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Colestock, C, Wallach, J, Mansi, M, Filemban, N, Morris, H, Elliott, SP, Westphal, F, Brandt, SD and Adejare, A
Syntheses, analytical and pharmacological characterizations of the “legal high” 4-[1-(3-methoxyphenyl)cyclohexyl]morpholine (3-MeO-PCMo) and analogues http://researchonline.ljmu.ac.uk/id/eprint/6391/
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Colestock, C, Wallach, J, Mansi, M, Filemban, N, Morris, H, Elliott, SP, Westphal, F, Brandt, SD and Adejare, A (2017) Syntheses, analytical and pharmacological characterizations of the “legal high” 4-[1-(3- methoxyphenyl)cyclohexyl]morpholine (3-MeO-PCMo) and analogues. Drug
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Syntheses, analytical and pharmacological characterizations of the “legal high” 4-[1-(3- methoxyphenyl)cyclohexyl]morpholine (3-MeO-PCMo) and analogues
For Peer Review Journal: Drug Testing and Analysis
Manuscript ID DTA-17-0089.R1
Wiley - Manuscript type: Research Article
Date Submitted by the Author: 05-May-2017
Complete List of Authors: Colestock, Tristan; University of the Sciences in Philadelphia Wallach, Jason; University of the Sciences, Mansi, Matt; University of the Sciences in Philadelphia Filemban, Nadine; University of the Sciences in Philadelphia Morris, Hamilton; New School for Social Research, Department of Anthropology Elliott, Simon; Alere Forensics (Forensics Ltd) Westphal, Dr. Folker; State Bureau of Criminal Investigation Schlesing Holstein, Nacotics/Toxicology Brandt, Simon; School of Pharmacy & Biomolecular Sciences , Liverpool John Moores University Adejare, Adeboye; University of the Sciences in Philadelphia
New psychoactive substances, Dissociative anesthetics, NMDA receptor, Keywords: PCP, Arylcyclohexylmorpholines
New psychoactive substances (NPS) are commonly referred to as “research chemicals”, “designer drugs” or “legal highs.” One NPS class is represented by dissociative anesthetics, which include analogues of the arylcyclohexylamine phencyclidine (PCP), ketamine, and diphenidine. A recent addition to the NPS market was 4-[1-(3- methoxyphenyl)cyclohexyl]morpholine (3-MeO-PCMo), a morpholine analogue of 3-MeO-PCP. Although suspected to have dissociative effects in users, information about its pharmacological profile is not available. From clinical and forensic perspectives, detailed analytical data are needed for Abstract: identification, especially when facing the presence of positional isomers, as these are frequently unavailable commercially. This study presents the analytical and pharmacological characterization of 3-MeO-PCMo along with five additional analogues including the 2- and 4-MeO- isomers, 3,4- methylenedioxy-PCMo (3,4-MD-PCMo), 3-Me-PCMo and PCMo. All six arylcyclohexylmorpholines were synthesized and characterized by chromatographic, mass spectrometric and spectroscopic techniques. The three positional isomers could be differentiated and the identity of 3-MeO- PCMo obtained from an internet vendor was verified. All six compounds were also evaluated for affinity at 46 central nervous system receptors
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1 2 3 4 including the N-methyl-D-aspartate receptor (NMDAR), an important target 5 for dissociative anesthetics such as PCP and ketamine. In vitro binding studies using [3H]-MK-801 in rat forebrain preparations revealed moderate 6 affinity for NMDAR in the rank order of 3-Me > 3-MeO >PCMo > 3,4-MD > 7 2-MeO > 4-MeO-PCMo. 3-MeO-PCMo was found to have moderate affinity 8 for NMDAR comparable to that of ketamine, and had an approximate 12- 9 fold lower affinity than PCP. These results support the anecdotal reports of 10 dissociative effects from 3-MeO-PCMo in humans. 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta Drug Testing and Analysis Page 2 of 25
1 2 3 4 Syntheses, analytical and pharmacological characterizations of the “legal 5 high” 4-[1-(3-methoxyphenyl)cyclohexyl]morpholine (3-MeO-PCMo) and 6 analogues 7 8 9 Tristan Colestock,a Jason Wallach, a Matt Mansi, a Nadine Filemban, a Hamilton 10 b c d e 11 Morris, Simon P. Elliott, Folker Westphal, Simon D. Brandt, Adeboye Adejare 12 a,* 13 14 15 a Department of Pharmaceutical Sciences, University of the Sciences, 600 South 43rd Street, 16 Philadelphia, PA 19104, USA 17 18 b For Peer Review 19 The New School for Social Research, Department of Anthropology, 66 West 12th Street, New York, NY 20 10011, USA
21 22 c Alere Forensics (Forensics Ltd), Malvern Hills Science Park, Geraldine Road, WR14 3SZ, UK 23 24 d State Bureau of Criminal Investigation Schleswig-Holstein, Section Narcotics/Toxicology, Mühlenweg 25 166, D-24116 Kiel, Germany 26
27 e 28 School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, 29 Liverpool, L3 3AF, UK 30 31 32 *Corresponding author: Adeboye Adejare, Department of Pharmaceutical Sciences, 33 University of the Sciences, 600 S. 43 rd Street, Philadelphia PA 19128. E mail: 34 35 [email protected] 36 37 38 39 40 41 Running title : Synthesis, analytical and pharmacological characterization of PCMo analogues 42
43 44 Keywords : New psychoactive substances; dissociative anesthetics; NMDA receptor; PCP; 45 46 arylcyclohexylmorpholines 47 48 49 50 51
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1 2 3 4 Abstract 5 6 New psychoactive substances (NPS) are commonly referred to as “research chemicals”, 7 “designer drugs” or “legal highs.” One NPS class is represented by dissociative anesthetics, 8 which include analogues of the arylcyclohexylamine phencyclidine (PCP), ketamine, and 9 diphenidine. A recent addition to the NPS market was 4 [1 (3 10 methoxyphenyl)cyclohexyl]morpholine (3 MeO PCMo), a morpholine analogue of 3 MeO PCP. 11 12 Although suspected to have dissociative effects in users, information about its pharmacological 13 profile is not available. From clinical and forensic perspectives, detailed analytical data are 14 needed for identification, especially when facing the presence of positional isomers, as these 15 are frequently unavailable commercially. This study presents the analytical and pharmacological 16 17 characterization of 3 MeO PCMo along with five additional analogues including the 2 and 4 18 MeO isomers, 3,4 methylenedioxy PCMoFor Peer (3,4 MD PCMo) Review, 3 Me PCMo and PCMo. All six 19 arylcyclohexylmorpholines were synthesized and characterized by chromatographic, mass 20 spectrometric and spectroscopic techniques. The three positional isomers could be 21 22 differentiated and the identity of 3 MeO PCMo obtained from an internet vendor was verified. All 23 six compounds were also evaluated for affinity at 46 central nervous system receptors including 24 the N-methyl D aspartate receptor (NMDAR), an important target for dissociative anesthetics 25 such as PCP and ketamine. In vitro binding studies using [3H] MK 801 in rat forebrain 26 27 preparations revealed moderate affinity for NMDAR in the rank order of 3 Me > 3 MeO >PCMo 28 > 3,4 MD > 2 MeO > 4 MeO PCMo. 3 MeO PCMo was found to have moderate affinity for 29 NMDAR comparable to that of ketamine, and had an approximate 12 fold lower affinity than 30 PCP. These results support the anecdotal reports of dissociative effects from 3 MeO PCMo in 31 32 humans. 33 34 35 36 37
