine (3-MXP) and 4-methoxy-diphenidine (4-MXP) as well as 2-Cl-diphenidine (2-Cl-DPH) ( Fig 1 ). The NMDAR antagonists, PCP, ketamine, (+)-MK-801 and memantine served as reference compounds. NMDAR selectivity was investigated using binding studies at an additional 45 CNS receptor sites including G protein-coupled receptors (serotonin, dopamine, norepinephrine, histamine, acetylcholine subtypes), monoamine reuptake transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT), mu (MOR), kappa (KOR) and delta (DOR) opioid receptors and sigma-1 and sigma-2 receptor sites. Compounds were also evaluated for inhibition of monoamine reuptake to establish the functional consequences of the observed interactions with monoamine reuptake transporters. To measure functional activity at central synapses in vitro, the target compounds along with reference compounds were assessed on NMDA receptor-mediated field excitatory postsynaptic potentials (NMDAR-fEPSP). Finally, in vivo pre-pulse inhibition (PPI) experiments were performed with DPH and 2-MXP.

Aside from some NMDAR binding studies [ 17 , 18 ] and a recent publication about the metabolism of DPH [ 19 ], little information has been published regarding the pharmacology of the dissociative ‘research chemicals’ DPH and 2-MXP. Due to the increasing appearance of 1,2-diarylethylamine based ‘research chemicals’ [ 11 – 13 ] along with reports of overdoses [ 20 ] and fatal intoxications [ 21 ] it is important to investigate the pharmacology of these compounds.

The phenomenology of the altered state induced by dissociative drugs is complex and dose dependent. However, key features of the dissociative state include sensory hallucinations, tactile distortions, euphoria, derealization and depersonalization [ 13 ]. A significant portion of the therapeutic and psychoactive effects of dissociative drugs is believed to be mediated through NMDAR antagonism [ 13 , 15 , 16 ]. Although NMDAR antagonism appears to be a common denominator involved in the dissociative pharmacology, additional receptors are likely to contribute to the effects of individual compounds [ 16 ].

Most recently, a variety of 1,2-diarylethylamines have appeared as ‘legal highs’ or ‘research chemicals’ from online vendors. These include the opioid MT-45 [ 9 , 10 ] and several dissociative agents such as diphenidine (DPH) and 2-methoxydiphenidine (methoxphenidine or 2-MXP) [ 11 – 13 ]. The dissociative diarylethylamines emerged as ‘legal highs’ or ‘research chemicals’, to circumvent regulations on human consumption, shortly following a United Kingdom ban (February 2013) on arylcyclohexylamine-based dissociative drugs such as methoxetamine (MXE) and 3-methoxyphencyclidine (3-MeO-PCP) [ 13 , 14 ]. Though DPH and 2-MXP were new to this market and had no previously documented history of human use, syntheses had been published as early as 1924 and 1989 respectively and both had undergone in vitro screening for NMDAR affinity.[ 13 ] Images of products sold online are provided as supporting information ( S11 – S13 Figs).

1,2-Diarylethylamines represent a structural class of organic molecules, which all share a core structure comprised of an ethylamine nucleus with vicinal aromatic substitutions. These compounds have diverse pharmacology and modifications of this structure have yielded analgesics, antidepressants, anticonvulsants and neuroprotective agents [ 1 – 3 ]. Their pharmacology appears to be mediated through a range of interactions including activation of opioid receptors [ 2 , 4 , 5 ], inhibition of monoamine transporters [ 6 , 7 ] and antagonism of glutamatergic N-methyl-D-aspartate receptors (NMDARs) [ 1 , 8 ]. Clinical interest in 1,2-diarylethylamines includes the well-tolerated NMDAR antagonist lanicemine, which exhibits antidepressant activity[ 1 ], and remacemide, which has shown promise in clinical trials for several therapeutic areas including neurodegenerative diseases, epilepsy and stroke [ 3 ]. Structures of some of these are given as supporting information ( S1 Fig ).

