Activation of μ‐opioid receptors by MT‐45 (1‐cyclohexyl‐4‐(1,2‐diphenylethyl)piperazine) and its fluorinated derivatives

Background and Purpose A fluorinated derivative (2F‐MT‐45) of the synthetic μ‐opioid receptor agonist MT‐45 (1‐cyclohexyl‐4‐(1,2‐diphenylethyl)piperazine) was recently identified in a seized illicit tablet. While MT‐45 is a Class A drug, banned in a number of countries, nothing is known about the pharmacology of 2F‐MT‐45. This study compares the pharmacology of MT‐45, its fluorinated derivatives and two of its metabolites. Experimental Approach We used a β‐arrestin2 recruitment assay in CHO cells stably expressing μ receptors to quantify the apparent potencies and efficacies of known (MT‐45, morphine, fentanyl and DAMGO) and potential agonists. In addition, the GloSensor protein was transiently expressed to quantify changes in cAMP levels. We measured Ca2+ to investigate whether MT‐45 and its metabolites have effects on GluN1/N2A NMDA receptors stably expressed in Ltk‐ cells. Key Results The fluorinated MT‐45 derivatives have higher apparent potencies (2F‐MT‐45: 42 nM) than MT‐45 (1.3 μM) for inhibition of cAMP accumulation and β‐arrestin2 recruitment (2F‐MT‐45: 196 nM; MT‐45: 23.1 μM). While MT‐45 and 2F‐MT‐45 are poor recruiters of β‐arrestin2, they have similar efficacies for reducing cAMP levels as DAMGO. Two MT‐45 metabolites displayed negligible potencies as μ receptor agonists, but one, 1,2‐diphenylethylpiperazine, inhibited the NMDA receptor with an IC50 of 29 μM. Conclusion and Implications Fluorinated derivatives of MT‐45 are potent μ receptor agonists and this may pose a danger to illicit opioid users. Inhibition of NMDA receptors by a metabolite of MT‐45 may contribute to the reported dissociative effects.

of chronic pain (Colvin et al., 2019). Furthermore, opioid exposure can be habit forming leading to addiction. Although prescription opioidrelated fatalities in the United States appear to have reached a plateau, illicit fentanyl and its analogues, including carfentanil, and other novel synthetic opioids (NSOs) are now contributing to a new wave of deaths (Socías & Wood, 2017). The addictive potential and profitability of opioids has driven a proliferation of novel synthetic opioids (Prekupec, Mansky, & Baumann, 2017). piperazine) is an novel synthetic opioid originally developed as a potential analgesic substitute for morphine (Fujimura, Tsurumi, Nozaki, Hori, & Imai, 1978;Nakamura & Shimizu, 1976). Research into MT-45 was discontinued shortly thereafter due to its side effect profile. However, a recent study demonstrated that MT-45 has a higher affinity for μ receptors than for either δor κ-receptors (Baumann et al., 2018). Opioids initiate their analgesic and adverse effects by activating μ-opioid receptors (Matthes et al., 1996). Activated μ receptors signal via G i/o -proteins to inhibit adenylate cyclase activity, causing a reduction in cAMP levels. Activation of G i/o -proteins also increases K + conductance, via activation of G protein activated inwardly rectifying K + channels and inhibits voltage-activated Ca 2+ channel activity (Williams et al., 2013). The resulting reduction in neuronal excitability, particularly in the pain pathways, underlies the analgesic effects of opioids. However, activation of μ receptors outside the pain pathways contributes to the detrimental effects of opioids, including respiratory depression and addiction, which are key contributors to rising drug death statistics in the United States and United Kingdom (Dwyer-Lindgren et al., 2018;Kimber, Hickman, Strang, Thomas, & Hutchinson, 2019;Middleton, McGrail, & Stringer, 2016).

