| Size | Price | Stock | Qty |
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| 250mg |
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| Other Sizes |
| Targets |
In life science research, 4-POBN primarily functions as a free radical detection reagent and does not possess a conventional "drug target"; instead, it directly reacts with reactive oxygen species and carbon-centered radicals via addition reactions to form spin adducts, enabling the identification and semi-quantification of various radical sources. Meanwhile, 4-POBN (with the CAS number 5351-17-7 when used as the MPO inhibitor 4-aminobenzohydrazide, noting that CAS number confusion exists in the literature) is also a potent irreversible inhibitor of myeloperoxidase, with an IC50 of 0.3 µM against this enzyme. It irreversibly binds to the heme cofactor in the MPO active site, blocking the production of oxidative species such as hypochlorous acid, thus demonstrating therapeutic potential in inflammatory diseases including subacute stroke.
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| ln Vitro |
At the in vitro level, 4-POBN acts as a spin trapping agent. In liver microsomes incubated with a NADPH regenerating system, ethanol and 4-POBN, an ESR signal characteristic of the α-hydroxyethyl radical adduct of 4-POBN can be detected; [¹⁷O] labeling techniques can further distinguish between superoxide and hydroxyl radical adducts. At the cellular level, 4-POBN protects CYP2E1-overexpressing HepG2 cells from arachidonic acid‑induced oxidative injury, improving cell viability and reducing apoptosis and necrosis. In isolated rat hepatocytes, high concentrations of PBN moderately impair cell integrity, whereas 4-POBN exhibits no comparable cytotoxicity under identical conditions, suggesting a relatively favorable cellular compatibility profile. As an MPO inhibitor, 4-POBN (CAS 5351-17-7) acts as an irreversible inhibitor, completely blocking the peroxidase cycle activity of MPO.
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| Enzyme Assay |
In vitro enzyme activity studies with 4-POBN primarily focus on MPO inhibition. Using 4-POBN (CAS 5351-17-7) as the test compound, recombinant human myeloperoxidase (MPO) is incubated with H₂O₂ and chromogenic substrates (e.g., Amplex Red, TMB, or taurine), and MPO activity is assessed by monitoring the rate of oxidative product formation. 4-POBN is dissolved in a suitable organic solvent (e.g., DMSO) and diluted to a series of target concentrations (e.g., 0–10 µM). Different concentrations of 4-POBN are pre-incubated with MPO, the reaction is initiated by adding H₂O₂, and the reaction products are monitored dynamically using a UV‑Vis spectrophotometer or a fluorescence microplate reader to calculate the IC₅₀. Washout or dilution experiments can further verify the reversibility or irreversibility of inhibition. In classical spin‑trapping assays, typical conditions include mixing 4-POBN (e.g., 20 mM) with a peroxidase system (e.g., horseradish peroxidase + H₂O₂) and a radical source (e.g., sodium formate); the characteristic hyperfine splitting pattern of the radical adduct is then detected by ESR spectroscopy to identify the trapped species.
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| Cell Assay |
A typical cell‑based protocol for using 4‑POBN in free radical research involves the following steps:
(1) Cell seeding and culture: target cells (e.g., CYP2E1‑overexpressing HepG2 cells) are seeded into multiwell plates and cultured to approximately 70–80% confluence in complete medium; (2) Treatment: 4‑POBN is dissolved in PBS or cell culture medium and pre‑incubated with cells (commonly 0.5–20 mM, pre‑incubation for 30–60 min), followed by exposure to a radical inducer (e.g., arachidonic acid, hydrogen peroxide, or sodium formate) to mimic oxidative stress; (3) Endpoint measurements: cell viability is assessed by CCK‑8/MTT assay, while apoptosis/necrosis is evaluated by Annexin V‑FITC/PI double staining coupled with flow cytometry; intracellular total ROS levels are measured using the DCFH‑DA probe; (4) Spin‑trapping ESR analysis: cells are lysed and incubated with 4‑POBN for spin‑trapping reactions, and the types of radicals generated are identified by analyzing the ESR spectra. All experiments include vehicle‑treated controls and multiple 4‑POBN concentration groups, with at least triplicate wells for each condition to ensure statistical validity. |
| Animal Protocol |
A representative in vivo animal protocol using 4‑POBN is as follows: (1) Administration: Fischer male rats (300–400 g) or mice are acclimated in standard housing conditions, then 4‑POBN (commonly 1.5 g/kg) is administered intraperitoneally (i.p.), optionally co‑administered with a radical inducer such as sodium formate (2 g/kg i.p.) or ethanol to provoke radical generation;