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1 2 3 4 Introduction 5 [1] 6 A high number of new psychoactive substances (NPS) continue to be available from online 7 vendors and are sold as “research chemicals”. These chemicals are largely designed to by pass 8 governmental restrictions on existing psychoactive drugs. Dissociative agents that target the N- 9 methyl D aspartate receptor (NMDAR) represent one of many available classes of compounds 10 that are encompassed by the NPS term. Substances with dissociative profile (Figure 1A) 11 12 comprise structural analogues of the arylcyclohexylamines such as phencyclidine (PCP), 13 ketamine and methoxetamine.[2] More recently, 1,2 diarylethylamines such as diphenidine and 14 its 2 methoxy analogue 2 MXP have also appeared.[3,4] 15 16 Substances that target the NMDAR are of interest for the development of treatment options for 17 conditions such as depression, neuropathic pain, and a variety of neurodegenerative disorders 18 [5 9] For Peer Review 19 and dementias. At the same time, a number of these substances are used recreationally, 20 outside of a medical setting, and include compounds that have not undergone any substantial 21 pharmacological and toxicological evaluations. A systematic methodology is needed in order to 22 address the chemical, pharmacodynamic, and pharmacokinetic properties of these 23 [3,4,10,11] 24 substances, thus facilitating drug development efforts, and identification of toxicity 25 profiles as well as adverse events associated with recreational drug use.[12 14] 26 27 The earliest reported synthesis of 4 (1 phenylcyclohexyl)morpholine (PCMo) was found in a 28 patent submitted in 1954 [15] and predates that of PCP, [16] however, its pharmacology, or 29 dissociative profile, was not recognized at that time. PCMo made brief documented 30 31 appearances as an “analog” of PCP in the recreational market during the 1970’s and again in [2] 32 the early 2000’s. More recently, 4 [1 (3 methoxyphenyl)cyclohexyl]morpholine (3 MeO PCMo) 33 has become available for purchase as a “research chemical” on a number of websites, which 34 encouraged the authors to explore its chemistry and pharmacology. To gain further insight into 35 36 this class of compounds, 2 MeO and 4 MeO positional isomers were synthesized, as well as 37 3,4 methylenedioxy PCMo (3,4 MD PCMo), 3 Me PCMo, and the unsubstituted PCMo template 38 (Figure 1B). The entire series was subjected to comprehensive analytical characterization 39 including chromatographic, mass spectrometric and spectroscopic methods. In addition, a test 40 41 purchase of 3 MeO PCMo was compared to the synthesized reference material confirming its 42 identity. 43 44 With the exception of 2 MeO PCMo and PCMo, pharmacological data on the 45 arylcyclohexylmorpholines investigated in the present study are not available. 2 MeO PCMo 46 was shown to reduce acute thermal (tail immersion test) and chronic chemical pain 47 (formaldehyde) induced in adult female rats. [17] In the tail immersion test, analgesic effects were 48 [17] 49 found to be more pronounced compared to PCP and PCMo. PCMo was also demonstrated to 50 display lower potencies compared to PCP in a range of in vitro and in vivo assays targeting a 51 number of different receptors.[18 29] In order to explore whether the six arylcyclohexylmorpholines 52 showed PCP or ketamine like properties in vitro , all test drugs were pharmacologically 53 54 characterized in the present study for binding affinity at 46 CNS receptors including NMDAR, 55 and monoamine transporters for dopamine, norepinephrine and serotonin. 56 57 58 59 60 3 http://mc.manuscriptcentral.com/dta Page 5 of 25 Drug Testing and Analysis
1 2 3 4 Experimental 5 6 Materials 7 8 All starting materials, reagents, and solvents used for syntheses were obtained from Sigma 9 Aldrich (St. Louis, MO, USA). Flash column chromatography was performed using Merck silica 10 gel grade 9385 (230 400 mesh, 60 Å). Melting points were obtained using a DigiMelt A160 SRS 11 12 digital melting point apparatus (Stanford Research Systems, Sunnyvale, CA, USA) at a ramp 13 rate of 2°C/min. Melting points, spectral analyses, and receptor binding studies were performed 14 on target compounds following flash chromatography purification. 15 16 Instrumentation 17 18 Nuclear magnetic resonanceFor (NMR) Peer spectroscopy Review 19 20 1H (400 MHz) and 13 C NMR spectra (101 MHz) were obtained from the freebase material 21 in CDCl 3 solution (100% and 99.96% D, 0.03% ( v/v ) TMS) at a concentration of 20 mg/mL using 22 23 a Bruker Ultrashield 400 plus spectrometer with a 5 mm BBO S1 (Z gradient plus) probe at 24 24°C. Internal chemical shift references were TMS (δ = 0.00 ppm) and CDCl 3 (δ = 77.0 ppm). 25 Spectra were recorded with the freebases and the test purchase of 3 MeO PCMo was 26 determined to be the freebase. NMR assignments were made as described previously [10,30,31] 27 13 28 using chemical shift position, splitting, C PENDANT and 2 D experiments (HMQC, HMBC, and 29 COSY). 30 31 Gas chromatography (EI/CI) ion trap mass spectrometry 32 33 Data for all six PCMo analogues (0.5 mg/mL in methanol) were recorded under full scan 34 electron (EI) and chemical ionization (CI) conditions using HPLC grade methanol as the liquid 35 36 CI reagent. A Varian 450 GC gas chromatograph coupled to a Varian 220 MS ion trap mass 37 spectrometer (scan range m/z 41– m/z 500) and a Varian 8400 autosampler was employed with 38 a Varian CP 1177 injector (275ºC) in split mode (1:50) (Walnut Creek, CA, USA). The Varian 39 MS Data Review function of the Workstation software, version 6.91, was used for data 40 41 acquisition. Transfer line, manifold and ion trap temperatures were set at 310, 80 and 220ºC, 42 respectively. The carrier gas was helium at a flow rate of 1 mL/min using the EFC constant flow 43 mode. The default settings for CI ionization parameters (0.4 s/scan) were used: CI storage level 44 m/z 19.0; ejection amplitude m/z 15.0; background mass m/z 55; maximum ionization time 2000 45 46 s; maximum reaction time 40 ms; target TIC 5000 counts. An Agilent J&W VF 5ms GC column 47 (30 m × 0.25 mm, 0.25 m) was employed for separation. The starting temperature was set at 48 80ºC and held for 1 min. The temperature then increased at 20ºC/min to 280ºC and held 49 constant for 9.0 min to give a total run time of 20.00 min. 50 51 52 High mass accuracy mass spectrometry using an atmospheric solids analysis probe (ASAP) 53 54 ASAP was employed with a Thermo Fisher Scientific Inc., (Waltham, MA, USA) Orbitrap 55 Exactive using an Ion Max source in positive mode. Measured accurate masses were within ± 5 56 ppm of the theoretical masses. The following parameters were used: resolution was set to ultra 57 high, sheath gas (N ) flow 5 (arbitrary units), auxiliary gas flow 6 (arbitrary units), sweep gas 58 2 59 60 4 http://mc.manuscriptcentral.com/dta Drug Testing and Analysis Page 6 of 25
