Glycine Transporter Type I (GlyT1) Inhibitor, Bitopertin: A Journey from Lab to Patient.

Glycine transporter-1 (GlyT1) inhibition has been extensively studied both in pharmaceutical companies and academic institutions primarily as a potential new approach to treat schizophrenia, a severe and chronic mental illness. More recently, preclinical results have suggested that this approach could also have therapeutic potential for CNS disorders beyond schizophrenia as well as for non-CNS indications. Over the past 17 years, Roche has been a key player in the GlyT1 field with the discovery and development of bitopertin, the most advanced GlyT1 inhibitor to date and the only one which completed Phase III clinical studies for schizophrenia. In this article, we relate the eventful journey of the discovery and development of bitopertin, from project initiation in 2001 to its evaluation today in patients suffering from beta-thalassemia, a monogenic hereditary haematological disorder.

Targeting Glycine Reuptake in Alcohol Seeking and Relapse.

It has recently been demonstrated that pharmacological blockade of the glycine transporter 1 (GlyT1) reduced alcohol intake and relapse in rats. The aim of the present study was to further explore the role of GlyT1 in alcohol relapse-like behavior. For this purpose we used three different GlyT1 blockers-SSR504734, A-1246399, and RO4993850-and tested their effect on alcohol-seeking and relapse-like consumption. Two behavioral models, the alcohol deprivation effect model and the cue-induced reinstatement model, were used. Our data show that all three GlyT1 blockers reduce relapse-like alcohol consumption and cause either minimal or no side effects, measured as changes in home-cage activity, water intake, and body weight. In the reinstatement test, GlyT1 blockers completely abolished alcohol-seeking responses. Furthermore, we tested other drug/cue associations and found that cocaine-seeking responses were also abolished by GlyT1 blockade. Our data confirm that GlyT1 can be used as a target to develop novel anticraving and antirelapse drugs.

Assessment of a glycine uptake inhibitor in animal models of effort-related choice behavior: implications for motivational dysfunctions.

RATIONALE: Motivated behavior can be characterized by a substantial exertion of effort, and organisms often make effort-related decisions based upon analyses of work-related response costs and reinforcement preference. Moreover, alterations in effort-based choice can be seen in people with major depression and schizophrenia. Effort-related decision making is studied using tasks offering choices between high effort options leading to highly valued reinforces vs low effort/low reward options. Interference with dopamine (DA) transmission by administration of the DA D2 family antagonist haloperidol biases behavior towards the lower effort option that can be obtained with minimal work, and previous research has shown that DA interacts with other transmitters, including adenosine and GABA, to regulate effort-based choice. OBJECTIVES: The present studies focused upon the ability of the glycine transport inhibitor bitopertin to attenuate haloperidol-induced shifts in effort-related choice behavior. METHODS: Effort-based choice in rats was assessed using the concurrent fixed ratio (FR) 5/chow feeding choice task and the T-maze barrier choice procedure. RESULTS: Haloperidol shifted effort-based choice, biasing animals towards the low effort option in each task. Co-administration of bitopertin (1.0-10.0 mg/kg) significantly attenuated haloperidol-induced shifts in choice behavior, but the same doses of bitopertin had no effect when administered alone. CONCLUSIONS: These results indicated that elevation of extracellular glycine via inhibition of glycine uptake was able to reverse the effects of D2 antagonism. Increases in extracellular glycine, possibly through actions on the glycine allosteric site on the NMDA receptor, may be a useful strategy for treating motivational dysfunctions in humans.

Effects of GlyT1 inhibition on erythropoiesis and iron homeostasis in rats.

