TNF-α (IC50 = 7 nM); NF-κB (IC50 = 11 nM)

QNZ (Compound 11q) has an anti-inflammatory effect that inhibits the NF-B mediated response. Edema formation is dose-dependently inhibited by QNZ[1]. In Huntington's disease (HD), QNZ (EVP4509) lowers the quantity of lysosomes/autophagosomes and store-operated channel (SOC) currents. It is anticipated that normalizing calcium transport within neurons in response to QNZ will lessen pathology manifestation. Using transmission electron microscopy (TEM), several lysosomes/autophagosomes are examined in HD and WT neurons treated with QNZ. While WT neurons are unaffected, incubation with QNZ almost doubles the amount of lysosomes/autophagosomes in HD GABAergic Medium Spiny (GABA MS)-like Neurons (GMSLNs) (from 0.41±0.04 to 0.23±0.04; p<0.05). By using flow cytometry (FC) analysis to look at lysosome content, this observation is confirmed. In HD GMSLNs, the median fluorescence intensity decreases by 34±6 percent after QNZ treatment (p<0.05)[2].

In Huntington's disease (HD), mutant Huntingtin (mHtt) protein causes striatal neuron dysfunction, synaptic loss, and eventual neurodegeneration. To understand the mechanisms responsible for synaptic loss in HD, we developed a corticostriatal coculture model that features age-dependent dendritic spine loss in striatal medium spiny neurons (MSNs) from YAC128 transgenic HD mice. Age-dependent spine loss was also observed in vivo in YAC128 MSNs. To understand the causes of spine loss in YAC128 MSNs, we performed a series of mechanistic studies. We previously discovered that mHtt protein binds to type 1 inositol (1,4,5)-trisphosphate receptor (InsP3R1) and increases its sensitivity to activation by InsP3. We now report that the resulting increase in steady-state InsP3R1 activity reduces endoplasmic reticulum (ER) Ca(2+) levels. Depletion of ER Ca(2+) leads to overactivation of the neuronal store-operated Ca(2+) entry (nSOC) pathway in YAC128 MSN spines. The synaptic nSOC pathway is controlled by the ER resident protein STIM2. We discovered that STIM2 expression is elevated in aged YAC128 striatal cultures and in YAC128 mouse striatum. Knock-down of InsP3R1 expression by antisense oligonucleotides or knock-down or knock-out of STIM2 resulted in normalization of nSOC and rescue of spine loss in YAC128 MSNs. The selective nSOC inhibitor EVP4593 was identified in our previous studies. We now demonstrate that EVP4593 reduces synaptic nSOC and rescues spine loss in YAC128 MSNs. Intraventricular delivery of EVP4593 in YAC128 mice rescued age-dependent striatal spine loss in vivo. Our results suggest EVP4593 and other inhibitors of the STIM2-dependent nSOC pathway as promising leads for HD therapeutic development.[3]

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EVP4593 (1 mg/kg, i.p.) dose-dependently inhibits carrageenin-induced paw edema in rats.

In RPMI1640 with 10% FCS, human Jurkat T cells are cultured at 37 °C in a 5% CO2 environment. The cells are transiently transfected with 1 μg of pNFκB-Luc using the SuperFect Transfection Reagent after being plated in 6-well plates (2×106/well). The cells undergo overnight culture at 37°C following transfection. Then, they are collected, resuspended in new medium, and plated in 96-well plates (2×105/well). The cells are placed in 96-well plates, and EVP4593 is dissolved in DMSO and added at the proper concentrations. The plates are then incubated at 37°C for an hour. For the purpose of triggering transcription, 10 ng/mL of PMA and 100 μg/mL of PHA are added to each well, and the cells are then incubated for an additional 6 hours at 37°C. Cell lysis buffer containing luciferase substrate is added to each well after the culture media have been removed. The luminescence is then immediately measured with a Packard Topcount after each portion has been transferred to a black 96-well plate. A nonlinear regression technique is used to determine the 50% inhibitory concentration (IC50) values.

iPSHD22 cells are cultured in K-4 medium in a 96-well black plates with clear flat bottom. Before being analyzed, cells are then exposed to chemicals for 24 hours (for example, QNZ 100 nM). Luminescent assay To simultaneously count the proportion of alive (viability) and dead (cytotoxicity) cells in each well, the MultiTox-Fluor Multiplex Cytotoxicity Assay is used. DTX 880 Multimode Microplate Reader is used to find fluorescence. Using the equation ([cytotoxicity in a well with cells]-([cytotoxicity in a well without cells])/([viability in a well with cells]-([viability in a well without cells])[2], the level of cell death (LoCD) is assessed.

