| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
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| 10mg |
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| 50mg |
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| Other Sizes |
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| Targets |
IC50: 27 nM (BRD4 BD1), 32 nM (BRD4 BD2)[1]
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| ln Vitro |
ZL0420 forms crucial interactions with Asn140 directly and Tyr97 indirectly through an H2O molecule when it is properly docked into the acetyl-lysine (KAc) binding pocket of BRD4. In cultured human small airway epithelial cells (hSAECs), ZL0420 demonstrates submicromolar potency of suppressing the TLR3-dependent innate immune gene program, including ISG54, ISG56, IL-8, and Groβ genes, with IC50s of 0.49-0.86 µM[1].
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| ln Vivo |
ZL0420 has a strong, low-toxicity effect on airway inflammation in a mouse model. ZL0420 exhibits remarkable efficacy and almost eliminates the deep-seated neutrophil build-up surrounding small and medium-sized airways that is brought on by the administration of poly(I:C)[1].
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| Enzyme Assay |
Time-resolved fluorescence energy transfer (TR-FRET) assays[1]
384 well plate-based commercial TR-FRET Assay kits were used to determine the binding ability of tested BRD4 inhibitors to the BRD4 and BRD2 bromodomains (BD) using the two recombinant BRD4 BDs or BRD2 BDs by time-resolved fluorescence energy transfer (TR-FRET) assays. A series of concentrations of BRD4 inhibitors from 0.01 nM to 100 μM were added into a 384 well test plate and mixed with other reaction components based on the instructions from vendor followed by incubation 1h at room temperature. The commercially available BRD inhibitors JQ1 and RVX208 were used as the controls. The plates were read in time-resolved format by exciting the sample at 340 nm and reading emissions at 620 and 670 nm, using a 100 μs delay and a 500 μs window at a Tecan M1000 pro reader. A plot of the TR-FRET ratio (670 nm emission/620 nm emission versus inhibitor concentration on semi-log axes results in a sigmoidal dose-response curve typical of competitive assays. These data were further calculated out with the IC50 values of tested BRD4 inhibitors to the bromodomains of BRD2 and BRD4 as well as other relevant target proteins, respectively.
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| Cell Assay |
Cell culture[1]
Immortalized human small airway epithelial cells (hSAECs) were previously described. hSAECs were grown in SAGM small airway epithelial cell growth medium in a humidified atmosphere of 5% CO2. Poly(I:C) was used at 10 μg/mL in cell culture. Compounds were solubilized in DMSO and added at the indicated concentrations. Quantitative Real-Time PCR (Q-RT-PCR)[1] For gene expression analyses, 1 μg of RNA was reverse transcribed using Super Script III as previously described. One μL of cDNA product was amplified using SYBR Green Supermix and indicated gene-specific primers. The reaction mixtures were subjected to 40 cycles of 15 s at 94 °C, 60 s at 60 °C, and 1 min at 72 °C in an iCycler. Quantification of relative changes in gene expression was calculated using the ΔΔCt method and expression as the fold change between experimental and control samples was normalized to internal control cyclophilin (PPIA). In vitro efficacy of BRD4 inhibitors on poly(I:C) induced innate immune response[1] hSAECs were first pretreated with a series final concentrations of BRD4 inhibitors from 0.01 nM to 100 μM for 24 hours and were then added poly(I:C) at 10 μg/mL for another 4 hours prior to harvesting the cells. The harvested cells were first washed with PBS twice and then the total RNA was extracted using acid guanidinium phenol extraction (Tri Reagent). The total RNA was further reverse-transcribed for gene expression analysis by Q-RT-PCR. The inhibitory effect of BRD4 inhibitors on poly(I:C)-induced innate immune gene expression was compared with that of poly(I:C) alone and inhibitory percentage of each treatment was obtained. For compounds 23, 28 and 35, in vitro efficacy of these BRD4 inhibitors on poly(I:C) induced innate immune response were presented as the IC50 values of these compounds. Compounds were dissolved in DMSO and further diluted at cell culture medium to appropriate concentrations. |
| Animal Protocol |
In vivo efficacy of BRD4 inhibitors on poly(I:C)-induced acute airway inflammation[1]