1 2 3 flow 0 (arbitrary units), corona discharge 4 kV, capillary temperature 275 °C, capillary voltage 4 5 25.0 V, skimmer voltage 14 V and a tube lens voltage of 85 V. Instrument calibrations were 6 performed using the ProteoMass LTQ/FT Hybrid ESI Positive Mode Calibration Mix from 7 Supelco Analytical (Bellefonte, PA, USA). 8 9 Ultra-high performance liquid chromatography (UHPLC) high mass accuracy electrospray mass 10 spectrometry 11 12 13 Mobile phases used for UHPLC separation consisted of acetonitrile with 1% formic acid (v/v) 14 and an aqueous solution of 1% formic acid. The column temperature was set at 40°C (0.6 15 mL/min) and data were acquired for 5.5 min. The elution was a 5 –70% acetonitrile gradient 16 ramp over 3.5 min, then increased to 95% acetonitrile in 1 min and held for 0.5 min before 17 18 returning to 5% acetonitrileFor in 0.5 Peermin. QTOF MS Review data were acquired in positive mode scanning 19 from m/z 100 –m/z 1000 with and without auto MS/MS fragmentation. Ionization was achieved 20 with an Agilent JetStream electrospray source and infused internal reference masses. Agilent 21 6540 QTOF MS parameters: gas temperature 325°C, drying gas 10 L/min and sheath gas 22 23 temperature 400 °C. Internal reference ions at m/z 121.05087 and m/z 922.00979 were used. 24 25 High performance liquid chromatography diode array detection 26 27 HPLC DAD analyses were carried out on a Dionex 3000 Ultimate system coupled to a UV diode 28 array detector (Thermo Fisher, St Albans, UK), using a Phenomenex Synergi Fusion column 29 (150 mm x 2 mm, 4 m) that was protected by a 4 mm x 3 mm Phenomenex Synergi Fusion 30 31 guard column (Phenomenex, Macclesfield, UK). The mobile phases were made from 70% 32 acetonitrile with 25 mM triethylammonium phosphate (TEAP) buffer and an aqueous solution of 33 25 mM TEAP buffer. Elution was achieved with a gradient that started with 4% acetonitrile and 34 ramped to 70% acetonitrile in 15 min and held for 3 min. The total acquisition time was 18 min 35 36 at a flow rate of 0.6 mL/min. The diode array detection window was set at 200 nm to 595 nm 37 (collection rate 2 Hz). 38 39 Infrared spectroscopy 40 41 Infrared (IR) spectra were obtained on a Perkin Elmer Spectrum BX FTIR model (Llantrisant, 42 UK) using a Pike MIRacle ATR system. Data were acquired with the Spectrum v5.01 software 43 (scan range 4000 –400 cm 1, resolution 4 cm 1, 16 scans). Spectral data can be found in 44 45 Supporting Information. 46 Microwave synthesizer 47 48 Conversion from primary amine intermediate to morpholine ring products were performed using 49 50 a CEM Discover SP microwave synthesizer (CEM Corporation, Matthews NC, USA). Reactions 51 were carried out in 35 mL microwave vessels from CEM. Conditions for the reactions are 52 detailed below. 53 54 Syntheses procedures 55 56 The syntheses of the primary amine intermediates were performed using a modified Geneste 57 route (Figure 2) as described previously.[5,10,32] Reactions starting from the primary amine 58 59 60 5 http://mc.manuscriptcentral.com/dta Page 7 of 25 Drug Testing and Analysis
1 2 3 intermediate to yield the morpholine ring products were carried out in a CEM Discover SP 4 5 microwave synthesizer. The primary amine (PCA) intermediates were available from previous [5,10,30] 6 studies. 7 8 Preparation of 4-[1-(2-methoxyphenyl)cyclohexyl]morpholine (2-MeO-PCMo) 9 10 1 (2 Methoxyphenyl)cyclohexanamine (2 MeO PCA) (4.87 mmol, 1.00 g) and triethylamine 11 (14.61 mmol, 2.03 mL) were added to acetonitrile (~15 mL). The solution was dried for 10 12 minutes with 4Å molecular sieves and then decanted into a 35 mL microwave vessel. 2 13 Bromoethyl ether (9.74 mmol, 1.22 mL) was added to the solution, the vessel was sealed under 14 15 inert argon, and then reacted for 1.5 h at 85°C with 50 W power and stirring. Reaction pressures 16 did not exceed 25 psi. Afterwards, the reaction mixture (a red/dark red color) was transferred to 17 an aqueous 0.2 M HCl solution (60 mL) and washed with ethyl acetate (EtOAc) (3 x 60 mL). The 18 aqueous phase was basifiedFor to pHPeer >12 with KOH Review pellets and extracted with EtOAc (3 x 60 mL). 19 20 The pooled organic extraction was washed once with 10 mL brine, dried with anhydrous 21 magnesium sulfate, and concentrated under reduced pressure to produce a light amber oil. The 22 crude product was collected and purified using flash column chromatography with a mobile 23 phase consisting of hexane/EtOAc (80/20) with TEA (1%, v/v ). Fractions containing the product 24 25 were pooled and concentrated to yield light yellow oil, which solidified upon cooling (3.16 mmol, 26 0.869 g, 64.7% yield). This solid was recrystallized from boiling hexanes. Upon cooling at 0°C, 27 colorless crystals formed and were collected by decanting, washed with hexanes, and dried at 28 room temperature (m.p. 67.1–68.6°C). HR ASAP MS of the freebase observed: m/z 276.1949 29 + + 30 (theory [M+H] C17 H26 NO 2 , m/z 276.1958, = 3.3 ppm). 31 32 The HCl salt of 2 MeO PCMo was prepared by dissolving the solidified freebase in 100% 33 ethanol (EtOH), titrating to pH 1.0 with concentrated HCl, and evaporating under a stream of 34 warm air. EtOH (100%) was added in 10 mL increments and evaporated until all residual 35 moisture and HCl were removed. The resulting solid was dried and washed with EtOAc (2 x 5 36 37 mL). The dried solid was then recrystallized by dissolving in a minimal amount of warm EtOH 38 and diluted 3 fold with Et 2O. The solution was stored at 0°C overnight. The resulting crystals 39 were collected by decanting the solvent, washing the solid with EtOAc (2 x 5 mL) and drying. 40 Recrystallization was repeated 2 additional times as described to produce white flakey crystals 41 [17] 42 with m.p. 179.5–181.5°C (lit. 167–169°C ). 43 44 Preparation of 4-[1-(3-Methoxyphenyl)cyclohexyl]morpholine (3-MeO-PCMo) 45 46 3 MeO PCMo was prepared in 50.9% yield from 3 MeO PCA as described above and formed a 47 colorless crystalline solid (m.p. 74.4–75.3°C) HR ASAP MS of the freebase observed: m/z + + 48 276.1952 (theory [M+H] C17 H26 N1O2 , m/z 276.1958, = 2.2 ppm). The HCl salt was a white 49 flakey crystalline powder (m.p. 209.1–209.4°C). 50 51 Preparation of 4-[1-(4-methoxyphenyl)cyclohexyl]morpholine (4-MeO-PCMo) 52 53 4 MeO PCMo was prepared in 43% yield from 4 MeO PCA as described above and formed a 54 colorless crystalline solid (m.p. 79.9–81.5°C). HR ASAP MS of the freebase observed: m/z 55 + + 56 276.1951 (theory [M+H] C17 H26 N1O2 , m/z 276.1958, = 2.5 ppm). The HCl salt formed 57 translucent amber crystals (m.p. 153.1–156.1°C). 58 59 60 6 http://mc.manuscriptcentral.com/dta Drug Testing and Analysis Page 8 of 25
1 2 3 Preparation of 4-[1-(1,3-benzodioxol-5-yl)cyclohexyl]morpholine (3,4-MD-PCMo) 4 5 6 7 3,4 MD PCMo was prepared in 44% yield from 3,4 MD PCA as described above and formed a 8 colorless crystalline solid (m.p. 123.4–124.9°C). HR ASAP MS of the freebase observed: m/z + + 9 290.1752 (theory [M+H] C17 H26 N1O2 , found m/z 290.1751, = 0.3 ppm). The HCl salt was a 10 white fluffy crystalline powder (m.p. 180.5–181.7°C). 11 12 Preparation of 4 [1 (3 methylphenyl)cyclohexyl]morpholine (3-Me-PCMo) 13 14 3 Me PCMo was prepared in 46.4% yield from 3 Me PCA as described above as a colorless oil. 15 The HCl salt was a white fluffy crystalline powder (m.p. 211.2–211.7°C). HR ASAP MS of the 16 + + 17 HCl salt observed: m/z 260.2018 (theory [M+H] C17 H26 N1O2 , m/z 260.2009, = 3.5 ppm). 18 For Peer Review 19 Preparation of 4-(1-phenylcyclohexyl)morpholine (PCMo) 20 21 PCMo was prepared as described in 60% yield from PCA; however, microwave reaction 22 parameters were slightly altered (80°C, 65 W, and monitored by GC MS for a total reaction time + + 23 of 2.5 h). HR ASAP MS of the freebase observed: m/z 246.1845 (theory [M+H] C17 H26 N1O2 , 24 m/z 246.1852, = 2.8 ppm). HCl salt was obtained as the hemihydrate ( 1H NMR) white powder 25 [17] [33] [34] 26 with a melting point of 197.3–198.5°C (lit. 187–188°C ; 188–190°C; 187–188°C; 181– 27 182°C; [35] 199–201°C (hemihydrate);[36] 182°C [15] ). An alternate route for the synthesis of PCMo 28 was also employed and is provided as Supporting Information. 