Novel GlyT1 inhibitor chemotypes by scaffold hopping. Part 2: Development of a [3.3.0]-based series and other piperidine bioisosteres

This Letter describes the development and SAR of a novel series of GlyT1 inhibitors derived from a scaffold hopping approach, in lieu of an HTS campaign, which provided intellectual property position. Members within this new [3.3.0]-based series displayed excellent GlyT1 potency, selectivity, free fraction, and modest CNS penetration. Moreover, enantioselective GlyT1 inhibition was observed, within this novel series and a number of other piperidine bioisosteric cores.     

Discovery and SAR studies of novel GlyT1 inhibitors

Inhibition of the glycine transporter GlyT1 is a potential strategy for the treatment of schizophrenia. A novel series of GlyT1 inhibitors and their structure-activity relationships (SAR) are described. Members of this series are highly potent and selective transport inhibitors which are shown to elevate glycine levels in cerebrospinal fluid.     

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 N-(2-aryl-cyclohexyl) substituted spiropiperidines as a novel class of GlyT1 inhibitors

Screening of the Roche compound library led to the identification of cis-N-(2-phenyl-cyclohexyl)-spiropiperidine 1 as 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.     

An octahydro-cyclopenta[c]pyrrole series of inhibitors of the type 1 glycine transporter

We describe a novel series of inhibitors of the type 1 glycine transporter (GlyT1) as an approach to relieving the glutamatergic deficit that is thought to underlie schizophrenia. Synthesis and SAR follow-up of a series of octahydro-cyclopenta[c]pyrrole derivatives afforded potent in vitro inhibition of GlyT1 as well as in vivo activity in elevating CSF glycine. We also found that a 3-O(c-pentyl), 4-F substituent may serve as a surrogate for the widely used 3-trifluoromethoxy group, suggesting its application as an isostere for future medicinal chemistry studies.     

Calpain Sensitive Regions in the N-terminal Cytoplasmic Domains of Glycine Transporters GlyT1A and GlyT1B

Glycine transporters are members of the Na⁺/Cl⁻ dependent transporter gene family and play crucial roles in regulating inhibitory as well as excitatory neurotransmission. In this report we show that calcium elevation in spinal cord synaptosomes decreases the levels of glycine transporter, GlyT1, N-terminal immunoreactivity, and that this decrease can be blocked by calpain inhibitor. Sequencing of GST fusion proteins containing the N-terminal domains of GlyT1A and B splice variants cleaved with rat recombinant calpain identified calpain cleavage sites after glycine 17 in GlyT1B and N-terminally of the first conserved arginine residue in both GlyT1A and GlyT1B. Expression in HEK293 cells revealed that truncation of the N-terminus of GlyT1 results in significant inhibition of glycine uptake. A syntaxin1A GST fusion protein was able to pull-down N-terminally deleted GlyT1, indicating that calpain cleavage does not eliminate syntaxin1A binding. These results suggest that calpain cleavage may regulate the transport activity/turnover of GlyT1 in vivo by cleaving its N-terminal domain.     

Synthesis and biological evaluation of (4H-1,2,4-triazol-4-yl)isoquinoline derivatives as selective glycine transporter 1 inhibitors

To identify novel glycine transporter 1(GlyT1) inhibitors with greater selectivity relative to GlyT2 and improved aqueous solubility, we synthesized a series of 4H-1,2,4-triazole derivatives with heteroaromatic rings at the 4-position and investigated their structure-activity relationships. Replacement of the 2-fluorophenyl group of lead compound 5 with various aromatic groups led to the identification of 5-(3-biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)isoquinoline (15) with 38-fold selectivity between GlyT1 and GlyT2. 15 also showed improved aqueous solubility and in vivo efficacy on (+)-HA966-induced hyperlocomotion in mice over the lead compound.     

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.     

Identification of N-(2-(azepan-1-yl)-2-phenylethyl)-benzenesulfonamides as novel inhibitors of GlyT1

A novel series of glycine transporter 1 (GlyT1) inhibitors is described. Scoping of the heterocycle moiety of hit 4-chlorobenzenesulfonamide 1 led to replacement of the piperidine with an azepane for a modest increase in potency. Phenyl sulfonamides proved superior to alkyl and non-phenyl aromatic sulfonamides, while subsequent ortho substitution of the 2-(azepan-1-yl)-2-phenylethanamine aromatic ring yielded 39 (IC₅₀ 37 nM, solubility 14 μM), the most potent GlyT1 inhibitor in this series. Favorable brain–plasma ratios were observed for select compounds in pharmacokinetic studies to evaluate CNS penetration.     

