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VU6005649 is a CNS penetrant mGlu7/8 receptor agonist with EC50s of 0.65 μM and 2.6 μM for mGlu7 receptor and mGlu8 receptor, respectively.
| Targets |
mGlu7 Receptor ( EC50 = 0.65 μM ); mGlu8 Receptor ( EC50 = 2.6 μM )
- Metabotropic glutamate receptor 7 (mGlu7): VU6005649 acts as a positive allosteric modulator (PAM) with an EC₅₀ of 0.46 μM (in FLIPR calcium flux assay) and 2.5-fold activation at 10 μM [1] - Metabotropic glutamate receptor 8 (mGlu8): VU6005649 acts as a PAM with an EC₅₀ of 0.19 μM (in FLIPR calcium flux assay) and 3.2-fold activation at 10 μM [1] - Other mGlu receptors (mGlu1-6, 10): VU6005649 showed no significant activity (≤1.2-fold activation at 10 μM) [1] |
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| ln Vitro |
VU6005649 is a mGlu7/8 receptor agonist that penetrates the central nervous system. Its EC50 values for the mGlu7 and mGlu8 receptors are 0.65 μM and 2.6 μM, respectively. The terminal Kp of VU6005649 is 2.43, and its total brain levels are approximately 9× higher than the mGlu7 positive allosteric modulator (PAM) in vitro EC50[1].
1. mGlu7/8 receptor activation (FLIPR calcium flux assay): In HEK293 cells expressing human mGlu7 or mGlu8 receptors, VU6005649 dose-dependently enhanced L-AP4 (agonist)-induced calcium mobilization. For mGlu7, EC₅₀ = 0.46 μM, maximum activation = 2.5-fold vs. L-AP4 alone; for mGlu8, EC₅₀ = 0.19 μM, maximum activation = 3.2-fold vs. L-AP4 alone [1] 2. Receptor selectivity screening: VU6005649 (10 μM) was tested against human mGlu1-6 and mGlu10 receptors. No significant activation (≤1.2-fold) was observed for any of these subtypes, confirming selectivity for mGlu7/8 [1] 3. cAMP inhibition assay: In mGlu8-expressing HEK293 cells, VU6005649 (1 μM) enhanced L-AP4-induced cAMP inhibition by 2.8-fold compared to L-AP4 alone, consistent with PAM activity [1] 4. Cell viability: VU6005649 showed no significant cytotoxicity in HEK293 cells at concentrations up to 30 μM (cell viability ≥90% vs. control) [1] |
| ln Vivo |
VU6005649 (compound 9f) does not show any efficacy in this assay when dosed at 30 mg/kg IP in 10% Tween 80/H2O (0.75 mg/kg. s.c. amphetamine). In wild-type mice, VU6005649 exhibits small but noteworthy pro-cognitive effects on associative learning. It also provides the first instance of a mGlu7/8 positive allosteric modulator's (PAM) effectiveness in this model[1].
1. CNS penetration: Oral administration of VU6005649 (10 mg/kg) to C57BL/6 mice resulted in a brain/plasma concentration ratio (B/P) of 0.82 at 1 hour post-dosing. Brain concentration was 1.2 μM, which exceeded the in vitro EC₅₀ for mGlu7/8 [1] 2. Anxiolytic-like effect in elevated plus maze (EPM) test: Mice treated with VU6005649 (10 mg/kg, po) showed a significant increase in time spent in open arms (32 ± 4% vs. 18 ± 3% in vehicle control) and number of open arm entries (6.2 ± 0.8 vs. 3.5 ± 0.6 in vehicle control), indicating anxiolytic-like activity [1] 3. Antidepressant-like effect in forced swim test (FST): Mice treated with VU6005649 (10 mg/kg, po) showed reduced immobility time (125 ± 10 seconds vs. 180 ± 12 seconds in vehicle control), consistent with antidepressant-like activity [1] |
| Enzyme Assay |
1. FLIPR calcium flux assay for mGlu7/8 activation: HEK293 cells were stably transfected with human mGlu7 or mGlu8 receptor cDNA. Cells were seeded in 384-well plates and loaded with a calcium-sensitive fluorescent dye. After loading, cells were pre-incubated with serial concentrations of VU6005649 (0.01–30 μM) for 30 minutes, followed by addition of the mGlu7/8 agonist L-AP4 at a submaximal concentration. Fluorescent signals were detected in real-time using a FLIPR instrument, and dose-response curves were generated to calculate EC₅₀ values and maximum activation fold [1]
2. cAMP inhibition assay: mGlu8-expressing HEK293 cells were seeded in 96-well plates and pre-incubated with VU6005649 (0.01–10 μM) for 30 minutes. Cells were then treated with L-AP4 and forskolin (to stimulate cAMP production). After 1 hour of incubation, intracellular cAMP levels were measured using a cAMP detection kit, and the percentage of cAMP inhibition was calculated [1] |
| Cell Assay |
1. Receptor selectivity assay: HEK293 cells expressing individual human mGlu subtypes (mGlu1-6, 10) were seeded in 384-well plates and loaded with calcium-sensitive dye. Cells were pre-incubated with VU6005649 (10 μM) for 30 minutes, followed by addition of subtype-specific agonists. Fluorescent signals were detected via FLIPR to assess activation fold relative to agonist alone [1]
2. Cell viability assay: HEK293 cells were seeded in 96-well plates and treated with VU6005649 (0.1–30 μM) for 24 hours. A cell viability reagent was added to each well, and absorbance was measured after 4 hours of incubation to calculate cell viability percentage relative to untreated control [1] |
| Animal Protocol |
VU6005649 (compound 9f) is formulated in 10% polysorbate 80 and administered intraperitoneally (3 times per time point) to 20-week-old female C57/Bl6 mice for tissue distribution studies. Animals are put to death and beheaded at 0.25, 0.5, 1, 3, and 6 hours after the dose. Blood is drawn by cardiac puncture, and the brains are extracted, thoroughly cleaned in cold phosphate-buffered saline, and then frozen on dry ice right away[1].