Male Wistar rats (Crl:Wi; Charles River, UK) aged 9–10 wk were sacrificed by neck vertebral dislocation (schedule 1 method) according to the United Kingdom (Scientific Procedures) Act of 1986. After their rapid removal, brains were placed in artificial cerebrospinal fluid (aCSF) consisting of 124 mM NaCl, 26 mM NaHCO 3 , 3 mM KCl, 1.4 mM NaH 2 PO4, 1 mM MgSO 4 , 2 mM CaCl 2 , and 10 mM D-glucose and continuously oxygenated with 95% O 2 and 5% CO 2 . The brain was cut parasagittally into 400 μm sections and hippocampal slices were removed, stored and placed in a submerged recording chamber at 28–30°C. Recordings of synaptic activity were made, analyzed and presented as described [ 29 , 30 ]. A bipolar electrode was used to deliver stimuli (0.03 Hz) to the Schaffer collateral pathway to enable recording of field excitatory synaptic potentials (fEPSPs) using a glass microelectrode positioned in the stratum radiatum of area CA1. The NMDA receptor-mediated component of the fEPSP (NMDAR-fEPSP) was revealed by adding 10 μM NBQX, 50 μM picrotoxin and 1 μM CGP 55845 to the aCSF, which abolished AMPA and GABA receptor mediated transmission. After 30 min of stable control responses, 1 and 10 μM solutions of the individual compounds were added to the perfusate for 3–12 h while recording the amplitude and area of the NMDAR-fEPSP on-line using WinLTP [ 31 ]. Single exponential curves were fitted to the graphs and curves extrapolated to show likely half-times and minimum plateau responses achieved with each concentration of the compounds. Raw data for all fEPSP experiments are provided as supporting information ( S2 , S3 and S4 Files ).

Monoamine reuptake inhibition assays were performed via the NIMH PDSP as previously described.[ 28 ] In brief, the neurotransmitter transporter uptake assay kit (R8174) (Molecular Devices) was used. Human monoamine transporters (DAT, NET and SERT) were stably expressed in HEK293 cells. Assay buffer consisted of 20 mM HEPES, 1x HBSS at pH 7.4. Cells were plated in Pol-L-Lys (PLL) coated 384-well black clear bottom cell culture plates with a density of 15,000 cells/well. Cells were incubated for at least 6 h prior to assay. Media was removed and replaced with 20 μL assay buffer and 5 μL of drug or positive control (cocaine or nisoxetine) as a 5X stock. Cells were incubated for 30 min at 37°C. 25 μL of dye solution was then added and following an additional 30 min incubation (at 37°C) fluorescence intensity was measured using a FlexStation II (bottom read mode, excitation wavelength = 440 nM, emission = 520 nM with a cutoff of 510 nM). Relative florescence units were exported and plotted against drug concentration to obtain 50% inhibition potencies (IC 50 ) using non-linear regression (Prism 5.0). Additional details are available in the NIMH PDSP assay protocol book [ 27 ].

Radioligands and concentrations used for additional 45 CNS receptor binding assays are listed as supporting information ( S1 Table ). This work was performed by the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH PDSP) as described previously.[ 26 ] Briefly target compounds were dissolved in DMSO and subjected to a primary screen at 10,000 nM concentration. Compounds exhibiting >50% inhibition were subjected to a secondary assay at varying concentrations to determine K i values. Additional experimental details are available in the NIMH PDSP assay protocol book [ 27 ].