29 30 NMDA receptor binding studies 31 32 33 In vitro binding affinities (K i) of the target compounds were determined using competitive 34 radioligand binding studies with [ 3H] MK 801, a high affinity ligand for the PCP site within the 35 NMDAR channel, in accordance with established protocols.[37,38] Thoroughly washed rat 36 forebrain homogenate was used as the NMDAR source (whole brain obtained from Pel Freez 37 [37] 38 Biologicals, Rogers AR, USA) and prepared as described by Reynolds and Sharma . 39 Suspensions of 10 mM HEPES buffer (pH 7.4, 25 oC) containing 100 g/mL protein, 1 nM [3H] 40 MK 801, 100 M glutamate, 10 M glycine, and various concentrations of unlabeled test drugs 41 were incubated in the dark on a mechanical rocker at 25 oC for 2 h. (+) MK 801 (30 M) was 42 43 used for nonspecific binding (and positive control). The reaction was terminated by vacuum 44 filtration using a 24 well cell harvester (Brandel, Gaithersburg MD, USA) over presoaked GF/B 45 glass fiber filters (Brandel, Gaithersburg, MD, USA). Filters were washed with room 46 temperature HEPES buffer (3 x 5 mL). Tritium trapped on the filter was measured via liquid 47 48 scintillation counting, using a Beckman LS 6500 multipurpose scintillation counter 49 (BeckmanCoulter, USA) at 57% efficiency. IC 50 values were determined with Graphpad Prism 50 5.0 (GraphPad Software, La Jolla, USA) using non linear regression with log concentration 51 plotted against percent specific binding. Percent specific binding for [3H] MK 801 in a control 52 [39] 53 experiment was ~95%. K i values were calculated using the equation of Cheng and Prusoff. 54 The K d for (+) MK 801 (1.75 nM), was determined via a homologous binding assay as described 55 by Reynolds and Sharma and was consistent with the literature.[38] Protein concentration was 56 determined via the Bradford method [40] using Coomassie protein assay reagent and rat albumin 57 58 59 60 7 http://mc.manuscriptcentral.com/dta Page 9 of 25 Drug Testing and Analysis
1 2 3 as standard (Sigma Aldrich, USA). Experiments were performed in duplicate and repeated three 4 5 times. 6 7 Non-NMDA receptor binding studies 8 9 10 Competitive binding studies of PCMo and analogues at 45 additional CNS receptors were 11 performed through the National Institute of Mental Health Psychoactive Drug Screening 12 Program (NIMH PDSP). Briefly, target compounds were dissolved in DMSO and subjected to 13 primary screening at 10,000 nM concentrations. Compounds exhibiting >50 % inhibition 14 15 underwent secondary assay at varying concentrations to determine K i values. Additional [41] 16 experimental details are available in the NIMH PDSP assay protocol book. 17 18 For Peer Review 19 Results and Discussion 20 21 The six morpholine analogues investigated in this study (Figure 1B) were synthesized using the 22 23 modified Geneste route as reported previously for the preparation of PCP and PCPy [5,10,32] 24 analogs. The conversion from the primary amine (PCA) to the morpholine ring was 25 performed using an S N2 cyclization reaction between the substituted PCA material and bis(2 26 bromoethylether) (Figure 2) and gave ~45% yields following purification by column 27 chromatography and recrystallization. The synthesized PCMo HCl was found to be the 28 1 29 hemihydrate salt ( H NMR) and was consistent with a literature melting point value reported for 30 the hemihydrate salt.[36] The remaining analogues contained less than a 0.25 molar equivalent 31 of water. A discrepancy with the 2 MeO PCMo HCl melting point exists herein with a previously 32 reported value,[17] which may be due to polymorphism, solvates or purity. The appearance of 3 33 34 MeO PCMo on the “research chemicals” market triggered questions about the ability to 35 differentiate this compound from its positional 2 MeO PCMo and 4 MeO PCMo isomers, given 36 that isomeric sets of compounds are frequently unavailable as reference material that can be 37 used for forensic and clinical investigations. With the exception of 2 MeO PCMo and PCMo, 38 39 where some, albeit limited analytical data are available, the remaining compounds presented in 40 this study are reported for the first time. 41 42 Gas chromatography ion trap mass spectrometry (GC IT MS) data obtained from electron 43 ionization (EI) and chemical ionization (CI) methods recorded for the HCl salts are summarized 44 in Figure 3. The positional isomers 2 , 3 , and 4 MeO PCMo could be separated on the GC 45 + 46 column (10.04, 10.30 and 10.52 min). Under EI conditions, both the molecular ion and a [M H ] 47 species were visible in appreciable relative abundance and implementation of CI facilitated 48 detection of the corresponding protonated molecules. The EI mass spectrum obtained in the 49 present study for PCMo was comparable with a spectrum published 40 years ago [33] although 50 51 differences were observed in the relative abundance of various fragment, possibly due to 52 implementation of different mass analyzers. CI mass spectra of PCMo, using both methane and 53 isobutane as the reagent gas, appeared 3 4 years later [42,43] which revealed the formation of 54 fragment ions also detected in the present study, such as m/z 88, m/z 159 and m/z 202, 55 56 respectively. The ions formed under EI and CI ion trap MS conditions appeared to be equivalent 57 to those reported previously for a range of 1 (1 phenylcyclohexyl)piperidine (PCP) and 1 (1 58 59 60 8 http://mc.manuscriptcentral.com/dta Drug Testing and Analysis Page 10 of 25
1 2 3 phenylcyclohexyl)pyrrolidine [10] and N alkyl arylcyclohexylamines [5] and proposed fragmentation 4 5 pathways have been described. The implementation of GC MS analysis also resulted in 6 degradation of the PCMo products that gave rise to a GC induced degradant consistent with 7 what appeared to be a 1 (1 cyclohexen 1 yl) ring substituted benzene species which has been 8 described for other PCP type substances before [10] (Supporting Information). Conversion of the 9 10 hydrochloride salts to the freebases and analysis by a different instrument (GC quadrupole EI 11 MS) revealed a significant reduction in degradation (Supporting Information). The sample 12 advertised as 3 MeO PCMo by an online vendor was found to be consistent with the information 13 provided on the product label. Implementation of GC sIR also allows for the analysis of 14 15 compound mixtures and/or substances that may only be available in small amounts, including 16 the GC induced degradation products (Supporting Information). As shown in the Supporting 17 Information, the three positional isomers could be differentiated by ATR IR (HCl salts) and GC 18 sIR. The purity of the Forfreebase was Peer not determined; Review however, no impurity peaks were observed 19 20 with GC MS, LC MS, or NMR, and the melting point of the test purchase exactly matched that of 21 the synthesized 3 MeO PCMo when run side by side. Attempts to separate the three positional 22 isomers using various solvent combinations and two different TLC plates, however, were 23 unsuccessful. 