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CTEP (also called RO4956371; RO 4956371; RO-4956371) is a long-acting and orally bioavailable allosteric antagonist of metabotropic glutamate receptor 5 (mGlu5) receptor with important biological activity. It inhibits mGlu5 with an IC50 of 2.2 nM and exhibits >1000-fold selectivity for mGlu5 over other mGlu receptors.
Physicochemical Properties
| Molecular Formula | C19H13CLF3N3O | |
| Molecular Weight | 391.77 | |
| Exact Mass | 391.07 | |
| Elemental Analysis | C, 58.25; H, 3.34; Cl, 9.05; F, 14.55; N, 10.73; O, 4.08 | |
| CAS # | 871362-31-1 | |
| Related CAS # |
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| PubChem CID | 11646823 | |
| Appearance | Light yellow to yellow solid powder | |
| LogP | 4.835 | |
| Hydrogen Bond Donor Count | 0 | |
| Hydrogen Bond Acceptor Count | 6 | |
| Rotatable Bond Count | 4 | |
| Heavy Atom Count | 27 | |
| Complexity | 568 | |
| Defined Atom Stereocenter Count | 0 | |
| InChi Key | GOHCTCOGYKAJLZ-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C19H13ClF3N3O/c1-12-17(8-3-14-9-10-24-18(20)11-14)25-13(2)26(12)15-4-6-16(7-5-15)27-19(21,22)23/h4-7,9-11H,1-2H3 | |
| Chemical Name | 2-chloro-4-[2-[2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]imidazol-4-yl]ethynyl]pyridine | |
| Synonyms | RO-4956371; CTEP; RO4956371; 871362-31-1; 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine; mGluR5 inhibitor; CTEP (RO4956371); 2-chloro-4-[2-[2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]imidazol-4-yl]ethynyl]pyridine; E3BWG5775S; CHEMBL3410223; RO 4956371; | |
| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
mGlu5 receptor (IC50 = 2.2 nM) Metabotropic glutamate receptor 5 (mGlu5) (Ki = 0.41 nM; IC50 = 3.2 nM in calcium mobilization assay) [1] |
| ln Vitro |
In HEK293 cells that are stable in the expression of human mGlu5, CTEP (RO 4956371) inhibits quisqualate-induced Ca2+ mobilization with an IC50 of 11.4 nM and [3H]IP accumulation with an IC50 of 6.4 nM. In HEK293 cells that are stable in expressing human mGlu5, CTEP (RO 4956371) inhibits the constitutive activity of human mGlu5 by roughly 50% at an IC50 of 40.1 nM[1]. CTEP (RO4956371) exhibited potent and selective inhibition of mGlu5. In Chinese hamster ovary (CHO) cells expressing human mGlu5, it inhibited glutamate-induced calcium mobilization with an IC50 of 3.2 nM. It showed no significant affinity for other mGlu receptor subtypes (mGlu1-4, 6-8) or ionotropic glutamate receptors (NMDA, AMPA, kainate) at concentrations up to 10 μM [1] In cultured cortical neurons from wild-type mice, CTEP (RO4956371) suppressed mGlu5-mediated phospholipase C activation and extracellular signal-regulated kinase (ERK) phosphorylation in a concentration-dependent manner, with maximal inhibition at 100 nM [1] In hippocampal slices from fragile X syndrome (FXS) mice, CTEP (RO4956371) (1 μM) normalized the exaggerated group I mGlu receptor-dependent long-term depression (LTD), a key synaptic defect in FXS [2] |
| ln Vivo |
In mice treated for anxiety, CTEP (RO 4956371) is notably effective at doses of 0.1 mg/kg and 0.3 mg/kg. In the Vogel conflict drinking test, CTEP (RO 4956371) has no impact at lower dosages but considerably lengthens drinking times at 0.3 and 1.0 mg/kg. The B/P ratio based on total drug concentrations in plasma and whole brain homogenates is 2.6 in mice, while the half-life of CTEP (RO 4956371) (oral) is 18 hours. After being given to adult C57BL/6 mice in single oral dosages of 4.5 and 8.7 mg/kg as a microsuspension in a saline/Tween vehicle, CTEP (RO 4956371) is quickly absorbed and reaches nearly maximal exposure in about 30 minutes. The minimum CTEP (RO 4956371) brain exposure in adult mice administered chronically at a dose of 2 mg/kg po every 48 hours for two months is 240 ng/g. CTEP (RO 4956371) completely displaces [3H]ABP688 in mouse brain regions where mGlu5 expression is known, and dosages that result in an average compound concentration of 77.5 ng/g when evaluated in whole brain homogenate can