It is now a Class
In addition to initiating G protein-mediated signalling, activated μ receptors also recruit the multifunctional scaffold protein, β-arrestin2 (Latorraca et al., 2018;Williams et al., 2013). β-arrestin2 participates in μ receptor internalisation and recycling, as well as in the recruitment of additional kinases. Recruitment of β-arrestin2 has been implicated in the detrimental effects of μ receptor agonists (Bohn et al., 1999;Raehal & Bohn, 2011). There is considerable interest in the idea that μ receptor agonists may be more or less prone to tolerance, respiratory depression and constipation when biased in favour or against β-arrestin2 recruitment, respectively (Ehrlich et al., 2019).
User experiences of MT-45 posted within online drug forums include analgesia, euphoria, tolerance, respiratory depression and constipation, consistent with other μ receptor agonists, as well as dissociative-like effects akin to those reported by users of ketamine (Kjellgren, Jacobsson, & Soussan, 2016). The cause of the apparent dissociative effects of MT-45 is unclear. It is not known whether MT-45 or its metabolites inhibit the activity of the NMDA receptor, the target of several dissociative drugs, including ketamine. The identification of metabolites of MT-45 reveals that some have structural similarities to another dissociative, NMDA receptor-inhibiting drug, diphenidine (McKenzie et al., 2018).
In this study, we characterised the pharmacology of MT-45, 2F-MT-45 and its regioisomers, 3F-MT-45 and 4F-MT-45, along with two of the major metabolites of MT-45, M1 and M17. We compared their potencies and efficacies with those of morphine, fentanyl and the synthetic peptide agonist DAMGO. We also examined the ability of MT-45 and its metabolites, M1 and M17, to inhibit NMDA receptors.

What this study adds
• Three fluorinated derivatives of MT-45 potently activate μ receptors and one metabolite inhibits NMDA receptors.

What is the clinical significance
• The pharmacology of MT-45, its derivatives and metabolites may contribute to the potential for harm. geneticin (500 μgÁml −1 ). All cells were cultured at 37 C in a humidified atmosphere in 95% air.

| β-arrestin2 recruitment assay
The PathHunter β-arrestin2 assay was used to assess recruitment of β-arrestin2 to μ receptors. CHO cells, stably expressing β-galactosidase fragment tagged human μ receptors and β-arrestin2, were suspended in OptiMEM (Invitrogen, UK), seeded onto 96-half well plates (Greiner, UK) at a density of 5 × 10 3 cells per well and left to settle overnight. Agonists were diluted into OptiMEM. Cells were incubated in the presence of agonists for 90 min at 37 C. The substrate reagent for luminescence production (DiscoverX, UK) was added according to manufacturer's instructions and incubated for 2 h at 37 C before the luminescence was recorded.

| Intracellular Ca 2+ imaging and Flexstation measurements
Ltk-cells stably expressing inducible NMDA receptor GluN1A and NR2A subunits were seeded at a density of 5 × 10 4 cells onto 16 mm glass coverslips or into each well of a black 96-well plate. Cells were incubated overnight in growth media supplemented with 1 μM dexamethasone (to induce expression of NMDA receptor subunits) and up to 200 μM AP-5 (to block activation of NMDA receptors by glycine and glutamate contained in growth media). The recording buffer composed of nominally Mg 2+ free HBSS, supplemented with 2 mM CaCl 2 , 20 mM HEPES and 2 mM probenecid. The pH was adjusted to 7.4 with NaOH. Cells were loaded with 2 μM Fura2-AM at room temperature for 1 h, followed by de-esterification in recording buffer for at least 30 min. All buffers during the loading and deesterification steps contain 200 μM AP-5. Coverslips were mounted in a chamber (RC-25, Warner Instruments) and constantly superfused with recording buffer at a rate of approximately 5 mlÁmin −1 using gravity feed. Intracellular Ca 2+ in single cells was measured using an inverted epifluorescence microscope with a 40× oil immersion objective. Fura-2 was excited at 340 or 380 nm, selected using a monochromator (Cairn, UK). Emission at 510 nm was selected using a dichroic filter (Chroma Tech. Corp., USA) and collected using a cooled intensified photometric camera (Photometrics CoolSNAP HQ, Roper Scientific). Fluorescence and images were acquired using MetaFluor software version 5.0r3 (Universal Imaging Corp., UK; RRID:SCR_014294). For Flexstation Ca 2+ measurements, agonists (NMDA and glycine) were added using the automated addition mode of the instrument from 10× drug stocks.
Fluorescence excited at 340 or 380 nm was collected at 510 nm every 5 s. A baseline of 2 min was recorded before the addition of agonists.

