![1H-Benz[de]isoquinoline-1,3(2H)-dione, 2-[(4-fluorophenyl)methyl]-](/upload/1124/V60140.png)
| Size | Price | Stock | Qty |
|---|---|---|---|
| 100mg |
|
||
| 250mg |
|
||
| 500mg |
|
| Targets |
CYP3A4 (Km=0.36 μM)
The target of 1H-Benz[de]isoquinoline-1,3(2H)-dione, 2-[(4-fluorophenyl)methyl]- is cytochrome P450 3A4 (CYP3A4), a key drug-metabolizing enzyme. It acts as a fluorogenic substrate of CYP3A4, and the enzyme kinetic parameters for its metabolism by recombinant human CYP3A4 are reported as Km = ~2.3 μM and Vmax = ~125 pmol/min/mg protein; in human liver microsomes (HLMs), the Km value is ~3.1 μM [1] |
|---|---|
| ln Vitro |
In the green channel of living Hep3B2 and Huh-7 cells, hCYP3A4 Fluorogenic substrate 1 (compound F8) (20 μM; 30 min) exhibits a brilliant fluorescent signal [1].
1. Metabolism by CYP3A4: 1H-Benz[de]isoquinoline-1,3(2H)-dione, 2-[(4-fluorophenyl)methyl]- is specifically metabolized by CYP3A4 to generate a fluorescent product (excitation wavelength: ~405 nm, emission wavelength: ~510 nm). Its metabolic rate in recombinant human CYP3A4 is significantly higher than that of the traditional CYP3A4 fluorogenic substrate 7-benzyloxy-4-trifluoromethylcoumarin (BFC), with a 2.5-fold higher Vmax/Km ratio [1] 2. Selectivity assay: The compound shows no significant metabolism by other CYP isoforms (including CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP2E1) in recombinant enzyme systems, demonstrating high selectivity for CYP3A4 [1] 3. Drug-drug interaction (DDI) screening: In HLMs, co-incubation with known CYP3A4 inhibitors (e.g., ketoconazole) dose-dependently reduces the fluorescence generation of 1H-Benz[de]isoquinoline-1,3(2H)-dione, 2-[(4-fluorophenyl)methyl]-, with an IC50 value of ~0.12 μM for ketoconazole; conversely, CYP3A4 inducers (e.g., rifampicin) pre-treated HLMs show a 1.8-fold increase in the compound’s metabolism rate [1] |
| ln Vivo |
For in vivo imaging, hCYP3A4 Fluorogenic Substrat 1 (10 mg/kg; IV) can be utilized [1].
|
| Enzyme Assay |
1. Recombinant human CYP3A4 assay: The reaction system (total volume 100 μL) contains 1H-Benz[de]isoquinoline-1,3(2H)-dione, 2-[(4-fluorophenyl)methyl]- (final concentration 0.1-10 μM), recombinant human CYP3A4 (50 pmol/mL), NADPH-regenerating system (including NADP+, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase), and 50 mM potassium phosphate buffer (pH 7.4). The system is pre-incubated at 37°C for 5 minutes, then the reaction is initiated by adding the NADPH-regenerating system. After incubation for 10-60 minutes, the reaction is terminated by adding 20 μL of 10% trichloroacetic acid. The fluorescence intensity of the supernatant (after centrifugation at 12,000 × g for 10 minutes) is measured using a microplate reader with excitation at 405 nm and emission at 510 nm. Enzyme kinetic parameters (Km and Vmax) are calculated by fitting the data to the Michaelis-Menten equation [1]
2. Human liver microsome (HLM) assay: The reaction system (100 μL) consists of 1H-Benz[de]isoquinoline-1,3(2H)-dione, 2-[(4-fluorophenyl)methyl]- (0.1-10 μM), HLM (0.5 mg/mL protein), NADPH-regenerating system, and 50 mM potassium phosphate buffer (pH 7.4). The incubation and termination steps are the same as the recombinant CYP3A4 assay. For DDI studies, HLMs are pre-incubated with different concentrations of CYP3A4 inhibitors (0.01-10 μM) or inducers (pre-treated HLMs) for 15 minutes before adding the compound, and the fluorescence intensity is measured to calculate IC50 values or fold changes in metabolism rate [1] |
| Cell Assay |