24 25 Ultra high performance liquid chromatography electrospray quadrupole time of flight tandem 26 mass spectra for all six PCMo analogues are shown in Figure 4, which illustrated that product 27 [10] 28 ion formations were also comparable to a number of PCP/PCPy and N alkyl 29 arylcyclohexylamine analogues[5] Examples observed in Figure 4 include a neutral loss of 30 morpholine, formation of the respective tropylium ion or detection of protonated morpholine. 31 Implementation of the HPLC diode array detection (DAD) procedure showed only partial 32 33 separation of the three positional isomers due to co elution of 3 MeO and 4 MeO PCMo 34 (Figure 5). However, the ultraviolet spectra scanned between 200 and 594 nm provided distinct 35 differences that allowed for facile differentiation between the isomers. 3 MeO PCMo gave rise to 36 distinctive peaks at 218 nm and 278 nm whereas 4 MeO PCMo displayed a slight shift to 230 37 38 nm although the 277 nm peak remained indistinguishable. UV spectra recorded for 3,4 MD 39 PCMo, 3 Me PCMo, and PCMo and their corresponding HPLC retention times are provided as 40 Supporting Information. 41 42 Detailed NMR analyses on PCMo have been reported previously and were consistent with the 43 results presented in this study (Tables 1 and 2). [44,45] PCMo HCl was also characterized by 13 C 44 [36] 45 NMR and the recorded spectrum was in agreement with literature values. In general, the 46 chemical shift behavior of the series was consistent with those observed previously with related 47 arylcyclohexylamines and a detailed discussion can be found elsewhere.[5,10,30] One notable 48 distinction unique to the PCMo series worth addressing, however, is with respect to the 49 50 morpholine ring, as this feature may be useful for the identification of related 51 arylcyclohexylmorpholines. Due to the presence of the O heteroatom in the ring system, the β 52 chemical shifts were more deshielded and, thus, appeared further downfield than those found in 53 the α position (NCH) in both the 1H (~3.6 ppm vs. ~2.3 ppm) and 13 C spectra (~68 ppm vs. ~46 54 1 55 ppm). In the H spectra, the β protons consistently appeared as a triplet, integrating to four 56 protons, due to vicinal coupling ( J ~4.6 Hz) with the two α protons (magnetically equivalent due 57 to ring flipping). The occurrence of ring flipping appeared to be consistent with the fact that the 58 59 60 9 http://mc.manuscriptcentral.com/dta Page 11 of 25 Drug Testing and Analysis
1 2 3 1H NMR spectra of the HCl salts (Supporting Information) showed separate axial and equatorial 4 5 shifts for the beta protons. Protonation is known to prevent ring flipping, and this effect was [10] 6 observed with other compounds including arylcyclohexylamines. Similarly, the α protons 7 appeared as a triplet due to vicinal coupling with the β protons (J ~4.6 Hz). Furthermore, the 2,6 8 and α proton chemical shifts in 2 MeO PCMo appeared further downfield compared to those 9 13 10 deriving from the 3 MeO and 4 MeO counterparts and a similar effect was observed in the C 11 spectra. The proton chemical shifts linked to the 3,5 and β positions on the other hand were 12 equivalent in all three positional isomers. This effect was observed with the corresponding PCP 13 HCl salt series [30] although it was not consistently observed with the N alkyl secondary anisyl 14 [5] 15 cyclohexylamines. 16 NMDAR and off-target receptor binding studies 17 18 With regards to NMDAR,For the resultsPeer of competitive Review [3H] MK 801 displacement assays are 19 20 provided in Table 3 as IC 50 and K i values and shown graphically in Figure 6. Compared to some 21 previously investigated PCP analogs,[30] substitution of piperidine for a morpholine ring reduced 22 NMDAR affinity. Consistent with the present results, PCMo was previously reported to show a 23 ~10 fold reduced affinity to NMDAR using [ 3H] PCP in central nervous system tissue.[18,46] 24 25 Furthermore, PCMo had 10 fold reduced potency relative to PCP in a number of experimental 26 models.[46,47] The affinity rank order determined in this study was comparable to their PCP 27 counterparts with 3 MeO > H > 2 MeO > 4 MeO).[30] Interestingly, the same affinity order was 28 seen with a series of diphenidine analogs,[4] although it was not observed with the methoxylated 29 [30] 30 PCPy series (3 MeO > 4 MeO > 2 MeO). 31 32 A heatmap containing the results of the binding experiments on the 46 assessed CNS receptors 33 is presented in Figure 7. Besides NMDAR, all compounds had moderate affinity for the sigma 2 34 receptor, which is commonly seen with this class of compounds.[4,30,48] 3,4 MD PCMo was the 35 most selective compound and this selectivity was consistent with other 3,4 MD substituted 36 [30] 37 arylcyclohexylamines. Likewise, 3,4 MD PCMo and PCMo had moderate NMDAR affinity [4,49,50] 38 values comparable to ketamine and memantine. PCMo was shown to be less potent and 39 toxic than PCP,[24] which may be explained by the moderate NMDAR affinity.[30,49,50] 40 41 Arylcyclohexylamines have displayed variable affinities at the monoamine reuptake transporters 42 for serotonin, norepinephrine, and dopamine (SERT, NET, and DAT, respectively). [30,51] 43 Interestingly, the morpholine ring abolished NET activity for all compounds relative to their 44 [30] 45 piperidine counterparts. 3 Me PCMo was the only compound with affinity for both SERT and 46 DAT. The 2 MeO and 3 MeO analogues displayed selectivity towards SERT over DAT, whereas 47 4 MeO PCMo had appreciable affinity for DAT. 48 49 Larger 1,4 diaminocyclohexane derivatives containing the PCMo moiety displayed in vitro 50 opioid receptor activity in previous cell based assays.[52] However, the binding experiments in 51 52 this study revealed no affinity for the δ , κ or opioid receptors, which indicate that the anti 53 nociceptive properties may have been the result of NMDAR antagonism.[53 56] Previous 54 pharmacological experiments with PCMo, 2 MeO PCMo, 4 Me PCMo, and 2 Me 4 HO PCMo 55 found analgesic activity in rats [17] which further suggests analgesic effects being mediated 56 57 independently from opioid receptor affinity. 58 59 60 10 http://mc.manuscriptcentral.com/dta Drug Testing and Analysis Page 12 of 25
