VU0361737_DataSheet_MedChemExpress

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Data Sheet

Product Name:Cat. No.:CAS No.:

Molecular Formula:Molecular Weight:Target:Pathway:Solubility:

VU0361737HY-144181161205-04-4C13H11ClN2O2262.69mGluR

GPCR/G ProteinDMSO: ≥ 43 mg/mL

BIOLOGICAL ACTIVITY: 

VU 0361737 is a selective positive allosteric modulator (PAM) for mGlu4 receptor with EC50 of 240 nM and 110 nM at human and ratreceptors, respectively, displays weak activity at mGlu5 and mGlu8 receptors, is inactive at mGlu1, mGlu2, mGlu3, mGlu6 and mGlu7receptors.

IC50 value: 110 nM (EC50, for rat ), 240 nM (EC50, for human)Target: mGlu4

in vitro: VU0361737 is inactive at mGlu1, mGlu2, mGlu3, mGlu6 and mGlu7 receptors and displays weak activity at mGlu5 and mGlu8receptors. [3]

in vivo: VU0361737 shows high in vivo CL in rat, a short half–life (T1/2 20 min), and demonstrates significant brain exposure(brain–to–plasma ratio of 4.1). [3]

PROTOCOL (Extracted from published papers and Only for reference)

Kinase assay [4]

Human mGluR4/Gqi5/CHO cells (30,000 cells/20 ol/well) are plated in blackwalled, clear–bottomed, TC treated, 384 well plates inDMEM containing 10% dialyzed FBS, 20 mM HEPES, 100 units/ml penicillin/streptomycin, and 1 mM sodium pyruvate. The cells aregrown overnight at 37°C in the presence of 5% CO2. During the day of assay, the medium is replaced with 20 μL of 1 μM Fluo–4, AMprepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F–127 and diluted in Assay Buffer (Hank'sbalanced salt solution, 20 mM HEPES and 2.5 mM Probenecid) for 45 minutes at 37°C. Dye is removed and replaced with 20 μL ofAssay Buffer. Test compounds are transferred to daughter plates using an Echo acoustic plate reformatter and then diluted into AssayBuffer. Ca2+ flux is measured using the Functional Drug Screening System 6000. Baseline readings are taken (10 images at 1 Hz,excitation, 470±20 nm, emission, 540±30 nm) and then 20 ol/well test compounds are added using the FDSS's integrated pipettor.Cells are incubated with compounds for approximately 2.5 minutes and then an EC20 concentration of glutamate is applied; 2 minuteslater an EC80 concentration of glutamate is added. For concentration–response curve experiments, compounds are serially diluted 1:3into 10 point concentration response curves and are transferred to daughter plates using the Echo. Test compounds are again appliedand followed by EC20 concentrations of glutamate. For fold shift experiments, compounds are added at 2X their final concentrationand then increasing concentrations of glutamate are added in the presence of vehicle or the appropriate concentration of testcompound. Curves are fitted using a four point logistical equation using Microsoft XLfit. Subsequent confirmations of

concentrationresponse parameters are performed using independent serial dilutions of source compounds and data from multipledays experiments are integrated and fit using a four point logistical equation in GraphPad Prism. Calcium assays are used to assessactivity of compounds at mGluRs 1, 4 and 5.Cell assay [1]

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VU0361737 (10 mM) stock solutions were prepared in DMSO and stored at –20°C. The UN– and RA –SH–SY5Y cells were treated withLY354740 (0.001–100 μM)); ACPT–I (0.001–100 μM); VU0361737 (0.001–10 μM); (S)–3,4–DCPG (0.01–100 μM); AZ12216052 (0.001–10μM) and AMN082 (0.001–1 μM). First, the effects of mGluR II/III ligands given alone for 24 h on UN– and RA–SH–SY5Y cell viabilitywere evaluated. Next, the agents were given to cells growing under normal conditions (medium with 10% FBS) for 24 and 48 h inorder to assess the impact of the above agents on cell proliferation rate. Subsequently, in order to test neuroprotective effects exertedby the investigated compounds against MPP(+)–toxicity, particular mGluR II/III agonists and PAMs were added into cell cultureconcomitantly with the neurotoxic agent. The effective concentrations of MPP(+) were established in our previous study where 1 and 2mM MPP (+) evoked about 50% cell damage (measured by MTT assay) in UN– and RA–SH–SY5Y cells, respectively.Animal administration [3]

VU0361737 was formulated as 10% tween 80 micro suspensions in sterile water at the concentration of 5 mg/ml and administeredintraperitoneally to male Sprague–Dawley rats weighing 225 to 250 g at the dose of 10 mg/kg. The rat blood and brain were collectedat 0.5, 1, and 8 h. Animals were euthanized and decapitated, and the brains were removed, thoroughly washed in cold phosphatebuffered saline and immediately frozen on dry ice. Trunk blood was collected in EDTA Vacutainer tubes, and plasma was separated bycentrifugation and stored at –80°C until analysis. Three animals were used for each time point.

References:

[1]. Jantas D, et al.   Neuroprotective effects of metabotropic glutamate receptor group II and III activators against MPP(+)–induced cell death in humanneuroblastoma SH–SY5Y cells: the impact of cell differentiation state. Neuropharmacology. 2014 Aug;83:36–53.

[2]. Jantas D, et al. Neuroprotective effects of mGluR II and III activators against staurosporine– and doxorubicin–induced cellular injury in SH–SY5Y cells:New evidence for a mechanism involving inhibition of AIF translocation. Neurochem Int. 2015 Sep;88:124–37.

[3]. Engers DW, et al.   Synthesis and evaluation of a series of heterobiarylamides that are centrally penetrant metabotropic glutamate receptor 4 (mGluR4)positive allosteric modulators (PAMs). J Med Chem. 2009 Jul 23;52(14):4115–8.

[4]. Engers DW, et al.   Discovery, synthesis, and structure–activity relationship development of a series of N–(4–acetamido)phenylpicolinamides as positiveallosteric modulators of metabotropic glutamate receptor 4 (mGlu(4)) with CNS exposure in rats. J Med Chem. 2011 Feb 24;54(4):1106–10.

Caution: Product has not been fully validated for medical applications. For research use only.

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