Animal experiments were performed according to the NIH Guide for Care and Use of Experimental Animals and approved by the University of Texas Medical Branch (UTMB) Animal Care and Use Committee (approval no. 1312058A). Male C57BL6/J mice (12 weeks old) were purchased from The Jackson Laboratory and housed under pathogen-free conditions with food and water ad libitum. C57BL/6 mice were pre-treated in the absence or presence of the indicated BRD4 inhibitors [10 mg/kg body weight, via the intraperitoneal route] one day prior to poly(I:C) stimulation. The next day, animals were given another dose of BRD4 inhibitor immediately followed by intranasal (i.n.) administration of phosphate-buffered saline (PBS, 50 μL) or poly(I:C) (300 μg dissolved in 50 μL PBS). One day later, the mice were euthanized. The bronchoalveolar lavage fluid (BALF) and lung tissues of treated mice were collected for further analysis. Compounds were first dissolved in DMSO and further diluted in 10% hydroxypropyl β-cyclodextrin in PBS to appropriate concentration prior to intraperitoneal administration. Evaluation of airway inflammation[1] Cellular recruitment into the airway lumen was assessed in the bronchoalveolar lavage fluid (BALF). Lungs were perfused twice with 1 mL of sterile PBS (pH 7.4) to obtain the BALF. Total cell counts were determined by trypan blue staining 50 μL of BALF and counting viable cells using a hemocytometer. Differential cell counts were performed on cytocentrifuge preparations stained with Wright-Giemsa. A total of 300 cells were counted per sample using light microscopy. Formalin-fixed lungs were embedded in paraffin, sectioned at a 4 μm thickness, and stained with hematoxylin and eosin or Masson’s trichrome. Microscopy was performed on a NIKON Eclipse Ti System. |
| References | |
| Additional Infomation |
A series of diverse small molecules have been designed and synthesized through structure-based drug design by taking advantage of fragment merging and elaboration approaches. Compounds ZL0420 (28) and ZL0454 (35) were identified as potent and selective BRD4 inhibitors with nanomolar binding affinities to bromodomains (BDs) of BRD4. Both of them can be well docked into the acetyl-lysine (KAc) binding pocket of BRD4, forming key interactions including the critical hydrogen bonds with Asn140 directly and Tyr97 indirectly via a H2O molecule. Both compounds 28 and 35 exhibited submicromolar potency of inhibiting the TLR3-dependent innate immune gene program, including ISG54, ISG56, IL-8, and Groβ genes in cultured human small airway epithelial cells (hSAECs). More importantly, they also demonstrated potent efficacy reducing airway inflammation in a mouse model with low toxicity, indicating a proof of concept that BRD4 inhibitors may offer the therapeutic potential to block the viral-induced airway inflammation.[1]
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| Molecular Formula |
C16H16N4O2
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|---|---|
| Molecular Weight |
296.32
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| Exact Mass |
296.127
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| Elemental Analysis |
C, 64.85; H, 5.44; N, 18.91; O, 10.80
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| CAS # |
2230496-80-5
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| Related CAS # |
(E/Z)-ZL0420;2229039-45-4
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| PubChem CID |
137285011
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| Appearance |
Typically exists as Yellow to orange solids at room temperature
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| LogP |
2.2
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
22
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| Complexity |
442
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
ANMQADUROYWADA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H16N4O2/c1-9-6-14(12(17)8-15(9)21)20-19-11-3-4-13-10(7-11)2-5-16(22)18-13/h3-4,6-8,21H,2,5,17H2,1H3,(H,18,22)
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| Chemical Name |
6-[(2-amino-4-hydroxy-5-methylphenyl)diazenyl]-3,4-dihydro-1H-quinolin-2-one
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| Synonyms |
6-[(2-amino-4-hydroxy-5-methylphenyl)diazenyl]-3,4-dihydro-1H-quinolin-2-one; 2230496-80-5; CHEMBL4176038; (E/Z)-ZL0420; 6-((2-Amino-4-hydroxy-5-methylphenyl)diazenyl)-3,4-dihydroquinolin-2(1H)-one; 6-[(2-Amino-4-hydroxy-5-methylphenyl)diazenyl]-3,4-dihydroquinolin-2(1H)-one;
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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: 125 mg/mL (421.84 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (7.02 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 20.8 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: ≥ 2.08 mg/mL (7.02 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 20.8 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.3747 mL | 16.8737 mL | 33.7473 mL | |
| 5 mM | 0.6749 mL | 3.3747 mL | 6.7495 mL | |
| 10 mM | 0.3375 mL | 1.6874 mL | 3.3747 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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