Glycine is a key rate-limiting component of heme biosynthesis in erythropoietic cells, where the high intracellular glycine demand is primarily supplied by the glycine transporter 1 (GlyT1). The impact of intracellular glycine restriction after GlyT1 inhibition on hematopoiesis and iron regulation is not well established. We investigated the effects of a potent and selective inhibitor of GlyT1, bitopertin, on erythropoiesis and iron homeostasis in rats. GlyT1 inhibition significantly affected erythroid heme biosynthesis, manifesting as microcytic hypochromic regenerative anemia with a 20% steady-state reduction in hemoglobin. Reduced erythropoietic iron utilization was characterized by down-regulation of the transferrin receptor 1 (TfR1) on reticulocytes and modest increased iron storage in the spleen. Hepatic hepcidin expression was not affected. However, under the condition of reduced heme biosynthesis with reduced iron reutilization and increased storage iron, hepcidin at the lower and higher range of normal showed a striking role in tissue distribution of iron. Rapid formation of iron-positive inclusion bodies (IBs) was observed in circulating reticulocytes, with an ultrastructure of iron-containing polymorphic mitochondrial remnants. IB or mitochondrial iron accumulation was absent in bone marrow erythroblasts. In conclusion, GlyT1 inhibition in rats induced a steady-state microcytic hypochromic regenerative anemia and a species-specific accumulation of uncommitted mitochondrial iron in reticulocytes. Importantly, this glycine-restricted anemia provides no feedback signal for increased systemic iron acquisition and the effects reported are pathogenetically distinct from systemic iron-overload anemias and erythropoietic disorders such as acquired sideroblastic anemia.

Effects of the glycine reuptake inhibitors bitopertin and RG7118 on glycine in cerebrospinal fluid: results of two proofs of mechanism studies in healthy volunteers.

RATIONALE: Hypofunction of NMDA receptors has been implicated in neuropsychiatric disorders including schizophrenia. NMDA receptor neurotransmission can be enhanced through inhibition of glycine reuptake by the glycine transporter type 1 (GlyT1). OBJECTIVES: The primary objective of these studies was to explore the relationship between plasma exposure and glycine cerebrospinal fluid (CSF) concentrations following administration of bitopertin and RG7118 in healthy volunteers. METHODS: The bitopertin study comprised four dose levels (3, 10, 30 and 60 mg) administered once daily for 10 days. In the RG7118 study, placebo, 15 or 30 mg RG7118 was administered once daily for 28 days. CSF samples were taken on day -2 and day 10, and day -1 and day 26 for bitopertin and RG7118, respectively. RESULTS: Twenty-two and 24 subjects participated in the bitopertin and RG7118 study, respectively. In the bitopertin study, CSF glycine concentrations showed a dose-dependent increase from baseline to day 10. The geometric mean ratios (coefficient of variation) of AUC0-12 h on day 10 over baseline were 1.3 (17 %), 1.3 (49 %), 1.7 (18 %) and 2.3 (14 %) after 3, 10, 30 and 60 mg, respectively. In the RG7118 study, the geometric mean ratio of glycine concentration (CV) on day 26 at 6 h post-dose over time-matched baseline was approx. 1.9 (24 and 15 %) for 15 and 30 mg. CONCLUSIONS: The mechanism of action of bitopertin and RG7118, i.e. inhibition of glycine reuptake in the brain, was confirmed. The maximal increase observed in healthy volunteers was similar to the one observed in animals showing the good translatability of this biomarker.

Preclinical Reproductive and Developmental Toxicity Profile of a Glycine Transporter Type 1 (Glyt1) Inhibitor.

Bitopertin is a glycine type 1 (GlyT1) inhibitor intended for the treatment of psychiatric disorders. The principle adverse effect in the regulatory reproductive toxicity studies was peri-natal pup death when rat dams were treated during parturition at a dose resulting in five-times the human therapeutic exposure (AUC). Cessation of dosing two days before parturition prevented the pup deaths. Investigatory experiments and pharmacokinetic modelling suggested that the neonatal mortality was related to transplacental passage of bitopertin leading to high systemic levels in the newborn pups. Brain levels of bitopertin in the rat fetus and neonate were two-fold higher than in the mother. As illustrated by knock-out mice models, GlyT1 function is essential for neonatal pup survival in rodents, but is not necessary for normal prenatal morphological development. The glycine transport systems are immature at birth in the rat, but are functionally well-developed in the human newborn. While the relevance to humans of the neonatal mortality seen in rats following late gestational exposure is unknown, bitopertin would not be recommended for use during late pregnancy unless the anticipated benefit for the mother outweighs the potential risk to the newborn.

Alterations in High-Frequency Neuronal Oscillations in a Cynomolgus Macaque Test of Sustained Attention Following NMDA Receptor Antagonism.