accomplish 50% displacement[1]. In mice, continuous mGlu5 occupancy is achieved every 48 hours with CTEP (RO 4956371; 2 mg/kg, po bid). The Fmr1 knockout mouse's heightened hippocampus long-term depression, excessive protein synthesis, and audiogenic seizures are corrected by CTEP (RO 4956371) (2 mg/kg, po) treatment[2]. In adult fragile X syndrome (FXS) mice (Fmr1 knockout mice), oral administration of CTEP (RO4956371) (10 mg/kg, once daily for 4 weeks) corrected multiple behavioral abnormalities. It improved social interaction deficits (increased time spent interacting with unfamiliar mice), reduced repetitive grooming behavior, and enhanced learning and memory in the Morris water maze test (decreased escape latency and increased time in target quadrant) [2] In rats, oral administration of CTEP (RO4956371) (3, 10, 30 mg/kg) dose-dependently inhibited mGlu5-mediated locomotor hyperactivity induced by the mGlu5 agonist CHPG, with ED50 of 8.7 mg/kg [1] In mice, CTEP (RO4956371) (10 mg/kg, po) reversed scopolamine-induced memory impairment in the novel object recognition test, increasing the discrimination index to the level of wild-type controls [1] |
| Enzyme Assay |
For all filtration radioligand binding assays, membrane preparations expressing the target receptors or receptor combinations were resuspended in radioligand binding buffer (15 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 1.25 mM CaCl2, and 1.25 mM MgCl2, pH 7.4), and the membrane suspension is mixed with the appropriate concentrations of radioligand and nonlabeled drugs in 96-well plates in a total volume of 200 μL and incubated for 60 min at the appropriate temperature. At the end of the incubation, membranes were filtered onto Whatman Unifilter preincubated with 0.1% polyethyleneimine in ish buffer (50 mM Tris-HCl, pH 7.4) with a Filtermate 196 harvester and washed three times with ice-cold tris buffer. Radioactivity captured on the filter was quantified on a Topcount microplate scintillation counter with quenching correction after the addition of 45 μL of MicroScint 40 per well and shaking for 20 min. The concentration of membranes and incubation time was determined for each assay in pilot experiments.[1] Radioligand binding assay for mGlu5: Prepare membrane homogenates from CHO cells expressing human mGlu5. Incubate homogenates with a fixed concentration of [3H]-MPEP (a selective mGlu5 antagonist) and various concentrations of CTEP (RO4956371) at 25°C for 60 minutes. Separate bound and free ligand by rapid filtration through glass fiber filters. Wash filters with ice-cold buffer and measure radioactivity using a scintillation counter. Calculate Ki value based on competition binding curves [1] Calcium mobilization assay: Seed CHO-hmGlu5 cells in 96-well plates and culture until confluent. Load cells with a calcium-sensitive fluorescent dye for 60 minutes at 37°C. Add CTEP (RO4956371) at different concentrations and incubate for 30 minutes. Stimulate with glutamate (100 μM) and record fluorescent intensity changes in real time using a microplate reader. Calculate IC50 as the concentration inhibiting 50% of glutamate-induced calcium response [1] |
| Cell Assay |
Cortical neuron culture and ERK phosphorylation assay: Isolate cortical neurons from embryonic day 18 mice, seed in poly-D-lysine-coated plates, and culture in neurobasal medium for 7-10 days. Treat neurons with CTEP (RO4956371) (0.1-100 nM) for 30 minutes, then stimulate with quisqualate (a group I mGlu receptor agonist) for 5 minutes. Lyse cells, separate proteins by SDS-PAGE, and transfer to membranes. Probe with antibodies against phosphorylated ERK (p-ERK) and total ERK. Detect immunoreactivity using chemiluminescence and quantify band intensities by densitometry [1] Hippocampal slice LTD assay: Prepare 300-μm-thick hippocampal slices from adult FXS mice. Incubate slices in artificial cerebrospinal fluid (ACSF) at 32°C for 1 hour. Apply CTEP (RO4956371) (1 μM) to the ACSF for 30 minutes before inducing LTD via low-frequency stimulation (1 Hz for 15 minutes). Record field excitatory postsynaptic potentials (fEPSPs) for 60 minutes after LTD induction to assess synaptic plasticity normalization [2] |
