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| Targets |
PU-48 specifically targets urea transporter A (UT-A), a member of the urea transporter family that is primarily expressed in the inner medullary collecting duct (IMCD) of the kidney. UT-A is responsible for urea reabsorption from the urine back into the medullary interstitium, which is critical for the urinary concentrating mechanism. PU-48 is a potent inhibitor of UT-A (IC50 = 0.32 microM). By inhibiting UT-A, PU-48 disrupts urea reabsorption, reduces medullary hypertonicity, and increases water excretion.
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| ln Vitro |
In vitro, PU-48 is a potent urea transporter A (UT-A) inhibitor with an IC50 of 0.32 microM. The compound significantly inhibits urea transport in perfused rat terminal inner medullary collecting ducts (IMCDs). PU-48 causes significant diuresis in UT-B null mice, which indicates that UT-A is the target of PU-48. The compound disrupts urea reabsorption in the kidneys, promoting increased urine output.
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| ln Vivo |
In vivo, PU-48 has a good diuretic effect in mouse models and shows little or no cytotoxicity. The diuresis caused by PU-48 did not change blood Na+, K+, or Cl- levels or nonurea solute excretion in rats and mice. The diuretic activity that PU-48 displays is mainly based on inhibiting the IMCD urea transporter UT-A isoforms, and it does not cause loss of electrolytes. PU-48 significantly increased urine output and decreased urine osmolality in an in vivo rat model without affecting electrolyte metabolism.
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| Enzyme Assay |
The specific protocol for assessing UT-A inhibition uses an in vitro urea transport assay in isolated terminal inner medullary collecting ducts (IMCDs). Male Sprague-Dawley rats are euthanized, and kidneys are removed. The terminal portion of the IMCD is isolated by microdissection and perfused in vitro. The perfused tubules are bathed in a solution containing varying concentrations of PU-48 (0.01-100 uM). Urea transport is assessed by measuring the concentration of a fluorescent urea analog (e.g., fluorescein isothiocyanate-labeled urea) or by using a microfluorescence method. The inhibition of urea transport is calculated, and the IC50 value (0.32 microM) is determined from the concentration-response curve.
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| Cell Assay |
For in vitro cellular assays, mouse inner medullary collecting duct (mIMCD-3) cells are cultured in DMEM/F12 medium in 24-well plates. Cells are treated with PU-48 at concentrations of 0.01-100 uM for 24 hours. Urea transport activity is assessed by measuring the uptake of [14C]-urea into the cells. Cells are incubated with [14C]-urea (50 uM) in the presence or absence of PU-48 for 10 minutes at 37degC. The cells are then washed three times with ice-cold PBS, lysed, and the radioactivity is measured. Cell viability is assessed using the MTT assay to ensure that the diuretic effect is not due to cytotoxicity.
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| Animal Protocol |
An in vivo protocol for PU-48 uses a mouse model of diuresis. Male C57BL/6 mice (8-10 weeks old, 20-25 g) are individually placed in metabolic cages. The mice are given free access to water and food. PU-48 is dissolved in a suitable vehicle (e.g., 10% DMSO/40% PEG400/50% saline or 0.5% methylcellulose) and administered intraperitoneally at doses of 1, 3, 10, and 30 mg/kg. Control mice receive vehicle only. Urine is collected over a 4-6 hour period, and urine volume is measured. Urine osmolality is measured using an osmometer. Serum electrolyte levels (Na+, K+, Cl-) are measured from blood collected at the end of the study. The diuretic effect is quantified as the increase in urine output compared to the vehicle control group.
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| ADME/Pharmacokinetics |
Detailed pharmacokinetic data for PU-48 is not available. As a small molecule UT-A inhibitor, it is expected to have moderate oral bioavailability and a half-life suitable for once- or twice-daily dosing. For in vivo studies, the compound is typically administered intraperitoneally to achieve systemic exposure. The diuresis caused by PU-48 does not affect blood electrolyte levels, confirming that its diuretic effect is specific to urea transport and does not cause electrolyte imbalances.
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| Toxicity/Toxicokinetics |
PU-48 shows little or no cytotoxicity in cell-based assays and exhibits no notable toxicity in animal models. The diuresis caused by PU-48 did not change blood Na+, K+, or Cl- levels or nonurea solute excretion in rats and mice, indicating that the compound is well-tolerated and does not cause electrolyte imbalances. Standard acute toxicity studies in mice would be required to determine the maximum tolerated dose (MTD) and LD50. PU-48 has the potential to be developed as a diuretic with a favorable safety profile.
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| References | |
| Additional Infomation |
PU-48 is a research-grade chemical and is not approved for clinical use. Its molecular formula is C14H12N2O5S with a molecular weight of 288.32. It is a potent urea transporter A (UT-A) inhibitor with an IC50 of 0.32 microM. PU-48 exhibits a significant diuretic effect in mouse models without notable cytotoxicity and shows little or no toxicity in animal models. Its diuretic activity is mainly based on inhibiting the IMCD urea transporter UT-A isoforms, and it does not cause loss of electrolytes. PU-48 has the potential to be developed as a novel diuretic agent for the treatment of edema, congestive heart failure, and other fluid retention disorders.
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| Molecular Formula |
C14H12N2O3S
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| Molecular Weight |
288.321681976318
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| Exact Mass |
288.056
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| CAS # |
335394-78-0
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| PubChem CID |
865472
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.6
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
20
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| Complexity |
381
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1(=C(SC2C1=CC1=CC(OC)=CC=C1N=2)C(OC)=O)N
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| InChi Key |
XRJKHUHLPVGTCL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C14H12N2O3S/c1-18-8-3-4-10-7(5-8)6-9-11(15)12(14(17)19-2)20-13(9)16-10/h3-6H,15H2,1-2H3
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| Chemical Name |
methyl 3-amino-6-methoxythieno[2,3-b]quinoline-2-carboxylate
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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: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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: 6.67 mg/mL (23.13 mM)
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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 | 3.4684 mL | 17.3418 mL | 34.6837 mL | |
| 5 mM | 0.6937 mL | 3.4684 mL | 6.9367 mL | |
| 10 mM | 0.3468 mL | 1.7342 mL | 3.4684 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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