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QNZ (also known as EVP-4593 or CAY-10470), a quinazoline derivative, is a novel and potent NF-κB inhibitor, which shows potent inhibitory activity toward both NF-κB activation and TNF-α production with IC50 of 11 nM and 7 nM in human Jurkat T cells, respectively. It was eliminated using human Jurkat T cells in a luciferase reporter gene-based assay. EVP4593 had neuroprotective effects on GMSLNs in HD and decreased the number of lysosomes/autophagosomes and SOC currents. EVP4593 might be an effective HD medication.
Physicochemical Properties
| Molecular Formula | C22H20N4O | |
| Molecular Weight | 356.42 | |
| Exact Mass | 356.163 | |
| Elemental Analysis | C, 74.14; H, 5.66; N, 15.72; O, 4.49 | |
| CAS # | 545380-34-5 | |
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| PubChem CID | 509554 | |
| Appearance | Light green to green solid powder | |
| Density | 1.3±0.1 g/cm3 | |
| Boiling Point | 602.0±55.0 °C at 760 mmHg | |
| Melting Point | 169-175ºC | |
| Flash Point | 317.9±31.5 °C | |
| Vapour Pressure | 0.0±1.7 mmHg at 25°C | |
| Index of Refraction | 1.714 | |
| LogP | 4.57 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 5 | |
| Rotatable Bond Count | 6 | |
| Heavy Atom Count | 27 | |
| Complexity | 434 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | 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] |
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| InChi Key | IBAKVEUZKHOWNG-UHFFFAOYSA-N | |
| InChi Code | 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) | |
| Chemical Name | 4-N-[2-(4-phenoxyphenyl)ethyl]quinazoline-4,6-diamine | |
| Synonyms | 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 | |
| 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 |
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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 | TNF-α (IC50 = 7 nM); NF-κB (IC50 = 11 nM) | ||
| ln Vitro | 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]. | ||
| ln Vivo |
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| Enzyme Assay | 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. | ||
| Cell Assay | 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. | ||
| Animal Protocol |
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| References |
[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. |
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| Additional Infomation |
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] 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] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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. 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. 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. Solubility in Formulation 4: 0.5% hydroxyethyl cellulose: 30 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8057 mL | 14.0284 mL | 28.0568 mL | |
| 5 mM | 0.5611 mL | 2.8057 mL | 5.6114 mL | |
| 10 mM | 0.2806 mL | 1.4028 mL | 2.8057 mL |