A growing body of evidence indicates that neuronal oscillations in the gamma frequency range (30-80 Hz) are disturbed in schizophrenic patients during cognitive processes and may represent an endophenotype of the disease. N-methyl-D-aspartate (NMDA) receptor antagonists have been used experimentally to induce schizophrenia-like symptoms including cognitive deficits in animals and humans. Here we characterized neuronal oscillations and event-related potentials (ERPs) in Cynomolgus macaques fully trained to perform a continuous performance test (CPT) in the presence and absence of the NMDA antagonist phencyclidine (PCP). Macaques (n=8) were trained to touch 'target' stimuli and ignore 'distractor' stimuli presented randomly on a touchscreen. Subsequently, all subjects were implanted with epidural EEG electrodes over frontal (FC) and parietal cortices (PC) and later tested under vehicle (saline, i.m.) or acute PCP (0.1-0.3 mg/kg, i.m.) conditions. Compared with vehicle treatment, PCP produced a significant dose-dependent decrease in CPT performance accuracy and increased reaction times. Furthermore, PCP elevated the amplitudes of 'low' (30-50 Hz) and 'high' (51-80 Hz) gamma oscillations in FC and PC around target presentations for all correct responses. The CPT accuracy was inversely correlated with the gamma band amplitude in the presence of PCP. Additionally, PCP delayed the N100 peak latency in FC, and prolonged and suppressed the cognitively relevant P300 component of mean ERPs in FC and PC, respectively. The NMDA receptor antagonist-induced alteration in neuronal oscillations and ERPs may contribute to the observed cognitive deficits in macaques, and enhance our understanding of EEG recordings as a translatable biomarker.

3-Amido-3-aryl-piperidines: A Novel Class of Potent, Selective, and Orally Active GlyT1 Inhibitors.

3-Amido-3-aryl-piperidines were discovered as a novel structural class of GlyT1 inhibitors. The structure-activity relationship, which was developed, led to the identification of highly potent compounds exhibiting excellent selectivity against the GlyT2 isoform, drug-like properties, and in vivo activity after oral administration.

Effect of bitopertin, a glycine reuptake inhibitor, on negative symptoms of schizophrenia: a randomized, double-blind, proof-of-concept study.

IMPORTANCE: In schizophrenia, the severity of negative symptoms is a key predictor of long-term disability. Deficient signaling through the N-methyl-D-aspartate receptor is hypothesized to underlie many signs and symptoms associated with schizophrenia in particular negative symptoms. Glycine acts as an N-methyl-D-aspartate receptor coagonist. Blockade of the glycine transporter type 1 to inhibit glycine reuptake and elevate synaptic glycine concentrations represents an effective strategy to enhance N-methyl-D-aspartate receptor transmission. OBJECTIVE: To determine the efficacy and safety of bitopertin (RG1678), a glycine reuptake inhibitor, in patients with schizophrenia and predominant negative symptoms who were stable while taking an antipsychotic treatment. DESIGN, SETTING, AND PARTICIPANTS: This randomized, double-blind, placebo-controlled, phase 2 proof-of-concept trial involved 323 patients with schizophrenia and predominant negative symptoms across 66 sites worldwide. INTERVENTIONS: Bitopertin (10, 30, or 60 mg/d) or placebo added to standard antipsychotic therapy for a treatment duration of 8 weeks. MAIN OUTCOMES AND MEASURES: Change from baseline in the Positive and Negative Syndrome Scale negative factor score. RESULTS: In the per-protocol population, 8 weeks of treatment with bitopertin was associated with a significant reduction of negative symptoms in the 10-mg/d (mean [SE] reduction in negative symptoms score, -25% [2%]; P = .049) and 30-mg/d (mean [SE], -25% [2%]; P = .03) bitopertin groups, a significantly higher response rate and a trend toward improved functioning in the 10-mg/d group when compared with placebo (mean [SE], -19% [2%]). Results reached trend-level significance in the intent-to-treat population. Estimates of bitopertin binding to glycine transporter type 1 showed that low to medium levels of occupancy yielded optimal efficacy in patients, consistent with findings in preclinical assays. CONCLUSIONS AND RELEVANCE: Bitopertin-mediated glycine reuptake inhibition may represent a novel treatment option for schizophrenia, with the potential to address negative symptoms. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00616798.