In vitro binding affinities (K i ) of the target compounds at the PCP site within the NMDAR channel were determined using competitive radioligand binding studies with [ 3 H]-MK-801 in accordance with established protocols published by Reynolds and Sharma [ 22 , 23 ]. Thoroughly washed rat forebrain homogenate were used as the NMDAR source (whole brain obtained from Pel-Freez Biologicals) and prepared as described by Reynolds and Sharma [ 22 ]. Suspensions of 10 mM HEPES buffer (pH 7.4 at room temperature) containing 100 μg/mL protein, 1 nM (+)-[ 3 H]-MK-801, 100 μM glutamate, 10 μM glycine, and various concentrations of unlabeled competitor or 30 μM (+)-MK-801 for nonspecific binding (and positive control), were incubated in the dark on a mechanical rocker at room temperature for 2 h. The reaction was terminated by vacuum filtration using a 24 well cell harvester (Brandel, Gaithersburg, MD) over presoaked GF/B glass fiber filters (Brandel, Gaithersburg, MD). Filters were washed with room temperature assay buffer (3 x 5 mL). Tritium trapped on the filter was measured via liquid scintillation counting, using a Beckman LS 6500 multipurpose scintillation counter (BeckmanCoulter, USA) at 57% efficiency. IC 50 values were determined in Graphpad Prism 5.0 using non-linear regression with log-concentration plotted against percent specific binding. Percent specific binding for [ 3 H]-MK-801 in a control experiment was ~95%. K i values were calculated using the equation of Cheng and Prusoff [ 24 ]. The K d for (+)-MK-801 (1.747 nM), was determined via homologous binding assay as described by Reynolds and Sharma and is consistent with the literature [ 22 ]. Protein concentration was determined via the Bradford method using Coomassie protein assay reagent (Sigma, USA) [ 25 ] with rat albumin (Sigma, USA) as standard. Experiments were performed in duplicate and repeated a minimum of three times. Raw counts per minute (CPM) for all NMDAR binding experiments are provided as supporting information ( S1 File ).

Synthesis and analytical characterizations of the target 1,2-diarylethylamines have been published elsewhere [ 11 , 12 ]. The exception is 2-Cl-DPH, which was not described previously. Details of the synthesis and analytical characterization of 2-Cl-DPH are provided as supporting information ( S6 File ).

The amount of PPI was calculated as a percentage score for each prepulse + pulse trial type: %PPI = 100−{[(startle response for prepulse + pulse trial)/(startle response for pulse-alone trial)] × 100}. Startle magnitude was calculated as the average response to all of the pulse-alone trials. PPI data were analyzed with two-factor analysis of variance (ANOVA) with treatment as the between-subjects factor and trial type (prepulse intensity) as a repeated measure. For experiments in which there was no significant interaction between drug and prepulse intensity, PPI data were collapsed across prepulse intensity and average PPI was used as the main dependent measure. ED 50 values were calculated using nonlinear regression. Startle magnitude data were analyzed with one-factor ANOVA. Post-hoc analyses were performed using Tukey’s studentized range method. The alpha level was set at 0.05.

Acoustic startle test sessions consisted of startle trials (pulse-alone) and prepulse trials (prepulse + pulse). The pulse-alone trial consisted of a 40-ms 120-dB pulse of broadband white noise. Prepulse + pulse trials consisted of a 20-ms acoustic prepulse, an 80-ms delay, and then a 40-ms 120-dB startle pulse (100-ms onset–onset). There was an average of 15 s (range = 6–22 s) between trials. During each inter-trial interval, the movements of the animals were recorded once to measure response when no stimulus was present (data not shown). Each startle session began with a 5 min acclimation period to a 65-dB broadband noise that was present continuously throughout the session. One week after arrival, animals were tested in a brief baseline startle/PPI session to create treatment groups matched for levels of startle and PPI. The startle test session contained 12 pulse-alone trials and 36 prepulse + pulse trials (12 prepulses each of 68-, 71-, and 77-dB [equivalent to 3-, 6-, and 12-dB above background]) presented in a pseudo-randomized order. Six pulse-alone trials were presented at the beginning and the end of the test session but were not used in the calculation of PPI values. Raw data for all PPI experiments are provided as supporting information ( S5 File ).