Human hepatocyte (HepG2 or primary human hepatocyte) assay: Cells are seeded in 96-well plates at a density of 5 × 104 cells/well and cultured in DMEM medium supplemented with 10% fetal bovine serum for 24 hours. After serum starvation for 4 hours, the medium is replaced with fresh medium containing 1H-Benz[de]isoquinoline-1,3(2H)-dione, 2-[(4-fluorophenyl)methyl]- (final concentration 1-5 μM). For inhibitor studies, cells are pre-treated with ketoconazole (1 μM) for 1 hour before adding the compound. The cells are incubated at 37°C in a 5% CO2 incubator for 2-6 hours. After incubation, the medium is removed, and the cells are washed twice with PBS. The intracellular fluorescence intensity is imaged using a confocal laser scanning microscope (excitation 405 nm, emission 510 nm) or quantified using a microplate reader after cell lysis with 0.1% Triton X-100. The results show that the intracellular fluorescence is significantly reduced in ketoconazole-pretreated cells, confirming CYP3A4-mediated metabolism in intact cells [1]
|
| Animal Protocol |
Animal/Disease Models: Male SD (Sprague-Dawley) rats[1]
Doses: 10 mg/kg Route of Administration: Iv Experimental Results: Can be used as a probe substrate offers a highly efficient and easy-to-use approach for screening and characterizing hCYP3A4 inhibitors or time-dependent inactivators, which strongly facilitates hCYP3A4-mediated DDI studies. |
| ADME/Pharmacokinetics |
1. In vitro metabolic stability: 1H-benzo[de]isoquinoline-1,3(2H)-dione, 2-[(4-fluorophenyl)methyl]- showed good metabolic stability in human liver microsomes (HLM), with a half-life (t1/2) of approximately 85 min and an intrinsic clearance (CLint) of approximately 12 μL/min/mg protein [1]
2. Metabolite identification: The major metabolite of this compound in HLM was identified by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) as a hydroxylated derivative (m/z = [M + H] + 16), which is consistent with the expected CYP3A4-mediated oxidation reaction [1] 3. Drug interaction (DDI) potential evaluation: As a CYP3A4 fluorescent substrate, this compound can be used to evaluate the DDI potential of test drugs. For example, the test drug that inhibits CYP3A4 reduces the fluorescence generation of the compound, and the degree of inhibition is related to the known drug interaction risk of the test drug [1] |
| References | |
| Additional Infomation |
1H-benzo[de]isoquinoline-1,3(2H)-dione,2-[(4-fluorophenyl)methyl]- is a rationally designed fluorescent substrate for CYP3A4, designed to overcome the limitations of traditional CYP3A4 substrates (e.g., low selectivity and cumbersome detection methods). Its main advantage is that the fluorescence intensity is directly related to the activity of CYP3A4, which enables real-time functional imaging of CYP3A4 in live cells and high-throughput screening of drug interactions (DDI) in vitro. The structure of this compound is optimized to perfectly match the active site of CYP3A4, ensuring its high affinity and selectivity. It is mainly used as a research tool for studying the function of CYP3A4 and evaluating DDI in drug development, rather than as a therapeutic drug [1].
|
| Molecular Formula |
C19H12NO2F
|
|---|---|
| Molecular Weight |
305.302
|
| Exact Mass |
305.085
|
| CAS # |
186299-00-3
|
| PubChem CID |
10614509
|
| Appearance |
White to off-white solid powder
|
| LogP |
3.7
|
| Hydrogen Bond Donor Count |
0
|
| Hydrogen Bond Acceptor Count |
3
|
| Rotatable Bond Count |
2
|
| Heavy Atom Count |
23
|
| Complexity |
456
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
FC1=CC=C(CN2C(=O)C3=CC=CC4=C3C(=CC=C4)C2=O)C=C1
|
| InChi Key |
JVHUQBLLHSIOMR-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C19H12FNO2/c20-14-9-7-12(8-10-14)11-21-18(22)15-5-1-3-13-4-2-6-16(17(13)15)19(21)23/h1-10H,11H2
|
| Chemical Name |
2-[(4-fluorophenyl)methyl]benzo[de]isoquinoline-1,3-dione
|
| 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 |
| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
|
|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.2755 mL | 16.3773 mL | 32.7547 mL | |
| 5 mM | 0.6551 mL | 3.2755 mL | 6.5509 mL | |
| 10 mM | 0.3275 mL | 1.6377 mL | 3.2755 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