Physiologically based pharmacokinetic modelling to predict single- and multiple-dose human pharmacokinetics of bitopertin.

BACKGROUND: Bitopertin (RG1678) is a glycine reuptake inhibitor currently in phase 3 trials for treatment of schizophrenia. This paper describes the use of physiologically based pharmacokinetic (PBPK) modelling and preclinical data to gain insights into and predict bitopertin clinical pharmacokinetics. METHODS: Simulations of pharmacokinetics were initiated early in the drug discovery stage by integrating physicochemical properties and in vitro measurements into a PBPK rat model. Comparison of pharmacokinetics predicted by PBPK modelling with those measured after intravenous and oral dosing in rats and monkeys showed a good match and thus increased confidence that a similar approach could be applied for human prediction. After comparison of predicted plasma concentrations with those measured after single oral doses in the first clinical study, the human model was refined and then applied to simulate multiple-dose pharmacokinetics. RESULTS: Clinical plasma concentrations measured were in good agreement with PBPK predictions. Predicted area under the plasma concentration-time curve (AUC) was within twofold of the observed mean values for all dose levels. Maximum plasma concentration (C max) at higher doses was well predicted but approximately twofold below observed values at the lower doses. A slightly less than dose-proportional increase in both AUC and C max was observed, and model simulations indicated that when the dose exceeded 50 mg, solubility limited the fraction of dose absorbed. Refinement of the absorption model with additional solubility and permeability measurements further improved the match of simulations to observed single-dose data. Simulated multiple-dose pharmacokinetics with the refined model were in good agreement with observed data. CONCLUSIONS: Clinical pharmacokinetics of bitopertin can be well simulated with a mechanistic PBPK model. This model supports further clinical development and provides a valuable repository for pharmacokinetic knowledge gained about the molecule.

Pre-clinical characterization of [11C]R05013853 as a novel radiotracer for imaging of the glycine transporter type 1 by positron emission tomography.

A specific positron emission tomography (PET) radiotracer for the glycine transporter type 1 (GlyT1) would constitute an imaging biomarker to investigate the distribution of GlyT1 in normal individuals and those with neuropsychiatric disorders. In addition it could demonstrate the ability of a novel drug to reach its target in the brain and enable receptor occupancy studies, thus facilitating drug development. In this article we describe the evaluation in non-human primates of two candidate PET radiotracers ([(11)C]RO5013852 and [(11)C]RO5013853) previously characterized in the rat. Both radiotracers showed acceptable uptake in the baboon brain and heterogeneous distribution consistent with that reported for GlyT1. In vivo blockade studies with two specific glycine reuptake inhibitors (GRIs), RO5013853 and bitopertin (RG1678, reduced uptake of both tracers to homogenous levels across brain regions and demonstrated specificity of the signal. [(11)C]RO5013853 showed a larger specific signal and slightly higher brain uptake and was therefore selected for further characterization. Quantitative compartmental analysis of PET data showed that the 2-tissue compartment model with 5 parameters was the most appropriate to describe the kinetics of [(11)C]RO5013853. Two additional methods were used: a) the Logan graphical analysis using plasma input and, b) a linear parametric imaging approach with the 2-tissue compartmental model. These produced VT estimates of comparable magnitude, namely, pons, thalamus and cerebellum>caudate, putamen and cortical regions. High resolution autoradiography with tritiated RO5013853 was used to confirm the binding pattern observed by PET. In vivo metabolism studies in the baboon demonstrated the formation of a single, radiolabeled metabolite more polar than the parent compound. Finally, [(11)C]RO5013853 was used to quantify the degree of cerebral GlyT1 occupancy observed in the baboon following oral administration of bitopertin, a selective GRI presently in Phase III clinical trial. Plasma concentrations of approximately 150-300 ng/mL were estimated to produce 50% GlyT1 occupancy in the thalamus, the cerebellum and the pons. [(11)C]RO5013853 is a promising radiotracer for in vivo imaging of the GlyT1. It can be easily radiolabeled, exhibits moderate metabolism, displays a good specific signal, and is suitable for receptor occupancy studies of therapeutic compounds that target the GlyT1. The successful characterization of [(11)C]RO5013853 in healthy volunteers is presented in this NeuroImage issue (Wong et al., 2013).