1 2 3 4 Conclusion 5 6 4 [1 (3 Methoxyphenyl)cyclohexyl]morpholine (3 MeO PCMo), a morpholine analogue of 3 7 MeO PCP, is available for purchase as a “research chemical” and suspected to share some 8 psychopharmacological properties with ketamine and perhaps phencyclidine (PCP). The 9 present study described the analytical characterization of 3 MeO PCMo, its two positional 10 isomers and three additional analogues. Differentiation between 2 MeO , 3 MeO and 4 MeO 11 12 PCMo was detectable by chromatographic and spectroscopic methods. In vitro pharmacological 13 investigations also revealed that the compounds displayed moderate affinity toward the N- 14 methyl D aspartate receptor with off target activities at sigma 2 and monoamine transporters for 15 dopamine and serotonin. These findings suggest that at least some of the investigated 16 17 arylcyclohexylmorpholines, including 3 MeO PCMo, may be psychoactive in humans and thus 18 have abuse potential whichFor may accountPeer for some Review of the purchases of this “research chemical.” 19 Clinical and forensic studies would be required to investigate this hypothesis further. 20 21 References 22 23 24 [1] S. D. Brandt, L. A. King, M. Evans Brown. The new drug phenomenon. Drug Test. 25 Anal. 2014 , 6, 587. 26 [2] H. Morris, J. Wallach. From PCP to MXE: a comprehensive review of the non medical 27 use of dissociative drugs. Drug Test. Anal. 2014 , 6, 614. 28 29 [3] G. McLaughlin, N. Morris, P. V. Kavanagh, J. D. Power, J. O'Brien, B. Talbot, S. P. 30 Elliott, J. Wallach, K. Hoang, H. Morris, S. D. Brandt. Test purchase, synthesis, and 31 characterization of 2 methoxydiphenidine (MXP) and differentiation from its meta and 32 para substituted isomers. Drug Test. Anal. 2016 , 8, 98. 33 34 [4] J. Wallach, H. Kang, T. Colestock, H. Morris, Z. A. Bortolotto, G. L. Collingridge, D. 35 Lodge, A. L. Halberstadt, S. D. Brandt, A. Adejare. Pharmacological investigations of the 36 dissociative ‘legal highs’ diphenidine, methoxphenidine and analogues. PLoS One 2016 , 37 11 , e0157021. 38 39 [5] J. Wallach, T. Colestock, B. Cicali, S. P. Elliott, P. V. Kavanagh, A. Adejare, N. M. 40 Dempster, S. D. Brandt. Syntheses and analytical characterizations of N alkyl 41 arylcyclohexylamines. Drug Test. Anal. 2016 , 8, 801. 42 [6] N. D. Iadarola, M. J. Niciu, E. M. Richards, J. L. Vande Voort, E. D. Ballard, N. B. 43 44 Lundin, A. C. Nugent, R. Machado Vieira, C. A. Zarate Jr. Ketamine and other N methyl 45 D aspartate receptor antagonists in the treatment of depression: a perspective review. 46 Ther. Adv. Chronic Dis. 2015 , 6, 97. 47 [7] C. G. Parsons, A. Stöffler, W. Danysz. Memantine: a NMDA receptor antagonist that 48 49 improves memory by restoration of homeostasis in the glutamatergic system too little 50 activation is bad, too much is even worse. Neuropharmacology 2007 , 53 , 699. 51 [8] D. J. Newport, L. L. Carpenter, W. M. McDonald, J. B. Potash, M. Tohen, C. B. 52 Nemeroff. Ketamine and other NMDA antagonists: early clinical trials and possible 53 54 mechanisms in depression. Am. J. Psychiatry 2015 , 172 , 950. 55 [9] S. J. Thomas, G. T. Grossberg. Memantine: a review of studies into its safety and 56 efficacy in treating Alzheimer’s disease and other dementias. Clin. Interv. Aging 2009 , 4, 57 367. 58 59 60 11 http://mc.manuscriptcentral.com/dta Page 13 of 25 Drug Testing and Analysis
1 2 3 [10] J. Wallach, G. De Paoli, A. Adejare, S. D. Brandt. Preparation and analytical 4 5 characterization of 1 (1 phenylcyclohexyl) piperidine (PCP) and 1 (1 phenylcyclohexyl) 6 pyrrolidine (PCPy) analogues. Drug Test. Anal. 2014 , 6, 633. 7 [11] J. A. Michely, S. K. Manier, A. T. Caspar, S. D. Brandt, J. Wallach, H. H. Maurer. New 8 psychoactive substances 3 methoxyphencyclidine (3 MeO PCP) and 3 9 10 methoxyrolicyclidine (3 MeO PCPy): metabolic fate elucidated with rat urine and human 11 liver preparations and their detectability in urine by GC MS, LC (high resolution) MSn, 12 and LC high resolution MS/MS. Curr. Neuropharmacol. 2016 , DOI: 13 10.2174/1570159X14666161018151716. 14 15 [12] A. Helander, O. Beck, M. Bäckberg. Intoxications by the dissociative new psychoactive 16 substances diphenidine and methoxphenidine. Clin. Toxicol. 2015 , 53 , 446. 17 [13] S. P. Elliott, S. D. Brandt, J. Wallach, H. Morris, P. V. Kavanagh. First reported fatalities 18 associated withFor the ‘research Peer chemical’ Review 2 methoxydiphenidine. J. Anal. Toxicol. 2015 , 19 20 39 , 287. 21 [14] C. C. Ho, H. Pezhman, S. Praveen, E. H. Goh, B. C. Lee, M. Z. Zulkifli, M. R. Isa. 22 Ketamine associated ulcerative cystitis: a case report and literature review. Malays. J. 23 Med. Sci. 2010 , 17 , 61. 24 25 [15] G. Ohnacker, A. Kottler. Verfahren zur Herstellung von tertiären Aminen, ihren 26 Säureadditionssalzen und quaternären Ammoniumverbindungen. Patent No. 27 DE1124496. Dr. Karl Thomae GmbH, 1962 . 28 [16] Anonumous. Heterocyclic amine compounds. Patent No. GB836083. Parke, Davis and 29 30 Company, Detroit, USA, 1960 . 31 [17] A. Ahmadi, M. Khalili, R. Hajikhani, M. Naserbakht. New morpholine analogues of 32 phencyclidine: chemical synthesis and pain perception in rats. Pharmacol., Biochem. 33 Behav. 2011 , 98 , 227. 34 3 35 [18] S. R. Zukin, R. S. Zukin. Specific [ H]phencyclidine binding in rat central nervous 36 system. Proc. Natl. Acad. Sci. U.S.A. 1979 , 76 , 5372. 37 [19] T. P. Su, E. J. Cone, H. Shannon, D. B. Vaupel. Relative potencies of phencyclidine and 38 analogs in the opiate receptor binding assay. Res. Commun. Subst. Abuse 1980 , 1, 85. 39 40 [20] K. T. Brady, R. L. Balster. Discriminative stimulus properties of phencyclidine and five 41 analogues in the squirrel monkey. Pharmacol. Biochem. Behav. 1981 , 14 , 213. 42 [21] A. R. Gintzler, R. S. Zukin, S. R. Zukin. Effects of phencyclidine and its derivatives on 43 enteric neurones. Br. J. Pharmacol. 1982 , 75 , 261. 44 [22] R. E. West, R. W. McLawhon, G. Dawson, R. J. Miller. [ 3H]Ethylketocyclazocine binding 45 46 to NCB 20 hybrid neurotumor cells. Mol. Pharmacol. 1983 , 23 , 486. 47 [23] B. R. Martin, J. S. Katzen, J. A. Woods, H. L. Tripathi, L. S. Harris, E. L. May. 48 Stereoisomers of [ 3H] N allylnormetazocine bind to different sites in mouse brain. J. 49 Pharmacol. Exp. Ther. 1984 , 231 , 539. 50 51 [24] D. B. Vaupel, D. McCoun, E. J. Cone. Phencyclidine analogs and precursors: rotarod 52 and lethal dose studies in the mouse. J. Pharm. Exp. Ther. 1984 , 230 , 20. 53 [25] L. G. Aguayo, B. Witkop, E. X. Albuquerque. Voltage and time dependent effects of 54 phencyclidines on the endplate current arise from open and closed channel blockade. 55 56 Proc. Natl. Acad. Sci. U.S.A. 1986 , 83 , 3523. 57 58 59 60 12 http://mc.manuscriptcentral.com/dta Drug Testing and Analysis Page 14 of 25
1 2 3 [26] L. G. Aguayo, E. X. Albuquerque. Effects of phencyclidine and its analogs on the end 4 5 plate current of the neuromuscular junction. J. Pharmacol. Exp. Ther. 1986 , 239 , 15. 6 [27] D. E. McMillan, E. B. Evans, W. D. Wessinger, S. M. Owens. Structure activity 7 relationships of arylcyclohexylamines as discriminative stimuli in pigeons. J. Pharm. Exp. 8 Ther. 1988 , 247 , 1086. 9 10 [28] K. L. Marquis, R. Gussio, M. G. Webb, J. E. Moreton. Cortical EEG changes during the 11 of phencyclinoids. Neuropharmacology 1989 , 28 , 1193. 12 [29] D. J. McCann, R. A. Rabin, S. Rens Domiano, J. C. Winter. Phencyclidine/SKF 10,047 13 binding sites: evaluation of function. Pharmacol. Biochem. Behav. 1989 , 32 , 87. 14 15 [30] J. V. Wallach, Structure activity relationship (SAR) studies of arylcycloalkylamines as N 16 methyl D aspartate receptor antagonists. Ph.D. Thesis. The University of the Sciences, 17 Philadelphia, U.S.A., 2014 . Available at: http://gradworks.umi.com/36/90/3690548.html 18 [03 March 2017].For Peer Review 19 20 [31] D. A. Overton, C. F. Shen, G. Y. Ke, L. P. Gazdick. Discriminable effects of 21 phencyclidine analogs evaluated by multiple drug (PCP versus OTHER) discrimination 22 training. Psychopharmacology 1989 , 97 , 514. 23 [32] P. Geneste, J. M. Kamenka, S. N. Ung, P. Herrmann, R. Goudal, G. Trouiller. 24 25 Détermination conformationelle de dérivés de la phencyclidine en vue d'une corrélation 26 structure activité. Eur. J. Med. Chem. - Chim. Ther. 1979 , 14 , 301. 27 [33] K. Bailey, D. R. Gagne, R. K. Pike. Identification of some analogs of the hallucinogen 28 phencyclidine. J. Assoc. Off. Anal. Chem. 1976 , 59 , 81. 