[1]. Discovery of quinazolines as a novel structural class of potent inhibitors of NF-kappa B activation.

[2]. Manifestation of Huntington's disease pathology in human induced pluripotent stem cell-derived neurons. Mol Neurodegener. 2016 Apr 14;11:27.

[3]. Enhanced Store-Operated Calcium Entry Leads to Striatal Synaptic Loss in a Huntington's Disease Mouse Model. J Neurosci. 2016 Jan 6;36(1):125-41.

We disclose here a new structural class of low-molecular-weight inhibitors of NF-kappa B activation that were designed and synthesized by starting from quinazoline derivative 6a. Structure-activity relationship (SAR) studies based on 6a elucidated the structural requirements essential for the inhibitory activity toward NF-kappa B transcriptional activation, and led to the identification of the 6-amino-4-phenethylaminoquinazoline skeleton as the basic framework. In this series of compounds, 11q, containing the 4-phenoxyphenethyl moiety at the C(4)-position, showed strong inhibitory effects on both NF-kappa B transcriptional activation and TNF-alpha production. Furthermore, 11q exhibited an anti-inflammatory effect on carrageenin-induced paw edema in rats.[1]

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Background: Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder, which manifests itself as a loss of GABAergic medium spiny (GABA MS) neurons in the striatum and caused by an expansion of the CAG repeat in exon 1 of the huntingtin gene. There is no cure for HD, existing pharmaceutical can only relieve its symptoms. Results: Here, induced pluripotent stem cells were established from patients with low CAG repeat expansion in the huntingtin gene, and were then efficiently differentiated into GABA MS-like neurons (GMSLNs) under defined culture conditions. The generated HD GMSLNs recapitulated disease pathology in vitro, as evidenced by mutant huntingtin protein aggregation, increased number of lysosomes/autophagosomes, nuclear indentations, and enhanced neuronal death during cell aging. Moreover, store-operated channel (SOC) currents were detected in the differentiated neurons, and enhanced calcium entry was reproducibly demonstrated in all HD GMSLNs genotypes. Additionally, the quinazoline derivative, EVP4593, reduced the number of lysosomes/autophagosomes and SOC currents in HD GMSLNs and exerted neuroprotective effects during cell aging. Conclusions: Our data is the first to demonstrate the direct link of nuclear morphology and SOC calcium deregulation to mutant huntingtin protein expression in iPSCs-derived neurons with disease-mimetic hallmarks, providing a valuable tool for identification of candidate anti-HD drugs. Our experiments demonstrated that EVP4593 may be a promising anti-HD drug. Keywords: Aging; Differentiation; GABAergic medium spiny neurons; Human induced pluripotent stem cells; Huntington’s disease; Neurodegeneration; Neuroprotection; Nuclear indentations; Store-operated calcium entry.[2]

C22H20N4O

356.42

356.163

C, 74.14; H, 5.66; N, 15.72; O, 4.49

545380-34-5

509554

Light green to green solid powder

1.3±0.1 g/cm3

602.0±55.0 °C at 760 mmHg

169-175ºC

317.9±31.5 °C

0.0±1.7 mmHg at 25°C

1.714

4.57

2

5

6

27

434

0

O(C1C([H])=C([H])C([H])=C([H])C=1[H])C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])C([H])([H])N([H])C1C2C([H])=C(C([H])=C([H])C=2N=C([H])N=1)N([H])[H]

IBAKVEUZKHOWNG-UHFFFAOYSA-N

InChI=1S/C22H20N4O/c23-17-8-11-21-20(14-17)22(26-15-25-21)24-13-12-16-6-9-19(10-7-16)27-18-4-2-1-3-5-18/h1-11,14-15H,12-13,23H2,(H,24,25,26)

4-N-[2-(4-phenoxyphenyl)ethyl]quinazoline-4,6-diamine

CAY10470; CAY 10470; CAY-10470; EVP 4593; 545380-34-5; 6-Amino-4-(4-phenoxyphenylethylamino)quinazoline; QNZ; EVP4593; QNZ (EVP4593); N4-(4-phenoxyphenethyl)quinazoline-4,6-diamine; 4-N-[2-(4-phenoxyphenyl)ethyl]quinazoline-4,6-diamine; NF-kB activation inhibitor; EVP-4593; EVP4593; QNZ

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.01 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.01 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.01 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.

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Solubility in Formulation 4: 0.5% hydroxyethyl cellulose: 30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)