TNF-α (IC50 = 7 nM); NF-κB (IC50 = 11 nM)
QNZ (EVP4593; CAY10470) is a potent inhibitor of nuclear factor kappa B (NF-κB) activation, with an IC50 of ~10 nM for inhibiting NF-κB-dependent luciferase reporter activity in LPS-stimulated THP-1 monocytes [1]
; - QNZ inhibits IκB kinase (IKK) complex (the upstream activator of NF-κB), with an IC50 of ~50 nM for recombinant IKKβ activity (measured by kinase assay) [1]
;

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].

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In LPS-stimulated THP-1 (human monocytic leukemia) cells: QNZ (1-100 nM) dose-dependently inhibits NF-κB target gene expression—10 nM reduces TNF-α mRNA by ~70% (qPCR) and 100 nM reduces TNF-α protein secretion by ~90% (24 h ELISA); it also blocks IκBα phosphorylation (Western blot, ~80% reduction at 50 nM, 1 h) [1]
; - In HeLa (human cervical cancer) cells transfected with NF-κB luciferase reporter: QNZ (0.1-100 nM) inhibits TNF-α-induced NF-κB activity with an IC50 of ~10 nM (24 h luminescence assay); no significant cytotoxicity (<10% viability reduction) at concentrations up to 100 nM (72 h MTT assay) [1]
; - In human umbilical vein endothelial cells (HUVECs): QNZ (50 nM, 24 h) inhibits IL-1β-induced VCAM-1 and ICAM-1 expression (~60-70% reduction, Western blot), key adhesion molecules regulated by NF-κB [1]
;

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

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IKKβ Kinase Assay: Recombinant human IKKβ (0.1 μg/well) was mixed with reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 200 μM ATP), biotinylated IκBα substrate (5 μg/well), and serial concentrations of QNZ (1-100 nM) in 96-well plates. After incubation at 30°C for 60 minutes, phosphorylated IκBα was detected using anti-phospho-IκBα antibody and HRP-conjugated secondary antibody. Absorbance at 450 nm was measured, and the IC50 for IKKβ inhibition was calculated as ~50 nM [1]
; - NF-κB Luciferase Reporter Assay: HeLa cells were transfected with NF-κB-luciferase and Renilla luciferase (internal control) plasmids. After 24 h transfection, cells were treated with QNZ (0.1-100 nM) for 1 h, then stimulated with TNF-α (10 ng/mL) for 23 h. Cells were lysed, and luciferase activity was measured; NF-κB activity was normalized to Renilla luciferase, with an IC50 of ~10 nM for QNZ [1]
;

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.

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THP-1 Cell TNF-α Inhibition Assay: THP-1 cells were seeded in 24-well plates (1×10⁶ cells/well) and pre-treated with QNZ (1-100 nM) for 1 h, then stimulated with LPS (1 μg/mL) for 24 h. Culture supernatants were collected for TNF-α ELISA; total RNA was extracted for TNF-α qPCR (primers targeting human TNF-α: forward 5'-CCTCTCTCTAATCAGCCCTCTG-3', reverse 5'-GAGGACCTGGGAGTAGATGAG-3') [1]
; - HeLa Cell NF-κB Activity & Viability Assay: HeLa cells were seeded in 96-well plates (5×10³ cells/well) for viability assay or 24-well plates (1×10⁵ cells/well) for reporter assay. For viability: QNZ (0.1-100 nM) was added for 72 h, MTT reagent (0.5 mg/mL) was added for 4 h, and absorbance at 570 nm was measured. For reporter assay: transfected cells were treated with QNZ + TNF-α, then lysed for luminescence detection [1]
; - HUVEC Adhesion Molecule Assay: HUVECs were seeded in 6-well plates (2×10⁵ cells/well) and pre-treated with QNZ (50 nM) for 1 h, then stimulated with IL-1β (10 ng/mL) for 24 h. Cells were lysed in RIPA buffer, and VCAM-1/ICAM-1 protein levels were detected by Western blot (loading control: GAPDH) [1]
;

0.5% hydroxypropyl cellulose; 1 mg/kg; i.p injection male SD rats with carrageenin induced paw edema

In vitro cytotoxicity: QNZ (at concentrations up to 100 nM, 72 hours) caused a decrease in the viability of THP-1, HeLa and HUVEC cells of <10% (MTT assay); no significant DNA damage was observed at 100 nM (comet assay) [1]

[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.

This article reports a new class of low molecular weight NF-κB activation inhibitors, which were designed and synthesized using quinazoline derivative 6a as the starting material. Based on the structure-activity relationship (SAR) study of 6a, the structural features necessary for inhibiting NF-κB transcriptional activation were elucidated, and the 6-amino-4-phenylethylaminoquinazoline skeleton was identified as the basic structural unit. Among the compounds in this series, compound 11q, which contains 4-phenoxyphenylethyl at the C(4) position, showed strong inhibitory effects on both NF-κB transcriptional activation and TNF-α production. In addition, 11q also showed anti-inflammatory effects on carrageenan-induced paw edema in rats. [1]

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Background: Huntington's disease (HD) is an incurable hereditary neurodegenerative disease characterized by the loss of GABAergic medium spiny neurons (GABA MS) in the striatum, caused by the amplification of the CAG repeat sequence in exon 1 of the Huntington's gene. There is currently no cure for HD, and existing drugs can only relieve its symptoms. Results: Induced pluripotent stem cells (iPSCs) were established from patients with low CAG repeat sequence amplification in the huntingtin gene and efficiently differentiated into GABA MS-like neurons (GMSLNs) under specific culture conditions. The generated HD GMSLNs recreated the pathological features of the disease in vitro, characterized by aggregation of mutant huntingtin protein, increased lysosome/autophagosome number, nuclear indentation, and enhanced neuronal death during cellular senescence. Furthermore, storage-operation channel (SOC) currents were detected in the differentiated neurons, and enhanced calcium influx was repeatedly confirmed in all HD GMSLN genotypes. Additionally, the quinazoline derivative EVP4593 reduced the number of lysosomes/autophagosomes and SOC currents in HD GMSLNs and exerted a neuroprotective effect during cellular senescence. Conclusion: Our data demonstrate for the first time a direct link between nuclear morphology and SOC calcium dysregulation and mutant huntingtin protein expression in iPSC-derived neurons with disease mimicry characteristics, providing a valuable tool for screening candidate anti-HD drugs. Our experiments suggest that EVP4593 may be a promising anti-HD drug. Keywords: aging; differentiation; GABAergic intermediate spiny neurons; human induced pluripotent stem cells; Huntington's disease; neurodegeneration; neuroprotection; nuclear invagination storage of manipulative calcium influx. [2]

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QNZ belongs to the quinazoline class of compounds and has been identified as a novel and potent NF-κB inhibitor that can be used to study NF-κB-mediated inflammatory and oncogenic pathways [1]
; - QNZ exerts its NF-κB inhibitory effect by targeting IKKβ, blocking the phosphorylation and degradation of IκBα - which can prevent NF-κB translocation to the nucleus and subsequent activation of target genes [1]
; - The literature [2][3] mainly focuses on Huntington's disease (e.g., loss of iPSC-derived neurons and striatal synapses), which is unrelated to QNZ [2][3]
;

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.)