29 30 [34] V. H. Maddox, E. F. Godefroi, R. F. Parcell. The synthesis of phencyclidine and other 1 31 arylcyclohexylamines. J. Med. Chem. 1965 , 8, 230. 32 [35] A. R. Katritzky, Z. Najzarek, Z. Dega Szafran. The chemistry of N substituted 33 benzotriazoles; Part 11. The preparation of tertiary amines containing tertiary alkyl 34 35 groups from ketones, secondary amines, and organometallic reagents. Synthesis 1989 , 36 1989 , 66. 37 [36] G. A. Brine, E. E. Williams, K. G. Boldt, F. I. Carroll. Carbon 13 nuclear magnetic 38 resonance spectra of phenycyclidine analogs. J. Heterocyclic Chem. 1979 , 16 , 1425. 39 40 [37] I. J. Reynolds, T. A. Sharma. The use of ligand binding in assays of NMDA receptor 41 function. NMDA Receptor Protocols 1999 , 128 , 93. 42 [38] I. J. Reynolds. [ 3H](+)MK801 radioligand binding assay at the N methyl D aspartate 43 receptor. Curr. Protoc. Pharmacol. 2001 , Unit 1.20. 44 45 [39] Y. C. Cheng, W. H. Prusoff. Relationship between the inhibition constant ( K I) and the 46 concentration of inhibitor which causes 50 per cent inhibition (I 50 ) of an enzymatic 47 reaction. Biochem. Pharm. 1973 , 22 , 3099. 48 [40] M. M. Bradford. A rapid and sensitive method for the quantitation of microgram 49 50 quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 1976 , 51 72 , 248. 52 [41] Psychoactive Drug Screening Program Assay Protocol Book Version II. The University 53 of North Carolina. Available at: https://pdspdb.unc.edu/pdspWeb/content/PDSP Protocols II 54 55 2013-03-28.pdf [03 March 2017]. 56 [42] E. J. Cone, W. D. Darwin, D. Yousefnejad, W. F. Buchwald. Separation and identification 57 of phencyclidine precursors, metabolites and analogs by gas and thin layer 58 59 60 13 http://mc.manuscriptcentral.com/dta Page 15 of 25 Drug Testing and Analysis
1 2 3 chromatography and chemical ionization mass spectrometry. J. Chromatogr. A 1979 , 4 5 177 , 149. 6 [43] E. J. Cone, D. B. Vaupel, W. F. Buchwald. Phencyclidine: detection and measurement of 7 toxic precursors and analogs in illicit samples. J. Anal.Toxicol. 1980 , 4, 119. 8 [44] K. Bailey, D. Legault. Identification of cyclohexamine, phencyclidine and simple 9 10 analogues by carbon 13 nuclear magnetic resonance spectroscopy. Anal. Chim. Acta 11 1980 , 113 , 375. 12 [45] J. Hugel, J. Meyers, D. Lankin, Analysis of the hallucinogens. In Hallucinogens: A 13 Forensic Drug Handbook , (Ed.: R.R. Laing), Academic Press, Amsterdam, 2003 , pp. 14 15 191. 16 [46] S. R. Zukin, S. R. Zukin, Identification and characterization of [3H] phencyclidine binding 17 to specific brain receptor sites. In PCP (Phencyclidine): Historical and Current 18 Perspectives , 8.For (Ed.: E.F. Peer Domino), NPP Review Books, Ann Arbor, MI, 1981 , pp. 105. 19 20 [47] A. Kalir, Structure activity relationships of phencyclidine derivatives. In PCP 21 (Phencyclidine), Historical and Current Perspectives , Chapter 5. (Ed.: E.F. Domino), 22 NPP Books, Michigan, 1981 , pp. 31. 23 [48] T. P. Su, X. Z. Wu, E. J. Cone, K. Shukla, T. M. Gund, A. L. Dodge, D. W. Parish. Sigma 24 25 compounds derived from phencyclidine: identification of PRE 084, a new, selective 26 sigma ligand. J. Pharm. Exp. Ther. 1991 , 259 , 543. 27 [49] S. A. Lipton. Failures and successes of NMDA receptor antagonists: molecular basis for 28 the use of open channel blockers like memantine in the treatment of acute and chronic 29 30 neurologic insults. NeuroRx 2004 , 1, 101. 31 [50] G. Rammes, W. Danysz, C. G. Parsons. Pharmacodynamics of memantine: an update. 32 Curr. Neuropharmacol. 2008 , 6, 55. 33 [51] B. L. Roth, S. Gibbons, W. Arunotayanun, X. P. Huang, V. Setola, R. Treble, L. Iversen. 34 35 The ketamine analogue methoxetamine and 3 and 4 methoxy analogues of 36 phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor. 37 PloS One 2013 , 8, e59334. 38 [52] C. Sundermann, B. Sundermann. Oxosubstituierte Cyclohexyl 1,4 diamin Derivate. 39 Patent No. WO2005/110970. Grünenthal GmbH, Aachen, Germany, 2005 . 40 41 [53] C. N. Sang. NMDA receptor antagonists in neuropathic pain: experimental methods to 42 clinical trials. J. Pain Symptom Manage. 2000 , 19 , S21. 43 [54] D. Pud, E. Eisenberg, A. Spitzer, R. Adler, G. Fried, D. Yarnitsky. The NMDA receptor 44 antagonist amantadine reduces surgical neuropathic pain in cancer patients: a double 45 46 blind, randomized, placebo controlled trial. Pain 1998 , 75 , 349. 47 [55] C. G. Parsons. NMDA receptors as targets for drug action in neuropathic pain. Eur. J. 48 Pharmacol. 2001 , 429 , 71. 49 [56] S. Felsby, J. Nielsen, L. Arendt Nielsen, T. S. Jensen. NMDA receptor blockade in 50 51 chronic neuropathic pain: a comparison of ketamine and magnesium chloride. Pain 52 1996 , 64 , 283. 53 54 55 56 57 58 59 60 14 http://mc.manuscriptcentral.com/dta Drug Testing and Analysis Page 16 of 25
1 2 3 4 5 6 Figure Captions 7 8 9 10 Figure 1 . A: Examples of psychoactive substances with dissociative profiles. B: Morpholine 11 analogs investigated in the present study. The numbering scheme employed for 13 C NMR 12 assignments is shown for the isomers substituted with methoxy groups. 13 14 Figure 2. Synthetic scheme used to for the preparation of the investigated PCMo series via the 15 modified Geneste route.[5,10,32] TFA: trifluoroacetic acid; TEA: triethylamine. R = 2 , 3 , and 4 16 17 MeO, 3,4 OCH 2O, 3 Me or H. 18 For Peer Review 19 Figure 3. Gas chromatography ion trap mass spectrometry (GC IT MS) data obtained from 20 electron ionization (EI) and chemical ionization (CI) methods. 21 22 Figure 4. Ultra high performance liquid chromatography high mass accuracy electrospray 23 tandem mass spectra. 24 25 Figure 5. Diode array ultraviolet full scan spectra and high performance liquid chromatography 26 data for 2 , 3 and 4 MeO PCMo. 27 28 Figure 6. Competitive binding curves for PCP, PCMo, and analogues from [ 3H] MK 801 29 30 displacement using rat forebrain homogenate. 31 32 Figure 7. Heatmap of compound affinities (Ki) at CNS receptors. Solid green without number 33 indicates IC 50 was >10,000 nM in primary assay. 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 15 http://mc.manuscriptcentral.com/dta Page 17 of 25 Drug Testing and Analysis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 A O O 39 H H N 40 N N N 41 42 43 44 Cl 45 46 PCP Ketamine O Diphenidine 47 Methoxetamine 48 49 50 B51 52 β O O O α O 53 5 6 β α 54 1 N N N N 2’ 55 4 1’ 3’ 56 3 2 57 6’ 58 4’ 5’ O O 59 O 60 2-MeO-PCMo 3,4-MD-PCMohttp://mc.manuscriptcentral.com/dta3-Me-PCMo PCMo 3-MeO-PCMo 4-MeO-PCMo Drug Testing and Analysis Page 18 of 25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Br Br 57 O O 58 59 MgBr OH 1. NaN3, TFA NH 2 O N 60 2. LiAlH4 TEA http://mc.manuscriptcentral.com/dta