| Materials
All chemical structures used in this study are depicted in Figure 1.

| Synthesis of the M1 metabolite of MT-45
The hydrochloride salt of 1,2-diphenylethylpiperazine (M1) was prepared using an adaption of the method reported by Geyer et al. (2016). Zinc dust (2.0 g, 30 mmol) was suspended in acetonitrile (40 ml) in a dried round-bottom flask, which was flushed with argon to create an inert atmosphere. To this mixture was added benzyl bromide (0.4 ml, 3.3 mmol) and trifluoracetic acid (0.2 ml) and stirred for 5 min. Subsequently, benzyl bromide (3 ml, 25 mmol), 1-Bocpiperazine (1.87 g, 10 mmol) suspended in acetonitrile and benzaldehyde (1.2 ml, 11 mmol) were introduced to the solution. This mixture was stirred for an additional hour at ambient temperature. The resulting solution was quenched with a saturated aqueous ammonium chloride solution (150 ml) and extracted with dichloromethane (2 × 100 ml). The organic layers were dried with magnesium sulphate and concentrated in vacuo. The resulting yellow oil was dissolved in diethyl ether (150 ml) and treated with sulfuric acid (0.75 ml) to remove the Boc-protective group. The mixture was stirred for 1 h, after which the ether was decanted off. The residue was re-dissolved in an aqueous sodium hydroxide solution (1.25 M, 150 ml). The solution was extracted with dichloromethane (2 × 100 ml) and the organic layers were combined, dried with magnesium sulphate and concentrated in vacuo. The resulting yellow oil was purified using gravity column chromatography, with a mobile phase of dichloromethanemethanol (90:10 v/v) and 0.8% ammonia. The resulting oil was dissolved in diethyl ether (200 ml) and treated with hydrogen chloride (4 M in dioxane, 6 ml, 24 mmol). This solution was left to stand for 5 min. The crystallised product was filtered and collected.
The synthesised product (yield 9.6%, purity 99.1%) was characterised using NMR and gas chromatography coupled to MS . Helium was used as the carrier gas at a constant flow rate of 1.0 mlÁmin −1 . Injection temperature was 265 C and the detection temperature was 300 C. The temperature program was 60-15-300 (2 0 ) with a split ratio of 50:1. Eicosane was used as an internal standard (t R = 11.79).

| Data analysis
Luminescence measurements, resulting from cAMP accumulation following the application of agonists, were expressed as a percentage of forskolin-stimulated maximum luminescence. Luminescence data from the β-arrestin2 assay were normalised to the maximum luminescence in the presence of DAMGO. Concentration-response relationships were plotted and fitted using a logistics equation: where apparent potency (EC 50 ), Hill slope (n H ) and efficacy (E max : Max − Min) parameters were derived.
Fura-2 emission fluorescence values collected from excitation at either 340 or 380 nm were expressed as a ratio (340 nm/380 nm).
Amplitudes were calculated from background subtracted peak ratio fluorescence induced by glutamate and glycine application.