Eight startle chambers (SR-LAB system, San Diego Instruments, San Diego, CA, USA) were used to measure startle reactivity in rats [ 32 , 33 ]. The startle test chambers were sound-attenuated, lighted, and ventilated enclosures containing a clear nonrestrictive cylindrical Plexiglas stabilimeter, 8.2 cm in diameter. A high-frequency loudspeaker mounted 24 cm above the Plexiglas cylinder produced all acoustic stimuli. The peak and average amplitudes of the startle response were detected by a piezoelectric accelerometer. At the onset of the startling stimulus, 100 1-ms readings were recorded, and the average amplitude was used to determine the magnitude of the startle response (measured in arbitrary units). A dynamic calibration system was employed to ensure comparable stabilimeter sensitivity across test chambers, and sound levels were measured using the dB(A) scale.

DPH hydrochloride and 2-MXP hydrochloride were dissolved in water containing 5% Tween-80 and administered subcutaneously, 10-min prior to the start of the startle test session. The injection volume was 1 ml/kg.

Male Sprague–Dawley rats from Harlan Industries (Indianapolis, IN, USA; initial weight 250–275 g) were housed in pairs under a 12-h reverse light/dark cycle (lights off at 0700 h). Use of a reversed light/dark cycle allows behavioral testing to be conducted during the awake phase of the light/dark cycle. Food and water were available ad libitum. Animals were acclimatized for approximately 1 wk after arrival prior to behavioral testing and maintained in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-approved facilities that meet all federal and state guidelines. Procedures were approved by the University of California San Diego institutional animal care and use committee. Principles of laboratory animal care were followed.

Results and Discussion

Receptor Binding affinities NMDAR binding affinities for the test compounds are presented in Table 1. [3H]-MK-801 displacement curves for the five 1,2-diphenylethylamines and reference compounds are provided as supporting information (S3 and S4 Figs respectively). Four of the five compounds had potent nM affinity for [3H]-MK-801 labeled NMDARs. 4-MXP had lower NMDAR affinity (461 nM) compared to the other members of the series. The rank order of potency of DPH and its three MeO- isomers (DPH = 3-MXP > 2-MXP > 4-MXP) is comparable with that seen with the equivalently substituted arylcyclohexylamines; with PCP = 3-MeO-PCP > 2-MeO-PCP > 4-MeO-PCP [34]. The parallel structure activity relationships (SAR) between aryl-methoxy substituted derivatives of PCP and DPH is not surprising given the structural overlap between the benzyl-piperidine portions of PCP and DPH (Fig 1). Therefore, the overlapping portions of arylcyclohexylamines and 1,2-diphenethylamines likely bind to the same region of the PCP binding site within the NMDAR channel. SAR studies on arylcyclohexylamines may have relevance for further optimization of 1,2-diarylethylamine-based NMDAR antagonists. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Table 1. NMDAR binding affinities for five target 1,2-diphenylethylamines and reference compounds https://doi.org/10.1371/journal.pone.0157021.t001 Binding affinities for the test compounds at NMDAR were described previously in a patent by Gray and Cheng [17]. Notably, in most cases the NMDAR binding affinities reported herein differed from those earlier studies. The rank order of potencies were, however, consistent. Surprisingly, Gray and Cheng reported 2-Cl-DPH to have pM affinity, an order of magnitude higher than other known NMDAR antagonists. This report of high affinity binding reported for 2-Cl-DPH prompted us to reinvestigate this compound in the present study. While in the current study 2-Cl-DPH had potent low nM affinity for NMDAR (9.3 nM), this is substantially less than the pM affinity reported previously. In a study by Berger et al., the two DPH enantiomers showed lower affinity than that reported both here and by Gray and Cheng ((S-)-DPH 120 nM, (R)-DPH 5,250 nM) [18]. Likewise, the reported affinity of PCP was ~4-fold lower than that observed here and reported by others [35]. These discrepancies may result from the fact that [3H]-TCP was used by Gray and Cheng while the more selective [3H]-MK-801 [36–39] was used in the current study. Berger et al. used [3H]-MK-801 to label NMDAR from a different tissue source (whole cell membranes prepared from rat cortex and hippocampus) than the current study (rat forebrain homogenate) [18]. Differences in affinities of the compounds for different NMDAR subunit combinations might be one possible explanation.