Calcium Dependent Interaction of Calmodulin with the GlyT1 C-terminus

The cytoplasmic regions of neurotransmitter transporters play an important role in their trafficking. This process is, to a high extent, tuned by calcium and calcium binding proteins, but the exact molecular connection are still not fully understood. In this work we found that the C-terminal region of the mouse glycine transporter GlyT1b is able to specifically interact with calmodulin in the presence of calcium. We found that several GlyT1 C-terminal mutations, including those in the ER retention signal, either eliminate or increase calmodulin interaction in vitro. In tissue-culture-expressed GlyT1 at least two of these mutations altered the sensitivity of GlyT1 surface expression and glycine uptake to calmodulin antagonists. These results suggest the possible involvement of calmodulin or calmodulin-like interactions in the regulation of GlyT1C-mediated transporter trafficking.     

PKCβ-dependent phosphorylation of the glycine transporter 1

The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [³²P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23–40%-inhibition on V_max was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKCα/β inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation.     

Transport activities and expression patterns of glycine transporters 1 and 2 in the developing murine brain stem and spinal cord

Glycine serves as a neurotransmitter in spinal cord and brain stem, where it activates inhibitory glycine receptors. In addition, it serves as an essential co-agonist of excitatory N-methyl-d-aspartate receptors. In the central nervous system, extracellular glycine concentrations are regulated by two specific glycine transporters (GlyTs), GlyT1 and GlyT2. Here, we determined the relative transport activities and protein levels of GlyT1 and GlyT2 in membrane preparations from mouse brain stem and spinal cord at different developmental stages. We report that early postnatally (up to postnatal day P5) GlyT1 is the predominant transporter isoform responsible for a major fraction of the GlyT-mediated [(3)H]glycine uptake. At later stages (≥ P10), however, the transport activity and expression of GlyT2 increases, and in membrane fractions from adult mice both GlyTs contribute about equally to glycine uptake. These alterations in the activities and expression profiles of the GlyTs suggest that the contributions of GlyT1 and GlyT2 to the regulation of extracellular glycine concentrations at glycinergic synapses changes during development.     

Sustained saturating level of glycine induces changes in NR2B-containing-NMDA receptor localization in the CA1 region of the hippocampus

Post-synaptic actions of glycine are terminated by specialized transporters. There are two genes encoding glycine transporters, GlyT1 and GlyT2. Glycine acts as a co-agonist at N -methyl- d -aspartate glutamatergic receptors (NMDARs). Blockage of GlyT1 enhances NMDAR function by controlling ambient glycine concentrations. Using whole-cell patch-clamp recordings of acute hippocampal slices, we investigated NMDAR kinetics of CA1 pyramidal neurons of mice expressing 50% of GlyT1 (GlyT1+/−). In this study, we report that the glycine modulatory site of the NMDAR at CA1 synapses is saturated in GlyT1+/− but not in wild-type (WT) mice. We also found that the effect of ifenprodil, a highly selective NR2B-containing-NMDAR antagonist, is significantly reduced at CA1 synapses in GlyT1+/− compared to WT mice while immunoblotting experiments do not show significant differences for NR1, NR2A-B-C-D subunits in both types of mice, suggesting alteration in NR2B-containing-NMDAR localization under a state of chronic saturating level of endogenous glycine. Using a pharmacological approach with MK-801 and DL-TBOA, we discriminated synaptic vis-à-vis extra-synaptic NMDARs. We found that NR2B-containing-NMDARs are expressed at a higher level in the extra-synaptic area of CA1 pyramidal neurons from GlyT1+/− compared to WT mice. Our results demonstrate that chronic saturating level of glycine induces significant changes in NMDAR localization and kinetic. Therefore, results from our study should help to gain a better understanding of the role of glycine in pathological conditions.     

Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition

The glycine transporter subtype 1 (GlyT1) is widely expressed in astroglial cells throughout the mammalian central nervous system and has been implicated in the regulation of N-methyl-D-aspartate (NMDA) receptor activity. Newborn mice deficient in GlyT1 are anatomically normal but show severe motor and respiratory deficits and die during the first postnatal day. In brainstem slices from GlyT1-deficient mice, in vitro respiratory activity is strikingly reduced but normalized by the glycine receptor (GlyR) antagonist strychnine. Conversely, glycine or the GlyT1 inhibitor sarcosine suppress respiratory activity in slices from wild-type mice. Thus, during early postnatal life, GlyT1 is essential for regulating glycine concentrations at inhibitory GlyRs, and GlyT1 deletion generates symptoms found in human glycine encephalopathy.     

PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination

Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1). Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40–50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications.     

Discovery of GlyT1 inhibitors with improved pharmacokinetic properties

Glycine transporter 1 (GlyT1) represents a novel target for the treatment of schizophrenia via the potentiation of glutamatergic NMDA receptors. The discovery of 4,4-disubstituted piperidine inhibitors of GlyT1 which exhibit improved pharmacokinetic properties, including oral bioavailability, is discussed.     

4-Substituted-8-(1-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-one as a novel class of highly selective GlyT1 inhibitors with superior pharmacological and pharmacokinetic parameters

A novel class of 4-substituted-8-(1-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. These molecules also exhibit superior pharmacological and pharmacokinetic parameters, relative to all GlyT1 inhibitors of the spiropiperidine family, culminating in the identification of 16b with an oral bioavailability of approximately 60%. In addition, a straightforward two-step procedure for the assembly of the target molecules is also presented.     

A novel, non-substrate-based series of glycine type 1 transporter inhibitors derived from high-throughput screening

The synthesis and structure-activity relationships (SAR) of a series of indane and tetralin inhibitors of the type 1 glycine transporter, derived from a high-throughput screening (HTS) hit, are described. Key modifications that reduced the 5HT1B receptor affinity of the HTS hit and the P450 2D6 inhibition of subsequent analogues are delineated. While these modifications led to potent and selective GlyT1 inhibitors, HERG affinity and human microsomal clearance remain an issue for this series of compounds.     

3H]-(R)-NPTS,a radioligand for the type 1 glycine transporter

The synthesis of NPTS, 6, a potent inhibitor of the type 1 glycine transporter (GlyT1) is described, as well as preparation of 6 in optically active and tritiated form for use as a radioligand for affinity displacement assay of GlyT1.     

Pre-synaptic glycine GlyT1 transporter--NMDA receptor interaction: relevance to NMDA autoreceptor activation in the presence of Mg2+ ions

Rat hippocampal glutamatergic terminals possess NMDA autoreceptors whose activation by low micromolar NMDA elicits glutamate exocytosis in the presence of physiological Mg(2+) (1.2 mM), the release of glutamate being significantly reduced when compared to that in Mg(2+)-free condition. Both glutamate and glycine were required to evoke glutamate exocytosis in 1.2 mM Mg(2+), while dizocilpine, cis-4-[phosphomethyl]-piperidine-2-carboxylic acid and 7-Cl-kynurenic acid prevented it, indicating that occupation of both agonist sites is needed for receptor activation. D-serine mimicked glycine but also inhibited the NMDA/glycine-induced release of [(3H]D-aspartate, thus behaving as a partial agonist. The NMDA/glycine-induced release in 1.2 mM Mg(2+) strictly depended on glycine uptake through the glycine transporter type 1 (GlyT1), because the GlyT1 blocker N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine hydrochloride, but not the GlyT2 blocker Org 25534, prevented it. Accordingly, [(3)H]glycine was taken up during superfusion, while lowering the external concentration of Na(+), the monovalent cation co-transported with glycine by GlyT1, abrogated the NMDA-induced effect. Western blot analysis of subsynaptic fractions confirms that GlyT1 and NMDA autoreceptors co-localize at the pre-synaptic level, where GluN3A subunits immunoreactivity was also recovered. It is proposed that GlyT1s coexist with NMDA autoreceptors on rat hippocampal glutamatergic terminals and that glycine taken up by GlyT1 may permit physiological activation of NMDA pre-synaptic autoreceptors.