| Statistics
The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). Summary data are presented as box and whisker plots, where the boundary of the boxes represent the interquartile range and the error bars represent the 5th to 95th percentile. Data median divides the boxes and data means are indicated as "+." All box plots are visually inspected for normality and skewedness. In-text summary data and concentration-response relationship data are presented as mean ± SEM, except EC 50 and IC 50 data, where mean and ranges are reported. Sample sizes represent independent experiments and statistical comparisons were performed using data on these independent values, and also where the sample size was at least 5. Pairwise comparisons (for intracellular Ca 2+ data) were performed using the Student's paired t-test. Comparisons of parameters derived from the logistics fit (pEC 50 or pIC 50 , n H and E max ) between different agonists were performed using one-way ANOVA. Post-hoc testing was conducted using the Bonferroni correction method if F in the one-way ANOVA had P < 0.05 and there was no significant variance inhomogeneity. Differences were considered statistically significant when P < 0.05.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 .  Table 1. A one-way ANOVA reveals a statistically significant difference in mean efficacies. Pair-wise comparisons between all mean efficacy values, with the Bonferroni correction reveal DAMGO to be significantly more efficacious than all of the other agonists . This is consistent with our previous finding that morphine is a partial agonist in terms of β-arrestin2 recruitment . Our data also suggest that fentanyl is a partial agonist in this assay. The analysis reveals that   3.2 | MT-45 and its fluorinated derivatives show differential apparent potencies as inhibitors of cAMP.
We used the GloSensor protein to assess μ receptor-mediated changes in intracellular cAMP levels utilising the PathHunter CHO cells. This provides consistency of μ receptor density when making comparisons of apparent potency and efficacy between the two assays.
PathHunter CHO cells transfected with the GloSensor protein were plated onto 96-half well plates and incubated in assay buffer containing forskolin (30 μM) to stimulate AC activity. Peak luminescence levels were subsequently measured, before addition of agonists.
Agonist-mediated reduction in luminescence was assessed after 30 min exposure. The luminescence values following agonist exposure were normalised to those prior to agonist addition and plotted against agonist concentrations. Similar to the β-arrestin2 assay data above,  Using a similar approach, we pre-applied MT-45, M1 and M17 (all at 30 μM). None of the drugs tested changed Fura2-mediated fluorescence immediately upon addition, indicating that they had no direct effects on intracellular Ca 2+ (data not shown). Figure  Indeed, the potencies of these fluorinated derivatives are similar to those of morphine and fentanyl. Furthermore, while MT-45 itself has no effect on NMDA receptor-mediated elevations of intracellular Ca 2+ , its major metabolite M1, caused a significant inhibition of NMDA-evoked responses.
Opioid analgesics, including morphine and fentanyl, remain a mainstay for pain relief despite their well-documented side effects such as opioid-induced hyperalgesia, respiratory depression, immunosuppression and constipation (Colvin et al., 2019). The use of opioids is further complicated by the development of analgesic tolerance, Logistics fit parameters of concentration-response data for β-arrestin 2 recruitment assay. RLU -relative luminescence units  can also be habit forming, potentially leading to misuse and addiction.
Activation of μ receptors and, to a lesser extent, δ-opioid receptors reduces inhibitory transmission in the ventral tegmental area (Bull, Baptista-Hon, Lambert, Walwyn, & Hales, 2017). The resulting disinhibition of dopaminergic neurones allows increased dopamine release into the striatum and prefrontal cortex, events implicated in opioidinduced reinforcement and reward (Fields & Margolis, 2015;Maldonado et al., 1997).