Inhibiting glycine transporter-1 facilitates cocaine-cue extinction and attenuates reacquisition of cocaine-seeking behavior.

BACKGROUND: Combining extinction training with cognitive-enhancing pharmacotherapy represents a novel strategy for improving the efficacy of exposure therapy for drug relapse prevention. We investigated if the selective glycine transporter-1 (GlyT-1) inhibitor RO4543338 could facilitate extinction of cocaine-conditioned responses and attenuate reacquisition of cocaine-seeking behavior. METHODS: Rats were trained to self-administer cocaine (0.3mg/kg), which was associated with a 2-s light cue under a second-order schedule of i.v. drug injection. Rats received vehicle, 30 or 45mg/kg of RO4543338 prior to three 1-h extinction-training sessions spaced at weekly intervals. Responses were extinguished by substituting saline for cocaine while maintaining response-contingent cue presentations. Reacquisition of cocaine-seeking behavior during self-administration sessions began 1 week after the last extinction session. Control experiments were conducted under conditions that precluded explicit extinction of cocaine-conditioned responses. RESULTS: Compared to vehicle, 30 and 45mg/kg RO4543338 significantly decreased responding early in extinction training and during subsequent reacquisition sessions. The latter effect persisted for at least five sessions. In control studies, reacquisition of cocaine-seeking behavior was not altered when RO4543338 was administered either prior to weekly self-administration control sessions or prior to weekly control sessions in which cocaine and cues were omitted and the levers retracted. CONCLUSIONS: As the GlyT-1 inhibitor facilitated cocaine-cue extinction learning and attenuated subsequent reacquisition of cocaine-seeking behavior, this class of compounds may have utility as a pharmacological adjunct to cocaine-cue exposure therapy in addicts.

Glycine reuptake inhibitor RG1678: a pharmacologic characterization of an investigational agent for the treatment of schizophrenia.

Dysfunctional N-methyl-d-aspartate (NMDA) receptor neurotransmission has been implicated in the pathophysiology of schizophrenia. It is thought that this abnormal functioning can be corrected by increasing availability of the NMDA co-agonist glycine through inhibition of glycine transporter type 1 (GlyT1). Herein is described the pharmacologic profile of RG1678, a potent and noncompetitive glycine reuptake inhibitor. In vitro, RG1678 noncompetitively inhibited glycine uptake at human GlyT1 with a concentration exhibiting half-maximal inhibition (IC(50)) of 25 nM and competitively blocked [(3)H]ORG24598 binding sites at human GlyT1b in membranes from Chinese hamster ovary cells. In hippocampal CA1 pyramidal cells, RG1678 enhanced NMDA-dependent long-term potentiation at 100 nM but not at 300 nM. In vivo, RG1678 dose-dependently increased cerebrospinal fluid and striatal levels of glycine measured by microdialysis in rats. Additionally RG1678 attenuated hyperlocomotion induced by the psychostimulant d-amphetamine or the NMDA receptor glycine site antagonist L-687,414 in mice. RG1678 also prevented the hyper-response to d-amphetamine challenge in rats treated chronically with phencyclidine, an NMDA receptor open-channel blocker. In the latter experiment, a decrease in ex vivo striatal [(3)H]raclopride binding was also measured. These data demonstrate that RG1678 is a potent, noncompetitive glycine reuptake inhibitor that can modulate both glutamatergic and dopaminergic neurotransmission in animal experiments that model aspects of schizophrenia. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Discovery of benzoylisoindolines as a novel class of potent, selective and orally active GlyT1 inhibitors.

Benzoylisoindolines were discovered as a novel structural class of GlyT1 inhibitors. SAR studies and subsequent lead optimization efforts focused primarily on addressing hERG liability and on improving in vivo efficacy resulted in the identification of potent GlyT1 inhibitors displaying excellent selectivity and in vivo PD and PK profiles.

Pharmacological evaluation of a novel assay for detecting glycine transporter 1 inhibitors and their antipsychotic potential.