(2) Sample collection: at designated time points (e.g., 1 hour post‑dose, or at 20‑min intervals), bile, blood, or target tissues (e.g., liver) are collected for subsequent ESR analysis; (3) Radical adduct detection: bile or tissue homogenates are processed and loaded into capillary tubes, and ESR spectroscopy is performed at room or low temperature to detect characteristic hyperfine splitting patterns of the radical adduct (e.g., a six‑line signal with aN ≈ 15.71 G and aβH ≈ 2.90 G); (4) For further structural identification of specific radical adducts, HPLC coupled with ESR can be employed. |
| ADME/Pharmacokinetics |
Pharmacokinetic studies of 4‑POBN have focused on its in vivo distribution. In male rats, after intraperitoneal administration of equimolar doses of 4‑POBN (165 mg/kg) and PBN (150 mg/kg), microdialysis sampling coupled with HPLC analysis showed that the steady‑state venous blood concentration of 4‑POBN was 210 ± 10 µM, whereas its steady‑state brain concentration was 149 ± 9 µM. Compared with PBN (brain concentration 331 ± 25 µM), brain penetration of 4‑POBN was significantly lower (p < 0.05), an observation attributed to the greater hydrophilicity of 4‑POBN. In addition, 4‑POBN exhibits significant biliary excretion; within one hour after intraperitoneal administration, a strong six‑line ESR signal of the 4‑POBN radical adduct can be detected in rat bile, indicating that 4‑POBN is metabolized and eliminated, at least in part, via the hepatic bile route. Once formed, 4‑POBN adducts are relatively stable under reducing conditions, allowing for detection over more extended time windows in both in vitro and in vivo settings.
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| Toxicity/Toxicokinetics |
In vitro and in vivo toxicity studies indicate that 4‑POBN has a relatively favorable safety profile. In isolated rat hepatocytes, high concentrations of PBN significantly impair hepatocyte integrity (reducing cell viability and GSH levels), whereas 4‑POBN under identical experimental conditions does not show comparable hepatocellular injury and does not interfere with intracellular ATP or cytochrome P‑450 content, suggesting that its hepatic cytotoxicity is much lower than that of PBN. In a rat model of endotoxin‑induced endotoxemia, intraperitoneal administration of 4‑POBN (0.85 M, 10 ml/kg) resulted in a 42% survival rate in mixed‑strain rats after 7 days (all vehicle‑treated animals receiving endotoxin alone died within 1 day), indicating that 4‑POBN provides protection against systemic inflammatory injury in vivo. In isolated, perfused norepinephrine‑preconstricted rat hearts, 4‑POBN showed dose‑dependent positive vasodilatory effects (an increased coronary flow of 40%), while at higher concentrations it induced a mild negative inotropic effect; within the dose range studied, it did not significantly affect heart rate (chronotropic effect). Of note, earlier studies have suggested that some nitrone spin traps may be sources of NO in biological environments, indicating that this potential effect should be considered in long‑term applications.
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| References |
| Molecular Formula |
C7H9N3O
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|---|---|
| Molecular Weight |
151.17
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| Exact Mass |
151.074
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| CAS # |
5351-17-7
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| PubChem CID |
21450
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| Appearance |
Light yellow to brown solid powder
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| Density |
1.3±0.1 g/cm3
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| Melting Point |
225-227 °C(lit.)
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| Index of Refraction |
1.641
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| LogP |
-0.75
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
11
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| Complexity |
141
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
WPBZMCGPFHZRHJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C7H9N3O/c8-6-3-1-5(2-4-6)7(11)10-9/h1-4H,8-9H2,(H,10,11)
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| Chemical Name |
4-Aminobenzoic acid hydrazide
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| Synonyms |
4 ABAH 4-POBN 4POBN 4 POBN4-ABAH NSC-6404-AminobenzohydrazideMyeloperoxidase Inhibitor 1 NSC640 NSC 640 4ABAH
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 : ≥ 25 mg/mL (~165.38 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1 mg/mL (6.62 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (6.62 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 10.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 6.6151 mL | 33.0753 mL | 66.1507 mL | |
| 5 mM | 1.3230 mL | 6.6151 mL | 13.2301 mL | |
| 10 mM | 0.6615 mL | 3.3075 mL | 6.6151 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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