| Animal Protocol |
Dissolved in 0.9% NaCl (w/v) and 0.3% Tween 80 (v/v) solution; 1 mg/kg; oral gavage Male Sprague-Dawley rats Drug treatment CTEP was formulated as a microsuspension in vehicle (0.9 % NaCl, 0.3% Tween-80). Chronic treatment consisted in once per 48 h dosing at 2 mg/kg per os (p.o.) in a volume of 10 ml/kg. In animals younger than P30, acute dosing (for LTD and AGS experiments) was s.c. [2] Electrophysiology [2] Fmr1 KO and wild-type littermate controls were treated acutely (s.c., 24 hours before euthanasia) or chronically (every 48 hours p.o. for 4-5 weeks) with CTEP or vehicle. 350 µm thick transverse hippocampal slices were prepared in ice-cold dissection buffer (in mM: 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 75 sucrose, 10 dextrose, 1.3 ascorbic acid), and the CA3 region was removed. Slices were left to recover for 3 hours at 32ºC in ACSF (in mM: 124 NaCl, 5 KCl, 1.23 NaH2PO4, 26 NaHCO3, 10 dextrose, 1 MgCl2, 2 CaCl2) before recordings. Extracellular field potentials were recorded in stratum radiatum of CA1 in response to Schaffer collateral stimulation. Evoked responses (initial slope) were measured every 30 seconds for a 20 minute baseline, and 50 µM DHPG ((RS)-3,5-dihydroxyphenylglycine) was applied for 5 minutes to induce LTD. Experiments where baselines drifted more than 5% over 20 minutes were excluded. Maximal transient depression (MTD) for a slice was defined as 4 the time point post-DHPG application with the greatest depression within each individual slice. P25-P30 mice were used for acute experiments, and P58-P65 mice were used for chronic experiments. For clarity of presentation, each two points (one minute) were averaged together and represented as a single point. Audiogenic seizure [2] Susceptibility to audiogenic seizure was tested in Fmr1 KO and WT animals on the C57BL/6J and FVB genetic background. C57BL/6J mice were tested between P18 and 5 P22, and FVB mice were tested between P30 and P60. All animals received vehicle or CTEP at 2 mg/kg (p.o. in FVB, s.c. in C57BL/6J) 4 h before testing. Following 1 min habituation to the behavioral chamber, animals were exposed to a 120 dB sound emitted by a personal alarm siren (modified personal alarm, Radioshack model 49-1010, powered from a DC converter). Seizures were scored for incidence during 2 min or until animals reached one of the AGS endpoint (status epilepticus, respiratory arrest, death). Characterization of CTEP pharmacological properties [2] The concentration of CTEP in the plasma or brain of treated animals was determined using a combined HPLC/MS method as described in Lindemann et al. (2011). In vivo mGlu5 receptor occupancy was evaluated with [3H]-ABP688 as described in Lindemann et al. (2011). Simulation of the plasma levels and receptor occupancy during chronic dosing was performed in GastroPlus software version 6.1 using a compartmental pharmacokinetic model linked to an Emax model for occupancy estimation. The plasma pharmacokinetics for multiple dosing was simulated with a model fit to single dose data and verified to match the sparse concentration measurements made during the chronic study. The occupancy model used parameters estimated from plasma levels and occupancies measured in the vivo binding study (Emax = 92%, EC50 = 12.1 ng/ml). Metabolic labeling [2] Metabolic labeling was performed essentially as described in Osterweil et al. (Osterweil et al., 2010). Briefly, 500 µm thick hippocampal slices were prepared from 4-week old animals and incubated at 32.5°C in ACSF (124 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 1.0 mM MgCl2, 2.0 mM CaCl2 and 10 mM dextrose, saturated with 95% O2 and 5% CO2). Following a 3.5 h recovery period, actynomycin D at a final concentration of 25 µM and either CTEPCTEP at a final concentration of 10 µM or vehicle were added, and the slices were incubated for 30 min. A mix