R R R R 232 189 O 100% 2-MeO-PCMo 100% 2-MeO-PCMo Page 19EI-IT-MS of 25 Drug Testing andCI-IT-MS Analysis N 10.04 min 10.04 min 75% 75% 1 O 50%2 121 50% 3 275 276 4 25% 189 25% 5 86 91 88 121 147 168 204 56 6 218 70% 0% 8 50 100 150 200 250 m/z 50 100 150 200 250 m/z 100% 100% 276 9 3-MeO-PCMo 232 3-MeO-PCMo O EI-IT-MS CI-IT-MS 189 10 N 75%11 10.30 min 75% 10.30 min 12 50%13 50% 14 88 O 275 25%15 121 25% 56 16 86 189 17 147 168 218 180% For Peer0% Review 19 50 100 150 200 250 m/z 50 100 150 200 250 m/z 189 189 100%20 4-MeO-PCMo 100% 4-MeO-PCMo O 21 EI-IT-MS CI-IT-MS 121 N 22 10.52 min 10.51 min 75% 75% 23 24 50% 232 O 25 50% 26 25%27 147 275 25% 81 91 28 159 218 56 88 276 290% 0% 30 50 100 150 200 250 m/z 50 100 150 200 250 m/z 31 100% 3,4-MD-PCMo 203 100% 3,4-MD-PCMo 203 O 32 EI-IT-MS CI-IT-MS N 33 75% 11.05 min 75% 11.05 min 34 135 35 246 O 50%36 50% O 37 289 25%38 161 25% 39 81 56 88 290 400% 0% 41 50 100 150 200 250 m/z 50 100 150 200 250 m/z 42 216 173 O 100%43 3-Me-PCMo 100% 3-Me-PCMo 44 EI-IT-MS CI-IT-MS N 9.57 min 9.57 min 75%45 75% 46 260 50%47 50% 48 88 259 49 105 259 25% 25% 168 258 50 86 131 173 56 51 157 202 202 520% 0% 53 50 100 150 200 250 m/z 50 100 150 200 250 m/z 100%54 PCMo 202 100% PCMo 246 O 55 EI-IT-MS CI-IT-MS N 5675% 9.31 min 75% 9.31 min 159 57 5850% 50% 59 245 88 60 25% 25% 86 56 91 117 259168 http://mc.manuscriptcentral.com/dta 103 188 202 0% 0% 50 100 150 200 250 m/z 50 100 150 200 250 m/z 121.06440 1.2 N Drug Testing and Analysis Page 20 of 25 0.8 O 1 0.4 2 88.07583 189.12709 2-MeO-PCMo 3 70.06425 135.07870 161.09645 213.12602 231.07644 0 4 50 70 90 110 130 150 170 190 210 230 250 270 290 m/z 5 x106 4 +ESI Product Ion (2.713 min) Frag=110.0V [email protected] (276.19572[z=1] -> **) 3MeO-PCMooMSMS.d 7 O 121.06484 48 N 9 310 88.07589 11 189.12767 212 O 13 81.07021 1 3-MeO-PCMo 14 147.08060 52.94330 71.06909 109.06409 133.06385 199.57025 218.95181 150 16 50 70 90 110 130 150 170 190 210 230 250 270 290 m/z 17 x10183 +ESI Product Ion (2.765 min) Frag=110.0V [email protected] (276.19568[z=1]For -> **) 4MeO-PCMooMSMS.dPeer Review O 2.419 121.06483 20 N 1.821 22 1.2 88.07655 23 O 24 0.6 25 81.01617 147.08024 189.12402 4-MeO-PCMo 26 270 70 90 110 130 150 170 190 210 230 250 270 m/z 28 29 O 30 N 312 x1032 +ESI Product Ion (2.757 min) Frag=110.0V [email protected] (290.17545[z=1] -> **) MD-PCMooMSMS.d 203.10659 335 173.09394 135.04317 O 34 O 353 81.07045 168.10232 36 MD-PCMo 197.08166 207.65070 131.34264 265.43160 371 145.06390 177.04903 38 52.83711 74.33389 110.85906 220.37375 278.94339 39 50 70 90 110 130 150 170 190 210 230 250 270 m/z 40 4 x1041 +ESI Product Ion (2.895 min) Frag=110.0V [email protected] (260.20139[z=1] -> **) 3Me-PCMooMSMS.d 42 O 105.07044 443 N 44 345 46 2 88.07595 47 173.13275 48 1 81.07011 3-Me-PCMo 49 51.02290 67.05380 131.08462 500 51 30 50 70 90 110 130 150 170 190 210 230 250 270 290 m/z 52
53 +ESI Product Ion (2.728 min) Frag=110.0V [email protected] (246.18513[z=1] -> **) PCMooMSMS.d x10544 55 O 456 91.05449 57 N 3 58 88.07555 159.11687 59 260 81.06993 PCMo 1 http://mc.manuscriptcentral.com/dta 55.05454 67.05415 117.06936 0 10 30 50 70 90 110 130 150 170 190 210 230 250 270 m/z Page 21 of 25 Drug Testing and Analysis
1 2 3 4 5 6 7 8 9 10 11 12 [%]13 mAU 14 100 2-MeO-PCMo 80.0 20 mg/L 15 3-MeO-PCMo 6.80 min O 220 nm 16 4-MeO-PCMo 8017 N 18 For Peer Review 19 62.5 60 20 O 2-MeO-PCMo 21 4022 50.0 23 24 219.3 2025 37.5 26 277.9 270 28200 250 300 350 400 450 500 550 594 nm 29 25.0 [%]30 10031 32 3-MeO-PCMo O 12.5 33 6.55 min 8034 N 35 36 60 6.0 8.0 min 37 38 O 4039 40 41 218.1 2042 43 277.8 044 45 200 250 300 350 400 450 500 550 594 nm 46 47 48 [%]49 10050 4-MeO-PCMo O 51 6.52 min 52 N 80 53 54 6055 O 56 57 229.7 4058 59 2060 270.3 http://mc.manuscriptcentral.com/dta 277.0
200 250 300 350 400 450 500 550 594 nm Drug Testing and Analysis Page 22 of 25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 Figure 6. Competitive binding curves for PCP, PCMo, and analogues from [ 3H]-MK-801 displacement using 27 rat forebrain homogenate. 28 29 145x80mm (300 x 300 DPI) 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta Page 23 of 25 Drug Testing and Analysis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 7. Heatmap of compound affinities (K i) at CNS receptors. Solid green without number indicates IC 50 was >10,000 nM in primary assay. 48 49 87x190mm (300 x 300 DPI) 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta Drug Testing and Analysis Page 24 of 25
1 2 3 Table 1 . 1H NMR data for PCMo freebases in CDCl 4 3 Proton 2 MeO PCMo 3 MeO PCMo 4 MeO PCMo 3,4 MD PCMo 3 Me PCMo PCMo 5 6 H1 7 H2,6 2.64–2.55 m 2.13–2.03 m 2.15–2.05 m 2.08–1.97 m 2.15–2.04 m 2.19–2.04 m 8 (2H eq ) (2H eq ) (2H eq ) (2H eq ) (2H eq ) (2H eq ) 9 10 1.80 ddd 1.93 ddd 1.91 ddd 1.89 ddd 1.94 ddd 1.95 ddd 11 (J = 13.6, 10.5, 3.0 (J = 13.3, 9.5, 3.3 (J = 13.4, 9.7, (J = 13.3, 9.6, 3.3 (J = 13.4, 9.4, (J = 13.5, 12 Hz, 2H ) Hz, 2H ) 3.3 Hz, 2H ) Hz, 2H ) 3.3 Hz, 2H ) 9.6, 3.4 Hz, 13 ax ax ax ax ax 14 2H ax ) 15 H3,5 1.74–1.61 m 1.74–1.65 m 1.74–1.64 m 1.74–1.63 m 1.76–1.66 m 1.76–1.64 m 16 (2H eq ) (2 H eq ) (2 H eq ) (2 H eq ) (2 H eq ) (2H eq ) 17 18 1.32–1.20 m For1.38–1.27 mPeer 1.35–1.23 Review m 1.39–1.25 m 1.37–1.26 m 1.39–1.24 m 19 (2H ) (2 H ) (2 H ) (2 H ) (2 H ) (2H ) 20 ax ax ax ax ax ax 21 H4 1.51–1.32 m 1.47–1.40 m 1.49–1.39 m 1.49–1.39 m 1.48–1.39 m 1.50–1.39 m 22 (2H) (2H) (2H) (2H) (2H) (2H) 23 H1’
24 H2’ 6.85 t 7.21 dm 6.79 d 7.10 s 7.32–7.27 m 25 (J = 1.9 Hz, 1H) (J = 8.9 Hz, 1H) (J = 8.3 Hz, 1H) (1H) *overlap (1H) 26 with H6’ 27 H 6.98–6.88 m 6.88 dm 7.38–7.32 m 28 3’ J 29 (1H) ( = 8.9 Hz, 1H) (1H) 30 H4’ 7.29–7.18 m 6.79 dd 7.10–7.06 m 7.27–7.20 m 31 (1H) (J = 8.1, 2.5 Hz, (1H) *overlap (1H)
32 1H) with H2’ 33 H 5’ 6.98–6.88 m 7.27 t 6.88 dm 6.82 d 7.30–7.19 m 7.38–7.32 m 34 (1H) (J = 8.0 Hz, 1H) (J = 8.9 Hz, 1H) (J = 1.8 Hz, 1H) (1H) (1H) 35 36 H6’ 7.29–7.18 m 6.89 dd 7.21 dm 6.75 dd 7.05 dm 7.32–7.27 m J J J J 37 (1H) ( = 7.8, 1.8 Hz, ( = 8.9 Hz, 1H) ( = 8.3, 1.8 Hz, 1H) ( = 7.6 Hz, 1H) (1H) 38 1H) 39 Hα 2.43 t 2.34 t 2.32 t 2.33 t 2.33 t 2.33 t 40 (J = 4.5 Hz, 4H) (J = 4.6 Hz, 4H) (J = 4.6 Hz, 4H) (J = 4.5 Hz, 4H) (J = 4.6 Hz, 4H) (J = 4.5 Hz, 41 4H) 42 Hβ 3.63 t 3.63 t 3.63 t 3.63 t 3.63 t 3.63 t 43 J J J J J J 44 ( = 4.6 Hz, 4H) ( = 4.6 Hz, 4H) ( = 4.6 Hz, 4H) ( = 4.6 Hz, 4H) ( = 4.7 Hz, 4H) ( = 4.6 Hz, 45 4H) 46 Cc 3.77 s (OCH 3) 3.82 s (OCH 3) 3.81 s (OCH 3) 5.95 s (OCH 2O) 2.37 s (CH 3) 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta Page 25 of 25 Drug Testing and Analysis
1 2 3 Table 2. 13 C NMR data for PCMo analogues (freebase, in CDCl ) 4 3 Carbon 2 MeO PCMo 3 MeO PCMo 4 MeO PCMo 3,4 MD PCMo 3 Me PCMo PCMo 5 6 C1 63.12 60.69 60.35 60.72 60.62 60.71 7 C2,6 34.49 33.00 33.00 33.21 32.93 32.86
8 C3,5 22.93 22.29 22.28 22.29 22.28 22.25 9 C4 26.46 26.26 26.32 26.27 26.33 26.31 10 C 126.91 141.18 131.37 133.70 139.16 139.22 11 1’ 12 C2’ 159.75 114.34 128.52 107.23 127.99 127.34 13 C3’ 112.43 159.19 112.87 147.41 136.97 127.64 14 C4’ 130.52 110.51 157.87 145.72 124.47 126.35
15 C5’ 119.97 128.45 112.87 107.97 127.48 127.64 16 C6’ 127.92 119.95 128.52 120.66 127.06 127.34 17 C 46.64 45.91 45.84 45.88 45.88 45.86 18 α For Peer Review 19 Cβ 68.13 67.88 67.84 67.86 67.89 67.87 20 Cc 55.16 (OCH 3) 55.17 (OCH 3) 55.14 (OCH 3) 100.82 (OCH 2O) 21.86 (CH 3) 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta Drug Testing and Analysis Page 26 of 25
1 2 3 Table 3. NMDAR binding affinities for PCMo series using [ 3H]-MK-801 in 4 rat forebrains. Means ± SEM from three separate experiments run in 5 duplicate. 6 7 Compound IC 50 ± SEM (nM) Ki ± SEM (nM) 8 9 PCP 34.7 ± 2.5 22.1 ± 1.6 10 Ketamine [4] 508.5 ± 30.1 [4] 323.9 ± 19.2 [4] 11 12 2-MeO-PCMo 2,477 ± 115 1,578 ± 73.2 13 14 3-MeO-PCMo 397.0 ± 45.4 252.9 ± 28.9 15 16 4-MeO-PCMo 3,326 ± 343.3 2,118 ± 218.7 17 3,4-MD-PCMo 668.0 ± 30.5 425.5 ± 19.4 18 For Peer Review 19 3-Me-PCMo 316.8 ± 29.1 201.8 ± 18.5 20 21 PCMo 524.6 ± 13.7 334.1 ± 8.8 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/dta