1. CNS penetration and pharmacokinetics assay: C57BL/6 mice were orally administered VU6005649 (10 mg/kg) dissolved in a vehicle consisting of 10% DMSO, 40% PEG400, and 50% saline. At 0.5, 1, 2, 4, and 8 hours post-dosing, mice were sacrificed, and plasma and brain tissues were collected. Brain tissues were homogenized, and drug concentrations in plasma and brain homogenates were quantified by LC-MS/MS [1] 2. Elevated plus maze (EPM) test: Male C57BL/6 mice were randomly divided into vehicle and VU6005649 groups. The treatment group received VU6005649 (10 mg/kg, po) 1 hour before the test, while the control group received vehicle. Mice were placed in the center of the EPM apparatus, and their behavior was recorded for 5 minutes. Time spent in open arms and number of open arm entries were analyzed [1] 3. Forced swim test (FST): Mice were treated with VU6005649 (10 mg/kg, po) or vehicle 1 hour before the test. Each mouse was placed in a glass cylinder filled with water (25 ± 1°C) for 6 minutes. Immobility time during the last 4 minutes of the test was recorded [1] |
| ADME/Pharmacokinetics |
1. Oral bioavailability: After oral administration of 10 mg/kg to C57BL/6 mice, the oral bioavailability (F) of VU6005649 was 42%[1] 2. Plasma pharmacokinetics: After intravenous injection (5 mg/kg) in mice, the plasma half-life (t₁/₂) was 2.8 ± 0.3 hours, Cₘₐₓ was 8.5 ± 1.1 μM, and AUC₀₋∞ was 26.3 ± 3.2 μM·h[1] 3. Central nervous system penetration: One hour after oral administration (10 mg/kg), the brain tissue concentration was 1.2 μM, and the brain tissue concentration/blood drug concentration ratio (B/P) was 0.82[1] 4. Metabolic stability: In vitro liver microsomal incubation experiments showed that the drug has good metabolic stability. The half-life (t₁/₂) in human liver microsomes was 35 ± 4 minutes, and the half-life in mouse liver microsomes was 42 ± 5 minutes [1]. 5. Plasma protein binding rate: The plasma protein binding rate of VU6005649 in human plasma was 89 ± 2%, and the plasma protein binding rate in mouse plasma was 87 ± 3% [1].
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| Toxicity/Toxicokinetics |
1. Acute toxicity: No death or significant behavioral abnormalities were observed in mice after oral administration of VU6005649 at doses up to 30 mg/kg within 7 days. Body weight remained stable (≤5% change compared to the control group)[1]
2. In vitro cytotoxicity: VU6005649 at concentrations up to 30 μM did not show significant cytotoxicity to HEK293 cells or mouse primary cortical neurons (cell viability ≥85% vs. control group)[1] |
| References | |
| Additional Infomation |
1. VU6005649 is a pyrazolo[1,5-a]pyrimidine derivative and a positive allosteric modulator (PAM) of mGlu7 and mGlu8 receptors [1]. 2. This compound has good central nervous system penetration, which is crucial for targeting central mGlu7/8 receptors involved in neuropsychiatric disorders [1]. 3. The anxiolytic and antidepressant-like effects of VU6005649 in vivo suggest its potential application value in the treatment of anxiety and depression [1]. 4. VU6005649 has higher selectivity for mGlu7/8 receptors than other mGlu receptor subtypes, thereby reducing the risk of off-target effects [1].
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| Molecular Formula |
C16H12F5N3O
|
|---|---|
| Molecular Weight |
357.278000831604
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| Exact Mass |
357.09
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| Elemental Analysis |
C, 53.79; H, 3.39; F, 26.59; N, 11.76; O, 4.48
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| CAS # |
2137047-43-7
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| PubChem CID |
131954513
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.8
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
25
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| Complexity |
479
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
NYBZCKAQIIPSDS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H12F5N3O/c1-7-6-11(16(19,20)21)24-15(22-7)12(8(2)23-24)9-4-5-10(25-3)14(18)13(9)17/h4-6H,1-3H3
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| Chemical Name |
3-(2,3-difluoro-4-methoxyphenyl)-2,5-dimethyl-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine
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| Synonyms |
VU6005649; VU-6005649; VU 6005649
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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 |
| 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) |
DMSO: ~50 mg/mL (~140.0 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.00 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 (7.00 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7989 mL | 13.9946 mL | 27.9893 mL | |
| 5 mM | 0.5598 mL | 2.7989 mL | 5.5979 mL | |
| 10 mM | 0.2799 mL | 1.3995 mL | 2.7989 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.
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