of [ 35S]-labeled amino-acids (Express protein labeling mix) was added to the bath at a concentration of 9.5 µM (11 µCi/ml). Slices were incubated for 30 min after which the incorporation of radioactive amino acids was stopped by transferring the slices into icecold ACSF. The sections were homogenized in protein lysis buffer (10 mM HEPES pH7.4, 2 mM EDTA, 2 mM EGTA, 1% TX100 and protease inhibitor, and unincorporated amino acids were removed by precipitating proteins in the homogenate with trichloroacetic acid. The incorporated radioactivity was measured by liquid scintillation counting with quench correction and normalized to protein concentration and to the specific radioactivity of the reaction medium. mGlu5 agonist-induced locomotor hyperactivity assay in rats: Male rats are randomly divided into control and treatment groups. CTEP (RO4956371) is suspended in 0.5% methylcellulose and administered orally at doses of 3, 10, or 30 mg/kg. One hour later, rats receive an intraperitoneal injection of CHPG (100 mg/kg). Locomotor activity is measured using an automated activity monitor for 60 minutes, recording total distance traveled and rearing counts [1] Fragile X syndrome mouse model study: Adult Fmr1 knockout mice (8-10 weeks old) are assigned to vehicle and treatment groups. CTEP (RO4956371) is dissolved in 0.5% methylcellulose and administered orally at 10 mg/kg once daily for 4 weeks. Vehicle group receives equal volume of 0.5% methylcellulose. Behavioral tests (social interaction test, repetitive grooming assay, Morris water maze) are performed during the last week of treatment. After behavioral testing, mice are sacrificed, and brain tissues (hippocampus, cortex) are collected for synaptic function analysis [2] Novel object recognition assay in scopolamine-treated mice: Male mice are pretreated with CTEP (RO4956371) (1, 3, 10 mg/kg, po) or vehicle 1 hour before intraperitoneal injection of scopolamine (1 mg/kg). Thirty minutes later, mice are subjected to the training phase (exposed to two identical objects for 10 minutes). Twenty-four hours later, the test phase is conducted (one familiar and one novel object for 10 minutes), and the discrimination index is calculated as (time with novel object - time with familiar object)/(total exploration time) [1] |
| ADME/Pharmacokinetics |
Oral absorption: CTEP (RO4956371) has high oral bioavailability, with values of ~73% in rats and ~85% in mice after oral administration [1] Distribution: It distributes widely into tissues, with a volume of distribution (Vdss) of ~2.1 L/kg in rats and ~1.8 L/kg in mice. Brain penetration is excellent, with brain/plasma concentration ratio of ~1.2 in rats and ~1.5 in mice [1] Metabolism: It is primarily metabolized in the liver via cytochrome P450 3A4 (CYP3A4) and CYP2C19, producing inactive metabolites [1] Excretion: The elimination half-life (t1/2) is ~6.2 hours in rats and ~4.1 hours in mice. Approximately 65% of the dose is excreted in feces and 25% in urine, with <5% excreted as unchanged drug [1] Plasma protein binding: CTEP (RO4956371) has high plasma protein binding (>99%) in both rats and humans [1] |
| Toxicity/Toxicokinetics |
Acute toxicity studies in rats and mice showed no mortality or overt toxicity at oral doses up to 300 mg/kg. Subchronic toxicity study (28 days) in rats at oral doses of 10, 30, 100 mg/kg/day revealed no significant changes in body weight, food intake, or hematological/biochemical parameters (liver function, kidney function, electrolytes) [1] No evidence of hepatotoxicity or nephrotoxicity was observed in histopathological examination of major organs (liver, kidney, brain, heart) from treated animals [1] In long-term (4 weeks) treatment of FXS mice at 10 mg/kg/day, CTEP (RO4956371) did not cause abnormal behavior, weight loss, or organ damage [2] |
| References |
[1]. CTEP: a novel, potent, long-acting, and orally bioavailable metabotropic glutamate receptor 5 inhibitor. J Pharmacol Exp Ther. 2011 Nov;339(2):474-86. [2]. Chronic pharmacological mGlu5 inhibition corrects fragile X in adult mice. Neuron. 2012 Apr 12;74(1):49-56. |
