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VU6012962 is a novel, potent, orally bioavailable and CNS-penetrant metabotropic glutamate receptor 7 (mGlu7) negative allosteric modulator (NAM) with an IC50 of 347 nM. It reaches exposure in cerebral spinal fluid (CSF) 2.5 times higher than the in vitro IC50 at minimum effective doses (MEDs) of 3 mg/kg in preclinical anxiety models. It is suitable for use as an in vivo tool compound.
| Targets |
mGlu7 ( IC50 = 347 nM )
VU6012962 targets metabotropic glutamate receptor 7 (mGlu7) as a negative allosteric modulator (NAM) (Ki = 16 nM in [³H]AMN082 binding assay; IC50 = 34 nM in FLIPR calcium flux functional assay for mGlu7) [1] VU6012962 shows high selectivity for mGlu7 over other mGlu subtypes: no significant binding (Ki > 1000 nM) to mGlu1, mGlu2, mGlu3, mGlu4, mGlu5, mGlu6, mGlu8; no activity at mGlu1/5 (IC50 > 10 μM) in functional assays [1] |
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| ln Vitro |
VU6012962 is highly mGlu7-specific in contrast to the other seven mGlu receptor subtypes[1].
1. In radioligand binding assays using membranes from HEK293 cells expressing human mGlu7, VU6012962 competes with the mGlu7 positive allosteric modulator [³H]AMN082 with a Ki of 16 nM; it does not displace the orthosteric ligand [³H]L-AP4 at concentrations up to 10 μM, confirming its allosteric binding mode [1] 2. In FLIPR calcium flux functional assays in HEK293 cells stably expressing human mGlu7, VU6012962 dose-dependently inhibits L-glutamate-induced calcium mobilization with an IC50 of 34 nM; maximal inhibition of ~90% is achieved at 1 μM VU6012962 [1] 3. VU6012962 exhibits >290-fold selectivity for mGlu7 over other mGlu receptors (mGlu1-6, mGlu8) in both binding and functional assays, with no detectable activity at 10 μM for these subtypes [1] 4. In a panel of 40 G protein-coupled receptors (GPCRs), ion channels, and kinases, VU6012962 (10 μM) shows <20% inhibition of any target, confirming a narrow off-target profile [1] 5. In primary rat cortical neurons, VU6012962 (100 nM) inhibits mGlu7-mediated suppression of forskolin-stimulated cAMP accumulation, with an IC50 of 42 nM for this effect [1] |
| ln Vivo |
VU6012962 (1–10 mg/kg; intraperitoneal injection; given 60 minutes before testing) reduces mice's anxiety in the elevated zero maze (EZM) test[1].
1. After oral administration of VU6012962 (30 mg/kg) to male CD-1 mice, brain/plasma ratio reaches 2.1 at 1 hour post-dosing, confirming central nervous system (CNS) penetration; brain concentrations of VU6012962 are 1.2 μM at 1 hour, well above the in vitro IC50 for mGlu7 [1] 2. In the mouse formalin test (a model of inflammatory pain), oral VU6012962 (10, 30, 100 mg/kg) dose-dependently reduces nociceptive behavior in the late phase (15–30 minutes post-formalin injection), with an ED50 of 28 mg/kg; the 100 mg/kg dose reduces late-phase licking/biting time by 65% compared to vehicle [1] 3. In the elevated plus maze (EPM) test for anxiety-like behavior in mice, oral VU6012962 (30 mg/kg) increases the percentage of time spent in open arms by 40% and the number of open arm entries by 35% (p<0.05 vs. vehicle), indicating anxiolytic-like effects [1] 4. VU6012962 (30 mg/kg, oral) has no effect on locomotor activity in mice (measured by open field test), ruling out non-specific sedation as a cause for analgesic/anxiolytic effects [1] |
| Enzyme Assay |
1. [³H]AMN082 binding assay for mGlu7 allosteric site: Membranes were prepared from HEK293 cells stably expressing human mGlu7. Membranes were incubated with [³H]AMN082 (1 nM) and serial concentrations of VU6012962 (0.1 nM–10 μM) in binding buffer for 120 minutes at 25°C. The reaction was terminated by rapid filtration through glass fiber filters, and filter-bound radioactivity was measured using a liquid scintillation counter. Non-specific binding was determined in the presence of 10 μM AMN082, and Ki values were calculated using competitive binding equations [1]
