The chemistry and pharmacology of putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS) 5F-PY-PICA, 5F-PY-PINACA, and their analogues

The structural diversity of synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS) has increased since the first examples were reported a decade ago. 5F-PY-PICA and 5F-PY-PINACA were identified in 2015 as putative SCRA NPS, although nothing is known of their pharmacology. 5F-PY-PICA, 5F-PY-PINACA, and analogues intended to explore structure-activity relationships within this class of SCRAs were synthesized and characterized by nuclear magnetic resonance spectroscopy and liquid chromatography–quadrupole time-of-flight–mass spectrometry. Using competitive binding experiments and fluorescence-based plate reader membrane potential assays, the affinities and activities of all analogues at cannabinoid type 1 and type 2 receptors (CB1 and CB2) were evaluated. All ligands showed minimal affinity for CB1 (pKi < 5), although several demonstrated moderate CB2 binding (pKi = 5.45–6.99). At 10 μM none of the compounds produced an effect > 50% of CP55,950 at CB1, while several compounds showed a slightly higher relative efficacy at CB2. Unlike other SCRA NPS, 5F-PYPICA and 5F-PY-PINACA did not produce cannabimimetic effects in mice at doses up to 10 mg/kg.

Experimental.General chemical synthesis details.All reactions were performed under an atmosphere of nitrogen or argon unless otherwise specified.Dichloromethane, N,Ndimethylformamide (DMF), methanol, and toluene were anhydrous and used as purchased.
Triethylamine was distilled over calcium hydride.All other commercially available reagents (Sigma-Aldrich) were used as purchased.Analytical thin layer chromatography (TLC) was performed using Merck aluminum-backed silica gel 60 F254 (0.2 mm) plates which were visualized using shortwave (254 nm) ultra-violet fluorescence.Flash chromatography was performed using Merck Kieselgel 60 (230-400 mesh) silica gel.Melting points were measured in open capillaries using an Laboratory Devices Mel-Temp II and are uncorrected.Nuclear magnetic resonance spectra were recorded at 298 K or 308 K using a Agilent 400 MHz spectrometer.The data are reported as chemical shift (δ ppm) relative to the residual protonated solvent resonance, relative integral, multiplicity (s = singlet, br = broad singlet, d = doublet, t = triplet, q = quartet, quin.= quintet, m = multiplet), coupling constants (J Hz) and assignment.Assignment of signals was assisted by COSY, DEPT, HSQC, and HMBC experiments where necessary.For compounds containing saturated heterocyclic amides (5-8, 11-14), severe exchange broadening of the amide α-and β-carbon signals was observed, consistent with previous characterization of analogous systems. 37eneral procedure for amidation of 1-alkyl-1H-indole-3-carboxylic acids.A solution of the appropriate carboxylic acid (0.5 mmol) in CH2Cl2 (1 mL) was treated with (COCl)2 (85 µL, 1.0 mmol, 2.0 equiv.)followed by DMF (1 drop).After stirring for 2 h, the solution was evaporated in vacuo, and the crude acid chloride was used immediately in the following step.
To a refluxing solution of potassium hydroxide (278 mg, 4.95 mmol, 3.3 equiv.) in methanol (0.55 mL) was added portionwise a solution of the crude 1-alkyl-3-trifluoroacetyl-1H-indole in toluene (1.25 mL) and the solution heated at reflux for 2 h.The solution was cooled to ambient temperature and partitioned between 1 M aq.NaOH (40 mL) and Et2O (5 mL).The layers were separated and the aqueous layer was adjusted to pH 1 with 10 M aq.HCl.The aqueous phase was extracted with Et2O (3 × 10 mL), dried (Na2SO4), and solvent evaporated under reduced pressure.The crude products were recrystallized from i-PrOH unless otherwise stated.In vitro cannabinoid receptor functional activity assay.Mouse AtT20 neuroblastoma cells stably transfected with human CB1 or human CB2 have been previously described. 43and were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U penicillin/streptomycin, and 80 µg/mL hygromycin (InvivoGen, San Diego, CA, USA).Cells were passaged at 80% confluency as required.Cells for assays were grown in 75 cm 2 flasks and used at 90% confluence.The day before the assay cells were detached from the flask with trypsin/EDTA (Sigma) and resuspended in 10 mL of Leibovitz's L-15 media supplemented with 1% FBS, 100 U penicillin/streptomycin and 15 mM glucose.The cells were plated in volume of 90 μL in black walled, clear bottomed 96-well microplates (Corning) which had been precoated with poly-L-lysine (Sigma, Australia).Cells were incubated overnight at 37 °C in ambient CO2.
Fluorescence was measured using a FlexStation 3 (Molecular Devices) microplate reader with cells excited at a wavelength of 530 nm and emission measured at 565 nm.Baseline readings were taken every 2 s for at least 2 min, at which time either drug or vehicle was added in a volume of 20 μL.The background fluorescence of cells without dye or dye without cells was negligible.
. CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint (which was this version posted October 3, 2018.; https://doi.org/10.1101/430959doi: bioRxiv preprint 20 Changes in fluorescence were expressed as a percentage of baseline fluorescence after subtraction of the changes produced by vehicle addition.The final concentration of DMSO was 0.1%.Data were analyzed with PRISM (GraphPad Software Inc., San Diego, CA), using four-parameter nonlinear regression to fit concentration-response curves.In all plates, a maximally effective concentration of CP 55,940 was added to allow for normalization between assays.
In vivo pharmacological assessment of 5F-PY-PICA and 5F-PY-PINACA.Two cohorts of 4 adult male C57BL/6J mice (Animal Resources Centre, Perth, Australia) were used for biotelemetric assessment of body temperature.The mice weighed between 20.5 and 28.5 g on arrival, and were singly housed in a climate-controlled testing room (23 ± 1 °C) on a 12 h light/dark cycle (lights on from 07:00 to 19:00).Water and standard rodent chow were provided ad libitum.All experiments were approved by The University of Sydney Animal Ethics Committee.
Biotelemetry transmitters (model TA-F10, Data Sciences International, St. Paul, MN) were implanted as previously described. 13The transmitter was implanted according to the manufacturers protocol into the peritoneal cavity following anesthetization with isoflurane (3% induction, 1-2% maintenance).The wound was sutured closed and data collection commenced after 10 days of recovery.
The mice were habituated over multiple days to injections of vehicle (a solution composed of 7.8% polysorbate 80 and 92.2% physiological saline).Injection always occurred at a set time of day .CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint (which was this version posted October 3, 2018.; https://doi.org/10.1101/430959doi: bioRxiv preprint 21 (10:00 am).Each cohort received injections of each compound in an ascending dose sequence (0.3, 1, 3, and 10 mg/kg).This ascending sequence was used in order to minimize the risk posed to the animals in assessing hitherto untested compounds.Two washout (drug free) days were given between each dose to limit development of tolerance or carryover effects.
Body temperature data was gathered continuously at 1000 Hz and organized into 15 minute bins using Dataquest A.R.T. software (version 4.3, Data Sciences International, St. Paul, MN), and analysed using PRISM (version 7, Graphpad Software Inc., San Diego, CA). 13sults and discussion.The synthesis of 5F-PY-PICA (5) and related analogues 7-15 is shown in Figure 2. Using a general procedure, indole (15) was first alkylated with the appropriate alkyl bromide, converted to the corresponding 3-trifluoroacetylindole, and hydrolyzed to the carboxylic acid (16-20).The relevant carboxylic acid 16-20 was converted to the corresponding acid chloride using oxalyl chloride, and treated with pyrrolidine, piperidine, azepane, diethylamine, or dimethylamine to furnish amides 5 and 7-14.
[APPROXIMATE PLACEMENT OF FIGURE 2]   The synthesis of 5F-PY-PINACA (6) required a slightly different route and is depicted in Figure 3. Methyl 1H-indazole-3-carboxylate (21) was deprotonated with potassium tert-butoxide, and then treated with 1-bromo-5-fluoropentane, to regioselectively yield the desired 1-alkylated indole-3-carboxylate (22) as previously described. 13Saponification of 22 afforded acid 23, which was subsequently coupled with pyrrolidine using EDC-HOBt to give 6.Attempts to form 6 by .CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint (which was this version posted October 3, 2018.; https://doi.org/10.1101/430959doi: bioRxiv preprint 22 converting 23 to the corresponding acid chloride and treating with pyrrolidine, as described for the indoles 5 and 7-14 above, were unsuccessful. [APPROXIMATE PLACEMENT OF FIGURE 3]   5F-PY-PICA, 5F-PY-PINACA, and analogues 7-14 were analyzed using melting point range determinations, 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy, and liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS).Full details of these analyses are provided in the experimental section.The mass spectra and fragment assignments for 5F-PY-PICA and 5F-PY-PINACA from LC-QTOF-MS/MS analysis are shown in Figures 4a and 4b, respectively.The QTOF mass fragmentation profiles of 5F-PY-PICA and 5F-PY-PINACA were similar, with molecular ions and fragments arising from scission of the amide C-N bond observed in each case.The base peak for 5F-PY-PICA was the molecular ion (m/z 303.1864, 100%), and an ion consistent with loss of pyrrolidine was next most abundant (m/z 232.1127, 55%).The reverse was observed for 5F-PY-PINACA, with the acylium ion resulting from amide C-N bond scission occurring as the base peak (m/z 233.1085, 100%), and the molecular ion as the next most abundant species (m/z 304.1799, 54%).For 5F-PY-PICA, the only other abundant fragment (>10%) was m/z 98.0595 (37%), likely formed from cleavage of the indole C3amide bond.Other minor peaks were consistent with amide cleavage and concomitant indole N1 dealkylation (m/z 144.0426, 8%) or defluorination (m/z 283.1804, 3%).5F-PY-PICA, 5F-PY-PINACA, and analogues 7-14 were screened in competitive radioligand binding assays and fluorescence-based functional assays against CB1 and CB2 receptors (Table 1).
. CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint (which was this version posted October 3, 2018.; https://doi.org/10.1101/430959doi: bioRxiv preprint 7.45 ± 0.03, and a maximum decrease in fluorescence of 32 ± 1%.Several ligands produced substantial hyperpolarizations of AtT20-CB2 cells, but none had a maximum effect greater than 75% of CP55,940 at 1 µM.The largest activation of CB2 was induced by 8 (72 ± 3%).
The in vitro pharmacological profiles of 5F-PY-PICA and 5F-PY-PINACA indicate that these putative SCRAs have very low affinity and efficacy at CB1 receptors, and are unlikely to be psychoactive in humans.Similar to many SCRA NPS, both 5F-PY-PICA and 5F-PY-PINACA had substantial activity at CB2 receptors, but this is unlikely to mediate any discernable effects on mood in people.
To further characterize these compounds, and determine if they could be metabolized into active cannabinoids, we examined the effects of 5F-PY-PICA and 5F-PY-PINACA in mice using biotelemetry.8][49][50][51] As anticipated from the in vitro pharmacological data, neither 5F-PY-PICA nor 5F-PY-PINACA produced physiological effects consistent with central CB1 activity, and hypothermic effects were not observed in mice at doses up to 10 mg/kg (Figure 5).Many SCRAs, including JWH-018, AB-FUBINACA, MDMB-FUBINACA, and 5F-CUMYL-P7AICA, elicit hypothermic effects at doses of 1 mg/kg or below in this rodent model via a CB1 receptor-mediated mechanism. 13,43,52Therefore, it can be concluded that neither 5F-PY-PICA nor 5F-PY-PINACA exhibit the in vitro and in vivo cannabimimetic profiles shared by SCRA NPS.This does not preclude, of course, the possibility of psychoactive effects generated through non-cannabinoid mechanisms.

QTOF
LC-MS/MS acquisition and analysis method.All samples were analyzed using an Agilent LC 1260 Infinity Binary Liquid Chromatograph (LC) System attached to an Agilent 6550 iFunnel Quadrupole Time-of-Flight Mass Spectrometer (QTOF/MS) 6550 (Agilent Technologies, Santa Clara, CA).An Agilent jet stream electrospray ionization (ESI) source with a dual nebulizer that allows constant introduction of reference mass during sample run was used to ionize sample organic components in positive mode.Each sample was prepared from a crystalline or resin aliquot of purified synthetic product and diluted to a final concentration of 100 and 500 ng/mL in 10% LC-MS grade acetonitrile (Honeywell B&J, Muskegon, MI); two concentrations were injected to elucidate potential solvent impurities or instrument artifacts.In each sample run, 2.5 µL was injected into an Agilent Poroshell 120 C-18 column (2.1 × 100 mm, 2.7 µm) maintained at 50 °C.Chromatographic separation was achieved by gradient elution using LC-MS grade water (Honeywell B&J, Muskegon, MI) with 0.05% formic acid and 5 mM ammonium formate as mobile phase A, and acetonitrile with 0.05% formic acid as mobile phase B. The elution gradient used was 0-0.5 min = 5% B; 1.5 min = 30% B; 4.5 min = 70% B; 7.5 min = 100% B; 7.5-10 min = 100% B; and 10.01-12 min = 5% B.Ionization of chromatographic eluates was induced on the QTOF/MS using an ESI source in the positive mode operated under the following conditions: gas temperature at 225 °C; sheath gas temperature at 350 °C; drying gas flow at 14 L/min; sheath gas flow at 11 L/min; nebulizer pressure at 40 psig; voltage cap at 3000 V; and nozzle voltage at 500 V. Data acquisition was run at 2 GHz in extended dynamic range mode.Both TOF/MS and MS/MS spectra were collected in automated MS/MS mode using 500 arbitrary units as threshold for inducing MS/MS data collection.An active exclusion was used after 1 spectra, with a release time of 0.05 mins.Each

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Despite their detection in drug markets in 2015, 5F-PY-PICA, 5F-PY-PINACA, and several analogues exhibited low affinity and efficacy at CB1 and CB2 receptors in vitro.Moreover, 5F-PY-PICA and 5F-PY-PINACA failed to elicit the hypothermic, bradycardic, and hypolocomotive effects potently induced by other SCRA NPS.Taken together, the in vitro and in vivo profiles of 5F-PY-PICA and 5F-PY-PINACA cast doubt on their classification as SCRAs, although NPS designation by non-cannabinoid mechanisms cannot be excluded.