Research in the 1970s seeking the next generation of opioidbased analgesics generated a number of synthetic opioids, including MT-45, originally developed as an alternative to morphine (Fujimura et al., 1978;Nakamura & Shimizu, 1976). However, the side effects of MT-45 were problematic and therefore development was halted. MT-   Activation of μ receptors modulates cellular function and affects receptor recycling through G proteins and β-arrestin2, respectively (Kang et al., 2015;Koehl et al., 2018). Mounting evidence suggests that β-arrestin2 recruitment to the μ receptor also engages additional signalling molecules, such as Src kinase, associated with the development of tolerance (Bull, Baptista-Hon, Sneddon, et al., 2017;Walwyn et al., 2007). User experiences of MT-45 in online forums recount the development of tolerance (Kjellgren et al., 2016). Other side effects are also mentioned, including nausea and vomiting, constipation, respiratory depression, and loss of motor skills. In addition to tolerance, opioid-induced respiratory depression and constipation have also been attributed to recruitment of β-arrestin2 (Bohn et al., 1999;Raehal & Bohn, 2011). However, a recent report suggests that β-arrestin2 may not participate in opioid-induced respiratory depression and constipation (Kliewer et al., 2019). It is unclear whether any of the self-reported users of MT-45 were also/alternatively consuming its fluorinated derivative.
We compared inhibition of cAMP accumulation (as an assay of that each receptor recruits one arrestin (Kang et al., 2015). Although in some cases arrestins may remain activated after their interaction with a GPRC has terminated, enabling amplification (Latorraca et al., 2018), this is unlikely in the case of the complementation assay, which is irreversible. The lack of amplification is advantageous in this case as it enables the assay to distinguish partial agonists from full agonists. By contrast, the assay of cAMP accumulation occurs at the culmination of several reversible events: first receptor activation, then G protein coupling and finally inhibition of adenylyl cyclase. Furthermore, the stoichiometry of receptors to G proteins will depend on the level of recombinant μ receptor expression. In PathHunter cells μ receptors are over-expressed, enabling partial agonists to fully inhibit adenylyl cyclase. However, despite these potential limitations, this assay has the advantage of being sensitive, even detecting the activity of weak partial agonists. Consistent with this idea, morphine, fentanyl and MT-45 and its fluorinated derivatives, partial agonists compared to DAMGO in the β-arrestin2 recruitment assay, were equally efficacious in the assay of cAMP accumulation. Our observation that 2F-  (Fels et al., 2017;Helander et al., 2014;Papsun et al., 2016).
The role of fentanyl and its analogues in the current opioid crisis in the United States is well documented (Prekupec et al., 2017;Socías & Wood, 2017). Our findings highlight the potential danger associated with novel fluorinated MT-45 derivatives, which exhibit higher potency as μ receptor agonists than the parent compound. In this regard, the work reveals the profound influence of minor modifications to the chemical structures of novel synthetic opioids. The recently reported deaths caused by MT-45 highlights the danger of novel synthetic opioid misuse (Fels et al., 2017;Papsun et al., 2016).
The concentration of MT-45 in one victim's femoral blood was more than sixfold higher than the EC 50 determined here for μ receptormediated inhibition of cAMP. The substantially higher apparent potencies of fluorinated derivatives of MT-45 may pose an additional danger to illicit opioid users.
Fluorination of existing psychoactive substances is a common strategy in both medicinal chemistry (Purser et al., 2008;Gillis et al., 2015) and the production of illicit drugs. Such a strategy has been particularly prevalent with synthetic cannabinoid receptor agonists (Banister et al., 2015;Chung et al., 2014). Bioisosteric substitution of a hydrogen atom for fluorine often increases drug potency as has been noted previously for the opioid analgesic viminol, the 2F-and trifluoromethyl-analogues of which are significantly more potent analgesics (Conti, 1979). In addition to increasing potency, addition of fluorine is also likely to increase bioavailability through alteration of physicochemical properties such as pKa. This effect was also noted for the fluorinated analogues of MT-45 (McKenzie et al., 2018).
Early in 2019, the State Council of the People's Republic of China announced controls for the production and export of fentanyl analogues; although it is too soon to establish the impact, one consequence could be a compensatory increase in the availability of nonfentanyl novel synthetic opioids on the illicit market. It will be important to rapidly isolate and identify novel synthetic opioids as they emerge and assess their mechanisms of action. This approach, which will require close cooperation between forensic drug researchers and pharmacologists, may provide indications of the potential harm of emerging novel synthetic opioids. In particular, it will be important to alert relevant health care practitioners and the communities exposed to risk, of chemical modifications to existing substances that cause significantly increased potency, such as we observed in the case of fluorinated MT-45.

CONFLICT OF INTEREST
The authors declare no conflicts of interest.