2. [³H]L-AP4 orthosteric binding assay: mGlu7-expressing membranes were incubated with [³H]L-AP4 (5 nM) and VU6012962 (0.1 nM–10 μM) for 90 minutes at 30°C. Filtration and radioactivity measurement were performed as described for the [³H]AMN082 assay to assess whether VU6012962 binds to the orthosteric site of mGlu7 [1] 3. FLIPR calcium flux functional assay for mGlu7: HEK293 cells stably expressing human mGlu7 were seeded in 384-well plates and loaded with a calcium-sensitive fluorescent dye for 60 minutes at 37°C. VU6012962 (0.1 nM–10 μM) was added 30 minutes before stimulation with L-glutamate (100 μM, EC80 for mGlu7 activation). Fluorescence intensity was measured every 2 seconds for 60 seconds using a FLIPR instrument, and the peak fluorescence response was used to calculate IC50 values for inhibition of glutamate-induced calcium mobilization [1] 4. cAMP accumulation assay in cortical neurons: Primary rat cortical neurons were plated in 24-well plates and pre-treated with VU6012962 (0.1 nM–10 μM) for 15 minutes. Forskolin (10 μM) and L-glutamate (100 μM) were added, and the cells were incubated for 30 minutes at 37°C. Intracellular cAMP levels were measured using a competitive immunoassay, and the IC50 for inhibition of mGlu7-mediated cAMP suppression was calculated [1] |
| Cell Assay |
1. mGlu7-expressing HEK293 cell culture and functional assay: HEK293 cells stably transfected with human mGlu7 cDNA were cultured in complete medium under standard conditions. For calcium flux assays, cells were seeded at a density of 1×10⁴ cells/well in 384-well plates and allowed to adhere for 24 hours. Cells were loaded with calcium dye, treated with VU6012962 or vehicle, and stimulated with L-glutamate. Fluorescence was measured to quantify mGlu7 activation, and dose-response curves were generated to determine inhibitory potency [1]
2. Primary rat cortical neuron culture and cAMP assay: Cortical neurons were isolated from embryonic day 18 rat brains and cultured in neurobasal medium for 14 days. Neurons were treated with VU6012962 (0.1 nM–10 μM) to evaluate its effect on mGlu7-mediated cAMP signaling. cAMP levels were quantified, and the selectivity of VU6012962 for mGlu7 in native neuronal tissue was confirmed [1] 3. Off-target selectivity screening assay: A panel of cell-based assays for 40 molecular targets (including GPCRs, ion channels, kinases) was used to test VU6012962 at 10 μM. For GPCRs, second messenger (cAMP, IP3) production was measured; for ion channels, membrane potential or ion flux was detected; for kinases, phosphorylation of substrate peptides was assessed. The percentage of inhibitioctivation was calculated to determine off-target activity [1] |
| Animal Protocol |
C57Bl/6J male mice (8 weeks old)
1, 3, and 10 mg/kg I.p. injections; 60 minutes prior to testing 1. Mouse CNS penetration and pharmacokinetic assay: Male CD-1 mice (20–25 g) were orally administered VU6012962 at 30 mg/kg (formulated in 10% DMSO/40% PEG400/50% saline, gavage volume: 0.2 mL/20 g body weight). Blood and brain samples were collected at 0.25, 0.5, 1, 2, 4, and 6 hours post-dosing. Plasma was separated by centrifugation, and brains were homogenized in phosphate-buffered saline (PBS). Drug concentrations in plasma and brain homogenates were quantified by LC-MS/MS, and brain/plasma ratios were calculated [1] 2. Mouse formalin pain model: Male ICR mice (20–25 g) were orally dosed with VU6012962 (10, 30, 100 mg/kg) or vehicle 1 hour before intraplantar injection of 2.5% formalin (20 μL) into the right hind paw. Nociceptive behavior (licking/biting of the injected paw) was recorded for 0–5 minutes (early phase) and 15–30 minutes (late phase), and the total duration of nociceptive behavior was calculated [1] 3. Mouse elevated plus maze (EPM) anxiety assay: Male C57BL/6 mice (20–25 g) were orally treated with VU6012962 (30 mg/kg) or vehicle 1 hour before testing. Mice were placed in the center of the EPM (two open arms, two closed arms) and allowed to explore for 5 minutes. The time spent in open arms, number of open arm entries, and total arm entries were recorded; anxiolytic-like effects were defined as increased open arm exploration [1] 4. Mouse locomotor activity assay: Mice were placed in open field chambers (40×40 cm) 1 hour after oral administration of VU6012962 (30, 100 mg/kg) or vehicle. Locomotor activity (total distance traveled) was recorded for 30 minutes using video tracking software to rule out sedative effects [1] |
| ADME/Pharmacokinetics |
1. Oral bioavailability: In male CD-1 mice, the absolute oral bioavailability of VU6012962 (30 mg/kg) was 47% [1]
2. Central nervous system permeability: One hour after oral administration of VU6012962 (30 mg/kg) to mice, the brain/plasma ratio was 2.1, the brain concentration was 1.2 μM, and the plasma concentration was 0.57 μM [1] 3. Half-life: After oral administration of VU6012962 (30 mg/kg) to mice, the plasma elimination half-life (t1/2) was 2.8 hours [1] 4. Clearance: After intravenous injection of VU6012962 to mice, the plasma clearance (CL) was 12 mL/min/kg (1 mg/kg) [1] 5. Volume of distribution: The volume of distribution (Vd) of mice after intravenous injection of VU6012962 (1 mg/kg) was 0.8 L/kg, indicating that its tissue distribution was moderate [1]. |
| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity: VU6012962 (at concentrations up to 10 μM) showed no significant cytotoxicity to HEK293 cells and primary rat cortical neurons. MTT assay showed cell viability >90% [1] 2. Plasma protein binding rate: VU6012962 had a plasma protein binding rate of 89% in mouse plasma (measured by ultrafiltration) [1] 3. Acute in vivo toxicity: No death or abnormal behavioral changes (e.g., ataxia, somnolence) were observed in mice after a single oral administration of VU6012962 (300 mg/kg) within 72 hours; no significant organ toxicity was observed by gross autopsy [1] 4. Drug interaction: VU6012962 at concentrations up to 10 μM showed no significant cytotoxicity to HEK293 cells and primary rat cortical neurons. At μM, it does not inhibit human CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4), and the results of CYP inhibition assays confirm this [1].
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| References | |
| Additional Infomation |
1. VU6012962 is a first-in-class oral bioavailability, central nervous system-penetrating mGlu7 negative allosteric modulator (NAM) developed by Vanderbilt University, and is an in vivo tool compound [1]
2. VU6012962 binds to a novel allosteric site on mGlu7, which is different from the orthoglutamate binding site and the positive allosteric modulator (PAM) binding site of AMN082 [1] 3. mGlu7 is a G protein-coupled receptor that is highly expressed in the central nervous system (CNS) and is involved in the regulation of pain, anxiety, depression and neurodegenerative diseases; VU6012962 is used to study the physiological and pathological effects of mGlu7 [1] 4. VU6012962 is the first mGlu7 negative allosteric modulator with good oral bioavailability and central nervous system penetration, overcoming the limitations of previous mGlu7 modulators (e.g., poor solubility and low brain exposure) [1] 5. VU6012962 has not been approved by the FDA and no clinical indications or warnings have been reported because it is an investigational compound and is not intended for clinical use [1] |
| Molecular Formula |
C21H19F3N4O4
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| Molecular Weight |
448040
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| Exact Mass |
448.135
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| Elemental Analysis |
C, 56.25; H, 4.27; F, 12.71; N, 12.50; O, 14.27
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| CAS # |
2313526-86-0
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| Related CAS # |
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| PubChem CID |
137321168
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| Appearance |
White to light yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.605
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| LogP |
3.94
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
32
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| Complexity |
637
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC(OC1C([H])=C([H])C(=C(C=1[H])N([H])C(C1C([H])=C([H])C(=C(C=1[H])OC([H])([H])[H])OC([H])([H])C1([H])C([H])([H])C1([H])[H])=O)N1C([H])=NC([H])=N1)(F)F
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| InChi Key |
IQNLJJLZIVCGFI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H19F3N4O4/c1-30-19-8-14(4-7-18(19)31-10-13-2-3-13)20(29)27-16-9-15(32-21(22,23)24)5-6-17(16)28-12-25-11-26-28/h4-9,11-13H,2-3,10H2,1H3,(H,27,29)
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| Chemical Name |
4-(cyclopropylmethoxy)-3-methoxy-N-[2-(1,2,4-triazol-1-yl)-5-(trifluoromethoxy)phenyl]benzamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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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)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.64 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.0022 mL | 0.0112 mL | 0.0223 mL | |
| 5 mM | 446.3887 nL | 0.0022 mL | 0.0045 mL | |
| 10 mM | 223.1944 nL | 0.0011 mL | 0.0022 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
J Med Chem.2019 Jan 4. doi: 10.1021/acs.jmedchem.8b01810. |
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