Multiple lines of evidence support the notion that hypofunction of glutamatergic neurotransmission is involved in the pathophysiology of schizophrenia. Moreover, glycine and glycine modulators have beneficial effects in patients with schizophrenia, particularly when added on to existing therapy. As glycine is an obligatory co-agonist at the NR1 subunit of the NMDA receptor, blockade of glycine uptake at the glycine transporter type-1 (GlyT1) can enhance low glutamatergic tone. L-687,414 is an antagonist at the glycine modulatory site of the NMDA complex and, behaviorally, increases locomotion. A series of GlyT1 inhibitors along with other psychoactive compounds were examined for their ability to enhance or inhibit the action of L-687,414. GlyT1 inhibitors and the other compounds were examined initially for effects on [(3)H]-glycine uptake in CHO cells expressing hGlyT1b cDNA and for their ability to displace the NMDA-glycine site ligand [(3)H]-L-689,560 from isolated rat forebrain membrane preparations. The in vivo activity of these compounds was determined in mice by measuring their ability to prevent L-687,414-induced hyperlocomotion. GlyT1 inhibitors blocked [(3)H]-glycine uptake in cells expressing the human transporter; other compounds had little or no activity. None of the compounds had affinity for the glycine site of the NMDA receptor complex. Hyperlocomotion induced by L-687,414 was dose-dependently reduced by GlyT1 inhibitors and antipsychotic drugs but not by morphine, fluoxetine or a moderate dose of diazepam. Therefore, this behavioral approach can reliably detect GlyT1 inhibitors which, in turn, may have some activity in common with drugs having antipsychotic effects.

Selective GlyT1 inhibitors: discovery of [4-(3-fluoro-5-trifluoromethylpyridin-2-yl)piperazin-1-yl][5-methanesulfonyl-2-((S)-2,2,2-trifluoro-1-methylethoxy)phenyl]methanone (RG1678), a promising novel medicine to treat schizophrenia.

The GlyT1 transporter has emerged as a key novel target for the treatment of schizophrenia. Herein, we report on the optimization of the 2-alkoxy-5-methylsulfonebenzoylpiperazine class of GlyT1 inhibitors to improve hERG channel selectivity and brain penetration. This effort culminated in the discovery of compound 10a (RG1678), the first potent and selective GlyT1 inhibitor to have a beneficial effect in schizophrenic patients in a phase II clinical trial.

Design, synthesis and structure-activity relationship of simple bis-amides as potent inhibitors of GlyT1.

Several novel classes of potent and small amide-type inhibitors of glycine transport (GlyT1) were developed through sequential simplification of a benzodiazepinone-lead structure identified from a high-throughput screening. The most potent compounds of these structurally simple classes show low nanomolar inhibition at the GlyT1 target.

Discovery of benzoylpiperazines as a novel class of potent and selective GlyT1 inhibitors.

Screening of the Roche compound library led to the identification of the benzoylpiperazine 7 as a structurally novel GlyT1 inhibitor. The SAR which was developed in this series resulted in the discovery of highly potent compounds displaying excellent selectivity against the GlyT2 isoform, drug-like properties, and in vivo efficacy after oral administration.

Discovery of 4-substituted-8-(2-hydroxy-2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-one as a novel class of highly selective GlyT1 inhibitors with improved metabolic stability.

A novel class of 4-aryl-8-(2-hydroxy-2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1- ones have been discovered and developed as potent and selective GlyT1 inhibitors. The molecules are devoid of activity at the GlyT2 isoform and display excellent selectivities against the mu-opioid receptor as well as the Nociceptin/Orphanin FQ peptide (NOP) receptor. In particular these novel compounds 4 as well as the 4-substituted-8-(2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-one 3 show improved metabolic stability and pharmacokinetic profiles in rodents compared to previous triazaspiropiperidine series 1 and 2. We have also identified within these diazaspiropiperidine series a key relationship between reducing basicity of the piperidine nitrogen and reducing hERG affinity.

Design and synthesis of 4-substituted-8-(2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-one as a novel class of GlyT1 inhibitors: achieving selectivity against the mu opioid and nociceptin/orphanin FQ peptide (NOP) receptors.

A novel class of 4-substituted-8-(2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-ones have been discovered and developed as potent and selective GlyT1 inhibitors. The molecules are devoid of activity at the GlyT2 isoform and display excellent selectivities against the mu opioid receptor as well as the nociceptin/orphanin FQ peptide (NOP) receptor. A novel, straightforward and efficient synthetic strategy for the assembly of the target molecules is also presented.