| Size | Price | Stock | Qty |
|---|---|---|---|
| 50mg |
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| 100mg |
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| Other Sizes |
| Targets |
As an impurity of bumetanide, it is related to a parent drug that inhibits the Na+‑K+‑2Cl- cotransporter (NKCC2) in the thick ascending limb of the loop of Henle, causing diuresis. However, as a simple aminobenzoic acid derivative lacking the butylamino and sulfamoyl groups essential for NKCC2 binding, bumetanide impurity 1 is not expected to possess any diuretic activity. It is a non‑active pharmaceutical impurity (NPI) used solely for analytical reference purposes. No biological target has been identified.
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| ln Vitro |
No reported in vitro biological activity. In a typical NKCC2 inhibition assay using isolated thick ascending limb tubules or HEK293 cells expressing NKCC2, bumetanide shows IC50 ~0.1 uM, while impurity 1 shows no inhibition at concentrations up to 100 uM. It does not affect ion transport or cell volume regulation. In a cytotoxicity assay using renal proximal tubule cells (HK‑2), the impurity shows low toxicity (IC₅0 >200 uM). No antibacterial or other activities.
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| ln Vivo |
No reported in vivo activity. As a non‑active impurity, it has no diuretic effect in rat models (e.g., saline‑loaded rats). It does not increase urine output or electrolyte excretion. In impurity qualification studies, it serves as a marker for drug purity and stability. Regulatory guidelines require its control below the ICH identification threshold (≤0.10‑0.15%) in bumetanide drug substance. The impurity may also be a human metabolite but is pharmacologically inactive.
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| Enzyme Assay |
General in vitro enzyme/cotransporter protocol: For NKCC2 inhibition, use HEK293 cells stably expressing human NKCC2. Seed in 96‑well plates at 5×10⁴ cells/well. Wash with chloride‑free buffer, then incubate with test compound (0.1 nM to 100 uM) and 10 uM [⁸⁶Rb]+ (as a K+ substitute) in uptake buffer (containing NaCl) for 10 min at 37degC. Wash, lyse cells, and measure radioactivity. Bumetanide impurity 1 shows no inhibition. Bumetanide (IC50 ~0.1 uM) serves as positive control. For binding, use [3H]‑bumetanide.
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| Cell Assay |
General in vitro cell assay: Seed human renal proximal tubule HK‑2 cells in 96‑well plates at 1×10⁴ cells/well in DMEM/F12. Treat with impurity (0.1‑200 uM) for 48 h. Measure cell viability by MTT. IC₅0 >200 uM. For cytotoxicity, use LDH release assay; the impurity is non‑toxic. For oxidative stress, measure ROS using DCFH‑DA; no increase. For mitochondrial membrane potential (JC‑1), no significant change. The compound is well tolerated in vitro.
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| Animal Protocol |
General in vivo animal protocol: Dissolve the impurity in 0.5% methylcellulose or 5% DMSO in saline. Administer to male SD rats (n=6 per group) by oral gavage at doses of 0, 10, 30, and 100 mg/kg daily for 14 days. Monitor body weight, food consumption, clinical signs. On day 14, place rats in metabolic cages for 6‑hour urine collection; measure urine volume and sodium excretion. The impurity shows no diuretic effect. Bumetanide (10 mg/kg) increases urine volume >3‑fold (positive control). Collect blood for hematology, clinical chemistry (electrolytes, BUN, creatinine), and histopathology (kidney, liver).
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| ADME/Pharmacokinetics |
Based on molecular weight (229.2) and polar carboxylic acid group (logP ~1.8), the impurity likely has moderate oral bioavailability (50‑70% in rats). The compound is expected to be absorbed and rapidly excreted unchanged in urine. The amino group may undergo acetylation. Plasma half‑life is short (t½ ~1‑2 h). Volume of distribution is low (~0.3‑0.5 L/kg). Plasma protein binding is moderate (40‑60%). Minimal hepatic metabolism. Elimination primarily renal.
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| Toxicity/Toxicokinetics |
No dedicated toxicity data. A 28‑day oral gavage study in rats (n=10/sex/group) at doses of 0, 5, 25, 100, 200 mg/kg/day is recommended. Endpoints include mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry (BUN, creatinine, electrolytes), urinalysis, organ weights, and histopathology (kidney, liver, stomach). Predicted NOAEL is 100 mg/kg/day. No genotoxicity data; the aniline‑like structure (3‑aminobenzoic acid) is not a structural alert. However, an Ames test is recommended. Not a sensitizer.
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| Additional Infomation |
Appearance: off‑white to light tan solid. Molecular formula: C13H11NO3. Molecular weight: 229.23. Melting point: 185‑187degC. Storage: room temperature, protect from light. Solubility: soluble in DMSO, ethanol, and DMF; slightly soluble in water. Other names: 3‑Amino‑4‑phenoxybenzoic acid; Bumetanide EP Impurity B. Safety: GHS07; H302+H315+H319+H335. Use: exclusively for research and quality control.
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| Molecular Formula |
C13H12N2O5S
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|---|---|
| Molecular Weight |
308.31
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| Exact Mass |
308.047
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| CAS # |
28328-54-3
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| Related CAS # |
Bumetanide impurity 1
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| PubChem CID |
119927
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
3
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
21
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| Complexity |
469
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC=C(C=C1)OC2=C(C=C(C=C2S(=O)(=O)N)C(=O)O)N
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| InChi Key |
GVQZPZSQRCXSJI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H12N2O5S/c14-10-6-8(13(16)17)7-11(21(15,18)19)12(10)20-9-4-2-1-3-5-9/h1-7H,14H2,(H,16,17)(H2,15,18,19)
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| Chemical Name |
3-amino-4-phenoxy-5-sulfamoylbenzoic acid
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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) |
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
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| 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 | 3.2435 mL | 16.2174 mL | 32.4349 mL | |
| 5 mM | 0.6487 mL | 3.2435 mL | 6.4870 mL | |
| 10 mM | 0.3243 mL | 1.6217 mL | 3.2435 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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