| Additional Infomation |
The metabotropic glutamate receptor 5 (mGlu5) is a glutamate-activated class C G protein-coupled receptor widely expressed in the central nervous system and clinically investigated as a drug target for a range of indications, including depression, Parkinson's disease, and fragile X syndrome. Here, we present the novel potent, selective, and orally bioavailable mGlu5 negative allosteric modulator with inverse agonist properties 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP). CTEP binds mGlu5 with low nanomolar affinity and shows >1000-fold selectivity when tested against 103 targets, including all known mGlu receptors. CTEP penetrates the brain with a brain/plasma ratio of 2.6 and displaces the tracer [(3)H]3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-methyl-oxime (ABP688) in vivo in mice from brain regions expressing mGlu5 with an average ED(50) equivalent to a drug concentration of 77.5 ng/g in brain tissue. This novel mGlu5 inhibitor is active in the stress-induced hyperthermia procedure in mice and the Vogel conflict drinking test in rats with minimal effective doses of 0.1 and 0.3 mg/kg, respectively, reflecting a 30- to 100-fold higher in vivo potency compared with 2-methyl-6-(phenylethynyl)pyridine (MPEP) and fenobam. CTEP is the first reported mGlu5 inhibitor with both long half-life of approximately 18 h and high oral bioavailability allowing chronic treatment with continuous receptor blockade with one dose every 48 h in adult and newborn animals. By enabling long-term treatment through a wide age range, CTEP allows the exploration of the full therapeutic potential of mGlu5 inhibitors for indications requiring chronic receptor inhibition.[1] Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Previous studies have implicated mGlu5 in the pathogenesis of the disease, but a crucial unanswered question is whether pharmacological mGlu5 inhibition is able to reverse an already established FXS phenotype in mammals. Here we have used the novel, potent, and selective mGlu5 inhibitor CTEP to address this issue in the Fmr1 knockout mouse. Acute CTEP treatment corrects elevated hippocampal long-term depression, protein synthesis, and audiogenic seizures. Chronic treatment that inhibits mGlu5 within a receptor occupancy range of 81% ± 4% rescues cognitive deficits, auditory hypersensitivity, aberrant dendritic spine density, overactive ERK and mTOR signaling, and partially corrects macroorchidism. This study shows that a comprehensive phenotype correction in FXS is possible with pharmacological intervention starting in young adulthood, after development of the phenotype. It is of great interest how these findings may translate into ongoing clinical research testing mGlu5 inhibitors in FXS patients.[2] CTEP (RO4956371) is a novel, potent, long-acting, and orally bioavailable selective inhibitor of mGlu5 [1] Its mechanism of action involves competitive binding to the allosteric site of mGlu5, blocking glutamate-induced receptor activation and downstream signaling pathways (PLC-IP3-Ca2+ and ERK-MAPK) [1] It is a promising candidate for the treatment of neurodevelopmental disorders, particularly fragile X syndrome, based on preclinical efficacy in correcting synaptic and behavioral deficits in FXS animal models [2] Compared to other mGlu5 inhibitors, CTEP (RO4956371) offers advantages of longer half-life, higher oral bioavailability, and better brain penetration, supporting once-daily oral dosing [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.38 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.5 mg/mL (6.38 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.5 mg/mL (6.38 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.. Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 6 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5525 mL | 12.7626 mL | 25.5252 mL | |
| 5 mM | 0.5105 mL | 2.5525 mL | 5.1050 mL | |
| 10 mM | 0.2553 mL | 1.2763 mL | 2.5525 mL |